ycT.dtf
JOURNAL OF
AGRICULTURAL
RESEARCH
Volume XX
OCTOBER i, 1920— MARCH 15, 1921
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE
WITH THE COOPERATION OF THE ASSOCIATION
OF LAND-GRANT COLLEGES
WASHINGTON. D. C.
XJ
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and A ssistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G: Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
f
At,
CONTENTS
Page
Fusarium-Blight (Scab) of Wheat and Other Cereals. Dimitr
Atanasoff I
Cause of Lime-Induced Chlorosis and Availability of Iron in the
Soil. P. L. Gile and J. 0. Carrero 33
An Experimental Study of Echinacea Therapy. James F.
Couch and Leigh T. Giltner 63
Investigations of the Germicidal Value of Some of the Chlorin
Disinfectants. F. W. Tilley 85
A New Avocado Weevil from the Canal Zone. H. F. Dietz and
H. S. Barber 111
Studies in Mustard Seeds and Substitutes: I. Chinese Colza
(Brassica campestris chinoleifera Viehoever). Arno ViE-
hoever, Joseph F. Clevenger, and Clare Own Ewing .... 117
Study of Some Poultry Feed Mixtures with Reference to Their
Potential Acidity and Their Potential Alkalinity. B. F.
Kaupp and J. E. IvEy 141
The Influence of Cold in Stimulating the Growth of Plants.
Frederick V. Coville 151
Composition of Normal and Mottled Citrus Leaves. W. P.
Kelley and A. B. Cummins 161
Control of Fluke Diseases by Destruction of the Intermediate
Host. Asa C. Chandler 193
Injury to Seed Wheat Resulting from Drying after Disinfection
with Formaldehyde. Annie May Hurd 209
Studies on the Life History and Habits of the Beet Leafhopper.
C. F. Stahl 245
Hypertrophied Lenticels on the Roots of Conifers and Then-
Relation to Moisture and Aeration. Glenn G. Hahn, Carl
Hartley, and Arthur S. Rhoads 253
Degree of Temperature to Which Soils Can Be Cooled without
Freezing. George Bouyoucos 267
Changes Taking Place in the Tempering of Wheat. E. L. Tague . 271
Vascular Discoloration of Irish Potato Tubers. H. A. Edson.. 277
Crownwart of Alfalfa Caused by Urophlyctis alfalfae. Fred
N Ruel Jones and Charles Drechsler 295
- Pathological Anatomy of Potato Blackleg. Ernst F. Art-
0 scitwager 325
(HI)
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IV Journal of Agricultural Research voi.xx
Sclerotinia minor, n. sp., the Cause of a Decay of Lettuce, Celery, page
and Other Crops. Ivan C. Jagger 331
Permanence of Differences in the Plots of an Experimental Field.
J. Arthur Harris and C. S. Scofield 335
Some Changes in Florida Grapefruit in Storage. Lon A. Haw-
kins and J. R. Magness 357
A Bacteriological Study of Canned Ripe Olives. Stewart A.
Koser 375
Relation of the Soil Solution to the Soil Extract. D. R. Hoag-
land, J. C. Martin, and G. R. Stewart 381
Effect of Season and Crop Growth on the Physical State of the
Soil. D. R. Hoageand and J. C. Martin 397
Carbon-Dioxid Content of Barn Air. Mary F. Hendry and Alice
Johnson 405
Rice Weevil, (Calandra) Sitophilus oryza. Richard T. Cotton. 409
Opius fletcheri as a Parasite of the Melon Fly in Hawaii. H. F.
Willard 423
Tamarind Pod-Borer, Sitophilus linearis (Herbst). Richard T.
Cotton 439
Influence of Temperature and Humidity on the Growth of
Pseudomonas citri and Its Host Plants and on Infection and
Development of the Disease. George L. Peltier 447
Daubentonia longifolia (Coffee Bean), A Poisonous Plant.
C. Dwight Marsh and A. B. Clawson 507
Fusarium-Wilt of Tobacco. James Johnson 515
Sugar Beet Top Silage. Ray E. Neidig 537
Nodule Bacteria of Leguminous Plants. F. Lohnis and Roy
Hansen 543
Correlation and Causation. Sewall Wright 557
Measurement of the Amount of Water That Seeds Cause to Become
Unfree and Their Water-Soluble Material. George J. Bouyou-
cos and M. M. McCool 587
Inheritance of Syndactylism, Black, and Dilution in Swine. J. A.
Detlefsen and W. J. Carmichael 595
Four Rhynchophora Attacking Com in Storage. Richard T.
Cotton 605
Concentration of Potassium in Orthoclase Solutions Not a Measure
of Its Availability to Wheat Seedlings. J. F. BreazEalE and
Lyman J. Briggs 615
Composition of Tubers, Skins, and Sprouts of Three Varieties of
Potatoes. F. C. Cook 623
Further Studies in the Deterioration of Sugars in Storage. Nicho-
las Kopeloff, H. Z. E. Perkins, and C. J. Welcome 637
Freezing of Fruit Buds. Frank L. West and N. E. Edlefsen . . 655
Oct. i, 1920-Mar. 15, 1921 Contents
Effect of Various Crops Upon the Water Extract of a Typical Silty page
Clay Loam Soil. G. R. Stewart and J. C. Martin 663
Another Conidial Sclerospora of Philippine Maize. William H.
Weston, Jr 669
Onion Smudge. J. C. Walker 685
Variations in Colletotrichum gloeosporioides. O. F. Burger 723
A Transmissible Mosaic Disease of Lettuce. Ivan C. Jagger. . 737
Leconte's Sawfly, an Enemy of Young Pines. William Middle-
ton 741
Amylase of Rhizopus tritici, with a Consideration of Its Secretion
and Action. L. L. Harter 761
A Comparative Study of the Composition of the Sunflower and
Corn Plants at Different Stages of Growth. R. H. Shaw and
P. A. Wright 787
Evaluation of Climatic Temperature Efficiency for the Ripening
Processes in Sweetcorn. Charles O. Appleman and S.V. Eaton 795
Some Lepidoptera Likely to Be Confused with the Pink Bollworm.
Carl Heinrich 807
Biology of the Smartweed Borer, Pyrausta ainsliei Heinrich.
George G. Ainslie and W. B. Cartwright 837
Effect of X-Rays on Trichinae. Benjamin Schwartz 845
Relation of the Calcium Content of Some Kansas Soils to the
Soil Reaction as Determined by the Electrometric Titration.
C. O. Sw anson, W. L. Latshaw, and E. L. Tague 855
Green Feed versus Antiseptics as a Preventive of Intestinal Dis-
orders of Growing Chicks. A. G. Philips, R. H. Carr, and
D. C. Kennard 869
Comparative Utilization of the Mineral Constituents in the Coty-
ledons of Bean Seedlings Grown in Soil and in Distilled Water.
G. Davis Buckner 875
Sunflower Silage Digestion Experiment with Cattle and Sheep.
Ray E. Neidig, C. W. Hickman, and Robert S. Snyder 881
Index 88q
ERRATA AND AUTHORS' EMENDATIONS
Page 9, line 23, "spikelet" should read "group."
Page 16, line 36, "Triticum" should read " Agropyron."
Page 68, after line 22 insert "picture."
Page 84, last line, omit " Not seen. "
Page 124, line 23, omit " is. "
Page 140, legend of Plate 15, omit " Late rosette stage of Chinese colza seedling. "
Pages 166, 168, 170, 175, 178, 180, and 181, Tables II, IV, VI, IX, XI, XIII, and XV, after column heading
"ash" insert "expressed as percentages of dry matter."
Pages 183 and 184, Tables XVI, XVII, and XVTII, column 2, omit "per cent. "
Page 193, line 16, "epidemic" should read "endemic."
Page 200, line 23, "0.005" should read "0.0005. "
Page 236, footnote to Table XII, after "42 days" insert "and 60 days. "
Page 246, line 23, after "apical portion " insert "of claval region. "
Page 413, Table I, cloumn 10, footnote reference "e" should read "I. "
Page 414, Table I, column 6, footnote reference "o " should be transposed to column 7.
Page 422, citation 4, omit "In press" and insert "no. 9, p. 235-243. "
Page 452, Table II, line i, footnote reference "a" should be inserted before all entries.
Page 479, line 32, "organism" should read "organisms. "
Page 481, "260" should read "250. "
Page 491, " Table XVII " should read " Table XVIII. "
Page 508, Table I, column 3, lines 13 to 22, ".283" should read ".028."
Page 607, line 13, "molas" should read "molar."
Page 614, Plate 72, figure E, and Plate 74, figure E, "ae" should read "al."
Page 810, line 25, " Kostelezkya" should read " Kosteletzkya. "
Page 815, line 32, "divini" should read "diveni. "
Page 816, line 18, "hessitans" should read "haesitans. "
Page 822, line 38, " Kosteleyzkya " should read "Kosteletzkya."
Page 828, line 32, " Kostelelzkya" should read " Kosteletzkya. "
(VI)
ILLUSTRATIONS
Fusarium-Blight (Scab) of Wheat and Other Cereals
Text Figures
Page
i. Conidia of Gibberella saubinetii 16
2. Special culture tube for maintaining moisture in culture 18
Plates
i. Gibberella saubinetii: Blighted ("scabbed ") wheat heads 32
2. Gibberella saubinetii: A. — Footrot of wheat caused by Fusarium. B. —
Seedling-blight of wheat caused by G. saubinetii 32
3. A. — Fusarium seedling-blight. B. — Tissue invaded by G. saubinetii in
causing the headblight of wheat 32
4. Gibberella saubinetii: A. — Kernels blighted and shriveled by Fusarium-
blight. B. — Perithecia development of G. saubinetii on an infected
wheat head 32
Cause op Lime-Induced Chlorosis and Availability of Iron in the Soil
Plates
5. A. — Rice grown in calcareous and noncalcareous soils and sprayed with
ferrous sulphate solution (experiment I). B. — Apparatus used in grow-
ing plants in experiment VII 62
0> a. — Effect of carbonate of lime in depressing the availability of iron (experi-
ment VII). B. — Effect of various substances on growth of rice in cal-
careous soil (experiment VIII) 62
A New Avocado Weevil from the Canal Zone
Plates
7. Heilipus perseae: A, B.— Adult, paratype. C. — An avocado fruit (re-
duced) showing feeding injury by the beetles 116
8. Heilipus perseae: Leaves showing the injury done by five beetles in 48
hours "6
9. Heilipus perseae, mature larva: A. — Ventral face of ventral mouthparts.
B. — Anterior part of head from above. C. — Lingua, hypopharynx,
hypopharyngeal bracon, and dorsal (buccal) face of maxilla. D. — Dor-
sal face of mandible. E. — Epipharynx. F. — Ventral face of mandible.
G.— Head capsule from above. H. — Thoracic spiracle from outside.
I. — Mature larva "6
Studies in Mustard Seeds and Substitutes: I. Chinese Colza (Bras-
SICA CAMPESTRIS CHINOLED7ERA VlEHOEVER)
Plates
IOi a. — Yellow seed of Chinese colza. B. — Brown seed of Chinese colza.
C. — Surface section of yellow seed of Chinese colza, showing lack of retic-
ulations. D. — Surface section of brown seed of Chinese colza, showing
reticulations. E. — Cross section of yellow seed of Chinese colza.
F. — Cross section of brown seed of Chinese colza 140
(VH)
vni Journal of Agricultural Research voi.xx
Page
ii. Seedling of Chinese colza, showing cotyledons and young leaves 140
12. Early rosette stage of Chinese colza seedling: A. — Plants from (1) brown
seed and (2) yellow seeds. B. — Usual form, showing almost entire
leaves 140
13. Early rosette stage of Chinese colza seedling: A. — Plant showing a variation
in lobing of the leaves. Two months old. B. — Plant showing a varia-
tion in lobing of the leaves. Three months old 140
14. Eate rosette stage of Chinese colza seedling: A. — Usual form. B. — Plant
showing a variation in lobing of the leaves 140
15. A. — Pe-tsai. B. — Cross between Pak-choi and Pe-tsai. C. — Pak-choi.... 140
16. Early flowering stage of Chinese colza: A. — Usual form, showing somewhat
enlarged stem base and stem-clasping leaves. B. — Plant without en-
larged stem base. C. — Usual form, showing glaucous leaves 140
17. Early flowering stage of Chinese colza: A. — Usual form, showing luxuriant
growth and long pedicels. B. — Flower cluster 140
18. A. — Fruiting stage of Chinese colza. B. — Mature fruit of Chinese colza .. . 140
19. A. — Herbarium specimen of Brassica chinensis L. B. — Herbarium speci-
men of Brassica campestris 140
The Influence op Cold in Stimulating the Growth op Plants
Plates
20. A. — Blueberry plants, V actinium corymbosum, made dormant without
cold. B. — Chilled and unchilled blueberry plants 160
21. A. — Chilled and unchilled plants of grouseberry, Viburnum americanum.
B. — Chilled and unchilled plants of tamarack, Larix laricina 160
22. A. — Chilled and unchilled plants of wild crab, Malus coronaria. B. —
Blueberry plant with one branch stimulated to growth by cold 160
23. Blueberry plant with one branch kept dormant by heat. A. — Dormant
indoor blueberry plant as it appeared on February 15, 1912. B. — Same
plant photographed May 21 160
24. A. — Blueberry cuttings starting to grow at 360 F. B. — Blueberry plant
growing in the dark at 360 F 160
25. A. — Dormant wild crab stimulated to growth by pruning. B. — Dormant
wild crabs stimulated to growth by girdling and by notching the stem . . 160
26. A. — Dormant blueberry buds stimulated to growth by chalking the stem.
B. — Dormant blueberry bud stimulated to growth by rubbing the stem . 160
27. A. — Normal spring growth on a blueberry stem. B. — Abnormal spring
growth on a blueberry stem, due to lack of chilling 160
28. Blueberry leaf exuding sugar from glands interpreted as osmotic-pressure
safety valves 160
29. A plant of bunchberry, Cornus canadensis, the seeds of which do not germi-
nate without chilling 160
30. A. — Trailing arbutus, Epigaea repens, flowering sparingly from lack of chill-
ing. B. — Trailing arbutus plant flowering normally after chilling. C. —
Blueberry plant forced into flower in September by artificial chilling. . 160
31. A. — Abnormal growth of an unchilled blueberry plant. B. — Awakening of
long dormant plants by artificial chilling 160
32. Plants brought out of dormancy at a specified time. A. — Blueberry plants
from a lot that had been kept in a dormant condition by warmth for
nearly a year. B. — Representative plants from each of the two chilled
lots described under A, from photograph made January 18, 1918 160
Oct. i, 1920-Mar. is, 1921
Illustrations IX
33> a— Plantation at Whitesbog, N. J., for the testing of blueberry hybrids. Page
B— Four-year-old blueberry hybrid in full fruit 160
34. The ordinary wild blueberry of New Jersey l6°
35. Fruit of a selected hybrid blueberry l6°
Injury to Seed Wheat Resulting from Drying after Disinfection
with Formaldehyde
Text Figures
1. Graph showing rate of evaporation of paraformaldehyde at room tempera-
ture, approximately 20° C • • • • 222
2. Graph showing the relation of humidity of the air to percentage of germina-
tion of stored seed in first experiment • • • • 226
3. Graph showing the relation of humidity of the air to percentage of germina-
tion of stored seed in second experiment 22&
4. Graph showing the relation between humidity of the air and seed injury
as indicated by rate of growth of germinated seedlings 229
5. Graph showing the diminution in the rate of evaporation of paraformal-
dehyde inclosed in a desiccator of 2,400-cc. volume 232
Plates
36. A.— Post-treatment seed injury occurring when wheat is dried after treat-
ment with a 0.1 per cent solution. B .—Germinating seedlings of Little
Club wheat, showing characteristic post-treatment injury when seed is
treated with a 0.1 per cent solution 244
37. A.— Pots showing germination of treated seed stored for 32 days after dis-
infection with a 0.1 per cent solution of formaldehyde. B.— Wheat
plants grown in soil from seed stored for 60 days after disinfection with
a 0.1 per cent solution of formaldehyde • • 244
38. A.— Wheat seedlings showing injury produced by allowing the seed to lie
in dry soil for 30 days after treatment with a 0.1 per cent solution of
formaldehyde. B — Desiccators with different degrees of atmospheric
humidity obtained by the use of mixtures of sulphuric acid and water in
different proportions 244
39. Germinating samples of wheat stored for 35 days after treatment in the
desiccators shown in Plate 38 B, illustrating the relation of seed injury
to humidity -• • 224
40. Varying injury to wheat treated with a 0.1 per cent solution of formalde-
hyde, and stored in sealed bottles. A.— Sealed immediately after treat-
ment, 100 per cent germination. B.— Sealed after drying 7 hours, spread
on towels in laboratory, no germination. C— Sealed after drying 24
hours, spread on towels in laboratory, no germination. D.— Sealed after
drying 3 days, spread on towels in laboratory, 14 per cent germina-
tion 244
41. Germinating wheat kernels, showing the prevention of post-treatment
injury by washing the seed with water immediately after treatment.
A.— Treated with 0.2 per cent solution, which was not washed off before
drying, 32 per cent germination. B.— Treated with 0.2 per cent solution
which was washed off before drying, 76 per cent germination. C—
Treated with 0.1 per cent solution, which was not washed off before
drying, 52 per cent germination. D.— Treated with 0.1 per cent solu-
tion, which was washed off before drying, 74 per cent germination 244
29667°— 21 2
Journal of Agricultural Research voi.xx
Studies on the Life History and Habits of the Beet Leafhopper
Plates
42. Eutettix tenella: A. — Adult, light form. B. — Adult, dark form. C. — Adult,
color gradation between A and B. D. — Nymph with protruding sac of page
dryinid parasite 252
43. Parasites of Eutettix tenella: A. — Pipunculus industrius: Adult, much en-
larged. B. — Polynema eutettixi: Adult, much enlarged 252
Hypertrophied Lenticels on the Roots of Conifers and Their
Relation to Moisture and Aeration
Plates
44. Section through a hypertrophied lenticel on root of Pinus rigida growing
in swampy situation 266
45. A. — Hypertrophied lenticels on the basal part of layering stem of Picea
mariana, which had been covered with sphagnum. B. — Tap root of a
Pinus ponderosa transplant, bearing an unusually large number of hyper-
trophied lenticels 266
46. A. — Cross section of the stem through one of the hypertrophied lenticels
shown in C. B. — Large patches of excrescences upon the tap root near
the root crown, on Pinus rigida. C. — Hypertrophied lenticels on root of
5-months-old Pinus ponderosa, grown in a loosely stoppered 2-ounce
bottle, in tap water which had not been changed since the germination
of the seed 265
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plates
47. Urophlyctis alfalfae: Drawing of alfalfa plant, showing abundance of crown-
wart, as found early in May, 1919, in northern California 324
48. Urophlyctis alfalfae: A-D. — Peripheral portions of actively growing thallus
of parasite dissected from living host. E. — Nearly mature resting spore
viewed from distil side, showing 11 haustoria in zonate arrangement.
F. — Mature resting spore viewed from distil pole, showing 13 pits that
mark former location of haustoria. G. — Mature resting spore viewed in
profile, showing pits in zonate arrangement and light concavity on
proximal side of spore 324
49. Urophlyctis alfalfae: A. — Section of epidermal region of young foliar struc-
tures, showing young primary turbinate cells ta-tg, the first products of
infection, within epidermal cells. B. — Section of young foliar element,
showing wall of invaded epidermal cell disrupted and advance of second-
ary turbinate cells tbc-tbe into underlying tissue. C. — Section of tur-
binate cell, showing 3 evacuated peripheral segments pa-pc. D. — Sec-
tion of maturing resting spore, showing 8 nuclei and a central vacuole
containing 4 granules staining red. E. — Section of mature resting spore,
showing numerous red-staining granules in center and 5 nuclei. F. —
Section of maturing resting spore, showing 11 normal nuclei and 4 en-
larged nuclei in center, the latter apparently degenerating 324
50. Urophlyctis alfalfae: Section of diseased bud scale of alfalfa, showing four
coalescing cavities, in three of which the large primary turbinate cells
taa, tba, and tc may be distinguished 324
51. Urophlyctis alfalfae: Section of diseased bud scale attacked by U. alfalfae,
showing a group of eight well-developed cavities a-h and their relation
to the host tissue 324
Oct. i, 1920-Mar. 15, 1921
Illustrations XI
52. A, C, B.—Urophlyctis pluriannulatus . B.—Urophlyctis alfalfac. A —
Portion of actively growing thallus of U. pluriannulatus dissected from
gall on leaf of Sanicula menziesii, including a turbinate cell ta with a
nearly mature resting spore ra. B.— Abnormally enlarged hyphae and
turbinate cells of U. alfalfae, showing conspicuous thickening of the
walls. C— Peripheral portion of actively growing thallus of U. pluri-
annulatus, similar to A, showing 8 turbinate cells of the second order,
of which 7 have produced turbinate cells of the last order as well as
resting spores. D— Nearly mature resting spore of U. pluriannulatus, page
viewed from polar end, showing 22 haustoria in zonate arrangement. ... 324
53. Urophlyctis pluriannulatis: Section of leaf of Sanicula menziesii, showing
development of parasite within gall 324
54. Crowns of alfalfa plants bearing galls caused by Urophlyctis alfalfae photo-
graphed at different stages of development. A.— A comparatively early
stage of development at which the origin of the gall structures from the
elements of developing buds can be traced. B— A later stage of devel-
opment at which the origin of the tissue has become obscured 324
55. A comparatively early stage of host reaction to invasion by Urophlyctis
alfalfae ." 324
56. A.— Late stage of development of host reaction to the invasion of Urophlyctis
alfalfae. B — Vertical section through a well-developed gall near its
central axis, showing its laminated structure arising from the thickening
of bud elements 324
Pathological Anatomy ok Potato Blackleg
Text Figure
1. Section of potato leaf, showing distribution of protein crystals 329
Plates
57. A.— Plant affected with blackleg. B— Section of single upper epidermal
cell of leaf and adjacent palisade cell. C— Section of pith cell which is
transformed into a sclereid adjacent to phloem fibers 33°
58_ a.— Pith cells of petiole transformed into sclereids with typically stratified
walls. B .—Vascular tissue of the petiole greatly increased by blackleg. . 330
Sclerotica minor, n. sp., the Cause op a Decay of Lettuce, Celery,
and Other Crops
Text Figure
1. Camera lucida drawings of 5. minor: A, Microconidia and conodiophores;
B, Ascospores; C, Germinating ascospores; D, Asci and paraphyses 332
Plate
59. A.— Sclerotia on hard potato agar: center, Sckrotina liberiiana, either end,
5. minor. B— Apothecia of S. liberiiana. C .— Apothecia of S. minor. . 334
Relation of the Soil Solution to the Soil Extract
Text Figures
1. Graph showing relation of freezing-point depressions in soil (calculated to
22 per cent moisture) to total solids extracted by 5 parts of water to 1 of
soil 382
Xii Journal of Agricultural Research voi.xx
2. Graph showing relation of freezing-point depressions in soil (calculated to
17 per cent moisture) to total solids extracted by 5 parts of water to 1 of Page
soil 383
Effect of Season and Crop Growth on the Physical State of the Soil
Text Figures
1. Effect of crop on physical state and electrolyte concentration of the water
extract of the soil 39^
2. Effect of crop on physical state and electrolyte concentration of the water
extract of the soil 399
3. Effect of crop on physical state and electrolyte concentration of the water
extract of the soil 400
4. Effect of crop on physical state and electrolyte concentration of the water
extract of the soil 4QI
Rice Weevil, (Calandra) Sitophilus oryza
Plate
60. Sitophilus oryza: A. — Egg. B. — Pupa, dorsal aspect. C. — Pupa, lateral
aspect. D. — Pupa, ventral aspect. E. — Adult. F. — Third-stage larva.
G. — First-stage larva. H. — Second-stage larva. I. — Fourth-stage larva. 422
Opius fletcheri as a Parasite of the Melon Fly in Hawaii
Text Figures
1 . Opius fletcheri: Egg just deposited 424
2. Opius fletcheri: Mature egg 424
3. Opius fletcheri: Larva, first instar, ventral aspect, showing head characters
and complete tracheal system, and the egg serosal cells 425
4. Opius fletcheri: Molted skin of first-instar larva, showing the absence of
egg serosal cells 42^
5. Opius fletcheri: New second-instar larva 427
6. Opius fletcheri: Mandible of second-instar larva 427
7. Opius fletcheri: Mandible of third-instar larva 427
8. Opius fletcheri: Larva, fourth instar, lateral aspect, showing general outline
and spiracles 428
9. Opius fletcheri: Spines on body of mature larva 428
10. Opius fletcheri: Mandible of fourth-instar larva 429
11. Opius fletcheri: Head of mature larva, dorso-cephalic aspect 429
12. Opius fletcheri: Pupa, female 43°
13. Opius fletclieri: Adult female 431
Tamarind Pod-Borer, Sitophilus linearis (Herest)
Plate
61. Sitophilus linearis: A. — Pupa, dorsal view. B. — Pupa, front view.
C. — Egg. D. — Mandible. E. — Mature larva. F. — Ventral view of
head. G. — Clypeus and labrum. H. — Pupa, lateral view. I. — Head,
face view. J. — Head, dorsal view. K. — Head, lateral view 446
Oct. i. 1920-Mar. is, 1921 Illustrations xin
Influence op Temperature and Humidity on the Growth op Pseudo-
monas citri and its host plants and on infection and development
of the Disease
Text Figure
1. Graph showing the rate of enzym action, as expressed in millimeters, at the page
various temperatures for a period of eight days on soluble starch agar ... 451
Daubentonia longifolia (Coffee Bean), A Poisonous Plant
Plate
6a. Herbarium specimen of Daubentonia longifolia, showing flowers, leaves,
and pods 5X4
Fusarium-Wilt of Tobacco
Text Figure
1. Camera-lucida drawings of spore forms of Fusarium oxysporum var. nicotianae,
n. var.: A, macroconidia; B, microconidia; C, chlamydospores; D, coni-
diophore of the sporodochial stage 521
Plates
63. A. — A typical spot in a field of Maryland Broadleaf tobacco infested
with Fusarium-wilt. Benedict, Md. 1916. B. — Uninoculated control.
C. — Plants grown in soil artificially inoculated with the tobacco-wilt
Fusarium and planted to White Burley 536
64. A. — Plant infected with Fusarium-wilt, showing wilting in vertical line
on stalk. B. — Last stages of Fusarium-wilt in Maryland Broadleaf
tobacco S36
65. A. — Result of plating out five pieces of infected vascular tissue from infected
plant, illustrating character of growth of mycelium on potato agar.
B. — vStem and midrib of plant, cut longitudinally to show the blackened
vascular system 53^
66. A. — Cross sections through vascular system of tobacco plant infected with
Fusarium-wilt, showing the fungus mycelium in the vessels. B. — Longi-
tudinal sections through the vascular system of plants infected with
Fusarium-wilt, showing the fungus strands in the vessels 536
67. I. — Plants illustrating the influence of soil temperature on degree of wilting
of plants in soil infested with Fusarium-wilt. II. — Plants grown in the
same soil uninfested and at corresponding soil temperatures.
III. — Plants illustrating the influence of varying soil reaction on the
amount of Fusarium-wilt in infested soil. IV. — Plants illustrating
varietal differences in resistance of tobacco to Fusarium-wilt 536
Nodule Bacteria of Leguminous Plants
Plates
68. A. — Soybean bacteria, J. K. Wilson's strain, 4 days old. B. — Vetch bac-
teria, 3 days old. C. — Bacillus radiobacter, 2 days old. D. — Soybean
bacteria, beef agar, 4 days old. E. — Red clover bacteria, beef agar,
4 days old. F. — Bacillus radiobacter, beef agar, 4 days old. G. — Cowpea
bacteria, potato, 6 days old. H. — Red clover bacteria, potato, 14 days
old. I. — B. radiobacter, milk, 7 days old. J. — Cowpea bacteria, man-
nite-nitrate agar, 8 days old. K. — Vetch bacteria, mannite-nitrate agar,
8 days old. L. — B. radiobacter, mannite-nitrate solution, 17 days old 556
XIV Journal of Agricultural Research vol. xx
69. A. — Mannite-nitrate agar slants, 8 days old, from left to right: soybean
bacteria, vetch bacteria, and Bacillus radiobacter. B. — Growth in milk,
4 weeks old, from left to right: soybean bacteria, vetch bacteria, and
B. radiobacter. C. — Growth on potato, 2 weeks old: vetch bacteria (left) Page
and B. radiobacter (right) 556
Correlation and Causation
Text Figures
1. Diagram illustrating the interrelations among the factors which determine
the weight of guinea pigs at birth and at weaning (33 days) 560
a. Diagram showing relations between two variables, X and Y, whose values
are determined in part by common causes, B, C, and D, which are in-
dependent of each other 565
3. Diagram showing relations between two variables, X and Y, whose values
are completely determined by common causes, B and C, which are in-
dependent of each other 565
4. A system in which the value of variable X is completely determined by
causes M and N, which are correlated with each other 566
5. A system in which the value of X is affected by a factor, B, along two differ-
ent paths, BMX and BNX 567
6. Diagram showing relations between two variables, X and Y, whose values
are determined in part by common causes, M and N, which are correlated
with each other 568
7. Simplified diagram of factors which determine birth weight in guinea pigs. 568
8. Path coefficients measuring the relations between birth rate (X), rate of
growth (Q), gestation period (P), size of litter (L), and other causes (A,
C) 570
9. Coefficients of determination. Symbols as in figure 7 570
10. Effect and one known cause 571
11. Effect and two correlated known causes 571
12 . Effect and three correlated known causes 571
13. Effect and four correlated known causes 572
14. Relations between wet-bulb depression (B), wind velocity (W), radia-
tion (R), and temperature (T) as assumed for direct analysis 576
15. Relations between factors of figure 14 and absolute humidity (H) expressing
causal relations better than figure 14 but adapted only to indirect analysis. 579
16. Relations between evaporations or transpiration (X) and the system shown
in figure 15 582
Inheritance op Syndactylism, Black, and Dilution in Swine
Plate
70. The four types of F2 segregates from a cross between mule-foot boar and
Duroc- Jersey sows. A. — Black mule-foot. B. — Black cloven foot.
C. — Red mule-foot. D. — Red cloven foot 604
Four Rhynchophora Attacking Corn in Storage
Plates
71. Araecerus fasciculatus . A. — Pupa, dorsal view. B. — Pupa, front view.
C. — Egg. D. — Mandible. E. — Mature larva. F. — Ventral view of head.
G. — Labium and clypeus. H. — Pupa, lateral view. I. — Head, face view.
J. — Head, dorsal view. K. — Head, lateral view 614
Oct. i, 1920-Mar. is. 1921
Illustrations XV
72. Caulophilus latinasus: A.— Pupa, dorsal view. B — Pupa, front view.
C. — Egg. D. — Mandible. E. — Mature larva. F. — Ventral view of
head. G— Labium and clypeus. H— Pupa, lateral view. I.— Head, page
face view. J.— Head, dorsal view. K— Head, lateral view 614
73. Sitophilus oryza: A. — Pupa, dorsal view. B. — Pupa, front view. C. —
Egg, D.— Mandible. E — Mature larva. F.— Ventral view of head.
G.— Labium and clypeus. H.— Pupa, lateral view. I.— Head, face
view. J. — Head, dorsal view. K. — Head, lateral view 614
74. Sitophilus granarius: A.— Pupa, dorsal view. B.— Pupa, front view.
C— Egg. D.— Mandible. E.— Mature larva. F.— Ventral view of
head. G.— Labium and clypeus. H.— Pupa, lateral view. I.— Head,
face view. J. — Head, dorsal view. K. — Head, lateral view 614
Freezing of Fruit Buds
Plate
75. Apparatus for freezing entire tree 66a
Effect of Various Crops upon the Water Extract of a Typical Silty
Clay Loam Soil
Text Figures
1. Decrease of water-soluble nutrients from the growth of various crops, as
shown by increases in specific resistance 664
2. Decrease of water-soluble nutrients from varying numbers of barley plants,
as shown by increase in specific resistance 664
3. Decrease of water-soluble nitrates from the growth of various crops. (Graphs
=K N03.) 665
4. Decrease of water-soluble nitrates from varying numbers of barley plants.
(Graphs=K N03.) 666
5. Decrease in the concentration of soil solution shown by freezing-point de-
pression 666
Another Conidial Sclerospora of Philippine Maize
Text Figure
1. Comparison of the sizes of 700 conidia of Sclerospora spontanea with 700
conidia of 5. philippinensis; A, variation of conidia in length; B, varia-
tion of conidia in diameter ; C, ratios of length to width of conidia arranged
in classes "75
Plates
76. Corner of a native-grown maize plot in the interior uplands of Cebu 684
77- a. — Clump of Saccharum spontaneum, showing characteristic size and
habit of healthy plants under natural conditions. B.— Clump of Sac-
charum spontaneum infected with Sclerospora spontanea 684
-8, A. — A young seedling (3 weeks old) of Saccharum spontaneum infected with
Sclerospora spontanea. B. — Conidiophores on the leaf of Saccharum
spontaneum. C— Young shoots of Saccharum spontaneum arising after
the primary stalk had been cut, and like it severely infected with Sclero-
spora spontanea 4
xvi Journal of Agricultural Research voi.xx
79. A. — Typical conidiophore, showing characteristically long, slender, un-
knobbed basal cell, relatively short main axis with its greatest diameter
about midway to the primary branches, and fairly well-developed branch
system bearing long, slender conidia. B. — Upper portion of a conidio-
phore which has a poorly developed branch system and hence bears
few conidia on sterigmata which are relatively large. C. — Portion of the
branch system of a conidiophore, showing the conidia germinating while
still attached to their sterigmata. D. — Stalk portion of a typical conidio-
phore, showing long, slender, unknobbed basal cell, and main axis which
is slender above the septum, expands rapidly to its greatest diameter
about midway, and contracts again below the branches. E, F. — Typical
basal cells of conidiophores. G. — Stalk portion of a conidiophore with
basal cell which, though unusually short, nevertheless is longer than the
extent of the main axis from septum to primary branches. H. — Typical
stalk portion of a conidiophore from sugar cane. I, J, K. — Typical
conidia showing variations in size and shape and method of germination Page
by hyphae 684
Onion Smudge
Text Figures
1. Conidia and appressoria of Colletotrichum circinans 689
2. Acervulus of Colletotrichum circinans on artificially inoculated onion scale . 690
3. Spores of Colletotrichum fructus (A) and C. circinans (B) 694
4. Relation of temperature to growth of Colletotrichum circinans on agar plates. 697
5. Relation of temperature to spore germination of Colletotrichum circinans. . . 698
6. Colletotrichum circinans: Stage of penetration of epidermal cell of onion
scale at 66 hours after inoculation 702
7. Cross section of epidermis, showing early stage of penetration by Colletotri-
chum circinans 7°3
8. Cross section of epidermis (A ) and underlying parenchyma cells (B) of onion
scale inoculated with a suspension of Colletotrichum circinans spores and
kept in a moist chamber at room temperature 704
9. Cross section of onion scale naturally infected with Colletotrichum circinans,
showing the mycelium developing first just beneath the cuticle and later
penetrating the subcuticular wall 7°5
10. Chart from data collected at Racine, Wis., during 1915 and 1916, showing
the daily mean soil temperature at a depth of 1 to 2 inches, and the rainfall . 708
Plates
80. Onion smudge: Onion sets (White Portugal variety) naturally infected with
Colletotrichum circinans 722
81. Onion smudge: A, B, E, D. — Advanced stages of smudge after several
months in storage. C. — Bulb inoculated in a moist chamber with a sus-
pension of Colletotrichum circinans conidia. F, G. — Macros porium sp. on
outer scale of white onion sets. H. — M. porrum and Phoma alliicola on
outer scale of white onion set 722
82. Relation of soil temperature to the development of smudge 722
83. Colletotrichum circinans and C. fructus: A. — Photomicrograph of cross section
of naturally infected onion scale. B. — Photomicrograph of cross section
of an infected onion scale held for several months in poorly ventilated
storage. C, D. — Photomicrographs of cross sections of C. circinans (C)
and C. frutus (D) on apple fruit 72»
Oct. i, i92o-Mar. is, w Illustrations xvii
84. Colletotrichum fructus and C. circinans: A.— Dilution plate from spores of
Colletotrichum fructus. B— Individual colony of C. fructus on potato
agar. C— Apple of Fameuse variety inoculated with mycelium from
pure culture of C. circinans. D.— Dilution plate from spores of C. cir- page
cinans. E — Individual colony of C. circinans on potato agar 722
85. Relation of curing conditions to the development of smudge: A, B.— Com-
parison of onion sets artificially dried immediately after harvest with
those not dried. C, D.— Comparison of white onion sets cured in shal-
low crates in the field under the best of natural conditions with part of
the same lot after exposure to moist conditions for one week 722
Variations in Colletotrichum gloeosporioides
Plate
86. A, B.— Variation occurring in strain 990. The cultures were not made
from a single spore. C— Variation occurring in a culture of strain 990
which was made from a single spore 736
Text Figures
1. Variability of strains of Colletotrichum gloeosporioides in spore length 728
2. I, culture 510: A, greenish black mycelium; B, white mycelium. II, cul-
ture 943: A, black mycelium; B, white mycelium; C, mycelium mostly
in medium, growth zoned, abundant spore production. Ill, culture
495: A, black mycelium; B, gray mycelium; C, white mycelium. IV,
culture 527: A, gray mycelium; B, greenish black mycelium; C, white
mycelium; D, black mycelium. V, culture 940: A, greenish black
mycelium; B, white mycelium, some greenish concentric circles; C,
black mycelium; D, white mycelium; E, white and black mixed 734
A Transmissible Mosaic Disease op Lettuce
Plate
87. a.— Leaves of Romaine lettuce. B.— Young expanding leaves of head
lettuce from experiment started March 22 74°
Leconte's Sawfly, an Enemy of Young Pines
Text Figures
1. Chart showing life and seasonal history of Neodiprion lecontei through the
active period of three years (November to March omitted, the insect
being in the cocoon during this period) 751
2. Position of end of abdomen of female when ovipositing, showing the various
parts and their position: 1, lance; 2, apical part of sheath; 3, basal part
of sheath; 4, nates or ninth tergite; 5, eighth sternite; 6, chitinized rods
at base of lancet ; 7, lancet 755
3. Distribution of Neodiprion lecontei. The larger dots indicate places from
which specimens have actual^ been received 759
Plates
88. Neodiprion lecontei: A.— Adult female. B.— Adult male 7°°
89. Neodiprion lecontei: A.— Larva. B.— Sixth-stage larva: The muscles of a
single abdominal segment distributed over several segments to show their
numbers, position, and attachment 760
29667°— 21 3
xviil Journal of Agricultural Research vol. xx
go. Neodiprion lecontei: Sixth-stage larva. A. — Front view of head. B. —
Ventral (or apical) view of head capsule. C. — Front view of head cap-
sule. D. — Lateral view of head. E. — Sagittal section of head. F. —
Antenna. G. — Frons, adfrons, and clypeus. H. — Mandibles. I. —
Epipharynx and labrum. J. — Internal view of hypopharynx maxillse,
and labium. K. — External view of maxillae, and labium. L. — Exter-
nal view of maxillae. M. — Interior and apical view of maxilla. N. — page
End view of maxilla. O. — End view of labium 760
91. Neodiprion lecontei: Sixth -stage larva. A. — External view of the thorax.
B. — External view of the second and third abdominal segments. C. —
External view of the ninth and tenth abdominal segments. D. — In-
ternal view of thoracic skin. E. — Internal view of the skin of the second
and third abdominal segments. F. — Diagrammatic cross section of the
abdomen showing the longitudinal areas of the body on its transverse
circumference 760
92. Neodiprion lecontei: A. — Some defoliated twigs showing feeding on bark
of stem. B. — Eggs within needles of Pinus virginiana 760
Evaluation of Climatic Temperature Efficiency for the Ripening Proc-
esses IN SwEETCORN
Text Figure
1. Comparison of early and late crops of sweet corn in respect to changes in per-
centage composition in equal lengths of time 798
Some Lepidoptera Likely to Be Confused with the Pink Bollworm
Plates
93. Male genitalia (Gelechiidae): A. — Gelechia trophella: Posterior part of
tegumen, showing uncus and gnathos, ventral view. B. — G. trophella:
Lateral view of male genitalia with eighth abdominal segment attached.
C. — G. hibiscella: Lateral view of male genitalia with eighth abodminal
segment attached 836
94. Male genitalia (Gelechiidae): A. — Telphusa mariona (type): Lateral view
of male genitalia. B. — T. mariona (type): Posterior part of tegumen,
showing uncus, ventral view. C. — Gelechia neotrophella (type): Aedoea-
gus and penis. D. — G. neotrophella (type) : Lateral view of male genitalia
with aedoeagus and eighth segment removed. E. — G. neotrophella
(type): Posterior part of tegumen, showing uncus and gnathos, ventral
view. F. — G. neotrophella (type) : Posterior half of harpes, ventral view.
G. — G. neotrophella (type) : Sternite and tergite of modified eighth abdom-
inal segment 836
95. Male genitalia (Gelechiidae, Stenomidae, and Oecophoridae): A. — Iso-
phrictis similiella: Ventral view of male genitalia, spread. B. — Aede-
moses haesitans: Ventral view of male genitalia, spread. C. — A. haesi-
tans: Enlargement of typical split hair on cucullus. D. — Borkhausenia
fasciata: Ventro-lateral view of male genitalia, spread, showing a sym-
metrical armlike projections from gnathos and costa of harpes 836
96. Male genitalia (Oecophoridae): A. — Borkhausenia minutella: Aedoeagus.
B. — B. minutella: Ventral view of male genitalia, spread, aedoeagus
omitted. C. — B. diveni (type): Ventral view of male genitalia, spread.
D. — B. diveni (type): Dorsal view of an abdominal segment showing
spinose condition of abdomen. E. — B. diveni (type) : Modified tergite of
eighth abdominal segment. F. — B. diveni (type): Modified sternite of
eighth abdominal segment 836
Oct. i, 1920-Mar. is. 1921 Illustrations xix
97. Male genitalia (Oecophoridae): A. — Borkhausenia conia: Portion of tergite
of seventh abdominal segment, showing spinose and chitinized character
of caudal margin. B. — B. conia: Ventral view of male genitalia, spread,
aedoeagus omitted. C. — B. conia: Aedoeagus. D. — B. conia: Modified
tergite of eighth abdominal segment. E. — B. conia: Modified sternite Page
of eighth abdominal segment 836
98. Malegenitalia (Blastobasidae): A. — Zenodochium citricolella: Aedoeagus.
B. — Z. citricolella: Lateral view of male genitalia, right harpe and aedoea-
gus omitted. C. — Z. citricolella: Right harpe. B.—Holcocera ochroce-
phala: Ventral view of male genitalia, spread, aedoeagus omitted. E. —
H. ochrocephala: Dorsum of an abdominal segment showing transverse
row of spines. F. — H. ochrocephala: Aedoeagus and penis 836
99. Male genitalia (Olethreutidae and Blastobasidae): A. — Crocidosema ple-
beiana: Ventral view of male genitalia, spread. B. — Eucosma discreti-
vana (type): Ventral view of male genitalia, spread. C. — Holcocera con-
famulella (type) : Ventral view of male genitalia, spread 836
100. Male genitalia (Phaloniidae and Pryalidae): A. — Phalonia cephalanthana
(type): Ventral view of male genitalia, spread. B. — Homoeosoma elec-
tellum: Ventral view of male genitalia, spread 836
101. Larval structures: A. — Pectinophoragossypiella: Head capsule , dorsal view,
showing arrangement of setae. B. — P. gossypiella: Head capsule, lateral
view, showing arrangement of setae. C. — Dicymolomia julianalis: Head
capsule, dorsal view, showing arrangement of setae. D. — D. julianalis:
Head capsule, lateral view, showing arrangement of setae. E. — Meskea
dyspteraria: Head capsule, dorsal view, showing arrangement of setae.
F. — M. dyspteraria: Head capsule, lateral view, showing arrangement of
setae 836
102. Larval structures: A. — Pyroderces rileyi: Head capsule, dorsal view, show-
ing arrangement of setae. B. — P. rileyi: Head capsule, lateral view,
showing arrangement of setae. C. — Crocidosema plebeiana: Head capsule,
dorsal view, showing arrangement of setae. D. — C. plebeiana: Head
capsule, lateral view, showing arrangement of setae. E.. — Z. enodochium
citricolella: Labium and maxillae. F. — Isophrictis similiella: Head cap-
sule, dorsal view, showing arrangement of setae 836
103. Larval structures: A. — Pectinophora gossypiella: Setal maps of first and
second thoracic and third, eighth, and ninth abdominal segments. B. —
Dicymolomia julianalis: Setal maps of first and second thoracic and third,
eighth, and ninth abdominal segments. C. — Pyroderces rihyi: Setal
maps of first thoracic and eighth and ninth abdominal segments. D. —
Heliothis obsoleta: Setal maps of first thoracic and third abdominal seg-
ments. E. — Crocidosema plebeiana: Setal maps of first and second tho-
racic and third, eighth, and ninth abdominal segments 836
104. Larval structures: A. — Platynoia rostrana: Setal maps of first and second
thoracic and third, eighth, and ninth abdominal segments. B. — Meskea
dyspteraria: Setal maps of first and second thoracic and third, eighth,
and ninth abdominal segments. C. — Z. enodochium citricolella: Setal
maps of first thoracic and third, eighth, and ninth abdominal segments.
D. — Aedemoses haesitans: Setal map of third abdominal segment. E. —
Moodna estrinella: Setal maps of second thoracic and eighth and ninth
abdominal segments 836
XX Journal of Agricultural Research vol. xx
105. Larval structures: A. — Platynota rostrana: Setalmaps of eighth and ninth
abdominal segments, dorsal view. B. — Eucosma helianthana: Setal maps
of eighth and ninth abdominal segments, dorsal view. C. — Pectinophora
gossypiella: Setal maps of eighth and ninth abdominal segments, dorsal
view. D. — Pyroderces rileyi: Setal maps of eighth snd ninth abdominal
segments, dorsal view. E. — Pectinophora gossypiella: Prothorax, ventral
view, showing position of legs. F. — Telphusa mariona: Ventro-caudal
view of tenth abdominal segment, showing anal fork. G. — Crocidosema
plebeiana: Ventro-caudal view of tenth abdominal segment, showing
anal fork. H. — Gelechia neotrophella: Ventro-caudal view of tenth ab-
dominal segment, showing anal fork. I. — Zenodochium citricolella: page
Prothorax, ventral view, showing position of legs 836
106. Larval structures: A. — Pectinophora gossypiella: Crochet arrangement of
abdominal prolegs. B. — Crocidosema plebeiana: Crochet arrangement of
abdominal prolegs. C. — Pyroderces rileyi: Crochet arrangement of ab-
dominal prolegs. D. — Dicymolomia julianalis: Crochet arrangement of
abdominal proleg. E. — Heliothis obsoleta: Crochet arrangement of ab-
dominal proleg g-jg
107. Pupal structures: A. — Pectinophora gossypiella: Ventral view of pupa.
B. — Pectinophora gossypiella: Caudal end of pupa, lateral view. C. —
Pectinophora gossypiella: Mature pupa, ventral view, shaded to show
eyes of imago visible through pupal skin and characteristic pubescence
of the pupa. D. — Pectinophora gossypiella: Dorsal view of pupa. E. —
Pyroderces rileyi: Ventral view of pupa. F. — Pyroderces rileyi: Dorsal
view of pupa 8,6
108. Pupal structures: A. — Crocidosema plebeiana: Abdomen of female pupa,
ventral view. B. — C. plebeiana: Abdomen of male pupa, ventral view.
C. — C. plebeiana: Lateral view of an abdominal segment, showing
arrangement and character of dorsal spines; one spine greatly enlarged
to show shape. D. — C. plebeiana: Abdomen of pupa, dorsal view. E. —
Dicymolomia julianalis: Dorsal view of pupa. F. — D. julianalis: Caudal
end of pupa, lateral view. G. — D. julianalis: Caudal end of male pupa,
ventral view. H. D. julianalis: Ventral view of female pupa 836
109. Pupal structures: A. — Meskea dyspteraria: Caudal end of female pupa,
lateral view. B. — M. dyspteraria: Abdomen of female pupa, ventral
view. C. — M. dyspteraria: Male pupa, dorsal view. D. — M. dyspter-
aria: Caudal end of male pupa, lateral view. E. — M. dyspteraria: Male
pupa, ventral view. F. — Amorbia emigratella: Abdomen of pupa, dorsal
view. G. — Telphusa mariona: Caudal end of pupa, ventral view,
showing peculiarly scalloped and fringed caudal margin of seventh
abdominal segment 836
Vol. XX OCTOBER 1, 1920 No. 1
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Pago
Fusarium-Blight (Scab) of Wheat and Other Cereals - 1
DIMITR ATANASOFF
( Contribution from Wisconsin Agricultural Experiment Station
and Bureau of Plant Industry)
Cause of Lime-Induced Chlorosis and Availability of Iron
in the Soil -------- 33
P. L. GILE and J. O. CARRERO
( Contribution from States Relations Service )
An Experimental Study of Echinacea Therapy 63
JAMES F. COUCH and LEIGH T. GILTNER
( Contribution from Bureau of Animal Industry )
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WASHINGTON I GOVERNMENT PRINTING OFFICE : 1920
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C,
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
JOURNAL OF AGRKMiAL RESEARCH
Vol. XX Washington, D. C, October i, 1920 No. 1
FUSARIUM-BUGHT (SCAB) OF WHEAT AND OTHER
CEREALS l
By Dimitr Atanasof?
Formerly Assistant in Plant Pathology, University of Wisconsin ~-
INTRODUCTION
The cereal crops — wheat, spelt, rye, and oats — and also some grasses
are subject to attack by a number of fungi belonging to the genus
Fusarium, of which the most common and most important is known, in
its ascigerous form, as Gibberella saubinetii (Mont.) Sacc. The organism
attacks each of the hosts named above in at least two different ways,
producing two distinct pathological conditions. The first condition
results from an attack on the root systems and the bases of the young
and later of the grown plants, occasionally causing partial or entire
wilting. The second condition results from an attack upon some of
the parts above ground. This may be a rotting of the nodes, found on
rye, wheat, and barley, or blighting of the heads of wheat, spelt, rye,
barley, and, less commonly, of oats and certain grasses. In all cases the
various attacks on the same host are independent of each other. A
wheat plant may be attacked underground or on the head only or on
both the roots and the head, and in some cases even on some of the
nodes; but in all cases these infections are quite independent.
Up to the present time little attention has been given to these two
forms of attack by Fusarium, and they have commonly been considered
two different diseases caused by one or more unknown species of Fusarium.
However, the results of the work reported here prove conclusively
that these two conditions are only two different phases of the same
problem. This is in accord with views previously held by Selby and
Manns (u),2 Schaffnit (8), and Naumov (5).
This report, which is of a preliminary nature, deals primarily with the
headblighting of wheat, spelt, rye, barley, and oats, as caused by
Gibberella saubinetii, comparatively little attention being given in this
paper to the rootrot caused by this organism. Nothing will be said
•In cooperation with the Office of Cereal Investigations, Bureau of Plant Industry. United States
Department of Agriculture.
1 Reference is made by number (italic) to " Literature cited," p. 31-32.
Journal of Agricultural Research, Vol. XX, No. 1
Washington, D. C Oct. 1, 1920
u V Key No. Wis.-i8
(l)
)
2 Journal of Agricultural Research vol. xx.No. i
here concerning other species of Fusarium connected with both phases
of this problem or of their possible relation to the similar diseases of corn.
While G. saubinetii is unquestionably the cause of headblighting of the
cereal crops under most conditions and throughout the greater part of
this country, it is equally true that under certain conditions and in some
parts of the country other species of Fusarium are also responsible for
the headblighting of cereal crops. The following organisms besides G.
saubinetii have been isolated from blighted wheat, rye, oats, and barley
heads or plants: Fusarium avenaceum (Fr.) Sacc., F. herbarum (Corda)
Fries, F. culmorum (W. G. Smith) Sacc, F. culmorum (W. G. Smith)
Sacc. var. leteius Sherb., F. arcuosporum Sherb., F. scirpi Lamb et Fautr.,
F. solani (Mart. pr. p.) Ap. et Wr., F. arthrosporioides Sherb., and F.
redolens Wr. These species, while very seldom responsible for the
headblighting of cereals, are not so unimportant in the rootrot problem
of these crops. Indeed, some of them (F. herbarum, F. avenaceum, F.
culmorum, and F. culmorum var. leteius) have, in my observations,
proved to be as important as G. saubinetii in causing rootrot of the
cereal crops.
There is extensive literature on this subject which can not be reviewed
in this brief paper. Only a few of the more important citations are
given.
THE DISEASE
COMMON NAME
In this country the headblighting of the cereal crops is generally
known under the faulty name of "wheatscab." It is not a wheat dis-
ease alone, because it also occurs on spelt, rye, barley, oats, and certain
grasses. And it is not "scab" because it causes no scabbing of the
heads or of any part of the various hosts but rather blighting of the
heads. The infected heads are perfectly normal and remain so except
that they are blighted, take on the color of bleached straw, and later
may be overgrown with the mycelium of the pathogen. Since the
name "wheatscab" is faulty in a number of respects, the name "Fusa-
rium-blight" is used in this paper.
GEOGRAPHIC DISTRIBUTION
The Fusarium-blight of cereals is more or less common throughout
the central and eastern cereal-growing sections of the United States. It
has been reported by the Plant Disease Survey for 191 7, 191 8, and 191 9
from the following States: Maine, New Hampshire, Vermont, Massa-
chusetts, Connecticut, New York, Pennsylvania, New Jersey, Delaware,
Maryland, West Virginia, Virginia, North Carolina, South Carolina,
Georgia, Alabama, Tennessee, Kentucky, Ohio, Indiana, Michigan,
Illinois, Wisconsin, Minnesota, Iowa, Missouri, Oklahoma, North Da-
Oct. i, 1920 Fusarium-Blight (Scab) of Wheat and Other Cereals 3
kota, South Dakota, Montana, and Oregon. It was looked for but was
not found in the following States: Washington, California, Wyoming,
Texas, Arkansas, Kansas, Louisiana, Mississippi, and Rhode Island.
It has been reported from various parts of Canada.
In Europe the disease has been found in England, France, Italy, Ger-
many, Austria, Holland, Denmark, Norway, Sweden, and Russia. In
Russia the disease is common throughout the wheat- and rye-growing
sections. In Asia it is very common in the Usurian provinces on the
Siberian Pacific coast. It has also been reported from Australia.
ECONOMIC IMPORTANCE
The Fusarium-blight of the cereal crops injures the plants in several
ways and is generally considered an important disease of these crops.
It lowers germination of the seed and causes dying off or weakening of
the young seedlings. Later it causes dying and wilting of fully grown
plants, and finally it blights the heads, wholly or in part, thus prevent-
ing them from filling. The severity of the headblighting varies
from a fraction of 1 per cent to 100 per cent, and the loss due to de-
crease in yield in individual fields and localities may vary from o to
over 50 per cent.
The data concerning the economic importance of the disease are in-
complete and inadequate. For some phases of the disease, and for
most of the crops, they are entirely lacking. The meager information at
hand on this subject is found in the Plant Disease Bulletin issued by the
United States Department of Agriculture.1 This covers only the losses
caused by blighting of the heads of wheat and is given for only a few of
those States where the disease is known to be present and common in
one form or another. No information is available concerning the
losses due to decrease in germination and the killing of seedlings and
grown plants.
The total loss due to the blighting of the wheat heads by Gibberella
saubinetii and various other species of Fusarium for the States report-
ing amounted to 10,620,000 bushels in 191 7, according to the Plant
Disease Survey. The States reporting highest losses were Ohio with
3,577,000 bushels, Indiana with 2,513,000 bushels, and Illinois with
2,288,000 bushels.
If the estimate of the Plant Disease Survey approximates the actual
loss due to the blighting of the wheat heads in the States reporting,
then the total annual loss for the United States is probably close to
20,000,000 bushels.
No definite information is available concerning the importance of the
disease in Europe, especially in Russia, where it is known to be one of
the most important and destructive of the cereal diseases.
1 U. S. Department op Agriculture. Bureau of Plant Industry. Plant Disease Survey.
Plant Disease Bulletin , Supplement 8, p. 21-27. May 1, 1920.
4 Journal of Agricultural Research vol. xx.no. t
description
In spite of the extensive literature on this subject, there is no de-
tailed description of any of the various phases of the disease. In some
discussions of the disease no symptoms are given; in others there is a
brief description of only the last stages of infection, or rather, of the
final results of infection. Because of this situation it seems necessary
to describe the disease in detail, giving special attention to some symp-
toms which previously have been overlooked.
BLIGHTED SEED
Wheat kernels obtained from heads blighted or partly blighted by
Gibberella saubinetii show marked evidence of the effect of the Fusarium
attack and can be easily distinguished in a sample of grain, even when
only a very small percentage of such kernels are present. Wheat seed
from blighted heads exhibits one of three more or less distinct and definite
pathological symptoms, depending upon the time of head infection.
(i) Kernels from heads infected early in their development, possibly
during or shortly after the blossoming period, are small in size, being
sometimes hardly two-thirds as long as the normal. They are pale
greenish gray in color, badly shrunken, not firm, and very light in weight.
As a rule, such kernels are never able to germinate. They may be
heavily infected or even covered with the mycelium of the fungus if they
developed near the point of infection, or they may be perfectly free from
any fungus mycelium, if they have developed far above the point of
infection where the food supply was cut off.
(2) Kernels from heads infected two or three weeks after the blos-
soming period may attain nearly a normal size, but they usually have
a slightly shrunken appearance. They are grayish white or cream-
white in color, soft and starchy in texture, and much lighter in weight
than the normal kernel. In this case, also, they may be infested and
even covered with mycelium, which is especially evident in the groove,
or they may be entirely free from mycelium, depending on their position
in the head with relation to the point of infection. The percentage of
germination of kernels in this class is very low.
(3) The third class of kernels consists of those which have been infected
shortly before or just after the head is ripe. Such kernels differ very
slightly from the normal, except that they are partly discolored, pinkish
spots being not uncommon on them. While it is true that Gibberella
saubinetii is the most common cause of pinkish red coloring on kernels
in all three of these classes, it must be remembered that other fungi,
Macrosporium and Alternaria for instance, may in some cases cause
this coloring of grain. Kernels of this last class usually germinate nor-
mally, but before the young plant has reached the surface of the soil,
or before it attains any considerable size, it not uncommonly wilts and
Oct. I, 1920
Fusarium-B light (Scab) of Wheat and Other Cereals
dies as a result of infection from the kernel. In many cases, however,
the seedling survives the attack and reaches full development.
Kernels of rye from blighted heads show symptoms similar to those
described for wheat. The kernels which are directly attacked by the
fungus in blighted barley heads become dirty brown in color and are
lighter in weight than the normal kernels if the infection takes place at an
early stage in development. Often barley kernels are found with salmon-
colored spots on which there are masses of conidia of Fusarium. Oat
kernels show much the same symptoms as barley, except that they remain
lighter in color. In all these cereals, symptoms similar to those caused
by Fusarium-blight may be caused by other agencies, such as the
exposure of the grain to rain.
SEEDLING-BLIGHT
Seedlings from seed naturally or artificially infected with Gibberella
saubinetii are subject to attacks by this organism at a very early stage of
their development, and the visible symptoms of the infection may
become evident at the time of the germination of the seed or only a few
days later. The first symptoms appear on the young coleorhiza and
coleoptile and consist of the browning and rotting of these parts. The
coleorhiza and coleoptile, which always die shortly after the formation
of the permanent roots and the appearance of the first foliage leaf, seem
to offer a good medium for the establishment of the various species of
Fusarium, which then penetrate into the tissues of the permanent roots
and the first foliage leaf, causing the browning and rotting of the invaded
portions. If the attack has proceeded successfully, the formation of
the two lateral roots, in the case of wheat, is either prevented or these
roots are destroyed before attaining any considerable size. The older
or basal portions of the roots are sometimes pink in color, but they are
usually brown to black. The lower portions of the roots continue normal
and healthy until their food supply is cut off by rotting of the upper parts.
Often the remnants of the kernel are heavily overgrown with the myce-
lium of the fungus, and in some cases they attain a dark carmine red
color. The leaves above the infected portion, which seldom extends
above the ground if the plant is still very small, become yellow and later
brown, the discoloration beginning at the tips. If the leaves are over
6 cm. long they usually take on a light-green color and then collapse and
wilt very rapidly, showing a blighted effect. In many cases the infection
may be restricted to the primary roots, the coleorhiza and coleoptile,
and even to the first foliage leaf. In such cases new roots are soon
formed, the second and third leaves develop, and the plant may recover
almost entirely from the attack, which is still restricted to the parts
originally infected. Such plants, if examined three or four weeks later,
will show no symptoms of the infection and will usually continue to
develop normally.
6 Journal of Agricultural Research vol. xx, No. i
FOOTROT
Careful examination of the underground portions of winter crops early
in spring and of spring crops somewhat later in the season shows partial
rotting of the roots, the bases, and, in some cases, the interior of the
stems just above the bases. Various fungi may be found associated
with this condition on the cereal crops, among which Gibberella saubi-
netii and species of Fusarium are common. No attempt has been
made to obtain definite data on the relative frequency of occurrence of
different species of Fusarium on root lesions and discolorations. This,
of course, would be necessary before their relative importance as or-
ganisms inducing rootrot under field conditions can be determined.
The first evidence of the pathological condition of the roots of the
cereals, whether the source of infection be the seed or the soil, is the same.
The organisms first appear on the remnants of the kernel and follow
some of the primary roots, causing rotting and browning as described
above. When the crown and the crown roots are formed, the primary
stem below the crown roots, now quite darkened and in some cases
beginning to die, is invaded by the organism from the remnants of the
kernel and the primary roots. Soon it, too, becomes brown and shows
evidence of rotting. When the invasion reaches the crown it may stop,
or, depending perhaps on the condition of the plant, it may continue,
invading the central woody portion of the primary stem above the crown
as well as the secondary stems and causing a browning of the woody
portions. Rotting and browning of the scale leaves and of the sheath
may also occur as a result of the invasion. How much of this rotting
and discoloration of the underground portion of the cereal crops due to
Fusarium species is parasitic and how much is saprophytic is not known.
That some of these organisms are parasites is shown conclusively by the
rotting of the roots next to the remnants of the kernel or next to the
crown while their lower portions continue to be normal. It is shown
also by the browning of the interior of the primary stem at and above
the crown. The separation of discolorations and rotting of underground
portions due to the parasitic and saprophytic action of the organisms
concerned is unusually difficult, as large portions of the original under-
ground parts of the plants eventually die even without any fungus in-
vasion, and the presence of parasitic organisms may have nothing to do
with it. Such is the case with the primary roots and the primary stem
below the crown, and later with some of the crown roots themselves.
The amount of damage, if any, due to this invasion of the roots and
other underground portions is even more difficult to determine. As a
rule, the plants so attacked are at first small and stunted, but with the
coming of sunny and warmer weather they usually recover and reach
normal development, even when very badly injured. With the coming
of favorable weather such plants may send out secondary roots or even
Oct. i, 1920 Fusarium-B light (Scab) of Wheat and Other Cereals 7
aerial roots, a development quite common in oats, and before long the
effects of the attack may largely disappear.
ON STEMS OF GROWN PLANTS
Occasionally full-grown plants are killed by Qibberella saubinetii or
by one of several Fusarium species just before or shortly after the
time of blossoming. The fungus attacks the roots and the stem close
to the ground, the first node usually being involved in the infected area.
The part of the stem in contact with the ground and the roots below are
rotted and are commonly pink or yellowish brown in color. This rotting
of the base interferes with the water and food supply of the plant, and
wilting of the entire plant is the result. Such plants become bent or
broken over soon after they wilt and hence are easily recognizable in
well-kept fields. When such plants are pulled up they break at the base,
the roots always remaining in the soil (PI. 2, A). It must be remembered,
however, that wilting of the whole plant in very much the same way is
caused by other fungi as well, for example by Colletotrichum sp., although
in attacks by this fungus the base of the dead plant is a much darker
brown or black in color.
This infection at the base of the plant may be due to any one of several
causes. It may be only a continuation of the attack upon the young
seedling or it may be the result of a new infection. Either the decline
in vigor or unfavorable weather conditions may be responsible for the
appearance of the disease at this time.
The succulent embryonic tissue just above the nodes of the various
cereals is especially susceptible to attack by Qibberella saubinetii. Here
the infection is usually restricted to the node or the area immediately
next to the node, seldom, if ever, extending more than 2% cm. in each
direction. In such cases the portion above the infected node usually
wilts and soon dies. Conidia may be formed under certain conditions
on the node itself and on the infected part of the sheath coming out from
it. This condition was first observed by McAlpine (4, p. 305) in 1 896.
BLIGHTING OF HEADS
Wheat. — The symptoms and effects of headblighting of different
varieties of wheat are, in general, the same. The blighted head usually
takes on the normal color characteristic of the ripe head of that variety
or a slightly lighter color.
Blighting of the wheat heads can be detected with absolute certainty
at a very early stage, three to four days after infection has taken place,
provided that weather conditions have been so favorable as to enable
the parasite to establish itself on the host and to begin its work of
destruction.
The symptoms of blight infection as they appear on Marquis or some
other of the beardless varieties are as follows : The very first sign of blight
8 Journal of Agricultural Research vol. xx, no. i
infection is a slightly brown and water-soaked spot, 2 to 3 mm. in length,
on the glumes. The veins appear more water-soaked and have a much
darker olive-green appearance than the area between them. The points
at which the infected glume or glumes are attached to the rachis soon show
the water-soaked appearance also. The water-soaked area increases
more or less rapidly, depending on weather conditions, until the whole
spikelet is covered. It then spreads to the neighboring spikelets.
If the weather is dry the infection may remain restricted to one spike-
let. At this time the glumes and the spikelets originally infected gradu-
ally begin to lose the water-soaked appearance, dry up, and take on the
typical color of the ripe head of the particular variety. This drying up
of the infected spikelets follows closely the advancing infection, which
usually proceeds downward, as was first observed by Freeman (2, p. 310)
in 1905. The healthy part of the head above the point of infection
usually dries up and dies without passing through the water-soaked stage,
because of the cutting off of the water and food supply by the fungus at
the point of infection. In some cases, however, one or more vascular
bundles of the rachis may remain free from the fungous invasion and con-
tinue to supply the uninfected portion of the head with water and food
until the head has ripened normally and has formed fairly normal kernels.
When infection proceeds down the stem, producing the same symptoms
as on the head, it may sometimes reach as far as the upper node. Here,
too, the whole or only one side of the stem may become affected, while
the other side with one or more vascular bundles still normal may con-
tinue to provide moisture and food for the living portion of the head.
Usually, however, especially in dry weather, the infection is restricted
to the head; and most commonly only a part of the head is destroyed.
This may be the upper, middle, or lower part, depending on the kind
and point of infection. Infection of the rachis causes blighting or dying
of the whole head above the point of infection. In such cases the dead
spikelets shrink and become more closely appressed to the rachis, while
the uninfected portions of the head continue their normal development to
maturity and become robust, with spikelets well filled, thus making the
difference between infected and uninfected parts still more striking.
The point of infection, even when the attack is in an advanced
stage, can easily be located, especially if the weather has been favorable.
It is usually covered at first with a short, cottony, slightly pinkish
fungous growth, while the rest of the infected area remains free from
such a growth. Later, if the weather is favorable, this growth extends
farther over the infected area and becomes the substratum on which a
layer of conidia develops. This layer of conidia may be smooth (pion-
notes) or more or less granular (sporodochia) , depending on the causal
organism and the age of the infection. The older it is the smoother it
becomes. The conidial masses, which were originally slightly pinkish,
now become dark salmon to grenadine in color, depending on the causal
Oct. i, igao Fusarium-B light (Scab) of Wheat and Other Cereals g
organism. The conidial masses tend to be more dense in the cases of
infection by Fusarium herbarum and F. avenaceum and less so in the
case of infection by Gibberella saubinetii and other Fusarium species.
Because of the fact that at the bases of the spikelets moisture from rain
or dew is held for a considerable length of time, the conidia are usually
formed here, extending along the furrow formed at the line where the
inner and outer glumes meet. In cases where the infection extends
down to the upper node, conidia may be produced on the node also.
They never form pionnotes but usually produce small sporodochia,
which are generally abnormal in size and shape.
Rye. — The symptoms of headblighting of rye are very much like
those of wheat, except that the water-soaked appearance is not so
prominent. The infection seldom extends as far down as the second
node before the plant naturally matures. Conidia are usually formed
only at the bases of the spikelets and in the furrow formed where the
inner and outer glumes meet and, to some extent, under the outer glumes.
In moist weather, however, conidia may be formed throughout the
infected area. Heads infected and killed at an early stage remain
straight, while normal heads are slightly bent.
Barley. — The symptoms of blight on barley heads are usually
different from those on wheat and rye, seldom resembling those on the
latter. Usually only one kernel is killed, or occasionally several kernels
in one row. In some cases the three kernels forming a spikelet are
attacked and later, if conditions are favorable, the rest of the head is
blighted. The first sign of infection is a small, water-soaked, somewhat
brownish spot appearing at the base or the middle of the glume or on the
rachis. The water soaking and browning spread in all directions from the
point of infection, soon including the whole glume, the whole spikelet, or
several spikelets, but the infection is by no means as uniform as it is in
wheat and rye.
Oats. — The symptoms of headblighting of oats resemble those of
wheat. Because of the structure of the panicle, however, the infection is
usually restricted to one spikelet and is therefore not so conspicuous as
it is in wheat or rye.
LIFE HISTORY OF THE CAUSAL ORGANISM IN RELATION TO
PATHOGENESIS
The life history of the parasite, so far as it is connected with that of
the hosts, has been followed by the writer through the entire year, and
is here briefly outlined.
PRODUCTION OF SPORES
CONIDIA
Production of conidia upon the host plant is more or less common in
all forms of Fusarium attacks on cereals. In many cases it may be
so abundant that it leaves no doubt as to the real source of inoculum for
subsequent infection in nature.
IO Journal of Agricultural Research voi.xx, No. i
On seedlings. — When a wilted seedling is pulled out and portions of its
partly decayed kernel or of the young stem are examined under the
microscope, a great number of normally developed conidia can fre-
quently be seen. In rare cases masses of conidia are also formed on the
rotted stem above the ground. The number of conidia so formed will
be still greater if any particles of organic matter like straw, old stems, or
stubble happen to be near the wilted or heavily infected plant, since the
conidia-forming growth will extend over them. This growth soon dis-
appears, however, leaving no evidence of its existence.
On nodes and bases. — Formation of conidia on the infected nodes or
bases of mature plants, while common, is never very abundant because
of the rapid drying out of these parts.
On heads. — The formation of conidia on the heads of cereal crops,
especially of wheat and rye, shortly after infection takes place is common
and so abundant as to give them a very distinct pinkish or salmon
color. In dry weather the formation of conidia is restricted to the area
where the infection originally took place, this being usually the base of
the spikelet where the rain drops collect and the moisture is held for a
longer time than on any other part of .the plant, except possibly in the
sheaths. The spore formation under such conditions extends up the
several furrows formed by the joining of inner and outer glumes and to
some extent even between the glumes. In moist weather the conidia are
formed in great abundance over the entire surface of the tissue through
which the hyphae of the parasite extend. The latter send out conidio-
phores through the stomatal openings, forming at first small balls of
conidiophores and conidia over each stoma. Soon these balls converge
into a uniform layer (pionnotes) of conidia extending over a large por-
tion of the head. The following observation in the field corroborates
this fact.
Before June 29, 191 8, the weather was dry and there were very few
conidia formed on the infected rye heads in the University experimental
plots. The last two days of the same month were rainy and compara-
tively cooler. Following this, conidia were formed in such abundance
that all the infected spikelets were practically covered with a layer of
conidia which gave them a distinctly pink or salmon color.
Dry, blighted rye, wheat, or barley heads without any conidia also
produced conidia in abundance when placed on the ground under a screen
and kept moist.
On dead organic matter. — Old straw and pieces of stems and corn-
stalks in fields where the year before the crop had been heavily infected
with the disease were often found to show large pinkish areas bearing
numerous conidia, some of which belonged to some of the species of
Fusarium which were found parasitizing wheat and corn. This condi-
tion was especially common on cornstalks and wheat heads left in the
field from the previous year and bearing the perithecia of Gibberella
Oct. i. i9*o Fusarium-B light (Scab) of Wheat and Other Cereals 1 1
saubinetii, thus confirming results obtained by Hoffer, Johnson, and
Atanasoff (j) in 191 8, when it was demonstrated that the hyphae present
in the previously infected heads or cornstalks remain viable till spring,
when they form new conidia and thus help the further propagation of
the fungus.
ASCOPORES
Whenever the cause of the disease is one of the species having a perfect
stage, as is the case with Gibberella saubinetii, the perithecia of this fungus
are produced in great number on all infected parts, but especially on
the pseudo-plectenchymatic structures, on which there has been more or
less formation of conidia. Perithecia are formed on seedlings and in-
fected kernels (observed only under greenhouse conditions), on the straw
and the heads of the various cereal crops, and on the stalks, sheaths,
and ears of corn. The ascospores play an important role in the life of
this organism, since they are likely to resist extreme weather conditions
and furnish inoculum for the first infection in the spring.
DISSEMINATION OF SPORES
The experimental work on this subject is limited to a study of the
agency of wind, and to some extent of rain, in distribution of conidia.
Other factors may also play some role in the dissemination of conidia
and ascospores, but time did not permit a study of other factors.
BY WIND
In a rye field slightly infected with blight, numerous spore traps l
were placed on stakes in vertical and horizontal positions, some on the
ground and some at various heights, ranging from 3 to 8 feet above the
ground, and exposed from 12 to 24 hours, then examined under the
microscope. The number of Gibberella saubinetii conidia caught was
very small when compared with the number of spores of other fungi,
especially rust spores, that was found on each spore trap. Gibberella
saubinetii conidia varied in number from none to eight on the traps set
closest to the ground and especially on those placed vertically and fac-
ing the prevailing wind. Most of the conidia of Gibberella saubinetii were
caught by the traps set on the ground. The statement that the conidia of
species of Fusarium are wind-borne is not new. Saito (7) , studying the at-
mospheric flora of Tokyo, found that Fusarium conidia are carried by the
air in small number. The same fact has been reported by a number of
other workers.
That the ascospores of Gibberella saubinetii are also wind-borne is
shown by the following observations in the field. One of the rye fields
under observation in 191 8, consisting of several acres, was located on
1 Common microscope slides were covered with a layer of glycerin, or glycerin with some vaseline, and
were used as spore traps.
12 Journal of Agricultural Research vol. xx, No. i
top of a hill. The field, which was only partly in rye, sloped at its west
end rather sharply to the south and at the east end sloped gently to the
south and east. The north side, the top of the hill, was fairly level and
protected by a wind-break of trees. To the east and west also there were
trees. The top or level part of the hill was sown with winter rye and the
sloping parts with second-year alfalfa in which barley had been the nurse
crop the preceding year. On the old barley stems left in the alfalfa
field were a considerable number of G. saubinetii perithecia with viable
spores. The only wind that could reach this field was from the south.
The rye field was as uniform as could be expected in all respects except
slope. The degree of head blight infection, however, was very different
in the different parts of the field, although it was only a small and narrow
strip of land. Blight was practically absent in the west part, which was
surrounded on the north and west sides by wind-breaks. However, on
the southwest edge there was considerable blight infection among the
plants that were immediately next to the alfalfa field in which, as stated
above, G. saubinetii was present and the slope was very steep. The
east part of the field, which was protected on the north and east sides
by wind-breaks, had, on the other hand, up to 5 per cent of blight, not
only among the plants next to the alfalfa field but also throughout its
south half, while its north half was free from blight. Knowing of no
other factors that could account for this difference, the writer is inclined
to think that the following is the possible explanation of the distribution
of the disease. The west end of the field bordering on the alfalfa field
where the slope was steep was infected only through the area next to
this field, because the wind, lifting the spores from the alfalfa field, could
not raise them into the upper air currents and so over the hill but deposited
them against the slope before they could reach the rye plants on the level
ground. Thus, only those rye plants were infected that were next to
the alfalfa field. In the east part of the field the situation was different.
The slope there was gradual and the spores needed to be lifted only
several feet in order to be on a level with the rye field. Thus they could
be easily carried to the rye plants even by the slightest air currents;
and for this reason, perhaps, the infection in this part of the field was
greater, although even here it was restricted to that half of the field
which bordered on the alfalfa field. This indicated that the source of
infectious material was the alfalfa field and that the infection extended
onlv as far as the topographical conditions permitted the wind to carry
the spores.
BY RAIN
The conidia produced at first are usually very loosely attached to the
mycelial growth and are easily detached from it by wind, insects, and
other agencies, while the conidia formed later and in pionnotes, as is
commonly the case, stick together. However, if a drop of water is
placed on the pionnotes the spores are set free with great rapidity and
Oct. i, 1920
Fusarium-Blight (Scab) of Wheat and Other Cereals 13
force, as shown by the fact that they are driven around in the drop
with considerable velocity. It is rather evident, therefore, that rain
assists in the liberation of conidia from the pionnotes, and thus they are
carried down to the ground or transmitted from plant to plant as the
plants wave in the wind.
Insects, no doubt, may also play some r61e in the dissemination of
Fusarium' conidia, but time did not permit a study of their importance.
TIME OF NATURAL INFECTION
The first blight infection in nature takes place during the latter part
of the blossoming period. It is, however, not the most severe one; the
secondary infections following shortly after the first being the ones that
are most destructive.
Several wheat, rye, barley, and oat fields, all located within 4 miles
of Madison, Wis., were selected for experimental purposes during the
spring and' summer of 191 8 and were examined every other day,
beginning about one week before the period of blossoming of rye and
two weeks before the blossoming of wheat, barley, and oats.
The following is a typical brief record of the observations on one of
the wheat fields :
Station No. 2. Town of Burke, Wis.
Field of Marquis wheat on corn ground. Field in level open country. Soil sandy
loam. vStand good. . ' . „,, « 1 it.
Tune 22 1918 Plants in blossom. No signs of blight infection. Throughout the
field there are numerous cornstalks with a great number of Gibberella saubinetn
perithecia with viable spores. ' ,
June 28, 1018. Wheat just passing blossoming stage. No signs of blight infection.
Ascospores in masses are oozing from Gibberella perithecia.
July 7 , 1918. First indication of blight infection apparent. It consists of a water-
soaked spot on single spikelets, usually on single glumes.
July 15, 1918. All suspected first infections have developed into distinct blighting
of the heads.
Following the first infection there may be as many successive infec-
tions as weather conditions permit.
This observation agrees with the results obtained with artificial inocu-
lations. Inoculation of plants before blossoming and following the
dough stage gave negative results. While the organism will attack
and penetrate the heads and the kernels in them during the latter part of
the dough stage and also after maturity, as demonstrated first by
Schaffnit (8) and later by Naumov (5), if there is abundant moisture and
warm weather, this can scarcely be spoken of as infection in the true
sense of the word. Wheat plants which were just heading out, others
which were just past blossoming, and a third lot which were in the late
dough stage were inoculated under exactly the same conditions, on the
same day, and with the same spore suspension. They gave the follow-
ing results: The first and third lots remained healthy during the first
14 Journal of Agricultural Research vol. xx.no. i
week, while the second lot showed ioo per cent severe infection and
the third lot remained free from the disease until full maturity. Some
of the plants in the first lot showed slight infection seven days from the
time of inoculation, during the time when they were in blossom. These
results show that the spores remain on the infected heads until the
heads reach a susceptible stage before infection takes place.
SOURCE OF NATURAL INFECTION
An important source of infection is the seed used for sowing. Cereal
seeds carry, externally, viable conidia of Gibberella saubinetii, as well as
of Fusarium spp., and many of the kernels are internally infected with
these fungi, as has been shown by Selby (9), Selby and Manns (u),
Schaffnit (<?), Bolley (1), Wollenweber (12), Naumov (5), and many
others. Many times the writer isolated G. saubinetii and several
Fusarium species from what seemed fairly normal wheat, barley, rye,
and oat kernels, as well as from kernels from blighted heads of the same
crops. In all cases G. saubinetii was the organism most commonly
isolated. Seed so infected carries the organism to the soil, where it
attacks the young seedlings if conditions are favorable. It passes the
winter in the soil, preferably on the killed seedlings or other organic
matter. In the spring it resumes its growth, producing new conidia
which when carried to other parts of the plant cause head or node
infection.
The perfect stage of this organism, which is formed in abundance on
infected heads, straw, or cornstalks, is an important source of natural
infection. The conidia of this organism, which are always produced in
abundance on the infected heads and stems, are the chief, if not the only,
source of secondary infection.
Whether Gibberella saubinetii, as well as the other Fusarium species
attacking the cereal crops, is present in the soil at all times and for long
periods of time, always ready to attack the susceptible hosts sown
on such soils, is an important phase of this problem to which the writer
has given no attention.
OVERWINTERING OF THE FUNGUS
The organism, because of its comparative resistance to cold and drying,
overwinters in various ways. When introduced into the soil with the
winter crops, it overwinters in the form of mycelium and conidia where
these are formed on the killed seedlings and on other organic substances.
It also overwinters in the form of mycelium in and on the seed, straw,
heads, and cornstalks that have been infected with the fungus the
summer before. The organism has been isolated from such plant parts
kept out of doors throughout the winter and spring. During the winter
Oct. i. i92o Fusarium-Blight (Scab) of Wheat and Other Cereals 1 5
of 1 91 8 it was frequently isolated from cornstalks fed to the cattle on the
University farm and from cornstalks that had been taken out into the
fields with the manure or for cattle feeding.
The mycelium of the organism present in infected straw and heads
of wheat, rye, and barley when stored in the laboratory at room tem-
perature and moisture was found viable after 12 months. In the
infected seed it remains viable even after the second year.
The undeveloped perithecia of the organism, which are often found in the
fall on the straw and heads of the cereal crops, on cornstalks and sheaths,
and on many grasses, are another form in which this organism over-
winters. In the spring these perithecia mature and form numerous
ascospores, which are later liberated from the perithecia and carried to
the various susceptible hosts. Mature ascospores in perithecia on wheat
heads and cornstalks preserve their viability for over 8 months when
kept in the laboratory at room temperature and moisture.
DESCRIPTION OF CAUSAL ORGANISM
TAXONOMY
The chief cause of headblight and one of the chief causes of rootrot
of the cereal crops in the United States is Gibber ella saubinetii (Mont.)
Sacc. The following is a list of synonyms :
Gibberella saubinetii (D. and M.) S., 1879, in Michelia, v. 1, p. 513.
Gibbera saubinetii Mont., 1856, Syll. Gen. Spec. Crypt., p. 252.
Botryospkaeria saubinetii (Mont.) Niessl, 1872, in Verhandl. Naturf.
Ver. Briinn, Bd. 10, p. 195, pi. 4, fig. 29.
Fusarium graminearum Schwabe, 1839, Fl. anhalt, v. 2, p. 285, pi. 6,
fig. 7; Sacc. Syll. v. 22, p. 1483-1484, 1913.
Gibbera pulicaris (Fr.) f. zeae maydis, Rehm: Ascomyceten 381. From
New Jersey, 8, 1875, J. B. Ellis.
Fusarium roseum Autorum.
Fusarium tropicalis Rehm, 1898, in Hedwigia, Bd. 37, p. 194. Is
probably a synonym of Gibberella saubinetii according to Wollenweber
{12).
Gibberella tritici P. Henn., 1902, in Hedwigia, Bd. 41, p. 301.
Fusarium rostratum App. and Wollenw., 1910, in Arb. K. Biol. Anst.
Land u. Forstw., Bd. 8, p. 30.
MORPHOLOGY
Perithecial stage. — The following description of the perfect stage
of this organism, given by Wollenweber (12), is adequate:
Diagnosis. — Perithecial stage : Perithecia scattered or gregarious, ovoid to subcorneal ,
free on the surface of the host as well as embedded in mycelium, or on a tubercular
plectenchymatic stroma, which may either push in sphaerostilbe-like bodies through
i6
Journal of Agricultural Research
Vol. XX, No. i
the surface of the host or remain endophytic, 150 to 250 by 100 to 250 m- Peridium
smooth and small-celled at the basal part, but large-celled, verrucose occasionally,
with protuberance-like projections of cell groups near the apical end, black to the
unaided eye (turning red-brown with acid reaction), dark blue with transmitted light
except the almost colorless often rather prominent beak; asci up to over a hundred
in each perithecium, intermixed with a few celled paraphyses; ascospores, 8 in one row
or irregularly in two rows, subdorsi ventral, fusiform, slightly curved, tapering at the
ends, ochreous in masses; largely 3-septate, 20 to 30 by 3.75 to 4.25 /u (up to 5 n in
diameter in germination, indicated by constrictions at the septa).
Conidial stage. — In shape the conidia (fig. 1) strongly resemble the
conidia of Fusarium culmorum. but they lack the constriction toward the
base so prominent in F. culmorum.
They differ also in being longer and
more slender and in having thinner
walls and less prominent septa; coni-
dia typically, sometimes up to 100 per
cent, 5-septate, 45 to 65 fj. by 4.2 to
5.5 m; 3-septate, 35 to 45 u by 5 to 5.5
n; seldom 4-septate; rarely 6-, 7-, or
more septate, 60 to 75 n by 4 to 5 /x;
ochreous in mass. Chlamy dospores
absent. Carmine red pigment on
starchy, neutral media.
Habitat. — This species is one of the
most widely distributed species of
Fusarium within the temperate zone,
causing headblight and rootrot of
wheat, emmer, rye, oats, spelt, and
corn in the United States, Germany,
Russia, Italy, Denmark, Sweden, and
probably elsewhere. Wollenweber iso-
lated it from berries of Solatium tuber-
osum near Berlin, Germany. C. A.
Ludwig isolated the same from Ipomoea batatas in storage at La
Fayette, Ind. The writer found the perithecia of the fungus on
Bromus, timothy stems, clover, and alfalfa, and also on Triticum repens
which had been plowed under. The fungus was also isolated from aspara-
gus stems collected at Baraboo, Wis., by Mr. E. H. Toole. According to
Saccardo (6, p. 313), the fungus occurs on dead stems of Angelica, Aspar-
agus, Beta, Clematis, Conium, Cannabis, Convolvulus, Cucurbita, Gyn-
eria, Phytolacca, Scirpus, and Stipa, and on branches of Buxus, Coronilla,
Fraxinus, Gleditschia, Juglans, Robinia, Rubus, Rosa, and Ulmus
in Europe, Algeria, North America, and Australia. A. D. Selby (10)
adds Emmer, Trifolium, and Medicago as new hosts. It has been found
also on Glyceria aquatica in Germany, on rice in Japan and Italy, and on
Triticum spclta in S. Paulo, Brazil.
Fig. i. — Conidia of Gibberella saubinelii.
Oct. 1. 1920 Fusarium-B light (Scab) of Wheat and Other Cereals 1 7
METHOD OF PERFECT STAGE DEVELOPMENT
In nature. — A limited study of the field conditions under which the
perfect stages of some Fusarium species which parasitize the cereal crops
and numerous grasses are formed showed that those conditions are as
follows :
(1) Successful parasitism of the fungus on some host. The perithecia
are formed usually and preferably on those dead parts of the host which
have been parasitized.
(2) Successful conidia production. Conidia production on the in-
fected substratum, root, stems, or heads always precedes the formation
of perithecia, since the latter are formed more readily on the crust or
plectenchymatic layer formed by the conidia- bearing hyphae and the
germinated masses of conidia themselves.
(3) Presence of moisture. No perithecia will ever be formed in the
absence of sufficient moisture, and their formation will be delayed until
moisture is sufficient.
(4) Suitable temperature also must play some role in the formation
of the perithecia. Formation of perithecia took place only during the
summer when the temperature was highest. Efforts to develop the per-
ithecia from infected material during October and November gave nega-
tive results.
When the foregoing conditions were established as factors in the for-
mation of perithecia, the following method of producing them was
worked out and has yielded good results. The infected parts of the various
cereals, including corn, such as stems, nodes, sheaths, heads, and ears,
were gathered from the field and laid on the ground during July and
August, 1 91 8, then covered with a wire screen, moistened thoroughly, and
covered with some dry grass and leaves to protect them from drying out.
During the first and second weeks, masses of conidia were formed over the
entire infected area of the various parts. Soon this extended even over the
uninfected area. Before long all conidia germinated and no others were
formed. During the third week the perithecia began to be formed. In
three or four more weeks numerous perithecia were formed, most of them
with matured ascospores.
The following is a record of one of the experiments for perfect-stage
development :
June 28, 1918. Rye heads infected with Gibberella saubinetii were placed under
screens so as to be exposed to the action of the weather. They were sprayed
thoroughly with water and covered with dry grass to protect them from drying out.
July 16, 1918. First perithecia beginning to appear.
August 2, 1918. Numerous perithecia formed, but asci not yet fully developed.
August 21, 1918. All perithecia have ripe ascospores. Heads taken to the laboratory
for study.
In laboratory. — Infected wheat kernels, when placed in a pot filled
with fine sand and only slightly covered with sand and kept moist at
187931°— 20 2
i8
Journal of Agricultural Research
Vol. XX, No. i
room temperature, produced numerous perithecia on their exposed
surfaces. These matured before the end of the fourth week from the time
of sowing. As soon as the ascospores in the perithecia were found to
be mature, the kernels were sifted from the sand and preserved in dry
condition until needed for study or inoculation.
The development, in the laboratory, of perfect stages of those species
of Fusarium which have a perfect stage was secured in the way originally
described by Appel and Wollenweber and later extended by Wollenweber.
It need only be emphasized that the perithecia of these fungi will rarely
be formed until the transfers and cultural work are begun from what
these authors call "normal" culture. Failure is bound to occur 95 times
out of 100 before the culture which is to be used
for development of the perfect stage is brought to
this condition.
Once the culture is in the proper condition, the
next step consists in transferring it to media that
are known to favor the development of the peri-
thecia, such as stems of any kind, but especially
those of Melilotus alba, bean pods, etc.
Care must be taken that the cultures on Melilo-
tus alba stems or other media are kept uniformly
moist until the perithecia are formed and the
ascospores in them are ripe. The presence of
certain bacteria in the cultures greatly favors the
formation and proper development of the perfect
forms of species of Fusarium. A certain bacte-
rium which was found in a contaminated culture
when added to cultures of Fusarium having per-
fect forms favored the formation of perithecia so
much that practically 100 per cent of the cultures
to which this organism was added developed
numerous normal perithecia, while even under best
conditions only a small number of the cultures to
which this bacterium was not added produced perithecia. What this organ-
ism is and whether other bacteria can produce the same result are not
known.
Heretofore the whole work of producing the perfect stage of any
ascomycete in pure culture has been handicapped to a certain extent
by the fact that the cultures of such fungi dried out long before the
formation and ripening of the perithecia. The addition of water to the
cultures from time to time exposes them to contamination and varies
the amount of moisture in the culture considerably. To avoid this the
writer designed a special culture tube. This consists of a common test
tube, to the lower end of which is attached a bulb (fig. 2). When the
bulb is filled with water it will drain into the test tube as rapidly as the
Fig. 2. — Special culture tube
for maintaining moisture in
culture. X Vi.
Oct. x. I92o Fusarium-B light (Scab) of Wheat and Other Cereals 19
water from the test tube evaporates or is used by the fungus. Such a
tube provides stem or potato plug cultures with uniform moisture for
four or five months without being refilled. This is as long a period of time
as is necessary for the formation of perithecia in any case. When stems
are used they can be placed directly in the test tube so as to reach the
bottom, but when potato plugs, bean pods, or other cultural substrata
are used it is better to place some cotton on the bottom of the test tube
so that the plugs will be just above the water level. Such test tubes are
handled in very much the same way as common test tubes, except that
more care should be taken in sterilizing them, since a sudden decrease
in the pressure in the sterilizer is likely to force the water out of the bulb
into the tube.
PATHOGENICITY
PREVIOUS INVESTIGATIONS
A large number of Fusarium species have been reported by various
workers as attacking the cereal crops in one way or another. In a large
number of cases the particular organisms have been wrongly identified
or not identified at all. The true relation of the various Fusarium
species to the different diseases on the cereal crops attributed to these
species is even less understood than their taxonomy. Indeed, there
are but few papers out of over 200 references in which proof of the
pathogenicity and true relation of some of these organisms to certain
cereal diseases is given. No papers except those most directly con-
nected with the problem can be mentioned here.
Selby (9) considered Gibber ella saubinetii and its conidial form which
he, following Saccardo, called Fusarium roseum, as the cause of the
blighting of wheat heads, but he failed to produce the disease by inocu-
lating heads with the conidia and ascospores of this organism. In
1909, Selby and Manns (11) succeeded in producing blighting of wheat
and oat heads by spraying them during moist weather with a suspension
of conidia obtained by washing samples of wheat, barley, oats, emmer,
and spelt. In this way they thought they obtained the conidia of
F. roseum and its perfect form, G. saubinetii. It is very likely that it
was the conidia of G. saubinetii that caused blighting of the heads in their
experiment, but it is incorrect to suppose that conidia of only this species
of Fusarium are found on samples of cereals. They also showed that
pure cultures of G. saubinetii from various sources when added to sterile
soil in which wheat and oats were sown caused severe rotting of the
roots and killing of the young seedlings.
Schaffnit (8), studying the cause of what is known as "snowmold"
in Europe, showed that while Fusarium nivale Ces., the conidial form of
Nectria (later Colonectria) graminicola, is the primary cause of "snow-
mold" of the cereal crops in Europe, the following organisms are also
more or less responsible for this disease: F. culmorum (F. rubiginosum),
20 Journal of Agricultural Research vol. xx. no. i
F. herbarum (F. metachroum) (13), F. didymium, F. avenaceum (F. subu-
latum), and F. lolii. He showed also that F. nivale causes not only the
snowmold but also rotting of the roots and killing of the young cereal
seedlings. Later it causes footrot of the grown plants, usually following
the wounding of the plants by insects or other agencies. F. nivale
attacks the heads of the cereals during the period beginning at blossom-
ing time and extending to the ripening of the crops and causes blighting.
In this connection he distinguished between primary infection, which
takes place before the ripening of the plants, and secondary infection,
during the period of maturity and harvest. In the secondary infection
he found that not only F. nivale but also less parasitic Fusarium species
play an important r61e.
Naumov (5), studying the cause of cereal headblighting, which is
reported to be severe throughout Russia, found that Gibber ella saubinetii
and Fusarium avenaceum (F. subulatum) are the cause of this disease in
Russia and Siberia, the first being common in the southern and the
second in the northern part of the country.
Studying the pathogenicity of these organisms, Naumov reported
that:
(1) Infection of the soil will result in the blighting of heads of wheat
and barley. How the organisms introduced into the soil under sterile
conditions reach the heads of the plants where they cause blighting is
not quite clear. Throughout the paper Naumov states that the myce-
lium of these Fusarium species is found in all parts of the plants, but it
is not very clear whether infection in the roots and the lower parts of
the plant proceeds up the stem, becoming systemic, or whether the
various parts are infected separately by external infections. Though
this view is not directly and plainly stated, in many cases the reader
will be led to believe that Naumov considers the infection systemic
and that it proceeds from the roots up to the heads, since in many places
in this paper he speaks of finding the mycelium of these organisms in
all the tissues of roots, stems, heads, leaves, and sheaths, but nowhere
causing any anatomical changes.
(2) Spores or conidia of the causal organisms when on the seed, or nat-
urally infected seed, can cause blighting of the seedlings.
(3) Conidia, ascospores, and mycelium of the organisms, when placed
on normal young plants, with or without wounding, cause infection.
(4) Spraying the heads of wheat, rye, and oats with a water suspen-
sion of conidia of these organisms produced typical blighting of the
infected heads as observed in nature.
(5) The results given under (4) were also obtained with ascospores of
Gibberella saubinetii.
(6) These organisms can invade the tissues of the seed, straw, and
heads of the cereal crops after ripening and harvesting if conditions are
favorable.
Oct. 1,1920 Fusarium-B light (Scab) of Wheat and Other Cereals 21
EXPERIMENTAL RESULTS
Isolations. — In the vicinity of Madison, Wis., where the writer secured
most of his material, Gibber ella saubinetii is the most common and most
important cause of the headblight of the cereals, and the writer believes
this to be true throughout the country. The following Fusarium species
were also isolated from blighted heads and other parts of the cereal plants :
Fusarium avenaceum, 10 times — 4 times from wheat heads from a field
near Madison and 6 times from a single sample of 10 blighted spelt heads
from Hawthorne, which is located in the extreme northwestern part of
Wisconsin; F. herbarum, 8 times — 3 times from blighted wheat heads
from a lodged wheat field near Madison, Wis., and 5 times from corn
stalks; F. culmorum, once from a blighted wheat head from Arlington,
Va. ; F. culmorum var. leteius, twice from blighted wheat heads from a
lodged wheat field near Madison, Wis.; F. arcuosporum, 10 times — once
from a blighted oat seedling and c) times from barley heads left in the
field late in the fall and cornstalks early in the spring; F. scirpi, four
times from blighted wheat heads from a lodged wheat field and once from
a blighted wheat head from one of the Experiment Station plots at Madi-
son, Wis., which was badly overgrown with weeds; F. solani, once from a
grown wheat plant showing footrot; F. arihrosporioidcs, 5 times — once
from a blighted wheat head from a lodged wheat field and 4 times from
blighted barley heads; F. redolens, 3 times — once from a discolored rye
stem near a node, once from a blighted wheat head from Knoxville, Tenn.,
and the third time from a blighted barley head from a weed-overgrown
plot in the Experiment Station field, Madison, Wis.
On the other hand, Gibber ella saubinetii was identified by the writer on
over 2,000 blighted wheat, barley, rye, oat, and spelt heads from various
parts of the following States: Wisconsin, Illinois, Minnesota, Indiana,
Maryland, Kentucky, Ohio, Virginia, West Virginia, South Carolina,
Georgia, Alabama, North Dakota, and Michigan. This shows that, from
the standpoint of headblight of the cereal crops, G. saubinetii is the most
important organism.
All species of Fusarium given here, including Gibberella saubinetii, were
isolated originally by poured-plate dilution of conidia from distinctly
blighted wheat heads. During the course of the work, however, some of
these species were often isolated from blighted rye, barley, and oat heads,
or stems, and from sheath, shank, root, and node rots of corn, or in a few
cases from other hosts. The organisms attacking the cereal crops above
the ground produce numerous conidia over the infected area. The conidia
so produced are often normal and uniform in size and shape, and the
trained student will not only have no difficulty in separating the various
species before he has grown them under artificial conditions but he will
be able also to determine in a general way the various species, at least
the various sections to which they belong.
22 Journal of Agricultural Research vol. xx.No. r
In order to prove that the Fusarium conidia produced on a blighted
wheat head are the conidia of the causal organism and not of a secondary
organism which has followed the first, parts of a large number of blighted
wheat heads were washed in distilled water to moisten them and then
disinfected by dipping them in i to 1,000 mercuric chlorid solution
(HgCl2) for two minutes. After this they were rinsed in distilled water
and then transferred with a sterile needle to cooled poured plates of a
suitable medium. In all cases only one organism was isolated from each
blighted head, and this was in all cases the same as the one obtained from
the conidia on this head. This is so true of the Fusarium organisms
causing headblight that the causal organism upon a clean, undiscolored
Fusarium-blighted head may almost surely, and even without micro-
scopic examination, be described as one and pure. In rare cases the
blighted heads may also be smutted, rusted, or brown spotted and dis-
colored; and in such cases, of course, more than one organism may be
found on a head. Such heads were discarded and never used for study
or isolation.
Plain water agar ' was used for diluting the conidia and for pouring
the plates. After 12 to 24 hours the plates were examined micro-
scopically, and single, germinating conidia were marked on the plate;
then with a sterile needle made for the purpose they were transferred to
test tubes containing suitable medium, usually hard oatmeal agar. In
all cases five single, germinating conidia were transferred, with only one
to each test tube. This was done to make sure that there was not more
than one species of Fusarium present. Except in rare cases when some
of the test tubes were contaminated during the manipulation with for-
eign organisms such as Penicillium or bacteria, all five test tubes yielded
the same species. To make certain, however, that the cultures were
free from bacteria they were transferred to plates, and second transfers
were made from the margins of the plate colonies. The pure cultures so
obtained were used as stock cultures for further study.
Inoculation work. — In this paper only the results of inoculation
with Gibberella saubinetii are given. The writer was able to produce
blighting of heads of wheat and rye by inoculation with several of the
species mentioned above and was able to produce more or less severe
seedling-blight by inoculation with nearly all of them, but the condi-
tions under which these species become pathogenic are not yet well
understood.
Seed and soil inoculation. — A number of methods have been used
in artificially infesting soil with species of Fusarium. Most of them
consist in growing the particular organism on a suitable medium and
then introducing the whole culture into sterilized soil. Such a method
is very good, except that it is an artificial one which does not reproduce
1 One liter of distilled water and 25 gm. of bacto-agar.
Oct. i, 1920
Fusarium-B light (Scab) of Wheat and Other Cereals 23
the conditions that actually exist in nature. It introduces into the
soil various substances, toxins perhaps, which may have some effect
upon the final results. In order to avoid this and to make conditions in
the greenhouse as natural as possible, only conidia were employed for
inoculation of the soils used for testing the pathogenicity of Gibberella
saubinetii on young seedlings. Practically all Fusarium species when
grown under proper conditions will produce large masses of conidia,
which can be gathered from the substratum with a flat needle, free from
any conidiophores or mycelial hyphae, and suspended in a test tube or
flask of sterile distilled water. If the conidia are not abundant, a fairly
heavy conidial suspension may be obtained by washing the culture
with sterile distilled water and straining the water through sterile cheese-
cloth. Suspensions of conidia thus obtained were used for inoculating
the seed by dipping the seed into it for a few minutes. Spore suspen-
sions thus obtained were used for artificially infesting sterilized soil
by pouring part of the suspension upon the soil in each of the pots and
mixing it with the upper layer of soil. By this method only a com-
paratively small number of conidia and only a negligible amount of
foreign matter were introduced into the soil.
In all the soil experiments the soil used was sterilized in pots in
an autoclave for 1 hour at 15 pounds pressure. All the seed used for
sowing was placed for several minutes in a weak solution of saponin }
and shaken hard, the object being to moisten the seed thoroughly and
to remove all air bubbles adhering to it. The seed was then soaked for
30 minutes in 1 to 1,000 mercuric chlorid solution. Seeds so treated
proved to be perfectly sterile on the outside. However, the fungi
present in their internal tissues are not affected by this treatment. For
this reason, only seeds that were comparatively free from such fungi and
healthy in appearance were used for experimental purposes.
Throughout the work 6-inch and 12-inch pots and garden soil were
used for sowing the seed. In each case two pots were planted with
infested soil or seed, and one pot was sown as a control. Each
experiment was repeated several times.
Seed of wheat, rye, barley, and oats naturally or artificially infected
with Gibberella saubinetii, or planted on sterile garden soil artificially
infested with this organism, showed a decrease in germination. In the
case of the seed naturally infected, the decrease in percentage of germi-
nation is greater and is variable, depending on the degree of infection
and percentage of seed infected. This may vary from 2 or 3 per cent
to as high as 50 per cent. Artificially infected seed or seed sown on
infested soil also shows a lower percentage of germination than the con-
trols similarly planted. Here, too, percentage of germination depends
on the kind and condition of the seed. It may vary from o to as high as
1 One hundred cc. of so per cent alcohol and i gtn. of saponin.
24
Journal of Agricultural Research
Vol. XX, No. i
15 per cent. Good, healthy, plump seed may show no decrease in
germination, while weak and shriveled seed may show considerable
decrease in germination.
Gibberella saubinetii, besides preventing some of the seeds from germi-
nating, attacked from 10 to 40 per cent of the young seedlings, causing
rotting and browning of their roots, bases, and sheaths (PI. 3, A). A
number of the plants so attacked, usually few under normal conditions,
rot and die before reaching the surface of the soil. Others wilt and die
after reaching the surface, while the large majority recover almost
entirely and attain practically normal development. Over 20 spring-
wheat plants which showed marked rotting and browning of the roots
and bases caused by this organism while they were grown on sterilized
soil from infected seed in pots out of doors, when transplanted to the
pathological garden recovered rapidly and reached full development,
producing heads as normal as those on the control plants. Only 2 of the
plants so transplanted wilted shortly after the transplanting, and the
writer is inclined to attribute the wilting more to the transplanting than
to the parasitism of the organism. This fact shows that, although G.
saubinetii when present on the seed will infect many of the seedlings, it is
not able to injure them materially unless the plants are growing under
extremely unfavorable conditions, as was the case with the plants shown
in Plate 2, B. In this case, the experiment was conducted during Febru-
ary, 191 8, at a time when there was a minimum of sunlight in the green-
house and when all the greenhouse plants were consequently weakened.
The results of the experiment are summarized in Table I.
Table I. — Average results of two inoculation experiments on each of 2 "wheat samples,
sample I consisting of hand-picked, healthy, plump kernels, and sample 2 consisting of
hand-picked, healthy, but average kernels sown May 23, 1919, in pots kept out of doors
Sample
No.
Number of kernels.
Germina-
tion.
Number of
healthy-
plants.
Number of
plants
showing
rotting
of
roots and
bases.
Number of
killed
plants.
/Control, 100. . .
\ Inoculated , 100
j Control, 100. . .
\ Inoculated, 100
Per cent.
90
76
69
89
75
7i
4?
IS
5
27
While it was shown by numerous experiments that Gibberella saubinetii
is able to decrease the percentage of germination of wheat, rye, barley,
and oats and to cause rotting and browning of the roots and bases of some
of the seedlings and even to cause wilting and dying of others, it was also
noticed that this varied considerably from time to time and that some
factors like light, temperature, moisture, and soil conditions have much
to do with the degree and severity of infection.
Oct. 1,1920 Fusarium-Blight (Scab) of Wheat and Other Cereals 25
Winter wheat, disinfected as described above, artificially inoculated
with conidia of Gibberella saubinetii, and sown October 20, 191 8, in
five 12-inch pots of sterile soil with 10 kernels in each pot, was left in the
greenhouse for 15 days and then taken out of doors, where it remained
till July, 1 91 9. A similar series of spring wheat similarly treated was
sown on April 2 1 in pots of the same size but was left out of doors from
the time of planting. Two pots sown with similarly treated but unin-
oculated seed were used as controls for each of the two series. In both
series the plants recovered rapidly from the primary attack and grew
normally, giving plants which were apparently normal, except that their
bases and roots were slightly rotted and browned. With the coming of
dry weather during the second half of June this rotting and browning of
the roots and especially of the bases was intensified somewhat, and the
plants began to wilt suddenly. In the field, wilting usually takes place
at the time of heading or shortly after. The general symptoms accom-
panying wilting of fully developed plants are somewhat similar to those
described for the footrot of the cereals in Europe and for "take-all" in
Australia. G. saubinetii was isolated from the browned and rotted bases
of the wilted plants in the foregoing experiments, as well as from those of
some of the similarly wilted plants in the field.
Head inoculation. — While much work must be done before the nature
and exact importance of the parasitism of Gibberella saubinetii on the
underground portions of the cereal crops and the factors influencing or
controlling it are fully understood, the question of headblighting due to
this organism is much easier to follow and is, therefore, better understood.
The methods used in testing the pathogenicity of Gibberella saubinetii
on wheat, rye, barley, oats, spelt, brome grass, quack grass, and timothy
are very simple. They consist in producing a heavy suspension of
conidia, either from heads already infected or from pure cultures, and
spraying it by means of a small atomizer on a number of heads, usually
10, of the various hosts mentioned above when they are in the proper
condition for infection. This method is successful when the weather is
moist and cloudy. In dry weather this method will give either no
results or only a very small percentage of infection. Certain results can
be obtained only when the infected heads are in some way kept moist for
at least three days after inoculation, and even this method will not give
good results during extremely dry and hot weather. In the work described
above the heads were kept moist by placing some moist absorbent cotton
around the stems of a group of heads, then covering both the heads and
the bundle of cotton around their stems with a glassine bag. The open
end of the bag was tied around the stems just below the bundle of cotton.
The heads so treated were heavy and required support. For this reason,
garden stakes 5 or 6 feet tall were driven into the ground near the plants,
and the bags covering the heads were tied loosely to them. The moist
cotton inside of the bag kept the air comparatively moist and created
26 Journal of Agricultural Research vol. xx. No. i
the condition desirable for successful infection. Since the glassine bags
were transparent, the heads were not seriously deprived of sunlight.
When the weather was very dry and warm the bags had to be opened
and the cotton again moistened to saturation. All controls were treated
in the same way as inoculated plants, except that they were sprayed
with water to which no spores had been added.
Since Gibberella saubinetii usually produces very few conidia in culture,
and since large quantities of spores were required for inoculations, it was
necessary to contaminate the cultures purposely with a certain bacterium
which has been found to bring about a great increase in sporulation. In this
way large quantities of spores could always be obtained. The bacterium
has not been identified, and the nature of its effect upon cultures of G.
saubinetii is not known. Further study of this relationship is planned
for the future.
The employment of such conidia for inoculation naturally raises the
question whether the bacterium present has some effect on the patho-
genicity of Gibberella saubinetii or whether it itself is pathogenic on
wheat. In order to establish this, numerous wheat heads were inocu-
lated at the same time with pure G. saubinetii conidia and others
with a suspension of a pure culture of the unidentified bacterium. In
all cases the heads inoculated with G. saubinetii conidia became blighted,
while all heads inoculated with the bacterium suspension remained per-
fectly free from blighting or other injury. This shows that the bacte-
rium favoring the sporulation and perithecia formation of G. saubinetii,
as mentioned before, is not pathogenic on the wheat heads and has no
effect upon the pathogenicity of G. saubinetii.
Wheat, spelt, rye, barley, and oat heads, as well as heads of Agropyron
repens when inoculated with a conidial suspension or an ascospore sus-
pension of Gibberella saubinetii became blighted. The blighting of A.
repens proceeded exactly as observed in nature. In over ioo inoculation
experiments in which over 3,000 heads of the various cereals, mostly
wheat heads, were concerned, some infections always resulted. The
number of blighted heads in each experiment varied from over 50 per
cent to 100 per cent. In the majority of the experiments, all inoculated
heads became infected and typically blighted. On many of these heads
conidia were formed, and on some even the perithecia of G. saubinetii
developed before the harvesting of the plants.
The inoculation experiments gave positive results from the time of
blossoming till the latter part of the dough stage. Inoculation made
before the first and after the second stage gave either negative or very
doubtful results.
PERIOD OF INCUBATION
On seedlings. — The period which elapses between the inoculation
and the time the first symptoms of attack on the seedling roots appear
varies so much that no definite incubation period can be given. It varies
Oct. i, 1920 Fusarium-B light (Scab) of Wheat and Other Cereals 27
considerably with the condition of the seed used. When light, shriveled
seed is sown on infested soil, or when such seed is inoculated by being
dipped in a suspension of conidia and then sown on sterile soil, the
seedlings will succumb to the attack of the parasite much more rapidly
than when healthy seed is used. Abundant watering of the plants also
increases to some extent the rapidity of the attack.
In general, under greenhouse conditions, the first symptoms of root
infection appear not earlier than the seventh day after sowing. Infec-
tion is usually abundant after the fourteenth day. When naturally
infected seeds have been used on sterile soil the symptoms of root infec-
tion may appear even before the seventh day.
On heads. — In head infection there is much less variation in the incu-
bation period. In damp weather, the period that elapses between
inoculation and the appearance of the first symptoms (water-soaking)
varies from three to six days. In dry weather, symptoms of infection
may not appear until after the first rain, or if heavy dew falls during the
night and lasts for the greater part of the forenoon, symptoms of infection
may appear from five to eight days later.
The rapidity with which the blight infection spreads from the point
of infection to the rest of the head varies greatly. It varies considerably
with different individuals and depends much upon the kind of weather.
On healthy, vigorous, and more succulent plants the infection spreads
much more rapidly than on plants of average vigor. Moist and cloudy
weather, followed by warm and clear weather, greatly accelerates the
rapidity of infection and killing, yet even under such conditions the
infection may be restricted on many heads to a single spikelet, the rest
of the head remaining healthy and developing perfectly normal, plump
kernels.
For the study of the rapidity of the spread of the disease from the
point of infection, heads showing primary infection were located daily
and marked with tags so that they could be located again. Heads so
tagged were examined every two or three days and the changes recorded.
In this way the effect of the various factors affecting the rapidity of
blight infection and killing were studied. The following are typical
records of some infected heads, made in 1918:
N 1009, July 11, 1 spikelet infected. Infection at base of head.
July 14, 4 spikelets infected.
July 17, whole head killed.
N 101, July 11, 5 spikelets infected. Infection at middle.
July 14, 8 spikelets infected.
July 17, whole head killed.
N 1038, July 9, third spikelet from bottom infected.
July 14, 4 spikelets infected.
July 17, Whole head killed.
N 1039, July 9, 1 spikelet infected. Infection at middle.
July 14, 4 spikelets infected.
28 Journal of Agricultural Research vol. xx.No. i
N 1039, July 17, 12 spikelets infected.
July 24, whole head killed.
N 1 1 56, July ii, uppermost spikelet infected.
July 24, 1 spikelet infected. Plant almost ripe. Infected spikelet covered
with Fusarium conidia.
There has been considerable discussion as to whether the headblighting
of the cereal crops caused by Gibberella saubinetii and some other Fusarium
species is the result of a systemic invasion of the host plants by these
organisms. Naumov (5) , as stated before, considers the invasion systemic.
He finds the mycelium of the fungus in all parts of the plants and even
in plants showing no blighting of the heads. He showed that infection
of the heads can also take place externally.
Since there is uncertainty in determining from its appearance the kind
and nature of any mycelium that may be present in the tissues of the
cereal plants, it was thought that the easiest and only reliable way to
show whether certain plants carry in their tissues the mycelium of
Gibberella saubinetii or any other Fusarium species would be to plate
out portions of such plants on some suitable artificial medium on which
the organisms are known to thrive well. If they are present in the tissues
of the plated plant they are sure to appear on the plates.
Wheat and rye plants with blighted heads where the infection from the
heads has extended to the upper part of the upper internode, as previously
described in this paper, were used for plating. Such peduncles were
cut in portions 1 inch long, beginning from the end next to the blighted
heads. These portions were disinfected on the outside by dipping them
in 1 to 1 ,000 mercuric chlorid for two minutes. They were then rinsed
in sterile distilled water and plated in order on hard potato agar. In all
cases colonies of Gibberella saubinetii were formed over the portion next
to the infected head and in some cases over the adjoining portion. The
portion of the peduncle which was farthest from the head and perfectly
green and free from discoloration never developed any fungous growth
(PI. 3, B). This shows very conclusively (1) that the infection on the
cereal heads is local, and (2) that it proceeds from the head down and not
from the roots up.
WEATHER CONDITIONS IN RELATION TO HEAD INFECTION
Weather is one of the important factors for the successful parasitism
of Gibberella saubinetii and the various Fusarium species on the cereal
crops. Indeed, it is the limiting factor for the occurrence of head-
blight under certain conditions, and its importance was noticed early
by students of the subject. Dry weather with slight winds during and
after the period of blossoming and extending well toward the dough
stage will practically eliminate blight infection though all the other
necessary conditions may be present. It was observed in many cases
that in fields where there have been only few blighted heads before the
Oct. 1. 1920 Fusarium-B light (Scab) of Wheat and Other Cereals 29
coming of rains and cloudy weather there was a marked increase in the
number of blighted heads only a week after the rain. This was shown
very plainly in experiment 22, one of the inoculation experiments in
1 91 8,
At 7 o'clock in the afternoon, July 2, 191 8, 60 wheat heads in one of
the Wisconsin Experiment Station plots were sprayed with a suspen-
sion of Gibber ella saubinetii ascospores and left uncovered.
On July 8, 1918, 12 heads, or 20 per cent, showed signs of first infec-
tion. Several days later there came a slight rain and the sky was cloudy
for over a day. By the twentieth of the same month 28 heads, or 45
per cent, showed symptoms of blighting.
On the other hand, an experiment, which differed from the fore-
going only in that the heads were kept moist artificially (see inocu-
lation experiments, p. 25), showed 70 per cent infection on July 7,
1 91 8. The number of the infected heads did not increase after the
rainy and cloudy weather that followed. All controls in both experi-
ments remained healthy. This case, which is one of several, shows
that in the absence of proper weather conditions there is much less
infection than when the weather is favorable. In experiment 20, in
which the heads were kept moist, all the heads that were successfully
infected showed infection within six days, and the coming of rain in this
experiment did not increase the number of infected heads.
Not only does rainy and cloudy weather favor blight infection but it
is also necessary for spore production, as already pointed out in this
paper.
CULTURAL CONDITIONS IN RELATION TO HEADBLIGHT
Even though they were well developed and still apparently healthy
and normal, the plants which were in shady places or overgrown by
weeds were attacked by headblight and noderot to a much greater
extent and by a greater number of the species of Fusarium than were
plants which had a normal amount of sunlight. This was especially
evident in one of the Wisconsin Experiment Station plots where a small
area sown with barley and wheat was allowed to be overgrown by weeds.
The blight infection on this plot was so abundant that in some small
areas practically all the plants were infected. In general, the whole
field had an average of 10 per cent of infection as compared with 5 per
cent from neighboring clean fields. Another interesting fact was that
nine different species of Fusarium, two of which have perfect stages,
were isolated from blighted heads gathered from this small plot cover-
ing not over 200 square yards. Gibber ella saubinetii was the most
common and most destructive species.
Lodging of the fields also gives a marked increase of headblight infec-
tion. This was brought out especially well in a wheat field located two
miles northeast of Madison, Wis., where the head infection among the
30 Journal of Agricultural Research vol. xx, No. i
standing plants even in the worst- infected portions of the field never
exceeded 15 per cent, while in the lodged portions of the field the head
infection was, in some small areas, as high as 100 per cent. Considering
that the field was not over two acres in extent, that the inoculum of
Gibberella saubinetii, which was responsible for over 90 per cent of the
infections in this field, was very uniformly distributed throughout the
field, and that there were no other explanations for this great difference
in degree of infection between the lodged and the standing plants, the
effect of lodging on the prevalence of headblight infection becomes more
striking.
VARIETIES IN RELATION TO THE DISEASE
During the summer of 191 8 more than 30 varieties of wheat, both
winter and spring, were grown by the Department of Agronomy, Uni-
versity of Wisconsin, on the University farm, and all were attacked
more or less by headblight. There was marked difference between
them in the degree of infection, but no variety was entirely free. As
will be seen from the list given in Table II, among the varieties ex-
amined were representatives of types having very different morpho-
logical characters, from those which have very fine and succulent chaff
to those which have hairy or very hard chaff.
Since the winter varieties examined were badly winter-killed, no
significant count could be taken which would indicate their relative
susceptibility to headblight. The spring varieties, on the other hand,
were in very good condition and uniform throughout the series of plots.
The 15 spring-wheat varieties were sown in small plots of the same
size, the plots being in one series which extended across the whole field.
The whole series of varieties was repeated so that the variety planted
on the first plot was repeated on the sixteenth plot, the variety planted
on the second plot was repeated on the seventeenth plot, and so on.
The plants in each plot were examined carefully and the blighted heads
counted. The number of blighted heads of each variety in the two
series was in many cases exactly the same. If there was a difference,
it did not amount to more than two or three heads. The results are
given in Table II.
These results, while not convincing, are very interesting, especially
when we consider that all plots had the same preparation and cultivation,
the same preceding crop, were on the same piece of land, that all varieties,
while not in exactly the same stage of development, were in a stage in
which they were susceptible to blight, and that the degree of infection of
a certain variety was the same in the two series located a considerable
distance apart.
One may suspect that the relative amount of infection of the seed used
for sowing is the cause both of the difference of infection between
different varieties and of the uniformity in degree of infection of the same
Oct. 1,1920 Fusarium-Blight (Scab) of Wheat and Other Cereals 31
variety in both series. While this seems possible, it does not seem
probable in this case. The plots were small and only 2 feet apart, so
that if some plots were more heavily infected because of the more heavily
infected seed sown on them the inoculum from them could easily have
served for the plants in the neighboring plots only 2 feet away. The
plot with the variety Preston X Kubanka cross (Wisconsin 101), which
had 22 blighted heads, was between plots that had only 1 and 3 blighted
heads, respectively.
Table II. — Averages of actual counts of blighted wheat heads in two series of different
varieties, arranged according to degree of infection
Variety.
Preston X Kubanka cross
Red Fife
Red Fife selection E. G. D. 9171. ..
Marquis
Marquis selection
Pedigree Marquis
Red Fife selection
Fife, Minn. 163
Spring Velvet Chaff
Haynes Bluestem X Kubanka cross
Spring- wheat selection
Bluestem
Bluestem
Spring- wheat selection
Wisconsin
No.
Number of
heads
blighted.
IOI
22
46
20
75
20
50
48
15
16
29
12
74
Pedigree 34
60
9
9
7
102
76
Pedigree 35
Pedigree 36
7
3
3
i
The differences between varieties in susceptibility to blight was brought
out more plainly in a field where two spring-wheat varieties, Marquis and
durum, were sown side by side on the same piece of land, following
corn. The infection of the Marquis wheat where the plants were standing
was less than 1 per cent and from 10 to 15 per cent among the lodged
plants, while the infection among the standing durum plants was from 9
to 10 per cent and as high as 100 per cent among the lodged plants.
Throughout the field there were numerous cornstalks with perithecia
containing viable spores of Gibberella sdubinetii and other parasitic species
of Gibberella, as well as numerous viable conidia of several blight-causing
Fusarium species. While we can doubt the result obtained with various
varieties on the University plots, the results obtained on this field indicate
clearly the existence of a difference in varietal susceptibility to head-
blight. Further observations and experiments in this direction will, no
doubt, be of great importance.
LITERATURE CITED
(]t) Bolley, H. L.
1913. wheat: soil troubles and seed deterioration; causes of soil
sickness in wheat lands; possible methods of control; crop-
PING methods with wheat. N. Dak. Agr. Exp. Sta. Bui. 107, 96 p.
45 fig-
32 Journal of Agricultural Research vol. xx. No. i
(2) Freeman, E. M.
1905. Minnesota plant diseases, xxiii, 432 p., front., illus. St. Paul. (Minn.
Geol.and Nat. Hist. Survey, Rpt. Bot. Ser. V.)
(3) Hoffer, G. N., Johnson, A. G., and Atanasoff, D.
1918. corn-root rot and wheat scab. [Preliminary paper.] In Jour. Agr.
Research, v. 14, no. 13, p. 611-612.
(4) Mc Alpine, D.
1896. Australian fungi. In Agr. Gaz. N. S. Wales, v. 7, pt. 5, p. 299-306,
2 pi.
(5) Naumov, N. A.
1916. l'ianyi khlieb [intoxicating bread]. Min. Zeml. [Russia] Trudy
Biuro Mykol. i. Fitopat., Uchen. Kom., no. 12, 216 p., 7 pi. 1916.
[Literature], p. 211-216.
(6) Saccardo, P. A.
1879. FUNGI GALLICI LECTI A CL. VIRIS P. BRUNAUD, C. C. GILLET ET ABB.
LETENDRE. In Michelia, v. 1, no. 5, p. 500-552.
(7) Saito, K.
1904. UNTERSUCHUNGEN UBER DIE ATMOSPHARISCHEN PILZKEIME. In Jour.
Col. Sci. Imp. Univ. Tokyo, v. 18, art. 5, 53 p., 5 pi.
(8) SCHAFFNIT, E.
1913. DER SCHNEESCHIMMEL UND DIE DURCH FUSARIUM NIVALE CES. HER-
VORGERUFENEN KRANKHEITSERSCHEINUNGEN DES GETREIDES. In
Landw. Jahrb., Bd. 43, Heft 4, p. 521-648, 5 pi.
(9) Selby, Aug. D.
1894. PROGRESS IN THE STUDY OF THE FUNGUS OF WHEAT SCAB. In 2& Ann.
Rpt. Ohio Acad. Sci., p. 33-34.
(10)
1910. A BRIEF HANDBOOK OF THE DISEASES OF CULTIVATED PLANTS IN OHIO.
Ohio Agr. Exp. Sta. Bui. 214, p. 307-456, 106 fig. Literature on
plant diseases referred to in this publication, p. i-vii.
(11) and Manns, Thos. F.
1909. STUDIES IN DISEASES OF CEREALS AND GRASSES. Ohio Agr. Exp. Sta.
Bui. 203, p. 187-236, illus., 2 col. pi.
(l2) WOLLENWEBER, H. W.
1914. IDENTIFICATION OF SPECIES OF FUSARIUM OCCURRING ON THE SWEET
potato, ipomoea batatas. In Jour. Agr. Research, v. 2, no. 4,
p. 251-285, pi. 12-16 (1 col.)
(13)
1917. fusarium autographicE dELINEata ... In Ann. Mycol., v. 15, no.
1/2, p. 1-56.
A set of 509 plates prepared to accompany this work, but not published, is on file in the Office of Cotton
and Truck Crop Disease Investigations of the United States Department of Agriculture.
187931°— 20 3
PLATE i
Gibberella saubinetii:
Blighted ("scabbed') wheat heads. Control plant on the right of others, showing
gTadation of blighting to completely blighted head on the extreme left.
Fusarium-Blight (Scab) of Wheat and Other Cereals
Plate I
Journal of Agricultural Research
Vol. XX, No. 1
Fusarium- Blight (Scab) of Wheat and Other Cereals
Plate 2
Journal of Agricultural Research
Vol. XX, No. 1
PLATE 2
Gibberella saubinetii:
A. — Footrot of wheat caused by Fusarium. The plants at the left were taken from
soil which had been inoculated with G. saubinetii. The control plant at the right gives
the comparative size of the normal wheat root system.
B. — Seedling-blight of wheat caused by G. saubinetii. The seed in the pot on the
left was inoculated with G. saubinetii conidia before planting. The control pot on the
left was planted with clean seed. Germination was reduced , and many of the seedlings
were killed.
PLATE 3
A.— Fusarium seedling-blight. The normal plant is on the left. The other five
show the gradations in blighting caused by Gibberella saubinetii.
B. — Tissue invaded by G. saubinetii in causing the headblight of wheat. Each
group includes the four consecutive sections which, after surface sterilization, were
cut from the upper internode of a wheat culm having a blighted head, the left segment
in each group being the upper. These were then incubated on agar plates. Note that
only the sections nearest the head were invaded by the fungus.
Fusarium-Blight (Scab) of Wheat and Other Cereals
Plate 3
Journal of Agricultural Research
Vol. XX, No. 1
Fusarium-Blight (Scab) of Wheat and Other Cereals
Plate 4
Journal of Agricultural Research
Vol. XX, No. 1
PLATE 4
Gibberella idubinetii:
A. — Kernels blighted and shriveled by Fusarium-blight. Wheat kernels above
are typical of Fusarium-blight. They are shriveled and much lighter than the normal
kernels below.
B. — Perithecia development of G. saubinetii on an infected wheat head.
CAUSE OF LIME-INDUCED CHLOROSIS AND AVAILABILITY
OF IRON IN THE SOIL
By P. L- GiLE, formerly Chemist, and J. O. Carrero, Assistant Chemist, Porto Rico
Agricultural Experiment Station
CAUSE OF LIME-INDUCED CHLOROSIS
INTRODUCTION
Some years ago a study was made of a chlorosis of pineapples that
occurred on certain soils in Porto Rico (12).1 The particular type of
chlorosis was confined to calcareous soils and seemed to be induced by a
disturbance in the mineral nutrition of the plant. This disturbance
appeared to consist in a lack of iron in the plant ash or in a diminished
amount of iron combined with an increased amount of lime. Con-
siderable work has since been carried on to determine more exactly the
manner in which carbonate of lime in the soil induces chlorosis in the
plant. The work comprises a number of direct experiments on the cause
and cure of chlorosis as well as general studies in plant nutrition under-
taken to gain information necessary for interpreting results obtained in
the experiments on chlorosis. Since the more general work on plant
nutrition has been published elsewhere, only the results will be referred
to here.
In the following pages the more important facts already established
concerning the cause of lime-induced chlorosis are given, together with a
full report of certain experiments on this subject hitherto unpublished.
EVIDENCE THAT CARBONATE OF LIME MAY INDUCE CHLOROSIS
Evidence that carbonate of lime produces chlorosis in certain plants
naturally falls into two classes, the results of soil surveys and the results
of direct tests with natural or artificial calcareous soils. These two
classes of evidence will be considered separately.
RESULTS OF SOIL SURVEYS
Ecological studies of calciphilous and calcifugous plants. —
Under the heading of soil surveys, reference should be made to the
extensive literature on calciphilous and calcifugous plants. This litera-
ture, of which Roux (39) gives a complete bibliography up to 1900,
consists chiefly of observations concerning the confinement of certain
plants to calcareous or noncalcareous soils. While most of these
1 Reference is made by number (italic) to "Literature cited," p. 59-61.
Journal of Agricultural Research, Vol. XX, No. 1
Washington, D. C Oct. 1, 1930
uz Key No. B-16
(33)
34 Journal of Agricultural Research voi.xx.No. x
observations do not deal directly with chlorosis, all are related to this
subject, since calcifugous plants are often chlorotic on calcareous soils
and since an exposition of the causes of chlorosis may afford an explana-
tion of the calcifugous character of some plants.
There are a few plants which are very generally classed as calcifugous.
Among these are the following: Maritime pine (Pinus pinaster) (9)
chestnut (Castanea vesca), blueberry (Vaccinium), yellow and blue lupines
(Lupinus luteus and L. angustifolius) , certain species of sphagnum moss,
etc. Cases have been recorded, however, where some plants generally
considered calcifugous have been found growing on calcareous soils (7).
Probably the unsuitability of calcareous soils for certain plants is due
not to carbonate of lime itself but to some soil characteristic usually
associated with carbonate of lime. This being so, calcifugous plants
might occur on certain calcareous soils provided some factor were oper-
ating to counteract the inhibiting characteristic usually associated with
carbonate of lime.
Studies of chlorotic plants. — Besides the soil surveys of calcifugous
plants, there are several soil surveys which deal directly with the appear-
ance of chlorosis in cultivated plants.
A case that has been the subject of much study is that of European
grapes grafted on certain American stocks. When these were introduced
on the calcareous soils of France and Germany they became chlorotic.
Several soil surveys and many observations prove that the chlorosis is
confined to calcareous soils and that there are varietal differences among
grapes with respect to their resistance to lime {22, 30, 33, 39 Viala and
Ravaz, 45). The accumulated data do not show, however, that all soils
containing more than a certain percentage of carbonate of lime produce
chlorosis in these varieties of grapes.
The chlorosis and failure of chestnut trees on most soils containing
more than 3 per cent of carbonate of lime has been well established
through soil surveys and through observations by Fliche and Grandeau
(10), Piccioli (36), Vallot (44), and others. Vallot (44, p. 202) states
that Dr. Bonnet reported that the chestnut failed to grow in a calcareous
soil of Dijon, but when it was grafted on an oak it grew superbly.
That yellow and blue lupines and serradella become chlorotic when
planted on calcareous soils is common knowledge in the calcareous
districts of France and Germany, 2 per cent of carbonate of lime usually
being sufficient to affect these plants.
A soil survey in Porto Rico showed that a chlorosis of pineapples was
confined to the calcareous soils (12, p. 8-18). The only calcareous soils
not producing chlorotic pineapples on which data could be obtained were
some from the Florida Keys. These contained an exceptional amount of
organic matter.
A chlorosis of sugar cane in Porto Rico was also found to be confined to
calcareous soils, although very many calcareous soils did not induce
Oct. i, 1920 Cause of Lime-Induced Chlorosis 35
chlorosis. Green cane was found growing on a soil containing 76.70 per
cent calcium carbonate (19).
Pears have frequently been reported as showing chlorosis on calcareous
soils (4, 6, 29, 38).
Instances have been noted where a great many other plants have become
chlorotic on calcareous soils (24). Many of these cases are doubtless
more or less exceptional, since some of the plants do not become chlorotic
on most calcareous soils. Roux (39), without attempting a complete
compilation, mentions some 50 genera and species of cultivated plants,
ranging from mosses and orchids to maples and citrus trees, which have
shown chlorosis when planted on soils containing calcium carbonate.
The results of the soil surveys and field observations seem to demon-
strate conclusively that this type of chlorosis is confined under field
conditions to calcareous soils. Probably no one species of plant, how-
ever, becomes chlorotic on all soils containing more than a certain per-
centage of calcium carbonate. Some plants are much more sensitive
to carbonate of lime than others — that is, they become chlorotic on soils
with lower lime contents and are less frequently found growing normally
on limy soils.
The fact that plants very subject to chlorosis have been found in
a few instances growing normally on markedly calcareous soils shows
that the ability of calcareous soils to induce chlorosis does not depend
entirely on the percentage of carbonate of lime in the soil. This fact
also lends credence to the idea that it is not the carbonate of lime itself
that induces chlorosis but some condition usually associated with the
presence of carbonate of lime.
RESULTS OF VEGETATIVE EXPERIMENTS IN WHICH CHLOROSIS WAS PRODUCED BY
NATURAL OR ARTIFICIAL CALCAREOUS SOILS
Compared with the mass of observations on the natural occurrence of
chlorosis, there has been little reported in regard to inducing chlorosis
by the use of calcium carbonate or in regard to direct tests of calcifugous
plants in calcareous soils. There have been several vegetative experi-
ments with yellow and blue lupines, however, where the addition of
carbonate of lime to the soils caused a marked depression in growth and,
in some cases at least, induced chlorosis. Concordant, positive results
were secured by Heinrich (23), Meyer (32), Pfeiffer, and Blanck {35), the
Agricultural Chemical Experiment Station at Breslau (2), Creydt (5),
and Roux (39, p. 147-183).
Biisgen (3) grew the calcifugous broom {Sarothamnus scoparius),
foxglove {Digitalis purpurea) , and heather {Calluna vulgaris) in artificial
calcareous and noncalcareous soils. The growth of all three plants was
moderately to greatly depressed in the calcareous soil, although only
broom was mentioned as showing chlorosis.
36 Journal of Agricultural Research voixx.No. 1
Roux (39, p. 147) grew some 20 species of calcifugous plants in cal-
careous soils. All species made diminished growth and became chlorotic
in certain calcareous soils, while none showed chlorosis in the noncalca-
reous soil.
Piccioli (36) planted many varieties of chestnuts, together with
Sarothamnus, Calluna, and Pteris, on soils with different additions of
carbonate of lime. Most plants eventually died on the soil containing
12 per cent calcium carbonate.
Experiments at this Station showed that the mere addition of carbo-
nate of lime to soils which normally produced green pineapples (12,
p. 20) or rice plants (13, p. 30) caused the soils to produce chlorotic
plants.
The preceding experiments seem to afford direct proof of the conclu-
sions derived from field observations and from soil surveys that a chlorosis
of some plants is caused by, or associated with, the presence of carbonate
of lime in the soil.
MANNER IN WHICH CARBONATE OF LIME IN THE SOIL INDUCES CHLOROSIS
IN THE PLANT
While it is quite generally conceded that carbonate of lime may induce
a chlorosis in certain plants, there is a great diversity of ideas regarding
the way the chlorosis is brought about. There are several classes of
evidence or kinds of data on which conclusions concerning the nature
of lime-induced chlorosis are based. These different kinds of evidence
will be considered under the following heads: Evidence from analyses
of plant ashes, effect of application of iron salts, effect of other lime
compounds in inducing chlorosis, and effect of an alkaline reaction in
inducing chlorosis.
RESULTS OP ASH ANALYSES OF PLANTS
In their work on the chlorosis of the chestnut and maritime pine
Fliche and Grandeau (9, 10) analyzed leaves and branches of green
and chlorotic trees. They concluded that the chlorosis and diminished
growth of the trees on the calcareous soils were the result of an undue
absorption of lime and a diminished absorption of other elements,
notably potash and iron.
Schulze (42) analyzed the wood and leaves of green and chlorotic
grapevines,1 determining only lime, magnesia, potash, and soda. Com-
pared with the green plants, the chlorotic ones had about one-half as
much potash and soda and slightly more lime and magnesia in the ash.
Biisgen (3) analyzed the broom plants grown by him in calcareous and
noncalcareous soils to determine lime and potash. The chlorotic and
1 Analyses by Mach and Kurmann {31 ) are often quoted in this connection. The results probably have
no bearing on this subject, however, as the chlorosis of their specimens seems to have been caused by too
much moisture or poor drainage.
Oct. 1. 1920 Cause of Lime- Induced Chlorosis 37
green plants from the two soils had almost equal percentages of lime and
potash in the ash, the percentage of total ash in the dry substance being
higher in the chlorotic plants.
Numerous ash analyses were made at this Station from chlorotic and
green pineapple plants grown in soils with and without carbonate of
lime (12). Compared with the green plants, the chlorotic ones in the
calcareous soils contained more lime and less iron in the ash ; differences
in other ash constituents were slight or inconstant, potash as a rule being
fully as high in the chlorotic plants as in the green ones.
Green and chlorotic rice plants were also analyzed at different ages for
their mineral constituents (13). In the case of rice grown 25 days, the
chlorotic plants from the calcareous soils contained much more lime, less
iron, and equal or greater percentages of potash in the ash than the green
plants from the soil containing no carbonate of lime; but in the case of
green and chlorotic rice of 84, 102, and 129 days' growth, the only con-
stant difference in the ash of the two kinds of plants was a greater per-
centage of lime in the chlorotic plants. These analyses and a special
study showed that the percentage of iron in the ash of rice diminished
very markedly as the plants became more mature (13). Since plants
affected with chlorosis matured much more slowly than normal plants,
probably the iron contents of the 84-, 102-, and 129-day samples were
affected more by the different maturities of the plants than by the char-
acter of the soils.
Four pairs of samples of green and chlorotic sugar-cane leaves were
analyzed for their ash constituents. The leaves were selected from canes
which were of the same size and age and which were growing on the same
calcareous soil. In each case the chlorotic leaves had a distinctly lower
percentage of iron in the ash than the corresponding green leaves (19). 1
A summary of the evidence from ash analyses in regard to the cause
of lime-induced chlorosis is as follows: Lime was determined in all seven
species of plants analyzed by the different investigators, and in five cases
it appeared that an excessive absorption of this element might be a cause
of chlorosis; in two cases it appeared that it was not. Potash was
determined in six of the different plants, and in only three cases did it
appear that a lack of potash might be a cause of chlorosis. Iron was
determined in five of the plants, and in all five cases it appeared that the
chlorosis might be due to a deficiency of this element.
The weight of the evidence from the ash analyses seems to be that a
deficiency of iron in the ash is at least one cause of the chlorosis and that
possibly an excess of lime is also a cause. Against this conclusion there is
the opinion of many physiologists, as Euler (8), Jost (28), and Sorauer
1 In a fifth comparison, leaves of green, slightly chlorotic, and chlorotic cane were analyzed, the canes
being of equal age but of markedly different size when grown on calcareous and noncalcareous soils. The
chlorotic leaves contained very slightly more iron than the green leaves. In this case, it is believed that
the maturities of the plants and the different ages of the leaves were the chief factors influencing the iron
content (19, p. is).
38 Journal of Agricultural Research voi.xx, no. i
{43), that lime-induced chlorosis is caused chiefly by a lack of potash in
the plant ash. This opinion is evidently based only on the analyses of
Fliche and Grandeau (9, 10, 11) and on those of Schulze (42). If a lack
of potash in the ash were the cause of the chlorosis, plants grown in non-
calcareous soils and in water cultures, under controlled conditions, with
an insufficient supply of potash, should show this type of chlorosis. In
such cases, however, the lack of potash is indicated by the appearance of
brown spots on the leaves and not by a yellowing.1 However, it has not
been shown that a combined excess of lime and deficiency of potash
would not produce chlorosis.
The reliability of ash analyses as the sole means of diagnosing the cause
of chlorosis is questionable. At the most, the results of ash analyses
should be taken as merely indicating the cause or as confirming other
evidence. The ash compositions of normal plants show such wide varia-
tions and are affected by so many conditions that it is sometimes unsafe
to assume that of two lots of plants those which have made the better
growth have an ash composition more nearly normal. •
Aside from difficulties in properly interpreting the results of ash
analyses, it is sometimes doubtful whether the samples selected for
analysis are truly comparable, even when whole plants are taken. This
uncertainty was demonstrated in the analyses of rice, previously referred
to. The practice of taking only a portion of a plant for analysis is also
susceptible to error, especially where iron is to be determined. Since
iron appears to be relatively immobile in the plant after it is once trans-
ported to the leaves, certain leaves of a plant might contain a sufficiency
of iron while other leaves and the plant as a whole might lack iron (16).
EFFECT OF APPLICATION OF IRON SALTS TO CHLOROTIC PLANTS
Eusebe Gris, in 1845 (20), and later Sachs (41) and other investigators
(12, 21, 25, 26, 27) showed that various plants which became chlorotic
on calcareous soils could be cured by applying ferrous sulphate to the
leaves. This treatment and the improved one of Rassiguier (57), that of
brushing cut surfaces of pruned vines with a concentrated solution of
ferrous sulphate, have been rather generally used on grapevines which
became chlorotic on the calcareous soils of France and Germany.
Various investigators have found that while iron salts were effective
in overcoming chlorosis when applied to the stems and leaves of plants,
they were ineffective when applied to the soil, even if used in considerable
quantity. Sachs (41), however, observed that where the roots of plants
were not completely surrounded by earth, as in the case of pot- bound
plants, applications of ferrous sulphate to the soil did cure the chlorosis.
1 If potash is concerned in the formation of starch from sugars, a low percentage of potash in chlorotic
plants might be a secondary result of the chlorosis. With insufficient iron, chlorophyll formation is
depressed, less sugar can be synthesized, and little potash would be needed.
Oct. i, 1920
Cause of Lime-Induced Chlorosis
39
Since ferrous sulphate is, of course, immediately transformed into ferric
carbonate in a calcareous soil, it seems evident that calcium carbonate
renders ferric carbonate unavailable, or less available, to certain plants.
It has been repeatedly demonstrated that the effectiveness of spraying
with ferrous sulphate is due only to the iron and that only soluble iron
salts are effective (12, 21, 25, 26, 27).
Experiment I.— The results in Table I show the effect of an iron spray
upon chlorotic rice growing in a calcareous soil. The plants were grown in
the open from February 29 to July 13, 191 2, in small brick compartments
with 36 plants to each compartment. Each compartment held about
200 pounds of heavy loam soil and received 5 gm. nitrogen, 3.4 gm.
phosphoric acid, and 5 gm. potash, derived from various commercial
fertilizers. The plants sprayed with ferrous sulphate were given 4 appli-
cations of a 0.5 per cent solution and 12 applications of a 1 per cent
solution.
Table I.— Effect of an iron spray upon chlorotic rice plants grown, on calcareous soils
Test
No.
Calcium
carbonate
content of
soil.
Per cent.
1
O
2
O
• 3
3°
4
3°
5
6
5°
5°
Treatment of plants.
Unsprayed
Sprayed with ferrous sulphate.
Unsprayed
Sprayed with ferrous sulphate.
Unsprayed
Sprayed with ferrous sulphate
Green weight of plants per compart-
ment.
Series A. Series B. Average.
Gm.
I, 022
1,088
4
946
242
874
Gm.
I, 071
I, 040
(a)
894
702
893
Gm.
1,047
I, 064
920
472
a Some plants were eaten by mole cricket, but according to comparative growths of plants before any
were eaten, the weight would have been about 250 gm.
Twenty-one days after planting, the plants in the calcareous soils were
markedly chlorotic, and spraying was begun at that time. Seven days
later, after nine sprayings, the sprayed plants in the calcareous soils were
much superior to the unsprayed in color and growth. All plants in the
noncalcareous soil had a good color at all times. (PI. 5, A.)
The results obtained by treating the leaves and stems of chlorotic
plants with iron salts show clearly that a lack of iron in the plant is at
least one of the causes of lime-induced chlorosis. This conclusion is
substantiated by the results of ash analyses of the plants. But this work
does not show: (i) whether the lack of iron in the plant is due to a low
availability of iron in the soil or to reactions in the plant rendering
ineffective the iron absorbed; (2) whether an increased absorption of
lime is a contributory cause of chlorosis ; or (3) whether the reaction of
the soil has any effect on the appearance of chlorosis, aside from affecting
the iron supply.
40
Journal of Agricultural Research
Vol. XX, No. i
EFFECT OF COMPOUNDS OF LIME IN INDUCING CHLOROSIS
To see whether lime salts in general induce chlorosis in certain plants,
experiments have been conducted with calcium carbonate, sulphate,
phosphate, and silicate. The effects of these compounds on the growth
of lupines have been determined by Heinrich {23), Pfeiffer and Blanck
(35), and Creydt (5). The calcium sulphate did not induce chlorosis
but depressed growth considerably, although much less than the calcium
carbonate, while calcium phosphate and silicate were markedly toxic.
The toxicities of the latter two substances were attributed to their acid
and alkaline reactions, respectively.
Large quantities of gypsum depressed the growth of pineapples about
20 per cent but did not cause chlorosis (12). Various experiments were
conducted to determine the effect on rice of large amounts of assimilable
lime in the form of gypsum.
Experiment II. — In this experiment, rice plants were grown from
December 17, 1912, to May 20, 1913, in small brick compartments, with
24 plants to each compartment. Each compartment held about 200
pounds of soil fertilized with 30 gm. sulphate of ammonia, 20 gm. nitrate
of soda, 30 gm. acid phosphate, and 18 gm. muriate of potash added in
two applications. The results are shown in Table II.
Table II. — Effect on the growth of rice of adding gypsum to the soil
Test
No.
Kind of soil.
Loam.
...do
Clay . .
do
Gypsum
(CaSO<.
2H2O)
added.
Per cent.
O
o
IS
Green weight of plants per compartment.
Series A. Series B. Series C Average
Gm.
I, 218
312
936
Gm.
1,229
446
808
958
Gm.
1,452
382
840
842
Gm.
1,300
380
824
912
During the first four weeks the plants were all of good color, but later
the plants in the loam soil containing gypsum became yellow, though not
typically chlorotic.
Experiment III. — A second experiment was conducted to see whether
large amounts of gypsum would depress the growth of rice if the plants
were sprayed with ferrous sulphate. The compartments contained about
200 pounds of a sandy soil and received 45 gm. sulphate of ammonia,
30 gm. acid phosphate, and 18 gm. muriate of potash. In each compart-
ment 22 plants were grown. The plants treated with ferrous sulphate
were sprayed twice with a 0.1 per cent solution, five times with a 0.15
per cent solution, once with a 0.2 per cent solution, three times with a
0.75 per cent solution, and seven times with a 1 per cent solution. The
results are given in Table III.
Oct. i, 1920
Cause of Lime-Induced Chlorosis
41
Table III. — Influence of spraying with ferrous sulphate on the depressing effect of gypsum
Gypsum
(CaSO*.
2H2O)
added.
Treatment of plants.
Air-dried weight of plants per compartment.
No.
Series A.
Series B.
Series C.
Average.
Per cent.
O
O
*5
15
Gm.
S21
478
324
395
Gm.
503
525
30O
364
Gm.
475
525
366
372
Gm.
500
2
3
4
Sprayed with ferrous
sulphate .
509
33°
Sprayed with ferrous
sulphate.
377
The plants in the soil to which gypsum had been added were markedly
behind the others in growth from the start, and they were at times of
poorer color, though they were never typically chlorotic. Plants in soil
without gypsum were of good color at all times. No effect from spraying
with ferrous sulphate was observable.
Experiment IV. — A further experiment with gypsum and ferrous
sulphate was conducted in pots in a glass house. Six rice plants per pot
were grown from July 7 to October 25, 191 3. Each pot contained 37
pounds of sandy soil, to which 13 gm. ammonium sulphate, 11 gm. acid
potassium phosphate, and 3.6 gm. sulphate of potash were applied.
The moisture content of the soil was maintained at the optimum. The
results appear in Table IV.
Table IV. — Influence of different treatments with ferrous sulphate on the depressing
effect of gypsum
Gvpsum
(CaSO*.
2H2O)
added.
Treatment of plants.
Air-dried weight of plants per pot.
Test
No.
Series
A.
Series
B.
Series
C.
Series
D.
Aver-
age.
Per cent.
O
15
15
15
Gm.
114
85
67
Gm.
113
92
5°
60
Gm.
"3
68
86
60
Gm.
118
67
85
78
Gm.
Ir5
78
3
4
Ferrous sulphate, 2.2 gm.,
added to soil.
Plants sprayed eight times
with 1 per cent solution of
ferrous sulphate.
74
66
The color of the plants grown in the soil to which gypsum was added
was as good as that of the controls up to the eighty-fifth day, but from
the eighty-fifth to the one hundred and tenth day the former were yellow.
The controls were always of a good green color. No effect from either
of the treatments with ferrous sulphate was observable.
Summary. — In all the tests, except that with the clay soil, calcium sul-
phate depressed the growth of rice and induced a certain amount of
yellowing. The yellowing, however, was not that typical of lime-induced
42
Journal of Agricultural Research
Vol. XX, No. i
chlorosis. Spraying with ferrous sulphate and adding ferrous sulphate
to the soil failed to increase the growth or improve the color of plants
growing in the soil containing calcium sulphate. That calcium sulphate
increased the amount of lime in the plants may be seen by the analyses
in Table V of plants 65 days old from experiment II.
Table V. — Ash analyses of plants from experiment II
Kind
of soil.
Gypsum
(CaSO*.
2HjO)
added.
Car-
bon-
free
ash
in dry
sub-
stance
of
plants.
Analyses of carbon-free ash.
Test
No.
Silica
(SiO,).
Lime
(CaO).
Mag-
nesia
(MgO).
Potash
(K20).
Soda
(Na20).
Iron
(FejOs).
Phos-
phoric
acid
(P2Os).
Sul-
phur
(SOs).
1
3
3
4
Loam..
...do...
Clay. . .
...do...
P.ct.
0
IS
0
IS
P.ct.
16.7s
13-92
14. 14
13- 08
P. ct.
54-7°
47. 24
SO. 77
45-04
P.ct.
3-63
6-45
3-87
6. 40
P.ct.
4. 20
5. 22
4.80
4- 77
P.ct.
24- 77
26.81
25-H
28.34
P.ct.
5.48
4- 54
4-65
7. 00
P. ct.
0-55
•53
.62
•38
P.ct.
2.58
4. 01
2-31
3-13
P.ct.
2. 19
6. 71
2-74
4- 76
It will be noted that the calcium sulphate increased the percentages
of lime and sulphur in the plant ash and diminished the percentage of
silicia but had little effect on the other constituents.
The injurious effect of calcium sulphate on rice might have been due
to several causes. A large amount of gypsum evidently maintains a
solution more concentrated than that existing in any except alkali soils.
There is also the possibility of hydrogen sulphid being formed by bacteria
reducing sulphates. This occurred with soil preserved in a sample jar,
although such a result was not to be expected in what appeared to be a
normally aerated soil. The fact that calcium sulphate did not depress
growth in the clay soil lends credence to the view that the injurious
effect might have been that of a too concentrated soil solution.
In order to make sure that an increased assimilation of lime is not a
cause of chlorosis, a test was conducted with lime salts applied to the
leaves. The results, given in experiments V and VI, to be described
further on, seemed to show definitely that an increased assimilation of
lime does not induce chlorosis.
Although excessive quantities of various lime compounds seem to be
more or less injurious, each one appears to act differently; there is no
evidence of a general "lime effect" in inducing chlorosis.
EFFECT OF AN ALKALINE REACTION IN INDUCING CHLOROSIS
Pfeiffer and Blanck (55) in their first work on the intolerance of lupines
for calcareous soils concluded that lupines are especially sensitive to an
alkaline reaction and that the carbonate of lime not only depresses the
absorption of nutrients but is directly injurious to the roots of the plants.
While the alkaline reaction of carbonate of lime is evidently a factor in
the chlorosis, it is very evidently not directly injurious to roots of even
calcifugous plants. It was found in experiments with pineapple and
Oct. i, 1920
Cause of Lime-Induced Chlorosis
43
rice at this Station that the ratio of root to top growth was much increased
in calcareous soils and solutions (12, 17). The stimulating effect of car-
bonate of lime on the root growth of plants which are not calcifugous
has been frequently noted.
In work with "pineapples it was shown that the alkalinity induced by
increasing amounts of carbonate of soda greatly depressed growth without
affecting the color of the plants (12, p. 31).
Work with rice in water cultures seemed to show definitely that the
alkalinity of carbonate of lime is not directly injurious to this calcifugous
plant, nor is the alkalinity in itself the cause of chlorosis (17). Rice was
grown with different quantities of iron from different sources in nutrient
solutions which were acid, neutral, and alkaline from carbonate of lime.
A summary of the relative growths made under the different conditions
is given in Table VI.
Table VI. — Relative growths of rice plants with different amounts of iron from various
sources in acid, neutral, and alkaline solutions
Source of iron in nutrient solutions.
Iron per
liter added
to nutrient
solutions.
Relative growths in-
Acid
solution.
Neutral
solution.
Alkaline
solution.
Ferrous sulphate.
Do
Gm.
o. 002
Do
Do
Do
Do
Do
Do
Ferric chlorid.
Do
Ferric citrate. .
Do
Do
Do
Ferric tartrate.
Do...
Dialyzed iron.
008
004
002
008
1 002
008
002
008
002
008
008
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
74
95
105
107
132
in
99
107
85
94
101
85
80
06
27
5i
95
26
26
86
97
104
58
76
100
Where growth was depressed to any extent the plants were more or
less chlorotic, and that this chlorosis was evidently due to lack of iron
was shown by analyses of the plants and by treatment of the leaves with
ferrous sulphate. The work showed quite definitely that rice is not
particularly sensitive to the reaction of carbonate of lime, except as the
reaction influences the availability of the iron. When the proper form
of iron was used in the proper quantity, the growth and appearance of
the plants were as good in the solutions containing carbonate of lime as
in the acid or neutral solutions.
The preceding results seem to show that neither increased assimilation
of lime nor mere alkalinity causes chlorosis. It remained to be seen
187931°— 20 i
44
Journal of Agricultural Research
Vol. XX, No. i
whether the combination of the two conditions would induce a typical
chlorosis. It was thought that this might be determined experimentally
by growing rice on soil to which sodium bicarbonate had been added to
render it alkaline and then inducing an increased assimilation of lime by
spraving the plants with calcium chlorid and gypsum" In case these
treatments should induce a chlorosis identical with that produced by
carbonate of lime, spraying with ferrous sulphate should cure it. Accord-
ingly, some plants grown in the soil with sodium bicarbonate were
sprayed with lime salts alone, with ferrous sulphate alone, and with both
lime and iron salts. Plants grown in a soil without sodium bicarbonate
were also sprayed as described above in order to check the results.
The experiment was carried out twice, once in the open, using small
brick compartments, and once in the glass house, using pots. Results
are given under the heads of experiments V and VI. The sodium bicar-
bonate was added in several doses until it became evident that sufficient
had been applied to affect growth. More was required for the soil in
the open than for that in the glass house, since the former was exposed
to leaching.
Experiment V. — This test was run from November 8, 1913, to Jan-
uary 20, 1914. Each plot containing 150 pounds of sandy soil received
45 gm. sulphate of ammonia, 30 gm. acid phosphate, and 18 gm. muriate
of potash. Thirty rice plants were grown on each plot. The results
are given in Table VII.
Table VII. — Effect of sodium bicarbonate, lime, and iron on Ike growth of rice:
Experiment V
Test
No.
Ap-
proxi-
mate
percent-
age of
sodium
bicar-
bonate
in soil.
O. 2
. 2
Treatment.
None
Sprayed 31 times with 0.5 to 2 per cent solutions
of calcium chlorid and gypsum
Sprayed 31 times with 0.5 to 2 per cent solutions
of calcium chlorid and gypsum and 8 times
with 0.5 to 1 per cent solutions of ferrous sul-
phate
None
Sprayed 31 times with 0.5 to 2 per cent solutions
of calcium chlorid and gypsum
Sprayed 31 times with 0.5 to 2 per cent solutions
of calcium chlorid and gypsum and 8 times
with 0.5 to 1 per cent solutions of ferrous sul-
phate
Sprayed 8 times with 0.5 to 1 per cent solutions
of ferrous sulphate. .. .
"
Air-dried weight of
plants per plot.
Series
A.
Gm.
144
142
163
IOO
Series
B.
Gm.
156
152
166
104
82
"3
Aver-
age.
Gm.
ISO
147
165
102
92
105
"3
Oct. i, 1920
Cause of Lime-Induced Chlorosis
45
The plants in the soils containing sodium bicarbonate became some-
what yellow, though the yellowing was not that of typical lime-induced
chlorosis. The yellowing, however, was not increased by the lime spray,
nor was it overcome by the iron spray. The lime and iron sprays also
had no effect on the appearance of the plants growing in the soil con-
taining no sodium bicarbonate.
Experiment VI. — In this test, conducted from November 4, 1913, to
March 12, 1914, 7 rice plants were grown per pot. Each pot contained
35 pounds of sandy soil and received 6 gm. ammonium nitrate, 1.3 gm.
potassium acid phosphate, and 2.5 gm. potassium sulphate. The mois-
ture content was maintained at 25 per cent of the dry weight of the soil.
The results are shown in Table VIII.
Table VIII.-
-Effect of sodium bicarbonate, lime, and iron on the growth of rice:
Experiment VI
Air-dried weight of
Ap-
proxi-
plants per pot.
Test
No.
percent-
age of
Treatment.
sodium
Series
Series
Aver-
bicar-
A.
B.
age.
bonate
in soil.
O
O
Gm.
83
Gm.
71
2
Plants sprayed 23 times with 0.=; to 2 per cent
77
solutions of calcium chlorid and sulphate
68
72
70
3
O
Plants sprayed 23 times with 0.5 to 2 per cent
solutions of calcium chlorid and sulphate and
7 times with 0.5 to 1 per cent solutions of fer-
65
44
70
68
4
5
O 08
56
50
.08
Plants sprayed 23 times with 0.5 to 2 per cent
solutions of calcium chlorid and sulphate
52
51
5°
6
.08
Plants sprayed 23 times with 0.5 to 2 per cent
solutions of calcium chlorid and sulphate and
7 times with 0.5 to 1 per cent solutions of fer-
42
39
40
7
.08
Plants sprayed 7 times with 0.5 to 1 per cent
49
60
55
The appearance of the plants in this test was the same as in experi-
ment V.
The plants from experiment V were analyzed for their ash constituents,
and the results appear in Table IX. The plants were washed imme-
diately after cutting, so no lime salts remained on the leaves. While it
. s believed that all iron applied as a spray was also removed by washing,
it is possible that some iron in the form of ferric oxid might have remained
adhering to the leaves; hence, in the case of the plants sprayed with
ferrous sulphate, it is possible that the analytical results may show more
iron than was actually present in the plants.
46
Journal of Agricultural Research
Vol. XX, No. i
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Oct. i, 1920 Cause of Lime-Induced Chlorosis 47
Summary. — None of the sprays affected the growth or color of the
plants, either in the normal soil or in the soil containing sodium bicar-
bonate. The amount of sodium bicarbonate required to depress growth
was rather surprising, and from this fact it was suspected that the availa-
bility of iron was not noticeably depressed by sodium bicarbonate, at
least not below the critical point. This was confirmed by the analyses
of the plants and by the fact that spraying with ferrous sulphate effected
■no improvement in either the growth or color of the plants planted in
the soil containing sodium bicarbonate.
The spraying with lime salts, however, notably increased the amount
of lime in the plants without affecting the quantity of iron, and spraying
with both lime and iron solutions increased the quantities of both ele-
ments in the plant. The yellowing and depression in growth produced
by the sodium bicarbonate were probably due to an injurious degree of
alkalinity, which must have been far greater than that which is produced
by carbonate of lime.
The results of these experiments, where a large amount of sodium
bicarbonate was required to depress growth, seem to show that the slight
alkalinity of carbonate of lime could not be directly injurious to rice,
nor could alkalinity in itself be the cause of chlorosis. While this exper-
iment failed to yield the decisive answer expected, it is felt that the
results point strongly to the conclusion that an increased assimilation of
lime is not the cause of chlorosis.
CHLOROSIS DUE SIMPLY TO A DEPRESSION IN AVAILABH.ITY OF IRON IN THE SOIL
An attempt was made to demonstrate directly that the only action of
carbonate of lime in inducing chlorosis lies in depressing the availability
of the iron. It was thought that this demonstration could be accom-
plished by growing rice plants with their roots divided between two
kinds of soil, one to contain carbonate of lime and all the mineral nutri-
ents except iron, and the other to contain only iron. The attempt was
not completely successful, due partly to a principle discovered later and
partly to difficulties in execution. The principle which tended to make
the results less striking than had been anticipated is the following:
Plants apparently are unable to attain a maximum absorption of any
one element with only a part of their roots (18).
Wire sieves were made which fitted into the tops of buckets. The
buckets were filled with soil to within 1 inch of the bottom of the
sieves, and the sieves were filled with about 2 inches of soil (PI. 5, B).
In this way an air space was left between the soil in the sieve and that in
the bucket; this prevented any soil solution passing by capillary attrac-
tion from the soil below to that above. It was the intention at first to
fill all except the control buckets with a calcareous soil containing all
the nitrogen, phosphoric acid, and potash, and to fill most sieves with
pure silica sand containing only iron. In conducting the experiment,
48
Journal of Agricultural Research
Vol. XX, No. i
however, it was found necessary to apply a small amount of nitrogen,
phosphoric acid, and potash to the sand in the sieve in order that the
plants might develop sufficiently for their roots to penetrate the soil
below.
The intention was that the plants should absorb practically all their
nutrients from the soil in the buckets (a calcareous soil except in control
buckets i to 4), but that in some cases the plants should be able to absorb
iron from a lime-free medium in the sieve. If carbonate of lime affected
the plants in any way except through depressing the absorption of iron,
all plants should make equally poor growth; but if, on the other hand,
the only action of the carbonate of lime lay in decreasing the availability
of the iron, those plants that could draw iron from a medium containing
no carbonate of lime should do much better than the others.
A preliminary test was run with two pots, No. 1 containing silica sand
in the sieve and a calcareous soil in the bucket, and No. 2 containing
silica sand plus carbonate of lime in the sieve and the same calcareous
soil in the bucket as No. 1. Four gm. of ferrous sulphate were applied
to both sieves (PI. 6, A). The yields from pots No. 1 and 2 were respec-
tively 169 gm. and 97 gm. of air-dried plants, the plants in No. 1 being
green in color and those in No. 2 chlorotic.
Experiment VII. — The results of a more extended test are given in
Table X. The plants were grown from October 22, 191 2, to March 3,
1913. A large number of seeds were planted, but the plants in each pot
were thinned to eight. The sieve of each pot contained 10 pounds of
silica sand to which were added 0.45 gm. ammonium nitrate, 0.1 gm.
acid potassium phosphate, and 0.2 gm. potassium sulphate. The
bucket of each pot contained 23 pounds of soil and received 12 gm.
ammonium nitrate, 3 gm. acid potassium phosphate, and 5.5 gm. potas-
sium sulphate, in two applications. The moisture content of the soil
was maintained at 31 per cent of the dry weight.
Table X. — Effect of carbonate of lime in the soil on the availability of iron
Treatment of soil in
bucket.
Treatment of sand in
sieve.
Green weight of plants per pot.
Pot No.
Series
A.
Series
B.
Series
c.
Series
D.
Aver-
age.
1 to 4. . .
5 to 8. . .
None
None
Gm.
206
137
204
112
Gm.
171
224
174
97
Gm.
204
156
236
"5
Gm.
161
187
84
Gm.
194
Calcium carbon-
ate, 15 percent.
do
None
170
200
Eight gm. ferrous
sulphate in four
applications.
Eight gm. ferrous
sulphate in four
applications; 15
per cent calcium
carbonate.
. . .do
102
Oct. i, i920 Cause of Lime-Induced Chlorosis 49
At 1 5 days after sowing the seed all plants were chlorotic except those
in pots 13 to 16, and many died because of their inability to establish
roots in the soil in the bucket. At 121 days the plants of pots 1 to 4
and 9 to 12 were green, while those of No. 5 to 8 and 13 to 16 were strongly
chlorotic.
The plants encountered some difficulty in establishing their roots in
the soil in the buckets; the roots after passing through the sieve often
grew for a time on the surface of the soil. This retarded growth con-
siderably, but when the roots once penetrated the soil, growth became
normal. At the end of the experiment the greater part of the roots were
in the soil in the bucket, where practically all the fertilizer was located.
The final yields of the plants and the chlorotic appearance of certain
plants during the latter stages of growth confirm the idea that the only
effect of carbonate of lime in inducing chlorosis lies in depressing the
availability of iron. The plants in pots No. 9 to 12 and those in No.
13 to 16 were exposed to the same conditions except that the plants in
No. 9 to 1 2 were able to draw part of their iron from a medium containing
no carbonate of lime; this difference was sufficient to double the growth
of plants. The plants of No. 9 to 12 had to assimilate practically all
their mineral nutrients, except iron, from the same calcareous soil as
the plants of No. 13 to 16; hence, if the carbonate of lime induced chlorosis
by depressing the availability of any nutrients other than iron, or if
an increased assimilation of lime were a contributory cause of chlorosis,
the yield from pots No. 9 to 12 should have been practically the same
as from No. 13 to 16.
The only apparent contradiction in this demonstration of the cause of
lime-induced chlorosis lies in the fact pots No. 5 to 8 yielded more than
No. 13 to 16. Plants in pots No. 5 to 8 evidently secured less iron than
those in No. 9 to 12, for they made less growth; but if the sand in the
sieve had been really iron-free they should have made no more growth
than plants No. 13 to 16. Later work showed that, although no iron
was added to the sieves of No. 5 to 8, doubtless the silica sand contained
enough iron to cause the unanticipated growth. In work with nutrient
solutions it was found that rice practically satisfied its iron requirements
in a solution containing no more than 1 part of truly soluble iron in
10,000,000 parts of solution (17, p. 5).
On repeating this experiment the same difficulties were encountered,
but the relative growths made by the differently treated plants were
similar to those in the preceding test.
AVAILABILITY OF IRON IN THE SOIL
INTRODUCTION
Since the preceding summary of facts and experiments seems to indi-
cate that lime-induced chlorosis is simply the result of insufficient avail-
able iron in the soil, evidently a knowledge of conditions affecting the
50 Journal of Agricultural Research voi.xx, No. i
availability of iron in the soil is essential to a complete understanding
of this chlorosis. If all the conditions affecting the amount of available
iron in the soil were known, it would doubtless be possible to explain why
some calcareous soils induce chlorosis when others do not; why in a sandy
soil a smaller percentage of carbonate of lime is required to induce chloro-
sis than in a clay soil; why a calcareous soil that produces chlorotic plants
at one time may not at another; and many other perplexing facts.
Since a method for determining the amount of available potash or
phosphoric acid in the soil is still unknown, in spite of years of work, the
prospect is not bright for even roughly determining the available iron by
direct means ; and to determine directly significant differences in amounts
of available iron seems hopeless when plants obtain their iron from such
exceedingly dilute solutions.
Soils which yield sufficient iron for the growth of plants may not show
a detectable amount of iron in the water extract. In some cases the
water extract of soils may show considerable iron, but the iron may be in a
colloidal state and not in true solution. Colloidal iron was found unavail-
able for rice in water culture (14).
While there are great difficulties in the way of determining the small,
significant quantities of soluble or available iron in the soil, it seems
from the work of Morse and Curry (34), Ruprecht (40), and Abbott (r)
that acid soils may contain much more soluble iron and aluminum than
neutral or calcareous soils and may even contain an injurious amount
of these compounds.
The following work on the availability of iron compounds is based on
the assumption that the chlorosis and the poor growth of rice in the
calcareous soils were caused by a lack of available iron. This assump-
tion seems justified by the results presented in the first part of this
report.
AVAILABILITY OF ORGANIC IRON COMPOUNDS
In work with pineapples it developed that in the presence of a great
amount of organic matter a large amount of carbonate of lime was
required to induce chlorosis (12). This suggested that in calcareous
soils organic iron compounds might be more available than the inorganic,
just as iron in solution as a complex ion is less completely precipitated by
the usual reagents. The idea seemed substantiated by tests with rice
in nutrient solutions containing carbonate of lime, where ferric tartrate
furnished much more available iron than equivalent quantities of ferrous
sulphate or ferric chlorid.
Experiment VIII. — Tests were accordingly conducted to determine
the effect of various iron compounds and organic materials on the growth
of rice in both calcareous and noncalcareous soils. In this experiment
the effects of certain pure organic compounds of iron were compared
with those of ferric chlorid and ferrous sulphate. A substance which
Oct. i, 1920
Cause of Lime-Induced Chlorosis
5i
may be called "ferric molasses" was also used. This was prepared by
boiling together 2 parts of ferrous sulphate and 10 parts of a final molasses.
It doubtless contained some ferric acetate, glucolate, laevulate, possibly
other organic iron compounds, and considerable inorganic iron. As a
control on the action of the "ferric molasses," the same quantity of
molasses which had been similarly boiled without addition of iron was
applied to two other lots of pots. To one of these lots ferrous sulphate
was applied after the boiled molasses had been mixed with the soil in
the pots designated as "molasses and ferrous sulphate" in Table XI.
Five rice plants were grown in each pot from September 28 to Decem-
ber 28, 1 91 4. In the noncalcareous series each pot contained 14 pounds
of loamy soil with the moisture content maintained at 23 per cent of the
dry weight; and in the calcareous series each pot contained 14 pounds of
loamy soil with the moisture content maintained at 27 per cent of the dry
weight. The calcareous soil contained 17.8 per cent of carbonate of lime.
A fertilizer consisting of 1.8 gm. ammonium nitrate, 4.2 gm. sodium nitrate,
3 gm. ammonium sulphate, 0.4 gm. acid potassium phosphate, 3.9 gm.
acid phosphate, and 3.8 gm. potassium sulphate was added to each pot
in four applications. The molasses and all the iron compounds were
mixed with the soil before the rice was planted. The iron was applied
at the rate of 0.25 gm. and the molasses at the rate of 6.25 gm. per pot.
The results of the experiment are summarized in Table XI.
Table XI. — Comparative availability to rice plants of organic and inorganic compounds
of iron in a calcareous and noncalcareous soil: Experiment VIII
Special additions to the
soil.
Oven-dried yield of plants per pot.
Calcareous soil.
Series Series
A. B.
Series
C
Series
D.
Series
E.
Aver-
age.
Noncalcareous soil.
Series
A.
Series
B.
Series
C
Series
D
Series
E.
Aver-
age.
Ferric chlorid
Ferric tartrate
Ferric citrate
Ferric valerianate
Ferric benzoate
Molasses
" Ferric molasses "
Molasses and ferrous sul-
phate
Ferrous sulphate
Gm.
20
16
12
18
Gm.
16
Gm.
Gm.
Gm.
13
Gm.
13
19
IS
Gm.
39
Gm.
41
Gm.
38
Gm.
4i
Gm.
Gm
Three weeks after planting, all plants in the noncalcareous soil were
green, while many plants in the calcareous soil were slightly chlorotic.
Those plants in the calcareous soil which received molasses alone or
molasses with ferrous sulphate were markedly chlorotic (PI. 6, B) . During
later growth the plants in noncalcareous soil remained green and those
in calcareous soil became more chlorotic, some plants eventually dying
from the top down.
52 Journal of Agricultural Research vol. xx.No. i
In the noncalcareous soil none of the special compounds affected
growth significantly, and in the calcareous soil none of the iron com-
pounds proved efficient sources of iron, although possibly the ferric
tartrate and benzoate increased growth slightly.
Molasses alone and molasses followed by ferrous sulphate depressed
growth markedly and intensified the chlorosis of plants in the calcareous
soil, but the "ferric molasses" had no effect. Probably the molasses
that had not been treated with iron still further depressed the availa-
bility of iron in the calcareous soils by promoting the formation of in-
soluble organic iron compounds.
Experiment IX. — Later a second test was conducted with pure
organic iron compounds and organic materials containing iron in cal-
careous and noncalcareous soils. The pure iron compounds were applied
so as to furnish 0.75 gm. or 1.50 gm. of iron per pot, the smaller applica-
tion being at approximately the same rate as in the preceding experiment,
if the sizes of the pots and quantities of soil used in the two experiments
are considered. In the tests with ferric citrate and ferric tartrate, a
comparison was made between the results obtained by mixing all the
material with the soil before planting and those obtained by applying
the material in small doses in solution during the growth of the plants.
This was done to see if the materials might not be available for a short
time in the soil although rendered unavailable in the course of time by
bacterial or other action.
The "ferric humate," which, it was thought, might contain some iron
compounds similar to those existing in a natural soil, was prepared by
extracting leaf mold with 4 per cent ammonia, acidifying with hydro-
chloric acid, washing the precipitate free from chlorids, and evaporating
the precipitate to dryness with sufficient ferric chlorid solution to furnish
25 per cent as much iron as dry matter. The "mixture" used per pot
was composed of 4 gm. dried blood, 40 gm. Stizolobium vines, 40 gm.
tobacco stems, and 0.90 gm. iron from equal parts of ferric citrate,
tartrate, "humate," tannate, oxalate, and benzoate. Velvet beans
(Stizolobium) were tested because they are extensively growrn as a green
manure crop. Both Stizolobium vines and tobacco stems were cut up
before mixing with the soil. Citric and tartaric acids were tried to see
whether an organic radical alone would have any effect in maintaining
available iron in the soil. The test was conducted from December 8,
1916, to February 19, 1917, with eight rice plants in each pot. The
pots contained 42 pounds of sandy loam soil, or 47 pounds of sandy soil
containing 10 per cent carbonate of lime. The moisture contents of
both soils were maintained at 18 per cent of the dry weight. The ferti-
lizer for each pot was given in two applications and consisted of 15 gm.
ammonium sulphate, 19.5 gm. acid phosphate, and 6 gm. potassium
sulphate. The special additions were mixed with the top 4 inches of
soil before the rice was planted, except the solutions of ferric citrate
Oct. i, 1920
Cause of Lime- Induced Chlorosis
53
and ferric tartrate which were applied to the soil every other day.
Results of the test are given in Table XII.
Table XII. — Comparative availability to rice plants of organic and inorganic iron com-
pounds in calcareous and noncalcareous soils: Experiment I
Special additions
to the soil.
Amount
added.
Oven-dried yield of plants per pot.
Calcareous soil.
Series
A
Series
B
Series
C.
Series
D.
Series
E.
Aver-
age.
Noncakareous soil.
Series
Series
Series
Series Series
A.
B.
C.
D.
E.
Gm.
Gm.
Gm.
Gm.
Gm.
60
67
68
6s
75
69
72
7o
69
76
7«
70
66
54
75
62
66
69
68
65
72
68
68
73
69
69
68
77
68
75
61
69
71
63
6s
59
61
66
63
56
63
64
65
71
64
75
75
79
77
69
7'
69
74
66
62
67
78
72
75
57
62
64
66
72
67
57
56
58
64
63
74
60
68
66
70
67
65
66
67
68
65
65
69
78
76
59
54
57
59
56
72
68
66
58
77
7'
67
71
68
73
Aver-
age.
None
None
Ferric oxalate
Do
Ferric tannate. . . .
Do
" Ferric humate".
Do
Ferric citrate
Solution of ferric
citrate
Ferric tartrate. . . .
Solution of ferric
tartrate
Tobacco stems
Do
Stizolobium vines.
Do
Dried blood
"Mixture"
Citric acid
Tartaric acid
Gm.
2-43
4.86
16.36
Gm.
28
30
18
19
19
38
Gm.
Gm.
23
Gm.
26
Gm.
26
28
24
24
25
28
16
16
24
Gm.
Gm.
After three weeks the plants in noncalcareous soil were about twice
the size of those in calcareous soil. Later the plants in calcareous soil
were all more or less chlorotic, but the plants in pots receiving the larger
applications of tobacco stems, cover crop, or "mixture" were less chlo-
rotic than others. All the plants in the noncalcareous soil were a good
green throughout growth.
In the noncalcareous soil none of the materials significantly affected
growth except the "mixture," which depressed the yield about 20 per
cent. In the calcareous soil the "ferric humate " was distinctly injurious,
while the larger applications of tobacco stems and Stizolobium vines were
plainly beneficial, although they did not induce a normal growth.
Summary. — All organic iron compounds tried in the two preceding
experiments failed to increase appreciably the growth of rice in the cal-
careous soils. It is, therefore, probable that organic iron is no more
available than inorganic iron in such soils.
While concentrated or soluble organic materials, such as dried blood,
citric and tartaric acids, molasses, and a humus extract, failed to ameli-
orate the chlorosis, bulky organic materials, such as tobacco stems and
velvet bean plants, when used in considerable quantities measurably
improved the growth and color of the plants. Also, in previous work with
pineapples and sugar cane large amounts of stable manure ameliorated
54 Journal of Agricultural Research vol. xx, no. i
or completely overcame the chlorosis, although small amounts were
without appreciable effect.
In view of the nonavailability of the concentrated organic iron com-
pounds, it seems probable that the beneficial effect of the bulky organic
materials was not due primarily to the addition of certain iron compounds
that were available in the calcareous soil as a whole. It is more probable
that the particles of organic material formed isolated centers or points
where iron was more available than in the rest of the soil. The plants
were not able to secure all the iron they needed from these points for the
reason that plants are apparently not able to absorb a maximum amount
of iron with only a portion of their roots (18).
It may seem that the results of the last two tests negative the con-
clusions arrived at in the experiments with rice grown in solutions con-
taining carbonate of lime where organic iron compounds supplied sufficient
available iron. Conditions in the nutrient solutions, however, were some-
what different from those in the soil. To begin with, in the nutrient
solutions the plants obtained their iron from an ordinary solution that
was more or less sterile and that was frequently renewed. In the soil,
on the other hand, the plants probably obtained their nutrients from
aqueous films surrounding the soil particles, and there is evidence that
in films reactions may occur which do not take place in ordinary solu-
tions. Furthermore, bacterial action in the soil might have destroyed
rapidly certain of the organic compounds supplied.1
EFFECT OF WATER CONTENT OF SOIL ON THE AVAILABILITY OF IRON
At present we know little of the true soil solution or film moisture. It
is evident, however, that the nature of the soil particles must influence
the composition of the solution or substances dissolved in the enveloping
film. In the films surrounding particles of calcium carbonate the amount
of iron in solution must be greatly reduced, since the iron would be pre-
cipitated as ferric oxid.
If it is assumed that each particle in the soil is isolated and that the
moisture films surrounding the individual particles are discontinuous, it
would follow that the larger the proportion of particles which were carbon-
ate of lime the less soluble iron there would be in the whole medium.
This assumption would explain why carbonate of lime is more effective
in inducing chlorosis the more finely divided it is and why a certain
quantity of carbonate of lime exerts a stronger influence in a sandy soil
containing relatively few particles than in a clay soil containing a large
number of particles.
However, the case is not so simple as is assumed above. The moisture
films are not discontinuous but more or less continuous, the continuity
1 The fact that ferric citrate and ferric tartrate were no more effective when applied in frequent small
doses than when applied all at once is some evidence against the idea that the organic iron compounds were
unavailable because they were destroyed by bacterial action.
Oct. i, i92o Cause of Lime-Induced Chlorosis 55
and thickness of the films depending somewhat on the amount of moisture
in the soil. The substances in solution in a film surrounding one particle
will therefore react with those in films surrounding adjacent films. One
particle of carbonate of lime would affect the soluble iron in the films of a
certain number of adjacent particles.
While the moisture films are to a certain extent continuous, we know
that the composition of the films is not uniform throughout the soil.
This is evident from certain well-established facts, such as the slight
lateral movement of fertilizers. If the composition of the films were
uniform and conditions were analogous to those in a solution with rela-
tively few solid particles, a slight amount of carbonate of lime would
have the same effect as a much larger amount. This, however, is not
the case.
It might be expected that the effect of carbonate of lime in depressing
the availability of iron and in inducing chlorosis would be influenced
somewhat by the amount of water in the soil, since the aggregation of
the soil particles and their moisture films would be affected by the water
content. It was, therefore, of interest to observe the manner in which
the growth and chlorosis of rice would be affected by different percentages
of moisture in calcareous soil.
A preliminary test was conducted with four pots, each of which held
36 pounds of soil containing 15 per cent of calcium carbonate. Twelve
rice plants were grown in each pot with abundant fertilizer. The plants
were grown 30 days with 22 per cent of moisture in the soil. Water was
then added to two of the pots until there were 2 inches of water above
the surface of the soil, and the other two pots were maintained unchanged
at 22 per cent moisture. After 67 days' growth the plants were cut.
The plants in all four pots were very slightly chlorotic at 30 days, but
a few days after the extra water was added the submerged plants became
intensely chlorotic and remained so for about 10 days. They then
quickly improved in color, and a few days later the submerged plants
were a perfectly normal green, while the plants in the soil with 22 per
cent moisture were markedly chlorotic. This difference persisted until
the plants were cut. The plants grown for the whole period with 22 per
cent moisture gave an average green weight of 175 gm. per pot, while
the plants grown for 30 days with 22 per cent moisture and then sub-
merged for 37 days yielded 424 gm. per pot.
Experiment X. — An extended test was conducted from January 2 to
March 22, 191 8, using one noncalcareous soil and two calcareous soils
(one a beach sand with practically no organic matter and the other a
loam) ,* The noncalcareous soil was used as a control to determine how
the growth of rice would be affected by different amounts of water in a
1 The calcareous loam was the same as the noncalcareous soil except for the addition of the carbonate
of lime some years before.
56
Journal of Agricultural Research
Vol. XX, No. i
soil adapted to its growth. Each pot received 9 gm. sulphate of potash,
6 gm. double superphosphate, and 22.5 gm. sulphate of ammonia divided
in two applications. Twenty rice plants were planted in each pot, but
these were thinned to 10 when growth was well established. The results
are given in Table XIII.
Table XIII. — Effect of varying degrees of moisture on the availability of iron to rice
plants in calcareous and noncalcareous soils
Opti-
mum
water
Per-
content
Soil
Kind of
centage
of cal-
of soil
ex-
No.
soil.
cium
carbon-
pressed
as per-
ate.
centage
of dry
weight
of soil.
1647
1648
25-5
23-2
...do...
8-53
1 194
Sand..
19.0
11. 6
Maxi-
mum
water
capac-
ity of
soil ex-
pressed
as per-
centage
of dry
weight
of soil.
Amount
of soil
per pot.
Amount of water main-
tained in soil during
growth of plant.
36. 2
Pounds.
69 !
69
22.3 per cent
26.3 per cent
30.3 per cent
34.3 per cent
Water at surface of soil.
Water 3 inches above
surface of soil
20.2 per cent
24.2 per cent ,
28.2 per cent
32.2 per cent
36.2 per cent
Water 3 inches above
surface of soil
1 1 per cent
18 per cent
25 per cent
Water 3 inches above
surface of soil
Oven-dried yield of plants
per pot.
Series
Series
Series
A.
B.
C.
Gm.
Gm.
Gm.
119- 8
122. 1
103.9
125-4
120.8
127.0
146. 1
128.9
137- 7
129. 2
142. 1
141. 6
159- 9
167.5
IS3-8
155-9
157-0
177.6
58.1
5i- S
57-5
74-9
68.9
79.1
53-7
70.8
78.8
66.1
74.2
67.1
87-3
72.9
77-8
112. 5
134.6
122.8
12.4
9.9
9.2
6.1
13-4
13-6
1.2
9-4
1.4
17.6
8.4
28.1
Aver-
age.
Gm.
U5-3
124.4
137-6
137-6
160.4
163.8
55-7
74-3
67.8
69- I
79-3
123.3
10.5
11. 1
4.0
18. 1
The different water contents maintained during the experiment were
made up when the plants were 4 days old, except that the pots to receive
3 inches excess water were made up with water at the surface at this
time, the water being raised to 3 inches when growth permitted it.
When n days old, the plants in soils No. 1647 and 1648, where water
was at the surface or above it, were markedly chlorotic, as well as all
the plants in soil No. 1194. After 31 days' growth, all the plants in soil
No. 1 1 94 were still markedly chlorotic; the submerged plants in soil No.
1647 were normal green and were growing rapidly, as were all other plants
in this soil; in soil No. 1648 the submerged plants and those in pots with
20.2 and 24.2 per cent water were normal green, while those in pots with
28.2, 32.2, and 36.2 per cent water were plainly chlorotic. At 72 days'
growth, when the plants were cut, the appearance in regard to chlorosis
was similar to that at 31 days, except that in soil No. 1 194 the few plants
that had not died in the pots with 3 inches excess water were normal
green and far larger than the others.
The temporary chlorosis affecting the plants where the excess water
was added is entirely distinct from the lime-induced chlorosis. A similar
yellowing takes place in the field when the fields are flooded following
Oct. i, i92o Cause of Lime-Induced Chlorosis 57
early growth without submergence. Several of the surplus plants in the
pots with excess water were brushed repeatedly with ferrous sulphate,
but the treatment did not improve the color of the plants in the slightest.
Evidently this particular chlorosis is not due to lack of iron. Doubtless
when the water content of the soil is raised above the point of saturation
the old roots are unable to function properly and the nutrition of the
plant is disturbed until new roots are sent forth which are able to function
under the new conditions.
It was thought that roots of the submerged plants might show morpho-
logical differences from roots of plants grown with ordinary amounts of
water in the soil. Samples of roots from plants grown in soil No. 1647
were therefore subjected to a preliminary examination by Dr. Albert
Mann, of the Bureau of Plant Industry, United States Department of
Agriculture, to whom thanks are due. A portion of Dr. Mann's report
of the preliminary examination follows:
The differences noted between No. 1805 with 24.2 per cent moisture, 1807 with
32.2 per cent, and 1809 with water standing three inches above the surface are slight.
There is in general more compactness and strength of tissue in 1805 than in the others.
The central fibrovascular bundle mass is larger in proportion to the cortex than in
1807 or 1809. The cells of all the tissues are slightly more robust. The light paren-
chyma, which makes up the cortex from the endodermal ring to the epiderm, is es-
pecially thinner walled and more developed in 1809. There is also a notable absence
of root hairs in this sample as compared with the other two, which is, of course, the
inevitable result of the roots growing submerged in water.
The series in the noncalcareous soil shows that the growth of rice
should increase regularly with increasing amounts of water in the soil
until a percentage near the saturation point of the soil is reached and
that, possibly because of a different root growth, there should be another
considerable increase when enough water is added for submergence. In
No. 1648, however, the series with the calcareous soil, there were two
maxima of growth, one at 24.2 per cent water and one at 3 inches excess;
and in the calcareous sand No. 1194 there were also two maxima. It is
believed that the first lower maximum was due to iron being a little
more available at that water content than at a higher content. The
great increase in growth in the calcareous soils produced by submergence ?
was probably due chiefly to the fact that the modified roots are better
able to assimilate iron than the ordinary type of root and was probably
not due to increased availability of iron in the submerged soil.
It is felt that the results substantiate the idea that the availability of
iron in the soil is affected somewhat by the amount of water in the soil,
the availability being slightly greater near the optimum water content
than with larger amounts.
The effect of the water content is probably due to its influence on
the extent to which reactions take place between the moisture films
1 It will be noted that in the calcareous soils the increase produced by submergence was much greater
than in the noncalcareous soil.
58 Journal of Agricultural Research vol. xx, No. 1
surrounding the calcareous particles and those surrounding the other soil
particles. With moisture contents above the optimum the moisture
films become more continuous and the sphere of influence of the particles
of carbonate of lime in reducing the availability of iron becomes- more
extended.
Incidentally the tests established a fact of considerable practical im-
portance— namely, that rice may be expected to make a practically
normal growth in certain calcareous soils if the soils are submerged.
SUMMARY
There are a few plants which are generally conceded to be calcifugous,
inasmuch as they are rarely found on calcareous soils.
Soil surveys of several species of cultivated plants show that a parti-
cular type of chlorosis affecting these plants occurs only on calcareous
soils. All calcareous soils, however, do not induce chlorosis in these
plants.
Addition of carbonate of lime to soils producing normal, calcifugous
plants causes the soils to produce chlorotic plants.
It is, therefore, evident that a chlorosis of some plants is caused by,
or is associated with, the presence of carbonate of lime in the soil.
The weight of the evidence from ash analyses of chlorotic plants seems
to point to a deficiency of iron in the ash as being one cause of the chlorosis,
with possibly an excess of lime as a contributory cause.
Treatment of chlorotic plants with iron shows that a lack of iron in
the plant is at least one of the causes of lime-induced chlorosis.
There is no evidence of a general "lime effect" in inducing chlorosis,
the different lime compounds affecting the plants differently.
Rice, one of the plants sensitive to lime, does not appear to be sensitive
to the alkalinity of carbonate of lime except as this alkalinity influences
the availability of the iron.
Lime-induced chlorosis seems to be due simply to a depression in the
availability of iron in calcareous soils.
A number of pure organic iron compounds and concentrated organic
preparations proved to be inefficient sources of iron for rice in calcareous
soils. Bulky organic compounds such as stable manure, velvet bean
plants, and tobacco stems, when used in considerable quantity, however,
enabled the plant to secure more iron.
The availability of iron in calcareous soils appears to be slightly
greater near the optimum water content of the soil than at higher per-
centages of water.
Although rice becomes chlorotic in calcareous soils with ordinary
percentages of water, it will grow normally in certain calcareous soils
if the soil is submerged. This is believed to be due to the growth, under
submerged conditions, of a new kind of root that is better able to assimi-
late iron than the root formed in the soil with less water.
Oct. i, 1920 Cause of Lime-Induced Chlorosis 59
LITERATURE CITED
(1) Abbott, J. B., Conner, S. D., and Smalley, H. R.
19 13. THE RECLAMATION OF AN UNPRODUCTIVE SOU, OP THE KANKAKEE
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(2) Breslau Agrikulturchemische Versuchsstation.
1902. dungungsversuch mit kohlensaurem kalk zu serradella
[1899.] In Landw. Jahrb., Bd. 30, 1901, Erganzungsbd. 2, p. 61.
(3) BtiSGEN, M.
1914. KIESELPFLANZEN AUF KALKBODEN. KULTURVERSUCHE ZUR PFLANZEN-
geographie. In Bot. Jahrb. [Engler], Bd. 50, Sup., p. 526-538,
pi. 10-11.
(4) Castle, R. Lewis.
1899. chlorosis in fruit Trees. In Gard. Chron., s. 3, v. 25, no. 652, p.
405; v. 26, no. 653, p. 4.
(5) Creydt, Bodo. •
1915. UNTERSUCHUNGEN UBER DIE KALKEMPFINDLICHKEIT DER LUPINE UND
ihre bekampfung. In Jour. Landw., Bd. 63, Heft 2, p. 125-191,
6 pi.
(6) Dauthenay, H.
1901. SUR LA CHLOROSE DES ARBRES FRUITIERS EN TERRAIN CALCAIRE. In
Rev. Hort. [Paris], ann. 73, no. 2, p. 50-51.
(7) Engler, Arnold.
I901. UBER VERBREITUNG, STANDORTSANSPRUCHE UND GESCHICHTE DER
CASTANEA VESCA GARTNER MIT BESONDERER BERUCKSICHTIGUNG DER
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(8) Euler-Chelpin, H. K. A. S. von.
1909. GRUNDLAGEN UND ERGEBNISSE DER PFLANZENCHEMIE. T. 3: DIE
CHEMISCHEN VORGANGE IM PFLANZENKORPER. Braunsch Weig .
(9) Fliche, P., and Grandeau, L.
1873. DE L 'INFLUENCE DE LA COMPOSITION CHIMIQUE DU SOL SUR LA VEGETA-
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(10)
1874. DE L'iNFLUENCE DE LA COMPOSITION CHIMIQUE DU SOL SUR LA VEGE-
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(11)
1879. RECHERCHES CHIMIQUES SUR LES PAPILIONACEES LIGNEUSES. In Ann.
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(12) Gtle, P. L.
191 1. THE RELATION OF CALCAREOUS SOILS TO PINEAPPLE CHLOROSIS. Potto
Rico Agr. Exp. Sta. Bui. 11, 45 p., 2 pi. (1 col.).
(13) and Ageton, C. N.
I914. THE EFFECT OF STRONGLY CALCAREOUS SOILS ON THE GROWTH AND ASH
composition of certain plants. Porto Rico Agr. Exp. Sta. Bui.
16, 45 p., 4 pi.
(14) and Carrero, J. O.
1914. assimilation of colloidal iron by rice. In Jour. Agr. Research,
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ds)
I915. ASH COMPOSITION OF UPLAND RICE AT VARIOUS STAGES OF GROWTH.
In Jour. Agr. Research, v. 5, no. 9, p. 357-364.
187931°— 20 5
60 Journal of Agricultural Research vol. xx, No. i
(16) Gile, R. L. and Carrero, J. O.
1916. immobility of iron in the plant. In Jour. Agr. Research, v. 7, no.
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1916. ASSIMILATION OK IRON BY RICE FROM CERTAIN NUTRIENT SOLUTIONS.
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(17)
(18)
I917. ABSORPTION OF NUTRIENTS AS AFFECTED BY THE NUMBER OF ROOTS
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(19)
1917. chlorosis OF sugar cane. In Porto Rico Agr. Erp. Sta. Rpt. 1917,
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(20) Gris.
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(21) GUTLLON, J. M.
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(22) and Brunaud, O.
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(27)
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Oct. 1. 1920 Cause 0} Lime-Indttced Chlorosis 61
(31) Mach, E., and Kurmann, Fr.
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(45) VERNEun,, A., and Lafond, R.
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PLATE s
A. — Rice grown in calcareous and noncalcareous soils and sprayed with ferrous
sulphate solution (experiment I).
1-4. Noncalcareous soil; plants in 1 and 3 unsprayed, those in 2 and 4 sprayed.
5-8. Soil containing 30 per cent carbonate of lime; plants in 5 and 7 unsprayed,
those in 6 and 8 sprayed.
B. — Apparatus used in growing plants in experiment VII.
(62)
Cause of Lime-Induced Chlorosis
Plate 5
Journal of Agricultural Research
Vol. XX, No. 1
Cause of Lime-Induced Chlorosis
Plate 6
Journal of Agricultural Research
Vol. XX, No. 1
PLATE 6
A.— Effect of carbonate of lime in depressing the availability of iron (experi-
ment vii):
i Calcareous soil in bucket, silica sand plus iron in sieve.
2 Calcareous soil in bucket, silica sand plus carbonate of lime and iron in sieve.
B — Effect of various substances on growth of rice in calcareous soil (experi-
ment VIII).
i. Noncalcareous soil.
2. Calcareous soil .
3. Calcareous soil with molasses added.
4. Calcareous soil with "ferric molasses" added.
5. Calcareous soil with molasses and ferrous sulphate added.
6. Calcareous soil with ferrous sulphate added.
AN EXPERIMENTAL STUDY OF ECHINACEA THERAPY
By James F. Couch and Leigh T. Giltner, Pathological Division, Bureau of Animal
Industry, United States Department of Agriculture
INTRODUCTION
The use of echinacea as a remedy for various disorders in both human
beings and animals is said to have originated with the American Indians,
from whom the early settlers in the West learned of the "virtues" of
the plant.1
In 1885 Dr. H. C. F. Meyer, of Pawnee City, Nebr., sent a specimen
of the plant to Prof. Lloyd. It was identified as Echinacea angustifolia
(DC). Dr. Meyer was using the root of this plant in a secret mixture
which he called "Meyer's Blood Purifier," and the claims which he made
for the curative properties of the root are described as " most exaggerated."
Indeed, he had such confidence in it that he offered to submit to repeated
bites of rattlesnakes, proposing to demonstrate the remedial power of
echinacea against this venin by using his preparation of the root as an
antidote. This offer was, of course, refused, but the drug was studied by
Dr. John King, Prof. H. T. Webster, and others, with the result that clinical
evidence was obtained which appeared to substantiate some of the claims
of Dr. Meyer. Preparations of the plant were placed on the market,
and the medicinal use of echinacea increased rapidly. Many physicians
have reported favorable results from its use in various diseases.
In 1909 a report (j) of the Council on Chemistry and Pharmacy of the
American Medical Association denied echinacea a place in "New and
Non-Official Remedies" and directed suspicion at the value of the drug,
stating :
In view of the lack of any scientific scrutiny of the claims made for it, echinacea
is deemed unworthy of further consideration until more reliable evidence is presented
in its favor.
In spite of this the use of echinacea has become extensive. Lloyd states
that it is used in largei quantities than any other American drug intro-
duced since 1 887. The fluid extract and tincture are made in enormous
quantities, and the root enters into the composition of a large number of
patent, proprietary, and nonsecret mixtures.
The last revision of the National Formulary includes a description of
echinacea and furnishes a formula for its fluid extract (r, p. 74, 294).
This amounts to a quasi official recognition of the drug. It has never
been official in the United States Pharmacopoeia.
1 The historical facts about echinacea have been obtained almost wholly from published accounts by
Meyer (75) and Lloyd (11, 12, 13). Reference is made by number (italic) to "Literature cited," p. 83-84.
Journal of Agricultural Research, Vol. XX, No. 1
Washington, D. C. Oct. 1, 1920
vb Key No. A-52
(63)
64 Journal of Agricultural Research voi.xx,No.i
Five species of echinacea are mentioned in works on botany (<5).
Brauneria purpurea (DC.) (Rudbeckia purpurea or Echinacea purpurea
[Moench]) is found from western Pennsylvania and Virginia to Michigan,
Iowa, and southward. B. angustifolia (DC.) (E. angustifolia [DC.]) is
found from Tennessee and Minnesota to Saskatchewan, Texas, and
Nebraska. B. pallida (Nutt.) occurs from Michigan and Illinois to Texas
and Alabama, while B. paradoxa (Norton) and B. atrorubens (Britton)
(R. pallida) are found from Missouri to Texas. The rays of the last two
species are bright yellow in color. The species which furnish the larger
proportion of the medicinal supplies are B. pallida and B. angustifolia.
It has been stated that the eastern species, B. purpurea, is inert.
CHEMICAL CONSTITUENTS
In 1897 Lloyd (//) reported the presence of a colorless alkaloid and a
colorless acid-reacting substance of intensely acrid properties. The root
has been subjected to analysis by Heyl and Staley (<?) and Heyl and
Hart (7), by whom the alkaloid was identified as betaine. Nothing of a
physiologically active nature, however, was isolated by these inves-
tigators.
THERAPEUTIC USES
General accounts of the various uses to which echinacea has been put
have been published by Ellingwood (4) and by Lloyd (13). Echinacea
is stated to be a corrective of "depravation" of body fluids, of septic,
fermentative, or zymotic conditions. It is said to antagonize infectious
processes and "blood poison," to be useful in puerperal sepsis, uremia,
pernicious malarial or septic fevers, typhoid fever, and all fevers caused
by absorption of septic material. It has been recommended as a specific
against the venins of rattlesnakes, other serpents, and insects (9) —
in crotalus it stands without a peer.
Pyemia, goiter, smallpox, anthrax, and hydrophobia are reported to have
been cured by echinacea. It is said to be an antidote for tetanus. It
has been used locally in erysipelas, bedsores, fever sores, chronic ulcers,
glandular indurations, syphilitic nodules, burns, and gangrene (14) and
is said to be an active sialogogue, diuretic, and diaphoretic. Jensen found
it useful in the treatment of carbuncles.
The uses of echinacea in veterinary practice have been discussed by
Fish (5), who found the root to increase the elimination of urea. In some
pharmacological experiments upon kittens he obtained evidence of nar-
cosis, and emesis was provoked by the fluid extract given per os. He
quotes five cases in which the administration of echinacea was followed
by improvement.
The compound of inula and echinacea prepared especially for parenteral
administration has been stated to be useful in the treatment of tuber-
culosis (18), has been designated "an effective treatment for canine
Oct. i, 1920 An Experimental Study of Echinacea Therapy 65
distemper," and is recommended in the treatment of equine influenza (10).
Slawson (16) does not consider this preparation satisfactory in the
treatment of canine distemper. He finds that its action does not differ
from that of nuclein, leucocyte extract, or plain serum.
PRESENT INVESTIGATION
The investigation of which the results are here reported was under-
taken for the purpose of determining, so far as the limits of laboratory
experiment permit, the usefulness of echinacea as a remedy in several
pathological conditions induced by bacteria, their products, or allied
toxins.
The animals used were guinea pigs bred at the Bethesda (Md.) Experi-
ment Station of the Bureau of Animal Industry, all in healthy condition
and apparently normal. The animals were kept under observation long
enough before experimental use to exclude any but the most remote
possibilities of accidental factors.
PREPARATIONS TESTED
The preparations of echinacea employed in the remedial work con-
sisted of the following:
1. A sample of "Specific Medicine Echinacea," manufactured by and
obtained from Lloyd Brothers, of Cincinnati, Ohio. This is a liquid
preparation which is stated to contain 480 gr. of echinacea root per fluid
ounce, or slightly more than a modern fluid extract. It contained 69
per cent of alcohol and conformed to the organoleptic tests for select
echinacea. It was identified and preserved free from change during
the whole course of the investigation. This remedy was diluted with
distilled water for administration per os. The treatment caused the
mixture to become cloudy because of the suspension of the resinous and
oily constituents of the plant. These mixtures were never allowed to
stand long enough for the insoluble matters to separate but were given
to the animals while still in the stage of emulsion. In this way it is
certain that the guinea pigs received all of the constituents of echinacea
which are soluble in 69 per cent alcohol.
2. A fluid extract of echinacea purchased on the open market. This
contained 70 per cent of alcohol and was identified, preserved, and
administered exactly as was the specific medicine mentioned above.
3. "Subculoyd Inula and Echinacea," manufactured by and obtained
from Lloyd Brothers. This liquid was used in the greater portion of
the parenteral administrations. It is stated to contain, in 3 mils, 1.33
mils of Inula helenium and 1 mil of echinacea. It does not contain
alcohol. This material was scrupulously preserved from contamination
and change. In certain of the experiments it was administered intra
muscularly; in other cases it was injected subcutaneously. Upon
66 Journal of Agricultural Research voi.xx.Nai
autopsy of animals treated with this liquid there was noticed some
necrosis of the tissues at the points of injection, but no other unfavorable
results from its administration were observed.
Certain other preparations of echinacea which are sometimes used
were not tested. A tincture of the green root is on the market, as is
also a variety of powdered and solid extracts of echinacea. These
preparations are all made with a menstruum of strong alcohol, and it is
therefore not to be supposed that they contain any components not
present in the fluid extracts which we used. The manufacturers of
certain green-root tinctures assert that this product is superior to prepa-
rations of the dried root; there is, however, not the slightest published
evidence to substantiate this assertion. The early settlers are said
to have used the green root bruised and in the form of infusion. In
the present work no such form of the remedy was used. It is quite
possible that an infusion would contain some substances which are absent
in the strongly alcoholic preparations and might, on this account, affect
the organism differently. The claims of the therapeutic efficiency of
echinacea have, however, been very largely made through the use of
alcoholic preparations, and we therefore felt justified in employing
these in determining its value as a remedy.
PATHOLOGICAL CONDITIONS TREATED
The acute experimental pathological conditions produced in the
guinea pigs were tetanus, botulism (in both of which the diseases were
produced by bacterial toxins), anthrax, septicemia (in both of which
the bacteria were injected into the animals), and crotalus poisoning (in
which the venin of rattlesnakes was injected). The chronic conditions
were those of tuberculosis, which was produced by inoculation with the
bacillus, and a trypanosomiasis (dourine), produced by inoculation with
the trypanosomes. The sources of these materials and the methods of
injection are described in the part of this paper which reports the experi-
mental work.
METHODS
The methods employed for testing the remedial powers of echinacea
against these several conditions were as follows :
i. Animals were injected with the pathogenic material and were
immediately afterwards treated with echinacea, in suitable doses, one
dose per diem, until the animal succumbed or became unable to swallow
(if the administration was per os).
2. Animals were dosed with echinacea for several days before they
were injected with pathogenic material, a protective treatment designed
to favor the drug as much as possible, and were given remedial doses
as long after the injection as possible. The treatment with the "Sub-
culoyd" followed the same course. Treatment was necessarily sus-
pended on Sundays and holidays, but in all except the chronic cases
the time was so chosen as to minimize breaks due to such cause.
Oct. i, i9*o An Experimental Study of Echinacea Therapy 67
DOSAGE
The dose of fluid extract echinacea is variously given as from 10
minims to 0.5 fluid ounce for adult human beings, and for the "Sub-
culoyd" preparation the parenteral dose recommended is 3 to 10 mils
daily. It has also been stated that large doses of echinacea do not
produce toxic effects upon healthy subjects, although this has been
contradicted. The doses chosen for our experimental animals ranged
from 0.25 to 1 mil daily of fluid extract and from 0.2 to 0.5 mil daily of
subculoyd, which, calculated on a kilogram-of-body-weight basis, would
correspond to from 40 to 160 mils daily of fluid extract and from 30 to
60 mils daily of the "Subculoyd" for man. It is well known, however,
that to produce a given effect in guinea pigs requires very much larger
doses per kilo than in larger animals. We decided upon a large dose
of the remedy so as to favor the echinacea as much as possible and to
remove any possibility of failure through administration of inadequate
amounts.
GENERAL RESULTS AND CONCLUSIONS
In no one of the diseases treated with echinacea was any evidence
obtained to show that the plant exerts any influence upon the course
of infectious processes under laboratory conditions. Daily feeding of
animals with echinacea preparations for several days before injection of
microorganisms or their toxins did not increase the resistance of the
animals to these agents. In the two chronic cases where the animals
were given doses of echinacea preparations for extended periods of time
nothing appeared in the autopsy pictures which could be attributed to
the action of the echinacea per se, except that in two cases a gastric
catarrh was present which may have been due to this plant. In all
cases the course of the disease was the same in the control animals and
in the animals which were given remedial treatment.
It does not appear, therefore, that echinacea or the preparation of
inula and echinacea are of value in the treatment of diseases produced
by microorganisms and their toxic products.
EXPERIMENTAL WORK
I. — TESTS OF ECHINACEA AS A REMEDY FOR TETANUS
In order to test the efficacy of echinacea as a remedy for tetanus a
total of 29 guinea pigs was used. The animals were injected with a
sample of standard tetanus toxin furnished by the Hygienic Laboratory
of the United States Public Health Service. This material was kindly
placed at our disposal by Dr. W. N. Berg, of our laboratory, who had
used a part of it in his work on the destruction of tetanus antitoxin by
chemical agents (2). It had been carefully standardized; the minimal
68 Journal of Agricultural Research voi.xx,No.i
lethal dose was 0.0007 mgm. for a 350-gm. guinea pig. The material
was preserved in vacuo in the dark and at low temperature. A fresh
solution of the toxin was prepared for use by carefully weighing out a
small quantity and dissolving this in just enough sterile normal salt
solution to furnish a liquid which should contain 6 minimal lethal doses
per mil. Each of the experimental animals received 0.5 mil of this
solution, an equivalent of 3 minimal lethal doses.
EXPERIMENT I. — ECHINACEA ADMINISTERED PER OS
Four guinea pigs were each given a 3-mil dose of a mixture of 1 mil of
the "Specific Medicine Echinacea" and 2 mils of distilled water once a
day for six days, a total of 6 mils of the remedy. The animals were
rested one day and on the eighth day were given another dose of the
remedial mixture, immediately followed by a subcutaneous injection of
0.5 mil of tetanus toxin solution (3 minimal lethal doses). On the fol-
lowing day all the animals received a dose of the remedy, so that each
guinea pig had then received a total of 8 mils of specific echinacea,
equivalent to somewhat more than 8 gm. of the root.
All of the animals exhibited the typical symptoms of tetanus and died
on the ninth day. The autopsies were negative; no evidence of any
ntercurrent disease was obtained. Three control guinea pigs which
were injected at the same time as the experimental animals died on the
same day with symptoms of tetanus and furnished the same post-mortem
EXPERIMENT 2. — ECHINACEA INJECTED INTRAMUSCULARLY
Echinacea injected intramuscularly was tested upon five guinea pigs.
The undiluted "Specific Medicine Echinacea" was injected into the
right and left thighs on alternate days. Each animal received four 0.5-
mil doses, one per day, a total of 2 mils. The treatment caused consider-
able swelling at the points of injection. On the fourth day the animals
were all given subcutaneous injections of 0.5 mil of the tetanus toxin
solution. They all exhibited the characteristic symptoms of tetanus
and died early in the morning of the third day after the injection. The
autopsy showed considerable local reaction of the tissues to the injection
of the echinacea. This consisted of a sero-sanguineous infiltration of
the subcutaneous and muscular tissues with small areas of degeneration
in the musculature at the point of injection. The internal organs
showed no gross lesions.
EXPERIMENT 3. — ECHINACEA AND TOXIN ADMINISTERED SIMULTANEOUSLY
In order to determine whether echinacea possesses properties similar
to the antitoxins, five guinea pigs were injected subcutaneously with 0.5
mil of the tetanus toxin solution and immediately received 0.5 mil of
undiluted "Specific Medicine Echinacea" injected intramuscularly into
Oct. i, 1920 An Experimental Study of Echinacea Therapy 69
the right thighs. On the following day 0.5 mil of the remedy was in-
jected into the left thighs of the animals. This treatment was wholly
remedial, no protective doses having been given as in experiments 1 and
2 of this series. In three days after the injection of the toxin all the
animals were dead after exhibiting typical tetanus. The autopsy pic-
ture was similar to that in experiment 2.
EXPERIMENT 4. — INULA AND ECHINACEA INJECTED INTRAMUSCULARLY
Protective doses of the "Subculoyd Inula and Echinacea" were in-
jected intramuscularly into five guinea pigs. The dose administered
was 0.5 mil per day for six days, a total of 3 mils, corresponding to 1
gm. of echinacea and 1.33 gm. of inula. On the eighth day after the
treatment was begun the animals were injected with 0.5 mil of tetanus
toxin solution, and a dose of 0.5 mil "Subculoyd" was given. The
total dose of the remedy was 3.5 mils. On the following day all the
guinea pigs showed typical symptoms of tetanus, and one died; the
remaining four died the next day. On autopsy there was found a mod-
erately severe local reaction of the tissues to the injection of the inula
and echinacea. The subcutaneous and muscular tissues at the site of
injection showed considerable hemorrhage and sero-sanguineous infiltra-
tion. No gross lesions were apparent in any of the internal organs.
EXPERIMENT 5. — INFLUENCE OF ALCOHOL ON TETANUS
Since the "Specific Medicine Echinacea" employed in the foregoing
experiments contained 69 per cent of ethyl alcohol, it was considered
desirable to study the influence of this factor upon tetanus under the
conditions of the echinacea experiments. Accordingly, a mixture of
alcohol and distilled water was made which contained exactly 69 per
cent of alcohol, and this was injected intramuscularly into four guinea
pigs in 0.5-mil doses. Each guinea pig received two doses, one into the
right thigh and, on the next day, one into the left thigh. Two days
afterwards all four received 0.5 mil of tetanus toxin solution subcuta-
neously. In three days two of these animals died, and the remaining
two died during the following night. All showed typical symptoms of
tetanus. The autopsy showed some congestion of the subcutaneous
tissues at the points of injection of the alcohol, hemorrhage in the mus-
culature, and evidence of local degeneration of the muscles. No gross
lesions were apparent in any of the internal organs.
EXPERIMENT 6. — CONTROLS
The six control animals were kept under the same conditions as the
experimental animals and received the same amounts of tetanus toxin.
They all developed the typical symptoms of tetanus and died in less
7o
Journal of Agricultural Research
Vol. XX, No. i
than three days. The autopsies were negative. No evidence of an
intercurrent disease was obtained.
The results of this series of experiments are given in Table I.
TABLE I. — Results of experiments with echinacea in the treatment of tetanus
6 (con-
trols).
/
Guinea
pig No.
Weight
cf guinea
pig-
Total
dose of
remedy.
Dose of
toxin.
Gm.
Mils.
M . 1. d. a
18
395
7
3
19
380
7
3
21
335
7
3
22
37°
7
3
1
445
2
a
2
455
2
3
\ 3
490
2
3
4
440
2
3
5
445
2
3
13
455
3
14
470
3
\ I5
435
3
16
45°
3
I 17
460
3
26
490
4
3
27
470
4
3
1 28
455
4
3
2Q
400
4
3
I 3°
445
4
3
9
59°
1
3
10
480
1
3
11
410
1
3
12
39°
1
3
6
415
0
3
7
480
0
3
8
440
0
3
23
475
0
3
24
435
0
3
I 25
395
0
3
Effect.
Tetanus. .
....do...
....do...
....do...
...do...
...do...
...do...
...do...
....do...
....do...
....do...
...do...
....do...
....do...
....do...
....do...
....do...
....do...
...do...
....do...
....do...
...do...
....do...
...do...
...do...
....do...
....do...
....do...
...do...
Termination.
Died.
Number of days
sick.
.do 2.
.do 2.
.do j 2.
.do Less than 3.
do.
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do..
..do..,
..do..,
Do.
Do.
Do.
Do.
Less than 3.
Do.
Do.
2.
" Minimal lethal dose.
SUMMARY OF EXPERIMENTS WITH TETANUS
"Specific Medicine Echinacea" was administered to guinea pigs both
per os and intramuscularly, the "Subculoyd Inula and Echinacea" was
administered to guinea pigs intramuscularly, and 69 per cent alcohol was
injected intramuscularly into guinea pigs, as a means of treatment for
tetanus. All of these animals were injected with 3 minimal lethal doses
of standard tetanus toxin in solution, some animals being injected sev-
eral days after they had been treated with echinacea, while others were
injected first and then treated with echinacea. Neither the protective
treatment nor the remedial treatment nor a combination of the two
appeared to influence the course of the disease, as all the experimental
animals acted in the same way and died in the same time as the controls.
From these results it does not appear that echinacea possesses remedial
value against experimental tetanus in laboratory animals.
Oct. i, i9io An Experimental Study of Echinacea Therapy 71
II. — TESTS OF ECHINACEA AS A REMEDY FOR BOTULISM
Since echinacea did not appear to influence the action of tetanus toxin,
it was thought desirable to compare its action against another bacterial
toxin. For this purpose botulinus toxin was chosen. The material used
to produce botulism in the experimental animals consisted of a germ-
free filtrate of a glucose beef infusion culture of Bacillus botulinus (Boise
strain) (17) incubated for one month at 370 C. The filtrate was diluted
with sterile normal salt solution in such amount that 1 mil was equivalent
to very nearly 10 minimal lethal doses for a 400-gm. guinea pig. This
toxin was not injected into the animals but was fed through the mouth
in order to duplicate the conditions under which this type of poisoning
usually occurs.
EXPERIMENT I. — ECHINACEA ADMINISTERED PER OS
Three guinea pigs only were used, because the results of the experiment
were so free from uncertainty that it was not considered necessary to
sacrifice more animals in order to determine the facts. The animals
were all given 2 -mil doses daily of a mixture of 0.5 mil fluid extract
echinacea and 1.5 mils distilled water for 6 days. The total protective
dosage was 3 mils of the fluid extract, equivalent to 3 gm of echinacea.
The animals were rested one day, and on the eighth day after the begin-
ning of the experiment all received 1 mil (10 minimal lethal doses) of
botulinus toxin immediately after receiving a 2-mil dose of the remedial
mixture. On the following day all the animals were sick. No. 78
received a remedial dose of 2 mils of the echinacea mixture. No. 79
received 1 mil of the same mixture, which was all that it could swallow.
With No. 80 the symptoms of pharyngeal paralysis were so marked that
it was considered inadvisable to drench the animal on account of the
danger of strangulation. This animal died during the afternoon. The
remaining two were found dead in the morning of the second day after.
The treated pigs and the controls showed no differences. The autopsy
showed general hyperemia of the internal organs; there was no evidence
of any intercurrent disease.
EXPERIMENT 2. — CONTROLS
Two guinea pigs were used as controls. These animals were fed a 1 -mil
dose of botulinus toxin (10 minimal lethal doses) on the same date as the
experimental pigs. In about 18 hours both animals showed symptoms
of botulism; one died in 23 hours after the dose; the other was found
dead in the morning of the third day after the dose. The post-mortem
findings were similar to those for the experimental animals. The results
are summarized in Table II.
72
Journal of Agricultural Research
Vol. XX, No. i
Table II. — Results of experiments with echinacea in the treatment of botulism
Experi-
ment
No.
Guinea
pig No.
Weight
of guinea
pig.
Total
dose of
remedy.
Dose of
toxin.
Effect.
Termination.
Num-
ber of
days
sick.
f ?8
79
I 8o
/ 8l
1 82
Gm.
345
37°
4°5
39°
365
Mils.
4
M. 1. d.
10
TO
Sick
Died
3
3
do
do
3- 5 1 IO
0 10
do
do
2 (con-
trols).
do
do
3
do
do . .
SUMMARY OF EXPERIMENTS WITH BOTULISM
Fluid extract echinacea was administered per os to guinea pigs for a
total of six protective doses. The animals were then fed botulinus toxin.
The treatment with echinacea was continued as long as the animals were
able to swallow. All the experimental animals developed positive
symptoms of botulism and died within three days after ingesting the
toxin. From this it does not appear that echinacea, possesses remedial
value against botulism.
III. — TESTS OF ECHINACEA AS A REMEDY FOR SEPTICEMIA
Twelve guinea pigs were used in testing the remedial value of
echinacea in septicemia. The pathogenic material was a 48-hour-old
glycerin-agar culture of Bacillus bovisepticus of only moderate virulence
for laboratory animals. A faintly cloudy suspension of the organisms
in sterile normal salt solution was prepared and used for inoculation.
While no attempt was made to determine the minimal lethal dose of
this organism for guinea pigs, a few preliminary tests undertaken indi-
cated that the dose employed in the following experiments was not
excessive.
EXPERIMENT I. — ECHINACEA ADMINISTERED PER OS (PROTECTIVE)
In order to determine whether the administration of echinacea would
increase the resistance of the organism to septicemia if given sufficient
time to develop immunity, two guinea pigs were given four daily doses
of 3 mils of a mixture of 1 mil of "Specific Medicine Echinacea" and 2 mils
of distilled water. The total protective dose was 4 mils, all administered
per os. The animals were then allowed to rest for 1 1 days, when they
were injected subcutaneously with 0.5 mil Bacillus bovisepticus suspen-
sion. Both animals became sick; one died in three days and the other
in five days. Two of the controls died in three days and the third
control survived. The autopsy showed septicemia.
Oct. i, 1920 An Experimental Study of Echinacea Therapy 73
EXPERIMENT 2. — ECHINACEA ADMINISTERED PER OS (REMEDIAL)
Two guinea pigs were given two daily doses per os of 3 mils of the
diluted echinacea mixture used in experiment 1. On the third day they
were injected with 0.5 mil of Bacillus bovisepticus culture, and imme-
diately afterwards were given a 3-mil dose of the echinacea mixture per
os. On the following day both animals were very sick. They were
given a fourth dose of 3 mils of the echinacea mixture per os. The total
dose was 4 mils of specific echinacea, equal to 4 gm. of the root. Case 65
died in 24 hours and case 66 in 48 hours. The autopsy showed septicemia ;
typical organisms were demonstrated in blood and organs.
EXPERIMENT 3. — INULA AND ECHINACEA INJECTED INTRAMUSCULARLY (PROTECTIVE)
This experiment was conducted exactly like experiment 1 of this
series except that the "Subculoyd" preparation was used instead of
the "Specific Medicine Echinacea." Three guinea pigs were given four
daily doses of the "Subculoyd" preparation, 0.5 mil being injected
intramuscularly, first into the right and then into the left thigh. The
total dose was 2 mils. The animals were allowed to rest 1 1 days and
then were injected subcutaneously with 0.5 mil of Bacillus bovisepticus
culture. All became sick. Case 62 died in 6 days after th° inoculation,
case 64 died in 12 days, and case 63 survived, being discharged as re-
covered 10 weeks after the injection. The autopsies on the fatal cases
revealed typical pictures of septicemia, and the organisms were demon-
strated in the blood and organs.
EXPERIMENT 4. — INULA AND ECHINACEA INJECTED INTRAMUSCULARLY (REMEDIAL)
Three guinea pigs were given daily injections of the "Subculoyd"
preparation, the injections being made alternately into the right and
left thighs. The dose used was 0.5 mil. After the third injection the
animals were all inoculated subcutaneously with 0.5 mil of Bacillus
bovisepticus culture. On the following day the animals were given a
fourth dose of the "Subculoyd." The total dose of remedy was 2 mils.
All these cases succumbed to the infection, the first in one day, the second
in two days, and the third in three days after the inoculation. The
autopsies showed the typical septicemia pictures, and the organisms
were demonstrated in the blood.
EXPERIMENT 5. — CONTROLS
Three control animals, each inoculated subcutaneously with 0.5 mil
of Bacillus bovisepticus culture, all became sick, and two succumbed to
the infection. The third survived and after 10 weeks' observation was
discharged as recovered. The autopsies on the fatal cases showed
septicemia, and the organisms were demonstrated in the blood and organs.
The experiments for septicemia are summarized in Table III.
187931°— 20 6
74
Journal of Agricultural Research
Vol. XX, No. i
Table III. — Results of experiments with echinacea in the treatment of septicemia
Experi-
ment
No.
5
(con-
trols)
Guinea
pig No.
Weight
Total
of guinea
pig.
dose of
remedy.
culture.
Gm.
Mils.
Mil.
540
4
°- 5
500
4
5
3»S
4
5
335
4
5
485
2
5
505
2
5
385
2
5
355
2
5
425
2
5
300
2
5
380
0
5
365
0
5
355
0
5 j
Effect.
Sick.
.do.
.do.
do.
.do.
do.
.do.
.do.
.do.
.do.
.do.
.do.
.do.
Termination.
Died
....do
....do
....do
....do
Recovered.
Died
....do
do
: do
Recovered
Died
do
Number
of days
sick.
12
3
SUMMARY OF EXPERIMENTS WITH SEPTICEMIA
"Specific Medicine Echinacea" and "Subculoyd Inula and Echinacea"
were used as protective and as remedial measures against septicemia
induced by Bacillus bovisepticus. The attempt was made to immunize
animals against septicemia by administration of the echinacea prepara-
tions several days before inoculation. In no case did it appear that
echinacea either increased the resistance of the organism to the infection
or served to modify it when given as a remedy.
IV. — TESTS OF ECHINACEA AS A REMEDY FOR ANTHRAX
The pathogenic material used to produce anthrax in the experimental
animals was a faintly cloudy suspension of Bacillus anthracis (48-hour-old
agar culture) in sterile normal salt solution. The remedial action of the
fluid extract only was investigated, and only five experimental animals
were used, the results of the experiment being so definite as not to
necessitate the sacrifice of any more animals.
Experiment i. — echinacea administered per os
Three pigs were given daily doses per os of 2 mils of fluid extract
echinacea diluted with 1.5 mils distilled water for 6 days. The total
protective dose was 3 mils, equal to 3 gm. of echinacea root. On the
eighth day the animals were given per os the same dose of echinacea and
immediately afterwards were inoculated with 0.4 mil of anthrax material
subcutaneously. On the following day they were given a second remedial
dose of echinacea. The total echinacea given was 4 mils of fluid extract.
All the animals became sick and all succumbed. No evidence was ob-
tained that echinacea has any influence upon the course of anthrax in
experimental animals. The autopsy was typical for anthrax; organisms
were demonstrated microscopically in the blood.
Oct. i, 1920
An Experimental Study of Echinacea Therapy
75
EXPERIMENT 2. — CONTROLS
Two controls were chosen at the beginning of experiment 1 of this
series and were kept under observation for 8 days, when they were
injected subcutaneously with 0.4 mil of the anthrax material at the same
time as the experimental animals. Both controls became sick, and one
died in 4 and the other in 8 days, having survived the experimental
guinea pigs by 1 and 5 days, respectively. The autopsy showed typical
anthrax.
Table IV summarizes the results of the experiments for anthrax.
Table IV. — Results of experiments with echinacea in the treatment of anthrax
Experi-
ment
No.
Guinea
pig. No.
Weight of
guinea
pig-
Total ~ ■ ,
dose of 1 D°seof
remedy. culture-
Efiect.
Termination.
Number
of days
sick.
f IX
Gm.
440
285
45°
355
35°
Mils. \ Mil.
4 | 0.4
4 1 -4
4 ! -4
0 j .4
O -4
Sick
do
Died
3
3
3
8
1 II 74
> 75
2 (con- J 76
trols) 1 77
do
do
do...
do
do
do
do....
4
SUMMARY OF EXPERIMENTS WITH ANTHRAX
Experimental animals were given protective and remedial doses of
fluid extract echinacea and were inoculated with Bacillus anthracis. All
the animals died, those which were treated dying before the control
animals. Echinacea does not appear to be of value as a remedy for
anthrax.
V. — TESTS OF ECHINACEA AS A REMEDY AGAINST RATTLESNAKE VENIN
Twenty-five guinea pigs were used in the experiments with rattlesnake
venin. The venin was furnished by Dr. Park Findley, of Des Moines,
Iowa, who had obtained it while with the United States Army on the
Mexican border. The venomous secretion of the rattlesnake was collected
and dried by inspissation in the sun. This treatment, of course, some-
what attenuated the venin. The product occurred in brittle, clear,
yellowish granules, much resembling dried egg albumen. The minimal
lethal dose was determined as 2 mgm. for a 400- to 450-gm. guinea pig.
The venin was hemolytic in a dilution of 1 to 1 ,000 against washed sheep
corpuscles. For injection, a quantity of the venin was carefully weighed
out and dissolved in sufficient sterile normal salt solution to furnish a
liquid which would contain 2 mgm. per mil.
EXPERIMENT I. — ECHINACEA ADMINISTERED PER OS
Each of three guinea pigs received daily 3 mils of a mixture of 1 mil
"Specific Medicine Echinacea" and 2 mils water for two doses, a total of
2 mils echinacea, as protective treatment. On the third day the animals
were given 2 mgm. of rattlesnake venin in 1 mil of salt solution injected
76 Journal of Agricultural Research voi.xx,No.i
subcutaneously into the ventral abdominal wall, and immediately after-
wards a dose of the echinacea was given per os. No. 50 was found dead
on the following morning. The surviving pigs were given a dose of the
echinacea mixture. The total amount of echinacea given in the first
case was 3 mils; in the second and third cases it was 4 mils. These latter
guinea pigs died on the third day after the injection of the venin. All
the animals showed the characteristic symptoms and local lesions of this
type of poisoning. On autopsy, the characteristic local lesions were
found, consisting of a marked inflammatory swelling with necrosis and
sloughing of the skin over a considerable area surrounding the point of
injection. In cases of early death from rattlesnake poisoning there is
usually some oozing of dark, incoagulable blood from the wound at the
seat of injection and extensive extravasation of blood into the
subcutaneous and muscular tissues. The inflammatory process in most
cases extends through the abdominal wall and involves the peritoneum.
If the animal survives for several days there may be complete sloughing
of the abdominal wall, allowing the viscera to protrude. The internal
organs are usually grossly normal in appearance, except in the case of
the kidneys, which may be somewhat enlarged and congested with
evidence of parenchymatous degeneration.
EXPERIMENT 2. — INULA AND ECHINACEA INJECTED INTRAMUSCULARLY
Each of three guinea pigs received 0.5 mil of the "Subcoloyd Inula and
Echinacea" in the right thigh on the first day; on the second day the same
dose was injected into the left thigh, both injections being made deeply
into the gluteal muscles. The total protective dose was 1 mil. On the
third day 1 mil of the venin solution, equal to 2 mgm. of dry venin, was
injected subcutaneously into the belly, and immediately afterwards 0.5
mil of "Subculoyd" was injected into the right thigh. On the following
day all the animals showed the characteristic symptoms and 0.5 mil of
the " Subculoyd " was injected into the left thigh of each animal. The
total dose was 2 mils. On the third day No. 51 died; on the fifth day
No. 53 died; and six weeks later No. 52 was discharged as recovered. The
autopsy was the same as in experiment 1 of this series. The guinea pigs
showed the usual local lesions produced by the injection of the inula and
echinacea.
EXPERIMENT 3. — CONTROLS
Three guinea pigs were used as controls and were injected subcutane-
ously into the belly with 1 mil of venin solution, corresponding to 2 mgm.
of dry venin. All the controls were sick on the following day. No. 57
and 58 died on the second day and No. 59 on the third day after the injec-
tion of the venin. The autopsy showed the same conditions as in experi-
ment 1 of this series. There was no apparent difference between the
controls and the treated animals in experiments 1 and 2.
The results are given in Table V.
Oct. i, 1920 An Experimental Study of Echinacea Therapy
77
Table V. — Results of experiments with echinacea as a remedy against rattlesnake venin
Experi-
ment
No.
Guinea
pig No.
Weight
of guinea
pig.
Total
dose of
remedy.
Dose of
venin.
Gm.
Mils.
Mgm.
31
37°
0
0. 1
32
39°
0
. 2
33
290
0
•3
34
297
0
• 4
35
36
315
260
0
0
• 5
.6
A°....
37
38
39
35°
290
265
0
0
0
.6
■7
•7
40
440
0
•5
41
34°
0
•5
42
0
1. 0
43
0
1. 0
44
0
2. 0
45
0
2. 0
f 48
395
4
2. 0 I
1
1 49
400
4
2. 0
5°
37°
3
2. 0
5i
440
2
2. 0
2
3 (con-
trols).
52
53
57
58
I 59
425
360
385
405
405
2
2
0
0
0
2. 0
2. 0
2. 0
2. 0
2. 0
Effect.
Sick.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
..do.
Termination.
Recovered .
...do
...do
...do
Died
Recovered .
Died
Recovered .
Died
do
do
Recovered.
do
Died
do
do
do
do
do
Recovered .
Died
do
do
do....
Number
of days
sick.
28
33
24
28
" To test toxicity of venin.
SUMMARY OF EXPERIMENTS WITH RATTLESNAKE VENIN
"Specific Medicine Echinacea" was administered to guinea pigs per os,
and "Subculoyd Inula and Echinacea" was injected as a means of treat-
ment against the venin of the rattlesnake. The venin had been standard-
ized and the minimal lethal dose determined. Neither of the echinacea
preparations appeared to influence the course of the poisoning. From
these results it does not appear that echinacea is of value in the treat-
ment of rattlesnake poisoning in experimental animals under laboratory
conditions.
VI. — TESTS OF ECHINACEA AS A REMEDY FOR TUBERCULOSIS
Jt has often been asserted that echinacea is a cure for tuberculosis, and
for this reason tuberculosis was chosen as one of the chronic diseases upon
which to test the remedial value of the plant. The type of organism used
to inoculate the experimental animals was strictly human (Igoe strain).
The immediate material used for our purpose was one-third of a tubercu-
lous spleen from a guinea pig, third passage of the original material,
finely triturated in mortar and suspended in 10 mils of normal salt solu-
tion. The dose was 1 mil per guinea pig, injected intraperitoneally.
78 Journal of Agricultural Research voi.xx.No.i
EXPERIMENT I. — ECHINACEA ADMINISTERED PER OS
Three guinea pigs were inoculated with tubercle bacilli November 20,
1919, and on the following day treatment was begun. Each animal re-
ceived a dose of a mixture of 0.25 mil of fluid extract echinacea and 0.75
mil distilled water per os each week day. The animals were weighed
three times a week. All the animals showed a progressive loss in weight
(see Table VII) and eventually succumbed. Case 85 died December 10,
(20 days after inoculation), after having received a total of 3.5 mils of
fluid extract echinacea as a remedy. The autopsy in this case was nega-
tive. Case 84 died December 22 (32 days after inoculation), having
received 6 mils of the echinacea. Case 86 was found dead in the morning
of December 26 (36 days after inoculation), having received 6.75 mils of
the echinacea. In the last two cases the autopsies revealed the typical
picture of generalized tuberculosis.
As these were probably the first experimental animals which had ever
received echinacea over an extended period of time, it was interesting
to observe the effects of the plant on the animals themselves, and espe-
cially upon the gastrointestinal tract. In case 85 there was found a
chronic catarrhal gastritis which was absent in cases 83 and 84. Apart
from the tubercular lesions there was no abnormality found in the other
organs upon macroscopic examination.
EXPERIMENT 2. — INULA AND ECHINACEA INJECTED SUBCUTANEOUSLY
Three guinea pigs were inoculated with the tuberculous material as
in experiment 1 on November 20, 191 9, and the treatment was begun on
the following day. Each animal received subcutaneously 0.2 mil of
the "Subculoyd Inula and Echinacea" each week day and was weighed
three times a week. All showed progressive loss of weight, as shown
in Table VII. Case 87 died December 19, 29 days after inoculation,
after having received 4.2 mils of the remedy. Case 88 died December
23, 33 days after inoculation, having received 5 mils of the remedy, and
case 86 died on December 28, 38 days after inoculation, having received
5.8 mils of the remedy. The autopsies in these cases showed great
emaciation, some necrosis at the points of injection, and generalized
tuberculosis. There was no evidence of systemic effects from the
remedy.
EXPERIMENT 3. — CONTROLS
Two control guinea pigs were inoculated with the same tuberculous
material as the animals in experiments 1 and 2, on November 20, 191 9,
and were weighed three times a week. They lost weight (see Table
VII). Case 89 died December 3, 13 days after inoculation, and case
90 died December 23, 33 days after inoculation. The autopsy showed
generalized tuberculosis.
The experiments are summarized in Table VI.
Oct. i, 1920 An Experimental Study of Echinacea Therapy
79
Table VI. — Results of experiments with echinacea in the treatment of tuberculosis
Ex-
peri-
ment
No.
Guinea
pig No.
Weight
of
guinea
P«g-
Total
dose of
remedy.
Effect.
Termination.
Number
of days
sick.
( 83
84
85
f 86
87
1 88
I 89
I 9°
Gm.
43°
425
495
470
45°
405
425
550
Mils.
6- 75
6. 00
3- 5°
5.80
4. 20
5.00
0
0
Sick
Died
36
32
I
do
do
do
do. ..
do
. do. ..
38
29
33
13
33
2
do
do. .. .
do
do
3 (con-
trols)
do
. .do. ..
do
do
Table VII. — Progressive loss of weight of guinea pigs in experiments with echinacea in
the treatment of tuberculosis
Date.
Weight of guinea pigs treated
with fluid extract echinacea.
Weight of guinea pigs treated
with "Subculoyd Inula and
Echinacea."
Weight of control
guinea pigs.
No. 83.
No. 84.
No. 85.
No. 86.
No. 87.
No. 88.
No. 89.
No. 90.
1919.
Nov. 18
20
24
26
28
Dec. 1
3
5
8
10
12
15
17
19
22
Gm.
43°
410
395
405
400
39°
385
380
365
355
35°
345
310
295
290
270
240
Gm.
425
43°
410
410
415
415
39°
395
385
365
355
345
325
290
270
Gm.
495
480
470
490
475
480
475
460
440
405
Gm.
475
455
440
460
460
470
470
470
460
465
460
445
405
405
400
355
340
320
Gm.
45°
45°
420
42 5
415
415
395
405
375
360
355
325
285
260
Gm.
405
405
380
360
370
37°
355
360
340
335
325
33°
300
280
280
245
Gm.
425
415
405
395
37o
325
285
265
Gm.
55°
535
520
530
525
525
5io
5i°
5io
485
485
480
400
380
355
305
24
26
SUMMARY OP EXPERIMENTS WITH TUBERCULOSIS
Fluid extract echinacea was administered per os, and "Subculoyd
Inula and Echinacea" was injected subcutaneously into experimental
guinea pigs daily for extended periods as remedies for tuberculosis pro-
duced by a human type organism. Neither of the preparations appeared
to influence the course of the disease. From these results it does not
seem probable that either fluid extract echinacea or the "Subculoyd
Inula and Echinacea" is of value in the treatment of tuberculosis.
The experimental animals did not show organic effects from echinacea
ingested in large doses for a long time.
80 Journal of Agricultural Research voi.xx.No. i
vii. — tests of echinacea as a remedy for trypanosomiasis
(dourine)
In connection with the experimentation with tuberculosis it was
considered of interest to study the remedial action of the echinacea
preparations upon another chronic condition. Trypanosomiasis induced
by Trypanosoma equiperdum and commonly called dourine was chosen.
This disease as produced under laboratory conditions in guinea pigs runs
a course of from 7 to 1 1 weeks, allowing ample time for the exhibition of
quantities of remedial agents and consequently favoring the remedy
more than a speedy, acute infection would.
The material used to produce the disease was kindly furnished by
Dr. H. W. Schoening, of this laboratory. It consisted of a normal salt
suspension of a sample of blood freshly drawn from rats which had been
inoculated three days previously with Trypanosoma equiperdum. Upon
microscopic examination this showed numbers of trypanosomes. The
dose given was 0.5 mil, injected subcutaneously.
EXPERIMENT I. — ECHINACEA ADMINISTERED PER OS
Three guinea pigs were inoculated with the dourine material on
December 1, 1919. On the following day treatment was begun, each
animal receiving 1 mil of a mixture of 0.25 mil fluid extract echinacea and
0.75 mil of distilled water. This dose was given each week day there-
after as long as the animal survived. All the animals were weighed three
times a week. The weights are reported in Table IX. At intervals the
blood of some of the animals was examined microscopically for the
presence of trypanosomes; on December 17 these were demonstrated in
the peripheral circulation of case 93, on January 6 in that of case 92, on
January 16 in cases 91 and 92, and on March 3 in case 93. Ail the animals
showed the typical symptoms of trypanosomiasis. Case 91 died on the
sixty-first day, after having received 12.5 mils of the fluid extract; case
92 died on the sixty-fourth day after having receiving 13 mils of fluid
extract; case 93 died on the ninety-third day, after having received
15.75 m^s °f flmd extract. Treatment of case 93 was suspended
February 14. The autopsies showed the usual picture of this type of
infection. In case 91 there was a chronic catarrhal gastritis; otherwise
no organic effects from the extended ingestion of the echinacea were
discovered.
EXPERIMENT 2. — INULA AND ECHINACEA INJECTED SUBCUTANEOUSLY
Three guinea pigs were inoculated and treated exactly as in experiment
1 , except that the remedy given was 0.2 mil of the "Subculoyd Inula and
Echinacea" each week day. The weights of the animals are given in
Table IX. On December 17 trypanosomes were demonstrated in the
peripheral circulation of case 94, on January 6 in that of case 96, and on
Oct. i, 1920
An Experimental Study of Echinacea Therapy
January 16 case 96 was positive, while cases 94 and 95 did not show
trypanosomes. Case 96 died on the forty-eighth day after inoculation,
having received 7.8 mils of the remedy; case 95 succumbed on the sixty-
sixth day, after receiving a total of 10.8 mils of the remedy, and case 94
died on the seventy-first day, having received 1 1 .4 mils of the remedy.
The autopsies in these cases showed a dirty, dark discoloration of the
subcutaneous and superficial abdominal muscular tissues over the area
where injections were made. Extreme emaciation was evident, the
spleen was greatly enlarged, and in general the typical dourine picture
was present.
Experiment 3. — controls
Four guinea pigs were used as controls. These were inoculated on the
same date as those in experiments 1 and 2 and were kept in separate cages.
One animal died in 17 days, another died in 30 days, and the remaining 2
died in 78 and 79 days, respectively, all with typical dourine symptoms.
These experiments are reported in Table VIII.
Table VIII.— Results of experiments with echinacea in the treatment of dourine
82
Journal of Agricultural Research
Vol. XX, No. i
Table IX.
-Progressive loss of weight of guinea piqs in experiments with echinacea in
the treatment of dourine
Weights of guinea pigs treated
■with fluid extract echinacea.
No. 91.
No. 92.
No. 93-
Weights of guinea pigs treated
■with "Subculoyd Inula and
Echinacea."
No. 94.
No. 95-
No. 96.
Nov. 29.
Dec. 1 .
3-
5-
8.
*5-
17-
19.
22 .
24.
26.
29.
3i-
Jan.
Feb.
3-
5-
7-
9-
12 .
14-
16.
19.
21.
23-
26.
28.
3°-
2 .
4-
6.
13
16.
19.
Mar.
24.
27.
1919.
1920.
Gm.
475
475
485
475
480
480
500
495
485
490
485
465
45°
460
460
45°
455
465
45°
455
435
415
410
395
385
400
39o
385
Gm.
5°5
505
5IO
500
5°5
51°
52O
5J5
510
520
5i°
505
495
480
485
470
475
485
470
465
455
435
43°
435
4i5
4i5
385
355
320
Gm.
445
45°
465
465
470
475
5°5
500
500
525
490
475
470
465
465
470
470
500
485
500
5io
495
500
520
5°5
5*5
5IQ
5J5
500
495
480
490
455
440
45°
455
455
4i5
39°
37°
345
Gm.
435
445
455
440
445
45°
445
45°
445
455
410
410
43°
420
405
410
420
435
415
43°
445
420
410
365
340
325
305
315
3i5
295
295
3°5
Gm.
400
410
415
425
420
415
440
42 5
43°
445
460
435
440
435
43°
445
455
465
45°
460
465
440
45°
465
445
440
440
440
450
405
560
57°
585
s6o
555
570
565
565
565
55°
560
535
495
500
485
480
480
480
470
460
435
395
365
SUMMARY OF EXPERIMENTS WITH DOURINE
Fluid extract echinacea and "Subculoyd Inula and Echinacea" were
tested as remedies in trypanosomiasis (dourine). Neither of these
preparations appeared to influence the course of the disease. They
certainly have no curative value.
Oct. i, 1920 An Experimental Study of Echinacea Therapy 83
GENERAL SUMMARY
Various preparations of echinacea — namely, the "Specific Medicine
Echinacea," the fluid extract echinacea, and the "Subculoyd Inula and
Echinacea" — were studied as remedies in several types of infectious and
allied diseases, both acute and chronic, in guinea pigs.
In both tetanus and botulism produced by the administration of
bacterial toxin the course of the disease was not modified by the echinacea.
In septicemia produced by injection of a culture of Bacillus bovi-
septicus, and in anthrax produced by injection of B. anthracis the
results indicated that echinacea had no influence.
In poisoning by the venin of the rattlesnake produced by injection
of a solution of the dry venom the echinacea preparations were without
curative effect.
In the chronic diseases, tuberculosis produced by injection of a
human strain of the bacillus and trypanosomiasis produced by injec-
tion of Trypanosoma equiperdum the remedy was exhibited over an
extended period of time without apparently influencing the course of
these diseases.
Definite evidence of organic effects from the echinacea itself was not
obtained.
LITERATURE CITED
(1) American Pharmaceutical Association.
1918. THE national Formulary, ed. 4, 394 p. Philadelphia.
(2) Berg, W. N., and KelsEr, R. A.
1918. DESTRUCTION OF TETANUS ANTITOXIN BY CHEMICAL AGENTS. In JoUT.
Agr. Research, v. 13, no. 10, p. 471-495, 4 fig. Literature cited, p.
cited, p. 494-495-
(3) Council on Pharmacy and Chemistry.
1909. echinacea considered valueless, report of the council on phar-
macy and chemistry. In Jour. Amer. Med. Assoc, v. 53, no. 22, p.
1836.
(4) Ellingwood, Finley.
1914. echinacea: the vegetable "antitoxin." its characteristics and
peculiar therapeutic effects. In Amer. Jour. Clin. Med., v. 21,
no. 11, p. 987-993.
(5) Fish, P. A.
1903. echinacea in veterinary practice. In Amer. Vet. Rev., v. 27, no. 8,
p. 716-726, 1 fig.
(6) Gray, Asa.
[1908.] new manual of botany . . . ed. 7, 926 p., illus. New York.
(7) Heyl, Frederick W., and Hart, Merrill C.
1915. some constituents of the root of brauneria ANGusTrFOLiA. In Jour.
Amer. Chem. Soc, v. 37, no. 7, p. 1769-1778.
(8) and Staley, J. F.
1914. analyses OF Two echinacea roots. In Amer. Jour. Pharrn., v. 86,
no. 10, p. 45°-455-
(9) KrLGOUR, J. C.
1897. lobelia and echinacea. In Eclectic Med. Jour., v. 57, no. 11, p.
595-598.
84 Journal of Agricultural Research voi.xx,No.i
(10) Little, George W.
191 7. AN EFFECTIVE TREATMENT FOR CANINE DISTEMPER. In Amer. JOUT.
Vet. Med., v. 12, no. 10, p. 691-694.
(11) Lloyd, John Uri.
1897. Empiricism — echinacea. In Eclectic Med. Jour., v. 57, no. 8, p. 421-
427, 2 fig.
(12)
1904. history OF echinacea angustifoi.ia. In Amer. Jour. Pharm., v. 76,
no. 1, p. 15-19.
(13)
1917. A treatise on echinacea. 32 p., 21 fig. Cincinnati. (Drug treatise,
no. XXX, issued by Lloyd Brothers.)
(14) Mathews, A. B.
1905. ECHINACEA — SOME OF ITS USES IN MODERN SURGERY. In Ga. Pract.,
v. 1, no. 5, p. 137-140.
(15) Meyer, H. C. F.
1887. echinacea angusTiFolia. In Eclectic Med. Jour., v. 47, no. 5, p.
209-210.
(16) Slawson, A.
1918. SERUM OR INULA AND ECHINACEA IN THE TREATMENT OF CANINE DIS-
TEMPER. In Jour. Amer. Vet. Med. Assoc., v. 53 (n. s. v. 6) no. 6,
p. 766-767.
(17) Thom, Charles, Edmundson, Ruth B., and Giltner, L. T.
1919. botulism from canned asparagus. In Jour. Amer. Med. Assoc, v.
73, no. 12, p. 907-912.
(18) Unruh, V. von.
I915. ECHINACEA ANGUSTIFOLIA AND INULA HELENIUM IN THE TREATMENT OF
tuberculosis. 14 p. [n. p.] Reprinted from the Nat. Eclect. Med.
Assoc. Quart. Sept. 1915. (Not seen.)
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Vol. XX OCTOBER 15, 1920 No. 2
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Page
Investigations of the Germicidal Value of Some of the
Chlorin Disinfectants -------85
F. W. TILLEY
( Contribution bom Bureau of Animal Industry)
A New Avocado Weevil from the Canal Zone - - - 111
H. F. DLETZ and H. S. BARBER
(Contribution from Bureau of Entomology)
Studies in Mustard Seeds and Substitutes: I. Chinese
Colza (Brassica campestris chinoleif era Viehoever) - 117
ARNO VIEHOEVER, JOSEPH F. CLEVENGER, and
CLARE OLIN EWING
(Contribution from Bureau of Chemistry )
Study of Some Poultry Feed Mixtures with Reference to
Their Potential Acidity and Their Potential Alkalinity - 141
B. F. KAUPP and J. E. IVEY
(Contribution from North Carolina Agricultural Experiment Station)
The Influence of Cold in Stimulating the Growth of
Plants ^ - 151
FREDERICK V. COVILLE
(Contribution from Bureau of Plant Industry)
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WAfHIHOTON t GOVERNMENT PRINTING. OFFICE t Itl*
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
FOR THE ASSOCIATION
KARL F. KELLERMAN, Chairman J. G. LIPMAN
Physiologist and A ssociate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
RAkOfcrt
JOURNAL OF AGRICDLTURAL RESEARCH
Vol. XX Washington, D. C, October 15, 1920 No. 2
INVESTIGATIONS OF THE GERMICIDAL VALUE OF
SOME OF THE CHLORIN DISINFECTANTS
By F. W. Tilley, Biochemic Division, Bureau of Animal Industry, United States
Department of Agriculture
SCOPE OF THE INVESTIGATION
During the great World War, which from the surgical standpoint was
distinguished by the frequency and intensity of wound infections, no
class of antiseptics was more extensively employed than the so-called
"chlorin antiseptics." When properly used they proved to be of very
great value, as may be seen by a perusal of the various publications of
Carrel and his colleagues and especially the book by Carrel and
Dehelly (2).1
In view of the great amount of work already done on the value of
these antiseptics in surgery no attempt has been made by the writer to
cover that field of work. The experiments herein described were intended
to furnish information regarding the value of the chlorin antiseptics for
general disinfection. The members of this group actually tested were:
(1) chloramin T, (2) Dakin's solution (NaOCl), (3) eusol (HOC1), and
(4) chlorin.
"Chloramin T" is the abbreviated name given by Dakin to sodium-
toluene-sulphon-chloramid (4) . It is described as a " white crystalline solid
with a faint chlorous odor" containing 12.6 per cent of chlorin and readily
soluble in water. The material used in the present work was obtained un-
der the trade name "Chlorazene." Its appearance corresponds to the
foregoing description, and titration of an aqueous solution with potas-
sium iodid and sodium thiosulphate showed it to contain 25 per cent of
"available chlorin," which corresponds to 12.5 per cent of actual chlorin,
since according to Dakin and Dunham (5) one molecule of chloramin T
liberates two atoms of iodin. The explanation they give is that each
atom of chlorin in chloramin T is equivalent to a molecule of hypo-
chlorous acid, which liberates two atoms of iodin from an acidified iodid
solution.
c : _
1 Reference is made by number (italic) to "Literature cited," p. 110.
— — .
Journal of Agricultural Research, Vol. XX, No. 2
Washington, D. C Oct. 15, 1920
vc Key No.A-53
(85)
LxJ
o
86 Journal of Agricultural Research vol. xx, no. 2
The term "Dakin's solution" as used in this paper signifies a neutral
solution of sodium hypochlorite. The methods of preparation were
essentially those given by Dakin and Dunham (5).
The details of the method with sodium carbonate are, according to
Dakin and Dunham, as follows: One hundred and forty gm. of dry
sodium carbonate (Na2C03), or 400 gm. of the crystallized salt, are dis-
solved in 10 liters of water, and 200 gm. of bleaching powder containing
24 to 28 per cent of "available chlorin" are added. The mixture is very
thoroughly shaken, and after it has stood half an hour the supernatant
fluid is siphoned off from the precipitate and filtered through a cotton
plug or through paper. Forty gm. of boric acid are added to the filtrate,
and it is then ready for use.
The details of the method with sodium carbonate and sodium bicarbo-
nate are, according to Dakin and Dunham, as follows: Two hundred
gm. of bleaching powder (containing 24 to 28 per cent of "available
chlorin") are shaken well with 5 liters of water and allowed to stand
for an hour or two. In a separate vessel 94 gm. of sodium carbonate
and 86 gm. of sodium bicarbonate (NaHC03) are mixed with 5 liters
of water, and this solution is added to the bleaching powder suspension.
The mixture is well shaken and allowed to stand until the precipitate
settles. The clear supernatant fluid is then siphoned off and filtered.
In actual practice the writer made the following modifications. The
amount of Dakin's solution made up at any one time was always smaller
than the amount indicated above, but the relative proportions of ingre-
dients were the same. The bleaching powder was rubbed up in a mortar
with a little water until it was of a creamy consistency. It was then
transferred to a graduated flask or cylinder and made up to volume with
more water. Dry sodium carbonate, or the solution of sodium carbonate
and sodium bicarbonate, was added in accordance with the directions of
Dakin and Dunham, and their further directions were followed except
that instead of the clear supernatant fluid being drawn off the entire
mixture was shaken up and filtered through paper. The bleaching pow-
der used contained approximately 28 per cent of "available chlorin."
In certain experiments Dakin's solution was also prepared by the
direct action of chlorin upon a solution of sodium carbonate, with the
use of the apparatus devised for the purpose by the Wallace & Tiernan
Co., of New York City.
The term " eusol, " as employed in this paper, signifies a solution pre-
pared from bleaching powder in aqueous solution by the addition of an
equal amount of boric acid. The originators of this solution (12) de-
scribe it as a solution of hypochlorous acid, but according to Dakin and
Dunham (5) the solution is alkaline to litmus and contains a balanced
mixture of calcium hypochlorite and calcium borate with an undeter-
mined amount of free hypochlorous acid.
Oct. is. 1920 Germicidal Value of Some Chlorin Disinfectants 87
The method of preparation as given by Dakin and Dunham is as
follows: To 1 liter of water add 12.5 gm. of bleaching powder and shake
vigorously. Add 12.5 gm. of powdered boric acid and shake again.
Allow the mixture to stand for some hours, preferably overnight, and
then filter. In actual practice the writer made the following modifica-
tions: The bleaching powder was rubbed up in a mortar with a little
water until the mixture had a creamy consistency. It was then trans-
ferred to a graduated flask or cylinder, the boric acid was added, and
then the amount of water necessary to make up the volume. The mix-
ture was shaken and then usually allowed to stand about two hours
before it was filtered through paper.
Chlorin was used in these experiments in the form of an aqueous solu-
tion, standardized by titration with potassium iodid and sodium thiosul-
phate.
Dilutions of these various disinfectants were made up for test as fol-
lows: Chloramin T dilutions were made by weighing the solid and dis-
solving it in the required amount of water. In certain experiments a
stock solution was made and titrated with potassium iodid and sodium
thiosulphate", and dilutions of the stock solution were then made so as
to contain specified amounts of "available chlorin."1 But for the most
part dilutions were made up to contain a given weight of the solid
chloramin T.
Dakin's solution and eusol were prepared according to the directions
previously given and were then titrated with potassium iodid and sodium
thiosulphate. Dilutions were then made from these original solutions
so as to contain a given amount of "sodium hypochlorite" or "hypo-
chlorous acid" for Dakin's solution and eusol, respectively. In certain
experiments the dilutions were made in both instances so as to contain
given amounts of "available chlorin."
It has already been noted that according to Dakin and Dunham (5)
eusol contains calcium hypochlorite with an indefinite amount of free
hypochlorous acid. In a similar way Dakin's solution may contain not
only sodium hypochlorite but also more or less hypochlorous acid, as
stated by Cullen and Austin (3). As regards "available chlorin"
Rosenau (11) states that this really represents available oxygen rather
than available chlorin. All three terms, however, are convenient as
conventional symbols and will be so used in this paper.
It should be stated further that for the purposes of certain experi-
ments it was necessary to modify the methods of preparing Dakin's
solution and eusol materially so as to secure more concentrated solutions.
In all such instances the changes made are indicated in connection with
the experiments.
1 The quotation marks used in this and the following paragraph are intended to indicate that the terms
are used in a conventional way for purposes of comparison, and not in their literal sense.
Journal of Agricultural Research
Vol. XX, No. a
EXPERIMENTS WITH STAPHYLOCOCCUS AUREUS, BACILLUS PYOCY-
ANEUS, AND B. TYPHOSUS AS TEST ORGANISMS
The cultures used in this series of experiments were stock cultures
which had been carried along in this laboratory for some time. They
had been previously examined in connection with other work and had
been found true to type. The cultures of Staphylococcus aureus and
Bacillus pyocyaneus were originally isolated from wounds and when re-
ceived in this laboratory were virulent for guinea pigs and rabbits.
The first three experiments of this series were made with chloramin
T only. The results are shown in Tables I and II.
Experiment i. — This was a preliminary experiment to test the value
of chloramin T against Staphylococcus aureus and Bacillus typhosus. The
technic was as follows: One-tenth cc. of 24-hour bouillon culture was
mixed with 25 cc. of blood serum.1 Then 2.5 cc. of this mixture were
mixed with 2.5 cc. of a dilution of chloramin T. After exposures of one
hour and two hours, respectively, subcultures were made with a 3-mm.
platinum loop into tubes of standard bouillon containing enough sodium
thiosulphate to neutralize the disinfectant carried over.
The number of organisms present in the test mixtures were calculated
to be 1,340,000 per centimeter for Staphylococcus aureus and 800,000 per
centimeter for Bacillus typhosus. Dilutions given in the table are, of
course, final dilutions, and the dilutions actually made up to begin with
were naturally just twice as concentrated. The results are given in
Table I.
Table I. — Germicidal efficiency of chloramin T against Staphylococcus aureus and
Bacillus typhosus, mixed with blood serum, using equal amounts of disinfectant and
of serum plus culture °
EXPERIMENT I
Concentration of chloramin T.
Staph, aureus.
B. typhosus.
Exposed Exposed i Exposed
1 hour. 2 hours. i hour.
Exposed
2 hours.
°+ Signifies growth; — , no growth.
Experiments 2 and 3. — The technic used in these experiments was
as follows : Each disinfectant dilution was mixed with an equal quantity
of blood serum (2.5 cc. each of disinfectant dilution and serum), and to
this mixture 2 drops of a 24-hour bouillon culture were added. The
mixture was then vigorously shaken, and at intervals of 15 minutes,
1 hour, and 2 hours subcultures were made with a 3-mm. platinum loop
1 Horse-blood serum was used In all experiments where blood serum is mentioned.
Oct. us. 1920 Germicidal Value of Some Chlorin Disinfectants
89
into tubes of standard broth. The mixtures were thoroughly shaken just
before subcultures were made.
In these experiments and in all others described in this paper, the test
mixtures were used at ordinary room temperatures.
The results of experiments 2 and 3 are given in Table II.
Table II. — Germicidal efficiency of chloramin T against Staphylococcus aureus, Bacillus
typhosus, and B. pyocyaneus when mixed with an equal quantity of blood serum before
culture is added a
EXPERIMENT 2
Staph, aureus.
B. typhosus.
Concentration of chloramin T.
Exposed
15 minutes.
Exposed
1 hour.
Exposed
2 hours.
Exposed
15 minutes.
Exposed
1 hour.
Exposed
2 hours.
+
+
+
+
+
4-
+ + 1 1
+
+
+
+
1 to 800
EXPERIMENT 3
B. pyocyaneus .
B. 'yphosus.
Concentration of chloramin T.
Exposed
15 minutes.
Exposed
1 hour.
Exposed
2 hours.
Exposed
15 minutes.
Exposed
1 hour.
Exposed
2 hours.
1 to 200
+
+
+
+
1 + + +
+ + + 1
+ + 1 1
+
4-
-
+
+
1 to 800
a+ Signifies growth; — , no growth.
Experiments 4 and 5. — These were preliminary experiments with
Dakin's solution, which was made up by the use of sodium carbonate
alone, as described in the first of the methods of preparation previously
mentioned. In order to determine what influence the boric acid exerts,
two portions were tested, one with and the other without the addition
of boric acid. The technic was the same as that described for experi-
ments 2 and 3. Dilutions given are based on the amount of sodium
hypochlorite. The results of these experiments are given in Table III.
The results given in Table III indicate that the boric acid adds some-
what to the germicidal power of Dakin's solution. This is probably due
to the small amount of hypochlorous acid set free by the boric acid.
Experiments 6, 7, and 8. — These experiments were made in order to
compare the germicidal powers of chloramin T and Dakin's solution
against Staphylococcus aureus, Bacillus pyocyaneus, and B. typhosus. The
technic was the same as that described for experiments 2 and 3, except
for the omission of the 15-minute exposures. The results are given in
Table IV.
9o
Journal of Agricultural Research
Vol. XX, No. a
Table III. — Germicidal efficiency of Dakin's solution against Staphylococcus aureus,
with and without boric acida
Concentration of NaOCl.
I to IOO
I tO 200
i to 400
i to 800
Without boric acid.
Exposed 15
minutes.
Exposed 1 Exposed 2
hour. hours.
+
With boric acid.
Exposed 15
minutes.
Exposed 1
hour.
4
+
Exposed 2
hours.
EXPERIMENT 5
1 to IOO
1 to 200
1 to 400
1 to 600
1 to 800
4
-U
+
+
+
+
4
a + signifies growth; — , no growth.
Table IV. — Comparative germicidal efficiency of chloramin T and Dakin's solution
against Staphylococcus aureus, Bacillus pyocyaneus, and B. typhosus, in the presence
of 50 per cent blood serum °
experiment 6
Staph.
lureus.
B. pyocyaneus.
B. typhosus.
Disinfectant and dilution.
Exposed
Exposed
Exposed
Exposed
Exposed
Exposed
1 hour.
2 hours.
1 hour.
2 hours.
1 hour.
2 hours.
Chloramin T:
1 to 200
—
—
—
—
—
—
1 to 300
—
—
4
—
—
—
1 to 400
—
—
4
+
—
—
1 to 500
—
—
4
+
+
—
1 to 600
—
—
+
4
+
+
1 to 800
4
—
4
4
+
+
Dakin's solution:
NaOCl 1 to 200
—
—
—
—
—
NaOCl 1 to 300
4
—
—
—
—
—
NaOCl 1 to 400
4
—
4
—
+
—
NaOCl 1 to 500
4
+
4
—
+
—
NaOCl 1 to 600
+
4
4
—
+
—
NaOCl 1 to 800
+
+
+
4
+
+
EXPERIMENT 7
Chloramin T:
1 to 200 . .
1 to 300 . .
1 to 400 . .
1 to 500.
1 to 600. .
1 to 800 . .
1 to 1,000.
No test
do...
..do...
+
+
+
+
No test ,
..do...
..do...
+
4-
+
No test .
..do...
4-
4-
No test
...do...
4-
4
No test
4
4
No test.
0+ signifies growth; — , no growth.
Oct. is, 1920 Germicidal Value of Some Chlorin Disinfectants
91
Table IV. — Comparative germicidal efficiency of chloramin T and Dakin's solution
against Staphylococcus aureus, Bacillus pyocyaneus, and B. typhosus, in the presence
of 50 per cent blood serum — Continued
Experiment 7 — continued
Staph.
aureus.
B. pyocyaneus.
B. typhosus.
Disinfectant and dilution.
Exposed
1 hour.
Exposed
2 hours.
Exposed
1 hour.
Exposed
2 hours.
Exposed
1 hour.
Exposed
2 hours.
Dakin's solution:
NaOCl 1 to 200
NaOCl 1 to 300
NaOCl 1 to 400
NaOCl 1 to 500
NaOCl 1 to 600
NaOCl 1 to 800
NaOCl 1 to 1,000. .. .
+
+
+
+
+
No test .
. . .do. .. .
+
4-
4-
No test .
...do....
No test .
+
+
+
+
No test .
+
+
No test.
+
+
+ •
+
+
No test.
+
-j-
EXPERIMENT 8
Chloramin T:
1 to 200
1 to 300
1 to 400
1 to 500
1 to 600
1 to 800
1 to 1,000
Dakin 's solution :
NaOCl 1 to 200
NaOCl 1 to 300
NaOCl 1 to 400
NaOCl 1 to 500 ... .
NaOCl 1 to 600 ... .
NaOCl 1 to 800 ... .
NaOCl 1 to 1,000. ..
No test .
No test.
. . .do. ...
. . .do. .. .
+
+
+
+
—
—
+
+
+
+
—
+
+
+
+
+
No test .
No test .
No test .
+
+
+
. ..do....
. ..do....
...do....
.. do. ...
...do....
+
—
—
—
No test.
+
—
+
—
+
+
—
j-
—
No test.
+
4-
+
—
+
+
+
+
+
+
No test.
No test.
+
+
+
+
+
+
No test.
Do.
No test.
No test.
4-
+
Experiments 9, 10, and ii. — These experiments were undertaken to
determine the efficiency of chloramin T and Dakin's solution against
Staphylococcus aureus, Bacillus pyocyaneus, and B. typhosus without the
addition of blood serum. They are in contrast with the three preceding
experiments, in which 50 per cent of blood serum was used.
The technic was as follows: Two drops of a 24-hour bouillon culture
were added to 5 cc. of disinfectant, and the mixture was well shaken.
After intervals of 1 hour and 2 hours, respectively, the mixtures were
again shaken and subcultures were made with a 3 -mm. platinum loop
into tubes of standard bouillon containing enough sodium thiosulphate to
neutralize the disinfectant carried over. The results are given in Table V.
In the results shown in Tables IV and V there is seen evidence of what
may be called "selective" action on the part of the two disinfectants
tested. For instance, the amount of chloramin T required to kill Staphy-
lococcus aureus is very much less than that required to kill Bacillus pyo-
cyaneus. In like manner in the presence of blood serum it requires more
92
Journal of Agricultural Research
Vol. XX, No. 2
Dakin 's solution to kill Staph, aureus than to kill B. pyocyaneus or B.
typhosus under like conditions. A comparison of the two disinfectants
shows that Dakin's solution is more effective than chloramin T against
B. pyocyaneus , while chloramin T is more effective than sodium hypo-
chlorite against Staph, aureus.
Table V. — Comparative germicidal efficiency of chloramin T and Dakin's solution against
Staphylococcus aureus, Bacillus pyocyaneus, and B. typhosus, without addition of
blood serum la
EXPERIMENT 9
Staph, aureus.
B. pyocyaneus.
B. typhosus.
Disinfectant and dilution.
Exposed
1 hour.
Exposed
2 hours.
Exposed
1 hour.
Exposed
2 hours.
Exposed
1 hour.
Exposed
2 hours.
Chloramin'T:
+ 1 1 1 1 ++ 1 1 1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
i to 50,000
+
+
+
+
Dakin 's solution :
NaOCl 1 to 1,000. .. .
NaOCl 1 to 10,000 . .
NaOCl 1 to 25,000 . .
NaOCl 1 to 50,000 . .
NaOCl 1 to 100,000 .
EXPERIMENT 10
Chloramin T:
1 to 1,000
1 to 10,000
1 to 30,000
1 to 50,000
1 to 100,000
Dakin 's solution :
NaOCl 1 to 1,000. .
NaOCl 1 to 10,000.
NaOCl 1 to 30,000.
NaOCl 1 to 50,000.
NaOCl 1 to 100,000
—
—
+
+
+
—
—
- +
+
+
+
+
+
+
+
+
+
+
+
+
-
-
+
+
-
+
—
+
+
+
+
+
+
+
EXPERIMENT II
Chloramin T:
1 to 1,000
1 to 10,000
1 to 30,000
1 to 50,000
1 to 100,000
Dakin's solution:
NaOCl 1 to 1,000. .
NaOCl 1 to 10,000.
NaOCl 1 to 30,000.
NaOCl 1 to 50,000.
NaOCl 1 to 100,000
+
—
—
+
+
—
—
—
+
+
+
—
—
+
+
+
+
+
+
+
+
-
-
+
+
-
—
—
+
+
—
+
+
+
+
+
+
+
a + signifies growth; — , no growth.
Oct. 15, 1920 Germicidal Value of Some Chlorin Disinfectants
93
Experiments 12 and 13. — These experiments were made for the pur-
pose of comparing the germicidal activity of chloramin T, Dakin's solu-
tion, and eusol against Staphylococcus aureus, Bacillus pyocyaneus, and
B. typhosus.
The technic was as follows : Each dilution of the disinfectant was mixed
with an equal amount of a 24-hour culture of the test organism, and the
mixture was thoroughly shaken. After intervals of 10 minutes and 30
minutes, respectively, the mixtures were again shaken, and subcultures
were made with a 3-mm. platinum loop into tubes of standard bouillon
containing enough sodium thiosulphate to neutralize the disinfectant
carried over. The amounts of culture and disinfectant used were 2.5
cc. of each. For purposes of comparison, tests were made with mer-
curic chlorid, and in these tests sodium sulphid was used to neutralize
the disinfectant carried over. The results are given in Table VI.
Table VI. — Comparative germicidal efficiency of chloramin T, Dakin's solution, eusol,
and mercuric chlorid against Staphylococcus aureus, Bacillus pyocyaneus, and B. ty-
phosus °
EXPERIMENT 12
Staph, aureus.
B. pyocyaneus.
B. typhosus.
Disinfectant and dilution.
Exposed
10 minutes.
Exposed
30 minutes.
Exposed
10 minutes.
Exposed
30 minutes.
Exposed
10 minutes.
Exposed
30 minutes;
Chloramin T:
1 to 1,000
1 1 1 1 1
-
+
+
+
-
1 to 2,000
Dakin 's solution :
NaOCl 1 to 2,000. . . .
Eusol:
HOC1 1 to 2,000. . . .
Mercuric chlorid:
1 to 2,000
-
EXPERIMENT 13
Chloramin T:
1 to 1,000
1 to 2,000
Dakin's solution:
NaOCl 1 to 2,000. . . .
NaOCl 1 to 4,000
Eusol:
HOC1 1 to 2,000. . . .
HOC1 1 to 4,000. . . .
Mercuric chlorid:
1 to 2,000
1 to 4,000
+
+
+
+
+
+
+
+
+
a + signifies growth; — , no growth.
Experiments 14 and 15. — In these experiments the same disinfectants
were compared as in experiments 12 and 13, but with the addition of
blood serum. The technic was the same also, except that a mixture of
equal parts of blood serum and culture was used instead of culture alone.
The results are given in Table VII.
94
Journal of Agricultural Research
Vol. XX, No. 2
Table VII. — Comparative germicidal efficiency of chloramin T, Dakin's solution,
eusol, and mercuric chlorid against Staphylococcus aureus, Bacillus pyocyaneus, and
B. typhosus in the presence of 25 per cent, blood seruma
EXPERIMENT 14
Staph, aureus.
B. pyocyaneus.
B. typhosus.
Disinfectant and dilution.
. Exposed
ioininutes
Exposed
30 minutes
Exposed
10 minutes.
Exposed
30 minutes.
Exposed
iominutes
Exposed
30 minutes.
. . — j.. -
Chloramin T:
-
+
+
+
f
+
+
+
Dakin's solution:
NaOCl 1 to 2,000. ... —
Eusol:
hoci 1 to 2,000 :..l -
Mercuric chlorid:
1 to 2,000 +
EXPERIMENT 15
Chloramin T:
1 to 1,000
1 to 2,000
Dakin 's solution :
NaOCl 1 to 2,000. . . .
NaOCl 1 to 4,000. . . .
Eusol:
HOCI 1 to 2,000
HOCI 1 to 4,000. . . .
Mercuric chlorid:
1 to 2,000
1 to 4,000
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
a 4- signifies growth; — no growth.
The four experiments shown in Tables VI and VII show that eusol is
decidedly superior to chloramin T, Dakin's solution, and mercuric chlorid,
especially in the presence of blood serum. Unfortunately, however,
eusol is very unstable and for that reason is not reliable, since it is im-
possible in practice to count on its containing any uniform amount of
active material. In the tests reported here the eusol was made up just
before the test and was used as soon as possible, but observations which
were made in connection with chemical work upon these various disin-
fectants would tend to show that there was probably a perceptible loss
of strength even in the time required for a test.
EXPERIMENTS WITH ANTHRAX SPORES
The experiments upon anthrax spores were performed by the Hill (6)
"rod" method, with some modifications. The method as modified is
as follows: Glass rods 3/i6-mch in diameter and 8 inches long are etched
at one end, the etched portion being about 1 inch long. Cotton is wrapped
Oct. iS, 1920 Germicidal Value of Some Chlorin Disinfectants
95
about the rods near the end not etched, and the rods are thrust into test
tubes so as to engage the cotton in the mouth of the tube. The tubes
containing the rods are sterilized by dry heat (1500 C.) for 1 hour or more.
In making tests the rods are removed from the tubes and the etched
portions are dipped into a suspension made from a culture of the organism
to be tested. They are then replaced in the tubes and dried in the
incubator for one hour.
Rods so infected are transferred to test tubes containing the disinfectant
to be tested, the amount of disinfectant being sufficient to cover all
the infected portion of the rod. They are exposed to the action of the
disinfectant for varying lengths of time. After exposure the rods are
washed with sterile water in order to remove traces of the disinfectant
and are then transferred to tubes containing bouillon or agar, which are
incubated for at least 48 hours at 37. 50 C. The suspension used in in-
fecting the rods is made from the surface growth on an agar tube by
rubbing up in several cubic centimeters of sterile water enough of the
growth to give a suspension of approximately the same density as a
24-hour bouillon culture of Bacillus typhosus. For an organism that
does not bear spores the culture should be 24 hours old, while for spore-
bearing organisms cultures 1 to 2 weeks old are usually the most suitable.
In making tests with a disinfectant containing mercury it is advisable
to dip the rods into a saturated solution of hydrogen sulphid or an aqueous
solution of some sulphid before placing them in subculture tubes. In this
connection it should be mentioned that media of acid reaction have been
found to exert an inhibitory action upon the growth of Bacillus anthracis
after exposure to disinfectants. For that reason the media used in these
experiments have been neutral or slightly alkaline.
Experiments 16 and 17. — In these experiments chloramin T was
tested in varying concentrations, both in water and in 50 per cent blood
serum. A sterile 10 per cent solution of sodium thiosulphate was used
for washing the rods before placing them in subculture tubes of exactly
neutral broth. The results are given in Table VIII.
Table VIII. — Germicidal efficiency of chloramin T against anthrax spores, with and
without the addition of blood serum a
EXPERIMENT 16
Concentration of chloramin T.
Amount of serum.
Exposed
2 hours.
Exposed
4 hours.
Exposed
24 hours.
IO. .
Per cent.
None . .
+
+
+
+
+
+
+
+
+
+
IO
50 per cent
None
5
5
50 per cent. . . .
None . . .
_
I
I
50 per cent. . . .
Control rod
+
n+ signifies growth; — , no growth.
96
Journal of Agricultural Research
Vol. XX. Xo. 2
Table VIII. — Germicidal efficiency of chloramin T against anthrax spores, with and
without the addition of blood serum — Continued.
EXPERIMENT 17
Concentration of chloramin T.
Per cent.
0.5.
0.5.
Control rod.
Amount of serum.
None
50 per cent.
None
50 per cent.
None
50 per cent.
Exposed
2 hours.
+
+
+
+
+
+
Exposed
4 hours.
Exposed
24 hours.
+
+
+
+
+
+
+
Experiment 18. — This experiment was undertaken in order to compare
chloramin T and Dakin's solution. The results are given in Table IX.
Table IX. — Comparative germicidal efficiency of chloramin T and Dakin's solution
against anthrax spores, with and without the addition of blood serum a
EXPERIMENT 18
Disinfectant and dilution.
Chloramin T:
2 per cent
Do
1 per cent
Do
0.5 per cent
Do
Dakin 's solution :
NaOCl 2 per cent . .
Do
NaOCl 1 per cent. . .
Do
NaOCl 0.5 per cent
Do
Control rod. . .
Amount of serum.
None
50 per cent.
None
50 per cent.
None
50 per cent.
None
50 per cent.
None
50 per cent.
None
50 per cent.
Exposed
2 hours.
+
+
+
+
+
+
+
+
+
Exposed
4-hours.
+
+
+
+
4-
+
+
+
+
+
Exposed
24 hours.
+
+
a + signifies growth; — , no growth
Experiment 19. — In this experiment eusol was used, the chlorin being
estimated as HOC1. In the dilutions there was approximately 0.128 gm.
HOC1 per ioo cc. The results are given in Table X.
Table X. — Germicidal efficiency of eusol against anthrax spores °
EXPERIMENT 19
Time of exposure.
30 minutes.
1 hour
2 hours ....
3 hours ....
4 hours ....
5 hours ....
24 hours. . .
50 per cent
No serum.
serum.
+
+
+
+
+
+
+
+
—
+
—
+
—
+
0+ signifies growth; — , no growth.
Oct. 15, 1920 Germicidal Value of Some Chlorin Disinfectants
97
Experiments 20 and 2 1 . — In these experiments comparison was made
between chloramin T, Dakin's solution, eusol, and mercuric chlorid.
The results are given below in Table XI. The results with mercuric
chlorid are included for comparison.
Table XI. — Comparative germicidal efficiency of chloramin T, Dakin's solution, eusol,
and mercuric chlorid against anthrax spores, with and without blood serum a
EXPERIMENT 20
Disinfectant and
dilution.
Amount of serum.
Exposed
2 hours.
Exposed
4 hours.
Exposed
1 day.
Exposed
2 days.
Exposed
4 days.
Chloramin T:
None. . .
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
50 percent. . .
None
50 percent. . .
None
+
+
Dakin 's solution :
NaOCl 1 to 100. - - .
NaOCl 1 to 100
NaOCl 1 to 200. . . .
50 per cent. . .
None
+
NaOCl 1 to 200
Eusol:
HOCl 1 to 200
50 per cent. . .
None
+
HOCl 1 to 200
HOCl 1 to 400
HOCl 1 to 400 ,
Mercuric chlorid :
1 to 2,000
50 per cent. . .
None
-
50 percent. . .
None
50 per cent . . .
+
+
EXPERIMENT 21
Chloramin T:
5 per cent
5 per cent
5 per cent
Dakin's solution:
NaOCl 1 per cent.
NaOCl 1 per cent.
NaOCl 1 per cent.
Eusol:
HOCl 0.75 per cent.
HOCl 0.75 per cent .
HOCl o. 75 per cent.
25 per cent
50 per cent
None
25 per cent
50 per cent
None
2 5 per cent
50 per cent
None
+
+
+
+
+
+
+
+
+
+
+
+
+° signifies growth; — , no growth.
It should be noted here that in the experiments upon anthrax spores
the strength of NaOCl and HOCl required was in most instances greater
than that obtained by preparing Dakin's solution and eusol by the
methods described at the beginning of this paper. So in these instances
the solutions were made with less water in proportion to the other
ingredients. Aside from this change the methods of preparation were
the same.
The experiments upon anthrax spores indicate that if comparison is
made on the basis of weight of chloramin T against weight of chlorin
98 Journal of Agricultural Research vol. xx, no. 2
as NaOCl or HOG, in Dakin's solution and eusol, respectively, chloramin
T must be regarded as less efficient than Dakin's solution or eusol against
naked spores. In the presence of blood serum it is more or less equal
to Dakin's solution, while eusol seems to be superior to both chloramin
T and Dakin's solution. Comparison on the basis of "available" chlorin
would, of course, be much more favorable to chloramin, since it contains
only 25 per cent available chlorin, or 12^2 per cent actual chlorin.
It is interesting to note that in experiments 16, 17, 18, and 2 1 chloramin
T was more efficient against anthrax spores in the presence of blood
serum than in the absence of serum. In experiments 16 and 17 this is
true only for the stronger dilutions (10 per cent and 5 per cent) and is
not true for the lowest dilution (1 per cent). In experiment 18 it is
true for 2 per cent and 1 per cent dilutions after 24 hours, but in experi-
ment 20 with dilutions of 1 to 100 and 1 to 200 and exposures of 2 days
there is greater efficiency without serum than with it. Experiment 21
confirms the results obtained in experiments 16 and 17 with a 5 per
cent dilution.
These experiments also seem more or less at variance with the widely
expressed opinion that chlorin compounds rapidly lose their activity
and soon become inert, especially in the presence of organic matter.
For example, in experiment 20, HOC1 1 to 200 did not destroy anthrax
spores until after an exposure of 2 days, the 4-day result serving as a
control to show the correctness of the result.
This usually accepted opinion is controverted by Rideal (9), who as
the result of his own experiments concludes that —
chlorin has a disinfectant value out of all proportion to that which would be expected
from the hitherto accepted theories, even in the presence of a chemical excess of
organic matter in certain forms.
The explanation which he gives is that the disinfecting action of chlorin
is not due merely to oxidation but also to the action of products formed
by its substitution for hydrogen in ammonia and organic compounds.
EXPERIMENTS WITH BACILLUS TUBERCULOSIS
In experiments upon the tubercle bacillus the method was as follows
Two and one-half cc. of disinfectant dilution were added to 2% cc. of a
suspension of culture (or a mixture of such suspension with an equal
quantity of horse-blood serum), and they were mixed thoroughly by
vigorous shaking. The suspension was made by rubbing up in sterile
distilled water enough of the surface growth from a bouillon culture to
give a suspension whose density was approximately equal to that of a
24-hour culture of Bacillus typhosus. After an exposure of 10 minutes
enough sterile sodium thiosulphate solution (or sodium sulphid where
mercuric chlorid was used) was added to insure complete neutralization,
and finally 1 cc. of each neutralized test mixture was injected subcutane-
ously into a guinea pig.
Oct. is, 1920
Germicidal Value 0} Some Chlorin Disinfectants
99
This technic was used in making a number of comparative experi-
ments with chloramin T, Dakin's solution, eusol, and mercuric chlorid.
The results are given in Tables XII and XIII.
Table XII —Comparative germicidal efficiency of chloramin T, Dakin's solution, eusol,
and mercuric chlorid against Bacillus tuberculosis, with and without the addition of 25
per cent blood serum
EXPERIMENT 22
Disinfectant and dilution.
Chloramin T:
1 to 1,000
Do
Dakin's solution:
NaOCl 1 to 1,000. ■ •
Do
Eusol:
HOC1 1 to 1,000
Do....
Mercuric chlorid:
1 to 1 ,000
Do
Tubercle bacillus suspen-
sion.
Do
Amount of serum.
Guinea
pig No.
None
25 per cent.
None
25 per cent.
None
25 per cent.
None
25 per cent.
+ serum .
S34I4
53415
534i6
53417
534i8
53419
53420
53421
53422
53423
Result.
Autopsy.
Died . . .
..do...
. . do . . .
...do...
Killed**
Died . .
...do...
...do...
...do...
...do...
Typical lesions.
Do.
Do.
Do.
Do.
Do.
Do.
Do.
Do.
EXPERIMENT 23
Chloramin T:
1 to 100
Do...
Dakin 's solution :
NaOCl 1 to 500 ... .
Do
Eusol:
HOC1 1 to 250
HOC1 1 to 500
Mercuric chlorid:
1 to 500
Do
Tubercle bacillus suspen-
sion.
Do
None
25 per cent.
None
25 per cent,
.do.
.do.
None
25 per cent.
+serum
54023
54024
54025
54026
54027
54028
54031
54032
54033
54034
Died . . .
..do....
..do....
..do....
..do....
. .do.;. .
Killed &
Died . . .
...do...,
...do...
Typical lesions.
Do.
Do.
Do.
Do.
Do.
Do.
Do.
Do.
a Killed after 33 days; perfectly normal.
b Killed after 2 months; perfectly normal.
Experiments 24 and 25.— In these experiments the disinfectants
were compared on the basis of the available chlorin contained, so these
experiments are grouped by themselves in Table XIII.
In the experiments upon Bacillus tuberculosis, as in experiments
upon anthrax spores, it was necessary to use Dakin's solution and eusol
of greater strength, and, as before, this result was obtained by lessening
the amount of water while the other ingredients and the method of
manufacture remained unchanged.
IOO
Journal of Agricultural Research
Vol. XX, No. 2
Table XIII. — Comparative germicidal efficiency of chloramin T, Ddkin's solution,
eusol, and mercuric chlorid against Bacillus tuberculosis, with dilutions based on
available chlorin
EXPERIMENT 24
Disinfectant and dilution.
Available
chlorin.
Amount of serum.
Guinea
pig No.
Result.
Autopsy.
Chloramin T. . . .
Do
Eusol
Do. . . .
Dakin's solution.
Do . .
Mercuric chlorid:
1 to 500
Do
Tubercle bacillus
suspension.
Do
1 to 200
1 to 200
1 to 200
1 to 200
1 to 200
1 to 200
None.
25 per cent.
None.
25 per cent.
None.
25 per cent.
None.
25 per cent.
None.
25 per cent.
54549
5455o
54551
54552
54555
54556
54557
54558
54559
5456o
Died . .
...do...
Killed**
Died . .
...do...
...do...
Killed"
Died . .
...do...
...do...
Typical lesions.
Do.
Do.
Do.
Not tubercu-
lous. °
Typical lesions.
Normal.
Typical lesions.
Do.
Do.
EXPERIMENT 25
Chloramin T.
Do
Eusol
Do
Dakin's solution.
Do
Mercuric chlorid:
1 to 500
Do
Tubercle bacillus
suspension.
Do
1 to 200
1 to 200
1 to 300
1 to 300
1 to 200
1 to 200
None.
25 per cent.
None.
25 per cent.
None.
25 per cent.
None.
25 per cent.
None.
25 per cent.
55552
55553
55554
Died . . .
...do....
...do....
55555
55558
...do....
...do....
55559
...do....
5556o
5556i
55562
Killed*
Died . . .
...do....
55563
...do....
Typical lesions.
Do.
Not tubercu-
lous.0
Typical lesions.
Not tubercu-
lous.d
Typical lesions.
Normal.
Typical lesions.
Do.
Do.
0 Killed after 10 weeks.
& Died after 7 weeks of an intercurrent pneumonia.
c Died after 2 months; no lesions observed; death probably due to scurvy.
<* Died after 1 month of an intercurrent pneumonia.
< Killed after 2 months; perfectly normal.
The results of trie experiments upon the tubercle bacillus would seem
to indicate that the chlorin compounds are entirely inefficient so far as
that organism is concerned. These are the results to be expected in
view of the use of antiformin for isolating tubercle bacilli.1
CARBOLIC-ACID COEFFICIENTS OF THE CHLORIN ANTISEPTICS
The results here given are those of a large number of tests made by
the Rideal- Walker method (10), modified only as stated below. Aside
from the use of Staphylococcus aureus and Bacillus pyocyaneus as test
organisms in addition to B. typhosus, the only modifications were the
use of bacto-peptone instead of Witte's peptone and a relaxation of the
rule that coefficients are to be deduced only where there is life after 5
minutes and death after 7^2 minutes.
On account of variation in the resistance of the cultures, especially
Staphylococcus aureus and Bacillus pyocyaneus , it was inconvenient to
1 Amounts actually found inefficient were as follows: Chloramin T, i to so; eusol, 0.5 percent; and Dakin's
solution. 0.5 percent.
Oct. is, 1920 Germicidal Value of Some Chlorin Disinfectants
101
adhere strictly to the rule; and coefficients were deduced at any time
within the 15-minute period, except that no coefficient was deduced
unless there was growth in the phenol subculture tubes after both 2%
and 5 minutes' exposure. This is really only a return to previous prac-
tice (7), and the results obtained are sufficiently accurate for all prac-
tical purposes.
In all these tests, dilutions were based on the amount of available
chlorin; and it should, therefore, be understood that the coefficients are
really, so to speak, those of available chlorin as it is present in chloramin
T, eusol, Dakin's solution, and chlorin water.
It should also be noted that in order to make the original solutions
more nearly equal in chlorin content the amount of bleaching powder
in proportion to water was the same for eusol as for Dakin's solution.
The amount used was 5 gm. to 250 cc, which follows the usual pro-
portion for Dakin's solution but varies from the usual proportion for
eusol. These original solutions were then diluted with distilled water to
obtain the desired amounts of available chlorin in the various dilutions.
The results are summarized in Table XIV, the successive figures
from top to bottom in each column being coefficients obtained at various
times. It will be noted that they do not always agree perfectly, but
they are not offered as examples of accuracy. On the contrary, they
are to be considered as approximate values to be taken for what they
are worth as illustrations of the general principles of selective action
already shown to a greater or less degree in previous experiments.
Table XIV. — Coefficients of chloramin T, Dakin's solution, eusol, and chlorin water,
based on tlie content of available chlorin
Chloramin T.
Dakin's solution.
Eusol.
Chlorin water.
Staphylococcus
aureus.
si
0
03
g
■0.°
^ .
*3
Ji
•3 a
G
0
i .
G
03
<->
3
if
?i
3 8
•3 0
a
03
0
a
03
3
|I
55
1"
0
03
i
03
114
92- 3
92-3
92- 3
92.3
8-3
8.8
8-3
66.6
66.6
66.6
57
57
57
66.6
55-5
66.6
66.6
55-5
66.6
66.6
66.6
66.6
114
92-3
92-3
92.3
I20
I20
I20
IOO
III
III
III
92- 3
92-3
92- 3
80
80
80
88
80
80
In connection with preceding experiments solutions of chlorin T,
Dakin's solution, and eusol were kept in a dark closet at room temperature,
and titrations were made at intervals to detect any changes that might
occur. It was found that Dakin's solution and solutions of chloramin T
will keep for a month or more without any great loss of available chlorin;
while, on the other hand, eusol deteriorates rapidly, there being a notice-
able change even within 24 hours. For example, in one instance a sample
of Dakin's solution showed only about 10 per cent loss after standing 6
187932°— 20 2
102
Journal of Agricultural Research
Vol. XX, No. 2
months, while a sample of eusol lost 10 per cent of its available chlorin in
24 hours.
In view of the instability of eusol an attempt was made to secure a more
stable product by reducing the amount of boric acid, and it was found
that by reducing the proportions from equal parts of bleaching powder
and boric acid to 10 parts of bleaching powder and 3 parts of boric acid a
product was obtained which was fully as stable as Dakin's solution. This
modified eusol was tested by the carbolic-acid coefficient method in com-
parison with the regular eusol. The results are given in Table XV.
Eusol made by the original formula is designated as eusol I, while that
made by the modified formula is designated as eusol II.
Table XV. — Carbolic-acid coefficients of eusol I {original formula) and eusol II {modified
formula), based on available chlorin
Feb. 4.
Do
Feb. 7.
Do
Solution.
Eusol I.
Eusol II
Eusol I.
Eusol II
Coefficient
Coefficient
with
with
Staphylo-
Bacillus
coccus
pyocy-
aureus.
aneus.
92-3
I20
77
80
92-3
I20
77
80
Coefficient
with
Bacillus
typhosus.
Ill
66.6
100
66.6
According to the results of these comparative tests it would seem that
eusol I is superior to eusol II in germicidal efficiency.
INFLUENCE OF AMMONIA UPON THE GERMICIDAL EFFICIENCY OF
CHLORIN DISINFECTANTS
It has been shown by Race (8) and Rideal (9) that the addition of
ammonia to electrolytic hypochlorite solutions greatly increases their
germicidal efficiency. Their explanation of this increase is that it is due
to the formation of chloramin (NH2C1). The experiments here discussed
were intended to verify these findings by the use of methods similar to
those employed in the experiments already discussed, without attempting
to ascertain the cause of the increased germicidal value.
The method first used was the Rideal- Walker method (jo), modified
by the use of an unadjusted culture medium as recommended by the
American Public Health Association Committee on Standard Methods of
Examining Disinfectants (//). The method was also modified by deduc-
ing coefficients at times other than 7X minutes, and in many instances no
coefficient was obtained.
By the use of this method experiments were first made upon Dakin's
solution, prepared from bleaching powder by the use of sodium carbonate
and bicarbonate as previously described. Ammonia was added so as to
furnish a molecular equivalent to the sodium hypochlorite of the Dakin's
solution. Experiment 26 (Table XVI) shows the comparative results
with no organic matter added, and experiments 27 and 28 (Table XVI)
show the results with blood serum added.
Oct. 15, 1920 Germicidal Value of Some Chlorin Disinfectants 103
Table XVI. — Effect of addition of ammonia upon germicidal activity of Dakin's solution
against Bacillus typhosus a
EXPERIMENT 26
without ammonia; no blood serum added
Concentration of NaOCl.
Ex-
Ex-
posed
posed
2P2 min-
S min-
utes.
utes.
+
+
+
+
+
+
+
+
Ex-
posed
7% min-
utes.
Ex-
posed
10 min-
utes.
Ex-
posed
min-
utes.
Ex-
posed
IS min-
utes.
I to 2,000
i to 4,000
1 to 6,000. . . .
1 to 8,000. . . .
Phenol 1 to 70
+
+
+
+
+
+
+
+
Coefficient 57,
4,000
with ammonia; no blood serum added
1 to 6,000. . . .
1 to 8,000. . . .
1 to 10,000 . . .
i to 12,000. . .
Phenol 1 to 70
+
+
1
+
+
+
—
—
—
+
+
+
—
—
—
+
+
+
+
—
—
+
+
"
"
"
Coefficient 86,
6j^2= 86.
EXPERIMENT 27
without ammonia; 5 PER cent blood serum added
1 to 500
1 to 1,000
1 to 2,000
1 to 4,000
Phenol ito8o&.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
—
with ammonia; s per cent blood serum added
1 to 500
1 to 1,000
1 to 2,000
1 to 4,000
Phenol 1 to 80 b.
+
+
+
+
+
-
-
EXPERIMENT 28
with ammonia; 10 PER CENT BLOOD serum added
1 to 500
1 to 1,000
1 to 2,000
1 to 4,000
Phenol 1 to 70 b .
-
-
-
-
-
-
+
+
+
+
_
_
_
+
+
+
+
+
+
+
"
"
~
WITH AMMONIA; 50 PER CENT BLOOD SERUM ADDED
1 to 500
1 to 1,000
1 to 1 , 500
1 to 2,000
Phenol 1 to 70 b .
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
~
°+ signifies growth; — , no growth.
b No blood serum added.
104
Journal of Agricultural Research
Vol. XX, No. 2
The experiments shown above in Table XVI indicate that the addition
of a molecular equivalent of ammonia to Dakin's solution not only greatly
increases its germicidal value against "naked" bacteria, but, to a large
extent, prevents depreciation of germicidal value due to the addition
of blood serum.
In Table XVII there are shown the results of a number of experiments
upon chlorin water, with and without a molecular equivalent of ammonia.
Table XVII. — Effect of ammonia upon the germicidal activity of chlorin in aqueous
solution against Bacillus typhosus a
EXPERIMENT 29
WITHOUT AMMONIA
Concentration of chlorin.
I to 4,000
I to 8,000
I to 12,000
i to 16,000
Phenol 1 to 80
Ex-
posed
2K
min-
utes.
+
Ex-
posed
5
min-
utes.
+
+
Ex- j Ex-
posed posed
min-
utes.
+
min-
utes.
Ex-
posed
12%
min-
utes.
Ex-
posed
IS
min-
utes.
WITH AMMONIA
1 to 4,000
1 to 8,000
1 to 12,000
1 to 16,000
Phenol 1 to 80
-
-
-
-
+
+
+
+
_
_
+
+
+
+
+
+
+
+
EXPERIMENT 30
WITHOUT AMMONIA; IO PER CENT BLOOD SERUM ADDED
i to 500
I to I.OOO
I to 2,000
1 to 4,000
Phenol 1 to 80 b
- .
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
4-
WITH AMMONIA; IO PER CENT BLOOD SERUM ADDED
I to 1,000
I to 2,000
1 to 4,000
I to 6,000
Phenol 1 to 80 b
a + signifies growth; — , no growth.
h No blood serum added.
Oct. 15, 19*0 Germicidal Value of Some Chlorin Disinfectants
105
The experiments shown in Table XVII indicate that the addition of a
molecular equivalent of ammonia to chlorin water decreases rather than
increases the germicidal value of the chlorin in the absence of organic
matter, but it does tend to prevent depreciation of germicidal activity on
the addition of blood serum.
The experiments shown in Table XVIII were designed to determine
the optimum amount of ammonia.
Table XVIII. — Effect of varying amounts of ammonia upon the germicidal value of
chlorin in aqueous solution °
EXPERIMENT 31
WITH MOLECULAR EQUIVALENT OF AMMONIA
Concentration of chlorin.
I to 5,000
i to 10,000. . . .
i to 15,000. .. .
1 to 20,000. . . .
Phenol 1 to 80
Ex-
Ex-
Ex-
Ex-
Ex-
posed
posed
posed
posed
posed
2M
s
1V2
10
12J4
min-
min-
min-
min-
min-
utes.
utes.
utes.
utes.
utes
+
+
—
—
—
+
1
+
+
+
+
-j-
+
+
+
+
+
+
—
—
Ex-
posed
min-
utes.
+
+
WITH ONE-HALP MOLECULAR EQUIVALENT OF AMMONIA
1 to 5,000
I to IO.OOO. .. .
1 to 15,000. .. .
I to 20,000. .. .
Phenol 1 to 80
+
EXPERIMENT 32
WITH MOLECULAR EQUIVALENT OF AMMONIA
1 to 5,000
I to IO.OOO. .. .
1 to 15,000. .. .
I tO 20,000. .. .
Phenol 1 to 80
—
—
—
—
+
+
+
+
-f
_
+
4-
+
+
+
+
+
+
WITH TWO MOLECULAR EQUIVALENTS OF AMMONIA
1 to 5,000
1 to 10,000. . . .
1 to 15,000. .. .
I to 20,000. . . .
Phenol 1 to 80
+
+
+
+
+
+
T"
+
+
+
+
+
+
+
+
+
+
+
+
+
+
"
a + signifies growth — , no growth.
io6
Journal of Agricultural Research
Vol. XX. No. 2
Table XVIII. — Effect of varying amounts of ammonia upon the germicidal value of
chlorin in aqueous solution — Continued
EXPERIMENT 33
WITH ONE-HALF MOLECULAR EQUIVALENT OP AMMONIA
Concentration of chlorin.
I to 10,000
i to 15,000
I to 20,000
i to 25,000
Phenol 1 to 80.
Ex-
Ex-
Ex-
posed
posed
posed
2*4
s
llA
min-
min-
min-
utes.
utes.
utes.
+
+
+
+
+
+
T"
+
_]_
+
+
Ex-
Ex-
posed
posed
10
12K
min-
min-
utes.
utes.
+
_
+
+
+
+
+
Ex-
posed
min-
utes.
+
WITH ONE-FOURTH MOLECULAR EQUIVALENT OF AMMONIA
I to 10,000 —
1 to 15,000 +
I to 20,000 4"
1 to 25,000 +
Phenol 1 to 80 +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
EXPERIMENT 34
WITH MOLECULAR EQUIVALENT OF AMMONIA; IO PER CENT BLOOD SERUM ADDED
1 to 1,000 +
1 to 2,000 + + + + + +
1 to 4,000 4- + 4- 4-
1 to 6,000 +
Phenol 1 to 80 +
+
4-
4.
_
+
+
4-
4-
+
+
_L-
4-
+
4-
4-
4-
+
WITH ONE-HALF MOLECULAR EQUIVALENT OP AMMONIA; IO PER CENT BLOOD SERUM ADDED
I to 1,000
1 to 2,000
1 to 4,000
I to 6,000
Phenol 1 to 80.
+
4-
+
4-
4-
4-
4-
4-
4-
-i-
4-
4-
4-
4-
4-
4-
4-
+
"
The experiments shown in Table XVIII indicate that the optimum
amount of ammonia is approximately one-half of the molecular
equivalent.
Experiments were next made with anthrax spores, using the following
method: Equal quantities (2% cc. each) of chlorin solution and spore
suspension, with or without blood serum added to it, were mixed in a
test tube and vigorously shaken. After it had stood at room temperature
for the required time of exposure the mixture was again shaken, and a
subculture was made by a standard platinum loop into a tube of nutrient
broth. No attempt was made to neutralize any excess of disinfectant.
The results of these experiments are shown in Table XIX.
Oct. 15, 1920 Germicidal Value of Some Chlorin Disinfectants
107
Table XIX. — Germicidal activity of chlorin against anthrax spores with and without
addition of ammonia °
EXPERIMENT 35
without ammonia; 10 per cent blood serum added
Concentration of
chlorin.
Ex-
posed
1
hour.
Ex-
posed
2
hours.
Ex-
posed
3
hours.
Ex-
posed
4
hours.
Ex-
posed
5
hours.
Remarks.
I to 1,000
+
+
+
+
+
+
+
+
+
+
+
+
+
+
The same dilutions without
blood serum killed the spores
in 30 minutes.
WITH ONE-HALF MOLECULAR EQUIVALENT OF AMMONIA; io PER CENT BLOOD SERUM ADDED
i to 1,000.
i to 2,000.
i to 4,000.
+
+
-
+
-
Number of spores 350,000, or
70,000 per cubic centimeter.
Experiment 36
WITHOUT AMMONIA; 10 PER CENT BLOOD SERUM ADDED
Concentration of
chlorin.
Ex-
posed
3
hours.
Ex-
posed
6
hours.
Ex-
posed
12
hours.
Ex-
posed
18
hours.
Ex-
posed
24
hours.
Remarks.
+
+
+
-J-'
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
The same dilutions without
blood serum killed spores in 15
minutes.
WITH ONE-HALF MOLECULAR EQUIVALENT OF AMMONIA; IO PER CENT BLOOD SERUM ADDED
1 to 2,000.
1 to 4,000.
1 to 6,000.
i to 8,000.
+
+
+
+
+
Number of spores 350,000, or
70,000 per cubic centimeter.
a + signifies growth; — , no growth.
The results shown in Table XIX seemed to show clearly that chlorin
with ammonia added had very great germicidal value, even in the
presence of organic matter in the form of blood serum.
Experiments were, therefore, undertaken to ascertain whether or not
such a solution could be used for disinfecting hides. The technic was
as follows: Small pieces of dry hide, cut to the same weight, were infected
by soaking them in a suspension of anthrax spores and then drying
them over sulphuric acid in a vacuum equal to about 5 mm. of
mercury for 48 hours. These pieces of infected hide were then treated
with the disinfectant solution in the proportion of 5 times as much
solution as hide by weight. At the end of the required period of ex-
posure the pieces of hide were transferred to a solution of sodium thio-
sulphate of sufficient strength to neutralize completely the disinfectant
carried over by the hide. After neutralization the hair and more or
io8
Journal of Agricultural Research
Vol. XX, No. a
less of the hide surface were scraped off with sterile instruments and
plated, using exactly neutral agar. The results of two such experiments
are given in Table XX.
It should be noted in this connection that the stock solutions of
chlorin water and Dakin's solution from which test dilutions were pre-
pared by the machine devised by the Wallace & Tiernan Co., of New
York, for the preparation of Dakin's solution, chlorin being run directly
into water or a solution of sodium carbonate, as the case might be.
Table XX. — Germicidal activity of chlorin water and Dakin's solution against anthrax
spores on pieces of hide
EXPERIMENT 37
CHLORIN WATER WITH MOLECULAR EQUIVALENT OF AMMONIA
Concentra-
tion of
chlorin.
Exposed 2 hours.
Exposed 6 hours.
Exposed 12 hours.
Exposed 24 hours.
I to 500. . .
1 to 1,000.
Plates over-
grown.
do
Colonies too
many to count.
do
16 colonies, 14
anthrax.
Many anthrax
colonies.
Plate overgrown .
Many anthrax
colonies.
Do.
1 to 2,000.
do
do
Do.°
CHLORIN WATER WITH ONE-HALF MOLECULAR EQUIVALENT OF AMMONIA
i to 500. .
I to 1,000
I to 2,000
Plates over-
grown.
....do
.do.
Colonies too
many to count.
...do
.do.
80 colonies, 70
anthrax.
Plates over-
grown.
do
20 anthrax col-
onies.
40 anthrax col-
onies.
Colonies too
many to count.
° No count possible on account of spreaders.
EXPERIMENT 38
DAKIN'S SOLUTION WITH NO AMMONIA ADDED
Concentra-
tion of
available
chlorin.
Exposed 6 hours.
Exposed 18 hours.
Exposed 24 hours
Exposed 48 hours.
I to 250.
I to 500.
/Plates all show heavy growth, spreaders plus discrete and confluent
\ colonies of anthrax.
DAKTN S SOLUTION WITH ONE-HALF MOLECULAR EQUIVALENT OF AMMONIA
i to 250. . .
i to 500. . .
1 colony °
Many anthrax
colonies.6
10 colonies0
4 colonies, 2 an-
thrax.
1 colony a
No colonies vis-
ible, c
4 colonies.0
2 colonies.0
DAKIN S SOLUTION WITH MOLECULAR EQUIVALENT OF AMMONIA
1 to 250. .
1 to 500. .
Many anthrax
colonies.6
do
4 colonies.
1 colony an-
thrax.
1 colony ° .
No colonies vis-
ible, c
4 colonies.0
1 colony.0
o Not anthrax.
6 No count possible on account of spreaders.
c Covered by spreaders.
Oct. is, 1920 Germicidal Value of Some Chlorin Disinfectants 109
The results shown in Table XX indicate that the results previously
obtained were too high, presumably because in the previous experiments
there was no attempt to neutralize the disinfectant. However, on the
addition of ammonia there is still evident a great increase in germicidal
activity and much less decrease in germicidal power when organic matter
is present.
A selective action upon the different types of organisms was also seen.
Where the concentration of available chlorin was low and the time of
exposure comparatively short the plates were overgrown by spreaders
and various types of colonies, of which the anthrax colonies made a very
small part. With greater concentration of chlorin and longer exposure
this proportion was reversed, and most of the colonies seen were those
of anthrax. In experiment 38 it was found that even after no anthrax
colonies were to be found there were still spreaders and colonies of organ-
isms other than anthrax. These organisms were not identified except
to make sure they were not anthrax but were evidently already present
on the hide and were more resistant than the strain of anthrax spores
employed.
CONCLUSION
(1) In the ordinary routine work of general disinfection, such as dis-
infection of cattle cars and pens, there is always a large amount of organic
matter present. It is evident, therefore, that because of the enormous
diminution in germicidal value on addition of organic matter as well as
because of the injurious effects on metals and fabrics the chlorin dis-
infectants as a class do not seem to be suited for use under the usual
conditions and by the usual methods of general disinfection. That is not
to say, however, that when properly used they are not efficient and
valuable in the treatment of infected wounds; in fact, the evidence
available goes to show that they are of great value when so used; and, of
course, chlorin and hypochlorites are being very widely and success-
fully used for the disinfection of drinking water.
(2) Compared on a basis of weight of chloramin T as against weight
of chlorin as sodium hypochlorite (Dakin's solution) or hypochlorous
acid (eusol), or as chlorin in aqueous solution, chloramin T is less efficient
than the others. But if the comparison is made on the basis of available
chlorin contained it is much more efficient against Staphylococcus aureus,
much less efficient against Bacillus pyocyaneus, and approximately equal
in efficiency against B. typhosus.
(3) The experiments upon Bacillus tuberculosis indicate that the
chlorin disinfectants are worth very little so far as that organism is
concerned. This is not surprising in view of the use of antiformin
(NaOCl + NaOH) in isolating tubercle bacilli.
(4) In the present work, considered as a whole, there is seen throughout
more or less "selective action" on the part of the various disinfectants.
The most clearly defined example of this is seen in the extremely high
no Journal of A gricultural Research vol. xx, No. 2
value of chloramin T against Staphylococcus aureus as compared with its
extremely low value against Bacillus pyocyaneus.
(5) The results of the experiments upon anthrax spores show that the
germicidal action of chlorin compounds is not always so speedy as is
commonly supposed but may extend over several days.
(6) The addition of ammonia to solutions of chlorin or hypochlorites
very greatly increases germicidal activity and tends to prevent deprecia-
tion in value on the addition of organic matter.
LITERATURE CITED
(1) American Public Health Association.
1918. report of the committee on standard methods of examining dis-
INFECTANTS. In Amer. Jour. Pub. Health, v. 8, no. 7, p. 506-521, 1 fig.
(2) Carrel, A., and Dehelly, G.
IQI7. LE TRAITEMENT DES PLAIES INFECTEES. Ed. 2, 201 p., 95 fig., 4 pi.
Paris.
(3) Cullen, Glen E., and Austin, J. Harold.
19 18. HYDROGEN ION CONCENTRATIONS OF VARIOUS INDICATOR END-POINTS
in dilute sodium hypochlorite solutions. In Jour. Biol. Chetn.,
v. 34, no. 3, p. 553-568, 1 fig. Bibliography, p. 568.
(4) Dakin, H. D., Cohen, J. B., and Kenyon, J.
1916. STUDIES IN ANTISEPTICS. II. ON CHLORAMINE, ITS PREPARATION, PROP-
ERTIES, and use. In Brit. Med. Jour., v. 1, no. 2874, p. 160-162.
(5) and Dunham, Edward Kellogg.
1917. handbook on antiseptics, ix, 129 p., 2 pi. New York.
(6) Hill, Hibbert Winslow.
1898. A METHOD OF PREPARING TEST OBJECTS FOR DISINFECTION EXPERIMENTS.
In Pub. Health Papers and Rpts., Amer. Pub. Health Assoc., v. 24,
p. 246-249, 1 pi.
(7) Partridge, William.
1907. THE BACTERIOLOGICAL EXAMINATION OF DISINFECTANTS. 66 p., illus.
London .
(8) Race, Joseph.
1916. THE USE OF AMMONIA IN THE CHLORINATION OF WATER. In Canad. Eng.,
v. 30, no. 11, p. 345-346.
(g) Rideal, Samuel.
1910. THE INFLUENCE OF AMMONIA AND ORGANIC NITROGENOUS COMPOUNDS
on chlorine disinfection. In Jour. Roy. Sanit. Inst., v. 31, no. 2,
P- 33-45-
(10) and Walker, J. T. Ainslie.
1913. approved technique OF THE rideal-walker TEST. In Amer. Jour.
Pub. Health, v. 3, no. 6, p. 575-581, 2 fig.
(11) Rosenau, M. J.
1917. preventive medicine and hygiene . . . ed. 3, xxviii, 1074 p., illus.
New York, London.
(12) Smith, J. Lorrain, Drennan, A. Murray, Rettie, Theodore, and Campbell,
William.
1915. experimental observations on the antiseptic action of hypo-
chlorous ACID AND ITS APPLICATION TO WOUND TREATMENT. In
Brit. Med. Jour., v. 2, no. 2846, p. 129-136.
(13) Taylor, Herbert D., and Austin, J. Harold.
1918. THE SOLVENT ACTION OF ANTISEPTICS ON NECROTIC TISSUE. In Jour.
Exp. Med., v. 27, no. 1, p. 155-164, pi. 5.
A NEW AVOCADO WEEVIL FROM THE CANAL ZONE
By H. F. Dietz,1 Entomological Inspector, with description of the species by H. S.
Barber, Assistant, Bureau of Entomology, United States Department of Agriculture
INTRODUCTION
The Federal quarantine against the avocado weevil (Heilipus lauri
Boheman) led Mr. James Zetek, Entomologist of the Panama Canal, and
the writer, during the service of the latter in the Canal Zone, to search for
the weevil in the native avocados in Panama. The weevil proves to be
a species previously unknown to science, but the results of investigations
of the breeding habits of these potential pests, still very imperfectly un-
derstood, supply the first records of field observations under natural
conditions.
Two closely related species of avocado weevils are known.2 As the
first, H. lauri Boheman, is indigenous to Mexico and the second, H.
pittieri Barber, is native in Costa Rica, the existence of this new form
had already been suspected.3 Its discovery is of special interest, how-
ever, since it has been recently intercepted entering the United States.*
FIELD OBSERVATIONS
Two males of this weevil had been found in June, 191 8, feeding on the
leaves of small seedling avocado trees in a nursery at Ancon, C. Z., by
Mr. Zetek, and further search was rewarded in April and May, 191 9, when
"wild" avocado fruits, the seeds of which contained Heilipus larvae, were
collected at the large avocado plantation at Frijoles, C. Z. These fruits
came from large trees growing wild at the edge of a plantation and at a
considerable distance from the cultivated, bearing trees. Attempts to
determine the previous history of these "wild" trees were unavailing.
Infested fruits were brought to the Board of Health Laboratory at Ancon,
and the adults reared from them did not differ from the two collected in 1 9 1 8
or from the large specimen which had been sent to the National Museum
by Mr. F. H. Jackson about 191 2. From the occurrence described above
and from the date of the last-mentioned specimen it would appear that
the species is endemic in Panama, but there remains a possibility that it
1 Resigned Nov. 3, 1919.
4 Barber, H. S. avocado seed weeviis. In Proc. Ent. Soc. Wash., v. 21, no. 3, p. 53-60, pi. 2. 1919.
3A very large specimen was received at the United States National Museum about 1912 from Las Cas-
cadas, C Z. (F. H. Jackson, cftllector), but it was not treated in the paper by H. S. Barber cited above
because of absence of data definitely associating it with avocado. Other close relatives with similar habits
will undoubtedly be found in other avocado-growing regions of tropical America.
* This interception was made by Mr. O. K. Courtney, Port Inspector of the Federal Horticultural Board
at New Orleans, La., in October, 1919. H. perseae Barber was found in an avocado seed in the baggage of a
steamship passenger arriving at New Orleans from Cristobal, C. Z.
Journal of Agricultural Research, Vol. XX, No. 2
Washington, D. C. Oct. 15, 1920
vd Key No. K-8S
(III)
112 Journal of Agricultural Research vol. xx, no. 2
might have become established there long ago through the importation of
avocados or their seeds from some other part of the American Tropics.
Miscellaneous information regarding the habits of the various stages of
the weevil was obtained in rearing it. Some notes regarding its economic
importance and distribution were also made.
At Frijoles only the "wild" fruits were infested, 17 out of 40, or 42.5
per cent, of such fruits containing from 1 to 4 larvae. Out of over 200
cultivated fruits examined here not one was found infested. Fruits
infested with Heilipus larvae have been found on fruit stands in Panama
City and Colon, in the Republic of Panama, and at Gatun and Ancon, in
the Canal Zone. The only information obtainable in these cases regard-
ing the origin of such fruits was that they came either from the Canal Zone
or neighboring parts of the Republic of Panama. From the data at hand
the species seems to be limited to the "Canal Zone region," though there
is little doubt that it occurs over a much wider area.
EGG PUNCTURES AND LARVAL HABITS
The egg punctures are somewhat crescent shaped, about 4 mm. long,
with the ends blunt. In a general way they resemble those of the plum
curculio. As many as 10 were found on a single fruit, but in 8 of these
the eggs had been crushed by the growing fruit and in 2 young larvae had
hatched. No eggs were found, but from the examinations of infested
fruits it is evident that the eggs are laid at the junction of the skin of the
fruit and the pulp. The exact time that oviposition takes place is not
known, but from the evidence at hand it is when the fruit is between
one-half and three-fourths mature.
After hatching, the larvae often wander through the pulp before entering
the seed, thus rendering a considerable part of the fruit inedible, especially
where more than one larva occur in it. Once the larvae enter a seed they
confine their activities to it. Mr. Barber has called attention to the fact
that seeds infested with H. lauri and H. pitiieri do germinate if the embryo
has not been injured by the tunnelling of the larvae, and the same thing
has been observed in the study of H. perseae; but when a seed becomes
infested with two or more larvae, it is usually so badly riddled that it can
not germinate. Furthermore, seeds infested with Heilipus larvae seem
to be subject to the attacks of several kinds of "dry rots" which follow
along the tunnels, invade the embryo, and kill it. Likewise, these fungi,
at least under laboratory conditions, seem to be indirectly responsible
for the death of a considerable number of larvae and pupae.
No natural migration of larvae from one seed to another, even when
these seeds are massed together, has been observed, but half -grown
larvae taken from infested seeds immediately tunnelled into uninfested
ones when these were provided.
The duration of the larval stage was not determined, but indications
are that it is not less than three months.
Oct. is. i&so A New Avocado Weevil from the Canal Zone 113
PUPATION
When the larvae are full grown, instead of leaving the seed they hollow
out a large spherical cell in which they pupate. Three such cells have
been found in one-half of a large avocado seed, and four adults have been
reared from a single seed. This is probably as large a number of adults
as can be obtained from one seed because of the quantity of food eaten
by the larvae and because of the fact that the larvae tunnel freely from one
cotyledon of the seed to the other. The minimum duration of the pupal
stage is 12 to 15 days.
HABITS AND INJURY BY ADULTS
The adults, on transforming from the pupal stage, rest in the pupal cell
from two to four days and then cut their way out. At the time they come
from the pupal cell the adults are decidedly reddish in color, with six
prominent yellowish spots, as given in the technical description. The
reddish color becomes darker with age and is finally blackish in reared
individuals that live over two months.
The adults readily drank water that collected on the sides of the glass
cages to which they were confined. They ate and seemed to flourish on
half-ripe fruit, young leaves, and stems of avocado and on fresh avocado
seeds. In one case an individual that had been starved for a week ate a
few holes in guaA^a leaves.
Injury to the fruit and to the leaves and stems is shown in Plate 7, C,
and in Plate 8. An interesting" thing about the fruit injury is that the
outer skin was first eaten off; then, as the surface of the pulp became
dry a day or so later, this in turn was eaten off, the result being that
within a week holes almost % inch deep were eaten out. On the
young stems the bark layers were gnawed off first and the woody areas
were then eaten through, so that all the parts above the injury col-
lapsed. Similar injury was done to the petioles of the leaves. In
inspection work at the Plant Inspection House of the Office of Foreign
Seed and Plant Introduction, Bureau of Plant Industry, at Washington,
D. C, avocado bud wood has repeatedly been received from Guatemala
showing insect scarring similar to that caused by the light feeding of
H. perseae on young stems. This injury on the Guatemala bud wood
may have been the feeding injury of H. pittieri that had "healed over."
In practically every way the feeding habits of H. perseae are similar to
those of H. lauri as recorded by Barber.
The shortest time that any individuals of this new species (H. perseae)
remained alive was 10 days, all of them without food. One male without
food but with copious and regular supply of water remained alive 23
days. It was observed that when individuals were kept in dry cages they
soon died, even in the presence of food. The longest time any individual
remained alive was 116 days, this being a female.
H4 Journal of Agricultural Research vol. xx, no. 2
Although five individuals (two males and three females) were kept
together 35 days, no mating was observed, nor did oviposition take place
on the half-ripe fruit that was provided for this purpose.
GENERATIONS
The apparently long duration of the larval stage and the known
longevity of the adults indicate that there is but a single generation in a
year. If this is true, then it is a long-drawn-out generation, for, from the
material obtained at Frijoles, adults emerged over a period of 40 days, and
in several cases a month elapsed between the emergence of the first and
last adults from the same seed. It is probable, however, that breeding
is controlled in the Tropics more by the activities of the host plants in
supplying the proper conditions for oviposition.
CONTROL
The control of all three species of the genus Heilipus now definitely
known to infest avocado seeds is comparatively simple, because pupation
takes place inside the seed. It consists of gathering up and burning the
fallen fruits and seeds. This control may be complicated, however, by
the presence of "wild" trees that are not readily accessible or easily
eliminated. In such cases it may be possible to protect cultivated fruits
by arsenical sprays, for the adults feed freely on the leaves and doubtless
in the field drink considerable water off the leaves when these are wet.
DESCRIPTION OF HEILIPUS PERSEAE
Heilipus perseae Barber, n. sp. (PI. 7, A, B.)
Closely related to H. lauri Boh., but more robust; the squamose fascia of elytra
larger, and, in addition, a similar squamose area on the sides of the pronotum. The
rostrum is short in both sexes, and the mesosternum is not prominent. The legs are
also much shorter than in either H. lauri or H . pittieri.
Ovate, shining, rufopiceous, clothed sparsely with scales which are white on legs
and under surface, pale ochreous in the seriate elytral punctures, and darker ochreous
on thoracic and elytral fasciae, the marginal scales of which appear paler. Frontal
fovea deep; eyes much larger than in H. lauri and separated above by less than half
the width of rostrum; the latter shorter (eye to apex) than the pronotum in both
sexes. Pronotum very coarsely sparsely punctate, median line impunctate but not
elevated; lateral squamose areas irregularly oval, usually a little produced downward
in the anterior constriction, but rarely extending to basal or apical margins.
Scutellum small, subtriangular, convex, impunctate, polished. Elytra sparsely
seriately foveolate, the foveas densely squamose; two large squamose areas in same
position as the small ones in H . lauri, the apical fascia usually extending from side
margin to suture, but sometimes nearly divided at suture'. Mesosternum a little
produced but not projecting beyond coxae. First and second ventral segments
feebly impressed at middle in the female, a little more strongly impressed in the male.
Tibial claws short and stout. Length (rostrum excluded) n to 15.5 mm., width 4.8
to 5.7 mm. Length of rostrum, males 2.9 to 3.4 mm., females 3.2 to 4.1 mm.
Oct. is, i92o A New Avocado Weevil from the Canal Zone 115
The sexes are extremely difficult to distinguish, unless the tip of the aedeagus or
the "palps" of the ovipositor can be seen. Nine males and seven females are before
me, all having been reared from avocado seeds at Frijoles, C. Z., by Mr. H. F. Dietz,
during May, June, and July, 1919, except a male taken at Ancon, June 20, 1918 (J.
Zetek No. Z1084), and a large undated female (the allotype) from Las Cascadas,
C. Z., received from F. H. Jackson about 19 12.
Type, allotype, and 14 paratypes, United States National Museum No. 22586.
One paratype retained by Federal Horticultural Board and one paratype sent to Mr.
Geo. C. Champion.
PLATK 7
Heilipus perseae:
A, B. — Adult, paratype. X 5-
C — An avocado fruit (reduced) showing feeding injury by the beetles.
(116)
A New Avocado Weevil from the Canal Zone
Plate 7
B
Journal of Agricultural Research
Vol. XX, No. 2
A New Avocado Weevil from the Canal Zone
Plate 8
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 8
Heilipus perseae:
Leaves showing the injury done by five beetles in 48 hours.
187932°— 20 3
PLATE 9
Heilipus perseae, mature larva:
A. — Ventral face of ventral mouth parts.
B. — Anterior part of head from above.
C. — Lingua, hypopharyiix, hypopharyngeal bracon, and dorsal (buccal) face of
maxilla.
D. — Dorsal face of mandible.
E. — Epipharynx.
F. — Ventral face of mandible.
G. — Head capsule from above.
H. — Thoracic spiracle from outside.
I. — Mature larva.
Drawings, from studies, by Dr. A. G. Boving.
A New Avocado Weevil from the Canal Zone
Plate 9
■$
.&
Journal of Agricultural Research
Vol. XX, No. 2
STUDIES IN MUSTARD SEEDS AND SUBSTITUTES:
I. CHINESE COEZA (BRASSICA CAMPESTRIS
CHINOLEIFERA VIEHOEVER)
By Arno ViehoEvER, Pharmacognosist in Charge, Joseph F. ClEVENgER, Assistant
Plant Histologist, and Clare Olin Ewing, Assistant Pharmacognosist, Pharma-
cognosy Laboratory, Bureau of Chemistry, United States Department of Agriculture1
INTRODUCTION
Shortly after the outbreak of the recent great war many products
which previously could be obtained from European countries were no
longer available, and as a result importers were obliged to seek other
sources of supply. One of the products thus affected was mustard seed.
It was soon apparent that much of the seed offered for entry as mustard
was quite different not only in quality but also in general appearance
and condimental character from that which had usually been imported.
Some of the shipments, for example, of Chinese mustard (Brassica
juncea (L.) Cosson), while not so satisfactory as the mustards formerly
recognized, consisted of seeds with condimental and medicinal qualities
which made them useful as substitutes. Others, consisting of Japanese
mustard (41) 2 {Brassica cernua Thunb.), proved to be very valuable
material. It is probably grown under more favorable climatic conditions
and is evidently collected more carefully than the Chinese seed.
Seeds from some other Brassica species which possessed no medicinal
or satisfactory condimental value, however, were imported (1, p. 469; 4.5;
46; 48), and among these was the one to which this article has reference.
The seed was first called to the attention of the authors because it had
been imported in large quantities as rape seed and subsequently was
introduced into interstate trade as mustard seed. Its appearance was
rather bright, though not shiny, and resembled in a way yellow or white
mustard (Sinapis alba L.) (31, p. 379). On account, however, of its
peculiar earthy flavor and lack of the pungency characteristic of mustard,
it did not meet with the unqualified approval of the trade.
1 During the progress of the botanical work the authors obtained valuable assistance from the Bureau
of Plant Industry, United States Department of Agriculture, and desire to acknowledge especially the
help of Messrs. Brown and Hillman, of the Seed- Testing Laboratories; Mr. Shoemaker, of the Office of
Horticultural and Pomological Investigations; Messrs. Fairchild, Bisset, Skeels, Stuntz, and Rankin, of
the Office of Foreign Seed and Plant Introduction; Messrs. Coville and Blake, of the Office of Economic
and Systematic Botany; and Messrs. Swingle and Tanaka, of the Office of Crop Physiology and Breeding
Investigations. Prof. Trelease, of the University of Illinois, also kindly gave his advice. For valuable
assistance in connect ion with the chemical work appreciation is due to Mr. Burnett, formerly of the Oil
Fat, and Wax Labor atory ; to Mr. Gowen, formerly of the Baltimore Food and Drug Inspection Station
and especially to Mr. Bornmann, of the Chicago Food and Drug Inspection Station, all of the Bureau of
Chemistry, United States Department of Agriculture.
2 Reference is made by number (italic) to "Literature cited," p. 137-139.
Journal of Agricultural Research, Vol. XX, No. a
Washington, D. C. Oct. 15, 1920
ve Key No. E-13
("7)
1 1 8 Journal of A gricultural Research vol. xx, no. 2
CLASSIFICATION
IDENTIFICATION
While a preliminary study seemed sufficient to exclude the seed from
the group of true mustards,1 much difficulty was encountered in defi-
nitely identifying it. The material had evidently not been imported
before, at least not in recent years, nor could similar authentic material
be located in this country in any of the larger museums. Since the
information on the subject in the literature was contradictory, insuffi-
cient, or entirely lacking, extended studies were undertaken to determine
the macroscopic and microscopic characteristics of the seeds, as well as
the chemical composition and certain physiological characteristics of
the volatile oil. Plants were also grown to maturity, and the charac-
istics at the different stages of growth were determined. These experi-
ments were correlated with data in the literature, as a result of which
identification of the seeds as those of Chinese colza, Brassica campestris
chinoleifera, n. var., was made possible. It should be mentioned here
that Chinese colza was first classified by us (/, p. 469; 45; 46) as Brassica
campestris chinensis oleifera, n. f. Upon suggestion of Messrs. Blake and
Coville the name was changed to Brassica campestris chinoleifera, n. var.,
in order to avoid the use of a polynomial.
TAXONOMY
Some confusion exists concerning the nomenclature of Brassicas, the
description of them in many instances being inadequate. This is espe-
cially true of the oriental species, of which the seed in question is a repre-
sentative. Linnaeus (26, p. 281) described Brassica chinensis (PI. 19, A)
as a plant having stem-clasping leaves and slightly compressed siliques.
It is obviously of the Brassica campestris type (PI. 19, B).
Iinouma (18) described among other vegetables two plants which he
called, respectively, Aburana (oil vegetable) and Tona (Chinese vegetable).
Tanaka and Ono (18) identified Aburana as Brassica chinensis var., and
Tona as Brassica chinensis L.
Ito and Matsumura (19, p. 290-301) include Brassica chinensis L. and
Brassica orientalis Thunb. under the species Brassica campestris var.
chinensis T. Ito. Kondo (21, 22) evidently accepted this classification
and described Aburana, used for oil production, and (4) other forms,
used for greens, as Brassica campestris chinensis T. Ito. Makino (18) t
apparently unaware of Ito's classification or Kondo's earlier work,
identified both Aburana and Tona as Brassica campestris L. var. chinensis
Makino. According to Georgeson (16), Abura-na, Nutum-na, and Chiri-
men-na2 are Japanese names for Chinese cabbage, Brassica chinensis L.,
1 Mustard seed is the ripe seed of Sinapis alba L. (white mustard), Brassica nigra (L.) Koch (black mus-
tard), Brassica juncea (L.) Cosson, or the varieties or closely related species of the types of Brassica nigra
and Brassica juncea, for example, Brassica cernua Thunb. (42).
- Free translation according to Georgeson: Na means green; abura, oil; nutum, rape seed; and chirimen,
crape, referring to the crimped leaves of certain varieties.
Oct. 15, 1920 Mustard Seeds and Substitutes: I. Chinese Colza 119
which he, in agreement with Miquel (29, p. 74-75), considers identical
with Brassica orientalis Thunb. He states (16, p. 652):
No other vegetable of this class is so universally grown, or is represented by so many
varieties. It is a kind of rape which has been transformed by cultivation. Certain
varieties of it are grown only for their seed, from which an oil is expressed, formerly
much used as lamp oil.
Judging by the illustrations and the very brief description given by
these authors, and considering the great variations observed in plants
grown from Chinese colza seed, it appears quite probable that both
Aburana and Tona may be included in the series of plants treated as
Brassica campestris var. chinensis by Lund and Kiaerskou.
Lund and Kiaerskou (28, p. 166-167), who carried on extensive grow-
ing and crossing experiments, classify under the name Brassica campestris
var. sativa annua chinensis two forms of Chinese vegetables, Pe-tsai and
Pak-choi.
Prain (32, p. 42, 45) gave to Pak-choi (Chinese cabbage), which he
found growing on the Indian plains, the name Brassica chinensis L., in-
cluding in this species also the plants described under the following names :
B. chinensis L. var.; B. campestris Forbes and Hensl. in part, not of L.;
B. juncea Forbes and Hensl. in part, not of H. f. et. Th.; B. oleracea
L.var. chinensis Prain; Sinapis brassicata L. ; Pak-choi Vilmorin; Pak-
tsoi Roxb. ; Yea-tsoi Roxb.
Vilmorin (40, p. 491) classifies under Brassica chinensis L-, in addition
to Pakchoi and Pe-tsai, a third form of less cultural interest which has
almost entire leaves with narrow petioles.
Stuart (38, p. 73) classifies only Pe-tsai or Pai-tsai under Brassica
chinensis and states that it is a most common variety of Brassica oleracea.
He points out, however, that Yu-tsai,1 undoubtedly Brassica rapa, yielding
rape seed from which rape-seed oil is manufactured, is also called Brassica
chinensis, possibly on account of its economic prominence in China.
Bailey (3) refers to Pak-choi and Pe-tsai as two different species, calling
the first Brassica chinensis L., and the latter Brassica pe-tsai. He con-
siders that Linnaeus' description for Brassica chinensis answers best for
Pak-choi.
Gagnepain (15) has renamed Pe-tsai, classified by Loureiro (27, p. 400)
as Sinapis pekinensis, Brassica pekinensis (Lour.) Gagnepain. Skeels
(43, p. 21), evidently unaware of Gagnepain's classification, renamed the
same form Brassica pekinensis (Lour.) Skeels.
Duthie and Fuller (10, p. 33-34) give the name Brassica chinensis to a
plant with many characteristics of Brassica juncea, but they point out
that they consider Brassica chinensis Duthie and Fuller synonymous
with Sinapis chinensis L- The choice of the name Brassica chinensis is
unfortunate for a plant with characteristics of Sinapis chinensis L. and
apparently identical with or closely related to Brassica juncea (L.) Cosson.
1 Dr. Yamei Kin, familiar with China and its agricultural products, suggested that the material which the
authors considered as Chinese colza was Yu-tsai. However, since the seeds examined by the authors dif-
fered from samples obtained as Yu-tsai from China, it appears that this name is not definite.
1 20 Journal of Agricultural Research vol. xx, no. 2
TERMINOLOGY
SCIENTIFIC NAME
While it is believed that the plants grown from material in the Phar-
macognosy Laboratory (PI. 18, A), show characteristics typical of
the plant described by Linnaeus as Brassica chinensis, and while they
apparently agree rather well, so far as the general morphology of the plant
is concerned, with Pak-choi (PI. 15, C), there are certain differences,
especially in the seeds, from Pak-choi as well as Pe-tsai. The seeds of
Pak-choi and Pe-tsai were generally found to be smaller, more spherical,
and usually of a brown color. As a rule, they show even less marked
reticulations than the brown seeds of the authors' material. The most
striking differences observed in the plants is the lack of broad petioles
(see also Vilmorin's description of one form) and the failure to form
heads in the rosette stage, so strongly developed in Pak-choi and especially
in Pe-tsai (Pi. 15, A). These differences, however, while distinct, are not
so marked that they might not be considered to fall within the latitude of
species character. It would, therefore, seem that the laboratory material
might be classified as a variety of the type species Brassica chinensis L.
were it not for the following reasons. The description which Linnaeus
gives is very brief, in fact so brief that much of the confusion in the use
of this specific name by different authors is probably due to this limited
species description. Bailey points this out, giving still another instance
where the name Brassica chinensis has been used, evidently not correctly
(3, P- 54-3) '
It is impossible to determine whether this particular plant [Pak-choi cabbage] is
the one that Linnaeus meant to distinguish by his Brassica chinensis, but it best answers
the description in his Amoenitates (Vol. IV). In Linnaeus's herbarium is a Brassica
marked " chinensis " in his own handwriting, but it shows purple fls. and has lyrate-
lobed lvs. , whereas Linnaeus described his plant as having yellow fls. and cynoglossum-
like lvs., probably not the original.
Linnaeus's description, nevertheless, indicates the close relation to
Brassica campestris, and Lund and Kiaerskou showed this close relation by
classifying both Pak-choi and Pe-tsai as Brassica campestris var. annua
sativa chinensis.
Bailey (2, p. 188) takes a different stand :
In common with all members of the genus Brassica, or cabbage and mustard tribe,
these Chinese plants are much confused respecting their botanical characters. Recent
writers have referred all the Chinese cabbages to Brassica campestris, the rutabaga;
but one who studies the plants carefully, both from herbarium and living specimens,
can not long hold this opinion. The genus Brassica divides itself naturally into two
groups — the cabbages and rape, characterized by thick leaves, very glaucous-blue
herbage and long flowers which are creamy white, and the mustards, with thinner
and green or lightly glaucous herbage and small, bright yellow flowers. The Chinese
cabbages belong to this latter group rather than to the former. Their flowers are those
of the mustards, and I have no hesitation in removing the plants from Brassica cam-
pestris.
Oct. is, 1930 Mustard Seeds and Substitutes: I. Chinese Colza 121
He thus takes a different stand from all other botanists who have given
attention to these "Chinese cabbages and mustards." The authors of
this paper also disagree with Bailey 's viewpoint and classification on the
basis of a rather extended investigation reported in the following para-
graphs. There is no doubt in their minds that the so-called Chinese
cabbages are not mustards but belong to the colza group, Brassica cam-
pestris L.
Concerning Brassica campestris L., Prain (52) states:
From the standpoint of commerce it is a matter of supreme indifference whether
campestris, napus, and rapa be treated as separate species or subspecies of one and the
same species.
Consequently, in his systematic synopsis he proposes a number of
groups :
(1) Brassica oleracea, cabbage group; and (2) Brassica campestris Linn. ampl. Sub-
species A, campestris (sp. Linn.), representing the colza group, subspecies B, napus
(sp. L.) representing the rape group, and subspecies C, rapa (sp. L.) representing the
turnip group.
As to the close relationship of the respective forms, Bailey states (j,
p. 544) that he —
ound no difficulty in crossing cabbage-kale-cauliflower and others.
Lund and Kiaerskou, especially, showed by extensive crossing experi-
ments the close relationship of Brassica oleracea, Brassica campestris,
and Brassica napus. Notwithstanding this close relationship, however,
it appears necessary to go further than Lund and Kiaerskou (28) in the
classification of some of these forms, for instance in the classification of
Pak-choi and Pe-tsai. Bailey states (2, p. 180) —
there is even good reason for separating the two types of Chinese cabbage . . .
into two species, for they differ widely in their leaf characters and pods; and the former
[Brassica pe-tsai] is truly annual, while the latter [Brassica chinensis] is evidently
normally biennial.
Although the authors did not study these forms extensively, Shoemaker
has shown that they can be readily crossed (PI. 15, B) and therefore
should not be considered as having species character (4). It is at pres-
ent impossible to state definitely the relationship of Chinese colza to
these forms. Since it has greater similarity to Pak-choi than to Pe-tsai,
it appears not unlikely that Chinese colza and Pak-choi have developed
from one common stock. Pe-tsai may present a further modification
of Pak-choi, since, it is said, plants with narrower petioles may develop
from Pe-tsai seeds. Pending further collection of data on Pe-tsai and
Pak-choi the following classification, based on that of Bailey, Lund and
Kiaerskou, Gagnepain, and others, appears satisfactory for the separa-
tion and identification of these horicultural and oil-yielding forms :
1. Pak-choi, Brassica campestris chinensis T. Ito.
2. Pe-tsai, Brassica campestris pekinensis (Lour.) Viehoever.
3. Chinese colza, Brassica campestris chinoleifera Viehoever.
122 Journal of Agricultural Research vol. xx, No. >
This classification appears the more satisfactory, at least so far as
Chinese colza is concerned, since it indicates clearly the very close rela-
tionship to Indian colza, Brassica campestris var. glauca Watt. This
relationship is evident from botanical characteristics of the plant, and
especially from the morphological and anatomical characters, as well
as from chemical characters of the seeds of both Chinese and Indian
colza.
POPULAR NAME
The popular name "Chinese colza" has been selected on the basis of
the findings enumerated. Furthermore, it appears preferable to "China "
or "Chinese cabbage," names often used for similar seeds, especially for
Pe-tsai or other related horticultural varieties. Tracy (59, p. 603) states:
The Chinese cabbage of this country is a wholly different species from the common
cabbages. Chinese cabbage does not form a compact and rounded head. . . .
Georgeson (16, p. 652) states:
The term cabbage is a misnomer, as its resemblance to that vegetable is quite
remote. The plants are merely bunches of large, smooth, more or less spreading
leaves, with broad fleshy midribs. They do not bear their leaves on a well defined
stem, as do the cabbage, the kale, etc., but look more like the Cos lettuce, the leaves
having their origin at the surface of the ground.
Learning also that certain forms of Brassica oleracea, apparently
peculiar to China, are grown there, the authors felt that the name
"Chinese cabbage" could properly be applied only to those.
The authors' form, although rather closely related to Brassica oleracea,
is primarily an oil-yielding form which does not head and which deserves
the designation "cabbage" even less than Pe-tsai and Pak-choi, both
more or less heading forms. Some consideration was given the name
"Chinese yellow rape," as the seeds resemble rape seeds in a way and
yield a fixed oil similar to rape oils. In order to avoid confusion in
horticultural nomenclature and to protect the agriculturist, however, it
was considered advisable to adopt the more specific name of "Chinese
colza."
BOTANICAL STUDIES
DESCRIPTION OF SEEDS
The seeds (PI. 10, A, B) of Chinese colza, Brassica campestris chinoleifera
Viehoever, are yellow or brown, and, if immature, green in color. In
mass they have a dull yellow color, due to the preponderance of yellow
seeds. In form they are somewhat compressed, oval,, and usually with
distinct ridges on one side. The size varies from 1.4 to 2.6 mm. in the
long axis. The weight varies from 1.4 to 6.4 mgm., with an average
weight (based on 1,000 seeds) of 2.865 mgm. The weight of 500 mils
(quantity filling a 500-mil measure cylinder to the 500-mil mark) was
352 gm.
Oct. is, 1920 Mustard Seeds and Substitutes: I. Chinese Colza 123
The surface usually appears smooth (PI. 10, A, B) but under a hand
lens shows very weak reticulations on the yellow seed and more distinct,
but by no means prominent, reticulations on the brown seed (PI. 10, C, D).
In cross section under the microscope the epidermis of the seed coat
(PI. 10, E, F, a) is striated tangentially, does not show any cell structure
as in the mustard seed, and is about 5 microns thick. It does not swell
appreciably when moistened and does not show crosses with polarized
light. The sclerenchymatic palisade cells (PI. 10, E, F, c) vary more in
height in the brown seed than in the yellow. This explains the
presence of the more pronounced reticulations in the brown seeds.
For the yellow seed the height is almost uniformly 20 microns, while
the average for the brown seed is about 25 microns, with a maximum
height of 31 microns. The limits found for all the seeds were 15 to 31
microns high by 8 to 15 microns wide. The cell walls are strongly
thickened at the base and sides, and the inner walls are smooth. The
lumen contains no color substance. The parenchyma, always developed
to one or more rows in the Brassicas (40, p. 615), is compressed to such an
extent that it appears to be almost entirely lacking (PI. 10, E, F, b).
In Brassica nigra one row and in Sinapis alba two rows of parenchymal
cells are clearly visible, even in the mature seeds.
The parenchyma (PI. 10, E, F, d), located below the palisade cells,
consists mainly of one row of cells which in the yellow seeds contain
no color substance but in the brown seeds are filled with pigment. The
endosperm (e) is characterized by the protein layer, a row of cells usually
one cell wide, but occasionally two cells wide, the cells varying in height
from 15 to 21 microns and in width from 15 to 42 microns and contain-
ing protein masses. The tissue (/) located below this layer is composed
of several layers of parenchyma cells which, especially in the mature
seeds, are strongly compressed. The embryo consists of two cotyledons
folded in a characteristic way around the radical. The tissue is paren-
chymatic or meristematic. The cells which form the cotyledon tissues
are not characteristic except that they contain globules of fatty oil,
protein masses, and, especially in the immature state, a limited number
of small starch grains which range in size up to about 6 microns in
diameter. Experiments to locate the glucoside as a crystalline body
have been unsuccessful. Studies to locate the enzym and glucoside
microchemically in the cells are being undertaken.
DESCRIPTION OF THE PLANT
Experiments in the growth of selected yellow and brown seeds were
made under greenhouse and field conditions. The field experiments
were made at Arlington, Va., during the summer of 191 6, and at Yarrow,
Md., during the summer of 191 7. The laboratory records, so far as
differences in stages of growth are concerned, are more complete for the
plants grown in the greenhouse.
124 Journal of Agricultural Research vol. xx, no. 2
The plants in all stages of their growth were generally smooth, with
entire leaves. The young leaves, however, especially if grown in humid
atmosphere, were more or less hairy, mainly on the margins (PI. 11).
In older leaves hairs were observed only occasionally. It was noted
that isolated plants showed variations in the lobing, the leaves in some
instances being deeply notched (PI. 13). Experiments are being carried
on to determine the latitude and significance of these variations. The
appearance of some of these lobed leaves was very similar to that de-
scribed for Brassica napiformis Bailey (Sinapis juncea var. napiformis
Paill. and Bois), an observation which has much significance in view of
Bailey's statement (3) that —
it is nearly related to pak-choi, and it may have sprung from the same species; but
it is clearly distinguished by its sharply toothed lvs. . . .
In the early stages the cotyledons had the same general appearance
but were somewhat larger and thicker than those of the following mus-
tards, Brassica nigra (L.) Koch, Brassica juncea (L,.) Cosson, and Bras-
sica cernua Thunb.1 They were about 1 cm. long and 1 cm. broad,
exclusive of the petiole, and are heart-shaped and smooth (PI. 11). The
first leaves were obovate, variously toothed, and somewhat crenate,
and were hairy, especially on the margin if the seedling had been grown
in very humid atmosphere. The leaves had a long petiole and a mid vein
extending at least one-third of the length of the blade (PI. 11).
In the late rosette stage (PI. 14, 15) the leaves were arranged in a loose
cluster, the wings of the leaf extending along the greater portion of the
petiole, with the margin of the leaf more or less wavy and almost entire.
The time required for the development of the full-grown rosette stage
varies with the conditions for growth, being on the average about two
months when grown under normal conditions in the field and about
three months in the greenhouse. This period is materially shortened
when there are conditions decidedly unfavorable for growth, such as
insufficient nutriment, insufficient moisture, or too high temperature.
The early flowering stage (PI. 16, 17) is characterized by a few erect
branches up to 1 foot in length. The early stem leaves are similar to
the rosette leaves, being almost entire, and obovate with long petioles.
The upper stem leaves are variously stem-clasping, entire, somewhat glau-
cous and somewhat lanceolate acuminate. Many of the leaves of the
secondary stems are not stem-clasping. The mature plant reaches a
height of about 2 or 2l/2 feet, branching, and often showing an enlarged
stem base (PI. 16, A).
The flowers (PI. 17, B), which are somewhat larger than those of Bras-
sica nigra, B. juncea, and B. cernua, are in dense wide corymbs, \% inches
1 Plants of Sinapis alba need not be considered in the comparison, since they are distinctly different from
the other forms and can readily be recognized by such characters as the abundance of typical hairs on the
entire young plant, as well as on the later plants, especially the pods, which themselves are readily distin-
guished by their typical shape.
Oct. i5l 1920 Mustard Seeds and Substitutes: I. Chinese Colza 125
long and 2 inches across when the flowers are open, subsequently elonga-
ting into racemes 6 to 18 inches long, with pedicels % to 2>£ inches long
in the extreme, slender, and without bracts or bractlets. The long pedi-
cel particularly distinguishes the flower from the flowers of the mustards,
which rarely have pedicels longer than K inch (17). Otherwise the flowers
do not differ essentially from the general type of the genus Brassica.
The mature fruit pods (PI. 18, B) are 2-valved, and are 2 to 3 inches
long, including the beak. The beak of the pod is rather thickly conical
and from 0.4 to 0.8 inch long. The valves are convex, rigidly leathery,
rather finely nerved, and beaded opposite the seeds. A cross section
of the pod is broadly elliptical throughout the entire length and about
% inch thick across the long axis. In some of the pods both yellow and
brown seeds have been observed, giving evidence that the yellow and
brown seeds are only variations in the same kind of seed. An examina-
tion of plants grown from brown and yellow seed will also prove this
statement to be correct (PI. 12, A). The green seeds are immature, as is
indicated by the abundance of small spherical starch grains occurring
in the cotyledons. From 8 to 12 seeds are found under each valve of a
fully developed fruit pod.
BOTANICAL CONCLUSIONS
On the basis of the descriptive data given, the authors' material must
be classified with the colzas and rapes rather than with the true mustards.
While some of the characteristics observed would have only a limited
diagnostic value if taken alone, they serve as additional means for the
differentiation. Considered together, they make the proper classification
the more certain. The botanical characteristics may be briefly recapit-
ulated as follows.
SEEDS
1. As is typical of the colza group, the seeds are rather smooth. True
mustards, except Sinapis alba 1,., show generally a more pronounced
reticulation of the seed coat.
2. As in the case of Indian colza (Brassica campestris var. glauca), the
seeds are more or less flat. True mustards are generally spherical, except
Brassica besseriana Andrews, which has large brown seeds of more or less
oval shape. Many rapes and Brassicas other than mustard, however, are
also spherical.
3. A very pronounced ridge can be found in almost every seed of the
Chinese and Indian colza, while it is scarcely developed in the mustard
seeds, with the possible exception of Sinapis alba.
4. The swelling and polarizing epidermis is lacking in the Chinese
colza seed, as usually also in other seeds of the colza group. While not
so distinct or appreciable in certain forms or variaties of Brassica juncea,
the swelling of the mucilaginous epidermis and the polarization are
126 Journal of Agricultural Research voixx, No. a
especially pronounced in Brassica nigra, Brassica besseriana, and Sinapis
alba. Swelling, however, has been observed in cabbage seed, Brassica
oleracea bullata gemmifera (40, p. 615 and table).
5. The form and size of the palisade cells of the seed coat are similar
to those of the general type found in the colza group and differ more or
less strikingly from the true mustards.1
PLANTS
1. The tendency to rosette-like growth of plants in the early foliage
stage, great in plants belonging to the colza group, was also observed in
the authors' material. With the exception of certain variations of Brassica
juncea, the authors have not observed a similar tendency in mustard
plants.
2. The almost entire lack of hairs, especially pronounced in more
advanced plants, has been noted on the plants studied, as well as on other
plants of the colza group, a possible exception being Brassica rapa,
reported by Bailey. In contrast, the plants of mustards are more or less
distinctly hairy.
3. The upper leaves of the flower stalk are stem-clasping, as is general
in the colza group ; no distinctly stem-clasping leaves have been observed
in plants of true mustards.
4. The pedicels (stalks of the flowers) of Chinese, as well as those of
other colzas, average well over % inch in length, while those of the
mustard flowers average less than % inch:
5. The greater length of the pods of Chinese and other colzas, often
more than 2 inches, including the beak, frequently distinguishes them
from the mustards, which, as a rule, have shorter pods, averaging usually
less than 2 inches. Bailey (2), however, reports short pods for Pe-tsai.2
CHEMICAL STUDIES
GENERAL COMPOSITION OF SEEDS
The chemical studies included the general composition of a number
of samples of the seed, as well as a more detailed examination of the
fixed and volatile oils. Table I shows the composition of typical samples
of the seed.
Judging from the composition of the seed and the low amount and
character of the volatile oil yielded, the authors believe that the pressed
oil cake will be a very good feeding material.
1 For further details and comparison with other cruciferous seeds, the key given in Winton (5/, p. 173-180)
may be consulted.
2 For further information and comparison, see Bailey (j), Howard et al. (77), and textbooks on taxonomy.
Oct. is, 1920 Mustard Seeds and Substitutes: I. Chinese Colza
127
Table I. — Analyses of seeds of Chinese colza (Brassica campestris chinoleifera Vie-
hoever) l
1
Sam-
ple \ Moisture.
No.
Ash.
Ether
extract.2
Protein
(NX6.2S).
Reducing
substances
as starch
by acid hy-
drolysis.
Crude
fiber.
Volatile
oil (croto-
nyl isothio-
cyanate).3
Iodin No.
on ether
extract
(Hanus).
Per cent.
1 ( 4I2
I 4-II
Per cent.
8.64
8.62
5-51
5-45
Per cent.
39-89
40. 22
42.19
42. 10
Per cent.
22. 76
22. 76
24.08
24.78
Per cent.
11. 41
n-34
11. 26
11.29
Per cent.
3-94
3-86
3-83
3-93
Per cent.
o-43
•43
•57
•54
Per cent.
4.07
2 \ 407
99.8
99.8
99.4
99.6
1/ 4- 07
I 4-04
8-45
8.49
8.41
8-39
5-14
40.2s
40-30
40- 35
40.42
42.4
22. 72
22.85
22.89
22. 76
24-38
11.64
11. 71
11.70
11-65
3-99
4-33
3-96
4- 03
4.09
•49
.50
•47
.48
•52
, I/ 3-86
4 \\ 3-88
98.6
'Analyses of samples 1 to 4 were made by J. H. Bornmann, of the Chicago Food and Drug Inspection
Station, Bureau of Chemistry, United States Department of Agriculture. Analysis of sample s was made
by P. L. Gowen, formerly of the Baltimore Food and Drug Inspection Station, Bureau of Chemistry.
determinations of ether extract on two other samples, made by L. B. Burnett, formerly of the Oil, Fat,
and Wax Laboratory, Bureau of Chemistry, showed 48.65 and 31-40 per cent, respectively.
3 Analyses of samples 1 to 4 were made by the method of Vuillemin (50); analysis of sample 5 was made by
the method outlined in this paper (p. 128); other determinations were made by the method given in the
Official Methods of the Association of Official Agricultural Chemists. Wiley, H. W., ed. official and
PROVISIONAL METHODS OF ANALYSIS, ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS. As Compiled by
the committee on revision of methods. U. S. Dept. Agr. Bur. Chem. Bui. 107 (rev.), 272 p., 13 fig. 1908.
Reprinted imgi 2.
ISOLATION AND IDENTIFICATION OF VOLATILE OIL
A chemical investigation of the volatile oil was made in order to de-
termine whether it should be properly classified with the volatile oil
obtained from rape seed or with that of true mustard. The following
procedure was employed to isolate the volatile oil :
Two kgm. of the seed in the form of a No. 20 powder were placed in
an 8-liter flask; 4 kgm. of water were added; and the mixture was
allowed to macerate for two hours at about 37 ° C. The mixture was
then distilled with steam, and the distillate was saturated with salt and
extracted with ether. The ethereal solution was dried over anhydrous
sodium sulphate, and the greater part of the ether was distilled off, the
last portions being allowed to evaporate spontaneously.
The volatile oil thus obtained had a specific gravity of 0.960 at
25°/25° C., and the distilled oil had a boiling point of between 1650
and 1720 C. (uncorrected) at 754 mm. These findings agree fairly well
with those for crotonyl isothiocyanate, the volatile oil previously reported
in rape by Sjollema (35, 36). The thiourea and phenylt.hiourea deriva-
tives were prepared, and their melting points and nitrogen content were
determined. The results are shown in Table II.
Table II. — Physical constants of allyl and crotonyl isothiocyanate
Specific gravity.
Boiling
point.
Thiourea.
Pheny lthiourea .
Substance.
Melting
point.
Nit^en. ™£«
Nitrogen.
Allyl isothiocyanate
Crotonyl isothiocyanate .
Oil in question
I.OIjto1 I.020 (25°/25°
C.)
2-9933(ll74°C.)
.960 (25°/2S0C.)
"C.
1 148 to 154. ■
2 174 (approx-
imate).
165 to 170. . . .
"C.
74
= 64
64
Per cent.
2 24. 12
2 21. 25
20.74
•c.
3 98. 5
3 53
54 to S5
Per cent.
3 14- 74
' 13-60
13-20
1 U. S. P. IX (1916).
2 Sjollema (1901).
3 Stein (1907)-
128 Journal of Agricultural Research vol. xx, no. a
From these data it may be seen that the oil consists largely of crotonyl
isothiocyanate, which, since it is the chief constituent of volatile oil of
rape, corroborates the botanical findings that the seed is related to the
rapes and not to the mustards. It was noted that the crotonyl isothio-
cyanate did not have the odor of volatile mustard oil (allyl isothiocya-
nate) but had an odor suggestive of turnip or cabbage. Furthermore,
it did not have the typical irritating effect of mustard oil on the mucous
membrane of the nose and on the eyes nor a blistering effect on the skin.
DETERMINATION OE VOLATILE OIL IN MUSTARD SEED AND MUSTARD
SUBSTITUTES
In the course of this work it became necessary to determine the amount
of volatile oil yielded by different varieties of mustards and mustard
substitutes. Reference to the literature showed that there had been
marked variation in the methods followed by different analysts in the
determination of volatile mustard oils, especially in regard to the time
of maceration and the conditions for distillation. Wehrmann, Wegener,
Braunwarth, and Meyer (57) made an extended study of a number of
these methods in order to arrive at a quick, convenient method for the
determination of the volatile oil. In general, the studies here reported
have corroborated their findings, except with respect to the effect of
alcohol added before maceration (51 , p. 325). Carles (7, 8) has also con-
tributed valuable data to the solution of this problem. As a result of
these studies, the following method, based largely upon that of Gadamer
(13, 14), is recommended.
METHOD
Place 5 gm. of the ground seed (No. 20 powder) in a 200-mil flask, add 100 mils of
water, stopper tightly, and macerate for 2 hours at about 370 C. Then add 20 mils
of U. S. P. alcohol (95 per cent), and distill about 70 mils into a 100-mil volumetric
flask containing 10 mils of 10 per cent ammonium-hydroxid solution and 20 mils of
Njio silver nitrate solution. Mix thoroughly, stopper, and set the distillate aside
overnight, heat to boiling on a water bath (in order to agglomerate the precipitate),
cool, make up to 100 mils with water, and filter, rejecting the first portions. Acidify
50 mils of the filtrate with about 5 mils of concentrated nitric acid and titrate with
Njio ammonium thiocyanate, using 2 mils of 10 per cent ferric-ammonium-sulphate
solution for an indicator. Each mil of Njio silver nitrate consumed is equivalent to
0.004956 gm. of allyl isothiocyanate or 0.005657 gm. of crotonyl isothiocyanate.
The method is based on the hydrolysis of the glucoside by an enzym,
both present in the seed. A volatile oil, glucose, and potassium hydrogen
sulphate are formed.
^^OSO,OK ^OYL
C— S — "- C6Hu05 + H20 = C SH + KHS04 + C6H1206
\NCH5 \NCJL
Sinigrin
>^OH
C SH > CSN - C3H5 +H20
\NC3H5 •
Allyl isothiocyanate
Oct. is, 1920
Mustard Seeds and Substitutes: I. Chinese Colza 129
The volatile oil, readily volatile with the alcohol and water vapor,
reacts with ammonia and silver nitrate. In the case of allyl isothiocya-
nate (the true volatile mustard oil) mainly allyl thiourea (thiosinamin'*
is first formed.
^^NHC3H5
CSNC3H5 + NH3 = C=S
^^NH2
Allyl isothiocyanate Thiosinamin
This reacts slowly in the cold but is completely decomposed by heating
with silver nitrate, silver sulphid and cyanallylamid being formed.
/^NHC3H5 ^=NH
C=S + Ag20 = Ag2 S + C
\NH2 ^NCA + H.O
Thiosinamin Cyanallylamid
Both are insoluble compounds. They are filtered off, and the silver not
used up in the reaction is determined volumetrically after Volhard.
1 atom of silver =K molecule of the volatile mustard oil.
Other compounds may also be formed in small amounts during the
process (13, 25).
NOTES ON METHOD
Carles (7) suggests a smaller sample, 3 or 4 gm., in the case of par-
tially defatted samples or others yielding especially large amounts of
volatile oil.
The seed used for analysis should, if possible, be freshly ground, as the
powdered material loses its strength through hydrolysis, especially if not
kept thoroughly dry— at or below 7 per cent moisture, according to
Carles, not exceeding 2 per cent according to Boutron (5).
Joergensen (20, p. 9) ana van Kampen (44. P- 63) in testing rape-seed
cakes recommended the addition of thymol; Brioux (6. p. 262-263)
recommended the addition of sodium fluorid to the rape-seed cake
when this is macerated and tested for the amount of volatile oil available.
They found that bacterial action would thus be largely inhibited in the
maceration and higher yields would be obtained. Brioux used 2 gm. of
sodium fluorid for 25 gm. of cake and 500 mils of water; van Kampen
used 10 mils of 1 per cent alcoholic thymol solution added to either 25
gm. of cake or .5 gm. of mustard seed and 300 mils of water. Joergensen
used a 1 per cent alcoholic solution of thymol and also in other experi-
ments mercuric chlorid, which, however, proved unsatisfactory. Raquet
(33) macerated the material in an aqueous alcoholic solution, adding 15
cc. of alcohol to the mixture before and 5 cc. after maceration, thus
obtaining seemingly higher yields. We (47) could verify his findings
but are still undecided whether this higher result is due to the formation
of other volatile reducing substances or, as Raauet claims, to the fact
130 Journal of Agricultural Research vol. xx, no. 2
that in the presence of alcohol during maceration no bacterial fermen-
tation causing a loss of volatile oil takes place.
A glycerin bath may be used to secure greater uniformitv in heating.
The use of ground glass joints in the distilling apparatus has been rec-
ommended in literature in order to avoid possible errors caused by the
use of rubber stoppers.
To insure complete absorption of the volatile oil, the tip of the con-
denser should always dip below the surface of the liquid in the receiving
flask. It is advantageous to have the condenser terminate in a tube of
small bore. A second receiver, containing ammonia and silver solution,
may be used in case the completion of the distillation is in doubt.
According to Kuntze {25), the mixture obtained after distillation may
also be heated directly without standing to ioo° C. for an hour, using
a reflux condenser or a long glass tube as a condenser. Possible further
formation of an urethane compound (allyl urethane in the case of allyl
mustard oil) can thus be avoided.
YIELD OF VOLATILE OIL
Examination by the method just given of a number of samples of seeds
obtained from Chinese colza showed that the content of crotonyl isothio-
cyanate varies from about 0.4 to 0.6 per cent. Various attempts were
made to increase the yield of volatile oil by addition of different chemicals
before maceration. Table III shows the results of these experiments.
Table III. — Effects of chemicals added before maceration upon the yield of volatile oil
.
Volatile oil
Substance added to 5 gin. of sample. (crotonyl iso-
thiocyanate).
Potassium hydroxid, 0.2 gm
Potassium fluorid, 0.2 gm
Tartaric acid, 0.2 gm
Tartaric acid, 5 gm
Tartaric acid, reflux 2 hours, 5 gm
Sinapis alba, as a source of myrosin, 5 gm
Alcohol, 20 cc
No chemical
Per cent
o
The results shown in Table III may be summarized as follows: Five
gm. of tartaric acid probably destroyed the enzym and no appreciable
yield of volatile oil was obtained; 0.2 gm. of tartaric acid slightly retarded
Oct. 15, 1920 Mustard Seeds and Substitutes: I. Chinese Colza 131
the reaction. Water alone gave results averaging about 0.6 per cent of
crotonyl isothiocyanate. The addition of 20 cc. of alcohol before the mac-
eration gave a higher percentage of volatile oil, the results reaching
almost 0.8 per cent. The formation of some allyl thiocyanate (34, p. 832) ,
allyl cyanid (13), and carbon bisulphid during the fermentation process of
sinigrin has been observed in experiments where no alcohol was present.
Other products may be formed in its presence and must be expected,
especially in the authors' material, where no sinigrin but another gluco-
side is present. It has been pointed out by Kuntze (25) that side re-
actions can occur between allyl isothiocyanate and alcohol with the
formation of allyl thiourethane, and it may be presumed that a similar
reaction might take place between crotonyl isothiocyanate and alcohol,
which will lead to erroneous but probably lower results. At the present
time the data obtained are insufficient to ascribe the discrepancy to any
of these causes, nor is it yet known whether, in the presence of alcohol,
potassium hydrogen sulphate has also an injurious effect upon the for-
mation of mustard oil through rendering myrosin largely ineffective by
causing its coagulation (12). It is believed, however, that the maximum
yield was obtained, since, even in the presence of large amounts of en-
zym (white mustard added), higher yields were not secured.
Shaking the maceration mixture at room temperature at intervals of
five minutes did not hasten the reaction sufficiently to give the total
amount of volatile oil in two hours.
It has been pointed out by Forster (11) that in the preparation of rape-
seed cake the material is heated to about 700 C, and that when it is so
treated a high yield of oil is obtained. In these experiments the authors
were unable to verify Forster's results. In order to see whether there
was any difference in the yield of oil from the brown and yellow seeds, a
separation of the two was made, and determinations were made on the
separated samples with the results (calculated as crotonyl isothiocyanate)
shown in Table IV.
Table IV. — Relative yield of volatile oil by brown and yellow seeds of Chinese colza.
Treatment.
Yield of volatile oil.
Brown
seed.
Yellow
seed.
I Per cent. Per cent.
2 hours' maceration at 370 C : o. 58 o. 55
% hour's heating at 700 C, followed by 2 hours' maceration at !
37° C I -44 ! -46
It will be observed that in both the brown and yellow seed a lower
yield was obtained by the preliminary heating at 700 C. for % hour.
187932°— 20 4
132 Journal of Agricultural Research vol. xx. No. »
CHARACTER OF FIXED OIL, AND CERTAIN OF ITS CONSTITUENTS
It has been reported that the seed of Chinese colza is used in China for
its fixed oil, and such use has also recently been made of the seed im-
ported into this country. A study of the fixed oil showed that it was
present in very large amounts, up to 50 per cent or more, and that its
composition was similar to that of rape oils. It was a light yellow oil
and apparently was of an excellent quality. The oil expressed from the
seeds showed the characteristics given in Table V.
Table V. — Characteristics of fixed Chinese colza oil l
Density, 250 C
Iodin No. (Hanus)
Saponification No
Percentage of insoluble acids and unsaponifiable matter
Percentage of soluble acids
Neutralization value of insoluble acids
Mean molecular weight of insoluble acids
Refractive index, 250 C
Iodin No. insoluble acids
Percentage of solid acids
Percentage of liquid acids
Iodin No. solid acids
0. 9097
100.3
173-8
96. 1
.07
172. 6
325-0
1. 4695
104. o
19-52
75.82
55-2i
'Analysis by L. B. Burnett, formerly of the Oil, Fat, and Wax Laboratory, Bureau of Chemistry
United States Department of Agriculture.
CHEMICAL CONCLUSIONS
i. The volatile oil yielded by Chinese colza has been identified as cro-
tonyl isothiocyanate, an oil formerly found in rape. Crotonyl isothio-
cyanate is slightly lighter and allyl isothiocyanate slightly heavier than
water. The boiling points of the oils and other physical constants re-
ferred to in Table II permit ready differentiation.
2. The yield of volatile oil varied from about 0.4 to 0.6 per cent, while
true mustards, with the exception of Sinapis alba, yielded from about
0.7 per cent up to more than 1 per cent of volatile oil of different com-
position.
3. The fixed or fatty oil expressed from the seed showed the general
characteristics of rape oils, these being slightly different from the fixed
mustard oils. The iodin value, for instance, was about 100 or below
in case of rape oils and the Chinese colza oil, while it was above 100 in
the case of oil expressed from different mustard species. These oils
were obtained under similar conditions and were unrefined. Methods of
refining may change the iodin value.
4. The yield of fixed oil varied from about 40 to 50 per cent, whereas
the true mustards examined usually contained less and rarely, if ever,
more than 40 per cent of a fixed oil.
Oct. iS> 1920 Mustard Seeds and Substitutes: I. Chinese Colza 133
PHYSIOLOGICAL DATA
GENERAL PHYSIOLOGICAL CHARACTERISTICS
Chinese colza seed when chewed has an earthy and slightly pungent
taste, the flavor being suggestive of cabbage or turnip rather than of
mustard. When a few grams of the freshly triturated seed macerated
with water have stood in a closed vessel at room temperature for a few
minutes, the odor of the volatile oil formed may readily be noted. This
odor gradually becomes weaker, however, and after the mixture has
stood for 24 hours, more or less, the odor is largely gone and is often
replaced by an odor of hydrogen sulphid. The mustards (except white
mustard) give the characteristic mustard oil flavor and irritating sensa-
tion to the membranes of nose and eyes much more strongly and for a
longer period. In fact, while the vapor of true mustard oil, even in
very small amounts, causes great discomfort to eyes, nose, and lungs,
the effect of the vapor of crotonyl isothiocyanate was by no means to
be compared in intensity and hardly in character.
The ground seed, with small amounts of water added, was applied in
the form of a plaster to the skin of the arm. No more than a reddening
was caused after 2 or more hours of application; no blistering whatsoever
was noted, and the reddening soon disappeared. It was necessary to
remove plasters prepared similarly with true mustards after shorter
application, and blisters were left. When applied to the skin the
isolated volatile oil itself caused only a burning sensation and a tempo-
rary reddening.
PHARMACOLOGICAL ACTION
While no pharmacological experiments were made in this investiga-
tion, those of Sjollema and others may be briefly mentioned. Sjollema
(35> P- 3I5) gave 0.212 gm. of the oil (crotonyl isothiocyanate isolated
from rape) to a rabbit in the form of an emulsion but observed no abnor-
mal symptoms. After about three hours the animal began to eat again,
appeared entirely normal, and lived. Allyl mustard oil, isolated from
black mustard, given in the same amounts and under comparable con-
ditions, caused death to a rabbit within a few hours. The experiments
were repeated with the same result. Stein (37) confirms Sjollema's find-
ings in general upon the basis of a larger series of experiments, also with
rabbits. He concludes that while the symptoms of poisoning are in a
way the same as those caused by allyl isothiocyanate, the general toxic
(resorptive) as well as the locally cauterizing properties are far less pro-
nounced in the case of crotonyl isothiocyanate. The toxic dosis may be
estimated to be 0.5 gm. of crotonyl, against less than 0.1 gm. of allyl
isothiocyanate per kilo body weight.
He experimented also with a goat (24 kgm. in weight) adding to the
feed of ground potato and sugar beets the following amounts of crotonyl
134 Journal of Agricultural Research vol. xx, no. t
isothiocyanate : i cc. the first day, 1.5 cc. the second day, and 2 cc. the
third day. Except for an increased urine excretion, no disturbance was
observed; the urine was free from albumen. In experimenting with
cattle, Moussu (30) used the pure oil of allyl isothiocyanate as well as
cakes containing crotonyl isothiocyanate. Concerning allyl isothio-
cyanate given internally, he concludes that it is very toxic and may cause
in doses of 2 gm. per 100 kilos speedy death, with the symptoms of an
acute inflammation of the intestines.
No injury was observed from feeding large and varying amounts of
rape-seed cakes, either dry or mixed with water, though cows of different
ages were fed with the cakes which, according to Brioux (6), contained
over % per cent of crotonyl isothiocyanate. One old cow, not in milking
condition, weighing about 400 kgm., was fed in four periods of five days
each the amount of 1 kgm. of cake per day, increasing to 3 kgm. per day.
Apparently no injury was caused, although the cow received in the final
period about 17 gm. of crotonyl isothiocyanate in the form of a cake,
yielding 0.57 per cent of this oil. Brioux, on the basis of Moussu's
experiments, concludes that allyl isothiocyanate is six or seven times as
toxic as crotonyl isothiocyanate. He pointed however, to the fact
that Moussu fed the volatile oil of allyl isothiocyanate directly, while
previous authors, using the mustard cake, could feed without causing
injury to the animal amounts which contained decidedly larger quanti-
ties of volatile mustard oil. For other experiments with allyl mustard
oil see Carlier (9).
BACTERICIDAL ACTION
The bactericidal effect, so strong in the case of allyl mustard oil and
so essential for the keeping qualities of prepared mustard, as Kossowicz
(24, p. 329) and others have pointed out, is lacking or very weak so far
as crotonyl isothiocyanate is concerned. Stein made the following inter-
esting experiments.
To a number of test tubes containing 10 cc. of raw milk a small amount
(on point of knife) of liver of sulphur and also increasing but definite
amounts of allyl isothiocyanate and crotonyl isothiocyanate, respec-
tively, were added. A test paper, moistened with lead salt solution,
was fastened in the opening of the tube. The opening was then closed
with cotton, and the tubes were set aside at from 380 to 400 C. for 24
hours. The blackening of the lead paper, caused by the bacterial
formation of hydrogen sulphid and subsequent formation of lead sulphid,
indicated in which experiments bacterial activity was not inhibited by
the addition of either of the volatile oils. His results are given in
Table VI.
The far greater bactericidal effect of allyl mustard oil is clearly evident.
Further interesting data on the bactericidal action of allyl mustard oil
and the amounts which prevent the growth of bacteria or yeasts belonging
to different species are reported by Kossowicz (23, p. 149-161).
Oct. 15, 1920
Mustard Seeds and Substitutes: I. Chinese Colza
135
Table VI. — Bactericidal action of ally I isothiocyanate and crotonyl isothiocyanate
Allyl mustard oil-
Crotonyl mustard oil.
Number of
drops.1
Intensity of
blackening.
Number of
drops.1
Intensity of
blackening.
1/20
1/10
1/5
1/3
1/2
I
IO
IO
8
I
O
O
1/20
1/10
1/5
1/3
1/2
I
2
3
4
6
8
10
IO
IO
IO
IO
IO
IO
IO
5
5
3
2
0
1 One drop= 0.05 cc.
Certain manufacturers of prepared mustard who unwittingly used the
Chinese colza seed as mustard in the usual proportions in preparing their
product noted extensive spoilage within a short time. The deficiency of
crotonyl isothiocyanate with respect to its bactericidal action is thus also
demonstrated in a practical and very impressive way.
PHYSIOLOGICAL CONCLUSIONS
Crotonyl isothiocyanate differs distinctly from true volatile mustard
oil (allyl isothiocyanate). Its flavor resembles that of cabbage or turnip
instead of that of onion. No appreciable effect on the eye or the mucous
membrane of the nose and no blistering effect on the skin were noted,
in contrast to such and other effects of allyl isothiocyanate. Crotony
isothiocyanate lacks also the pronounced bactericidal qualities of true
volatile mustard oil. Moreover, crotonyl isothiocyanate is distinctly dif-
ferent from the nonvolatile mustard oil, para-oxybenzyl isothiocyanate,
of white mustard, which has no odor but has a strong biting taste and a
strong blistering effect on the skin.
SUMMARY
Material imported as rape seed and sold as mustard seed was identified
as Chinese colza, Brassica campestris chinoleifera Viehoever, n. var.
The characteristics of the seed have been established, and those which
permit the identification and differentiation from true mustard seed have
been pointed out.
Plants have been grown from the seed, and the characteristics have
been established, especially with reference to their close relationship to
the colza group, Brassica campestris.
The volatile oil obtained from the material was identified as crotonyl
isothiocyanate, which is not a suitable substitute for mustard oil, in
respect to condimental, bactericidal, or medicinal value.
136 Journal of Agricultural Research vol. xx, no. 2
The fixed oil proved to be of the general composition of the rape oils,
and the quantity of the oil present, amounting to more than 40 per cent,
characterized the seed as a very valuable oil seed.
On the basis of the general composition of the seed and the character
of the volatile oil, it is suggested that the pressed oil cake may well be
used as a stock feed.
The leaves are succulent and should be of value as greens.
The plants, which are very vigorous and apparently hardy, seem to
offer possibilities as a forage crop. Experiments along this line have
been undertaken in cooperation with the Bureau of Plant Industry.
CONDENSED DESCRIPTION OF CHINESE COLZA (BRASSICA CAMPESTRIS
CHINOLEIFERA VIEHOEVER)
Basal or radical leaves first single, later numerous, arranged in cluster,
large glossy green, usually smooth, obovate or round, obovate in general
outline, entire or obscurely wavy, variously toothed, sometimes crenate,
tapering into a distinct thin petiole, which is more or less margined,
showing sometimes a few leaflike lobes.
Leaves of flowering stem more or less glaucous, clasping, obovate,
oblong, or somewhat lanceolate acuminate; leaves of secondary stem
not always clasping.
Flowers light yellow, of medium size (generally that of mustard flowers),
pedicels averaging well over % inch.
Pods rather large and long, tapering into conical beak (0.4 to 0.8 inch
long) ; pod and beak together from 2 to 3 inches long ; from 8 to 1 2 seeds
in pod.
Seeds yellow and brown, yellow greatly predominating; somewhat
compressed, oval, usually distinct ridge on ventral side, usually smooth
brown, slightly reticulated, varying in size (from 1.69 to 2.07 mm.),
weighing from 1.4 to 6.4 mgm. (1,000 seeds weighed 2.865 gm- and 500 cc.
weighed 352 gm.).
Seed coat. — Epidermis about 5 microns thick ; when it is moistened
shows no swelling, no polarization of light, or cell structure. Paren-
chyma almost completely compressed. Sclerenchyma (palisade cells),
from 15 to 31 microns high and from 8 to 15 wide, strongly thickened at
base and side, without pigment, inner wall smooth. Pigment layer
consists of one row of cells containing pigment only in brown seeds.
Protein layer is formed usually by one row of large cells (from 15 to 21
microns high and from 15 to 42 microns wide).
Seed. — Composition averages as follows: Over 40 per cent fatty oil
(colza or rape oil type); about 23 per cent protein (N = 6.25); 11.5 per
cent reducing substances ; 4 per cent crude fiber; 0.5 per cent by hydroly-
sis of volatile oil consisting of crotonyl isothiocyanate.
Oct. i5> 1920 Mustard Seeds and Substitutes: I. Chinese Colza 137
LITERATURE CITED
(1) Alsberg, Carl L., ViehoevER, Arno, and Ewing, Clare Olin.
1919. SOME EFFECTS OF THE WAR UPON CRUDE DRUG IMPORTATIONS. In Jour.
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(2) Bailey, L. H.
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(3)
1914. brassica. In his Standard Cyclopedia of Horticulture, v. 1, p.
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(4) Biological Society of Washington.
1919. what kind of characteristics distinguish a species from a sub-
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(5) BOUTRON.
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(6) Brioux, Ch.
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(8)
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(13)
1897. UBER DIE BESTANDTEH.E DES SCHWARZEN UND DES WEISSEN SENF-
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(14)
1899. PRUFUNG DES SENFOLES UND DES SENFSPIRITUS. In Arch. Pharm.,
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1917. JAPANESE MUSTARDS; TRANSLATIONS FROM "O MOKU DZU SETSU, BY Y.
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(19) Ito, Tokutaro, and Matsumura, J.
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Japan, v. 12, pt. 4, p. 263-541.
(20) Jorgensen, Gunner.
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Landw. Vers. Sta., Bd. 72, Heft 1/2, p. 1-14.
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(22)
1917. UNTERSUCHUNG DER SAMEN DER IN JAPAN VERTRETENEN BRASSICA-
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(24) —
1914. LEHRBUCH DER CHEMIE, BAKTERIOLOGIE UND TECHNOLOGIE DER NAH-
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(26) Linne, Carl von.
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(27) Loureiro, Juan de.
1793. FLORA cochinchinENSis ... v. i. Berolini.
(28) Lund, Sams0e, and Kiaerskou, Hjalmar.
1884. EN monografisk skildring af havekaalens, rybsens og rapsens
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(29) MiquEL, F. A. Guil.
1866-1867. prolusio florae iaponicae. viii, 392 p., 2 pi. Amstelodami.
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(31) pharmacopoeia of the united STATES of America. 9th decennial revision . . -
1916. Official from September 1, 1916. lxxx, 728 p. Philadelphia.
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1898. A NOTE ON THE MUSTARDS CULTIVATED IN BENGAL. In Agr. Ledger, V. 5
no. 1, p. 1-78, pi. 1-10, maps 1-2. Reprinted from Dept. Land Rec.
and Agr. Bengal Bui. 4, 78 p., 10 pi., 2 maps.
Oct. is, 1920 Mustard Seeds and Substitutes: I. Chinese Colza 139
Raquet, d.
1912 . DOSAGE DE L'ALLYLSENEVOL DANS LA FARINE DE MOUTARDE. In Rupert.
Pharm., s. 3, t. 24, no. 4, p. 145-148.
Schmidt, Ernst.
1910. AUSFUHRLICHES LEHRBUCH DER PHARMACEUTISCHEN CHEMIE. Aufl. 5,
Bd. 2, Abt. 1. Braunschweig.
SjOLLEMA, B.
1900. ENTWICKELUNG UND SCHADLICHE WIRKUNG VON SENFOL AUS RAPSKUCH-
EN. In Landw. Vers. Sta., Bd. 54, Heft 3/4, p. 311-318.
1901. l'isosulfocyanat'e des graines de brassica napus. In Rec. Trav,
Chim. Pays-Bas et Belg., t. 20 (s. 2, t. 5), no. 3, p. 237-242.
Stein, E. H.
1907. UEBER DIE GIFTIGKEIT INDISCHER RUBKUCHEN ... 32 p. Berlin
Stuart, G. A.
1911. Chinese materia medica; vegetable kingdom. . . 5<;8p. Shanghai
Tracy, W. W.
1914. Cabbage. In Bailey, L,.H.,ed. Standard Cyclopedia of Horticulture .
v. 2, pp. 603-608. New York.
Tschirch, A.
1905. KLEINE beitrage zur pharmakobotanik und pharmakochemie.
In Schweiz. Wchnschr. Chem. u. Pharm., Jahrg. 43, No. 45, p. 614-618.
U. S. Department of Agriculture. Bureau of Chemistry.
1915. USE OF SINAPIS (BRASSICA) CERNUA IN MUSTARD PREPARATIONS. In
U. S. Dept. Agr. Bur. Chem. Serv. and Reg. Announc. no. 14, p. 12.
1917. MUSTARD SEED STANDARD AND ASSAY METHOD. In U. S. Dept. Agr.
Bur. Chem. Serv. and Reg. Announc. no. 20, p. 58-59.
Bureau of Plant Industry.
(33
(34
(35
(36
(37
(38
(39
(40
(4i
(42
(43
(44
(45
(46
(47
1920. report ON medicinal plants. In Jour. Assoc. Offic. Agr. Chemists,
v. 3, no. 3, p. 381-386.
(48) Ewing, C. O., and Clevenger, J. F.
1917. studies on mustards and mustard substitutes. In Science, n. s.
v. 46, no. 1196, p. 545-546.
(49) Vilmorin-Andrieux, et CompagniE.
1904. LES plantes potagErES . . . ed. 3, xx, 804 p. Paris.
(50) VurLLEMiN, Armand.
1904. beitrage zur kenntnis der senfsamen ... 95 p., 2 pi. Zurich.
(51) Wehrmann, Fr., Wegener, K., Braunwarth, Fr. H., and Meyer, K.
1915. VERGLEICHENDE UNTERSUCHEN UBER DIE WERTBESTIMMUNG VON
SEMEN SINAPIS, SPIRITUS SINAPIS, OLEUM SINAPIS AND CHARTA SINAPI-
SATA NACH DEN VERSCHIEDENEN DAFUR AUSGEGEBENEN MATHODEN.
In Arch. Pharm., Bd. 253, Heft 4, p. 306-320; Heft 5, p. 321-327.
(52) Winton, A. L., Moeller, Josef, and Winton, Kate Barber.
1916. MICROSCOPY OF VEGETABLE FOODS . . . ed. 2, xiv, 701 p., 635 illUS.
New York. General bibliography, p. 671-674.
1909. SEEDS AND PLANTS IMPORTED DURING THE PERIOD FROM OCTOBER I TO
DECEMBER 31, 1908. U. S. Dept. Agr. Bur. Plant Indus. Bui. 153, 58p.
Van Kampen, Ir. G. B.
1917. de stand van het mosterdolievraagstuk. In Verslag. Landbouwk,
Onderzoek. Rijkslandb. Proefssta. no. 20, p. 53-70.
ViEHOEVER, Arno.
1919. Chinese colza — a valuable new oilseed. In Oil, Paint and Drug
Reporter, v. 96, no. 10, p. 53.
1919. THE PHARMACOGNOSY LABORATORY, ITS ACTIVITIES AND AIMS. In Jour.
Amer. Pharm. Assoc, v. 8, no. 9, p. 717-724.
PLATE 10
A. — Yellow seed of Chinese colza. Approximately X 5.
B. — Brown seed of Chinese colza. Approximately X 5.
C. — Surface section of yellow seed of Chinese colza, showing lack of reticulations.
Approximately X 103.
D. — Surface section of brown seed of Chinese colza, showing reticulations. Ap-
proximately X 103.
E. — Cross section of yellow seed of Chinese colza. Approximately X 289.
F. — Cross section of brown seed of Chinese colza. Approximately X 289.
E and F show the following :
(a) Tangentially striated epidermis.
(b) Almost obliterated parenchyma.
(c) Sclerenchymatic palisade cells.
(d) Parenchyma, which in the brown seed (F) contains a pigment.
(e) Protein layer of the endosperm.
(t) Compressed parenchyma of the endosperm.
(140)
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 10
Journal of Agricultural Research
Vol. XX, No. 2
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 1 1
Journal of Agricultural Research
Vol. XX, No. 2
PLATE ii
Seedling of Chinese colza, showing cotyledons and young leaves. The leaves show
hairs, especially on the margin. Twenty-three days old. Grown in greenhouse.
Planted March 23, 1917.
PLATE 12
Early rosette stage of Chinese colza seedling:
A. — Plants from (i) brown seed and (2) yellow seeds. Three weeksold. Grown in
greenhouse. Planted March 14, 191 7.
B. — Usual form, showing almost entire leaves. Three monthsold. Grown in green-
house. Planted January 20, 1017.
Mustard Seeds and Substitutes: I.Chinese Colza
Plate 12
Journal of Agricultural Research
Vol. XX, No. 2
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 13
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 13
Early rosette stage of Chinese colza seedling:
A. — Plant showing a variation in lobing of the leaves. Two months old. Grown
in greenhouse. Planted February 20, 1917.
B. — Plant showing a variation in lobing of the leaves. Three months old. Grown
in greenhouse. Planted February 20, 1917.
PLATE 14
Late rosette stage of Chinese colza seedling:
A. — Usual form. Three and one-half months old. Grown in greenhouse. Planted
September 27, 1916.
B. — Plant showing a variation in lobing of the leaves. Two months old. Grown
in field at Yarrow Station, Md. Planted May 16, 1917.
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 14
Journal of Agricultural Research
Vol. XX, No. 2
lustard Seeds and Substitutes: I. Chinese Colza PLATE 15
;
Journal of Agricultural Research
PLATE is
Late rosette stage of Chinese colza seedling:
A. — Pe-tsai. Grown in field at Arlington, Va., by D. N. Shoemaker. The rule is
17X inches in length.
B. — Cross between Pak-choi and Pe-tsai. Grown in field at Arlington, Va., by D.
N. Shoemaker. The rule is 15 inches in length.
C. — Pak-choi. Grown in field at Arlington, Va., by D. N. Shoemaker. The
marked portion of the rule is 12 inches in length.
PLATE 1 6
Early flowering stage of Chinese colza:
A. — Usual form, showing somewhat enlarged stem base and stem-clasping leaves.
Almost 5 months old. Grown in greenhouse. Planted September 27, 1916.
B: — Plant without enlarged stem base. Almost 5 months old. Grown in green-
house. Planted February 20, 1917. The rule is 5 cm. in length.
C. — Usual form, showing glaucous leaves. Two months old. Grown in field at
Arlington, Va. Planted about May 1, 1916.
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 16
Journal of Agricultural Research
Vol. XX, No. 2
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 17
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 17
Early flowering stage of Chinese colza:
A. — Usual form, showing luxuriant growth and long pedicels. Two and one -halt
months old. Grown in field at Yarrow Station, Md. Planted May 16, 1917.
B. — Flower cluster. Plant about 5 months old. Grown in greenhouse. Planted
October 4, 1916. Natural size.
187932°— 20 5
PLATE 18
A. — Fruiting stage of Chinese colza. Plant about 3 months old. Grown in field
at Arlington, Va. Planted May 1, 1916.
B. — Mature fruit of Chinese colza. • From a plant 7 months old . Grown in green-
house. Planted October 4, 1916. Natural size.
Mustard Seeds and Substitutes: I. Chinese Colza
Plate 18
Journal of Agricultural Research
Vol. XX, No. 2
Mustard Seeds and Substitutes: I.Chinese Colza
Plate 19
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 19
A.— Herbarium specimen of Brassica chinensis h. Approximately X Vs*
B.— Herbarium specimen of Brassica campeslris. Approximately X ll&.
STUDY OF SOME POULTRY FEED MIXTURES WITH
REFERENCE TO THEIR POTENTIAL ACIDITY AND
THEIR POTENTIAL ALKALINITY: I1
By B. F. Kaupp, Investigator and Pathologist, and J. E. IvEy, Assistant in Poultry
Husbandry, Research Laboratory of the Office of Poultry Investigations and Pathology,
North Carolina Agricultural Experiment Station
HISTORICAL REVIEW
Interest in the acid-base balance of dietaries has increased greatly in
recent years. Sherman and his collaborators pointed out the basis for
work of this kind when they made more accurate determinations than
had hitherto been available of the ash constituents of the common feeds.
Sherman 2 has shown that meats and cereals have a preponderance of
acid-forming elements, whereas, on the other hand, fruits and vegetables
have an excess of base-forming elements.
It has been shown that ash has an influence on the reaction of the
urine. Acid-forming feeds lead to the formation of more acid urines,
and base-forming feeds cause the excretion of less acid or of alkaline
urines. However, it has been found in studies carried on with men that
certain exceptions were found — namely, plums, prunes, and cranberries,
which, although yielding a basic ash, nevertheless increase the acid
excretion because of the presence of benzoic acid, excreted as hippuric
acid.3
Although the question whether or not an acid-forming diet eaten for
some period of time is productive of undesirable results is debatable,
probably the concensus of opinion is in favor of diets in which the acid-
forming and base-forming elements are approximately balanced. The
possibility that the continued use of acid-forming diets may lead to a
greater susceptibility to disease of the less infectious type has seemed
worthy of investigation.4 Work along this line is in progress in this
laboratory. Sour milk and buttermilk function as base because when
used by the body the lactic acid is oxidized to carbonic acid, which is
thrown off by the lungs, leaving a base residue of mineral salts. A
common defect is the use of quantities of proteins and fats far in excess
of the needs of the body. Proteins and fats are relatively expensive
materials.
> This paper deals with the flocks for the first 24 weeks. Part II will deal with the first laying year.
s Sherman, Henry C food products, ix, 594 p., illus. New York, 1919.
8 Blatherwick, N. R. the specific role of foods in relation to the composition op thr urine.
In Arch. Int. Med., v. 14, p. 409-450. 1914.
'Htndhede, M. protein and nutrition ... p. 8. London, 1913.
Journal of Agricultural Research, Vol. XX, No. 2
Washington. D. C Oct. 15, 1920
vf Key No. N. C-14
(HI)
142 Journal of Agricultural Research voi.xx. No. 2
THE PROBLEM
Profitable production of broilers begins with the baby chicks and
extends over a period of about eight weeks, at the end of which time
the birds should weigh, as a flock, approximately 1% pounds each.
Our problem consisted of two parts:
1. To ascertain the mineral content of poultry feeds and, from this as
a basis, to determine the potential acidity and potential alkalinity of
these feeds.
2. To determine the acid-base balance of the feed mixtures used in
our experimental feeding work in the production of broilers, giving some
of the feeding results.
EXPERIMENTAL METHODS
The baby chicks were produced from a single flock of pure-bred
Single-Comb White Leghorns, bred at the station and college poultry
plant, and were hatched in the same incubator. Each lot was housed
under similar oil-burning hovers of 100-chick capacity.
The experiment was carried on in periods of 8 weeks each and extended
over three periods, or 24 weeks. The samples of feeds for analyses were
obtained, as composite samples, from the various bags of feed used in
the experiment. The potential acidity and potential alkalinity were
estimated from the mineral analyses of the feed samples.
The caloric values of all the animal food rations — No. 2, 3, 4, and 7 —
are about the same, being 27 to 31 per cent protein calories. The soybean
meal ration is in the same column so far as protein calories are concerned.
The grain feeds have less protein caloric value, having only 12 per cent.
Dried buttermilk functions as base. The meat scrap and bone meal and the
digester tankage are base on account of the large amount of calcium in the
bone. Blood is normally alkaline because of the sodium salts. The grain
ration is 61.96 cc. acid per pound. The mashes are all alkaline or base.
Those containing milk or bone — rations 2,3, and 4 — run high in base ele-
ments, while the blood meal runs only 41.21 cc. base and compares
favorably with the two rations containing no animal food but contain-
ing protein from leguminous sources.
It will be noted that ration No. 6, the peanut-meal mixture, is low in
protein caloric value. This is due to the fact that the peanut meal
used was ground peanuts and hulls, not fat-extracted, and showed 40.4
per cent fat content.
Many interesting things are brought out by Table IV. The greatest
gain in weight in chicks is during the first eight weeks, or the first period.
Following the first period the increase in weight is gradually less during
the remainder of the two periods.
The amount of feed required to produce a pound of gain gradually
grows greater as the bird becomes older.
Oct. is. i93o Acidity and Alkalinity of Poultry Feed Mixtures 143
8
"fe.
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144
Journal of Agricultural Research
Vol. XX, No. 2
Table II. — Potential acidity and potential alkalinity of southern poultry feeds
Kinds of feed.
Acid.a
Corn meal , bolted . . .
Pinhead oats
Whole wheat
Wheat middlings. . . .
Bone meal
Meat and bone meal .
Rolled oats
Whole corn
Hulled oats
Velvet bean meal . . .
Soybean meal
Peanut meal
Skim milk
Rape, green
Limestone grit
Oyster shell
Dried buttermilk. . . .
Digester tankage
Dried blood
Egg, including shell.
Peas, dried
Potatoes, sweet
Potatoes, Irish
Rice
Spinach
Turnips
Beans, dried
Beets
Bread, hard
Cabbage
Carrots
Fish, dried
Hominy
Lettuce
206. 58
2, 104. 60
565- 13
2i-75
121. 94
79.09
62. 17
156.01
94.90
23.18
15-33
11, 943- 80
8, 782. 10
803. 96
789. 89
63.68
108. 21
44.76
79.17
2-55
24. 00
24. 00
26. 00
122. 00
25.00
79. 00
50.00
22. 00
38.00
27. 00
43. 00
38.00
36.00
24. 00
° Expressed in excess cubic centimeters per pound of feed.
The bird gradually, in these tests, consumed more grain as it grew
older. Likewise it was found that as a pullet comes into laying it con-
sumes a greater proportion of mash and again slackens in its mash
consumption as it goes into a nonlaying period.
The consumption of more grain and less mash has a tendency to lessen
the base balance or, if the balance is already acid, has a tendency to
increase the acid balance.
It was noted that the cereals are decidedly acid while the by-products
from the legumes are of a base reaction. Feeds containing by-products,
such as soybean meal and peanut meal, have a tendency to add to the
base balance.
It is noted that rations containing buttermilk, digester tankage, and
meat scrap and bone meal give a base balance in all cases.
The soda in the blood makes the blood meal base but not so strongly
so as digester tankage or meat and bone meal containing much bone.
Oct. 15, 1920 Acidity and Alkalinity of Poultry Feed Mixtures
145
Bone is rich in calcium and also contains other bases such as sodium.
As stated before, sour milk functions as base.
Table III.- — Acid-base balance of rations 1 to 7
Ration
No.
Kinds of feed.
Amount.
Acid.a
Base."
Percentage
of protein
calories.
I
[Scratch ieed :
\ Corn
Pounds.
IOO
IOO
35
30
35
35
35
3°
35
20
35
3°
35
18
35
3°
35
24
35
3°
35
41
35
3°
35
U
,
Oats
61. 96
(Mash feed:
Wheat middlings
2
I Corn meal
Ground oats
Dried buttermilk
23I-4
27
Wheat middlings
Corn meal
3
Ground oats
Meat and bone meal
125-9
3°
Wheat middlings
Corn meal
4
Ground oats
Digester tankage
153-7
3°
Wheat middlings
Corn meal
5
1 Ground oats
Soybean meal
63-9
3°
Wheat middlings
Corn meal
6
Ground oats
Peanut meal
54-5
19
Wheat middlings
Corn meal
7
1 Ground oats
Blood meal
41. 21
31
0 Expressed in excess cubic centimeters per pound of feed.
In this work no account has been taken of the amount of calcium
entering the crop as grit and oyster shell. This will, in all probability,
overcome the acid reaction, though without definite data this is a mere
guess.
In a large table of studies of rations furnished by the medical staff of
the United States Army, the percentage of protein calories ran from 10 to
18. In the present work the percentage of protein calories ran from 13
to 22, according to the estimate of the actual intake in each period.
There is a possibility, however, that we will need to pay more attention
to the source or kinds and quantities of protein calories, since we have
shown in this work that those birds that received animal food, including
milk, tankage, meat and bone meal, and dried blood were prepared by
the storing up of the proper potential energy to begin heavy egg pro-
duction very young, while those birds that did not receive animal food
of any kind were not prepared and did not go into early heavy egg
production.
146
Journal of Agricultural Research
Vol. XX, No. 2
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Acidity and Alkalinity of Poultry Feed Mixtures
147
Table V. — Acid-base balance 0/ rations 8 to 11
Ration
No.
Kinds of feed.
Amount.
Aeid.°
Base.3
Percentage
of protein
calories.
(Rolled oats
Pounds.
8
8
2
1
3
2
1
6
3
3
1
3
2
1
1 Wheat middlings
8
I Meat and bone meal
[Bone meal
128.3
29
(Cracked wheat
0
\ Cracked corn
[Pinhead oats
67. 2
13
(Wheat middlings
J Corn meal
10
| Meat and bone meal
[Bone meal
382.6
30
(Whole wheat
11
I Cracked corn
[Hulled oats
67. 2
13
0 Expressed in excess cubic centimeters per pound of gain.
Table VI.— Relation of protein calories, amount of feed, and acid-base balance, to pound
of gain during first eight weeks
Kinds of feed.
Amount.
Excess acid."
Excess base."
Milk
Pounds.
52-3°
2. 42
8.42
4-3°
5-98
7.81
I, 212. 31
3IO. 48
Ration No. 8
Ration No. 9
565- 82
Ration No. 10
1,645. l8
Ration No. 11
401.85
Rape
119. 72
0 Expressed in excess cubic centimeters in each feed consumed.
Amount of feed per pound of gain, 3.4 pounds.
Percentage of gain, 686.
Excess base intake, 2,320 cc.
Excess base intake per pound of feed, 85.5 cc.
SUMMARY
From Table II we note that corn, wheat, and oats, as well as egg with
the shell, rice, bread, fish, and hominy are acid. The wheat middlings,
in six analyses, on account of the high content of sodium and potassium,
are base. Green feeds, such as rape, cabbage, carrots, beets, turnips,
potatoes, spinach, and lettuce, are base. Seeds of the legumes, such as
velvet bean, soybean, peanut, and peas, are base. Bone meal, on account
of its high calcium content as well as other base-forming elements, is
highly base. Limestone grit is very highly base, and also to a less extent is
crushed oyster shell. The animal feeds containing bone, such as meat
and bone meal and digester tankage, are base. The calcium of the egg
shell does not quite overcome the acid of the albumin of the egg. Dried
148 Journal of Agricultural Research vol. xx, no. 2
milk functions as base because the lactic acid is oxidized to carbonic acid,
which is thrown off by the lungs, leaving the basic residue of mineral
salts. Dried skim milk or dried buttermilk is therefore quite base in
function. Dried blood, on account of its magnesium, calcium, and sodium
content, is moderately base.
In these studies there have been arranged 1 1 feed mixtures for acid-
base studies. The first 7 are North Carolina Experiment Station for-
mulae and the last 4 are those of Prof. Rice. The mixtures that contain
considerable amounts of either dried milk, meat and bone meal, or digester
tankage are quite base. The mixture containing soybean meal is approxi-
mately as much base as the grain mixture is acid, so that equal amounts
would approximately balance so far as acid-base content is concerned.
The peanut meal mixture is slightly below the soybean meal mixture,
and the blood meal comes slightly below the peanut meal.
We note from Table III that the grain mixture contains 12 per cent
protein calories and the ground feed mixtures contain from 19 per
cent in the mixture containing peanut meal in which peanut meal not
fat-extracted to 31 per cent in the ration in which blood meal was used.
We note by a study of Table IV, which gives the total intake of each
mixture for each period, that the final percentage of protein calories
runs from 13 to 22. For comparison with rations for human beings
we may again refer to the study of army rations during the late World
War, in which the percentage of protein calories ran from 10 to 18.
Dr. Osborne1 found that 12.5 per cent protein calories produced maxi-
mum growth in rats. The indications are that the kinds as well as the
quantities of proteins are essential factors. While the kinds of amino
acids and vitamins are important factors in addition to kinds and quan-
tities of minerals, there is a possibility that there are other factors un-
discovered which have a profound bearing on growth, egg production,
and the preparing of pullets, by aiding the storing up of potential energy,
for early and heavy egg production. Data which will be published later
show that pullets grown on range or in confinement without animal food
of any kind, though the protein calories were above those indicated in
comparison rations,1 were not prepared for early heavy egg production
and did not show high egg yields until animal food of some kind had
been added. In this instance this was the soybean meal and peanut-
meal lots. In the second and third periods the balances of intake was
acid.
Further studies are being made to determine whether acid feeds will
in any way interfere with either growth or egg production. In these
studies rations 5 and 6 can be made base by the addition of ground
limestone or gound oyster shell. The amounts to be added would
depend upon the proportions in which the mash and grain were fed.
1 Osborne, Thomas B., and Mendel, Lafayette B. a quantitative comparison of casein, lactal-
bumin, and edestin for GROWTH or MAINTENANCE. In Jour. Biol. Chem., v. 36, no. 1, p. 9. 1916.
Oct. is, 1920 A cidity and A Ikalinity of Poultry Feed Mixtures 1 49
We find, by a study of Table V, that the grain rations 9 and 11 are
acid and that the mash is base. In these mash mixtures there has been
added both bone meal and meat and bone meal. Wheat middlings also
aid in overcoming the acidity of corn meal and of rolled oats. In this
test the total intake excess was base. The percentage of protein calories
was 22.
CONCLUSIONS
Grain mixtures as ordinarily used in poultry feeding are acid.
Mash mixtures containing sufficient quantities of digester tankage,
meat and bone meal, dried milk, or dried blood will be base.
Acid balances of feed mixtures can be overcome by the addition to
mashes of dried milk, digester tankage, meat and bone meal, bone
meal, dried blood, or ground limestone or oyster shell. Green feed,
milk to drink, and limestone and oyster-shell grit also aid in overcoming
the acid balance of grain mixtures.
THE INFLUENCE OF COLD IN STIMULATING THE
GROWTH OF PLANTS *
By Frederick V. CovrtLE
Botanist in Charge, Office of Economic and Systematic Botany, Bureau of Plant
Industry, United States Department of Agriculture
In regions having a cold winter like ours, with prolonged or repeated
freezing, the native trees and shrubs become dormant in autumn. Ac-
cording to the general belief this condition is brought about by the cold.
It is also the general belief that warm weather is of itself the sufficient
cause of the beginning of new growth in spring. Both these ideas are
erroneous. It is the object of the present address to show : first, that
in our native trees and shrubs dormancy sets in before cold weather, and
that cold weather is not necessary for the establishment of complete
dormancy; second, that after such dormancy has begun, the exposure of
the plants to an ordinary growing temperature does not suffice to start
them into growth; third, that these plants will not resume normal growth
in the warm weather of spring unless they have been subjected pre-
viously to a period of chilling; and, finally, a theory will be advanced
to explain this paradoxical effect of cold in stimulating growth instead of
retarding it.
The subject will be presented in a series of numbered statements, each
followed by supporting evidence.
i. Trees AND SHRUBS of cold climates become dormant at the
END OF THE GROWING SEASON WITHOUT THE NECESSITY OF EXPOSURE
TO COLD WEATHER.
A little more than 10 years ago, while engaged in a series of greenhouse
experiments, the speaker came upon a strange phenomenon which was
wholly unexpected and which threatened to interfere seriously with the
success of the experiments. Healthy blueberry plants, intended to be
used during the winter for breeding purposes, were brought into the
greenhouse at the end of summer and were kept at an ordinary growing
temperature. They refused to continue their growth during the autumn,
gradually dropped their leaves, and went into a condition of complete
dormancy. They did this at a greenhouse temperature which in spring
and summer would have kept the plants in a condition of luxuriant
growth. The completeness of the condition of dormancy which such
plants reach can be best appreciated from photographs (PI. 20, A).
Since 1910 this experiment has been repeated many times and with
many species of plants, and without exception those trees and shrubs
1 An address delivered before the National Academy of Sciences Apr. 27, 1920.
Journal of Agricultural Research, Vol. XX, No. 2
Washington, D. C. Oct. 15, 1920
vg Key No. G-205
(151)
152 Journal of Agricultural Research vol. xx, No. a
native of our northern cold-winter region which were tested went dor-
mant in fall or winter regardless of temperature. In comparing outdoor
plants with indoor plants of the same species the most that can be said
in favor of outdoor conditions is that dormancy progresses a little faster
in outdoor plants, evidently because their foliage is injured by freezing
weather, and they drop their leaves somewhat earlier than indoor plants.
2. Trees and shrubs that are kept continuously warm during
the winter start into growth much eater in spring than those
that have been subjected to a period of chilling.
In the late winter and early spring of 1910 I waited patiently, and
then impatiently, for my indoor plants to bloom, and at last I was forced
to realize that they never would bloom. When compared with plants of
the same kind that had been outdoors during the winter and had been
brought into the greenhouse in early spring, the difference was astonish-
ing. The outdoor plants burst into leaf and flower luxuriantly, while
the indoor plants remained completely dormant and naked. The exper-
iment was repeated many times and with various species of plants, some
of which may be used in illustration. (See PI. 20, B; 21 ; 22, A.)
At first it was supposed that the plants needed to be frozen to start
them into growth, but a single freezing proved not to be effective. And
then it was found that the dormant plants would start into growth
without any freezing whatever. It was necessary only that they be
subjected to a period of prolonged chilling, usually two or three months,
at a temperature a few degrees above freezing.
If plants are kept continuously in a warm place without chilling, the
dormant condition often continues for an extraordinary length of time.
In some instances plants have remained dormant for a whole year under
conditions of heat, light, and moisture that ordinarily would make the
same plant grow with the greatest luxuriance.
3. The stimulating effect of cold is limited to such portions
of the plant as are subjected to the chilling.
The conspicuous difference in spring growth between chilled plants
and plants not chilled has already been shown. These differences,
furthermore, can be produced experimentally upon different parts of
the same plant. Plants thus treated present a very curious and remark-
able appearance, as shown in Plate 22, B, and Plate 23.
On February 3, 1912, a blueberry plant (PI. 22, B) 44 inches in height,
which had shed its leaves and become dormant in a warm greenhouse
maintained at a temperature of 6o° to 700 F., was subjected to the
following experiment: It was repotted in a 7-inch pot and set in the south
end of a greenhouse at the temperature already mentioned. A small
opening was made in the glass, and through this opening one of the two
stems of the plant was pushed. The open space about the stem where
it passed through the glass was carefully plugged with moss. During
Oct. is, 1920 Influence of Cold in Stimulating Growth of Plants 1 53
the rest of the winter the plant remained in the same position, the pot
and the stem, shown at the left in the illustration, continuing in the
warm temperature of the greenhouse, while the stem at the right,
projecting through the glass, was exposed to the rigors of winter,
with its alternate freezing and thawing. The illustration, from a photo-
graph made April 18, shows that when spring came the outdoor branch
started into normal growth while the indoor branch continued dormant.
A second illustration (PI. 23) shows a modification of the first experi-
ment. In this case the plant was set on a shelf outside the greenhouse,
and a single branch was passed through the glass wall into the warm
interior. When spring came it was this interior branch that remained
dormant, all the outside branches putting out leaves promptly and
normally.
From a comparison of the two experiments it is evident that the
difference in behavior of the indoor and outdoor branches could not
have been caused by any special action of the root system, for in one
experiment the roots were inside, in the other outside. It is clear that
the causes that stimulated growth in the exposed stems operated in the
stem itself, not in the roots. This principle is still further exemplified
and confirmed by the behavior of cuttings taken from blueberry plants
in the first stages of their dormancy. Such cuttings if kept warm con-
tinue their dormancy into late spring or summer, but if chilled for two or
three months they start into growth at the normal time in early spring.
It should be stated here that the difference in the amount of light
inside and outside the greenhouse had nothing to do with the stimulation
to growth, for chilled plants are ready to start into growth promptly
whether the chilling is done in the full light of an outdoor situation, or
in the partial light of a greenhouse, or in the complete darkness of an
ordinary refrigerator.
4. The stimulating effect produced on dormant plants by cold
is intimately associated with the transformation of stored
starch into sugar.
In most of our wild species of trees and shrubs the reserve carbo-
hydrate material is stored away during summer and autumn in the form
of starch. At the beginning of dormancy the twigs and sap wood are
gorged with this material, the starch grains being stored ordinarily in the
cells of the medullary rays and sometimes in the pith. As the process of
chilling goes on, this starch little by little is transformed into sugar.
The presence of large quantities of starch in the fall and early winter
may be observed by applying to freshly cut surfaces of the twigs the well-
known starch test of a 2 per cent solution of iodin in a 1 per cent solution
of iodid of potassium. With a strong hand lens the starch is readily
observed, if present, by the deep blue color it assumes under this treat-
ment. The intensity of the coloration gives roughly an idea of the
187932°— 20 6
1 54 Journal of Agricultural Research vol. xx, no. a
number of starch grains present, and thus by this simple means anyone
may observe in the twigs of trees and shrubs the gradual disappearance
of their starch as spring approaches.
The measurement of the increasing amount of sugar is more difficult
and must be done by chemical analysis. Through the courtesy of the
Chief of the Bureau of Chemistry, exact data can be presented on this
point from analyses by Mr. Lorin H. Bailey. In samples of dormant
blueberry wood taken in early spring when growth was about to begin
the ratio of sugar to starch proved to be seven times what it was in
similar dormant wood taken in autumn.
I desire at this time to comment on the fact that one of my colleagues
reading the manuscript outline of this address criticized the use of the
word "stimulate" as applied to the effect which chilling produces on
these dormant plants. His idea was that the chilling induced certain
physiological changes in the cell contents but that the actual stimulation
to growth came from the temperatures that followed the chilling. I
defend, however, the propriety of the language I have used, for although
the later stages of growth admittedly can not take place without warm
temperatures, not only does the transformation from starch to sugar
take place at the chilling temperature but the buds actually swell and
push if the chilling temperature is continued for several months. In
illustration I may cite the following experiments.
On March 3, 191 5, 286 cuttings were made from dormant outdoor
blueberry plants. They were stored in bundles, some in moist sphagnum
moss, others in moist birch sawdust, at a contemplated temperature of
31 ° F., just below freezing. The cuttings remained in cold storage until
December 6, a little more than nine months. An examination of the
cuttings on that date showed that one or more buds had begun to swell
on every cutting with the exception of a small number which were
mildewed and dead. In other words, growth had already begun to take
place at the cold-storage temperature. The thermograph record for the
278 days was as follows:
Hours.
290 to 320 F 5, 591
32°to33°F 990
330 to 340 F 91
The temperature record did not go above 340 F. It is an astonishing
fact that temperatures so very near freezing will start dormant plants
into growth.
On March 3, 1915, 58 cuttings from dormant, outdoor blueberry plants
were placed in moist birch sawdust in commercial cold storage at 330 to
360 F. On December 4, nine months later, buds on every cutting had
begun to grow. Not one of these cuttings gave a starch reaction when
tested with iodin. The transformation of their stored starch into sugar
was complete. (See PI. 24.)
Oct. is, 1920 Influence of Cold in Stimulating Growth of Plants 155
5. The; theory advanced in explanation of the formation of
sugar during the process of chilling is that the starch grains
stored in the cells of the plant are at first separated by the
living and active cell membranes from the enzym that would
transform the starch into sugar, but when the plant is chilled
the vital activity of the cell membrane is weakened so that the
enzym "leaks" through it, comes in contact with the starch,
and turns it into sugar.
I have stated the theory in these words out of regard for simplicity
and general understanding, but if anyone should require that it be pre-
sented in orthodox technical language it might be restated as follows:
The reserve amylum carbohydrate bodies are isolated from the amylo-
lytic enzym by semipermeable protoplasmic living membranes of high
osmotic efficiency, but under the influence of low temperatures the pro-
toplasmic membranes are proximately devitalized, they become per-
meable to the amylolytic enzym, and amylolysis ensues. I may add,
however, that the use of such terminology seems to me to involve a cer-
tain degree of unnecessary cruelty.
From the evidence already presented, no one, presumably, will question
that the chilling of dormant trees and shrubs is followed by growth and
that the growth is associated with the transformation of starch into sugar.
But the hypothesis that this transformation is brought about by the
weakening of the cell membrane and the consequent leakage of starch -
transforming enzyms into the starch chambers may very properly be
challenged. In the Tropics there is no chilling weather, yet trees and
shrubs spring into growth after the dormant period of the dry sea-
son just as they do in temperate climates after the dormant period of
winter. The critical scientific man will therefore ask, "Are there not
other agencies than chilling which will start dormant trees and shrubs
into growth even in our latitude?" It must be said in reply that there
are. And it will be worth while to consider some of these causes, for
not only are they of interest in themselves but also, instead of weakening
the hypothesis here presented, they serve to strengthen and confirm it.
The data may best be presented through a series of illustrations.
The pruning of a long-dormant plant will often start it into growth
(PI. 25, A). Girdling produces a similar result (PI. 25, B, at left).
Notching the stem does the same (PI. 25, B, at right). Rubbing the
stem also starts the plant into growth (PI. 26).
In all these examples of the stimulation of growth by injury it is con-
ceived that the enzym is brought into contact with the starch as a direct
result of the breaking and straining of the cells. Sugar is then formed
and growth begins.
It should be observed that when a normal chilled plant starts growing
it grows from many buds (PI. 27, A), for the effect of the chilling on sugar
formation is general. When a dormant plant starts growing as the result
1 56 Journal of Agricultural Research vol. xx, No. 2
of injury, however, it usually starts, as shown in several illustrations
already presented, from a single bud, the one nearest the point of injury-
The injury is local, and both the sugar formation and the growth that
follows it are local.
We are now brought to the consideration of a phenomenon which I
take to be of special significance — namely, the procedure by which the
dormant plant starts itself into growth in the absence of chilling. After
a blueberry plant has remained dormant at a warm temperature for a
very long period, sometimes a whole year, the tips of the naked branches
begin to lose their vitality. Just before or just after the death of the tip
a single bud, or sometimes two buds, situated next below the dead or
dying part starts growing (see PI. 27, B; 31, A). The new growth of the
stem is confined to the one or two buds, just as it was in the case of
growth induced by injury. My interpretation of the phenomenon is
that, as death approaches, the cell membranes become weakened in
much the same way as when chilled, the enzym passes through into the
starch storage cells, sugar is formed, and the adjacent bud begins to
grow. The process going forward here in a restricted portion of the
stem, and due to a local cause, is essentially the same as that taking place
generally over the plant, from a general cause, when the plant is chilled.
In the Tropics some plants are able to grow continuously; others be-
come dormant in the dry season and start into growth again at the com-
ing of the rainy season. Tropical plants probably have various methods
of coming out of their dormancy, and there is every reason to expect
that some of them will be found to accomplish this act in the same way
as our long dormant greenhouse plants, by the weakening of their cell
membranes. This, I have endeavored to show, is in its effect substan-
tially identical with chilling.
6. The twigs of trees and shrubs after their winter chill-
ing AND THE TRANSFORMATION OF THEIR STARCH INTO SUGAR MAY BE
REGARDED AS MECHANISMS FOR THE DEVELOPMENT OF HIGH OSMOTIC
PRESSURES WHICH START THE PLANT INTO GROWTH.
Food in the form of starch can not be utilized by a plant directly.
The starch must be changed into sugar before it can be used in making
new growth. But this transformation does more than make the starch
available as food for the growing plant. It serves also to increase the
tendency of the cells to swell and enlarge. In the form of starch the
material is inert in the creation of osmotic pressures, but when trans-
formed into sugar it becomes exceedingly active. According to the rigid
experimental tests of H. N. Morse and his associates, a normal solution
of cane sugar at 320 F. has an osmotic power of 25 atmospheres of pres-
sure. It has been demonstrated that there sometimes occur in the cells
of plants osmotic pressures as high as 30 atmospheres, or 450 pounds to
the square inch, a pressure sufficient to blow the cylinder head off an
Oct. iSl 1920 Influence 0} Cold in Stimulating Growth of Plants 157
ordinary steam engine. It can hardly be questioned that these or even
much lower osmotic pressures take an important part in forcing open
the buds of once dormant plants.
We have evidence that there sometimes arise within the plant osmotic
pressures of such intensity as to threaten the rupture of the cells. Con-
sider the case of the exudation of drops of sugar solution from certain
specialized glands. When this exudate of sugar occurs in flowers it is
known as nectar, and it serves a useful purpose to the plant by attract-
ing sugar-loving insects which unconsciously carry pollen from flower to
flower and accomplish the beneficial act of cross-pollination. But sugar
solution is often exuded outside the flower, in positions, or at times, that
preclude any relation to cross-pollination. For example, a blueberry
plant during its spring growth, when a leaf has reached nearly full size,
is sometimes observed to exude drops of sugar solution from certain
glands on the margins of the leaf and on the back of the midrib (PI. 28).
It is physically impossible for the sugar to have left the cells by osmosis.
The sugar serves no useful purpose to the plant through the attraction
of insects. The exudate certainly can not represent the elimination of a
waste product, for sugar is one of the substances most used by plants in
forming new tissues. I can conceive of no reason why the plant should
exude sugar except to relieve a dangerous physiological condition —
namely, the development of excessive osmotic pressures which would
burst the cells of the plant or in some other way derange its physiological
activities. I look upon such sugar glands as safety valves for the relief
of excessive osmotic pressures that are dangerous to the internal economy
of the plant. And not only is this conception applicable to extra-floral
nectaries in general, but it may serve also, in the case of floral nectaries,
to explain their origin. Having once arisen as osmotic safety valves,
the usefulness of the floral nectaries as an aid to cross-pollination would
then tend strongly to bring about their natural selection and perpetuation.
7. The establishment of a dormant condition before the ad-
vent OF FREEZING WEATHER AND THE CONTINUATION OF THIS DOR-
MANCY THROUGH WARM PERIODS IN LATE FALL AND EARLY WINTER ARE
PROTECTIVE ADAPTATIONS OF VITAL NECESSITY TO THE NATIVE TREES
AND SHRUBS.
A little consideration will show how important the principle of chill-
ing is to those species of trees and shrubs which are subjected each
year to several months of freezing weather. If they were so consti-
tuted as to start into growth as easily in the warm days of late fall as
they do in the warm days of early spring many species would come into
flower and leaf in those warm autumn spells that we call Indian summer,
and the stored food that the plant required for its normal vigorous growth
in the following spring would be wasted in a burst of new autumn growth,
which would be killed by the first heavy freezes and would be followed
by a winter of weakness and probable death. But when two or three
1 58 Journal of Agricultural Research vol. xx, no. 2
months of chilling are necessary before a newly dormant plant will
respond to the usual effect of warmth, such plants are protected against
the dangers of growth in Indian summer. It is probable that all our
native trees and shrubs are thus protected.
Any member of this audience may make a simple and instructive
experiment next fall and winter with such early spring blooming plants
as alder, hazelnut, pussy willow, yellow bush jasmine, forsythia, Jap-
anese quince, peach, and plum. In mid-autumn bring into your living
room and set in water freshly cut, dormant, leafless branches of these
plants. They will not bloom. At intervals of a few weeks during late
autumn and winter try the same experiment again. You will find that
the branches cut at later dates will come into bloom under this
treatment. They will not do so, however, until the expiration of the
period of chilling appropriate to the various kinds of plants included in
the experiment. The required period of chilling varies greatly. For
some of the cultivated shrubs about Washington, especially the yellow
bush jasmine (Jasminum nudiflorum), so brief a period of chilling is
required that an extraordinarily cold period in late October or early
November may chill them sufficiently to induce them to bloom if a period
of warm weather follows in late November. The period of chilling re-
quired for the peach is so short that in Georgia unusually warm weather
in December sometimes brings the trees into flower, and their crop of
fruit is destroyed by the freezes that follow.
From these facts it appears that our native trees and shrubs are so
intimately adjusted to the changes of the climate to which they have
been long subjected that they are almost completely protected from
injury by freezing, but some of the cultivated species brought from parts
of the world having a climate different from ours are only imperfectly
adapted to our climatic changes. They grow at times when our native
species have learned to hold themselves dormant, and they often suffer
severely in consequence.
Chilling, as a protective adaptation, has become a physiological
necessity in the life history of cold-winter trees and shrubs. So fixed
indeed, is the habit that it appears to be a critical factor in determining
how far such plants may go in the extension of their geographic distri-
bution toward the Tropics. In the Tropics our common northern fruit
trees, apples, pears, peaches, cherries, grow well for a time and then
become half dormant. In the absence of chilling they never fully recover
from their dormancy; they grow with weakened vitality and finally die.
If these fruits are to be grown successfully in the Tropics they must be
given artificially the periodic chilling they require.
When it became evident from the earlier observations and experi-
ments that chilling played so essential a part in the behavior of our trees
and shrubs, it was clear that additional experiments ought to be con-
ducted in which actively growing plants might be subjected to chilling
Oct. iSl 1920 Influence of Cold in Stimulating Growth of Plants 1 59
temperatures without being put in a dark place like the ordinary
refrigerator. To meet the requirement of both cold and light a glass-
covered, outdoor, brick chamber was constructed in 191 2. It was kept
above freezing by heating with electric lights, which were turned on and
off automatically by a simple thermostat. In summer the chamber
was kept cool, though not really cold, by means of ice and electric fans.
Although much was learned with this apparatus, it was crude and inade-
quate. To provide for more exact experiments a glass-covered com-
partment chilled by a refrigerating machine was constructed in one of
the Department of Agriculture greenhouses. The refrigerating appa-
ratus is a sulphur-dioxid machine having a refrigerating power equivalent
to 1,000 pounds of ice a day. It is run by a 2 -horsepower electric motor,
and it furnishes ample refrigeration for the lighted compartment, which
is a glass-covered frame 25 feet long, 3 feet wide, and 14 to 20 inches in
depth. The first of these refrigerated frames was devised and con-
structed in 1 91 6. In this enterprise I had the valued advice and assist-
ance of Dr. Lyman J. Briggs. The usefulness of this refrigerated frame
in experimental work with plants was so great that another similar
equipment was installed in 1918.
With the aid of this apparatus many of the experiments described in
this address have been carried on or verified, as well as other experiments
of a related character. For example, at ordinary summer temperatures
many kinds of seed will not germinate but remain dormant until death
overtakes them. Under the influence of chilling, however, these seeds
are stimulated to prompt germination. (See PI. 29.)
The experiments thus far made indicate the importance of a much
wider use of the principle of chilling in many lines of experimentation
bearing on the improvement of horticultural and agricultural practices.
I commend the subject of chilling to experimenters in these lines, and
I wish to call especial attention to the desirability of determining" proper
temperatures for the storage of seeds, bulbs, cuttings, and grafting
wood, proper temperatures for the treatment of plants which are to be
forced from dormancy to growth at unusual seasons, and proper tempera-
ture for the storage of nursery stock so that the nurseryman may have
plants in proper condition for shipment on any date he desires. (See
PI. 30; 31, B; 32.)
The whole question of the effect of chilling on herbaceous perennials
is an open field.
An understanding of the process of chilling explains the reason of
some of the practices of gardeners, which they as well as botanists have
erroneously ascribed to the need of "resting." What a gardener calls
"resting" is often in reality a period of chilling, characterized not by
physiological rest but by pronounced internal activity. Rest alone would
not, in the case of our cold-climate trees and shrubs, accomplish the
purpose the gardener has in mind. It is chilling, not rest merely, that
1 60 Journal of A gricultural Research vol. xx , no. 2
is required. The practice of gardeners and nurserymen known as the
"stratification " of seeds is probably to be explained as in reality a process
of chilling.
As a single example of the application of the principle of chilling let
me cite the case of the blueberry. For several years we have been
trying at the Department of Agriculture to domesticate this wild plant.
We have raised many thousand hybrids and have set them out in waste
sandy lands in the pine barrens of New Jersey ( PI. 33, A) . We have grown
the bushes to fruiting age and have brought them into highly productive
bearing (PI. 33, B). We have made them fruit so lusciously and so abund-
antly that they have brought returns to the grower at the rate of more
than $1,000 an acre. In a word, we have changed the blueberry from a
small wild fruit the size of a pea to a fruit the size of a Concord grape, and
we have made its culture a profitable industry. (See PI. 34, 35.) These
things we should not have been able to do unless we had first worked out
the principle of chilling, an understanding of which was essential to our
work of breeding and propagation.
In conclusion, I wish to express the opinion that the chilling of dormant
trees and shrubs of temperate climates as a prerequisite to their resump-
tion of normal growth in the spring ought to be recognized in books on
plant physiology as one of the normal processes in plant life. These
works should contain chapters on chilling, just as they now contain
chapters on other fundamental factors and principles relating to the life
history of plants. And especially in books on plant physiology in relation
to agriculture should the subject of chilling be dealt with in detail, for
when in the pursuit of agriculture we take plants from one part of the
world to another, or undertake to grow them out of season, or attempt
to propagate them in quantity by grafting or by other processes unknown
in nature, we are greatly handicapped and limited in our operations if we
do not understand the principles of a process so widely existent in nature
and so indispensable to a large proportion of the plants of temperate
agriculture as the process of chilling.
PLATE 20
A. — Blueberry plants, Vacciniuin corymbosum, made dormant without cold. These
blueberry seedlings, in 2-inch pots, were kept during the fall and winter in a greenhouse
at a temperature of 550 to 700 F. Although this is a very favorable temperature for
the growth of the blueberry, these plants shed their leaves and became completely
dormant, just as they ordinarily do when exposed to the frost and cold of an outdoor
fall and winter. The photograph was taken on January 25.
B. — Chilled and unchilled blueberry plants. The six blueberry plants at the left,
after an outdoor winter chilling, were brought indoors on March 25, into a greenhouse
having a temperature of 550 to 700 F., and were repotted. On April 20, when the
photograph was taken, they had developed both leaves and flowers, while the six
plants at the right, which had been in the same greenhouse at the same temperature
all the fall and winter and were repotted on the same date as the others, were still
completely dormant.
Influence of Cold in Stimulating Growth of Plants
Plate 20
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 21
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 21
A. — Chilled and unchilled plants of grouseberry, Viburnum americanum. The
illustration shows two i-year-old seedlings with the same history', except that the one
at the right was kept during the winter in a warm greenhouse at a temperature of 550
to 700 F., while the one at the left was wintered in a cold greenhouse at a temperature
of 320 to 400. When spring temperatures warmed up this coldhouse, the plants in it
began to grow, and on April 7, 1914, when the photograph was taken, they had reached
the stage shown in the left-hand figure , while the plants in the warmhouse , as illustrated
by the right-hand figure, were still completely dormant.
B. — Chilled and unchilled plants of tamarack, Larix laricina. These two seedlings,
grown from seed procured in Alaska, have had the same history except that the one at
the left was wintered in a cold greenhouse at a temperature of 32 ° to 400 F., the one at
the right in a warm greenhouse at a temperature of 550 to 700. When the photograph
was taken, on April 10, 1914, the chilled plant had put out new growth in the warm
spring weather, while the unchilled plant still showed only its leaves of the year before.
PLATE 22
A. — Chilled and unchilled plants of wild crab, Malus coronaria. The plant at the
left had been outdoors during the fall and winter, leafless and dormant, exposed to
the frost and cold . The plant at the right had been in the warm greenhouse during the
fall and winter at a temperature of 550 to 700 F. When the outdoor, chilled plant was
brought into the greenhouse in early spring, it promptly began to put out new leaves
and twigs, but the indoor, unchilled plant continued its dormancy. The photograph
was taken April 24, 1917.
B. — Blueberry plant with one branch stimulated to growth by cold. The right-
hand branch has been stimulated to growth by chilling; the left-hand branch has been
kept dormant by heat. For a detailed description of this experiment see p. 152-153.
Influence of Cold in Stimulating Growth of Plants
Plate 22
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Sti
mulating Growth
Of
Plants
Plate 23
Uj
%JL 4
4
^Pp*
CD
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 23
Blueberry plant with one branch kept dormant by heat.
A. — Dormant indoor blueberry plant as it appeared on February 15, 1912. On that
date the pot containing the plant was placed on a shelf outside a greenhouse, and a
single branch was passed through the glass wall into the warm interior.
B. — Same plant photographed May 21. When spring came, all the outside branches,
which had been chilled, burst into normal leaf, while the branch inside the greenhouse,
which had been kept warm, still remained dormant.
PLATE 24
A. — Blueberry cuttings starting to grow at 360 F. These cuttings were placed in
cold storage while still completely dormant. Although the temperature did not go
above 360 F., buds on each of the cuttings finally began to grow. It is to be noted
that although growth took place in the buds the other kind of growth which results
in the formation of a callus, or healing-over tissue, at the severed base of the cutting
is wholly lacking. Callusing can not take place at so low a temperature.
B. — Blueberry plant growing in the dark at 360 F. This plant was in cold storage
in the dark in a commercial refrigerating establishment from March 30 to December
4, 1915. The temperature ranged from 330 to 360 F. vSome of the plants in this
experiment made new growth to the length of 32 mm.
Influence of Cold in Stimulating Growth of Plants
Plate 24
•
-
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 25
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 25
A. — Dormant wild crab stimulated to growth by pruning. This plant had remained
dormant in the warm greenhouse during the fall and winter at a temperature of 550
to 700 F. On April 5 three branches were pruned, and on April 24, when the photo-
graph was taken, the uppermost bud on each of the pruned branches had begun to
grow. On other, unpruned plants no bud growth had taken place.
B. — Dormant wild crabs stimulated to growth by girdling and by notching the stem.
These plants had had the same preliminary treatment as the one illustrated in A —
that is, they had been kept in the warm greenhouse all winter, without chilling.
On April 4 a ring of bark was removed from the plant shown in the left-hand figure,
and the soft cambium was carefully scraped away, down to the hard wood. On
April 24, when the photograph was made, the bud next below the girdle had begun
to push. The stem of the right-hand plant was notched in early April. The bud
next below the notch soon began to grow. The photograph was taken on May 2.
PLATE 26
A. — Dormant blueberry buds stimulated to growth by chalking the stem. This
plant was brought into the greenhouse February 4, 1913, to be used in breeding experi-
ments. It flowered, but since it had been insufficiently chilled only a few of the
uppermost leaf buds on each stem grew. In order to keep small ants from crawling
up the stems and interfering with the pollination experiments the stems were chalked
near the middle. The dormant buds in and just below the chalked areas started
growing. The photograph was taken April 5, the stems being rechalked over the same
areas that were originally chalked. After numerous repetitions of the experiment
it was found that if the chalking was done lightly the buds would not grow, but if
the stems were rubbed hard in the process of chalking, as commonly happened in the
case of very smooth stems, the buds grew. It was the hard rubbing, not the chalk,
that stimulated the growth.
B. — Dormant blueberry bud stimulated to growth by rubbing the stem. The
photograph, which was taken June 14, 1913, shows a single bud starting into growth
on a dormant blueberry plant. The dark area just above the bud is a brown band
on an otherwise green stem. It shows the position of a rubbing that was given the
stem with a smooth knife handle a few weeks earlier. This bud afterwards grew into
a long, vigorous branch, while all the other buds remained dormant.
Influence of Cold in Stimulating Growth of Plants
Plate 26
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 27
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 27
A. — Normal spring growth on a blueberry stem. This illustration is from a photo-
graph taken April 24, 1909. In the preceding season the plant had sent up an
unbranched shoot. After an outdoor chilling through the winter and early spring
it put out flowers and new twigs as shown in the illustration. The fact to be especially
noted is that the new growth on this stem took place from numerous buds.
B. — Abnormal spring growth on a blueberry stem, due to lack of chilling. This
photograph was taken on May 19, 1913. Growth is taking place from only one bud,
the third from the tip. The uppermost bud is a flowering bud, the second a leaf
bud. Both are dead or dying. This plant had stood in the warm greenhouse all
winter and spring. If it had had the usual two to three months' chilling its starch
would have been transformed into sugar and the stem would have flowered and put
out new twig growth from numerous buds in the same manner as the stem shown in A.
187932°— 20 7
PLATE 28
Blueberry leaf exuding sugar from glands interpreted as osmotic-pressure safety
valves.
This is a leaf of the highbush blueberry, Vaccinium corynibosum. The photograph
was taken May 19, 1916. The sugar-secreting glands, sometimes called extra-floral
nectaries, are situated in this plant on the back of the midrib and along the margins
of the leaf, toward its base. The drops of sugar solution have been wiped away from
the glands on the left-hand margin and from two glands on the midrib at the base of
the second and fourth lateral veins above the sugar drop shown near the middle of
the picture. X 4.
Influence of Cold In Stimulating Growth of Plants
Plate 28
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 29
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 29
A plant of bunchberry, Cornus canadensis, the seeds of which do not germinate
without chilling.
Bunchberry seeds sown October 9, 1912, and chilled during the winter germinated
promptly the following spring. Another lot of the same seeds sown on the same date
but kept in a greenhouse at a temperature of not less than 55° F. showed no germina-
tion in 12 months. These seeds were then chilled for 2 months at a temperature of
350 to 400 F., and when brought back into the greenhouse they germinated within
a month. The very healthy plant shown in the illustration grew from one of these
long-dormant seeds. The exposure of seeds to winter weather is sometimes practiced
by gardeners, but they usually attribute its beneficial effect to freezing, which in all
the cases tried in these experiments is unnecessary.
PLATE 30
A. — Trailing arbutus, Epigaea repens, flowering sparingly from lack of chilling.
This plant of trailing arbutus was grown from seed. In the autumn, when about
a year old, it laid down clusters of flowering buds. It was kept in a warm green-
house all winter, but when flowering time came most of its flower buds were dead
and brown. Only a single flower opened.
B. — Trailing arbutus plant flowering normally after chilling. This plant had the
same history as the plant described under A, except that it was kept outdoors during
the winter and brought back into the greenhouse in the spring. At the age of 14
months, when the photograph was taken, March 27, 1011, the plant was in full flower,
healthy and normal.
C — Blueberry plant forced into flower in September by artificial chilling. This
plant was brought indoors in late winter. It made new growth, and during the cool
weather of May it laid down flowering buds for the next year, as a blueberry plant
ordinarily does in autumn. During the summer, however, the plant was given an
artificial winter by chilling it for three months in an artificially refrigerated glass-
covered frame exposed to daylight. When brought out of the frame, in September,
the plant promptly flowered, as shown in the illustration.
Influence of Cold in Stimulating Growth of Plants
Plate 30
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 31
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 31
A. — Abnormal growth of an unchilled blueberry plant. This plant became dormant
in the autumn in a warm greenhouse, and since it was not chilled it continued its
dormancy through spring and summer for a period of nine months. Then three of its
stems began to die at the tips and, following this, growth began to take place from a
single bud next below the dying tip on each stem. For the explanation of this ab-
normal activity see p. 156. The photograph was taken October 12, 1916.
B. — Awakening of long dormant plants by artificial chilling. The illustration
consists of two photographs of the same plant. At the left is shown the condition of
the plant on December 26, 1916, after more than a year of warmth and dormancy.
The figure at the right, from a photograph taken April 27, 1917, shows the appear-
ance of the plant after it had been subjected to artificial chilling for a period of
three months and then had been returned to the warm greenhouse. It began to
put out new growth from 10 or more of its leaf buds. Even after its extraordinarily
long period of dormancy the plant had been brought back to normal activity by a
suitable period of chilling.
PLATE 32
Plants brought out of dormancy at a specified time.
A. — Blueberry plants from a lot that had been kept in a dormant condition by
warmth for nearly a year. On October 30, 1917, plants from this lot were placed
under chilling conditions at a temperature of about 350 F. At the end of a month's
chilling eight plants were taken out, repotted, and brought into a greenhouse main-
tained at a temperature of 500 to 700 F., and after two months' chilling eight other
plants were brought out.
B. — Representative plants from each of the two chilled lots described under A, from
photograph made January 18, 1018. The plant at the left, which was kept under re-
frigeration for a month, was only imperfectly chilled, and although it started growing
the growth was from abnormally few buds. But the plant at the right, under refrigera-
tion for two months, was adequately chilled and started into growth from many buds
in a normal manner. It is evident that by the proper application of this procedure
a plant of this nature can be brought into proper condition for shipment and planting
on any date desired.
Influence of Cold in Stimulating Growth of Plants PLATE 32
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 33
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 33
A. — Plantation at Whitesbog, N. J., for the testing of blueberry hybrids. From
very carefully selected wild blueberry plants hybrid seedlings are raised in the green-
houses of the Department of Agriculture at Washington. In order to bring them into
fruit under favorable outdoor conditions so that selections of the best hybrids can be
made for further propagation, the young seedlings are sent to a plantation at Whitesbog,
4 miles east of Browns Mills, in the pine barrens of New Jersey. In the photograph
2-year-old hybrids are shown at the right and 3-year-olds in the row at the left.
B. — Four-year-old blueberry hybrid in full fruit. This illustration shows the
vigor, beauty, and productiveness of a hybrid blueberry bush when it is given the
proper and peculiar conditions which by its nature it requires for successful growth.
From a ^3-acre patch of hybrid bushes a yield of berries was secured in 1919 at the rate
of 96 bushels per acre. They sold at a little over $10 a bushel, bringing gross receipts
at the rate of $966 per acre. In 1920 this planting yielded at the rate of 117 bushels
per acre, which sold at a little less than $11 a bushel, yielding gross receipts at the
rate of $1,280 per acre.
PLATE 34
The ordinary wild blueberry of New Jersey.
This is a photograph, natural size, of a quart box of wild New Jersey blueberries,
rather better than the average. It was taken for the purpose of comparison with the
selected hybrid blueberries shown in Plate 35.
Influence of Cold in Stimulating Growth of Plants
Plate 34
Journal of Agricultural Research
Vol. XX, No. 2
Influence of Cold in Stimulating Growth of Plants
Plate 35
Journal of Agricultural Research
Vol. XX, No. 2
PLATE 35
Fruit of a selected hybrid blueberry.
This illustration shows in natural size a quart box of blueberries from a hybrid
produced at Washington and fruited at Whitesbog. The photograph represents the
average product of the bush, for it was taken from a clean picking, including the
small berries as well as the large ones. Hybrids with berries of still larger size have
been fruited at Whitesbog.
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Vol. XX NOVEMBER 1, 1920 No. 3
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Fag*
Composition of Normal and Mottled Citrus Leaves - 161
W. P. KELLEY and A. B. CUMMINS
(Contribution from California Agricultural Experiment Station )
Control of Fluke Diseases by Destruction of the Inter-
mediate Host ----- _ 193
ASA C. CHANDLER
(Contribution from Oregon Agricultural Experiment Station 1
Injury to Seed Wheat Resulting from Drying after Dis-
infection with Formaldehyde ----- 209
ANNIE MAY HURD
(Contribution from Bureau of Plant Industry)
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE.
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, L>. C.
WASHIMOTOtt I COVeUKWCKT PBINTINQ OfFMB : I MM
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Station*
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS,
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
*■*»***?
JOURNAL OF AGRKMIAL RESEARCH
Vol. XX Washington, D. C, November i, 1920 No. 3
COMPOSITION OF NORMAL AND MOTTLED CITRUS
LEAVES1
By W. P. KELLEY and A. B. Cummins, Citrus Experiment Station, College of
Agriculture, University of California
INTRODUCTION
Knowledge concerning the composition of a plant is essential to an
understanding of its growth. The amounts and proportions of the
different constituents absorbed from the soil or other nutrient medium,
as revealed by accurate analysis of the several parts of plants, undoubt-
edly give some indication concerning their nutritional requirements. If
determined progressively, such data may contribute to a clearer under-
standing of fundamental physiological processes of growth.
The interpretation of plant analyses, so far as growth processes and
requirements are concerned, demands great caution, however. Many
plants undoubtedly have the power of adapting themselves to a wide
range of soil variations; and the composition of the plant, owing to
selective absorption, commonly bears little direct relation to the com-
position of the nutrient solution. It is well known that the concentration
of a given constituent in the nutrient solution may be varied considerably
without producing any material change in the composition of the plant.
The effect of an excess or deficiency of one ion on the absorption of
other ions, and especially the effects of nonessential salts on the absorption
of essential ions, have not been sufficiently studied. Despite the many
investigations during recent years on antagonism, comparatively few
analyses have been made showing the effects on absorption. Likewise,
investigations on the so-called nutritional or physiological diseases have
not dealt with absorption specifically, except to a very limited extent.
Previous studies on the rate of absorption of nutrients have been con-
ducted mainly with annual plants, chiefly cereals, very limited study
having been devoted to trees. There is much need for accurate data on
the several phases of absorption as related to the growth of fruit trees.
1 Paper No. 67, University of California, Graduate School of Tropical Agriculture and Citrus Experiment
Station, Riverside, Calif.
Journal of Agricultural Research, Vol. XX, No. 3
— Washington, D. C Nov. 1, 1920
vh Key No. Calif.-»4
(161)
1 62 Journal of Agricultural Research voi.xx,No.3
In connection with investigations on the nutrition of different species
of citrus trees, especially as related to that condition known as mottle-
leaf, we have determined the composition of different parts of the tree,
such, for example, as the roots, old wood, young wood, leaves, leaf sap,
and fruit. This work has extended over a period of several years, and
further study is contemplated. Some of the results already obtained
have proved to be of special interest. The present paper will be devoted
mainly to a discussion of the composition of the leaves.
It is not necessary to review the many published analyses of citrus
fruits. Most of the publications on this subject have dealt mainly with
the organic constituents and total ash, with an occasional analysis of
the ash. Comparatively few analyses have been published showing the
composition of portions of citrus trees other than the fruit.
The earliest investigation we have been able to find, and perhaps the
best known, is that of Rowney and How {15) 1, published in 1848. Anal-
yses were reported of the roots, stems, leaves, and fruit of orange trees,
Citrus aurantium, grown on the island of St. Michael. The variety was
presumably that now known as St. Michael.2 The analyses were ex-
pressed as percentages of the carbon-dioxid-free ash. The results were
similar to our analyses of California orange trees, when calculated to the
same basis.
In 1 891 Oliveri and Guerrieri (13) published an extended study on the
composition of the wood, leaves, and different portions of the fruit of
the orange, Citrus aurantium Riss;2 Mandarin, C. nobilis var. deliciosa,
Swingle; and lemon, C. limonia Osbeck, grown in Palermo, Italy. This
investigation, extending over a period of three years, is the most com-
plete study yet published on the composition of different parts of citrus
trees. They recorded the number and weights of fruits produced by
different classes of trees and the number and weights of leaves and the
weights of wood pruned from the trees during a period of three years,
representative samples of which were analyzed. Some of their analyses
also agree reasonably closely with our data.
In 1 001 Alino (1) determined the phosphoric acid, potash, and nitrogen
content of orange wood, leaves, and fruit; and in 1909 Muller (12) pub-
lished complete analyses of seedling orange leaves from healthy and
diseased trees grown in South Africa.
In 1 910 Blair (2) analyzed orange leaves and stems grown in Florida.
His samples represented the new growth taken in October from certain
plots of a fertilizer experiment. In 1917 Jensen (7) published a paper
on the composition of normal and mottled orange, lemon, and grape-
1 Reference is made by number (italic) to "Literature cited," p. 190-191.
2 In this case, the sweet orange. Citrus sinensis Osbeck, is doubtless the species studied. W. T.
Swingle's revision of citrus nomenclature, as given in the "American Standard Cyclopedia of Horticulture,"
is followed in this paper.
Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 163
fruit (Citrus grandis Osbeck) leaves grown in California. Further
reference will be made to this paper later.
As is well known, the composition of annual herbaceous plants depends
on their age. It has been shown that the ash content and the proportions
of the individual constituents absorbed from the soil change as growth
proceeds. Of the changes in perennials much less is known. It seems
reasonable to suppose, however, that the growth processes are similar.
The periodically developing new shoots may be likened to the portion
of annual plants growing above ground.
New shoots appear on citrus trees several times each year. The tree,
being evergreen, bears leaves at all seasons. Consequently, the foliage
is composed of leaves of different ages. A given leaf ordinarily remains
on the tree for a period of from two to three or more years.
SELECTION OF SAMPLES
Special care has been taken to secure representative samples of leaves
of known age. Familiarity with the appearance of developing citrus
leaves proved to be a material aid in selecting the samples. A consider-
able portion of the samples were obtained from trees growing near the
laboratory where daily observations were made. The leaves of the
Washington Navel and Valencia orange, the Eureka lemon, and the
Marsh seedless grapefruit have been analyzed. Each sample was com-
posed of several hundred leaves, collected from six or more adjacent
trees, all of which were reasonably uniform in appearance and the culture
and fertilization of which had been the same. The trees were 10 or more
years of age. The entire leaf, including the petiole, was analyzed as a unit.
The samples were picked from the trees, placed in tight bags and
immediately taken to the laboratory and weighed. In most cases this
procedure did not require more than 30 minutes. In order to remove
dust and other adhering foreign material, the leaves were thoroughly
cleaned by wiping each leaf with a moist cloth, but washing with water
was necessary with a few samples heavily coated with dust or showing
evidences of residues from previous spraying. Early in this work it was
found that the samples from which the dust had not been completely
removed contained abnormally high percentages of silica, alumina, iron,
and inorganic materials not soluble in dilute hydrochloric acid.
METHODS OF ANALYSIS
The samples were dried at 1050 C. for 24 hours, and the loss in weight
was calculated as moisture. The dry samples were ground to a powder in
a small hand mill, were thoroughly mixed, and were then stored in
tightly stoppered bottles for analysis.
Total nitrogen was determined by the official Kjeldahl method, modified
to include nitrates. Total sulphur was determined by the sodium-peroxid
164 Journal of Agricultural Research voi.xx,No.3
fusion method. The fusions were made over alcohol flames, and the
sulphate was precipitated as barium sulphate, usually from the solution
of the entire mass used in making the fusion. Total phosphorus was
determined by treating 1 to 2 gm. of the dry material with a solution of
magnesium nitrate, evaporating to dryness, igniting, and proceeding
in the usual manner. Chlorin was determined in a special portion of the
ash made by igniting at a low heat 5 to 10 gm. of the dry material, dis-
solving the residue in dilute nitric acid, and proceeding with the Volhard
volumetric method. In some cases chlorin was also determined by per-
forming the incineration in the presence of an excess of sodium carbonate
in order to avoid the possible loss of chlorin, but the results of the two
methods were similar.
For the determination of total ash, 10 to 20 gm. of the dry samples
were incinerated in porcelain dishes over Bunsen burners. The material
charred easily and burned quietly upon the application of low heat and
was reduced to a gray ash without approaching dull redness. The
residue was then allowed to cool, was taken up with hot water, trans-
ferred to a filter, and washed thoroughly. The insoluble material with
its filter paper was transferred to a platinum dish, dried, pulverized
with an agate pestle, and heated to full redness. When the platinum
dish cooled, the filtrate from the previous leaching was added and
evaporated to dryness. Ten to 20 cc. of strong ammonium-carbonate
solution were then added, and the treatment was repeated until the
ash was completely carbonated, as was indicated by constant weight
upon evaporating to dryness and heating gently. The results are re-
corded as percentages of ash. It should be stated that the ash thus
obtained differs from that reported by other investigators in that we are
dealing with completely carbonated ash, whereas previous analyses of
citrus leaf ash have been calculated to a carbon-dioxid-free basis.
The ash was dissolved in water and dilute hydrochloric acid, and the
solution was evaporated to complete dryness on the water bath in order
to dehydrate the silica. The amount of uncombined carbon found in
the ash was always entirely negligible. The residue was taken up with
warm water and dilute hydrochloric acid. The silica was determined
by the loss in weight occasioned by treating the incinerated residue with
hydrofluoric acid. The material nonvolatile in hydrofluoric acid usually
amounted to only 0.1 to 0.2 per cent of the ash and was neglected in
this work. The filtrate from the silica separation was made up to a
definite volume, usually 500 cc, and the various constituents were
determined in aliquots representing from 0.2 gm. to 0.4 gm. of the ash.
The methods of the Association of Official Agricultural Chemists '
were used with slight modifications, as noted. Iron, aluminum, and
■Wiley, H. W., ed. official and provisional methods of analysis, association of official agri-
tural chemists. As compiled by the committee on revision of methods. U. S. Dept. Agr. Bur. Chem.
Bui. 107 (rev.), 272 p., 13 fig., 190S. Reprinted in 1912.
Nov. 1. 19:10 Composition of Normal and Mottled Citrus Leaves 165
phosphoric acid were precipitated collectively by adding a weighed
excess of ferric chlorid, neutralizing with ammonia, filtering, redissolving
in dilute hydrochloric acid, and repeating the process. Iron was pre-
cipitated with ammonia from a separate aliquot and determined volu-
metrically by reduction with zinc and titration with permanganate.
This method was occasionally supplemented by the ferrocyanid colori-
metric method with fairly satisfactory results. Aluminum was calculated
by difference after the phosphoric acid was gravimetrically determined
in a separate aliquot. Calcium, magnesium, potassium, and sodium were
determined in the filtrate after the ammonia precipitate was removed,
and in some cases manganese was determined by bromin oxidation.
Sulphate was determined gravimetrically in an aliquot of the original
solution. Carbon dioxid was not determined.
COMPOSITION OF NORMAL MATURE ORANGE LEAVES
A considerable number of analyses have been made of mature orange
leaves representing both the Washington Navel and Valencia varieties.
Owing to the absence of previous records showing the age of the leaves
available for analysis, and in view of the fact that orange leaves, when
from 4 to 6 months of age, assume an appearance not unlike that of
leaves 1, 2, or more years of age, it is highly probable that random sam-
ples will always represent mixed ages.1 Most of our samples of mature
leaves were taken at random, always avoiding immature or abnormal
individuals. The samples were gathered at different seasons of the
year and from a considerable number of different sets of trees, some of
which were growing in different localities. Typical analyses are sub-
mitted in Tables I and II.
It is interesting to note that the composition of the different samples
was found to be reasonably uniform despite the fact that their average ages,
although they were mature in appearance, probably varied considerably.
Other samples not reported above showed a similar composition. The data
also afford but little evidence of seasonal variation in composition.
Except in calcium and potassium content, the different samples of
the same variety differed almost as widely in composition as the samples
of different varieties. The samples from different localities were also
similar in composition, although those from Riverside were grown on
sandy loam soil, that from Anaheim on light sandy soil, and the one from
Whittier on heavy adobe.
It will be noted that the average calcium content of Valencia leaves
was found to be somewhat higher than that of Navels, while the reverse
is true for potassium.
'Ensign (<5) has recently shown that the size of the vein islets of Citrus grandis is directly correlated with
the maturity of the leaf. From the most immature to fully matured leaves there is a gradual increase in
the size of the vein islets. If further investigation prove that similar relations occur in other species of
citrus, a direct means will be afforded by which the age of the leaves can be determined.
i66
Journal of Agricultural Research
Vol. XX, No. 3
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Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 1 67
Throughout this work we have determined the aluminum. Quali-
tative tests usually indicated this element to be present, but the quantity-
was never more than a few tenths of 1 per cent of the ash. Frequently
the amount was undeterminable. The manganese was also determined in
several samples. The amount was found to vary from 0.1 per cent to
0.2 per cent of the ash.
The size of the leaves as gauged by their average weights was recorded,
but there appears to be no consistent difference in composition referable
to the size of the leaf. As is well known, the size of apparently normal
orange leaves may vary widely. Even on a given tree, the fully mature
leaves of certain cycles of growth may be at least twice as large as others.
From the analysis of many other samples in this laboratory it may be
said that the composition of mature orange leaves when grown in Cali-
fornia is remarkably uniform, provided, however, that the leaves be
borne on vigorous trees. On the other hand, the composition of the leaves
of improperly nourished and diseased trees may vary widely. If the
supply of available nitrate be deficient, the content of nitrogen in the
leaves may be considerably below that reported above, but there seems
to be some doubt whether the reverse is true.
COMPOSITION OF LEMON AND GRAPEFRUIT LEAVES
The analysis of mature Eureka lemon and Marsh seedless grapefruit
leaves is submitted in Tables TTI and IV.
Two of the samples of lemon leaves were collected in midwinter and
the other on August 29. They were grown on widely different types
of soil. The Riverside sample grew on sandy loam, the Whittier sample
on heavy adobe, and the Tustin sample on highly calcareous sandy loam
soil. The grapefruit leaves were grown on sandy loam.
The composition of the different samples of lemon leaves is fairly
uniform, the average being similar to the average composition of Valencia
orange leaves. On the other hand, the composition of the grapefruit
leaves closely resembles that of Navel orange leaves.
The composition of the leaves of the different varieties and species of
citrus has been found to be remarkably uniform from the standpoint of
both the ash and the dry matter. A more detailed discussion of the
composition will be given below.
1 68
Journal of Agricultural Research
Vol. XX, No. 3
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Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 169
COMPOSITION OF ORANGE LEAVES AT DIFFERENT STAGES OF GROWTH
The results obtained from the analysis of samples of leaves approxi-
mately one month of age, gathered on May 11, 1917, were found to be
considerably different from previous analyses of mature leaves. Samples
representing the new spring growth and that of the previous year, gathered
from the same trees on May 21, 191 7, also proved to be widely different
in composition. These results, together with the discordance between
the analyses previously made in this laboratory and those published by
Blair (2) from Florida and by Jensen (7) from California, suggested the
desirability of making a study on the composition of orange leaves at
different stages of growth.
Samples were collected at four different intervals in the growth cycle.
The first represented leaves approximately 1 week old; the second, those
6 to 8 weeks old; the third, leaves at full maturity, the ages of which
ranged from 6 months to approximately 2 years; the fourth, old leaves
that were about to be shed, as indicated by their yellowish brown color.
Each sample was picked from six normal, vigorously growing trees of
plot V at the Citrus Experiment Station, Riverside, Calif. The samples
representing different ages were all taken from the same trees, and those
representing the first three periods of growth were gathered on the same
day, November 9, 1917. These trees support an abundant foliage; and,
as frequently occurs, they at that time bore numerous shoots of varying
ages, ranging from a few days to 2 or more years of age, which made it
possible to secure samples of widely different ages on a given day. The
samples of old leaves were gathered December 10, 1917.
The data expressed as percentages of the ash show that notable
changes take place in the relations of certain constituents as growth
proceeds. Especially prominent among these changes are the decreases
in the percentages of phosphate and potassium, on the one hand, and the
increases in calcium on the other. For example, the ash of navel
leaves at the age of 1 week was found to contain 16.83 Per cent phosphate
(P04), at 6 weeks 7.10 per cent, at maturity 2.47 per cent, while the ash of
old leaves contained only 1.32 per cent.
The changes in the percentages of potassium were quite parallel to
those of phosphate. When navel leaves were 1 week of age, the ash
contained 19.87 per cent potassium, when 6 weeks of age, 10.32 per cent,
when mature, 5.68 per cent, while the old leaves contained only 1.66
per cent.
The percentages of calcium underwent changes quite opposite to those
of potassium. With the ash containing 20.72 per cent calcium when
the leaves were 1 week old there was an increase to 28.44 Per cent
at 6 weeks, to 33.21 per cent at maturity, and finally to 34.41 per cent
in the very old stage.
170
Journal of Agricultural Research
Vol. XX, No. 3
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Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 171
Among the other necessary nutrients, the percentages of iron, magne-
sium, and sulphate decreased with age, although to a lesser degree than
potassium and phosphate. The ash of the youngest leaves contained
approximately twice as much iron as that of the mature leaves, and
differences almost as great occurred in the percentages of magnesium
and sulphate.
As was anticipated, the changes that take place in Valencia orange
leaves are quite similar to those of navel leaves.
The percentages of phosphorus and sulphur refer to the total amounts
as determined by the magnesium-nitrate and sodium-peroxid fusion
methods, respectively, and are somewhat higher than the corresponding
data calculated from the ash analyses. As is well known, organic
materials usually lose a portion of their phosphorus and sulphur in the
ashing process.
It will be noted that the content of water decreased considerably as
growth took place. At 1 week of age the navel leaves contained 72.31
per cent water, at 6 weeks 70.81 per cent, at maturity 60.98 per cent,
and the very old leaves still contained 60.73 Per cent. The content of
total ash, on the other hand, increased markedly with age, rising from
6.54 per cent of the dry matter at the age of 1 week to the very high
content of 21.39 Per cent in the old leaves.
The nitrogen decreased from 3.01 per cent at the age of 1 week to 2.39
per cent at maturity, and finally to 1.31 per cent in the old stage. The
percentage of phosphorus decreased still more rapidly during the actively
growing period, but later the phosphorus content remained approxi-
mately constant. The percentage of potassium also decreased rapidly
during the early period of growth but remained almost constant after
the second period until the period of senility approached, when a still
further decrease took place.
The percentage of iron in the dry matter was found to be reasonably
constant at all stages of growth. However, in considering the iron con-
tent of these and all other samples reported herein, it is important to
bear in mind that the analytical error involved in the determination of
small amounts of this element is likely to be relatively great. For this
reason small variations in the results are probably not significant.
The percentages of sulphur and magnesium each increased somewhat
as growth took place.
The constituent of the dry matter of orange leaves that undergoes the
greatest percentage change as a result of growth is calcium. At 1 week
of age, the navel leaves contained 1.36 per cent calcium, at 6 weeks
2.62 per cent, at maturity 5.63 per cent, and the very old leaves contained
7.36 per cent.
Of the supposedly unessential constituents, the greatest concentration
of sodium was found in the young leaves; but the amount was always
small, while the data for silica and chlorin show no consistent variation.
172 Journal of A gricultural Research vol. xx, no. 3
It is interesting to note that in certain respects the composition of
orange leaves changes with growth, somewhat as is the case with the
vegetative portion of other plants. With certain cereals a considerable
portion of the potassium, magnesium, phosphorus, and nitrogen migrate
from the leaves into other parts of the plant as maturity approaches (9,
10). The potassium tends to accumulate in the straw of rice, while the
magnesium, phosphorus, and nitrogen are translocated to the grain.
The composition of citrus leaves differs markedly from that of cereals
in certain other respects. The ash content of the former increases much
more rapidly and reaches a very high point in the old leaves. The cal-
cium content increases very rapidly during the most actively growing
period and continues to be deposited in the leaves, although at a some-
what slower rate, almost until the time the leaves fall off.
While it is probable that the composition of normal orange leaves
varies to some extent when grown in different parts of the world or on
different soils in a given locality, careful study of the analyses of the
Florida-grown leaves published by Blair (2) and those reported from
Italy by Olivieri and Guerrieri (13) suggests that these were immature
leaves. From Jensen's results (7), it is evident that his samples were
not composed of mature leaves. Recognition of the relationships be-
tween the age and the composition of orange leaves is especially im-
portant in the study of the composition of mottled leaves, as will be
pointed out more fully later.
It does not necessarily follow from the preceding discussion that a por-
tion of a given element .potassium, for example, migrates back into other
parts of the tree after the leaves reach a certain stage of development.
Increase in the size of a leaf, owing to the elaboration of carbonaceous
matter, may dilute the nutrients present and, therefore, lower the per-
centage without there being an actual loss. To establish this point, it is
necessary to determine the weights of the constituents present per leaf at
different periods. From the average weights of the individual leaves at
each period we have calculated the content of the different constitu-
ents, expressing the results in grams per 1,000 leaves. (Table VII.)
The old Navel leaves were considerably smaller on the average than
either those representing maturity or 6 weeks of age, while the mature
Valencia leaves were larger than the old leaves of the same variety. In
addition, the leaves of each sample of the Valencia variety were consider-
ably larger than the corresponding Navel leaves.
Despite these irregularities in the size of the leaves, the data show that
the content of calcium in a given orange leaf increases very rapidly during
the early part of the growth period. In the Navel leaves, approximately
a tenfold increase in calcium content took place between the first and the
sixth week of age. From the sixth week to maturity a further increase,
more than twofold, took place, and finally the calcium content increased
still further as the leaves approached the time of normal dropping.
Nor. i. iswo Composition of Normal and Mottled Citrus Leaves 1 73
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174 Journal of Agricultural Research voi.xx,No.3
The rates of increase in magnesium and sulphur are also rapid during
the early part of the growth period, and each of these constituents con-
tinues to accumulate in the leaves up to maturity, but the absolute
amounts never become high. Since irregularities occurred in the size
of the leaves, it is doubtful whether any important amount of either
magnesium or sulphur is translocated to other portions of the tree after
maturity has been reached.
The maximum amounts of potassium, phosphorus, and nitrogen were
deposited before the leaves were 6 weeks of age. The rates of increase
of each were considerably less than that of calcium. The data show that
a considerable portion of these elements migrates away from the leaves
after certain periods. With potassium and nitrogen the loss takes place
after maturity has been reached, while the phosphorus begins to recede
even before maturity is attained.
Similar data for iron are omitted because of the magnitude of the ana-
lytical error involved in its determination.
Samples representing more frequent intervals in the growth cycle
would certainly afford more detailed information regarding absorp-
tion. It is possible that the analysis of such samples when plotted
might show breaks in the curves not indicated by the existing data.
For example, the exact period in the growth cycle when the leaves con-
tained the maximum amount of potassium might be shifted to some
extent and other fluctuations might also be found. However, other
analyses of immature orange leaves at different seasons of the year show
a fairly close agreement with those reported above. On the whole, we
are inclined to believe that the main features of the composition of the
orange leaf have been determined.
It seems appropriate to emphasize the fact that citrus leaves are ex-
tremely calcareous, and much more so than most of the economic plants.
As is well known, the ash of some of the legumes contains high percentages
of calcium, but relatively few have been reported to contain as high per-
centages of calcium as citrus leaves. Not only is the ash of citrus leaves
high in calcium but the total ash content is high also. It is unusual to
find dried plant material that contains from 5 to 7 per cent calcium.
COMPOSITION OF MOTTLED ORANGE LEAVES
The condition of citrus trees known as mottle-leaf has been widely
discussed. Much study has already been devoted to it, and several
hypotheses have been advanced concerning the disease. The symptoms,
mode of occurrence, and general distribution were fully discussed in a
paper by Briggs, Jensen, and McLane (5). The disease is commonly
thought to result from some nutritional disturbance, but the cause has
not been definitely determined.
Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 175
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We have analyzed different portions of orange and lemon trees affected
with mottle-leaf, as well as grapefruit leaves and samples representing
different degrees of mottling. Most of the samples were collected from
the fertilizer plots of the Citrus Experiment Station. In all cases the
leaves were collected from shoots 6 or more months of age. The analysis
of orange leaves in an advanced stage of mottling is presented in Tables
VIII and IX.
Comparison of the data with the previously submitted analyses shows
at once that the composition of mottled leaves differs considerably
from that of average mature normal leaves. The principal differences
are found in the greater percentages of potassium and phosphate, on
the one hand, and the lesser percentages of calcium on the other. The
ash of mottled leaves also contains greater percentages of magnesium
and sulphate, while the iron, silica, sodium, and chlorin do not differ
materially.
Considerable variations will also be noted among the different samples
of mottled leaves. This is probably due to the varying degrees of
mottling represented by the samples. However, every sample of
mottled leaves that has been analyzed in this laboratory has been found
to vary from the normal in the same general direction.
The average content of water in mottled leaves was found to be
slightly higher than in normal leaves and the ash content somewhat
lower. Considering the dry matter, the most pronounced differences
are found in the lesser calcium content, on the one hand, and the abnor-
mally high percentages of potassium and phosphorus in mottled leaves,
on the other. The average nitrogen content of mottled leaves is also
considerably above normal, as was previously pointed out by McBeth (//).
From his analyses of normal and mottled citrus leaves, Jensen (7)
failed to find any consistent difference in composition. In order to
insure uniformity in the age of his samples, he collected the leaves from
the current season's growth. On the dates two of his samples were
collected, April 18 and May 11, the current season's growth is probably
never mature at Riverside. Furthermore, the calcium content, which
he reported, was very much below that of any mature normal orange
leaf we have been able to find. It seems safe to conclude, therefore, that
Jensen's studies were made with immature leaves. It is possible, of
course, that the variations in composition incident to mottling may
not occur until after the leaves have reached a certain stage of growth,
although recent analysis of a sample of leaves about 10 days of age,
taken from severely mottled trees, indicates that the composition may
begin to diverge from the normal at a very early period.
It is well known that, with the exception of severe cases of mottle-leaf,
the discoloration ordinarily does not become apparent until the leaves
have reached an age of 2 to 3 months. Subsequently, the degree of
discoloration becomes increasingly intense until the period of normal
Nov. i, 1930 Composition of Normal and Mottled Citrus Leaves 177
maturity. In addition, mottle-leaf is usually most pronounced from
September to February, when it becomes very noticeable on the leaves
of the previous spring and summer cycles of growth.1
Some light may be thrown on mottle-leaf by comparing the compo-
sition of mottled leaves with that of normal leaves at different stages
of growth. By reference to Tables V and VIII it will be seen that the
composition of the ash of the former is quite similar to that of normal
leaves approximately 6 weeks of age, although the total ash content of
mottled leaves is considerably higher (compare Tables VI and IX).
It is especially interesting to note that the nitrogen content of mottled
leaves is somewhat higher than that of normal leaves at the age of 1
week and much greater than that of normal leaves at the age of 6 weeks.
The data indicate, therefore, that the essential nutrients are depos-
ited in mottled orange leaves at abnormal rates. A satisfactory expla-
nation of this fact can not now be given. The rising sap is itself probably
abnormal in composition.
By calculating the weights of the several constituents contained in
a unit number of mottled leaves, it is found (Table X) that notwith-
standing the fact that the average size of the mottled leaves was less
than one-half that of normal leaves they contained as great amounts of
potassium and approximately as much phosphorus per leaf (compare
Tables VII and X). On the other hand, the content of calcium was
less than one-third as great as normally occurs, while the magnesium,
sulphur, and nitrogen were intermediate in amount.2
The preceding analyses represent extreme cases of mottling. Sam-
ples of Valencia orange leaves at a less advanced stage have also been
studied. These latter were of an intermediate size, showing the typical
yellowish spots between the veins. They were selected from trees a
considerable portion of whose foliage was normal and some of which
bore a fair crop of fruit. The results are recorded in Table XI
The percentages of calcium and potassium closely approach those of
severely mottled Valencia leaves (Tables VIII and IX), but the phos-
phorus content is more nearly normal. The percentage of nitrogen was
found to be no greater than occurs in normal Valencia leaves.
Thus, it appears that the early stages of mottling are first attended by
the absorption of subnormal amounts of calcium 3 and supernormal
amounts of potassium and phosphorus, and that modifications in the
absorption of nitrogen occur later.
'Mottled leaves fall off in large numbers during the latter part of the -winter and early spring. New
shoots developing at this season give the trees the appearance of having recovered from the disease. These
latter, however, may become mottled the following fall. It is never safe to pass judgment on the state
of the disease in the spring or early summer. We have never known of a leaf once severely mottled which
became normal later. New leaves grown later, however, may be entirely normal.
2These data were calculated for only a portion of the samples of mottled leaves, because the average
weight of the leaves was not determined for all the samples.
» Jensen (7) found that the yellow spots of mottled orange leaves, similar to those discussed here, contain
less calcium than the remaining portion of the leaf.
9507°— 20 2
i7»
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Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 1 79
Severely mottled lemon and grapefruit leaves have also been analyzed
(Tables XII and XIII).
The results show that the composition of mottled lemon and grape-
fruit leaves is similar to that of mottled orange leaves. As was found
from the analysis of normal leaves, the composition of lemon leaves
closely resembles that of Valencia orange leaves, while the composition
of grapefruit leaves was found to be like that of Navel leaves. How-
ever, the different varieties and species do not vary greatly in com-
position.
The fact that the composition of the leaves of one species of citrus is
affected in the same general way as that of other species is not surprising,
since their appearance when mottled is also similar.
As is well known, it is rare that all the leaves on a given orange tree
are mottled. As a rule, those growing on the outer portions of the tree
are the most severely affected, as sometimes, although not invariably,
is the case with the leaves borne on the south and southeastern portion
of the trees. The leaves of severely affected trees, however, may be
mottled throughout the tree. Frequently the greater portion of the
leaves borne by the shoots of a given growth cycle may be mottled, while
those immediately preceding and following this cycle may be entirely
normal in appearance. It is interesting, therefore, to compare the com-
position of normal and mottled leaves from the same tree.
With this end in view, samples of normal-appearing leaves were col-
lected from the same trees from which some of the previously discussed
samples of mottled leaves were drawn and on the same days. The
analyses are reported in Tables XIV and XV.
The data are concordant with the previously reported analyses of
normal leaves (Tables I and II). The results suggest that the leaves of
different cycles of growth are mutually independent in composition and
that the peculiarities in the composition of mottled leaves are not due to
any special peculiarity of the tree upon which they have grown. A leaf
of normal appearance borne by an orange tree the major portion of
whose foliage is severely mottled, as were some of these samples, has
approximately the same composition as any other normal orange leaf.
Some study has also been devoted to citrus trees affected by chlorosis1
and injured by alkali, the results of which will be presented elsewhere.
The composition of albino and etiolated plants is of interest in this
connection. Church (4, 5) analyzed the normally green and albino por-
tions of the maple (Acer negundo), holly {Ilex aquifolium) , ivy (Hedera
helix), and several other species. He found that the albino portions
uniformly contained greater amounts of water than the green portions.
The ash of the former contained greater amounts of potash and phos-
phoric acid and lesser amounts of lime than the latter, while the content
of iron was approximately the same.
1 Chlorosis of citrus, as it occurs in California, is distinguishable from mottle-leaf by a general fading of
the chlorophyl over the entire mesophyl tissue, while mottle-leaf, as the name implies, denotes the lack of
chlorophyl in spots between the veins.
i8o
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Nov. i. i9ao Composition of Normal and Mottled Citrus Leaves 181
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Journal of Agricultural Research
Vol. XX, No. 3
Palladin (14) also found that the composition of the normal green and
etiolated specimens of Vicia faba, the latter having been grown in the
absence of light, differed in composition in the same general way as the
normal and albino plants reported by Church. Weber (16) studied the
effects of different parts of the spectrum on the composition of plants
and found similar effects. Jensen (7) has recorded similar observations
on the leaves of the privet plant, Ligustrum aurea.
While the fundamental cause of vegetable albinism is not known,
the fact that light of certain wave lengths is essential to the formation of
chlorophyl is well known; but in mottled citrus leaves the deficiency
of chlorophyl certainly can not be caused by an insufficiency of light.
The fact that the composition of albino and etiolated plants differs
from that of normal specimens in the same general way as is the case
with mottled and normal citrus leaves shows that different causes
may bring about similar effects in different species of plants. This fact
also suggests at once that the composition of a plant may not afford a
safe basis for forming a judgment as to the cause of a particular phenom-
enon. A satisfactory elucidation of these questions is not possible at
present owing, in part at least, to the lack of definite knowledge con-
cerning the fundamental principles underlying the growth processes of
plants. The formation of chlorophyl is undoubtedly the result of a
number of interdependent factors, and it is highly probable that either
the absence or the inhibition of any one of these factors may prevent the
formation of chlorophyl or ultimately lead to its decomposition.
COMPOSITION OF THE SAP OF ORANGE LEAVES
Some study has also been devoted to the sap of orange leaves. The
sap was obtained by first subjecting the leaves to a temperature a few
degrees centigrade below zero for a period of several hours. Im-
mediately after the leaves were removed from the freezing chamber
they were quickly ground to a pulp with an ordinary meat grinder.
The juice was then pressed from the pulp by the use of a hand-screw
press. A portion of the juice was filtered through folded filter paper,
and its specific gravity was determined by the pycnometer. Partial
analysis was made on weighed portions of the juice by first evaporating
to dryness and then using the methods previously described. Special
investigations were also made on unfiltered portions of the sap as described
below.
Mature normal leaves, collected from healthy navel orange trees on
May 29, 1 91 8, were first studied. A sample of 861 gm. of leaves yielded
approximately 1 50 cc. of sap. Partial analysis gave the following results :
Specific
gravity.
Ca.
K.
P.
1. 08
Per cent.
I. 07
Per cent.
0. 54
Per cent.
O. 036
Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 1 83
These data show that the expressed sap of mature orange leaves is
comparatively rich in solids, calcium, and potassium, but the ratio of
calcium to potassium in the sap is widely different from the ratio of the
total amounts of these elements in the leaf. (Table II.)
On June 5, 191 8, three sets of samples of Valencia orange leaves were
collected. One of these was composed of normal leaves about 6 weeks
of age; another sample obtained from the same trees consisted of healthy
mature leaves; whereas the third sample was chosen to represent severely
mottled leaves of the previous year's growth. Each of the samples was
divided into three parts, one of which was used to study the sap, another
to determine the water-soluble constituents, and the third for total
analysis.
The sap was pressed out after freezing as described above. The water-
soluble constituents were extracted by first grinding 100 gm. of the fresh
leaves in a meat grinder, shaking with 1 ,000 cc. distilled water for one hour,
and filtering through filter paper. Total acidity was determined by titra-
tion with N/io sodium hydroxid, using phenolphthalein as indicator.
It was necessary to dilute the sap considerably because of its dark color,
and a high degree of accuracy is not claimed for the results. They are
rather approximations. The acidity is expressed for convenience as
anhydrous citric acid.1 The results are presented in Tables XVI, XVII,
and XVIII.
Table XVI. — Composition of Valencia orange leaves at the age of 6 weeks
Specific
gravity.
Ash.
Ca.
K.
P.
N.
Acid.
Per cent.
Sap ... .... t . ofi c
Per cent.
3-J7
J3-23
Per cent.
O. 67
1-57
3-56
Per cent.
0. 72
1. 69
I.99
Per cent.
O.045
■ 13
. 21
Per cent.
Per cent.
Water extract a
Total leaf 0
1.005
2-45
I. 64
a Expressed in terms of dry matter.
Table XVII. — Composition of normal mature Valencia leaves
Specific
gravity.
Ash.
Ca.
K.
P.
N.
Acid.
Sap
Per cent.
I.097
I. 008
Per cent.
4-32
17- 56
Per cent.
I. 41
2.85
5-78
Per cent.
O. 42
.64
•94
Per cent.
0-°35
•063
• J3
Per cent.
Per cent.
Water extract a
Total leaf a
I. 92
I- 15
° Expressed in terms of dry matter.
1 The nature of the acid constituents of the leaves has not been investigated sufficiently to justify a
definite statement as to their identity.
1 84
Journal of Agricultural Research
Vol. XX, No. 3
The results show that the sap of Valencia orange leaves at the age of
6 weeks contains smaller amounts of dissolved solids and total ash ma-
terial than mature leaves. The calcium content increases more than two-
fold, and the potassium and phosphorus content decreases in passing to
maturity. On the other hand, the sap of mottled leaves has a higher
specific gravity and a higher ash content than that of mature normal
leaves. The calcium content, however, is considerably less, while the
potassium and phosphorus content is much higher.
It is evident from these data, therefore, that the sap of mottled Valen-
cia orange leaves is materially different from that of normal leaves,
either when they are 6 weeks of age or mature.
The water-soluble constituents were found to diverge in the same
general direction as the sap. It is interesting to note that a very high
percentage of the potassium, phosphorus, and calcium of orange leaves
is soluble in water.
Samples of fully mature normal leaves and of severely mottled leaves
of the previous year's growth were collected from Navel orange trees of
the fertilizer plots at Riverside in August, 1918. The sap was expressed
and used for more complete chemical study. (Tables XIX and XX.)
Table XVIII. — Composition of mottled Valencia leaves
Specific
gravity.
Ash.
Ca.
K.
P.
N.
Acid.
Sap
Per cent.
I. 118
I. 009
Per cent.
4-85
15.06
Per cent.
I- 13
2.85
4-OS
Per cent.
0. 91
1. 64
I.98
Per cent.
O. Ill
. 180
•243
Per cent.
Per cent.
Water extract °
Total leaf «
3.00
a. 75
<» Expressed in terms of dry matter.
The results are fairly concordant with those reported above for Valencia
leaves. It is again shown that the composition of the sap of mottled
orange leaves differs widely from that of normal leaves. The data also
show that the ash of the sap of each sample contained considerably
smaller percentages of calcium and higher percentages of iron than
those reported above for the ash of the leaf as a whole, while the per-
centages of the other constituents are not materially different from
those of the entire leaf. The calcium content of the sap of Navel
orange leaves appears to be lower than that of Valencia leaves. (Com-
pare Tables XVII and XX.)
Upon studying the preceding data, it seems difficult to escape the con-
clusion that there must be some important physiological significance
attached to the fact that the sap of mottled orange leaves contains only
about one-half as much calcium and approximately twice as much
potassium and nitrogen and three times as much phosphorus as normal
leaves.
Nov. i, 1920 Composition of Normal and Mottled Citrus Leaves 185
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i86
Journal of Agricultural Research
Vol. XX, No. 3
The hydrogen-ion concentration of the sap was also determined by the
use of the hydrogen electrode. Mature normal-leaf sap was found to
give a PH value of 5.816 and mottled-leaf sap a value of 5.647, which
implies hydrogen-ion concentrations of 0.153 X 10 ~5 and 0.226 X 10 ~5,
respectively. These determinations are probably within the range of
variation of different samples of the same leaves.
After the determination of the hydrogen-ion concentration, total acidity
was determined by titration, using the hydrogen electrode to determine
the end point. It was found that 10 cc. of the normal sap required 3 cc.
N/10 alkali and the mottled-leaf sap 7.05 cc. In other words, the actual
acidity (hydrogen-ion concentration) of mottled-leaf sap is approximately
the same as that of normal leaves, but the latter sap is more nearly sat-
urated with base. It is probable that in each case the ionization of the
acids is held at approximately the same level by the buffers present.
Samples of normal Navel orange leaves approximately one week of
age, fully mature leaves, and severely mottled leaves of the previous
year's growth were collected in April, 191 9. The sap was expressed,
and the hydrogen-ion concentration and total acidity were determined
by the hydrogen electrode. Freezing-point depressions were also deter-
mined in portions of the unfiltered sap. The acidity is expressed in cubic
centimeters of N/10 sodium hydroxid required to neutralize 10 cc. of the
sap.
Table XXI. — Acidity and freezing-point depression of orange- leaf sap
Condition of leaves.
Hydrogen-ion con-
centration.
Total acid-
ity.
Freezing-
point de-
pression.
Normal, 1 week old
Normal , mature
Mottled
Mottled
6. 069
5.664
5-647
5- 63°
D. 852 X IO
. 217 X 10"
. 226 X 10
• 235 X 10-
Cc.
1.80
3.80
7. 00
8.25
°c.
1. 258
1.588
1-734
These data show that the actual acidity (hydrogen-ion concentration)
of mature orange-leaf sap is approximately two and one-half times as
great as that of leaves at the age of 1 week; but again it is shown that
the acidity of mottled leaves is approximately the same as that of nor-
mal leaves. The capacity to neutralize base — that is, total acidity —
however, was fully twice as great in mature leaves as in those 1 week of
age, while the mottled-leaf sap neutralized about twice as much base
as the normal mature leaf sap.
The freezing-point depressions show that while the normal mature-
leaf sap is more concentrated than that of young leaves the sap of mot-
tled leaves is more concentrated than either.
The results of the preceding investigation on the sap of orange leaves
are very suggestive. They are in harmony with the preceding ash
Nov.. 1. 19-0 Composition of Normal and Mottled Citrus Leaves 187
analyses in that they indicate that the composition changes materially
as growth proceeds and that the composition of mottled leaves differs
from that of normal leaves.
It is interesting to note that the total water content of mottled and
normal mature leaves is roughly correlated with the concentration of
the sap, but this correlation does not hold when immature leaves are
compared with mature leaves.
GENERAL DISCUSSION
It has been shown that the composition of orange leaves changes
rapidly as growth takes place. The relationships between the several
constituents drawn from the soil undergo important alterations. The
percentages of potassium and phosphorus, when expressed on the basis
of either the ash or the dry matter, decline rapidly during the early part
of the growth cycle and continue to decline, although at reduced rates,
during the latter part of the growth period. The percentages of nitro-
gen in" the dry matter also decrease as growth proceeds. The percent-
age of calcium, on the other hand, increases rapidly at first, and later
more slowly. The concentration of iron is greatest in very young leaves,
but later its concentration decreases slowly, while no very pronounced
changes take place in the percentages of the other constituents. The
concentration of the different constituents probably remains practically
constant throughout the period of normal maturity.
As the leaves approach senility just preceding the time of normal
dropping, notable amounts of potassium and nitrogen are translocated
back into the stem or other portions of the tree. A part of the phos-
phorus also appears to leave the leaf sometime preceding the period of
normal maturity. In contrast to certain cereals, the absolute content
of magnesium does not decrease as maturity approaches.
It has been shown that a given orange leaf normally contains the
maximum amounts of potassium, phosphorus, and nitrogen by the time
it is approximately 6 weeks of age. It is interesting that the leaf also
reaches its maximum size about the same time. On the other hand, the
absolute content of calcium continues to increase until full maturity is
reached.
Mature orange leaves are extremely rich in certain nutrients. The con-
tent of carbonated ash ranges from 14 to 18 per cent of the dry matter, and
the nitrogen content is usually above 2 per cent. The most pronounced
characteristic of the orange leaf, however, is found in its highly calcareous
nature. When the leaf is mature, the dry matter contains from 5 to 6
per cent of calcium.
Lemon and grapefruit leaves are similar in composition to orange leaves.
The composition of mottled citrus leaves is widely different from that
of normal leaves. The difference lies mainly in the smaller calcium
content, on the one hand, and the greater content of potassium and
1 88 Journal of Agricultural Research voi.xx.No.j
phosphorus, on the other. Usually the nitrogen content of mottled
leaves is also abnormally high. The composition of mottled orange
leaves resembles that of immature leaves, although the percentages of
ash and nitrogen in the former are materially greater than in the latter.
It has been shown that the absolute amounts of potassium and phos-
phorus contained in mottled orange leaves are fully as great as ordi-
narily occur in normal leaves that are two or three times as large, while
the calcium content is not more than one-third that occurring in average
normal leaves.
The sap of normal orange leaves becomes increasingly concentrated and
acidic as growth proceeds. When mature it is especially rich in calcium
and contains fully twice as much of this element as of potassium.
The abnormalities of mottled leaves noted above also occur in the sap
and among the water-soluble constituents. The sap of mottled leaves
contains subnormal amounts of calcium and fully twice as high concentra-
tions of potassium and phosphorus as mature normal leaves. The
hydrogen-ion concentration of mottled leaves is not materially different
from that of normal leaves, but the sap is less nearly saturated with base.
In other words, abnormally large amounts of unionized acids occur in
mottled-leaf sap.
Limited study of portions of citrus trees other than the leaves in-
dicates that the composition of the leaf spurs of severely mottled trees
varies from the normal in much the same way as the leaves. The compo-
sition of the older wood, however, is more nearly normal. On the other
hand, both the large roots and small rootlets of severely mottled trees
appear to contain considerably less potassium and phosphorus than nor-
mal roots, while the calcium content is approximately normal.
Should more extended study confirm these latter observations, it
would seem that the excessive proportions of potassium and phosphorus
occurring in mottled leaves may have been drawn, in part at least, from
the supply normally stored in the roots.
The results of these investigations suggest that mottled citrus trees
are deficient in calcium, but the cause of the subnormal content of
calcium can not be definitely stated.
While we recognize that growing plants have the power, through
selective absorption, of regulating their composition to a marked degree,
and that a given variation in the composition of a plant does not neces-
sarily reflect a corresponding deficiency in the nutrient medium, the above
data suggest that the abnormalities in the composition of different parts
of mottled citrus trees may be due, in part at least, to the inability of the
tree to satisfy its normal calcium requirements at critical periods.
It is well known that manure and other forms of decaying organic
matter exert an ameliorating effect on mottle-leaf. It is interesting in
this connection that the concentration of soluble calcium in the soil
Nov. 1. 1920 Composition of Normal and Mottled Citrus Leaves 189
becomes materially increased as a result of the decomposition of such
materials (8). On the other hand, the occurrence of heavily compacted
layers of soil (plowsole) around the roots, especially when present im-
mediately below the depth of cultivation, and of soils of low organic
content {3) and low natural solubility afford conditions that are con-
ducive to mottle-leaf. Where such conditions occur, it is possible that
the supplies of those nutrients which are normally absorbed at relatively
high rates may become inadequate. The nature and extent of the root
system of citrus trees must also be considered in this connection. It is
interesting that the absorbing roots of citrus trees are not provided with
the usual root hairs. Consequently, they may possess less absorbing
surface than is afforded by other plants that normally absorb relatively
large amounts of nutrients. These and other related questions will be
more fully discussed elsewhere.
The fact that mottle-leaf sometimes appears on trees that have been
injured by alkali suggests the possibility that alterations in permeability
occasioned by the presence of excessive concentrations of salts, or pos-
sibly toxic substances of other kinds in the soil moisture, may prevent
the roots from taking up normal amounts of calcium.1
If we may judge from the composition of normal leaves, the calcium
requirements during the period when mottle-leaf develops most pro-
nouncedly are extremely heavy. The leaves at that stage normally absorb
calcium at a high rate.
Just why subnormal concentrations of calcium accompanied by super-
normal concentrations of potassium and phosphorus in the leaves should
afford conditions that tend to limit chlorophyl production is not known,
if indeed further investigations prove that such is the case. There may,
of course, be no causal relationship between these facts, but rather each
may be the result of causes not yet suggested.
It is recognized that calcium is not a normal constituent of chlorophyl.
In addition, while iron is essential to the formation of chlorophyl yet
does not enter into its final composition, we are not aware that a similar
relationship exists between calcium and chlorophyl formation. Conse-
quently, even though further study should prove that mottle-leaf can be
produced as a result of an inadequate supply of available calcium, it is
probable that the lack of chlorophyl and its disappearance from the
localized areas of the leaves would be found to be indirect rather than
direct effects of a shortage of calcium. In any event, whether the
shortage of calcium or some other factor conditions the deficiency of
chlorophyl, photosynthesis is doubtless reduced by the lack of chlorophyl.
With an adequate supply of nitrogen, phosphorus, and potassium pres-
ent in the soil moisture, osmosis might bring about the absorption of
1 As is well known, the occurrence of mottle-leaf is sometimes correlated with the species of root stock,
but this phase of the subject has not been systematically investigated in California. Mr. H. Atherton
Lee has called the writer's attention to his studies on this phase of mottle-leaf in the Philippine Islands.
190 Journal of Agricultural Research voi.xx,No.3
greater or lesser amounts of them, despite the deficiency of chlorophyl
in the leaves; but the reasons why excessive amounts of these elements
accumulate in mottled citrus leaves are not clear. It seems probable
that some physico-chemical principle not elucidated by the preceding
data must be fundamentally involved.
Before any explanation of mottle-leaf can be safely accepted, it is
necessary to show that the disease can be produced experimentally, and
that too under conditions admitting of scientific analysis. Additional
studies already projected may throw further light on this subject.
Whatever may ultimately be found to be the primary cause of mottle-
leaf, the preceding investigations strongly suggest that the leaves are
not suffering from inadequate supplies of potassium, phosphorus, or
nitrogen. We have also found little, if any, indication of a deficiency of
iron.
LITERATURE CITED
1) Alino.
1901. the cultivation of oranges. In Jour. Roy. Hort. Soc. [London], v.
25, pt. 3, p. 341-352-
2) Blair, A. W.
[1910.] report of chemist. In Fla. Agr. Exp. Sta. Rpt. [19091/10, p. xxv-
xxxiv.
3) Briggs, Lyman J., Jensen, C. A., and McLanE, J. W.
1916. mottle-leaf of citrus trees in relation to soil conditions. In
Jour. Agr. Research, v. 6, no. 19, p. 721-740, 4 fig., pi. H, 96-97.
4) Church, A. H.
1879. A CHEMICAL STUDY OF VEGETABLE ALBINISM. In Jour. Chem. Soc.
[London], v. 35, p. 33-41.
5)
1886. A CHEMICAL STUDY OF VEGETABLE ALBINISM. PART III. EXPERIMENTS
with quErcus rubra. In Jour. Chem. Soe. [LondonJ, v. 49, p.
839~843-
6) Ensign, M. R.
1919. venation and senescence of polyembryonic citrus plants. In
Amer. Jour. Bot., v. 6, no. 8, p. 311-329, 6 fig. Bibliography, p. 329.
7) Jensen, C. A.
191 7. composition of citrus leaves at various stages of mottling. In
Jour. Agr. Research, v. 9, no. 6, p. 157-166. Literature cited, p. 166.
8)
191 7. EFFECT Oi DECOMPOSING ORGANIC MATTER ON THE SOLUBILITY OF CER-
TAIN inorganic constituents OF the soil. In Jour. Agr. Research,
v. 9, no. 8, p. 253-268.
9) Jones, W. J., Jr., and Huston, H. A.
1914. composition of maize at various stages of its growth. Ind. Agr.
Exp. Sta. Bui. 175, p. 599-629, 10 fig., 1 fold. pi. (col.).
10) Kelley, W. P., and Thompson, Alice R.
1910. a study of the composition of The rice plant. Hawaii Agr. Exp.
Sta. Bui. 21, 51 p.
11) McBeth, I. G.
1917. RELATION OF THE TRANSFORMATION AND DISTRIBUTION OF SOIL NITRO-
GEN To the nutrition of citrus plants. In Jour. Agr. Research,
v. 9, no. 7, p. 183-252, 19 fig. Literature cited, p. 251-252.
Nov. x. igao Composition of Normal and Mottled Citrus Leaves 191
(12) MuLLER, John.
1909. yellowing OF citrus TREES. In Agr. Jour. Cape Good Hope, v. 34,
no. 2, p. 149-157. 2 %•
(13) OliviEri, V., and GuErrieri, F.
1895. ricErche suc-li agrumi. In Staz. Sper. Agr. Ital., v. 28, fasc. 5, p.
287-301.
(14) Palladin, W.
1892. aschEngEhalt DER ETiolirten blatter. In Ber. Deut. Bot. Gesell.,
Bd. 10, p. 179-183.
(15) RownEy, Thomas H., and How, Henry.
1848. analysis op the ashes op the orange-tree (citrus aurantium).
In Mem. and Proc. Chem. Soc. London, v. 3 (1845/48), p. 370-377.
(16) Weber, Rudolf.
1875. ueber den einfluss farbigen lichtes auf die assimilation und
die damit zusammenhangende vermehrung der aschenbestand-
THEiLE in ErbsEn-keimlingEN. In Landw. Vers. Stat., Bd. 18,
p. 18-48.
CONTROL OF FLUKE DISEASES BY DESTRUCTION OF
THE INTERMEDIATE HOST1
By Asa C. Chandler
Instructor in Biology, Rice Institute, Houston, Tex.; formerly Assistant Professor of
in Zoology and Physiology, Oregon Agricultural College and Experiment Station
Flukes have long been known as causative agents of disease in ani-
mals, especially sheep; in fact the loss resulting from their ravages in
some sheep-raising countries can be estimated in millions of dollars an-
nually. Within comparatively recent years flukes have been discovered
to play an important role in some countries in the production of human
disease. At present human fluke infections are known to be more or
less prevalent in nearly all tropical and subtropical countries and in
some countries of temperate climate. The blood flukes, Schistosoma,
occur in the oriental countries and throughout most of Africa and trop-
ical America. Human liver flukes, Clonorchis, and the lung flukes,
Paragonimus, are primarily diseases of the Orient, but epidemic cases
have been reported from other countries. The various species of intes-
tinal flukes which are habitual or accidental human parasites occur in
both Asia and Africa and probably in other tropical countries, but these
are of minor importance.
The important relation of fluke infections to the public health in en-
demic countries is not generally realized. In Egypt, for instance, over
half the population is said to suffer from schistosomiasis, and in an ex-
amination of 54 boys in the village of El Marg, near Cairo, Leiper {id) 2
found 49 to be infected. Cawston (j) states that in some districts in
South Africa 80 per cent of the school boys and 10 per cent of the girls
are infected and that Schistosoma infections seriously retard both the
physical and mental development of the school children. Troops oper-
ating in endemic regions are much affected by the disease unless strin-
gent preventive measures are taken. The British army suffered severely
in the Boer war, and in 1914 the British Government was still under
heavy expense for pensions for soldiers invalided by schistosomiasis.
Laning, of the United States Navy, states that it is not uncommon for
large proportions of the crews of patrol gunboats operating on the Yangtze
River to be completely disabled by Schistosoma japonicum infections.
Nakagawa (13) states that lung flukes are harbored by as high as 50 per
cent of the population in some districts in Formosa, and in parts of
Japan the infection is hardly less prevalent. Clonorchis, a human liver
1 Contribution from the Zoological Laboratory, Oregon Agricultural Experiment Station, Corvallis,
Oreg., and from the Biological Laboratory, Rice Institute, Houston, Tex.
2 Reference is made by number (italic) to "Literature cited," p. 208.
Journal of Agricultural Research, Vol. XX, No. 3
Washington, D. C Nov. 1, 1920
vi Key No. Oreg.~5
(193)
9507°— 20 3
194 Journal of Agricultural Research vol. xx,No.3
fluke, is even more prevalent in Japan and is said by Kobayashi (9) to
affect as many as 60 per cent of the inhabitants of some endemic areas.
Like malaria and hookworm disease, fluke diseases are comparatively
seldom fatal in themselves but are particularly injurious in causing loss
of efficiency, reduced vitality, and lowered resistance to other diseases.
The long duration and relative incurability of fluke infections are a very
serious factor. In this respect fluke infections are far more to be feared
than are infections with intestinal parasites, most of which are relatively
easy to expel. Of the numerous drugs which have been tried in the
treatment of extra-intestinal fluke infections, only tartar emetic, re-
cently shown by Christopherson (4, 5, 6, 7) to be more or less specific in
its action on Schistosoma, gives promise of being of any great value.
No unquestionably effective remedy for lung or liver flukes has been
found, and even the use of tartar emetic for schistosomiasis is far from
satisfactory, since the serious symptoms of the disease are caused by the
eggs of the worms deposited in the tissues and often continue to exist
long after the worms are dead.
With regard to fluke diseases of domestic animals the situation is no
less serious. The common liver fluke of sheep and cattle, Fasciola hepatica,
is found almost all over the world in temperate climates, being prev-
alent wherever these domestic animals are grazed on wet or marshy
pastures. In the British Isles, France, Germany, and other parts of
Europe and in some parts of the United States, notably western Oregon
and Washington and the humid districts of Texas, Louisiana, Florida,
and other southern States, the losses from flukes in sheep, cattle, and
goats amounts to millions of dollars annually, on account of loss of vi-
tality among the animals, depreciation in quantity and quality of meat,
and the loss of the infested livers themselves. In the Tropics Fasciola is
largely replaced by other flukes — for example, the intestinal Amphistoma
and Gastrodiscus, and various blood flukes, Schistosoma. As with hu-
man flukes, the extra-intestinal flukes of animals can not be reached
readily by drugs, and as pointed out by Ransom and Hall (16) there is
still much doubt about the efficacy of drugs which have been recom-
mended for use against them, though there is room for much more
experimentation.
On account of the difficulty encountered in treating or curing fluke
diseases, preventive measures loom up with even greater importance
than they do in dealing with hookworm or other intestinal parasites.
The working out of preventive measures based on scientific knowl-
edge has only recently become possible, for, although the life history and
mode of infection of the common liver fluke, Fasciola hepatica, of sheep and
cattle have been well known for a number of decades, such knowledge of
human flukes has been acquired only in the past three or four years.
Leiper's work on Schistosoma in Egypt in 191 5-1 6 (10), Kobayashi's
work on Clonorchis in Japan in 191 5 (9), and Nakagawa's work on
Nov. i, 1920 Control 0} Fluke Diseases 195
Paragonimus in Formosa in 191 6 (13) have given a definite basis for
preventive measures against all these parasites of man and of the related
parasites of domestic animals.
In every case of fluke infection of man or domestic animals in which
the life cycle of the parasite has been worked out it has been shown that
fresh-water snails act as necessary intermediate hosts. It appears, there-
fore, that if some efficient and practical method of destroying the snails
could be found, this would furnish a logical point of attack in the control
of all fluke diseases. Other preventive measures are, of course, valuable
also and could be used as supplementary measures — for example, the
impounding of water before use for drinking or bathing as a preventive
against Schistosoma infections, the discouragement of the habit of eating
improperly cooked meat of crabs in the case of Paragonimus and of fish
in the case of Clonorchis, and care in the disposal of feces and urine in all
cases. The last, exclusive of individual mechanical protection against
infection, is the only preventive measure that can be adopted against
hookworm and many other intestinal parasites. To accomplish this in
some warm countries where there has never existed anything approach-
ing sanitation and where the very idea of sanitation is so strange and
foreign to the habits of life and thought of the natives is well nigh impos-
sible. The fact, therefore, that fluke infections may possibly be con-
trolled by attack upon an intermediate host instead of by reliance upon
the enforcement of sanitary regulations makes the ultimate eradication
of these infections, in spite of their relative incurability, a matter of
brighter prospect than is the case with many other verminous parasites.
Already a number of suggestions for the destruction of the snails which
act as intermediate hosts of flukes have been made. Thomas (18) ad-
vised the extensive scattering of salt on pastures where sheep were known
to become infected by flukes, and he commented on the absence of fluke
infestations among sheep grazing on salt marshes. The effect of the
salt, of course, was to destroy the snail, Limnaea, which acts as the inter-
mediate host. Leiper (10) suggested the eradication of the disease in
agricultural districts in Egypt by the intermittent flow of water in the
irrigation ditches, the water being turned off for 1 5-day periods, thus
drying up the ditches and destroying the snails by desiccation. Such
a procedure is, of course, very limited in its application, and in view of
the remarkable resistance which many snails have to drouth it is doubtful
whether all the implicated species could be killed by this method even if
it were feasible. Leiper suggested that ammonium sulphate be applied
to pools which were inhabited by the intermediate hosts of Schistosoma.
Lime has been recommended by a number of writers, particularly
Japanese, as the cheapest and best method of destroying snails. One
Japanese writer, Ando (1) states that 1 per cent lime water killed 6 of 10
snails in seven hours, and a 1 per cent solution of copper sulphate would
kill them in six hours. It is obvious that none of the above methods of
196 Journal of Agricultural Research vol. xx, N0.3
exterminating snails would be practical on a large scale, either on account
of the prohibitive cost or on account of the excessive amounts of the
material used and consequent injury to the water for drinking, bathing,
or irrigation purposes.
In the hope of finding some effective means of destroying disease-
carrying fresh-water snails a series of experiments was undertaken by the
writer. The original purpose of the investigation was to find a solution
to the liver fluke problem among sheep and cattle raisers in the Willa-
mette Valley of Oregon, but it was realized that if a means of controlling
all fresh-water snails could be found, the results would be of infinitely
greater value than the solution of the local problem, and the experiments
were carried on with this in mind.1
It was obvious that any chemical which could be used on a large scale
for the destruction of snails in ponds, marshes, or streams must not be
toxic to man or domestic animals in the dilutions used and must not be
expensive. An attempt, therefore, was made to find a cheap chemical
substance, readily soluble in water, which would be destructive to snails
in relatively weak solutions and which would not render water either
injurious or unpalatable for man or domestic animals.
The chemicals which were selected for preliminary experiments, the
dilutions which were made, and the results obtained are shown in Table I.
The snails, Limnaea (Galba) bulimoides,2 were immersed in each solution,
using chemically pure salts and tap water, Corvallis tap water being
unusually clear, pure, and soft. The sign — indicates no evident effect,
± slight noticeable effect in behavior, + distinct illness without com-
plete prostration, + + complete prostration, and © death. It was found
later that snails which were apparently dead would sometimes revive if
placed in fresh, aerated water; therefore the results shown in this table
are not absolutely dependable. They do, however, demonstrate beyond
question one striking thing — the fact that copper salts have an extremely
toxic effect on these snails, even in such great dilutions as one part to a
million of water. Mercuric bichlorid is the only other salt experimented
with which approaches the salts of copper in its toxicity to snails, but
since it is evidently not so effective as copper, is more toxic to higher
animals, and is more expensive, no further experiments with it were
carried out.
The salts of copper being evidently the most promising substance with
which to attack aquatic snails all subsequent work was concentrated on
them. Experiments with various copper salts (CuCl2, CuS04, Cu[N03]2)
were tried, and it was found that with equivalent concentrations of the
Cu++ ion their toxicity was approximately the same. Copper sulphate,
1 The writer has been unable to get access to the following paper: Germain, L. de l'EFFET des poisons
min£raux sur quelques moiausques terrestres et fluviatiles de France, In Bui. Soc. Amis Sci.
Nat. Rouen, s. 4, ann. 34, 1898, sem. 1, p. 71-78. 1899.
2 Snails specifically named in this paper were kindly identified by Dr. H. A. Pilsbry, Dr. F. C. Baker,
or Mr. Bryant Walker.
Nov. i, 1920
Control 0} Fluke Diseases
197
being the cheapest copper salt, was therefore selected for further experi-
mentation.
Chemical.
Dilution.
1 hour.
4
hours.
8
hours.
f?*°
};
+ +
+ +
+
+ +
+ +
+ +
e
©
++
±
+ +
++
-
±
{?-
-
-
±
_
-
-
24
hours.
As203
Ba(N03)2
CaOCl2
CaOCl2
Ca(OH)2
CuCl2
CuS04
CuS04
HgCla
NaCl
NaCN
NaCN
(NH4)2S04
(NH4)2S04
Pb(CH2COOH)2
Pb(CH2COOH)„
ZnCl2
ZnS64
1 to 1,000,000
1 to 100,000
1.3 available chlorin per
1,000,000.
2.6 available chlorin per
1,000,000.
1 to 10,000
1 to 100,000
1 to 100,000
1 to 1,000,000
1 to 1,000,000
1 to 1,000
1 to 100,000
1 to 1,000,000
1 to 100,000
1 to 1,000,000
1 to 100,000
1 to 1,000,000
1 to 1,000,000
1 to 1,000,000
7©
3 +
\2 + +
±
o Figures beside symbols indicate number of snails out of the 10 used in the experiment.
The effect of copper salts on various kinds of organisms is extremely
variable. Their highly toxic effect on algae, first demonstrated by
Moore and Kellerman (ji), is well known, and copper sulphate is exten-
sively and successfully used in eliminating algae from ponds and reser-
voirs. Copper sulphate is effective against some algae in dilutions up
to i part in 25,000,000 or more parts of water but is commonly used in
the proportion of 1 part to from 1,000,000 to 3,000,000 parts of water.
Its bactericidal action is less marked and varies greatly with temperature.
At 200 C, in water relatively free from organic matter, all pathogenic
bacteria are destroyed in 24 hours at a dilution of 1 part to 400,000
parts of water. Peters (14) showed that the concentration necessaiy
to kill instantly certain protozoa was 12 to 60 X io-8 gram molecular
parts per cubic centimeter of water (about 3 to 15 parts per million).
The toxic effect of copper on fungi is as striking as its effect on algae
and is taken advantage of commercially in the use of Bordeaux mixture
for spraying trees and vines.
Curiously enough the effect of copper salts on both higher plants and
higher animals is in general far less toxic than it is on lower animals
and plants. In dilute solutions copper sulphate has a stimulating action
on the growth of many higher plants, having been tested particularly
on various grains. In the animal series, copper salts are usually
198 Journal of Agricultural Research vol. xx. N0.3
harmless in the dilutions which are lethal to the single-celled organisms.
Copper is, in fact, a normal constituent of their tissues and replaces iron
in the blood of some invertebrates. Experiments by the writer, as well
as by others, show that copper, 1 part per million, is not injurious, at
least within 48 hours, to annelids, crustaceans, or aquatic insect larvae.
Of vertebrate animals, fish are highly susceptible, various species being
affected by 1 part of copper sulphate in from 500,000 to 10,000,000
parts of water. Amphibians are immune to dilutions of 1 to 1 ,000,000.
Contrary to popular opinion, copper is not highly toxic to mammals and
can, in fact, be taken by the mouth in considerable quantities without
injury. Five to 10 gr. (0.32 to 0.64 gm.) can be taken as an emetic.
Horses and cattle can take 3.9 to 7.7 gm. and sheep 1.3 to 2.6 gm. It
is evident, therefore, that copper salts in high dilution have a selective
effect on various organisms, being particularly destructive to single-
celled organisms, certain molluscs, and fishes. For destroying aquatic
snails, therefore, copper sulphate can be used in perfect safety so far
as any possibility of injury to man or domestic animals from drinking
or bathing is concerned, without injuring the water for irrigation pur-
poses, and without destroying other higher organisms, except certain
species of fish.
After it was found that very dilute solutions of copper salts are specifi-
cally toxic to Limnaea (Galba) bulimoides, experiments were carried out to
determine their effect on other species of snails and also to ascertain as
accurately as possible the effect of varying concentrations of the salts.
In all of these experiments only chemically pure copper sulphate was used.
Preliminary experiments showed that there was no appreciable difference
in effect whether distilled water or the local tap water was used in the
experiments ; therefore the tap water was used except for making up the
stock 0.1 per cent and 0.01 per cent solutions. All the local species of
snails of which sufficient numbers could be obtained were tried. It was
not practicable to experiment with the species of snails which have actually
been incriminated as. the intermediate hosts of important flukes of man
and domestic animals, but a greater variety of snails than those which
have been incriminated were used, including representatives or close
allies of all the incriminated families and in some cases genera.
Of the species used, Planorbis callioglypius belongs to the family Plan-
orbidae, to which belong Bullinus, Planorbis, and Physopsis, intermediate
hosts of Schistosoma haematobium and 5. mansoni; Goniobasis, according
to Pilsbry, is closely akin to Melania, intermediate host of Paragonimus,
Metagonimus, and Clonorchis; Fluminieola belongs to the family Amni-
colidae in common with Blanfordia, intermediate host of Schistosoma
japonicum; several species of Iyimnaea serve as intermediate hosts for
Fasciola hepatica.
Some difficulty was encountered in correctly reading the effect pro-
duced on the snails, and all earlier experiments had to be discarded.
Nov. i, 1920 Control of Fluke Diseases 199
It was found that snails which were prostrate and would not respond to
stimuli, and were therefore apparently dead, would frequently revive
after being placed in fresh, aerated water for from 12 to 24 hours. The
only criterion for death which was used, therefore, was failure to revive
within 24 hours after being placed in fresh water.
Experiments, using 10 snails of a species in one liter of the solution
at approximately 180 to 200 C, were made as follows:
DILUTION. SPECIES TESTED.
i to 100,000 Goniobasis plicifera, Limnaea bulimoides, Physa occidentalis .
1 to 500,000 Fluminicolafusca, Goniobasis plicifera, Physa occidentalis .
1 to 1,000,000 Ancylus caurinus, Fluminicolafusca, Goniobasis plicifera, Limnaea
bulimoides, L. proxima rowelli, Physa nuttalli, P. occidentalis,
Planorbis callioglyptus.
1 to 1,500,000. . .Fluminicolafusca, Goniobasis plicifera, Physa occidentalis .
1 to 2,000,000. . .Fluminicolafusca, Goniobasis plicifera, Limnaea bulimoides, L. prox-
ima rowelli, Physa occidentalis, P. nutalli, Planorbis callioglyptus.
1 to 2,500,000. . .Fluminicola fusca, Goniobasis plicifera, Limnaea bulimoides, L. prox-
ima rowelli, Physa occidentalis.
1 to 3,000,000. . .Fluminicola fusca, Goniobasis plicifera, Limnaea bulimoides, Physa
occidentalis.
1 to 4,000,000. . .Physa occidentalis.
i to 5,000,000. . .Fluminico la fusca, Goniobasis plicifera, Limnaea bulimoides, L.
Proxima rowelli, Physa nuttalli, Physa occidentalis .
1 to 10,000,000. .Limnaea bulimoides, Physa occidentalis.
The results of these experiments may best be summarized as follows:
1. All species of snails experimented with, eight in all, belonging to
six genera and as many families, are similar to each other in their suscepti-
bility to copper sulphate. There is, in fact, much more individual
variation shown than there is difference between species. Ancylus,
Fluminicola, Limnaea, and Planorbis become prostrate a little more quick-
ly than do Goniobasis and Physa. Goniobasis has a little more recupera-
tive power than the other species after being placed in pure water.
2. All species die within 48 hours, many specimens sooner, in solutions
of 1 to 500,000 and 1 to 1,000,000. Fluminicola, Limnaea, Physa, and
Planorbis die within 48 hours in solutions of 1 to 1,500,000 and 1 to
2,000,000, but a few specimens of Goniobasis and one specimen of Limnaea
proxima rowelli revived slightly after being placed in fresh water for 24
hours but died within 48 hours.
3. A 1 to 500,000 solution appeared to be no swifter in its action than
was a solution of 1 to 2,000,000. In all dilutions between 1 to 500,000
and 1 to 2,000,000 some specimens revived after exposure for 24 hours,
whereas after 48 hours none revived except as noted in the preceding
paragraph.
4. Solutions ranging between 1 to 2,500,000 and 1 to 5,000,000 killed
50 per cent or more of the specimens, but not all, whereas all specimens
revived after 48 hours in a 1 to 10,000,000 solution, although they became
sick or prostrate while immersed in it.
200 Journal of Agricultural Research vol. xx, No. 3
5. In the 1 to 100,000 dilution, which was tried merely to ascertain
whether this concentration would kill quickly, the snails became prostrate
immediately upon being immersed and remained motionless, but they
almost all revived after immersion for one hour.
• The actual physiological effect of the copper salts on the snails has not
been determined. Within a few minutes the snails immersed in a dilute
copper-sulphate solution lie prostrate, being apparently unable to cling
to the sides of the jar. A mucous albuminoid substance is exuded, and
frequently feces, eggs, and even the penis, are extruded. It is highly
probable that the poisoning effect is due at least in part to inactivation
of enzyms necessary to life. Peters and Burres (15) showed that the
concentrations of copper sulphate necessary to kill Paramoecium and
Stentor were approximately the same as those necessary to inactivate
their normal enzyms. It was thought that possibly there was a special
tendency in snails to absorb copper, since this metal is an important con-
stituent of the blood and is found only in minute traces in the normal
environment. However, analyses of snails killed in copper-sulphate
solutions, compared with normal snails, failed to show appreciably
greater quantities of copper. Furthermore it was found that the snails
succumbed as quickly in a few cubic centimeters of the solutions as they
did in large quantities. Five specimens of Limnaea bulimoides were
killed in 10 cc. of a 1 to 1,000,000 solution, yet the total amount of
copper present was only about 0.0025 mgm> or 0.005 rngm. per snail.
By analogy with Helix pomatia, which was shown by Dubois (8) to con-
tain 6. 1 1 mgm. of copper per 100 gr. of body weight, a specimen of
Limnaea should normally contain several milligrams of copper. If
the mode of action of the copper salts is by inactivation of enzyms, the
similarity in effect of such varying dilutions as 1 to 500,000 and 1 to
2,000,000 is more readily explained.
The effect of a 1 to 1,000,000 copper-sulphate solution was also tried
on the eggs of Physa nuttalli and of Limnaea bulimoides. Eggs in intact
gelatinous masses were apparently uninjured by the copper solutions in
14 days, though the inclosed embryos seemed to grow more slowly than
the controls.
There are a number of factors which influence the effect of copper
sulphate on organisms in water, the most important being temperature,
presence of algae, alkalinity, and organic matter in solution. As regards
temperature, no extended experiments were carried out, but experiments
with a 1 to 1,000,000 solution were carried out at temperatures of from
1 50 to 270 C, and the snails apparently succumbed as quickly at the
lower as at the higher temperature. Water in which snails were to be
destroyed would probably not fall below 150 C. in temperature. Alkalin-
ity of water, to the extent normally found in natural ground waters,
appears to have little effect on the action of the copper salts, although
copper sulphate is precipitated as basic sulphates or carbonates in
Nov. iri9*> Control of Fluke Diseases 201
alkaline solutions. The tap water at Rice Institute, which is strongly
alkaline because of the presence of sodium carbonate, when used in a
1 to 1,000,000 solution of copper sulphate was apparently as effective
as distilled water, even after standing for 24 hours to allow time for
possible precipitation of the copper.
Since organic matter in solution rapidly precipitates copper, water
containing considerable quantities of it should receive larger quantities of
copper sulphate to make up for loss by precipitation. The concentration
of copper sulphate necessary to destroy typhoid bacilli, according to
Rettger and Endicott (17), was four times as great in water containing
0.01 per cent peptone as in distilled water and 40 times as great in the
presence of 1 per cent peptone. Moore and Kellerman (12) advise an
increase of 2 per cent in the concentration used to kill algae for each
part per 100,000 of organic matter. It is probable that a similar
increase in the amount of copper used against snails would be sufficient
to counteract the effect of the organic matter.
The presence of algae in the water has a marked effect on the action
of copper salts on snails, since the algae, which are killed by the salts,
absorb them. Bado (2) has demonstrated considerable quantities of
copper in the ash of algae which had been exposed to copper sulphate in
dilute solution, and he states that it is absorbed at different rates by
different species. In a preliminary experiment, the writer found that
snails placed in one liter of a 1 to 1 ,000,000 solution of copper sulphate,
together with a large handful of algae (Vaucheria and attached diatoms)
although they showed symptoms for a few hours after immersion, sub-
sequently revived and on the following day were as active as the controls.
To test more accurately the effect of algae, a quantity of fresh green
algae was rinsed and then squeezed like a sponge until water was no
longer expelled by moderate pressure. Quantities of this weighing 0.25,
0.5, 1, 2, 3, 4, and 5 gm. were placed in liters of a 1 to 1,000,000 copper-
sulphate solution, and snails (Physa occidentalis) were placed in each.
The snails in the jars containing up to 1 gm. of the wet algae (1 gm. = 150
mgm. dry weight) died as quickly as did the controls in a simple copper-
sulphate solution. Those in jars containing 2, 3, and 4 gm., although
prostrate within 24 hours, still responded weakly to stimuli at the end of
48 hours but did not revive when placed in fresh water. One-third of
the snails in the jar with 5 gm. of algae partially revived in the solution.
A second experiment, similarly conducted, but with the use of Spirogyra,
one of the algae most susceptible to copper salts, was tried. In this ex-
periment only 1, 2, and 3 gm. quantities were used. The snails with 1
gm. of Spirogyra did not die within 48 hours but failed to revive in fresh
water and died within 48 hours after being refreshed. Of those with 2
gm. 50 per cent revived after being refreshed, whereas of those with 3 gm.
all revived.
202 Journal of Agricultural Research voi.xx,No.3
As shown in the preliminary experiments with various chemicals,
chlorinated lime up to double the amount used for sterilizing drinking
water does not affect snails at all. It was found, furthermore, that the
presence of chlorinated lime in the proportion of i to 250,000 (about 1.3
parts available chlorin per million) had an inhibiting effect on the action
of copper sulphate on snails to such an extent that some specimens did
not even become prostrate in the solution. The mode of interaction of
the copper sulphate and chlorinated lime was not investigated, but it is
possible either that the liberated oxygen from the chlorinated lime may
counteract the effect of the copper sulphate on the enzyms, or that a
chemical reaction takes place which precipitates the copper. If the
latter is true it might be feasible to remove copper sulphate from solution
in water by the use of chlorinated lime, in case this should for any reason
be desirable after using it in destroying algae, snails, or other organisms.
A number of practical field experiments were carried out to demonstrate
the effectiveness of copper-sulphate treatment for snails in actual practice.
The first experiment was conducted on a pool in the vicinity of Corval-
lis estimated to contain about 113,000 liters of water. This pool was a
portion of a stream which dries up during the summer, leaving isolated
bodies of water, probably connected by seepage through the sandy sub-
stratum. The pool contained patches of Spirogyra here and there
together with a number of higher aquatic plants (Veronica, Cicuta, and
others). The fauna included frogs, newts, and stickle-backs among
vertebrates, and a great variety of insect life, the most abundant forms
being Notonectids, Corisids, damsel fly larvae, neuropterous larvae, and
beetles of various kinds, both adults and larvae. Five species of mol-
luscs were present. Physa occidentalis and the small bivalve Musculium
walkeri were abundant in the aquatic vegetation. Fluminicola fusca
was abundant, and Goniobasis plicifera was fairly common on the sandy
bottom, especially around the edges of the pool, and an unidentified
Planorbis occurred sparingly in the vegetation.
On August 26, 113 gm. of commercial copper sulphate were dissolved
in about 10 liters of water and sprinkled on the surface of the pool by
means of a watering pot, making approximately a 1 to 1,000,000 solution,
but without making any allowance for impurity of the copper sulphate,
absorption of algae, combination with organic matter in solution, or
dilution by seeping in of fresh water.
The effect of the experiment was studied 48 hours later. The masses
of algae had been killed, but the higher plants, vertebrates, including
the stickle-backs, and the various kinds of insects were apparently
unharmed. No living specimens of Fluminicola or Planorbis could be
found, though hundreds of dead ones were seen lying on the bottom.
The majority of the Physae were dead, but a few seemed to be merely
prostrate. Some specimens of Goniobasis were withdrawn into their
shells and were evidently not dead. All the Musculium were lying on
Nov. i, 1920 Control of Fluke Diseases 203
the bottom with their shells tightly closed. Another examination was
made on August 30, and at this time all specimens of Physa, Flumini-
cola, Planorbis, and Musculium and the majority of the Goniobasis
were dead, but about one-third of the last had revived and were appar-
ently well again. This fact was evidence that practically all the copper
sulphate had been removed either by absorption by the algae or by
the dissolved organic matter, increased by the disintegration of thou-
sands of snails or by seepage through the sandy substratum, since it
had previously been fully demonstrated that Goniobasis remained pros-
trate even in a 1 to 5,000,000 solution of copper sulphate.
A similar experiment was carried out on another pool of similar kind
and with practically the same fauna and flora; this pool was, in fact,
another isolated portion of the same stream. This time a copper-
sulphate solution of 1 to 500,000 was made. All molluscs were appar-
ently dead in 48 hours, and none subsequently revived. No other
higher animals were affected at all.
To test the use of copper sulphate for destroying snails in a flowing
stream, an experiment was attempted in Oak Creek, near Corvallis,
Oreg. The water in this creek is cold and clear and flows rapidly.
The stream is very uneven as to width, depth, and speed, consisting,
in fact, of a series of sluggish pools connected by rapids and cascades.
At this season of the year, September 1, the stream was very low, and
was found to flow only about 550 liters per minute. The stream con-
tained enormous numbers of Goniobasis plicifera, the bottom in some
places being fairly covered with them.
To treat this stream a 7-gallon keg fitted with a drawn-out glass
spigot which would feed a solution into the stream at an average rate
of 1.5 liters per hour was filled with a copper-sulphate solution strong
enough to make a 1 to 500,000 solution in the stream. This strength
of solution was used to make allowance for combination with organic
matter, precipitation in other ways, and error in estimation of the vol-
ume of the stream. The experiment ran smoothly for about 14 hours,
and at the end of this time the snails for at least a mile down the stream
were prostrate and apparently dead. Meanwhile, however, a rain storm
came up which in the following 10 hours approximately tripled the
volume of water in the stream. An attempt was made to strengthen
the solution fed into the water at a corresponding rate, and this seemed
to be successful. Pressure of other duties made it impossible to visit
the experiment again until 48 hours later. At this time it was found
that the spigot had been plugged by a particle of debris, though pre-
cautions had been taken to keep the solution as clear as possible. The
cessation of flow had evidently occurred shortly after the experiment
had last been visited, consequently the stream had been treated little
more than 24 hours. A few of the snails were dead, but the majority
had revived and were as active as ever.
204 Journal of Agricultural Research vol. xx, No.3
On account of the writer's moving from Corvallis, Oreg., to Houston
Tex., a few days later, this experiment could not be repeated on Oak
Creek, but a similar experimemt was made on a small stream or "bayou"
a short distance from Houston. This stream, flowing about 1,500 liters
per minute, is sluggish, fairly even in width and depth, and contains
water moderately alkaline and rich in lime. The only abundant snail
in the stream was a small Ancylus which occurs on dead leaves on the
bottom. A few specimens of Physa anatina were obtained at each
dredging.
To treat this stream a 10-gallon barrel was used, fitted with a glass
spigot as before but protected from plugging up by the use of a glass
funnel with the large end inside the barrel, this being covered with
cheesecloth to strain the solution as it flowed out. The addition of a
few cubic centimeters of sulphuric acid prevented the flocculent pre-
cipitation of iron sulphate, which is present as an impurity in commer-
cial copper sulphate. The diminution in rate of flow from the spigot
resulting from a lowering of the level of the fluid in the barrel follows a
parabolic curve, in this case decreasing fairly steadily from 50 cc. to
30 cc. per minute until the barrel was half empty. To prevent a greater
fall In pressure a 20-liter jar was placed above the barrel and connected
with it by an automatic siphon, so that the contents of the jug would
be utilized when the barrel was half empty. A simpler method would
have been the utilization of a tube equal to the height of the barrel to
give a greater head. By this method the entire contents of the barrel
could be utilized before refilling without too great a change in the rate
of flow of the copper solution. The experiment was allowed to run for
72 hours, although 48 hours' exposure to the copper solution had been
found experimentally to be sufficient to kill snails. However, in a
flowing stream it was thought advisable to give an extra day to make
up for uneven flow and dilution in the deeper portions of the stream
during the early part of the experiment and to give time for diffusion
into the "dead" water along the sides of the stream. At the end of
the experiment — that is, for the last 12 hours — the lower half of the
barrel was allowed to run itself out, thus gradually diminishing the
strength of the solution in the stream. It was thought that in this
way the actual time during which the stream was treated by a full
1 to 500,000 solution would be at least 48 hours. Three days after the
completion of the experiment the stream was again dredged at intervals
of about one-third of a mile at the same points at which dredgings
were made prior to the experiment. A few empty Physa shells were
found, but no living snails of any kind were obtained at any point
along the length of the stream (about 1% miles). It was unfortunate
that the stream was not longer so that the actual distance over which
the treatment was effective could be determined, but since this would
Nov. i, 1920 Control of Fluke Diseases 205
obviously vary greatly in different streams, according to evenness of
width and depth, strength of current, purity of water, and possibly
other factors, it would be necessary in treating any stream for the
destruction of snails to determine, after the experiment, the distance
over which it is effective and to repeat the experiment at a point on
the stream a little above where the first live snails were found. ,
By utilizing a 50-gallon barrel and filling it at 12-hour intervals with a
10 per cent solution, streams running as much as 3,500 gallons per
minute could be treated by this method, and, of course, by the use of
several such barrels, still larger streams could be treated. Repeated
attempts were made to find a method by which the copper salt could be
fed into a stream at a constant rate without first being put into solution.
This would, of course, save much time and labor in the treatment of
large streams. A method was finally worked out by which it was hoped
that this could be accomplished. Cylinders of sheet metal were care-
fully lined with paraffine inside to prevent any chemical action with the
copper sulphate. Wooden tubes could be used as well but are not so
readily obtainable as are the sheet metal tubes, which, in diameters of
from 2 inches up, can be obtained from any tinsmith. A copper or
bronze screen is tied over the end of the tube, and the tube is filled with
copper-sulphate crystals of more or less uniform size. Commercial
"pea" crystals could be used, or crystals of desired size can be obtained
by sifting through two screens. The screened end of the tube is immersed
about 1 cm. in the stream to be treated, and the copper sulphate is dis-
solved out from the bottom of the tube, a fresh supply being constantly
furnished by gravity in the tube. Theoretically the copper salt should
go into solution at a fairly constant rate, determined by the area exposed
to the water, the speed of the stream, and the temperature of the water.
Up to the present, however, it has not been found possible to make this
simple apparatus work satisfactorily in practice, because of the fact that
all the water in the vicinity of Houston is strongly alkaline. The alka-
linity precipitates the iron sulphate contained as an impurity in com-
mercial copper sulphate and also forms, in the course of two or three
hours, considerable deposits of copper carbonates. These two substances
together tend to clog the screen through which the copper sulphate is
taken into solution, thus causing a rapid diminution in the rate of solu-
tion. If this difficulty could be overcome by some feasible method of
keeping the water at the mouth of the tube slightly acidified, or if the
water to be treated were not alkaline, large streams could be treated
with comparatively little trouble by this method, using several tubes
of suitable diameter at intervals across the streams. It would, of course,
be preferable to treat streams at a comparatively shallow, rapid-flowing
point, since this would facilitate a rapid diffusion throughout the water.
206 Journal of Agricultural Research vol. xx, No3
SUMMARY AND CONCLUSIONS
(i) Fluke diseases of both man and domestic animals are of great
importance in many parts of the world. They are debilitating diseases of
long duration and difficult to treat or cure. Preventive measures, there-
fore, are of great importance. The working out of preventive measures
based on scientific facts has only recently become possible , since the life
histories and modes of infection of the human flukes have been discov-
ered only in the last three or four years.
(2) In all known cases fresh water snails act as intermediate hosts for
the important flukes of man and domestic animals. A practical and
efficient method of destroying these snails would make the ultimate
eradication of fluke diseases, in spite of the difficulty in treating them,
a matter of brighter prospect than the eradication of hookworm and
other intestinal parasites, in which the sanitary disposal of feces must be
relied upon.
(3) Experiments by the writer, carried out to find some cheap, harm-
less method of treating water to destroy snails, demonstrated that copper
salts exert a powerful toxic effect upon snails even in very high dilution.
In an experiment upon eight species of six families it was demonstrated
that copper sulphate in proportions of 1 part to from 500,000 to 2,000,000
parts of water destroys snails of all these species within 48 hours; 50 per
cent or more are destroyed in dilutions up to 1 to 5,000,000. From the
point of view of expense, harmlessness, and convenience in use copper
sulphate is preferable to any other substance which has been tried or
suggested for destroying snails. The eggs of the snails are not destroyed
by the copper salts.
(4) Copper salts are also highly toxic to algae, fungi, and other lower
organisms but are apparently harmless, in the dilutions used, to higher
plants and animals, except fish. Water treated with copper sulphate,
therefore, is uninjured for drinking, bathing, or irrigation purposes.
(5) The effectiveness of copper sulphate in water is modified more or
less by temperature, alkalinity, dissolved organic matter, and living
algae. Some allowance should be made for these factors in estimating
the amount of copper to be used in any given body of water. The pro-
portion should vary from 1 to 1 ,000,000 in relatively pure water at 200 C.
or above to 1 to 500,000 in water which is very cold, is alkaline, contains
dissolved organic matter, or harbors an abundance of algae. If the
growth of algae is very luxuriant, it would probably be advisable to kill
these algae by a preliminary treatment with a 1 to 1 ,000,000 solution of
copper sulphate, following this in the course of a few days or a week by a
second treatment.
(6) Copper sulphate can be administered to ponds, reservoirs, or
other bodies of standing water in the way advised by Moore and Keller-
man for the destruction of algae in water. This method provides for the
Nov. i, 1920 Control of Fluke Diseases 207
solution of the correct amount of the salt from a sack attached to the
back of a canoe or boat, or, in very small pools, to the end of a pole.
Dissolved copper sulphate can conveniently be sprayed on small pools
from a spray pump or even an ordinary garden watering pot. In most
cases Bullinus, Physopsis, Planorbis, and Limnaea could be destroyed by
these methods.
(7) For the treatment of running streams the use of a barrel of suitable
size, fitted with a screened spigot, is recommended. The barrel is filled
with water, and sufficient copper sulphate is dissolved into it so that the
desired amount will be fed into the water per hour. Inasmuch as no two
spigots will flow at exactly the same rate and since the rate of flow will
diminish as the level of the fluid in the barrel is lowered, it is necessary
to determine beforehand the rate of flow at different levels and to cal-
culate the amount of copper sulphate to be dissolved according to the
average rate of flow. By the use of a tube of equal or greater length than
the height of the barrel, so that the head is increased, the diminution in
rate of flow can be greatly lessened. The addition of a few cubic centi-
meters of sulphuric acid to the solution in the barrel prevents the pre-
cipitation of iron sulphate, which is present as an impurity in commercial
copper sulphate and tends to clog the filter. Melania and Blanfordia
would probably have to be attacked by this method, since they live in
flowing water.
(8) In water which is not alkaline, large streams could be treated
more easily by allowing the copper sulphate, in the form of uniform
crystals, to dissolve directly into the stream through the screened end of
a tube. The amount of salt which would go into solution per unit of
time would depend on the diameter of the tube, the speed of the stream,
and the temperature of the water. If some feasible method could be
devised for slightly acidifying the water at the point where solution of
the salt is taking place, this method could be used advantageously in all
but very small streams.
(9) It is believed that by attacking the intermediate hosts of the
various pathogenic flukes of man and domestic animals by the use of
copper sulphate as herein outlined trematode diseases can successfully be
brought under control and can either be greatly reduced or entirely
eliminated in endemic areas, and this with comparatively little expense
and without active cooperation on the part of natives. With Govern-
ment aid and supervision, the work being carried out under the direction
of scientifically trained men or commissions, it seems entirely possible
that entire States or countries, at least in the vicinity of towns and
villages, could be freed of human fluke diseases, and that seriously
affected districts where sheep and cattle are raised could have the fluke
scourge wiped out in a short time with little expense.
208
Journal of Agricultural Research
Vol. XX, No. 3
LITERATURE CITED
i) Ando, R.
1915. PARAGONIMUS WESTERMANII — SUGGESTIONS AS TO PROPHYLAXIS.
(Abstract.) In China Med. Jour., v. 31, no. i, p. 73-74. 1917.
Original in Med. News, Dom. and For., no. 856, p. 202-203,1915.
2) Bado, Atilio A.
1916. LA ACCI6N DEL SULFATO DE COBRE SOBRE LAS ALGAS DE LAS AGUAS
potables. 15 p., illus., 2 col. pi. Buenos Aires.
3) Cawston, F. G.
1918. bilharziasis in south Africa. In Jour. Amer. Med. Assoc, v. 70,
no. 7, p. 439-441-
4) Christopherson, J. B.
1918. intravenous injections of antimonium tartaratum in bilhar-
ziosis. In Brit. Med. Jour., 1918, v. 2, p. 652-653.
1918. the successful use of antimony in bilharziosis. In Lancet, v. 195,
no. 4958 (1918, v. 2, no. 10), p. 325-327.
1919. antimony in bilharziosis. In Lancet, v. 196, no. 4976 (1919, v. 1,
no. 2), p. 79.
1919. ANTIMONY TARTRATE IN BILHARZIOSIS AND TACHYCARDIA. In Brit.
Med. Jour., 1919, v. 1, no. 3042, p. 480-481.
8) Dubois, R.
1901. DU CUIVRE NORMAL DANS LA S&RIE ANIMALE (ANIMAUX MARINS ET
terrestres). In Ann. Soc. Linn. Lyon, n. s. t. 47, p. 93-97.
9) Kobayashi, Harujiro.
1915. ON THE LrFE-HISTORY AND MORPHOLOGY OF CLONORCHIS SINENSIS. In
Centbl. Bakt. [etc], Abt. 1, Orig., Bd. 75, Heft 4, p. 299-318, 4 pi.
(10) Led?er, Robert T.
1915-18. REPORT ON THE RESULTS OF THE BILHARZIA MISSION IN EGYPT, I915.
In Jour. Roy. Army Med. Corps, v. 25, no. 1, p. 1-55, fig. 1-22; no. 2,
p. 147-192. %• 23-39; no. 3, p. 253-267, fig. 40-55. i9J5: y- 27> no. 2,
p. 171-190, fig. 56-85, 1916; v. 30, no. 3, p. 235-260, illus., 1918.
Bibliography, v. 25, no. 1, p. 48-55; no. 2, p. 182-192; no. 3, p.
261-267.
(n) Moore, George T., and Kellerman, Karl F.
1904. A METHOD OF DESTROYING OR PREVENTING THE GROWTH OF ALGAE
AND CERTAIN PATHOGENIC BACTERIA IN WATER SUPPLIES. U. S.
Dept.Agr. Bur. Plant Indus. Bui. 64, 44 p.
(12)
1905. COPPER AS AN ALGICIDE AND DISINFECTANT IN WATER SUPPLIES. U. S.
Dept. Agr. Bur. Plant Indus. Bui. 76, 55 p.
(^13) Nakagawa, Koan.
1917. HUMAN PULMONARY DISTOMIASIS CAUSED BY PARAGONIMUS WESTER-
manni. In Jour. Exp. Med., v. 26, no. 3, p. 297-323, pi. 22-31.
(14) Peters, Amos W.
1908. THE BIOCHEMICAL ACTION OF COPPER SULPHATE ON AQUATIC MICRO-
ORGANISMS. In Science, n. s. v. 27, no. 702, p. 909-910.
(15) and Burres, Opal.
1909. STUDIES ON ENZYMES. II. THE DIASTATIC ENZYME OF PARAMECIUM
IN RELATION TO THE KILLING CONCENTRATION OF COPPER SULPHATE.
In Jour. Biol. Chem., v. 6, no. 1, p. 65-73.
(16) Ransom, Brayton Howard, and Hall, Maurice C.
1912. THE ACTION OF ANTHELMINTICS ON PARASITES LOCATED OUTSIDE OF THE
alimentary canal. U. S. Dept. Agr. Bui. 153, 23 p. Bibliography,
p. 20-23.
(17) Rettger, Leo F., and Endicott, H. B.
1906. THE USE OF COPPER SULPHATE IN THE PURIFICATION OF WATER. In
Engin. News, v. 56, no. 17, p. 425-426.
(18) Thomas, A. P.
1883. THE LIFE HISTORY OF THE LIVER-FLUKE (FASCIOLA HEPATICA). In
Quart. Jour. Micros. Sci., n. s. v. 23, no. 89, p. 99-133.
INJURY TO SEED WHEAT RESULTING FROM DRYING
AFTER DISINFECTION WITH FORMALDEHYDE
By Annie May Hurd j
Assistant Pathologist, Office of Cereal Investigations, Bureau of Plant Industry, United
States Department of Agriculture
INTRODUCTION
Much has been written on the use of formaldehyde as a fungicide
for wheat and other grains infested with smut, but relatively little has
been carefully done on the effect of such treatment on the seed. The
usual recommendation has been a dip of about 10 minutes in a solu-
tion consisting of i part of commercial formaldehyde solution to 320
parts of water, followed by a 10-minute drain. Almost without excep-
tion instructions are given to dry the seed thoroughly before storing
it. The frequent advice that it be sown immediately after treatment
and not stored indicates that it has been learned by experience that
injury to the grain occurs not so much from the treatment as from
holding it in storage afterward. However, it has been almost univer-
sally concluded, without experimental evidence, that damp storage
causes the injury. Thus, practically every publication dealing with
seed treatment carefully warns against the storage of formaldehyde-
treated seed that has not been thoroughly dried after treatment.
The present investigation of the post-treatment action of formalde-
hyde on seeds was begun in 191 8 in the plant pathology laboratories
of the University of California as a part of the cereal-smut eradication
campaign carried on by the United States Department of Agriculture
and was continued through a period of nine months. The major con-
clusion reached is that it is extremely hazardous to dry seed which
has been treated with formaldehyde solution,2 and that, contrary to
common belief, seed wheat is absolutely uninjured by a 0.1 per cent
solution (1 to 40) and, if kept moist, may be held indefinitely without
injury, unless attacked by molds. We believe that the data here pre-
sented will contribute to our knowledge of the physical and chemical
properties of formaldehyde and the relation of these properties to
physiological processes in the seed. Such knowledge will undoubtedly
1 The writer wishes to acknowledge with gratitude the helpful suggestions of Dr. C W. Porter and
Dr. G. R. Gray, of the University of California, and the hearty cooperation of Prof. W. W. Mackie during
this study of formaldehyde. To Dr. H. B. Humphrey she is indebted for assistance in the preparation
of this report, and to Mr. A. A. Potter for cooperation in the preparation of the bibliography.
3 Reports sent in to Dr. H. B. Humphrey and to Prof. W. W. Mackie of occasional poor stands of wheat
from treated seed sown by farmers in the dry regions of California and Oregon indicate that field results
confirm those arrived at through these experiments.
Journal of Agricultural Research, Vol. XX, No. j
Washington, D. C Nov. i, 1920
vj Key No. G-206.
(209)
9507°— 20 4
2 1 o Journal of A gricultural Research vol. xx, No. 3
be helpful in any consideration of the more practical problems con-
nected with the use of this chemical as a fungicide.
Certain investigators working on this problem have shown that
injury to formaldehyde-treated seed occurs when the seed is allowed
to dry after treatment. The earliest report we have found of such
work is that of McAlpine (u),1 whose experiments showed that seed
treated with a solution of i pound of formaldehyde in 40 gallons of
water just prior to sowing under conditions favoring immediate germi-
nation grew as well as untreated seed. If, however, the seed was
allowed to dry for a day or more before germinating or if it remained
in dry soil some days before a rain, it suffered extreme injury. He
gives instances of such injury reported by farmers who from experience
had learned to sow formaldehyde-treated seed in moist soil immediately
after treating. McAlpine attributed this injury to the hardening effect
of formaldehyde on the seed coat. He claimed that by soaking the
dried treated seed in water prior to sowing this injury was averted.
He further stated that the injury after a dip in a 1 to 40 solution was
most pronounced when the seed had been kept a week after treatment.
After two weeks it began to improve until, when sown a month after
treatment, it was practically as good as 24 hours after treatment. He
stated also that this recovery did not occur when the solution used
was twice as concentrated.
In 1908, Shutt (14) found that a delay of three days in sowing after
the formaldehyde treatment reduced the percentage of germination and
increased the proportion of weak and slender plants. In opposition to
this are the results reported by Hurst (8), who states that seed may be —
treated and kept for any reasonable length of time without affecting its vitality.
Some of his samples, he says, had been treated 12 months before and ger-
minated as well as the untreated seed. Stewart and Stephens (16)
found that after the use of a 1 to 50 solution their samples were uninjured
by 6 weeks' dry storage, which was the longest storage period tested.
Brittlebank (3) noted a falling off in the germination of seed treated with
formaldehyde solution after being kept dry a week, the decrease contin-
uing to the sixth week, after which the percentages rose and fell with
various fluctuations through the remainder of the 54 weeks. Giissow
(6, p. 21-22) reported some figures obtained by Dr. C. H. Saunders,
Dominion Cerealist, showing that treated seed which originally germinated
75 per cent was entirely killed after being stored dry a year. Some barley
and oats treated similarly were almost wholly killed after standing
dry a year.
The first investigators to connect this storage injury with that property
of formaldehyde by virtue of which it forms a solid condensation product
or polymer upon evaporation were Darnell-Smith and Carne (5), who
1 Reference is made by number (italic) to " Literature cited," p. 243-244.
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 2 1 1
attributed the conflicting reports of the injury resulting from the for-
maldehyde treatment to variations in the deposit of this polymer on the
seed as it dried. They found low germination percentages and defective
seedlings to result from the drying of treated seed. Their results do not
agree with those of McAlpine, which were responsible for the latter's
conclusion that soaking in water prior to sowing removed the cause of
injury. They did find, however, that washing immediately after treat-
ment prevented subsequent injury in storage by removing the source of
the deposit. They thought that there was no internal poisoning of the
seed before germinating but that there was some deleterious chemical
action of a formaldehyde salt in the pericarp, which was alleviated by
soaking. Miiller and Moltz (12) proved that the polymer, paraformalde-
hyde, when mixed with the soil was very injurious to wheat sown in it.
An interesting and comprehensive report on the secondary effects of
formaldehyde treatment is the recent article by Kiessling (9). He ob-
tained severe injury upon storing treated seed which had been dried, and
this injury he found to be cumulative as the duration of the storage period
continued. He also was unable to confirm McAlpine's statement that
soaking the dried seed before sowing prevented the injury. Although
giving adequate and convincing proof that dry storage is more fatal than
damp storage, he does not advance any explanation.
Coons (4) also found that it is unwise to hold formaldehyde-treated
grains any length of time and that the injurious action is cumulative
when the treating solution is dried on the seed. He suggests that this
injury may be due to the formation of the solid condensation product,
paraformaldehyde, which might persist on the grain even after months of
drying.
POST-TREATMENT ACTION OF FORMALDEHYDE ON DRYING SEED
WHEAT
Except the studies of Coons (4) and those of Stewart and Stephens (16),
it will be noted that all the work on dry-storage injury to wheat has been
done outside the United States. This no doubt accounts for the fact
that it has been generally overlooked in this country or at least has not
resulted in any modification of the widespread instructions relative to
drying formaldehyde-treated wheat before storage. It was to investi-
gate this supposed formaldehyde injury to damp stored seed that the
studies here recorded were begun. These experiments resulted in the
rediscovery of the fact that so long as the seed treated with a 0.1 per
cent (1 to 40) solution remains damp there is no injury from the chemical
but, when dried, the seed is variously injured, depending upon the man-
ner of drying and upon the moisture content of the atmosphere surround-
ing the seeds.
2 1 2 Journal of Agricultural Research Vol. xx, no. 3
In the following experiments the seeds were left for 10 minutes in a
o.i per cent solution of formaldehyde followed by a draining period of 10
minutes. This strength is equivalent to i pint in 40 gallons of solution,
varying in small degree from that commonly referred to as 1 to 40, which
means 1 pint of standard formaldehyde solution in 40 gallons of water.
As the formaldehyde solution used in the laboratory contained 36.2 per
cent formaldehyde, such a dilution would be 1 part of formaldehyde in
884 parts of solution, or 0.113 per cent. Unless otherwise stated, the
wheat used was Little Club with a low percentage of thrashing injury.
After treatment the seed was spread on towels for an hour in order to
remove excess surface moisture. The damp seed was then divided into
two lots. One lot was put into three Mason fruit jars, holding about a
quart each, and sealed. The other lot was put into three boxes, 4 by 5
by 6 inches, and left uncovered. They were stirred frequently through-
out the experiment. These boxes each contained the same quantity of
wheat as did the jars. The original idea in having three samples of each
seed lot was to determine the relation of temperature to the injury which
was expected to appear in the damp samples. One box and one sealed
jar were left in the refrigerator at a temperature of io° C, one of each in
the laboratory at 200, and one in the greenhouse, where the tempera-
ture averaged about 300. For each of the six samples, as in all subse-
quent experiments, there was a control of seed dipped in water instead of
formaldehyde.
The following germination tests were made on blotters placed in
square pans, 12 by 12 inches, 1% inches deep, kept at room temperature.
The pans were covered with square pieces of glass, which made it easy
to observe the progress of the germinations. The depth of the pans
gave the seedlings a chance to grow erect and more normally than
would be the case if they were grown between blotters. Only those
seeds were called germinated which produced both a root and plumule.
Many which did so were too severely injured to produce plants in soil,
but the approximate percentage of these was obtained by contempo-
raneous soil germinations (Table II). Soil germinations have the advan-
tage of approximating more closely field results. The many advantages
in the use of blotters, however, lead the writer to emphasize the fact
that they are just as valuable to show the occurrence and comparative
degrees of seed injury. In view of the possibility of earlier detection
and easier study of such injury, they even may be preferable. The
results of the blotter germination tests are given in Table I,
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
213
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214 Journal of Agricultural Research vol. xx, no. 3
The outstanding fact shown by these experiments is that all the seed
which was treated with the formaldehyde solution and then dried by
being allowed to stand open to the air was either killed or seriously
injured after three to six days, while that treated at the same time and
stored at the same temperatures, but kept damp by being sealed in
jars, was practically uninjured up to the time it was destroyed by molds.
Later experiments have shown that injury may appear in dry-stored
seeds in less than three days, depending on the manner of drying. The
dry controls maintained the original germination throughout, and the
wet ones did also until they were killed by the development of fungi in
the jars. It will be noticed that molds appeared more slowly in the
damp, treated seed than in the damp controls, giving evidence of the
fungicidal action of the formaldehyde remaining on the seed. The
reason for the more extreme injury in the lots stored at room temper-
ature and in the refrigerator compared with those in the greenhouse
will be discussed later. These percentages also show most strikingly
that the injury to dried seeds is cumulative and that there is no recovery.
This is borne out by all subsequent experiments and refutes the claim
of McAlpine (11) and Darnell-Smith and Carne (5) that there is a steady
improvement after the extreme injury which appears after a week or so.
In addition to low germination percentages, the injured samples
showed a characteristic deformity and extreme retardation of the injured
seedlings. The earliest appearance of injury in the dried seeds was simply
a noticeable retardation of germination in the samples after being stored
three and six days, the plumules and roots never catching up with those
of the uninjured seedlings. The retardation became more extreme as
storage continued, with an ever-increasing number of short plants which
grew very slowly and resulted in stunted and misshapen plumules and
underdeveloped roots. After 10 days' storage all the seeds of the three
treated and dried lots were thus inhibited, so that upon germinating they
presented the appearance shown by those in Plate 36, A. The character-
istic deformity by which this extreme formaldehyde injury can always be
detected is the curving of the plumule as it emerges until it is sickle-
shaped (PI. 36, B). The growth of the sheath is inhibited so that it
never grows more than a few millimeters, leaving the young leaves to
push out unprotected, spindling, and weak, unable to push their way
through soil. The roots are underdeveloped but show no deformity.
It has been noted throughout these experiments that the greater the
retardation of germination in any injured seed lot, the greater the pro-
portion of weak, spindling plants produced. Whether the effect of the
formaldehyde on the sheath is to stop growth by stopping cell division
or by inhibiting the growth of the cells after they have divided was
not determined.
Anyone observing the seeds of the injured dry lots, the uninjured damp
ones, and the controls, germinating in blotters where invasion by Rhizopus
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 215
was possible, would notice at once the luxuriant growth of mycelium on
the injured seeds and its comparative rarity on the uninjured ones. He
might be inclined to ask whether the injury of the former samples was
not the result of fungous activities instead of action of formaldehyde
which might by its presence simply stimulate the growth of the mold.
This question is easily answered by disinfecting some of the dried, treated
seeds by a 10-minute dip into a 1 to 1,000 solution of mercuric chlorid
and germinating them on sterile blotters. The seedlings show the same
characteristic injury, but the percentages of germination are higher,
though not normal. This is because when they escape infection some
of the injured seeds succeed in germinating and produce weak plants.
These seeds, had they not been disinfected, would have been killed by the
invading fungus before the retarded root and plumule could emerge. The
extent of the development of this fungus on the various lots of germinat-
ing seeds serves as a fairly accurate index of the injury done to the seed by
the treatment. It is concluded from such experiments and many others
showing the same fact, which will be reported in detail in a subsequent
paper, that injury from drying after the formaldehyde treatment predis-
poses the seed to attack by molds, especially Rhizopus, the chemically
injured embryo being unable to resist infection.
It is commonly believed that blotter germinations are worthless so far
as being an indication of the viability of seeds in soil. Therefore, along
with the blotter germinations summarized in Table I, occasional tests of
the stored seeds were made in pots of sandy loam soil in the greenhouse.
It was found that with the uninjured samples the soil germinations gave
the same results as those made at the same time in blotters. With injured
seeds they were lower, as was to be expected, for in the blotters all those
seeds were counted germinated which produced both root and plumule
even though these were stunted or deformed. In the soil such seedlings
would never reach the surface, and so the count of germinated plants
from injured seed lots would be lower. Consequently, the injury pro-
duced by drying the formaldehyde treated seeds appeared even more
strikingly in the soil and would more closely approximate actual field
results. This is shown in Table II.
These figures do not indicate the full extent of the injury suffered
by the dried treated seed. Many of the seedlings from the injured
samples are short and spindling, while none of this sort are found in
the controls or in the samples which had been stored damp (PI. 37, A).
This same extreme injury was shown by the seeds stored dry in the
laboratory, but the figures are not included in Table II, because the
damp controls of both the untreated and the treated seed were destroyed
very quickly by the rapid development of Penicillium and Aspergillus
at that temperature. Plate 37, B, shows the seedlings produced by
these three seed lots injured by drying and the seedlings produced by
two of the controls.
2l6
Journal of Agricultural Research
Vol. XX, No. 3
Table II. — Percentage of germination in potted soil of wheat treated with o.i per cent
formaldehyde and stored for various periods
Treatment and storage.
Stored in refrigerator at io° C:
Treated, stored dry
Control, stored dry
Treated, stored damp
Control, stored damp
Stored in greenhouse ati5°to35°C
Treated, stored dry
Control, stored dry
Treated, stored damp
Control, stored damp
Stored 10
Stored 32
days.
days.
62
28
IOO
IOO
100
a g2
IOO
a g2
60
54
98
IOO
96
96
96
a 94
Stored 56
days.
18
IOO
090
o76
74
98
100
"82
°The germination of these samples is lowered by the development of molds in the jars. As will be
reported in a subsequent paper, saprophytic fungi attack stored wheat whenever the humidity is 70 per
cent or more, the treated seeds being attacked more slowly because of the slight protection afforded by the
formaldehyde.
After it had been determined that wheat stored and allowed to dry
after treatment was seriously injured, the next question which arose
was whether the same injury would be produced if seed sown immedi-
ately after treatment in dry soil remained there for some time before
sufficient rain fell to dampen the soil and induce germination. In dry
regions wheat often lies in the soil for weeks before germinating. To
duplicate these conditions, seed was treated in the usual manner with
a 0.1 per cent solution of formaldehyde and sown, 50 seeds in a pot, in
air-dry soil. On one series, a 0.2 per cent solution was used to show
more strikingly the cumulative nature of the injury. One pot of each,
with a control of seed treated similarly with water, was watered after
predetermined intervals such that the first pot was watered and started
to germinate immediately after planting while the last one remained
dry for a month. The results of the experiments with wheat are given
in Tables III and IV.
Table III. — Percentage of germination of Little Club wheat after lying in dry soil ( Yolo
clay loam) following treatment with 0.1 per cent formaldehyde solution
Water applied after —
Treatment.
0
days.
1
day.
2
days.
3
days.
4
days.
5
days.
7
days.
10
days.
14
days.
98
IOO
94
98
94
IOO
86
98
94
98
64
98
52
98
52
98
42
Controls, soaked in water
IOO
The data in Table III indicate that it is not safe to treat wheat with
formaldehyde, even when the strength of solution is as weak as 0.1 pei
cent, if the seed must be sown in very dry soil without certainty of rain
within a few days.1 Besides a lower percentage of germination, the ger-
1 Field reports are found to be in agreement with these laboratory tests. The hitherto unexplainable
poor stands of wheat from treated seed obtained by the farmers of the dry regions of California can now be
safely attributed to the fact that the seed lay in the dry soil for some time before rain.
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
217
mination of the injured seed lots was retarded, often several days, and
they produced a considerable number of spindling or short plants which
apparently never would be strong (Pi. 38, A).
The injury from drying, either in storage or in the soil, is greater the
more concentrated the solution used. The data given in Table IV demon-
strate this fact, the experiment differing from that summarized in Table
III only in the use of sandy-loam soil instead of the heavy Yolo clay loam
and in the fact that a parallel experiment was run at the same time in
which some of the treated seed was kept in a box in the laboratory and
a sample was germinated in blotters after drying for periods correspond-
ing to those in the soil experiment (Pi. 38, A).
Table IV.-
-Perceniage of germination of Little Club wheat treated with formaldehyde
and dried, both in the soil and in the air
Length of drying period.
Sown in dry soil.
o. 1 per
cent for-
maldehyde
solution.
o. 2 per
cent for-
maldehyde
solution.
Control,
dipped
in water.
Dried in the air and germinated
in blotters.
o- 1 per
cent for-
maldehyde
solution.
o. 2 per
cent for-
maldehyde
solution.
Control,
dfpped in
water.
Days
O
2
5
7
10
14
20
30
IOO
84
84
80
86
74
88
62
92
68
66
60
4S
34
44
90
84
86
90
62
80
60
66
So
52
54
44
96
100
IOO
IOO
96
96
96
96
In none of the experiments summarized in Tables I to IV was there
any injury to seed germinated at once after the dip into either 0.1 per
cent or 0.2 per cent formaldehyde. This fact is not in agreement with
results reported by many experimenters. Stewart and Stephens (16),
for instance, found that an immersion of 10 minutes in a 1 to 40 solution
(0.1 per cent) caused almost a 50 per cent loss. Kiessling (9), for ex-
ample, notes the great variation in the results reported on the effect of
formaldehyde on germination of seed. He concludes from the work
of others and from his own experiments that formaldehyde produces a
serious effect on the seed, the degree of injury depending on the sensi-
tiveness of the different varieties and the condition of the sample. None
of the wheat varieties tested in this laboratory (Little Club, Early Baart,
Marquis, Defiance, Sonora, and White Australian) was ever found to be
injured in the least by the recommended treatment, or by one twice as
strong, whether germinated in blotters or in the soil, so long as it was
sown immediately after treatment. Not only will the seed be uninjured
by the usual 20-minute exposure to a 0.1 per cent solution but it will
2l8
Journal of Agricultural Research
Vol. XX, No. 3
stand an immersion of 8 hours without injury. It can remain i hour
without injury in a solution twice as strong. Table V shows the result
of an experiment to determine the resistance of Little Club wheat to
long exposures to various strengths of formaldehyde solutions.
Table V. — Relation between strength of solution, duration of exposure, and seed injury
Soaked 20
minutes.
Soaked 1 hour.
Soaked 6 hours.
Soaked 8 hours.
Soaked 24 hours.
Strength of solution.
Germi-
nation.
Height
of
plants.
Germi-
nation.
Height
of
plants.
Germi-
nation.
Height
of
plants.
Germi-
nation.
Height
of
plants.
Germi-
nation.
Height
of
plants.
Per ct.
0
100
95
100
100
100
Cm.
3-5
3-5
3-5
3- 5
3- 5
Per ct.
0
25
90
95
95
95
Cm.
0. 2
.6
3-5
3- 5
3-5
Per ct.
0
0
0
40
100
100
Cm.
1. 0—
3-5
3-5
Per ct.
0
0
0
15
95
95
Cm.
1.0—
3-5
3-5
Per ct.
0
0
0
0
85
95
Cm.
4.50 per cent
0.45 per cent
0.10 per cent
Control, untreated . .
2-5
3- 5
Table V shows that Little Club wheat, thrashed with little injury, will
stand an 8-hour exposure to ao.i per cent solution., a i-hour exposure to
a 0.2 per cent solution, or a 20-minute exposure to 0.45 per cent and 4.5
per cent solutions.
The post-treatment injury from dry storage after subjection to a 0.1
per cent solution as well as to stronger ones has been demonstrated not
only with Little Club and Early Baart wheat but with Sonora, Marquis,
Defiance, and White Australian.
PHYSICAL PROPERTIES OF
FORMALDEHYDE AND PARAFORMALDE-
HYDE
After the fact had been established that a 0.1 per cent solution is
innocuous but that the drying of this solution on the seed is harmful, the
next step was to investigate the physical and chemical properties of formal-
dehyde in order to find a cause for the injury and a means of avoiding it.
The natural supposition was that the injury is due either to a concentra-
tion of the solution on the seeds as they dry or to a coating of paraformal-
maldehyde left upon them as the solution evaporates. It seemed at first
inexplicable, however, that the seeds stored damp, or even wet, should
remain absolutely uninjured indefinitely. In an effort to connect these
facts with the possible persistence and disappearance of the chemical on
the seed some qualitative tests for formaldehyde in washings of the
damp and dried seed were undertaken. It was the result of these first
qualitative tests which led to the intensive study of the behavior of
formaldehyde solution and paraformaldehyde and the possible determina-
tion of the cause of seed injury reported in this paper.
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 2 1 9
To detect the presence of formaldeyde on treated seed, Tollen's "sil-
ver mirror" aldehyde test 1 was used. To obtain comparable watei
extracts of the seed lots a uniform procedure was adopted which con-
sisted in extracting 15 cc. of the wheat sample with 10 cc. of distilled
water for two minutes in a 100-cc. graduated cylinder which was ro-
tated and shaken constantly to wash all the seeds as thoroughly as pos-
sible. Five cc. of the washings were then transferred to a test tube by
means of a pipette. Extracts of all the wheat samples to be studied
were thus prepared before proceeding. This is because it was necessary
to add the reagent to all at as nearly the same instant as possible in
order that results given by color changes might be comparable, since it
is by the relative rapidity of their appearance that the relative quantities
of precipitate formed by the presence of formaldehyde are shown. One
cc. of Tollen's reagent was then added quickly to each tube by means of
a pipette, and the tubes were watched for the appearance of the black,
or, at first, dark brown precipitate indicating the presence of formal-
dehyde. The relative quantities of formaldehyde present in the tubes
were shown by the rapidity of formation and by the density of this
precipitate.
Several interesting facts were disclosed by the application of this test
to the washings of treated seed. In the first place, distinct and positive
reactions were invariably obtained from seed which had been drying for
weeks, thus giving a clue to the reasons for the cumulative injury suffered
by seeds in drying. Positive reactions were given by extracts of samples,
the germinations of which were reported in Tables I and II, after the
seed had dried nine weeks in the laboratory. This, however, was longer
than thcaverage persistence of the paraformaldehyde, which, on account
of its volatility, usually disappeared in a month, depending on the con-
ditions of drying. It is understood, of course, that, in the presence of
moisture, paraformaldehyde at once breaks down and is again formal-
dehyde in solution.
In addition to this proof of the persistence of formaldehyde on the
seed in the form of paraformaldehyde, the qualitative tests showed in-
variably that about 24 hours after treatment there was more formalde-
hyde on the seed stored damp in a sealed jar than on that treated at the
same time and stored dry, showing a diminution in the quantity as the
seed dried. After 48 to 72 hours, the amount on the seeds in the sealed
jars had diminished at a more rapid rate, so that extracts from them gave
weaker and slower reactions than those from the dried seed. Within a
1 Tollen's reagent is an ammoniacal solution of silver nitrate which when added to a dilute aldehyde
solution produces a black precipitate or, upon standing and in the presence of a sufficient amount of the
aldehyde, forms a silver mirror by the precipitation of metallic silver on the sides of the test tube or other
container. It is made by dissolving 3 gm. of silver nitrate in 30 gm. of water and 3 gm. of sodium hydroxid
in 30 gm. of water, the two solutions being kept separate until ready for use, when they are mixed in equal
parts by volume and the resulting precipitate of silver oxid is dissolved by the addition, drop by drop, of
ammonia (specific gravity 0.923).
220 Journal of Agricultural Research voi.xx.No. 3
week, or at most two weeks, the damp seed ceased entirely to give any
formaldehyde reaction. An odd reddish brown color resulted when
Tollen's reagent was added to these extracts, but there was no black
precipitate. The question was to determine where the formaldehyde
had gone, for it seemed extremely inconsistent that it should disappear
in a sealed jar and yet remain on seed open to the air. The answer was
suggested by Dr. C. W. Porter, organic chemist at the University of
California, who said that it probably was absorbed by bacteria and mold
growing in the damp wheat.
To determine whether this were the case, some treated seeds were
divided into several lots. Part were inoculated with the spores of Penicil-
lium and sealed in small jars. The rest were left uninoculated and stored
similarly. Within a few days extracts of the former samples ceased
giving the formaldehyde reaction and produced the peculiar reddish
brown color noted above. The uninoculated lots continued to show the
presence of the chemical for some days longer but eventually became
moldy and then gave the same reddish brown color with the ammoniacal
silver nitrate.
Having demonstrated the persistence of formaldehyde on drying seed
and its disappearance from seed stored damp, and having evidence
pointing to the fact that seed injury from this fungicide may be dependent
on the formation of paraformaldehyde on the seed, we next undertook a
more critical study of the evaporation and polymerization of formalde-
hyde solutions.
It was found, upon evaporating the undiluted commercial solutions,
that a surprisingly large quantity of the solid, white, condensation product
was produced from comparatively small volumes. The percentage by
weight of the solid formed varied greatly in different determinations
because of variations in the conditions affecting the rate of evapora
tion — namely, quantity of solution, area of free surface, atmospheric
humidity, temperature, etc. Even with these factors controlled, the
same percentage could not be obtained with successive determinations
because there is continuous evaporation of the solid paraformaldehyde
after it has formed, as well as of the moisture in the, at first, waxy residue.
In our determinations a procedure as nearly uniform as possible was
always followed — that is, 50 cc. of undiluted 36.2 per cent formaldehyde
solution were evaporated by exposure to the air in a 100-cc. evaporating
dish, the residue being allowed to dry until the yellow color and waxy
texture had disappeared. The dry residue was weighed as soon as
possible after it became pure white, brittle, and easily powdered. A
solution analyzed at the Insecticide Laboratory of the University of
California and found to contain 36.2 per cent formaldehyde (specific
gravity 1.090) produced under these conditions an average of 9.85 gm.
of paraformaldehyde per 50 cc. This is 18.07 Per cent °f the weight of
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 221
the solution ( ^— - — = 18.07 ) and 49.92 per cent of the weight of the
V50X 1.090 '/ r
formaldehyde present ( — , ' r = 49.92 Y A 20-cc. volume
of undiluted formaldehyde solution gave 16.1 per cent paraformaldehyde
by weight of the solution and 44.6 per cent by weight of formaldehyde
originally present in it. A 10-cc. volume, evaporated under the same
conditions as the other two, gave only 7.8 per cent by weight of the
solution and 21.5 per cent by weight of formaldehyde. From this and
other data we know that the quantity of paraformaldehyde appearing
as residue upon the evaporation of a formaldehyde solution depends on
the original volume evaporated. Rate of evaporation is probably the
determining factor, the extent of the evaporating surface being small in
proportion to the volume as the latter is increased.
It has been shown (10, 14) that dilute formaldehyde solutions grow
stronger as evaporation proceeds. Notwithstanding this fact, published
statements to the contrary occur in literature relating to the use of
formaldehyde as a fungicide. The weakest solution analyzed by the writer
was a o. 113 per cent dilution. It was found by quantitative analyses1
of solutions before and after evaporation that the amount of formalde-
hyde per cubic centimeter of solution steadily increased as evaporation
proceeded. Some was lost with the water, as, otherwise, the amount in
the last 5 cc. would have been considerably larger than it was. The
increased concentration was great enough to indicate a deposit of para-
formaldehyde upon complete drying. As shown by the following test,
this proved to be the case. A 0.1 per cent solution of formaldehyde was
made with distilled water, and 50 cc. were put in each of two 8-cm.
evaporating dishes and evaporated by leaving them exposed to the air of
the laboratory, together with two controls containing 50 cc. each of
distilled water. As soon as the dishes were absolutely dry (in 12 days)
each dish was rinsed with 5 cc. of hot distilled water, and the washings
were poured into test tubes. To each was added 1 cc. of Tollen's reagent.
Results were distinct and decisive, a dark brown color appearing in the
1 The mosc accurate and convenient method found for determining quantitatively the amount of formal-
dehyde in a solution is that of Romijn (13). To 5 cc. of the formaldehyde solution are added 5 cc. N/io
iodin solution and so much strong sodium hydroxid solution, drop by drop, that the liquid assumes a light
yellow color. After a period of 10 minutes the solution is acidified with hydrochloric acid and the free
iodin is titrated back with Njio sodium thiosulphate solution. Every cubic centimeter of the iodin which
has been used up in the reaction with formaldehyde (the difference between the original 5 cc. added and the
amount left to react with the sodium thiosulphate) represents 0.001501 gtn. of formaldehyde present In
the solution.
The analyses, repeated several times with approximately the same results, were obtained by evaporating
100 cc. of a 0.1 per cent solution at room temperature in an 8-cm. evaporating dish. The quantity of solu-
tion used, atmospheric humidity, and other factors determine the degree of concentration of the evaporat-
ing solution at any point in the process. In the first analysis the amount of formaldehyde per cubic centi-
meter of solution increased from 0.0055 Sm- to 0.0069 gm. after the solution had evaporated from an original
volume of ioocc.to6cc. (in8 days). Inasecond analysis the increase was from 0.0058 gm. to 0.0069 gm. per
cubic centimeter, the evaporating solution decreasing in volume from 100 cc. to 10 cc. in an equal length of
time.
222
Journal of Agricultural Research
Vol. XX, No. 3
washings of the formaldehyde dishes, while the controls remained color-
less. This showed that paraformaldehyde is left as a residue on the
evaporation of solutions as weak as o. i per cent.
By successive weighings of the same sample it was found that para-
formaldehyde is volatile, gradually breaking down and escaping as
formaldehyde gas. To this property we may safely look for a large part
of the seed injury following treatment with formaldehyde. Figure i
illustrates graphically the rate of decreasing weight of 10.54 gm- °f para-
formaldehyde exposed to the air of the room in an 8-cm. evaporating
dish in which it was originally formed by the evaporation of 50 cc. of a
36.2 per cent solution.
r/ME /A/ WEEKS
2 3 4- & € 7 8 9 /O // /2 13 /4 /S /€ /7 /&
to
9
ie
V
*s
If
/
0
Fig. i
—Graph showing rate of evaporation of paraformaldehyde at room temperature, approximately
ao"C
INJURIOUS EFFECT OF PARAFORMALDEHYDE ON SEEDS
After it had been demonstrated that a solid residue is left upon the
evaporation of a formaldehyde solution and that this substance is con-
stantly breaking down to form formaldehyde gas, it seemed probable that
the cause of injury to treated seeds upon drying was the production of
an atmosphere of concentrated gas adjacent to the seed as a result of the
constant evaporation of this coating of paraformaldehyde. The gas,
being heavier than air, would tend to remain around the seeds, especially
when they are dried in heaps so that diffusion is not rapid. This idea
was borne out by the results of an experiment showing the deleterious
effect on the seed of contact with the dry, powdered, paraformaldehyde.
Dry, untreated seeds were put in Syracuse watch crystals and covered
with powdered paraformaldehyde which was packed closely around them.
The watch crystals were left uncovered and placed in a dry place. At
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
223
intervals 25 seeds were removed and germinated, with the results shown
in Table VI.
Table VI. — Percentage of germination of wheat kept in contact with powdered para-
formaldehyde a
Length of contact.
i^hours
24 hours
2 days
3 days
5 days
6 days
8 days
14 days
20 days
42 days
Control .
Experi-
ment 1 ,
Little Club,
harvester-
thrashed.
80
5°
Experiment 2,
Little Club.
Harvester-
thrashed .
5°
30
Hand-
thrashed.
OO
00
70
Experiment 4, Early
Experi- I Baart, hand-thrashed.
ment 3 , Eai -
ly Baart,
hand-
thrashed. Uninjured.
100
80
3°
o
Seed coats
broken over
embryo.
90
80
80
70
30
20
IO
O
a No germination tests were made at the intervals represented by blank spaces.
The data in Table VI show that dry paraformaldehyde powder kills
seed in contact with it, even those with unbroken seed coats. Those
with the testa injured, either by the thrashing machine or by breaking in
the laboratory with a needle, were injured and killed most quickly, as was
to be expected. It is noteworthy that the appearance and progression of
the seed injury was similar to that previously noted as occurring in the
successive germinations of treated seed being dried. The first sign of
injury was the retardation of the development of the plumule, which
became gradually more extreme. Finally, it was so injured that it did
not elongate at all after emerging from the seed, the sheath breaking
prematurely and showing the same curved, sickle-shaped deformity
previously found so characteristic of dried formaldehyde-treated seeds.
As it would be difficult to conceive of any absorption of solid para-
formaldehyde, the only plausible explanation of such "paraformalde-
hyde injury" is the penetration of formaldehyde gas through the seed
coat, the gas being concentrated in the interstices of the powder as a
result of the evaporation of the latter. Later experiments in which it
was found that absolutely dry seeds were uninjured by formaldehyde
fumes make it appear probable that the gas is dissolved in the cells of the
seeds and really diffuses into them as a solution.
HUMIDITY AS THE DETERMINING FACTOR IN SEED INJURY
The first hint that the humidity of the atmosphere surrounding the
seeds at the time of drying determined the amount of seed injury from
treatment with formaldehyde — by controlling the evaporation of the
224 Journal of Agricultural Research vol. xx, No. 3
solution on the seed and the formation of paraformaldehyde — came from
the difference in the degrees of injury sustained by the original samples
of treated wheat dried in the greenhouse, laboratory, and refrigerator
(see Tables I and II and PI. 37). The dried seed from the greenhouse,
where the atmosphere was warmest and most humid, was the least
injured. From our knowledge of the unstable constitution of para-
formaldehyde it seemed probable that it would form but slowly if at all
in the presence of moisture. Work, therefore, was undertaken to deter-
mine whether the degree of this seed injury resulting from drying after
treatment depended on the humidity of the atmosphere at the time of
drying.
The moisture content of the three dried samples of treated seed from the
greenhouse, laboratory, and refrigeratorw as determined after six weeks of
storage. By drying the seed to constant weight in an electric oven at a
temperature of 95 ° C. it was found that the seed dried in the laboratory
contained 13.28 per cent moisture, that from the refrigerator 15.35 Per
cent, and that from the greenhouse 16.63 per cent. Samples of each
lot were then tested qualitatively by means of Tollen's silver-mirror
aldehyde test for the presence of formaldehyde. A distinct difference
was obtained. The precipitate appeared most rapidly and was most
dense in the laboratory-stored seed which had the small moisture con-
tent, while it was decidedly least in the greenhouse-stored sample with
highest moisture percentage. These facts then suggested that the for-
mation of paraformaldehyde is dependent on the dryness of the atmos-
phere. Since all evidence points to the fact that seed injury upon dry-
ing after treatment is dependent on the formation of paraformaldehyde
on the seeds as the solution evaporates, it follows that seed injury may
vary inversely with the moisture content of the surrounding atmosphere.
So far as the three seed lots of this original experiment were concerned,
this was true, for the greenhouse where least injury occurred was most
humid and the laboratory where injury was most extreme was driest.
However, more evidence was necessary, and this could be obtained only
by storing treated seed under controlled and definitely known moisture
conditions.
Atmospheric humidities varying by 10 per cent intervals from satura-
tion over water to dryness over concentrated acid were produced in
desiccators by the use of sulphuric acid dilutions.1 Given the specific
gravity of the solutions necessary to produce the desired atmospheres
(PI. 38, B), they are easily made up in quantity by means of specific
gravity spindles and kept in stock bottles (17, p. 114).
'Since these experiments were completed, a paper written by Neil E. Stevens (is) has come to the
writer's attention in which a table is given showing the approximate humidities obtained in desiccators
containing aqueous solutions of sulphuric acid of various specific gravities. These differ somewhat
from those given by Woodworth (17, p. 114), and the method is described more fully and the data
given are more complete.
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
225
Some of the same machine-thrashed Little Club seed used in all these
experiments was treated with a 0.1 per cent solution, and, after the sur-
plus liquid was removed by spreading on towels for a half hour, the seed
was divided into n lots, each lot nearly filling a rectangular glass dish
6 by 8 cm. and 3 cm. deep. One of these dishes of wheat was then placed
in each of the 1 1 desiccators containing 100 cc. of their respective sul-
phuric acid and water mixtures. These solutions were changed at the
end of the first, second, third, fifth, and tenth days, so that they were
kept at the proper strength. Samples of wheat were removed after
various intervals, and the injury was determined by germinating on blot-
ters at room temperatures.
Table VII. — Relation between seed injury from drying after treatment with a 0.1 per
cent formaldehyde solution and the humidity of the atmosphere
Specific gravity of sulphu-
ric acid and water mix-
tures.
Ap-.
proxi-
mate
per-
cent-
age of
hu-
midity
pro-
duced
in des-
icca-
tors
cj.
Percentage of germination after storage in desiccators for —
day.
days.
5
days.
days.
days.
16
days.
22 26
days. days.
28
days. a
days.
OOO 1 OO
070 90
130 80
206 70
273 60
334 5°
400 40
47° 3°
53° 20
604 10
840 o
Control
98
96
94
96
96
96
94
96
96
94
98
96
96
98
96
94
98
92
100
94
96
92
90
74
84
«4
9S
96
94
90
90
82
70
74
84
80
<5>
(b)
94
96
82
76
76
So
(b)
96
90
96
88
74
78
80
(b)
(b)
(6)
98
90
86
78
70
84
72
82
96
(b)
(b)
(6)
90
96
92
82
b)
b)
(b)
88
86
94
74
72
70
72
64
100
(6)
(6)
100
92
88
82
76
72
76
86
96
a Germinated in soil.
b Attacked by molds.
A study of these germination percentages reveals several most interest-
ing facts. It is at once obvious that they show the existence of a close
relationship between the seed-treatment injury caused by drying and the
humidity of the atmosphere. They show that there is no injury in the
damper atmosphere of 70 per cent humidity and above, so long as the
seed is not attacked by molds. They show also that there is less injury
in the dryest desiccators, those containing from 20 per cent moisture to
none at all, than in those of intermediate humidities. These compara-
tive injuries are made clearer by a graph (fig. 2) the points on which
represent the averages of all the percentages obtained for each sample,
beginning with those obtained after five days' storage. The data for the
9507°— 20 5
226
Journal of Agricultural Research
Vol. XX, No. 3
80
HUMIDITY PERCENTAGE
70 60 SO 40 30 ZO /O
samples in 8o, 90, and 100 per cent humidities were not included because
of the small number of germinations obtained before the seed was par-
tially destroyed by molds. The curve shows graphically that there was
a decrease in germination from the uninjured samples in the high hu-
midities to those in 30 per cent humidity, after which it increased in the
successively drier desiccators but did not reach normal.
It is also noteworthy, in connection with the data given in Table VII,
that no injury appeared, as indicated by the germinated samples, until
at some time between two and five days after treatment. Thus, a test
of all samples after three days, the results of which were not included
in the table because of complications from an unusual growth of Rhizopus
in the germinators, showed no visible evidence of formaldehyde injury.
The harmful effects were first apparent after five days' storage, where,
however, molds again interfered with
the germination of four of the samples.
It will also be noticed in Table
VII that the successive percentages
obtained show no increasing injury
between the 5 -day and 42 -day germi-
nations. They differ in this from
those of many other experiments
(Tables I to IV).
Some months later this experiment
was repeated with some of the same
lots of wheat. This second experi-
ment differed from the first, so far
as was known, only in the smaller
quantities of treated wheat placed in
each desiccator and in the amounts
of sulphuric acid and water mixtures
used. Approximately 20 cc. of wheat
were put in each desiccator, which was about one-fourth of the quantity
used before. One hundred cc. of the desiccating solutions were left in
the desiccators for the first 24 hours, at the end of which period they
were changed, and 200 cc. quantities of the fresh solutions were sub-
stituted and left unchanged for the rest of the experiment. As will be
seen from Table VIII, the resulting seed injury was more extreme than in
the first experiment and reached its maximum in a more humid atmos-
phere (Pi. 39). The explanation for the difference may be the greater or
lesser effectiveness of the desiccating solutions, owing to the difference in
the quantities used and in the amount of seed dried over each.
The data in Table VIII show, as do those of the preceding experiment,
that the highest humidities allow no injury and that in the lowest the
germination percentages are normal also, only the retarded growth giving
Fig. 2. — Graph showing the relation of humidity
of the air to percentage of germination of stored
seed in first experiment.
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
227
evidence of some deleterious effect of the treatment. There is a very
definite point of maximum injury — the 70 per cent humidity. This
is somewhat different from the situation in the preceding experiment,
where the maximum injury was at approximately 30 per cent humidity,
with none at all occurring at 70 per cent.
Table VIII.
-Data from the second experiment on the relation between humidity and
seed injury after formaldehyde treatment
Specific gravity of sulphuric
acid and water mixtures.
Ap-
proxi-
mate
per-
centage
of hu-
midity
pro-
duced
in des-
icca-
tors
(2°°C).
Stored 10 days.
Germi-
nation.
Height
of
plants."
Stored 21 days.
Germi-
nation.
Height
of
plants."
Stored 35 days.
Germi-
nation.
Height
of
plants."
Stored 42 days.
Germi-
nation.
Height
of
plants."
I. OOO
I-I30
I.206
1-273
1-334
1.400
i-53°
1.604
1.840
Control
100
80
7°
60
5°
40
20
10
Per ct.
96
94
6
18
96
90
90
98
96
98
Cm.
5
5
Per ct.
98
IOO
4
38
70
84
IOO
Cm.
8.0
7.0
I. o —
I. o—
I. o
i-5
5°
4.0
6.0
7.0
Per ct.
Cm.
20
45
80
90
80
100
IOO
Per ct.
Cm.
92
88
1. 0-
1. o-
i-5
i-5
3-5
3-5
3-5
6.0
"The average heights of the plumules after 6 days are given for each germinating sample, because a
comparison of these for all the samples of any one test shows any injury indicated by retardation which
sometimes would not be shown by the germination percentage alone. A height of less than one centi-
meter (1 — ) indicates extreme injury, with usually stunted, deformed plumules which could not reach
the surface of the soil.
Figure 3 shows more plainly the comparative germinations given in
Table VIII. As in figure 2, each point was obtained by averaging all
the germination percentages given by the sample stored at each indicated
humidity.
Since all germinations were made in blotters without temperature or
humidity control, the rate of growth of seedlings of successive 6-day
germinations of the same sample varied in a meaningless way and so
were valueless except for comparisons of the injury shown by the dif-
ferent samples in the same germination test. However, as noted in the
discussion of the first experiment with the desiccators, the growth meas-
urements follow closely the germination percentages and are more deli-
cate indicators of harmful effects of treatment than the latter.
If the averages of the heights of the seedlings from each desiccator
for all the germination tests of both experiments be plotted with the
humidities in which the respective seed samples were stored, a graph
such as figure 4 is obtained. These heights were measured after six and
seven days' growth, but the conditions of germination in successive
228
Journal of Agricultural Research
Vol. XX, No. 3
tests were so variable that accurate comparisons of growth can not be
made. However, the graph, in its similarity to the germination graphs,
illustrates the close correlation between viability of the sample and the
retardation of seedling growth. Being an average of the two experi-
ments, it brings the maximum growth retardation to 60 per cent.
Since the relation between degree of seed injury and the moisture
content of the atmosphere in which the seed was stored had been shown,
it was surmised that a similar correlation could be shown to exist be-
tween humidity and the formation of paraformaldehyde. After the
seed samples of the first experiment were removed from the desiccators,
a Syracuse watch glass containing 10 cc. of commercial formaldehyde
solution was placed in each. The solid polymer first appeared after
three days as a white suspen-
sion in the dishes in humidities
of 20 and 10 per cent, and in
the very dry atmosphere over
concentrated acid. Two davs
later only the dry, white solid
was left in these dishes, and a
white precipitate made the
solutions opaque in the 30,
40, and 50 per cent atmos-
pheric humidities. The den-
sity of the suspensions was in
inverse proportion to the hu-
midity in these desiccators.
Xot until 10 days had passed
did any paraformaldehyde ap-
pear in the 60 per cent hu-
midity, at which time all that formed earlier in the dishes in the dryer
atmospheres was dry. No sign of the white solid ever appeared in the
more humid desiccators, although the solution in 70 per cent eventually
evaporated to dryness (PI. 38, B). It was very interesting thus to find
that the highest humidity permitting the formation of paraformaldehyde
was also the highest in which seed injury occurred after treatment with
the 0.1 per cent solution of formaldehyde — that is, the germination of
wheat was lowered in the same desiccators in which paraformaldehyde'
formed upon the evaporation of formaldehyde solutions in them.
Again, at the end of the second experiment, after the wheat was re-
moved from the desiccators, dishes containing equal quantities of un-
diluted formaldehyde solutions were placed over the sulphuric acid
dilutions, and the appearance and rapidity of formation of paraformal-
dehyde were noted. In this case 5-cc. instead of 10-cc. quantities were
used. After two days, the first white suspension appeared in the desic-
HUM/D/TY PERCENTAGE
/OO SO SO 70 60 SO -40 30 ZO /O O
95
Hi6*
,
1
1
\
^50
\
/
I
/
I
/
\
/
^ 40
16
IO
5
O
it
/
/
/
I
/
zr
/
1
j
f
\
/
id
/
/
Fig. 3. — Graph showing the relation of humidity of the air
to percentage of germination of stored seed in second
experiment.
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
229
HUMIDITY PERCENTAGE
IOO 90 80 70 60 SO 40 30 ZO /O O
cators having humidities varying from 40 per cent to dryness, being very
faint in the former and increasing to a considerable quantity in the
latter. The next day a faint opaqueness showed in the dishes of solution
in the 50 per cent, and on the day following in those in the 60 per cent
humidity, at which time all those in the drier chambers were entirely
dry. It is indeed interesting that although no solid ever formed in the
70 per cent humidity, this dish, as in the preceding experiment, evap-
orated to dryness but left no residue. The volume of liquid left unevap-
orated in the dishes in the damper atmospheres was greater the higher
the humidity (Table IX).
When the residue of paraformaldehyde left after the evaporation of
the solutions in the drier desiccators was weighed, it was found in both
experiments that, in general,
the quantity formed varied in-
versely with the humidity of
the atmosphere (Table IX). &
Since the degree of injury to y^tg
the stored treated wheat was
in the opposite order, it was at
once evident that the factor
causing the progressive varia-
tion in seed injury in the des-
iccators was not the quantity
of paraformaldehyde formed on
the seeds. Before this point is
considered further, however,
the results of a contempora-
neous experiment should be
presented. When the dishes
of formaldehyde solution were
placed in the desiccators to be evaporated , small quantities of untreated
seed were inserted at the same time to determine if formaldehyde
gas would evaporate in each humidity to produce sufficient concen-
trations in the different atmospheres to kill the wheat exposed to them.
When samples of this wheat were germinated at the end of the
experiment, surprising results were obtained. It was found after both
experiments that there was no germination of this seed from desiccators
of 70 per cent humidity and above and that the germination percentages
of seed from the drier atmospheres varied inversely with the moisture
percentage, the seed being least injured by the formaldehyde fumes from
the solution over concentrated acid. All these secondary experiments
on the dependence of the behavior of formaldehyde and its solutions on
atmospheric humidity are summarized in Table IX.
In brief, then, the facts are these: The seed injury resulting after
treatment with a 0.1 per cent solution, which occurs as the result of drying
\
\
1
/
\
■
to
2D
I.S
Fig. 4. — Graph showing the relation between humidity
of the air and seed injury as indicated by rate of growth
of germinated seedlings.
230
Journal of Agricultural Research
Vol. XX, No. 3
in atmospheres of such moisture content as permit the formation of
paraformaldehyde in evaporating solutions, is greatest in intermediate
humidities, becoming less as the moisture percentage decreases. This
is in spite of the fact that there is an increase in the quantity of para-
formaldehyde formed in these successively lower humidities. Secondly,
the degree of injury to untreated seed placed in desiccators alongside
of evaporating formaldehyde solutions in closed chambers is least in the
driest atmosphere and increases with increased humidity. It therefore
seems probable that the seeds in the lower humidities were so dry that
penetration of the seed coat by formaldehyde was difficult because of
the lack of sufficient moisture to permit solution of the gas on or in the
testa and its subsequent diffusion to the embryo.
Table IX. — Relation of the humidity of the atmosphere to the evaporation of formalde-
hyde solutions, the formation of paraformaldehyde, and the effects of the fumes on un-
treated wheat
Length of time
before appear-
Weight of
paraformalde-
hyde formed.
Volume of solution
left unevaporated.
Germination of un-
treated wheat left in
desiccators during
evaporation of
formaldehyde.
Humidity.
formaldehyde
in the solutions.
After
40 days.
After
24 days.
Exp. 1
(10-cc.
quan-
tity).
Exp. 2
(s-cc.
quan-
tity).
Exp. 1
( IO-CC.
quan-
tity).
Exp. 2
S-cc.
quan-
tity).
Exp. 1
(original
volume
10-cc.).
Exp. 2
(original
volume
S-cc).
Exp. 1
(with 10-cc.
quantity).
Exp. 2
(with 5-cc.
quantity).
Per cent.
Days.
Days.
Gm.
Gm.
Cc.
9.6
7.2
5-4
. 0
Cc.
5-°
1.8
. 0
Per cent.
O
O
O
O
O
O
O
4
36
54
Per cent.
O
O
80
O
70
O
00. .
10
5
5
3
3
3
4
3
2
2
2
2
0. 07
• 40
i-33
2. 25
1. 12
1. 12
0. 07
.96
1. 14
1. 36
I. 42
I.36
O
50
O
O
10
l6
O
30
In presenting this explanation, we are assuming that formaldehyde
does not penetrate seed coverings easily, if at all, as a gas but must be
dissolved. A small quantity of moisture in the cells of the seed covering
therefore would perhaps be necessary to permit injury from formaldehyde
fumes. This is consistent with the statement of Humphrey and Potter
(7) that—
disinfection with formaldehyde gas seems to require some moisture.
This supposition would explain the relation found between the degree
of injury resulting from drying treated seed and the humidity of the at-
mosphere in which the seed is dried. With the atmosphere sufficiently
dry to allow the formation of the "formaldehyde reservoii " — the coating
of paraformaldehyde on the seed — the ease of penetration of the formalde-
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 231
hyde gas constantly formed next to the seed by its decomposition would
be determined by the moisture in the seed coat. It would follow, as was
actually found, that there would be a point where maximum seed injury
would occur — at a humidity low enough to permit the solid polymer to
form on the seed as the solution evaporates, yet high enough to permit
diffusion in solution of the gas formed from it through the cells of the seed
coat to the embryo. Thus may be explained the gradual lessening of
the degree of injury from the point of maximum injury to practically
normal germination in dry atmospheres.
The work of Arcichovskij (1) on the effect of graded concentrations of
formaldehyde solutions ranging from 0.125 to 40 per cent supports the
assumption that the ease of penetration of formaldehyde is dependent on
the dilution of the solution as it passes through the cells into the seed.
He found that, for any given duration of exposure, seed injury did not
increase directly with the concentration of the solution. After a definite
point of maximum injury, the harmful action of the solution decreased
with increased concentration, until in all exposures over four hours the
undiluted 40 per cent formaldehyde solution caused less injury than the
0.125 per cent dilution. For instance, after 256 hours 37.5 per cent of
the seeds from the 40 per cent solution germinated, while those in the
0.125 per cent solution were entirely killed after 32 hours' exposure.
The curve he has drawn showing the relation between concentration of
the solution and the percentage of germination is similar to the curves
in this report which show the relation between humidity and formalde-
hyde injury to seeds upon drying after treatment.
The preceding paragraphs merely offer a suggestion of an explanation
of the observed facts. This interpretation of these facts is based on
several assumptions which have not been proved by direct evidence.
One is that paraformaldehyde, as a solid, does not injure seeds but only
upon its breaking down into formaldehyde gas and forming a toxic vapor
about the seed. Another is the assumption that this formaldehyde does
not penetrate seed coats as a gas but that it must enter in solution.
It should be pointed out here that in experiment 2 the maximum injury
occurred in the atmosphere of 70 per cent humidity (Table VIII) in the
desiccator in which it was found that the formaldehyde solution evapo-
rated to dryness without the formation of paraformaldehyde (Table IX).
This indicates that seeds may be injured by the concentration of a o. 1
per cent solution on the surface as evaporation proceeds, without the
formation of the solid polymer.
RELATION OF DEGREE OF INJURY TO MANNER OF DRYING
In the course of the experiments it was noted that the drying injury
was not always of the same severity, and it was finally found that it
depended on the aeration of the drying sample, thinly spread seed escap-
ing the injury suffered by that dried in heaps. This observation was
232
Journal of Agricultural Research
Vol. XX, No. 3
decided to be consistent with our previous conclusions as to the manner
in which formaldehyde solutions injure the treated seeds upon which
they dry. If injury occurred as the result of the close adherence to the
seed of concentrated formaldehyde gas formed by the decomposition of
paraformaldehyde deposited on the surface as the seed dried, then it
would follow that well-aerated seeds might very probably escape injury
by virtue of the rapid breaking down of the polymer and its escape by
diffusion into the air. Formaldehyde gas is heavier than air, so that if
seeds were dried in large quantities in sacks or in boxes, diffusion would
be slow and the air around the seeds would become saturated with gas,
which would be held around them long enough to cause seed injury.
The evaporation of but a relatively small quantity of paraformaldehyde
in a closed space saturates the atmosphere so that further breaking down
T/ME //V WEEKS
O I 2 3 4 S 6 7 8 9 fO // 12 i3 fit JS /6 17 tS
8
I7
K4
Fig. 5. — Graph showing the diminution in the rate of evaporation of paraformaldehyde inclosed in a
desiccator of 2,400-cc. volume.
of the solid is inhibited by the partial pressure of the formaldehyde gas.
This was shown experimentally by placing some paraformaldehyde in
desiccators at the same time that dishes containing approximately the
same quantities were evaporating in the open air of the room. The rate
of evaporation of each sample was measured by the loss in weight after
successive weekly intervals. Figure 5 illustrates the initial rapid rate of
evaporation of a sample in the open air and the slowing up of that rate
when it was placed in a 2 ,400-cc. desiccator containing calcium chlorid as
a drying agent. When the sample was removed from the desiccator the
rate increased again, and the curve representing this period shows a
steady , even fall, until after 1 8 weeks the solid had practically disappeared.
If we compare the curve with figure 1, we note that whereas when the
sample is exposed to the open air it disappears entirely, when it is inclosed
and hence unaerated its evaporation practically stops. The exact weight
v
PIACSO /N
DESICCATOR
\
\
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 233
of the solid which when evaporated in a space of 2 ,400 cc. checked by its
partial pressure further decomposition of the sample is not shown. It
would appear to be approximately o. 1 gm., the average decrease in weight
found upon successive weekly weighings of the inclosed sample. The
slight fall of the curve for this period in the desiccator is explained by the
fact that when the dish was removed each time for weighing the con-
centration of gas within would be diluted and so the sample would con-
tinue to lose weight. A parallel control experiment gave the same curve
and the same total loss in weight, 0.21 gm., during the two weeks in the
desiccator.
The significance of this curve for the problem of post-treatment injury
of dried seeds is that when there is no aeration the formaldehyde gas
from the evaporating paraformaldehyde on the seeds easily saturates the
atmosphere in the interstices of the sample and inhibits the evaporation
of more of the solid. The slower the outward diffusion of the gas the
longer will the paraformaldehyde remain on the seed surfaces and the
longer will a toxic atmosphere exist about them. As the penetration of
the seed coat and subsequent injury by formaldehyde is comparatively
slow, usually occurring in from 3 to 5 days with a 0.1 per cent solution
(Table VII), it is entirely conceivable that with rapid drying and thinly
spread seed any paraformaldehyde formed can be completely evaporated
and its dissipation effected so rapidly that it can not enter and injure the
embryo.
Seeds treated with a 0.2 per cent solution, twice as strong as the usual
treatment, were dried without injury when spread in a single layer on
towels, while such seeds dried in quantity in an open box were prac-
tically all killed. That it was the time required for the formaldehyde to
penetrate the testas which saved the former lot of seed was shown by the
fact that some of the same sample which had the seed coats broken over
the embryos were dried beside the others and were severely injured after
24 hours. In the former case the paraformaldehyde evaporated and
diffused before it could penetrate the sound seed coat. But when a 4.5
per cent solution was used, even the seeds with unbroken coats were
found to be injured after 24 hours' drying under these conditions. The
quantity of paraformaldehyde formed presumably was too great to
escape before seed injury occurred. The broken seeds dried at the same
time showed proportionately greater and more rapid injury than the
broken seeds treated with the weaker solutions. It will be noted in
Table X that embryos exposed by broken testas are not injured by a
10-minute dip into formaldehyde as strong as 0.2 per cent but that a
4.5 per cent solution is injurious. It is significant that with rapid drying
and aeration even the seeds with broken seed coats were not injured by
a o. 1 per cent solution. Yet it has been found repeatedly that when
perfect seeds thus treated are dried without aerating they are injured or
killed.
234
Journal of Agricultural Research
Vol. XX, No. 3
Table X. — Relation between strength of formaldehyde solution, condition of seed coat,
and the cumulative injury to Early Baart wheat well spread during the drying period a
Length of
drying period.
iM hours
4 hours.. .
24 hours..
3 days
6 days
14 days. . .
45 per cent formaldehyde
solution.
Seed coats
unbroken.
Ger-
mina-
tion.
Per
cent.
ioo
65
Height
of
plants.
Seed coats
broken over
embryo.
Ger-
mina-
tion.
Per
cent.
60
Height
of
plants
Cm.
5
1.2 per cent formaldehyde
solution.
Seed coats
unbroken.
Ger- Height
mina- j of
tion. plants
Per
cent.
95
95
95
100
95
95
95
Cm.
5-0
3- 5
3-5
4-0
2. o
2-5
2. o
Seed coats
broken over
embryo.
Ger-
mina-
tion.
Per
cent.
1. 1 per cent formaldehyde
solution.
Seed coats
unbroken .
Height Ger-
of I mina-
plants.l tion.
Cm
Per
cent.
95
100
100
100
100
100
90
Height
of
plants.
Cm.
5-o
4-o
5-o
5.0
5-o
5.0
3-5
Seed coats
broken over
embryo.
Ger-
mina-
tion.
Per
cent.
95
95
100
Height
of
plants.
Cm.
4-5
4.0
5-o
5-o
5-o
3-0
3-5
a The average heights of the plumules after 6 days are given for each germinating sample, because a
comparison of these for all the samples of any one test shows any injury indicated by retardation which
sometimes would not be shown by the germination percentage alone. A heighth of less than one centi-
meter (1-) indicates extreme injury, with usually stunted, deformed plumules which could not reach
the surface of the soil.
In brief, Table X shows that when treated seed is dried rapidly by being
thinly spread in the laboratory, it is uninjured by a o. i per cent solution
even if the embryos are exposed by broken seed coats ; that seed treated
with a 0.2 per cent solution is uninjured if the seed coat is perfect, but
severely injured after 24 hours if it is broken; and that, with a 4.5 per
cent solution, perfect seeds are slowly injured and that seeds with broken
testas are injured by the dip into the treating solution, which injury
rapidly increases upon drying. The cumulative nature of this seed
injury is well shown by the germination data for all these injured
samples.
Lest there be any misunderstanding, it may be well to consider again
the case of treated seed which is sealed damp. It may be asked at this
point that if aeration is necessary to prevent injury from formaldehyde
fumes, how can seed stored damp in sealed jars remain uninjured? The
answer is probably to be found in the fact that paraformaldehyde does
not form on damp seeds; hence the damp seeds are not surrounded by
concentrated formaldehyde vapor. The moisture in the jar is a weak
dilution, and neither it nor the amount of formaldehyde in the air
in the presence of so much water is strong enough to injure the seed.
Moreover, the formaldehyde does not remain on damp seeds indefinitely,
owing to the activity of microorganisms which decompose it. The case
is different with solutions stronger than o. 1 per cent, however. Damp
seed is slightly injured by a 0.2 per cent solution after 24 hours' storage,
and a 4.5 per cent solution is fatal in a sealed jar. Whether in these
instances it is the solution on the seed which injures or the resulting
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
235
formaldehyde fumes was not determined ; but according to Auerbach and
Barschall (2), the partial pressure of formaldehyde gas above solutions
in a closed space increases with the concentration of the formaldehyde
solution ; hence the fumes may be the cause of injury.
Several experiments showed clearly the varying degrees of injury
resulting from drying the seed at different rates. The usual procedure
was to treat some wheat with a 0.2 per cent solution and some barley
with a 4.5 per cent solution, the latter being more resistant to drying
injury and therefore requiring the use of a strong solution to produce it.
Some of each lot was then spread thinly over towels on the laboratory
table, while the rest was put in an open tumbler or a slender, uncovered
bottle. For comparison, a third lot usually was placed in a similar
bottle. and sealed while damp. Samples were removed after various
intervals and were germinated in the usual way to determine the degree
of injury. The data on the germination of wheat are shown in Table XI
and those on the germination of barley in Table XII.
Table XI. — Percentage of germination of Little Club wheat treated with 0.2 per cent
solution of formaldehyde and dried under different conditions and during periods of
varying lengths
Experiment i.
Experiment 2.
Control,
untreated.
Length of drying period
Spread on
towels.
In open
bottle.
In sealed
bottle.
Spread on
towels.
In open
bottle.
Days.
O
96
84
96
74
96
82
94
94
98
94
76
88
96
98
96
I
2
3
70
64
40
52
40
16
84
80
82
6
76
88
74
56
64
52
52
5°
98
98
94
92
18
28
60
50
8 £n
Wheat treated with a o. i per cent solution was dried overnight in a
sealed jar, in an open jar, and in a thin layer on towels. After drying
24 hours, equal samples were washed in equal volumes of water, and the
washings were subjected to Tollen's aldehyde test for the presence of
formaldehyde. Comparison of the density and rapidity of formation of
the silver precipitate showed that there was least formaldehyde on the
thinly spread seed and greater amounts on the other two samples. At
the end of the second 24-hour period the experiments were repeated.
It was found that the amount of formaldehyde on the sealed seed had
diminished until it gave a much less dense precipitate than either of the
dried samples. Of the latter, the extract from the seed dried in the
bottle showed the presence of more formaldehyde than that from the
236
Journal of Agricultural Research
Vol. XX, No. 3
well-spread seed. Throughout subsequent tests, continued almost daily
for two weeks, the dried samples gave stronger reactions than the damp
ones, which, after about six days, showed no more than the extract from
the untreated control. The dried samples soon gave about equal reac-
tions. The results of the first two tests, which showed that there was
more formaldehyde on the seed dried in the open bottle than on that
spread on towels, confirm the conclusion already drawn from the germi-
nation data — that is, that more paraformaldehyde remained on the seed
dried without aeration because the formaldehyde gas could not escape
readily from around the seed. Gradually, however, this gas escaped
and the quantity present, as shown by the reaction, decreased to that
of the aerated sample.
Table XII. — Percentage of germination of Coast barley treated with 4.5 per cent formalde-
hyde solution and dried under different conditions and for varying periods of time
length of drying
period .
Experiment 1.
Experiment 3
Spread on
towels.
In open
bottle.
In sealed
bottle.
Control,
untreated.
Spread on
towels.
In open
bottle.
Control,
untreated.
Days.
O
98
88
98
66
98
64
98
94
80
86
82
5°
82
52
80
74
44
O
10
2
88
I
90
92
92
96
92
6
80
68
36
a 80
20
0
0
8
4
0
0
4
94
90
84
96
17
28
60
0 70
10
90
"These increased germinations after 42 days, though they apparently indicate recovery, are probably
due to more favorable germination conditions.
From these data it appears that any prediction or explanation of post-
treatment injury must be based on the humidity of the atmosphere
immediately surrounding the seed and on the manner of drying the seed
as affected by its aeration. Temperature may also be an important
factor, but its relation to the problem has not yet been determined.
Temperature or some other variable must account for the fact that,
with all the foregoing conditions controlled, repetitions of experiments
do not always give the same results. For instance, in Table XI injury
is shown to thinly spread wheat after a subjection to a 0.2 per cent
solution of formaldehyde, while less injury is shown in the data in
Table X. In these cases the differences may possibly be chargeable to
the fact that different samples of different varieties of wheat were used.
In one experiment no greater injury occurred to seed dried in an open
bottle than to that thinly spread on the table beside it. Such excep-
tions are only occasional, but they indicate that certain apparently
minor factors have not yet been ascertained.
Nov.
Effect of Drying Disinfected Seed Wheat
23?
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238 Journal of Agricultural Research vol. xx. N0.3
Interesting data were obtained as the result of an experiment origi-
nally intended to show the relation between the moisture content of the
seed and the degree of injury upon drying. Samples of wheat and
barley were treated with a 0.1 per cent solution for 10 minutes, drained
10 minutes, and allowed to dry partially by spreading on towels for an
hour. At the end of that time about 70 cc. were sealed in a small screw-
top bottle, and the rest were allowed to continue drying. Equal quan-
tities were removed from the drying lot daily for four days and sealed,
the object being to get samples of different moisture content so stored
as to insure constant humidity in the bottles. It was found that constant
weight was reached after two or three days' exposure to laboratory air.
The moisture percentage of each sealed sample was obtained by drying
in an electric oven at 95 ° C. Samples from each bottle were germinated
after various intervals, and the injury shown by each was compared by
means of germination percentages and rate-of-growth observations.
Tables XIII and XIV summarize the results of several experiments.
The data in Table XIII show that none of the samples were injured
by the drying period which preceded their being sealed. This was due,
no doubt, as explained earlier in this paper, to the fact that they were
spread thinly and, therefore, were well aerated. In the second place, it
shows, surprisingly enough, that the subsequent injury from being sealed
did not bear a direct relation to the moisture content of the seed, as had
been expected. After 21 days' storage samples sealed wet immediately
after treatment and those sealed after 1 hour's drying were uninjured.
This was to be expected, for they contained too much moisture to permit
the formation of paraformaldehyde. But in all germination tests made
after 6 or more days' storage those samples dried for 10, 20, and 30 hours
before sealing showed extreme injury, while those dried longer were less
injured. Seed dried 72 hours before sealing was nearly as free from injury
as the uninjured, damp seed. This lesser injury to the samples dried
for the longer periods seemed so puzzling that the experiment was re-
peated, twice with wheat and once with barley, with the same results.
The data in Table XIV again show that, although no injury resulted
from these various drying intervals, yet when the seed was sealed there
was extreme injury after 5^2 , 9, and 24 hours' drying, after 48 hours
slight injury (retarded plumules), and after 72 hours practically no injury.
The maximum injury occurred in seed dried 5^2 and 9 hours, respectively,
decreasing steadily with the longer drying periods of the other samples.
This shows particularly well in the germinations of seed in soil, where the
weak and injured seedlings, called "germinated" on the blotters, did
not reach the surface of the ground and so particularly emphasized the
injury to the 5^- and 9-hour samples. Elsewhere in this paper it has
been noted that blotter germinations suffice to show comparative injuries
and to indicate the deformity and retardation of the seedlings; but,
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
239
except to one trained to distinguish the weakened and injured seedlings,
the germination counts will not give an accurate measure of field results.
In soil, the percentage of germination of injured samples will be much
lower, of course, depending on the nature of the soil and the difficulty
encountered by the seedling in emerging from it.
Table XIV. — Percentage of germination of Little Club wheat and Coast barley treated
with O.I per cent formaldehyde solution and sealed in bottles after drying for various
periods
LITTLE CLUB WHEAT
Length of
storage
period after
drying.
Dried
1 hour,
20.96 per
cent
moisture
when
sealed.
Dried
$yi hours,
18.36 per
cent
moisture
when
sealed.
Dried
9 hours,
16.34 per
cent
moisture
when
sealed.
Dried
24 hours,
14.81 per
cent
moisture
when
sealed.
Dried
48 hours,
12.94 Per
cent
moisture
when
sealed.
Dried
72 hours,
13 -3 7 per
cent
moisture
when
sealed.
Dried
96 hours,
12.15 per
cent
moisture
when
sealed.
Control,
untreated,
12.06 per
cent,
moisture
when
sealed.
Days.
100
100
92
92
96
58
14
48
88
60
2
42
98
82
70
74
98
94
88
92
100
94
100
92
98
96
88
94
96
96
7°
96
COAST BARLEY
7...
7a-.
96
a Germinated in soil; all others germinated on blotters.
This second experiment also demonstrated that this phenomenon is
shown by barley as well as wheat. For barley, as for wheat, the maxi-
mum injury was to those samples dried for 5K and 9 hours, with de-
creased injury to the samples dried longer before sealing.
In subsequent experiments on wheat treated with both 0.1 per cent
and 0.2 per cent solution, then dried and sealed, there was always this
upward gradation in injury from a maximum below 24 hours of drying
to almost normal germination in samples dried for several days and then
sealed. However, in the experiment illustrated in Plate 40, there was
severe, though lessened, injury to the sample dried three days before
being sealed in the bottles.
For a long time after the first results of this nature were obtained they
seemed inexplicable. After the later studies of the behavior of formalde-
hyde and the manner in which it injures seeds through the volatilizing
of its polymer, paraformaldehyde, an explanation suggested itself.
In the first place, it is obvious from what we know of paraformalde-
hyde that it did not form on the dampest seeds. Hence, those seeds
sealed after one hour showed no injury because at the end of that time
they were still damp. Paraformaldehyde formed on those dried more
thoroughly, and the gas resulting from its evaporation at once began to
diffuse away from around the seeds because they were thinly spread.
240
Journal of Agricultural Research vol. xx. No. 3
As a result of this steady evaporation of the paraformaldehyde from
these seeds those spread the longest before sealing had the smallest
quantity on them when put in the bottles, while those sealed earlier had
increasingly greater quantities. Since evaporation of the solid would
continue to a certain extent after the seeds were in the bottles, it would
seem plausible that the concentration of formaldehyde gas in the atmos-
pheres of the sealed bottles would vary, being greatest where seed had
previously dried for but a few hours and least where it had had a longer
time to dissipate into the air before sealing. It follows that the seed
injury in each bottle is proportionate to the quantity of paraformalde-
hyde left on the seed at the time of sealing, which, upon evaporation in
the bottle, cannot escape and is held around the seed.
SUSCEPTIBILITY OF OTHER GRAINS TO POST-TREATMENT INJURY
In laboratory experiments it was found that barley is much less sensitive
than wheat to dry-storage injury after treatment with ao. 1 percent solution
and often escapes injury altogether. Retardation or a slight lowering of
the germination percentage usually results, however, from drying the
seed in bulk or from sowing it in dry soil. In experiments where the seed
was allowed to lie in dry soil for varying intervals one experiment showed
rather severe injury, while the two repetitions showed none at all. If
a 0.2 per cent solution or a 4.5 per cent solution is used the characteristic
cumulative post-treatment injury occurs markedly, just as in wheat.
The latter strength is especially destructive when the seed dries (Table
XII). The germination percentages shown in Table XV (on blotters,
with one exception) are typical of the results obtained in the laboratory
when Coast barley was dried in tumblers after treatment.
Table XV. — Percentage of germination shown by Coast barley when dried in the labora-
tory after formaldehyde treatment
Length of drying period.
0.1 per cent
solution.
Exp. 1. Exp.
y2 hour 94
7 days 90
2 1 days 84
42 days 84
56 days 88
70 days a 76
92
94
So
0.2 per cent
solution.
Exp. i. Exp. 2.
92
74
82
32
70
34
04
86
52
40
4-5 per
cent so-
lution,
Exp. 2.
52
6
Control,
untreated.
Exp. 1. Exp. 2
96
90
90
92
90
90
88
90
92
° Germinated in soil.
The presence of the glumes on the barley grains probably affords the
protection which makes them more resistant than wheat to the harmful
effects of treatment and subsequent drying.
Nov. i, 1920
Effect of Drying Disinfected Seed Wheat
241
Three sorghums, Brown durra, Honey sorgo, and Sudan grass, were
found to be uninjured by either a 0.1 per cent or a 0.2 per cent solution
of formaldehyde even after weeks of drying. When the seed was stored
dry in the same manner as was the severely injured wheat, no effects of
the treatment ever appeared. This probably is due to protection af-
forded by the glumes in some instances, and in others by the thick seed
coats.
PREVENTION OF POST-TREATMENT INJURY RESULTING FROM
DRYING
McAlpine (11) thought that soaking the seed which had been held
some time before sowing prevented the appearance of formaldehyde in-
jury, but neither Darnell-Smith and Carne (5) nor Kiessling (9) was
able to confirm this. The writer also has been unable to show that the
injury can be avoided in this way. Soaking the seed hastened the
germination, as it always does even with untreated wheat. But the
characteristic injury to the seedling remained, and the percentage of
germination, although occasionally somewhat augmented, was far from
normal. It seems probable, therefore, that the hardening of the pericarp
is not the primary injury.
It has been shown in this paper that thorough aeration of the treated
seed as it dries retards and lessens storage injury but does not always
prevent it (Tables X, XI, and XII). Neither is rapid drying possible
where large quantities of wheat are handled. However, it was found
that dry-storage injury can be entirely avoided by simply washing the
seed with water after treatment (PI. 41). The extent to which this
simple procedure would do away with the danger in the use of formalde-
hyde solutions is shown by the data in Table XVI.
Table XVI. — Percentage of germination of wheat treated with o.l and 0.2 per cent
formaldehyde solutions and washed with water, compared with percentage of germina-
tion of unwashed samples
Length of drying period.
Days
O
7
14
30
60
0.1 per cent solution.
Seed not
washed in
water.
78
62
58
52
36
Seed
washed in
water.
78
74
74
74
0.2 per cent solution.
Seed not
washed in
water.
72
So
30
32
Seed
washed in
water.
76
74
82
76
72
Control, un-
treated."
70
72
76
72
74
"This seed had been injured by fumigation with carbon bisulfid, hence the low germination of the
untreated control and washed samples.
9507°— 20 6
242 Journal of Agricultural Research vol. xx, No. 3
SUMMARY
(i) No seed injury was produced by treating wheat with either a o.i
per cent (i to 40) or a 0.2 per cent (1 to 20) solution of formaldehyde if
the seed was germinated immediately after treatment.
(2) If treated seed is held several days or more before sowing, it is
severely injured if allowed to dry without thorough aeration during the
storage period. If, however, the seed remains damp, it suffers no injury
from a 0.1 per cent solution and can be so kept indefinitely or until
attacked by molds.
(3) Post-treatment injury is usually cumulative, increasing in degree
the longer the seed is stored.
(4) This seed injury upon drying apparently is due to a deposit of
paraformaldehyde on the seed, which forms as the formaldehyde solution
evaporates. The solid paraformaldehyde, being volatile, is constantly
breaking down into formaldehyde gas. This gas, being thus concentrated
and held so close to the seed, penetrates it slowly, probably going into
solution in the testa.
(5) The degree of post-treatment injury depends primarily on atmos-
pheric humidity during the storage period. In atmospheres damper than
70 per cent humidity the treated seed can be kept indefinitely without
ill effects. In those of 70 per cent and less there is decided injury, which
is most severe in the intermediate humidities, gradually decreasing in the
lower ones until seed stored in an absolutely dry chamber is almost unin-
jured.
(6) No paraformaldehyde formed upon the evaporation of formalde-
hyde solutions placed in these damper chambers in which no seed injury
occurred, but it did form in all solutions evaporated in desiccators of 60
per cent humidity and less, the quantities by weight increasing as the
atmosphere became drier. Therefore, seed injury in the desiccators was
not determined by the quantity of paraformaldehyde formed on the seeds
in each.
(7) Untreated wheat, when placed in desiccators of varying atmos-
pheric humidities alongside of evaporating, undiluted 36.2 per cent
formaldehyde solutions, was least injured in the absolutely dry chamber
and was entirely killed by the formaldehyde vapor in all the chambers
damper than 30 per cent humidity.
(8) In view of the facts that treated seed is less injured in very dry
atmospheres than in intermediate ones and that untreated seed is least
injured by formaldehyde fumes in the dry atmosphere of desiccators, it
is considered probable that formaldehyde does not enter seeds as a gas or
in the solid polymeric form but in solution in the seed coats. For the
maximum seed injury to occur as a result of drying after formaldehyde
treatment, therefore, there must be an optimum atmospheric humidity
Nov. i, 1920 Effect of Drying Disinfected Seed Wheat 243
to permit, first, the formation of paraformaldehyde, and second, the solu-
tion of formaldehyde gas in the seed.
(9) This post-treatment injury is minimized by spreading the seed as
it dries so that maximum aeration occurs, thus hastening the evaporation
of paraformaldehyde and the escape of the gas from around the seed.
(10) Barley is less susceptible to post-treatment injury upon drying
after soaking in a o. 1 per cent solution, probably because of the protection
afforded by the glumes; but when stronger solutions are used the injury
is very severe.
(n) Seed dried for an hour by being thinly spread on towels in the
laboratory and then sealed in bottles is uninjured after weeks of storage;
but seed dried longer, although uninjured by the rapid drying, is injured
upon being sealed, presumably because of the concentration of gas in the
bottle as a result of decomposition of the paraformaldehyde on the seed.
Treated seed dried from 5 to 24 hours was more injured upon being sealed
than when dried for a longer time.
(12) The sorghums, Brown durra, Honey sorgo, and Sudan grass, are
uninjured upon being stored dry after treatment, even when a 0.2 per
cent solution is used.
(13) Post-treatment injury from dry storage is entirely prevented by
washing the seed with water immediatelv after treatment.
LITERATURE CITED
(1) Arcichovskij, V.
1913. DIE WIRKUNG DER GIFTSTOFFE VERSCHIEDENER KONZENTRATIONEN AUF
die samen. In Biochem. Ztschr., Bd. 50, Heft 3/4, p. 233-244, 5 fig.,
pi. 1.
(2) Auerbach, Friedrich, and Barschall, Hermann.
1905. STUDIEN UBER FORMALDEHYD. I. MITTEILUNG. FORMALDEHYD IN WAS-
siger losung. In Arb. K. Gsndhtsamte, Bd. 22, Heft 3, p. 584-629,
(3) Brittlebank, C. C.
19 13. EFFECT OF FORMALIN AND BLUESTONE PICKLE ON THE GERMINATION O*
wheat. In Jour. Dept. Agr. Victoria, v. 11, pt. 8, p. 473-476.
(4) Coons, G. H.
1918. THE USE OF FORMALDEHYDE TO CONTROL CEREAL SMUTS. In Mich. Agf.
Exp. Sta. Quart. Bui., v. 1, no. 1, p. 11-14.
(5) Darnell-Smith, G. P., and Carne, W. M.
1914. THE EFFECT OF FORMALIN ON THE GERMINATION OF PLANTS. In 3d Rpt.
Govt. Bur. Microbiol. [N. S. Wales], 1912, p. 178-180.
(6) Gussow, H. T.
1913. smut diseases of cultivated plants. Canada Cent. Exp. Farm Bui.
73, 57 p., illus.
(7) Humphrey, H. B., and Potter, A. A.
1918. cereal smuts and the disinfection of seed grain. U. S. Dept. Agr.
Farmers' Bui. 939, 28 p., 16 fig.
(8) Hurst, R. J.
1911. bunt and germination experiments. . . . In Agr. Gaz. N. S. Wales,
v. 22, pt. 9, p. 749-752-
244 Journal of Agricultural Research voLxx.no. 3
(9) Kiessling, L.
1918. UBER SCHADLICHE NEBENWIRKUNGEN DER FORMALINBEIZUNG DBS SAAT-
GUTES AUF DIE KEIMUNG. In Jour. Landw., Bd. 66, Heft i, p. 7-51.
(io) I/ADD, E. F.
1904. ANALYSIS OP FORMALDEHYDES SOLD IN NORTH DAKOTA. In N. Dak. Agr.
Exp. Sta. 15th Ann. Rpt., [19041/05 , pt. 1, p. 18-30.
(11) McAlpine, D.
1906. EFFECT OF FORMALIN AND BLUESTONE ON THE GERMINATION OF SEED
wheat. In Agr. Gaz. N. S. Wales, v. 17, pt. 5, p. 423-439.
(12) Muller, H. C, and Molz, E.
1914. VERSUCHE ZUR BEKAMPFUNG DES STEINBRANDES BEI DEM WINTER-
WEIZEN MITTELS DES FORMALDEHYD-VERFAHRENS. In Fuhling's
Landw. Ztg., Jahrg. 63, Heft 23, p. 742-752.
(13) Romijn, G.
1897. ueber die bestimmung DES formaldehyds. In Ztschr. Anal. Chem.,
Jahrg. 36, Heft 1, p. 18-24.
(14) Shutt, F. T.
1908. report of the chemist, insecticides and fungicides. In Canada
Exp. Farms Rpts. [i907]/o8, p. 165-173.
(15) Stevens, Neil E.
1916. A METHOD FOR STUDYING THE HUMIDITY RELATIONS OF FUNGI IN CULTURE.
In Phytopathology, v. 6, no. 6, p. 428-432.
(16) Stewart, Robert, and Stephens, John.
1910. THE EFFECT OF FORMALIN ON THE VITALITY OF SEED GRAIN. Utah Agr.
Exp. Sta. Bui. 108, p. 145-156.
(17) WOODWORTH, C. W.
1914. entomology. In Cal. Agr. Exp. Sta. Rpt. 1913/14, p. 109-118.
PLATE 36
A. — Post-treatment seed injury occurring when wheat is dried after treatment
with a 0.1 per cent solution. Sample No. 1 was stored dry during the 28 days pre-
ceding this germination test, and sample No. 2 was stored damp in a sealed jar, the
latter germinating at the end of that time as v/ell as the untreated control.
B. — Germinating seedlings of Little Club wheat, showing characteristic post-
treatment injury when seed is treated with a 0.1 per cent solution. The upper row
shows the usual deformity — curved, sickle-shaped plumule and prematurely broken
sheath. Below are seedlings from untreated seed, showing normal germination.
Effect of Drying Disinfected Seed Wheat
Plate 36
Journal of Agricultural Research
Vol. XX, No. 3
Effect of Drying Disinfected Seed Wheat
Plate 37
B . V m
Journal of Agricultural Research
Vol. XX, No. 3
PLATE 37
A. — Pots showing germination of treated seed stored for 32 days after disin-
fection with a 0.1 per cent solution, of formaldehyde: No. 1, stored dry in labora-
tory; No. 2, stored damp in laboratory; No. 3, stored dry in refrigerator; No. 4,
stored damp in refrigerator; No. 5, stored dry in greenhouse; No. 6, stored damp in
greenhouse; No. 7, control, untreated.
B. — Wheat plants grown in soil from seed stored for 60 days after disinfection with
a 0.1 per cent solution of formaldehyde: No. i, stored dry in refrigerator, germina-
tion 18 per cent; No. 2, stored dry in laboratory, germination 34 per cent; No. 3,
stored dry in greenhouse, germination 70 per cent; No. 4, stored damp in greenhouse,
germination 100 per cent; No. 5, control, untreated, germination 100 per cent.
PLATE 38
A. — Wheat seedlings showing injury produced by allowing the seed to lie in dry
soil for 30 days after treatment with a 0.1 per cent solution of formaldehyde: Left,
control, dipped in water, 100 per cent germination; center, dipped in 1 to 320 (0.1
per cent) formaldehyde, 62 per cent germination; right, dipped in 1 to 160 (0.2 per
cent) formaldehyde, 48 per cent germination.
B. — Desiccators with different degrees of atmospheric humidity obtained by the
use of mixtures of sulphuric acid and water in different proportions. The dishes
containing formaldehyde were not placed in the desiccators until after the degree of
injury to the treated seeds had been determined. The atmospheric humidities were as
follows: No. 1, saturated; No. 2, 90 per cent; No. 3, 80 per cent; No. 4, 70 per cent;
No. 5, 60 per cent; No. 6, 50 per cent; No. 7, 40 per cent; No. 8, 30 per cent; No. 9,
20 per cent; No. 10, 10 per cent; while No. 11 was absolutely dry, over undiluted
acid. Note the white paraformaldehyde formed in these dishes in the drier cham-
bers, beginning with No. 5. (See Table VII for specific gravity readings of sulphuric
acid and water mixtures.)
Effect of Drying Disinfected Seed Wheat
Plate 38
Journal of Agricultural Research
Vol. XX, No. 3
Effect of Drying Disinfected Seed Wheat
Plate 39
Journal of Agricultural Research
Vol. XX, No. 3
PLATE 39
Germinating samples of wheat stored for 35 days after treatment in the desiccators
shown in Plate 38 B, illustrating the relation of seed injury to humidity.
Sample A, 100 per cent humidity, 95 per cent germination.
Sample B, 80 per cent humidity, 100 per cent germination.
70 per cent humidity,
60 per cent humidity,
50 per cent humidity,
40 per cent humidity,
20 per cent humidity,
10 per cent humidity,
Sample C,
Sample D,
Sample E,
Sample F,
Sample G,
Sample H,
Sample I,
Sample J,
o per cent germination.
20 per cent germination.
45 per cent germination.
80 per cent germination.
90 per cent germination.
80 per cent germination,
o per cent humidity, 100 per cent germination,
control, untreated, 100 per cent germination.
PLATE 40
Varying injury to wheat treated with a 0.1 per cent solution of formaldehyde, and
stored in sealed bottles:
A. — Sealed immediately after treatment, 100 per cent germination.
B. — Sealed after drying 7 hours, spread on towels in laboratory, no germination.
C. — Sealed after drying 24 hours, spread on towels in laboratory, no germination.
D. — Sealed after drying 3 days, spread on towels in laboratory. 14 per cent ger-
mination.
The control germinated 96 per cent.
Effect of Drying Disinfected Seed Wheat
Plate 40
Journal of Agricultural Research
Vol. XX, No. 3
Effect of Drying Disinfected Seed Wheat
Plate 41
Journal of Agricultural Research
Vol. XX, No. 3
PLATE 41
Germinating wheat kernels, showing the prevention of post-treatrnent injury by
washing the seed with water immediately after treatment. Susceptibility of seeds
injured by treatment to Rhizopus and other saprophytes is also shown. This seed
had been kept in open tumblers for 30 days after treatment.
A. — Treated with 0.2 per cent solution, which was not washed off before drying, 32
per cent germination.
B. — Treated with 0.2 per cent solution, which was washed off before drying, 76 per
cent germination.
C. — Treated with 0.1 per cent solution, which was not washed off before drying, 52
per cent germination.
D. — Treated with 0.1 per cent solution, which was washed off before drying, 74
per cent germination.
Control germinated 74 per cent.
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Vol. XX NOVEMBER 15, 1920 No. A
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Page
Studies on the Life History and Habits of the Beet Leaf-
hopper --_-_--_. 245
C. F. STAHL
( Contribution from Bureau ol Entomology)
Hypertrophied Lenticels on the Roots of Conifers and
Their Relation to Moisture and Aeration - 253
GLENN G. HAHN, CARL HARTLEY and ARTHUR S. RHOADS
(Contribution from Bureau of Plant Industry)
Degree of Temperature to Which Soils Can Be Cooled
without Freezing _______ 267
GEORGE BOUYOUCOS
(Contribution from Michigan Agricultural Experiment Station)
Changes Taking Place in the Tempering of Wheat - 271
E. L. TAGUE
(Contribution from Kansas Agricultural Experiment Station)
Vascular Discoloration of Irish Potato Tubers - - 277
H. A. EDSON
( Contribution from Bureau of Plant Industry)
Crownwart of Alfalfa Caused by Urophlyctis alfalfae - 295
FRED RUEL JONES and CHARLES DRECHSLER
(Contribution from Bureau of Plant Industry)
Pathological Anatomy of Potato Blackleg - 325
ERNST F. ARTSCHWAGER
( Contribution from Bureau of Plant Industry)
Sclerotinia minor, n. sp., the Cause of a Decay of Lettuce,
Celery, and Other Crops - - - - - -331
IVAN C. JAGGER
(Contribution from Bureau of Plant Industry )
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WASHINGTON : GOVERNMENT PRINTINQ OPFI0E I ItM
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and A ssociate Chief. Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, Slate CoVexje of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellcrman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment -Station, New
Brunswick, N. J.
JC OF AGRICULTURAL RESEARCH
Vol. XX Washington, D. C, November 15, 1920 No. 4
STUDIES ON THE LIFE HISTORY AND HABITS OF THE
BEET EEAFHOPPER1
[PRELIMINARY PAPER]
By C. F. Stahl
Scientific Assistant, Truck-Crop Insect Investigations, Bureau of Entomology, United
States Department of Agriculture
INTRODUCTION
Much has been published concerning the distribution and history of
the beet leafhopper and its relation to the curly-top disease of sugar
beets, but no complete account of its life history and habits has appeared.
The present paper gives a brief summary of observations bearing on
these points made during the past few years at Jerome, Idaho, and in
the sugar-beet growing regions of California.
DESCRIPTION
EGG
The egg when first laid is transparent, elongate, and slightly curved. The pos-
terior end tapers gradually almost to a point. Length 0.0612 to 0.0696 mm. ; average
width 0.0182 mm.
As the embryo develops, faint spots which later become conspicuous eye spots
appear on either side of the anterior end. During development the color of the egg
changes from white to lemon yellow with a slight tinge of green.
NYMPH
The recently hatched nymph is nearly transparent, with a light yellow tinge in
the thorax and abdomen. The antennae are hairlike and more than half as long as
the body. The head is wider than the thorax or abdomen and is the most distinc-
tive characteristic of this instar.
After the first molt the nymph is more slender and the head and antennae are not
nearly so conspicuous. Average length 1.40 mm.; width 0.45 mm. Color usually
milky white with a green tinge. Faint brown blotches may be distinguished on
the thorax.
In the third instar there is more variation in the coloring. General color varying
from yellow with light brown markings to almost black. The pattern made by the
brown blotches does not seem to be constant, but the denser coloration on the thorax
has been designated as a "saddle" (j, p. 21).2 Length 1.99 mm.
1 Euletlix lenella Baker, suborder Homoptera. family Jassidae.
2 Reference is made by number (italic) to " Literature cited," p. 252.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C Nov. 15, 1920
vk Key No. K-86
^ (245)
246 Journal of Agricultural Research vol. xx, No. 4
The color variations in the fourth instar are similar to those of the third. A red
coloration is often observed. The spines on the legs are more conspicuous than
formerly, and the wing pads extend to the dorsal margin of the third abdominal
segment. Length 2.30 mm.
After the fourth molt the nymph has a slender appearance and is nearly the size
of the adult. The wing pads extend approximately to the dorsal margin of the
fourth abdominal segment. Length 3.2 mm.
ADULT
In California, during the summer, adults of this species may be collected showing
a gradation in color from light green with no markings to dark gray with numerous
markings on the elytra (PI. 42, A-C). In the fall the percentage of dark forms
is much larger, and during the winter it is unusual to find a light form. Some of
the winter forms appear almost black.
The following color details are given to show, to some extent, the extreme contrast
in coloration:
Light form (PI. 42, A). — Front yellow, with faint, light brown, transverse stripes.
Eyes gray, with occasional brown spots. Vertex green and lemon yellow, the yellow
predominating. Pronotum green. Scutum deep yellow. Elytra hyaline with light
brown venation. No pigment in the elytra. Tergum appearing as dark bands
through the folded elytra.
Dark form (PI. 42, B). — Frontyellow, with irregular, testaceous, transverse bands.
Eyes a mixture of red and brown, red usually predominating. Vertex fulvous,
apical portion with a white band cut in center by a narrow dark band. Pronotum
olive, except for ivory anterior band with several black spots. Scutellum with two
square, black spots at basal angles. Elytra subhyaline, marked with, black about
as follows: Two large, almost circular spots on corium; apical portion and irregular
black blotches on claval region. Nervures dark brown, with dark pigment on each
side forming irregular bands.
RESEMIU.AXCE TO OTHER SPECIES
There should be little difficulty in distinguishing the beet leaf hopper
from other leafhoppers commonly found on sugar beets in California.
Occasionally the darkest forms resemble some species of Agallia in
coloration, but even a superficial examination will be sufficient to sepa-
rate these two genera. These species of Agallia do not have the slender
appearance of the beet leafhopper and are much slower in their move-
ments. After a little experience in collecting it is possible to distinguish
between the two genera by their movements. Eutettix tenella rarely,
if ever, feigns death when disturbed; but some of the species of Agallia
are almost certain to fall over on their backs and lie for some time as
if dead. This habit is often an aid in collecting when the leafhoppers
are not abundant and a careful search is necessary. One species, Cica-
dula 6-notata Fallen, may often be confused with the beet leafhopper,
especially when individuals of the latter are mainly of the green colora-
tion. The six spots on the vertex of C. 6-notata are usually plainly
evident, however, and will serve to distinguish this species from
E. tenella.
Nov. is, 1920 Life History and Habits of the Beet Leafhopper 247
LIFE HISTORY AND HABITS
' REPRODUCTION
During the summer season mating occurs within a few days after the
last molt is accomplished, but during the fall this period is greatly pro-
longed. In Idaho adults were observed copulating in cages during the
late fall as well as during the summer season. At Spreckels, Calif.,
mating continued throughout the winter. Unfertilized females have
been known to lay sterile eggs under certain conditions, but partheno-
genesis has never been observed.
The preoviposition period is comparatively long. In all experiments
15 to 17 days elapsed between the date the female reached maturity and
the date the first eggs were laid. A much longer period is common,
especially during the winter and early spring.
OVEPOSITION
Under normal conditions the eggs of the beet leafhopper are usually
placed in the petiole or midrib of the sugar-beet leaf, beneath the fibrous
strands and at a slight angle. They are invariably deposited one at a
time, but often they are arranged in rows of from two to five, placed end
to end so that they give the appearance of overlapping. It is almost
impossible to find the recently deposited eggs in the petioles; but after
the embryo has developed a little and the eye spots have appeared they
are comparatively conspicuous. When, deposited in the leaf tissue the
eggs are more easily detected by the raised areas on the leaf surface. By
transmitted light eggs in this position appear as small, transparent slits.
While apparently preferring the sugar beet as a plant in which to
deposit its eggs, this leafhopper will oviposit in a large number of other
plants. Fleshy or succulent species offer the most suitable conditions
for oviposition. Russian thistle (Salsola kali var. tenuifolia), filaree
(Er odium cicutarium and E. moschatum) , Chenopodium spp. (especially
murale), and Atriplex spp. are plants from which eggs have been most
commonly noted hatching under natural conditions. Most perennial
plants are too tough and woody to be suitable for this purpose, and it is
doubtful if they are of any great importance as hosts during the egg-
laying period.
Ball (2, p. 40) records the number of eggs deposited by a single female
of this species as about 80. At Spreckels, Calif., the maximum number of
eggs deposited by one female was 237, while at Riverside, Calif., the maxi-
mum was 247. Many difficulties were encountered in the conduct of
these experiments, and it is probable that, given more favorable condi-
tions, the females might have deposited a larger number of eggs.
Meteorological conditions influence greatly the incubation period. A
maximum period of 52 days has been observed during the early spring
and a minimum of 10 under most favorable conditions. During the
248 Journal of Agricultural Research voi.xx, N0.4
height of the egg-laying season the incubation period ranged from 10 to
15 days.
Seasonal variations in the development of the nymph are wide, due
mainly to differences in temperature and food supply. The entire
nymphal period ranged from 25 to 52 days, while from 4 to 10 days
were required for the completion of each instar.
NUMBER OF GENERATIONS
Ball (1, p. 95; 3) states that the beet leaf hopper is a single-brooded
species and implies that such is the case for conditions even as far south
as Glendale, Ariz. Experiments conducted at Spreckels, Calif., demon-
strated that there were unquestionably at least two generations annually
in that locality. Under conditions more favorable than was usual for
this part of the Salinas Valley, a third and even a fourth brood were
obtained. There was only one brood on sugar beets in southern Idaho,
but it seems probable that further investigation would reveal an addi-
tional brood, possibly on the wild vegetation.
LONGEVITY OF ADULTS
Under natural conditions it is doubtful if the normal length of life
of the adult is more than 4 or 5 months. Fall-brood adults are not
found in the fields during the summer, and the spring brood is rarely
noted in the fall. Females have been kept alive in cages for 19 months,
but it is doubtful if they would ever survive so long under field conditions.
SEASONAL HISTORY
IN SOUTHERN IDAHO
Although persistent effort was made to locate adults of the beet leaf-
hopper during the winter and early spring in southern Idaho, they were
not observed until their appearance on the sugar beets. The earliest
record for this was June 6, 1914, when several individuals were collected
on volunteer sugar-beet plants at Jerome. Apparently the leafhoppers
are in the cultivated fields as soon as the beets are up.
Oviposition begins in the field as soon as the adults appear. Records
have been made as early as June 22, when the beets were still young
and had not yet been thinned. June 28 was the earliest hatching record
obtained in cage experiments. Starting thus, early in June, oviposition
continues throughout the season until late in October.
During 191 3 adults were not observed copulating until late in the fall.
On October 12, a large number of adults confined in a lantern globe were
noted copulating for several days. During the one winter spent by
the writer in this district only a few adults placed in cages in the fall
survived the winter, and all of these were females. These observations
indicate that the females are fertilized in the fall before hibernation and
that a large percentage of males perished during the winter.
Nov. 15, 1920 Life History and Habits of the Beet Leaf hopper 249
Weather conditions were severe enough during the winter in this dis-
trict to necessitate hibernation. All attempts to determine the method
of hibernation, however, as well as the places in which it takes place
were failures. Adults in cages survived the winter underneath dead
beet leaves and in the crown of the plant.
EN CALIFORNIA
Under California conditions adults and nymphs are most abundant
in the field during August. At harvest time they are scattered, and no
doubt a large number perish. After the beets have all been removed
from the fields the leafhoppers seem to be greatly diminished in numbers,
although they may be collected from certain weeds growing in the fields
and along the irrigating canals. No indications of a general migration
have been noted at such times, so it is assumed that the surviving individ-
uals scatter over wild vegetation, selecting that which is most suitable
for food and protection. Later they may congregate in certain spots
which furnish especially favorable conditions during winter.
There is no true hibernation in the districts of California that have
been under observation. Adults have been collected every week in the
winter under conditions indicating that they were feeding when captured.
Under cage conditions food must be available at all times. As a rule, all
individuals kept without food died within 48 hours.
The characteristic dark-colored individuals of the fall brood that leave
the beet fields could hardly be confused with the light-colored adults
that appear the next spring. A small percentage of the fall-brood
adults may remain in or near the beet fields during the winter and be
responsible for the early injury in the spring, but it is usually not until
the light forms appear in considerable numbers that attention is directed
to the damage. The striking difference in coloration between the fall
and spring forms suggests at once the possibility of a new brood on wild
vegetation before migration into the beet fields. Observations and cage
experiments have proved that such a brood occurs.
The time when the leafhoppers first appear in fields in spring in Cali-
fornia varies with the seasonal conditions in different localities, being
from April 1 to June 1. The condition of wild vegetation in the natural
breeding areas is an important factor in determining when migration to
the beet field will take place. As long as this vegetation is abundant and
succulent it is doubtful if there is any general movement into the culti-
vated areas.
Oviposition begins as soon as the adults appear in the field and con-
tinues throughout the season. There is an overlapping of broods
which makes it impossible to determine the exact number under field
conditions. Cage experiments, however, have demonstrated that there
may be from one to three each year on the beets. Thus the maximum
number of broods in one year would be four.
250 Journal of Agricultural Research vol. xx, No. 4
NATURAL ENEMIES
EGG PARASITES
The following three species of egg parasites have been reared from the
beet leafhopper and studied to some extent. They are given in the order
of their importance.
Polynema EUTETTixi GiraulT (4, p. 18) (Pl. 43, A). — This small
brown or black species was first reared from eggs of Eutettix tenella at
Spreckels, Calif., early in 1915 and has proved to be the most effective
parasite of this group in the Salinas Valley. Eggs parasitized by this
species are conspicuous in the petioles of the beets because of the black
color of the parasite pupae. Development is rapid, the life cycle from
adult to adult covering about 35 days on an average, and there are at
least nine generations annually.
Abbella subflava Girault. — Concerning this parasite W. J. Hartung
(5) writes as follows:
Hyper-parasites were bred from parasitized eggs of Eutettix. These were deter-
mined by Girault as Abbella subflava Girault.
This species 1 was never found among the parasites reared from ma-
terial collected at Spreckels, Calif., but at Riverside it w7as reared in
about equal numbers with Polynema eutettixi.1 It is a primary parasite,
ovipositing readily in eggs of the beet leafhopper. It has also been
reared from eggs of Empoasca sp.
Anagrus giraulti Crawford. — This common orange or red jassid
egg parasite has been reared in each locality where parasite studies have
been conducted. It oviposits readily in eggs of the beet leafhopper and
is usually reared along with Polynema eutettixi, but not in such large
numbers. The presence of this species in the petioles of the beet can be
detected by the red or orange color found in both larva and pupa.
PARASITES OF THE NYMPHS AND ADULTS
As previously reported by Hartung and Severin (6), two species of the
dipterous family Pipunculidae are known to be parasitic on the nymphs
and adults of the beet leafhopper. These have been described (7) as
Pipunculus industrius Knab and Pipunculus vagabundus Knab. The
former is the more common species in the Salinas Valley.
Pipunculus industrius Knab (Pl. 43, B). — Eggs of this species are
deposited in both nymphs and adults of the beet leafhopper, but mature
larvae have never been knowrn to emerge from a nymph. There are no
indications that the adult female prefers either the mature or immature
stages of the host in which to deposit her eggs, very small parasitic
larvse having been dissected in about equal numbers from both stages.
It is known, by dissection, that eggs may be deposited in small nymphs
1 Specimens identified by Mr. A. B. Gahan.
Nov. 15, 1920 Life History and Habits of the Beet Leaf hop per 251
no further developed than the third instar. In all instances, however,
where an action thought to be oviposition was observed, the adult host
was the victim.
The adult is very graceful in flight, darting here and there so suddenly
that it is impossible to follow the movements with the eye. The beet
leaf hopper, also, is very quick in its movements, but none is quick enough
to avoid this active little parasite.
Pipunculus vagabundus Knab. — This species is not common in the
Salinas Valley and is of little importance. Its habits are similar to those
of Pipunculus industrius, and, with the exception of the conspicuous
stigma which is absent in the wings of P. vagabundus, the two species
are similar in appearance.
Dryinidae. — Occasionally beet leafhoppers, both adults and nymphs,
are found with a dark brown sac or pouch protruding from the abdomen
(Pi. 42, D). This pouch contains the larva of a dryinid parasite. Har-
tung and Severin (6) report a parasite of this family, Gonatopus contort-
ulus Patton, from the Salinas Valley. Although the writer has reared
many specimens of this family, none has been determined. Judging from
the number of parasitized leafhoppers collected, these dryinids are not
of much economic importance. It has been observed, however, that the
adults devour a larger number of the leafhoppers, especially nymphs,
than they parasitize. In this way they may be of more importance than
would at first appear.
SUMMARY
Eggs of Eutcttix tcnella are deposited in a wide range of cultivated and
wild plants, but the sugar beet seems to be preferred for this purpose
during the summer season. A maximum record of 247 eggs was obtained
for a single female. The incubation period covered from 10 to 15 days
during the height of the egg-laying season and the nymphal period from
25 to 52 days.
One generation only was observed in southern Idaho, while from two
to four were observed under California conditions.
In southern Idaho the beet leafhopper appears in the beet fields in
June and starts reproducing at once, oviposition continuing throughout
the season. After harvest the leafhoppers enter a true hibernation pe-
riod.
In California the adults appear in the beet fields soon after April 1 and
remain until harvest time, when they disperse to wild vegetation suitable
for food and protection. No true hibernation was noted in California.
Three species of egg parasites were reared and studied. Two of these
are very effective. Two species of Pipunculus, internal parasites of the
nymphs and adults, were reared; and one of these was quite effective.
Dryinid parasites, also, were reared but are not considered very efficient.
252 Journal of Agricultural Research vol. xx, no. 4
LITERATURE CITED
(1) Ball, E. D.
1907. THE GENUS EUTETTIX, WITH ESPECIAL REFERENCE TO THE BEET LEAF
hopper. In Proc. Davenport Acad. Sci., v. 12, p. 27-94.
(2)
1909. THE LEAFHOPPERS OF THE SUGAR BEET AND THEIR RELATION TO THE
"curly-leaf" condition. U. S. Dept. Agr. Bur. Ent. Bui. 66, pt.
4. P- 33-52-
(3)
1917. THE BEET LEAFHOPPER AND THE CURLY-LEAF DISEASE THAT IT TRANS-
MITS. Utah Agr. Exp. Sta. Bui. 155, 56 p.
(4) Girault, A. A.
1917. DESCRIPTIONES STELLARUM NOVARUM. 22 p. [n. p.]
(5) Hartung, W. J.
1919. ENEMIES OF THE LEAFHOPPER; NATURAL FOES OF EUTETTIX TENELLA
in California and their usefulness. In Facts about Sugar, v.
8, no. 24, p. 470-471.
(6) and Severin, H. H. P.
191 5. NATURAL ENEMIES OF THE SUGAR-BEET LEAFHOPPERS IN CALIFORNIA.
In Mo. Bul. State Com. Hort. [Cal.J, v. 4, no. 5/6, p. 277-279.
(7) Knab, Frederick.
1915. Two new species of pipunculus. Proc. Biol. Soc. Washington, v.
28, p. 83-86.
PLATE 42
Eutettix tenella:
A.— Adult, light form.
B. — Adult, dark form.
C. — Adult, color gradation between A and B.
D— Nymph with protruding sac of dryinid parasite.
All much enlarged.
Life History and Habits of the Beet Leafhopper
Plate 42
^L
r
:&h:
V
Journal of Agricultural Research
Vol. XX, No. 4
Life History and Habits of the Beet Leafhopper
Plate 43
FR C
Journal of Agricultural Research
Vol. XX, No. 4
PLATE 43
Parasites of Eulettix tenella:
A.—Pipunculus industrius: Adult, much enlarged.
B.—Polynema eutettixi: Adult, much enlarged.
HYPERTROPHIED LENTICELS ON THE ROOTS OF CONI-
FERS AND THEIR RELATION TO MOISTURE AND
AERATION
By Glenn G. Hahn, Scientific Assistant, Carl Hartley, Pathologist, and Arthur
S. Rhoads,1 Assistant in Forest Pathology, Investigations in Forest Pathology,
Bureau of Plant Industry, United States Department of Agriculture
INTRODUCTION
At the Bessey Nursery of the United States Forest Service at Halsey,
Nebr., warty excrescences were observed upon the roots of coniferous
seedling stock during the shipping season of 1915. Such excrescences
occurred on all pine species grown there. They were so abundant on
western yellow pine (Pinus ponderosa) 2 that the possibility of a parasite
as the causal agent was suggested, and the forest officers properly ques-
tioned the advisability of shipping the stock to other regions.
Attempts were made by the writers to obtain evidence of a pathogenic
organism, but always with negative results. This experimentation con-
sisted of (a) incubation in moist chambers of portions of roots bearing
excrescences, (b) insertion of the interior portion of the excrescences,
removed with aseptic precautions, into nutrient agar, and (c) inocu-
lation of portions of the excrescences into roots of healthy 2-year-old
and 4-year-old Pinus ponderosa stock.
After the failure to obtain evidence of a pathologic organism, a histo-
logical examination was made, which showed that the excrescences had
the structure of the hypertrophied lenticels (PI. 44) so commonly seen in
many dicotyledonous plants.
DESCRIPTION
The hypertrophied lenticles are found both upon the main tap root
(Pi. 45, B) and upon the lateral roots, not only close to the ground level
and upon the stems proper but also on the tap roots as far as 14 inches
(36 cm.) below the surface of the soil.3 On the stems of conifers the
hypertrophied lenticles usually occur only on the basal portions of trees
growing in abnormally wet situations (PI. 45, A) or on parts otherwise
submerged. In exceptionally humid situations they may occur occa-
sionally on parts of the stems above the soil surface.
1 The writers wish to acknowledge helpful suggestions from Dr. B. E. Livingston, of the Johns Hopkins
University, and Dr. T. H. Goodspeed, of the University of California.
2 All the western yellow pine referred to in this paper was the type sometimes referred to as Pinus pon-
derosa var. scopulorum, from eastern Rocky Mountain seed.
3 In all probability hypertrophied lenticels will be found at much greater soil depths on the roots of older
trees.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C. Nov. 15, 1920
vi Key No. G-207
(253)
254 Journal of Agricultural Research vol. xx, no. 4
On the small roots the hypertrophied lenticels occur most commonly,
but not always, at the junction of a lateral root or rootlet with its parent
root, usually originating immediately above the point of origin but also
subtending, at the sides or immediately below, the root or rootlet in
question. This agrees with the findings of De Vaux (5)1 on normal lenti-
cels, who reports that primary lenticels on roots are always at the bases
of root branches, though secondary lenticels are sometimes formed later
at other points. It was this coincidence of lenticels and root branches
that caused some botanists during the early part of the nineteenth
century to believe lenticels equivalent to buds, a doctrine attributed to
De Candolle (7; 13, Vorwort) and overthrown by Majer (13),2 Unger (22),
Terras (19), and others.
The excrescences vary greatly in size and shape, from minute circular
areas 0.5 mm. in diameter to bands nearly encircling the larger roots in
cases where two or more lenticels have become laterally confluent.
Around the root crowns and the bases of the submerged stems large,
wartlike patches may occur, 5 to 8 mm. in diameter and projecting 1 to
3 mm. above the surface of the bark. Examination with a dissecting
microscope shows these excrescences to be made up of a very loosely
piled mound of pale yellowish tissue. As a general rule these mounds
of loosely piled cells split in a stellate manner, the segments recurving out-
ward, occasionally leaving a few filamentous columns standing by them-
selves in the center. Such structure is evident only when the young
trees have been removed from the ground with great care, for the slightest
touch upon these loose-lying columns causes them to crumble instantly
to a flat, powdery mass, especially when they are dry. On the bases of
still older stems 1 to 2 inches (2.5 to 5 cm.) in diameter that stand for
a large part of the growing season in water or poorly drained soil, the
bark, which is here considerably thickened, exfoliates in patches of varying
size, revealing irregularly connected flattened masses of cells, or, more
rarely, unbroken areas of such cells 1 inch (2.5 cm.) broad. On some
pines these excrescences frequently become so abundant that con-
siderable areas of the lower stem and the tap root are covered by them
(PI. 46, B). After the cessation of growth in the lenticels, these excre-
scences become dark root-brown and gradually slough off.
The lenticellular excrescences vary in different conifers from loosely
connected, more or less divergent, columnar masses crumbling at the
slightest touch, common in the pines, to fairly compact, corky masses
usually seen in the trees of other coniferous genera.
Histological examination of the excrescences at once proves the white,
spongy tissue to consist of more or less loosely connected masses of cells
developed from the phellogen. Plate 44 illustrates a cross section of
1 Reference is made by number (italic) to " Literature cited," p. 264-265.
2 This seems to be the 1836 paper attributed to Mohl by Haberland (7). Mohl apparently directed the
work of Majer and wrote a preface for the dissertation, but Majer was the author of the paper itself.
Nov. 15, 1920 Hypertrophied Lenticels on the Roots of Conifers 255
one of these hypertrophied lenticels on a root of Pinus rigida. The out-
growths consist of homogeneous parenchymatous elements, more or less
radially elongated, sometimes very much so. The individual cells are
thin-walled with a thin layer of cytoplasm.
SPECIES AFFECTED
Stahl (i<?) states that all trees which have lenticels on the stems also
have them on the roots. De Vaux (5) reports the presence of lenticels
on the roots of a large number of tree species, including a number of
conifers. For one species of Ephedra he states that lenticels are found
only on the roots. He states that especially in Pinus maritima the
lenticels on the roots are larger than those on the stems. This author
was able to find or to produce lenticel hypertrophy on some part of the
plant in 60 per cent of the 155 plant species considered but was unable
to secure any hypertrophy on the representatives of the several conif-
erous genera which he studied. On roots less than 3 mm. in diameter
he found the normal lenticels so small that the microscope was usually
necessary in demonstrating them. Tubeuf (20) lists a small number of
species, of which he was able to secure lenticel hypertrophy on some part
of 12 nonconifers. He, however, failed to get this hypertrophy on
species of Sequoia, Thuja, and Taxus, or on Gingko biloba and 14 other
nonconifcrous species. Zach (23) later secured hypertrophy of lenticels
on stems of G. biloba under certain conditions. However, a rather
careful search in the earlier literature appears to justify the statement
by the reviewer of Zack's paper (16) that no hypertrophy of lenticels
had been up to that time reported on conifers.
The present writers have found hypertrophied lenticels on the roots of
the following conifers : Pinus ponderosa, Pinus coulteri, Pinus rigida, Pinus
resinosa, Pinus banksiana, Pinus virginiana, Pinus syhestris, Pinus cari-
baea, Pinus strobus, Pinus monticola , Pinus excelsa, Picea canadensis , Picea
rubens, Picea mariana,1 Picea pungens, Abies balsamea,2 Tsuga canaden-
sis, Larix laricina, Taxus cuspidata, Taxus brevifolia, and Araucaria
bidwellii.
Several of the species of Pinus on which the hypertrophy was found
were growing in the greenhouse of the United States Department of
Agriculture at Washington, D. C. It was noteworthy that plants of
Juniperus virginiana under the same conditions in the same greenhouse
apparently were free from such growths so far as could be determined.
In a swamp in which the hypertrophied lenticels were found on Abies
balsamea, Picea rubens, and Tsuga canadensis none could be discovered
on Taxus canadensis. Among the pines the hypertrophied lenticels
were frequent mainly on the 3-needled species, Pinus ponderosa and Pinus
1 Material furnished by Dr. H. P. Brown, of The New York State College of Forestry at Syracuse
University.
2 Dr. James R. Weir advises the writers that he has frequently found hypertrophied lenticels on the
rcots of Abies grandis in the Northwest.
256 Journal of Agricultural Research vol. xx, No. 4
rigida, while on the strictly 2-needled Pinus virginiana, Pinus banksiana,
and Pinus resinosa they were very difficult to find. Klebahn (10, p. 582,
586) states that up to the time of his publication he had not been able to
find lenticels on Pinus sylvestris, nor had he satisfactorily demonstrated a
substitution for lenticels.
Excrescences like those just described on the conifers are common
and widespread occurrence on a number of dicotyledonous plants,
particularly upon swamp plants such as Sambucus canadensis, Rhus
copallina, Decodon verticillatus , and Cephalanthus occidentalis . Such
excrescences on dicotyledonous plants have long been known under the
term "water lenticels. "
CONDITIONS UNDER WHICH HYPERTROPHY HAS BEEN FOUND
The lenticel hypertrophy observed on roots has been generally limited
to plants growing in wet soil. Affected hemlock, balsam fir, red spruce,
and black spruce have already been noted as growing under swamp
conditions. All the pitch pines found with hypertrophied lenticels in
the vicinity of Washington were in heavy, wet soil. There hypertrophy
was very frequent on Pinus rigida and P. virginiana growing in
swampy locations. The pines found so affected in the greenhouse at
Washington were all growing in soil very much wetter than that in which
they are usually found. The only Scotch pines found with hypertrophied
lenticels were growing at the edge of an irrigation ditch in especially
wet soil at a Michigan nursery. The same has been true in the most
striking cases of hypertrophy at the Bessey Nursery. In a bed, a portion
of which was repeatedly flooded from a leaking irrigation ditch, approxi-
mately 20 per cent of the plants showed marked cases of hypertrophy,
while less than i}4 per cent of the plants showed hypertrophy in parts
of the neighboring beds which were not affected by the leakage. Infor-
mation has been received from Mr. W. H. Schrader that at the Monu-
ment Nursery of the United States Forest Service in Colorado the only
conspicuous occurrence of root lenticel hypertrophy was during an
unusually wet season. The hypertrophy here considered has been found
both in heavy and in very sandy soils; in the latter case there was ap-
parently more hypertrophy in parts of the beds to which clay had been
added.
The youngest seedling observed with lenticel hypertrophy was one
of Pinus ponderosa which was raised from the seed with its roots in a
2-ounce bottle of tap water in the laboratory. This water was not
changed during the entire period of growth. The bottle was stoppered
but was not absolutely sealed at the point of passage of the stem through
the stopper. At the end of approximately five months the plant, which
still seemed fairly vigorous, had developed a single root, which, after
reaching the bottom of the bottle, had coiled itself around two or three
times close to the peripheral limit of the bottle. On this tap root were a
Nov. is, 1920 Hypertrophied Lenticels on the Roots of Conifers 257
number of conspicuous, glistening, mound-shaped excrescences, as is
shown, slightly magnified, in Plate 46, C. A microscopic examination
of sectional preparations of these excrescences (Pi. 46, A) showed clearly
their lenticellular structure. The outgrowths were so loose and delicate
that the outer portions were necessarily lost in sectioning, but the figure
shows enough of the bases to indicate the type of structure.
In general, root-lenticel hypertrophy has been found especially frequent
not only on species like western yellow pine, which are somewhat inclined
to lack fine fiberous roots, but also on individuals of other species when
a strong tap root has been developed with relatively little development
of laterals. Whether or not the larger lenticels are of advantage to such
plants in fulfilling part of the functions that the missing laterals might
have performed is of course uncertain. In this connection it is of some
interest to note the finding of root-lenticel hypertrophy in Michigan on
white and Colorado blue spruce (Picea canadensis and P. pungens)
whose roots had been injured by May beetle larvae. It is also especially
interesting that nursery trees that have not been transplanted or that
are in their second season in the transplant beds show decidedly less
hypertrophy than recently transplanted stock. The recently trans-
planted trees have, of course, lost most of their absorbing roots, while
the trees transplanted the preceding season have had a chance to develop
normal root system again after transplanting.
IRRIGATION EXPERIMENTS
Trees of Pinus ponderosa in their third year in the nursery, and two
months following transplanting, were given river water from the irri-
gating ditch frequently during a three months' period, beginning July 1 1 ,
1 91 7. All the tests considered in this and the following section were
conducted at the Bessey Nursery in cooperation with Forest Supervisor
Jay Higgins and his assistants. The water added at each irrigation was
approximately equivalent to 2.2 inches (5.6 cm.) of rainfall. A bed
which received 31 such irrigations during these three months showed at
the end of the period 31 per cent of the trees with 8 or more distinctly
hypertrophied lenticels each and a total of 57 per cent with some evi-
dence of hypertrophy. The figures are based on an examination of 255
trees. This amount of watering was sufficient to cause more or less
chlorosis, especially of the shoots which arose after the watering began.
Another bed in the same section, on which the frequent watering was
not started until a month later and which received during the entire
three months a total of 17 irrigations, showed at the end of the period
eight or more enlarged lenticels each on approximately 13 per cent of
the plants examined. Other beds used as controls received the usual
amount of water given at this nursery, involving six irrigations in addi-
tion to the j.j inches (20 cm.) rainfall during the period of three months.
9508°— 20 2
258
Journal of Agricultural Research
Vol. XX, No. a
These showed less than iK per cent of the plants with abundant hyper-
trophied lenticels and a total of less than 13 per cent showing any evi-
dence of hypertrophy. The results in the most heavily watered bed
and in the controls are given in Table I. The results with the pruned
trees shown in the table lead to the same conclusions as the results cited
above on the unpruned trees — namely, that heavy watering increased
the amount of lenticel hypertrophy.
Table I. — Effect of watering and top pruning on root-lenticel hypertrophy of third-year
western yellow pine at Bessey Nursery, Halsey, Nebr., pruned in early July and examined
September 10 to 15
Plot.
ABC
DE
Part removed by pruning.
All the secondary needles b
All the secondary needles b and
tip of third season terminal shoot.
All the secondary needles & and
entire third-season shoot
Third season terminal shoot only
Half the secondary needles only b
Unpruned
Additional unpruned rows scat-
tered among the different series.
Heavily pruned.
Lightly pruned .
Unpruned
Number of
trees
examined.
Heav- Nor- Heav
ily mally ily
watered watered watered
series, series, series.
Percentage of
trees with
hypertrophy.
185
182
32
108
58
206
49
399
166
255
42
47
Si
48
o
72
7i
140
143
Nor- Heav- Nor-
mally ily I mally
watered watered watered
series, series, series.
6
41
3i
58
5i
9
37
57
Percentage of
trees with
strong hyper-
trophy.0
17
C3
2.9
17
19
17
33
24
2-3
18
3i
1.4
a Having 8 or more noticeably hvpertrophied root lenticels per tree.
b Including the needles that had appeared on the third-season shoot as well as those produced in earlier
years. Cut back to sheath but portion of needle remaining in the sheath left intact.
PRUNING EXPERIMENTS
Pruning experiments w.ere conducted in an effort to throw a little more
light on the factors controlling the lenticel hypertrophy. The tops of a
number of rows of western yellow pine transplants at the Bessey Nursery
were pruned with different degrees of severity during the first week in
July, 1 91 7. This is about the middle of the season of vigorous growth
at this nursery. The results of a root examination three months later
appear in Table I. The most heavily pruned plants showed the least
lenticel hypertrophy, with the exception of plot E in the normally watered
series. The percentage in this case is based on only 48 trees, only one-
third as many as furnished the basis for each of the other figures in the
three lower lines of the table. The pruning did not so injure the plants
as to prevent growth entirely, for even those most heavily pruned reacted
by sending out new shoots.
Nov. 15, 1920 Hypertrophied Lenticels on the Roots of Conifers 259
CAUSES OF LENTICEL HYPERTROPHY
Schenck (15) attributed lenticel growth on roots to oxygen hunger.
However, the association which has been observed between moist condi-
tions and abnormal lenticel growths, as well as experience in artifically
producing lenticel hypertrophy by placing cuttings in water or moist air,
have led more recent writers to suppose that for dicotyledonous plants
the hypertrophies are directly due to the presence of an unusual amount
of water (5; 11, p. 72-80; 17). It is reasoned, in the first place, that
water or constantly moist atmosphere on the outside of the lenticels
allows the steady growth of the lenticels, while dry or intermittently dry
air tends to dry out the superficial cells of the lenticels or to increase their
solute concentration, with resultant chemical changes, including cork and
lignin formation. According to this idea the growth of the lenticel tissue
is controlled by transpiration through the lenticels; with intense trans-
piration the tissues become dried and the hypertrophy is checked. The
suberized or lignified layers thus formed are supposed to restrain mechan-
ically further proliferation on the part of the cells beneath them from
which the lenticel structures arise. So far this supposition seems logical,
though there is as yet no basis for a quantitative estimation of the im-
portance of tissue drying in the phenomenon.
DeVaux has advanced another theory, based on the fact that the sup-
plying of abundant water to the absorbing surfaces and the reduction of
transpiration have both been found to be followed by lenticel hypertrophy
in experiments with dicotyledons. This writer supposes that both
these treatments result in increased sap pressure in the plant as a whole
and exert their influence entirely through increased sap pressure. He
does not apparently give sufficient weight to the possibility that both
decrease in transpiring surface and increase in soil moisture may involve
decreased oxygen supply as well as increased sap pressure. The limited
aeration of wet soils is a matter on which there is general agreement.
The necessity of soil oxygen for the normal development of mesophytic
plants, as indicated by common observation, has been recently confirmed
by direct experiments by Cannon and Free (3) and by Livingston and
Free (12). It is by no means certain that over-wet soil results in in-
creased sap pressure in mesophytic plants, especially since the last-named
authors find that a deficiency of oxygen in the soil results in some cases
in decreased water absorption. The association between swampy soil
and lenticel hypertrophy is at least as easily explained on the basis of
oxygen hunger as by DeVaux's " hyperhydrose " doctrine.
The argument which Tubeuf (20, 21) seems to consider strongest
against oxygen hunger as the stimulus for lentical enlargement is the
fact that enlargement can be produced in cuttings in a moist chamber.
By placing cuttings with paraffined ends in moist chambers he secured
lenticel overgrowth, even in cases in which an atmosphere of oxygen was
260 Journal of Agricultural Research vol. xx, No. 4
provided. This seems at first glance to dispose of the oxygen-hunger
hypothesis quite effectively. However, an atmosphere of oxygen would
not necessarily insure an oxygen supply to the interior of a woody stem
unless the lenticels were already open at the time the cutting was placed
in the chamber. A section of stem removed from the plant and therefore
deprived of the oxygen that it would normally get from the leaves and
perhaps also from the roots, if its lenticels were closed, might easily by
oxidation of stored food materials develop abnormal partial pressures
of carbon dioxid in its interior tissues which would not be relieved till
the lenticels were opened by the stimulated growth which Tubeuf de-
scribes. The experience reported in his later paper, in which he records
interesting cases of lenticel stimulation secured by covering bark with
impervious materials, and observation of lenticel hypertrophy on the
swelling above a heat lesion lead him to consider the stimulation
lenticel growth too complicated to be explained by any single factor so
simple as humidity. He still appears to consider oxygen hunger as
excluded from further consideration. However, his observation of
numerous lenticels on the stem of a heart-rotted spruce is the only refer-
ence that has been found concerning abnormal lenticel growth on any
part of a coniferous tree.
The intumescences produced by Atkinson on tomato (1) and by Douglas
on potato (6) were clearly related in some way to excessive general sap
pressure. They are not analogous cases to the root lenticels here con-
sidered, since the hypertrophy in the intumescences was, so far as can
be judged from the illustrations given, mainly due to the stretching of
soft tissue cells already present rather than to the formation of large
masses of new cells.
It may be of some interest to note in passing that Cowles (4, p. 553-554)
expresses himself as inclined to regard lacunar tissue in submerged parts
of water plants to be a response to lack of transpiration rather than
to oxygen deficiency.
The present writers' findings bearing on the factors causing hypertro-
phy of subterranean lenticels on young conifers are as follows:
1. Hypertrophy is apparently limited to trees with their roots in water
or very wet soil. This may indicate either increased sap pressure or
decreased aeration as among the effective stimuli. It seems rather
improbable that there should be a significantly greater sap pressure in
a mesophyte like Pinus rigida or a semixerophyte like P. ponderosa
(Rocky Mountain type) in an excessively wet soil than in a plant in more
normal condition. This seems especially improbable in view of the slow
water absorption by the mesophytes in soil deficient in oxygen in the
experiments already referred to (12).
2. While lenticel hypertrophy seems to be most common in soils con-
taining clay, it has been frequently found in one nursery (at Halsey,
Nov. 15, 1920 Hypertrophied Lenticels on the Roots of Conifers 261
Nebr.) having a very sandy, well-drained soil, with a wilting coefficient1
in the neighborhood of 3.4 per cent for the nursery as a whole, and an
unusually high proportion of the soil (79 per cent) in particles between
0.25 and 0.05 mm. in diameter. The results of a mechanical analysis of
this soil have already been published (8, p. 2). This, at first thought,
indicates sap pressure rather than deficient aeration as the cause of
hypertrophy. It is worthy of note, however, that in this case there was
frequent artificial watering in addition to considerable rainfall, and it is
therefore entirely possible that even in this case aeration was insufficient.
Buckingham (2) found that both diffusion and molar movement of gas
were slower in a wet sand than in any of the other soils, wet or dry, with
which he experimented.
3. Reduction of the transpiring surface by removal of a large part of
the needles, or of the terminal growth, or both, resulted in distinctly
reducing the tendency to lenticel hypertrophy. (Table I.) The un-
pruned plants presumably had, at least part of the time, a lower general
sap pressure than the pruned. The result of the experiment therefore
tends to diminish the probability that there is any important causal
relation between general excessive sap pressure and the hypertrophy
in question.
4. The finding of the most abundant hypertrophy on roots which are
deficient in fibrous laterals or whose absorbing surface has been greatly
reduced by insect injury or by transplanting also tends to weaken the
hypothesis that excessive general sap pressure throughout the plant is
the chief cause of the hypertrophy. It is possible that roots which have
little absorbing surface will take less oxygen from the soil than would
better-developed root systems. An indication that this is the case is
seen in the experience of Livingston and Free (12, p. 185) with the oxygen
requirements of roots with different amounts of surface area. This
association between deficient root surface and lenticel hypertrophy may
therefore be an indication of a relation between oxygen deficiency and
lenticel production.
The fact that lenticel hypertrophy was actually less in plants whose
leaf surfaces had been reduced by pruning not only tends to decrease
the probability of the " hyperhydrose " explanation; it is suggested that
it is perhaps a further support for an oxygen-hunger (or carbon-dioxid
excess) hypothesis. Plants with their leaf surfaces reduced during the
latter part of the summer will of necessity produce less carbohydrate.
The smaller amount of carbohydrate reaching the roots in consequence
of the pruning might conceivably result in less respiration in the root
tissues and therefore in a decreased need for oxygen. If this were the
case the decreased oxygen hunger might furnish a partial explanation of
the slight lenticel growth in the pruned plants.
1 Determined by the Office of Biophysical Investigations, Bureau of Plant Industry.
262 Journal of Agricultural Research vol. xx,no.4
Another possible connection between leaf pruning and oxygen hunger
of root and stem is suggested by Prof. Livingston in a personal communi-
cation to one of the writers. A reduction of the transpiring surface by
pruning should result in less absorption by the roots. If it be supposed
that oxygen dissolved in water absorbed from the soil is important as a
source of oxygen supply for the root tissues, a decrease in the amount of
water absorbed might result in oxygen deficiency in the root tissues.
This suggestion might help to explain the earlier reports of the stimulated
growth of lenticels on stems of dicotyledons whose transpiration has been
experimentally reduced. It obviously complicates any attempt to
explain on an oxygen-hunger basis the effects of pruning on lenticel
growth described in the present paper.
Of course it does not seem likely that any part of a plant accustomed
to the presence of free oxygen would be likely to make much growth in
the entire absence of oxygen. However, the condition existing in the
soil in which the hypertrophies occurred certainly did not involve the
entire absence of oxygen. Pfeffer concludes (14, p. 115), in spite of some
conflicting evidence, that experiments have shown that reduction of the
proportion of oxygen, at least in some cases, acts as an accelerating
'stimulus to growth.
It is of course true that any strong local growth is probably dependent
on high local sap pressure. However, it is well known that such local
high pressures are not necessarily dependent on excessive turgidity of the
plant as a whole. Unusual chemical conditions, such as might conceiv-
ably result from local oxygen hunger, might easily cause them. The
writers do not consider that oxygen hunger is established as the main
cause of the lenticel hypertrophy found. They can not, however, agree
with De Vaux in attributing the effect of increased soil moisture on
lenticel growth entirely to increased water supply, excluding oxygen
hunger as a possible factor in stimulating lenticel growth.
Experiments in which the oxygen, carbon-djoxid, and water supplies
in the soil are independently controlled, as by the technic of Livingston
and Free (12), and perhaps also with temperature control, will be needed
to make a beginning on determining the relative importance of these
various environmental factors in causing hypertrophy of root lenticels.
Since conifers are rather difficult to handle in experimental work, poplar
would perhaps be a better subject for preliminary experimentation.
It seems likely, as has been suggested for hypertrophied lenticels in
general by Tubeuf (21) and for intumescences by Hasselbring (9), that
these unusual lenticel enlargements on the roots of conifers depend on a
complex of conditions rather than on any one simple stimulus, and that
with different species the conditions which call forth lenticel hypertrophy
may be found to differ in relative importance.
Nov. 15, 1920 Hypertrophied Lenticels on the Roots of Conifers 263
RELATION BETWEEN LENTICEL HYPERTROPHY AND HEALTH OF
PLANTS
Sorauer (17, p. 210-219) has used the name "tan disease" for lenticel
hypertrophy on roots and stems of fruit trees. His use of the term
"disease" appears justified in view of the association in many cases
between the lenticel hypertrophy and a general pathological condition of
the trees. The large lenticels described in the foregoing paragraphs as
occurring on conifers are undoubtedly abnormal and in that sense are
pathological. Since they occur only in abnormally wet situations, it is
to be expected that in many cases the pines on which they have been
found are unused to very moist surroundings and under the unfavorable
conditions are subnormal in general vigor. The hypertrophies were first
noted in a part of a nursery in which general vigor was unsatisfactory.
Comparisons of the less vigorous and more vigorous plants in the section
in which the hypertrophy was common showed lenticel hypertrophy
present in both the weaker and stronger plants. The first examina-
tion, made by Hartley on about 200 3-year-old transplants of Pinus
ponder osa, showed lenticel hypertrophy on a larger proportion of the weak
trees than of the stronger trees. Later examinations made by Hahn on
about 2,000 plants showed, particularly on P. ponder osa, that the greatest
number of hypertrophied lenticels were associated with vigorous growth,
This was true of plants in which the terminal root was rapidly advancing
and the roots were large and stocky but correspondingly undeveloped as
to lateral root surface. In one particular instance, however, where 2 -year-
old transplants of P. ponderosa had been badly affected by yellowing
due to excessive irrigation, 50 per cent of 95 vigorous plants examined
showed light occurrence of lenticel formation, while of no weakened and
dying plants 80 per cent were found to exhibit light occurrence, and 10
per cent showed pronounced occurrence. This same bed examined a
month later showed that the majority of the weak plants had died, while
the vigorous plants, or those beginning to show renewed terminal growth,
were alone showing freshly proliferating lenticels, those upon the dying
plants becoming darkened and sloughing off. It therefore appears that
lenticel hypertrophy is found on both weak and strong plants and that
the conditions which bring on their formation may, if sufficiently pro-
longed, eventually cause the weakening and death of the plant. There
is, however, so little direct connection between lenticel hypertrophy and
the pathology of the conifers that it seems logical to recommend that any
further investigation of the factors stimulating lenticel growth should be
made from the point of view of physiology rather than from that of
pathology.
264 Journal of Agricultural Research vol. xx, No. 4
SUMMARY
(1) Unusual excrescences on the roots of a number of different pines,
spruces, and other conifers are found to have the structure of lenticels,
much enlarged. They are produced in various kinds of soil in the pres-
ence of excessive moisture. Hypertrophy may occur on either weak or
vigorous plants. Hypertrophy was decreased by top pruning and was
increased by root injury. Such overgrowths have apparently not been
previously reported on conifers.
(2) Conclusions of certain writers, based on work with dicotyledons,
that excessive soil moisture stimulates lenticel hypertrophy mainly by
increasing general sap pressure and that oxygen hunger is of no impor-
tance as a stimulus are not supported by the experience here set forth with
conifers. Experiments in which the oxygen supply to the roots is varied
without varying the water supply are believed necessary to settle the
relative importance of these two factors.
LITERATURE CITED
1) Atkinson, G. F.
1893. oedema of THE tomato. N. Y. Cornell Agr. Exp. Sta. Bui. 53, p.
77- 108, 8 pi. Also in N. Y. Cornell Agr. Exp. Sta. 6th Ann. Rpt. 1893,
p 99-128. 1894.
2) Buckingham, Edgar.
1904. CONTRIBUTIONS TO OUR KNOWLEDGE OF THE AERATION OF SOILS. U. S.
Dept. Agr. Bur. Soils Bui. 25, 52 p.
3) Cannon, W. A., and Free, E. E.
1917. THE Ecological significance of soil aeration. In Science n. s. v.
45, no. 1 156, p. 178-180.
4) Coulter, John Merle, Barnes, Charles Reid, and CowlES, Henry Chandler.
1911. a textbook OF botany ... v. 2. New York, Cincinnati.
5) Devaux, Henri.
1900. recherches sur LES lEnticellES. In Ann. Sci. Nat. Bot., s. 2, t. 12,
p. 1-240, pi. 1-6.
6) Douglas, Gertrude E.
1907. THE FORMATION OF INTUMESCENCES ON POTATO PLANTS. In Bot.
Gaz., v. 43, no. 4, p. 233-250.
7) Haberlandt, Gottlieb.
1875. beitragE zur kenntniss der lenticellen. In Sitzber. K. Akad.
Wiss. [Vienna], Math. Naturvv. Kl., Bd. 72, Abt. 1, p. 175-203.
8) Hartley, Carl.
1915. INJURY BY DISINFECTANTS TO SEEDS AND ROOTS IN SANDY SOIL. U. S.
Dept. Agr. Bui. 169, 35 p., pi.
9) Hasselbring, H.
1905. [review of papers on intumescences.] In Bot. Gaz., v. 40, no. 5.
P- 39o-39 i-
(10) Klebahn, H.
1884. DIE RINDENPOREN . . . In Jenaische Ztschr. Naturw., Bd. 17
(n. F. Bd. 10), p. 537-S92- pl- I2-
(11) Kuster, Ernst.
1903. pathological plant anatomy. Translation by Frances Dorrance.
258 p. [n. p.] Multigraphed.
Nov. is. 1920 Hypertrophied Lenticels on the Roots of Conifers 265
(12) Livingston, B. E., and Free, E. E.
191 7. THE EFFECT OF DEFICIENT SOIL OXYGEN ON THE ROOTS OF HIGHER PLANTS.
In Johns Hopkins Univ. Circ. 293 (n. s. 3), p. 182-185.
(13) Majer, Carl Eduard.
1836. untersuchungen uber die LEnticellen. 19 p. Tubingen. Inaug.
Diss., Hugo von Mohl, praeses.
(14) Pfeffer, W.
1900-06. the physiology of plants . . . ed. 2, rev., transl. and ed.
by Alfred J. Ewart ... 3 V., illus. Oxford.
(15) Schenck, H.
1889. UEBER DAS AERENCHYM, EIN DEM KORK HOMOLOGES GEWEBE BEl
SUMPFPFLANZEN. In Jahrb. Wiss. [Pringsheim], Bd. 20, p. 526-574,
pi. 23-28.
(16) Simon.
1912. [review of] zach, fr. zur kenntnis hyperhydrischer gewebe. In
Just's Bot. Jahresber., Jahrg. 37 (1909), Abt. 1, Heft 5, p. 832.
(17) SorauER, P.
1914-17. manual of plants diseases, ed. 3, transl. by Frances Dorrance,
v. 1, p. 1-8. Wilkes-Barre, Penn.
(18) Stahl, E.
1873. ENTWICKELUNGSGESCHICHTE UND ANATOMIE DER LENTICELLEN. In
Bot. Ztg., Jahrg, 31, No. 35, p. 565-567; No. 37, p. 577-586; No. 38,
P- 593-601; N°- 39. P- 609-617. pi. 5-6.
(19) Terras, James A.
T900. THE RELATION BETWEEN THE LENTICELS AND ADVENTITIOUS ROOTS
of solanum dulcamara. In Trans. Bot. Soc. Edinburgh, v. 21, pt.
4, P- 34i-353> 2 pi. Literature referred to, p. 352-353.
20) Tubeuf, K. von
1898. UEBER LENTIZELLEN-WUCHERUNGEN (AERENCHYM) AN HOLZGEWACHSEN.
In Forstl. Naturw. Ztschr., Bd. 7, p. 405-414, illus.
(21)
1914. ERKRANKUNGEN DURCH LUFTABSCHLUSS UND UBERHITZUNG. /nNaturw.
Ztschr. Forst u. Landw., Jahrg. 12, Heft 2, p. 67-88, 2 fig.; Heft 4,
p. 161-169.
(22) Unger.
1836. UEBER DIE bedeutung DER LEnticellen. In Flora, Jahrg. 19, Bd. 2,
p. 577-606.
(23) Zach, Fr.
1908. zur kenntnis hyperhydrischer gewebe. In Osterr. Bot. Ztschr.,
Jahrg. 58, No. 7/8, p. 278-284, 2 fig.
PLATE 44
Section through a hypertrophied lenticelon root of Pinus rigida growing in swampy
situation. Approximately X 59.
(266)
Hypertrophic:! Lenticels on the Roots of Conifers
Plate 44
Journal of Agricultural Research
Vol. XX, No. 4
Hypertrophied Lenticels on the Roots of Conifers
Plate 45
Journal of Agricultural Research
Vol. XX, No. 4
PLATE 45
A. — Hypertrophied lenticels on the basal part of layering stem of Picea mariana,
which had been covered with sphagnum. Approximately X iK-
B. — Tap root of a Pinus ponderosa transplant, bearing an unusually large number of
hypertrophied lenticels. Approximately X i-H-
PLATE 46
A. — Cross section of the stem through one of the hypertrophied lenticels shown in C.
In embedding and sectioning most of the loose outer tissues are unavoidably lost.
Approximately X 112.
B. — Large patches of excrescences upon the tap root near the root crown, on Pinus
rigida. Approximately X iM-
C. — Hypertrophied lenticels on root of 5-months-old Pinus ponderosa, grown in a
loosely stoppered 2-ounce bottle, in tap water which had not been changed since the
germination of the seed. The entire structure of the lenticel, which is too delicate
to recover in digging roots from the soil, is here preserved. Approximately X iK-
Hypertrophied Lenticels on the Roots of Conifers
Plate 46
Journal of Agricultural Research
Vol. XX, No. 4
DEGREE OF TEMPERATURE TO WHICH SOILS CAN BE
COOLED WITHOUT FREEZING
By George Buoyoucos
Michigan Agricultural Experiment Station
The general impression seems to be that when the temperature of soils
falls slightly below the freezing point (o° C. or 320 F.) they freeze, that
is, the soil moisture is converted into ice. This is hardly the case, how-
ever. In conducting investigations to study and measure the different
forms of water in the soil by means of the dilatometer method l and to
study and measure the concentration of the soil solution directly in the
soil by means of the freezing-point method, 2 it was discovered that it is
almost impossible to freeze the soils when they are cooled only slightly
below the freezing point. This is true even when the concentration of
the soil solution is exceedingly small and the freezing-point depression
consequently negligible. Indeed, it was found that it is difficult
to start solidification in the soils unless they are supercooled at
about i° C. below their true freezing point. Even at this degree of
undercooling freezing begins only with vigorous agitation. If the soil
is not vigorously agitated or disturbed it will remain at this temperature
indefinitely without freezing. As the degree of undercooling is increased,
however, the ease with which solidification is induced is also increased.
Finally a temperature is reached where freezing starts automatically
without agitation of the soil mass. This critical temperature is sur-
prisingly low for all soils, as will be observed from the experimental data
presented in Table I. This table shows the amount of cooling which
the soils are able to withstand without freezing. The procedure by which
these experimental results were obtained consisted in placing a i-inch
column of wet soil in a freezing-point tube, inserting the bulb of a Beck-
mann thermometer into this column of soil, and cooling the soil in
different low temperatures until a temperature was reached where
freezing would readily take place automatically. The figures rep-
resent approximately the limit of supercooling which these soils can
resist without freezing. At this maximum degree of supercooling
the soils can be maintained indefinitely if they are not disturbed or
agitated. With a slight disturbance or agitation, however, they will
1 Bouyoucos, George J. measurement of the inactive or unfree moisture in the son. by means
of THE dilatometer method. In Jour. Agr. Research, v. 8, no. 6, p. 195-217, 1 fig. 1917.
classification measurement of the different forms of water in the soil by means op the
dilatometer method. Mich. Agr. Exp. Sta. Tech. Bui. 36, 48 p., s fig. 1917.
2— and McCool, M. M. further studies on the freezing point lowering op soils. Mich.
Agr. Exp. Sta. Tech. Bui. 31, 51 p. 1916.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C. Nov. 15. 1920
vm Key No. Mich. -11
(267)
268
Journal of Agricultural Research
Vol. XX, No. 4
readily freeze. Again, if the temperature of cooling is only slightly
lowered the soils will immediately freeze. These numerical data, there-
fore, represent just about the maximum cooling which the soils can
withstand indefinitely without freezing when they are kept quiet.
For the sake of an interesting comparison, Table I also presents the
limit of supercooling without freezing of several artificial materials.
Table I. — The degree of cooling which soils and artificial materials can witlistand without
freezing when they are kept quiet and with the water content at about the saturation
point
Material.
Degree
of
super-
cooling
without
freezing.
Quartz sand...
Coarse sand
Fine sand
Very fine sand
Stony loam
Loam
Silt loam
Clay loam
Humus loam. .
Clay
Red clay
Dark clay
Brick clay
Clay subsoil...
Peat
Muck
Water
Silica
Carbon black. .
Gelatin
Agar
°C.
-4.2
-4.2
-4.2
-4. 2
-4.2
-4.2
-4. 2
-4.2
-4. 2
-4. 2
-4.2
-4. 2
-4. 2
-4.2
-5-o
-5-.o
-6.0
-6.0
-6.0
-6.0
-6.0
An examination of the foregoing experimental results reveals at once
the fact that the amount of cooling which the soils are able to withstand
without freezing is considerable, being about -4.20 C. (7.56^.) for the
mineral soils and about -50 C. (90 F.) for the peats and mucks.
It is of interest to observe that the maximum supercooling is still
greater for the water and for the artificial materials, amounting in all
cases to about -6° C. (10.80 F.). Since water freezes at about the same
degree of supercooling as the artificial materials, it would logically seem
that it is the water which limits the degree of supercooling of those
materials and that they themselves have no influence on the degree of
supercooling of water in one way or the other.
The question now rises, why do the soils withstand a smaller degree of
supercooling than the artificial materials?
Nov. 15, 1920 Temperature Which Soils Can Reach without Freezing 269
No definite explanation can be offered for this phenomenon. It
would appear, however, that the true explanation is to be found in the
difference in the size of particles of the two classes of materials. The
artificial materials possess, of course, incomparably finer-sized particles
than the soils do, and it would seem that when the division of a substance
approaches the molecular state it ought not to affect the freezing of water
materially. However, in a series of experiments conducted to ascertain
if clay soils could withstand a greater degree of supercooling than coarse
sands, it was found that sands with infinitely larger-sized particles re-
sisted freezing equally as well as clays. It is possible, therefore, that
other factors, such as the nature of the material, its cohesive and ad-
hesive properties, its specific gravity, etc., also come into play in affect-
ing the degree of supercooling.
In order to ascertain if the degree of moisture content exerts any
influence upon the resistance of soils to freezing, different water contents
were employed in all the various soils. The results failed to show,
however, that moisture had any appreciable influence on the resistance
of soils to freezing. Soils at a very low moisture content could not be
supercooled any further than at a very high moisture content.
The foregoing experimental results afford a new and significant in-
sight into the temperature of soils during the cold seasons. In the first
place, they go to show that mineral soils may be cooled down to -4.20 C.
(7. 540 F.) and peats and mucks down to -50 C. (90 F.) without freez-
ing. This being the case, the conclusion naturally follows that during
mild winters and in mild climates in the winter the soils may not freeze
even though they are cooled below their freezing point.
In the second place these findings prove quite conclusively that the
method now in vogue for measuring temperature in soils in cold seasons
may not give entirely the true facts. The thermometers will be re-
cording the temperature to be several degrees below the freezing point
and yet the soils may not be actually frozen.
The foregoing experimental results are very significant from still
another standpoint. As it is well known, water in the liquid state has
twice the specific heat that ice has. As long as the soil moisture remains
in the liquid state the temperature fluctuations in the soil will be corres-
pondingly slower and smaller.
Indeed, the ability of soils to resist freezing even when their tem-
perature is much below the freezing point throws considerable new
light on questions regarding the temperature of soils in cold seasons and
consequently upon the physical, chemical, and bacteriological pro-
cesses going on in the soils during those seasons.
CHANGES TAKING PLACE IN THE TEMPERING OF
. WHEAT
By E. L. Tague
Department of Chemistry, Kansas Agricultural Experiment Station
In milling wheat it has been found advisable to "temper," "dampen,"
or "condition" the grain before grinding. This process consists in add-
ing a certain amount of water to the wheat, then thoroughly mixing and
allowing it to stand for a time. The treatment toughens the bran coat
oi the kernel, thus making possible a closer separation of the bran and
the flour, and increases the desirable milling qualities of the wheat in
other ways. The yield of flour is increased, and a flour is obtained from
which better bread can be made. All practical millers are well acquainted
with the fact that tempering improves the milling qualil y.
That Jago l recognizes the fact is shown by the following quotation :
On making baking tests with the flours from such slightly dampened wheats, com-
pared with those of the wheats milled dry, the dampened wheat flours fall off less
during fermentation, yield bread of a better color and flavor, and in practically the
same quantity. The slight damping of very dry wheats enables the miller to pro-
duce a better quality of flour.
Swanson2 observes that conditioning not only toughens the bran of
the wheat and makes it easier to crush the endosperm but it also affects
the quality of the gluten and the baking quality of the flour. Temper-
ature, moisture, and time play an important part in this process. Im-
provement through conditioning is similar to that brought about by
natural ageing.
The changes in the flour are probably either physical or chemical, or
more likely a combination of the two. The thorough elimination of the
bran gives a flour of better color, and the closer separation of the bran
and the endosperm produces a flour of higher gluten content. It is pos-
sible that the quality of the gluten is also affected. If so, this would
indicate a chemical change during tempering or a physical change of
such a nature as to make possible a more pronounced chemical change
during fermentation and baking.
Since the experience of practical millers indicates that the physical
changes mentioned above ao occur, the subject is one which calls for
accurate investigation. Millers often ask the question whether the
obvious physical changes are accompanied by chemical changes. If so,
a standardization of the factors which govern the tempering of wheat
would lead to a more uniform product.
1 Jago, William, and Jago, William C technology of bread making, p. 360. London, 1911.
2 Swanson, C O. wheat conditioning. In Amer, Miller, v. 41, no. 6, p. 467-469, illus.
Journal of Agricultural Research, Vol. XX, Nc. 4
Washington, D. C Nov. 15, 1920
vn Key No. Kans.-22
(271)
9508°— 20 3
272 Journal of Agricultural Research voi.xx,No.4
The principal factors involved in the tempering of wheat are (i) time,
(2) amount of water added, and (3) temperature. These factors vary
somewhat with different varieties of wheat. The general practice of
millers seems to be to temper from 12 to 48 hours and to add sufficient
water to make the total moisture content 15^2 per cent. There does
not seem to be any fixed temperature used. Some millers pay no atten-
tion at all to this factor, while others "warm" the water before adding
it to the wheat.
EXPERIMENTAL WORK
Three varieties or lots of wheat were used for the experimental work —
a variety of hard wheat known as Kanred, developed recently by the
Kansas Agricultural Experiment Station; a hard, red wheat (Turkey or
Kharkof) from central Kansas; and a soft wheat from Colorado. This
latter variety came to the department as Arizona White wheat.
The only chemical changes considered in this study were changes in
the (1) hydrogen-ion concentration, (2) total acidity, (3) water-soluble
phosphorus, and (4) titrable nitrogen. Yields of straight flour were also
computed, and the milling qualities were judged as nearly as possible.
Other investigations under way at the present time will be reported in a
later paper.
Preliminary experiments were first conducted, from the results of which
it seemed advisable to compare different periods of time, different tem-
peratures, and different moisture contents as follows: (1) Time, 24 hours,
48 hours, and 72 hours; (2) temperature, 50, 200, and 400 C; and (3)
moisture content, 15X and 18 per cent. The preliminary experiments
seemed to indicate that the best results would be secured within these
limits.
APPARATUS AND METHODS
The wheat was ground in a small burr mill driven by an electric motor.
This mill was so made that it could be taken apart easily and cleaned.
In addition, it was fitted with bran and flour sieves of silk bolting cloth.
The wheat was tempered and extracted in a large water thermostat
fitted with a stirring device run by a small water motor. The thermo-
stat was heated by a gas burner, and the temperature was kept constant
(within i° C.) by means of a mercury gas regulator.
The same hydrogen-ion apparatus was used as that described in a
former paper,1 excepting that the saturated potassium-chlorid electrode
was used instead of the normal potassium-chlorid electrode.
The original moisture content of each lot of wheat was determined by
drying in the air oven at 110° C. to constant weight. This was found to
be 12.65 Per cent Ior Kanred, 10.86 per cent for the Hard Red winter
wheat, and 10.80 per cent for the Arizona White. In preparing the
wheat and flour samples 200 gm. of wheat were weighed out into a
500-cc. bottle. To this was added sufficient distilled water to bring
1 Sw anson, C O., and Tague, E. L. determination of acidity and titrable nitrogen in wheat
with the hydrogen electrode. In Jour. Agr. Research, v. 16, no. i, p. 1-13, 6 fig. 1919.
Nov. is, 1920 Changes Taking Place in the Tempering of Wheat 273
the total moisture content up to the desired percentage. The bottle was
corked tightly and the mixture was well shaken. The bottle was then
placed in the thermostat, which had been brought to the desired tem-
perature, and the mixture was allowed to remain in the thermostat for
the desired length of time. At the end of the time the wheat was ground
as rapidly as possible in the mill. The mill was set to grind to the same
fineness for each lot of wheat, and each lot was put through the mill the
same number of times. During the grinding the milling qualities were
judged as nearly as possible, and after grinding the yields of straight
flour were calculated.
Sufficient flour to equal 50 gm. on a moisture-free basis was imme-
diately weighed out. This was placed in a fruit jar, and sufficient dis-
tilled carbon- dioxid-free water was added to make the ratio of moisture-
free flour to water 1 to 10. This water had been previously heated to
400 C. To this mixture 2 cc. of toluene were added as a preservative,
the jar was tightly closed by means of a rubber and a screw cap, and
the contents were thoroughly mixed by shaking. The jar was then
placed in the thermostat, where the temperature was 400 C. The flour
was extracted for 2 hours at this temperature, the jar being well shaken
every 15 minutes. At the end of 2 hours the jar wras removed and the
contents were poured into a centrifuge cup. The cup was then placed
in the centrifuge and whirled for 5 minutes at a speed of 2,500 revolu-
tions per minute. Finally the supernatant liquid was poured through a
folded filter, and the filtrate was used for the determinations of hydrogen-
ion concentration, total acidity, water-soluble phosphorus, and titrable
nitrogen. For the determination of the hydrogen-ion concentration and
total acidity 100 cc. of the filtrate were pipetted into an electrode vessel.
The vessel was then placed in the hydrogen-ion apparatus, and hydrogen
gas was passed through until the potential remained constant (within
1 millivolt) for 15 minutes. During the entire time the vessel was shaken
60 times per minute. After this constant potential was noted, N/10
alkali was run in from a burette in small portions at a time until the
constant potential indicated a PH value of 7, which is the absolute neutral
point. The number of cubic centimeters of N/10 alkali used were then
taken to represent the total acidity.1
The water-soluble phosphorus was determined from a second 100-cc.
portion from the same filtrate. The phosphorus was determined by the
usual method after the organic matter had been destroyed by boiling
with nitric acid. The titrable nitrogen was determined in a third 100-cc.
portion by the formaldehyde method of Sorensen, using thymolphthalein
as an indicator. The number of cubic centimeters given in Table I
multiplied by 1.4 gives the number of milligrams of titrable nitrogen
in 100 cc. of the extract.
For a control, a portion of each variety of wheat, untempered, was
ground, and an extract was made of each in exactly the way described
1 For fuller description see Swanson, C. O., and Tague, E. L. op. cit.
274
Journal of Agricultural Research
Vol. XX. No. 4
above. The same determinations were then made on these extracts as
on the tempered lots.
The results for each variety of wheat are presented in Tables I, II,
and III.
Table I.
■Yield of flour, hydrogen-ion, concentration, total acidity, water-soluble phos-
phorus, and titrable nitrogen of the flour from Kanred wheat
Time
tempered.
Temper-
ature.
Hydro-
gen-ion
concen-
tration.
Total
acidity.0
Water-
soluble
phos-
phorus.
Titrable
nitrogen."
Yield of
flour. 6
Remarks.
Hours.
"C.
6. 20
6. 20
6. 22
6. 20
6. 17
6. 19
6.13
6.06
6.00
6.00
1-7
1-7
1.8
1-7
1-7
1-7
1.8
2- O
2.0
2-0
Per cent.
4.2
4.2
4.1
4-3
4.4
4.6
4-5
4.6
4.6
4.8
68
67
69
68
7°
72
71
72
72
70
-'4
4«
24
48
-'4
48
72
5
5
5
20
20
20
40
40
40
0359
0360
0361
0361
0362
0380
0376
0379
0378
Ground fairly well.
Do.
Do.
Do.
Ground well.
Do.
Do.
Do.
Sticky.
0 Expressed as number of cubic centimeters A'/io sodium hydroxid required to titrate 10 gm. flour.
6 Expressed as number of grams of flour obtained from 100 gm. of wheat.
Table II. — Yield of flour, hydrogen-ion concentration, total acidity, water-soluble
phosphorus, and titrable nitrogen of the flour from Hard Red winter wheat {Turkey or
KhorkoJ )
Time
tempered.
Temper-
ature.
Hydro-
gen-ion
concen-
tration.
Total
acidity.0
Water-
soluble
phos-
phorus.
Titrable
nitrogen."
Yield of
flour. b
Remarks.
Hours.
"C.
Ph-
6.13
6.13
6- IS
6. 10
6.06
6.06
6.06
5-92
5-91
5-92
1.9
1.8
1.9
1.9
1.9
1.9
1.9
2- 2
2. I
2. 2
Per cent.
3-6
.3-8
3-7
3-8
3-9
3-8
4.0
4.0
4.1
4.0
65
67
67
68
68
69
70
7i
"o
68
24
48
72
24
48
72
24
48
72
5
5
5
20
20
20
40
40
40
0460
0468
0472
0472
0471
0476
0479
0483
0482
Somewhat softer.
Do.
Do.
Ground fairly well.
Do.
Ground well.
Do.
Do.
Sticky.
0 Expressed as number of cubic centimeters of Nlw sodium hydroxid required to titrate 10 gm. flour.
b Expressed as number of grams of flour obtained from 100 gm. of wheat.
Table III. — Yield of flour, hydrogen-ion concentration, total acidity, water-soluble
phosphorus, and titrable nitrogen of the flour from Arizona White wheat
Time Temper-
tempered, ature.
Hydro- |
gen-ion Total
concen- | acidity."
tration.
Water-
soluble
phos-
phorus.
Titrable
nitrogen."
Yield cf
flour.''
Hours. \ "C.
24 5
48 5
72 5
24 20
48 20
72 ' 20
24 40
48 40
5-92
5-92
5-89
5-89
5-86
5-86
5-82
5-82
1.6
1.6
Per cent.
0177
0176
0176
0179
0179
0181
0182
0186
0185
67
Brittle.
67
Ground well
68
Do.
68
Do.
70
Do.
72
Do.
70
Do.
7i
Do.
70
Sticky.
6-)
Do.
" Expressed as number of cubic centimeters of Njio sodium hydroxid required to titrate 10 gm. flour.
6 Expressed as number of grams of flour obtained from 100 gm. of wheat.
Nov. 15, 1920 Changes Taking Place in the Tempering of Wheat 275
The addition of sufficient water to make the total moisture content 18
per cent was tried with each variety of wheat. In every case the result-
ing flour was sticky, the sieves became clogged, and the yields were
reduced below that for the untempered grain. For this reason the
analyses of the flour from this treatment were not completed.
It will be noted that when the wheat was tempered at 50 C. there was
practically no chemical change as compared with the untempered wheat.
As a general rule the yields were slightly higher and the milling qualities
were considerably better than those secured from the control or un-
tempered wheat. In each case the bran was tougher, and a cleaner sepa-
ration of the bran and endosperm was possible. The length of time
appeared to have very little influence on either the physical or chemical
composition of the flour.
When the wheat was tempered at 200 C, a small but definite chemical
change took place. The hydrogen-ion concentration was increased, as
was shown by a lower PH value. The total acidity, the water-soluble
phosphorus, and the titrable nitrogen were also higher. Both the yield
and the milling quality were better than when the wheat was tempered
at 50 C. The time of tempering appeared to be a factor in the chemical
changes but had very little if any relation to the physical qualities.
The chemical changes were still more pronounced when the grain was
tempered at 400 C. The physical changes appeared to be detrimental to
the milling qualities of the grain. In other words, increasing the time of
tempering increased the chemical changes but proved detrimental after
48 hours so far as the milling value of the wheat was concerned.
In general the milling qualities of the drier wheats were improved by
tempering more than were those of the wetter wheats, and the hard wheats
were improved more than the soft wheats.
It may be concluded from these experiments that slight chemical
changes take place during the tempering process and that these changes
increase with time and temperature. Improvement in the milling qualities
is confirmed also, excepting in cases where the time of tempering exceeded
48 hours and where the temperature exceeded 200 C. It would appear
from this that (1) the improved milling quality of tempered wheat is due
chiefly to physical changes, (2) a temperature of 20 to 250 C. is best, (3)
i$/4 per cent moisture appears to be about the best, (4) the maximum
improvement takes place in 48 hours, (5) hard wheats are improved more
than soft wheats, and (6) dry wheats are improved more than wet
wheats.
VASCULAR DISCOLORATION OF IRISH POTATO TUBERS
By H. A. Edson
Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of
Plant Industry, United States Department of Agriculture
INTRODUCTION
The exact significance of vascular discoloration in the stem-end
tissues of Irish potato tubers has never been fully determined. Various
types of both flesh and vascular necrosis are iecognized, some of which
are associated with the presence of Fusaria of various species or with
Verticillium albo-atrum. Others, however, at least in the initial stages,
yield no organisms when subjected to culture, nor does the microscope
reveal the presence of organisms. It is also recognized that a superficial
necrosis may develop in the stem tissues of apparently perfectly normal
stock. There is no such perfect natural abscission of the potato tuber
from the stolon as is common with fruits. Moreover, they are frequently
harvested before the plants are mature, and the tubers are then broken
off from green stolons. It has been assumed that suberization of the
wound thus made normally follows in two or three days, so that not
more than a few layers of dead cells should appear unless some aggressive
parasite gains entrance to the wound. A popular impression has pre-
vailed that any except the most superficial stem-end discoloration might
be taken as a trustworthy indication of the presence of Fusarium, or, at
least, that the stock was grown on vines affected with Fusarium or
Verticillium.
Somewhat extensive preliminary observations and cultural studies,
made by the writer both at the time of harvest and during or at the
close of the rest period, on stock grown in sections where Fusarium blight
and wilt do not occur, as well as in sections where they are known to be
general, show that, while Fusarium and Verticillium undoubtedly do
cause vascular discoloration of potato tubers, discoloration can not be
accepted as proof of the presence of Fusarium or, indeed, of any other
organism, nor can the absence of discoloration be confidently accepted
as proof of the sterility of the vessels near the stolon attachment. There
seems to be reason to think that vascular necrosis may often arise from
purely physiological causes and that it need not necessarily be seriously
abnormal, though frequently it is. A more complete discussion of this
question must await the outcome of studies at present incomplete, but
it seems advisable to present some available data regarding the fungous
flora of potato stem ends.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C. Nov. 15, 1920
vo Key No. G.-208
(277)
278 Journal of Agricultural Research voi.xx.no.4
The notes from which these data have been compiled were obtained
jointly by Venus W. Pool, M. B. McKay, H. G. MacMillan, R. D. Rands,
and the writer during the spring and summer of 191 5. The writer wishes
to make full acknowledgment to these associates and to assume the
entire responsibility for the construction placed on the notes and the
deductions made from them, as well as for the accuracy of the tabula-
tions and compilations presented.
OUTLINE OF METHODS AND WORK
The general plan followed in the work may be outlined briefly as
follows: Material for experimental plantings, involving about 4 acres
of plots, was secured from various sources, as reported below. It was
treated 30 minutes in 1 to 1,000 mercuric chlorid solution and allowed
to dry, after which each tuber was examined for vascular discoloration
by removing with a flamed and cooled scalpel a shallow cone of tissue
with the stolon attachment at the center of its base. A record was
made of the presence or absence of discoloration and of the general
character of the discoloration when present, as slight, medium, brown,
dark, etc. When discoloration was found, the depth to which it pene-
trated in the tuber was determined by removing a wedge of tissue.
When browning was confined to a shallow area around the removed cone
it was designated by recording the symptom A. If the discoloration
extended to a greater depth, involving up to one-fourth the length of the
tuber, symptom B was recorded. A deeper discoloration was designated
by C. Discolored tubers were submitted to culture. In general one
planting of tissue was made from each region involved in discoloration.
As a rule, therefore, one planting was made from tubers showing symp-
tom A, twro from those showing symptom B, three from tubers showing
symptom C, and none from those showing no discoloration. In the
actual prosecution of the work, however, certain deviations from the
general rule were introduced, either to check the dependability of re-
sults or to secure additional information. The tubers of each lot were
weighed and numbered consecutively in the order of their respective
weights, which were recorded. With the exception of lot No. 3, the
tubers of each lot weighing less than 3 ounces were divided into two
groups, one comprising all the even numbers and the other all the odd
numbers. Those weighing 3 ounces or more were halved from stem to
apex, one half being placed with the small tubers of even number and
the other half with the small tubers of odd number. When the half
tubers weighed 3 ounces or more they were cut into stem and apex
portions. In a few cases the half tubers were so large as to yield stem,
middle, and apex pieces, or even stem, two middle, and apex pieces —
four in all from each half. The minimum seed piece for cut tubers was
\\i ounces.
Nov. is, 1920 Vascular Discoloration of Irish Potato Tubers 279
The two lots of seed stock were planted and grown in widely separated
regions and under distinctly different environmental conditions of soil
and climate, one lot being planted on a light, sandy soil, under rainfall,
at Waupaca, Wis., and the other on a heavy clay loam under irrigation
at Greeley, Colo. The identity of each plant was preserved, and fre-
quent records were made by the same observers in rotation in each and
in both regions to secure all the data possible regarding the influence
of the seed piece and environment and of the interrelations of these
upon individual plant performance, with special reference to the develop-
ment of pathological conditions.
DESCRIPTION OF MATERIAL
The material may be divided advantageously for consideration into
three groups, each containing several lots. The first group comprises
stock affected with tuber-borne diseases of undertermined origin; the
second lot is from healthy parentage; and the third is from diseased par-
entage where the malady is regarded as of parasitic origin. For brevity
in presentation many lots which were held separate during the investiga-
tion have been combined, so as to appear as a unit, whenever their origin
and performance made such treatment feasible.
A brief description and index of the lots presented in the tables follows.
A. — Obscure disease group.
1. Thirty-four seedling varieties originated by Prof. Wm. Stuart,
of the Department of Agriculture, and originally regarded as promising
but ultimately discarded because of the persistent reappearance of
destructive but imperfectly understood hereditary diseases. This
material had been grown at Jerome, Idaho, in 191 3 and 191 4, in the
pathological plots there.
2. The progeny of 31 hills of Western Peach Blow, grown at Greeley,
Colo., which were suspected of Fusarium infection. This stock is now
known to be affected also with leafroll and mosaic and is therefore placed
in this group.
3. A miscellaneous collection of 21 lots from the pathological collection
of the field station at Presque Isle, Me. Both seedling and commercial
varieties affected with leafroll. mosaic, and dwarfing diseases were
included. This lot was grown only at Greeley, Colo., and the tubers
were either planted whole, or, if they weighed over 3 ounces, they were
cut once crosswise into stem and apex halves.
B. — Healthy group.
4. A representative commercial lot of the variety Late Ohio, grown at
Greeley, Colo., in 191 4 and obtained from the grower.
5. An exceptionally good commercial strain of the variety Pearl,
grown in Greeley, Colo., in 1914, obtained from the grower and col-
lected from the field at harvest time.
280 Journal of Agricultural Research, voi.xx, no.4
6. A fine commercial strain of the variety Pearl, grown at Crandon,
Wis., in 1914 and reported to be free from wilt, leafroll, and similar
diseases.
7. Wisconsin certified seed potatoes, variety Pearl, secured from the
grower.
8. Culls from two lots of Maine-grown stock of the variety Pearl.
One of these lots was reported healthy and the other as diseased with
leafroll. There was no difference in the performance of the two lots in
either locality where they were grown, and disease was absent. They
are therefore grouped together as healthy.
9. Certified seed potatoes of the variety Rural New Yorker, grown at
Boss Lake, Wis. A second lot of similar, though uncertified, material
of the same variety but from another grower near Racine, Wis.
10. A small lot of Wisconsin-grown stock of the variety Pearl, com-
posed of tubers on the stolons of which Colletotrichum pycnidia were
developing.
1 1 . Four so-called types of commercial stock of the variety Rural New
Yorker, supplied by a local grower of Greeley, Colo., who had used his
own home-grown seed for a series of years. These types were really only
rather imperfectly established size grades, evidently obtained by bin
selection from the general field run of his stock.
C. — Parasitic disease group.
12. The progeny of representative hills from a typical "Fusarium-
blight" field of the variety Early Ohio, grown at Greeley, Colo., in 1914,
dug in August and stored in a mass lot.
13. Ten hill lots of the variety Early Ohio, grown at Greeley, Colo.,
in 1914. The physical condition of the soil of the field was poor, and the
plants were small and dwarfed.
14. A representative lot from a field of choice stock of the variety
Sir Walter Raleigh, grown in 191 4 on a field at East Lansing, Mich.,
which was heavily infected by Fusarium. Every plant in the field, with
the exception of about one-quarter of 1 per cent, wilted and died three
or four weeks before frost.
15. Sixty-one hill lots of the variety Pearl, grown in Wisconsin in 1914.
The hills selected were from vines with more or less rolled foliage and a
brown discoloration of the vascular tissue of the stems. Cultures from
the discolored stem tissue failed to yield Fusarium.
16. Eighteen hill lots of the variety Pearl, grown from Wisconsin seed
at Greeley, Colo., in 1914. Cultural tests at digging time showed unusual
infection of the vines with Fusarium oxysporum.
17. Six hill lots of the variety Red McClure, grown at Greeley, Colo.,
in 1 914 on vines shown by isolations to be infected with Fusarium
oxysporum.
Nov. is, 1920 Vascular Discoloration of Irish Potato Tubers 281
18. Forty hill lots of the variety Rural New Yorker, grown on dis-
eased vines at Waupaca, Wis., in 1914. Cultural tests of the vines for
Fusarium at digging time yielded a Fusarium and a Colletrotrichum
culture in about equal numbers, but these did not appear to be general.
19. Twenty-five hill lots of the variety Rural New Yorker, grown at
Greeley, Colo., in 1914 on vines infected with Fusarium oxysporum, as
shown by isolation tests from the vascular tissue of the stem9 at digging
time.
PRESENTATION OF RESULTS
VASCULAR DISCOLORATION
The number of tubers in each lot of material and the number having
discolored vascular bundles, grouped according to the relative depth
of penetration below the stolon attachment, are shown in Table I. A
column for miscellaneous symptoms is included to provide for a variety
of incidental occurrences, such as net necrosis, decay, mechanical injury,
and the like; and following this, the number of tubers of each lot with
no vascular discoloration is shown.
It has already been stated that, in general, tubers with stem-end
vascular tissue of normal appearance were not submitted to culture and
that one, two, or three cultures were made from tubers with discolored
vessels, the actual number being determined by the depth of the necrosis.
No regular procedure was adopted with respect to the tubers belonging
to the miscellaneous group. The figures in the column marked "theo-
retical," under "number of cultures," have been obtained by adding
the number of shallow discolorations, twice the number of deep discol-
orations, three times the number of very deep discolorations, and what-
ever number the notes show to be correct to provide the cultures made
from tubers with miscellaneous symptoms. The actual number of
cultures made and reported upon follows in the next column. Under
"duplicates" are included the number of cultures made from discol-
ored tubers in excess of the number theoretically required. The num-
ber of cultures made from tubers with no discoloration of the stem-end
tissue is next recorded, and last of all is given the number of cases in
which a culture was theoretically called for but was not reported. In
some cases, for one reason or another, these cultures were not made,
while in others they were made and discarded before being studied,
because of broken tubes, loss of identifying label, and similar accidents.
If the number given in the last column is subtracted from the sum of
the numbers in the two preceding columns and the difference is added to
the theoretical number of cultures, the actual number is obtained.
282
Journal of Agricultural Research
Vol. XX, No. 4
Table I. — Appearance of vascular tissue and origin of cultures
OBSCURE DISEASE GROUP
Num-
ber of
tubers.
Nature of discoloration.
Number of cultures.
Lot No. and
designation.
Shal-
low.
Deep.
Very
deep.
Miscel-
laneous.
None.
Theo-
retical.
Actual.
Dupli-
cate.
From
tubers
not dis-
colored.
Theo-
reti-
cally re-
quired
but
lacking.
i Id
2 WPB
3 Me
i>73i
3«7
636
474
159
206
26
1
2
4
1
0
5
2
10
1, 222
224
418
544
164
217
590
162
215
44
8
6
57
13
14
55
23
22
HEALTHY GROUP
4CLO
5 PC
6PW
7PSW...
8PMe
9RW
10RWC0II.
11CRC.
12AEO.. .
13 EO
14M
15DPW...
16DPC...
17 RMc ...
18 DRW..
19DRC...
Total
335
233
21
2
9
70
290
373
85
5
957
563
0
O
2
392
564
572
19
11
537
80
0
I
5
451
84
89
I
12
65
10
. 0
O
0
55
10
9
0
0
133
14
0
O
0
119
14
16
0
2
360
58
0
O
I
301
58
59
2
5
7
0
0
O
7
0
7
7
O
0
664
262
3
O
2
397
269
280
13
9
PARASITIC DISEASE GROUP
212
132
17
0
0
63
166
181
24
2
69
17
1
1
0
5°
22
22
I
1
546
289
6
I
1
249
3°5
298
10
8
391
85
0
0
3
303
88
80
1
7
152
88
0
0
0
64
88
106
16
5
47
14
0
0
0
33
14
16
1
2
222
51
0
0
0
171
51
55
0
5
145
61
3
0
0
81
67
73
8
3
7-596
2, 796
80
10
47
4-663
3,022
3.203
239
161
ISOLATION AND IDENTIFICATION OF FUNGI
Isolations were made by transferring a small piece of tissue removed
under aseptic conditions from the region of discoloration directly to a
test tube containing sterilized nutrient material prepared in the usual
way. Melilotus stems, potato cylinders, and steamed rice were used,
the number of each diminishing in the order named. Identifications
were made direct from the original tube in some cases, while subcultures
were resorted to in others. Except in part of the Fusarium cultures, no
attempt was made to identify the species. Two hundred and ninety-
one out of the 718 cultures of Fusarium secured were identified as F.
discolor var. sulphureum or F. oxysporum, but it is not to be supposed
that the remaining 499 cultures were all of other species. Indeed, it is
probable that F. oxysporum and F. radicicola predominated among the
cultures reported as Fusarium spp. The summarized results of the cul-
tural studies are presented in Table II. Two columns of figures appear
under each genus reported. In the first column is given the number of
instances when the culture was either pure or so nearly so as not to give
Nov. is, 1920 Vascular Discoloration of Irish Potato Tubers 283
visible evidence of the presence of other organisms at the time of identifi-
cation. In the second column is recorded the number of times the genus
in question was found in a tube associated with some other organism.
Each tube containing a mixed culture is reported twice, once for each
organism. In no case were more than two organisms identified from a
single tube. The total number of identifications reported is therefore
the sum of all the columns marked ."pure" plus the sum of all the col-
umns marked "mixed," while the total number of plantings reported is
the sum of all the columns marked pure plus one-half the sum of all the
columns marked "mixed."
One very significant thing shown in Table II is the fact that out of
3,203 plantings, all but 161 of which were made from discolored tissue,
1,352 gave no growth. There is good reason to believe that in the great
majority of these cases the tubes yielded no growth because the tissue
transplanted was sterile, or at least free from filamentous fungi. These
results are in entire accord with those obtained by the writer in numerous
other cases where cultural tests of discolored vascular tissue of potatoes
have been carried out. In some instances the discoloration may be a
response to parasitic attack on some other portion of the plant, though
the tissues of the tuber are not actually attacked. In such cases it may
be regarded as a parasitic phenomenon of a secondary character. From
the physiological point of view, however, it matters little whether a
lethal dose of toxin diffuses from some point in the stem back oi the
stolon or from a point within the tuber itself. Likewise, the result is
the same whether the tissue is killed by the action of fungi, primary or
secondary, or through the operation, directly or indirectly, of malign
environment of whatever nature. Conclusions based on field experi-
ments with many factors uncontrolled must not be accepted without
reserve, but the writer has secured deep vascular discoloration which he
believes to be the direct result of too rapid respiration induced in the
soil at high temperatures such as prevail during the summer months in
the vicinity of Washington and which are occasionally experienced at
more northern and western points. This was the case with stock grown
at Arlington Farm during the summer of 1917, in which vascular dis-
coloration was universal and pronounced, extending throughout the
tuber in most cases. While certain lots of this material yielded
Fusarium or other fungi from a certain portion of the plantings, other
lots yielded only an occasional saprophytic growth out of hundreds of
plantings. The results were confirmed by repeated trials, which gave
uniformly identical results.
There seems, therefore, to be good reason to regard some of the stem-
end browning of vascular tissue as physiological, even in the cases in
which it extends well into the tubers.
284
Journal of Agricultural Research voi.xx,No.4
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\r. k O
ain^
*0 Ov O
id
ft; a
paxij^
M O 0
■ainj
"•.00
•paxij^
0 0 H
aJtij
0 ~>^o
'£ a
s &
•paxijv
-O H M
ajnj
u-. t C-)
.= 4 .
a si
a "■ ■-
paxiK
000
3JHJ
00 >- 0
Fusarium
discolor
snip bur-
nt m.
•paxij^
0-0
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M 0 vC
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0 M t
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6
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a; n o m « 00 ** '
o>« "tnmom*
o*ac *o OO O ^" <
O M O O M o o
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r~ m o O O O r-
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m o O O O O O
t- o o o o o «r
O O N O O 1-
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oo»o«
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r^wOOwOO^f"
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0000 -<t-tM VIM -e}-
5
-0
CI OOOOOOO
«
00
mO0OO""OO
00
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a
■*
MMOONMl/-, OvO
■o
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M
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H
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1-
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HOOOtHOM
0
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■"J-OkjwOOmi-i
£
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a
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w O f. in M 0 m O
If
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n O N OiO 0 1 •*
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(
Nov. is, 1920 Vascular Discoloration of Irish Potato Tubers 285
Another striking thing brought out in Table II is the frequency with
which Alternaria was recovered from the vascular tissue. Almost 20
per cent of the discolored tubers carried this genus, in most instances
unmixed with other fungi. This proportion is so high as to suggest that
it may possess some significance hitherto unsuspected or at least undis-
covered. Similar results have frequently been secured with other
material. As high as 50 per cent of some lots of tubers have yielded
Alternaria in cultural tests, even from stock presenting an attractive
appearance on superficial examination.
FIELD STUDIES
The general manner in which the stock was handled in planting has
already been indicated (p. 277-278). In taking notes in the field a full
description of each plant was recorded at each reading, including such
matters as size, habit, character, color, and orientation of stems and
foliage, as well as the general appearance as to vigor. At least three
sets of notes, and in the case of some lots more, were made on each plant
during the season. Successive sets of notes were taken by different
members of the staff, and no reference to the previous notes was made
while preparing the new set. In the preparation of the present article
the writer has endeavored to translate these descriptions into the ex-
pressions "diseased" and "healthy." Every plant has been placed in
one group or the other, even though in some cases the assignment had
to be more or less arbitrary. Consistency has been maintained, however,
and the writer has been able to bring to his aid thorough familiarity
with the appearance of the material throughout the season. One of
the three principal sets of notes is his own.
Plants whose description at any given note taking indicates probable
suspicion in the mind of the observer of the presence of disease have been
recorded as diseased, even though at previous or subsequent note takings
they may be recorded as healthy. It is certain that many cases of
recorded disease at the first note taking represent only delayed germi-
nation, but as this may be correlated with reduced vitality or fungous
attack on the sprout or tuber, it seems important to record it. Records
of recovery as well as of disease have been made and will be considered
later, but it is of interest first to inquire into the general relation of
vascular discoloration to fungous invasion and the correlation of these
within the tuber with disease in the plants produced. For the purpose
of this consideration plants once reported as diseased have been counted
as diseased whether later reported as diseased or healthy.
286
Journal of Agricultural Research
Vol. XX, No. 4
RELATION OF VASCULAR DISCOLORATION TO FUNGOUS INVASION AND
DISEASE
Table III is designed to show the performance in the field of all the
tubers studied, arranged according to the character of the tubers. The
tubers are grouped under four headings :
i. Tubers with vascular discoloration yielding a culture.
2. Tubers with vascular discoloration yielding no culture.
3. Tubers without vascular discoloration yielding no culture.
4. Tubers without vascular discoloration yielding a culture.
The tubers under each heading are arranged in two columns, according
as they yielded plants which were healthy or diseased. In case a tuber
was cut into two 01 more pieces at least one of which produced a diseased
plant, the tuber has been reported in the disease column. As is to be
expected, most of the plants in the progeny of the lots carrying obscure
tuber-borne diseases are diseased. The results presented in the remain-
ing two groups, however, seem to indicate that vascular discoloration
does not necessarily imply f ongous invasion ; nor is either of these in the
tuber a guarantee of disease in the plant, or their absence a guarantee of
health.
Table III. — Number of healthy and diseased plants from tubers eramined
OBSCURE DISEASE GROUP
Lot No.
Discoloration ;
fungus present.
Discoloration ;
fungus absent.
No discolora-
tion; fungus
absent.
NO discolora-
tion; fungus
present.
h
0
X
•6
3
y
5
"3
H
•6
u
a
u
5
>•
h
w
0
x
s
•d
til
u
0
Total.
15
22
8
147
90
46
23
14
22
324
37
142
86
42
73
I, 124
171
339
0
2
2
12
9
4
J> 731
387
636
Total
45
283
59
5°3
201
1,634
4
25
HEALTHY GROUP
128
263
28
5
8
19
3
82
65
118
33
4
0
25
0
46
53
131
12
1
6
6
0
74
19
53
13
0
0
9
4
65
55
261
249
22
114
I51
0
216
10
122
190
33
3
146
0
175
4
6
8
0
2
2
0
5
1
3
4
0
0
2
0
1
335
c
957
6
537
7
65
8
*33
360
7
664
Total
536
291
283
i63
1,068
679
27
11
Nov. is, 1920 Vascular Discoloration of Irish Potato Tubers 287
Table III. — Number of healthy and diseased plants from tubers examined — Continued
PARASITIC DISEASE GROUP
Lot No.
Discoloration;
fungus present.
Discoloration;
fugus absent.
No discolora-
tion; fungus
absent.
No discolora-
tion; fungus
present.
Total.
13-
14-
is-
16.
i7-
18.
19.
Total.
Grand total.
113
2
76
20
17
3
17
5
5
62
39
43
6
16
4
76
7
10
3
4
23
83
52
34
133
29
12
93
29
9
IS
"3
182
31
20
74
5°
2 53
143
497
494
834
784
830
1, 766
2, 807
40
M
212
69
546
39i
JS2
47
222
145
5° 7» 596
INFLUENCE OF ENVIRONMENT
Influence of environment upon the development of disease and recovery
is a subject of much interest and importance. Table IV brings out some
interesting facts regarding the development of disease in Wisconsin and
in Colorado in cut and uncut seed. It should be borne in mind that the
plants grown in the two States from cut seed are from the same individual,
since, as has already been stated, the tubers were halved lengthwise and
one half was planted in each place. The seed under 3 ounces was not cut
but was divided into two approximately equal portions for planting.
For the cut seed the total number of tubers cut and the total number of
seed pieces appear in each line. One-half the number of seed pieces
is the number planted in each State, except from lot 3. This lot was
planted in Colorado only, and it was halved crosswise into stem and apex
pieces instead of lengthwise.
The third and fourth columns give the number of diseased plants
developing in Wisconsin and Colorado, respectively, and the following
column gives the number of cases in which corresponding portions of a
given tuber yielded diseased plants in both places. These plants are
referred to as pairs. In No. 3 only, the pairs are from stem and apex
halves of the same tuber. Of the 197 diseased plants recorded, 106 were
from stem-end seed pieces and 91 were from apex or seed ends. As
shown in the table, 79 pairs occurred.
For the uncut seed the number of tubers planted in each State and
the number developing disease in each State appear.
9508°— 20 4
288
Journal of Agricultural Research
Vol. XX, No. 4
Table IV. — Distribution of diseased plants
OBSCURE DISEASE GROUP
Cut seed.
Whole seed.
Lot No.
Num-
ber of
tubers.
Num-
ber of
seed
pieces.
Num-
ber of
dis-
eased
plants
in Wis-
consin.
Num-
ber of
dis-
eased
plants
in Colo-
rado.
Num-
ber of
dis-
eased
pairs.
Num-
ber of
tubers
in Wis-
consin.
Num-
ber of
dis-
eased
plants
in Wis-
consin.
Num-
ber of
tubers
in Colo-
rado.
Num-
ber of
dis-
eased
plants
in Colo-
rado.
757
ii
143
1,636
22
286
729
8
698
9
197
650
7
79
483
194
43 2
181
491
I§2
493
454
Il6
2
414
o
Total
911
1,944
737
904
736
677
613
1, 166
984
HEALTHY GROUP
184
631
482
46
10
300
7
84
376
i,5S2
1, 076
114
20
718
14
202
52
i°5
177
17
0
104
3
10
45
201
141
35
0
153
2
73
25
42
62
12
0
64
1
8
75
163
27
9
60
3°
0
290
12
24
4
3
1
11
0
82
76
163
28
10
62
30
0
290
42
3
3
6
8
5
142
Total
i,744
4,072
468
650
214
654
137
658
208
PARASITIC DISEASE GROUP
12
13
14
15
16
17
18
19
Total..
Grand total
23
5
484
125
28
15
96
61
837
3,492
46
10
1, i36
272
56
30
214
150
1,914
7,93°
"3
56
19
10
40
3i
270
i,475
1
197
74
17
4
37
45
377
i,93i
52
41
14
3
17
23
150
96
34
3°
121
63
17
52
45
458
1,789
13
13
11
75
41
13
25
14
205
955
93
3°
32
145
62
15
74
39
490
2,3*5
13
14
8
9i
3i
4
25
26
1,404
The figures given in Table IV indicate no conspicuous relation between
the character of the tuber used for seed and the occurrence of disease,
since the number of pairs of diseased plants is only equal to from one-half
to one-third the total number of diseased plants in either locality. It is
to be noted that in general the Colorado conditions resulted in more dis-
ease than did those of Wisconsin, particularly when cut seed was used,
and this, too, notwithstanding the fact that the cut tubers were well
suberized when planted.
Nov. iS> 1920 Vascular Discoloration of Irish Potato Tubers
289
These results seem to indicate that the soil and not the tubers should
be considered the most potent source of disease, a fact substantiated for
the Greeley section by the more recent studies of Dr. MacMillan. Addi-
tional indication of this probability is given in Table V, where the
behavior of stem and apex seed pieces is presented and the number of dis-
eased plants per tuber is shown. The obscure disease group, of course,
shows a majority of cases in which all the plants from a tuber were dis-
eased, when any of them were; but the combined results from the healthy
and the parasitic disease groups show that out of 283 quartered tubers
yielding diseased plants, 123 yielded 1 such plant only, 99 yielded 2,
33 yielded 3, while only 28 yielded 4.
Table V. — Number of tubers yielding diseased plants
OBSCURE DISEASE GROUP
Lot No.
From
stem
pieces.
From
apex
pieces.
From
both
stem
and
apex
pieces.
Total
num-
ber of
tubers.
Total
num-
ber of
tubers
yield-
ing
dis-
eased
plants.
Num-
ber of
tubers
yield-
ing
1 dis-
eased
plant.
Num-
ber of
tubers
yield-
ing
2 dis-
eased
plants.
Num-
ber of
tubers
yield-
ing
3 dis-
eased
plants.
Num-
ber of
tubers
yield-
ing
4 dis-
eased,
plants.
51
51
49
a57
0
6 143
53
3
5
3
42
106
91
79
118
39
79
HEALTHY GROUP
1
7i
48
11
1
33
25
9
0
27
23
9
c4
d 141
c56
en
O
59
0
C17
2
77
5°
11
1
44
17
1
1
20
18
6
0
5
12
1
0
e
8
6
3
3
7
8
0
46
23
21
48
20
14
11
3
16
13
12
17
5
12
0
0
PARASITIC DISEASE GROUP
0
0
C84
11
0
0
11
C14
41
9
26
7
18
6
49
10
24
2
16
6
3
0
6
It
2
16. .
18
6
9
3
10
3
6
6
13
2
7
3
3
0
1
1
10
2
° Two tubers were cut into eight pieces each. All yielded diseased plants. Other tubers were cut into
four pieces each.
& Tubers were cut into two pieces each.
e Tubers were cut into four pieces each. ,
<* Four tubers were cut into six pieces each. All produced healthy plants, except one stem ana one
middle piece from the same side of one tuber. These are both recorded as stem plants. Otner tuDers
were cut into four pieces.
290 Journal of Agricultural Research voi.xx.No. 4
It appears further from the data given on the second and third groups
in Table V that a tuber from healthy parentage or from fungous-invaded
parentage is more likely to yield a diseased plant from a stem-end seed
piece than from the apex. Two hundred and fifty-eight tubers yielded
diseased plants from stem ends and 1 50 yielded diseased plants from apex
ends. One hundred and twenty-five of these tubers yielded diseased
plants from both stem and apex. The ratios, therefore, of stem, apex,
and pairs were approximately 10:6:5. The fact that the proportion of
diseased stem plants to diseased apex plants is slightly higher in the
healthy group than in the parasitic disease group is not inconsistent with
other data presented in this paper.
The facts seem to indicate that the greater liability of stem-end plants
to disease results not because the vascular tissue of the seed piece is more
often infected by fungi but because it is more often endowed with less
physiological resistance.
DISEASE AND RECOVERY
Data dealing with disease and recovery are presented in Table VI.
The total number of plants reported at the first note taking as diseased
is recorded in the first column. Following this is recorded the number
of these plants which subsequently appeared to recover and to remain
healthy. The next column gives the number of additional plants re-
ported diseased at the second note taking, followed similarly by the
number of those which subsequently recovered. The next column
records the number of hitherto healthy plants which appeared to be
diseased at the third note taking.
In the lower portion of the table the Rural New Yorker and the Pearl
varieties have been summarized in juxtaposition for purposes of con-
venient comparison. The outstanding feature of this table is the re-
markable degree of recovery shown, particularly in Colorado. This is
especially noticeable with the Pearl stock in Colorado. It is, possibly,
the ability of the Pearl to recuperate in that section which accounts for
the popularity of this variety in the G: seley region.
A summary of the data on disease and on recovery for the entire
experiment in total and by States is given in Table VII. Table VIII
shows percentage data figured from information shown in Tables IV
and VII. Attention is directed to the figures in Tables IV and VII in
connection with the percentage averages in Table VIII, because per-
centage figures may be misleading when the numbers from which they
are computed are small. A striking example of this is shown in Table
VIII, where one plant in Wisconsin was diseased and did not recover,
while two were diseased in Colorado and both recovered. This appears
in the respective columns on recovery as o and 100 per cent. In the
larger groups and in the aggregates, however, reduction to percentage
gives a clearer presentation of the facts.
Nov. 15, 1920
Vascular Discoloration of Irish Potato Tubers
291
Table VI. — Disease and recovery
OBSCURE DISEASE GROUP
Number
Wisconsin.
Colorado.
not recov-
1
.2
"a
•a
01
a
•3
ered.
Lot No.
Number
in first
note
taking.
Number
added in
second
note
taking.
Number
added
in third
note
taking.
Number
in first
note
taking.
Number
added in
second
note
taking.
Number
added
in third
note
taking.
a
•6
•6
■6
u
•0
01
u
41
•a
01
0
•d
01
V
•o
a
•3
a
41
3
>
0
0
fi
a
01
5
U
O
P4
M
01
s
01
s
>
0
u
01
fi
q
01
s
0
u
01
a
5
0
1
0
2,313
314
6ll
943
15
67
1
170
112
31
IS
48
62
824
93
372
97
73
41
263
32
237
24
32
29
6s
0
3
1,063
173
1)031
541
Total
3,238
958
68
282
46
no
1,289
211
532
85
67
1,236
i>S92
HEALTHY GROUP
120
372
32s
58
3
273
s
307
34
29
67
0
ss
2
3
II
14
18
I
0
26
I
I
19
54
13
18
I
51
0
75
3
16
5
13
0
41
0
62
11
46
101
0
0
9
I
14
53
177
127
32
2
135
2
208
23
"3
5°
12
2
74
2
182
0
28
13
S
0
22
0
4
0
14
8
3
0
18
0
3
3
38
4
1
0
I
0
3
SO
99
158
6
1
48
2
29
33
116
86
23
66
10
3°
Total
1,463
192
72
231
140
182
736
458
72
46
5°
393
354
PARASITIC DISEASE GROUP
29
28
329
296
108
31
127
116
14
1
26
25
35
1
20
6
12
1
8
2
1
0
4
0
0
9
87
65
6
22
44
38
0
0
63
3
0
7
35
12
0
3
II
41
19
0
1
I
5
3
184
35
34
2
43
55
3
3
143
22
13
2
24
49
9
I
12
125
8
6
16
2
6
1
4
101
3
3
16
I
1
II
9
S
6
0
3
14
12
53
126
59
16
26
33
6
S8
14
*
42
32
3
Total
1,064
128
28
271
120
76
36l
2 59
179
I3S
49
327
195
758
404
98
60
33
3
86
7i
34
3
147
60
338
69
177
3S
46
133
25
104
43
11
264
185
225
74
1. 162
158
60
26
36
28
4
157
37
207
407
212
179
129
54
449
299
Healthy Rural New
585
243
126
82
I°3
47
24
2
345
98
258
73
26
18
21
17
4
17
79
S9
96
Diseased Rural New
43
Total
828
86
32
208
150
26
443
331
44
38
21
138
139
292
Journal of Agricultural Research
Vol. XX, No. 4
Table VII. — Summary of disease and recovery
OBSCURE DISEASE GROUP
Colorado and Wisconsin.
Wisconsin.
Colorado.
Lot No.
Num-
ber of
tubers.
Num-
ber of
seed
pieces.
Num-
ber of
diseased
plants.
Num-
ber of
recov-
ered
plants.
Num-
ber of
seed
pieces.
Num-
ber of
diseased
plants.
Num-
ber of
recov-
ered
plants.
Num-
ber of
seed
pieces.
Num-
ber of
diseased
plants.
Num-
ber of
recov-
ered
plants.
1,731
387
636
2,610
398
779
2,313
314
611
219
121
70
1,301
205
1, 161
189
98
16
1,309
193
779
1,152
125
611
Total
2,754
3,787
1, 506
i'35°
114
2,281
1,888
HEALTHY GROUP
335
957
53 7
6S
133
360
7
664
5i6
1,878
1,131
133
275
778
M
782
120
372
325
58
3
2 73
5
307
37
157
81
34
I
159
3
248
263
939
565
66
70
389
7
391
64
129
181
20
1
"5
3
92
M
3°
23
14
0
67
1
63
264
939
566
67
72
389
7
391
56
243
144
38
2
158
2
215
23
6
58
15
8
185
Total
• 3,058
5' 5°7
1,403
720
2,690
605
212
2,695
858
S°4
PARASITIC DISEASE GROUP
212
69
546
39i
152
47
222
145
235
74
1,198
538
181
62
340
234
29
28
329
296
108
31
127
116
21
5
218
128
17
12
79
62
119
39
598
257
91
32
159
120
14
13
124
13*
60
23
65
45
12
71
39
12
116
35
600
281
90
30
181
114
15
15
205
i6S
48
8
62
7i
9
4
147
«3
16
16
5
40
SO
18
Total
1,784
2,862
1,064
542
1,415
475
148
1,447
589
394
7,596
12,156
5,765
1,672
5, 611
2,430
474
6,423
3,335
1,194
Healthy Pearl
Diseased Pearl
1,692
543
3-417
719
758
404
2 73
145
1,640
348
331
191
67
6
1,644
371
427
213
202
139
Total
2,235
4.134
1. 162
418
1,988
522
73
2.015
640
341
Healthy Rural New
1,031
367
i,574
574
S8S
243
410
141
787
279
210
no
131
51
787
295
375
133
279
90
Diseased Rural New
Total
1,398
2,148
828
551
1,066
320
182
1,082
508
369
Nov. is, 1920 Vascular Discoloration of Irish Potato Tubers
293
Table VIII. — Summary of disease and recovery in percentage
OBSCURE DISEASE GROUP
Percentage diseased.
Percentage re-
Lot No.
Cut seed.
Whole seed.
All seed.
covered.
Wis-
consin.
Colo-
rado.
Wis-
consin.
Colo-
rado.
Wis-
consin.
Colo-
rado.
Wis-
consin.
Colo-
rado.
89.12
72- 73
85-33
81.82
66.88
89.44
93-30
92.46
63-74
83.98
89.24
92. 20
88.01
64.77
78.43
8.44
8-47
10. 52
84. 00
Total
88.90
81.08
90. 55
84-39
89.64
82. 77
8.44
15. 69
HEALTHY GROUP
27. 66
13-53
32.90
29. 82
0. 00
28.97
42.86
9.90
23-94
25.90
26. 21
61.40
0. 00
42. 62
28.57
72. 28
16. 00
14.72
14.81
33-33
1.67
36.67
0. 00
28.28
14.47
25- 77
10. 71
30. 00
3-23
16.67
0. 00
48.97
24-33
13-74
32.04
30-3°
1-43
29.56
42.86
23-53
21. 21
25-88
25.44
56- 72
2.78
40. 62
28.57
54-99
21
88
41. 07
23
12
70
O
58
33
68
26
71
00
00
26
33
,18
52. 26
6
40. 28
39-47
8
100. 00
58-23
100. 00
86.05
Total
22. 99
31- 93
20.95
31-56
22.49
31.84
35-04
58-74
PARASITIC DISEASE GROUP
Grand total .
Healthy Pearl..
Diseased Pearl .
Total.
Healthy Rural New Vorker.
Diseased Rural New Yorker.
Total.
4-35
o. 00
19.89
41. 18
67.86
66.67
37-38
41-33
Total 28. 21
38.59
21.65
45- 73
25-05
39.01
8. 70
20. OO
34-68
54.41
60. 71
26. 67
34-58
60. 00
39-39
27.30
55-48
48.82
45-05
13-54
38.24
36.67
61.98
65.08
76.47
48.08
3I-H
44. 76
53-38
12. 36
63.04
29. 06
31-65
13.98
46.67
25.00
62. 76
50.00
26. 67
33-78
66.67
43-27
60.65
19. 01
58.94
36.60
45-94
45-13
II. 76
33-33
20. 74
5C97
65- 93
71.88
40.88
37-SO
33- 57
20. 18
54-89
26.68
39-43
12-93
42.86
34-17
58.72
53-33
26. 67
34- 25
62. 28
40. 70
25-97
57-41
31- 76
47-65
45.08
46-95
85-71
7.69
57-26
3-82
I.67
30.43
60. 00
26. 67
31. 16
20. 24
3- 14
62.38
46.36
56.87
60. 00
26. 67
71.71
74-55
33-33
62. 50
64. 52
70.42
66.89
47-31
65.26
74.40
67. 67
72.64
SUMMARY
In the material studied, vascular discoloration of stem-end tissues of
Irish potato tubers was not found to be proof of the presence of para-
sitic fungi. Discolored bundles were often sterile, and fungi were fre-
quently isolated from tissues which appeared normal.
The organisms recovered, in the order of their greatest frequency,
were Fusarium 720, Alternaria 615, bacteria 241, Verticillium 147,
Penicillium 104, Colletotrichum 91, Rhizoctonia 12, miscellaneous 87.
294 Journal of Agricultural Research voi.xx.No.-,
Out of 3,203 plantings, all but 161 of which were from discolored tissues,
1,352 gave no growth.
The field trials indicate that neither vascular discoloration nor fungus
invasion of the tissues of the mother tuber is a guarantee of disease in
the resulting plants, nor is their absence a guarantee of health. The soil
and not the tuber appeared to have been the more potent source of
disease.
Stem-end seed pieces yielded slightly higher percentages of disease
than eye-end pieces, evidently because the stem end is endowed with
less physiological resistance.
The plants showed a marked capacity for recuperation, which varied
with the variety, with the environment, and with the interaction of
the two.
CROWNWART OF ALFALFA CAUSED BY UROPHLYCTIS
ALFALFAE
By Fred Reuee Jones, Pathologist, and Charles DrechslEr, Assistant Patholo-
gist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant
Industry, United States Department of Agriculture
INTRODUCTION
When between the years 1909 and 1914 the so-called erownwart of
alfalfa was found scattered through several important alfalfa-growing
regions on the Pacific slope of the United States, much interest was
aroused. The earliest publication dealing with the disease in South
America indicated that it might become of considerable economic im-
portance. The fact that it had attained but limited distribution sug-
gested that prompt study might reveal the possibility of effective
measures against further spread as well as means of averting serious loss
in the regions already invaded. In 191 5 this interest formulated itself
in a petition 1 framed by the American Phytopathological Society
addressed to the United States Department of Agriculture calling atten-
tion to existing conditions and urging work upon this interstate problem.
In 1 91 7 it became one of the duties of the senior author to begin work
upon this disease. The junior author was associated with the work in
1919, making the field observations, giving especial consideration to the
taxonomy and morphology of the causal organism, and preparing all the
drawings. This paper is a report of the progress that has been made in
the study of this disease.
THE DISEASE
COMMON NAMES
In the United States the disease is commonly know bv either of two
names, crowngall and erownwart. As will be shown later, the structure
of the diseased tissue is that of a true gall, and it was called such in the
earlier reports of its occurrence. Later the name erownwart was sug-
gested in order to distinguish the disease from the bacterial crowngall
caused by Pseudomonas tumefaciens , though it had not then been shown
that this disease occurs upon alfalfa in the field. Recently, however,
galls have been found by Mr. H. L. Westover on alfalfa in Arizona
which appear to be true crowngalls, though complete proof is lacking.
In view of the fact that a gall similar in appearance to that caused by
Urophlyctis alfalfae (Lagerh.) P. Magnus is found upon alfalfa, it is even
1 Phytopathology, v. 5, no. 2, p. 130-131. 1915.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C Nov. 15, 1920
Vp Key No. G-209
(295)
296 Journal of Agricultural Research vol. xx, no.4
more desirable than formerly that the disease caused by Urophlyctis
should have a distinctive name. The fact that the name crownwart is
well established in usage is much in its favor. It will be seen, however,
from facts presented later in this paper that this name is somewhat
misleading, inasmuch as the galls are not typical warty growths, nor are
they formed from the tissue of the so-called crown of the plant in a manner
comparable with that in which crowngalls are formed. A name more
truly distinctive is suggested by the French name used by Arnaud (j),1
"La Maladie des tumeurs marbrees de la Luzerne." An English equiv-
alent, marbled gall of alfalfa, the word marbled referring to the mot-
tled effect produced by the brown spore masses seen when any of these
galls are cut, would call attention to the one distinctive character of
these galls observable at any time and would be accurately descriptive.
HOST PLANTS
Of the many species of the genus Medicago introduced into the United
States, Medicago sativa is the only one on which the disease has been
found commonly. McKee (77) found it also on M. falcata. The two
species, grown near together at the Plant Introduction Field Station at
Chico, Calif., seemed to be about equally infected.
Spegazzini (32) records the fungus as occurring on Medicago denticulata
and species of Adesmia in Argentina. Hauman-Merck (11) also records
the fungus on M. denticulata from the same locality and further states
that it does not occur upon alfalfa. In view of the fact that search has
not revealed the fungus upon M. denticulata in the United States even
when the plant is growing abundantly close in association with diseased
alfalfa, it seems advisable to hold it an open question whether the fungus
found in Argentina upon M. denticulata and Adesmia spp. is identical
with that which causes the disease of alfalfa.2 Thus, the evidence at hand,
while it is inadequate for the formation of final conclusions, appears to
indicate that the species of Urophlyctis occurring on M. sativa is prob-
ably limited to that species and to M. falcata.
DISTRIBUTION AND ECONOMIC IMPORTANCE
The only available information regarding the economic importance of
the disease consists of expressions of opinion based on a larger or smaller
amount of field observation. The trend of the opinion that has devel-
oped from this observation is that the disease is, or becomes locally, very
destructive to alfalfa plants.
The first report of the disease by von Lagerheim (14) from Ecuador
gave inception to this trend. He states that diseased plants can easily
1 Reference is made by number (italic) to " Literature cited," pp. 321-323.
2 A portion of a collection of Urophlyctis alfalfae var. adesmiae on Adesmia bicolor, sent by Spegazzini to
the Office of Pathological Collections, Bureau of Plant Industry, has been examined and been found to
contain a Synchitrium rather than a Urophlyctis.
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 297
be distinguished in the field, and his illustrations of diseased plants with
crowns encrusted with large galls contributed effective support to his state-
ments. However, von Lagerheim states that he did not see the disease
in the field himself, though he sought for it in fields near Quito. He
received his specimens from the owner of an estate in the Andes, and his
description of the effects of the disease in the field was gathered from
several observers.
In Europe, Magnus (20) reports a destructive outbreak of the disease
in Alsace, basing his report on the observations of two farmers. Later,
from an adjoining Province of Germany, Grimm and Korff (10) report the
disease as present in an alfalfa field without causing much apparent harm.
In fact, the diseased plants seemed somewhat more vigorous than the
others. Nevertheless, they think measures should be taken to elimi-
nate it.
Peglion (25) finds the disease in Italy, and raises the question whether
or not it may be a factor in producing alfalfa sickness in some fields. He
suggests that experimental work should be undertaken to determine the
matter. In France Arnaud (1) reports the disease as apparently doing
considerable damage in a single field in the Department of Seine-et-Oise.
In 19 1 6 Salmon (27) found a single field infested with wart in England
and urged further search for the disease. No reports of serious infesta-
tions have followed, though the writers have been told that occasional
specimens are found. The disease has been found in Holland (8) and
Sweden, but no apparent damage has been reported.
A critical reading of these reports of the destructive action of the dis-
ease calls attention to the fact that the two most important reports,
those of von Lagerheim and of Magnus, are not based on first-hand obser-
vation. In all cases damage is noted only in small areas. Therefore
we must still hold it an open question whether this disease has been
primarily responsible for any serious or widespread injury to alfalfa in
either South America or Europe.
In the United States the disease has been found abundant only west
of the Sierra Nevada and Cascade Mountains, though it occurs in a few
regions east of these mountains. It has not been found east of the
Rockies. However, in view of the fact that the disease when not abun-
dant is often completely concealed unless a plant is uprooted, it is pos-
sible that its distribution is more widespread than records show.
The first report of the disease by Smith (31) gives no clue to its im-
portance. O'Gara (22) finds the disease very common and occasionally
destructive in fields in the Rogue River Valley in Oregon. Jackson (12)
later reports the disease from the same region, making no comment
regarding its importance. Again O'Gara (23) is first to report the disease
present in the Salt Lake Valley in Utah, though he has not in this case
determined to what extent it causes injury. McCallum (16) reported
the disease present in Arizona. McKee (17), who has had an opportunity
298 Journal of Agricultural Research voi.xx,n0.4
to observe the disease extensively, concludes that crownwart decreases
the yield and shortens the life period of plants. He says:
Alfalfa fields that had the crown wart in abundance in 19 14 produced good crops of
hay in that year and in 1915. In one field sown in 1910 that has been under obser-
vation the past two years, practically every plant has galls. This field has produced
apparently normal crops of hay, but more critical observation shows decreased vigor
in the plants and a corresponding decrease of yield.
McKee believes the disease much more widespread than is commonly
supposed and urges work to determine its importance in alfalfa culture.
Thus it appears that although the disease is scattered through large
alfalfa-growing areas in the United States, yet it does not appear at any
place to have become regarded as a serious limiting factor in the growth
of the crop, except during years of severe attack and even then in small
areas.
The writers have not attempted to determine the present limits of
spread of the disease in the United States. A limited amount of time has
been spent in the spring of three years observing the disease, chiefly in
the river valleys where it is known to be most abundant, the Sacramento
River Valley in California and the Rogue River Valley in Oregon. The
second of these years, 19 18, appears to have been distinctly unfavorable
for the development of the disease, especially in California. The winter
rainfall was below normal, in consequence of which the Sacrafnento
River did not overflow its flood plain where McKee observed the disease
to be most abundant. The disease was commonly present on a larger or
smaller percentage of plants, but nowhere did observation bring con-
viction that considerable damage was being done. In the San Joaquin
Valley that year only occasional diseased plants could be found, though
in some localities there was excellent testimony from farmers of the
abundance of the disease in previous years.
In 19 1 9 there was much more winter rain, especially in the Sacra-
mento River Valley, and a greater amount of disease was found. Even
then it was only rarely that the disease was sufficiently abundant to appear
to be of serious economic importance. Plants could be found whose
early buds had become so completely infected that few were left to form
the second and later cuttings, but such plants were usually widely scat-
tered among others less severely infected. Rarely indeed does the dis-
ease appear to be solely responsible for the killing of entire plants, though
it must often weaken them. A significant estimate of the actual damage
done can be made only after careful observation has extended over a
period of years when the varying intensity of the annual attacks can be
studied and the behavior of the diseased plants followed throughout the
year.
DESCRIPTION OF THE DISEASE (PL. 47)
The disease is more easily described by stating briefly the origin and
method of development of the galls. So far as the writers can discover
Nov. 15. 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 299
all galls as they occur naturally in the field result from the infection of
buds in early stages of development as they emerge from the crown of
the plant. It is well known that there is an almost continuous succes-
sion in the development of buds from the so-called alfalfa crown during
the entire year. A portion of those buds which will produce the shoots
furnishing the first crop in the spring have begun development as early
as the preceding autumn. Generally speaking, the first buds to be formed
in the seasonal succession have a point of origin deeper in the soil than
those which are formed later, so that many of the buds from which the
shoots of the third cutting arise develop from positions quite above the
surface of the soil. Buds produced below the soil level in cool weather
appear to have a meager protection of scaly covering, and it is for the
most part such buds that become infected and give rise to galls. Thus,
galls are swollen and distorted bud elements, scales, leaves, and stipules.
Unless overwintered galls which are described later are discoverable,
the disease is first evidenced in the spring by a slight thickening and round-
ing of the young buds. During two years this has been observed near
Chico, Calif., in the latter part of March or early in April. The diseased
buds become more and more rounded as growth progresses and are glis-
tening white in color (PI. 54, A). Then, as the infected structures begin
to push apart, some of them grow much more rapidly than others until
the structure as a whole assumes a conspicuously irregular form. In
most cases, however, an examination of the gall will show that it is made
up of thick, scalelike layers about a central growing axis (PI. 36, B).
Sometimes this axis continues growth in spite of the demands of the mass
of developing gall tissue and produces a weak shoot. The earlier and
more vigorous buds produce the larger galls. Smaller galls often appear
to be developed from smaller buds along the stems below ground that
would ordinarily remain dormant. The origin of galls that appear on
stems several inches above the surface of the ground in wet weather
appears to be due in part to the infection of axillary buds that would
never develop in the ordinary course of events and in part to the elon-
gation of the stems and petioles which force infected tissue upward.
Since a large part of the infected buds are developed at a depth of
2 or 3 inches below the surface of the soil, the majority of the galls are
so far below ground that they escape observation unless the soil is
removed from around the plant. If they are of large size some of them
come to the surface, where they take on a green color and in extreme
cases form a crust of diseased tissue around the base of the healthy
stems.
Another type of gall that is not common results from local infections
on young leaves. Such infections give rise to small blister-like galls
much like those produced on Sanicula spp. by another species of this
fungus which will be mentioned later.
3<X) Journal of Agricultural Research vol. xx, No. 4
The galls reach full development (PI. 54, B) early in the summer, in
early June in northern California. From this time on the majority of
them begin to decay if moisture is abundant, or to shrivel and dry
with the coming of drouth. However, in almost all fields a few galls
more deeply situated become covered with a corky layer and survive
the winter.
When plants are subjected to dry conditions in late summer, as they
usually are in the Rogue River Valley in Oregon, many of the galls do
not decay but remain living throughout the autumn and winter. It
does not appear that such galls make appreciable growth during the
following year. Nevertheless, gall tissue may accumulate around old
plants in considerable mass. The exterior becomes covered with a
brown, corky layer that has a much warted appearance. This accumu-
lation of gall tissue has not been found on plants that have grown in
well-irrigated fields.
At whatever age or state of development these galls are found, they
possess one distinctive character that is discovered when they are cut
open. The interior of the galls contains many small, irregularly shaped
brown masses of fungus spores which are easily visible (PI. 56, B). In
old dried galls the host tissue has shrunken so much that the spore
mass often occupies a large portion of the mass of the gall. Even in
decayed galls that have not yet been broken to fragments the spore
masses can be recognized by their golden brown color.
CAUSAL ORGANISM
NOMENCLATURE
Some difference of opinion concerning the identity of the parasite
causing crownwart of alfalfa has prevailed. Von Lagerheim (24) seems
first to have regarded it as a new and distinct species, which he cited
as Cladochytmim aljaljae. Later, however, he (14) identified it with
Urophlyctis (Physoderma) leproidea, a parasite causing conspicuous mal-
formations on the beet, originally described from Algeria by Trabut (34)
and assigned by him as well as by Saccardo and Mattirolo (26) to a
new Ustilaginous genus, Oedomyces. In making this disposition, von
Lagerheim opposed the views of both Vuillemin (55), who had identi-
fied Trabut's beet organism with Urophlyctis (Cladochytrium) pulposa
(Wallroth), long known to be parasitic on species of Chenopodium and
Atriplex, and of Magnus (18-20), who later came to regard the
parasites on Chenopodium spp., on the beet, and on alfalfa as three dis-
tinct species. None of these views appear to be based on evidence alto-
gether conclusive; nor can we adduce such evidence here, because the
lack of fresh diseased material of beet and of Chenopodium spp. have
made it impossible to attempt cross-inoculation experiments.
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 301
Provisionally, it appears advisable to follow Magnus in recognizing
the alfalfa parasite as a distinct species, not, perhaps, so much on
account of some differences in morbid host anatomy as because of the
general improbability that two unrelated plants serve as hosts to a
parasite which shows in general no omnivorous tendencies. The beet
disease has not been reported in the regions where crownwart is preva-
lent; and Chenopodium spp. with every chance for infection have not
been observed to be attacked. Reference has been made in another
connection to Spegazzini's (32) report of crownwart on Medicago denti-
culata and its absence from alfalfa in the same range. This condition
could most readily be attributed to the existence of another species
producing similar galls.
DEVELOPMENT AND MORPHOLOGY OF THE FUNGUS
The morphology of the crownwart organism has not hitherto received
much attention. Magnus (20) made some observations regarding en-
larged hyphae frequently found in old material and referred to the pres-
ence of a hyaline cell attached to the concave side of the resting spores;
but in the main his specific details concern the pathological anatomy of
the host. In more recent years, Wilson (37) published a cytological
account of Urophlyctis alfalfae, arriving at conclusions considerably at
variance with those of Magnus. The utilization of old material by both
these writers may largely account for their failure to observe important
details of development and morphology, as well as explain interpre-
tations that it appears impossible to reconcile with conditions as found
in young material much more favorable for study.
GERMINATION OF THE RESTING SPORES
As has long been recognized, the fungus passes through the prolonged
periods of summer drouth by means of the resting spores contained
within cavities in the galls of the host. In the course of the rainy season
the galls disintegrate completely, thus setting free the spores; and it is
not improbable that the exposure incident to this method of liberation
may be necessary for germination. However, the conditions that may
favor germination remain more or less obscure; for although many at-
tempts were made by the writers with spores from freshly gathered
material both old and young, as well as with limited supplies of material
that may, in addition, have suffered deterioration in transit, the results
obtained have been so meager and dubious that this phase of the life
history of the fungus must be reserved for a later paper. In a number of
preparations an appearance was noted as of resting spores producing a
number of subspherical bodies varying from 1 to 9, by the passage of
protoplasm through pores in the spore wall. The vesicles that usually
attained half the linear dimensions of the spore in some cases were seen
3<D2 Journal of Agricultural Research vol. xx. No. 4
to produce endogenous motile bodies resembling zoospores that later
escaped through a number of openings on the distil side of the vesicular
wall. As the Van Tiegham cultures in which this process was noticed
were usually several days old, the development of bacteria and various
protozoa brought into the observations a considerable measure of un-
trustworthiness. Indications that similar contaminations may have
affected the observations of Wilson (37) on Urophlyctis alfaljae and of
Bally (2) on U . rilbsaameni are not entirely wanting. Both of these
writers describe the resting spore as functioning directly as zoosporan-
gium.1
PENETRATION OF THE HOST
Because of difficulties encountered in efforts to bring about infection
under artificial conditions, it has not been possible to observe directly the
penetration of the host by the germinating zoospore. However, as an
abundance of conditions immediately following the entrance of the
parasite were found in stained sections of buds, the course of events
during the time of invasion can be followed in incipient stages in the
same manner as during advanced stages.
Bodies measuring 3 to 4 n in diameter were frequently found attached
or adhering to the scales or developing axis of the bud. They appear to
have made their way under the bud scales very close to the most rapidly
growing meristem. Unfortunately, no clear figures showing the immedi-
ate development of these bodies were observed — a failure attributable
apparently to the fact that by the time the galls became noticeable
many weeks had seemingly elapsed since the period during which infection
took place abundantly. As a result, the earliest demonstrable stage of
invasion was represented by the presence of small turbinate bodies (the
"Sammelzellen," "corps centrals," "vesicules collectrices," or "vesi-
cules collectives" of other writers) within the epidermal cells of the outer
foliar or scale elements of buds exposed to attack, and attached to and
perforating the cuticular wall by an elongated beak (PI. 49, A, ta-tg).
More than one body may be present in the same epidermal cell, two or
three being not unusual; and occasionally a considerable number of
contiguous cells may show such evidence of multiple and concentrated
attack. The beak manifestly represents the tube proliferated by the
zoospore through which the contents of the latter were conveyed into
the host cell after the manner prevailing very generally throughout the
Chytridiales.
1 In an article that has appeared since this paper was prepared, Wilson {38) gives a more detailed ac-
count of his findings. So far as his account concerns the germination of the resting spores, it appears to
differ very considerably from that more recently published by C Emlen Scott (30), according to whom
each resting spore proliferates from 1 to 15 sporangia, the zoospores escaping through a number of tubes
in the hyaline wall. With the latter account the observations recorded above are not at variance.
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 303
GROWTH OF THE PARASITE
The fungus cell thus produced is first uninucleated and bears at its
apex a short, cylindrical projection. As it becomes older it increases in
size, the single nucleus divides, giving rise to a multinucleated condition,
and the short apical projection proliferates more or less successively
three or four terminal branches which are directed nearly at right angles
to the primary axis. These branches subsequently proliferate usually
three to five secondary branches directed in the same plane or forward.
As a result of this continued ramification, the larger cells may be seen
to bear at their apices an apparatus consisting of a short axial stalk
branching to form a score of ultimate terminations. There can be little
doubt that these processes function as absorbing organs and may thus
be regarded as haustoria. In stained sections they are often too badly
obscured by host protoplasm to be readily distinguishable; but in prep-
arations of material dissected from fresh, living host plants, they may be
studied with ease and certainty.
In the meantime the turbinate cell has increased considerably in size
and in number of nuclei, the latter usually ranging from 10 to 20 or even
more. As no septa have appeared, the parasite is represented at this
stage by a simple coenocyte. With the cessation of growth by enlarge-
ment, this condition is altered by the appearance of a number of delicate
septa, the ultimate number usually ranging from 3 to 5 but occasionally
even reaching 7, each of which delimits a peripheral uninucleated mass
of protoplasm. As the septa do not appear altogether simultaneously,
the first to be inserted represent convex membranes united to the periph-
eral wall of the turbinate cell along an elliptical line of juncture, the long
axis being parallel with the axis of the turbinate cell. The septa in-
serted later, when the surface of the turbinate cell has been appropriated
in considerable measure, are more likely to be in relation to septa pre-
viously laid down as well as to the peripheral wall itself. While the
protoplasts first delimited thus tend to approach a double-convex, ellip-
tical lenticular shape, the later ones may be more irregular and have
several concave facets (Pi. 49, B).
The further development of each of the peripheral protoplasts thus
delimited takes place independently of the other protoplasts similarly
derived from the same turbinate cell and follows in the main the course
described by Maire and Tison (21) for Urophlyctis hemisphaerica (Speg.)
Syd. {U. kriegeriana Magnus) and by Vuillemin (j<5) for U. leproidea.
Material embedded in paraffin, sectioned, and stained shows the proto-
plasm very slightly contracted away from the septum along the inner sur-
face, and indications of such contraction are present also in freshly
dissected material mounted in water (PI. 48, B, tb). This slightly con-
tracted protoplast now pushes out a protuberance from the outer periph^
eral wall bounding it (PI. 48, C, D, tbx). In those peripheral segments
95080— 20 5
304 Journal of A gricultural Research vol. xx. No. 4
occupying a position on the side or toward the base of the turbinate cell,
the protuberance will invariably take place at some point along the edge
closest to the apical end of the turbinate structure ; while in the segments
on the apical end the protuberance usually occupies a middle position.
By the movement of the nucleus and part of the cytoplasm into the pro-
tuberance, the tip of the latter becomes somewhat distended. The con-
stricted position now rapidly elongates, resulting in the formation of an
attenuated hypha, uniform in thickness and approximately 0.5 fx'va diam-
eter (PI. 48, A-D). The transfer of protoplasm from the peripheral
segment to the distended termination continues for some time, until the
former has been completely evacuated (Pi. 48, B, D, la).
The elongation of the hypha involves a translatory movement of the
termination in a forward direction, from which, however, it may be de-
flected by a host cell wall, or even reflected back toward the cuticular
wall (PI. 49, B, tba). Ultimately elongation ceases, and the terminal
distension develops into a turbinate cell entirely similar to the original
product of infection, the single nucleus dividing repeatedly to reproduce
the coenocytic condition and the branching haustorial process developing
from the apical projection, which becomes observable at an early stage
during the period of hyphal elongation.
The proliferation of secondary turbinate cells, which tends to be more
abundant from the expanded apical end than from regions more nearly
basal, thus involves a certain number of lenticular uninucleated masses
of protoplasm, always peripheral in position. The larger remaining por-
tion of the contents of the original turbinate cell is consequently not
concerned in this process. It may conveniently be designated as the
sporogenous cell and always embraces the contents along the longitudi-
nal axis of the spore and as much peripheral protoplasm as is not involved
in the peripheral segments. The contents of the sporogenous cell func-
tions in giving rise to a resting spore in the manner described in the fol-
lowing paragraph.
vSooner or later after the segmentation of the turbinate cell has been
initiated, the axial haustorial prolongation buds terminally to produce
a small globose swelling, which, when it first becomes noticeable, has no
demonstrable irregularities on its surface. Later when the swelling or
young resting spore has attained a diameter of perhaps 5 fi (PI. 48, D,
rb), there are proliferated along a zone midway between the equatorial
region and the distil pole from 9 to 15 slender, unbranched, minute proc-
esses. The swelling continues to increase in size until it attains the
dimensions of the resting spore (about 25 to 35 by 40 to 50 /*), growth in
the earlier stages being due mainly to the transfer of protoplasmic con-
tents from the sporogenous cell through the axial haustorial element
but later quite largely by the assimilation of food material from the host.
Although the surface of the resting spore is rendered impervious by the
deposition of a thick wall during the later stages of enlargement, such
Nov. IS. 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 305
assimilation is made possible by the zone of haustorial processes, each
of which has in a manner similar to the apical process become branched
to form a ramifying apparatus (PI. 48, A-D, ra, rb).
DETAILS OF MORPHOLOGY AND CYTOLOGY
The branched haustorial processes with their unusually definite local-
ization, either as a solitary apparatus at the apical end of the vegetative
cell or arranged in a well-defined zone between the equator and the dis-
til pole of the resting spore, constitute perhaps the most striking mor-
phological feature of the parasite. Although the literature regarding
these structures, especially with reference to their development and
orientation on the resting spore, is unsatisfactory, there seems to be
good reason to believe that all the other species usually referred to Uro-
phlyctis, as well as many species commonly assigned to related genera,
will show complete similarity to U. alfalfae in this respect. Thus De-
Bary (3) in his account of Physoderma (Protomyces) menyanthis states
that—
Auf demselben (distil) Ende der Blasen finder man sehr haufig ein Biischelchen
sehr feiner und kurzer in ein Kopfchen endigender Faden, welche bald verschwinden
und iiber deren Bau und Zweck ich nichts Naheres angeben kann ;
and in the figure referred to the appendages are clearly represented at
the apices of the obovoid vesicles ("verkerteiformige Blasen"). Ludi
(15), who later studied the same fungus, figured a number of unbranched
processes arising independently but in close proximity to each other
from the apex of the "Sammelzelle" ; and in a few cases he represented
a hypha arising also independently from the midst of this cluster. Biis-
gen (4) observed the same structure in Physoderma (Cladochytrium)
butomi at the apex of the swellings less rich in contents. Like DeBary
this author remained uncertain as to their function but considered it
probable that the apparatus consists of budding hyphae together with
granular host protoplasm. He reported, too, the presence in this spec-
ies of —
irregular cylindrical projections which appear early on the spore, and later are not
greatly inferior in length to the diameter of the spore. Stained with iodin, a mem-
brane and hyaline contents with a few granules may be recognized. When the spore
matures, these break down.
These structures he designated as haustoria and related their function,
in our judgement altogether correctly, to the assimilation of food mater-
ial. His figures, however, with the exception of figure 19, a, which shows
a detached branching rhizoid, lack clearness and lead one to believe that
probably groups of newly proliferated young turbinate cells were con-
fused with the rhizoids. On the other hand, the haustoria he shows
associated with the resting spores of Physoderma {Cladochytrium) flam-
mulae suggest a good possibility of a zonate arrangement similar to that
306 Journal of Agricultural Research voi.xx,No.4
found in the alfalfa parasite corresponding, for example, to Plate 48,
A-D, ra, rb; although, to be sure, the attachment of the "Sammelzellen"
to the convex haustoria-bearing side would be at variance with any close
homology. It appears not unreasonable, however, to suspect that Biis-
gen was in error in regard to this point and that the resting spore may
be attached by its concave side, the concavity, as in Urophlyctis spp.
generally, very probably being opposite the side bearing the haustoria.
Clinton (5) noted the presence of a rhizoid-like process on the side of
the "Sammelzellen" toward the young sporangium in Physoderma
(Cladochytrium) macular e and figured it both as a terminal apical struc-
ture before the development of the resting sporangium has been initiated
and as a median whorl after the latter has been formed. Regarding its
function he states that —
The exact nature of these processes is not clearly shown, though they seem to bind
the sporangium cell to the Sammelzellen.
In his figure 32 he shows a similar process attached to an element that
appears to be a young resting spore, although he makes no reference to
this condition in the text.
Schroeter (28) observed the apical apparatus on the vegetative cells of
Urophlyctis pulposa, designating it as —
ein Kronchen, ein Schopf feiner und kurzer, oft verzweigter Protoplasma Anhangsel.
Vuillemin (35), who later studied the same species as well as the beet
parasite, appears to have recognized the apparatus as consisting of a
"tronc" bearing terminally a "houppe" of short ramifying processes —
the "panache terminate. " To these processes and to the haustoria on
the resting spores, as well as to the "appareil nourricier" generally, he
(36) assigned a structure identical to that of the striated muscle fiber of
animals. We have not been able to distinguish anything suggesting
striation in any portion of the thallus of U. alfalfae. The haus-
toria here, moreover, appear to have a membrane that seems to persist
after the contents have been withdrawn by plasmolysis or have degenerat-
ed. The history of the development of the haustoria on the resting
spore as given for the beet parasite again is at variance with their develop-
ment as observed in U. alfalfae. For the resting spore, according to
Vuillemin, ccmes about by the swelling of the "sommet du tronc du pan-
ache" in such a way that —
Les branches se trouvent dissociees en plusiers buissons et entrainc'es a diverses
hauteurs sur la boule terminale, tandis que d'autres fragments sont restes a la base.
Whereas in U. alfalfae the resting spore is initiated as a bud from the tip
of the axial haustorial element, never involving translocation of any
haustorial ramification. And as has been pointed out, the haustoria on
the resting spores are subsequently developed as new structures in a well-
defined zone and are not portions of the apical haustorium distributed
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 307
in a miscellaneous manner over the surface of the resting spore by the
enlargement of the latter.
The time of proliferation of the resting spore seems to be rather vari-
able. It may follow immediately after the septum delimiting the last
peripheral segment has been laid down, before the proliferation of the
new order of turbinate cells has begun (PI. 48, B, rb), or more usually
somewhat later when one or more of the peripheral segments have pro-
liferated secondary turbinate cells (PI. 48, C, rb). Or, as is not infre-
quently the case with the unusually large primary turbinate cells, the
immediate product of infection, the resting spores may not be formed until
three or four successions have intervened and the original lesion has be-
come a well-developed cavity (Pi. 50, tba). The protoplasm in the spo-
rogenous cells of such primary turbinate structures as well as the host
protoplasm of cells or cavities that have long harbored the fungus fre-
quently take a dense uniform stain with safranin — a result that might
readily be attributed to the diffusion of a deep-staining substance.
Where this abnormal condition becomes very pronounced, it is not im-
probable that no resting spore is produced at all, the deep-staining
protoplasm finally disintegrating in place. With perhaps this occasional
exception, every turbinate cell produces always one resting spore. Ac-
cording to Maire and Tison (21), Urophlyctis hemisphaerica (Speg.) Syd.
produces first a succession of "vesicules collectives," each of these in
turn giving rise to several others of the next order, until ultimately
each "vesicule collective" produces only a single resting spore. Such
separation of vegetative and reproductive stages is not discernible in U.
alfalfae, the production of resting spores being common to each order
of turbinate cells; and, although toward the end of the season, when
conditions for growth become poor, the proliferation of turbinate cells
may be considerably reduced, as may be inferred from the relatively
small number of young conditions in old galls containing an abundance
of mature resting spores, it is questionable whether their production is
ever entirely stopped so long as the host tissue is alive and growing.
In this connection it may be mentioned that the presence of unfavorable
conditions for development is indicated usually by a very pronounced
enlargement of the hyphae. When the parasite is growing vigorously the
hyphae, by which the youngest turbinate cells are attached, do not ordi-
narily exceed 0.5 ll in diameter. Tater their diameter ordinarily increases
to 0.8 to 1 fi, the increase being, as Vuillemin (35) has pointed out, in
the wall, the lumen remaining the same and, indeed, soon appearing
devoid of protoplasmic contents. In old, overwintering galls, however,
there may be found usually an abundance of hyphae measuring 3 to
5 /x, the surface of which may be marked with irregularities which give
the structure a granular appearance, especially in stained paraffin sec-
tions. Within these hyphae the turbinate cells occur as loculi in dis-
tensions occupying junctional or terminal positions and are connected
-208 Journal of Agricultural Research vol. xx, no. 4
with each other by the persisting, very narrow, central lumina (Pi. 52, B).
Magnus (20) designated this as encysted mycelium and regarded it as
being probably viable; although the degenerated condition of the pro-
toplasm where this is present, and more particularly the very frequent
absence of any contents whatsoever, would not argue for a high degree
of vitality. However this may be, the appearance of such swollen
mycelium suggests a pathological condition of the parasite rather than a
normal one.
Beyond a statement by Wilson (37), quite impossible of interpreta-
tion in the light of the life history here presented, that the —
content, cytoplasm, and the nuclei of the resting spores in the dormant condition cor-
responds to that of the plasmodium in the stage immediately preceding spore forma-
tion,
there appear no cytological allusions in the literature on the alfalfa
parasite. However, certain details regarding the nuclear behavior in
Urophlyctis rilbsaameni have been given by Bally (2) , and the valuable
paper on U. hemisphaerica by Maire and Tison (21) contains a
brief account of nuclear changes in the congeneric parasite on Carum
incrassatum and Kundmannia sicula.
The variability in size of the nucleus pointed out by these authors is
well exemplified also in Urophlyctis alfalfae, the larger and smaller dimen-
sions being here generally characteristic of certain stages in the develop-
ment of the organism. Thus, in the young primary turbinate cell, the
nucleus, which is subspherical in shape, commonly measures about 2 m in
diameter and is composed largely of refringent, nonstainable material and
a single, very conspicuous, deep-staining body (PI. 49, A, ta-tg). Later,
the nuclei may increase appreciably in size, even before their migration
into the secondary turbinate cells or into the young spore (Pi. 49, B, ta).
Considerable increase, however, appears to take place quite invariably
in the single nucleus of the young secondary turbinate cell, a maximum
diameter of 5 to 6 n being here attained before division occurs (Pi. 50,
tb-bx) . Division is initiated by the deep-staining body becoming elongated
and being drawn out into a spindle-shaped figure, which may be either
straight or distinctly cresentic, depending on the curvature of the portion
of the nuclear membrane to which it is laterally applied (Pi. 50, tbd).
This spindle-shaped structure appears to divide in the middle, yielding
two bodies similar to the original, which assume positions separated from
each other. A membrane is now formed between the two granules,
dividing the nonstainable material about equally; and when the two
hemispherical division products have rounded up, the structure of the
parent nucleus is reestablished, although pairs of sister nuclei can usually
be distinguished for some time by their nucleoles facing each other — a
figure that is by no means uncommon (Pi. 50, tab).
We have never been able to make out in the nucleus at any stage in
the development of turbinate cells anything that would need to be inter-
Nov. 15, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 309
preted as a chromatin network. Occasionally in nearly evacuated sporo-
genous cells, where the attenuated condition of the cytoplasm permits
of more accurate study, strands were observed close to the periphery of
the refringent nonstainable portion; however, from their general appear-
ance and staining reaction, it is much more probable that these represent
overlying strands of cytoplasm. The chromatin material here seems to
be very largely if not completely concentrated in the conspicuous, densely
staining body, which may thus be regarded as a karyosome or chromatin-
nucleole. This mode of division presumably constitutes a type of ami-
tosis; and, indeed, with a nucleus of the structure described, mitosis of
the regular type is manifestly out of question. And yet the enlongated
spindle shape assumed by the nucleole suggests that perhaps division
here may involve some mechanism resembling in a rudimentary way the
apparatus associated with mitosis. The whole process bears consider-
able resemblance to that described by Kusano (13) as occurring in the
zoosporangia of Olpidium viciae.
By repeated divisions the nuclei in the turbinate cells reach a number
of 10 to 20 before the latter has attained its final dimensions; and this
increase in number seems to involve usually a decrease in size, which may
sometimes be quite insignificant, or again quite considerable, but is
nearly always perceptible. Nutrition seems to have some influence on
the size of the nuclei at this stage, the turbinate cells found in recently
invaded tissues rich in protoplasm generally remaining relatively large
throughout, while those farther toward the origin of the cavity appear
to suffer the greatest reduction.
The cytoplasm of the growing turbinate cells stains moderately deeply
and seems to have a uniform, finely granular or reticulate structure.
During the earlier stages of growth, a relatively large vacuole may usually
be distinguished near the proximal end. Perhaps this is later associated
with the insertion of a septum near the base of the cell that is probably
not always concerned in delimiting a uninucleated protoplast but appears
to serve more frequently in shutting off the protoplasm from the
evacuated hypha. Although the number of vacuoles of a size readily to
be observed may be increased during the later stages of growth to several,
the difference between the basal and distil ends never becomes consider-
able, the structure of the cytoplasm at the time of the insertion of the
peripheral septa being generally rather uniformly granular or finely
reticulate. The progressive evacuation of contents of both the peripheral
segments and the sporogenous cell brings about an attenuation of the
cytoplasm which, especially in the sporogenous cell, is associated with
the appearance of large vacuoles that ultimately, with the exception of a
few strands of cytoplasm, coalesce to fill the entire cell.
As the isthmuses between the peripheral segments and the anlagen of
the young turbinate cells, as well as that between sporogenous cell and
resting spore, are considerably narrower than the nuclei, the latter
310 Journal of Agricultural Research vol. xx,No.4
undergo some distortion in their passage through these communications.
The achromatin passes into the lumen of the connecting element as a
beaked extension followed by the chromatin-nucleole, which, too, is
drawn out in a conspicuous manner (PI. 49, C). The normal nuclear
structure is recovered when the material has reached, for example, the
flaring portion of the isthmus at the proximal end of the resting spore.
The result of the total protoplasmic movement is that in Urophlyctis
alfalfae the penultimate cells are either evacuated or in the process of
evacuation and that all elements more basal in position, hyphae as well
as peripheral segments and sporogenous cells, are always quite empty of
living material.
Within the young, growing resting spore, the nuclei increase somewhat
in size; but much more marked is the immediate increase in size of the
chromatin-nucleoles, which at this stage measure 2 /1 in diameter, or
approximately half the linear dimensions of the nucleus. It is not
improbable that some nuclear divisions may take place. In living
material the resting spores show a beautifully vacuolate structure, the
vacuoles being numerous and relatively large (PI. 48, A-D, ra, rb). This
structure is apparently poorly preserved in the processes of killing,
embedding, and staining. Microtome sections stained with Flemming's
triple combination show the cytoplasm as having a dense reticulate
structure readily distinguishable, however, even in the earliest stages
from the cytoplasm of the turbinate cells by its greater affinity for
gentian violet.
Later, during the maturation period, the cytoplasm of the resting
spores appears more loosely reticulate, and the nuclei assume still
greater dimensions, finally measuring 6 to 8 n in diameter (Pi. 49, D-F).
This increase in size is associated with the appearance of very minute
granules of chromatin more or less irregularly disposed near the periphery
of the achromatin mass and easily distinguished from the surrounding
cytoplasm by a marked difference in staining properties. In many cases
the arrangement in a definite reticulum is particularly pronounced
(PI. 49, F). Maire and Tison (21) report that in the resting spore of
Urophlyctis hemisphaerica certain nuclei become enlarged, their nucleoles
becoming vacuolated and giving rise to large masses of a substance
staining red with safranin which accumulate in the center of the spore.
Something similar seems to occur in the maturing resting spores of
U alfalfae. Plate 49, F, represents an early stage in the process, the
three nuclei shown in the center having become conspicuously enlarged,
the achromatin having partly lost its refringency, and the nuclear
contours having become less distinct. Later, as in Plate 49, E, the
chromatin masses are no longer distinguishable but appear to have been
transformed or replaced by vacuolate cytoplasm somewhat more attenu-
ated than at the periphery and inclosing in its meshes the numerous
granules of red-staining material that have presumably been derived
Nov. 15, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 311
from the chromatin. Plate 49, D, shows a condition that frequently
appears in spores that probably have been poorly nourished. The degen-
eration of the central nuclei leads to the origin of a large vacuole that
ultimately develops into a cavity near the periphery of which a variable
number of red-staining granules are always to be found.
Maturation involves, too, a conspicuous transformation and thicken-
ing of the wall of the resting spore. Even while growth is still pro-
ceeding, the spore wall becomes increasingly thick; and during the later
stages of enlargement, although still capable of further distension, in all
probability it no longer permits of an easy passage of food materials.
After final size is attained, thickening proceeds rapidly. The mature
spore wall is a structure about 1.5 n in thickness, of a yellow, vitreous
appearance, inelastic and brittle; when the wall is fractured by pressure
applied in manipulation, fragments may break out like pieces of shell
from a nut, often leaving the contents quite intact.
When the spore has attained maturity, the haustorial processes dis-
appear, whether by retraction, degeneration, abscission, or accidental
fracture could not be definitely determined. However this may be,
a circle of pits or scars, corresponding in number and position to the
haustoria (PI. 48, F, G), is always left, because the thickening of the
spore wall never involves the places of attachment of the haustoria.
In examinations of herbarium material, in which turbinate cells and
hyphae are only too frequently quite unrecognizable, these pits serve as
a morphological feature of no mean taxonomic value.
GENERAL, TAXONOMIC CONSIDERATIONS
The taxonomic relations of the plants included under the genera
Urophlyctis, Physoderma, and Cladochytrium remain in need of study.
Schroeter (20) saw in the association of the "Oosporangium " of U. pulposa
with the "leere Blase" a sexual apparatus consisting of two conjugating
"Fruchtkorper," one of which has yielded its contents to the other.
On the basis of this interpretation he erected the genus Urophlyctis,
including it with Diplophysa and Polyphagus in the Oochytriaceae,
which family he distinguished from all the other families in the Chytri-
dineae not excluding the Cladochtriaceae, under which were brought
Physoderma and Cladochytrium by the presence of sexuality in the
origin of the resting spores. Fischer (9), on the other hand, denied the
existence of sexuality in Schroeter's genus and placed it with Physoderma
as a subgenus under Cladochytrium. Schroeter's views received support
from Magnus, who described a number of forms — U. kriegeriana (18),
U. leproidea (18), U. rilbsaameni (19), and U. alfalfae (20) — as con-
generic with U. pulposa and exhibiting the same type of oogamy. The
later investigations on U. leproidea by Vuillemin (55) , on U. rubsaameni
by Bally (<?), and on U. hemisphaerica by Maire and Tison (21) have
312 Journal of Agricultural Research voi.xx, No. 4
not confirmed Magnus' assumption of sexuality in these forms; and from
the present account it is obvious that in the formation of the resting
spores of U. alfalfae there is no indication of any process of conjugation.
In order to determine more nearly in what measure the development
and morphology of the alfalfa parasite might be common to related
forms, the writers examined herbarium material of various species of
Urophlyctis, Physoderma, and Cladochytrium. Fresh living material of
a species other than U. alfalfae was obtained only from U. pluriannulatus
(B. and C.) Farlow (7), occurring in the Pacific States on Sanicula men-
ziesii, on which host it was collected in excellent condition near Philo-
math, Oreg., on April 7 and May 16, 191 9. As its range extends over
the region in which crownwart is known, suspicion has arisen now and
then that the two parasites might be identical. This suspicion may
now be definitely dismissed.
Urophlyctis pluriannulatus may very easily be dissected from the
cavities in the wartlike protuberances on the stems and leaves of dis-
eased plants of Sanicula menziesii (PI. 53). Mounts of thalli consisting of
hundreds of turbinate cells and resting spores in a good state of preserva-
tion were obtained in this way. Plate 52, A, C, shows two small portions
of such a thallus. The general method of development corresponds
exactly to that described for U. alfalfae, yet morphological differences
sufficient to separate the two as distinct species are readily recognizable.
Greater dimensions are characteristic of U. pluriannulatus, both of
turbinate cells (which measure approximately 22 /x in length and 18 /x
in major diameter, against 19 /x length and 15 /x major diameter for
U. alfalfae), and of resting spores, the equatorial diameter here ranging
from 45 to 60 /x, as contrasted with 40 to 50 xx for U. alfalfae. The
turbinate cells of U. alfalfae produce usually a maximum of four to five
secondary turbinate cells, a greater number being occasionally produced,
however, by the very large primary turbinate structures; whereas in
U. pluriannulatus, turbinate cells not infrequently produce seven or eight
turbinate cells of the next order, five or six being the rule. An interesting
but rather inconspicuous difference in the structure of the rhizoids on the
resting spores may be noted. Since the primary branches are inserted
at nearly right angles in U. alfalfae while the corresponding angles tend
to be much smaller in U. pluriannulatus, there is brought about a differ-
ence that might crudely be compared, for example, to the difference in
habit between a palm and an elm. In U. pluriannulatus, too, the haus-
toria are inserted slightly nearer the equator than in the alfalfa parasite.
But the most unmistakable specific difference is to be found in the
number of haustoria on each resting spore, which in U. alfalfae varies
from 9 to 15 and in U. pluriannulatus ranges from 14 to 24. (Compare
PI. 48, E, with PI. 52, D.)
In this connection it may be mentioned that resting spores from herba-
rium material of all the other species of Urophlyctis examined, after being
Nov. 15,1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 313
boiled with caustic potash and cleared with chloral hydrate, reveal a ring
of pits altogether similar to those observed on spores of U. alfalfae and U.
pluriannulatus . That this implies the presence of haustoria in the fol-
lowing species can hardly be doubted:
Urophlyctis bohemica Bubak on Tri folium montanum, Rabenhorst-
Pazsche, Fungi Europaei et extraeuropaei, No. 4378.
Urophlyctis kriegeriana Mag. on Carum carvi, Jaap. Fungi sel. exs.
No. 126.
Urophlyctis kriegeriana Mag. on Pimpinclla nigra, Bubak, F. Fungi
Bohemici June 9, 1901.
Urophlyctis magnusiana Neger on Odontites rubra, Vestergreen, Mic.
rar. sel. No. 1614.
Urophlyctis major Schroeter on Rumcx britannica, Davis, J. J., Wiscon-
sin fungi. Aug. 27, 1 91 3.
Urophlyctis pulposa (Wallr.) Schroeter on Cheno podium glaucum, Sydow
Myc. ger. No. 1086.
Urophlyctis rubsaamcni Magnus on Rumex scutatus, Jaap, O Fungi
sel. exs. No. 402.
Seventeen species of Physoderma and Cladochytrium were also exam-
ined by the same method, and of these at least 2 species — namely, Physo'
derma menthae Schroeter on Mentha aquatica, Vestergreen, Mic. rar.
sel. No. 1609, and P. zeae-maydis on Zea mays, material furnished by
W. H. Tisdale — revealed a zone of pits, although no direct evidence could
be obtained that these had served as places of attachment for haustoria.
It is interesting to note that a certain range in number of pits was found
to be characteristic of species and that even numbers seemed to predomi-
nate. Thus Urophlyctis rilbsaameni showed either 6 or 8. Pronounced
and constant disparity in number of pits may, indeed, be interpreted as
indicating rather clearly that forms assigned to the same species because of
close relationship of their hosts may belong to quite different species. It
appears hardly admissible, for example, to designate the parasite on Pim-
pinclla nigra with 10 to 14 pits as U. kriegeriana, when this species of
Carum carvi shows only from 6 to 10; and the identity of U. kriegeriana
and U. pluriannulatus, suggested by Farlow (7) as a fair possibility, would
seem to be equally improbable.
In a number of species as, for example, Physoderma maculare (5),
P. butmio (4), and P. zeae-maydis (33), the germination of the resting
sporangium involves the lifting off of a circumscribed portion of the
spore wall by the expanding endosporangium. Although this "lid" is
usually not apparent in the spore wall, its presence on the resting
spores of P. comari, P. eleochardis, P. gerhardti, P. iridis, P. menthae,
P. schroeteri, P. vagans, and P. graminis could be determined from an
examination of herbarium material with moderate certainty. It remains
a question whether the resting spores of those species in which nothing
resembling a lid could be made out, including for example, P. agrostidis,
314 Journal of Agricultural Research vol. xx.No.4
P. calami, P. hipuridis, P. spargani, and P. speciosum, germinate, per-
haps, in a manner similar to P. menyanthis , in which, according to Clinton
(5) , the outer wall is ruptured by the elongating protoplast, dehiscence of
the zoospores taking place at the tip of the protrusion. The absence of
any indication of lids from the spores of all species of Urophlyctis exam-
ined may be of taxonomic significance, although this can not be deter-
mined until more reliable results have been obtained in the germination
of the spores. It would be interesting, too, to determine from living
material the positional relation between the zone of haustoria and the lid
in those species where both appear to be present, as seems to be the case,
for example, in P. menihae and P. zeae-maydis.
The more striking recorded departures of a number of species of Physo-
derma from the general thallus structure of the two species of Urophlyctis
investigated by us remain in need of explanation. One of the departures
is found in the septation of turbinate cells and in the fate of the different
segments. As has been pointed out, in Urophlyctis alfalfae and U. pluri-
annulatus the production of secondary turbinate cells always starts with
the delimitation of peripheral segments that involve portions of the
parent cell wall, most frequently subapical or lateral and occasionally
subbasal. The distinction between a smaller basal cell and a larger
distil cell, made by Biisgen for Physoderma butomi (3) and by Clinton
(5) for P. maculare, is thus without significance here; while their
accounts of the origin of the resting spore from the proximal cell are
directly at variance with developments in U. alfalfae and U. pluriannu-
latus, in which the resting spore is invariably developed from the large
multinucleate residue not involved in peripheral segments. Ludi (15)
figured the " Sammelzellen " of P. menyanthis with 1 or 2 transverse
septa and represented the resting spore as being attached to the
distil segment thus delimited by a filament of considerable length.
According to this writer's account, the resting spore here is not always
terminal, but by itself proliferating a "Sammelzelle" it often appears
as an intercalary structure associated with two "Sammelzellen." Tis-
dale's (jj) account of P. zeae-maydis presents even more points of
difference, showing structures consisting of two to four lobulate seg-
ments set off by transverse septa, these segments, with the exception of
one, capable of forming a resting spore either directly or at the end of a
fiber. In this form, organization and development would appear to be
of a rather miscellaneous type, contrasting sharply with the definite
sequence of growth found in the two plants figured in this paper.
Reference has been made elsewhere to Biisgen's figures of Physoderma
(Cladochytrium) flammidae, in which the resting spore is represented as
being attached to the "Sammelzelle" by the side bearing the haustoria.
Another detail worthy of note in the same figure of Biisgen's is the
length of the hypha connecting "Sammelzelle" and resting spore, ap-
proximating as it does half the length of the resting spore. In Cornu's
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlychs alfalfas 315
(6) figures of P. maculate (Melanotaenium alismatis) , the hypha con-
necting "corps central" and spore is even longer, exceeding here the
length of the "corps central"; and, as has been indicated above, an
entirely comparable figure is given by Ludi to illustrate conditions in
P. menyanthis. If these writers have not mistaken turbinate cells (or
their homologues) for resting spores and have not erred in relating the
latter to the wrong turbinate cells, it would appear that conspicuous
variability in length is characteristic of the connecting isthmus which in
Urophlyctis alfalfae and U. pluriannulatus is extremely short.
Magnus emphasized the difference in anatomical effects produced by
species he referred to the genus Urophlyctis and by those he assigned to
Physoderma. The former cause hypertrophy and thickening of host
cell wall, while the latter leave the host tissue in an approximately normal
condition. Perhaps a distinction on such grounds would make the
classification of parasitic forms contingent in too large a measure on
reactions of the host plant to be admissible in a taxonomic sense. It
seems not improbable that further study of the plants now referred to
Urophlyctis, Physoderma, Cladochytrium, and perhaps a few other
related genera will reveal possibilities in generic regrouping based on the
more significant similarities and differences in morphology and develop-
ment.
PATHOLOGICAL MORPHOLOGY
It has already been stated that the fungus attacks primarily leaf scales
and leaves at a very early stage of development in the growing bud.
Only rarely has it been found to have penetrated to the axis in the
dividing undifferentiated tissue of the bud. The stimulative effect of
the fungus is limited strictly to the structure which has been invaded,
while other structures in the vicinity of the main axis and the axis itself
show retardation and often cessation of development.
The first morphological change in the host consequent upon invasion
consists in a slight enlargement of the first cell entered so that it comes
to project both outwardly and inwardly against the underlying cells.
These underlying cells may also show a slight enlargement before they
are actually entered by the advancing fungus. The nuclei of the affected
cells enlarge notably, and the large deep-staining nucleoles persist for a
long time in the fungus cavities, their number serving as an index to the
number of host cells that have been destroyed.
The fungus evidently gains access to new cells by the solution of thin
cell walls in advance of the growing turbinate cells. In early develop-
ment when a number of these fungus cells are advancing close together
in the same direction, the walls of the host cells are found dissolved
before the fungus comes in contact with them (PI. 55), thus precluding
the possibility of mechanical pressure as a factor in effecting the advance.
In later stages, however, when turbinate cells are fewer and more scattered,
316 Journal of Agricultural Research vol. xx, No.4
the host wall does not always yield until the advancing cell is in contact
with it, suggesting that mechanical pressure may here be a factor.
The enlargement of cells under the stimulus of the fungus is the smaller
factor in the production of galls. As soon as the fungus has begun its
advance into the tissue, cell division is stimulated in the vicinity, and
even at a considerable distance if the fungus is making rapid growth.
The first notable divisions take place in the cells just beneath the epi-
dermis in the region of the point of invasion (PI. 55). Walls are inserted
tangentially to the outer surface of the structure, and the increase in
tissue at this point surrounds and may even bury deeply the base of the
fungus cavity so that it no longer leads to the exterior of the gall. The
thin-walled parenchyma in which the fungus forms its cavities may
show little morphological change near the invader in the early stages of
its progress, especially if these cells have matured and are not readily
capable of division. However, the older part of the surrounding wall of
the fungus cavity is soon greatly thickened with a layer which is very
brittle when cut and which is therefore poorly preserved in stained
preparations. The peculiar structure and markings sometimes found in
these walls has been noted by Magnus (20), though his assumption that
the window-like openings between fungus cavities are due to the local
absorption of these walls seems less probable than that they are the
partly filled openings through which the fungus advanced at an earlier
stage. As soon as this thickening is well under way, the host cells
adjoining the cavity begin to divide with walls tending to be oriented
tangentially to the wall of the cavity. Such divisions proceed further in
the vicinity of vascular bundles than elsewhere, giving rise to a con-
siderable mass of cells in parallel rows, almost cubical in shape, with walls
a little thicker than those of the normal parenchyma (PI. 56, A). But
these processes are rarely rapid enough to surround the newer portions
of the cavity where the fungus is slowly breaking into cells and extending
its ramifying maze. Perhaps the larger bulk of the cells that make up
the gall are developed from the vascular bundles where division, especially
in later stages in development, becomes very active. Sometimes a
bundle becomes much broadened, and from the active cambial region a
large mass of parenchyma on one side and a few leaf tracheids on the
other are set off. Tissue from this source is likely to be richer in proto-
plasmic contents than that from the other sources mentioned and is more
extensively penetrated by the advancing fungus. Thus, it may be said
that the response of the cells to the stimulation of the fungus is in pro-
portion to their capability for meristematic activity and to their nearness
to the source of stimulation. Cells near the exterior of the gall divide
with walls tangential to the surface of the gall; those in close proximity
to the older portions of the fungus cavity divide with walls tangential
to the wall of the cavity; while vascular bundles function in division
Nov. i5, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 317
like stem bundles in giving rise to secondary thickening, producing ir-
regular masses of leaf elements. Thus, the normal limitation in the
direction of cell division and growth which produces thin, laminated
structures is removed, and thick, fleshy amorphous masses of tissue
inclosing ramifying cavities filled with the fungus in all stages of de-
velopment are produced. On irrigated land these structures are not
usually well protected by epidermis or cortex and readily dry out or decay,
but in dry regions many become covered with a corky layer that pro-
tects them from destruction.
In partial contrast to the galls upon alfalfa is the gall upon Sanicula
menziesii (PI. 53) caused by Urophlyctis pluriannulatus previously men-
tioned, a contrast indicated by Magnus (79) in his classification of Uro-
phlyctis galls into two types, those upon underground parts of plants
and those upon aerial parts. Although the earliest stages in the forma-
tion of these galls have not been traced, evidence from more mature
stages indicates that the general development is similar to that of galls
formed on alfalfa and in fact is exactly like that of the blister-like galls
sometimes found on alfalfa leaves. In the attack of the fungus on Sani-
cula, infection of the leaf, petiole, and stem structures takes place at a
later stage of host development than is common on alfalfa, and the re-
sponse of the host tissue to the stimulus of the fungus is not nearly so
great, extending only to a distance of a few cells. Apparently a small
number of cells are rapidly invaded soon after the fungus enters the host.
Thickening of the host cell walls around the cavity formed, especially its
basal portion, soon occurs ; and thereafter it appears that a part at least
of the enlargement of the fungus cavity is accomplished by the pressure
of the growing fungus mass against the surrounding cells, which become
flattened and distorted. Thus, each infection produces one partly cham-
bered cavity in the parenchymatous tissue which has become hypertro-
phied to form a small blister-like gall.
INOCULATION EXPERIMENTS
In order to avert any possible danger of spread of the disease from
experimental plots, inoculation experiments were limited to a few potted
plants in a greenhouse at Washington and to plants in the greenhouse
and on the trial grounds of the United States Plant Introduction Garden
at Chico, Calif. At the latter place, perhaps because of the limited time
during which work was done there, no success was attained in producing
infection. Since one of these failures may be significant, it will be men-
tioned. On April 15, 191 8, nine days after wart was first found devel-
oping on plants in the field, an inoculum was prepared by shaking soil
and the fragments of decomposed warts from the crowns of a large num-
ber of plants which had been badly diseased the previous year and adding
a small amount of crushed warts which had been found not yet decayed.
3 1 8 Journal of Agricultural Research vd. xx, no. 4
A square yard of vigorously growing alfalfa plants in the corner of a 2-
year-old plot was selected for inoculation. These plants were already
producing shoots 1 foot or more in height. The soil and debris were
carefully scraped away from around the crowns of these plants, exposing
a large number of developing buds and shoots. The inoculum was care-
fully packed around these crowns, the growing tops of which were finally
sprinkled and dusted with crushed galls. Sphagnum was packed over
and around the plants to a depth of 2 or 3 inches, water was sprayed
over the plot, and the sphagnum and soil beneath were kept thoroughly
wet for 10 days. On June 1 the material was removed from around the
plants, but no trace of any infection was discovered. Whether the rapid
growth which the plants were already making at the time when inocu-
lation was made prevented infection or whether some other circumstance
was responsible for the failure can not be told until further work is done.
From observations which were made in the field, it appears probable
that most of the warts which developed that spring resulted from infec-
tions which had taken place previous to the date at which the inocula-
tion was made. Thus it is possible that at the late date at which the
experiment was begun the spores of the fungus had in large part ceased
to germinate, or the plant itself might have passed its period of greatest
susceptibility.
Inoculations of plants in the greenhouse at Washington gave two
instances of successful infection. In one case a pot of seedling plants
about 6 inches tall were inoculated by replacing the dirt around the
crowns with crushed diseased tissue and debris from plants recently
received from California. Inoculation was made October 1, and on
January 3 three plants with very young infections were found.
Attempts to obtain infected plants by sowing seed in soil to which
crushed warts had been added usually resulted in the destruction of the
young plants by Rhizoctonia and possibly other fungi introduced with
the inoculum. In one case, however, among nine plants from seed
mixed with Urophlyctis spores and sown in April there were found in
the following January three infected plants, two of which were dwarfed
and much injured by the disease. If it were possible to obtain a large
percentage of plants in the field as badly infected as those in this experi-
ment, this disease would be capable of much harm. As a matter of
fact, however, only a relatively small percentage of young plants have
been found infected in the field even under what would appear to be the
most favorable conditions.
When germination of spores can be obtained with some degree of cer-
tainty or when field experiments under suitably controlled conditions
can be freely undertaken, opportunity will be open for further infection
studies that should add to our meager knowledge of the conditions
necessary for infection in the field.
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 319
CLIMATE IN RELATION TO THE DISEASE
The fact that the disease has apparently remained so long limited in
its distribution to certain regions in the western portion of the country
without invading the larger alfalfa-growing areas in the central portion
of the country raises the question whether this limitation is due to cer-
tain climatic conditions which favor the development of the fungus in
these localities or to some factors which have prevented the spread of the
causal organism. That the spread of the organism has been inhibited by
lack of facilities for distribution is hard to imagine. Even if it should be
found that the spores are incapable of withstanding the drying incident
to being transported with seed or hay, still a considerable number of
plants have been and still are transported by individuals for trial or
experimental purposes, and it is hard to believe that no warted plants
have been sent at some time into the central and eastern States. On the
other hand, it is not easy to discover any common factors of climate in
the regions where the disease now occurs which do not exist in the larger
eastern regions. For the most part, the disease exists in valleys where
the winter is very mild and where there is at least a slight growth of the
plant during every month of the year. Such conditions would seem to
furnish a long period favorable for infection. However, the disease also
occurs in the Salt Lake Valley in Utah and in certain high mountain
valleys where the winter is severe. The mere fact of severe winter does
not seem to be the sole limiting factor. Thus, it is not possible to answer
with an opinion based upon suitable evidence the most important ques-
tion from an economic point of view that is being asked regarding the
disease. Of course it might be determined decisively whether the dis-
ease can develop in the central and eastern portions of the country by
bringing diseased plants into these regions and observing their behavior.
Fear that such experiments might result in a destructive spread of the
disease has prevented the initiation of such experiments thus far.
CONTROL MEASURES
Thus far no experimental work bearing directly upon control meas-
ures has been undertaken. The direction which such experimental work
should take appears to be clearly indicated by the observation of the
field conditions under which the disease now becomes most abundant.
The one condition which more than any other appears to favor the
development of the disease is an excess of moisture in the soil in the
early spring when it appears that infection must take place if at all.
Any measure which will avert this excess, as by drainage or a dimin-
ished supply of irrigation water, should bring about a reduction in the
amount of disease.
Under some conditions deep cultivation may reduce the disease. In
the spring of 191 8 some fields which had received a thorough and deep
95080— 20 6
320 Journal of Agricultural Research vol. xx.no.
cultivation in February were observed to have less of the disease than
neighboring fields which had not been so treated. There was ample
evidence that the disease had been severe in these fields in the previous
season. However, in the following spring the difference between culti-
vated and uncultivated fields had disappeared.
There is a limited amount of field evidence that the amount of dis-
ease is increased when alfalfa is planted directly after alfalfa. Fortu-
nately, such succession is rarely practiced. Thus, on the whole, it can
be said that when conditions are made most favorable for the develop-
ment of the alfalfa plant the disease is diminished, perhaps not so much
because the plant is better able to withstand its attacks as because
abundant infection is dependent upon conditions which are not of them-
selves most favorable for plant development.
Search has been made in vain for any evidence of conspicuous cases
of apparent resistance to the disease. In one instance in 191 9 a plot
of alfalfa was found conspicuously freer from the disease than the adjoin-
ing plots which appeared to be under exactly the same conditions. It
was found that the seed used in this plot was from a different source
than that used in the other plots, and in fact the type of plant was
different. An effort to obtain seed from this field for experimental
work was frustrated by the ravages of grasshoppers. During the fol-
lowing year observation failed to discover any material difference in
the amount of disease in this field as compared with its neighbors, and
therefore efforts to obtain seed from it were abandoned.
It hardly need be said that until it is known for a certainty whether
the disease can be troublesome in the eastern alfalfa-growing regions,
care should be taken to prevent its introduction. At least living plants
from fields where the disease is known to occur should not be trans-
ported to other localities.
SUMMARY
The disease of alfalfa caused by the fungus Urophlyctis alfalfae, com-
monly known as crownwart, has been found to have its origin in the
infection of very young buds, the foliar elements of which develop into
abnormalities not involving the mature structures of root or stem.
Infection appears to take place only early in the spring, becoming
easily discoverable in the latter part of March or in early April in
northern California.
In irrigated regions, or in regions where there is abundant moisture
during the entire season, most of the galls reach full development early
in the summer and thereafter decay rapidly, only a few surviving until
the next spring.
The thallus of the fungus consists of two types of structures, turbinate
cells and resting spores. In the first turbinate cell that is the imme-
diate development of the infecting fungus are inserted a number of septa
Nov. is, 1920 Crownwart of Alfalfa Caused by Urophlyctis alfalfae 321
which delimit uninucleated peripheral segments from a polynucleated
central sporogenous mass. A hyphal structure of limited growth devel-
ops from each of these segments and carries the nucleus in its expanded
termination, the latter constituting a young turbinate cell of the next
succession. At its mature stage the turbinate cell bears a branched
apical haustorium, the short axial element of which proliferates at its
tip a globose terminal expansion into which the polynucleate sporogen
ous mass of protoplasm migrates to produce the resting spore. This
is characterized by 9 to 15 branched haustoria in zonate arrangement
between the equator and the distal pole.
. A solution of the thinner cell walls in proximity to the young, advancing
turbinate cells leads to the development of cavities in the hypertrophied
tissue in which the resting spores are finally found inclosed.
The abundant development of the disease in the regions where it now
occurs is apparently associated with excessive moisture during the period
when infection is taking place. Any measures which can be taken to
reduce the moisture near the surface of the soil at this time should reduce
the disease.
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(33) Tisdale, W. H.
1919. physoderma disease of corn. In Jour. Agr. Research, v. 16. no. 5,s
p. 137-154. pl- A-B, 10-17.
(34) Trabut, M. L.
1894. SUR UNE USTILAGIN'EE PARASITE DE LA BETTERAVE (OEDOMYCES
LEPROIDES). In Rev. Gen. Bot., t. 6, p. 409-410, pl. 16.
(35) VuillEmin, Paul.
1896. LE cladochytrium pulposum parasite des betteraves. In Bui.
Soc. Bot. France, t. 43 (s. 3, t. 3), p. 497-505.
(36)
1897. SUR L'APPARErL NOURRICIER DU CLADOCHYTRIUM PULPOSUM. In
Compt. Rend. Acad. Sci. [Paris], t. 124, no. 17, p. 905-907.
(37) Wilson, Orville T.
1915. the crown gall OF alfalfa. In Science, n. s. v. 41, no. 1065, p. 797.
(38)
1920. crown-gall of alfalfa. In Bot. Gaz., v. 70, no. 1, p. 51-68, pl. 7-10.
Literature cited, p. 65-66.
PLATE 47
Uroph lye t is a If a l/ae:
Drawing of alfalfa plant, showing abundance of crownwart, as found early in May,
19 19, in northern California. The dotted line gl represents the ground level. Varying
degrees of abnormality in the development of the buds are shown. X %i.
(324)
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plate 47
Journal of Agricultural Research
Vol. XX, No. 4
Crownwart of Alfalfa Caused by Urop.hlyctis alfalfae
Plate 48
Journal of Agricultural Research
Vol. XX, No. 4
PLATE 48
Urophlyctis alfaljae:
A-D. — Peripheral portions of actively growing thallus of parasite dissected from
living host: ta, tb, tc, turbinate cells of successive orders; ra, rb, resting spores pro-
duced by successive orders of turbinate cells; tbx, peripheral segments beginning to
proliferate turbinate cells by budding. Note the single apical haustorium on the
developing turbinate cells tc; its median position on the isthmus connecting turbinate
cell tb and developing resting spore rb; its absence from isthmus between evacuated
turbinate structure B, D, ta, and maturing resting spore B, D, ra. Note also zonate
arrangement of haustoria between equator and distil pole of resting spore, A-D, ra, rbs.
E. — Nearly mature resting spore viewed from distil side, showing 11 haustoria in
zonate arrangement.
F. — Mature resting spore viewed from distil pole, showing 13 pits that mark former
location of haustoria.
G. — Mature resting spore viewed in profile, showing pits in zonate arrangement and
light concavity on proximal side of spore.
Drawn with the aid of the camera lucida. Approxiately X 847.
PLATE 49
Urophlyctis alfalfae:
A. — Section of epidermal region of young foliar structures, showing young primary
turbinate cells ta-tg, the first products of infection, within epidermal cells. Note
attachment to cuticular wall by attenuated beak, increase in number of fungus nuclei
during growth of turbinate cells, and pathological enlargement of host nuclei hn,
in invaded cell, knx being normal host nucleus.
B. — Section of young foliar element, showing wall of invaded epidermal cell dis-
rupted and advance of secondary turbinate cells tbc-tbe into underlying tissue. One
of the other secondary turbinate cells, tbb, is forcing its way down along the host cell
wall, while another, tba, has been reflected toward the cuticular wall; ta, sporogenous
cell of primary turbinate structure.
C. — Section of turbinate cell, showing 3 evacuated peripheral segments pa-pc. A
nucleus is passing through the narrow isthmus connecting the nearly evacuated sporo-
genous cell with the resting spore, the elongated nucleole following the achromatin.
D. — Section of maturing resting spore, showing 8 nuclei and a central vacuole con-
taining 4 granules staining red.
E. — Section of mature resting spore, showing numerous red-staining granules in
center and 5 nuclei.
F. — Section of maturing resting spore, showing 1 1 normal nuclei and 4 enlarged nuclei
in center, the latter apparently degenerating.
Drawn with the aid of the camera lucida. X &60.
Crown wart of Alfalfa Caused by Urophlyctis alfalfae
Journal of Agricultural Research
Vol. XX, No. 4
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plate 50
Journal of Agricultural Research
Vol. XX, No. 4-
PLATE 50
Urophlyctis alfalfae:
Section of diseased bud scale of alfalfa, showing four coalescing cavities, in three of
which the large primary turbinate cells taa, tba, and tc may be distinguished; taa has
not started to proliferate any resting spore, while the resting spore produced by tba is
moderately young, although turbinate cells of later orders tab, tbc, and others have
produced resting spores further along in development. The thickening of the host cell
walls bounding the cavity and the enlargement of the host nuclei hn lying free within
the cavity are conspicuous. Note also the large dimensions of the nucleus in the
uninucleated turbinate cell tbbx and the relatively larger proportions of the nucleoles
in the nuclei of the resting spore rs. Drawn with the aid of the camera lucida. X 860.
PLATE 51
Urophlyctis alfalfae:
Section of diseased bud scale attacked by U. alfalfae, showing a group of eight well-
developed cavities a-h and their relation to the host tissue. Many of the cells adjacent
to the cavities have divided at unusual angles, giving the tissue a characteristic appear-
ance. In b the host cytoplasm and fungous material stain unusually deeply, as the
result perhaps of general infiltration with some diffusing substance. Drawn with
the aid of the camera lucida. Approximately X 417.
Crownwart of Alfalta Caused by Urophlyctis alfalfae
Plate 51
Journal of Agricultural Research
Vol. XX, No. 4-
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plate 52
Journal of Agricultural Research
Vol. XX, No. 4
PLATE 52
A, C, D. — Urophlyctis pluriannulatus . B. — Urophlyctis alfalfae.
A. — Portion of actively growing thallus of U. pluriannulatus dissected from gall on
leaf of Sanicula menziesii, including a turbinate cell ta with a nearly mature resting
spore ra; ta is completely evacuated, having produced 7 turbinate cells of the next
order, in one of which tba peripheral segments have been delimited, another, tbb,
has produced two turbinate cells of the tertiary order tea and tcb, as well as a developing
resting spore rb. Approximately X 847.
B. — Abnormally enlarged hyphae and turbinate cells of U. alfalfae, showing con-
spicuous thickening of the walls. X 860.
C. — Peripheral portion of actively growing thallus of U. pluriannulatus, similar to
A, showing 8 turbinate cells of the second order, of which 7 have produced turbinate
cells of the last order as well as resting spores. Approximately X 847.
D. — Nearly mature resting spore of U. pluriannulatus, viewed from polar end,
showing 22 haustoria in zonate arrangement. Approximately X 847.
Drawn with the aid of the camera lucida.
PLATE 53
Urophlyctis pluriannulatus:
Section of leaf of Sanicula menziesii, showing development of parasite within gall.
Some of the fungus thallus appears to have dropped out of the section in the course of
manipulations, as is indicated by the large unoccupied gaps; la, primary turbinate cell;
tb, tc, turbinate structures or cells of successive orders, the former completely evacu-
ated, the latter in early first or second nucleated stage; ra, rb, resting spores produced
by turbinate cells of successive orders: hn, host nuclei considerably enlarged as result
of influence of parasite. Note the similarity in development of parasite to U. alfalfae
and the relatively slight influence of parasitism on host anatomy. Drawn with the aid
of the camera lucida. X 860.
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plate 53
Journal of Agricultural Research
Vol. XX, No. 4
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
PLATE 54
Journal of Agricultural Research
Vol. XX, No. 4
PLATE 54
Crowns of alfalfa plants bearing galls caused by Urophlyctis alfalfae photographed at
different stages of development.
A. — A comparatively early stage of development at which the origin of the gall
structures from the elements of developing buds can be traced.
B. — A later stage of development at which the origin of the tissue has become
obscured. The tap root of this crown was cut off and the photograph taken from
below. Galls usually become considerably larger than this before they begin to
disintegrate if the plant continues vigorous growth.
PLATE 55
A comparatively early stage of host reaction to invasion by Urophlyctis alfalfae.
The cavities produced by the invading fungus can still be traced from the exterior
into the parenchymatous tissue. A few of the cells which are about to be entered
by the advancing fungus show some hypertrophy. The division of the cells beneath
the epidermis has begun.
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plate 55
Journal of Agricultural Research
Vol. XX, No. 4
Crownwart of Alfalfa Caused by Urophlyctis alfalfae
Plate 56
:^>:*:W'~
-.1.
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■BESS
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v> «
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Journal of Agricultural Research
Vol. XX, No. 4
PLATE 56
A. — Late stage of development of host reaction to the invasion of Urophlyctis
alfalfae. Infections have taken place near the tip of a growing point. The division
of exterior cells has gone on extensively. The cells around the older portions of the
cavity formed by the fungus have also been stimulated to division. The vascular
bundle at the center of the mass of tissue has begun to produce parenchyma toward
which the fungus is inclined to direct its course.
B. — Vertical section through a well-developed gall near its central axis, showing
its laminated structure arising from the thickening of bud elements. The cavities
containing the dark-colored spore masses are seen distributed through the tissue, a
condition that causes the mottled appearance of the interior of the living gall.
PATHOLOGICAL ANATOMY OF POTATO BLACKLEG
By Ernst F. Artschwager
Scientific Assistant, Office of Cotton, Truck, and Forage Crop Disease Investigations,
Bureau of Plant Industry, United States Department of Agriculture
Blackleg, as has been shown by the researches of numerous investi-
gators, notably Appel (2),1 Smith (7), and Morse (6), is a bacterial dis-
ease affecting the underground part of the potato stem and the tubers.
In its typical form the base of the potato stem shows a pronounced
blackening which may extend several inches above the ground. The
seed piece from which the diseased plants have been grown is found in a
state of decay or is already completely destroyed by rot. The external
symptoms are sufficiently striking to enable one to recognize diseased
plants even at a distance. Such plants are of a lighter color and usually
exhibit a xerophytic texture. They may be normal in size, but most
often they are dwarfed and stocky, so that the disease is easily mistaken
for leafroll. But while leafroll plants are firmly anchored in the ground,
blackleg stems are easily pulled and always show the characteristic
lesions on the underground part. The ease with which diseased plants
are removed is so striking that one unconciously looks for a contributing
mechanical cause, and that such a supposition is not altogether unfounded
is shown in a note by Heygi (4) who reports wire-worm injuries in almost
all the blackleg material that came under this observation. From the
results of his investigations, Hegyi is inclined to consider the presence of
the bacteria a secondary factor which has nothing to do with the original
cause. Yet while it is true that wire worms may cause a loss of many
potato hills, they are probably not responsible for the death of plants
suffering from blackleg.
The xerophytic texture of the diseased plants is exhibited by stems
and leaves alike. The foliage is discolored, usually light and of a metallic
luster. The leaflets are folded, and the petiole and midrib are woody
and lacking the elasticity and softness which characterize the normal
organs. Not all the stems of a diseased plant are necessarily affected.
Healthy sprouts may appear side by side with diseased ones (PI. 57, A),
and the diseased sprouts may exhibit various degrees of injury. Plants
which have been attacked rather early often continue to live for a con-
siderable period. These plants remain naturally dwarfed, the stalks
are spindling, the internodes shortened, and the leaves small. Plants
attacked at a more mature age may attain full size, though they usually
succumb more quickly to the attack of the parasite than do many of the
1 Reference is made by number (italic) to " Literature cited," p. 330.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C. N»v- *5. 1920
vq Key No. G-210
9508°— 20 7
(325)
326 Journal of Agricultural Research voi.xx,No.4
plants affected earlier in their life. In the last stages of the disease,
when the rot has progressed far enough to cut off completely the water
supply, the entire plant turns brown and sooner or later, depending on
weather conditions, falls prey to the attack of saprophytic bacteria and
fungi.
MATERIAL AND METHOD OF STUDY
The field from which the material for study was obtained is located in
a clearing of the river bottom land near the Fort Lewis Mesa, Colo. The
altitude is 7,500 feet. The soil is a sandy loam containing some organic
matter and a water table sufficiently high to insure the growing of a crop
without the customary irrigation. The tubers used were of Green
Mountain and Rural New Yorker types. They were cut before planting,
and because of the apparent soundness of the tubers no surface steriliza-
tion was attempted. The season was a normal one. The months of
May and June were characterized by excessive dryness. Throughout
July and August frequent showers insured a rapid growth of the plants.
During the first week of August a severe hailstorm injured the foliage
and stems so badly as to make further observations impracticable.
The first diseased plants appeared early in July. Their number
increased during the following two weeks and then showed a decline on
account of the death of a number of early infected plants and the reduc-
tion in number of new infections. Tubers of the same lot which had
been disinfected and grown on irrigated mesa soil remained free from
disease. The observations made at Fort Lewis were extended on mater-
ial obtained from other parts of the State, especially the San Louis
Valley. In every case the symptoms were similar, the only real differ-
ence being in the number of diseased plants per acre.
The plants taken for study were examined while fresh. For the pur-
pose of completing microchemical work and checking results, suitable
material was killed in Flemming's weaker solution and embedded in
paraffin in the usual way. The principal reagents used were Haiden-
hein's haematoxylin-safranin stain for histological structures, Devaux's
stain for pectic degeneration, phloroglucin-hydrochloric acid for lignifi-
cation, and Altmann's acid fuchsin stain for protein crystals.
While all previous investigations on the blackleg disease deal with the
morphology of the causal organism and its pathogenicity, this study
has for its object a consideration of the pathological changes concomitant
to the presence of the organism.
PATHOLOGICAL ANATOMY
In general, the histological changes consist in an increase of strongly
lignified vascular tissue and in a transformation of part or most of the
parenchyma cells of pith and cortex into sclereids (Pi. 58, B). Cyto-
logical abnormalities lie mainly in the occurrence of protein crystals in
the parenchyma cells of the leaves, the stems, and the tubers.
Nov. 15, 1920 Pathological Anatomy of Potato Blackleg t>27
The elements of the xylem are normal in size, though occasionally
they appear smaller. The lumen is reduced; the walls are thicker and
more strongly lignified. Even in unstained sections and without the mi-
croscope the xylem appears to be darkened. The discoloration often ex-
tends to the stem apex and into the petiole, but it is most pronounced in
the underground parts of the stem where the external symptoms are most
striking. Usually the cell wall alone is discolored, but sometimes a
brown, gummy deposit is found in the lumen of the cells, especially of
the larger vessels. In typical cases, only the primary xylem is affected;
in advanced stages, however, a part of the secondary xylem may also
show the browning of the walls. This discoloration of the elements of the
xylem is not necessarily a symptom limited to blackleg, since it is asso-
ciated with numerous other pathological disturbances and is commonly
observed in plants which are suffering from excess of water.
The phloem fibers are more abundant. They, too, show a general in-
crease in wall thickening and intensity of lignification. The secondary
wall often is so thick as completely to fill the lumen (Pi. 57, C; 58, A); it
is distinctly layered and contains numerous simple pits.
While one occasionally finds sclereids in the cortex of the under-
ground stem of the normal plant, there is nothing that could compare
with their relative abundance in plants suffering from blackleg. These
sclereids are typical parenchyma cells with strongly lignified secondary
walls (PI. 57, C; 58, A). They are either scattered or form solid masses
of tissue, often completely replacing the pith and part of the cortex.
The transformation of pith cells into sclereids is most pronounced in the
apical stem region and in the petiole. In the midrib and in the stem
region close to the base, where the browning of the xylem is most pro-
nounced, relatively few sclereids are found.
In the small parenchyma cells of the perimedullary zone similar changes
occur. The cells show at first pectic degeneration, which is followed by
lignification. The peripheral pith cells, especially in the interfascicular
region, are sometimes completely transformed so that they form a
sclerenchymatous sheath on the inner face of the vascular tissue.
The phloem elements are mostly normal at the base of the stem but
show increasingly advanced pathological changes toward the apex and
in the petiole. The cell walls are swollen, occasionally necrotic. The
cells of the pericycle undergo similar changes which are more severe and
are noticeable even in the lower stem regions.
Plants which are infected early but do not succumb to the attack of the
parasite very readily show the most typical and pronounced symptoms.
In plants in which the course of the disease has been a rapid one, relatively
few changes are exhibited. It will be understood, however, that indi-
vidual plants vary and that the environment, the age of the plant, and
its physiological constitution will in a large measure determine the degree
of anatomical changes in tissues and organs.
328 Journal of Agricultural Research voi.xx,No.4
The presence and activity of the blackleg organism results in a gradual
or rapid cutting off of the water supply from the roots and in a break in
the path of translocation for plastic materials in the lower stem region.
As a consequence of the decreased water supply, we have a decrease in
growth activities, especially a check in elongation. The newly formed
cells seem to mature more rapidly; in fact, mature and already strongly
lignified cells are found close to the growing apex. As long as the leaves
remain green and a minimum of water is insured synthesis of foods will
go on, though less extensively than in healthy plants. There is not,
however, an accumulation of starch as is commonly found in plants
suffering from leafroll, but there is a utilization of the food in the laying
down of extensive secondary thickenings in the cells of the xylem and
fibers and in a transformation of parenchyma cells into thick-walled
sclereids. Morse (6) reports that when the progress of the disease is
slow —
numerous aerial tubers will be formed on the stalks at the surface of the ground or in
the axils of the leaves above.
It would be of interest to know whether in such a case the same ana-
tomical changes occur which normally accompany blackleg.
Just as the formation of sclereids is the most pronounced histological
symptom, the appearance of protein crystals in all organs of the plant,
the leaves in particular, is a cytological phenomenon always associated
with the disease. Protein crystals have been found in the tubers of
normal plants. Bailey (1) reports the occurrence of cubical crystals in
the tubers of Solarium tuberosum. A few years later Cohn (5) by the
use of protein reactions identified the crystals of Bailey as belonging
to the typical group. Heinricher (5) observed that in potato plants
where the root system had been destroyed by decay the basal portions
of the plant contained cubical protein crystals which were especially
abundant in the cells of the phloem but were altogether absent from
the cells of the epidermis and the collenchyma. Crystals have not been
found in the aerial parts of the normal plant, and in the researches of
the writer on the anatomy of the potato plant and the pathological
anatomy of the leafroll disease they have not been observed. However,
crystals have been noted by Stock (8) in aerial, axillary tubers, where
they show the same distribution in peripheral cells of the cortex as do
normally developed underground tubers. Protein crystals occur in great
abundance in all organs of "blackleg" plants, especially in the leaves
(PI. 57, B; fig. 1). The crystals are usually cubical and vary in size from
minute bodies to large structures with a diameter of two-thirds the size
of a palisade cell. They are normally found in the cell sap or in the
cytoplasm, very rarely inside the nucleus, although nuclear crystals,
according to the extensive researches of Zimmermann (9), are not at all
uncommon.
Nov. is, 1920
Pathological Anatomy of Potato Blackleg
329
Nothing definite may be said in regard to the physiological importance
of these structures. Crystals have been observed in many plants, in
the fungi as well as the highly specialized angiosperms; but, while
certain groups of plants show them in great abundance, other plant
groups show just as conspicuous a
lack. Heinricher (5) believed that
the interception of the movement
of plastic material to the roots
causes a forcible deposition of the
protein in the basal parts of the
stem. This, however, could in
itself not account for their forma-
tion as has already been pointed
out by Stock (8), who observed
protein crystals in aerial tubers
but failed to find them in the stem,
although the cells in the latter are
completely filled with starch. The
crystals probably constitute transi-
tory food which may be used again
in the metabolism of the plant FlG. , —Section of potato leaf, showing distribution
and may accumulate when growth of protein crystals"
is inhibited unless an excess of photosynthetic products (as starch in
the case of leafroll plants) stops protein synthesis altogether.
SUMMARY
(1) Potato plants affected with blackleg show an increase in strongly
lignified vascular tissue and a transformation of part or most of the
parenchyma cells of cortex and pith into sclereids.
(2) Associated with blackleg is the occurrence of protein crystals,
especially in the cells of the leaves. Under normal conditions protein
crystals have been observed only in the peripheral cell layers of the
cortex of the potato tubers.
(3) Only diseased plants grown in the arid western parts of Colorado
have been studied. It is possible that plants grown in the eastern
United States and at a lower altitude do not exhibit the anatomical
changes reported in this paper.
330 Journal of Agricultural Research vol. xx.No. 4
LITERATURE CITED
(i) Amadei, Giuseppe.
1898. UEBER SPINDELFORMIGE EIWEISSKORPER IN DER EAMILIE DER BALSA-
mineen. In Bot. Centbl., Bd. 73, No. 1, p. 1-9; No. 2, p. 33-42, pi. 1-2.
(2) Appel, O.
1903. UNTERSUCHUNGEN UBER DIE SCHWARZBEINIGKElT UND UBER DIE DURCH
BAKTERIEN HERVORGERUFENE KNOLLENFAULE DER KARTOFFELN. In
Arb. Biol. Abt. K. Gsndhtsamt., Bd. 3, Heft 4, p. 365-432, 15 fig., pi. 8
(col.).
(3) Cohn, Ferdinand.
1859. UEBER PROTEinkrystallE IN DEN KARTOFFELN. In 37. Jahresber. Schles.
Gesell. Vaterland. Kidt., 1859, p. 72-82.
(4) Hegyi, D.
1910. EINiGE BEOBACHTUNGEN BETREFFS DER SCHWARZBEINIGKElT DER KART-
offel. In Ztschr. Pflanzenkrank., Bd. 20, Heft 2, p. 79-81.
(5) Heinricher, E.
1891. UBER MASSENHAFTES AUFTRETEN VON KRYSTALLOIDEN IN LAUBTRIEBEN
der kartoffelpflanze. In Ber. Deut. Bot. Gesell., Bd. 9, p. 287-291,
2 fig.
(6) Morse, W. J.
1917. STUDIES UPON THE BLACKLEG DISEASE OF THE POTATO, WITH SPECIAL
REFERENCE TO THE RELATIONSHIP OF THE CAUSAL ORGANISMS. hi
Jour. Agr. Research, v. 8, no. 3, p. 79-126. Literature cited, p. 124-126.
(7) Smith, Erwin F.
1905. bacteria in relation To plant DISEASES, v. i. Washington, D. C.
(Carnegie Inst. Washington Pub. 27, pt. 1.)
(8) Stock, Georg.
1892. EIN BEITRAG ZUR KENNTNISS DER PROTEINKRYSTALLE. In Beitr. Biol.
Pflanz., Bd. 6, Heft 2, p. 213-235, pi. 1 (col.).
(9) ZlMMERMANN, A.
1893. BEITRAGE ZUR MORPHOLOGIE UND PHYSIOLOGIE DER PFLANZENZELLE. V. I,
Heft 3, 6 pi. Tubingen.
PLATE 57
A. — Plant affected with blackleg. One stem is healthy, while the other is severely
diseased.
B. — Section of single upper epidermal cell of leaf and adjacent palisade cell. The
epidermal cell is filled with granular tanniferous material ; the palisade cell shows dis-
organized protoplasm, starch grains, and crystals. A small crystal is seen inside the
nucleus.
C. — Section of pith cell which is transformed into a sclereid adjacent to phloem
fibers. The walls of the latter are very thick and strongly lignified.
Pathological Anatomy of Potato Blackleg
Plate 57
Journal of Agricultural Research
Vol. XX, No. 4
Pathological Anatomy of Potato Blackleg
Plate 58
•^^v:
B
Journal of Agricultural Research
Vol. XX, No. 4
PLATE 58
A. — Pith cells of petiole transformed into sclereids with typically stratified walls.
B. — Vascular tissue of the petiole greatly increased by blackleg. A number of
sclereids are seen in the pith.
SCLEROTINIA MINOR, N. SP., THE CAUSE OF A DECAY
OF LETTUCE. CELERY, AND OTHER CROPS
By Ivan C. Jagger
Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of
Plant Industry, United States Department of Agriculture
Smith 1 (1900) recorded the occurrence of a fungus similar to Sclerotinia
libertiana Fuckel, which, however, produced much smaller sclerotia
(PI. 59, A) in greenhouses of Massachusetts, where it was causing a
destructive rot of lettuce. Duggar 2 (1909) states that a similar fungus
occurs on lettuce in the vicinity of both Boston and New York City.
In 191 1 the writer3 obtained what appeared to be the same fungus from
decayed lettuce grown in the vicinity of New York. It was collected
in 1 91 2 and again in 191 4 at South Lima in western New York, where it
seemed to be well established and was causing considerable injury to
lettuce grown on muck soil. In 1 914 it was also collected on lettuce in a
greenhouse at Rochester, N. Y., but the fungus was not again found in
that vicinity, although numerous collections of diseased lettuce were
made during the next three years. In the fall of 191 9 Dr. W. S. Beach
of the Pennsylvania Agricultural Experiment Station advised that the
fungus is frequently found on celery and lettuce in the vicinity of Phila-
delphia. During the winter season of 1919-20 the writer observed the
fungus in destructive amounts in a single field of lettuce at Sanford, Fla.
In numerous inspections of lettuce in that vicinity throughout the
season the fungus was observed in no other fields, although 5. libertiana
was more or less abundant in all fields. This suggests that the fungus
forming small sclerotia may have been recently introduced into that
section.
The fungus causes a very rapid decay and collapse of growing lettuce
plants. The disease produced is almost identical with that caused by
S. libertiana. A soft, watery decay may begin at any point on the plant
but usually on the lower leaves, which rest on the ground, or on the
stem near the ground. The rot spreads very rapidly, and usually the
main stem and bases of the leaves are soon involved. The result is a
rather sudden collapse of the whole plant. The plant is rapidly con-
verted to a soft, watery mass. When the decayed mass is pulled apart
the spaces between and around the decayed leaves and stem are found to
1 Smith, Ralph E. botrytis and sclerotinia: their relation to certain plant diseases and to
Each other. In Bot. Gaz., v. 29, no. 6, p. 369-407, pi. 25-27. 1900.
2 Duggar, Benjamin Minge. fungous diseases of plants ... p. 198. Boston, [1909].
3 Jagger, Ivan C the small LETTUCE sclerotinia, an undescribed species. (Abstract.) In Phyto-
pathology, v. 3, no. 1, p. 74. 1913.
Journal of Agricultural Research, Vol. XX, No. 4
Washington, D. C Nov. 15, 1920
vt Key No. G-211
(331)
332
Journal of Agricultural Research
Vol. XX, No. 4
be filled with white wefts of mycelium, which in a few days are replaced
by numerous small black sclerotia. General observations indicate that
the fungus possibly causes a rather more rapid decay and collapse of
plants than is caused by S. libertiana. The wefts of white mycelium in
decaying plants are less conspicuous, and the sclerotia are much smaller
and much more numerous than in plants attacked by 5. libertiana.
On several occasions bits of culture media covered with mycelium of
the fungus have been placed on growing lettuce plants. When moist
conditions have followed the inoculation, characteristic rapid decay has
invariably resulted. Prof. H. H. Whetzel has found that the fungus is
capable of attacking a large number of plants, data on which will be
Fig. i. — Camera lucida drawings of S. m,inor: A, microconidia and conodiophores; B, ascospores; C,
germinating ascospores; D, asci and paraphyses.
published in connection with his studies of the genera Sclerotinia and
Botrytis.
Strains of the fungus isolated from lettuce grown in the vicinity of
New York, Rochester, and South Lima, N. Y., Philadelphia, Pa., and
Sanford, Fla., have been grown in parallel cultures on various media
and have in every case appeared to be identical. Apothecia produced
by the three strains from New York State have shown neither macro-
scopic nor microscopic differences.
Apothecia (PI. 59, B, C) have several times developed from sclerotia
which had been allowed to age on unsterilized sand for from 4 to 12
months and which were then held under moist and well-lighted condi-
tions. Studies of fresh mature apothecia were made in 191 2, 191 4, and
1917 (fig. 1). Measurements of spores, asci, and paraphyses in the
Nov. is, i92o Sclerotinia minor, n. sp. 333
description are from the combined data of the three years, since the
three sets of data agree very closely. Microconidia (fig. 1) have appeared
in abundance on a medium consisting of a 2 per cent agar flour in dis-
tilled water. Cultures have been obtained repeatedly from single asco-
spores which have shown the apothecia to be the fruiting stage of the
sclerotia-producing fungus.
Smith (1900) 1 in studies of this fungus was unable to obtain apothe-
cia, although apothecia of S. libertiana were obtained in abundance. In
hundreds of cultures the fungus developed only the characteristic small
sclerotia, but in a single culture the small sclerotia at first appeared, and
later the characteristic large sclerotia of 5. libertiana appeared among
the small ones. Smith believed that 5. libertiana developed directly
from the small sclerotia and, therefore, concluded that the fungus is —
a degenerate form of S. libertiana which has almost entirely lost the ability to repro
duce by spores.
The repeated development during several years of characteristic
apothecia and the fact that during 10 years numerous cultures of sev--
eral strains of the fungus have shown very uniform characteristics seem
sufficient grounds for considering the fungus a distinct species. As it
seems to agree with no described species, the following description is
given.
Sclerotinia minor, n. sp.
Apothecia one, rarely more, from a single sclerotium; disc saucer-shaped, 0.5 to
2 mm. in diameter; stalk cylindrical, slender, flexuous, attenuated downward, 5 to
12 mm. long; asci cylindrical to cylindro-clavate, 125 to 175 n by 8 to 11 m. average of
30 measurements 141 by 8.9 n; spores 8, ellipsoid to ovoid, hyaline, 5 to 8.8 /t by 8.3
to 19.9 n, average size of 200 spores 7 by 14. 1 n with over 80 per cent 6 to 8 ^ by 12 to
it n; paraph yses filiform to cylindro-clavate, septate, rarely branched, same length
as asci, 3 to 4 n in diameter; microconidia globose, hyaline, 3 to 4.2^, borne apically
on short obclavate conidiophores; appressoria abundant; sclerotia black, irregular,
0.5 to 2 mm. in diameter, often anastomosing to form irregular flattened bodies several
millimeters in length.
Parasitic on lettuce (Lactuca sativa L.), celery (Apium graveokns L.), and other
plants; distribution, Massachusetts, New York, Pennsylvania, and Florida.
SUMMARY
Sclerotinia minor, n. sp., produces a decay of lettuce and other plants
similar to that produced by 5. libertiana. It is known to occur in Massa-
chusetts, New York, Pennsylvania, and Florida.
1 Smith, Ralph E. op cit.
PLATE 59
A. — Sclerotia on hard potato agar: center, Sclerotinia libertiana, either end, S.
minor.
B. — Apothecia of S. libertiana.
C. — Apothecia of S. minor.
Note relative size of apothecia in B and C.
(334)
Sclerotinia minor, n. sp.
Plate 59
™m&^
Journal of Agricultural Research
Vol. XX, No. 4
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V
Vol. XX DECEMBER 1; 1920 Uo. 5
JOURNAL OP
AGRICULTURAL,
RESEARCH
CONTENTS
Page
Permanence of Differences in the Plots of an Experi-
mental Field - . - - - - - - - 335
J. ARTHUR HARRIS and C. S. SCOFIELD
(Contribution from Bureau of Plant Industry)
Some Changes in Florida Grapefruit in Storage - - 357
LON A. HAWKINS and J. R. MAGNESS
(Contribution from Bureau of Plant Industry)
A Bacteriological Study of Canned Ripe Olives - - 375
STEWART A. KOSER
( Contribution from Bureau of Chemistry )
Relation of the Soil Solution to the Soil Extract - - 381
D. R. HOAGLAND, J. C. MARTIN, and G. R. STEWART
(Contribution from California Agricultural Experiment Station )
Effect of Season and Crop Growth on the Physical State
of the Soil - -397
D. R. HOAGLAND and J. C. MARTIN
(Contribution from California Agricultural Experiment Station)
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WASHINQTOM : GOVERNMENT PRINTING OFFIOE : IMO
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania Stale College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
•
JOURNAL OP AGRIOJITIAL RESEARCH
Vol. XX Washington, D. C, December i, 1920 No. 5
PERMANENCE OF DIFFERENCES IN THE PLOTS OF AN
EXPERIMENTAL FIELD
By J. Arthur Harris, Investigator, Station for Experimental Evolution, Cold Spring
Harbor, N. Y ., and Collaborator, Office of Western Irrigation Agriculture, and C. S.
SCOFIELD, Agriculturist in Charge, Office of Western Irrigation Agriculture, Bureau
of Plant Industry, United States Department of Agriculture
I.— INTRODUCTION
Agronomists have long recognized the fact that the plots of an experi-
mental field may differ considerably among themselves. This varia-
bility is the source of the greatest difficulty in the interpretation of
comparative cultures. A recent analysis (j)1 of the available data by
adequate biometric formulae (7) has shown that heterogeneity is a
practically universal characteristic of experimental fields and that it
must be considered in the interpretation of the results of all plot tests.
With the demonstration of this characteristic of experimental areas
the questions naturally arise: Are the differences between plots tran-
sient or are they relatively permanent from year to year? Do these
differences tend to increase or to decrease with cultivation ?
Presumably the differences which obtain in the soil of an experi-
mental field are in part permanent and in part transient. Lyon (5)
suggested that tillage and other factors will change the plots so that the
results will not be comparable from year to year. Unfortunately he
does not present data to show to what extent this may be true. He
gives a series of yields for successive years on the same plots, which
measured 33 by 66 feet or V20 °f an acre in area, at the Nebraska Agri-
cultural Experiment Station and shows that the rank of the yield of
these plots differs greatly from year to year. Thus he concludes that if
they differ among themselves in their capacity for crop production this
difference is very little constant from year to year.
Smith (6) took advantage of the breaking up of a piece of land which
had lain 16 years in pasture to investigate the effect of cultivation on
the uniformity of a series of plots. Any influence of 1 or 2 years pre-
ceding cultures on the variation or correlation of yields should, he
assumed, be apparent in the statistical constants deduced from these
■ — — ■ — — ■
1 Reference is made by number (italic) to " Literature cited," p. 356.
Journal of Agricultural Research, Vol. XX, No. 5
Washington, D. C. Dec. 1, 1920
vr Key No. G-313
2: (335)
336 Journal of Agricultural Research vol. xx.no. s
data. He gives a table which indicates that there is such a change.
He says:
It is noticeable that the variability as measured by the standard deviation becomes
less in each succeeding year. This suggests the question as to whether continued
cropping might not tend to induce uniformity. The records of a few of these plots
which were continued in corn for three years longer do not support such a conclusion.
It must be noted that in Smith's experiments seasonal conditions
varied greatly from year to year. Thus 1895, which was exceedingly
dry and also cool in the early part of the season, was highly unfavor-
able. The two following years were unusually favorable for corn. As
a result the yields were, respectively, 31.6, 91.6, and 71.4 bushels per
acre in the three years.
Lehmann in his work at the experimental farm near Bangalore at-
tempted to use the experience of previous years in the standardization of
experimental plots. His data will be considered in some detail below.
II.— METHODS AND RESULTS
The permanency of the differentiation of plots in their capacity for
crop production may be measured in terms of correlation. If the plots
of a field differ among themselves in a more or less permanent way
there will, with reasonably uniform climatic conditions, be a correla-
tion between the yields of the plots of a series in two or more successive
years — in short, an intera nnual correlation (2).
The problem of the correlations between the yields of identical plots
in different years is one of very great interest. If this correlation be
high it should be possible to standardize a field of plots by one or more
sowings to the same variety. A chief difficulty in the standardization
of the field by the carrying out of a preliminary test in which the pro-
ductive capacities of the plots are determined once and for all lies in
the fact that the factors which determine yield are in part edaphic —
that is, pertaining to soil conditions — and in part meteorological. For
example, in a very dry year sections of a field which are lower may pro-
duce the heaviest crops because adequate moisture is longer retained
in these places. In a wet year the case may be just the reverse, for the
crops in the lower-lying portions may be too wet for the best plant
growth. Thus, it is quite possible that in cases in which there is a
profound influence of environmental factors there may be a negative
correlation between the yield of the same plots in different years.
It is conceivable, therefore, that the interannual correlation for yield
per plot may range from negative to positive values, zero correlation
being found in cases in which edaphic and meteorological factors exactly
counterbalance each other in their influence upon the yield of the plots
of a heterogeneous field.
Dec. i, 1920 Permanence of Differences in Experimental Plots 337
A. — PUBLISHED DATA
Unfortunately few data are available for analysis from the literature.
Lehmann has given (4, p. 6) yields of paddy on the 17 plots of ranges
B and C, respectively, of the wet tract of the Experimental Farm at
Hebbel. Grouping the yields for the two ranges, we find for the corre-
lations between the yields of the same plots in the two years 1905 and
1906:
Range B, r = 0.834 ±0.050, r/Er= 16.7.
Range C, f = 0.799 ±0.059, r/Er= 13.5.
Stockberger (7) gives data for the extremes of a series of hill yields
for hops. The interannual correlations deduced from these data have
been shown (2) to be as follows:
Vwrs Lowest Highest
hills.
1909 and 1910 " o. 29±o. 17 o. 59±o. 13
i9ioandi9n 55± . I3 . $2± . I4
1909 and 1911 43± .15 . 30± . jg
Stockberger has also given (8) the yields for 30 rows, each 210 feet"
in length, from hop fields of several hundreds of acres in the Sacramento
Valley of California :
The plants in these rows averaged well in number and uniformity of growth with
the plants on several hundreds of acres of hops in the midst of which the experimental
area was located . •
Data are available for the years 1909 to 1914. Calculating the corre-
lation between the yields in the different years, we have the results
set forth in Table I. It appears that with one single exception the con-
stants are positive throughout. In general they are significant in com-
parison with their probable errors, indicating a superiority in a subsequent
year if. a superiority is shown in a given year.
The constants in the table are arranged in a way to show the change
in the coefficient of correlation as the years become more widely separated
in time. Thus, in the case of the correlation for the 1909 yields, the
constant for "first and second" is that showing the relationship between
the 1909 and 1910 yields, while "first and third" indicates the constant
measuring the relationship between the yields of 1909 and 191 1. Simi-
larly, in the series of coefficients for 1910 "first and second" designates
the correlation between 1910 and 191 1, etc.
For the series beginning with 1909 we note a marked decrease in the
magnitude of the constants as the yields correlated become more widely
separated in time. The same is true for the series beginning with 1910.
The other series are more irregular.
338
Journal of Agricultural Research
Vol. XX, No. 5
Table I. — Interanmial correlations for yield of hops
Beginning of series.
1909
1910
1911
1912
1913
First and
second years.
First and
third years.
First and
fourth years.
+0. 768±o-05i j +o-622±o.o75 . +o-38o±o. 105
+ • 577± -082 j + -447± • °99 + -45'i -098
+ .o62± .123 j + -3I3± -in — .I26± .121
+ -3ii± .111 + .7°5± -062
+ • S97± • 079
First and
fifth years.
+0. 259±o. 115
+ -274± .114
First and
sixth years.
+o.o6i±o. 123
The most reasonable explanation of the higher correlation of more
closely associated years is that both field conditions and the productive-
ness of the individual vines change more or less as time goes on. The
result of such changes would be a lower correlation between the yields
of periods more widely separated in time.
The data for the dry-land experiments in Mysore State have been
discussed elsewhere (j) in relation to the problem of field hetero-
geneity. It was shown there that in two dry years the field showed
marked hetereogeneity, but that in one unusually wet season there was
marked abnormality of yield with little correlation between the yields of
adjacent plots.
It seems of unusual interest, therefore, to determine to what extent
the differences between these plots are permanent from year to year.
Correlating between the yields of ragi, we find the following correlation
coefficients for the whole series of 105 plots for which data are available.
Grain.
1905 and 1906 o. 591 ±0. 043
1905 and 1907 ! . 693 ± . 034
1906 and 1907 . 4501b • 052
o. 777 ±0. 026
■ 85S± .018
.678± .036
Total.
o. 757 ±0. 028
.852± .018
. 6io± .041
The correlations are of very substantial order, and without exception
they are clearly significant in comparison with their probable errors.
They show that the differences in the plots are to a high degree per-
sistent during the three years of this experiment.
For grain, straw, and total yield the correlations between the yield for
1905 and 1907 are higher than those for 1905 and 1906 or for 1906 and
1907. If there were a progressive change in the field one might have
expected that the correlations would be higher between consecutive
years. Apparently the influence of the abnormal conditions of 1906 has
been to lower the correlations for this year.
The results show that the capacity for production is to a high degree
persistent from year to year, notwithstanding great diversity in
meteorological conditions.
A series of records of unusual interest is provided by Smith (6) for
yields of corn in three successive years, 1895, 1896, 1897. It has been
Dec. i, 1920 Permanence of Differences in Experimental Plots
339
shown elsewhere (3) that this field, which had lain in grass for many
years, is highly heterogeneous, showing correlations between adjacent
plots of r = 0.61 to ^ = 0.83.
The conditions for corn production differed very greatly in the three
years. Thus the constants for yield were :
1895
1896
1897
Mean.
31-7
91. 6
71.4
Standard
deviation.
7.91
IO. 64
6. 27
Coefficient
of varia-
tion.
25.0
11. 6
8.8
Yield is over twice as heavy in the second and third years as in the
first. The variability in yield as measured by the coefficient of variation
is far lower in the second and third years than in the first.
Computing the correlations between the yields for the three years,
we have the following results:
For 1895 and 1896, r= -o.354io.054, r/Er=6.6.
For 1895 and 1897, r= — -0.221 + 0.059, r/Er=3-&-
For 1896 and 1897, r= +0.818 ±0.020, r/£r=4o.i.
There is a negative correlation for 1895 and 1896 and for 1895 and
1897 but a high positive correlation for 1896 and 1897. Thus the plots
which were better in the highly unfavorable year 1895 were poorer in
the two favorable years 1896 and 1897. Plots which were better in the
favorable year 1896 were also better in the favorable year 1897.
B. — THE HUNTLEY UNIFORM CROPPING EXPERIMENT
The most extensive series of records available is that for a uniform
cropping experiment conducted for the past several years at the Field
Station of the Office of Western Irrigation Agriculture, at Huntley, Mont.
The Huntley field lies in the Yellowstone Valley on land having a very
slight and uniform slope to the north. The detailed history of the field
prior to 19 10 is not known definitely. It was probably first broken
from the native prairie sod in the spring of 1908. In 1909 it was planted
to sugar beets, but the crop was destroyed in the late summer. It came
under experimental control in 1910, when the major portion of it was
sown to oats and yielded a crop of 66 bushels per acre. In that season
a small tract in the northeast corner of the field was used as a machinery
park or stack yard and was not put into crop. This tract occupied
about two-thirds of the length of the first five plots in series II. It is
possible that this difference of treatment in 1910 may have been reflected
in the crop yields of 191 1 , but it seems hardly probable that any material
effects could have persisted longer.
34-0 Journal of Agricultural Research vol. xx, No. 5
In the spring of 191 1 this field was laid out into 46 plots, each measur-
ing 23K by 317 feet and containing 0.17 acre, arranged in two parallel
series of 23 plots each. The two series of plots were separated merely
by a temporary irrigation ditch. In 191 1 it was planted to sugar beets,
and in the spring of 191 2 it was seeded to alfalfa, and one cutting was
harvested that year. This stand remained on the ground during 191 3
and 1914, when the entire field was fall-plowed. In 1913 three cuttings
were made, but the third cutting was lost in a heavy wind which scat-
tered and mixed the crop before weighings from the various plots could
be made. The first cutting, designated as alfalfa I, was made on plots
one-half the original size. The second cutting was harvested from
plots one-quarter the original size. The first and second cuttings in
1914 were weighed for plots one-quarter the original size — that is, 0.0425-
acre plots — while the third cutting was recorded for plots one-third the
original size. These furnish the data for alfalfa I, II, and III for 1914.
Total yields for the first and second cuttings in 1913 and 1914 and for
the first, second, and third cuttings in 1914 are also considered.
In 191 5 and 191 6 ear corn was grown. In 191 7 1 the fields were
planted to oats, and records were made of grain, straw, and total yield.
In 1 91 8 silage corn was grown. In 1919 the land produced a crop of
barley.
It has been the practice each season to treat the whole field as a unit
until harvest time, when the plot boundaries are established in order to
measure the crop yields. In other words, all cultural operations, includ-
ing irrigation, are carried out on a field scale and uniformly throughout
the field. No manuring has so far been attempted. An effort has been
made to avoid any artificial causes of heterogeneity.
The crop yields each year have been satisfactory — that is, they have
not been abnormal — as is shown in Table II, where the mean yields per
plot and per acre are set down. Fortunately, this experiment has also
escaped injury from insect pests, plant diseases, and storms, which so
often interfere with the success of long-term field experimentation.
1 Because of other activities the plots could not be harvested in halves and quarters in 1917-1919.
Dec. i, 1920 Permanence of Differences in Experimental Plots 341
Table II. — Mean yields of Die Huntley uniform cropping experiment
Crop.
191 1, sugar beets
19 12, total alfalfa. . . .
1913, alfalfa I
1913, alfalfa II
19 13, alfalfa I and II
1914, alfalfa I
1914, alfalfa II
19 14, alfalfa I and II
1914, alfalfa III
1914, alfalfa I to III .
1915, ear corn
1916, ear corn
1917, oat grain
1917, oat straw
1917, total yield . . . .
1918, silage corn
1919, barley grain
1919, barley straw. .
19 19, total yield. . . .
Number of
pounds
per plot.
4, 179-
356-
54i-
483.
1, 024.
489.
499.
988
47i
1, 460.
522.
396.
555'
521
i>°77
3.175'
358.
230.
Number of
tons or bushels
per acre.
12. 29
1. 04
!-59
1.
4-
52-
41.
102.
3. 10
9-34
43-8
.67
i-73
The data furnished by this series of records are of particular value, since
(a) they are based on irrigated plots and (b) it is possible to compare the
correlations between the same crop and different crops in the different
years. t
The correlations between the yields of the various crops in the different
years may be considered in three series.
(1) The first comprises the yields for the whole plots. In this series
we determine the correlation between the crop produced on the 46 plots
in one year and that produced on the same 46 plots in another year.
(2) In the study of certain crops the plots were divided into two sub-
plots, and we may determine the relationship between yield of individual
subplots in different years. Then the number of observations is twice
what it was in the preceding correlation, that is, N = 92 instead of 46.
(3) Finally, in a more limited series of cases the 46 original plots were
harvested in 4 subplots each, thus increasing the number of units which
may be entered in the correlation tables to 184.
The data for determining the correlations between yields of various
crops for the 46 whole plots are given in Table III. The data for half
plots and quarter plots may be obtained from the diagrams in an earlier
paper by Harris (3) on the practical universality of field heterogeneity
as a factor affecting plot yields. The correlation coefficients and their
probable errors for whole plots are shown in Table IV.
342
Journal of Agricultural Research
Vol. XX, No. 5
Table III. — Yield of plots of field B at the Huntley (Mont.) Field Station «
Plot No.
II, xv.
2. . ,
3--
4...
5--
6...
7...
8..,
9-.
io. .
II. .
12. .
I3-.
I4-.
IS..
16...
17...
18...
19...
20. . .
31.". .
22. . .
23...
Ill, X*.
2. . .
3-..
4...
5---
6...
7...
8...
9...
10. . ,
11. .
12. .
13- •
14..
IS- ■
16..
I7-.
18..
19..
20. .
21. .
22. .
23 ••
1911,
suear
beets.
1912,
total
alfalfa.
1913.
alfalfa
I.
1913.
alfalfa
II.
12. 78
260
S95
600
12. 70
39S
530
560
10. 04
397
640
630
10.35
43 S
640
650
9-33
442
625
540
9- 40
419
62s
59S
II- S3
438
640
575
12. 40
410
555.
57o
IO.28
418
570
470
II. 8l
393
S4Q
47o
13-99
405
585
435
12.28
435
530
450
II. 91
385
S65
485
11.42
395
555
5io
12.28
40s
655
S6S
13- 76
305
650
475
II- 73
312
590
43 5
12.49
290
635
425
15-55
3IS
635
455
H-93
3IO
605
440
13-52
330
625
455
14.36
32S
625
500
16.81
3IO
590
425
13- 93
405
53 S
42S
13-04
3SO
47o
430
10. 55
400
5io
405
H.63
435
475
425
10. 56
350
460
445
10. 00
36S
510
5io
10.54
390
500
440
10.00
32S
455
425
8.85
360
490
375
10.48
360
440
415
12. 61
335
485
390
II. 22
350
470
400
12.08
370
500
450
11. 91
25S
470
485
12.65
3/0
485
455
11. 71
32S
460
440
12. 19
280
460
445
12. 62
280
430
500
13-45
320
480
515
15.60
2/S
520
565
16. 25
29c
460
5io
14. 70
345
530
535
16. 52
337
505
530
191^.
alfalfa
I and
II.
1, i9S
1,090
1,270
1, 290
1,165
1,220
1,215
1,125
1,040
1,010
1,020
980
1,050
1,065
1,220
1,125
1,025
1,060
1,090
1,045
1,080
1,125
1,01s
960
900
9iS
900
905
1,020
940
880
865
85s
875
870
950
955
940
900
905
930
995
I, 085
970
1.065
1.03S
1914,
alfalfa
I.
1914,
alfalfa
II.
585
550
610
605
605
690
640
660
59o
700
645
73 5
625
775
555
725
590
615
545
505
580
430
555
425
465
44S
S40
480
535
5IS
545
440
540
435
540
52S
54S
490
540
50S
580
53S
610
525
490
445
420
470
430
395
395
435
440
450
435
420
430
375
410
400
415
380
425
410
365
390
360
420
360
430
390
SOS
370
495
380
470
360
455
395
425
395
425
450
515
435
480
435
4S0
475
SIS
475
475
1914.
alfalfa
I and
II.
1914,
alfalfa
III.
1,135
1,215
1)295
1,300
1,290
1,380
1,400
1,280
1, 205
1,050
1,010
980
910
1,020
1,030
985
975
1, 065
1,035
1,04s
1, "S
I, I3S
935
890
82s
830
890
855
80s
810
795
83S
755
780
790
895
86S
850
8iS
820
820
965
915
915
990
950
580
60s
S9S
610
595
510
500
500
475
450
400
445
455
485
540
475
460
45S
465
475
5io
520
380
645
520
495
440
455
415
44S
415
385
360
385
37o
43 5
425
455
410
430
38s
475
445
465
495
475
1914.
alfalfa
I to
III.
i,7is
1,820
1,890
1,910
1, 885
1,890
1,900
1,780
1,680
1,500
1,410
1,425
1,365
1-505
i- 59o
1,460
1-435
i- 520
1,500
1,520
1,625
1,655
I-3I5
1,535
1-345
1.325
1.330
1,310
I, 220
1,255
1,210
I, 220
1,115
I. 165
I, 160
I • 330
I. 29O
I-305
1,225
1,250
1,205
I,440
I • 36o
I.380
1,485
1,425
0 All yields are given in pounds per plot with the exception of that for sugar beets, which is given in tons
jeracre.
Dec. i, 1920 Permanence of Differences in Experimental Plots 343
Table III. — Yield of plots of field B at the Huntley (Mont.) Field Station a — Con.
Plot No.
1915.
ear
corn.
II, 1.
Ill, 1.
2.
3-
4-
5-
6.
7-
SS6
598
526
S58
5°9
521
499
502
515
513
S24
507
S28
5°7
5"
324
520
479
455
489
519
573
578
545
552
5°4
547
544
533
505
519
513
5°9
493
496
5°3
496
518
499
483
469
477
490
55i
628
654
1916,
ear
corn.
513
514
481
495
487
45°
489
441
434
415
399
379
376
372
398
409
389
408
404
383
455
413
414
404
376
337
318
338
312
3"
345
353
337
322
357
343
333
360
372
353
367
410
407
426
423
401
1917,
oat
grain.
580
593
606
598
614
596
572
574
553
614
574
S48
537
540
518
564
499
538
637
579
567
553
509
S63
560
5ii
523
532
536
538
552
515
521
473
520
645
525
557
578
549
563
562
56l
486
573
561
1917,
oat
straw.
574
631
588
414
59°
584
458
524
495
606
578
510
523
522
616
57°
481
518
605
497
513
477
391
547
522
511
497
516
552
544
556
535
545
479
462
377
469
485
504
515
517
512
4S1
456
571
573
1917.
total
yield.
1918,
silage
corn.
I,
154
3,65S
I
224
3,285
I
194
3,290
I
012
3,390
I
204
3,570
I
180
3,240
I
030
3,005
I
098
3,010
I
048
3,060
I
220
2,885
I
152
2.955
I
058
3.055
I
060
3,125
I
062
3.210
I
134
3-155
I
.134
2.870
980
2,950
I
.056
3,235
I
1 242
3.330
I
.076
3.150
I
,080
3.180
I
.030
3-075
900
3-375
I
, no
3.68s
I
,082
3.36s
I
.022
3.315
I
,020
3-170
I
,048
3.240
I
,088
3.290
I
.082
2.855
I
,108
2.905
I
,050
2,965
I
,066
2. 760
952
2,640
982
2,850
I
,022
2,880
994
3,190
I
.042
3,100
1
,082
2.975
1
.064
2,995
i,c8o
1,074
1.042
942
1,144
1. 134
1919.
barley
grain.
3.315
3-540
3,280
3.37o
3.625
3.705
392
349
377
352
414
426
463
424
425
422
386
36s
350
368
344
3SI
333
309
313
304
316
306
288
332
362
375
342
416
460
410
400
400
386
403
305
296
290
301
335
317
320
293
323
331
362
341
1919,
barley
straw.
288
251
253
218
246
264
262
276
265
298
224
240
220
222
191
204
127
241
177
221
229
199
257
238
218
260
183 J
284 I
250
330
260
260
274
262
255
199
130
174
185
188
190
187
177
259
218
249
I9'9.
total
yield.
680
600
630
570
660
690
725
700
690
720
610
605
57o
590
535
5S5
460
550
490
525
545
505
545
570
58c
635
710
740
660
660
660
665
560
495
420
475
520
505
580
400
590
580
« All yields are given in pounds per plot with the exception of that for sugar beets, which is given in
tons per acre.
3/]/] Journal of Agricultural Research volxx.no. 5
From the series of correlations as a whole it appears that of the 152
coefficients showing the relationship between crop yields in different
years, 133 are positive while only 19 are negative in sign. If the differ-
ences in capacity for crop production demonstrated in different years
were due to purely transient causes, one would expect to find an approxi-
mately equal number of positive and negative correlations with the gen-
eral average value sensibly zero. Instead we find the proportion of 133
to 1 9. This is a deviation from the ratio 76 to 76, which one might ex-
pect on the assumption that there is no correlation between the yields
of plots in a series of years, of
57±°-6745VI52X5Xo.5 = 57±4.i6.
The deviation from equality is 13.7 times as large as its probable error
and is unquestionably significant.
If we consider that coefficients which are 2.5 times or more as large as
their probable errors represent statistically significant interrelationships,
we find that of the 82 relationships which may be regarded as falling in
this class 78 are positive whereas only 4 are negative in sign.
Averaging the values of the coefficients considered in Table IV, we
note that the average for the 133 positive values is + 0.3346, whereas that
for the 19 negative values is — 0.1475. Taking the constants altogether,
the average value is + 0.2743.
There is, therefore, an overwhelming body of evidence to show that
plots, even of the small size and the apparent uniformity of those of
the Huntley Station, which yield higher in one year will yield higher
persistently throughout a series of years.
It is now desirable to determine whether the same relationships hold
when these plots are divided into smaller subplots. It is possible to
subdivide a number of the plots into 2 subplots, each one-half the original
size. Correlations may be determined for the 92 yields of these half
plots in the same manner as for the total yields on the 46 original plots.
The results appear in Table V.
The constants are positive throughout. In general, they are statis-
tically significant in comparison with their probable errors. As a matter
of fact, only 2 of the 22 constants are less than twice as large as their
probable errors. Thus, they indicate a real biological relationship
between the productions of the half plots in different years. Those
which give a higher yield in one year give a higher yield in another year.
For a smaller number of the crops it is possible to divide the original
plots into quarter plots, thus securing 1 84 subplots to be used as a basis of
calculation. The coefficients of correlation between the yields in the
several years are shown in Table VI.
1917.
oat straw.
1917.
total oats.
1918,
silage orn.
1919.
barley grain.
1919.
barley straw.
L
•ain.
1919.
total barley.
—0. n6±o. 098
— 1. 18
—0. 098 ±0. 098
— 1. 00
+0. 348 ±0. 087
+4.00
—0. 539 ±0. 070
-7.66
— 0. 262 ±0. 092
— 2.82
to. 099
1911, sugar beeti24
— 0. 449 ±0. 079
-5.66
;±-095
1912, alfalfa. ... 1. 26
+. i66±. 097
+ 1. 71
+ . 229±- 094
+ 2-44
— .o7i±. 099
— 0. 72
+ . 527±. 071
+ 7-33
+ -34i±.o87
+3-89
+ -483±-076
+6-34
,±.087
1913, alfalfa I. ... n
+ . 190J;. 096
+ 1.98
+. 3i7±.o89
+3-56
+ . I5i±.097
+ 1.56
+.076±. 098
+ .78
— .003d:. 099
—•03
+.043 + . 099
+ •43
,±.080
1913, alfalfa II . , 54
+. 2o8±. 095
+ 2. 19
+-372±-o86
+4-33
. +. 45i±. 079
+ 5- 7i
+.203 ±.095
+ 2. 13
+ . 025±. 099
+.26
+ . 131 ±.097
+ 1-34
.±.078
1 913, alfalfa I a». 01
+ . 233±.094
+ 2.48
+-404±. 083
+4.87
+. 35°±-o87
+4. 02
+. 163 ±. 096
+ 1.68
+. 012 ±. 099
+•13
+. ioi±.098
+ 1.03
(±.080
1914, alfalfa I. .J. 60
+• 28i±.092
+3- OS
+. 429±. 081
+ 4.29
+. 209 ±. 095
+ 2. 20
+■ 255 ±.092
+ 2- 75
+ . I39±-°97
+ 1-43
+ . 22I±.094
+ 2-33
>±-074
1914, alfalfa II. k. 88
+.079 ±.099
+.80
+.308 ±.090
+ 3-42
+ . 237±.Q94
+ 2. 52
+ . 268±. 092
+ 2. 90
+ . I43±-Q97
+ I-47
+ . 230±.094
+2-44
)±-o73
1914, alfalfa I up. n
+. i88±.<396
+ 1.96
+ -395±-o84
+4. 70
+ . 242 ±. 094
+ 2- 57
+ . 283±.09i
+ 3- 10
+ . I53±.097
+ 1- 57
+. 244±.093
+2.61
> ± . 080
1914, alfalfa ITI{. 42
+ . 3ii±. 098
+3.46
+.446±. 079
+ 5- 60
+ . 579±- 066
+ 8-77
+.o86±.098
+.87
+ . 066 ± . 099
+ .68
+ .o84±.098
+.86
t±.07i
1914, alfalfa I tq. 52
+ ■ 239±.093
+ 2-55
+. 44i±.oSo
+ 5- 50
+.361 ±.086
+ 4-18
+ . 246 ± ■ 093
+ 2.64
+ . i39±-°97
+ 1 42
+ . 2is±.094
+2. 27
;±-099
1915, ear corn. .. 25
+ . II2±. 098
+ 1- 14
+.o72±- 099
+•73
+ .459±-°7S
+ 5-88
+.042 ±.099
+.42
+ . l84±. 096
+ 1. 91
+ . ii9±.098
+ 1. 22
r±-075
1916, ear corn. 0. 63
+ . 220±-095
+ 2-32
+ -407±-o83
+4-9°
+. 439±.o8o
+ 5-49
+ . I04±- 098
+ 1.06
+ . I44±097
+ 1.48
+ • i35±-097
+ 1 39
+ . 227±.094
+ 2. 41
+. i8g±-096
+ 1-97
+.253±.093
0 +2. 72
+.<>34±.o99
+•35
+.372±-o8s
+4-34
+. 294 ±- 090
+3- 24
— . i66±. 096
-1.72
— . 020± O99
— . 20
+. 225 ± O94
+ 2 38
+ . I58±.096
+ 1 63
— 063 ±.099
-.64
1917, oat grain.
+.10
+ -333±-o88
1917, oat strawy , . ,
+3-76
+. 253±.093
+ 2. 72
+ . 253 ±. 093
1917, total oats,
+ 2. 71
?±.D94
191S, silage conj. 41
+. i89±. 096
+ 1-97
— . I29±- 097
— 1.32
t±-099
1919, barley gra. 35
^±.099
1919, barley stri. 2o
3±. 099
+4-34
+ . 22S±-094
+2-38
+ .333±-o88
+3-76
+ . 294±-°90
+3-24
+. is8±.096
+ 1.63
+-2S3±-093
+2. 71
— . i66±.og6
-1. 72
—.063 ±.099
-.64
— . I29±. 097
-1.32
16916
Table IV. — Inlcrannual correlations for yield of 46 plots in the Huntley uniform cropping experiment
,
I 4j*8±
■** •**
Dec. i, I920 Permanence of Differences in Experimental Plots 345
to
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346
Journal of Agricultural Research
Vol. XX, No. 5
Unfortunately the number of crops which can be included in Table VI
is rather small. The coefficients are positive in sign throughout, and in
all cases they are statistically significant in comparison with their probable
errors. The individual constants will receive attention in the following
discussion.
The fact that the yields are correlated in the different years for whole
plots of 0.17 acre, for half plots of 0.085 acre, and for quarter plots of
only 0.0425 acre emphasizes the permanence of the substratum differences.
We now have to compare the correlations secured for these three divisions.
The difference in the actual magitudes of the correlations appear in
Table VII. The three entries, when all comparisons are possible, show:
(1) the difference between the correlation for whole plots and half plots,
(2) the difference between the correlation for whole plots and quarter
plots, and (3) the difference between the correlation for half plots and
quarter plots.
The signs are positive when the correlations are larger for the larger
areas.
The comparisons show that in general the correlations decrease in
magnitude as the areas upon which they are based are subdivided. Thus
16 of the 22 comparisons of the correlations deduced from whole plots
and from half plots (first entry) show a lower correlation in the half plots
as compared with 6 which show higher correlations in the half plots.
Table VII. — Differences in interannual correlations for whole plots, half plots, and
quarter plots
■' I
1913, alfalfa I.
1913, alfalfa !
1913, alfalfa I and II,
1914, alfalfa I
1914, alfalfa II
1914, alfalfa I and II.
1913, ' 1913.
alfalfa I. alfalfa II.
1913.
alfalfa I
and II.
1915, ear corn.
f— 0.0863
(— o. 0514
J-0.0558
f + o. 0910
1916, ear com.
+0.0387
— o. 1622 — o. 1442
— • 2276
— • 0653
— • 1335
— -3615
— . 2280
— . 1462
— • 2794
— • 1332
— . 0267
— .0623
— -°3S5
— . 102 1
— . 2081
— . 1059
1914.
alfalfa I.
— o. 0863
. 1622
. 2276
•0653
1914.
alfalfa II.
■ 1335
•3615
.2280
1914.
alfalfa I
and II.
1915.
ear corn.
—0.0558 ' +0.0910
- . 1462
- . 2794
- -1332
+ .0388
— -0150
— °S39
— .0560
— -1578
— . 1018
+ -0705
+ .0676
— .0029
— -0493
— • 1599
— .1105
. 0649
.0385
.0264
•0377
.1388
. 1011
— .0267
— • 0623
— -035S
+ -0403
+ .0388
— .0150
— 0539
+ .0705
+ .0676
— . 0029
+ . 0649
+ -0385
— .0264
— .0492
+ .04X7
— • 0004
1916,
ear corn.
+0.0387
. 102 1
2081
.1059
.0560
•1578
.1018
•0493
.1599
• 1 105
•0377
.1388
. ion
.0492
.0487
.0004
Dec. i, 1920 Permanence of Differences in Experimental Plots
347
Of the 12 comparisons possible between the interannual correlations
deduced from whole plots and from quarter plots (second entry), 9 show
lower correlations for quarter plots as compared with 3 which show
higher correlations for the quarter plots. Finally, all 12 of the correla-
tions deduced from quarter plots are lower than the correlations deduced
from half plots.
It appears, therefore, that 0.085 and 0.0425 acre are rather too small
to give the highest values of the interannual correlations. On areas
of this size other factors than the peculiarities of the plots themselves
have too large an influence upon variation of yield to allow the indi-
viduality of the plots to express itself fully in its influence upon the yields
of successive years.
In support of the conclusion that the lower value of the correlations
for half and quarter plots is due to the greater variability of the yields
of these plots we note that the coefficients of variation for subplots are
without exception larger than those for the plots of the original size.
The coefficients of variation are as follows for the years in which the plots
were subdivided.
Crop.
1913, alfalfa I
1913, alfalfa II
1913, alfalfa I and II
1914, alfalfa I
1914, alfalfa II
1914, alfalfa I and II
1915, ear corn
1916, ear corn
Whole
plots.
12. 52
13.60
II. II
17.94
19.81
17-47
7.29
13-43
Half plots.
*5
Quarter
plots.
21.87
23.68
25.87
21.88
9-23
17.68
It is now desirable to examine the results for the individual crops.
In doing this it may be noted that there are two factors to be taken into
account. First, there is the possibility of an inherent difference in the
plots which is persistent from year to year and is quite independent of
the crop grown. Second, it is conceivable that the crop itself may exert
an influence upon the soil such that the yields of subsequent crops will
be influenced by variations in its growth which are measured in terms of
yield.
The first of these factors would influence all correlations between
plots — those between the yields of given years and the yields of both
preceding and subsequent seasons. The second would influence only
correlations with subsequent years.
In a series of only 46 plots it will probably be impossible to distinguish
between the influences of these two factors.
We note that the higher yields of beets are followed by lower yields of
alfalfa in 191 2, but that there is practically no relationship between the
yields of sugar beets in 191 1 and the yield of other crops on the same
348
Journal of Agricultural Research
Vol. XX, No. 5
plots from 1913 to 1918. Possible exceptions are ear corn in 191 5 and
silage corn in 191 8, for which the correlations are positive and perhaps
statistically significant in comparison with their probable errors. The
correlations for yields of sugar beets in 191 1 and yields of barley in 1919
are negative in sign and apparently statistically significant in compari-
son with their probable errors. We have no explanation to offer con-
cerning this apparent relationship. The average value, with regard
to sign, of the correlations between the yield of sugar beets and other
crops is —0.077.
The correlations between the 9 different cuttings of alfalfa made during
1912^0 1914 and all other yields are generally positive and statistically
significant in comparison with their probable errors. The only excep-
tions are the negative correlation with sugar beets in 191 1 which have
already been noted and the slight and statistically insignificant correla-
tion for the 1 91 2 yield of alfalfa and the yield of silage corn in 191 8.
vSince it is quite reasonable to assume that in a crop harvested more
than once a year thickness of stand and variation in the size of the indi-
vidual plants will have a large influence on the yields of the different
plots in the same year, the correlations between the different cuttings
of the same year as well as those between single cuttings and totals of
two or more cuttings in the same year have been omitted from the tables.
The correlations between different cuttings in the same year are given
in Table VIII.
Table VIII. — Comparison of correlations between different cuttings of alfalfa in the
same year
Cuttings of alfalfa.
Whole plots.
Half plots.
Quarter plots.
1913, first and second cuttings. .
1914, first and second cuttings.' .
1914, first and third cuttings. . . .
1914 (first plus second) and
+0. 454±o. 079
-f . 7ii± .049
+ • 595 ± ■ o64
+ -653± .057
+ O.442 ±0.057
+ . 633 ± . 042
+ 0. 558 ±0.034
We shall now consider the correlations between the yields of alfalfa
and between the yields of alfalfa and of other crops on the same plots in
different years. The individual constants may be studied in the funda-
mental table (Table IV). The averages are given in Table IX. This
shows that the correlations between different cuttings of alfalfa are on
the average larger throughout than those between the yield of alfalfa
and the yields of other crops on the same plots.
Dec. i, 1920 Permanence of Differences in Experimental Plots 349
Tablb IX. — Comparison of correlations between different yields of alfalfa with correla-
tions between yields of alfalfa and yields of other crops
Cuttings of alfalfa.
1912, single cutting
1913, first cutting
1913, second cutting
19 13, first and second cuttings ,
1914, first cutting ,
1914, second cutting
19 14, first and second cuttings
1914, third cutting
19 1 4, first, second, and third cuttings
With other With
cuttings of yields of
alfalfa. other crops.
+0. 331
+ .611
+ -604
+ • 72°
+ .666
+ .629
+ -699
+ .524
+ -7°6
+0.
+
+
+
+
+
+
+
+
Difference.
171
+0.
187
+ •
282
+ •
274
+ •
■295
+ •
.244
+ •
. 290
+ •
■ 303
+ •
.316
+
160
424
322
446
371
385
409
221
390
It is clear, therefore, that either stand or specific adaptation of the
individual plots to alfalfa influences to an unusual degree the closeness
of correlation between the yields of the plots of alfalfa in the different
years.
In the first crop of ear corn (191 5) we find higher yields of ear corn in
1916, a negligible difference in the yield of oat grain and straw and total
yield in 1917, higher yield of silage corn in 1918, and slightly but not
significantly higher yield of barley grain, straw, and total yield in 1919
following higher yield of corn in 191 5.
Turning to the constants for ear corn in 191 6, we note that higher
yields of grain in this year are followed by higher yields of oat straw
and grain in 191 7 and of silage corn in 191 8, and by slightly higher
yields of barley grain and straw in 191 9.
The average value of the correlation between the yield of ear corn
in 1 91 5 and the yield of other crops during the eight years is +0.167,
whereas that for ear corn in 191 6 and other crops is +0.486. These
averages include the correlations for alfalfa, which are, as shown by
Table VIII, high for the crop of 191 6.
Considering the correlations for- oat straw, grain, and total crop on
the several -plots in 191 7 and the yields of silage corn in 191 8, we find
that higher values of each of these measures of capacity for oat produc-
tion are on the average followed by slightly, but perhaps not signifi-
cantly, higher yields of silage corn in 191 8 and generally by higher
barley yields in 191 9.
For the oat yields the average correlations with other crops are: for
straw, +0.202; for grain, +0.289; and for total yield, +0.293.
The correlations of the yields of silage corn with the yields of the
preceding crops are, with one exception, positive in sign. The average
value for the eight years is +0.226.
The averages of the correlations between barley yields and the yields
of other crops on the same plots during the eight years of the experiment
are +0.141 for grain, +0.086 for straw, and +0.126 for total yield.
350 Journal of Agricultural Research voi.xx, No. 5
Summarizing this discussion of the results for the individual crops,
we have the following average values of the correlation coefficients :
1911, sugar beets — o. 077
1912, total alfalfa + ■ 242
1913, alfalfa I + - 346
1913, alfalfa II + • 403
1913, alfalfa I and II + - 441
1914, alfalfa I + . 401
1914, alfalfa II + . 354
1914, alfalfa I and II + . 407
1914, alfalfa III + . 366
1914, alfalfa I to III + .428
1915, ear corn +0. 167
1916, ear corn + . 486
1917, oat straw + . 202
1917, oat grain + . 289
1917, total oats + . 293
1918, silage corn -j- .226
1919, barley grain + . 141
1919, barley straw + . 086
1619, total barley -f- . 126
General average + . 274
With the exception of the sugar beets the average correlation for
every crop is positive in sign, and in many cases it is of a very material
value.
Returning to the averages for the individual crops, we note from
Table IX that the lowest correlation for alfalfa, whether with other
cuttings of alfalfa or with the yield of other crops, is that for the single
cutting of 191 2.
It might be suggested that the 191 2 yields of alfalfa are less likely
to reflect the real producing capacity of the plots than the yields of the
later cuttings of this crop, for the reason that the first cutting of alfalfa
when sown without a nurse crop is subject to much variation because
of slight differences in surface condition of the soil at seeding time and
also because of differences in weediness of different plots. Both these
conditions would become relatively less important in their effect on
crop yield after the first cutting.
Because of its nitrogen-fixing capacity and the resistance to decay of
the roots and stubble of alfalfa the correlation between the various
yields of this legume and the yields of subsequent crops is of especial
interest. Fortunately two crops of ear corn were grown immediately
after the alfalfa, which was broken up in the fall of. 191 4.
A comparison of the correlations of these two series of corn yields
with the preceding yields of alfalfa is made in Table X. These coefficients
indicate a positive correlation between all the yields of alfalfa and the
yields of ear corn in both 191 5 and 191 6.
Of the 19 correlations determined between the yields of alfalfa for 191 2
to 1 914 and the yields of ear corn in 191 5 only 9 may be looked upon
as probably significant in comparison with their probable errors. Of
the 19 correlations between the yields of alfalfa in 191 2 to 1914 and the
yields of ear corn in 1916 only one coefficient — that for the 1912 yield of
alfalfa and the 191 6 yield of corn — can not be considered as represent-
ing a real agronomic relationship between yield of alfalfa and yield of corn.
The constants for 191 6 are without exception larger and with two
exceptions significantly larger in comparison with their probable errors
than those for 191 5.
Dec. i, 1920 Permanence of Differences in Experimental Plots 351
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352 Journal of Agricultural Research Vol. xx, no. s
The average value for the nine pairs of correlations deduced from
the yields of whole plots is +0.159 for alfalfa and corn yield in 1915
but +0.708 for alfalfa and corn yield in 191 6. For the six pairs of
correlations which may be deduced for half plots the average of the
coefficients for the various yields of alfalfa in 191 3 and 1914 and the
yield of ear corn in 1915 is +0.181, whereas the average correlation of
the same yields of alfalfa with corn one year later is +0.729. Finally,
in the four cases in which it was possible to calculate correlations between
alfalfa and corn yields on the basis of data for quarter plots the average
for the correlations with ear corn in 191 5 is +0.159, whereas the con-
stants showing the relationship between the yield of alfalfa in 191 3 and
1914 and ear corn in 1916 give an average of +0.626.
This more intimate relationship between the yields of alfalfa and the
second crop of ear corn does not necessarily mean that the corn crop of
1 91 6 was larger than that of 191 5 but merely that the variations in the
individual plot yields in 1916 are more dependent than those of 191 5
upon the yields of alfalfa during 191 2 to 191 4. As a matter of fact the
average yield in 191 5 was 522.6 pound per plot, while in 191 6 it was
396.2 pounds per plot. The greater yield in 191 5 may have been, and
probably was, due to factors other than soil conditions as such.
It is of interest in this connection to turn back to the table of coeffi-
cients of variation of yield (p. 347) and to note that for whole plots, half
plots, and quarter plots the coefficients of variation of plot yield are
distinctly lower in 191 5 than in 191 6. This result is quite in line with
what one would expect if the fixed nitrogen of the varying growths of
alfalfa were not yet fully available in 191 5.
There is also another possible explanation for the lower correlation
between the alfalfa yields and the yields of corn in 191 5. It is always
a difficult matter on the heavy soils at Huntley to break up alfalfa sod
and to get the soil into good tilth for the succeeding crop. It may be
that some of the plots in this field include heavier soil which ordinarily
ogives good yields but which was harder to get into good condition in
time for the 191 5 corn crop. If this were the case, these differences in
tilth might have been smoothed out by the season's cultivation so as
not to be expressed in the 191 6 crop yields.
Some light may be thrown upon the problem of the residual influence
of alfalfa in the following manner.
If the correlations between the plot yields of later crops be in a large
degree determined by differences in fertility referable to differences in
stand and yield of the preceding alfalfa crops, one might expect a closer
correlation between the yields of ear corn in 191 6 and oats in 191 7 than
between ear corn in 1916 and ear corn in 1915, since, as is shown above,
variations in the alfalfa yields have little influence until 191 6. This
will be true, provided there be a residual influence of the variations in
the yields of alfalfa such that these variations in fertility due to varia-
Dec. 1. 1920 Permanence of Differences in Experimental Plots 353
tions in yield from 191 2 to 1914 inclusive will influence not merely the
yield of corn in 191 6 but the yield of oats in 191 7, etc. The correlations
between corn yields in 191 5 and corn yields in 1916 and the yields of
subsequent crops are shown side by side in Table XI.
Table XI. — Comparison of correlations of the yields of ear corn in 1915 and in 1916
with the yields of subsequent crops
1917.
Oat grain
Oat straw
Total yield
1918.
Silage corn
1010.
Barley grain
Barley straw
Total yield
Con. 1915.
Corn, 1916.
— O. 02 5 ±0. 099
-f- • H2± . O98
+ -o72± .099
+ -459± -°78
+ • 042 ± • 099
+ . i84± . 096
+ . ii9± . 098
+ 0.497 ±0.075
+ • 220± . O95
+ -407± .083
+ -439± -o80
+ • io4± . 098
+ . I44± -097
+ . I35± .097
Difference.
+0. 522 ±0. 124
4- . lo8± . 136
+ -335± -129
. 020± . 112
4- .062+ .139
— . 040± . 136
+ .oi6± . 138
These comparisons show that the yields of oats in 1917 are much
more closely correlated with the yields of corn in 191 6 than with the
yields of ear corn in 191 5. No such relationship is apparent in the
correlations for silage corn in 191 8 or for barley in 191 9. The after
effect of the alfalfa crops of 1912 to 1914 is, therefore, apparently largely
limited to an influence on the yield of oats in 191 7.
Turning from this indirect to a more direct method of comparison,
we have determined the averages of the correlations between the several
individual cuttings of alfalfa and the yields of the single antecedent
and of the five subsequent crops. The results are given in Table XII.
Table XII. — Averages of the correlations between the cuttings of alfalfa in IQI2 to 1914
and the antecedent and succeeding crops
Crop correlated with alfalfa.
Grain.
Straw. Total yield.
Sugar beets, 191 1.
Ear corn, 1915 +0. 159
Ear corn, 1916 + . 708
Oats, 1917 -I- .437
Silage corn, 1918.
Barley, 1919 + .234
+0. 210
+ • 113
— o. 082
+ -371
+ -279
+ -195
There should be no correlation between the yield of sugar beets and
alfalfa except that due to the initial heterogeneity of the field. The
354 Journal of Agricultural Research voi.xx.No. s
insignificant negative correlation observed may be due to some pecu-
liarity of the crop. The comparison of the correlation for the 191 5 and
1 91 6 corn crops has already been made (Table XI). Inspection of the
averages in Table XII shows that on whatever character they are based
the correlations decrease from the maximum relationship observed in
1 91 6 to the lowest values in 191 9. #
Whether the residual influence of alfalfa per se has any influence on
the 1 91 9 or later crops can only be determined by further experimenta-
tion in which the interannual correlations can be deduced from the
yields of plots upon which alfalfa has not been grown.
III.— DISCUSSION AND RECAPITULATION
The purpose of this paper has been to present the results of a new
method of attack upon the problems of (a) the permanency of the differ-
ences which are found in the plots of an experimental field, and of (b)
the influence of variations in the yields of certain crops in the rotation
upon the yields of subsequent crops.
The data upon which the studies were primarily based comprise the
yields of 46 plots — subdivided in several cases into half plots and quarter
plots — each of 0.17 acre in area at the Huntley (Mont.) Field Station of
the Office of Western Irrigation Agriculture for the nine years between
191 1 and 1 91 9, inclusive.
The uniform cropping experiment, involving sugar beets, alfalfa,
corn, oats, and barley, was initiated merely to determine the variation
in the yields of plots of a given size when homogeneously planted and
uniformly treated. The experimental procedure was, therefore, deter-
mined in advance and was wholly independent of the statistical analysis.
This is in certain regards fortunate. It frees the data absolutely fronf
any suspicion of an influence of preconceptions or of personal equation
on the biometric results. On the other hand, it is quite possible after
the statistical analyses have been made to recognize ways in which the
experiments could have been improved and made to yield more valuable
results. This is, however, a feature of research in general. The dis-
covery of inadequacies in a first set of experiments makes possible their
elimination in subsequent work. The most unfortunate defect in the
data was that the harvesting and weighing could not be done by half
and quarter plots in 1917, 1918, and 1919, but this curtailment could
not be avoided under existing conditions.
The results of a previous study (3) have shown that fields selected for
plot tests of all kinds are practically without exception heterogeneous
to a degree that influences profoundly the yields of the crops grown
upon them. It was there pointed out that the correlation between
the yields of adjacent plots might either be due to initial physical and
chemical differences in the soil or be referable to the influence of previ-
ous crops upon the composition, texture, or tilth of the soil-.
Dec. i, 1920 Permanence of Differences in Experimental Plots 355
The first purpose of the present study has been to determine whether
such differences in fields selected for their apparent uniformity by skilled
agronomists are of a purely transitory nature or whether they are of a
relatively permanent character.
This problem can be solved by determining whether in such series of
uniformly treated plots the yields of the same plots in different years
are correlated.
The results of the present study show that of the 152 correlations be-
tween the yields of the plots in different years, 133 are positive as com-
pared with 19 which are negative in sign. The average value of the
positive correlations is + 0.335, whereas the average of the negative
constants is — 0.148. The general average is + 0.274. With the excep-
tion of the 191 1 crop of sugar beets the correlation between the yields
of each individual crop and the yields on the same plots in the eight
other years of the experiment are on the average positive.
The data available for half and quarter plots fully substantiate the
results for whole plots.
The results show conclusively, therefore, that plots, even of the small
size and apparent uniformity of those at the Huntley Station, are
characterized by differences which may persist throughout a period of
years. Thus, in general, plots which produce more in one year will
produce more in another year.
This is, of course, a well-recognized principle for large tracts. Its
validity for small plots has apparently not been recognized heretofore.
It is probably not a principle of universal applicability, because of the
fact that meteorological as well as soil conditions play a large part in
determining yield. It is quite probable that certain soil characteristics
would result in maximum yields with one set of meteorological con-
ditions but in minimum yields with another complex of aereal con-
ditions.
The determination of the proximate factors to which these corre-
lations are due presents a problem of considerable difficulty. Unfor-
tunately (for this phase of the problem only) alfalfa was introduced
early in the rotation and occupied the ground for three of the nine years
covered by the experiment. It seems quite possible that the correlations
between certain of the yields is due in part to the variation in nitrogen
content of the soil referable to the variation in thickness of stand and
strength of growth of the alfalfa crops.
The results show that there is but little correlation between the alfalfa
yields of 1912 to 1914 and the ear corn yields of 191 5, whereas the corre-
lations for ear corn in 1916 are high. Thus the influence of alfalfa
upon the yield of a subsequent crop is not fully evident until the second
year after it is turned under.
There is a definitely demonstrable residual influence of the variation
of alfalfa yields upon the yields of subsequent crops. The influence of
356 Journal of Agricultural Research vol. xx, No. s
the alfalfa upon the yield of subsequent crops decreases with the lapse
of time from the maximum correlation found for ear corn in 191 6. The
residual influence of the alfalfa is clearly marked in the oat crop of 191 7
and may still be evident in the silage corn and barley crops of 191 8
and 1 91 9.
In view of the early introduction of alfalfa into the rotation, it is
impossible to determine whether the correlations between yields other
than those for alfalfa are due to the variation from plot to plot of the
amount of nitrogen fixed by the alfalfa or whether it is to a considerable
extent due to the original heterogeneity of the plots. This and other
problems which will suggest themselves to the reader can be solved only
by the analysis of further experimental data. The illustrations of the
present paper are sufficient to show the value of the application of the
interannual correlation method to agronomic problems.
LITERATURE CITED
(1) Harris, J. Arthur.
1915. ON A CRITERION OF SUBSTRATUM HOMOGENEITY (OR HETEROGENEITY)
IN FIELD experiments. In Amcr. Nat., v. 49, no. 583, p. 430-454.
(2)
1915. the value OF inter-annual correlations. In Amer. Nat., v. 49, no.
587, p. 7°7-/12-
(3) —
1920. practical universality of field heterogeneity as a factor in-
fluencing PLOT YIELDS. In Jour. Agr. Research, v. 19, no. 7, p. 279-
314. Literature cited, p. 313-314.
(4) Lehmann, A.
1907. SEVENTH ANNUAL REPORT OF THE AGRICULTURAL CHEMIST FOR THE YEAR
1905-1906. [Department of Agriculture, Mysore State.] 53 p.
Bangalore .
(5) Lyon, T. L.
1912. SOME EXPERIMENTS TO ESTIMATE ERRORS IN FIELD PLAT TESTS. In
Proc Amer. Soc Agron., v. 3, p. 89-114, 5 fig.
(6) Smith, Louie H.
1910. PLOT ARRANGEMENTS FOR VARIETY EXPERIMENTS WITH CORN. In ProC.
Amer. Soc. Agron., v. 1, 1907/09, p. 84-S9.
(7) Stockberger, W. W.
1912. A STUDY OF individual performance in hops. In Ann. Rpt. Amer.
Breeders' Assoc, v. 7/8, p. 452-457.
(8)
1916. relative precision of formulae for calculating normal plot
yields. In Jour. Amer. Soc. Agron., v. 8, no. 3, p. 167-175.
SOME CHANGES IN FLORIDA GRAPEFRUIT IN
STORAGE 1
Lon A. Hawkins, Plant Physiologist, and J. R. Magness,2 Scientific Assistant, Office
of Horticultural and Pomological Investigations, Bureau of Plant Industry, United
States Department of Agriculture
INTRODUCTION .
Zoller (n),3 in his paper on the constituents of the grapefruit (Citrus
decumana) , has pointed out that very little attention has been paid to tht
chemical constituents of this important fruit. This statement might
also be made concerning the physiology of the fruit and the changes
which go on in it after it is removed from the tree and held at storage
temperatures. Some analyses of grapefruit have been made, however,
by various investigators.
Chace, Tolman, and Munson (4) in their work on tropical fruits analyzed
several different varieties of grapefruit. Rose (8) and others connected
with the Florida Agricultural Experiment Station have made many
analyses of citrus fruits in working out a basis for the regulation of the
shipping of them. These last-mentioned analyses were for the most
part determinations of the acid and sugar content of the pulp or juice and
of the soluble solids present in the juice.
Collison (5) determined the acids and sugars in the juice of several
varieties of grapefruit picked at various times during the season. He
found, in general, that there was a decrease in acidity and an increase in
sugar content as the season advanced and that after the fruit matured the
sucrose was gradually broken down to reducing sugars. The fruits were
analyzed shortly after removal from the tree.
Shamel (9) quotes a number of analyses of Florida and California
grapefruit by E- M. Chace. Zoller (11) found that the acid of the pulp
decreased during storage and records a marked increase in sugars after
the fruit is removed from the tree. He found also that the content of
the glucoside naringin, which is the bitter principle of grapefruit, was
less in the peel after storage. This writer apparently used only a small
number of fruits in his storage experiments, the work being for the most
part a chemical study of the various constituents of the fruit.
Chace and Church (j) recently made a chemical study of some different
types of grapefruit grown in California and Arizona. They determined
1 This paper gives the result of a portion of the work carried on under the project " Factors Affecting the
Storage Life of Fruits."
2 The writers' thanks are due Mr. L. B. Scott, formerly Pomologist, Office of Horticultural and Pomologi-
cal Investigations, for advice and helpful criticism while this work was in progress.
3 Reference is made by number (italic) to " Literature cited," p. 372-373.
Journal of Agricultural Research, Vol. XX, No. 5
Washington, D. C Dec. 1, 1920
vs Key No. G-213
(357)
358 Journal of Agricultural Research vol. xx,No. s
the acid-solids ratio of grapefruit picked at intervals throughout the
season from a number of localities. Some little work was also done on the
effect of cold storage and storage in lemon curing rooms on the acid-solids
ratio of the juice as compared to that of similar fruit direct from the tree.
The data given seem to show that there is an increase in the acid-solids
ratio during storage.
While other investigations have been carried out on certain chemical
phases of the composition of grapefruit, the articles mentioned above are
apparently all that are of interest in connection with the present work.
It is evident from the brief review of the literature here presented that
the longer the fruit is held on the tree the lower the acid content. The
acid content also apparently decreases during storage. The sugar con-
tent increases in fruit on the tree as the season advances, and some
evidence is brought out that it increases during storage.
The present investigation was taken up to determine the effect of
storage at various temperatures on the fruit and particularly on the
sugar and acid content of the pulp, since these substances make up the
major portion of the dry matter of the pulp or interior of the fruit, with-
out the seeds.
METHODS OF EXPERIMENTATION
The fruit used in these experiments was from single trees of two
named varieties, Silver Cluster and Davis, and ' 'common Florida. "* Most
of the work was done with the two varieties last mentioned, the fruit of
these varieties all being from three trees, one Davis and two "common
Florida."2
The fruit from each tree was packed separately and was shipped to
Washington, where the storage experiments were carried out. The first
season's experiments, those of 191 7-1 8, were preliminary, and only
Silver Cluster fruit was used. All the fruit was obtained from one tree.
It was shipped to Washington, where part of it was stored at 86° F. and
the rest placed in a commercial cold storage at 32 °. In the experiments
with this fruit, the juice alone was analyzed, though the comparative
percentage of peel and pulp was determined in some cases. The method
followed was to peel the fruit, grind the pulp, and press out the juice
through thin muslin. The acid-solids ratio was determined according to
the usual method (8), and samples were, in most cases, made for sugar
determinations. The samples for sugar determinations were pipetted
into 250-cc. volumetric flasks, cleared with neutral lead acetate, made up
to volume, filtered, and the excess lead removed with sodium oxalate.
The reducing substances in this solution were determined. For total
1 The writers are indebted to Mr. W. J. Krome, of the Medora Grove, Homestead, Fla., for his kindness
in picking, packing, and shipping the fruit from these three trees at various times during the season, and to
Mr. F. S. Poole, of Lake Alfred, for the Silver Cluster fruit used in the first season's work.
2 " Common Florida" is the name applied in Florida to fruit of seedling grapefruit trees or trees budded
from seedlings to which no distinctive varietal name has been applied. The term, therefore, may include
fruit which represents a rather wide range in some of its characteristics.
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage 359
sugars a 50-cc. aliquot was pipetted into a 100-cc. volumetric flask, the
sucrose inverted by adding 5-cc. of concentrated hydrochloric acid and
allowing it to stand overnight at room temperature. This solution was
made up to volume, neutralized, and the reducing substance in it was
determined.
Matthews's modification of Bertrand's method (7, p. 994) was followed
in the determination of the sugars. The sugars were calculated as dex-
trose according to Munson and Walker's tables (10).
PRELIMINARY EXPERIMENTS, 191 7-18
Table I shows the results obtained from several experiments in which
fruit was placed in the incubator maintained at 86° F\ In these experi-
ments a sample consisting of six or more fruits was analyzed when the
fruits were placed in the incubator, and analyses were made at the
dates indicated in the first column. In experiment 2 fruit of the same
lot as that used in experiment 1, which had been kept in cold storage
since November 28, was placed in the incubator on January 6. The
analyses on this latter date give data as to the effect of storage at 320
on the acid and sugar content of the fruit. The change in the acid-
solids ratio of this fruit maintained at 86° for 15 and 28 days is shown
in the table.
Table I.
-Changes in the composition of Silver Cluster grapefrui t during storage att
as indicated by the change in acidity and sugar content of juice
F.
EXPERIMENT I
Date sampled.
Nov. 28, 1917
Dec. 8, 1917.
Jan. 5, 1918.
Acid as
citric.
Per cent.
I. 16
I. 14
1. 09
Soluble
solids
(Brix).
9-i5
9.81
10.97
Acid-
solids
ratio.
7-9
8.6
10. 06 : 1
Sugar as dextrose.
Reducing.
Per cent.
2-95
3-03
3-49
Per cent.
2.76
2.79
2-59
2.28
Total.
Per cent.
5-7i
5- 77
5. 62
5-77
EXPERIMENT 2; FRUIT PLACED IN INCUBATOR
Jan. 6, 1918.
Jan. 21, 1918
Feb. 3, 1918.
1. 14
•83
.76
8-95
9-83
9.29
7.8 : 1
11. 8 : 1
12. 2 : 1
2. 90
2.87
2.68
2-59
5-58
5-46
From Table I it is evident that there is a decrease in acidity when the
fruit is stored at warm temperatures, while there is little, if any, de-
crease in the total sugar content of the juice. The reducing sugar is
increased somewhat, but there is a corresponding decrease in the cane
360
Journal of Agricultural Research
Vol. XX, No. 5
sugar. The acid-solids ratio increases markedly in storage at 86° F.,
but there is no evidence of change at 320 in 38 days.
Some idea of the shrinkage in grapefruit and the change in acid-solids
ratio was obtained in another experiment in which eight grapefruits
which had been stored for 13 days were removed from storage, weighed,
four of them peeled, and the percentages of peel and pulp determined.
Acid, sugar, and soluble solids were determined in the juice of these
four fruits. The other four fruits were placed in the incubator at 86° F.
and allowed to remain 12 days. They were then removed, weighed,
and the percentage of shrinkage, the percentage of peel, and the acid
and soluble solids determined according to the usual method. The data
obtained from these determinations are shown in Table II.
Table II. — Acids, soluble solids, acid-solids ratio, shrinkage, and peel in single Silver
Cluster grapefruit
When placed in storage.
Fruit
No.
After 12 days' storage at 86° F.
Fruit
No.
Acid as
citric.
Soluble
solids
(Brix).
Aeid-
solids
ratio.
Peel.
Acid as
citric.
Soluble
solids
(Brix).
Acid-
solids
ratio.
Shrink-
age of
fruit.
Peel.
Per cent.
1. 07
1. 17
1. 12
9-75
9- IS
9.09
9. 1 : 1
7-7:i
8. 1 : 1
9. 1 : 1
Per cent.
26
28
27
27
1
2
3
4
Per cent.
o-95
•93
r. oo
I. 02
11. 67
11. 61
11. 07
11. 61
12. 27 : 1
12.49 : 1
11. 07 : 1
11.37 = 1
Per cent.
24
32
26
35
Per cent.
23
6
18
21
8
18
The data in Table II show that the acidity decreases markedly and
that the acid-solids ratio is much higher after storage for 12 days at
86° F. Much of this apparent increase in soluble solids is probably due
to a concentration of the juice by the loss of water from the fruit.
Inasmuch as the average shrinkage of the fruit is 29 per cent, while the
average percentage of peel dropped from 27 to 20 per cent, obviously
much of the water given off comes from the pulp.
EXPERIMENTS IN 1918-19
In the second season's work, Davis and "common Florida" grape-
fruits were obtained from Mr. W. J. Krome, Homestead, Fla. The
entire crop from three trees was used in the storage experiments, one
picking being made from the Davis tree and two from the "common
Florida" trees. The fruit was shipped to Washington by express and
stored at the cold-storage plant at Arlington Farm. Cold-storage tem-
peratures of 320, 360, and 400 F. were used as well as common storage
at a mean temperature of about 500, probably fluctuating 50 above and
below that temperature, and two warm storage temperatures which were
about 700 and 86°, respectively. In most cases the fruit was weighed
when placed in storage so that the skrinkage could be determined.
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage 361
The structure of citrus fruit makes the study of the physiological
changes taking place in it rather difficult. Considering the peel, pulp,
and seeds of the fruit, there are then three structures which have very
different water contents and water-holding powers. It is impossible to
grind the entire fruit and weigh out comparable samples. It would be
impossible to slice the fruit and expect the various slices to be compar-
able because of loss of juice from the pulp in slicing and the fact that the
seeds are not necessarily evenly distributed. If fruits are sliced and
the seeds removed, the operation is liable to be attended with a con-
siderable loss of juice. After a number of experiments, the following
method of sampling was decided upon. After the fruit was weighed it
was peeled by making two cuts through the skin completely around the
fruit, the cuts crossing each other at right angles at the stem and blossom
ends. The peel was removed, and the thickness of each quarter was
measured midway along the side by means of callipers. Such por-
tions of the rag as adhered to the fruit were removed, and the fruit was
weighed again. The percentage of peel was calculated from the weights
before and after peeling. The fruit was divided into segments, and the
seeds were removed, care being taken that no appreciable amount of
juice was lost. Duplicate samples were made from segments from
opposite sides of the fruits. One segment from each of the 10 fruits was
used for each sample. While this method is not the most accurate, the
results of analyses of duplicate samples indicate that it is sufficiently
accurate for the work. It must always be taken into account that no
two grapefruits have precisely the same chemical composition and that
while in this work lots of 10 fruits were commonly used in each set of
analyses, some variation will occur between any two lots no matter how
carefully the fruits are selected.
In preparing the samples for analysis, the samples for sugar deter-
minations were placed in beakers and covered with 95 per cent alcohol.
A few drops of ammonia were added to neutralize the acidity, and the
sample was brought to a boil. It was then transferred to extraction
thimbles, the alcohol extract was separated at the same time by filtra-
tion, and the residue was subjected to continuous extraction for about
14 hours with alcohol in a soxhlet apparatus. The extract was added to
the filtrate, the whole was made up to 1 ,000 cc. in a volumetric flask, and
two 50-cc. aliquots were pipetted off for analysis. Sugar determinations
were made according to the method already described.
For the acid determinations, the pulp was brought to a boil in water
and was placed in liter volumetric flasks under toluol and allowed to
stand with frequent shakings for about 10 days. It was then strained
through linen, and two aliquots were titrated against sodium hydroxid,
using phenolphthalein as an indicator. The dry-weight determinations
were made by covering the samples with 95 per cent alcohol, driving off
362 Journal of Agricultural Research vol. xx, No. 5
the alcohol on a steam bath, and drying in a vacuum oven until there
was no appreciable loss in weight between successive weighings. The
results of the sugar, acid, and dry-matter determinations were cal-
culated to percentage of wet weight of pulp. The percentage of peel
was determined by weighing before and after peeling.
COLD AND COMMON STORAGE
As mentioned earlier in this paper, two pickings were made from the
two "common Florida" trees, while all the fruit from the Davis tree
was picked at the same time as the first lots from the other two trees.
The first fruits were harvested October 31 and, as the cold-storage rooms
were not yet completed, were allowed to remain in common storage at
mean temperature of about 550 F. until November 21, when they were
sampled. The fruit was then placed An the various storage chambers.
The results of the analyses of the fruit held at 320, 360, 400 at various
times during the storage season appear in Tables III and IV. The time
in days after they were first sampled, when they were placed at the
various storage temperatures, is given in the first columns, and the percent-
age of acid, sugar, dry matter, and the shrinkage of peel and percentage
and thickness of peel appear in order. The second lots of fruit from the
two "common Florida" trees were picked November 26, and the fruit
was placed in the three cold-storage chambers December 4. Some of
this picking was also placed in common storage, and the results of
analyses of the fruit held in this type of storage are included with the
data from the three cold-storage temperatures in Table III.
An inspection of Tables III and IV shows that there is a general de-
crease in tritratable acids during storage. This decrease would be more
marked if it were possible to take into account the shrinkage of the fruit
in storage. The actual decrease in acid would be somewhat more than
that shown in the table.
In comparing the acid content of the fruit held at the three different
cold-storage temperatures, 32 °, 360, and 400 F., it is evident that there
is no constant difference in the rate at which the acid decreased. In
most cases, however, at comparable samplings the fruit from the 400
storage is somewhat lower in acid content. This is especially noticeable
in the Davis fruit (Table IV), where the fruit from the 32 ° storage is in
all four samplings higher in acid content than that fruit from the other
two cold storages.
The ' 'common Florida" fruit in common storage was in general lower
in acid than comparable lots in cold storage, with the exception of the
second sampling which was made 42 days after the fruit was placed in
storage. There was undoubtedly a greater shrinkage in the fruit in com-
mon storage, as was evidenced by the fact that the peel was thinner and
the percentage of dry matter increased in the latter part of the season.
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage
363
Table III. — Percentage of sugars, acids, dry matter, shrinkage of fruit, peel, and thick-
ness of peel of "common Florida" grapefruit at various times during storage season
. TREE I, first pick; placed IN STORAGE NOV. 21, 1918
STORED AT 32° F.
Acids as
citric.
Sugar in pulp as dextrose.
Dry
matter.
Shrink-
age of
iruit.
Peel.
Thick-
Time of sampling.
Reduc-
ing.
Sucrose.
Total.
ness ol
peel.
When placed in stor- / 1.02
} 2.16
J 2.71
} 2.72
} 2.84
2. 17
2-35
2-15
2. 14
4-33
5.06
4.87
5.06
f 8.20
I 8.14
8.06
/ 8.26
\ 8.01
/ 8.40
\ 8.53
} "...
24.8
24. 2
23-4
24- S
Mm.
age
/ -93
I -94
/ -97
I -94
/ -89
I -89
)
6.13
5-64
4-50
Alter 102 days
After 178 days
} „
| 8.0
STORED AT 36 F.
After 102 days.
After i7Sdays.
J 0.94 |l
I -97 l/
{ :S)
2-35
2.27
4. 62
{
8.51
8.14
>
3-8
24.4
2.86
2.04
4.90
8.6
5-9
22. 7
6. 22
4-3°
STORED AT 40 F.
After 60 days. .
After 102 days.
After 1 78 days.
1
( 0.98
\ -97
}
2. 76
2. 20
4.96
8.9
2. 2
24.0
/ -91
I -97
}
2.49
2.3O
4-79
/ 8.42
1 8.54
}
4.1
21. 2
.87
2. 69
2. l6
4.85
/ 8.13
\ 8.04
}
5-5
21. 1
5-31
5- 30
4.00
TREE I, SECOND PICK; PLACED IN STORAGE DEC. 7, I918
STORED AT 32° F.
When placed in stor-
age
After 61 days
After 109 days. .
After 165 days ;<
{
1.28
1-23
\
2. 6l
2.82
1.02
2.84
2.49
1.07
2- 91
2-33
{
.89
.90
}
3. 26
2.6,
8.90
\
8. 78
1
8.79
3-1
24.8
9. 20
9. 16
\
4-5
23.0
9.48
9-77
}
8-5
24-5
5-56
5.87
5- 40
4. 60
STORED AT 36° F.
After 109 days.
After 157 days.
f 1.00 1
I .98 J
( 1.00 1
I -98 J
2.8s
3-39
2.66
2. 76
5- SI
9. 22
3-6
23.0
6. is
{
9.44
9.91
}
5-8
23.0
5- 90
4.00
STORED AT 40 F.
After 61 days. .
After 167 days.
{
1.06
.98
}
2.58
2-43
5- 01
9. 00
2.6
22. 7
!
1. 01
.96
}
3- OI
2.66
S-67
{
9.04
9.44
}
6.6
19. 6
5-57
3- 90
COMMON STORAGE
After 42 days. .
After 121 days.
After 179 days.
1. 11
1. 22
1.02
1.09
■92
•77
5-69
6.17
24- S
24.4
24.6
5-33
4.60
4.60
364
Journal of Agricultural Research
Vol. XX, No. s
Table III. — Percentage of sugars, acids, dry matter, shrinkage of fruit, peel, and thickness
of peel of "common Florida" grapefruit at various times during storage season — Con.
TREE 2, SECOND PICK; PLACED IN STORAGE NOV. 21, 1018.
STORED AT 32° F.
Sugar in pulp as dextrose.
Dry
matter.
Shrink-
age of
fruit.
Peel.
Thick-
Time of sampling.
citric.
Reduc-
ing.
Sucrose.
Total.
ness of
peel.
When placed in stor-
/ 1-03
j 2.44
} 2.63
j 2.76
} 2.89
2.32
2-43
2.44
2.40
4.76
5.06
5.20
5-29
/ 8.36
\ 8.20
/ 8.60
\ 8.22
J 8.86
\ 8.97
J 8.70
\ 8.60
\
24.6
24
23-9
26.3
Mm.
I 1
{
{
{
11
92
92
98
94
go
89
I
5.98
After 104 days
After 179 days
} 5"7
j 6.s
6. 20
4- 30
STORED AT 36° F.
After 104 days .
After 153 days.
/ -97
I .98
!
2.68
2.47
5- IS
{
8.70
8.68
)
5-7
24. 2
/ -89
\ -91
}
3- 03
I.78
4.81
8-77
5-7
24. 2
6. 20
4. 20
STORED AT 40 F.
After 61 days. .
After 106 days .
After 153 days.
.96
3-9°
2.44
5-34
8-7S
2.6
26.4
J -92
\ -90
}
2.87
2.08
4- 95
/ 8.88
I 8.67
) '•'
25.6
/ -89
I -91
I
3- 03
2. IS
S-l8
/ 8.70
\ 8.60
} 1.
28.3
6-73
5.10
TREE 2, SECOND .PICK; PLACED IN STORAGE DEC. 7, IQl8
STORED AT 32° F.
When placed in stor-
age
After 62 days
After 109 days
After 169 days
•94
■93
2.77
3-38
3- °7
3.06
2.43
2.94
2.86
2. 90
5- 20
6.32
5-93
5.96
8.96
9. 12
9.4
9.49
9. 12
9.84
2.9
5- 1
7.8
24.8
22. 6
24. 1
19
6.08
6. 09
STORED AT 36 F.
After 109 days .
After 157 days.
(
1.02
•99
)
2-99
2.70
5-69
{
9-59
9.28
\ 4-8
.96
3-32
2-59
5-91
{
9.80
9.89
} *.
24.8
STORED AT 40 F.
After 62 days. .
After 134 days.
0.94
/ .93
I .91
) 3- 03
2.36
5-39
9.6
9.44
9.71
2-5
6-5
24- S
24. 2
6.09
4.60
COMMON STORAGE
After 42 days. .
After in days.
After 179 days.
{ I
3-S5
3-23
3-56
2. 23
2. 12
2.28
5-78
5-35
S-84
9-43
10. 10
9-77
9.84
24-3
22. 7
20-4
4-74
3- 7°
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage
365
Table IV. — Percentage of sugars, acids, dry matter, shrinkage of fruit, peel, and thick-
ness of peel of Davis grapefruit at various times during storage season
PLACED IN STORAGE NOV. 21, 1918
STORED AT 32° F.
Sugar in pulp as dextrose.
Dry
matter.
.Shrink-
age of
fruit.
Peel.
Thick-
Time of sampling.
citric.
Reduc-
ing.
Sucrose.
Total.
ness of
peel.
When placed in stor-
{ '
{
{
{
93
96
91
97
86
83
«3
84
84
| 2.69
} 3- 06
3-os
| 3-25
} 3- °9
1.66
2. 29
2.09
2. 02
2.03
4-35
S-35
5- 14
5- 27
5. 22
J 7-94
\ 8. 10
{ 8^
8-37
f 8.23
\ 8.09
8.91
\
23- 7
23-1
23-4
25
26. s
Mm.
5- ic
/
} 3-5
3-9
} 3-3
5-5
4- 73
5- 16
4-8
5-6
After 118 days
After 140 days
STORED AT 36 F.
After 58 days. .
After 88 days . .
After 1 18 days.
After 139 days .
After 58 days . .
After 88 days. .
After 1 18 days.
After 139 days.
0
87
3- 11
2. 23
5-34
{
8
8
43
42
}
2.6
23-5
{
79
76
\
3.10
2.I4
5-24
{
8
8
39
25
\
5-3
22. 1
{
80
75
)
3.00
2. IO
5.10
{
8
8
27
69
)
6
22. 1
{
78
80
}
3- 16
2-73
5-99
{
8
8
02
26
)
8-7
22.5
STORED AT 40 F.
4.72
4-56
4- 7
0.8s
3-62
2. 04
5-66
{
8-53
}
1. 2
23-3
■ 79
3- 05
2. 10
5- 15
8- 5
8.41
7.6
19.9
{
• 77
• 73
>
3-14
2. 09
5-23
{
8-43
8.13
}
8.1
18.9
{
•73
• 75
}
3- 18
2.14
5-31
8.47
7-5
19. 2
4- 7
4. 01
3-9
3-9
A comparison of the acid content of the fruit from the two different
pickings, when placed in storage, showed that the fruit picked last has a
somewhat higher acid content, probably because the fruit of the first
picking stood in common storage 22 days before the first analyses.
The sugar content of stored fruit is in rather striking contrast to the
acid content. With few exceptions, the percentage of total sugar is higher
in the stored fruit than in the samples analyzed when the fruit was placed
in storage. In some cases, as in the Davis fruit (Table IV), which had
been stored 139 days at 360 F., the sugar content is more than 30 per cent
higher than in the analyses made when the fruit was placed in storage.
The difference is as marked in other cases. In general, however, the in-
crease in total sugar content is more apparent than real and is probably
due to the loss of water from the fruit. The shrinkage of the fruit is in
many cases sufficient to account for the apparent increase in sugar con-
tent. It is, however, undoubedly true that there is no appreciable dim-
inution of the sugar content during storage at the four temperatures here
considered.
The sucrose content, when calculated as percentage of pulp, remains
about the same during storage. Apparently the breaking down of the
sucrose just about keeps pace with the shrinkage of the fruit. This
366
Journal of Agricultural Research
Vol. XX, No. 5
increase in total sugars, then, as the storage season advances, is due to
an increase in free-reducing substances.
The dry-matter determinations are not particularly conclusive in the
analyses here shown. A careful inspection of the data obtained from
the 17 storage experiments shown in Tables III and IV indicates that
there is, in general, an increase in dry matter. This is probably due to
the loss of water from the fruit as well as to losses from respiratory
activities, both of which are included in shrinkage.
The shrinkage increases with the length of time the fruit remains in
storage and is in general around 5 per cent for the first 100 days in cold
storage. Only in two cases is it more than 8 per cent for the entire time
the fruit was stored. There is no marked difference in shrinkage in the
three temperatures. That the shrinkage is from the pulp as well as the
peel is shown by the fact that the decrease in the percentage of peel is
not sufficient to account for the loss in weight.
In general, the peel is from 19 to 25 per cent of the fruit used in these
experiments, and there is no wide variation between the two varieties.
The decrease in thickness of the peel during storage is about 30 per cent,
due, probably for the most part, to loss of water.
WARM STORAGE
As mentioned in the earlier part of this paper, in addition to the three
cold-storage and one common-storage temperatures, grapefruits were
placed in two warm storages at temperatures of about jo° and 86° F.
Some lots of fruit were stored in boxes and others in lard cans with tight-
fitting lids, the lids being removed from the cans occasionally for a short
time to aerate the fruit. The storage season for this fruit was, of course,
not so long as for that stored in the cold- or common-storage temperatures,
The results of analyses of fruit stored at 700 are shown in Table V, while
data obtained from the 86° storage are given in Table VI.
Table V. — Percentage of sugars , acids, dry matter, peel, and thickness of peel of "common
Florida" grapefruit stored at about yo° F. in ventilated and unventilated packages
TREK I, FIRST PICK
Acids, as
citric.
Sugar in pulp as dextrose.
Dry
matter.
Peel.
Thick-
Time of sampling.
Reduc-
ing.
Sucrose.
Total.
ness of
peel.
/ 1.02
1 .98
\ i- 03
| 2.16
| 2- 17
2.17
1.82
4-33
4.69
24.8
Mm.
After 61 days, unventilated
1 8.68
\ 8.47
TREE 2, FIRST PICK
When placed in storage
After 50 days, unventilated.
After 50 days, ventilated
/ I- 03
\ I. II
}
2. 44
2.32
476
/ 8-36
\ 8.20
24. 6
/ 1. 02
\ 1. 01
I
2. 96
1.95
4.91
/ 8.9
I 8.4s
24.5
/ -94
\ -93
1
2.98
2. 40
5-38
9.90
17. 12
6.00
6. 13
3- 03
Dec. i, J920 Some Changes in Florida Grapefruit in Storage
367
Table VI. — Percentage of sugars, acids, dry matter, shrinkage of fruit, peel, and
thickness of peel of grapefruit stored at about 86° F. in ventilated and unventilated
packages
TREE I, "COMMON FLORIDA," FIRST PICK
Time of sampling.
When placed in stor-
age
After 30 days, unven-
tilated
After 30 days, venti-
lated
Acids as
citric.
1. 05
1.04
Sugar in pulp as dextrose.
Reduc-
ing.
Sucrose.
2. 17
2. 01
2.09
Total.
4-33
4.78
4-97
Dry
matter.
8.20
8.14
9. 12
9- 30
9.01
9-33
Shrink-
age of
fruit.
Peel.
24.8
24.6
Thick-
ness of
peel.
Aim.
S-77
3-12
TREE 1, "COMMON FLORIDA," SECOND PICK
When placed in stor-
age
After 60 days, venti-
lated
After 86 days, venti-
lated
{
1.28
I 23
}
2.61
2.82
5-43
{
8.90
8.78
\
/
{
1. 25
1. 10
}
XO.44
26.9
12. s
{
1.16
1. 18
}
3-98
2- 02
6.00
12.09
34-6
11. s
5- 56
2.41
2.08
TREE 2, "COMMON FLORIDA," FIRST PICK
When placed in stor-
age
After 30 days, unven-
tilated
1.03
1. 11
1. 14
1. 12
4.76
8.96
9. 12
24.6
24.3
TREE 2, COMMON FLORIDA, SECOND PICK
When placed in stor-
age
After 61 days, venti-
lated
After 86 days, venti-
lated
1. 12
1. 12
.96
i- IS
1. 26
1.20
6-55
8.96
9. 12
10. 10
9- 75
12.23
24.8
13-8
11.4
6.08
2.42
2.34
When placed in stor-
age
After 19 days, unven-
tilated
After 24 days, venti-
lated
0-93
.96
.96
2.69
2.78
1.66
2.32
2.29
4-35
5- 20
7-94
8.10
8-33
8.21
9.76
23-7
23.7
In an inspection of the tables it may be seen that in general there is
very little, if any, decrease in titratable acids in the fruit stored in cans,
that is, in unventilated packages, at either of the two temperatures.
In some cases there is an apparent increase, as in tree 1 of "common
Florida," first pick (Table VI), which had been stored 30 days at 86°
F. and again in tree 2 of the same variety, pick, temperature, and length
of storage period. The increase in total sugar content is more, com-
paratively, in both these cases than is the increase in acid. In all other
cases the fruit in the unventilated package has an acid content about
16916°— 20 3
368 Journal of Agricultural Research vol. xx, no. s
the same as when placed in storage and an increased sugar content.
There is some loss of water from the fruit even in the cans which are
closed most of the time, and it is possible that the acid decreases, the
decrease in most cases being as rapid as the shrinkage of the fruit. It
is, of course, always possible that at these high temperatures and under
the low oxygen pressures some acid is formed in respiration.
With the stored fruit in ventilated packages the analyses made j#ter
24 or 30 days, as shown in Table VI, gave an acid content as high as or
higher than when the fruit was placed in storage. At the longer storage
periods in both temperatures the acid content was usually lower than at
the beginning of the storage period. In every case there was a marked
increase in sugar content, as calculated to wet weight of pulp. This
increase was greater where the fruit had been in storage more than 30
days.
While no exact data are at hand, it seems probable that the increase
in acid is due, for the most part, to loss of water from the fruit. Cases
in which the shrinkage was determined show that it was over 34 per
cent in 86 days at 86° F., the higher storage temperature. The thick-
ness of the skin of the fruit and the percentage of peel decrease markedly
in ventilated warm storage. This, of course, makes impossible the cal-
culation of the actual shrinkage of the pulp. The percentage of total
sugar in the pulp is in all cases higher after storage. This increase is
due in most cases to an increase in the reducing-sugar content, for the
percentage of cane sugar remains about the same in all analyses. It
is quite possible, in spite of the apparent increase in sugar content, that
some of the sugar originally present in the fruit actually disappears
during storage.
Another series of experiments was carried out in which fruit from the
second picking of the two "common Florida" trees was placed in the
warm room at 700 F. after it had remained in common storage 51 days.
The fruit was stored in cans and boxes, as in the experiments just de-
scribed. The results are given in Table VII.
From Table VI it is apparent that fiuit removed from common storage
and placed at a higher temperature behaves the same as fruit stored
at the higher temperature throughout the season. The findings in this
series are then mostly corroborative.
In ventilated packages there was, in some cases, an apparent increase
in acids, and in others the acid content was a little less. If the exceed-
ingly high percentage of shrinkage is taken into account, the results
seem to indicate that there is no actual increase in the amount of acid
during storage and that there may be a decrease as compared with the
amount originally present. The sugar content of the fruit stored in
unventilated packages shows always a decrease in the percentage of
total sugars present, while in ventilated storage the increase in sugar
content is in no case more than sufficient to account for the probable
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage 369
shrinkage of the pulp. In one case, tree 1, stored 67 days, the sugar
content is less after storage, probably because of variation in the samples.
The results indicate that there may be a slight decrease in sugar at the
higher storage temperatures.
Table VII. — Percentage of sugar, acids, dry matter, shrinkage of fruit, peel, and thickness
of the peel of "common Florida" grapefruit stored 5/ days in common storage then
placed in warm storage
TREE I, SECOND PICK
Time of sampling.
After 42 days in com-
mon storage
After si days in com-
mon storage; 29
days at 70° F., ven-
tilated
After 51 days in com-
mon storage; 29
days at 700 F., un-
ventilated
After 51 days in com-
mon storage; 67
days at 700 F., ven-
tilated
After 51 days in com-
mon storage; 67
days at 700 F., un-
ventilated
Acids as
citric.
1. 02
1. 01
Sugars as dextrose.
Reduc-
ing.
3.62
Sucrose.
2-45
2.56
2.38
Total.
5-69
s-ss
5-56
Dry-
matter.
9.44
9-34
9-45
9.64
Shrink-
age of
fruit.
Thick-
ness of
peel.
Mm.
5-33
5- 76
TREE 2, SECOND PICK
After 42 days in com-
mon storage
r 1.06 \
1.07 /
3-55
2. 22
5-7-S
9-43
24-3
After 51 days in com-
mon storage; 2S
days at 700 F., ven-
1.12 \
1. 10 f
3- 76
2.28
6.04
{
9. 61
9.40
} -
19.9
3-35
tilated
After si days in com-
mon storage; 28
days at 700 F., un-
,.02 |
I- 03 )
3-32
1.99
5-31
{
10. 1
10. 41
| ,.,
22.8
4.88
ventilated
After 51 days in con-
mon storage; 67
{
} -
days at 700 F., ven-
3-65
2.17
5.82
10. 01
7-5
2. 50
tilated
After si days in com-
mon storage; 67
days at 70° F., un-
•99 I
■96 J
3-26
2. 20
5- 46
{
965 \ 4-S
9-32 1 4
24.4
5-3°
There is a marked difference in the shrinkage of the fruit and percent-
age of peel as well as in thickness of the peel in the ventilated and unventi-
lated packages, the shrinkage being around. 4 per cent in the unventilated
fruit for 67 days and from 20 to 23 per cent for comparable lots stored
in ventilated packages. The peel, as would be expected, becomes very
much thinner in the fruit stored in ventilated storage.
There is a marked increase in the percentage of dry matter in the pulp
of the fruit stored in ventilated storage, while that of fruit in unventilated
packages remains practically constant.
37o
Journal of Agricultural Research
Vol. XX, No. 5
To determine the effect of cold storage followed by warm storage upon
the keeping quality of the fruit and also to obtain more data as to the
acid-sugar changes, "common Florida" grapefruits of the first pick,
which had been maintained at 32 ° F. for 61 days were removed, weighed,
and placed in boxes at 700. The analyses of this fruit after 46 days at
700, as compared with the analyses of comparable lots from 320 at the
time the fruit was placed in the warm chambers, are given in Table VIII.
Table VIII. — Percentage of sugars, acids, dry matter, shrinkage of fruit, peel, and
thickness of peel of "common Florida" grapefruit stored in cold storage 61 day s and
removed to warm storage for a period
TREE I, FIRST PICK
Acids as
citric.
Sugar in pulp as dextrose.
Dry
matter.
Shrink-
age of
fruit.
Peel.
Thick-
ness of
peel.
Time of sampling.
Reducing.
Sucrose.
Total.
After 60 days at 320 F.
After 61 days at 320;
46 days at 70°, ven-
tilated.
J 0.92
•93
J -99
•97
| 2.71
J 2.91
2-35
a- 39
S-o6
S-3I
8.06
I 9-86
\ 9-8o
Mm.
6.13
J 24.1
17-3
2.70
TREE 2, FIRST PICK
After 61 days at 320. .
After 61 days at 320;
46 days at 700, ven-
tilated.
0.92
.92
1. 01
i. 05
)
2.63
3- 18
2-43
2.19
S-oo
5-37
8.60
8.22
9.24
9- 32
S-98
3.10
It is evident from Table VIII that there is an apparent increase in
acidity, as was the case in most of the other warm-storage experiments.
The total sugar content is somewhat increased, though less propor-
tionally than the acid content. The percentage of dry matter is
increased markedly, the shrinkage at the high temperature is very
marked, the percentage of peel decreases, and the peel becomes thinner,
the fruit behaving much as in all the warm-storage experiments.
It seems probable that there was in these experiments a decrease in
the sugar during the period of warm storage, while the amount of acids
remained about the same. The fruit compared very favorably in
analyses with the grapefruit from the warm-storage experiments, the
results of which are given in Tables V to VII.
GENERAL DISCUSSION
While this investigation is primarily concerned with the acid and
sugar changes in the fruit, some data were obtained as to the general
appearance and attractiveness of fruit stored at the various cold-storage
temperatures and also at common storage.
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage 371
The fruit will apparently keep for a longer period in cold storage than
in either common or warm storage. In the first place, the losses from
decay caused by microorganisms are much less in the cold-storage
temperatures. In the second place, the shrinkage in cold storage is
much less than in warm, ventilated storage or in common storage.
A high percentage of the fruit rotted in warm, unventilated packages.
A high degree of humidity is necessarily maintained in this storage,
which is very favorable to the growth of various fungi which break down
the fruit. There is, therefore, much loss. The fruit which does survive
this treatment is, however, very attractive in appearance and has an
excellent flavor. In the third place, the life of the fruit is apparently
lengthened in cold storage — that is, the average fruit apparently tends
to break down more quickly when maintained at temperatures above
400 F. than when stored at lower temperatures.
An undesirable feature of cold storage is the breaking down or pitting
of the peel at the temperature of 400 F. or lower. This breaking down of
the peel begins as a slightly sunken spot, which increases in size and
becomes brown in color. The sunken portions are usually not more than
% inch in diameter, but several may coalesce, making a large sunken area
of dark-brown tissue. This does not extend into the pulp, and the flavor
is apparently unaffected, but the fruits are rendered unsightly. In these
experiments no pitting was noticeable on the fruit stored at the two
warm-storage temperatures or in common storage. It occurred only
on the fruit stored in the three cold storages. In these* temperatures
the fruit at 400 was most seriously affected. There was somewhat less
pitting on the fruit in the 360 storage and only a little on fruit at 32 °.
The flavor of the fruit improves in cold storage. The fruit is sweeter,
as is obvious from the fact that the sugar content of the pulp is higher
and the acid content lower. The fruit is apparently not so bitter after
storage, which may be due to the breaking down of the naringin in the
pulp. Zoller (11) has shown that this glucoside breaks down in the peel
during storage. The fruit improves in taste more rapidly at high storage
temperature than in cold storage, which is to be expected, inasmuch as
the changes are more rapid in warm storage. After longer storage,
however, the fruit in cold storage attains the excellence brought about
more quickly at a higher temperature.
The experiments in which the fruit was removed from storage at 320 F.
after 60 days and stored at 700 for 46 days (Table VIII) indicate that
the grapefruit does not deteriorate rapidly after removal from cold
storage. The fruit compared very favorably with fruit that had been
stored at 700 from the beginning of the storage period.
From the data shown in Tables I to IX, there is no question but that
the titratable acids in the fruit decrease after the fruit is removed from
the tree and placed in cold storage, which is in accord with the behavior
372 Journal of Agricultural Research vol. xx.no. s
of the acids in apples, as found by Bigelow, Gore, and Howard (2), and
others, Bigelow and Gore on peaches, (1) and in pears by Magness (6).
The sugar content apparently does not decrease appreciably in cold
storage, though definite evidence on this point is lacking. The shrinkage
of the peel and pulp may not be proportional, so that an accurate deter-
mination of the original weight of the pulp is impossible. There is indi-
cation that the sugar content decreases slightly in warm storage if the
shrinkage of the fruit is taken into consideration. There was in no case
evidence of a markedly increased sugar content in the fruit, mentioned
by Zoller (11). There is considerable variation in individual fruits, and
it is possible that this would account for the increase in sugar content
which he found. In the preliminary experiments, the results of which
are given in Tables I and II, it is shown that there is a marked increase
in the acid-solids ratio after storage at 86° F. This increase in amount
of soluble solids is undoubtedly due mainly to the loss of water from the
pulp and a concentration of the juice. While acid-solids determinations
were not carried out in the later experiments, the results of the sugar
and acid determinations show that a similar condition would hold for
fruit stored at the cold-storage temperatures, though possibly not for
fruit stored for long periods at the higher temperatures used.
In conclusion, it has been shown in this investigation that the acid
content of grapefruits decreases in cold storage. There is an apparent
increase in sggar content in cold storage, calculated to percentage of
pulp, which seems to be due to loss of water from the fruit. The dry
matter increases during storage. The shrinkage of the fruit runs from
5 to 8 per cent in cold storage to around 23 per cent in warm, ventilated
storage.
Fruit was kept in cold storage for about six months. The best storage
temperature seemed to be 320 F, for at this temperature the pitting was
much less marked. Pitting of grapefruit does not apparently develop
at high temperatures but occurs only on the cold-storage fruit. Grape-
fruits do not keep so long in common storage or warm storage as in cold
storage. There is much more loss from decay at the higher temperatures.
LITERATURE CITED
(1) Bigelow, W. D., and Gore, H. C.
1905. studies on peaches ... U. S. Dept. Agr. Bur. Chera. Bui. 97, 32 p.
(2) and Howard, B. J.
1905. studies ox apples ... U. S. Dept. Agr. Bur. Chera. Bui. 94, 100 p.,
30 fig., 5 pi.
(3) Chace, E. M., and Church, C. G.
I918. NOTES ON CALIFORNIA AND ARIZONA GRAPEFRUIT. Calif. CitTOgraph, V.
3, no. 9, p. 200-201.
(4) Tolman, L. M., and Munson, L. S.
1904. CHEMICAL COMPOSITION OF SOME TROPICAL FRUITS AND THEIR PRODUCTS.
U. S. Dept. Agr. Bur. Chem. Bui. 87, 38 p.
Dec. i, 1920 Some Changes in Florida Grapefruit in Storage 373
(5) COLLISON, S. E.
1913. SUGAR AND ACID IN ORANGES AND GRAPEFRUIT. Fla. Agr. Exp. Sta.
Bul. 115, p. 1-23.
(6) Magness, J. R.
1920. investigations in the ripening and storage of pears. In Jour.
Agr. Research, v. 19, no. 10, p. 473-500, 8 fig. Literature cited, p.
499-500.
(7) Mathews, Albert P.
1916. physiological chemistry . . . ed. 2, 1040 p. New York.
(8) Rose, R. E.
1914. report of THE chemical division [1913]. In Fla. Quart. Bul. Dept.
Agr., v. 24, no. 1, p. 1-218.
(9) Shamel, A. D.
1916. California grapefruit. In Mo. Bul. State Com. Hort. [Calif.], v. 5,
no. 7, p. 239-249, fig. 77-79.
(10) Wiley, H. W., ed.
1912. OFFICIAL AND PROVISIONAL METHODS OF ANALYSIS, ASSOCIATION OF OFFI-
CIAL agricultural chemists. As compiled by the committee on
revision of methods. U. S. Dept. Agr. Bur. Chem. Bul. 107 (rev.),
272 p., 13 fig.
(11) ZollER, Harper F.
1918. SOME CONSTITUENTS OF THE AMERICAN GRAPEFRUIT (CITRUS DECUMANA).
In Jour. Indus, and Engin. Chem., v. 10, no. 5, p. 364-374, 2 fig. Bib-
liography, p. 374-375-
A BACTERIOLOGICAL STUDY OF CANNED RIPE OLIVES
By Stewart A. Koser '
Bacteriologist, Microbiological Laboratory, Bureau of Chemistry, United States Depart-
ment of Agriculture
As a result of the first of the recent series of outbreaks of botulism trace-
able to the consumption of ripe olives infected with Bacillus botulinus,2
many lots of canned ripe olives were collected by inspectors of the Bu-
reau of Chemistry for bacteriological examination. These were obtained,
for the most part, from various retail and wholesale houses in all parts
of the country and bore the label of the same company as did those re-
sponsible for the fatalities. While the primary object of the investiga-
tion was the detection of the presence of Bacillus botulinus, this object
was extended to include a study of the types of microorganisms respon-
sible for the spoilage and also to determine whether viable microorganisms
might be encountered in apparently normal containers. The containers
subjected to examination included all sizes of both cans and glass jars.
Some were apparently normal while others were swelled or obviously
spoiled.
In the bacteriological examination of these samples the following pro-
cedures were adopted as a routine. All containers were opened with usual
aseptic precautions, and 1.5 to 2 cc. of the liquor were withdrawn by
means of a sterile pipette. Approximately 0.5 cc. of this was spread
over a dextrose agar slant (for aerobes) , and the remainder was then run
into a tube of infusion broth under oil. This medium was a 0.2 per cent
dextrose beef infusion broth (PH 7.4 to 7.6). It was covered before auto-
claving with a layer of liquid petrolatum. In place of this medium
there was occasionally used a 2.0 per cent dextrose-beef infusion broth,
similarly covered with a layer of oil and containing a small piece of meat.
In most cases a piece of olive was removed with sterile knife or forceps
and was transferred to the dextrose broth tube. Incubation was at 370
C. In addition, notes were kept on the condition of the container,
whether normal, swelled, etc., and also on the odor. Cans which were
obviously leaking were discarded.
It is realized that for the sake of completeness it would have been
desirable to have included a greater variety of culture media and several
1 The author wishes to express his appreciation of the valuable criticism and suggestions given by Dr.
Charles Thorn, of the Microbiological Laboratory.
* Armstrong, Chas., Story, R. V., and Scott, Ernest, botulism from eating canned ripe olives.
In Public Health Rpts., v. 34, no. 51, p. 2877-2905, 5 fig. 1919.
Jennings, Charles G., Haass, Ernest W., and Jennings, Alpheus F. an outbreak op botulism,
report OF cases. In Jour. Amer. Med. Assoc, v. 74, no. 2, p. 77-80. 1920.
Sisco, Dwight I,, an outbreak of botulism. In Jour. Amer. Med. Assoc, v. 74, no. 8, p. 516-521.
1920.
Journal of Agricultural Research, Vol. XX, No. 5
Washington, D. C. Dec. 1, 1920
Vy Key No. E-14
(375)
376 Journal of Agricultural Research vol. xx, no. s
temperatures of incubation. The number of samples, as well as the
urgency of the examination, however, forbade any elaborate series of
tests. The total number of cans and glass jars, both normal and spoiled,
which were cultured by various members of this laboratory, together
with the number showing the presence of living organisms, is summar-
ized as follows:
Experiment with cans
Exp. No.
I. Number of normal cans cultured 181
Of this number 173 were sterile, while 8, or 4.4 per cent, were found to
contain viable microorganisms.
II. Number of "swelled" or "springy" cans cultured . . 157
Of these 154 contained living microorganisms, while 3 were apparently
'sterile (2 of these 3 were "springers," the other was a "hard swell").
Total number of cans cidtured 338
Experiment with glass containers
Exp. No. . .
I. Number of containers normal in appearance and odor 116
Of this number 105 were sterile, while 11, 'or 9.5 per cent, revealed the
presence of living microorganisms.
II. Number of containers obviously spoiled or of bad odor 26
All of these 26 gave positive cultural results.
Total number of glass containers cultured 142
Total number of cans and glass containers cultured 480
Thus, it is seen that all the obviously spoiled glass jars, and, with one
exception, all the swelled cans revealed the presence of living micro-
organisms. On the other hand, the normal containers were, for the
most part, sterile. In this connection it is interesting to note that 4.4
per cent of the normal cans were found to contain viable organisms,
while in the normal glass containers the proportion was decidedly higher,
namely, 9.5 per cent. Of the 157 swelled or "springy" cans, all but
three gave positive cultural tests. Two of these three were "springers,"
due probably to imperfect exhausting, and were no doubt otherwise
normal. The third was a "hard swell." Whether the failure to obtain
living organisms from this one can was due to lack of a greater diversity
of culture media or whether the causative organisms had been killed as
a result of their own metabolic products is not known.
Of the total of 480 containers examined bacteriologically, 117 of those
which had yielded positive cultural tests were studied further to gain some
knowledge of the types of organisms present. As a rule, extensive cul-
tural and biochemical tests were omitted, and merely the general type or
group to which the organisms belonged was determined. A summary
of the types obtained from the 1 17 containers thus studied is shown below.
The figures indicate the number of times each was encountered.
Dec. i, 1920 A Bacteriological Study of Canned Ripe Olives 377
Types of organisms fou nd
Colon group 81
Colon group, sluggish liquefaction of gelatin {Bacillus cloacae) 4
Bacterium fl uorescens (liquefying) 2
Proteus 3
Other Gram-negative, non-spore-forming bacilli, not identified 5
Gram-positive, aerobic, spore-forming bacilli, gelatin liquefiers —
Bacillus cereus type 3
Bacillus mycoidcs type 4
Bacillus mesentericus type 6
Type not determined 19
Slender, Gram-positive, aerobic or facultative anaerobic bacilli, oval terminal
spores, gelatin not liquefied 10
Gram-positive diplococci 31
Gram-positive staphylococci 10
Spore-forming, obligate anaerobes 6
Yeasts 3
Mold (.4 spergillus terreus) ' 1
In addition to these, Bacillus bottdinus was found in 7 of the spoiled
glass jars. A report of the findings of this laboratory with respect to
Bacillus botulinus, both from the material obtained in the open market
and from specimens received from the poisoning cases, has been made
the subject of another paper.2
The large proportion of non-spore-forming organisms, particularly of
the colon group and the Coccaceae, was indeed surprising. Many of the
cultures of the colon group when first isolated exhibited a delayed
fermentation of lactose somewhat similar to that reported by Bronfen-
brenner and Davis,3 though not so marked. In lactose broth, acid
formation was delayed from 48 to 72 hours, while gas was produced
only after 3 to 5 days' incubation. After several successive transplants
in lactose broth, fermentation of this sugar was markedly accelerated.
Although some of the organisms obtained were placed without diffi-
culty in their proper groups, others were not identified by the limited
number of cultural tests employed, and these are designated in the fore-
going list by their chief cultural characteristics or morphology. Many
Gram-positive diplococci were encountered. These exhibited a distinct
lance-shaped appearance in liquid media, with occasional short chains of
three or four elements. On dextrose agar slants the individual colonies
appeared as minute white pin points. In beef infusion broth under oil
1 For identification of this species the writer is indebted to Dr. Margaret B. Church, of the Microbio-
logical Laboratory.
2DeBord, G. G., Edmondson, R. B., and Thom, Charles, summary of bureau of chemistry
investigations of poisoning due To ripe olives. In Jour. Amer. Med. Assoc, v. 74, no. 18, p. 1220-
1221, 1920
3 Bronfenbrenner, J., and Davis, C R. on methods of isolation and identification of the mem-
bers OF THE COLON-TYPHOID GROUP OF BACTERIA. LATE FERMENTATION OF LACTOSE. In Jour. Med.
Research, v. 39 (n. s. v. 34), nc. 1, p. 33~37- 1918.
378 Journal of Agricultural Research vol. xx, No. ^
the growth was fairly luxuriant, producing a distinct cloudiness after 24
hours' incubation. The other type of Gram-positive coccus encountered
grew more luxuriantly on plain agar slants and was found upon staining
to occur in irregular clusters. The several obligate anaerobes were inoc-
ulated into milk and into the meat medium of Holman.1 One culture
digested the meat with a distinct putrefactive odor. The remaining five
caused neither putrefaction of meat nor stormy fermentation of milk.
Dextrose was attacked with acid and gas production. Up to the present
time they have not been studied further.
Flora oe swelled cans. — The flora of swelled cans was found to con-
sist largely of members of the colon group, for of 85 swelled cans studied
this group was obtained from 75, and from 40 of these in apparently pure
culture. In the others they were found in mixed culture with the several
types of Coccaceae, the aerobic, Gram-positive, spore-forming bacilli, or,
more rarely, with an obligate anaerobe, with Proteus, or with a yeast.
In three instances spoilage, with resultant swelling of the can, was
evidently due to spore-forming anaerobes only. In one instance Proteus
was found in pure culture. A few of the swelled cans yielded cultural
results from which no evidence could be gathered as to the type of
organism causing gas formation within the can. Thus, an aerobic,
spore-forming, Gram-positive rod was the only type obtained from 2
swelled cans, while from 2 others Gram-positive cocci were obtained in
pure culture. Since none of these organisms attacked carbohydrates2
with gas production, it is evident that the gas-producer had disappeared
or was overlooked.
Normal containers. — As previously shown, 8 normal cans and 11
normal glass containers were found to contain living microorganisms.
Four of these 8 normal cans yielded cultures of the colon group. The
others contained cocci and several types of aerobic, spore-forming bacilli.
The finding of members of the colon group in 4 of the normal cans was
rather surprising. Evidently for some unknown reason the bacilli failed
to multiply to any extent in these cans. Without exception, the types
encountered in the normal glass jars were aerobic, spore-forming, Gram-
positive rods. Several were identified as Bacillus mesentericus and one
as Bacillus cereus.
Spoiled glass jars. — The flora of the spoiled glass jars was as a rule
more varied and complex than that of the swelled cans. The contents
of several jars were obviously spoiled and disintegrated to a mushy con-
sistency with a disagreeable odor, unrecognizable as that of olives. These
yielded a diversity of types of which the following are illustrative :
" Holman, W. L. the value of a cooked meat medium for routine and special bacteriology.
In Jour. Bact., v. 4, no. 2, p. 149-155. 1919- References, p. 155.
8 Chemical analyses by the Food Control Laboratory of the Bureau ol Chemistry showed the liquor in
which the olives were packed to contain from o. 16 to 0.23 per cent reducing sugars after inversion, expressed
as percentage of invert sugar.
Dec. i, 1920 A Bacteriological Study of Canned Ripe Olives 379
1 . Putrefactive anaerobe which digested a cooked meat medium with
a putrefactive odor, an aerobic Gram-positive, spore-forming rod, and an
unidentified Gram-negative bacillus.
2. Bacterium fluorescens liquefaciens , Proteus, aerobic Gram -positive,
spore-forming bacillus, and an unidentified non-gas-producing Gram-
negative bacillus.
3. Staphylococcus, a yeast, and Gram-positive, sporing bacillus.
4. Gram-positive diplococci, colon group, Aspergillus terreus, and a
Gram-positive, spore-forming rod.
No definite correlation between the odor of the spoiled samples and the
type of organism contained therein was noted. The swelled cans from
which the colon group only was obtained were recorded as possessing
either a flat or slightly "off" odor — that is, they lacked the character-
istic fragrant aroma of the first-class product. Since many of the sterile
normal cans, particularly of certain brands, had a similar odor, it is
doubted whether this condition can be ascribed solely to the metabolic
activities of the colon group. Three cans containing spore-forming
anaerobes possessed a disagreeable or rancid odor. The liquor, together
with portions of the olives from several of the most offensive cans, was
fed to guinea pigs without ill effects.
The large numbers and diversity of types encountered, particularly of
the non-spore-formers, point to insufficient heating of the product. While
it is realized that there may be a slight leakage along the seam of the can
immediately after heating, and with subsequent closure, it would seem
improbable that this could account entirely for the results obtained in
this investigation.
SUMMARY
(1) In the bacteriological examination of 480 commercial containers
of ripe olives, living microorganisms were obtained in practically every
instance from samples which were abnormal, as indicated either by a
swelled condition of the container or a bad odor.
(2) Viable microorganisms were found in a small percentage of normal
containers. These were either aerobic, spore-forming bacilli, cocci, or
apparently dormant members of the colon group.
(3) A study of the organisms encountered in the spoiled samples
showed a great diversity of types, among which the colon group pre-
dominated.
RELATION OF THE SOIL SOLUTION TO THE SOIL
EXTRACT
By D. R. Hoagland, J. C. Martin, and G. R. Stewart
Division of Agricultural Chemistry, California Agricultural Experiment Station
Modern views of soil fertility recognize the general principle that
plants derive their immediate supply of inorganic elements entirely from
the soil solution. It has also been proved that the soil solution is subject
to highly significant fluctuations. The concentration and composition
of the soil solution may undergo very great alterations as a result of
seasonal changes, crop growth, activities of microorganisms, rainfall, fer-
tilization, etc. The evidence supporting this point of view is now too
strong to admit of any doubt. It is justifiable to assume, therefore, that
further progress in the study of the soil as a medium for plant growth
will depend upon an increased knowledge of the soil solution, particularly
in its dynamic relations to the soil mass, to the plant and microorganisms,
and to the application of fertilizing materials.
Experimental work on the soil solution immediately encounters a
formidable obstacle in the difficulty of separating from the soil the solu-
tion which provides nutriment to the plants. When the soil contains
moisture in percentages most suitable for plant grov/th, the solution is
held by the soil particles with such force that no ordinary means will
serve to effect a separation. This fact is well recognized, and various
attempts have been made to overcome the difficulties involved and to
obtain the soil solution in an unmodified state. The most important
developments in this phase of the work have been described by Morgan
(8) l and by C. B. L,ipman (7). It is yet too early to state any final con-
clusions based on data obtained by these methods, but their further per-
fection may lead to the attainment of most essential information. A
considerable advance in our ideas concerning the soil solution has already
resulted from the application of the freezing-point method to soils, as
first described by Bouyoucos and McCool (4). However, the study of
the soil solution in its relation to plant growth is so fundamental that it
should be approached from every possible angle, with the hope that
eventually we may possess an adequate understanding of the nature of
the nutrient medium in the soil and of the modifications produced in this
medium by various treatments.
Since the soil solution is constantly undergoing modification, the inves-
tigator is often required to make numerous determinations at frequent
1 Reference is made by number (italic) to " Literature cited," p. 394~395-
Journal of Agricultural Research, Vol. XX No. 5
Washington, D. C Dec. 1, 1920
w Key No. Calif. -25
(38l)
382
Journal of Agricultural Research
Vol. XX, No. 5
intervals during the growing season at least. This introduces certain
practical difficulties in the application of presssure methods. The
freezing-point method is most rapid and convenient as a means of study-
ing the approximate total concentrations , but it can not give any informa-
tion concerning the individual solutes. The method of water extraction
has been used rather frequently in . past investigations with the in-
tent to determine the amounts of plant foods available to the plant.
One of the writers (9) has carried out an extensive investigation in which
.02s
JOZO
.o/s{ r-
.0/0
.005
\
\
,-°"
P
ws
b-
• -o-
--0-.
-a
*
<o 3 3 sr
Fig. i. — Graph showing relation of freezing-point depressions in soil (calculated to 22 per cent moisture)
to total solids extracted by 5 parts of water to 1 of soil. Individual data from six soils composited.
the very significant effect of season and crop growth on water extracts
of soils was made clear. At the same time the freezing-point method of
Bouyoucos and McCool was applied to the soils under investigation, and
a general agreement was noted between the values obtained by this
method and by the water-extraction method (6). Thus the effect of the
crop in diminishing the concentration of the soil solution was definitely
shown by both methods. At the present time the study of water ex-
tracts offers such promise that it has seemed highly important to attempt
Dec. 1. 1920 Relation of the Soil Solution to the Soil Extract
383
to throw further light on the relation between the soil extract and the
soil solution. The value of the determination made by the water-
extraction method rests primarily on the assumption that a logical rela-
tionship exists between water extracts and the soil solution.
In the articles referred to above considerable data were presented to
show that in general the larger fluctuations in the total solids found in
1 to 5 water extracts occurred coincidently with similar fluctuations in
the freezing-point depressions of the moist soil. Later much more ex-
.020
.0/5
jO/O
.005
.00+
^
\ ^_ o^Zj^^o
./
\
^Sato
\ \
J*
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co w jo
Fig. 2. — Graph showing relation of fre,ezing-point depressions in soil (calculated to 17 per cent moisture)
to total solids extracted by 5 parts of water to 1 of soil. Individual data from seven soils composited.
tensive observations were made on this important question, and the data
have been plotted in two graphs (figs. 1,2), one for the group of silty
clay loam soils and the other for the various fine sandy loams, the soils
being the same as those described in a previous article by Stewart (9).
The data for the individual soils have been composited for the present
paper. In these graphs the determinations of total solids and freezing-
point depressions are plotted for various time intervals, for the soils in
both the cropped and uncropped condition. The correlation between
16916°— 20 i
384 Journal of Agricultural Research voi.xx.No. s
the curves for total solids and freezing-point depressions is on the whole
excellent, considering the technical difficulties involved. Chief among
these is the uncertainty concerning the free and unfree water in the soil,
which, as Buoyoucos (1) has clearly shown, markedly affects the concen-
tration of the soil solution. While all the values have been calculated
to the same moisture basis, it is not to be expected that this can be done
with a high degree of accuracy, since the percentage of unfree water may
vary with different moisture contents and perhaps with different concen-
trations of the soil solution. In both groups of soils there is a somewhat
marked divergence between the curves for total solids and freezing-
point depressions at a period beginning about 10 weeks after planting
the crop. This can reasonably be explained on the basis of certain
observations reported in former articles (6, 9). It was shown in these
that a larger quantity of very slightly soluble material was dissolved
from a soil by a given proportion of water when the soil solution had
reached a low concentration as a result of absorption of solutes by the
plant. At a certain period, therefore, the cropped soil will yield a higher
percentage of dissolved material (not part of the actual soil solution) as
compared with earlier periods. This means that the extractions of the
cropped and uncropped soils are not on exactly the same basis at all
times, and it might be predicted that at the period of low concentration
in the cropped soil the proportion of dissolved substances would increase.
The inference is substantiated by the experimental data. This generally
neglected phenomenon of the effect of the solutes already present in the
soil solution in depressing the solubility of substances dissolved from the
soil mass by water is thought to be of considerable importance in all
studies on soil equilibria by means of water extracts. Finally, it should
be emphasized that at no time is there any indication that conclusions
based on the water extracts would lead to an erroneous estimate of the
general relation between the soil solutions of cropped and uncropped
soils. As the authors have pointed out before, the actual differencse
would tend to be of greater magnitudes than those calculated from the
results on water extracts.
When a 1 to 5 extract of soil is made with distilled water, the quan-
tity of total solids is from 1.5 to 5 times that present in the soil solution,
as calculated by the freezing-point method. By the latter method we
can calculate the total concentration in the soil solution; but this does
not enable us to determine whether or not the relation between the ele-
ments in the soil solution is at all similar to that in the soil extracts.
Another type of experiment is necessary to give evidence on this point.
It war suggested that such evidence might possibly be obtained by
determining the concentration and composition of a solution which
would remain unchanged when in contact with the soil mass. In other
words, if one passed through a sample of moist soil a solution having the
Dec. i, 1920 Relation of the Soil Solution to the Soil Extract 385
same concentration and composition as the soil solution already present,
then it may be assumed that the resultant extract would have the same
composition and concentration as the original solution. On the other
hand, if the solution used were of different concentration or composition
a readjustment of the equilibrium should take place so as to produce a
different extract. It was decided to attempt an experiment based on
this hypothesis.
Obviously, the preparation of a solution having the same composition
and concentration as the soil solution is a matter of great difficulty.
The only feasible scheme seemed to be the use of a soil extract con-
centrated to a point where it would have the same concentration as the
soil solution, this concentration being determined by the method of
Bouyoucos and McCool. It was reasonable to assume that in such a
solution there would exist, between some of the most important elements,
a relation very similar to that found in the actual soil solution— that is,
the solution of the free water with the soil at approximately optimum
moisture content. In order to limit as far as possible the quantity of
solutes dissolved from the soil mass, an extract was made with cold
water, and only X part of water was used to 1 part of soil. The time of
contact was limited to that necessary for complete admixture. Filtra-
tion was made through a Buchner funnel, and final clarification was
effected with the use of a Pasteur filter. A separate portion of the soil
was then made up to its optimum water content, and the freezing-point
depression was carefully determined. The extract of the soil made in
the manner described was then concentrated on a hot plate, meanwhile
passing through the solution a stream of carbon-dioxid gas in order to
prevent any precipitation. Finally the volume of the concentrated ex-
tract was adjusted with distilled water so that it had exactly the same
freezing-point depression as that of the moist soil. This solution was
used in extracting the moist soil (1 part of soil to }4 part solution). Care-
ful analyses were made of the extract before and after contact with the
soil, and the results were compared.
Before the data are considered it should be recalled that ordinarily in
a water extraction from 2 to 5 times as much total solids are dissolved
as are actually present in the soil solution, and this is true with the
extractions now considered. Under certain conditions, however, it is
possible to obtain an extract which contains a comparatively small
quantity of dissolved substances in addition to that originally present
in the soil solution, as indicated by the method of Bouyoucos and McCool.
For example, a sample of soil 9, having a freezing-point depression of
0.1480 C. at 17 per cent moisture gave in a 1 to yi extract only about 1.16
times the quantity of total dissolved solids equivalent to this depression.
In this case the unfree water was determined directly by dilatometer
measurements (1). Such a result apparently can be obtained only with
386 Journal of Agricultural Research vol. xx. no. s
a soil having a low percentage of colloidal material and having a fairly __
high concentration in its soil solution, which exercises a repressive effect
on the solubility of certain soil constituents as previously explained.
In Table I the results of the equilibrium studies with three different
soils are presented. Comparisons are made between the composition of
the concentrated extracts and the same extracts after treatment with
the soil. It will be noted that the total concentration has suffered prac-
tically no change, as shown by the freezing-point depressions, conductivity
determinations, and proportion of total solids. Also, the concen-
trations of potassium, magnesium, calcium, nitrate, and sulphate agree
within the limits of experimental error. The agreement for sodium is
less perfect, but considering the small quantities involved the differences
are also probably within the limits of error. In one case more phosphate
is found in the re-extract, and in two cases the agreement is fairly close.
In one case the two silica determinations agree almost perfectly, and in
two cases silica seems to have been retained by the soil. It is very
difficult to explain the action of this radicle, first because of the chance of
contamination of the solution from glass vessels and secondly because of
the numerous types of silicates possible with varying proportions of
silica.
While the agreement between. the extracts and re-extracts is on the
whole remarkably close, it might be objected that the conditions for the
attainment of equilbrium were inadequate and that another extract
having a different composition might also remain unchanged by the soil.
In order to test this possibility extracts were made of soils 9 and 15 in
the previously described manner, and then potassium sulphate was added
to the extracts so as to double approximately the concentration of
potassium. These modified extracts were then concentrated until they
had the same osmotic value as the soil solutions, and re-extracts were
made as in the first experiment. The composition of the different
solutions is given in Table II. It is evident that in this experiment the
soil has had a marked effect on the extract. There is very much less
potassium in the re-extract than in the original extract, but the decrease
of potassium is accompanied by an increase in the quantity of calcium
and in one case of sodium. In one case there is a slight decrease of
sulphate. The other elements are not greatly changed, nor is the total
concentration very different in the two cases. It seems clear that a
rearrangement of the solutes has taken place in this case which did not
occur in the first experiment. In other words, the extract introduced
was different in composition from the soil solution already present, with
the result that certain chemical reactions took place forming an entirely
new soil mass — soil solution svstem.
Dec. i, 1920 Relation of the Soil Solution to the Soil Extract
387
1
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388
Journal of Agricultural Research
Vol. XX, No. 5
Table II. — Extraction of soil with concentrated extract containing added potassium
sulphate (K2S04)
Soil
No.
Description of extracts.
Composition of extracts.
Total
solids.
Potas-
sium
(K).
Cal-
cium
(Ca).
Mag- So-
nesium dium
(Mg). (Na).
Ni-
trate
(NOs).
Phos-
phate
(POO.
Sul-
phate
(SO<).
Silica
(SiOj).
Concentrated extract plus
potassium sulphate
Same after passing through
soil
Increase or decrease in con-
centration
Concentrated extract plus
potassium sulphate
Same after passing through
soil
Increase or decrease in con-
centration
P. p.m. P.p.m.
1,652
1,896
+ 244
2.408
2,592
+ 184
76
52
- 24
277
119
P.p.m.
212
280
+ 68
236
300
P.p.m.
33
31
— 2
41
41
-158 + 64
P.p.m.
122
129
+ 7
72
109
+ 37
P.p.m.
312
333
+21
P.p.m.
+ 1
4
3
— 1
P.p.m.
166
161
- 5
413
369
— 44
P.p.m.
40
40
All the experiments just discussed would seem to justify the conclusion
that in a concentrated extract the relation between the various elements
may be very similar to that existing in the soil solution. In seeking
an explanation of the results it is first essential to describe the components
which probably enter into a water extract of a soil. In the first place,
these would include the constituents of the soil solution diluted by the
added water. This diluted soil solution would then tend to bring into
solution constituents which were not present in the soil solution itself.
Thus, in no case would the solvent be pure water but rather a solution
the composition and concentration of which would vary with the soil
solution. It is plausible to assume that the solvent thus formed would
bring into solution principally either "adsorbed" salts or easily soluble
chemical compounds, originally derived from the more resistant minerals.
Finally, a certain quota of this very slightly soluble material would come
into solution, and the total quantity dissolved would depend at least in
part on the total volume of water as well as on time and temperature.
This latter fraction of the soil extract would ordinarily form only a small
portion of the total dissolved material. Evidence for this view has been
presented previously (6, g) and is also upheld by certain experiments of
Bouyoucos with regard to the solubility of soil minerals (2). It would
follow, therefore, that if the adsorbed or immediately soluble material
has the same relative composition as that already present in the soil
solution, then the water extract might also retain similar relations. It is
impossible at present to obtain direct evidence to this effect, but an
experiment was carried out from which certain inferences may be drawn.
A large quantity of moist soil (silty clay loam 1) was placed in a
Buchner funnel and leached with the least possible proportion of distilled
water, about y& part of water to 1 part of soil. Two subsequent leach-
ings were made with similar proportions of water. These three extracts
were then analyzed for the most important elements, and the ratios
Dec. i, 1920
Relation of the Soil Solution to the Soil Extract
389
between them were calculated by dividing the concentration of each
element by the sum of the concentrations of all the elements determined
(Table III). The ratios were found to be very similar in the three
extracts, the agreement for several elements being especially close in the
first two extracts. In the first extract the larger proportion of solutes
present were probably derived from the soil solution, while the subsequent
extracts represented to a greater degree previously undissolved fractions
of the soil. The results would, therefore, seem to indicate that in con-
centrated extracts there is a great similarity in composition between the
soil solution and the extract containing the substances which immediately
go into solution on the addition of a slight excess of water. Even with
nitrate, which might be supposed to have such a high degree of solubility
that the total quantity present would be contained in the soil solution,
it is probable that a certain proportion is held in some adsorbed or
undissolved form. If an extract of the soil be made, a readjustment
takes place because of the great dilution of the soil solution, and the
total quantity of adsorbed nitrate would be greatly diminished, even
though the partition ratio between solution and soil remained constant.
Thus, it is possible to extract nearly all the nitrate present but difficult
or impossible to remove the last traces.
Table III— Composition of successive teachings of soil
[8 parts soil to 1 part water.]
Solute.
First leaching.
Second leaching.
Third leaching.
Concentra- individuai
tionot flutes to
solution. totaL
^° A Concentra- JS£&£, Concentra-
Nitrate (NOs) . .
Calcium (Ca) . . .
Magnesium (Mg)
Potassium (K). .
Sulphate (SO,).
P. p. m.
42 5
90
Per cent.
57- °
12. 1
11. 8
3-°
16. 2
tion of
solution.
P. p. m.
195
45
43
15
67
individual 1 tkm of
solutes to s^ution.
total.
Per cent.
53- °
12.3
11. 8
4.1
18.4
P. p. m.
133
46
34
13
66
Ratio of
individual
solutes to
total.
Per cent.
46. O
IS-8
11. 6
4.4
22.6
With regard to phosphate the case is not so clear. Most of the extrac-
tion studies described in previous articles have indicated that the various
extracts are saturated with respect to phosphate. Thus, if the extract
were concentrated without precipitation the concentration of phosphate
should be considerably greater than in the soil solution. Since, however,
the adsorption or precipitation of phosphate by the soil is a relatively
slow process, in the present experiment the time may have been insuffi-
cient for readjustment of the equilibrium. From our previous experi-
ments we should be inclined to infer that the concentration of phosphate
in the soil solution is usually very low, but that immediate replacement
occurs as phosphate is absorbed by the plant, thus producing a constant
concentration of phosphate over long periods of time.
390 Journal of Agricultural Research vol. xx. No. s
That there exists some sort of definite and reversible state of equilib-
rium between the soil mass and the soil solution for any given set of
conditions is suggested by another experiment. Two soils were treated
with water in the proportion of i part of dried soil to i part of water.
After the soil and water were thoroughly mixed the resultant mixtures
were allowed to dry at room temperature until they reached the optimum
moisture content. Freezing-point depressions were then made and com-
pared with determinations made on samples of the same soils simply
moistened to optimum water content. The data given below show
that the agreement is, at least in these two cases, almost perfect.
Table IV. — Freezing-point depressions of soil at optimum moisture content and of
treated soil evaporated to optimum moisture content
Description of soil.
Soil iC at optimum moisture content
Soil iC after mixing i to i with water and allowing to evaporate to optimum
moisture content
Soil 9 at optimum moisture content
Soil 9 after mixing i to i with water and allowing to evaporate to optimum
moisture content
Freezing-
point
depressions.
' C.
O.063
. 062
•045
.047
In other words, although several times as much material was brought
into solution as was contained in the soil solution at optimum water
content when the excess water was added, these dissolved substances
were immediately removed from solution on lowering the moisture con-
tent. This, of course, does not mean that the concentration of the soil
solution may not easily be altered by the addition of soluble salts, as will
be discussed presently.
If the general method of studying soils by means of their water extracts
is of value, then it becomes of considerable importance to determine the
most suitable conditions for making the extract. The technic might be
based on either one or two general objectives, first the attainment of
equilibrium (as nearly as possible final) for a given proportion of water,
and, second, the limitation of the extract as far as was practicable to the
material actually existing in the soil solution. In the first case a long
period of contact and continuous shaking would be essential; in the
second case the time would be limited to that necessary for complete
admixture of soil solution and added water. In order to determine the
magnitudes of dissolved substances under varying conditions, extracts of
3 soils were made by various methods as follows : (a) 1 part soil to 5 parts
water, as described by the Bureau of Soils of the United States Depart-
ment of Agriculture; (b) 1 part soil to 5 parts water, shaking for 1 week;
(c) 1 part soil to 1 part water, as in (a) ; (d) 1 part of soil to 1 part water,
shaking for 1 week.
Relation of the Soil Solution to the Soil Extract
39i
In Table IV the results on extracts obtained by these different methods
are presented, all calculated to parts per million of the dry soil, so that
comparisons may be made on the same basis.
If the total solids are considered, it will be noted that the magnitudes
are very similar except in the case of the 1 to 5 extract shaken for 1 week.
More potassium is extracted by a 1 to 5 extract than by a 1 to 1 extract,
but the quantities are essentially the same whether the time is 40 minutes
or 1 week. The calcium, magnesium, and sulphate may be appreciably
increased during the week's contact when the proportion is 1 to 5 but
not when the proportion is 1 to 1 . Nitrate is not greatly changed in a 1
to 5 extract by the increased time of extraction. In the 1 to 1 extract in
one case of a heavy- textured soil there is a decrease after 1 week, and in
another case of a light-textured soil there is an increase. Very probably
biological action is concerned in these changes. Phosphate is increased
markedly in the 1 to 5 extract as compared with the 1 to 1 extract.
Several fairly definite deductions may be drawn from the data just
presented. When a smaller proportion of water to soil is used, as 1 to 1 ,
there is only slight increase in dissolved substances with the period of 1
week as compared with a shorter period, although some changes in nitrate
may result from biological action. There would not seem, therefore, to
be any advantage in the longer period of contact; in fact the biological
changes would make such a procedure undesirable. In the 1 to 5 ex-
tracts there is a significantly increased solution of various elements (par-
ticularly calcium and magnesium) during the period of a week. This
must be due to the solution of soil minerals, more of which are dissolved
in the 1 to 5 extract because of the greater dilution of the solvent, as
previously explained. Phosphate is in a somewhat different category
from the other elements in that the total quantity dissolved is some-
what directly dependent upon the volume of the solvent. As was stated
before, to a certain extent the solution is always saturated with respect
to phosphate.
Table V. — Comparison of extracts of soil prepared by various methods
Soil
No.
Time of extraction.
Ratio of
soil to
water.
Composition of extracts calculated to basis of water-free soil.
Total
Solids.
Potas-
sium
(K).
Cal-
cium
(Ca).
Magne-
sium
(Mg).
Nitrate
(NOs).
Phos-
phate
(PO4).
Sul-
phate
(SO4).
40 minutes
1 week
40 minutes
1 week
40 minutes
1 week
40 minutes
1 week
40 minutes
1 week
40 minutes
1 week
P. p. m.
680
612
636
l>°34
SIO
562
5°3
806
532
524
S82
836
P. p.m.
24
26
38
P. p. VI.
62
47
69
P. p.m.
26
P. p. m.
80
54
128
126
68
114
114
128
78
63
116
145
P. p. m.
P. p. m.
60
79
392 Journal of Agricultural Research vol. xx. no. $
The application of the foregoing conclusions would seem to indicate
that soil extracts should be made with a small proportion of water and
for a short period. It would probably be desirable to use not more than
i part of water to i part of soil, but in many cases this may be imprac-
ticable, so that i to 5 extracts must suffice. It is true that special studies
of soil equilibria must take into account long-continued solvent action,
but in attempts to gain some idea of periodic changes in the soil solution
the technic should be directed toward lessening the solution of material
not actually present in the soil solution. This aim is less possible of
attainment in proportion as the volume of water or time of contact with
the soil is increased. It is not evident that attempts to reach approxi-
mate final equilibrium by large excess of water or long shaking are likely
to result in more accurate knowledge of the soil solution as it exists at
any given moment. On the contrary, the increase in solutes is derived
from substances not actually present in the soil solution, and their
solubility is in part conditioned on the concentration of the soil solution,
the variable under investigation.
In concluding this discussion it may be well to summarize briefly our
present point of view with regard to the soil solution based on recent
researches in this and other laboratories. All the evidence supports the
general views expressed by Cameron (5) a number of years ago to the
effect that soil phenomena must be considered as dynamic. His criti-
cisms of the older methods of study by means of hydrochloric-acid
extracts of soils, analyses of total quantities present in the soil, etc., are
found to be entirely justified. It is now generally recognized, however,
that Cameron's conclusions with regard to the nature of the soil solution
were not sufficiently far-reaching. It is certain that the soil solution is
not simply a solution saturated with respect to all the original mineral
components of the soil and tending to approach a constant composition.
The original soil minerals themselves doubtless have a very slight solu-
bility in pure water, but the soil solution of a normally occurring soil is
something quite different. The solvent is never pure water, but rather
a solution of salts and organic matter, accompanied by carbon dioxid,
oxygen, and other gases. The soil solution at any given moment is the
resultant of the cumulative effect of the continuously varying solvent on
the soil minerals. The actual concentration of the solution is governed
principally by the equilibria existing between the dissolved substances
and the immediately soluble or absorbed substances. It is possible that
these latter may be removed almost completely from the soil mass by an
excess of water. The soil solution in contact with the residual soil has a
very low concentration, and this is not readily increased by the solvent
action of pure water. To a lesser degree a similar state of affairs results
when the dissolved or immediately soluble components of the soil are
removed by a crop. This effect may be of long duration, or, on the other
hand, the concentration of the soil extract with respect to many solutes
Dec. i, 1920 Relation of the Soil Solution to the Soil Extract 393
may easily be increased by the addition of soluble salts. Bouyoucos and
Laudeman (j) have shown, moreover, that this increase of concentration
occurs immediately and in most cases is not altered over a long period
of time.
Theoretically, also, it is very apparent that the soil solution or extract
may be increased in its concentration of a given element by the addi-
tion of a soluble salt. A simple case will illustrate this fact. A saturated
solution of slightly soluble silicates of potassium, for example, can be
prepared by shaking the finely divided minerals with water. The con-
centration of potassium in the solution is limited by the solubility of
the components of this particular system. However, the addition of
another component of different solubility, such as potassium chlorid,
will increase the concentration of potassium in the solution, although
the solubility of the potassium silicate may possibly be diminished
because of the increased concentration of the potassium ion. In the
same way the soil solution is saturated only with respect to the particular
system existing at any given moment. In general it is not saturated
with respect to any particular ion, so from theoretical considerations
there is no reason to accept the earlier statements of Cameron that the
chemical equilibria would require the precipitation of added salts with
a tendency to maintain a constant composition in the soil solution.
The fact that water extracts of soils become more dilute with each
increase in the proportion of water used gives evidence to show that
the solubility of the original soil minerals is not the chief factor govern-
ing the concentration of the soil solution.
Presumably in the actual soil solution the increase of concentration
due to the addition of soluble salts will in part be limited by the removal
from the dissolved to the absorbed phase. When an excess of water
is employed, however, as in making an extract, nearly all of the added
solutes will appear in solution or be represented by equivalent quantities
of other substances, as is shown, for example, in the well-known exchange
of bases. The total quantity of absorbed substances would be a func-
tion of the concentration of the surrounding solution, which would
vary with the moisture content of the soil or volume of water used in
making an extract. In extraction procedures there would occur, of
course, a very great dilution of the soil solution. While the latter
would be increased in concentration by the addition of soluble salts,
the evidence at hand does not indicate that all the added salt would
necessarily be effective in increasing the concentration of this soil solu-
tion even when the water extracts contained the total or equivalent
quantities of the elements added. It is reasonable to assume, however,
that the "adsorbed" substances are capable of easily replenishing the
soil solution when its concentration is decreased as a result of withdrawals
by the plant, new soil solution-adsorption systems being formed con-
tinuously during the season.
394 Journal of Agricultural Research vol. xx, no. 5
SUMMARY
(i) Seasonal studies on cropped and uncropped soils have shown that
water extracts reflect the principal fluctuations taking place in the soil
solution as indicated by the freezing-point method.
(2) A soil extract is composed chiefly of the solutes present in the
soil solution plus substances dissolved from "adsorbed" or easily soluble
components of the soil. This latter fraction of the soil extract is depend-
ent in part on the concentration and composition of the soil solution,
since the solutes of the latter exert a depressing effect on the solubility
of certain soil constituents. This fact is believed to be of great impor-
tance in studies of chemical equilibria in soils.
(3) A new method is suggested for indicating the relations between
the chemical elements in the soil solution. Extracts were prepared
which did not change appreciably in composition or concentration on
contact with the soil. The consideration of the equilibria involved sug-
gests the probability that the ratios between most of the important
elements are very similar in concentrated soil extracts and in the soil
solution. It is concluded that analyses of suitable soil extracts and
determinations of freezing-point depressions may frequently permit a
calculation of the concentration and approximate composition of the
soil solution.
(4) Various methods of making water extracts have been compared.
The data obtained suggest that in seasonal studies extracts should
be made with the smallest proportion of water to soil practicable and
with the time of contact limited to that necessary for thorough admix-
ture. In routine work 1 to 1 or 1 to 5 extracts are convenient and
satisfactory.
(5) Further experimentation has confirmed previous conclusions that
the soil solution fluctuates in composition and concentration with every
environmental change and with crop growth.
LITERATURE CITED
(1) Bouyoucos, George J.
1917. CLASSIFICATION AND MEASUREMENT OF THE DIFFERENT FORMS OP WATER
IN THE SOIL BY MEANS OF THE DILATOMETER METHOD. Mich. Agr. Exp.
Sta. Tech. Bui. 36, 48 p., 5 fig.
(2)
1919. RATE AND EXTENT OF SOLUBILITY OF SOILS UNDER DIFFERENT TREAT-
MENTS and conditions. Mich. Agr. Exp. Sta. Tech. Bui. 44, 49 p.
(3) and Laudeman, W. A.
1917. the freezing-point method as a new means of studying velocity
OF reaction between soils and chemical agents and behavior
of equilibrium. Mich. Agr. Exp. Sta. Tech. Bui. 37, 32 p.
(4) and McCool, M. M.
1916. the freezing-point method as a new means of measuring the con-
centrations OF THE SOIL SOLUTION DIRECTLY IN THE SOIL. Mich. Agr.
Exp. Sta. Tech. Bui. 24, p. 592-631,-2 fig.
Dec. i, 1920 Relation of the Soil Solution to the Soil Extract 395
(5) Cameron, F. K.
191 1. the son. solution. 136 p., 3 fig. Easton, Pa.
(6) Hoagland, D. R.
1918. THE FREEZING-POINT METHOD AS AN INDEX OF VARIATIONS IN THE SOIL
solution due To season and crop growth. In Jour. Agr. Research,
v. 12, no. 6, p. 369-395, 8 fig. Literature cited, p. 394-395.
(7) Lipman, Chas. B.
1918. A NEW method of extracting the soil solution, (a preliminary
communication.) In Univ. Cal. Pub. Agr. Sci., v. 3, no. 7, p. 131-134.
(8) Morgan, J. Franklin.
1916. THE SOIL SOLUTION OBTAINED BY THE OIL-PRESSURE METHOD. Mich.
Agr. Exp. Sta. Tech. Bui. 28, 38 p., 5 fig.
(9) Stewart, Guy R.
1918. EFFECT OF SEASON AND CROP GROWTH IN MODIFYING THE SOIL EXTRACT.
In Jour. Agr. Research, v. 12, no. 6, p. 311-368, 24 fig., pi. 14. Litera-
ture cited, p. 364-368.
EFFECT OF SEASON AND CROP GROWTH ON THE PHYSICAL
STATE OF THE SOIL
By D. R. Hoagi.and and J. C. Martin, Division of Agricultural Chemistry, California
Agricultural Experiment Station
Investigations previously reported by this laboratory x have shown
definitely that the soil solution is extremely variable in its composition
and concentration, as indicated by water extracts or by the freezing-
point method of Bouyoucos and McCool.2 Recently McCool and Millar3
in an extensive series of field studies have upheld this conclusion. In all
these investigations it has been demonstrated that the absorption of
solutes by the plant may have a very pronounced influence on the soil
solution at certain periods and may bring about a very striking decrease
in the concentration of nitrates and other constituents. Moreover, this
condition may persist for a long time. During the course of our experi-
ments it was noted that the state of dispersion of the colloidal matter in
the various soils fluctuated in a most decided manner under the influence
of the different treatments. It was decided, therefore, to make a sys-
tematic series of observations relating to this point.
The soils used were kept under controlled conditions in tanks as
described by Stewart.4 Both cropped and uncropped soils were com-
pared under otherwise identical conditions. The principal measurements
were made on a number of tanks of silty clay loam soil, clay in which vari-
ous crops were grown — namely, corn, barley, potatoes, beans, and beets.
There were three tanks of barley, containing, respectively, 24, 50, and 71
plants. All soils were kept at approximately optimum moisture con-
tent by the addition of distilled water. At frequent intervals during
the growth of the crops samples of soil were taken for examination.
In order to study conveniently the changes in the water-soluble con-
stituents, conductivity measurements were made on water extracts of
the soil. These were made by thoroughly mixing 1 part of moist soil
with 2 parts of distilled water and filtering through filter paper. This
1 HOAGLAND, D. R. THE FREEZING-POINT METHOD AS AN INDEX OF VARIATIONS IN THE SOU, SOLUTION
DUE TO season and crop growth. In Jour. Agr. Research, v. 12, no. 6, p. 369-395, 8 fig. 1918. Literature
cited, p. 394-395-
McCool, M. M., and Millar, C. E. soluble salt content of soils and some factors affecting
IT. Mich. Agr. Exp. Sta. Tech. Bui. 43, 47 p., 4 pi. 1918.
Sharp, L. T. salts, soil-colloids, and soils. In Proc Nat. Acad. Sci., v. 1, no. 12, p. 563-568. 1915
Stewart, Guy R. effect of season and crop growth in modifying the son. extract. In Jour.
Agr. Research, v. 12, no. 6, p. 311-368, 24 fig., pi. 14. 1918. Literature cited, p. 364-368.
2 Bouyoucos, George J., and McCool, M. M. the freezing point method as a new means of meas-
uring the concentration of the son. solution directly in the son,. Mich. Agr. Exp. Sta. Tech.
Bui. 24, p. 592-631, 2 fig. 1916.
8 McCool, M. M., and Millar, C E. op. cit.
4 Stewart, Guy R. op. err.
Journal of Agricultural Research, Vol. XX, No. 5
Washington, D. C Dec. 1, 1920
v w Key No. Calif. -26
(397)
398
Journal of Agricultural Research
Vol. XX, No. s
method gives results of the same relative values as those obtained by
determining the total solids in water extracts or by estimates based on
depressions of the freezing point in the soil itself. It is justifiable to
assume that the conductivity measurements give at least a rough idea of
the changes taking place in the soil solution under the various conditions.
8
16
32
40
48
56
64
72
80
88
96
/04-
m
tzo
24
32
40
48
56
64
72
80
88
36
/04
112.
/SO
C/?OZ=> TURN /PS
CROR HORSE BERNS
»» w <»> $ <0
WEEKS
Fig. i. — Effect of crop on physical state and electrolyte concentration of the water extract of the soil.
Unfortunately no methods exist which permit the determination of the
exact kinds or quantities of colloidal matter in the soil. We can measure
only approximately certain resultant effects by the use of empirical pro-
cedures. Of these, turbidity observations are doubtless as valuable as
any others. In the present experiments the samples of soil were mixed
with water, in the proportion of i part of soil to 2 parts of water, and the
Dec. i, 1920
Effect of Season and Crop Growth on Soil
399
soil suspensions were poured into burettes. After 24 hours the upper
10 cc. were carefully pipetted off into weighed dishes, and the total
residue was estimated after evaporation and drying at ioo° C. While
such a method unquestionably leaves much to be desired, it is neverthe-
less apparent that considerable changes in the colloidal state of the finer
24-f=L/9A/TS
*"» (\j O $ 'Q ^ 0) N
Fig. 2. — Effect of crop on physical state and electrolyte concentration of the water extract of the soil.
soil particles are reflected in the quantities of suspended material obtained
in this manner.
The data have been expressed in the form of graphs with the time (in
weeks) plotted against values expressing the magnitudes of the suspended
material and also against the resistances of the extracts in ohms. Since
the concentration of the solution varies inversely as the resistance, the
scale has been inverted to bring out the relations more clearly. (Fig. 1-4.)
16916—20 5
400
Journal of Agricultural Research
Vol. XX, No. s
It is evident that there exists a very good general correlation between
the quantity of soluble constituents in the soil and the quantity of sus-
pended material and that in both cases the magnitudes undergo very
marked variations coincidentally with seasonal changes and crop growth.
These fluctuations are far more pronounced, however, in the cropped
e
16
24-
"f-0
4-8
56
64-
72
SO
66
96
/Of-
112
J 20
pPS?"0
XT'
CROP B/9RLEY
7/ PL/9 NTS
TUf?B/DtTY T//Q
VJSC--0
CfPOR A/ON£
Fig. 3. — Effect of crop on physical state and electrolyte concentration of the water extract of the soil.
soils than in the uncropped soils. In other words, it is a fair conclusion
that the absorption of solutes by the plant has lowered the concentra-
tion of the soil solution at a period of 8 or 10 weeks after planting and
that the physical state of the soil has undergone an equally definite
change. It can scarcely be doubted that there is some definite relation
between the concentration and composition of the soil solution and the
Dec. i, 1920
Effect of Season and Crop Growth on Soil
401
physical state of the soil. That this correlation is only approximate is
not difficult to explain, even if we assume that the factors mentioned
above are the only ones to be considered. The quantity of suspended
material obviously can not bear an exact relation to the concentration
of the solution throughout all ranges. At a certain point the supernatant
liquid will become almost clear, and while further increases in the con
8
-
-0-^-0
/6
-
24
32
- --0...
r~ '■0-
+8
S6
-
V
6+
72
_
A """"
SO
86
96
-
\
\ /
b'
CROP POTATOES
/Of-
'
//2
(T) /20
-
. , > . 1 t
16
24
32
4-0
\ / * ^s.
48
64<
72
/> — 0-
A \/ '' N>
80
38
96
tOQ.
CROP COPA/ N^=
\
\
112
f20
\
D
111 1 i _ J.
Fig. ,
WEEKS
-Effect of crop on physical state and electrolyte concentration of the water extract of the soil.
centration of electrolytes will diminish the resistance almost proportion-
ally, no further change can be shown in the physical state of the soil as
measured by the present method. Moreover, the conductivity is a re-
sultant measurement expressive of the concentration or mobility of all
the ions present. It is not true, however, that equivalent quantities of
different ions have equal effects on the colloidal state of the soil.
4-02 Journal of Agricultural Research vol. xx, no. 5
The effect of various salts on the flocculation of soils has been studied
in many investigations. Sharp * in an extensive series of observations
has presented evidence to show that a remarkable change is produced in
the physical state of the soil by the addition of various salts and sub-
sequent washing of the soil with water. This change in the degree of
dispersion of the colloids is attributed to the formation of new silicate
compounds which give to the soil its new properties.
These investigations by Sharp have all dealt with rather extreme salt
effects, such, for example, as might occur in "alkali" soils or heavily
fertilized soils. So far as known, no study has been made of the changes
which may take place in soils because of the normal fluctuations in the
soil solution under varying conditions of cropping and season. In such
cases the total quantity of salts dissolved in the soil solution is extremely
small, and it might be questioned whether these could have any appre-
ciable effect on the physical state of the soil. However, an analysis of
the data presented by Stewart 2 and Hoagland 3 brings out the fact that
relatively enormous fluctuations may take place in the soil solution.
The growth of a crop, for example, in certain instances may reduce the
concentration of the soil solution to an extremely low point. Recently
McCool and Millar 4 have presented extensive data to show that in the
field very profound changes may occur in the soil solution as a result of
cropping, moisture variations, biological activities, rainfall, etc. Appar-
ently all of these fluctuations in the soil solution may be reflected in the
physical state of some at least of the soil constituents.
Since the effect of cropping is to reduce the water-soluble constituents
of the soil and the concentration of the soil solution, it might be pre-
dicted on the basis of the foregoing discussion that soils which had been
cropped would show a physical condition distinctly different from the
same soils kept uncropped. In order to decide this point more definite
turbtditv determinations were made on a number of different soils.
Except that one tank of each soil had been cropped for four years and one
tank had been kept without crop for three years, the soils were maintained
under identical conditions. Originally both portions of the soil were from
one sifted, homogeneous mass. The details of treatment have already
been described in an article by Stewart.5 Chemical analyses and conduc-
tivity measurements on water extracts, as well as freezing-point depres-
sions on the moist soil, all pointed to the fact that the uncropped soil
yielded a soil solution of higher concentration than did the cropped soil.
The data contained in Table I give evidence that these differences were
reflected in the physical state of the soils. It is particularly easy to
demonstrate this relation for the silty soils, but even the sandy soils
display the same tendency.
1 Sharp, L. T. op cit. 4 McCool, M. M., and Millar, C. E. op. err.
5 Stewart, Guy R. op. cit. 6 Stewart. Guy R. op. err.
3 Hoagland, D. R. op. err.
Dec. i, 1920 Effect of Season and Crop Growth on Soil
403
Table I. — Relation of physical state to the electrolyte concentration of the soil extract
Soil
No.
Condition of soil.
14
iC
f Cropped. . .
(.Uncropped .
/Cropped. . .
\ Uncropped .
f Cropped. . .
1 Uncropped .
f Cropped. . .
\ Uncropped .
f Cropped. . .
1 Uncropped .
(Cropped. . . ,
1 Uncropped.
/Cropped. . . .
[Uncropped .
f Cropped. . . .
\ Uncropped .
/Cropped. . . .
1 Uncropped .
f Cropped. . . .
\ Uncropped .
/Cropped. . . .
(Uncropped .
f Cropped. . . .
\ Uncropped .
f Cropped. . . .
1 Uncropped .
/Cropped. . . .
[Bin
June 3.
Turbid-
ity fl
170
80
33°
190
230
160
170
90
290
IOO
270
180
, 120
95°
340
300
,090
,070
970
i33°
.730
130
220
050
320
, 140
Specific
resistance.
Ohms.
4,
3.
6,
5.
5,
3,
8,
7,
7,
7,
5>
3>
12,
6,
500
900
000
200
300
700
300
900
800
700
500
400
500
900
800
700
000
500
400
©00
800
300
400
500
700
300
July 26.
Turbid-
ity."
I, 400
640
6, 620
220
IOO
60
410
160
Specific
resistance.
6,800
5, IOO
6, 900
4, 100
3>9°o
3,000
5,800
4, IOO
1, 060
610
2, IOO
160
1,320
I, 160
I, 260
I, 420
2,OIO
T5°
10, 400
4, 400
5>90°
3.4oo
16, 900
8, 100
9, 200
6,500
9, 000
4.300
a Expressed in milligrams per 100 cc.
It has already been pointed out that under certain conditions of storage
a soil may accumulate a large amount of soluble constituents. It was
thought to be of interest to compare a sample of soil which had been kept
in a bin for several years in a slightly moist condition with a sample of
the same soil cropped for several years. The two samples displayed
widely different concentrations of electrolytes, and the turbidity measure-
ments indicate that 20 times as much material was kept in suspension in
the cropped soil. These samples demonstrate the extreme effects which
may occur, even without fertilization or leaching.
Sharp 1 has shown that salt-treated soils washed with water are made
far more impervious than soils washed with water without previous
treatment. If, however, a soil is very completely leached with distilled
water after stirring, an extremely impervious condition of the soil results.
At the same time the final leachings are exceedingly dilute, and the con-
centration of solution in the leached soil is so small as to be scarcely
determinable. In Sharp's experiments the impervious condition of the
1 Sharp, L. T. op. cit.
404 Journal of Agricultural Research vol. xx. No. 5
soil is considered to be the result of the formation of certain new silicates.
Possibly in soils leached with water and not containing an excess of salts
the dispersed condition may be the result of the almost complete removal
of electrolytes from the films of solution surrounding the soil particles.
To a lesser extent the s?,me thing occurs when the soil solution is depleted
through absorption of solutes by the plant. None of the data presented
in this paper, however, are of such a nature as to permit of any conclu-
sions with regard to these very difficult questions concerning the colloid
chemistry of the soil.
Neither is it possible to state definitely the effects of the fluctuating
soil solution on the physical state of the soil under field conditions.
A sample of soil may be maintained in a relatively pervious state even
after long washing, provided the compound particles of soil are not dis-
turbed by stirring or mixing while the soil is saturated with moisture.
Nevertheless, it is probable that the soil in the field is subject to certain
modifications in its physical state which are merely accentuated when the
laboratory tests are carried out.
It is interesting to speculate on the indirect effects of the changes in the
physical condition of the soil noted in these experiments. It is entirely
possible that such changes may be of considerable importance. The
aeration, resistance to root penetration, ease of cultivation, percentage
of unfree water, etc., are very probably affected to a greater or less degree,
and these alterations in the soil conceivably may have an important
influence on the growth of microorganisms or plants. In any case, it is
highly desirable to make observations on all the effects, direct and indi-
rect, which may be correlated with the changing concentration or com-
position of the soil solution. It should be strongly emphasized that in
studies of soil fertility the whole system of soil, soil solution, and plant is
so constituted that all the components must be considered as interrelated.
Thus, the plant may exhaust the soil solution with a resultant change in
physical condition of the soil which may be unfavorable to the growth of
microorganisms, and this inhibition in time may influence the concentra-
tion of certain solutes in the soil solution. It is believed that the greatest
advances in theories of soil fertility will come with an extension of our
knowledge of the soil solution in its dynamic aspects.
CONCLUSIONS
The physical state of certain soil constituents is influenced to a marked
degree by the concentration of the soil solution. The colloidal state of the
soil suspension undergoes significant alterations during the season. A
large increase in colloidal matter is noted when the soil solution is de-
pleted as a result of absorption of solutes by the plant.
Vol. XX DECEMBER 15, 1920 No. 6
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Page
Carbon-Dioxid Content of Barn Air - 405
MARY F. HENDRY and ALICE JOHNSON
(Contribution from New Hampshire Agricultural Experiment Station)
Rice Weevil (Calandra), Sitophilus orza - - - - 409
RICHARD T. COTTON
( Contribution from Bureau of Entomology )
Opius fletcheri as a Parasite of the Melon Fly in Hawaii - 423
H. F. WILLARD
( Contribution from Bureau of Entomology )
Tamarind Pod-Borer, Sitophilus linearis (Herbst) - - 439
RICHARD T. COTTON
( Contribution from Bureau of Entomology )
Influence of Temperature and Humidity on the Growth
of Pseudomonas citri and Its Host Plants and on In-
fection and Development of the Disease - 447
GEORGE L. PELTIER
(Contribution from Alabama Agricultural Experiment Station)
Daubentonia longifolia (Coffee Bean), A Poisonous Plant - 507
C. DWIGHT MARSH and A. B. CLAWSON
( Contribution from Bureau of Animal Industry )
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WA8HINOTOM : OOVEBNMENT PRINTINQ OFFICE : f»M
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Expert-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
~0
JOIMAL OP AGMCULTURAL RESEARCH
Vol. XX Washington, D. C, December 15, 1920 No. 6
CARBON-DIOXID CONTENT OF BARN AIR
By Mary F. Hendry and Alice Johnson, Carnegie Nutrition Laboratory, Boston,
Mass., and New Hampshire Agricultural Experiment Station
In connection with the construction and establishment of a respiration
chamber 1 for large domestic animals in the dairy barn at the Agricul-
tural Experiment Station, Durham, N. H., the question as to the carbon-
dioxid content of barn air and its probable influence upon respiration
experiments, in case such air should inadvertently leak into the chamber,
assumed considerable importance. Recent information with regard to
the carbon-dioxid content of barn air is extremely scarce, and the earlier
work is practically unrecognized. The extensive investigations of
Pettenkofer on ventilation, unfortunately published in a number of small
and wholly inaccessible journals, have been cited from time to time by
various writers, and to him have been ascribed carbon-dioxid percent-
ages in stable air of 0.105 and 0.21 per cent. The most extended serious
study of the carbon-dioxid content of barn air was that made by Schultze
in the experiment station at Gottingen-Weende, the results of which
have been reported by Marcker.2 Employing the Pettenkofer method,
Schultze made nearly 200 analyses of stable air in the vicinity of Gottingen
and found that the carbon-dioxid content varied enormously, depending
upon the number of animals in the stable, the volume of space available,
and the degree of ventilation. The values for the carbon-dioxid per-
centages in the air of stables at Weende are as high as 0.435 Per cent m a
number of instances, and a maximum of 0.594 Per cent is recorded. For
outdoor air the usual value of not far from 0.03 per cent to 0.034 Per cent
was found. Marcker concludes that the ventilation of a stable should be
such that the carbon dioxid in the air is not greater than 0.25 to 0.30 per
cent. Angus Smith cites two analyses of the carbon-dioxid content of
air in stables showing but 0.0833 and 0.0875 Per cent.3
After our analyses of the air in the dairy barn at Durham were made,
our attention was called to the report of the Committee on Farm
1 Benedict, F. G., Collins, W. E., Hendrv, Mary F., and Johnson, Alice, a respiration chamber
For large domestic animals. N. H. Agr. Exp. Sta. Tech. Bui. 16, 27 p., 7 fig. 1920.
2 Marcker, Max. tjeber den kohlensaure-gehalt der stallluft und den luftwechsel in stal-
LUNGEN. In Jour. Landw., Jahrg. 17 (F. 2, Bd. 4), p. 224-275. 1869. We have seen this remarkably
complete paper cited but once and then erroneously. It deserves careful study.
3 Smith, R. A. air and rain. p. so. London, 1872.
Journal of Agriculture Research, Vol. XX, No. 6
Washington, D. C Dec. 15, 1920
vx Key No. N. H.-5
(405)
406 Journal of Agricultural Research voi.xx, no. 6
Building Ventilation.1 In this report are given the results of analyses of air
samples taken at various points in five different barns. Mr. Clarkson
has called our attention to the fact that although the amounts of carbon
dioxid per 10,000 parts of air are correctly expressed in the tables, the
conversions to percentages are erroneous, because of misplaced decimal
points, and the percentage values should accordingly.be multiplied by 10.
The results published in this report show that in the five barns exam-
ined, which were presumably of reasonably modern construction, the
carbon-dioxid content of the air might be as high as 1.231 per cent, but
for the most part was not higher than 0.2 to 0.3 per cent.
The dairy barn at Durham is admirably lighted and is, so far as one
can judge by the senses at least, well ventilated. The stock room is
approximately 100 feet long, 35 feet wide, and 8 feet 8 inches high, is
provided with windows on both sides, and has a concrete floor. The
ventilating ducts withdraw the air from near the floor, and outdoor air
can blow in on either side through screened openings. Practical experi-
ence indicates that this barn is admirably adapted for maintaining stock
in good health with a negligible amount of disease.
Our study of the air in this barn did not include an examination of the
ventilation conditions, so far as draft, temperature, and psychrometric
measurements are concerned, but consisted solely of gas analysis made
in connection with the possibility of leakage of barn air into the respi-
ration chamber. To study the carbon-dioxid content of the air in a mod-
ern, well-ventilated dairy barn seemed a justifiable procedure. Being
unfamiliar at the time of our tests with the earlier series of observa-
tions cited above, we were astonished at our first results, which showed
on the average an amount of carbon dioxid in the barn air not far from
8 to 10 times the normal carbon-dioxid content of outdoor air. The
analyses were all made with the small Haldane gas analysis apparatus2
by both authors at different times and after many years' experience
with the use of this type of apparatus.3
To obtain a general picture of the distribution of the carbon dioxid
in the air, samples were taken at different parts of the barn, but unfor-
tunately not simultaneously. Four samples were taken at 8.50 a. m.,
four at 10.05 a- m-i f°ur at 11 a. m., and three at 11.40 a. m., all in
different locations. Subsequently the samples were taken at three
positions only, but variations in the time of day were studied under
these conditions.
Approximately 40 milch cows were in the barn at the time. Of the
15 different positions at which air samples were taken, locations 1 to 5
were in the feed alley between the two rows of stalls and therefore in
1 Clarkson, W. B., Smith, L. J., and Ives, F. W. [report of the] committee on farm building ven-
tilation. In Trans. Amer. Soc. Agr. Engin. Rpt. 12th Ann. Meeting, 1918, p. 282-306, illus. 1919-
2 Haldane, J. S. methods of air analysis, ed. 2, p. 68. London, 1918.
3 Special mention should be made here of the intelligent cooperation in our work of the dairyman, Mr.
Mario Quaregno, who collected samples for us at night with the greatest fidelity.
Dec. 15, 1920
Carbon-Dioxid Content of Bam Air
407
front of the animals. Locations 6 to 15 were in the two outer alleys and
therefore at the rear of the animals. Locations 1 and 6 were nearest
the respiration chamber. The results of the analyses are presented in
Table I. All samples were taken approximately 4 feet from the floor.
Table I. — Carbon dioxid in air of barn at Durham, N. H., during January and
February, IQIQ
Time of day.
Location.
Percentage of
carbon dioxid.
8. so a. m
i, beginning of feed alley. . . .
Do
2, feed alley, about 15 feet from No. 1
3, center of feed alley. . . .
.225
.214
.228
• 194
. 106
.089
.098
Do
Do
4, feed alley, about 15 feet from No. 3
5, end of feed alley. . . .
10. 05 a. m
Do
6, beginning of right-hand outer alley ] . . . .
7, outer alley, about 15 feet from No. 6
8, center of right-hand outer alley
Do
Do
11. 00 a. m. .
9, outer alley, about 15 feet from' No. 8
10, end of right-hand outer alley
Do
.097
. 107
.089
Do
11, beginning of left-hand outer allev
12, outer alley, about 15 feetfrom No. 11. .. .
13, center of left-hand outer alley
Do
11.40 a. m
Do
14, outer alley, about 15 feet from No. 13. . . .
15, end of left-hand outer alley
.116
•"5
• 149
Do :...
1, beginning of feed alley
5. 20 p. m
2, feed alley, about 15 feet from No. 1
3, center of feed alley
5. 30 p. m
• 2I9
. 207
• i77
• i32
. 109
1;. 00 a. m
1, beginning of feed alley
10. 30 p. m
do
15. 00 a. m
do
Do
do
do
11. 30 p. m
3, center of feed alley. . .
do
.187
. 167
. 184
• 130
•*39
168
5. 00 a. rn
do
11. 30 p. m
do
=;. 00 a. m
do
11. 20 p. m
do
5. 00 a. m
do
11. 15 p. m
do
.178
.094
. 209
5. 00 a. m
do
11. 50 p. m
do
1 This position was nearest the respiration chamber.
Since under the conditions of experimentation the amount of carbon
dioxid inside the respiration chamber varies from o. 1 to 0.7 per cent,
being usually not far from 0.35 to 0.40 per cent, and since the method of
experimentation depends upon the supplying of pure outdoor air with a
carbon-dioxid content of 0.03 per cent, it can be seen that any leakage
of barn air into the respiration chamber would be detrimental to the
success of the experiment. The fact that all the control tests of this
respiration chamber have shown most satisfactory agreement of results,
when the technic is properly carried out, testifies to the care with which
this chamber was constructed by the mechanician, Mr. W. E. Collins.
408 Journal of Agricultural Research vol. xx, No. 6
The production of carbon dioxid by dairy cows is very large because
of several factors, among others the high metabolism of the animal itself
and the conversion of carbohydrate into fat, which of itself results in a
large splitting off of carbon dioxid (so-called "atypical" carbon dioxid).
While the cows are not given any exercise when in the barn they are
very energetic during feeding periods, striving to gather in every particle
of food. At other times they are, for the most part, extraordinarily
quiet and placid.
It is clear from the table that even in this modern barn there is a large
percentage of carbon dioxid in the air. That the presence of this amount
of carbon dioxid has, for two decades, had no apparent influence upon
the health of the animals is worthy of special notice. The excellent
health of the animals in this barn leads us to believe that what is true of
men is likewise true of animals — that is, that carbon dioxid per se, even
in percentages 8 or 10 times the normal percentage, has no serious effect
upon the animal itself.
RICE WEEVIL, (CALANDRA) SITOPHITUS ORYZA
By Richard T. Cotton, Scientific Assistant, Stored-Product Insect Investigations,
Bureau of Entomology, United States Department of Agriculture
INTRODUCTION
As early as 196 B. C. mention is made. of the ravages of weevils
in stored wheat (9).1 Whether the species referred to was Sitophilus
oryza L. or the closely allied granary weevil 5. granarius L. we do not
definitely know. The latter species, however, is thought to be the older
and is presumably the one referred to. However that may be, since
about the middle of the eighteenth century, when it was discovered in
Europe, 5. oryza has everywhere attracted the attention of scientists,
and innumerable accounts have been written concerning its ravages.
It is not the purpose of the writer to review at this time the extensive
literature relating to this weevil; it will suffice to state that the early
accounts are very general in character and the majority of the later
ones little more than repetitions of the earlier observations. The publica-
tion of Hinds and Turner (6) in 191 1 on the biology of the rice weevil
gives us the only really definite information that we had regarding the
life and habits of this species. A general presentation of the economic
problem centered in the rice weevil was given in 191 9 by Back (1) in a
publication of the Department of Agriculture. It is with the purpose of
adding to our knowledge of this cosmopolitan insect that this paper is
presented.
ORIGIN AND DISTRIBUTION
The rice weevil, Sitophilus oryza, so called because of its discovery
breeding in rice, is thought to have originated in India. It was carried
by commerce to Europe at an early date, where it was subsequently
found and described by Linnaeus in 1763 (7, p. 395).
At present it is perhaps the most widely distributed of known insects,
being found in all parts of the world where grain is used. In North
America it is reported from Florida to Alaska, though it is found in its
greatest abundance in the South Atlantic and Gulf States.
DAMAGE CAUSED
From time immemorial the rice weevil has taken its yearly toll of the
grain crops of man. The total amount of rice, corn, wheat, barley, rye,
etc., that has been destroyed by this weevil alone is almost beyond con-
ception.
1 Reference is made by number ("italic) to "Literature cited," p. 422.
Journal of Agricultural Research, Vo1- xx- No- s
Washington, D. C P.ec- Jf' r*°_
vu
(409)
Key No. K-87
4io Journal of Agricultural Research vol. xx, no.o
In the eight southern States of North America where the weevil is
most abundant and destructive, 350,000,000 bushels of corn were pro-
duced in the year 191 8. Of this vast amount it is estimated that approxi-
mately $28,000,000 worth was destroyed by the rice weevil alone. This
represents only a small portion of the annual world crop of corn and a
considerably smaller portion of the world crop of grains that are attacked
by this weevil.
To cite another instance of the ravages of this weevil, Fitch (2) records
that from 145 tons of American corn, \% tons of weevils were screened
out or, in round numbers, about 4,056,729,600 weevils, a truly enormous
number. Such an occurrence as this was by no means rare in earlier
times when cargoes of grain were transported long distances in slow-
going vessels; in fact, it was not uncommon for whole cargoes to be
destroyed by the weevil or rendered unfit for use.
At present losses are particularly severe in India, Mexico, South
America, and other tropical countries where the weather conditions are
such that the weevil can breed unchecked the year round.
Loss is occasioned by the feeding activities of both the grubs or larvae
and the adult beetles. The feeding of the larvae is confined chiefly to
the seeds of our common grains, but the adults feed on a great variety of
seeds, fruits, and .other foodstuffs. In addition to the loss in weight
caused by the feeding of the larvae and weevils, infested grain is often
rendered unfit for consumption and has poor powers of germination.
FOOD OF ADULT WEEVILS
The adult weevils feed on a great variety of seeds and seed products.
The following list has been compiled from the numerous reports of the
feeding habits of this weevil: Rice, wheat, corn, barley, rye, hulled oats,
buckwheat, maize, chickpeas, table beans, millet, chestnuts, cashew
nuts, bird seed, seed of Nebulium sp., hemp seed, Job's tears (Coixa
lachryma), packages of "feuilles de sagon," packages of cereals, tobacco,
peaches, grapes, apples, mulberries, bags of meal, yeast cakes, biscuits,
macaroni, cakes, crackers, wheat flour, rice flour, and white bread and
other wheat products. The author has found the adult weevils burrowing
and feeding in the berries of the Chinaberry tree, in both Irish and sweet
potatoes, and in the seed of the avocado. In the laboratory they showed
a liking for most kinds of ripe fruits, and it was found that they would
live indefinitely on a majority of the wild berries growing in the vicinity
of the laboratory. With such adaptable food habits as this long list
would indicate it is little wonder that this weevil is so widespread and
causes so much damage.
FOOD OF LARVAE
The larvae or grubs of the rice weevil are much more restricted in their
diet than are the adult beetles, owing to the fact that they pass the entire
larval period within a single seed and are limited to seeds that contain
Dec. is, 1920 Rice Weevil, {Calandr a) Sitophilus or yza 411
sufficient food to enable them to develop to maturity. They have been
reported to breed in rice, wheat, corn, hulled oats, millet, barley, rye,
buckwheat, chickpeas, Job's tears {Coixa lachryma), acorns of several
species of oak, galls of Phylloxera devastatrix on Hicoria pecan, and old
cotton bolls.
LIFE HISTORY
The observations from which the following data are taken were made in
Orlando, Fla., during the year 1919 and the early part of 1920. Since
this weevil is of more importance in the southern States as a pest of corn,
the life-history records were taken from weevils breeding in corn.
All stages of the rice weevil are active throughout the year in Florida.
The egg, larval, and pupal stages are somewhat prolonged during the
winter months, but there is no hibernation period, and oviposition con-
tinues throughout the year.
The adult weevils appear on corn in the field as soon as it reaches the
roasting-ear stage and are often to be found in the markets at this time on
the ears presented for sale. It is not until the corn has become a little
firmer, however, that oviposition begins. When it has reached the firm
stage the female weevils oviposit in all parts of the grain that can be
reached with the proboscis and ovipositor, for at this time it is a simple
matter for the weevil to excavate an egg cavity, and the rate of oviposition
is much greater at this time than later when the corn has hardened. As
the kernels of corn become harder the majority of the eggs are laid in the
white starch part of the kernel that is found at the outer end as the kernel
is attached to the cob. With shelled corn the majority of the eggs are
deposited in the soft germ part near the tip of the kernel where excava-
tion is relatively easy.
In the field the ears with tips protruding from the shucks, those with
loose, open shucks, or those with shucks that have been injured by the
corn earworm or some other agency are the first to be infested. Ears that
have a long, tight-fitting shuck that extends well beyond the tip of the ear
at the period when the corn is ripening are practically immune from weevil
attack. The weevils encounter great difficulty in penetrating a well-
developed, tight-fitting shuck and therefore congregate on the ears with
the damaged or poorly developed shucks. The kernels at the exposed
tip are the first to be infested, but the weevils soon work their way to all
parts of the ear.
METHOD OF OVIPOSITION
The female weevil after selecting a favorable spot on a kernel of corn
proceeds to excavate the egg cavity. This she accomplishes with her
powerful though slender proboscis or beak, oscillating her body in such a
manner as to impart a combined up and down and rotary motion to the
proboscis. The mandibles attached at the end of the beak chew away at
the corn until finally a hole is excavated equal to the length of the
412 Journal of Agricultural Research voi. xx, no. 6
proboscis. The cavity is trimmed and enlarged and the sides smoothed off
until the weevil is satisfied that all is as it should be. She then with-
draws her proboscis and turning around swings the abdomen about until
the egg cavity is located. The ovipositor is then thrust into the cavity
and a single egg is deposited.
Before the ovipositor is withdrawn a translucent mass of material is
discharged on top of the egg and is tamped down level with the surface of
the kernel of corn, forming a protective cap to the egg. This cap, because
of its translucent character, assumes the color of the portion of the kernel
in which it is located, thereby making the discovery of the egg difficult at
times. Occasionally one or more extra discharges are made on top of the
first cap, causing the cap to protrude above the surface of the kernel.
These latter discharges are usually irregular, opaque, and mixed with
fecal matter.
The time taken to excavate the egg cavity varies with the condition of
the grain. When the corn is soft the cavity may be completed in less
than 30 minutes, whereas in hard corn the operation may take as long as
2 hours. The actual time of depositing the egg after the cavity is finished
is short, from 3 to 4 minutes on the average.
WHERE THE EGGS ARE PLACED
The egg cavities are made usually in some part of the soft starch of the
grain or in the germ, where the work of excavation is easier and the
young larva upon hatching will have an abundance of food ready for
instant use. Frequently in kernels of corn that have not sufficiently
hardened numerous excavations will be made only to be abandoned by the
weevil as unfit for use, the weevils apparently having the instinct of
knowing when the corn is unfit to maintain larval life. Several eggs are
often deposited in the same kernel of corn, though when the supply of
grain is abundant it is not usual for a weevil to deposit more eggs in a
single kernel than can mature in the limited amount of food present.
When weevils are confined with only a few kernels, however, the instinct
to continue laying eggs predominates and eggs are deposited in all parts
of the grain.
The egg itself is somewhat flexible in character and conforms to the
shape of the egg cavity. It is placed with the top just below the surface
of the kernel and with the larger end toward the inner end of the cavity.
RATE OF OVIPOSITION
The rate of oviposition varies with the condition of the grain, the age of
the weevil, and the temperature. During the warm weather of summer,
with young female weevils and with corn in the "hard gum" stage, the
oviposition rate reaches its maximum. Under such conditions from 8 to
10 eggs are laid per day, though as many as 20 to 25 may occasionally be
laid in a like period.
Dec. 15 1920
Rice Weevil, (Calandra) Sitophilus oryza
413
As the weevils get older the oviposition rate gradually decreases until, a
few weeks before death, egg laying ceases altogether. With the approach
of cold weather the rate of oviposition also decreases, and especially is
this true of the older weevils. The younger female weevils are more
vigorous and are much less affected by the cold.
Normally eggs are laid every day during the spring, summer, and fall
months, but during the winter egg laying is sporadic and is controlled
chiefly by the daily temperatures.
In Florida the winter temperatures are very variable, short periods of
cold weather occur frequently, and during these oviposition usually
ceases.
During the warmer months the weevils normally lay from three to six
eggs per day in hard corn.
Table I shows the rate of oviposition at various times of the year.
It contains abstracts from the oviposition records of 14 weevils that are
representative of the species. The number of eggs laid by each weevil
on two consecutive days in each week from June, 1919, to March, 1920,
is given, together with the daily mean temperatures and the dates of
emergence and death of each individual weevil. The corn was at its
most favorable stage for oviposition during the latter part of June and
the early part of July.
Table I. — Rale of opposition of Sitophilus oryza; extracts from oviposition records
at Orlando, Fla., June, IQIQ, to March, IQ20
Mean
tem-
pera-
ture.
Number of eggs laid by weevil No. —
Date.
A2
a3
B2
B6
C3
c7
Di
D2
D4
E3
E6
Fi
F2
F4
1919.
June 22
23
"F.
82. s
80
78-s
79- S
79
78. S
81
82. S
81.5
81. s
81
81
85
8s-5
81. s
83
81.5
81. s
82. s
82
80. s
81.5
84
84
82.5
83
77- S
73- S
10
5
12
13
11
11
12
14
6
6
5
2
7
8
16
23
IS
11
12
9
(6)
29
July 3
4
(O)
9
13
8
5
6
6
9
7
8
8
7
7
8
4
6
4
3
6
4
3
3
10
8
6
5
8
10
7
10
10
6
S
7
4
8
7
7
S
s
4
4
17
25
Aug. 4
5
9
(c)
2
8
7
7
8
6
4
S
6
5
4
(d)
19
27
3
4
t
6
4
6
6
4
4
Sept. 3
4
19
w
4
3
w
3
4
(/)
4
S
29
.
'Weevil emerged July 5, 1919.
1 Beetle escaped.
> Weevil emerged. Aug. 10, 1919.
d Weevil emerged Aug. 18, 1919.
« Weevil emerged Sept. 18, 1919-
/ Weevil emerged Sept. 19, 1919.
4i4
Journal of Agricultural Research
Vol. XX, No. 6
Table I.-
-Rate of oviposition of Sitophilus oryza; extracts from oviposition records
at Orlando, Fla., June, IQIQ, to March, 1920 — Continued
Mean
tem-
pera-
ture.
Number of eggs laid by weevil No. —
a. : a3
B2
B6
c3
c7
Di
D2
r>4
E3
E6
Fi
F2
F4
1919-
Oct. 1
°r.
77-5
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81
80
80
80.5
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76.5
76. s
76
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70
70
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69
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67
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76
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67
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57
57
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62
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69
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Nov. 3
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(*)
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4
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28
(0
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1920.
Jan. 3
4
1
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(m)
1
2
2
:::::: ::::::
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2
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3
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Feb. 3
:::::::::::
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26
(0)
(p) (••>
(7)
(0)
(o)
(0)
(0)
(0)
1
0 Female died Oct. 25, 1919.
A Weev'! emerged Oct. 30. 1919.
i Weevil emerged Nov. 6. 1919.
; Weevil died Nov. 26, 1919.
* Weevil emerged Nov. 30, 1919.
1 Female died Dec. 30, 1919.
m Weevil emerged Dec. 9, 1919.
« Weevil emerged Dec. 15, 1919.
o Female living,
p Female died Mar. 5, 1920.
q Female died Feb. 25, 1920.
NUMBER OF EGGS LAID
The largest number of eggs laid by a single weevil was 576. These
were laid during a period of 149 days. The weevil in question emerged
July 5, 1919, began laying eggs on July 12, and continued oviposition
until December 7, 191 9. Egg laying was apparently stopped by the
cold weather and the exhaustion of the weevil, and death occurred
December 30, 191 9. This oviposition record is in all probability longer
than the average, though it does not represent the maximum period, for
when winter intervenes, a period during which few eggs are laid, the
oviposition period may be considerably longer.
Dee 15, 1920
Rice Weevil, (Calandra) Sitophilus oryza
415
Table II contains data concerning the preoviposition period, the ovi-
position period, and the number of eggs laid. The records of the 10
individuals cited were selected as being representative.
Table II. — Data concerning ovi position and longevity of Sitophilus oryza at
Orlando, Fla., 191Q
Weevil No.
Average.
Date
weevil
emerged.
July
14
18
Aug. 10
Date
first egg
was
laid.
July
Aug. 19
26
Length of
preovi-
position
period.
Days.
Date
last egg
was
laid.
Oct. 5
24
Dec. 7
Oct. 3
Sept. 19
Nov. 7
Oct. 22
Nov. 5
¥$? Nrber
position ° Jfs
period.
Days.
89
108
149
80
67
no
270
552
576
288
420
445
339
237
389
284
380
Date of
death.
Oct.
-'5
Dec.
Oct. 5
Sept. 23
Nov. 20
Oct. 23
Nov. 28
Dec. 6
Nov. 26
Length
of life.
Days.
95
"3
179
93
78
130
98
in
119
101
From Table II it will be seen that the average preoviposition period
is about 7 days, the average oviposition period during the warm months
of the year is 93.9 days, and the average number of eggs laid per female
is 380, or about 4 per day. >
DESCRIPTION OF EGG
Egg opaque, shining, white, ovoid to pear-shaped in form, widest below middle,
bottom broadly rounded, neck narrowing sharply towards top, which is somewhat
flat and bears a small protuberance that fits into a cap or plug which cements the
egg into place. Length 0.65 to 0.70 mm.; width 0.28 to 0.29 mm.
INCUBATION PERIOD
The eggs usually hatch in from 3 to 5 days during the warm months
of the year, although by far the majority of them hatch in 4 days.
During the colder weather of winter the incubation period is somewhat
longer and may last 10 or more days. The variation in the length of
the incubation period at different times of the year may be seen in
Table III.
EARVAL PERIOD
The embryo develops within the egg with its head toward the top,
the darker color of the mandibles showing through the thin, transparent
shell some time before the egg hatches. The eggshell undulates with
the movements of the newly formed larva but is finally ruptured and
the young larva begins to feed on the tissues of the corn.
The egg is usually placed so that at least part of it is embedded within
the soft white starch of the grain so that the young larva is at once sup-
plied with a readily available food supply. Occasionally the egg is sur-
rounded entirely by the horny portion of the seed, and in this case growth
of the larva is somewhat slower until it makes its way to the softer
white part.
4i6
Journal of Agricultural Research
Vol. XX, Xo. 6
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Length
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Dec. is, 1920 Rice Weevil, (Calandra) Sitophilus oryza 417
DESCRIPTION OF LARVA
Mature larva from 2.5 to 3 mm. in length. A pearly white, fleshy grub, very thick-
bodied, the ventral outline being approximately straight while the dorsal outline is
almost semicircular. Head light brown in color, the anterior margin and mandibles
much darker. Head longer than broad and somewhat wedge-shaped, the sides
broadly rounded from middle to apex, which is slightly angular. Sides nearly straight
from middle to the anterior angles, and lateral area with an oblique, longitudinal,
lighter stripe or area. Epicranial and frontal sutures distinct and light in color; also
two oblique, longitudinal, light stripes rising from frontal sutures and coalescing with
epicranial suture near base of head. Frons subtriangular with a distinct, dark,
median line indicating the carina running from the posterior angle to beyond the
middle. Sutural margins irregular or sinuate. Frons provided with five pairs of
large setse, the sutural margins each bearing a large seta. Each epicranial lobe bearing
the following sets: One close to posterior angle of frons and located within the oblique,
longitudinal stripe rising from the frontal suture; one very small seta posterior to this
and near occiput; two anterior to it on disk of epicranium; two opposite middle of
frons; one opposite middle of mandible; one opposite hypostomal angle of mandible;
and one 011 hypostoma near base of mandible. Epistoma represented by thickened
anterior margin of front, distinctly darker in color, with anterior margin declivous and
slightly curving and lateral angles slightly produced and elevated where they sup-
port the dorsal articulation of the mandibles. Pleurostoma represented by the darker
declivous area surrounding the mandibular foramen. Mandibles stout, triangular,
with the apex produced into a broad apical tooth; inner edge toward the apex pro-
vided with a subapical tooth and a small medial tooth; no molar part. Dorsal
area of mandible provided with a pair of stout bristles set apart. Eye represented
by a well-defined black spot beneath the exoskeleton. Clypeus attached in front of
frons and broadly transverse, broad at base, sides narrowing toward the apical angles,
slightly longer and broader than labrum, and bearing on epistomal margin two fine
setse on each side. Labrum distinctly broader than long, with two small lateral and
a larger, rounded, median lobe. Labrum provided with six large setse behind mid-
dle, two marginal, short, thickened setae on each lateral lobe, and six similar marginal
setae on median lobe.
Maxilla with cardo present and distinct, stipes not divided into stipes proper, sub-
galea, and palpifer but one continuous piece, with the anterior inner angle produced
into a single setose lobe. Palpus 2-jointed, bearing a single seta near apex of first
segment. There are three other setae on maxilla, two located on the vaginant
membrane between palpus and palpifer and one stouter and longer midway between
palpus and cardo. No articulating maxillary area between maxilla and mentum-
submental region.
Labium: Submentum and mentum fused and represented by a broad lobe bearing
three pairs of stout setae. Stipes labii posteriorly enforced by a median, triangular
chitinization, the anterior, median section produced anteriorly between the palpi
into a small lobelike ligula which is fused with the lingua. Each stipes labii bearing
a single seta. The short, conical, 2-jointed palpi are situated on the anterior angles
of the stipites. The ligula bears four small setae.
Prothorax dorsally not divided, but two areas, praescutal and scuto-scutellar, are
roughly indicated by rows of setae. The mesothoracic and metathoracic segments
are above divided into two distinct areas, the anterior of which represents the prae-
scutum and the posterior the scuto-scutellum and alar area. The thoracic spiracle
is located on a lobe pushed into the prothorax from the epipleurum of the mesothorax.
It is bifore, elongate, larger than abdominal spiracles, and placed with the fingerlike
air tubes pointing dorsad.
41 8 Journal of Agricultural Research vol. xx, n0.6
Ten abdominal segments; ninth small, tenth reduced. Each tergum of the first
three abdominal segments is above divided into three distinct areas, praescutum,
scutum, and scutellum. Each tergum of the fourth to eighth abdominal segments is
above divided into but two areas, the first of these containing the praescutal and
scutal elements, the second representing the scutellum. Below these two areas and
adjacent to the epipleurum is the alar area. The abdominal spiracles are placed ante-
riorly and in a small, separate corner piece probably of the alar area; they are bifore
and are found on abdominal segments i to 8, that on the eighth being located slightly
more dorsad than the rest. Below a very indistinct and abrupt dorso-lateral suture
and above a well-defined ventro-lateral suture is a large, not subdivided epipleurum.
The abdominal epipleura are located considerably higher than the thoracic lobes.
Below the ventro-lateral suture is the hypopleurum, subdivided into three lobes, one
right under the other. Below the hypopleurum is the coxal lobe, and below that is
the sternum, consisting of eusternum and a posterior triangular area representing the
parasternum or parasternum fused with sternellum. Abdominal segments provided
with setae as follows: One on praescutum, a long and two short ones on scutellum,
two on alar area located just above spiracle, two on epipleurum, one on coxal lobe,
and two on eusternum. One of the setae on scutellum is usually missing on abdominal
segments 5 to 9.
LARVAL STAGES
First-stage larva: Similar in appearance to mature larva but smaller; width of head
capsule 0.22 mm.
Second-stage larva: Width of head capsule 0.32 mm.
Third-stage larva: Width of head capsule 0.48 mm.
Fourth-stage larva: Width of head capsule 0.64 mm.
NUMBER OF LARVAL STAGES
After hatching the larva feeds rapidly, molting three times at more
or less regular intervals. Previous writers have stated that there are
only three larval stages. This is erroneous; there are invariably four,
as is the case with other weevils of this genus. Owing to the fact that
the larva passes its entire existence buried within the seed and obscured
from view it is somewhat difficult to observe all the changes that take
place. The writer, however, with the aid of binoculars and dissecting
instruments has followed through the life histories of several hundred
individuals at various times of the year, making daily observations on
each individual.
The first three larval stages average four days each, while the fourth
stage varies from four to nine days. During the cooler weather the
periods are all lengthened. Table III gives a good idea of the varying
length of the larval stages at different times of the year.
LARVAL HABITS
The larva occasionally bores near the surface of the grain, forming
elongate mines filled with white frass, but it more often bores directly
down into the heart of the seed. As it feeds and moves along, the frass
and debris are kept packed behind it. The space around it is kept
Dec. is, 1920 Rice Weevil, (Calandra) Sitophilus oryza 419
clear and free and is slightly larger than the grub, so that the latter can
readily turn around if it desires. If it is disturbed, the grub will turn its
head toward the point of attack, gnashing its mandibles.
PREPUPAL STAGE
When it is fully grown, the larva constructs a pupal cell. It uses the
end of its burrow for this purpose, strengthening the weak and soft
sides of the cavity with a cement formed from a larval secretion mixed
with frass and waste material of the burrow. This forms a hardened
shell around the larva. After it is completed the larva becomes sluggish,
lengthens out, and loses its plump appearance. This prepupal stage
invariably lasts for one day except during the winter months when it
usually lasts for two days; then the pupal form is assumed.
PUPAL STAGE
The pupal stage normally lasts for five days. On the fourth day the
mouth parts begin to color, then the tips of the inner wings. Spots of
color show on the prothorax, the beak, and the appendages and finally
on all parts of the body. On the fifth day the adult form is assumed.
DESCRIPTION OF PUPA
Pupa uniformly pearly white when first formed. Length 3.75 to 4 mm.; width
about 1.75 mm. Tips of wing pads attaining seventh abdominal segment, tips of
metathoracic tarsi extending beyond tips of inner wings. Head rounded, beak
elongate and slender. Head with two prominent spines toward vertex, a group of
two small spines and two spinules on each side above eyes, two pairs of small spines
near anterior margin, and one on each side of front between the eyes. Three pairs
of spines on beak between frontal ones and base of antenna, a pair of small ones on
beak midway between base of antenna and tip of beak, a pair on sides of beak between
latter pair and tip of beak, and two pairs of smaller ones on tip of beak.
Prothorax provided with one pair of anteromarginal setigerous tubercles, one pair
of anterolateral, two pairs of mediolateral, and four pairs of dorsal setigerous tubercles.
Mesonotum and metanotum each provided with three pairs of spines.
Abdomen with seven distinct dorsal tergites, the seventh being much larger than
the rest, dorsal area of each armed with a pair of large and a pair of smaller spines.
Lateral area of each tergite armed with a spine, at the base of which is a small seta.
Epipleural lobes each armed with two minute spines. Ninth segment as usual
armed with two prominent spines.
ADULT
The mature weevil measures from 2.1 to 2.8 mm. in length and is a
dull brown. It has the thorax densely pitted with round punctures,
and the elytra are marked with four reddish spots.
The adult weevil on first transforming is soft and is light in color and
stays within the pupal cell until it has hardened and become darker.
It usually emerges from the grain within a few days after transforming
A2o Journal of Agricultural Research voi.xx, no.6
but may sometimes remain within to feed. In winter months individuals
have been observed to remain within the grain for as long as a month
before cutting their way out.
NUMBER OF MALES AND FEMALES
Males and females are apparently produced in very nearly equal
numbers. Of i ,000 bred specimens examined 52 per cent were females
and 48 per cent males. The majority of the specimens examined were
bred during the later months of the year when the percentage of females
produced was slightly higher. During the early months of summer
more males were bred than females. Whether these conditions hold
true always can not be determined until many more specimens have
been reared and examined.
COPULATION
Copulation takes place within a day or two after emergence, one
female weevil being observed in copula two days after assuming adult
form. Copulation is frequent. It occurs rather often during the day-
time but more frequently at night.
PARTHENOGENESIS
Unfertilized female weevils, as previously reported by Hinds and Turner
(2), do deposit eggs that are fertile. The rate of oviposition is very
much lower, however, than with fertilized females, and very few of the
eggs hatch and produce grubs.
LIFE CYCLE AND NUMBER OF GENERATIONS
The period from egg to adult during the warm months of the year
averages 28 days, which together with a preoviposition period of 7 days
gives a life cycle of approximately 35 days. In some cases the life cycle
is completed in a much shorter period, one reared individual completing
the cycle in 30 days. On the other hand, the life cycle may be very
considerably prolonged on account of unfavorable food and weather
conditions.
Table III presents the life-history data of 30 weevils bred at various
times of the year and shows the variation in the length of the stages
from egg to adult at different seasons.
In Florida there are usually about seven full generations a year, six
during the period from April to November and one from December to
March.
MULTIPLICATION
Several calculations have been made and published of the theoretical
number of the progeny of a single pair of weevils. Owing to lack of
information on the rate of oviposition, the number of eggs laid, and the
length of the life cycle, the number has in some cases been greatly under-
estimated and in other cases greatly overestimated. From the data
given in Table II it is to be seen that the average female weevil lays
Dec. is, 1920 Rice Weevil, (Calandra) Sitophilus oryza 421
about four eggs a day for a period of nearly 100 days. Taking 35 days
as the length of the average life cycle, we find that by the time the female
weevil has stopped laying eggs, or in about three months' time, the
progeny from a single pair of weevils would theoretically amount to
approximately 100,000 weevils. From this time on during warm weather
the increase would be extremely rapid and is left to the imagination of
the reader.
LONGEVITY
The length of life of the adult weevils is variable and depends upon
a number of different factors. With weevils that emerge during the
spring and summer months the average length of life is from three to six
months. In this case the weevils mate almost immediately after
emergence, and egg laying ensues. The female weevils continue deposit-
ing eggs until exhausted and then die. With weevils that emerge in the
fall and winter months, mating and oviposition are less frequent, the
weevils do not become exhausted so rapidly, and life is consequently
prolonged. Several female weevils that were kept segregated and were
not allowed to mate laid only a few eggs, did not become exhausted, and
were still alive eight months from the date of emergence. In another
case several weevils of both sexes were kept segregated for a period of
four months and were then allowed to mate. Of these, several weevils
of both sexes were still alive and active eight months from date of
emergence.
Weevils deprived of food do not live long. In cold weather when they
are somewhat sluggish specimens have lived for 30 days without food.
In warm weather, however, they are very active and soon become ex-
hausted, seldom surviving for more than a week without food.
FEIGNING DEATH
When suddenly disturbed, the adult weevils often feign death, drawing
their legs up close to the body and dropping. This state does not last
long, and the weevils are soon hurrying off as active as ever. It is inter-
esting to note that the habit of feigning death is not nearly so well devel-
oped in this species as it is in the closely allied species Sitophilus granarius.
Weevils of the latter species feign death at the slightest disturbance and
remain motionless for a considerable length of time. The fact that S.
oryza possesses functional wings with which to escape, while S. granarius
does not, may have some bearing on the explanation.
PARASITES
Parasites of Sitophilus oryza are numerous and attack all stages of
this insect. A predaceous mite, Pediculoides ventricosus Newport, is
often found in weevil-infested corn in the southern States and attacks
and kills eggs, larvae, and pupse.
Two hymenopterous parasites, Cercocephala elegans Westwood and
Aplastomorpha vandinei Tucker, are found in great abundance in Florida
attacking the larvae.
16917°— 20 2
422 Journal of Agricultural Research vol. xx. No. 6
In addition to the parasites mentioned above, Pierce (8, p. 8o) reports
the following Hyraenoptera as being parasitic on Sitophilus oryza:
Meraporus calandrae Howard,1 M. utibilis Tucker, ' M . requisitus Tucker,
and Catolaccus incertus Ashmead.
From Australia Mr. G. F. Hill (5) reports that he bred the two chalcids
Spalangiomorpha fasciatipennis Girault and N eocatolaccus australiensis
Girault l from grain infested with Sitophilus oryza. T. B. Fletcher (3)
reports that the adult beetle Tenebroides mauritanicus L> preys upon
adult weevils of Sitophilus oryza.
CONTROL MEASURES
Of the vast number of remedies that have been advocated for the con-
trol of this weevil the most effective agents now known are carbon
disulphid and heat.
Infested grains should be fumigated in a gas-tight container or crib.
Four to 6 pounds of carbon per 1 ,000 cubic feet used in such a crib has
proved to be very effective in ridding the grain of the weevils.
Where it is practicable to apply heat to the infested grain, this method
of control will prove very effective. A temperature of 11 6° F. main-
tained for two hours will kill all adults, and a temperature of 1240
maintained for two hours will kill all stages from egg to adult.
LITERATURE CITED
(r) Back, E. A.
1919. CONSERVING CORN FROM WEEVILS IN THE GULP COAST STATES. U. S.
Dept. Agr. Farmers' Bui. 1029, 36 p., 21 fig.
(2) Fitch, Ed. A.
1880. GRANARY WEEVILS: SITOPHILUS GRANARIES AND S. ORYZAE. In Amef.
Ent., v. 3, no. 2, p. 41.
(3) Fletcher, T. B.
1916. agricultural entomology. In Ann. Rpt. Bd. Sci. Advice India,
1914-15, p. 148-162.
(4) Gahan, A. B.
1921. on the identity of several species of chalcidoidea. In Proc. Ent.
Soc. Wash., v. 22. In press.
(5) Hill, G. F.
1915. INSECT PESTS OF PLANTS, NORTHERN TERRITORY OF AUSTRALIA. Bui.
North Ter., Aust., 13, 16 p.
(6) Hinds, W. E., and Turner, W. F.
191 1. LIFE HISTORY OF THE RICE WEEVIL (CALANDRA ORYZA L.) IN ALABAMA.
In Jour. Econ. Ent., v. 4, no. 2, p. 230-236, 1 pi.
(7) Linnaeus, Carolus.
1763. amoenitates academicae ... v. 6. Lugduni Batavorum.
(8) Pierce, W. D.
1912. THE INSECT ENEMIES OF THE COTTON BOLL WEEVIL. U. S. Dept. Agr.
Bur. Ent. Bui. 100, 99 p. , 26 fig.
(9) Plautus.
t96 b. c? curculio, or the forgery.
1 Gahan (4) has pointed out that Meraporus utibilis Tucker and Meraporus calandrae Howard are both
identical with Lariophagus dislinguendus Foerster, and he also states that Girault has reduced Neocatolaccv s
australiensis Girault to synonymy with Aplastomorpha vandinei.
A.— Egg.
B. — Pupa, dorsal aspect.
C. — Pupa, lateral aspect.
D. —Pupa, ventral aspect.
E— Adult.
F. — Third-stage larva.
G. — First-stage larva.
H. — Second-stage larva.
I . —Fourth-stage larva.
PLATE 60
Sitophilus oryza:
Rice Weevil, (Calandra) Sitophilus oryza
Plate 60
Journal of Agricultural Research
Vol. XX, No. 6
OPIUS FLETCHERI AS A PARASITE OF THE MELON
FLY IN HAWAII
By H. F. WlLLARD
Assistant Entomologist, Mediterranean Fruit-Fly Investigations, Bureau of Entomology,
United States Department of Agriculture x
INTRODUCTION
The braconid parasite Opius fletcheri Silvestri was introduced into the
Hawaiian islands from India in May, 1916, by D. T. Fullaway, represent-
ing the Board of Commissioners of Agriculture and Forestry of the
Territory of Hawaii. It was brought in as a parasite of the melon fly
(Bactrocera cucurbitae Coquillett) which had been causing great losses
to the vegetable growers of the islands. The only host here which it
attacks freely under field conditions is the melon fly, although it can be
bred freely in the laboratory from the Mediterranean fruit fly (Ceratitis
capitata Wiedemann). From many thousands of Mediterranean fruit-
fly puparia, secured from fruits collected in the field, only four adult
O. fletcheri have been reared. One was bred from fruit-fly larvae devel-
oping in fruits of Chrysophyllum oliviformae, one from larvae in fruits of
tropical almond (Terminalia catappa), and two from larvae secured from
coffee (Coffea arabica). The first two were collected in Honolulu, and
the last two were from the Kona district of the island of Hawaii.
A clear conception of the biology of this parasite and a record of its
activities since its introduction into Hawaii are the two principal objects
of this paper.
DESCRIPTION AND LIFE HISTORY
EGG
The egg is always deposited in the larva of the host, just beneath the
skin. Its pointed, attenuated end becomes firmly glued to the inner
surface of the larval integument by a dark, almost black substance;
and its free end projects obliquely into the body cavity of the larva.
The spot receiving the egg soon becomes darkened; and the dark sub-
stance by which the egg is attached to the host larva may be a darkened
clot of larval fluids which originally exuded when the wound was made
by the insertion of the ovipositor.
Immediately after deposition (fig. 1) the egg is cylindrical, bluntly
pointed at both ends, slightly more convex dorsally than it is concave
ventrally, and translucent white with a smooth, glistening surface. Its
average length is 0.54 mm. and it is about one-sixth as broad as long.
Just before hatching (fig. 2), its width is a little over one-third the length,
I Credit is due C E. Pemberton, formerly with the Bureau of Entomology, for the drawings contained
in this paper and for the greater part of the microscopic work performed during its preparation.
Journal of Agricultural Research, Vo1- xx- No- 6
Washington, D. C *>ec. '*• ^2°
wa Key No. K-88
(423)
424
Journal of Agricultural Research
Vol. XX, No. 6
Fir., i. — Opius flelcheri: Egg just deposited,
length 0.54 mm.
which averages 0.66 mm., the cephalic end being drawn out into a dis-
tinct tubercle while the caudal end retains the blunt point. At this
time magnification renders the embryo plainly visible.
Only by careful dissections of host larvae into which many eggs of
Opius fletcheri have been deposited during a short period is it possible
to ascertain accurately the duration of the egg stage. In the month of
July, 1918, 439 eggs were under observation, all of which hatched be-
tween 37 and 40 hours after oviposition. The eggs may hatch while
the host is still a larva, or after it has formed a puparium. Even though
a host larva contains several parasite eggs or newly hatched larvae, it is
not killed but continues to feed in
an apparently normal manner and
eventually leaves the fruit and forms
its puparium. In fact, the parasite
seems to have no effect upon the
development of the fly until a com-
plete histolysis of the larval tissues within the puparium has taken place.
At this time all development of the parasitized fly ceases. No histo-
genesis occurs, and the young parasite larva develops rapidly by feeding
upon the liquid mass of the broken-down larval tissues of its host which
surround it.
LARVA
During this period of development there are four distinct instars, dur-
ing which many interesting changes occur. The first instar (fig. 3) is
easily distinguished by a large, chitinized head bearing the strong, pointed
mandibles, and by the chitinized ventral plate of the head which has a
distinct U-shaped cephalic line. In this stage a tracheal system is
present, but no open spiracles
can be seen, even with high
magnification. The two longi-
tudinal, lateral trunks throw
out branches into each body
segment, including the head,
and are connected at their
cephalic and caudal extremi-
ties by a transverse connecting branch. When first hatched, the
larva is surrounded by a mass of egg serosal cells, which cling to
it until it is almost ready to molt into the second instar. This mass,
however, has never been observed clinging to the first larval molt
(fig. 4), as it does in the case of the three Mediterranean fruit-fly para-
sites (Opius humilis Silvestri, Diachasma iryoni Cameron, and D. fulla-
wayi Silvestri).1 The digestive tract, which is a simple tube the greater
portion of which consists of the large intestine, is closed at the caudal
end, although an apparently open anus is present.
1 Pemberton, C. E., and WlX.LA.RD, II. F. A CONTRIBUTION TO THE lilOLOOY of fruit-fly parasites
in Hawaii. In Jour. Ayr. Research, v. \-. no. 8, p. 419-465, 41 fig., pi. 32. 1918. Literature cited; p. 465.
Opius fletcheri: Mature egg. Length 0.66 nun.
Dec. 15, 1920 Optus fletcheri, Parasite of the Melon Fly in Hawaii 425
This is the active stage of the larva, in which it is specially equipped
with long, sharp mandibles for its struggle for survival over other larvae
of the same species, which it often finds in the same host individual.
This struggle takes place immediately after hatching, and usually within
four hours all but one of the larvae of Opius fletcheri have been killed.
Many cases have been observed where there were only one living and
from two to eight dead parasite
larvae in the same host individual.
Thus, having all the food material
of its host available for itself, the
surviving larva is able to proceed
with its development to the adult
stage.
The duration of this instar
varies greatly and depends upon
the development of the host. The
larva never molts into the second
instar until the parasitized host
larva has formed its puparium.
Several instances have been ob-
served where larvae of Opius
fletcheri have developed to adults
while other individuals, from eggs
laid at the same time, still re-
mained first-instar larvae. The
host larvae of the former formed
their puparia soon after they were
parasitized, while those of the lat-
ter were still in the larval stage
when examined. In all the ex-
periments to prove this point the
host was Ceratitis capitata,\a.rvgd of
which were feeding in the fruits of
Mimusops elengi. These fruits
become rather dry soon after fall-
ing from the tree, so that fruit-
fly larvae within them find diffi-
culty in obtaining sufficient food for rapid development. This results
in retarding pupation, sometimes for over three weeks beyond the normal
period. On June 1 1 eggs of O. fletcheri were deposited into fruit-fly larvae,
which were examined with the following results: On June 18, 10 of these
larvae contained living first-instar larvae of O. fletcheri, and 3, that had
formed puparia, each contained a fourth-instar larva of O. fletcheri. On
June 22, 3 more larvae and 2 of the puparia of this lot were examined.
Each of the larvae contained a well-developed living larva of O. fletcheri
Fig. 3. — Opius fletcheri: Larva, first instar, ventral as-
pect, showing head characters and complete tracheal
system, and the egg serosal cells. Length 0.88 mm.
426
Journal of Agricultural Research
Vol. XX, No. 6
in the first instar, and each puparium contained a well-formed pupa of
O. jletcheri. C. capitata larvae into which O. jletcheri had deposited eggs
on June 12 were examined on June 24. Each of 7 which were still in
the larval stage contained a strong, living, first-instar larva of O. fletcheri;
while 7 of the host larvae, which
had formed puparia, each con-
tained a mature pupa of 0.
fletcheri about to emerge. Eggs
that were deposited on June 13
produced, on June 27, ro adult
male O. jletcheri, and on June 28,
4 males and 2 females. On June
28, also, 2 of the host larvae that
had not yet pupated each con-
tained a living first-instar larva
of O. jletcheri. On June 14 eggs
of O. jletcheri were deposited in
fruit-fly larvae. On June 27, 1
adult male O. jletcheri had de-
veloped from this lot, while 4
of the host larvae, that had not
formed puparia, each contained
a living first-instar larva of 0.
jletcheri.
These results indicate that the
first instar of Opius jletcheri is
controlled to a great extent by
the development of its host, since
it never molts into the second
instar until the host has formed
its puparium, and that the first
instar may extend over a period
of 10 to 12 days. When the host
forms a puparium shortly after
being parasitized, the first instar
may be as short as 1^ days.
The second-instar larva (fig. 5)
is very much without distinctive
characters. The mandibles (fig.
6) are very small, soft, and indis-
tinguishable even under high magnification, except upon occasions where
the position and lighting are most favorable. They are 0.045 mm- m
length and so far as can be seen serve no purpose. No tracheal system is
present. None can be detected under the best of lighting and the highest
of magnification. No part of the head or body is chitinized. The entire
Fig. 4.— Opius jletcheri: .Molted skin of first-instar larva,
showing the absence of egg serosal cells. Length
0.8 mm.
Dec. is, 1920 Opiu9 fletcheri, Parasite of the Melon Fly in Hawaii 427
body is very delicate and can be easily crushed beyond recognition with
a very slight pressure on the coverglass. The digestive tract is simple
and tubular and is closed caudally as in the first instar. In this stage the
larva is sluggish in its movements, although it rapidly ingests a quantity
of fat into its mid-intestine. Toward the latter part of this instar the
mandibles of the third
instar can be seen
pushing at the bases
Fig. 5. — Optus fletcheri: New second-instar larva. C.reatly enlarged.
Fig. 6.— Optus fletch-
eri: Mandible of
second-instar larva.
Length 0.045 mm.
of the mandibles.
The third instar,
when first formed, is
without a vestige of
tracheae. Tracheae
can be seen developing beneath the surface of the integument toward
the latter part of this stage, but they are of the last instar and
serve no purpose in the third. Few differences can be detected between
this and the preceding instar, except an increase in size and a change in
the shape of the mandibles. The third-instar larva measures 2.5 to 3
mm. in length. The mandibles (fig. 7) are somewhat more pointed and
strong than those of the second instar; they bear no
colored chitinization and measure 0.047 mm- in length.
Toward the latter part of this instar the strong, chitin-
ized mandibles of the last instar can be seen pushing
at the bases of the mandibles.
The mature, fourth-instar larva (fig. 8) averages 4
mm. in length and at its greatest width is about three-
eighths as wide as long. When first molted into this instar it is 3 to 3.5
mm. long. The body is slightly curved, being concave ventrally, and,
including the head, is composed of apparently 14 segments, although
segment 14 is not clearly defined. A rather large, distinct spiracle is
present on each side of segments 3, 5, 6, 7, 8, 9, 10, 11, and 12, counting
the head as segment No. 1. These spiracles are joined
on each side by a large lateral trunk extending nearly
the length of the body. The trunks are connected near
their caudal and cephalic extremities by a single, trans-
verse, connecting trunk, these being the only connections
between the two lateral systems. Branches from the lat-
eral trunks extend dorsally and ventrally into each body
segment, and prolongations of the lateral trunks extend
into the head region. Portions of the body are covered by minute, strong,
wide-based spines (fig. 9), which are closely set and abundant on the dorsal
and lateral portions of body segments 2 and 3, counting the head as seg-
ment No. 1, and on the lateral areas of segments 4 to 12, inclusive. No
spines occur on the head, on the articulation areas between the segments,
or on the ventral portion of any segment of the body, and very few occur
Fig. 7. — O pius fletch-
eri: Mandible of
third-instar larva.
Length 0.04 ymm.
428
Journal^pj Agricultural Research
Vol. XX, No. 6
Fig. 8. — Opius ftelcheri: Larva, fourth instar, lateral aspect, showing
general outline and spiracles. Length 4 mm.
on the last segment. The only colored ehitinized parts occur in the
head, where a pair of strong, pointed mandibles (figs. 10, n) — of which
the distal half only is ehitinized — and the tentorial structures are ehitin-
ized a yellowish brown color. Small maxillae bearing minute papillae are
present, together with
a well-defined labrum
and suboval labium.
The most important
changes that take
place, then, during the
larval development of
Opius fletcheri occur in
such a manner as to
adapt it to the chang-
ing environment with-
in its host. Larvae of
the first instar are very active and have long, sickle-like mandibles, which
enable them to search out and destroy other parasite larvae which occur
in the same host individual. Second and third instar larvae live in and
feed upon the liquid or semiliquid medium contained in the host pupa-
rium. The mandibles, therefore, being useless, are small and incon-
spicuous, and there is no tracheal system whatever. In the fourth instar
the liquid within the host puparium has been nearly all consumed, and
the mature larva is
found with fairly
strong mandibles and
a well-defined tracheal
system connected with
easily observed spi-
racles.
Two species of opi-
ine parasites of the
Mediterranean fruit
fly hibernate as mature
larvae for varying
lengths of time during
the cooler seasons of
the year.1 NO hiber- Fig. 9. — Opius fletckeri: Spines on body of mature larva. Length
nation of Opius fletch- °'°13 mm
eri has been observed during any stage of its development, although
thousands of parasitized puparia have been under observation. In Sep-
tember, 1 91 8, 592 parasitized melon-fly pupae were held in a refrigerator,
where the temperature was constantly about 65 ° F., until two weeks
after all adults had emerged. All unhatched puparia were then exam-
ined and no hibernating larvae were found. One hundred and sixty
1 PembErton, C H., and V.'ii.lard, H. F. op. cit.
Back, E. A., and Pemberton, C. E. the mediterranean fruit fi,y in Hawaii. U. S. Dept.
Agr. Bui. 536, 119 p., 24 fig., 21 pi. 1918.
Dec. 15, 1920 Opius fletcheri, Parasite of the Melon Fly in Hawaii 429
Fig. 10. — Opius fletcheri: Mandible of fourth-
instar larva. Length 0.075 mm.
adults of Opius fletcheri emerged in the refrigerator, and each of
the remaining 432 unhatehed puparia contained a well-developed,
dead pupa of Opius fletcheri. A control lot of 500 parasitized
puparia that were held at the same time
at normal temperatures, 750 to 850 F.,
produced 487 adult parasites and 13
dead pupae of Opius fletcheri. Seventy-
two and six-tenths per cent of the para-
sites developing in the refrigerator and
2.6 per cent of those developing at nor-
mal temperatures died while in the pupa
stage. These data seem to indicate that
it is difficult for Opius fletcheri to develop
through the pupal stage at a temperature
as low as 650 F. This mortality of pupae,
however, is not evident under field con-
ditions. While records of parasitism of the melon fly, which was devel-
oping in cucurbits collected in the field at all seasons of the year, were
being obtained, thousands of unhatehed melon-fly puparia were opened.
Although some of these records were secured when the temperature
ranged from 6o° to 700 F., less than 3 per cent mortality of Opius fletcheri
pupae was found. The cause of the high mortality of pupae in the refrig-
erator has not been determined.
pupa '
In the process of transforming from the mature larva to the pupa
(fig. 12) this insect passes through a prepupal state of from one to two
days. The larva be-
comes motionless.
The anterior portion
of the body, which is
to form the head and
thorax of the pupa,
becomes slightly con-
tracted, so that it
is somewhat smaller
than the remainder of
the body. The eyes
can be seen, forming
beneath the integu-
ment, as indistinct
reddish brown spots;
these become more
distinct and darker in
color until, just before the moltinto the pupal stage, they can beplainly seen.
In the last larval molt the skin is split from the head backward and,
by slight expansions and contractions of the body, it is pushed back over
FlG. 11. — Opius fletcheri: Head of mature larva, dorso-cephalic aspect.
Width 0.63 mm.
43°
Journal of Agricultural Research
Vol. XX, No. 6
the tip of the abdomen and finally comes to rest on the dorsal portion of
the pupa. This exuvium often adheres to the antennae of the male or
the ovipositor of the female for a short time after the adult has emerged
from the puparium of its host. The length of the pupa is 3.8 mm.
When first formed it is pale
white, excepting the eyes, which
are a very dark reddish brown;
but within a few hours it begins
to acquire a yellowish tinge and
continues to assume the colora-
tions of the adult until ready to
emerge.
The length of this stage varies
from four to eight days, even
though it is passed under the
same temperature and other con-
ditions. During the month of
July, 1 91 8, when the tempera-
ture ranged from 75 ° to 85 ° F.,
90 parasitized puparia were
under observation. Adults of
Opius fletcheri emerged from
these puparia from 80 to 200
hours after pupation. Emer-
gence was taking place at fre-
quent intervals between these
two extremes but was most fre-
quent between 1 30 and 1 50 hours
after pupation. This would in-
dicate that the length of the
pupal stage in the majority of
cases was about six days. Be-
tween 80 and 100 hours after pu-
pation, 17 males emerged, but
it was between 100 and no hours
before the first two females
emerged. Thelastmaleemerged
after a period of from 1 70 to 1 80
hours, and the last two females
emerged between 190 and 200 hours after pupation. The pupal stage of
the male is usually about 24 hours shorter than that of the female.
Fl<;. 12. — Opius fletcheri: Pupa, female.
3.8 mm.
Length
ADULT
The following description of the adult by Silvestri is translated from
the Italian :
Dec. iS> 1920 Opius fletcheri, Parasite of the Melon Fly in Hawaii 431
Opius fleicheri, n. sp.
Female. — Body ochreous yellow or testaceous, with the anterior part of tergites
2-6 of the abdomen brownish. Antennas, except at the apex, where they are brown-
ish, and legs, except the pale brown hind tarsi, of the same color as the body. Wings
hyaline, with the nervures in great part brown. The stigma brown, except the
middle part, which is yellowish white. Length of body 4.5 mm.; width of thorax
1.05 mm.; length of antennae 6.5 mm.; of the wings 5 mm., width of same 2 mm.,
length of ovipositor (the part protruding) 2 mm.
Head just a little wider than the thorax, about two-fifths wider than high, with
eyes large, convex, nude, reaching below almost to the level of the margin of the
clypeus. Face, excepting at the base of the antennae, full, and subcarinate in the
middle. Antennae longer than the body, attenuate, composed of 42 to 48 segments,
of which the scape is about five-eighths longer than the second segment.
Thorax. — esothoracic scutum with parapsidal grooves, indistinct, nude. The
transverse prescutellar groove furnished with a series of about ten pits, not very deep.
Metanotum lightly con-
vex, and smooth in the
middle for the greater part
of its length, and carinate
for a short space behind,
pitted in the sides; pro-
podium provided with a
median longitudinal ca-
rina which divides be-
hind, with a sublateral
carina near the side, but
within the stigmata,
which are sufficiently
large and round . The sur-
face between the carinas
smooth . Mesopleura with
the longitudinal groove
crenulate.
Anterior wings with the
discoidal cell and the first
cubital very large , subrec-
tangular, longer than the
second cubital, with the
recurrent nervure long,
arcuate as seen in the
figure.
Abdomen suboval, with
the first tergite lightly
carinate at the side and lightly rugose in the middle,
with a few long hairs, second suture rather distinct,
and straight, about as long as the abdomen.
Male. — Similar to the female but a little smaller.
Observations. — This species of Opius is quite distinct from the numerous species
I know from Palaeartic and Ethiopian faunas by the shape of the recurrent nervure,
and by the length of the discoidal and first cubital cells.
Habitat. — India. Prof. Fletcher obtained examples of this species from the
pupae of Chaetodacus cucurbitae Coquillett, the larvae of which live in the fruits of
Momordica charantia L.
Fig. 13. — Opius fletcheri: Adult female. Length 4.5 mm.
The rest smooth and furnished
Ovipositor, which is very sharp
432 Journal of Agricultural Research voi.xx.No. 6
The adult (fig. 13) liberates itself from the host puparium by gnawing
a transverse slit near the end and by pushing with its head until the entire
end of the puparium breaks off, allowing it to emerge. Immediately
after emergence the meconium is discharged. This meconium is an
ovoid, hard pellet, consisting of all the waste material which has collected
in the digestive tract during the larval stage. No waste material is
voided before this time, although many braconids discharge it just prior
to pupation.
Copulation may occur frequently, and at any time, from immediately
after emergence to the death of the adult. Two newly emerged females
were put into a glass tube with one male that had just emerged, and the
male successfully copulated with both females within 10 minutes. Nine
females that emerged May 18 to 20 were put into a tube with males,
where several instances of successful mating were observed. On July 1 ,
when these females were 6 weeks old, they were put into a glass tube
with 30 newly emerged and vigorous males. Within 45 minutes 12
successful matings were observed, and one of the females mated four
times within 15 minutes. In all of these instances the females made no
great effort to escape from the males. The period of coitus lasts from
yi to 2 minutes, although in the majority of instances it is less than 1
minute. In six of eight cases under observation the duration was be-
tween 30 and 45 seconds, while in the other two cases it was extended to
\]/2 and 2 minutes, respectively. As far as it has been possible to ob-
serve, all of the sex attraction is produced by the male. When within
about 2 inches of the female, the male becomes greatly excited and
while slowly approaching her, and during coitus, vibrates the wings
vigorously and spasmodically. No strong, sweet odor, such as is emitted
by the males of the fruit-fly parasites Opius humilis Silvestri and Dia-
chasma tryoni Cameron,1 has been detected during work with this species.
Opius fletcheri is capable of parthenogenetic reproduction, and the ab-
sence of mating does not influence oviposition. Large numbers of adults,
all of which were males, have been reared from unmated females. The
fact that mated females will produce a considerably larger percentage
of females than males is of much interest. Eight females that were
observed mating within two hours after emergence were put into individual
glass tubes, where host larvae were available at all times. From these
females 39 males and 72 females were reared, giving 35.1 per cent males
and 64.9 per cent females. Under field conditions about 10 per cent
more females than males are produced. While records of parasitism of
the melon fly developing in cucumbers collected in the field during 191 8
and 1919 were being secured, 7,746 adult 0. fletcheri were reared. Of
this number 4,273, or 55.2 per cent, were females, and 3,473, or 44.8 per
cent, were males. Many species of opiine parasites consistently produce
more males than females. For example, the parasites of the Mediterra-
1 Pembbrton, C E., and Willard, H. F. op. cit.
Dec. 15, 1920 Opius fletcheri, Parasite of the Melon Fly in Hawaii 433
nean fruit fly, D. tryoni and O. humilis, that were reared from material
collected in the field, produced 37.6 per cent and 43.5 per cent females,
respectively. Since the females are responsible for all the parasitism of
the host, the ability of 0. fletcheri to produce so many more females than
males greatly enhances its value as an enemy of the melon fly.
The longevity of the adult depends largely upon the conditions under
which it lives and may extend from a few days to 16 weeks. When con-
fined without food it will not live much over 5 days. Of 6 males and 17
females that were confined in a glass tube without food, 3 females died
before they were 3 days old, and 3 more lived to be a few hours over 5
days old, but the majority of both males and females died between the
ages of 3% and 4 days. The life of females that have had continual
access to host larvae is much shorter than that of those which have had
no opportunity to oviposit; and the life of males is considerably shorter
than that of the females. Of 9 females that were allowed to oviposit at
will, 2 died at the end of 2 weeks, 2 at the end of 8 weeks, and the other
5 lived 3, 5^, 6, 6^2, and 7 weeks, respectively. With no opportunity
to oviposit, 85 females, together with 43 males, were confined in a glass
tube and kept in partial darkness, with daily feedings of a mixture of
one-fourth honey and three-fourths water. Three of these females
lived to be 16 weeks old, 33 of the males died between the ages of 6 and 8
weeks, while 1 male lived to be 11 weeks old. The majority of the
females died between the ages of 11 and 13 weeks, while 15 lived a little
beyond this period.
OVIPOSITION
Oviposition takes place in only the larva of the host and may occur
at any time after the larva is one-half grown; but it is most frequent
in well-developed larvae. Observations of the female, just prior to
oviposition, indicate that she locates the host larva beneath the skin
of the containing fruit by a sense of touch. She walks rapidly over the
surface of an infested fruit, stopping at frequent intervals, evidently
endeavoring to detect vibrations caused by a feeding host larva. While
searching for the host, and during the act of oviposition, the female
often vibrates her wings rapidly and spasmodically, although this does
not always happen. When a favorable spot is found, she elevates her
abdomen and pierces the skin and pulp of the fruit with her ovipositor,
raising and lowering it until the host is located. She then inserts the
ovipositor into the larva and deposits an egg just beneath the skin.
Then she withdraws the ovipositor from the fruit and usually begins to
search for another larva; but occasionally, after a short rest, she will
oviposit again in the same one. The female is unable to discern between
parasitized and unparasitized larvae.
Although mating may occur immediately after emergence, oviposition
does not begin until 2 days later and, in the majority of cases, 3 to 5
days after emergence. Eight fertile females were given constantly
434
Journal of Agricultural Research
Vol. XX, No. 6
available host larvae from the time of emergence. Two of these began
ovipositing in 2 days, one in 3, three in 5, and two in 7 and 9 days,
respectively. None of these females oviposited after they were 30 days
old, excepting one, which deposited one egg at the age of 33 days. The
majority of eggs are deposited within the first 3 weeks after oviposition
begins. As noted before, females that have had daily opportunity to
oviposit do not live so long as those that have had no opportunity; but
they frequently live from 4 to 5 weeks after oviposition has ceased.
IMPORTANCE AS A PARASITE
Opius fletcheri, in the three years since its introduction into the
Hawaiian Islands, has become firmly established on all the large islands
of the group. While this parasite alone will never exercise a complete
control over the melon fly in Hawaii, it has already proved of much value
by decreasing the numbers of this pest considerably. Good examples of
the most abundant melon-fly host plants are cucumber, squash, pump-
kin, and watermelon. The fruits of these plants are large and fleshy,
and melon-fly larvae that develop in them feed so far from the surface
that a larval parasite, such as O. fletcheri, that oviposits entirely from
the outside, finds it impossible to parasitize enough of the larvae to exert
a control over the pest.
Table I gives data showing the extent of parasitism by Opius fletcheri
of melon-fly larvae developing in cucumbers collected in and about
Honolulu during the last eight months of 191 8 and the first eight months
of 1 91 9.
Table L-
-Percentage of parasitism by Opius fletcheri of larvce of Bactrocera cucurbitae
in cucumbers
Month of collection.
Number of larvae
emerging during
first two to four
days.
1918
Percentage of parasit-
ism.
1918
January. .
February.
March. .. .
April
May 1,014
June 2, 719
July 2,052
August 43 1
September 3, 594
October 2, 516
November 8, 282
December ' 4, 319
1,031
539
6, 442
3,i92
1,481
1,318
5.255
19. 321
5-9
10. o
21. 9
21.8
29. 8
16.6
22. 1
7-3
2.9
14-5
9.0
2. 2
6.4
10. 6
7-3
The highest percentage of parasitism existed in September, 191 8,
when 1,070 out of 3,594 melon-fly larvae under observation were parasit-
ized. This shows a parasitism of 29.8 per cent, while the parasitism
Dec. iS, 1920 Opius fletcheri, Parasite of the Melon Fly in Hawaii 435
from all cucumbers collected during 191 8 was 18.1 per cent. Parasitism
from larvae developing in cucumbers collected in the first eight months of
1 91 9 amounted to 7.3 per cent. These records were secured from only
those larvae that emerged from the cucumbers the first two to four days
after collection. Larvae emerging after this time would not give a true
representation of parasitism under field conditions, because at the
time they were collected they were comparatively small and had been
subject to parasitism only a short time. These cucumbers were specially
selected by the collector as being the most heavily infested ones in the
fields. Considering the fleshy nature of cucumbers and the fact that
those from which these data were obtained were from 4 to 10 inches
long, it is remarkable that Opius fletcheri is able to destroy such a high
percentage of the melon-fly larvae developing in them.
Considerable effort has been made to establish a series of records a
comparison of which would show the amount of infestation by the melon
fly from time to time and which would determine the extent of control
exerted by Opius fletcheri. Infestation records of the Mediterranean
fruit fly have been secured by recording the average number of larvae
per fruit, this average being obtained from a large number of fruits of
the same species. The great variation in size of cucumbers made this
method impracticable, and the following method was used: All cucum-
bers that were collected for records of parasitism were weighed and then
held until all the melon-fly larvae had emerged. Accurate records of
these larvae were kept, and at the end of December, 191 8, and of August,
191 9, the average number of larvae per pound of host fruit was obtained.
From July to December, 191 8, inclusive, 200 pounds of cucumbers were
collected, which contained 47,888 melon-fly larvae, or an average of 239.4
per pound. From 337 pounds of cucumbers, collected during the first
eight months of 1919, 57,921 melon-fly larvae were secured, giving an
average of 172 larvae per pound. These averages indicate that the
melon-fly infestation of cucumbers in and about Honolulu was approxi-
mately 28 per cent less during the period from January 1 to August
31, 1919, than it was between July 1 and December 31, 1918.
It appears from observations of melon-fly infestation in Hawaii made
during the past several years that this decrease in the numbers of the
melon fly is due to a great extent to the activities of Opius fletcheri.
Before this parasite was introduced into Hawaii in 19 16 it was almost
impossible to find a cucumber in the Honolulu markets that did not
show more or less evidence of attack by the melon fly. From observa-
tions made by them in 1915 and 1916, Back and Pemberton state '
that one rarely sees cucumbers offered for sale in the Honolulu markets
that do not show some evidence of attack, even when carefully selected,
and that during midwinter 150 out of 152 cucumbers ready for market
1 Back, E. A., and Pemberton, C E. the melon ply in Hawaii, U. S. Dept. Agr. Bui. 491, 64 p.,
24 pi., 10 fig. 1917. Bibliography, p. 57-64.
16917°— 20 3
436 Journal of Agricultural Research vol. xx, No. 6
at Moiliili were found variously infested. They state also that the
ordinary cucumber, when very young, is the most resistant to melon-fly
attack of all the cucurbits cultivated in Hawaii, but that inasmuch as
the fly has been permitted to increase unchecked since its introduction
it has become so abundant that slight differences in inherent resistance
to attack are not evident among host fruits growing in the field. The
condition of cucumbers offered for sale in Honolulu during the first
eight months of 191 9 indicates that O. fletcheri, while not being able
completely to control the melon fly on the island of Oahu, has been able
to reduce its numbers to such an extent that the infestation of cucumbers
has been greatly decreased. During this period there have been good
quantities of this vegetable on the market at all times, a very small
portion of which has shown evidences of melon-fly attack. The writer
has observed on several occasions at different plantations wagon loads
of cucumbers that had been selected for market, among which it was
difficult to find any great number that had been attacked. While col-
lecting cucumbers during the past year from the different gardens for
parasitism records, it has often been difficult to get a sufficient quantity
of well-infested fruits. These observations, as compared with those
made previous to the establishment of O. fletcheri, would lead to the
conclusion that this parasite has already become of much value, even
while attacking its host in the larger cucurbits.
The ability of Opius fletcheri to reach and parasitize the majority of
host larvae developing in the smaller fruits is clearly shown by data
collected during the past five years in the Kona district of the island of
Hawaii. In this district it comes nearer to controlling the melon fly
completely than in any other locality that has been observed. This
great degree of control is without doubt due to the great abundance of
the wild Chinese cucumber (Momordica sp.). The fruits of this plant
are small, about % to \l/i inches in diameter by 1 to 2 inches long. The
following observations give a good conception of their susceptibility to
melon-fly attack and of the ability of Opius fletcheri to decrease their
infestation greatly by parasitizing a large percentage of the larvae de-
veloping in them.
From observations made in this district, Back and Pemberton state '
that—
From Momordica vines covering a patch of pasture land 6 feet square, 331 fruits
were gathered during November, 1914, of which only 12 had not been infested. These
fruits, which were of all sizes up to \% inches in diameter, averaged between three and
four punctures per fruit, with a maximum of 15 punctures on the more exposed fruits.
From 7 feet of stone wall 442 fruits were gathered, and of these 193 were so badly-
affected that they had dried up without developing seeds, and only 11 were not
affected. From 250 fruits placed over sand 1,586 larvae, or an average of 6.5 larvae
per fruit, were reared.
1 Back, E. A., and Pemberton, C. E. the melon fly in Hawaii. U. S. Dept. Agr. Bui. 491, 64 p.,
24 pi., 10 fig. 1917. (See p. 17-18.)
Dec. 15, 1920 Opius fletcheri, Parasite of the Melon Fly in Hawaii 437
A careful examination of 442 fruits of Momordica, collected at random
over an area of % square mile in the Kona district, made by C. E. Pem-
berton on May 8, 1916, gave the following results: 194 were not in-
fested, and the 248 that were contained a total of 559 eggs and 1,222
larvae of the melon fly. This is an average infestation per fruit for the
442 fruits of 4 flies either in the egg or larval stage.
The first adults of Opius fletcheri were liberated in this district in the
summer of 19 16. Data secured by C. E. Pemberton during the latter
part of April and the first part of May, 19 18, showed that it had
become widely established, was parasitizing a very high percentage of the
melon fly developing in Momordica, and that it had so greatly reduced
the number of flies that cultivated cucurbits were being raised with
little or no infestation. Out of 1,706 Momordicas collected by him on
April 25 and 26, 19 18, 347 fly larvae emerged the first two days after col-
lection, of which 299, or 86.2 per cent, produced parasites. On April
28 and 29, 700 Momordicas were collected, from which 226 melon- fly
larvae emerged during the first two days. Of these 226 larvae 219, or
96.9 per cent, produced parasites. From these two lots 103 larvae
emerged after the first two days, making a total of 676 larvae developing
in 2,406 ^fruits. This is an average of less than 0.3 larva per fruit, as
compared with an infestation of from 4 to 6.5 larvae per fruit before the
liberation of O. fletcheri.
Further observations made at the same time of 1 ,706 ripe Momordicas
collected in the same locality showed that only 36 of this number con-
tained either eggs or larvae of the fly. Thirty ripe fruits of the same
plant, collected at Honaunau, about 12 miles from Kealakekua, showed
no infestation whatever. On May 10, 1918, 400 cucumbers, both large
and small, 28 young watermelons, 20 young muskmelons, and 21 young
pumpkins were carefully examined in a garden in Kealakekua. This
garden was bounded on one side by a coffee plantation and on the other
three sides by pasture land that was overrun with heavily-fruiting vines
of wild Momordica. Only one cucumber was found that had been
punctured by the melon fly. None of the other vegetables or melons
that were examined had puncture scars, either new or old, and none of
the blossoms of any of the plants were stung.
In June, 19 19, this same low degree of infestation still existed in this
district. From 890 Momordicas collected at that time the average
infestation was less than 0.2 larva per fruit. In several gardens less
than 3 per cent of the cucumbers and melons that were examined showed
evidences of attack, and none of the blossoms were found that had been
stung.
When the vines of wild Momordica are abundant on pasture lands,
their ability to cover and kill large patches of grass has caused them to
be considered a pest, and consequently they have not been allowed to
438 Journal of Agricultural Research voi.xx.No. e
become abundant in many localities in Hawaii. When Momordica is
abundant and Opius fletcheri is present, it has proved of considerable
value as a trap plant for the melon fly. Infestation records made before
the parasite was liberated show that Momordica is much favored as a
host by the melon fly, while subsequent records of parasitism show that
its size and texture permit the parasite to kill about 90 per cent of the
larvae developing in its fruits. Whether or not it would be of advantage
to plant these vines around vegetable gardens as a catch plant is a
problem open to further investigation.
Opius fletcheri, besides becoming firmly established on all the larger
islands of the group, has shown itself capable of reducing the number of
melon flies by at least 25 per cent, even when the host larvae are devel-
oping in fruits the size and nature of which make parasitism difficult.
In a location where the fruits and conditions are most favorable to its
reproduction it has reduced the flies so greatly that they have almost
ceased to be a pest. While O. fletcheri is far from being able to control
the melon fly in Hawaii completely, the benefits derived from its activ-
ities since its establishment there have been sufficient to warrant the
efforts connected with its introduction.
TAMARIND POD-BORER, SITOPHILUS LINEARIS
(HERBST)1
By Richard T. Cotton, Scientific Assistant, Stored-Product Insect Investigations,
Bureau of Entomology, United States Department of Agriculture
The literature of North American entomology contains occasional ref-
erence to the curculionid beetle, Sitophilus linearis (Herbst), but nothing
definite has been published regarding the biology of this interesting weevil
or the extent of its distribution in the United States.
HISTORY AND DISTRIBUTION
The tamarind pod-borer was described in 1797 by Herbst under the
name of Rhynchophorus linearis. The specimens described were ob-
tained from the West Indies, where the weevil had been introduced with
its food plant, the tamarind. It undoubtedly is native to India but has
now spread to all places where the tamarind is grown. In 181 5 it was
described by Thunberg as the variety striata, and again in 1834 by
Christy under the name of Calandra tamarindi, and finally in 1837 by
Dejean under the specific name of frugilega. All of these later names
have since been reduced to synonymy.
In 1 892 Casey 2 noted the occurrence of Sitophilus linearis in North
America, but in 1895 Chittenden3 stated that in his opinion 5. linearis
should not be inserted in our faunal list until it could be ascer-
tained that the species actually bred in some plant within our faunal
limits. Up to the present time all records of its occurrence in the United
vStates refer to occasional specimens picked up in the southern Atlantic
and Gulf States which had undoubtedly been imported in shipments of
tamarind pods. The writer has found it to be exceedingly abundant in
southern Florida where the tamarind is now grown; therefore there is no
longer any doubt that it is well established within our faunal limits.
In 1916 A. H. Ritchie4 recorded this species as causing considerable
damage to the pods of the tamarind in Jamaica, and T. B. Fletcher 5
has recorded similar damage in India.
1 The writer was enabled to make a study of this species through the courtesy of the Federal Horticultural
Board, whose representative, Mr. O. K. Courtney, intercepted at the port of New Orleans a shipment ol
infested tamarind pods from Guatemala, which was forwarded for study to the division of Stored-Product
Insect Investigations of the Bureau of Entomology. The writer wishes to acknowledge his indebtedness
to Dr. Adam G. BSving, of the Bureau of Entomology, for his valuable aid and advice in the study of the
larval characters of this weevil.
2 Casey, Thos. L. coleopterological notices iv. In Ann. N. Y. Acad. Sei., v. 6, 1891-92, p. 359-71;.
1892. [Calandra linearis, p. 686.]
'Chittenden, F. H. on the distribution of certain imported beetles. In Insect Life, v. 7, no.
4, p. 326-332. 1895-
4 Ritchie, Archibald H. report of entomologist for year 1915-1916. In Ann. Rpt. Dept. Agr.
Jamaica [1915] 16, p. 31-34. 1916.
5 Fletcher, T. Bainbrigge. one hundred notes on indian insects. In Agr. Research Inst. Pusa
Bui. 59 39 p., 20 fig. 1916. Weevils in tamarind fruits, p. 10.
Journal of Agricultural Research, Vol. XX, No. 6
Washington, D. C Dec. 15, 1920
wb Key No. K-89
(439)
44-0 Journal of Agricultural Research vol. xx, no. 6
This weevil is now known to occur in the United States. India, Brazil,
Mexico, Ecuador, Jamaica, Montserrat, St. Bartholomew, Cuba, and
Costa Rica. It occurs, undoubtedly, wherever the tamarind is grown.
NATURE OF INJURY
The injury is confined entirely to the seed pods of the tamarind. The
adult weevils feed little, but the larvae or grubs bore in the seeds or beans
and reduce them to powder. The entire crop is frequently completely
destroyed unless promptly harvested and protected.
For those not familiar with the tamarind a few descriptive and histor-
ical notes are here inserted.
The tamarind, Tamariyulus indicus, although attributed to India, is
positively asserted to be indigenous to Africa and Australia. It was
introduced into the West Indies by the Spaniards soon after the dis-
covery of those islands, and was naturalized at an early date in Brazil,
Ecuador, Mexico, and other parts of the tropical world. A few trees have
been introduced into the United States in Florida and California. Al-
though a tropical plant it does well in southern Florida.
The seeds are borne in large pods and are embedded in a sweet, sticky,
reddish pulp. This pulp has mild laxative properties and is found on
the market usually mixed with sugar or syrup. In tropical countries
the pulp is used extensively for the preparation of a cooling beverage
and as a flavoring for ice cream. In European countries it is said that
the pods and seeds when roasted are considered a delicacy. The bark,
seeds, and leaves are used to a limited extent by natives of the Tropics
as therapeutic agents.
The wood is heavy and hard and is used for making furniture on ac-
count of its fine grain and color. It is used also in making tools, axles,
wagon wheels, and similar articles.
LIFE HISTORY AND BIOLOGY
Since the tamarind grows only in tropical or subtropical climates, the
activities of the weevil are not stopped by winter. It breeds throughout
the year. In Florida the seeds of the tamarind usually mature in May,
but a few may be found maturing in almost all months of the year, thus
providing a more or less continuous food supply for the weevils. As the
pods mature they quickly become infested.
The adult weevils enter the tough-shelled pods through the stem end.
The swaying of the pods in the wind causes small breakages in the pod
rind to occur at the juncture of the stem, and through these breaks the
weevils find an easy entry. The female weevils bore directly through the
pulpy covering and into the tough seeds. In the seeds they excavate a
cylindrical cavity about 3 mm. deep and 1.5 mm. in diameter. If the
shell of the pod is broken away the weevils may be seen at work, the top
Dec. i5, 1920 Tamarind Pod-Borer, Sitophilus linearis (Herbst) 441
of the abdomen alone showing above the surface of the pulpy covering,
the rest of the body being concealed within the cavity. This cavity
is usually completed in from two to three days. The individual egg
cavities are then bored in the seed all around the interior of this larger
cavity, an egg being deposited as soon as a hole is finished. The eggs are
all placed as close together as possible, so that the interior of the large
cavity has the appearance of being lined with rows of egg-caps. From
12 to 50 eggs are laid in one group, the time taken for the completion of
the group varying from one to two weeks, according to the number of
eggs laid. By the time the last egg is laid the first eggs have hatched
and the grubs have become half grown. This habit of the female weevil
of grouping a number of eggs together in one seed exhibits an interesting
difference from the egg-laying habits of the grain weevils belonging to
this genus. One would naturally conclude that it was developed to save
energy, since it would be no mean undertaking to bore through the
pulpy covering and the tough seed coat for each individual egg.
The operation of excavating the egg cavities is accomplished by a
combined up and down and rotary motion of the proboscis, effected by
turning the head from side to side while the thorax is oscillated back and
forth. As soon as an individual egg cavity is completed and the sides are
smoothed to the satisfaction of the weevil the proboscis is withdrawn.
The weevil then reverses its position and, inserting its ovipositor into the
cavity, deposits an egg, sealing it in with a plug of opaque, yellowish
material resembling faecal matter. In a few days this plug turns to a
dark yellowish brown.
It is interesting to note that, so far as observations go, the female
weevil does not leave the egg cavity from the time it is started until the
last egg has been laid. She works day and night until the operation has
been accomplished unless disturbed by outside agencies. Whenever
she rests it is without leaving her position in the cavity. As soon as one
group has been finished the weevil immediately seeks out another loca-
tion and begins operations again. For sheer industry and continuous
application to the object of perpetuating its kind this weevil would be
hard to surpass.
The eggs hatch at the end of three days. Previous to hatching the
larva may be distinctly seen through the thin outer shell of the egg.
This shell or skin is very flexible and undulates with the movements of
the young grub. It becomes somewhat wrinkled and finally breaks at
the bottom, allowing the grub to escape. The young larvse begin at
once to feed and bore through the seed, their burrows radiating from
the large cavity to all parts of the seed, and usually ending near the
shell of the seed, through which, however, they never break.
As in other species of this genus, there are four larval instars, although
previous writers have erroneously attributed but three larval instars to
442
Journal of Agricultural Research
Vol. XX, No. 6
the grain weevils of this genus. The lengths of the various stages are
regular and are given in Tables I and II.
Table I. — Life history data of the tamarind pod-borer 1
Weevil No.
Egg laid.
Hatched.
First molt.
Second
molt.
Third
molt.
Prepupa.
Pupa.
Adult.
June 19
23
2$
26
29
July 2
2
4
4
11
June 22
26
28
29
July 2
5
5
7
7
14
June 24
29
3°
July 1
4
7
7
9
9
16
June 26
July 1
2
3
6
9
9
11
11
18
June 28
July 3
4
S
8
11
11
13
13
20
July 5
11
12
13
14
16
17
21
21
24
July 6
12
13
14
IS
17
iS
22
22
25
July 13
19
2
21
6
24
8
29
31
1 Data included in tables were secured at Orlando, Fla., during June and July, 1919. The mean tem-
peratures for period were as follows: June, average mean temperature 79.4 F., high mean 90. 50, low
mean 68.3°; July, average mean temperature 81. 4° , high mean 92.4°, low mean 70.3°.
Table II. — Length of stages of tfie tamarind pod-borer
Weevil No.
Egg.
First
larval
stage.
Second
larval
stage.
Third
larval
stage.
Fourth
larval
stage.
Prepupal
stage.
Pupal
stage.
Days.
3
3
3
3
3
3
3
3
3
3
Days.
2
3
2
2
2
2
2
2
2
2
Days.
2
2
2
2
2
2
2
2
2
2
£>a.v.s.
2
2
2
2
2
2
2
2
2
2
Days.
7
8
8
8
6
5
6
8
8
4
I
I
I
I
I
I
I
I
I
I
jDay.s.
7
7
7
7
6
6
7
7
6
8
7
6
The pearly white larvae, when full grown, construct pupal cells within
the seed by lining the cavities at the end of their larval burrows with a
mixture of frass and borings cemented together with a secretion that
gives it when dry the appearance and consistency of a dark brown
shellac.
As shown in Table II the larval stage usually requires from 12 to 14
days. After a prepupal stage of about 1 day the pupal form is assumed,
and 7 days later the adult is formed. The adult does not immediately
leave the seed but remains within to harden and feed for a few days.
It then makes its way to the original cavity made by the mother weevil
when laying her eggs and emerges, rarely if ever forcing its way through
the shell at any other point.
After the adults have all emerged little is left of the seed but the empty
shell and a mass of powder.
Dec. is, 1920 Tamarind Pod-Borer, Sitophilus linearis (Herbst) 443
PREOVIPOSITION PERIOD
Copulation takes place soon after emergence, and the females deposit
their first eggs in from 7 to 10 days after attaining adult form. Copu-
lation is frequent and often takes place while the female is at work on the
egg cavity.
OVIPOSITION PERIOD
The longest oviposition period recorded lasted for 84 days, and during
this time 183 eggs were deposited. Toward the latter part of this
period fewer eggs were laid than at first, the female becoming more and
more feeble and exhausted. Three weeks after the last egg was laid the
female died. The male died a few days later.
Other female weevils in captivity deposited from 126 to 165 eggs.
It seems probable that under natural conditions with an abundant
supply of fresh seed the oviposition period would be longer and the
number of eggs deposited would be correspondingly larger.
HABITS OF ADULT
The males are, as a rule, slightly more abundant than the females.
Of 488 bred specimens, 258, or about 53 per cent, were males. The
males apparently feed but seldom, spending their time in constant
attendance on the working females or in fighting among themselves for
the females. They are of a very combative nature, and it is not un-
common to see two and sometimes three males fighting together for
hours at a time with apparently great ferociousness. As they have no
efficient or deadly weapons, however, little damage is done; and long
before a decision is reached another male has assumed the care of the
female, who, intent only on her work, is oblivious to the struggles of the
aspiring males. The males are readily distinguished from the females
by their shorter, thicker beaks. The beak of the male is considerably
broader at the base than that of the female. The adults in captivity
have fed on acorns, sweet potatoes, and various fruits. Normally,
however, they do not attack anything but the tamarind seeds.
PARASITES
No parasites have been reared from any of the stages of Sitophilus
linearis. Larval and pupal stages in the laboratory were attacked and
killed by a predacious mite, Pediculoides ventricosus Newport. It seems
very doubtful, however, that -this mite would be able to penetrate to the
larval burrows under field conditions.
DESCRIPTION OF IMMATURE STAGES
EGG
The egg is opaque, white, shining, ovoid to pear-shaped, rounded at
the bottom; the top is slightly flattened and narrower, fitting into a plug
or cap that cements it into place. The shell of the egg is very delicate
and flexible, conforming to the shape of the egg cavity. Its length is
0.60 to 0.64 mm., the width 0.31 to 0.35 mm.
444 Journal of Agricultural Research voi.xx,No. 6
MATURE LARVA
The mature larva measures from 2.5 to 3.5 mm. in length and is pearly
white in color. It is a footless, fleshy grub, very thick-bodied, the ven-
tral outline being approximately straight while the dorsal outline is
almost semicircular. The head is light brown in color, the anterior
margin and mandibles are much darker, the head is longer than broad
and somewhat wedge-shaped, and the sides are broadly rounded from middle
to apex. The apex is slightly angular. The sides are nearly straight
from the middle to the anterior angles, and the lateral area has an
oblique, longitudinal, lighter stripe or area. The epicranial and frontal
sutures are distinct and light in color; there are also two oblique, longi-
tudinal, light stripes rising from the frontal sutures and coalescing with
the epicranial suture near the apex. The frons is subtriangular with a
distinct dark median line from the posterior angle to the middle, indi-
cating a carina. The sutural margins are irregular or sinuate. The frons
is provided with five pairs of large setae, and each sutural margin bears a
large seta. Each epicranial lobe bears the following setae: One close
to the posterior angle of frons and located within the oblique, longitu-
dinal stripe rising from the frontal suture; one very small seta posterior
to this and near occiput, two anterior to it on disk of epicranium; two
opposite middle of frons ; one opposite middle of mandible ; one opposite
hypostomal angle of mandible; and one on hypostoma near base of
mandible. The epistoma is represented by the thickened anterior
margin of the front. It is distinctly darker in color, with the anterior
margin declivous and slightly curving and the lateral angles slightly
produced and elevated where they support the dorsal articulation of the
mandibles. The pleurostoma is represented by the somewhat darker
declivous area surrounding the mandibular foramen. The mandibles
are stout, triangular, with the apex produced into an acute apical tooth.
The inner edge toward the apex is provided with a subapical tooth and a
small medial tooth, no molar parts present. The dorsal area of the
mandible is provided with a pair of bristles set apart. The eye is repre-
sented by a well-defined black spot beneath the exoskeleton.
The clypeus is attached in front of the frons and is broadly transverse.
It is broad at the base, the sides narrowing toward the apical angles,
and is slightly longer and broader than the labrum. It bears on the
epistomal margin two fine setae on each side. The labrum is distinctly
broader than long, with two lateral and a larger median lobe. It is
provided with six large setae behind the middle, two marginal, short,
thickened setae on each of the lateral lobes, and six similar marginal
setae on the median lobe.
The cardo is present and distinct in the maxilla; the stipes is not
divided into stipes proper, subgalea, and palpifer but is one continuous
piece, with the anterior inner angle produced into a single setose lobe.
Dec. is, 1920 Tamarind Pod-Borer, Sitophilus linearis (Herbst) 445
The palpus is 2 -jointed and bears a single seta near the apex of the first
segment. There are three other setae found on the maxilla, two located
on the vaginant membrane between the palpus and palpifer, and one
stouter and longer seta midway between the palpus and cardo. There is
no articulating maxillary area between the maxilla and the mental-
submental region.
The submentum and mentum are fused and are represented by a broad
lobe bearing three pairs of stout setae. The stipes labii are posteriorly
enforced by a median, trangular chitinization ; the anterior median sec-
tion is produced anteriorly between the palpi into a small lobe-like ligula'
which is fused with the lingua. Each stipes labii bears a single seta. The
short, conical, 2 -jointed palpi are situated on the anterior angles of the
stipites. The ligula bears four small setae.
The prothorax is dorsally not divided; but two areas, the praescutal
and scutoscutellar areas, are roughly indicated by rows of setae. The
mesothoracic and metathoracic segments are above divided into two
distinct areas, the anterior of which represents the praescutum and the
posterior the scuto-scutellum and alar area. The thoracic spiracle is
located on a lobe pushed into the prothorax from the epipleurum of the
mesothorax. It is bifore, elongate, larger than the abdominal spiracles,
and placed with the finger-like air tubes pointing dorsad. The meta-
thoracic spiracle is rudimentary.
There are 10 abdominal segments, the first 7 similar, the last 3 smaller
and reduced. Each of the abdominal segments 1 to 8 is supplied with
a spiracle, that of the eighth being located more dorsally than the rest.
Each tergum is divided above into two distinct areas. The first contains
praescutal and scutal elements; the second represents the scutellum.
Below these two areas and adjacent to the epipleurum is the alar area.
The abdominal spiracles are placed anteriorly and in a little separate
corner piece, probably of the alar area.
Below a very indistinct and abrupt dorso-lateral suture and above a
well-defined ventro-lateral suture is a large, not subdivided epipleurum.
The abdominal epipleura are located considerably higher than the
thoracic, and the ventro-lateral suture makes an S-shaped line between
metathorax and first abdominal segment. Below the ventro-lateral
suture is the hypopleurum subdivided into three lobes, one right under
the other. Below the hypopleurum is the coxal lobe, and below that is
the sternum, consisting of the eusternum and a posterior triangular area
representing the parasternum or the parasternum fused with the ster-
nellum.
The setae on the abdominal segments are arranged as follows : One on
the praescutum, a long and two shorter ones on the scutellum, two on
the alar area located just above the spiracle, two on the epipleurum, one
on the middle lobe of the hypopleurum, one on the coxal lobe, and three
on the eusternum. One of the hairs on the scutellum is sometimes
missing on the last few abdominal segments.
446 Journal of Agricultural Research vol. xx, No. 6
LARVAI, INSTARS
First-instar larva 0.53 to 0.60 mm. long, 0.37 to 0.43 mm. wide;
pearly white; head about 0.25 mm. wide, 0.26 long.
Second-instar larva 0.65 to 0.80 mm. long, 0.5 to 0.65 mm. wide; head
0.32 mm. wide, 0.36 mm. long.
Third-instar larva 0.75 to 1.3 mm. long, 0.6 to 1 mm. wide; head 0.42
to 0.45 mm. wide, about 0.52 mm. long.
Fourth-instar larva 1.5 to 3.5 mm. long, 1 to 2.5 mm. wide; head about
0.57 mm. wide, about 0.80 mm. long.
PUPA
The pupa is uniformly white when first transformed, 3.5 to 4.25 mm.
long, and about 1.65 mm. wide. The tips of the wing pads attain the fifth
abdominal segment; the tips of metathoracic tarsi extend beyond the tips
of the inner wings. The head is oval, the beak elongate and slender. The
head has two prominent spines towards the vertex, a group of two spines
and two spinules on each side above the eyes, two pairs of small spines
near the anterior margin, and a small one on each side of the front
between the eyes. There are three pairs of spines on the beak between
the frontal ones and the base of antenna, a pair of small ones on the beak
midway between the base of antenna and tip of beak, a pair on the sides
of the beak between the latter pair and the tip of the beak, and two pairs
on the tip of the beak.
The prothorax is provided with one pair of antero-marginal, setigerous
tubercles, one pair of antero-lateral, two pairs of medio-lateral, and
four pairs of dorsal setigerous tubercles. The mesonotum and meta-
notum are each provided with two pairs of spines. The abdomen has
seven distinct dorsal tergites, the seventh being somewhat larger than
the rest. The dorsal area of each is armed with a pair of large spines
and a pair of smaller ones. The lateral area of each tergite is armed with
a spine at the base of which is a small seta. The epipleural lobes are each
armed with two minute setae. One pair of the dorsal spines of the seventh
abdominal segment is much larger than the rest and is usually directed
cephalad; the second pair is small and slender and is directed caudad.
The ninth abdominal segment is armed with two fleshy processes.
A. — Pupa, dorsal view.
B. — Pupa, front view.
C-Egg.
D. — Mandible.
E. — Mature larva.
F. — Ventral view of head.
G. — Clypeus and labrum.
H. — Pupa, lateral view.
I. — Head, face view.
J. — Head, dorsal view.
K. — Head, lateral view
PLATE 61
Sitophilus linearis:
Tamarind Pod-Borer, Sitophilus linearis (Herbst)
Plate 61
B
%
Journal of Agricultural Research
Vol. XX, No. 6
INFLUENCE OF TEMPERATURE AND HUMIDITY ON
THE GROWTH OF PSEUDOMONAS CITRI AND ITS
HOvST PLANTS AND ON INFECTION AND DEVELOP-
MENT OF THE DISEASE1
By George L. Peltier
Plant Pathologist, Alabama Agricultural Experiment Station, and Agent, Bureau of
Plant Industry, United States Department of Agriculture
INTRODUCTION
In the writer's investigations on the susceptibility and resistance of
a large number of rutaceous plants to citrus-canker (Pseudomonas citri
Hasse) he has been impressed {7-9Y by a number of factors which appear
to play an important role in these studies. The factors may be briefly
stated as follows :
1 . The anatomical structure of the plants.
2. The reaction of the host plants to their environment.
3. The influence of external conditions on the organism and on
the susceptibility to infection of the host.
4. The influence of the host on the virulency of the organism. 3
THE PROBLEM
The problem was attacked from the standpoint of the influence of
temperature on the growth of the organism and its hosts and on infection
and development of the disease and from the standpoint of the influence
of humidity on the growth of the organism and its hosts and on infection
and development of the disease.
1 Published with the approval of the Director of the Alabama Agricultural Experiment Station as a
report on cooperative investigations between the Department of Plant Pathology, Alabama Agricultural
Experiment Station, and the Bureau of Plant Industry, United States Department of Agriculture.
2 Reference is made by number (italic) to "Literature cited," p. 503-506.
3 To determine more definitely just what part some of these factors play in governing the susceptibility
and resistance of rutaceous plants to canker, leave of four months was granted the writer by the Director
of the Alabama Agricultural Experiment Station to carry on this investigation in the Plant Physiology
Laboratory at the University of Illinois during the winter of 1918-19. Through the cooperation of Dr. K. F.
Kellerman, Associate Chief, Bureau of Plant Industry, United States Department ol Agriculture, a second
four months' investigation was made possible the following winter. It is indeed with great pleasure that
the writer acknowledges his indebtedness to the University of Illinois for the privileges and facilities of
the Plant Physiology Laboratory. The writer is especially indebted to Prof. C F. Hottes for the
suggestions, methods, and advice offered during the course of the work and for the time spent by him in
preparing, setting up, and regulating the apparatus used. He also wishes to thank Prof. F. L. Stevens
for the use of the Plant Pathology Laboratory. The plants used in the experiments were kindly furnished
by Mr. W. T. Swingle, in Charge, Office of Crop Physiology and Breeding Investigations, Bureau of Plant
Industry, United States Department of Agriculture.
Journal of Agricultural Research. Vol. XX, No. 6
Washington, D. C Dec. 15, 1920
wc Key No. Ala.-7
(447)
448 Journal of Agricultural Research vol. xx, No. 6
APPARATUS USED
A complete description of the temperature and humidity cases used
in this investigation will soon be published by Prof. Hottes. It is
sufficient to state here that the cases were large, well ventilated, well
lighted, and most important of all, supplied with accurate and reliable
controls. The temperature cases remained constant to within 0.50 C.
and were controlled at 50 intervals from 50 to 300. For work above 300
ordinary bacteriological incubators and one large case held at 35 °, but
varying several degrees, together with constant-temperature water baths,
were used. The cases used for the humidity work were accurate to
within 2 to 4 per cent and could be regulated for any desired percentage
of relative humidity. The temperature of these cases could also be
readily regulated and controlled. Thus, the writer has had the extreme
good fortune of working with well-regulated temperature and humidity
controls, which were not a continual worry or source of error.
INFLUENCE OF TEMPERATURE ON GROWTH OF THE ORGANISM
Little work has been done on the temperature relations 01 Pseudomonas
citri. Doidge (1) states that —
it grows well at 3o°C, rather more slowly at 250 C, and very slow progress is made
at 200 C.
Wolf (17) in preliminary tests found that —
the thermal death point was between 580 C. and 700 C.
and further that —
no growth occurred in tubes exposed for temperatures above 650 C.
Stevens (12) reports that —
bacteria (P. citri) have been killed by temperatures ranging from 550 C.-600 C,
when exposed for a period of five minutes.
Three types of culture media were tested — a liquid, a liquefiable solid,
and a solid. These furnished a means of comparing the growth of the
organism on different types of media, and if any differences existed
between the rate and amount of growth on the different media at various
temperatures they could be easily detected. Beef bouillon was used as
the liquid, soluble starch agar as the liquefiable solid, and steamed
potato cylinders as the solid. Since the most comparable results were
obtained with soluble starch agar, they will be taken up first.
Soluble starch agar.— Hasse (2), Wolf (iy), and Jehle (5) have
noted the characteristic growth of Pseudomonas citri on potato plugs,
and especially the formation of a narrow white zone along the margin of
the bacterial growth. Doidge (7), however, says:
I have failed to perceive, except in one or two doubtful instances, the narrow white
zone on the uninfected surface following the line of the streak in young cultures,
which have been recorded both by Hasse and Wolf.
Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 449
The writer has always noticed this zone on potato plugs, especially in
young cultures.
Preliminary tests on inoculated potato plugs with iodin solution
showed that the narrow white zone was completely free from starch,
while it was surrounded by a small light band of red and blue, indicating
that the decomposition of the starch was slowly taking place. In old
cultures the cell walls were separated, showing that the middle lamella
had been attacked and dissolved. Wolf (77) and Doidge (z) have re-
ported similar observations. Thus, by the use of soluble starch agar
and potato cylinders, the growth of the organism as well as the rate of
enzym action at different temperatures could be measured directly.
The soluble starch agar was made up as follows : x
12.0 gm. shredded agar.
5.0 gm. soluble starch (Merck), according to Lintner.
.5 gm. potassium phosphate (dibasic).
.5 gm. magnesium sulphate.
.5 gm. sodium chlorid.
i.ogm. ammonium sulphate,
i.ogm. calcium carbonate.
1,000 cc. distilled water.
Two methods of measuring the growth of the organism presented
themselves: first, the pouring of dilution plates and measuring the growth
formed from a single bacterium by means of an enlarged projection
through a fixed camera, and second, the placing of a definite amount of
inoculum on the agar and measuring the increased diameter of the colony.
The most serious objection to the first method was that the plates
could not be poured at the temperatures to which they were subsequently
exposed. The minimum temperature for the growth of the citrus-canker
organism is approximately 20 to 40 C. lower when this method is used.
Also the initial growth at temperatures between 50 and 150 is greater.
This is due to the fact that all materials are at room temperature when
the inoculations of the plates are made and, furthermore, there is a
definite time limit required to bring the plates or tubes to the tempera-
ture of that of the case.
In the second method a 2-mm. loop was pressed gently on the hardened
agar at three or four points on the plate, so that the inoculum remained
on the spot made. The increase in the diameter of the colonies was then
measured from day to day. This method is not so accurate from the
standpoint of measurement as the first, but it gives much more compara-
ble results, when the temperature and time factors are considered.
All the plates were poured at the same time, care being taken to get
the agar in the plates of the same thickness. They were then placed in
the various temperature cases overnight, so that at the time of inocula-
1 A modification of the starch agar used by McBeth and Scales. (McBeth, I. G., and Scales, F. M. the
DESTRUCTION OF CELLULOSE BY BACTERIA AND FILAMENTOUS FUNGI, U. S. Dept. Agr. Bur. Plant Indus.
Bui. 266, p. 26-28, 1913.)
16917°— 20 4
450
Journal of Agricultural Research
Vol. XX, No. 6
tion they were at the temperature of the cases. The inocolum used in all
instances was from a 48-hour-old culture of Pseudomonas citri in beef
bouillon. While the plates were being inoculated precautions were taken
to maintain them at the same temperature as that of the case. At the
end of every 24 hours two plates were taken from each case, and the
increased diameter of the colonies was measured.
In studying the rate of enzym action, an iodin solution1 was poured
over the plate to be tested, was allowed to remain a few moments, and
was then poured out. The result was that the colonies stood out as a
lemon- yellow color, surrounded by a clear zone which came next showed
the disappearance of the starch and its conversion into maltose and
achroo-dextrin. This was followed by a reddish band, indicating erytho-
dextrin, an intermediate product, which merged into a light blue band
and finally into the dark blue color of the remaining agar. Thus, on
one plate, both the growth of the colonies and the rate of the enzym
action, as indicated by the iodin test, could be measured.
Table I gives the diameter of the colonies in millimeters for each day
and temperature. Each reading represents an average of 28 measure-
ments.
Table I. — Diameter in millimeters of colonies of Pseudomonas citri on soluble starch
agar at various temperatures
Temperature.
After 1
After 2
After 3
After 4
After 5
After 6
After 7
After 8
day.
days.
days.
days.
days.
days.
days.
days.
°C.
Mm.
Mm.
Mm.
Mm.
Mm.
Mm.
Mm.
Mm.
5
O
O
O
O
O
O
O
O
10
O
O.25
o-75
O. 94
I. 24
I.32
I.50
I. 63
15
O
°-5i
I. OO
I.44
I.94
2.38
2- 75
3-38
20
O. 50
1.50
2. OO
2.86
3-2S
3-76
4- 13
4- 5°
25
1.25
2-37
2.8l
3-3°
4. 06
4.84
5-3°
5-8i
3°
1.38
2.63
3. OO
3-5°
4- 5°
5-3°
6. 00
6.38
33*035
O
0
O
0
0
0
0
0
38 to 40
O
0
O
0
0
0
0
0
When the time factor, or length of exposure, is considered, the opti-
mum temperature for the growth of Pseudomonas citri on soluble starch
agar is between 200 and 300 C. There is evidence of a decided lag in the
growth of the organism between 150 and 200. In other words, while the
amount of growth at 200 is just one day behind that produced at 250
and two days behind that at 300, the growth at 150 is much slower, being
two days behind the growth made by the organism at 200. At 200, growth
starts the first day, while at 150, growth is just starting at the end of the
second day. This point is very well brought out in figure 1 , where the
rate of enzym action at the various temperatures is plotted.
1 The solution was composed of 0.5 gm. potassium iodid and 1.0 gm. iodin, allowed to stand overnight
together in 10 ce. of water. It was then diluted to 100 ec. (stock solution). As needed, the stock solution
was diluted to abmit one-half or less, depending on the material tested.
Dec. is, 1920 Effect of Temperature and Humidity on Citrus-Canker 45 1
Growth is inhibited at 50 C. and again at 330 to 350. At io° some
growth occurs. That the organism is not killed at 50, but is merely
inhibited, was shown when plates kept at this temperature for eight days
were transferred to the 300 case. Growth immediately took place at
the normal rate for that temperature. The same was true when plates
held at 330 to 350 for eight days were transferred to 300; the organism
started growing. However, when plates held at 380 to 400 for 24 hours
FlG. i. — Graph showingthe rate of enzym action, as expressed in millimeters, at the various temperatures
for a period of eight days on soluble starch agar.
were placed in the 300 case, no growth took place, showing that the
organism had been killed by the higher temperatures. Thus, in working
out the temperature relations of Pseudomonas citri, the temperature at
which growth is inhibited must be clearly distinguished from the tem-
perature at which the organism is killed.
Table II gives the rate of enzym action at the various temperatures.
E?ch reading represents an average of 28 measurements.
452
Journal of Agricultural Research
Vol. XX, No. 6
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Dec. i5. 1920 nfject of Temperature and Humidity on Citrus-Canker 453
At 50 C, 330 to 350, and 380 to 400 a light blue color was given with
the iodin, indicating that only a partial hydrolysis of the starch took
place. At io° the light blue zone persisted for several days, followed by
a wide reddish zone. It was not until the fourth day that a clear zone
was formed. Likewise, at 150 no clear zone was formed until the third
day. At 200, 250, and 300 the clear zones were present at the end of 24
hours, increasing in diameter in proportion to an increase in temperature.
The curves for the rate of enzym action are shown in figure 1 . Especially
noticeable are the differences in the rate of enzym action represented by
the 1 50 and 200 curves. The l#g mentioned under the rate of growth of
the organism at these temperatures is very well shown. Further in-
vestigations must be carried out before the explanation of this lag can
be given.
Potato plugs.— The first trial with the growth of the organism on
potatoes was attempted with blocks of raw potatoes cut under aseptic
conditions and placed in Petri dishes with plain agar poured into the
dishes even with the top of the blocks t^keep them moist. However,
this method had to be abandoned because the surface of the blocks
oxidized and dried out too rapidly. Therefore in the following trials,
steamed potato cylinders were used. The same procedure was followed
as in the tests with soluble starch agar to bring the cylinders to the tem-
perature of the cases prior to and during inoculation. They were inocu-
lated by means of a shallow stab, and the organism was allowed to grow
out over the surface. The inoculum was taken from a 5-day-old culture
of Pseudomonas citri on potato plugs. The results are not as comparable
as those obtained for starch agar because of the variation in the amount
of inoculum and the physical differences in the potato cylinders them-
selves. However, in general the growth of the organism and the rate of
enzym action, as determined by the iodin test, followed the curves shown
in figure 1. As the red and blue zone was very narrow on the potato
cylinders, the total diameter of the zone is represented in Table III,
together with the growth of the organism. This table gives the average
of two trials of four readings each.
454
Journal of Agricultural Research
Vol. XX, No. 6
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Dec. i5, 1920 Effect of Temperature and Humidity on Citrus-Canker 455
At 50 C. a very small zone was noticed after several days, which in-
creased very slowly until at the sixth day the colony was just visible to
the naked eye. Growth at io° was first observed on the third day and
increased slowly with time. Growth at 200, 250, and 300 was, of course,
much more pronounced. No visible growth occurred at 330 to 350 and
380 to 400, although some enzym action took place.
The surfaces of the cylinders were slightly depressed at 200 C, the
depression increasing in depth at 250 and 300. When the cylinders
were cut open, it was found that the clear zone proceeded down in the
shape of a cone, and its progress was almost as rapid as that of the zone
on the surface.
At 250 and 300 C, where the organism grew over the whole surface and
down the sides, the decomposition of the upper half of the plug took
place. Examination for starch grains under the microscope showed that
none were present, while the middle lamella was completely dissolved,
the cells standing apart. From the results of the study of the enzym
action of Pseudomonas citri on soluble starch agar and steamed potato
plugs, we can conclude that it is a strong diastase secretor. Cytase is
also produced abundantly.
The organism appeared to thrive longer and produce more enzym near
the critical temperatures on potato plugs than it did on the starch agar.
At 50 C. a small white zone was produced with a trace of growth. No
growth was visible at 330 to 350 or at 380 to 400, although a rather large
depressed zone was distinctly noted. Potato plugs with no visible growth
in the 50 and 330 to 350 cases at the end of 8 days produced abundant
growth when transferred to 300. However, plugs kept for 24 hours in
the 380 to 400 case when transferred to 300 produced no growth, nor
did the white zone increase in size.
BEEF bouillon. — All the beef bouillon used in the experiments was
adjusted to +8 Fuller's scale, since it was found that the organism de-
veloped very well at this acidity. During the course of the work with
beef bouillon, no counts were made of the bacterial growth in cultures
at the different temperatures.
By means of a bulb burette 10 cc. of the bouillon were placed in each
tube. The tubes were kept in the various cases overnight and were inoc-
ulated the next morning with a 2-mm. loop from a 48-hour-old culture of
Pseudomonas citri. Each day two tubes were withdrawn and a reading
was taken.
Pseudomonas citri makes a very characteristic growth in beef bouillon.
Growth is first noticed by the clouding of the medium. After a few days,
flakes appear, followed by a yellow ring at the surface of the bouillon;
later, the flakes precipitate to the bottom. Thus, in Table IV, the
readings are based on the characteristic behavior of the organism.
456
Journal of Agricultural Research
Vol. XX, No. 6
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Dec. is, 1920 Effect of Temperature and Humidity on Citrus-Canker 45 7
No visible growth occurred at 50 C. or at 380 to 400. Growth was
visible at io° the third day, at 150 the second day, and at 200, 250, 300,
and 330 to 350 at the end of 24 hours. However, at 330 to 350 no increase
in growth was noted after the first day.
Growth proceeded at a regular rate at 200 C, 250, and 300, each of the
lower temperatures being about one day behind. Growth at 150 never
approached that produced at 200. The growth of Pseudomonas citri in
beef bouillon at the different temperatures proceeded at the same relative
rate as on agar and potato plugs.
Cultures placed in the 50 C. and the 330 to 350 cases were viable
at the end of 8 days, while no organisms were viable in tubes held
at 380 to 400 for 24 hours. In constant water baths, the organisms
were viable at 37. 6° to 380 at the end of 24 hours and at 41 ° to 41. 50
at the end of 2 hours. At 43. 20 to 43. 6° all organisms were dead at the
end of 2 hours. The interesting point brought out here is that no organ-
isms were viable in the incubators run at 38 ° to 400 C. at the end of 24
hours, but at 37. 6° to 380 in the water bath they were viable at the end
of 24 hours. Thus, the point at which growth is completely inhibited
at the higher temperatures is very sharp when the time factor is constant.
In determining the thermal death point by the usual laboratory
methods, all the organisms were killed at a temperature of 520 C, while
all the trials at 490 yielded positive results. Thus, the thermal death
point of the citrus-canker organism is above 490 and below 520.
Distilled water. — To determine the length of life of Pseudomonas
citri in water, ordinary distilled water, supplied by the Department of
Chemistry, University of Illinois, was used, as it was impossible at the
time to obtain good well or rain water. The distilled water contained
traces of organic matter, but no mineral substances were present. No
attempts were made to determine the conductivity of this water.
The sterilized water blanks were placed in the cases at the various
temperatures overnight and were inoculated with a loop from a 48-
hour-old culture of the organism in beef bouillon. At the end of each
day, two tubes were withdrawn from each case and were reinoculated
into beef bouillon to test for growth. In Table V the results of the
experiment are given.
Table V. — Viability of Pseudomonas citri in sterilized distilled water at various temper-
atures
Temper-
After
After
After
After
After
After
After
After
ature.
1 day.
2 days.
3 days.
4 days.
5 days.
6 days.
7 days.
8 days.
0 C
IO
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
15
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
20
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
3°
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
35
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
458 Journal of Agricultural Research vol. xx, no. 6
The results show very clearly that Pseudomonas citri can remain viable
in the distilled water used for a period of eight days at temperatures
ranging from io° to 350 C. They suggest that the citrus-canker organism
under certain field conditions may remain viable in rain and surface water
for some time at a range of temperatures much larger than is usually
found in the field.
Comparative tests with the organism in beef bouillon and in distilled
water at temperatures higher than 35 ° C. gave the same results. For
example, the thermal death point of the organism in the distilled water
was between 490 and 520, just as in beef bouillon.
CONCLUSIONS ON THE TEMPERATURE RELATIONS OF THE ORGANISM
(1) The optimum temperature for the growth of Pseudomonas citri on
soluble starch agar, potato cylinders and in beef bouillon lies between
200 and 300 C.
(2) There is a decided lag between the rate of growth at 150 C. and that
at 200 in all media.
(3) The minimum temperature for the growth of Pseudomonas citri
is 50 C. on potato plugs. However, growth on soluble starch agar and
in beef bouillon is inhibited at this temperature, so that the minimum
temperature for the growth on these media must be slightly above 50.
(4) The maximum temperature for the growth of Pseudomonas citri
in beef bouillon is 430 C. for periods of less than 2 hours, 41 ° for a period
of 2 hours, 380 for a period of 24 hours, and 330 to 350 for periods longer
than 24 hours. Growth on potato cylinders and soluble starch agar
was, in all cases, inhibited at temperatures of 330 to 350, so that the
maximum temperature for the growth on these media must be slightly
below 330 to 350.
(5) The thermal death point of the organism is above 490 and below
52° C.
(6) The temperatures at which growth is inhibited must be clearly
distinguished from the temperatures at which the organism is killed.
This is especially important near the critical temperatures at or above
the maximum. The point at which growth is completely inhibited at
the higher temperatures is very sharp with a constant length of exposure.
(7) The production of diastase by Pseudomonas citri on soluble starch
agar and potato cylinders follows the well-known chemical law of Van't
Hoff, between temperatures of 200 and 300 C. As in the growth of the
organism, there is a decided lag between the rate of enzym action at 150
and that at 200. This fact has not been pointed out heretofore. Only
partial hydrolysis of the starch in the agar and the potato cylinders
occurs at 50 and again at 330 to 350 and 380 to 400.
(8) The citrus-canker organism is viable in ordinary distilled water
at temperatures ranging from io° to 350 C. for a period of eight days.
Dec. i5. 1920 Effect of Temperature and Humidity on Citrus-Canker 459
INFLUENCE OF TEMPERATURE ON GROWTH OF THE HOST
PLANTS
The literature on the influence of the environmental conditions on the
growth and development of Citrus plants is very meager. What litera-
ture is available concerns itself chiefly with the injury to Citrus orchards
caused by low temperatures, with an occasional reference to the maxi-
mum temperatures at which the Citrus plants can thrive.
The most complex factor entering into the study of the temperature
relations of Citrus plants is the fact that they have rest and growth
periods which vary to some extent with each group, although they are
more or less definite within the group itself. Under greenhouse condi-
tions, the rest and growth periods are variable. However, as a general
rule, most Citrus plants can be forced into active growth within short
periods of time. An exception to this statement must be made for
deciduous plants like Poncirus trifoliata. With plants of this type,
external conditions in the greenhouse have no influence on the rest
period, within certain limits.
Three types of plants were used — Poncirus trifoliata (L.) Raf. and
Rusk citrange (a hybrid between P. trifoliata and Citrus sinensis Osbeck,
Florida sweet orange), plants which are deciduous, hardy, susceptible to
citrus-canker, and having a very definite dormant period; C. grandis
(L.) Osbeck, grapefruit, an evergreen and nonhardy plant, extremely
susceptible to citrus-canker and having a dormant period of variable
nature; and C mitis Blanco, calamondin, an evergreen and nonhardy
plant, somewhat resistant to citrus-canker, and native of the Philippine
Islands.
The plants were grown from seed in the Crop Physiology greenhouses
at Washington, D. C. The seedlings ranged from 6 to 14 inches in
height and were shipped from Washington from time to time, both in
pots and balled. Several shipments of Poncirus trifoliata were made of
seedlings growing outside, from Auburn, Ala., during the month of Jan-
uary. The plants were kept under greenhouse conditions until needed.
In the experiments reported below, the plants were placed under large
bell jars in the various temperature cases. During the course of the
experiments, a saturated atmosphere was maintained in the bell jars.
Observations and readings were made of the condition of the plants
from time to time.
Experiment 1
Two plants of each species were placed in the cases at the various
temperatures, while one set was kept under greenhouse conditions where
the temperature range was considerable, varying from 200 to 300 C.
All plants, with one or two exceptions, were either in a dormant state or
had completed their growth. In Table VI are given the observations
made on the plants at intervals for a period of six weeks.
460
Journal of Agricultural Research
Vol. XX, No. 6
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Dec. is, 1920 Effect of Temperature and Humidity on Citrus -Canker 461
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462 Journal of Agricultural Research voi.xx.No. t
It will be noted that, even in a saturated atmosphere, the various
temperatures had no influence whatsoever on the dormant plants of the
trifoliate orange. No growth of the calamondin plants occurred except
at 300 C. and in the greenhouse.
At io° C. no growth of the grapefruit plants took place. It is very
evident that at 150 the growth of grapefruit is not only slow but that the
growth matures very rapidly. Leaves which mature at this temperature
are small, being from one-fourth to one-half the size of the normal grape-
fruit leaf. Good growth of the grapefruit plants took place at 200 C.
However, the shoots did not grow so rapidly and the maturation of the
leaves was faster than at the higher temperatures of 250 and 300. At
these temperatures, where the grapefruit plants were in good condition,
a rapid growth took place, the new shoots were longer, and the period
over which the maturation of the leaves took place was extensive. To
illustrate, at 150 it required from 7 to 8 days for a new shoot to complete
its growth, while at 300, 16 to 20 days were necessary.
EXPERIMENT 2
In this experiment plants of the Rusk citrange were substituted for
the trifoliate orange. Three plants of the citrange, three of the cala-
mondin, and one of the grapefruit were used. One plant each of the
citranges and calamondins, in a good growing condition, was chosen for
the first group ; one set in which the growth was complete, but with a
new bud starting, was selected for the second group; and dormant plants
were placed in the third group. Grapefruit plants in good growing con-
dition were used. The experiment was carried through in the same way
as experiment 1, except that at the end of 15 days the plants in the 50,
io°, and 1 50 C. cases were all transferred to the 300 case under their
original bell jars.
During the 15-day period no growth of the citrange, calamondin, and
grapefruit plants occurred at 50 and io° C. (Table VII.) An extremely
slow growth was recorded for the grapefruit plants at 150. Measure-
ments of two grapefruit leaves showed an increase in growth of 3 mm.
and 9 mm. in length and 1 mm. and 4 mm. in width, respectively, for a
period of 15 days. As noted in experiment 1, leaf maturity increased
very rapidly at these temperatures, the leaves reaching about one-fourth
to one-half the size of those at higher temperatures.
When the plants held at temperatures of 50 and io° C. were placed in
the 300 case, a normal growth for that temperature immediately took
place in most instances. The rate of growth of the growing citranges
was about 25 mm. per day. The behavior of the dormant plants when
transferred to the higher temperatures was erratic. Some immediately
responded and started growth, while others remained dormant.
Dec. is, 1920 Effect of Temperature and Humidity on Citrus-Canker 463
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464
Journal of Agricultural Research
Vol. XX, No. 6
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Dec. is, 1920 Effect of Temperature and Humidity on Citrus-Canker 465
In no instance were the small, undersized leaves, which were pushed to
maturity at the low temperatures, affected when transferred to a higher
temperature. Thus, a leaf that has once reached its maturity can not
be made to increase in size by a change of environment.
At the temperatures of 200, 250, and 300 C, growth responded at a
normal rate where the citrange and grapefruit plants were in active
condition. In general, no differences were noted in the rate of growth
at these temperatures. Thus, the optimum temperature is between these
points for the plants named above. With one exception, all dormant
grapefruit and citrange plants were forced into active growth. The
plants and leaves also made a rapid and large growth and reached
maturity rather slowly. Not much difference was noted between the
plants kept as controls under greenhouse conditions and those grown at
the temperatures named. Apparently, calamondin has a little higher
optimum temperature, since little or no growth occurred at 200.
EXPERIMENT 3
This experiment was carried out with a view of determining the rate
of growth under a varying day and night temperature. Thus, plants
were exposed during the day at 300 C.,and during the night three different
sets of plants were placed at temperatures of io°, 150, and 200. The
bell jars with the plants were shifted from the 300 case at 5 p. m. and
replaced at 8 a. m. the next day.
Two plants each of the trifoliate orange, calamondin, grapefruit, and
one of the Rusk citrange were used in each set. The experiment was
carried out under the same conditions as the others described above.
As will be noted in Table VIII, the plants held at 300 C. throughout the
experiment produced the most growth. Where a day temperature of
300 and a night temperature of 200 were used, there was a very slight
slowing down of the growth in all plants except the grapefruit. When
night temperatures of 150 and io° were used, there was a decidedly
slower growth. However, growth was not checked, especially with the
rapidly growing grapefruit plants. The maturation of the leaves was
also more rapid at the low night temperatures. Thus, a night temper-
ature lower than that at which growth normally occurs merely slows up
the growth somewhat so long as a high day temperature prevails; it does
not completely stop the growth of the trifoliate orange, citrange, and
calamondin plants. Little or no difference could be detected in the rate
of growth of the grapefruit plants at the different night temperatures.
Leaf maturity was hastened somewhat by low night temperatures.
16917°— 20 5
466 Journal of Agricultural Research vol. xx, No. 6
EXPERIMENT 4
It was found in experiment 3 that an alternating day and night temper-
ature inhibited the growth of the trifoliate orange, Rusk citrange, and
the calamondin plants, while little or no difference could be detected in
the rate of growth of the grapefruit at the different night temperatures.
To determine the effect on growth of an alternating temperature for longer
periods, plants were started at a high temperature, then placed at a low
temperature for about three weeks, and then transferred back to the higher
temperature
Two sets of plants in approximately the same condition consisting of
one Rusk citrange, one calamondin, and two grapefruit plants (one large
plant and one just starting new growth) were used. The first set was
retained at 300 C. as a control. The second set after being held at 300
for 24 hours was placed in the 150 case for 18 days and then was finally
transferred back to the 300 case for approximately 2 weeks. The results
of the experiment are given in Table IX.
Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 467
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Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 469
The plants which served as controls all made a rapid growth. At
1 50 C. the plants were all inhibited in their growth, there being practi-
cally no change during the interval the plants were held at this tem-
perature. The leaves of the large grapefruit plant grew slowly and
began to mature.
Immediately on being transferred back to the 300 C. case, all but one
plant proceeded to grow rapidly at the normal rate for this temperature.
Thus, a temperature of 150 has a very decided inhibiting effect on the
growth of Citrus plants, much more so than in experiment 3, where the
plants were subjected to a temperature of 300 during the day and 150
and lower at night.
EXPERIMENT 5
In the preceding experiments, it has been clearly demonstrated that
the most growth of all Citrus plants tested occurs at 300 C. Likewise,
the best development of the organism in culture occurred at this same
temperature. Above this temperature, growth of the organism was more
or less inhibited. Thus, to determine what effect temperature higher
than 300 would have on the growth of the plants, the following experi-
ment was carried out. Plants in various stages of growth, as shown in
Table X, were divided into four sets and placed in a saturated atmosphere
under bell jars at a temperature of approximately 350. The results show
very decidedly that grapefruit and the other plants of this same type
were distinctly inhibited by this temperature, even though actively
growing plants were used. However, after they were transferred to the
300 case, the young growth started out at the normal rate for that tem-
perature.
On the other hand, the trifoliate orange and limequat 1 plants made
a good growth at 350 C. It is interesting to note that this is just the
opposite of the result obtained at lower temperatures. Grapefruit was
able to make a slow growth at 150, while the trifoliate orange and
calamondin plants were unable to develop at all.
1 A hybrid between Citrus aurantifolia, West Indian lime, X Fortunclla japonica, round kumquat.
47©
Journal of Agricultural Research
Vol. XX, No. 6
u
0
Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 47 1
It can be concluded from this experiment that the growth of grape-
fruit and plants of a similar type is decidedly inhibited at a temperature
of 350 C, while the trifoliate orange and limequat can make a normal
growth, at least for the period of time covered by the experiment.
CONCLUSIONS ON THE TEMPERATURE RELATION OF THE HOST PLANTS
(1) With actively growing Citrus grandis plants in a saturated atmos-
phere the optimum temperature lies between 200 and 300 C. The lower
limit of the optimum temperature is a little higher for C. mitis, while for
Poncirus irifoliata and allied plants the upper range of the optimum
temperature is above 300.
(2) No temperature used was able to force the dormant Poncirus iri-
foliata plants into active growth.
(3) The minimum temperature for the growth of Citrus grandis is 1 50 C,
and for the others tested it was 200.
(4) Citrus grandis plants kept at a temperature of 150 C. matured their
foliage very rapidly and in most instances within a week's time. At
temperatures of 200 and above, growth was more rapid and extensive.
The period of maturation of the leaves was extended so that 16 to 20 days
or more were required, which is twice as long as at 150.
(5) Leaves that have once reached their maturity at low temperatures
can not be forced to increase their size by a change of environment.
(6) A low night temperature checks the growth of plants held at a
high temperature during the day and also hastens maturation of the
leaves. This is especially noticeable with the Poncirus trifoliata, cit-
range, and Citrus mitis plants. C. grandis, on the other hand, is not so
easily influenced.
(7) Plants grown at a high temperature are inhibited in their growth
when transferred to low temperatures. Citrus grandis is only slightly
inhibited, while Poncirus trifoliata, Rusk citrange, and Citrus mitis plants
are completely checked.
(8) Growth of Citrus grandis and plants of a similar type is decidedly
inhibited at a temperature of 350 C, while Poncirus trifoliata and limequat
make a normal growth, at least for the period of experiment.
INFLUENCE OF TEMPERATURE ON INFECTION AND DEVELOPMENT
OF THE DISEASE
In discussing infection and development of citrus-canker, two factors
have been stressed by the workers in this field. Both have been given
equal prominence and can not very well be dissociated. These factors
are weather conditions and the condition of the host plant. In discussions
of weather conditions, most of the emphasis has been placed on humidity
as favoring the more rapid development of the disease, while little has
been said regarding the influence of temperature. However, it has
usually been inferred that a favorable temperature for infection existed.
472 Journal of Agricultural Research vol. xx.no. 6
Since the literature on the influence of temperature can not be discussed
separately from that of humidity, a brief review of the literature on the
relation of weather conditions and the condition of the host plant on in-
fection and development of citrus-canker will be given at this point.
Both Hasse (2) and Doidge (1) found that the disease developed most
rapidly on inoculated plants in a saturated atmosphere kept at 300 C.
Stevens (11, 12) makes the following statements:
In this experiment, it was found that considerable moisture must be present before
infection took place, and in many cases, the small trees thus treated had to be kept
drenched and under bell-jars for two or three days. Infections developed slowly
under greenhouse conditions, and were fewer in number than those obtained in the
open.
Warm humid weather favors rapid development of the disease and thus it is more
destructive during the rainy season.
The disease develops and spreads rapidly during rainy weather but it is more or less
retarded during periods of drought or in dry weather.
High temperatures and high humidity favor a rapid development and spread of
Citrus-canker and these are the prevailing factors of the Florida climate.
Stirling (15) states that —
during warm, wet periods, the disease infects quickly and matures in a few days.
Further that—
during a time when the atmosphere is humid, in the rainy season, it spreads rapidly.
I have found that during the early part of the season, it requires two or three months
for the canker to infect and mature so as to reproduce itself, owing, no doubt, to the
dryness and coolness of the weather. Under favorable conditions, however, the canker
will infect and mature in a much less time.
Wolf (17) observed that —
the most rapid development of the disease occurred under humid conditions.
Jehle (4, 5) in a number of articles states :
Citrus-canker is one of the most destructive diseases of citrus plants . . . and espe-
cially where the climate is warm and moist during part or all of the year, — as the
disease develops most rapidly when the humidity is high ... it was most severe
and the incubation period shortest during warm moist weather. The disease does net
develop as rapidly in cool, dry weather as it does in warm, damp weather.
He finally summarized his observations as follows —
it is much more prevalent and severe, and the incubation period is much shorter
during the summer than during the winter. In Florida, the humidity and tempera-
ture are usually high during the summer, humidity averaging from 50% to 95%
and temperature from 65 to 95 degrees F. at the Tropical Laboratory. Local
showers are very prevalent and frequently follow one another with such rapidity that
the trees do not dry off for long periods of time. During the winter, the opposite
conditions prevail, the air being dry and cool and showers few with long intervals
between them. At Redland, the temperature usually ranges from 45 to 85 degrees F.
and the humidity from 20% to 90%. Swingle learned that the disease was much
more destructive and prevalent in Japan during warm moist seasons than it was
during cool dry ones.
In discussing citrus-canker in the Philippines, Mackie (6) states that —
during the dry season, which occurs from January until the monsoon changes in June,
the disease is apparently more or less quiescent, cankers being numerous on the leaves
Dec. i5, 1920 Effect of Temperature and Humidity on Citrus-Canker 473
but not seeming to show very much on the twigs, except on the young growth and on
nursery stock. However, after the rains begin, trees send out new growth and it is
on this new growth the canker appears, coming into evidence in about a week. In
some species, it will fairly cover the new foliage, while there also appears an abundance
of canker on the twigs. Throughout the rainy season, the disease thrives, infecting
practically all the young growth. This season (1917) would seem to offer ideal con-
ditions as to climate, the weather being warm, the humidity varying from 60 to 88.
Tanaka (16), quoting Abe, of Japan, states that —
The severity of the organism is more pronounced in the wet years and spreads more
rapidly at such times.
It can be clearly seen from the foregoing excerpts from the literature
that the greatest development of canker occurs during warm, humid
weather, which in some localities can be translated into the term rainy
season, which in turn is usually associated with high temperatures. On
the other hand, these same weather conditions stimulate the rapid growth
of Citrus plants. The relation of the development of canker to the con-
ditions of the host has been reported on by the various workers.
Stevens (11) says that —
young and succulent growth under humid conditions is very susceptible.
According to Wolf (17) —
new infections appear in spring shortly after the new growth has begun. Under
favorable conditions, new infections may appear at any time throughout the growing
season of the host.
Mackie (6), in the Philippines, says:
However, after the rains begin, trees send out new growth and it is on this new
growth the cankers appear. Throughout the season, the disease thrives, infecting
practically all the young growth.
Jehle (4, 5) reports:
Citrus canker develops more rapidly on trees which are in a thrifty, healthy,
growing condition than it does on those which are semi-dormant, unthrifty, or un-
healthy. Trees in a neglected condition may harbor the disease for months before
it becomes conspicuous enough to be recognized.
The vitality and vigor of the host have a marked effect upon the prevalence and
severity of Citrus canker as well as upon the period of incubation. The disease is
much more prevalent and severe upon trees which are in an otherwise thrifty, healthy,
growing condition than it is upon those which are unthrifty and unhealthy. The
period of incubation is much longer when the trees are unthrifty and unhealthy and
the disease may remain on such trees in a dormant condition without becoming visible
for long periods of time. . . . If a tree has become infected with the organisms,
they apparently do not die, no matter how long the tree is kept in a semi-dormant or
neglected condition, but persist until active growth does occur, when the canker lesions
become visible.
Tanaka (16), quoting Bakura, of Japan, says —
it seems to attack young plants mostly.
Tanaka (16), quoting Nishida, of Japan, says —
I do not claim the entirely resistant nature of the Satsuma variety. It is a matter
which largely depends upon the environmental condition and habit of growth of the
474 Journal of Agricultural Research vol. xx, no. 6
twigs. Satsuma does not produce as much summer growth as others, which is another
reason for escaping from the severe summer infection.
All writers agree that the young and tender growth of trees in a good
growing condition favors the development of the disease. Some few
go so far as to give the age of the parts most susceptible. Thus, Jehle
(4) found that —
medium sized, thrifty leaves seem to be most susceptible, and canker is seldom found
on those which are yellowish, unhealthy, very young or very old.. . . The young
tender twigs and thorns are more subject to citrus canker than are the older more
corky ones. . . . As the fruit matures, it seems to become less and less susceptible
to citrus canker, and mature picked fruits seem to be immune.
Other investigators have also noted the absence of infection on the mature
fruits.
The writer (7) has stated that —
even though ideal conditions of temperature and humidity were supplied for infec-
tion, few or no canker spots developed if the plant was not in good growing condition.
The largest number of spots naturally occurred on mature leaves which were still
tender and of a light-green color. Few spots appeared on the young leaves, while
spots developed on the old foliage of the more susceptible plants only.
The writer (7) has gone one step further in discussing the relations of
the condition of the plant to infection when he stated that —
apparently resistance is in part mechanical — for example, the texture of the leaf
determines to a large extent the size and character of the spot. Leaf texture plays an
important role in the resistance of the host plant to Citrus-canker and seems closely
related to the rapidity with which the leaves mature. There is a considerable varia-
tion in the time required for the maturation of the leaves of the various Citrus plants.
Thus, the leaves of the kumquat, which are rather thick and highly resistant, reach
maturity much sooner than the thin, extremely susceptible leaves of the grapefruit.
Weather conditions which influence not only the growth of the organ-
ism but the trees themselves, are also responsible for retarding growth,
both of the organism and the host. Thus, Jehle (5) finds that —
the disease has a peculiar faculty for lying dormant for long periods without producing
any visible symptoms, but sooner or later making its appearance in a typical form.
There are numerous instances on record in which it has remained dormant in this
way for many months on trees which have been shipped from an infected nursery.
Examples of dormancy of the organism have been encountered in the
field, especially with nursery stock. The writer with Neal (8) proved
experimentally under field conditions that the canker organisms could
remain dormant through the winter in the outer bark tissue of some of the
hardy hybrids for a period of 6>£ months.
It is clearly evident from the facts brought out that it is extremely
difficult to separate the influence of weather conditions on the develop-
ment of the disease from its relations to the growth and development of
the host. Even experimentally it is impossible to separate the influ-
ence of temperature and humidity. Thus, in the following experiments
Dec. iS, 1920 Effect of Temperature and Humidity on Citrus-Canker 475
the temperature was varied, but a saturated atmosphere was main-
tained.
Prior to placing the plants under bell jars at the various temperatures
in the experiments reported on under the heading "Influence of temper-
ature on growth of the host plants" they were thoroughly sprayed with
a 48-hour-old culture of Pseudomonas citri in beef bouillon. All in-
oculations were made at 10 a. m., about which time the stomata have
reached their maximum opening. As readings and observations were
made on the growth of the plants notes were taken on the development
of canker. Thus, a correlation could be obtained on the condition of
the plant and its relation to infection and development of the disease.
In Tables XI to XVII, the total number of spots and the part attacked
are given. On consulting Tables VI to X it will be noted that all new
spots are starred. Thus, a double check was obtained between the con-
dition of the plant, infection, and development of the disease.
EXPERIMENT IA
On consulting Table VI it will be seen that no spots developed on any
of the dormant plants of Poncirus trifoliata, nor on any plants subjected
to temperatures below 200 C. Thus, in Table XI, only the positive re-
sults with Citrus mitis and C. grandis are included.
No spots occurred on the calamondin plants at 200 C. Canker first
appeared on these plants held at 250. At 300 the spots were more
numerous, while at greenhouse temperature the number fell off. Canker
was not general on these plants because they are somewhat resistant.
The spots in all cases were small, unruptured, and occurred for the most
part on the mature or old leaves.
Even though an extremely slow growth of grapefruit occurred at 150 C.
no canker was produced. On the grapefruit plants canker first devel-
oped at 200, the spots increasing in numbers at 250. At 300 the number
of spots dropped off considerably, while under greenhouse conditions
the disease was more severe. It should be noted, however, that the
grapefruit plants at 250 and those kept at the greenhouse temperature
were in much better condition for infection.
476
Journal of Agricultural Research
Vol. XX, No. 6
■ft.
S
■ft,
^
■s
1
s
G
• 6
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s
4 spots on leaf, 4 spots on 2
leaves.
18 spots on 3 mature leaves.
1 spot on old leaf.
1 spot on twig, s spots on 4
old leaves.
d
!zi
a
a
7 small spots on 2 mature
leaves, 1 spot at tip of twig.
Shoot 1: 3 spots on leaf, 31
spots on 4 leaves, 6 spots on
petioles, 2 spots on twigs.
Shoot 2: 4 spots on 1 mature
leaf, 7 spots on leaf, 3 spots
on 3 old leaves.
6 spots on s mature leaves . .
60 spots on 9 old leaves, 12
spots on 3 mature leaves.
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Dec. ,s. i92o Effect of Temperature and Humidity on Citrus-Canker 47 7
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478 Journal of Agricultural Research vol. xx. no. 6
At 200 C. the spots which developed were more or less typical of
those produced under natural conditions. At 250 and 300, however,
they were extremely soft, loose, and spongy. These differences were
due to the stimulating influence of the high humidity and temperature.
EXPERIMENT 2A
In reality, this experiment is made up of two parts: First, the influ-
ence of temperature on infection of the plants at 50, io°, and 150 C,
with the subsequent transfer of the bell jars, together with the plants,
to the 300 case; and secondly, the infection of the plants at tempera-
tures between 200 and 300.
At the end of a 15-day period, the plants held at the temperatures
of 50, io°, and 150 C. were transferred to the 300 case to see, first, if
the shock would force growth of the dormant plants, and secondly, if
canker would develop, for during this period no spots appeared at any
of these temperatures. The appearance of new spots after the transfer
is noted in Table VII, while the number and type of spots, with the
part and age of the host attacked are given in detail in Table XII.
All the actively growing citranges became diseased soon after the
transfer. The two plants which remained dormant stayed clean. In
all cases, canker was confined to the new growth. It will be seen that
most of the citrange plants developed at a normal rate after the transfer
to the higher temperatures. The spots after breaking out were not
scattered over the new leaves and twigs but on definite portions of
the leaves, principally at the tip, along the midrib of the leaf and petiole,
and, in case of twig infection, along one side in regular arrangement.
Unpublished experiments with grapefruit seedlings and plants, in
both the greenhouse and field, on the time required for initial infection
have shown that the organism was able to enter the leaves within 20
minutes. Apparently, when the organisms were sprayed on the plants,
they were able to enter the stomata and there lie quiescent. The cit-
range plants were either just starting growth or were dormant when
inoculated and remained so until transferred. When the plants were
shifted from the 50, io°, and 150 C. cases to 300, the majority of them
pushed out into rapid growth, and the organisms also started to develop.
As the leaves unfolded and the twigs grew in length the spots broke
out where the organism had entered the tissues, which, as is stated
above, occurred at definite points on the new growth. The spots
appeared on the plants in from 5 to 8 days after they were placed in
the 300 case.
No canker developed on the calamondin plants when they were taken
from the 50 Cease and kept at a temperature of 300. Only two plants
making a rapid growth after being placed at a temperature of 300 from
the io° and 150 cases became diseased. The others remained free
from canker. Both the plants which later became diseased were in a
Dec. i5> 1920 Effect of Temperature and Humidity on Citrus-Canker 479
good growing condition when first inoculated, while the others had com-
pleted their growth or were dormant. Even though some of these
plants developed new growth when transferred to the higher tempera-
ture, they remained free from canker.
As on the citranges, just the new foilage was attacked in the majority
of instances. The spots were present at the base of the new growth or
petioles, and when present on the leaves most of them were on the
midrib or near the tip of the leaves. The majority of the spots were
elonagted rather than round and became visible in from five to eight
days after the plants were placed in the 300 C. case.
The grapefruit plants were all in excellent condition for infection
when inoculated and placed in the 50, io°, and 150 C. cases. However,
in no instance did the disease appear at these temperatures. Immediately
after the plants were transferred to the 300 case, growth proceeded at
the normal rate for that temperature, and all plants showed visible spots
within five days of the transfer. Canker was much more severe than on
the citranges and calamondin However, the spots were limited
to the young growth and were usually grouped at the tips of the young
leaves. Very few spots were found scattered over the leaves in general.
Thus, while no canker occurred on any of the plants held at 50, io°,
and 1 50 C. for a 15-day period, it did develop on those plants irrespective
of species which were in good growing condition when inoculated, after
they were all transferred to a temperature of 300. Even though the
plants did start growing after they were transferred, no canker occurred
at this temperature on any which had completed their growth or were
dormant when inoculated, except that one elongated spot developed at
the base of the new growth on one citrange plant. Apparently, the
organisms were able to enter the stomata of the very young growth
and remain inactive at the lower temperatures, but when the plants
were placed at a higher temperature the organisms became active and
produced canker. From the location and type of the spots there is no
doubt that the organism entered the tissues and remained quiescent
until a higher temperature was available.
In Table XIII are given the results obtained between temperatures of
200 and 300 C. for a period of approximately four weeks. At 200 all the
citrange plants became diseased. However, the spots were limited to
the new growth and did not become visible until 15 days after inocula-
tion. Only a few spots occurred on the twigs, and no mature or old
leaves were attacked.
Canker was much more severe at 250 C, causing some defoliation and
producing numerous spots on all plants. The spots were first visible
eight days after inoculation, which is one week earlier than at 200.
The majority of the spots occurred on the young foliage. Twig canker
was much more general than at 200, and some spots were formed on
the old leaves.
480
Journal of Agricultural Research
Vol. XX, No. 6
42
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Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 48 1
At 300 C, canker on old leaves and twigs was general and was much
more severe on the new growth than at the lower temperatures. On one
plant, the spots were visible four days after inoculation, on the others at
eight days.
No consistent results were obtained at the greenhouse temperature.
Very little canker occurred on the new foilage or twigs, while spots on the
old leaves were common. Canker did not develop until 15 and 20 days
after inoculation.
The results with the calamondin plants were rather variable. No
canker occurred on these plants at 200 C. Only one rapidly growing
plant inoculated at 25 ° became diseased, even though the other two
plants made some growth later on. At 300, canker was general on the
mature and old leaves of all three plants, only two spots occurring on
the new growth. One plant kept at the greenhouse temperature devel-
oped canker, and the spots here were limited to the new growth. Canker
was visible 12 days after inoculation. At the other temperatures, the
spots were visible in eight days. The spots produced on the calamondin
plants were small and unruptured.
With the exception of the grapefruit plants kept at the greenhouse
temperature, all developed canker within four days. Only two leaves
were attacked at 200 C, and in both cases the spots were localized at the
tip of the leaves or along the midrib. The plants held at 250 did not
grow nvell, so that only a few spots developed on some of the mature
leaves. At 300, canker was fairly well distributed over the new foliage
and twigs. Several leaves were defoliated by the severe attack, but no
spots occurred on the old leaves. This is in contrast to the general dis-
tribution of canker on the plant held at the greenhouse temperature.
The spots produced on the grapefruit varied with the temperature. At
200, the spots were more typical of those found under natural conditions,
while at 260 and 300 they were extremely spongy and corky. The same
was true for the spots on the citranges and calamondin.
Experiment 6
In this experiment, another attempt was made to obtain infection at
1 50 C. There were two plants each of the trifoliate orange, Rusk cit-
range, calamondin, and one of grapefruit. All plants chosen were in
*good condition for infection. As a control a similar set was included at
200. The plants were inoculated with a 6-day-old culture of Pseudo-
monas citri in beef bouillon, grown at 150 and 200, respectively, set
under bell jars, and kept in a saturated atmosphere for 1 month. Obser-
vations on the condition of the plants were made from time to time. It
was noticed that at 150, the young growth matured rapidly, especially
that of the grapefruit plant. No spots were found at the end of the
month. At 200, on the other hand, spots were visible on the grapefruit
16917°— 20 6
482 Journal of Agricultural Research voi.xx, N0.6
plant at the end of 8 days, and on the trifoliate orange and citrange plants
within 20 days. One month after inoculation several tiny spots ap-
peared on the leaves of one ealamondin plant. This was the only suc-
cessful infection of this species at 200 during the course of the work.
At the end of the first month, the plants held at 150 C. were transferred
to the 300 case, and the set kept at 200 was abandoned. Four days
after the plants were transferred to the higher temperature all were
diseased, having from several to many spots. By the end of two weeks
the disease was general on all the plants. The spots were more or less scat-
tered and typical and not at all like those described in experiment 2 a.
However, this was due, in part, to the fact that the leaves of the plants
used in this experiment were from one-half to three-fourths grown,
while the foliage of the others was mature except for the small unfolding
buds. The results obtained are the some as those reported on in experi-
ment 2 a, except that in this case the plants were held at the lower tem-
perature 1 month instead of 15 days. Table XIV gives the total number
of spots with part of the plant attacked at the temperature of 200 for
one month and for two weeks after transferring the plants to the 300 case
from a temperature of 150.
EXPERIMENT 3A
According to the results of experiment 3, a varying day and night
temperature had no appreciable effects on the development of the grape-
fruit plants. On the other hand, the effect was noticeable on the growth
of the other plants used. Thus, in this experiment, canker occurred at
all temperatures on the grapefruit plants, as can be seen in Table XV.
On the ealamondin plants held at the constant temperature of 300 C.
considerable canker developed. However, only one spot (on new growth)
occurred at the varying night temperatures. In other words, the eala-
mondin plant does not respond to so wide a temperature range for in-
fection as grapefruit.
The citranges and the trifoliate orange plants differ from the grape-
fruit in their reaction to sudden changes. On the citrange, canker
developed at a constant temperature of 300 C. , while no spots whatever
were produced on the others, in spite of the fact that they were all in the
same condition when inoculated. Only a few spots occurred on a few"
of the trifoliate orange plants. However, the majority remained free
from canker at the varying temperatures. Thus, except on grapefruit
plants, a low night temperature has a tendency to inhibit infection and
the development of the disease.
Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 483
^
2
i
s
o S
U
8
"ft.
X
Citrus grandis.
Shoot 1, 50+ small
corky spots on i leaf,
1 spot on twig. Shoot
2, leaf 2, 25+ small
corky spots; leaf 3
25+ small corky
spots; leaf 4, 25+
small corky spots.
Shoot 3, leaf 1, 10
small corky spots.
Shoot 1, 10 small spots
on 2 leaves. Shoot
3, 25+ small spots on
2 leaves. Shoot 4. 30
small spots on 2
leaves.
1
|
0'
d
S
5
3 small spots
on 3 leaves
at wound on
tip.
25 spots on
new leaves
above.
6
1
s
1 small spot on
leaf above.
25 spots on
new leaves
above.
u
a
a
g
'v
3
6
B
5
Leaf 4, 19 small spots at
tip; leaf 5. 2 small
spots at tip.
7 small spots on leaf
above.
6
d
a
5
7 small spots on 3 leaves
above, 6 small corky
spots in row on twig.
6 small spots on two
upper leaves.
c
1
i
a,
6
d
a
5
(
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U
u
V .
■*-* V)
<« 2
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O cu
= 3
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\l
6
d
OS
5
3 small spots on 2 leaves
above.
Clean
a
c
15° C.,
transfer-
red to 300.
484
Journal of Agricultural Research
Vol. XX, No. 6
8
"Is
r<> -G
§ 3
■o
s
o
g
G
d
4J
a
S
S
Leaf i, 2 corky spots;
leaf 2, 4 corky spots;
leaf 3, ioo-l- corky
spots near tip, i spot
on twig.
Shoot i, is small spots
on twig, petiole, and
midrib of tip leaf.
Shoot 3, 2s+ spots on
twig, petiole, and
midrib of tip leaf.
ioo+ small spots on
leaf above, bad.
Shoot i, leaf i, 25 corky
spots; leaf 2, 100+
corky spots; leaf 3,
100+ corky spots,
bad; leaf 4, 25+
corky spots; leaf 5,
6 small spots, 20
spots on twigs.
Shoot 2, leaf 1, 11
small spots; leaf 2,
10 small spots, 6
spots on twig.
0
a
«
5
.Shoot 1, leaf 1, 50 corky
spots; leaf 2, 50+ corky
spots; leaf, 3, 3 corky
spots, 1 spot on old
leaf. Shoot 2, leaf 1,
25+ corky spots; leaf
2, 25+ corky spots, 1
spot on twig, 2 spots
on old leaf.
Shoot 1, 1 spot on tip of
leaf, 1 corky spot on
twig. Shoot 2, leaf 1,
6 small spots; leaf 2,
25 small spots; leaf 3,
50+ corky spots, 6
spots on twig.
Shoot 1, 6 small spots
on tip leaf. Shoot 3,
200+ spots on tip leaf,
bad.
Shoot 1, leaf 1, 100+
small spots, leaf 2, 5
small spots; 3 spots
on twigs. Shoot 2,
leaf 1 , 50+ corky spots;
leaf 2, 50+ corky spots;
leaf 3, 25+ corky spots,
8 spots on twig.
1
G
d
S3
a
a
Shoot 2, 20+ small spots
on tip of leaf. Shoot
3, 10+ spots on tip of
leaf, 1 spot on old leaf.
Shoot 1, 10 small spots
on tip of leaves.
Shoot 2, 1 spot on old
leaf.
Clean
d
a
3
S
Shoot 1, 1 spot on leaf
above. Shoot 3, leaf
leaf 1, 2 small spots;
leaf 2, 5 small spots.
Shoot 4, 10 small spots
on twig.
Clean
■z
c
t:
0
u
be
a
a
g
'o
S
Shoot 1, leaf 1, 1 small
spot; leaf 2, 6 small
corky spots; leaf 3, 25
small corky spots; leaf
4, 5 small corky spots;
leaf 6, 1 spot on petiole;
leaf 7, 1 spot on petiole,
defoliated, 6 spots on
twig. Shoot 2, 2 spots
on 2 leaves above.
Clean
•5
.2
.1
3
i
d
!
5 small spots
on tip of leaf.
Clean
2 spots on 2
leaves above,
1 spot on
twig.
Clean
d
a
a
5
1 spot on twig .
1 small spot on
leaf above.
*
Clean
0
•0
Q.u
30° C , con-
trol.
200 C. ,
trans-
ferred to
30°.
15° C.
trans-
ferred to
io° C.
trans-
ferred to
30°.
*
Dec. iS. 1920 Effect of Temperature and Humidity on Citrus-Canker 485
EXPERIMENT 4A
In experiment 4, it was pointed out that where plants were held for a
short time at 300 C. and then placed at 150 a marked inhibition of growth
occurred, although the grapefruit leaves made an extremely slow growth
and the younger leaves matured to some extent. However, when
transferred back to the 300 case, growth of all the plants except one
proceeded at a regular rate for that temperature.
When the two sets of plants were placed in the 300 C. case, both were
inoculated in the usual way. At the end of 24 hours set 2 was transferred
to the 1 50 case to determine whether canker would develop at this tem-
perature. No doubt the organisms were able to enter the host plants
during the 24-hour interval, for canker was observed on the grapefruit
plants of the control 48 hours after they were inoculated.
At 1 50 C. all the plants remained free from canker, with the exception
of the larger grapefruit plant. Nine days after the transfer of the plants
a few small, unruptured spots occurred on one grapefruit leaf (Table XVI) .
However, after the plants were transferred back to the 300 case, the
severity of canker was as great as on the control plants, except on the
one citrange plant which did not produce new growth. These results
indicate quite clearly that the organisms were able to enter the plants
during the interval they were held at 300 in as great a number as in the
control plants, but when the plants were transferred to the 150 case,
growth of the plants and likewise the development of the organism were
inhibited, although in culture at this temperature a fairly good growth
is made by the organism. When the. plants were again placed in the 300
case and normal growth for that temperature was resumed, as much canker
subsequently appeared on these as on the control plants. All experi-
ments so far presented along this line indicate quite clearly that the
development of the disease is primarily dependent upon the activity of
the plant.
Table XVI. — Percentage of infection on plants at an alternating high and low temperature
EXPERIMENT 4A
Tempera-
ture.
Rusk citranse.
Cilrus milis.
Citrus grandis.
Plant No. 1.
Plant No. 2.
100 per cent leaf in-
fection; spots few,
small, and corky;
1 spot on twig
at base of new
growth.
300 C,
transferred
to 150.
is'C,
transferred
to 30°.
Clean.
Few small, scatter-
ing, compact spots
on lower leaves.
Clean .
Clean; no
growth.
Spots plentiful at
old leaf scars.
100 per cent leaf in-
fect i on ; spots
many, small to
medium, corky; 2
spots at tip of 2
twigs, large and
corky.
Few small, scatter-
ing, unruptured
spots on one leaf.
100 per cent leaf in-
f e c t i o n ; spots
many, small to
large, corky; 2
twig spots at tip.
100 per cent leaf in-
fection ; spots small
to medium, few
corky; 1 twig
spot, large and
corky.
Clean.
100 per cent leaf in-
fection; spots few
small to medium,
corky.
486
Journal of Agricultural Research
Vol. XX, No. 6
EXPERIMENT 7
Heretofore, in all the experiments at low temperature no attempt was
made to bring either the plants or cultures to the temperature of the case
to which they were subsequently exposed. To check this phase of the
work one set of plants was inoculated in the usual way. In the second
set, the plants and cultures were held at 150 C. for 24 hours before the
inoculations were made, to insure that both the plants and the organisms
in culture were at the temperature desired. As will be noted in Table
XVII, no canker developed on the plants of either set at 150 during the
18-day period they remained at this temperature. However, when both
sets were transferred to the 300 case, canker appeared on the citrange
and grapefruit plants in about the same proportion. The first method of
inoculation which was more generally used compared favorably with the
second method herein described. A similar experiment was carried out
at 200. The period of incubation, amount of infection, and growth of
the plants were the same in the two experiments.
Table XVII. — Comparison of methods of inoculating plants at low temperatures
EXPERIMENT 7
Tempera-
ture.
Duration of
experiment.
Rusk citrange.
Citrus:
mitis.
Citrus grandis.
Plant No. 1.
Plant No. 2.
I5°-I5°C..
"R-i5°C
I5°-I5°C,
transferred
t0 3o°.
R-150 C,
transferred
to 300.
Dec. 10
to
Dec. 29, 1919.
do
Clean . .
...do.. .
do
.. do. ...
Clean
Clean.
Do.
1 spot on 1 leaf.
Bud attacked and
killed by can-
ker; no new
growth.
do
do
Dec. 29, 1919,
to
Jan. 10, 1920.
do
Few small spots
on 1 leaf.
Shoot i, leaf i. 10 tiny-
spots; leaf 4, 1 small
spot. Shoot 2, leaf i,
10 small spots; leaf 2,
2 large corky spots;
leaf 3, 2 small corky
spots.
Shoot i, leaf 2, 5 small
spots; leaf 3, 10 small
spots; leaf 4, 3 small
spots. Shoot 2, leaf 1.
defoliated by canker;
leaf 3, 1 small spot.
nR= greenhouse temperature.
EXPERIMENT 5A
The results obtained in experiment 5 seemed to indicate clearly that
at 350 C. the growth of grapefruit and plants of the same type was
practically inhibited, whereas the trifoliate orange and limequat were
both able to make a normal growth. It will be noted that four sets of
plants were used in this experiment. After the four sets of plants
remained at this temperature overnight, they were inoculated with
5-day-old cultures of the organism grown at temperatures of io°, 150,
250, and 350 C, respectively.
Dec. is. I92o Effect of Temperature and Humidity on Citrus-Canker 48 7
Because of the limited amount of infection the results are not tabulated.
No sign of canker developed on any of the plants in set 4, which had been
inoculated with a culture of the organism grown at 35 ° C. As was to be
expected, only three spots (two on grapefruit and one on sweet lemon)
occurred on this type of plant in the other three sets. This extremely
light infection was due to the distinctly inhibitive influence of the high
temperature on the growth of these plants.
Many spots occurred on both the limequat and trifoliate orange plants
in the remaining sets. Incubation required from 5 to 1 1 days on the
trifoliate orange and 11 or more on the limequat plants. The spots
were medium-sized, ruptured, and very corky. In no case did any of the
trifoliate orange plants, which were dormant when inoculated, become
infected when new growth appeared later. Furthermore, where a new
shoot had started prior to inoculation, many spots developed on this
shoot, but no canker appeared on any shoots which developed after
inoculation. Evidently, at this temperature, the organism is unable to
survive for any length of time and is only able to infect the actively
growing tissue of the plant.
CONCLUSIONS ON THE INFLUENCES OF TEMPERATURE ON INFEC-
TION AND THE DEVELOPMENT OF THE DISEASE
(1) No canker whatsoever has been produced on dormant plants.
(2) The minimum temperature for the successful inoculation of
Poncirus trifoliata, Rusk citrange, and Citrus grandis plants is 200 C.
Apparently, it is a little higher for plants of C. mitis.
(3) The optimum temperature for infection of the Citrus plants used,
which were in an active growing condition, lies between 200 and 300 C,
with the possible exception of C. mitis.
(4) A low night temperature has a decidedly inhibiting effect on
infection and development of the disease on citrange and Citrus mitis
plants. This does not hold true for C. grandis.
(5) At 200 C. only the new growth was attacked with few or no twig
cankers; not only the new growth but twigs developed cankers at 250,
and there were few spots on old leaves; while at 300 all of these parts
were readily attacked.
(6) The period of incubation varied not only with the host plant but
also with the temperature. With citrange and Citrus mitis, the period of
incubation was shortest at 300 C. With grapefruit, the period^of incu-
bation was very short at all temperatures between 200 and 300.
(7) At 200 C. the spots produced on the plants are more typical of
those found under natural conditions, while at 25 ° and 300 they are
extremely loose, soft, and spongy.
(8) Judging from the location, parts of the plant attacked, and type of
spots produced on growing plants when transferred to a temperature of
300 C. after being held from two weeks to one month at 50, io°, and 15 C,
488 Journal of Agricultural Research vol. xx, No. 6
there can be no doubt that the organism entered the tissues of the host
shortly after inoculation and remained quiescent until a higher tem-
perature was available. This fact may explain the many cases of
inactivity of the disease met with under field conditions.
(9) Plants held at 300 C. for 24 hours after inoculation and then trans-
ferred to a lower temperature failed to produce infection except on one
grapefruit plant. However, when returned to a higher temperature,
most of the plants showed 100 per cent infection.
(10) At a temperature of 350 C. infection took place only on the plants
which made a normal growth, while little or no disease occurred on plants
of the Citrus grandis type. However, all successful inoculations even
on the Poncirus trijoliata type of plants were made with cultures of the
organism grown at temperatures below 350.
INFLUENCE OF HUMIDITY ON THE ORGANISM
The influence of humidity on bacteria resolves itself principally into a
question of drying or desiccation. Bacterial growth takes place only in
the presence of free moisture. Thus, in a study of the influence of
humidity on bacteria, one must consider the viability of the organism
and not the growth.
The common methods used heretofore have been the drying of the
organisms on silk threads, glass beads, or glass slides. Some few investi-
gators have used seeds. The method ordinarily followed by the pathol-
ogist is to smear with a sterile platinum needle on sterile microscopic
slides bacteria from vigorous pure cultures and to set these slides away
in the dark in a dry-air room. After a few days they are tested for
viability, either by pouring nutrient agar over the slides in Petri dishes
or by dropping cover glasses, which are sometimes used, into a suitable
culture medium.
In the work on the resistance to drying of bacteria, no one has deter-
mined the temperature or the humidity at which the prepared slides have
been kept. Again, no attention has been paid to making a uniform
smear of the organism on the slides. The only factor which has been
considered necessary has been that the smear be taken from young,
vigorous cultures.
A brief review of the literature reveals the fact that organisms dried
on seeds or on silk threads remain alive much longer than those dried on
glass slides, cover glasses, or beads. However, since conditions varied
with each experiment, no comparisons can be drawn.
Using the prescribed method for testing resistance to drying, Stevens
(12) found that —
bacteria (P. citri) from young and old cultures exposed for two weeks on glass slips
to dry in the air of the laboratory failed to germinate.
Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 489
Wolf (17), varying the method somewhat, states that:
The organism seems to exhibit a very considerable resistance to drying. In the
desiccation experiments bacteria from vigorous pure cultures on potato plugs were
smeared by means of a sterile platinum needle on clean microscopic slides in moist
chambers. The moist chambers containing the microscopic slides were sterilized
prior to transferring the bacterial smear to the slides. These preparations were made
on June 1, and placed in a wall closet in the laboratory. On July 1, August 1, and
September 1, several of the microscopic slides were removed from the moist chambers
and placed in the sterilized Petri dishes, using proper aseptic precautions in making
the transfers. Tubes of melted nutrient agar which had been cooled almost to the
point of solidification were poured upon these smeared slides. No growth occurred
in the case of those tested on September 1 , but those tested on July 1 and August 1
were still alive. From this, it is believed that the organism can retain its viability for
about two months.
Stevens (12) later carried out the following experiment:
Pieces of sterilized cloth were wetted with suspensions of bacteria (P. citri) from
cultures of different ages, from four days old to seventy-five days old . The pieces were
then allowed to dry in the air of the laboratory in the dark. Germination tests from
these pieces of cloth showed a very large number of the organisms alive after a drying
period of five weeks.
He also states:
That the bacteria may live for a month or more in the dried canker spots, is shown
by the disease having been transferred to healthy citrus tissue fronf dried leaves that
had been kept in the laboratory for a month.
On the other hand, Wolf (77) states that :
Unsuccessful attempts, however, have been made to recover the organism from the
leaves kept in the laboratory from September, 1014, to May, 191 5; nor has recovery
been possible in the case of twig cankers kept under laboratory conditions from March
to October, 1915.
Stevens (75) concludes from his experiments with the growth of Pseu-
domonas citri in dry sterilized soil that —
P. citri can propagate and remain alive and virulent when kept in soil for a
period of twenty-six months, and that the organisms are capable of surviving long
periods of desiccation without complete loss of vitality and with little apparent loss
of virulence.
The following experiments, which are to be considered of a preliminary
nature only, were undertaken to determine the viability of the organism
at different temperatures and under various humidities.
The method used was essentially as follows: Eighteen silk threads 2
inches long were stretched across an aluminum wire frame 2}i inches
square, with legs 1^ inches high , inclosed in glass stockings of the same
height. These frames were then placed in ordinary moist chambers 2
inches high and 3^ inches wide and sterilized in the autoclave. Larger
Koch moist dishes, with ground-glass lids, were then sterilized. Under
sterile conditions, the threads were immersed in a 48-hour-old culture of
Pseudomonas citri in beef bouillon for 5 minutes. In the meantime, a
49° Journal of Agricultural Research vol. xx, No. 6
sulphuric-acid solution was added to the two dishes. The smaller dish,
set in the larger one, was filled to within i inch of the top, and the larger
dish was filled to the same height, so that about i inch of the smaller
dish projected out from the liquids. The frames were then replaced in
the smaller dishes, so that the threads were ]/A inch from the surface of the
liquid. The lids of the outer dishes were then vaselined and made air-
tight. At the end of each 24 hours, two silk threads were cut off and
placed in tubes of beef bouillon to test for the viability of the organism.
The reason for the use of two dishes, both filled with the solution, will be
explained by Prof. Hottes in a forthcoming article. It is sufficient to
say that this method gives a very accurate vapor pressure, which in turn
could be translated into terms of relative humidity. For the sulphuric-
acid concentrations, vapor pressure, and relative humidity the tables
published by Stevens (14) were used. The specific gravity of all solu-
tions was determined with a Twadell hydrometer when the temperature
of the solution was 150 C. The dishes were set in the different tempera-
ture cases, so that they were exposed to a rather strong diffused light.
The writer wishes to point out one difficulty that had to be overcome
and which caused him more or less trouble during the course of this
experiment. The citrus-canker organism, as has been pointed out before,
makes a very characteristic growth in beef bouillon. One of its character-
istics is to produce flakes after a certain time, depending on rapidity of
growth. Whenever a beef-bouillon culture of the organism which was
used to inoculate the threads showed any signs of flaking, no consecutive
results were obtained. Thus, several sets had to be discarded and re-
peated on this account. The reason is perfectly obvious and needs no
further explanation. Thus, it is imperative that strictly uniform sus-
pensions of the organism be used to inoculate the threads in order to
obtain consistent results.
The results of the experiment given in Table XVIII clearly demonstrate
that there is a distinct influence between temperature and humidity on
the viability of the organism on the threads. At the medium humidities
(49 t<3 70.4 per cent) the organisms were alive for the duration of the
experiment at all temperatures. No organisms were viable at the end of
24 hours at the higher humidities (80.5 to 100 per cent) at 300 C. How-
ever, with each drop of 50 in the temperature more of the organisms
remained viable at these humidities, until at io° the organisms were
viable at all humidities for the duration of the experiment. The same
thing held true for the lower humidities. Here more or less variation
existed, but there is a more or less regular sequence in the increase of
viability at these humidities with each drop of 50 in temperature, until
we reach io°, where again, as is the case of the higher humidities, they
are viable for eight days.
Because of the preliminary nature of this phase of the investigation
no explanation of these results can be made at this time, except to point
Dec. i5. 1920 Effect of Temperature and Humidity on Citrus-Canker 49 1
out that there is a distinct relation between temperature and humidity
on the viability of the citrus-canker organism, so far as this experiment is
concerned. It is interesting to note that at the low temperatures humid-
ity apparently has little influence on the viability of the organism on the
threads, while at the higher temperatures it is the limiting factor, espe-
cially at the higher humidities. At the humidities generally encountered
in the field in Alabama the organisms are viable at all temperatures on
the threads for eight days. Another puzzling fact is that in ordinary
distilled water the organism remained viable at temperatures between
io° and 350 C. for a period of eight days, while at the higher tempera-
tures (300) the organisms are dead at the end of 24 hours in a saturated
atmosphere.
Table XVII. — Viability of Pseudomonas citri on silk threads at varying humidities over
sulphuric acid
Ap-
Tem-
Specific
proxi-
mate
Satura-
tion
deficit.
After.
After.
After.
After.
After.
After.
After.
After.
ture.
gravity.
relative
humid-
1 day.
2 days.
3 days.
4 days.
5 days.
6 days.
7 days.
8 days.
ity.
Per
°c.
1. 00
1. 14
1. 20
cent.
ICO
89.9
80.5
0
3-2
6.1
-
1. 25
70-4
9-3
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
30
1.29
60. 7
12.4
+
+
+
+
+
-f
+
+
+
+
+
+
+
+
+
+
1-344
49.0
16.0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.398
38.0
19-5
+
+
+
+
+
+
+
+
I-S03
18.5
25.4
+
+
+
+
—
—
—
—
+
+
—
—
+
+
+
—
I 1.82
1. 00
+
+
+
+
-
—
100. 0
0
I. 14
89.9
2.4
1. 20
80.5
4.6
+
+
+
+
1-25
70. 4
7.0
+
+
+
+
+
+
+
+
+
+
+
+
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+
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25
1. 29
60. 7
9-3
+
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49.0
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+
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1.398
38.0
14. 7
+
+
+
+
+
+
+
+
+
+
+
+
—
—
—
—
I- 503
18.5
19.4
+
+
+
+
+
+
—
—
+
+
+
+
—
+
—
—
1 1-82
1. 00
+
+
+
+
100. 0
0
1. 14
89-9
1.8
+
+
1. 20
80.5
3- 4
+
+
+
+
—
+
+
+
—
—
—
+
+
+
+
1. 25
70.4
5- 2
+
+
-+
+
+
+
+
+
+
+
+
+
+
+
+
+
20
1. 29
60. 7
6.8
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1-344
49.0
8.9
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.398
38.0
io. 9
+
+
+
+
+
+
+
+
+
+
+
+
—
—
— —
I-S03
18.5
14.4
+
+
+
+
+
+
+
+
+
+
+
{ 1.82
[ 1. 00
+
+
+
+
+
+
+
+
+
+
+
+
+
100. 0
0
I. 14
89.9
1-3
+
+
+
+
+
+
+
+
+
+
+
+
—
—
—
-
I. 20
80.5
2- 5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
70. 4
3-8
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
IS
I. 29
6o- 7
5- 0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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I- 344
49.0
6-5
+
+
+
+
+
+
+
+
+
+
+
+
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+
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+
1-398
38.0
7-9
+
+
+
+
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+
+
+
+
+
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—
—
1.503
18.5
10. 4
+
+
+
+
+
+
+
+
+
+
I I.82
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
89.9
•9
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
80.5
1.8
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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3-6
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+
+
+
+
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+
+
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+
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49.0
4- 7
+
+
+
+
+
+
+
+
+
+
+
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1.398
38.0
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+
+
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+
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18.5
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+
+
+
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+
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{ I.82
+
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+
+
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+
+
492 Journal of Agricultural Research vol. xx, No. 6
INFLUENCE OF HUMIDITY ON GROWTH OF THE HOST PLANTS
The preliminary experiments reported below are indicative of what
might be expected. Before placing the plants in the cases, all the pots
were wrapped with a double layer of parraffin paper, so that no moisture
could escape from the soil.
EXPERIMENT I
Two plants each of Poncirus trifoliata, Citrus mitis, and C. grandis
were used in each case. For the most part, the plants were dormant or
had completed their growth.
Three cases with humidities of 90 to 95 per cent, 82 to 86 per cent,
and 73 to 77 per cent were used. The temperature (dry bulb) in the
cases varied between 210 and 23°C. As can be seen in Table XIX,
with the exception of two grapefruit plants held at 90 to 95 per cent
humidity, none of the plants were pushed into active growth. However,
it will be remembered that at no temperature in a saturated atmosphere
did the trifoliate oranges produce new growth, and likewise no results
were obtained with the calamondin plants at 200 in a saturated
atmosphere. The grapefruit plant did make a rapid growth at 200,
in fact much more so than those held at 90 to 95 per cent humidity and
at approximately the same temperature. Thus, with dormant plants
which have completed their growth, the temperature and humidities
used did not stimulate the production of new growth
EXPERIMENT 2
In this experiment, three plants each of the Rusk citrange, calamondin,
and grapefruit were used. One plant of each species had sufficient new
growth for infection, a second had mature leaves, while the third was in
a dormant condition. The results of the experiment are reported in
Table XX.
Of the plants used, calamondin appeared to thrive and grow best at
the humidities used in this experiment. In the experiment on the
influence of temperature in a saturated atmosphere, little or no growth
occurred at 2o°C, but here with approximately the same temperature a
good vigorous growth was made, even the dormant plants of this species
starting. The results with grapefruit and citrange were not so clear-cut.
Their behavior was decidedly different from that at 200 in a saturated
atmosphere. Growth at the humidities used was faster, and the leaves
were much smaller. Apparently, then, low humidities have the same
influence as low temperatures on the maturation of the leaves of some
of the Citrus plants. The cause for the decided difference in the growth
of the calamondin plants is not known.
Dec. is, 1920 Effect of Temperature and Humidity on Citrus-Canker 493
I
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494 Journal of Agricultural Research volxx.no. 6
EFFECT OF HUMIDITY ON INFECTION AND DEVELOPMENT OF THE
DISEASE
The literature on this subject has already been discussed thoroughly,
and the consensus of opinion has been that citrus-canker developed best
and spread most rapidly in a warm, humid climate. It has also been
pointed out that the host plants themselves thrive best under these
influences. It has likewise been shown that the greatest number of
plants are infected at 300 C. in a saturated atmosphere, while even at
200 infection takes place, particularly on grapefruit and citrange plants.
Just before the plants were placed in the humidity cases already
reported on, they were thoroughly sprayed with a 48-hour-old culture
of Pseudomonas ciiri in beef bouillon, which was almost allowed to dry
on the foliage before they were placed in the cases.
No infections of any kind occurred on the plants listed in Table XIX
during a period of 18 days.
In the second experiment (Table XX), only two infections occurred
during the 15 days the plants were in the cases. Both of these occurred
at the higher humidity. In one case, one spot developed on a young
leaf of a calamondin plant, and several corky spots were found on the
tip leaf of one grapefruit plant. No doubt, in these instances, the
organism was able to enter before the plants had adjusted themselves
to the humidity of the case. On January 31, 1919, the plants in both
cases were removed to a saturated atmosphere and approximately the
same temperature. Within eight days, one plant of the Rusk citrange,
two of the calamondin, and one of the grapefruit became infected as
shown in Table XXI. Only two spots on two mature leaves of one
of the grapefruit plants developed on those held at the lower humidity
before being transferred to a saturated atmosphere.
Dec. i5l 1920 Effect of Temperature and Humidity on Citrus-Canker 495
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Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 497
CONCLUSIONS ON THE INFLUENCE OF HUMIDITY ON GROWTH OF
THE ORGANISM AND HOST PLANTS AND ON INFECTION AND
DEVELOPMENT OF THE DISEASE
(1) The results of the silk thread experiment prove very conclusively
that there is a distinct relation between temperature and humidity on
the viability of Pseudomonas citri.
(2) The citrus-canker organism is very susceptible to a combination
of high temperature and humidity. Medium humidities at all tempera-
tures are not injurious to the organism. With all humidities at low
temperatures none of the organisms are killed.
(3) Apparently, at the humidities and temperatures used there is an
inhibiting action on the growth of the Citrus plants, with the exception
of Citrus mitis.
(4) Little or no infection occurred at the humidities and temperatures
used.
DISCUSSION
The writer realizes keenly the imperfections and incompleteness of
the experimental data presented, because of the complexity of the pro-
blem with its numerous and diverse factors. However, he feels that
enough qualitative data have been accumulated to indicate that a most
excellent field of endeavor lies in investigations of this nature. Several
fundamental principles have been uncovered, which, with further study,
should lead to promising results. Thus, with the incompletness of the
experimental work in mind, the writer will attempt to discuss his results
as a whole and correlate them with actual field conditions as he has
observed them during the past four years.
A superficial study of the temperature relations, in culture, of the
bacteria causing plant diseases shows that, in the main, the temperature
relations of Pseudomonas citri agree very well with those which have
been studied from time to time. One point which pathologists have not
considered in their studies of temperature relations of bacteria in culture
has been the time element. However, when this factor is considered,
the plant-disease bacteria belonging to the Pseudomonas group have a
minimum temperature of approximately 50 C. or slightly higher. By
the use of former methods, lower minimums have been obtained in some
cases. They have an optimum between 200 and 300, a maximum varying
with the time factor, but between 350 and 380 for a period of 24 hours,
and a thermal death point between 490 and 510. The plant-disease
organisms of the bacillus group, with but few exceptions, have a maxi-
mum temperature and thermal death point several degrees lower than
the Pseudomonas group. ,
Temperatures below the minimum simply inhibit the growth of the
bacteria, so that low temperatures within reasonable limits and with
the length of exposure considered do not cause their death. It should
16917—20 7
498 Journal of Agricultural Research voi.xx, no.6
be noted that all the active plant-disease bacteria can develop in cultures
at temperatures lower than that of their host plants. The writer wishes
to point out here again the pronounced lag in the growth of Pseudomonas
citri on media between temperatures of 150 and 200 C. To him, this
difference is of marked significance. No explanation of this phenomenon
can be offered at this time.
In most cases, the optimum temperature for the growth of these
organisms is approximately the same as that of the host plant. Thus,
the temperatures at which the best development of the host plant occurs
are the same as those which yield the best growth of the bacteria in culture.
For extended periods of time, the host plant develops at temperatures
slightly higher than the bacteria in culture, although the plant's develop-
ment is likewise retarded at the high temperatures. The extent to
which the growth of the bacteria at or near the maximum is retarded or
inhibited depends on the length of exposure.
While studies of the temperature relations of the bacteria in cultures
are necessary, the results can not be strictly interpreted in the light of
field conditions. They serve only in indicating an approximation,
especially where minimum and maximum temperatures are concerned.
Our present methods of determining the resistance of bacteria to drying
have been exceedingly crude, and with but few exceptions no attention
has been paid to conditions which might influence the results. At best,
the usual methods do not even have an empirical value, in that the
results are not comparable. A glance at the literature on the subject
will reveal this fact.
Different investigators have obtained widely divergent results with
the same orgainsm. To illustrate, Stevens (12) states that —
bacteria (P. citri) from young and old cultures exposed for two weeks on glass slips
to dry in the air of the laboratory failed to germinate,
while Wolf (17) comes to the conclusion that —
the organisms seems to exhibit a very considerable resistance to drying
and further that —
the organism can retain its viability for about two months.
Smith (10) —
found this organism (P. campestris) much more resistant to dry air than Harding's
first report would indicate, to wit; in Harding's experiments, invariably destroyed
in 45 hours, and 7 out of 8 cover-slips sterile at the end of 2 1 hours. In my own tests,
the organism on 8 out of 24 cover-slips was alive after 34 days, when inoculated from
a potato culture 2 days old and on 2 out of 23 cover-slips when inoculated from bouillon .
Later Harding, Stewart, and Prucha (j) found that Pseudomonas cam-
pestris could live on cabbage seed for a year under certain conditions.
In the experiments carried out by the writer, strict attention has been
paid to the amount of the inoculum on the threads, as well as to tempera-
ture and humidity. The most striking results obtained indicate that
at low temperatures humidity has little influence on the viability of the
Dec. is, 1920 Effect of Temperature and Humidity on Citrus-Canker 499
organism, while at high temperatures it is the limiting factor. It is
extremely interesting to note that at the medium humidities the organ-
ism is alive at all temperatures for the period of the experiment. Even
at the extremely low humidities the organism is viable for varying lengths
of time, depending somewhat on the temperature.
No attempt will be made at this time to explain the results obtained,
nor to compare them with those showing that in ordinary distilled water
the organism is alive at the end of eight days at temperatures between
io° and 350 C. It is sufficient to state here that the death rate of the
organism on the silk threads is not due to the rapidity with which drying
takes place, since at the low humidities where drying is most rapid, the
death rate is slow, while at high humidities where the rate of drying
is slowest the death rate is most rapid. At the medium humidities,
where the organism is alive at all temperatures for the duration of the
experiment, some other factor or factors must enter in other than the
rapidity of drying. It should be noted that the medium humidities
used in these experiments are the ones most generally prevalent under
field conditions in Alabama during the greater part of the year.
The life of a plant-disease bacterium in culture in the laboratory and
in the field outside of the host plant is ruled by entirely different factors
from those which govern when it is parasitically active in the host tissues.
Thus, a sharp distinction must be drawn between these conditions.
It is extremely difficult to compare the results obtained in the green-
house experiments with observations in the field, because of the widely
divergent conditions which exist. In the greenhouse work constant
temperatures and humidity controls were used, while in the field all sorts
of conditions are met. After the problem has been studied from all
angles, it appears that only general statements can be made at this time.
For the purpose of this discussion, two types of rest periods can be
distinguished without entering into a long explanation of the probable
causes of rest periods in horticultural plants — namely, winter dormancy
brought about by either the approach of cold weather or freezing tem-
peratures and the short rest periods which occur during the growing
season. During winter dormancy the cell activities cease to a great
extent, while during the short rest periods which occur in the growing
season some of the cell functions merely slow up.
In Alabama, as a rule, grapefruit and allied plants usually grow on
into the winter, until temperatures of 50 C. or lower are reached. At
this time, the plant is thrown into a state of dormancy, which persists
until a period of higher temperatures occurs and active growth is resumed.
This may happen several times during the winter. With Satsuma
{Citrus nobilis var. unshiu, Swingle) and other mandarin oranges growth
proceeds until low temperatures occur and after that no growth takes
place until suitable temperatures prevail. Kumquats (Fortunella marga-
rita (Lowr.) Swingle) go into dormancy and cease their growth with the
5<x> Journal of Agricultural Research vol. xx, no. 6
approach of low temperatures and remain dormant for a longer period
in the spring than any other of the Citrus plants. The trifoliate orange,
being deciduous, has a very fixed dormant period. The plants become
dormant in the fall with the approach of cold weather and do not start
growing until a period of favorable temperature is reached in the spring.
To summarize, the Citrus plants in Alabama become dormant in the fol-
lowing order, trifoliate orange, kumquat, Satsuma, and grapefruit. New
growth starts out in almost the reverse order, grapefruit, Satsuma, tri-
foliate orange, and kumquat.
Thus, with other factors eliminated, grapefruit plants develop at the
lowest range of temperatures, both in the fall and spring, in the field.
In all cases, the leaves formed late in the fall and early in the spring are
much smaller in size and mature in a shorter period than those which are
formed later in the season.
In the greenhouse experiments under control conditions it was found
that grapefruit could develop very slowly at 150 C. and also that in no
instance could any of the other plants used be pushed into growth at this
temperature. However, at 200 all plants became active, although the
calamondin, which resembles the kumquat in some respects, did not de-
velop rapidly until a temperature of 250 was reached. The differences in
the size of the leaves and time required for their maturation in comparison
with those obtained at 300 were also noticed at the lower temperatures,
grapefruit leaves being one-fourth to one-half the size of those produced
at 300. It was likewise observed that 16 to 20 days were required at 300
to complete the maturation of the grapefruit leaves, while at 150 7 to 8
days were sufficient.
Thus, a mean temperature of 150 C. or thereabouts is sufficient for
starting active growth of grapefruit plants in the field, while tempera-
tures of 200 or slightly less are needed for the trifoliate orange and
Satsuma. Kumquat does not start until a slightly higher mean is
reached. These figures are borne out by the weather records and obser-
vations of the conditions of the plants in the field for the past four years.
The optimum temperature for the growth of the Citrus plants used in
the greenhouse experiment lies between 200 and 300 C. Some differ-
ences were noted in the behavior of the different plants at these tempera-
tures. There is no question but that at 300 the best development of all
the plants occurred. Above 300 grapefruit was inhibited, while plants
like the trifoliate orange seemed to make as good a growth as they did
at 300.
The short rest periods of Citrus plants during the growing season are
in all probability a maturation phase, following the period of elongation
of the new growth. Field observations have shown that temperature
and humidity play an important part in the rate and amount of growth
made during these periods; in fact, they determine to some extent the
number of growth periods which occur during a season.
Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 50 1
Because of the preliminary nature of the greenhouse experiments on
the influence of humidity on Citrus plants, no statements can be made
at this time, except to point out that there is a definite relation between
the development of the plant and humidity.
The first prerequisite for infection of Citrus plants by Pseudomonas
citri is the presence of free moisture on the plant. The second condition
is a suitable temperature. However, with both these conditions fulfilled,
no infection can take place unless the plant is in an active, growing con-
dition. In other words, no infection of a dormant plant is possible.
This fact has been clearly demonstrated by the greenhouse experiments
and is borne out by observations under field conditions. During the
short rest periods in summer, it is infrequent that new infections occur.
This is due to the fact that the shoots have completed their growth and
the period of maturation is at hand. In other words, canker is most
abundant during the growth periods, the severity of the disease de-
creasing during the short rest period. Thus, we have cycles of infection
which in turn correspond to the growth periods of the plants themselves.
In speaking of infection one must distinguish between the period of
initial infection and the period of incubation. By the period of initial
infection is meant the time required by the organism, after it reaches a
a leaf, to enter the stomata or, in the case of wounds, the tissue of the
plant. The period of incubation, on the other hand, is the period ex-
tending from initial infection until the disease is visible. As has been
stated before, experiments have clearly shown that the period of initial
infection is short, the organism getting into the stomata within 20
minutes. The period of incubation, on the other hand, may be short
(48 hours) or long (several months), depending on external conditions.
The presence of free moisture is necessary for limited periods only in
order that initial infection may take place. Initial infection does not
occur at high humidities, but because of the stimulating influence of high
humidities on the active growth of the plant, when accompanied by
suitable temperatures, they are more conducive to the disease. As has
been noted before, all investigators agree that the greatest development
of canker occurs during warm, humid weather. However, in all local-
ities where warm, humid weather prevails, we have alarge rainfall. Thus,
so far as initial infection and, incidentally, the development of the disease
is concerned, it is not the high humidity that must be considered but the
frequency of the rains. The temperature factor must not be overlooked,
in that, even though frequent rains occur, no canker will develop unless
a suitable temperature for the development of the organism and growth
of the host is at hand. Thus, without question, even though the same
amount of rain occurred in the orange districts of Japan as falls in the
Gulf coast section, canker would not be so severe, because of the lower
mean temperature prevailing in that country.
502 Journal of Agricultural Research voi.xx. no. 6
On the other hand, conditions are met with where a suitable temper-
ature for growth and infection is present, but there is a decided deficiency
in rainfall. The conditions existing in the Philippines can be cited as a
typical example. Thus, Mackie (6) states that —
during the dry season which occurs from January until the monsoon changes in June,
the disease is apparently quiescent. * * * However, after the rains begin, the
trees send out new growth and it is on this new growth that the canker appears,
coming into evidence in about a week. * * * Throughout the rainy season, the
disease thrives.
Initial infection can take place under conditions which do not favor
the development of the disease. Furthermore, it may occur and the
organisms may remain quiescent in the tissues for long periods of time
without any signs of the disease being manifested. In fact, we may
assume that there are occasions when initial infection takes place without
the subsequent developent of the disease because of unfavorable con-
ditions for its development after the organism enters the tissues of the
host plant.
The writer has shown that initial infection did occur at low temper-
atures, although no canker developed until the plants were transferred
to a higher temperature. These experiments were repeated under green-
house conditions several times with the same results. In the field,
plants were inoculated in September, 191 7, and no canker became visible
until the following April, when it developed very rapidly and was ex-
tremely severe on the twigs and stems of some of the hardy hybrids.
No doubt, in the case of kumquat, the organism is able to enter the
stomata but is unable to develop because of the resistance offered by
the tissues. Where the tissues are broken, kumquat leaves can be
readily infected. Thus, initial infection requires a definite set of con-
ditions entirely different from those required for the development of the
organism after it enters the host plant.
No canker whatsoever has been obtained under any conditions at
150 C. or lower on any of the plants experimented with in the greenhouse.
At 200 the disease has been produced on all plants, although the amount
of canker and the period of incubation varied greatly with the different
plants. Thus, only one calamondin plant was successfully inoculated at
200 in all the greenhouse experiments ; all growing plants became diseased
at 25 ° ; while at 300 the number of spots increased very rapidly in number
over those produced at 25 °.
On the trifoliate orange only a few leaf spots occurred at 200 C. after
15 days. At 250 spots were more numerous on the young leaves; a few
old leaves became diseased and a few twig spots were formed. The
period of incubation at this temperature was only 8 days. Canker was
general on all the plants at 300, with the period of incubation shortened
to 4 days. It is interesting to note in this connection that in the eradi-
cation of canker in Alabama practically all canker on trifoliate orange
Dec. i5l 1920 Effect of Temperature and Humidity on Citrus-Canker 503
stock has been found during the months of July and August, months
with the highest mean temperature. In other words, the trifoliate orange
is not very susceptible at temperatures of 200, but when temperatures
of 300 are reached the period of incubation is as short as that of grape-
fruit, and the plants themselves are as susceptible as grapefruit, or more so.
This fact can be still more clearly shown by stating that in Japan, where
the temperatures are rather low and uniform during the growing season,
cankers on the trifoliate orange are rare, though grapefruit and navel
orange in the same orchard or nursery may be badly infected. 'The slow
growth of the trifoliate orange, then, at temperatures around 200 makes
it more or less resistant to canker, though when grown at temperatures
of 300 it becomes extremely susceptible.
Grapefruit, which grows at a much lower range of temperature than
any of the other Citrus plants tested, is the first plant to become infected
in the spring and the last in the fall. The greenhouse experiments showed
that the period of incubation at 200, 250, and 300 C. was 4 days. How-
ever, the spots produced at 200 were not so large or so numerous as those
produced at 300.
Thus, it has been found that the optimum temperature for the growth
of the organism in culture media in the laboratory lies between 200 and
300 C. Since the same optimum has been found for the host plants, it
should be expected that the same optimum should prevail for infection
and development of the disease. That such is the case has been proved
in the experiments reported.
At a temperature of 350 C. or thereabouts, the maximum for the
growth of the organism in culture is approached, especially when the
length of exposure is included. This same temperature also inhibited
the growth of some plants in the greenhouse experiment. No canker
was obtained on any of the plants when a culture of the organism grown
at 350 was used to inoculate plants kept at this temperature in the
greenhouse. Only one spot was formed on grapefruit when plants were
inoculated at 35 ° with cultures grown at lower temperatures. The tri-
foliate orange appears to make a good growth at 350, and general infections
were obtained on these plants at this temperature. In the field, temper-
atures of 350 prevail for portions of some days over periods of several
months. The question naturally arises whether the organism can exist
outside the host plant for extended periods, especially if high humidities
prevail at the same time. On the other hand, we know that the disease
develops during these periods.
The influence of temperatures below 150 C. in the field will be dis-
cussed more fully in a forthcoming article on the overwintering of the
disease. It is sufficient to state here that although a temperature of 200
is necessary for infection, the disease after it is once produced can keep
on developing at temperatures lower than 200 and is fully dependent on
the growth of the host plant. In other words, the canker organism is
504 Journal of Agricultural Research voi.xx, no.6
active in the tissues so long as the host cells are active, and when the
plant is forced into dormancy the organism becomes inactive and the
disease is then quiescent.
From the present extent of our knowledge of this disease, it can be
concluded that environmental conditions play an exceedingly important
rdle in the susceptibility and resistance of Citrus plants. Thus, environ-
mental conditions determine to some extent the anatomical structure of
the plant parts attached by canker, by influencing the size and rapidity
of maturation of the new growth and the leaf texture. Apparently, each
species studied has a definite reaction to its environment and differs
from other species in its behavior under a given set of conditions. There-
fore, one should be able to forecast the susceptibility and resistance of a
given plant under certain environmental conditions. Lastly, the in-
fluence of humidity and temperature on the host favors to some extent
the increased or decreased virulency of the organism toward a definite
species. It appears that it will be necessary to study the behavior of
the host plant in its environment before any scientific selection or breed-
ing for disease resistance can be made.
SUMMARY
(1) The temperature relations of Pseudomonas cilri Hasse in culture
are similar to those of the plant-disease bacteria of the Pseudomonas
group. With the time factor included, the minimum temperature for
growth in culture is about 50 C, the optimum between 200 and 300, the
maximum about 35 ° for a period of 24 hours, and a thermal death point
between 490 and 520.
(2) The influence of humidity on the viability of the organism is very
distinct and is closely associated with temperature. At low temperatures,
humidity appears to have little or no influence, while at high temperatures
and high humidities it is the limiting factor. At medium humidities at
all temperatures the organism is viable for the period of the experiment.
Some factor or factors other than the rapidity of drying are responsible
for these results.
(3) The Citrus plants used in the greenhouse experiments vary mark-
edly in their reaction to temperatures and humidity, especially at low
and high temperatures. However, with the time factor included, the
optimum temperature for all the plants used lies between 200 and 300 C.
With some slight variations, the same temperature relations hold in the
field.
(4) Three conditions are essential for infection — the presence of free
moisture on the plant, a suitable temperature, and an actively growing
plant.
(5) The life of the organism in culture and outside the host plant
is ruled by an entirely different set of conditions from those which
Dec. iS, 1920 Effect of Temperature and Humidity on Citrus-Canker 505
control it when it is parasitically active in the host plant. Likewise,
the conditions necessary for initial infection of the plant differ.
(6) The period of initial infection must be clearly distinguished from
the period of incubation and subsequent development of the disease.
(7) The conditions which bring about the most active growth of the
host plant are also responsible for the most rapid development of the
disease.
(8) The difference between host plants in their temperature and
humidity relations, in both the greenhouse and field, is further brought
out in their behavior toward infection and the development of the
disease.
(9) The organism is active in the tissues so long as the host cells are
active, and when the plant is forced into dormancy the organism becomes
inactive and the disease is then quiescent.
(10) Environmental conditions play an exceedingly important role in
the susceptibility and resistance of Citrus plants to canker.
(11) The results indicate that it will be necessary to study the behavior
of the host plant in its environment and its relation to the causal organism
before any scientific selection or breeding for disease resistance can be
made.
LITERATURE CITED
(1) DoidgE, Ethel M.
1916. THE ORIGIN AND CAUSE OP CITRUS-CANKER IN SOUTH AFRICA. Sci. Bui.
Union So. Afr. Dept. Agr. no. 8, 20 p., illus., 10 pi. Literature cited,
p. 18-19.
(2) HassE, Clara H.
191 5. PSEUDOMONAS CITRI, THE CAUSE OP CITRUS-CANKER. A PRELIMINARY
report. In Jour. Agr. Research, v. 4, no. 1, p. 97-100, pi. 9-10.
(3) Harding, H. A., Stewart, F. C, and Prucha, M. J.
1904. VITALITY OF THE CABBAGE BLACK ROT GERM ON CABBAGE SEED.
N. Y. State Agr. Exp. Sta. Bui. 251, p. 175-194, 1 pi.
(4) JEHLE, R. A.
1916. means OF identifying citrus-canker. In Quart. Bui. State Plant
Bd. Fla., v. 1, no. 1, p. 2-10, 12 pi. (partly col.)
(5)-
191 7. CHARACTERISTICS OF CITRUS-CANKER AND OF THE CAUSAL ORGANISM.
In Quart. Bui. State Plant Bd. Fla., v. 1, no. 2, p. 24-27, illus.
(6) MackiE, D. B.
1918. some observations ON citr us-cankER. In Cal. Citrograph, v. 3,
no. 10, p. 231, 244-245.
(7) Peltier, G. L.
1918. susceptibility and resistance to citrus-canker of the wild
relatives, citrus fruits, and hybrids of the genus citrus,
preliminary paper. In Jour. Agr. Research, v. 14, no. 9, p. 337-357.
pi. 50-53. Literature cited, p. 356-357.
506 Journal of Agricultural Research vol. xx, no. 6
(8) Peltier, G. L-, and Neal, D. C.
1918. OVERWINTERING OF THE CITRUS-CANKER ORGANISM IN THE BARK
tissue op hardy citrus hybrids. In Jour. Agr. Research, v. 14,
no. 11, p. 523-524, pi. 58.
(9) and Frederich, W. J.
1920. relative susceptibility to citrus-canker of different species
and hybrids of the genus citrus, including wild relatives.
In Jour. Agr. Research, v. 19, no. 8, p. 339-362, pi. 57-68. Literature
cited, p. 361-362.
(10) Smith, Erwin F.
1911. bacteria in relation To plant diseases, v. 2. Washington, D. C.
(Carnegie Inst. Washington Pub. v. 27, pt. 2.)
(11) Stevens, H. E.
1914. studies of citrus-canker. Fla. Agr. Exp. Sla. Bui. 124, p. 31-43.
fig. 7-11.
(12)
1915. citrus-canker — in. Fla. Agr. Exp. vSta. Bui. 128, 20 p., 6 fig.
(13) 1
1918. report of plant pathologist. In Fla. Agr. Exp. Sta. Rpt. [i9i6]/i7,
p. 66R-75R, illus.
(14) Stevens, Neil E.
1916. A METHOD FOR STUDYING THE HUMIDITY RELATIONS OF FUNGI IN
culture. In Phytopathology, v. 6, no. 6, p. 428-432. Literature
cited, p. 432.
(15) Stirling, Frank.
1914. eradication of citrus-cankek. Fla. Agr. Exp. .Sta. Bui. 124, p. 44-53.
fig. 12-14.
(16) Tanaka, T.
1918. A brief history of the discovery of citrus-canker in japan
and experiments in its control. In Quart. Bui. State Plant Bd.
Fla., v. 3, no. 1, p. 1-15. Bibliographical footnotes.
(17) Wolf, F. A.
1916. citrus canker. In Jour. Agr. Research, v. 6, no. 2, p. 69-100, 8 fig.,
pi. 9-1 1. Literature cited, p. 98-99.
DAUBENTONIA LONGIFOUA (COFFEE BEAN), A
POISONOUS PLANT
By C. Dwight Marsh and A. B. Clawson
Physiologists, Bureau of Animal Industry, United States Department of Agriculture
Daubentonia longifolia, known in some localities as the "coffee bean,"
was first brought to the attention of the Department of Agriculture when,
in February, 191 8, Inspector J. B. Reidy, of Houston, Tex.*, sent in a
sample of the plant and stated that a sheepman who had lost several
hundred sheep thought this plant was the cause. He reported also the
result of a post-mortem examination of one of the animals.
Preliminary experiments showed that the plant is toxic, and further
work has made it clear that it is very poisonous and may be the cause
of considerable losses of live stock.
DESCRIPTION OF THE PLANT
Daubentonia longifolia D. C. (PI. 62), called by some authors Sesbania
cavanillesii Watson, is a shrub or small tree of the pulse family (Legumi-
nosae), which includes the locusts, mesquites, etc. The leaves are alter-
nate and pinnate, with 12 to 60 leaflets, which are oblong and pointed.
The flowers, varying in color from scarlet to yellow, are in racemes which
are shorter than the leaves. The pods are oblong, compressed, with four
wings rising from the margins of the valves and produced beyond the
sutures. The seeds are separated from one another by transverse par-
titions.
The plant is found on sandy soils from Florida to central Texas and as
far north as the northeastern border of Texas. In some places, as in the
lower Rio Grande and San Antonio regions, it is very abundant. In
Houston and vicinity it is common along the roadsides and in waste
places. Farther east it is confined rather closely to the Gulf region.
While this species does not appear to have been considered poisonous —
in fact it is said by Havard l that the seeds have been used for coffee —
it is an interesting fact that at various times some closely related plants
have been said to be poisonous.
EXPERIMENTAL WORK
The experimental work on this plant was done in the summers of 191 8
and 1 91 9. Excluding the animals that received extracts in various forms
and those which were offered the plant and refused to eat, 42 experiments
were made with sheep. Table I gives a summarized statement of these
experiments.
1 Havard, V. report on the flora op western and southern Texas. In Proc. U. S. Nat. Mus.,
v. 8, no. 32, p. 500. 1885.
Journal of Agricultural Research, Vo1- ~sx< No- 6
Washington, D. C Dec- «• I9A20
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(507)
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All the experimental work was done with sheep. The cases differed
somewhat in detail, but on the whole they gave a fairly good picture of
the symptoms and effects. The symptoms were not so marked nor
the effects so striking as in some other forms of plant poisoning.
Sheep No. 533 may be considered as a typical case. She was a ewe
that had been used in another feeding experiment by which no ill effects
were produced. She was in good normal condition and weighed 105
pounds at the time of the Daubentonia experiment.
On July 25, 1919, at 11. 11 a. m. she was given by the balling gun 0.22
pound of ground seed per hundredweight of animal. No symptoms
were noted during the day or during the next morning, but at 3.25 p. m.,
July 26, the pulse was rather rapid (104) and somewhat irregular. Two
hours later it was still more rapid (128) and the sheep showed distinct
depression. At 8.30 p. m. the pulse was 180, and the depression con-
tinued. This general condition continued with little change until 4.15
p. m., July 27, when she was down, groaning with each respiration but
still able to get upon her feet. The pulse was rapid and weak. At
5.12 p. m. the sheep was down, her breathing labored, pulse impercepti-
ble, and temperature 104.80 F. About an hour later, after no marked
change, she kicked a few times and died.
The autopsy showed the heart in diastole, the lungs congested, more
or less inflammation in the fourth stomach, jejunum, ileum, and cecum,
the pancreas congested, and the blood vessels of the brain unusually full.
SYMPTOMS
The symptoms of Daubentonia poisoning are not very characteristic.
In very light cases of poisoning little except depression is noticed. This
is more marked in the severe cases. The pulse is rapid, sometimes
weak and irregular, and the respiration is usually labored. The tem-
perature in some cases was rather high, in one case being 104. 8° F.
This, however, would not be considered as necessarily abnormal. Diar-
rhea was a common symptom and may be considered as characteristic of
Daubentonia poisoning. Death occurred with little or no struggling.
The experimental work showed that, in the animals which recovered,
the depression and diarrhea might continue for several days. In han-
dling sheep poisoned by Daubentonia it is important to recognize this fact
and to know that recovery is likely to be a slow process.
DELAY IN PRODUCTION OF SYMPTOMS
It is somewhat difficult to determine when the first symptoms of
Daubentonia poisoning are exhibited, as much depends on the acuteness
of the observer in detecting changes in the behavior of the animal.
Depression is the first real symptom, and it is not always easy to deter-
mine whether a sheep is slightly depressed. In determining the time
elapsing between the feedings of the plant and the appearance of the
first symptoms the estimate was made very conservatively, and the
Dec. 15, i02c
Daubentonia longifolia
5ii
actual time for toxic effects to appear was probably rather less than the
figures which have been tabulated.
The time elapsing between a single feeding and the appearance of
symptoms is shown in Table II.
Table II. — Time elapsing between single feeding and appearance of symptoms
Sheep No.
Dry weight of
plant eaten per
100 pounds of
animal.
525
528
548
533
55°
Pounds.
O.882
.440
.066
Time before
symptoms
appeared.
Result.
Hours.
8V5
29V2
26V,
20V4
Death.
Do.
Recovery.
Death.
Do.
It is seen that the time varies from 8'/5 hours to 29V2 hours, with an
average of slightly more than 21 hours. Excluding sheep 528, the
average would be nearly 24V2 hours. From the experimental work it
appears that in most cases the symptoms appear in approximately 24
hours.
AUTOPSY FINDINGS
There was a fairly good general agreement in the pictures presented
in the autopsies of the five sheep that died. The heart was generally in
diastole and the lungs were congested. The fourth stomach in all cases
showed more or less congestion. This was true also of the duodenum,
jejunum, ileum, and cecum. Congestion in the colon was noted in only
one case. The spleen and kidneys were congested, and this condition
was found in the pancreas in two cases. The brain and spinal cord
showed an unusual fullness of the blood vessels.
PATHOLOGICAL CHANGES IN TISSUES
In the animals poisoned, degenerative tissue changes occur principally
in lymphoid tissues, smooth muscle, and in the red blood corpuscles.
The more delicate cells of the lymph nodules are almost universally found
to have undergone degeneration. Tissues composed of smooth muscle
fibers are similarly though perhaps not so conspicuously affected. In
the blood stream are many thrombi containing degenerated erythrocytes,
granular material, and often fibrin.
Probably the degenerative changes in the erythrocytes and lymphoid
tissues are the most important causes of the thrombus formation. Small
hemorrhages due to ruptured vessels are not uncommon. Weakening
of the muscle layers of the vessels, together with thrombi in the vessels,
would appear to be a sufficient cause for the rupture of the vessel walls.
Degenerative changes also may occur in various glands, as the kidney
and liver, but they are less severe than those in the tissues described.
512 Journal of Agricultural Research vol. xx, no. 6
TOXIC AND LETHAL DOSES
The smallest dose producing death in the experimental work was that
given to sheep 550, which received o. 11 pound (49.89 gm.) per hundred-
weight of animal. The smallest dose producing symptoms was that
given to sheep 548, 0.066 pound (29.9 gm.) per hundredweight of animal.
Inasmuch as sheep 523 received 0.066 pound (29.9 gm.) per hundred-
weight without effect, it appears that this quantity is about the lowest
limit of toxicity.
Sheep 463 is noted in Table I as receiving on September 25, 1918, 0.928
pound per hundredweight of animal without effect. However, there is
no doubt that its illness on September 26, followed by death, was really
the result of the feeding of September 25, for, as is shown elsewhere,
the toxic symptoms ordinarily do not appear until about 24 hours after
the feeding.
CUMULATIVE EFFECT
The experiments show clearly that the toxic substance of Daubentonia
is excreted very slowly, so that poisoning may result from repeated
administration of quantities somewhat below the toxic dosage. Sheep
520 and 518 received three doses each of 0.044 pound (19.95 gm-) Per
hundredweight of animal, administered on alternate days. These
doses produced illness in both cases. Since the smallest single dose
producing illness was 0.066 pound (29.9 gm.) per hundredweight, it is
evident that there was a cumulative effect in these animals.
In this connection it should be noted that sheep 372 received on alter-
nate days from July 31 to August 20, 0.022 pound (9.9 gm.) per hundred-
weight with no bad results.
COMPARATIVE TOXICITY OF PARTS OF THE PLANT
Only two experiments were made in feeding dry leaves. In sheep
556, mild symptoms were produced by 0.661 pound per hundredweight
of animal. This indicated a much lower toxicity than that in the seeds.
The experimental work with extracts on guinea pigs showed that the
toxicity was also present in the dry pods. The experiments of feeding
pods to the sheep, however, were entirely negative, although as much as
1.653 pounds (716 gm.) per hundredweight was fed. It is evident that,
as compared with the seeds, the pods are only slightly toxic and are
not likely to cause any damage to live stock.
ANIMALS AFFECTED BY THE PLANT
Dr. Reidy's report was in regard to the loss of sheep, and the experi-
mental work of the department has confirmed the toxicity of Dauben-
tonia for these animals. Dr. Dwight H. Bennett, of the Texas Agricultural
Experiment Station, has reported a case of the loss of 500 goats which
were probably killed by this plant. At the present time there is no
Dec. I5, 1920 Daubentonia longijolia 513
experimental evidence of its effect on cattle and horses, but certainly
it would be wise for stockmen to be very cautious about letting any
domestic animals feed largely upon the fruit of the plant.
TREATMENT AND PREVENTION
No suggestions can be made for treatment other than that which
would be indicated for most forms of plant poisoning. Doubtless the
administration of laxatives or purgatives like linseed oil or Epsom salt
would be helpful. Reliance should be placed upon prevention rather than
treatment. If the plant is recognized as dangerous, stock can, with
proper care, be kept from eating any considerable quantity of it. As
with other poisonous plants, it is unlikely that animals eat it from choice,
and they are not likely to take a quantity sufficient to produce bad
results except when there is a lack of suitable forage.
So far as present knowledge goes, it appears that cases of poisoning may
occur in the winter when stock, because of scarcity of other forage, are
induced to eat the pods and seeds. It is at such times that animals will
seize upon anything that can be eaten.
The peculiar form of the pods makes it possible for anyone to recognize
the plant without difficulty, and the careful and observant stockman
should be able to avoid any large losses.
16917°— 20 8
PLATE 62
Herbarium specimen of Daubentonia longifolio, showing flowers, leaves, and pods.
(5i4)
Daubentonia longifolia
Plate 62
Journal of Agricultural Research
Vol. XX, No. 6
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OF THIS PUBLICATION MAY BE PKOCTJBED FROM
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Vol. XX JANUARY 3, 1921 No. 7
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Page
Fusarium-Wilt of Tobacco ------ 515
JAMES JOHNSON
(Contribution from Bureau of Plant Industry and Wisconsin Agricultural
Experiment Station)
Sugar Beet Top Silage ------- 537
RAY E. NEIDIG
(Contribution from Idaho Agricultural Experiment Station)
Nodule Bacteria of Leguminous Plants - 543
F. LOHNIS and ROY HANSEN
( Contribution from Bureau of Plant Industry and Illinois
Agricultural Experiment Station)
Correlation and Causation - - - - - 557
SEWALL WRIGHT
(Contribution from Bureau of Animal Industry)
Measurement of the Amount of Water That Seeds Cause
to Become Unfree and Their Water-Soluble Material - 587
GEORGE J. BOUYOUCOS and M. M. McCOOL
(Contribution from Michigan Agricultural Experiment Station)
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WASHINGTON : GOVERNMENT PRINTING OFFICE i 1(31
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, OMce of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Expert'
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of lite University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director;
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
LfRRAR*
JOURNAL OF ACRICET1ML RESEARCH
Vol. XX Washington, D. C, January 3, 1921 No. 7
FUSARIUM-WILT OF TOBACCO1
By James Johnson
Associate Professor of Horticulture, University of Wisconsin, and Agent, Office of
Tobacco Investigations, Bureau ■ of Plant Industry, United States Department of
Agriculture
INTRODUCTION
During the summer of 191 6 the writer's attention was called to a wilt
disease of tobacco occurring near Benedict, Charles Co., Md. The dis-
ease occurred on the Maryland Broadleaf variety of tobacco, which was
nearing maturity, and showed all the appearances of a typical wilt dis-
ease. Plants in all stages of wilting were found, from those showing the
first signs of infection to those in which all the tissues of the plant were
dead. When the stalks or midribs of the leaves were cut, the fibro-
vascular bundles were found to have a distinctly brown to black color in
place of the normal white. It was at first suspected that the bacterial
wilt due to Bacillus solanacearum Erw. Smith had been introduced into
the Maryland tobacco fields. Although the general symptoms of the
disease were very similar to those of bacterial wilt, the absence of bacterial
ooze, the uniform occurrence of Fusarium on plated out material, the
absence of vessels filled with bacteria, and the presence of fungus strands
in the vessels gave strong evidence that bacteria were not concerned.
Considerable difficulty was at first encountered in getting good infection
with the Fusarium isolated. When artificial infection was finally secured,
however, further study of this disease became of special interest, since no
Fusarium-wilt disease of tobacco has apparently been proved to exist,
although, as will be shown, in one case it seemingly had been reported
erroneously, and in another case a Fusarium disease, apparently not a
wilt, has been described. The present paper is intended primarily to
establish the occurrence of a Fusarium-wilt of tobacco, with a description
of the causal organism and a discussion of certain matters bearing on the
control of the disease under practical conditions.
1 Cooperative investigations of the Office of Tobacco Investigations, Bureau of Plant Industry, United
States Department of Agriculture, and the Wisconsin Agricultural Experiment Station.
Published with the permission of the Director of the Wisconsin Agricultural Experiment Station.
Journal of Agricultural Research, Vol. XX, No. 7
Washington, D.C. Jan. 3, 1921
we Key No. G-214
^ (515)
CO
516 Journal of Agricultural Research vol. xx.No. 7
OCCURRENCE OF THE DISEASE
In the summer of 191 6 the disease was found only on the plantation of
Mr. James H. Boiling near Benedict, Charles Co., Md. It was serious in
only one field of about 6 acres on this farm, where perhaps 10 to 20 per
cent of the plants were dead or showed symptoms of the disease, although
in smaller areas in the field it is estimated that 50 to 75 per cent of the
plants were damaged (PI. 63, A). According to Mr. Boiling and the
tenant on the farm this disease had occurred at intervals for many years
on this farm but not so seriously as in 1916.
During the summer of 19 17 Charles County was again visted, with the
result that the disease was found on two other farms near Newport, Md.
The disease was not apparently so serious this season as in the previous
one. This region was not visited during the seasons of 191 8 and 1919,
and nothing further is known of the disease in that section.
In the summer of 191 9 a "new" disease of tobacco was called to the
writer's attention by correspondence from Clermont Co., Ohio, and speci-
mens were received through the courtesy of Mr. David Geesner of Owens-
ville on September 20, which showed typical symptoms of Fusarium-wilt
on mature plants of the White Burley variety. Sixty-six pieces
from diseased portions were plated out, practically all of which yielded
Fusarium, from which artificial infection was later secured. The disease
is also said to have occurred previously in the vicinity of Owensville.
The symptoms of the disease are so evident that growers could not
fail to note and report its occurrence. On account of the scarcity of
such reports either from the farmers or experiment station workers in
the tobacco-growing regions outside of the Granville (bacterial) wilt areas
it is believed that the Fusarium-wilt is not a serious disease and probably
will never become of great economic importance. If, however, it becomes
more generally introduced into the White Burley districts it may become
a serious parasite, since this variety, as will be shown, is very suscepti-
ble to the wilt.1 In North and South Carolina, Georgia, and Florida
where the Granville wilt occurs, it is possible that the Fusarium-wilt is
also present, but growers as well as plant pathologists would be likely to
report such cases as Granville wilt unless a special examination of the
diseased tissue were made. It is not believed that there is much danger
that this disease will become serious in the northern cigar tobacco growing
regions on account of the resistance of the varieties grown and the climatic
conditions prevailing.
REVIEW OF THE LITERATURE
The occurrence of Fusarium-wilt diseases of a considerable number of
plants are now reported in literature. The Fusarium-wilt of tobacco
possesses much in common with these diseases in that it is a vascular
disease. However, it is not proposed here to enter into a review and
1 During the summer of 1920 specimens of Fusarium-wilt were received from the White Burley dis-
trict of Kentucky.
Jan. 3,1921 Fusarium-Wilt of Tobacco 517
comparison of these diseases. The Fusarium problem viewed as a whole
or even as that part which has to do with the nomenclature of the vascular
parasites, is recognized as being in a rather unsatisfactory state. Rather
uncertain precedent in naming forms, together with the plasticity in
physiology, and, one is tempted to say, in morphology of the forms
themselves, is the cause of the greatest difficulties encountered in this
problem. It is felt, therefore, that until a more detailed study of the
Fusaria causing wilt of tobacco and related plants can be made, it will
not be profitable to enter upon a review preliminary to discussion of this
subject. The review here presented, therefore, includes only the evi-
dence which we now have relating to Fusarium as a probable cause of
disease in the tobacco plant.
McKenney (7)1 in 1903 described a wilt disease of tobacco in North
Carolina as due to Fusarium. No proof of pathogenicity was obtained.
This disease was soon afterward studied by Stevens and Sackett (11)
and by Smith (10, p. 220-271) and was found to be a bacterial wilt
(Bacillus solanacearum), so that Fusarium could no longer be associated
with the disease. According to Smith no good evidence for a Fusarium-
wilt existed; but, reasoning from the universal distribution of Fusarium
and its occurrence as a vascular parasite in plants closely related to
tobacco, he predicted that a Fusarium-wilt of tobacco would be found.
Judging from the description of McKenney's disease and the virulence
attributed to it, the writer believes it could not have been Fusarium-wilt.
Lounsbury (6) in 1906 reports a wilt disease of tobacco in the Kat
River Valley, Cape of Good Hope, which he states is, in his opinion,
not similar to the American (Granville) wilt. Bacteria, fungi, and
insects are all said to be concerned. Smith (10, p. 220-271) places it as a
doubtful bacterial wilt. To judge from the description, this may have
been a Fusarium-wilt disease, at least the South African disease should
again be checked up, if it still occurs.
Petch (9) in 1907 reported a disease of tobacco in Dumbara, Ceylon,
which is said to be a "root-disease" causing "sudden and premature
ripening," killing out plants in patches. The stem is said to be dis-
colored at the base. A Fusarium was isolated from the roots. This
description may fit one or more diseases of tobacco. The isolation of a
Fusarium from the roots is, of course, of no significance. The "'sudden
and premature ripening in patches," however, suggests a wilt disease.
Delacroix (2) in 1906 reported a disease of tobacco occurring around
Perigneux and Razoc, France, as due to a species of Fusarium which he
named Fusarium tabacivorum. The disease is said to resemble super-
ficially a bacterial cancer localized at the collar of the plant, and the
port of entry of the parasite is believed to be always an insect puncture.
The mycelium of the fungus was found to be present throughout the whole
1 Reference is made by number (italic) to "Literature cited," p. 534-535.
51 8 Journal of Agricultural Research vol. xx, no. 7
base of the stalk when the disease was well established. The fungus is
said to lose its virulence in culture after the "first generation." The
conidia are described as straight or slightly curved, round obtuse at both
extremities, possessing usually three septa, their size varying from 25 to
35 microns by 4 to 6 microns.
Delacroix's Fusarium disease is probably not a true wilt disease, since
it is not described as such. The description and illustration of the
causal organism are, furthermore, too fragmentary and unsatisfactory to
permit of comparison. The new species created (Fusarium iabacivorum
Delac.) has apparently not been credited by any recent workers with the
Fusaria. It is interesting to note that Delacroix knew of McKenney's
Fusarium disease but could not say whether his disease was identical
with it or not. In view of the fact that Delacroix's description may fit
other diseases of tobacco as far as symptoms are concerned, and since we
have only the statement that infection has been secured with an organism
of such universal occurrence as Fusarium, together with the unreliable
description of the causal organism, it is difficult to see how at the present
time we can accept either the disease as such or the species described
as authentic.
A brief abstract was published by the writer in 1918 (4) calling atten-
tion to the wilt disease in Maryland and giving reasons for believing it
was due to Fusarium, although artificial infection had not been secured
at that time.
SYMPTOMS OF THE DISEASE
The symptoms of the disease may first become evident upon very
young or on nearly mature plants. Under the field conditions observed
it is evident that plants may succumb at any stage in their growth, al-
though it is not clear as to what time the original infection of the plant
occurs. It seems probable that infection may take place at any time,
but that it is more likely to occur when the plants are young, the parasite
remaining in a more or less latent stage until favorable environmental
conditions for the further development of disease occur. In full-grown
plants the earliest symptoms seem to be the sudden wilting of only one
or more leaves on the plant, accompanied by yellowing and finally brown-
ing and death, but not decay of the leaf. In some cases this symptom
at first may be localized on only one side of the leaf. At other times all
the leaves in a narrow vertical band, comprising about one-fourth or one-
eighth of the leaves of the plant, may become wilted while the others
remain apparently free from the disease (Pi. 64, A). If the stalks of
such plants are cut, it will be found that the discolored bundles are con-
fined to only a part of the circumference of the vascular ring. All de-
grees of wilting from those described, to complete collapse of all the
leaves on the plant, however, may occur (Pi. 64, B). If the plants are
pulled up, large or small dead roots may be found, while others are appar-
jan. 3,1921 Fusarium-Wilt 0} Tobacco 519
ently healthy. If, now, the diseased stalk, roots, suckers, midribs, or
veins of the leaves are cut either in cross section or longitudinally (PI.
65, B) the vascular system will be found to be brown or distinctly black,
but upon pressure no "ooze" appears. The vascular decay is distinctly
"dry."
On young plants in the greenhouse where the writer has had an oppor-
tunity to note the symptoms of the disease more carefully th'ey are essen-
tially the same so far as the vascular system is concerned, but the leaves
first lose their chlorophyll, becoming yellow and somewhat wrinkled but
distinctly turgid and "brittle," as compared with healthy leaves. This
condition may obtain for some time previous to wilting unless excep-
tionally high transpiration occurs. The leaves, of course, finally dry up
as they do in the field (PI. 63, C). In the greenhouse the symptoms are
most likely to appear first on the youngest leaves, and this may be
more or less characteristic in the field.
So far as has been noted the parasite is not able to cause any rotting
of the living parenchymatous tissues of the plant. In heavily infested
soil where the cortical layers of the plant have been severely wounded or
a leaf petiole has been broken off below the surface of the soil, the para-
site may enter the vascular system readily and cause the death of the
aerial portion without in any way affecting the parenchyma of the stem
or roots at or below the surface of the soil.
Histological studies of the disease were made by various methods,
but best results were secured by killing and fixing young tissue in Gilson's
fixative, imbedding in paraffin, and staining with the Pianese stain, as
described by Vaughan (12). Transverse sections of infected stems or
midribs of leaves (PI. 66, A) showed that all the vessels in local areas of
the vascular ring were more or less invaded, sometimes almost completely
"clogged" with mycelium. Longitudinal sections (PI. 66, B) showed in
an even more striking manner the general occurrence and the "bunch-
ing" of mycelium in the vessels. Nevertheless, from the behavior of the
diseased plants, especially with regard to yellowing and early turgidity,
it is not believed that death of the plants is due to clogging of the vessels
but rather to toxic materials formed by the parasite or as a result of
the parasitic action on the host.
ISOLATION AND INFECTION EXPERIMENTS
In the first isolations pieces of the discolored portions of the stem,
together with some surrounding healthy tissue, were cut out and treated
with 1 to 1,000 mercuric chlorid for 30 to 120 seconds, rinsed in sterile
water, and placed on hard potato agar in Petri dishes. Growth of fun-
gus mycelium from the diseased tissue was slow and not uniform. Pure
cultures of Fusarium, however, were secured. Isolations were later
made by cutting off the cortical layers with a hot blade and cutting out
520 Journal of Agricultural Research voi.xx.No. 7
fairlv large pieces under as sterile conditions as possible, rinsing these
through 5 to 10 sterile water blanks, transferring to a sterile Petri dish,
where they were further cut up into small pieces and transferred to 10 cc.
of potato agar in a Petri dish acidified with two to three drops of 25
per cent lactic acid. Out of hundreds of pieces plated out in this manner
apparently pure cultures of the causal organism were rapidly secured in
practically all cases. Mercuric chlorid treatment apparently resulted
in part of the chlorid entering the bundles, from which it was not readily
washed out, and consequently did not prove useful for plating out in
this case.
Single spore isolations were made from the Maryland Fusarium, and
these have been used in some but not in all infection experiments, cul-
tural studies, and spore measurements.
Infection experiments during the summer of 1917 consisted chiefly
in inoculating large plants in the field with pure cultures of the Fusarium
through wounds in the stalk. No infection was secured except in one
instance which was questionable. In the fall of 1917 sterilized soil was
inoculated with mycelium from pure cultures of the wilt Fusarium, and
very young White Burley tobacco plants were transplanted into it.
After about five weeks several of the plants wilted and died. Infection
thereafter was intermittently secured on White Burley through the
medium of the soil. The inoculum was usually grown on a mixture of
100 gm. of sand, 10 gm. of corn meal, and about 1 gm. of glucose to 50 cc.
of water in i-pint milk bottles or mason jars. This culture medium
was cooked for one hour in the autoclave, then stirred up so as to render
the medium "spongy," and again sterilized. After being cooled, the
medium was inoculated with the Fusarium and incubated at 250 to 300
C. for four or five weeks, after which the inoculum was allowed to dry
sufficiently to permit pulverizing, when it was thoroughly mixed with the
soil. Good infection was also secured from mycelium and spores directly
from potato agar tubes and also from a suspension of conidia alone.
The latter method was usually not so successful as the former. Failure
to secure as high precentage of infection at one time as at another led to
a preliminary study of environmental and other conditions favoring the
disease, and these will be reported upon briefly in this paper. It should
be stated here, however, that as soon as the plants were intentionally
wounded more uniform results in infection were secured. Ordinarily
this consisted simply in pulling or pinching off one or two of the lower
leaves and setting the plant deep enough to bring the resulting wound
below the surface of the soil. Although it can not be said with certainty
that infection would never occur in a plant perfectly free from wounds of
any sort on the root or stem, it is quite certain that infection is
greatly enhanced by wounding. The first signs of infection on leaves
have been secured in as short a time as two days after exposing wounded
stems to heavily infested soil.
Jan. 3. 1921
Fusarium-Wilt of Tobacco
521
CAUSAL ORGANISM
The causal organism can be readily isolated from diseased tissue by-
plating out on acid potato agar. The mycelium ordinarily imparts only
a dull pinkish tinge to the substratum and seemingly has a more or less
characteristically sparse growth and "powdery" surface (PI. 65, A) as
compared with the dense cottony development of some forms of Fusarium.
The "powdery" appearance is due to microconidia which are formed in
abundance, as is characteristic on a number of other media where
similar growth is made. "Strains" bearing sporodochia may or may
not occur. Where fruiting "strains" have been secured sporodochia
have usually been produced in abundance, especially on Melilotus stems,
oatmeal agar, and occasionally on potato agar and other media. True
Fig. 1. — Camera-lucida drawings of spore forms of Fusarium ozysporum var. nicotianae, n. var: A, macro-
conidia; B, microconidia; C, chlamydospores; D, conidiophore of the sporodochial stage.
pionnotes have not been observed in the cultures during a period of
four years on various kinds of media. " Pseudo pionnotes " or reduced
pionnotes could, however, be made to appear. Blue sclerotia and some-
times salmon-colored sclerotia are produced.
An examination of the conidia from well-developed sporodochia of the
Maryland strain ordinarily shows a preponderance of 3-septate conidia,
which, together with the shape and size of the spores (fig. 1) and the fact
that the fungus produces a wilt disease, placed it readily in the section
Elegans, according to Wollenweber's classification (13). A more careful
study of the size and shape of the conidia brings out a close resem-
blance to Fusarium oxysporum Schlecht., according to recent descrip-
tions of this species. Studies were therefore undertaken to establish
whether the tobacco-wilt Fusarium is identical with Fusarium oxysporum
522 Journal of Agricultural Research vol. xx, No. 7
as described. After the conclusion was reached that the tobacco-wilt
Fusarium is related morphologically to Fusarium oxysporum but is not
identical with it, several methods of study were undertaken with the
hope of furnishing further evidence. These consisted of (1) infection
experiments with the tobacco-wilt Fusarium on the potato and certain
other plants, (2) comparative cultural studies with Fusarium oxysporum
strains secured from other sources, and (3) infection experiments with
strains of Fusarium oxysporum from potato upon tobacco.
Several attempts at producing infection with the Maryland strain
of the tobacco-wilt Fusarium on the potato vine failed. Potatoes were
grown in artificially infested soil, and in several instances the stems were
wounded immediately below the surface of the soil. This, however, is not
regarded as conclusive evidence that infection is unobtainable.
Authentic cultures of Fusarium oxysporum were sought from various
recent workers on this species. Cultures of Dr. Wollenweber's strain
(No. 207) were received through Dr. W. A. Orton, and also a strain
(No. 208) isolated by Dr. H. A. Edson from potatoes. From Minnesota,
Bisby's culture No. 3315 and a reisolation from inoculation on potato
were secured (j). From the stock cultures in the Department of Plant
Pathology at the University of Wisconsin two strains were received,
numbered 226 and 227, both apparently originally from Dr. Wollenweber
to Link at Nebraska and thence to Goss at Michigan, who brought them
to Wisconsin. Finally a culture of MacMillan's strain (8) from potatoes
in Colorado, which was sent by MacMillan to the Department of Plant
Pathology at Wisconsin, was obtained. None of these cultures was
apparently in a good normal growing condition when transferred to my
media, as compared with more recently isolated forms on the same
media. The growth may be best expressed as "slimy" in nature, as if
bacterial contamination had occurred — that is, aerial growth was sparse
or absent and a rather thin mycelial growth was formed on the surface
of the media only. Many microconidia and some macroconidia were
produced. Repeated trials on various media failed to bring about the
sporodochial fruiting stage, without which a satisfactory comparison
with the septate conidia of the tobacco-wilt Fusarium could not be made.
Therefore, the cultures were at first used largely for comparison of cul-
tural characteristics on different media, especially on Melilotus stems,
cooked rice, oatmeal agar, potato plugs, and potato agar. The various
strains of Fusarium oxysporum from the various sources did not behave
in a similar manner on the same media, and consequently it was felt that
the significance of the cultural comparisons obtained was much reduced.
Whether this condition was due to differences in age or condition of the
strains or to actual physiological differences inherent in the strains can
not be said.
The following notes were taken on the growth of the tobacco-wilt
Fusarium on various media in an early trial. Not much emphasis can be
Jan. 3,i92i Fusarium-W ilt of Tobacco 523
placed on the shade of the pigment given, because comparisons were not
made with Ridgeway's color standards and nomenclature at this time.
Acid potato agar. — Good but rather light and "fluffy" aerial growth,
pure white, no agar coloration to a pale pink coloration, and formation
of blue-green sclerotial masses at margin of agar in older cultures.
Potato plug. — Excellent growth, mycelium becoming faintly salmon-
colored and plug deep blue in parts; abundant formation of small bluish
black sclerotia in older cultures.
Oatmeal agar. — Good growth, pale salmon-colored mycelium, medium
changing to pale lilac. Large sclerotial masses form at base of agar.
Rice. — Good growth of white to pink mycelium.
MELiLOTus stem. — Fair growth of white to pink mycelium. Sporo-
dochia formed abundantly after 15 to 30 days. Sporodochia forming
singly or in large masses. Pale to deep salmon in color. Abundant
production of small bluish black sclerotia.
String-bean plug. — Excellent growth with production of lilac-
colored mycelium.
Carrot plug. — Good growth with faint lilac coloration.
Lima-bean agar. — Fair to poor growth only, hardly any pigment
production.
Corn-meal agar. — Poor growth, practically no pigment production.
Synthetic agar. — Good growth of white mycelium.
Gelatin (BEEF). — Fair growth with some liquefaction.
Tobacco agar (from green leaves). — Very poor growth.
The cultural differences between the various strains of Fusarium
oxysporum used and those of the tobacco-wilt Fusarium are not believed
to be of sufficient importance to warrant presentation in detail, and only
the more striking differences will be mentioned. On cooked rice the
pigment of MacMillan's F. oxysporum was uniformly of a deeper color,
appearing usually as a blue violet to jasper red as compared with light or
shell pink with the tobacco- wilt Fusarium. The same was more or less
characteristic on oatmeal agar, while on the other media pigment differ-
ences were insignificant. A fairly striking difference appeared with
respect to the formation of sclerotial masses which came on early and
in abundance on potato plugs with the tobacco-wilt Fusarium but only
slowly or not at all with the F. oxysporum strains on hand. Sporo-
dochia were also produced in abundance with the tobacco-wilt Fusarium
on Melilotus stems but did not appear in any of the F. oxysporum strains,
although they had, no doubt, occurred previously in these strains. In
the absence of sporodochia in the cultures of F. oxysporum a satisfactory
detailed comparison from a morphological standpoint could not, of
course, be made. On the basis of certain morphological and cultural dif-
ferences— that is, pigmentation and sclerotial formation, together with the
failure to obtain wilt of the potato, it was at first believed that we were
524 Journal of Agricultural Research voLxx.no. 7
dealing with a form on tobacco sufficiently distinct from F. oxysporum
to warrant the creation of a new species. These conclusions were upset,
at least for the time being, by the appearance of signs of wilt in one
plant of the White Burley tobacco, out of six or eight planted, in soil
inoculated with MacMillan's F. oxysporum. Several pots of soil were
now prepared in December, 191 9, and were again infested with several
strains of F. oxysporum in comparison with my own strains secured from
Maryland and Ohio, one of them being a 191 6 isolation of the tobacco-
wilt Fusarium which had been transferred from an old, dried culture.
Good infection (about 80 per cent) was obtained with the tobacco-wilt
strains and with MacMillan's strain but not with Bisby's strains (cultures
in better growing condition than MacMillan's) nor with Wollenweber's
strains (cultures in poorer condition than MacMillan's strain). Mac-
Millan's strain did not, however, prove as virulent as the strains from
tobacco, and the symptoms were not identical — that is, the leaves did
not uniformly lose their color but presented more of a mottled appear-
ance in the early stages of the disease, and the vascular system was not
so distinctly discolored. On plating out the stem and midrib of the
infested plants from MacMillan's strain in comparison with the others,
the characteristic "sub-normal" condition of MacMillan's strain re-
appeared, showing that the strains producing the disease were the ones
inoculated into the soil. A third series of inoculations was made, using
all the strains of F. oxysporum at hand. Infection was again obtained
with MacMillan's strain and with two of Wollenweber's original strains
but not with the others.
In view of these results it appears that strains of Fusarium oxysporum
may vary considerably as regards pathogenicity, but whether this is a
true strain difference or merely one resulting from culturing can not be
stated. It was evident, however, that the tobacco-wilt Fusarium had
not suffered any loss in virulence from four years in culture, existing for
a large part of this time under unfavorable cultural conditions. If F.
oxysporum is as common in potato fields as a parasite and as common a
soil saprophyte as literature would lead us to believe, it is quite surpris-
ing to us that wilt of tobacco has not been more generally noted, provided
we assume the tobacco-wilt may be caused by F. oxysporum, since
tobacco and potatoes are frequently grown in close proximity and are
frequently rotated. This would be even more surprising when we add
that certain varieties of tobacco are apparently more susceptible to the
wilt than is the potato.
As has been stated, no infection has been secured on potato with the
tobacco-wilt Fusarium, although this may sometime be accomplished.
In the early work attempts were also made to get infection on tomato,
cowpeas, and cabbage, but without results. Excellent infection has,
however, been secured upon Nicotiana glauca (California tree-tobacco)
Jan. 3,1921 Fusarium-Wilt of Tobacco 525
which is very dissimilar to ordinary tobacco. N. glauca has, in fact,
been the most susceptible plant to tobacco-wilt with which we have
worked. Infection has also been secured upon N. rustica.
On the basis of this study of the tobacco- wilt Fusarium, it is believed
that although Fusarium oxysporum from potato is to be regarded as
being able to cause a wilt of tobacco, it is not to be regarded as identi-
cal with the tobacco- wilt Fusarium as regards pathogenicity. That
certain cultural differences exist has also been indicated. The final
justification for placing the tobacco-wilt Fusarium as a variety of F.
oxysporum lies in the small but nevertheless significant morphological
differences which have been found to exist. These morphological
differences are to be found in the somewhat larger conidia but more
particularly in the higher percentages of 4- and 5-septate conidia.
One Fusarium has already been described on tobacco — Fusarium
tabacivorum, Delac. — and although this species can not be regarded as
authentic, it is thought best not to confuse the nomenclature by deriving
the variety name from the specific name of tobacco. Furthermore the
tobacco-wilt Fusarium is not limited to Nicotiana tabacum alone but
attacks other members of this genus. It is therefore proposed to derive
the third member of the trinomial from the generic name of tobacco.
Accordingly the name Fusarium oxysporum (Schlecht.) Wr. var. nicotianae,
n. var. is proposed. The following description is presented.
Fusarium oxysporum (Schlecht.) Wr. var. nicotianae, n. var.
Fusarium nicotianae isolated from wilting tobacco plants {Nicotiana tabacum L.)
from Maryland and Ohio agrees quite closely morphologically with Wollenweber's
diagnosis of F. oxysporum (Schlecht.) Wr. except in certain details not readily de-
termined. Mycelium on most media pure white to a light pinkish tinge, of a rather
"powdery" appearance, due to presence of numerous microconidia. Blue and light
ochraceous salmon-colored sclerotia formed early on steamed potatoes. No true
pionnotes observed. Reduced pionnotes or " pseudopionnotes " obtained. Sporo-
dochia produced in abundance on Melilotus stems and on oatmeal agar. These are
salmon-colored and when "normal" contain almost entirely 3- to 5-septate conidia,
slightly larger than those of F. oxysporum. Three-septate conidia up to 100 per cent
34.9 by 4.2 microns (25.0 by 3.7 microns to 45.4 by 4.6 microns). Four-septate up to
40 per cent, '39.3 by 4.0 microns (29.6 by 3.7 microns to 46.3 by 4.6 microns). Five-
septate up to 18 per cent 44.3 by 4.0 microns (38.9 by 3.7 microns to 51.1 by 4.1 mi-
crons). Six-septate very rare. Non-septate conidia in old sporodochia rare (7.1 by
2.4 microns). One-septate equally rare (10. 1 by 2.7 microns). Two-septate up to
4 per cent (18.5 by 3.7 microns). Non-septate spores from mycelium 8.1 by 3.4 mi-
crons (10.2 by 3.7 microns to 3.7 by 2.7 microns). Chlamydospores terminal, inter-
calary and conidial, smooth, round, frequently in masses 8.2 microns (6 to 10.2
microns).
Pigment production not so deep as in most descriptions of Fusarium oxysporum.
Habitat. — Parasitic in fibro-vascular bundles of Nicotiana tabacum in Maryland
and Ohio, causing a decided wilting of plants followed by death. Also produces a
similar disease of N. glauca and N. rustica by artificial inoculation.
cj26 Journal of Agricultural Research voi.xx.No. 7
CONDITIONS INFLUENCING THE DISEASE
A thorough study of the environmental conditions influencing the
occurrence and extent of injury by the Fusarium-wilt disease has not
been undertaken. Some evidence has been obtained, however, through
experimental work and observation which is of interest in this con-
nection. The progress of experimental work along this line was inter-
fered with by the difficulty of obtaining a high percentage of infection
even under favorable conditions, so that a considerable number of plants
would have to be grown to obtain good data. This very fact should in
itself stimulate further research along this line, since it is evidence that
the environmental conditions most conducive to parasitism are not fully
understood. Where a number of factors are involved, however, this
subject becomes exceedingly complex. The introduction of such a factor
as wounding, which may occur "naturally" or may vary in considerable
degree when produced artificially, is a complicating factor in the tobacco-
wilt disease, which in some respects renders it unfavorable for such a
study.
The evidence for the conclusions presented in this paper will not be
given in detail. The methods of investigation were essentially the same
as those which were used in a study of the influence of soil environment on
the rootrot of tobacco as described by Johnson and Hartman (5). The
soil used was, however, artificially infested from pure cultures following
steam sterilization. The object of the soil sterilization has been partly
to secure better infestation of the soil following inoculation. In practi-
callv all cases the inoculum has been grown on 100 parts sand, 10 parts
of corn meal, and 1 part of glucose to 50 parts of water. A heavy
growth of mycelium and an abundance of spores on this medium un-
doubtedly suffice to inoculate the soil thoroughly, as is shown by instances
in which "100 per cent infection" occurs (Pi. 63, B. C). Where a uni-
form infestation of the soil is not required, rapid infection can be secured
by using conidia and mycelium from ordinary cultures placed in the soil
about the wounded stems.
As will be shown later, the White Burley variety of tobacco was
found to be the most susceptible to the Fusarium-wilt disease; therefore,
this variety was used in all cases in the environmental studies. The
use of other more resistant varieties would have rendered the securing
of results far more difficult. It is assumed, however, that the same
relative results would have been secured with the more resistant
varieties.
The seedlings were in all cases transplanted into the infested soil from
steam-sterilized soil. The root systems especially were therefore in all
cases more or less wounded in their removal from the soil. Although
infection has been observed in seedlings not transplanted, it is quite
certain that the tobacco-wilt organism is largely dependent upon wounded
Jan. 3, i9=i Fusarium-W lit of Tobacco 527
host tissue for initial infection. However this may be, it was found
that wounding the plant greatly increased the possibilities of infection,
and in some of the later experiments the plants were all wounded,
usually by pinching or pulling off two basal leaves, in addition to the
"natural" wounding resulting from transplanting.
Soil temperature. — Four series of experiments were run in the soil
temperature control "tanks" during the winters of 191 8-1 9 and 1919-20
in a manner similar to that which has been described for the Thielavia
rootrot studies (5). Two plants in uninfested soil and two in infested
soil were grown at each temperature. The temperatures usually ranged
from 150 to 380 C, with intervals of 2° — that is, 12 different temperatures
were used. The results in two of the trials were not convincing on
account of a low percentage of infection, although the later results were
approximated. In one series wilt occurred only at the approximate
temperatures of 280 and 32 ° and not at the intermediate temperature
used. In the other case, wilt occurred only at 260 to 270 and 300 to 320.
These results can only be said to indicate roughly that the higher soil
temperatures are more favorable than the lower temperatures.
In the third experiment, however, more uniform infection was secured.
Signs of disease were first evident at 280 to 290 and 250 to 260 C, and
these were soon followed by disease at 260 to 270, 300 to 31 °, 230 to 240,
and 2i° to 220. Eighteen days later all plants in the infested soil were
dead at all temperatures between 210 to 22 ° and 300 to 310 and also at
320 to 330. One plant was dead and one diseased at 310 to 320, 340 to
350, 190 to 200, and 17° to 180, and two were slightly diseased at 150 to
1 6°. The most favorable temperature for infection and progress of the
wilt appeared to be between 250 and 300, but it seemed evident that a
wide range of temperature existed within which the disease could occur.
The surface soil of the pots in the first three experiments was not
insulated, though it should be, particularly in diseases of this sort where
the parasite is systemic; therefore, it is quite likely that infection may
have occurred near the surface where for short periods the temperature
varied considerably from those given, particularly at temperatures
above 300 C. Difficulties are encountered in controlling soil tempera-
tures sufficiently accurately at all points in the soil containers in dealing
with a systemic disease, though these difficulties do not play so large
a role in cases where a parasite is limited entirely to subterranean parts.
In a fourth test, using soil temperatures 30 apart, in which special attempts
were made to keep the temperature at the surface of the soil constant
by means of glass covers and shading of the jars in the tanks, wilt
occurred first and most abundantly at 300 to 31 °, and no wilting occurred
at 1 30 to 14° or at 350 to 360. We feel confident in concluding from
the results of these experiments that the optimum temperature for the
disease lies between 2 8° and 310 — that is, the Fusarium-wilt organism is
528 Journal of Agricultural Research voi.xx, No. 7
a warm-weather parasite, and at lower temperatures the likelihood of
its occurrence is diminished. (PI. 67, I and II.)
It is significant that the optimum for the growth of the wilt Fusarium
in culture was also found to be between 280 to 300 C. Growth was
very slow at io°, and no growth was obtained at 70 and 35 °. Since no
growth occurred in culture at 350, it is seemingly quite evident that no
infection should occur above 350 and that chances of infection probably
are considerably reduced before that temperature is reached.
It may now be recalled that the Fusarium-wilt of tobacco was first
brought to our attention in 1916, when it apparently was assuming
serious proportions, although it had been previously noted by the growers
in lesser amounts. It will also be remembered that the summer of 1916
was one of the warmest seasons recorded by the Weather Bureau stations
throughout the country, and that the soil temperature was correspond-
ingly high that season, as shown, for instance, by records taken at
depths of 2, 4, and 8 inches at Wisconsin (5). The season of 191 9, when
the disease occurred in Ohio, was also relatively warm. Though the
evidence is scanty for the occurrence of the disease under field conditions,
there is no doubt a correlation with high soil temperatures.
Soil reaction. — In experiments with Thielavia a series of soil cul-
tures was prepared and described (5) in which the reaction of a soil
of very high acidity was changed by adding varying amounts of calcium
carbonate so as to give different reactions ranging from high acidity
(9.38 tons lime required per acre) to one of high alkalinity. These soils
have changed somewhat in reaction during the two years in which they
have been used, but, as shown by the Troug color test, the same relative
reaction was probably maintained. The determination of the reaction
of these soils by the hydrogen-ion method, however, indicated that high
alkalinity was apparently not obtained, the PH value ranging from 5.4 to
7.2. These soils were sterilized, and one series in duplicate was inoculated
with Fusarium oxysporum var. nicotianae, the other series being left as
uninfested controls. Tobacco seedlings of the White Burley variety
were then transplanted into them. Three separate trials were run,
two of which gave reliable results, and one yielded unreliable results
because of poor infection. In one experiment all the plants died at the
three highest soil acidities, one died in each of the next three lower
reactions, and none died in the three jars at the alkaline end, although
finally they all became infected. In another experiment all plants
died in the first five grades of reaction from the acid end and one in
each of series 6, 8, and 9, but none in the seventh, although they were
both infected. The evidence seems fairly conclusive that an acid soil
favors the wilt disease, although it may occur in neutral or alkaline
soils. The crocks were, however, watered from the top, and part of the
soluble salts were washed downward. The soil may not have been of
the same reaction throughout for this reason, but the difference could
jan. 3, 1921 Fusarium-Wilt of Tobacco 529
not have been great, since the soils were repeatedly mixed and stirred.
Infection apparently occurred within a wide range of soil reaction,
although it was strikingly more pronounced at the higher acidities
(PI. 67, III). For this reason we can not agree with MacMillan (8)
that infection with Fusarium is favored by alkaline soils. The behavior
of Fusarium in the experiments described is also in line with the results
secured in the culture of F. oxysporum var. nicotianae in culture media
of varying reaction.
The Fusarium-wilt organism was inoculated in tubes of beef broth
at reactions ranging from — 5 per cent to + 5 per cent. After 5 days
the best growth was at + 1 . After 1 2 days it was apparently growing
best at +3 and poorest at +5, but after about 40 days the fungus
growth seemed most profuse at +5. On potato agar, however, the
best growth was obtained at neutral to +0.7. After 8 days there was
decidedly poorer growth as alkalinity was increased as well as a retarded
growth at + 1 per cent. This fungus, in common with most forms, is not
favored by alkaline media, and there seems to be no good reason for
expecting it to be more virulent in alkaline soils.
Other environmental conditions. — With respect to other environ-
mental conditions, we are able to say very little. Observation seems
to indicate that high soil moisture is not especially favorable to the
disease. Infection has been noted incidentally in both relatively dry
and moist soils, but the writer has been of the opinion that the soil
should be kept relatively dry to get good artificial infection. The disease
in Maryland occurred on high, sandy land, and the two years, 191 6 and
1919, in which the disease was called to the writer's attention were both
both notably hot and dry.
A single trial with soils ranging from no organic matter to pure leaf
mold did not indicate any decided preference on the part of the disease
for the presence of organic matter in the soil.
In the soil-inoculation experiments it has appeared that the highest
infection has always been secured by planting to tobacco soon after the
inoculation of the soil. Later plantings in the same soil usually resulted
in a lower percentage of infection. The parasite apparently does not
find the soil a very favorable medium for maintaining itself, even in
the presence of host plants, and in their entire absence it probably
gradually dies out completely. Nothing definite is known, however,
as to how long the fungus may persist in the soil.
To summarize briefly, the conditions which seem most necessary for
good infection and progress of the disease are :
1. Heavy soil infestation.
2. Wounded host tissue, particularly of stems below the surface of
the soil.
3. A relatively high temperature.
4. A susceptible variety.
530 Journal of Agricultural Research voi.xx.No. ?
VARIETAL RESISTANCE TO WILT
The early infection experiments indicated that a difference in varietal
resistance to the Fusarium-wilt probably existed in tobacco, but facilities
for carrying out varietal tests under field conditions were not readily
obtainable. It was therefore decided that preliminary tests would be
carried out on a small scale, using artificially inoculated soil in green-
house "flats" (boxes 16 inches by 24 inches and 3 inches deep). Twelve
to 14 of these flats were filled with greenhouse soil and sterilized at ioo° C.
for two hours. When cooled, each flat was inoculated by mixing into
it a sand-cornmeal culture previously referred to, after which the soil in
all the' flats was dumped together and again thoroughly mixed to obtain
uniform infestation, and the flats were again filled. Twenty plants of
each variety used were then transplanted into each flat from the sterilized
soil in which they had been grown. Three series of tests were carried out,
two out of doors in the summers of 1918 and 1919 and one in the green-
house in November, 191 8. In the first two tests no special attempt at
artificial wounding was made. In the last series the plants were wounded
by pinching off two basal leaves from each plant. Relatively higher in-
fection was obtained in this manner. The varieties tested represent
practically all the types grown commercially in the United States, and a
few others, including two other species, Nicotiana glauca and N. rustica,
and in one instance also an Fj of a cross between a White Burley resistant
to Thielavia rootrot and Fusarium-wilt and one susceptible to these
diseases. The experiments were terminated about one month after
transplanting. In taking notes on the results it was found convenient
to grade the individual plants into one of four classes: 1, dead; 2, badly
diseased; 3, slightly diseased; 4, healthy.
If a plant was completely wilted and dried it was classed as dead.
All remaining plants showing any exterior symptoms of disease were
classed as badly diseased. The remainder of the plants were then cut
off close to the root system, slit longitudinally, and examined for dis-
colored vascular systems. If any discoloration occurred attributable
to infection, the plant was listed as slightly diseased, and if none occurred
it was placed in the healthy class. In this manner the classification in-
cluded the important conditions and yet was not wholly arbitrary.
The results of the three tests are shown in Table I. In order to average
these data and to arrive at a fair average figure for relative resistance
expressed on the percentage basis, a more or less arbitrary formula
was established. This method may be briefly described as follows:
If a plant remained healthy it was credited with three points; if slightly
diseased, 2 points; if badly diseased, 1 point; and if dead it was rated at
zero. Twenty seedlings in one flat all healthy would be credited with
60 points (20X3), which is the maximum given and corresponds to 100
per cent resistance. Twenty seedlings in one flat all dead would receive
Jan. 3,i92i Fusarium-Wilt of Tobacco 531
no credit (20X0), which is the minimum and equals o per cent resistance,
or 100 per cent susceptibility. On the other hand, if, out of 20 plants
in a flat, 5 were dead, 5 badly diseased, 5 slightly diseased, and 5 healthy,
30 points would result (5 X 0 = o, 5 X 1 = 5, 5 X 2 = 10, 5 X 3= 15, total 30)
which is 50 per cent resistance.
It is only in some such manner, in fact, that resistance could be fairly
recorded in figures. Comparative yield of plants would give no better
criterion, since a plant might be infected and show no depreciation of
yield and might even reach maturity and be badly diseased without
appreciably influencing yield.
The average resistance given is on the basis of only 60 plants, except
in a few instances when it is on a basis of only 40 or 20 plants. Though
the numbers are small, they are believed to be more significant than
could be obtained under field conditions with a greatly increased num-
ber of plants, because of the uniformity of the soil and of infestation.
From these calculations it will be noted that none of the varieties
tried were absolutely immune. The most resistant varieties are the
Connecticut Havana, Cuban, and Sumatra, with 98 per cent resist-
ance. Since the figures are not regarded as significant within about 5 per
cent, the Pennsylvania Broadleaf and the Wisconsin binder selection
Hi 2074, a strain selected for resistance to rootrot due to Thielavia basi-
cola, should be included in this group. The least resistant of the Nico-
tiana tabacum varieties is the ordinary White Burley (32 per cent) (Pi. 67,
IV). Strangely enough, N. glauca, perhaps the species farthest removed
from N. tabacum in similarity, is the least resistant (23 per cent) to
F. oxysporum var. nicotianae of all plants tried. The varieties listed have
been repeatedly tried out for their resistance to the rootrot of tobacco
due to T. basicola (5), and it is interesting to note the correlation in
resistance to the two parasites. N. rustica is immune to Thielavia but
may be attacked by Fusarium. Shade-grown Cuban, Little Dutch, and
Wisconsin selection Hi 2074 are very resistant to Thielavia, but, while
Little Dutch is not very resistant to Fusarium, the other two are de-
cidedly resistant. The Pryor and Oronoco types are very susceptible
to. Thielavia but relatively resistant to Fusarium. The White Burley,
which is most susceptible to Thielavia, is also most susceptible to Fu-
sarium. A strain of White Burley selected for resistance to Thielavia
is also fairly resistant to Fusarium. The Ft generation of a cross be
tween resistant and susceptible Burley is seemingly intermediate in
resistance to Fusarium- wilt, as it is to Thielavia. The figures for the
latter are, however, not large enough to be of much significance. The
cases cited seem to be sufficient to warrant the statement that the cor-
relation between resistance in tobacco to Thielavia basicola and to F.
oxysporum var. nicotianae is low.
17777°— 21 2
532
Journal of Agricultural Research
Vol. XX, No. 7
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Jan. 3,1921 Fusarium-Wilt of Tobacco 533
No work has been done upon the selection of resistant strains within
individual varieties with the object of controlling the Fusarium-wilt
disease of tobacco. From the data presented, however, it is obvious
that this is a logical procedure in the control of this disease, should its
economic importance warrant the undertaking. The evidence at hand
indicates that the White Burley which was selected for its resistance to
Thielavia rootrot also shows marked resistance to Fusarium-wilt, as
compared with the ordinary White Burley, although the selection was
made, of course, without reference to resistance toward Fusarium. This
is, in fact, a step in the direction of control should the Fusarium-wilt
become serious in the White Burley section, where, because of the sus-
ceptibility of the ordinary strains grown, it is most likely to become of
economic importance. Selections in the Maryland Broadleaf variety,
which is the next most susceptible of the commercial types, seems en-
tirely feasible. Since it is on this type grown in Maryland that the dis-
ease has apparently been most common, it may be advisable in the near
future to undertake to select a resistant strain of this variety unless
other control measures are found which are more readily applicable.
CONTROL MEASURES
In the absence of the use of resistant varieties or strains, there appear
to be only the ordinary measures of control applicable to plant para-
sites infesting the soil. Since the disease is due to a living organism
which is carried over in the soil from year to year either as a parasite
on the tobacco plant or existing as a saprophyte in the vegetable mat-
ter of the soil for possibly a limited number of years, the most evident
measure of control seems to be the avoidance of infested soil. Espe-
cially when planting on new ground free from disease, it is advisable to
be certain also that the seedlings to be used have not been grown on in-
fested soil, since the parasite may be transmitted to the new soil in this
manner. Using new ground for seed beds or thoroughly sterilizing old
ground by means of steam is therefore desirable. New fields or seed beds
receiving surface drainage water from old, infested fields should also be
avoided, as should any unnecessary farm operation capable of carrying
even relatively small amounts of soil from infested fields to uninfested
ones. Where relatively few plants in a field are infected and show the
disease, it is a good precaution to remove these plants together with the
roots and to burn them so as to decrease the amount of infestation.
SUMMARY
(1) A disease of tobacco, apparently previously undescribed, has been
found to occur in Maryland and Ohio. The disease is characterized by
a yellowing and wilting of the leaves of the plant, usually followed by
death of the entire plant. The fibro-vascular system of infected plants
is characteristically brown or black.
534 Journal of Agricultural Research vol. xx, No. 7
(2) A species of Fusarium can be readily isolated from the discolored
area, and infection of seedlings can be produced by inoculating the soil
with this fungus. The causal organism is shown by stained paraffin
sections to exist throughout the fibro-vascular system of infected parts.
(3) The Fusarium concerned seems to be closely related to Fusarium
oxysporum (Schlecht.) Wr. but differs somewhat from this species in
morphology, physiology, and pathogenicity.
(4) Infection has been secured with two strains of Fusarium oxysporum
from potato on tobacco but has not been secured with the tobacco strain
on potato.
(5) The trinomial Fusarium oxysporum (Schlecht.) Wr. var. nicotianae,
n. var., is proposed for the tobacco-wilt organism.
(6) The conditions favoring infection with the tobacco-wilt organism
are heavy soil infestation, wounded host tissue, a relatively high soil
temperature (280 to 31 ° C), and a susceptible variety.
(7) It has been found that varieties of tobacco differ markedly in their
resistance to Fusarium-wilt. The White Burley variety is most sus-
ceptible, and the Havana Seed and Cuban varieties are among the most
resistant.
(8) Where the disease threatens to become serious, growers are advised
not to grow tobacco on the infested soils and to avoid the danger of
infested seed beds. The most hopeful means of control appears to lie in
the development of strains resistant to the disease within the various
susceptible varieties.
LITERATURE CITED
(1) Bisby, G. R.
1919. STUDIES ON PUSARIUM DISEASES OF POTATOES AND TRUCK CROPS IN
Minnesota. Minn. Agr. Exp. Sta. Bui. 181, 58 p., illus. Bibliography,
p. 40-44-
(2) Delacroix, Georges.
1906. RECHERCHES SUR QUELQUES MALADIES DU TABAC EN FRANCE. In Ann.
Inst. Nat. Agron., s. 2, t. 5, p. 141-232. Bibliographic, p. 203-205.
(3) Johnson, James.
1916. resistance in tobacco to the root-rot disease. In Phytopathology,
v. 6, no. 2, p. 167-181, 6 fig.
(4)
1918. wilt disease of tobacco attributed to fusarium. (Abstract.) In
Phytopathology, v. 8, no. 2, p. 76-77. 1918.
(5) and Hartman, R. E.
1919. the influence of soil environment on the rootrot of tobacco. In
Jour. Agr. Research, v. 17, no. 2, p. 41-86, 8 pi.
(6) Lounsbury, C. P.
1906. tobacco wilt in kat river valley . . . In Agr. Jour. Cape of Good
Hope, v. 28, no. 6, p. 784-803, illus.
(7) McKenney, R. E. B.
1905. THE WILT DISEASE OF TOBACCO AND ITS CONTROL. In U. S. Dept. Agr.
Bur. Plant Indus. Bui. 51, p. 5-8, illus.
Jan. 3,1921 Fusarium-Wilt of Tobacco 535
(8) MacMtllan, H. G.
1919. Fusarium-bught OF potatoes under irrigation. In Jour. Agr. Re-
search, v. 16, no. 11, p. 279-303, pi. 39-41- Literature cited, p. 301-303.
(9) Petch, T.
1907. diseases of tobacco in dumbara. Circ. and Agr. Jour. Roy. Bot. Gard.
Ceylon, v. 4, no. 7, p. 41-48.
(10) Smith, Erwin F.
1914. bacteria IN relation To plant diseases, v. 3. Washington, D. C.
(Carnegie Inst. Washington Pub. 27, v. 3.)
(n) Stevens, F. L., and Sackett, W. G.
1903. THE GRANVILLE TOBACCO WILT: A PRELIMINARY BULLETIN. N. C. Agr.
Exp. Sta. Bui. 188, p. 77-96, illus.
(12) Vaughan, R. E.
1914. A METHOD FOR THE DIFFERENTIAL STAINING OF FUNGOUS AND HOST CELLS.
In Ann. Mo. Bot. Gard., v. 1, no. 2, p. 241-242.
(13) WOLLENWEBER, H. W.
1913. studies ON the FUSARIUM problem. In Phytopathology, v. 3, no. 1,
p. 24-50, pi. 5.
PLATE 63
A. — A typical spot in a field of Maryland Broadleaf tobacco infested with Fusarium
wilt. Benedict, Md. 1916.
B. — Uninoculated control.
C. — Plants grown in soil artificially inoculated with the tobacco-wilt Fusarium and
planted to White Burlev.
(536)
Fusarium-Wilt of Tobacco
PLATE 63
Journal of Agricultural Research
Vol. XX, No. 7
Fusarium-Wilt of Tobacco
Plate 64
Journal of Agricultural Research
Vol. XX, No. 7
PLATE 64
A. — Plant infected with Fusarium-wilt, showing wilting in vertical line on stalk.
B. — Last stages of Fusarium-wilt in Maryland Broadleaf tobacco.
PLATE 65
A. — Result of plating out five pieces of infected vascular tissue from infected plant,
illustrating character of growth of mycelium on potato agar.
B. — Stem and midrib of plant, cut longitudinally to show the blackened vascular
system.
Fusarium-Wilt of Tobacco
Plate 65
Journal of Agricultural Research
Vol. XX, No. 7
Fusarium-Wilt of Tobacco
Plate 66
Journal of Agricultural Research
Vol. XX, No. 7
PLATE 66
A. — Cross sections through vascular system of tobacco plant infected with Fusarium-
wilt, showing the fungus mycelium in the vessels. Pianese stain.
B. — Longitudinal sections through the vascular system of plants infected with
Fusarium-wilt, showing the fungus strands in the vessels. Pianese stain.
PLATE 67
I. — Plants illustrating the influence of soil temperature on degree of wilting of
plants in soil infested with Fusarium-wilt. The plants were grown at the following
soil temperatures:
iA, i5°to 170 C.
2A, iq° to 200 C.
3A, 22° tO 24° C.
4A, 260 to 280 C.
5A, 290 to 310 C.
6A, 320 to 340 C.
The upper limit for infection is close to 350. Infection has occurred at 190 to 200,
but the progress of the disease is very slow.
II. — Plants grown in the same soil uninfested and at corresponding soil
temperatures.
III. — Plants illustrating the influence of varying soil reaction on the amount of
Fusarium-wilt in infested soil. A, highest acidity (medium to strong) to E near
neutral and I alkaline end. Same soil (selected for high acidity) in all crocks but
brought to various reactions by addition of precipitated calcium carbonate.
IV. — Plants illustrating varietal differences in resistance of tobacco to Fusarium-
wilt. Soil artificially inoculated, uniformly mixed, and transplanted with 20 plants
each of the following varieties: A, Connecticut Havana; B, Little Dutch; C, Mary-
land Broadleaf; D, White Burley.
Fusarium-Wilt of Tobacco
Plate 67
JMUUIJUL
Journal of Agricultural Research
Vol. XX, No. 7
SUGAR BEET TOP SILAGE
By Ray E. Neidig
Chemist, Idaho Agricultural Experiment Station l
The growing of sugar beets in the Pacific Northwest for the manufac-
ture of sugar is rapidly becoming a major occupation, but the beet root
from which the sugar is produced is not the only source of revenue when
sugar beets are grown. There remains for the farmer a considerable
portion of the crop in the form oi sugar beet tops, which represent a
large amount of value as a feed for stock. In recent years the farmer
has utilized this source of feed, thereby securing additional revenue in
the form of live stock and also in increased fertility of the soil.
It is estimated that a normal crop of sugar beets produces from 50
to 60 per cent of the weight of the crop in the form of salable beets and
the remaining percentage in beet tops. This being true, it is evident
that beet tops furnish no mean supply of feeding stuff for the farmer,
and the careful preservation of this by-product of the beet-growing
industry should be practiced.
The older countries many years ago realized the food value contained
in the by-products of the sugar beet industry. Many methods have been
used for the preservation of the sugar beet tops, but the siloing has re-
ceived the popular choice because more food value is retained bv this
method than by any other. In the United States, siloing sugar beet tops
has been practiced for many years. Recently the United States Depart-
ment of Agriculture 2 has estmated that beet tops, when properly siloed
and when fed with alfalfa hay, will reduce the hay requirement by ap-
proximately one-half. With the high prices of hay that have prevailed
for the past few years, it is evident that the proper preservation of beet
tops is a subject of no little economic importance.
During the past two years, numerous instances have come to the
writer's notice of stock dying when fed beet top silage, and the cause of
their death was attributed to the feeding of this product. However,
since thousands of head of stock are successfully fed on this silage, it
appeared to the writer that the fatalities were due mainly to the feeding
of abnormal rather than normal silage. With the idea in mind of secur-
ing knowledge of the chemical nature of the average beet top silage as
found on the average farm in the sugar beet districts, several samples of
1 Published by the permission of Director E. J. Iddings, Idaho Agricultural Experiment Station.
2 Jones, James W. beet-top silage and other by-products of the sugar beet. U. S. Dept. Agr.
Farmers' Bui. 1095, 24 p., 12 fig. 1919.
Journal of Agricultural Research, Vol. XX, No. 7
Washington, D. C Jan- 3. 1921
wf Key No. Idaho-4
(537)
538
Journal of Agricultural Research,
Vol. XX, No. 7
silage were collected and sent in to the chemistry department of the
Idaho Agricultural Experiment Station.1
In the fall of 1918, four samples of beet top silage were collected from
the southern part of the State by Mr. Rinehart. In 1919, six samples
were collected by Mr. Aicher. All samples are representative of the
average silage made in Idaho. An approximate analysis was made on
each of these samples. In addition volatile and nonvolatile acid deter-
minations were made on several of these samples of silage. The results
of the approximate analysis are given in Tables I and II. Table I gives
the results on the wet basis — that is, on the basis of the original moisture
content — and Table II the results on the anhydrous or moisture-free
basis.
Table I. — Analysis of 100 gm. sugar beet fop silage containing moisture
Sample No.
3
4
5
6
Moist-
ure.
Per ct.
81. s
76
59
80
49-5
68.5
70
70
78.2
74-4
Dry
mate-
rial.
Per ct.
18. s
24
21.8
25.6
Total
residue
left on
ignition
(dirt and
ash).
Per cent.
7.04
5-29
17.27
10.51
25-65
11.80
19. 08
Dirt.
Per ct.
5- °9
2.32
12. 79
8.42
18.39
7.09
14.46
9-79 5-13
14-13
12. 22
11.65
8.26
Ash.
P. ct,
1-95
2.97
4.48
2. 09
7. 26
4. 71
4. 62
4-66
2.48
3- 96
Pro-
tein.
Per ct.
2.18
4.40
1.88
6.51
4-38
1.38
Ether
extract.
Per ct.
48
8s
92
48
68
14
53
Carbohy-
Crude
drates
fiber.
(by dif-
ference).
Per el.
Per cent.
I- 52
7.28
3-05
11.86
3-44
14.97
1-43
5- 70
3-89
13-77
2. 70
11.57
1. 91
6-44
2. 60
12-39
I. 10
4-93
2.00
8.38
Quality
of silage.
Poor.
Fair.
Poor.
Do.
Do.
Fair.
Poor.
Fair.
Poor.
Do.
Table II. — Analysis of 100 gm. moisture-free sugar beet top silage
Sample No.
Total
residue
left on
ignition
Dirt.
Ash.
Protein.
Ether
extract.
Crude
fiber.
(dirt and
ash).
Per cent.
Per cent.
Per cent.
Per cent.
Per cent.
Per cent.
38.04
27-51
10.53
11.80
2-57
8.22
22.04
9- 65
12.39
12.28
3-54
12.69
42- 12
31-20
10.90
10.74
2-24
8.38
52-55
42. 10
10.45
9.40
2.40
7-15
50- 81
36.41
14-37
12.89
1-35
7.70
37-45
22.50
14-95
13.61
3.62
8-57
63-59
48. 19
I5-38
6.79
1.76
6.36
32.60
17.08
15-52
14-59
2.80
8.66
64.82
53-44
11-38
6-35
I. 17
5-17
47- 73
32. 26
15-47
10.58
I. 12
7-8i
Carbohy-
drates
(by dif-
ference).
Per cent.
39-37
49-45
36-51
28.50
27. 26
36.75
21.50
41-35
22-19
32.76
An examination of the results shows that only three of the samples
were classed as fair silage. The remaining seven samples were classed as
beet top silage of poor quality. A noteworthy fact seen from the inspec-
•The collecting of the samples was made possible through the kind cooperation of Mr. E. F. Rinehart,
Field Animal Husbandryman for Idaho, and Superintendant L,. C. Aicher, of the Aberdeen substation.
The writer wishes to thank these men for their careful notations of general conditions and their interest and
cooperation in the work.
Jan. 3,i92i Sugar Beet Top Silage 539
tion is the high percentage of dirt or sand found in the residue after
ashing. The real or true ash of the beet top silage was separated from
the total residue after igniting in an electric furnace, the difference repre-
senting sand or dirt. Even on the basis of the silage containing the
original moisture it is seen that the percentage of dirt is high in the three
samples classed as fair silage, the amount ranging from 2.32 pounds to
7.09 pounds on the basis of the wet silage. When calculated on the
moisture-free basis these samples contain dirt and sand to the amounts of
9.65 and 1 7. 1 pounds per 100 pounds of moisture-free silage. On the other
hand, the amount of dirt found in the poorer grades of silage ranges on
the wet basis from 8.26 pounds to 18.39 pounds per 100 pounds of wet
silage, while on the basis of 100 pounds moisture-free silage there are
found from 22.50 to 53.44 pounds. These figures are all the more striking
when applied to the average daily amount of beet top silage eaten by
stock. An animal consuming an average ration containing 35 pounds of
beet top silage must necessarily consume from 2.89 pounds to 6.44 pounds
of dirt. It is not unfair to assume that such quantities of dirt, which in
most localitie's engaged in growing sugar beets is a light, sandy, volcanic
ash, would tend to produce serious digestive disturbances which in turn
might produce the death of the animal. In samples 4 and 9, death of
stock did actually take place while the silage was being fed. An inspec-
tion of the dirt content of these two silages shows a dirt content of 8.42
and 11.65 pounds in every 100 pounds of wet silage and 42.1 and 53.44
pounds, respectively, in every 100 pounds of moisture-free silage.
The reasons, for the presence of such a large quantity of dirt in the
silage are many. A brief summary of the methods used by the average
farmer when siloing sugar beet tops will be given, since it will tend to
explain the large quantities of sand and dirt that are present. In the
first place, the type of silo is very crude. Usually it is a shallow dirt
trench or pit of sufficient size to accommodate the crop of beet tops.
The beet tops are thrown into piles in the field and scooped upon wagons.
More or less dirt clings to the beet tops, especially if this work is carried
on in rainy weather. The wagons are driven into the trench and dumped,
each load tending to pack the beet tops previously unloaded. Such
procedure does not hinder but rather aids in the carrying in of some dirt.
It is readily seen that the whole process of siloing sugar beet tops is one
where dirt is collected in all steps of the process from the time of topping
the beets until the tops are actually siloed, unless extreme care is used
to keep out excess dirt. Without extreme care a good silage can not
be obtained. The United States Department of Agriculture has recently
issued a bulletin * which sets forth the best methods of siloing sugar
beet tops and describes the best types of pit silos. Pit silos with con-
crete side are recommended. Many good suggestions as to the proper
1 Jones, James W. op. cit.
540 Journal of Agricultural Research vol. xx,No. 7
methods that a farmer should use are given. From the study of Tables
I and II of this paper it is plain that more care is needed on the part of the
average farmer before he can expect to secure a silage of good quality.
If the suggestions embodied in farmer's bulletins of the United States
Department of Agriculture are followed, the farmer will not only be
rewarded with a silage of good quality and high feeding value but he
will also avoid the loss of stock.
ACIDITY OF SUGAR BEET TOP SILAGE
Investigations of many types of silage by the writer x and others have
indicated that in practically all silages that have undergone a normal
fermentation there results an acidity in which the chief acids are lactic,
acetic, and propionic, their relative importance decreasing in the order
named. In the sugar beet top silage it was desired to study the acidity
of several samples to learn what types of acids were formed in the silage
found on the average farm. With this idea in mind, several of the
samples sent in to the experiment station were examined. The Duclaux
method 2 was used for estimating the volatile acids, and the zinc lactate
method was used for the nonvolatile or lactic acid. The algebraic and
graphic methods described by Gillespie and Walters 3 were used in
calculating the individual volatile acid after they were identified by the
qualitative tests suggested by Dyer.4 The results on the volatile and
nonvolatile acids are given in Tables III and IV. Table III gives the
results on the wet basis and Table IV gives the results on the moisture-
free basis.
An inspection of Tables III and IV shows that the acids developed in
the sample of sugar beet top silage are not similar to those usually found
in the corn silage. Corn silage contains lactic, acetic, and propionic
acids. The proportion of lactic to the two volatile acids is usually about
1 part to 75 hundredths, while the proportion of acetic to propionic is
usually 1 part to one-tenth. Butyric acid was never found in silage that
was classed as good corn silage. It was found, however, in partially spoiled
samples. Hence the conclusion was reached that silage containing buty-
ric acid has undergone an abnormal fermentation.
'Neidig, Ray E. acidity of silage made from various crops. In Jour. Agr. Research, v. 14, no.
10. P- 395-409- 1918. Literature cited, p. 408-409.
2 Duclaux, E. recherches sur les vtns. deuxif.me mf.moire: sur les acides volatils du vin.
In Ann. Chirn. et Phys., s. 5, t. 2, p. 289-324. 1874.
— traite de microbiologie. t. 3, p. 388. Paris, 1900.
'Gillespie, L. J., and Walters, E. H. the possibilities and limitations op the duclaux method,
for the estimation of volatile acids. In Jour. Amer. Chem. Soc, v. 39, no. 9, p. 2027-2055, 3 fig. 1917.
Literature cited, p. 2055.
<Dyer, D. C a new method of steam distillation for the determination of the volatile
FATTY ACIDS, INCLUDING A SERIES OF COLORIMETRIC QUALITATIVE REACTIONS FOR THEIR IDENTIFICATION.
In Jour. Biol. Chem., v. 28, no. 2, p. 445-473, 2 fig. 1917.
Jan. 3, 1921
Sugar Beet Top Silage
54i
Table III. — Acidity of 100 gm. sugar beet top silage containing moisture
Sample No.
Moisture.
Dry ma-
Acetic
Propi-
Butyric
Valeric
Total vol-
Lactic
terial.
acid.
onic acid.
acid.
acid.
atile acid.
acid.
Per cent.
Per cent.
Gm.
Gm.
Gm.
Gm.
Gm.
Gm.
81.5
18. s
0.51
0
o-73
0
1
24
0-59
76.0
24.0
.42
0
■17
0
m
.69
49-5
50-5
•17
0. IS
•25
0
57
Trace.
69- S
30. S
•71
• 05
I- 13
0
1
*9
«1-71
70. 0
30.0
0
0
•54
0.04
58
Trace.
70. 0
30.0
.29
. 10
0
0
39
.41
78. S
21-5
•31
•05
•44
0
So
.26
Total
acids.
5
6
7
8
9
Gm.
1-83
1.28
•57
3-6o
•58
.80
1.06
Table IV. — Acidity of 100 gm. sugar beet top silage on dry basis
Sample No.
Acetic
acid.
Propi-
onic acid.
Butyric
acid.
Valeric
acid.
Total vol-
atile acid.
Lactic
acid.
Gm.
Gm.
Gm.
Gm.
Gm.
Gm.
2-73
0
3-92
0
6.6s
3- °7
1-74
•35
0
0.29
.70
•SO
0
0
2.44
1. 14
2.79
Trace.
2.32
0
•17
0
3-70
1.79
0
O- 12
6. 19
1. 91
Trace.
.96
•34
0
O
1-30
1-35
1.46
•25
2. 04
O
3-7S
i. 21
Total
acids.
Gm.
5-23
1. 14
11.80
1. 91
2.6s
4.96
In the samples of sugar beet top silage, sample 8 is the only one that
contains the same acids that are found in corn silage. This silage was
classed as a fair quality of silage by experts when it was sent to this
station. The remaining samples of silage all contained some butyric
acid. The quality of- the silage ranged from fair to poor, depending
chiefly upon the amount of dirt that was in the silage. While the
amount of butyric acid present indicates in a degree the type of fermen-
tation, it does not seem to prevent stock from eating the silage. Some
samples contained butyric acid in quantities that made drying the mate-
rial in an oven very unpleasant unless the process was carried on under a
hood, and yet cattle ate the silage with relish. It is not known how
much effect the abnormal fermentation has on the feeding value of
silage, but no doubt some loss occurs. Such losses could be greatly re-
duced by carefully packing the beet tops when siloing and by covering
the tops in such manner that all the air is excluded.
The mere presence of butyric acid in silage is not in itself harmful, but
it is the fact that the presence of butyric acid indicates an abnormal fer-
mentation, resulting in a partial decomposition of silage, which tends
to lower its feeding value.
It is hardly to be expected that beet tops can be packed sufficiently
to exclude all air, because of the nature of the tops, but possibly cutting
the tops in a silage cutter would solve the problem. Experiments are
planned for the coming year to determine the best n_ethods of siloing
sugar beet tops.
542 Journal of Agricultural Research vol. xx, No. 7
Lactic acid is present in very small amounts in many of the samples.
It is possible that more lactic acid is present in the early stages of fermen-
tation and that it is either changed into other acids or is decomposed.
An additional investigation is needed to explain fully the reason for the
small amounts of lactic acid in abnormal silage. The lactic acid present
is the racemic mixture.
The fact that sample 8 contains the characteristic acids of normal
silage indicates that sugar beet tops can be successfully siloed if proper
precautions are taken to pack the tops well and exclude air. The sam-
ples of silage analyzed came from a pit silo ranging from iy2 feet to 8
feet in depth. Without question, depth of the pit silo is an important
factor in the production of good silage. Where shallow silos are used,
air gains access to the greater portion of the beet tops and a poor silage
results, whereas, in the deeper silos there is less chance for the entire
silage to be partially spoiled on account of access of air. It is important,
then, to have a deep silo to eliminate dirt, and to pack thoroughly so as
to exclude air. These precautions will insure a better average silage
throughout the Northwest than is now found.
SUMMARY
(i) It is evident that the quality of sugar beet top silage put up by the
average Idaho farmer is very poor.
(2) Large quantities of dirt are present, which could be eliminated in a
large measure by careful handling of the product during siloing.
(3) To improve the quality of silage, pit silos should be deep and the
silage should be packed thoroughly and covered sufficiently to exclude
air. Excess dirt should be eliminated.
(4) More care should be taken by the average farmer in siloing sugar
beet tops. While stock will eat silage that is very poor, there is a loss
of food value in improperly made silage as well as danger of mortality.
NODULE BACTERIA OF LEGUMINOUS PLANTS
By F. Lohnis, Soil Biologist, Bureau of Plant Industry, United States Department of
Agriculture, and Roy Hansen, Professor of Soils, University of Saskatchewan,
Saskatoon, Sask.1
INTRODUCTION
Despite the fact that the nodule bacteria of the leguminous plants
have been made the subject of numerous publications, it is not to be
disputed that their true morphological and physiological character, as
well as their correct systematic position, are by no means sufficiently
known. This is especially clearly demonstrated by the fact that they
are still proclaimed by several writers to be the representatives of a
special genus Rhizobium, once established by A. B. Frank as the result
of rather inadequate studies upon this subject. In the new classification
of bacteria, adopted by the Society of American Bacteriologists, the
nodule bacteria again are widely separated from closely related species,
and the error concerning the so-called genus Rhizobium has been re-
vived once more.
Comparative investigations upon the symbiotic and the nonsymbiotic
nitrogen-fixing bacteria of the soil, published in 1905 by the senior author,
have proved conclusively that the nodule bacteria are not representatives
of a special genus Rhizobium, but that they are closely related to Bacillus
radiobacter Beijerinck and further to B. lactis viscosum Adametz, B.
pneumoniae Friedlander, and B. aerogenes Escherich. The last three
organisms are immotile, while the first one is motile; but here again the
very close relationship between the immotile B. aerogenes and the motile
B. coli has to be kept in mind. In fact, there can be easily isolated from
every soil numerous varieties of B. radiobacter, which lead gradually up
to B. coli, acquiring the power of fermentation and that type of growth
on solid substrates which is characteristic of the last-named species.
It has been pointed out in detail that all species mentioned above differ
only gradually, not principally, in their physiological and morphological
qualities, and especially that those branched or otherwise changed cell
forms which are frequent in the root nodules are equally common with
all members of this group of capsule bacteria, if these are tested ade-
quately.2 The ability to fix the atmospheric nitrogen was shown to be
common in this group of organisms.
1 Most of the experiments discussed in this paper were made in the summer of 1919, at the University of
Illinois, where at that time the junior author held the position of Associate in Soil Biology. The photo-
graphs accompanying the paper were made by Mr. F. L. Goll, of the Bureau of Plant Industry, United
States Department of Agriculture.
2 It is not superfluous to emphasize once more that persistence in calling these forms " bacteroids " is by
no means to be recommended. They are true bacteria, not foreign bodies looking like bacteria, as Frank's
pupil Brunchorst erroneously believed. To speak of a "bacteroid" growth of bacteria is no less absurd
than it would be to speak of a "fungoid" growth of fungi.
Journal of Agricultural Research, Vol. XX, No. 7
Washington, D. C Jan. 3, 1921
wg Key No. G-215
17777°— 21 3 (543)
544 Journal of Agricultural Research vol. xx.No. 7
Bacillus radiobacter was found to be peritrichic, and the same paper
also indicated (12, p. 592, footnote)1 that in all probability B. radicicola
has the same kind of flagellation. But no faultless preparates were
obtained at that time.
In the same year, 1905, G. T. Moore wrote concerning the nodule
bacteria (14, p. 26):
There does not seem to be any necessity for creating a new group to include these
organisms, as has been done by Frank, under the name of Rhizobium, for although
there is a certain amount of polymorphism, it is no greater than frequently occurs in
the bacteria.
With regard to the flagellation, however, Moore himself evidently
made no special studies, and, accepting Beijerinck's statement that the
"swarming bodies " (gonidia) of Bacillus radicicola are monotrichic as being
valid for the bacteria too, he proposed to call the nodule bacteria Pseu-
domonas radicicola. Numerous authors have followed this suggestion,
and experiments made by Harrison and Barlow (8) apparently confirmed
the view that the flagellation of these organisms is indeed monotrichic.
However, these experiments are, in fact, not convincing, as has been
emphasized especially by Kellerman (o). This author and also G. de
Rossi (16,17), Zipfel (ig), and Prucha (75) secured results all of which
demonstrated more or less clearly that the senior author's assumption
was correct : Bacillus radicicola is peritrichic ; it is no " Pseudomonas."
But this seemed again to be contradicted by certain results obtained
by the junior author while working with the late T. J. Burrill (6). Nu-
merous tests made with the bacteria isolated from cowpea, soybean,
Japan clover, and other plants showed clearly and invariably monotrichic
flagellation, and, therefore, the designation Pseudomonas radicicola was
restored once more. Additional results, however, indicated that there
are other features which differentiate the bacteria of the cowpea-soybean
group from those living in the roots of clover, alfalfa, pea, and vetch.
Especially the slime production and the speed of growth appeared to be
different, and the organisms studied were arranged into two groups,
"slow growers" and "fast growers" Both, however, were supposed to
be merely varieties of P. radicicola.
This point remained to be investigated more thoroughly. In addition,
another "fast grower" presented itself for detailed study, which quite
regularly appeared on thickly sown plates of the "slow growing" groups,
and which, indeed, has been mistaken by several investigators as the
true nodule organism of cowpea, soybean, Japan clover, etc. Repeatedly
such cultures were sent to and tested by the junior author. They were
all unable to produce nodules.
The data given on the following pages make it evident that this "fast
grower" is Bacillus radiobacter, which plays in this case, also, a very
1 Reference is made by number (italic) to " Literature cited," p. 554-555.
Jan. 3) 1921 Nodule Bacteria of Leguminous Plants 545
interesting r61e. As this same species undoubtedly takes part in
many processes occurring in soil and in water, it was thought useful to
give another more detailed description of it, especially because, despite
its ubiquity, B. radiobacter is much too little known. In addition to
the rather short description given by Beijerinck, only the more complete
one published by the senior author in 1905 exists thus far. On account
of its great similarity to B. radicicola, B. radiobacter should be very well
known to all bacteriologists working with the nodule bacteria in order
to avoid mistakes which may otherwise not be discovered until only
negative results are obtained in the inoculation tests.
Concerning the flagellation of the nodule bacteria three statements
have been published more recently which also will have to be discussed
presently. According to J. K. Wilson (18) the soybean bacteria are
peritrichous ; Barthel (2) declared lupine and alfalfa bacteria to be
lophotrichous; Fred and Davenport (7) found the alfalfa organism
peritrichous, but they found the lupine bacteria characterized by having
one, rarely two, flagella.
EXPERIMENTAL RESULTS
The following strains of nodule bacteria were studied after having
been tested with positive results in regard to their ability to produce
nodules on the host plants from which they were isolated.
1. Cowpea. 6. Red clover.
2. Peanut. 7. Sweet clover.
3. Japan clover. 8. Vetch.
4. Beggar weed. 9. Strophostyles.
5. Soybean.
There were also included in our investigations two strains isolated
from:
10. Black locust. 11. Lupine.
No positive inoculation test could be made on black locust. The
lupine culture was kindly furnished by Dr. E. B. Fred, of the University
of Wisconsin, who had tried it with positive results on this plant. Our
tests were equally successful.
Two noninfectious "fast growing" cultures isolated from legume
nodules and identified as Bacillus radiobacter were studied in comparison
with six Radiobacter strains which originated from soil and which were
kept in the senior author's collection since the years given in parentheses.
12. Fast grower from cowpea. 16. Bacillus radiobacter from soil (1908).
13. Fast grower from soybean. 17. Same (1908).
14. Bacillus radiobacter from soil (1904). 18. Same (1908).
15. Same (1907). 19. Same (1916).
No. 14 is the strain which in 1904 had been acknowledged by Prof.
Beijerinck as being identical with his Bacillus radiobacter and which was.
used by the senior author for the original description published in 1905
{12).
546
Journal of Agricultural Research
Vol. XX, No. 7
Table I. — Development of cow pea-soybean bacteria, Bacillus radicicola {from clover,
■vetch, etc.), and B. radiobacter
Substrates.
Cowpea-soybean bacteria.
Mannite-nitrate agar slant.
Macroscopic examination. — Raised whitish to porcelain white, glossy
layer.
Microscopic examination. — After 3 days slender rods, sometimes curved;
after 7 to 10 days unstained, irregular sheaths, with 1 to 4, most frequent-
ly 2, darkly stained granules; after 2 to 3 weeks many small globules,
ovals, and short rods outside of the unstained sheaths, also small globular
regenerative bodies.
Beef agar slant.
Macroscopic examination. — Fairly good whitish growth.
Microscopic examination. — After 3 days weakly stained, irregular, thin,
short rods; after 7 to 10 days irregular rods, producing gonidia and glob-
ular regenerative bodies, which may multiply as such; after 2 to 3 weeks
very variable appearance, rather long slender rods, often branched, or
club shaped, globular regenerative bodies, also unstained, irregular
sheaths with dark granules, and large globular gonidangia.
Beef gelatin stab.
Macroscopic examination. — Very small, gray, nonliquefying disk on the
surface, hardly any growth in the stab.
Microscopic examination. — Thin rods, sometimes branched or swollen,
producing gonidia and small globular regenerative bodies; in old cultures
gonidia and regenerative bodies frequently predominating.
Beef broth.
Macroscopic examination.— Broth at first clear, with little sediment;
later (after about 2 weeks) slightly turbid.
Microscopic examination.— After 3 days slender rods, sometimes curved;
after 2 weeks granular rods producing gonidia, also budding and branch-
ing, small globular regenerative bodies, and symplasm; after 3 to 4 weeks
very irregular forms, branching, swelling.
Milk.
Macroscopic examination. — During the first weeks no change visible,
later slow digestion, no clear serum zone.
Microscopic examination.— Mostly small globules and ovals, few short,
slender rods.
Potato.
Macroscopic examination. — Very meager, transparent, thin layer.
Microscopic examination. — After 7 days slender rods, sometimes branch-
ed, or with terminal swelling; after 4 weeks small globules and ovals,
irregular rods (frequently branched), globular regenerative bodies, and
symplasm with very variable new development.
Jan. 3, 1921
Nodule Bacteria of Leguminous Plants
547
Table I. — Development of cow pea-soybean bacteria, Bacillus- radicicola (from, clover,
vetch, etc.), and B. radiobacter — Continued
Substrates.
B. radicicola (from clover, vetch, etc.).
B. radiobacter .
Mannite-nitrate
agar slant.
Macroscopic examination. — Slimy, trans-
parent growth, with or without whitish
streaks.
Microscopic examination. — Small ovals
and short rods, producing after 1 to 2 weeks
gonidia and small globular regenerative
bodies. Also unstained slime threads
with dark granules and large globular, or
oval gonidangia; irregular pale forms from
symplasm.
Macroscopic examination.— Thick, slimy
transparent layer, with whitish streaks.
Microscopic examinations.— After 7 days
small ovals and short rods, imbedded in
slime; after 14 days some rods with thick
unstained capsules forming symplasm;
after 3 to 4 weeks normal cells, stars, and
large globules and clubs from symplasm.
Beef agar slant.
Macroscopic examination. — Meager, flat,
grayish growth.
Microscopic examination. — Mostly small
ovals and short rods, the latter sometimes
curved, budding and branching; later
also large rods, and large globular, oval, or
club-shaped gonidangia.
Macroscopic examination. — Flat, whit-
ish slimy layer, thick sediment below.
Microscopic examination. — As on man-
nice-nitrate agar.
Beef gelatin
stab.
Macroscopic examination. — Small, gray,
nonliquefying disk on surface, very little
growth in stab.
Microscopic examination. — Small ovals
and short rods, gonidia, and small globu-
lar regenerative bodies.
Macroscopic examination. — Grayish, flat,
round, nonliquefying surface growth, lit-
tle growth in stab; after 2 to 4 weeks gela-
tine sometimes brown on top.
Microscopic examination. — Typical ovals
and short rods, these sometimes curved or
branched, also unstained slime threads
with dark granules, later symplasm with
stars.
Beef broth.
Macroscopic examination. — Broth either
clear or very slightly turbid, little whitish
sediment.
Microscopic examination. — Small ovals
and short rods, budding and branching,
occasionally threads; after 1 to 2 weeks
many gonidia and small, globular regen-
erative bodies.
Macroscopic examination. — Broth tur-
bid, white ring, whitish film, much whit-
ish sediment.
Microscopic examination. — Small ovals
and short rods, budding and branching;
later gonidia, globular regenerative bodies,
threads, and fine stars from symplasm.
Milk.
Macroscopic examination.— After 1 to 4
weeks clear serum zone on top, 2 to 5 mm.
thick; milk below nearly unchanged, very
fine flocculation.
Microscopic examination. — Small ovals
and rods, later also granular threads and
symplasm.
Macroscopic examination. — First slime
ring and serum zone on top; later whole
milk turning brown.
Microscopic examination, — After 7 days
typical ovals and rods; later small and
large cells from symplasm.
Potato.
Macroscopic examination. — Meager, trans-
parent, slimy growth.
Microscopic examination. — Small slen-
der rods, budding and branching, some
ovals, globular regenerative bodies; later
gonidia predominant.
Macroscopic examination.— First gray,
later coli-brown slimy layer, potato turns
frequently gray.
Microscopic examination. — First small
ovals and short rods, budding and branch-
ing, later also large oval and globular
gonidangia and symplasm with stars.
548 Journal of Agricultural Research vol. xx, No. 7
The results of our investigations leave no doubt that the earlier find-
ings of the junior author were correct so far as the polar flagellation and
the peculiar morphological and cultural features of the cowpea-soybean
organisms are concerned. On the other hand, it could now be ascer-
tained with equal certainty that the bacteria producing nodules on
clover, alfalfa, vetch, and other plants originally cultivated in Europe
are all peritrichic and exhibit all the characteristics of Bacillus radici-
cola, as described by Beijerinck and other authors.
Those findings which were obtained most frequently and which may
be considered as being typical for the two groups of nodule bacteria are
compiled in Table I, together with analogous data pertaining to Bacillus
radiobacter. Photographs of the most characteristic details are repro-
duced on Plates 68 and 69.
When grown from the root nodule on Harrison and Barlow's ash agar,
mannite agar, or similar agar of low nitrogen content, the two groups of
nodule bacteria are best characterized and differentiated by the very
slow growth of colonies in the cowpea-soybean group and the com-
paratively quick growth of those of Bacillus radicicola (6, pi. 11, fig. 1-11).
Frequently, but not always, the development of B. radiobacter is still
somewhat more rapid than that of B. radicicola; in the macroscopical
as well as in the microscopical aspects, however, the colonies of these
two species on such media are so very much alike that it is almost im-
possible to distinguish them with certainty. Both, when developing on
the surface, are perfectly round, drop-like, soft, watery or slimy, glisten-
ing, transparent. Often a whitish center or whitish streaks become
visible within the more transparent mass, especially if the surface colony
is the outgrowth of an imbedded colony. Sometimes it appears as if
this whitish center were regularly to be seen only with certain strains of
Radicicola and Radiobacter. This is not the case, however. Its pres-
ence or absence is erratic and can not be used for differentiation. The
imbedded colonies are always small, white, opaque, mostly lentiform,
less frequently circular. Under the microscope the surface colonies
present themselves as sharp-edged disks, pure white at the outside with
yellowish-grayish granulation in the center. In a few cases a radiate
structure becomes visible. The colonies of the cowpea-soybean group
appear macroscopically, as well as microscopically like young colonies
of the Radicicola type. The presence of Radiobacter colonies on the
plate stimulates their growth markedly.
In cell morphology there is again a more pronounced relationship be-
tween Radiobacter and Radicicola than between the nodule bacteria of
the clover-vetch group on the one side and of the cowpea-soybean group
on the other. This holds true with the regular rod forms as well as with
the very pleomorphic, curved, swollen, branched, or otherwise changed
types of growth characteristic of these groups. The photographs on
Plate 68, D-L, represent the pictures we have seen most frequently, but
they do not pretend to give a complete illustration of the wide pleomor-
Jan. 3, i92i Nodule Bacteria of Leguminous Plants 549
phism of these organisms. Before their complete life history can be
given much additional material will have to be collected, especially with
regard to the form of gonidangia, regenerative bodies, and the various
cells developing from the symplastic stage. At present we intend only
to bring out as clearly as possible those points which are important
for a correct differentiation and diagnosis. As far as one may judge
from the microscopic appearance, it is the inclination of Bacillus radio-
bacter to form stars which is most characteristic (Pi. 68, L), and this was
used, therefore, by Beijerinck for its denomination. With B. radicicola
the frequent occurrence of the clear-cut, compact Y forms is the most
conspicuous feature (Pi. 68, H) ; whereas the bacteria of the cowpea-
soybean group present themselves in most cases, when stained with aqueous
aniline dyes in the usual manner, as short or long, unstained sheaths
with one or more darkly stained granules (Pi. 68, J). Of course Y forms,
as well as unstained sheaths with darkly stained gonidia, can be ob-
served not infrequently with the other organisms, too, and the star for-
mation is by no means solely to be found with Radiobacter; but we feel
sure that those pictures, as shown on Plate 68, G-L, will be found most
valuable for diagnostical purposes.
The flagellation is the same with Radiobacter (PI. 68, C) and Radici-
cola (PI. 68, B), while the bacteria of the cowpea-soybean group are
characterized by one coarse, fairly straight polar flagellum ( Pi. 68, A).
Just before fission one cilium may be seen at each end; as a rare excep-
tion a tuft of polar flagella was observed occasionally. Frequently a
darkly stained body becomes visible within the rod just at that point
where the flagellum springs forth, which may be considered to be a flag-
ellated, not yet liberated, gonidium, such as can be seen occasionally
with many other bacteria, especially with Bacillus radicicola, too. When
liberated this becomes the monotrichic small "swarming body" described
by Beijerinck in 1888 (4).
The growth on mannite-nitrate agar, as well as on beef agar slants, as
described in Table I, is quite characteristic, and after the eyes have been
sufficiently trained, one seldom makes a mistake in guessing the group
to which a culture presented foi inspection may belong. But it must be
admitted that occasionally and temporarily a strain of the cowpea-
soybean group can show the flat, transparent growth characteristic of
Radicicola, whereas it is a very rare occurrence that a member of the
last-named group simulates the former one. The growth of Radiobacter
is always very typical, except when a very weak strain is encountered,
which does not frequently occur within this group. Plate 69, A, demon-
strates the characteristic differences noticeable on mannite-nitrate agar
as clearly as they can be shown in a photographic reproduction.1
1 As was the case with Azotobacter, for which the mannite-nitrate agar was first used (13, p. 686), so also
the nodule bacteria and Bacillus radiobacter grew very readily on this substrate. Allen (/, p. jj) asserted
recently that he could not get any growth of Azotobacter on a dextrose agar, which he erroneously called
"Lohnisand Smith's medium." But not even the formula used by us has been quoted correctly by Allen,
and it is, of course, quite obvious that on account of the alterations made by Allen his agar must indeed
have been quite unsuitable.
550 Journal of Agricultural Research vol. xx.No. 7
Cultures on beef gelatine and in beef broth differentiate clearly Radio-
bacter and nodule bacteria, while, as stated in Table I, the two groups
of nodule organisms grow very much alike on these substrates. Micro-
scopic tests, however, made from gelatine and broth furnish, in most
cases, especially characteristic pictures, provided that the growth has
not been altogether too poor to get a satisfactory preparate.
The growth in milk and on potato, as described in Table I and illus-
trated on Plate 69, is very characteristic and can be used to great advan-
tage for diagnosis. It is not to be denied that with old stock cultures
atypical results may sometimes be obtained in this direction also.
Especially cultures rich in or entirely made up of the globular regenera-
tive bodies, which are produced by these as well as by all other bacteria,
furnish whitish, yellowish, or only slightly brownish growth on potato
in the case of Bacillus radiobacter and B. radicicola. But we have never
seen such atypical growth with new isolations. Here the coli-brown
color of the potato cultures separates Radiobacter sharply from the
nodule bacteria, and these in turn are equally sharply to be distinguished
by the behavior of their milk cultures. It is true that sometimes milk
cultures of the B. radicocola group also leave the milk unchanged, but
the microscopic test of such abnormal cases probably will always show,
as it did in the cases studied by us, that the abnormality was simply
caused by the fact that the bacteria which were inoculated did not multi-
ply at all. Furthermore, no alteration may be seen if milk is used which
has been kept for a long time and has become concentrated by evapora-
tion of part of its water.
To determine on a larger scale whether this different behavior of the
two groups of nodule bacteria, when grown in milk, can be correctly
accepted as of real diagnostic value, all cultures of nodule bacteria at
our disposal were tested simultaneously with the following results:
MILK WAS CHANGED AS TYPICAL FOR MILK WAS LEFT UNCHANGED BY THE
BACILLUS RADICICOLA BY THE FOLLOWING CULTURES:
FOLLOWING CULTURES:
5 from red clover. 10 from cowpea.
4 from sweet clover. 8 from soybean.
6 from navy bean. 5 from peanut.
1 from vetch. 4 from Japan clover.
2 from lupine. 2 from beggar weed.
3 from black locust. 2 from Cassia chamaecrista.
3 from Amorpha.
2 from Strophostyles.
If kept for longer than four weeks milk cultures of the cowpea-soybean
organisms usually become more or less transparent on account of partial
decomposition of the casein; but they never show the perfectly clear zone
characteristic of the other group.
The bacteria were also tested on other media besides the standard
substrates, of which sterilized soil, moistened with 0.5 per cent mannite
Jan. 3, 1921 Nodule Bacteria of Leguminous Plants 551
solution, mannite-nitrate solution as used for studying the life cycle of
Azotobacter, tap water plus 0.5 per cent beef broth, and 2 per cent salt
agar furnished the most satisfactory results, especially with regard to a
more complete knowledge of the cell morphology of the organisms. For
diagnostic purposes, however, these substrates are of minor importance,
as they do not bring out anything which is not already to be seen on
the standard media. Nevertheless, it should be pointed out that cultures
of the nodule bacteria in soil are to be recommended for two reasons.
First, they are useful in keeping the organisms in a normal state of
virility for a long time, and, in the second place, they demonstrate very
clearly, when studied microscopically, that it is erroneous to believe —
though numerous authors have promoted such hypotheses — that the
nodule bacteria behave very differently in soil and could, therefore, not
be isolated in their typical form from their natural habitat. Our results
are in complete agreement with those recently obtained by Barthel (j)
concerning the growth of bacteria in sterilized soil.
Tap water containing 0.5 per cent beef broth gave also very good
development and proved repeatedly helpful in reviving old, weakened
strains which refused to grow on solid substrates.
DISCUSSION
Our experimental results leave no doubt that the nodule bacteria of
the leguminous plants are to be divided at least into two distinct groups,
differing morphologically as well as culturally. It is equally beyond
dispute that these differences are so marked and constant that one might
be inclined to establish the nodule organism of the cowpea-soybean group
as a new species. On account of its behavior in the inoculation test O.
Kirchner has considered the soybean organism a distinct species, which
he named in 1895 Rhizob deter ium japonicum(io). According to the
rules of priority, this species name would have to be given preference,
despite the fact that different behavior in the inoculation test generally
can not be accepted as a distinguishing mark of such quality as to vali-
date the creation of a new species. The generic name Rhizobacterium,
used by Kirchner, is, of course, equally as untenable as the generic name
Rhizobium. According to the two tmost requently used modes of clas-
sifying the bacteria, one might name the cephalotrichic non-sporulating
rod of the cowpea-soybean group Pseudomonas japonica or Bacterium
japonicum, while the name Bacterium or Bacillus radicicola would have
to be retained for the peritrichic organisms to be found with clover,
alfalfa, sweet clover, vetch, pea, etc.
However, we do not advocate such a procedure. We are firmly of
the opinion that much more must be known of the complete life history
of a bacterium than is obtainable along the standardized lines of cus-
tomary bacteriological research, before one can safely proceed to estab-
lish a genuine species on a truly scientific basis. Undoubtedly many if
552 Journal of Agricultural Research vol. xx, no. 7
not most of the commonly used so-called species names of bacteria are
no species names at all, but merely denominations more or less correctly
applied to organisms about whose complete life history and, accord-
ingly, about whose true systematic value and position comparatively
little is known at present.
It is by no means impossible that future systematic investigations may
demonstrate the peritrichic and the cephalotrichic nodule bacteria to
be relatively constant types of growth of one species. There are a few
reports in the literature indicating that occasionally cross inoculations
have been obtained, which might support this hypothesis. While O.
Kirchner never found nodules on soybeans grown in Germany and there-
fore thought his Rhizobacterium japonicum to be the active agent in the
Far East, F.. Cohn said in a note appended to Kirchner's report that
soybeans grown for the first time in his experimental garden in Breslau
did produce nodules, though these were not of the normal type and con-
tained only a few rodlike bacteria. Kellerman reported upon a case
where a culture originally isolated from alfalfa was found to be infective
on alfalfa and lupine as well as on soja when tested by Leonard after
six years' cultivation on artificial substrates. It may be mentioned also
in this respect that cross inoculations between navy bean and cowpea
seem to be equally possible, under circumstances, however, which need
further elucidation.
But just as negative results in cross inoculation experiments can not
be accepted as sufficient basis for establishing different species, so also
such rather exceptional positive results can not be used as valid support
of the hypothesis that monotrichic and peritrichic nodule bacteria are
only two types of growth of the same species. First of all, it would have
to be ascertained whether in such cases the peritrichic organism has
really changed into the monotrichic one, or vice versa. The possibility
remains, of course, that occasionally the one type of organisms may
invade a host plant whose nodules are normally caused by the other
type of bacteria.
Changes in flagellation from peritrichic to cephalotrichic have been
observed, according to Lehmann and Neumann (n, p. 256, 357, 371),
with Bacillus coli and B. alcaligenes. Both species are related to B. ra-
diobacter and B. radicicola, and under this aspect an analogous change
should not be rejected prematurely as a priori improbable.
At the end of the introduction three statements have been quoted from
the more recent literature which one might be inclined to accept as con-
firmative evidence in this direction. However, on account of the follow-
ing reasons we do not feel justified in advocating such an interpretation.
J. K. Wilson says that in his preparations of soybean organism —
The flagella were peritrichous, the highest number found being four.
As no photomicrographs had been made, Dr. Wilson was kind enough
to furnish, on request of the senior author, one of his slides for examina-
Jan. 3,i92i Nodule Bacteria of Leguminous Plants 553
tion. The flagella visible therein were all very weakly stained, so that
no definite conclusion could be drawn. A culture, for which we are also
indebted to Dr. Wilson, behaved in our hands like all those tested before;
practically all cells were distinctly monotrichous. A comparison of
Plate 68, A, with the pictures published on Plates IV and V of Bulletin
202, Illinois Agricultural Experiment Station (6), leaves no doubt about
this point.
In Barthel's paper (2, p. 16) two drawings and one photomicrograph
are to be found which clearly illustrate the following statement :
Bei den Lupinenbakterien sind die Geisseln ziemlich lang, wellig geformt und an
einem Pole befestigt. Ihre Anzahl variiert von 1 bis 6. Ihre Placierung ist recht
eigentiimlich. Sie sitzen namlich ofters nicht gerade an der Spitze des Zellleibes,
sondern sozusagen an den "Ecken" und oft etwas von dem Hinterende entfernt.
Oft findet man auch eine Geissel an der einen "Hinterecke" und mehrere andere
zusammen an der anderen. . .
Bei den Luzernebakterien waren die Geisseln meist weniger und kiirzer, am hauf-
igsten 1 oder, seltener 3 oder 4, aber auch hier deutlich lophotrich. . .
Fred and Davenport (7), on the other hand, saw only one or two cilia
with the lupine bacteria, while several strains of alfalfa organisms left no
doubt as to their peritrichic flagellation.
We believe that these conflicting views are in fact not so irreconcilable
as they seem to be. If well-made slides are examined carefully, some
cells will always be discovered which clearly show that on account of the
primary swelling and the following shrinking of their capsules, the flagella
are often more or less dislocated. Some of the cells shown in Plate 68,
A-C, exhibit this phase as clearly as it is possible in such reproductions.
The flagella of the monotrichous bacteria of the cowpea-soybean group are
to be seen in an exactly polar position only when the cells themselves are
lying lengthwise within the "drift," as indicated by the floating flagella.
In all other cases dislocations may take place, removing the cilia to the
corners or even to the side of the cells, where they should not be viewed,
however, as remnants of a peritrichic flagellation.
On the other hand, analogous disturbances may cause the occurrence
of apparently cephalotrichic bacteria among the peritrichic cells of Ba-
cillus radicicola and B. radiobacter. That there exists no truly polar
flagellation in these cases, however, is evidenced by the fact that the cilia
composing such an apparently polar tuft do not protrude exactly from the
same spot, as they do, for example, in the cell with several polar flagella
shown in Plate 68, A. They are always more or less separated and are
only accidentally drawn together in the course of the shrinking of the
capsule. A thorough examination of well-made preparations leaves no
doubt that the original position of the flagella is peritrichic.
SUMMARY
(1) The nodule bacteria of the leguminous plants are to be divided into
two groups, differing morphologically as well as physiologically.
554 Journal of Agricultural Research vol. xx.No. 7
(2) The first group shows all features characteristic of Bacillus radi-
cicola Beijerinck. It is peritrichic, grows relatively fast on agar plates,
and changes the milk in a very characteristic manner. It produces
nodules on the roots of the following plants: clover, sweet clover, alfalfa,
vetch, pea, navy bean, lupine, black locust, Amorpha, and Strophostyles.
(3) The second group is characterized by monotrichic flagellation,
comparatively very slow growth on agar plates, and inability to cause a
marked change in milk. It has been isolated from cowpea, soybean,
peanut, beggarweed, Acacia, Genista, and Cassia.
(4) According to the customary manner of classifying bacteria, this
second group of nodule bacteria would have to be considered to be a new
species, and according to the rules of priority, it would have to be named
Pseudomonas japonica or Bacterium japonicum (Kirchner). But we do
not advocate such a procedure, because only a complete study of the life
history of these two groups of organisms would make it possible to say
definitely whether they are, indeed, two distinct species or merely differ-
ent types of growth of the same organism.
(5) Bacillus radicicola is closely related to B. radiobacter. The generic
name Rhizobium is to be rejected. The correct systematic position of
both species is in the neighborhood of B. aerogenes and B. coli.
(6) Bacillus radiobacter seems to be regularly present in the root
nodules of leguminous plants, stimulating development and activity
of the nodule bacteria. On account of its similarity to B. radicicola,
it has been repeatedly mistaken for the nodule-producing organism in
the cowpea-soybean group, whose bacteria it outranks very considerably
in the development on the plates made from the nodules. By its brown
growth on potato, B. radiobacter can be easily differentiated from B.
radicicola.
LITERATURE CITED
(1) Allen, E. R.
1919. SOME CONDITIONS AFFECTING THE GROWTH AND ACTIVITIES OF AZOTO-
bacter chroococcum. In Ann. Mo. Bot. Gard., v. 6, no. 1, p. 1-44,
1 pi. Bibliography, p. 42-43.
(2) Barthel, Chr.
1917. die geisseln des bacterium radicicola (beij). In Ztschr. Garungs-
physiol., Bd. 6, No. 1, p. 13-17.
(3)
1919. cultures de bacteries sur terre sterilisee. In Meddel. K. Vetensk.
Nobelinstitut, bd. 5, no. 2, 13 p., 1 pi.
(4) Beijerinck, M. W.
1888. DIE BACTERIEN DER papilionacEEN KNOLLCHEN. In Bot. Ztg., Jahrg.
46, No. 46, p. 725-735, pi. 11; No. 47, p. 741-750; No- 48, p. 757-771;
No. 49, p. 781-790; No. 50, p. 797-804.
(5) and van Delden, A.
1902. UEBER DIE assimilation des freien STICKSTOFFS durch BAKTERIEN.
In Centbl. Bakt. [etc.], Abt. 2, Bd. 9, No. 1/2, p. 1-43.
Jan. 3, 1921 Nodule Bacteria of Leguminous Plants 555
(6) Burrill, Thomas J., and Hansen, Roy.
191 7. IS SYMBIOSIS POSSIBLE BETWEEN LEGUME BACTERIA AND NON-LEGUME
plants? 111. Agr. Exp. Sta. Bui. 202, p. 115-181, 17 pi. Biblio-
graphies, p. 161-181.
(7) Fred, E. B., and Davenport, Audrey.
1918. influence of reaction on nitrogen-assimilating bacteria. In
Jour. Agr. Research, v. 14, no. 8, p. 317-336. Literature cited, p.
335-336.
(8) Harrison, F. C, and Barlow, B.
1907. THE NODULE ORGANISM OF THE LEGUMINOSAE . . . In Centl. Bakt.
[etc.], Abt. 2, Bd. 19, No. 7/9, p. 264-272; No. 13/15, p. 426-441, 9 pi.
(9) Kellerman, K. F.
1912. the present status OF soil inoculation. In Centbl. Bakt. [etc.],
Abt. 2, Bd. 34, No. 1/3, p. 42-50, 2 pi. Bibliography of American
studies, p. 46-50.
(10) Kirchner, O.
1895. die wurzelknollchen DER SOJabohnE. In Beitr. Biol. Pflanzen,
Bd. 7, Heft 2, p. 213-223.
(11) Lehmann, K. B., and Neumann, R. O.
1912. ATLAS UND GRUNDRISS DER BAKTERIOLOGIE . . . Aufl. 5, Teil 2, xiv,
777 p. Miinchen.
(12) IyOHNIS, F.
1905. BEITRAGE ZUR KENNTNIS DER STICKSTOFFBAKTERIEN. I. UEBER STICK-
STOFFFixiERENDE BAKTERIEN. In Centbl. Bakt. [etc.], Abt. 2, Bd.
14, No. 18/20, p. 582-597.
(13), and Smith, N. R.
1916. LIFE CYCLES OF the bacteria. (Preliminary communication.) In
Jour. Agr. Research, v. 6, no. 18, p. 675-702, 1 fig., pi. A-G. Literature
cited, p. 701-702.
(14) Moore, George T.
1905. soil inoculation for legumes ... U. S. Dept. Agr. Bur. Plant Indus.
Bui. 71, 72 p., 10 pi.
(15) Prucha, Martin J.
191 5. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA.
N. Y. Cornell Agr. Exp. Sta. Mem. 5, 83 p. Bibliography, p. 79-83.
(16) Rossi, Gino de.
1907. UEBER DIE MIKROORGANISMEN, WELCHE DIE WURZELKNOLLCHEN DER
LEGuminosEn Erzeugen. In Centbl. Bakt. [etc.], Abt. 2, Bd. 18,
No. 10/12, p. 289-314; No. 16/18, p. 481-488, 2 pi. Literatur, p.
483-488.
(17)
1909. STUDI SUL MICROORGANISMO PRODUTTORE DEI TUBERCOLI DELLE LEGUMI-
NOSE. In Ann. Bot., v. 7, fasc. 4, p. 618-652, pi. 23.
(18) Wilson, J. K.
1917. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF SOYBEAN (SOJA
MAX PD?ER) AND OF FACTORS INFLUENCING NODULE PRODUCTION. N.
Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig. 80-94.
(19) Zipfel, Hugo.
191 1. BEITRAGE ZUR MORPHOLOGIE UND BIOLOGIE DER KNOLLCHENBAKTERIEN
DER LEGuminosEn. In Centbl. Bakt. [etc.], Abt. 2, Bd. 32, No. 3/5
P- 97-!37- Literatur, p. 136-137.
PLATE 68
A. — Soybean bacteria, J. K. Wilson's strain, 4 days old.
B. — Vetch bacteria, 3 days old.
C. — Bacillus radiobacter, 2 days old.
D. — Soybean bacteria, beef agar, 4 days old.
E. — Red clover bacteria, beef agar, 4 days old.
F. — Bacillus radiobacter, beef agar, 4 days old.
G. — Cowpea bacteria, potato, 6 days old.
H. — Red clover bacteria, potato, 14 days old.
I. — B. radiobacter, milk, 7 days old.
J. — Cowpea bacteria, mannite-nitrate agar, 8 days old.
K. — Vetch bacteria, mannite-nitrate agar, 8 days old.
L. — B. radiobacter , mannite-nitrate solution, 17 days old.
A-C Loeffler's stain; D-L aqueous fuchsin. X 1,000.
(556)
Nodule Bacteria of Leguminous Plants
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Nodule Bacteria of Leguminous Plants
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Journal of Agricultural Research
Vol. XX, No. 7
PLATE 69
A. — Mannite-nitrate agar slants, 8 days old, from left to right: soybean bacteria,
vetch bacteria, and Bacillus radiobacter .
B.— Growth in milk, 4 weeks old from left to right: soybean bacteria, vetch bac-
teria, and B. radiobacter.
C. — Growth on potato, 2 weeks old: vetch bacteria (left) and B. radiobacter (right).
CORRELATION AND CAUSATION
By Sew all Wright
Senior Animal Husbandman in Animal Genetics, Bureau of Animal Industry, United
States Department of Agriculture
PART I. METHOD OF PATH COEFFICIENTS
INTRODUCTION
The ideal method of science is the study of the direct influence of one
condition on another in experiments in which all other possible causes
of variation are eliminated. Unfortunately, causes of variation often
seem to be beyond control. In the biological sciences, especially, one
often has to deal with a group of characteristics or conditions which are
correlated because of a complex of interacting, uncontrollable, and often
obscure causes. The degree of correlation between two variables can be
calculated by well-known methods, but when it is found it gives merely
the resultant of all connecting paths of influence.
The present paper is an attempt to present a method of measuring the
direct influence along each separate path in such a system and thus of
finding the degree to which variation of a given effect is determined by
each particular cause. The method depends on the combination of
knowledge of the degrees of correlation among the variables in a system
with such knowledge as may be possessed of the causal relations. In cases
in which the causal relations are uncertain the method can be used to
find the logical consequences of any particular hypothesis in regard to
them.
CORRELATION
Relations between variables wThich can be measured quantitatively are
usually expressed in terms of Galton's (4)1 coefficient of correlation,
2X'Y' ,
fxY = (the ratio of the average product of deviations of X and Y to
the product of their standard deviations), or of Pearson's (7) correlation
ff( * \
ratio, rjx.Y= \Y x' (the ratio of the standard deviation of the mean values
of X for each value of Y to the total standard deviation of X), the
standard deviation being the square root of the mean square deviation.
Use of the coefficient of correlation (r) assumes that there is a linear
relation between the two variables — that is, that a given change in one
variable always involves a certain constant change in the corresponding
average value of the other. The value of the coefficient can never exceed
1 Reference is made by number (italic) to " Literature cited," p. 585.
Journal of Agricultural Research, Vol. XX, No. 7
Washington, D. C Jan. 3, 1921
wh Key No. A-55
17777°— 21 4
(557)
558
Journal of Agricultural Research
Vol. XX, No. ;
+ i or — i . For many purposes it is enough to look on it as giving an
arbitrary scale between + i for perfect positive correlation, o for no corre-
lation, and — i for perfect negative correlation.
The correlation ratio (tj) equals the coefficient of correlation if the rela-
tion between the variables is exactly linear. It does not, however, depend
on the assumption of such a relation, and it is always larger than r when
the relations are not exactly linear. It can only take values between
o and + i , and it can be looked upon as giving an arbitrary scale between
O for no correlation and i for perfect correlation.
The numerical value of the coefficient of correlation (r) takes on added
significance in connection with the idea of regression. It gives the aver-
age deviation of either variable from its mean value corresponding to a
given deviation of the other variable, provided that the standard devia-
tion is the unit of measurement in both cases. The regression in terms
of the actual units can, of course, be obtained by multiplying by the
ratio of the standard deviations. Thus, for the deviation of X correspond-
ing to a unit deviation of Y, we have regx.Y = rXY— • This formula may
be deduced from the theory of least squares as the best linear expression
for X in terms of Y. The formula for what Galton later called the coeffi-
cient of correlation was, in fact, first presented in this connection by
Bravais (i) in 1846. Any such interpretation is of course impossible
with the correlation ratio.
The numerical values of both coefficients, however, have significance in
another way. Their squares (rj2, or r2 if regression is linear) measure the
portion of the variability of one of the variables which is determined by
the other and which disappears in data in which the second is constant.
Thus if Y(r2x is the mean square deviation of X for constant Y, Pearson
has shown that:
y<r2x = o-2x(i-T?2x.Y)
or yc2x = 0'2x(i— -y2xy) ^ regression is linear.
It often happens that it is desirable to consider simultaneously the
relations in a system of more than two variables. For such cases, involv-
ing onlv linear relations between the various pairs of variables, Pearson (6)
has devised the coefficient of multiple correlation.
R,
x(abc • • • n)
-4
in which
A =
1
Jan. 3. 1921 Correlation and Causation 559
and Axx is the minor made by deleting row X and column X.
•K2x(abc • • • n) measures the degree of determination of X by the whole
A
set of other factors, and 1— R2X(ABC. • -n)= t — is the maximum possible
squared correlation between X and a factor independent of those con-
sidered. This formula for multiple correlation leads to one for multiple
regression. Letting X', A', B' ', etc., be the deviations of variables X,
A, B, etc., from their mean values, Pearson has shown that the most
probable value of X' for known values of the other variables is given by
the formula
Xi = *M^+AM& A^AT
°X Axx °A AxX ^B Axx <TN
^
0X — JV • • • BAaX ~ 0"X
Analogous but more complex formulae have recently been published
by Isserlis (5) for the multiple correlation ratio for use in cases in which
the regressions are not necessarily linear.
CAUSATION
In all the preceding results no account is taken of the nature of the
relationship between the variables. The calculations thus neglect a very
important part of the knowledge which we often possess. There are
usually a priori or experimental grounds for believing that certain factors
are direct causes of variation in others or that other pairs are related as
effects of a common cause. In many cases, again, there is an obvious
mathematical relationship between variables, as between a sum and its
components or between a product and its factors. A correlation between
the length and volume of a body is an example of this kind. Just because
it involves no assumptions in regard to the nature of the relationship, a
coefficient of correlation may be looked upon as a fact pertaining to the
description of a particular population only to be questioned on the grounds
of inaccuracy in computation. But it would often be desirable to use a
method of analysis by which the knowledge that we have in regard to
causal relations may be combined with the knowledge of the degree of
relationship furnished by the coefficients of correlation.
The problem can best be presented by using a concrete example. In
a population of guinea pigs it will be found that the birth weights, early
gains, sizes of litters, and gestation periods are all more or less closely
correlated with each other. The influence of heredity, environmental
conditions, health of dam, etc., are also easily shown. In a rough way,
at least, it is easy to see why these variables are correlated with each other.
These relations can be represented conveniently in a diagram like that
in figure 1 , in which the paths of influence are shown by arrows.
560
Journal of Agricultural Research
Vol. XX, No. 7
The variety and complexity of the relations which may be back of a
correlation are well illustrated in this case. Thus, the weight at weaning
(33 days of age) should be correlated with the birth weight and with the
gain between birth and weaning simply because it is their sum. The
relations of birth weight with gestation period and the prenatal rate of
growth are also essentially mathematical rather than causal. Birth
weight is necessarily fully determined by the character of the prenatal
growth curve and the time at which this is interrupted by birth.
In the relation between gestation period and size of litter we come to
a case in which there is no necessary mathematical relationship. We
naturally attempt to account for the high negative correlation by the
hypothesis that a large number in a litter in some way causes early
&cr/n
O-JJdoys
fVe/afyfat
*i
Rate, of
Growf/7
(Sesfdf/on
Pe>r/od
Fxternal
conditions
Conc//'f/on
of Dasn
//erecf/'fq
of Panr
Fig. 1. — Diagram illustrating the interrelations among the factors which determine the weight of guinea
pigs at birth and at weaning (33 days).
parturition. Similarly, a large number in a litter might be expected to
be a cause of slow growth in the foetuses.
Birth weight and gain after birth are highly correlated. Here neither
variable can be spoken of as the cause of variation in the other, and the
relation is not mathematical. They are evidently influenced by common
causes, among which heredity, size of litter, and conditions which affect
the health of the dam up to the time of birth at once come to mind.
Most of the variables are connected with each other through more than
one path. Thus, weight at birth is correlated with weight at weaning
both as a component of a sum and as the effect of common causes.
There may be a conflict of the paths. Thus, a large number in a litter
has a fairly direct tendency to shorten the gestation period, but this is
probably balanced in part by its tendency to reduce the rate of growth
of the foetuses, slow growth permitting a longer gestation period. Large
litters tend to reduce gestation period and rate of growth before and
after birth. But large litters are themselves most apt to come when
jan. 3,1921 Correlation and Causation 561
external conditions are favorable, which also favors long gestation periods
and vigorous growth.
The coefficient of correlation is a resultant of all paths connecting the
two variables. It would be valuable in many cases to be able to deter-
mine the relative importance of each particular path. The usual method
in such cases is to calculate the partial correlation between two variables
for a third constant, using Pearson's well-known formula
c ab n n — r-. S ;
-V(I-^AC)(I-^BC)
for correlation between A and B for constant C. Such partial correla-
tions, however, must be interpreted with caution. It is true that by
making constant a connecting link between two variables, whether it is
a common cause or the cause of one and effect of the other, we eliminate
the path in question. This elimination of connecting paths in which the
constant factor is a link is not, however, the only way in which correlation
is affected. If an effect of a number of causes is made constant, spurious
negative correlations appear among the causes and their other effects.
Thus, if weight at 33 days is made constant, the correlation between
birth weight and gain necessarily becomes — 1 . We are simply picking
out a population in which any deficiencies in birth weight happen to be
exactly balanced by excess in gain after birth. This is an extreme case,
but where the relations of cause and effect are at all complex it is evident
that the correlation between two variables may be changed in more than
one way by making a third variable constant, making the interpretation
doubtful.
Where there is a network of causes and effects, the interrelations could
be grasped best if a coefficient could be assigned to each path in the
diagram designed to measure the direct influence along it. The following
is an attempt to provide such a coefficient, which may be called a path
coefficient.
DEFINITIONS
We will start with the assumption that the direct influence along a
given path can be measured by the standard deviation remaining in the
effect after all other possible paths of influence are eliminated, while
variation of the causes back of the given path is kept as great as ever,
regardless of their relations to the other variables which have been made
constant. Let X be the dependent variable or effect and A the inde-
pendent variable or cause. The expression ax.A will be used for the
standard deviation of X, which is found under the foregoing conditions,
and may be read as the standard deviation of X due to A . In a system
in which variation of X is completely determined by A, B, and C we
have o-x.A = CT CB(rx representing the constant factors, B and C, and
also the variation of A itself (aA) by subscripts to the left. The path
562 Journal of Agricultural Research vol. xx, No. 7
coefficient for the path from A to X will be defined as the ratio of the
standard deviation of X due to A to the total standard deviation of X.
Px-a = -
"x
0",
Just as the regression of X on A is expressed by r^— the deviation
0"a
of X directly caused by a unit deviation of A is given by the formula
. °X °"x-A
Px-A „ = rr~'
°A &A
Another coefficient which it will be convenient to use, the coefficient
of determination of X by A, dx.A, measures the fraction of complete
determination for which factor A is directly responsible in the given
system of factors. This definition implies that the sum of such coefficients
must equal unity if all causes are accounted for.
SYSTEMS OF INDEPENDENT CAUSES
The degree of determination of one variable by another is most easily
found where the variables are connected by a mathematical relationship.
The simplest mathematical relationship is that between a sum and its
components. For the standard deviation of a sum the following relation
is well known :
o"a+b = — -=* A + <r2B+2<rA0-BrAB.
If A and B are independent of each other, rAB = 0, and we have
0-2A+B = 0-2A + ^2B-
The degree to which variation of the sum is determined by that of each
component is obvious.
^x-A = _2^ and dx.B = -^> where X= A +B,
<* x ' o" X
giving dx.A + dx.B = 1, as required by definition.
For the standard deviation of X due to A we have in this case, cx.A = crA.
Thus, £x-a = — = — bv definition.
°x °x '
. . 2(A' + B')A' 2A'2 crA
Again, rXA=-L- '- — = =— •
n<xx(TK naxaA ax
Summing up, px.A = ^dx.A = rXA.
It can easily be shown that the same formulae hold in case we are
dealing with the sum of multiples of a number of independent factors
instead of with their own sum.
We can pass at once from this case to cases in which variation of X is
caused in the physical or physiological sense by variation in several causes
Jan. 3,1921 Correlation and Causation 563
provided that these causes are independent of each other, have linear
relations to the dependent variable X, and that the deviations which they
determine are additive. They are independent of each other if there is
no correlation between their variations. A cause has a linear relation to
the effect and is combined additively with the other factors if a given
amount of change in it always determines the same change in the effect,
regardless of its own absolute value or that of the other causes. The con-
clusion is that, under these conditions, the path coefficient equals the
coefficient of correlation between cause and effect, and the degree of
determination equals the square of either of the preceding coefficients.
CHAINS OF CAUSES
If we know the extent to which a variable X is determined by a cer-
tain cause M, which is independent of other causes, combines with them
additively, and acts on X in a linear manner, and if we know the extent
to which M is determined by a more remote cause A , the degree of deter-
mination of X by A must be the product of the component degrees of
determination.
Let X = M+N, and M = A+B
O^m , _^!a a j _0~2A
° X u M °X
Thus dx.A = dx.Mdu.x
and px.A = Px-mPk-a-
NONADDITIVE FACTORS
In cases in which a factor does not act additively with the other factors
in determining the variations in the dependent variable, its influence on
the latter can not be completely expressed apart from the other factors,
at least in terms of the ordinary measures of variability. This can be
made clearer by an illustration. Multiplying factors are among the most
important of those which do not combine by addition.
I^et X=AB and assume that rAB = o
v\ = M \o\ + M\a\ + —f-
where A' and B' are deviations of A and B from their mean values MA
and MB. Putting B constant, we have o"2x.A = M\<r2A; and similarly
putting A constant, we have o"2x.B = M2A<r2B. There remains a portion of <r2x
which is due to A and B jointly and which can not be separated into parts
M2 <72
due to each alone. If we write dx.A = — f — as the degree of determi-
0 X
M2K<T2„
nation of X by variation of A alone, and dx.B = —2 — ~ as the corre-
0 x
sponding degree of determination of X by variation of B alone, we must
2Af2B'2
recognize an additional term 4-ab = 2 — ' in order that the sum of the
5°4
Journal of Agricultural Research
Vol. XX, No. 7
coefficients of determination may equal unity. Regression is linear and
M2 a2
— I — -• Thus dx.A = r\A as in the case of independent
additive factors. The term
)A'2B'
is small unless the amounts of
variation in A and B are large in comparison with the mean values. In
many cases it is safe to deal with path coefficients and degrees of deter-
mination in the case of multiplying factors just as in the case of addi-
tive factors.
As a concrete illustration of these points take two independent vari-
ables, for each of which the values 1, 2, and 3 occur in the frequencies
1,2, and 1, respectively. Below is the correlation table between one of
these factors and their product.
Product (X).
I
2
1
2
3
4
5
6
7
8
9
<
I
2
2
I
I
4
8
4
V
4
2
2
3
I
IS
ft
I
4
2
4
0
4
O
O
I
16
MA = 2 aA=^JTf2 rAX=-y[8/Tj
</>
8/17
., ,— -2A'2B'2 . dx.B=8/i7
v ^ n<r2x 1 i/i7
"X-AB
I
In this case the amounts of variation in the factors are relatively large
compared with their mean values, making the distribution surface mark-
edly heteroscedastic, yet the degree of determination by either factor
comes out only slightly less than one-half.
NONLINEAR RELATIONS
<t( m )
Pearson's definition of the correlation ratio, t?x.a= > has already
fx
been given. The variations of the mean value of X for different values
of A are the variations which can be attributed to the direct influence of
A, assuming that A is cause, X effect, and that other causes are com-
bined with A additively. Thus o-x.a = o-(amx) and we have at once
Again, as the total variation of X is composed of the variation of its
mean values for different values of A, plus the variation about these
mean values, we have o-2x = <r2(AMx) +A(T2X, giving Aor2x = o-2x (1— v2x.a), as
already noted.
Thus r?2x.A measures the portion of a\ lost by making A constant, so
that as before dx.A = v2x-A = p2x-A.
Jan. 3, 1921
Correlation and Causation
565
Unfortunately we can not deal with chains of factors which involve
nonlinear relations by mere multiplication of the path coefficients of the
component links. In the present paper, unless otherwise stated, it will
be assumed that all correlations are m
essentially linear. >^ rt
EFFECTS OF COMMON CAUSES
Suppose that two variables, X and Y,
are affected by a number of causes in
common, (B, C, D). Let A represent
causes affecting X alone and E causes
affecting Y alone (fig. 2).
Let px-A = a
Px-b = b
px-c = c
px-D = d
px-E = o
B, C, and D are assumed
dependent of each other — thai
etc.
Py
•A =
-0
h
•B =
■b'
h
■0 =
■c'
pr.
D =
d'
h-
E =
e'
to
be in-
is,
rKi
- = o,
Hence
px-B = rXB, etc.
rxY—bb'
r
B XY
Fig. 2. — Diagram showing relations be-
tween two variables, X and Y, whose
values are determined in part by com-
mon causes, B, C, and D, which are in-
dependent of each other.
V(i-&2) (i-6/2)
B^XY B^XC B^YC
fxY — bb' — cc'
■yl(l-b2-C2)(l-b'2-c'2).
X
V(l— B^XC) (I — B^yc)
When all common causes have been made constant, dcb^xy = o
rxY = bb' + cc' + dd' = 2px.BpY.B.
Thus, in those cases in which the causes are independent of each other,
the correlation between two variables equals the sum of the products of
the pairs of path coefficients which con-
nect the two variables with each common
cause. An illustration of the use of this
principle was given in an earlier paper
(c?) in analyzing the nature of size factors
X^ y \ ^~ in rabbits.
^^> C £y It may be deduced from the foregoing
Fig. 3.— Diagram showing relations be- formula that two variables may even be
tween two variables, x and y, whose completelv determined by the same factors
values are completely determined by
common causes, b and c, which are in- and yet be uncorrelated with each other.
dependent of each other. j^t variation of X be completely deter-
mined by factors B and C, the path coefficients being b and c, respectively.
Let Y be completely determined by the same factors, the path coeffi-
cients being b' and c' (fig. 3). Then rxY = bb' + cc'. The condition
566
Journal of Agricultural Research
Vol. XX, No. 7
under which rXY may equal zero is evidently that bb' = —cc'. An
example may be found in the absence of correlation between the sum
and difference of pairs of numbers picked at random from a table.
In many cases a small actual correlation between variables will be
found on analysis to be the resultant of a balancing of very much more
important but opposed paths of influence leading from common causes.
SYSTEMS OF CORRELATED CAUSES
The discussion up to this point has dealt wholly with causes which
act independently of each other. It is necessary to consider the effects
of correlation among the causes.
Let us consider the sum of £wo correlated variables (fig. 4).
Let X=M + N
o-2x = c2M + <r2N + 2ailai{rMN.
We have defined o-x.M as the standard deviation of X when factors
other than M are constant, but M varies as much as before. The latter
qualification is important in the present case, since the making of N
constant tends to reduce the variation of M, reducing <rM to c^V1 ""Aim-
The definition of <rx.M implies that
not only is N made constant but
that there is such a readjustment
among the more remote causes, A,
B, and C, that o-M is unchanged.
Under the definition it is evident
that in this case <xx.M = <rM and (rx.N
On
o"x °"x"
In attempting to find the degrees
of determination of X by M and N
we meet a difficulty somewhat similar to that met in the case of non-
additive factors. The squared standard deviation is made up in part
of elements due wholly to M and N, respectively, but in part to a portion
which can not be divided between them. The term 2<riSaNrWN is due
solely to the fact that the variations of X, which M and N determine,
tend to be in the same direction and so have greater effect than if varia-
tions M and N were combined at random. It seems best to define dx.M
2
as the degree of determination of X due to M alone. Thus dx.M=-^>
Fig. 4. — A system in which the value of variable
X is completely determined by causes M and N,
which are correlated with each other.
Thus px.M = ~* and />,
dx-N =
The remaining term may be considered as determination by
M and N jointly and may be written dx-^= 2px.Mpx.Nrm,.
These rules can be extended at once to the sums of more than two
variables, to sums of multiples of variables, and hence, as before, to
Jan. 3, 1921
Correlation and Causation
567
X'A+"X'B+^X*C+"x-D !•
X-M + ^X-N + 2rX-MPx-NrMN "T"
linear relations of cause and effect in which the influence of the causes is
combined additively. It is also easy to show that the formulae apply
approximately for multiplying factors.
Summing up, px.M = V^x-m™-^
2dx.M + 22£s.M£x.NrMN= 1.
The next problem is to find the
degree of determination of X by a
factor such as B, which is connect-
ed with X by more than one path
(fig- 5).
Assume that A, B, C, and D are
independent and completely deter-
mine X. d
But also d
dx.D=i.
"x-B = "x-M — "x-A + "x-N — ^X-C + piG. s _a system in which the value of X is af-
2/'x-M/,X-N/)M-B/'N-B)rememrjeringthat fected by a factor, B, along two different paths,
_ , BMX and BNX.
*MN— PM-BrN-B-
Since d1A.x + dM.B=i, etc., we have dx.M=dx.Mdu.K + dx.Mdu.B = dx.A +
dji-ud-UL-BJ and rfX'K=<^X'C + (*X-N(*N>B-
Therefore c?x-b= ^x-m^m-b +^x-A-b + ^Px-uPx-nPh-bPn-b
= P2x-mP\vb + P2xsP\-b + 2Px-uPx-nPu-bPx B
= (Px-mPm-b + Px-kPx-b)2
Px-B~ Px-T&PtA-B + Px-NrN-B-
These results are easily extended to cases in which B acts on X through
any number of causes. If a path coefficient is assigned to each com-
ponent path, the combined path coefficient for all paths connecting an
effect with a remote cause equals the sum of the products of the path
coefficients along all the paths. Since B is independent of A , C, and
D, rx.B= px.B = Px-mPm-b+Px-nPn-b-
GENERAL FORMULA
We are now in a position to express the correlation between any two
variables in terms of path coefficients. Let X and Y be two variables
which are affected by correlated causes M and N. Represent the various
path coefficients by small letters as in the diagram. Let A , B, and C be
hypothetical remote causes which are independent of each other (fig. 6).
*'xy=/,x-a/,y-a + />x-b/,y-b + />-c/Vc
= mam' a + (mb+nb') {m'b + n'b') + ncn'c
— mm' + mbb'n' + nn' + nb'bm' .
568
Journal of Agricultural Research
Vol. XX, No. 7
Thus, the correlation between two variables is equal to the sum of the
products of the chains of path coefficients along all of the paths by
which they are connected.
If we know only the effects, X and Y, and correlated causes, such as
M and N, it will be well to substitute rMN for bb' in the foregoing formula.
>xy = Px-mPy-u + />x-m*mn/>y-n+ Px-sPy-s + />x-n*WVm-
We have reached a general formula expressing correlation in terms of
path coefficients. This is not the order in which knowledge of the coeffi-
cients must be obtained, but, nevertheless, by means of simultaneous
equations the values of the path coefficients in a system can often be
calculated from the known correlations. Additional equations are fur-
nished by the principle that the sum of the degrees of determination must
Fig. 6. Diagram showing relations between two
variables. A' and V, whose values are de-
termined in part by common causes, M and
N, which are correlated with each other.
Fig. 7. — Simplified diagram of factors which
determine birth weight in guinea pigs.
equal unity. The fundamental equations can be written in general form
as follows:
^X-A = PVa
^X'ab = 2 Px- Arx-B^AB
APPLICATION TO BIRTH WEIGHT OF GUINEA PIGS
As a simple example, we may consider the factors which determine
birth weight in guinea pigs (fig. 7).
Let X be birth weight; Q, prenatal growth curve; P, gestation period;
L, size of litter; A, hereditary and environmental factors which deter-
mine Q, apart from size of litter; C, factors determining gestation period
apart from size of litter.
For the sake of simplicity, it will be assumed that the interval between
litters (if less than 75 days) accurately measures the gestation period
Jan. 3, 1921
Correlation and Causation
569
and that the variables are connected only by the paths shown above.
In a certain stock of guinea pigs the following correlations were found:
Birth weight with interval, rxp = +0.5547.
Birth weight with litter, rXL = — 0.6578.
Interval with litter, r^ = — 0.4444.
We are able to form three equations of type rx? = '2px.xpY.K and three
of type 2/>2x.A + 22/>x.A/>x.BrAB= 1. These six equations will enable us
to calculate six unknown quantities. The six path coefficients in the
diagram in figure 7 can thus be calculated from the information given
here, but no others.
The equations are as follows:
(I)
rxp= +0.5547 = p + qll'.
(2)
rXL= - 0.6578 = ql+ pi'.
(3)
rn.=— 0.4444=/'.
(4N
• q2 + p2+2qpir=i.
(5)
a2 + l2=i.
(6)
/'2 + C2=I.
From (3),
Pv-l=1'= -0.4444 dr
From (6),
Pv-c = c = 0. 8958 dp
From (1) and (2), px.r = p = o. 3269
ql=—o. 5125
From (4), Px-Q = q = 0.8627
Pq-l=1 =—0.5941
pQ.\ = a = o. 8044
dp.L =/'2
= 0. 1975
d —r2
aP-c — c
= .8025
1. 0000
dx.v =p2
= 0. 1069
dx.Q =q2
= -7442
dx.7Q = 2pqH'
= . 1489
1. 0000
dQ.L=l2
= 0.3530
dQ.A = a2
= .6470
1. 0000
^•q-l =q2i2
= 0. 2627
dx.P.L =p>l'2
= . 02 1 1
dx-^-L=2pqW
= . 1489
dx.L =(ql+pl')2
= • 4327
dx.A = q2a2
= .4815
dx.c=p2c2
= .0858
1. 0000
57°
Journal of Agricultural Research
Vol. XX, No. 7
Assuming that the diagrams in figures 7, 8, and 9 accurately represent
the causal relations, it appears that birth weight is determined to a very
much greater extent by variations in the rate of growth of the foetuses
than by variations in the length of
the gestation period (^x-q==°-74»
dx.P = o.ii). Size of litter has much
more effect on birth weight by re-
ducing the rate of growth of the
foetuses than by causing early partu-
rition (dx.Q.L = 0.26, </x.p.l = o.02). The
difference in birth weight caused
by a difference of a day in gestation
period can be calculated from the path
coefficient and the standard deviations
Fig. 8. — Path coefficients measuring the rela-
tions between birth rate (AT), rated growth
(Q), gestation period (P), size of litter (L),
and other causes (.4 , C).
by the formula for path regression, p. regx-p = px.r--^' The result, 3.34
Ox
0p
gm. per day, should measure the average rate of growth just preceding
parturition. The actual regression, 5.66 gm. per day of delay in parturi-
tion, is larger because a long gestation period means not merely a longer
time for growth but also, in general, a smaller litter and hence more
rapid growth.
On introducing other data the analysis can be carried much farther.
There are other paths of influence which should be recognized, positive
paths connecting A, C, and L, representing the favorable effects of good
health in the dam on rate of growth, gestation period, and size of litter,
and a negative path from Q to P
to represent the tendency of rapid
growth to induce early parturition.
The relations between the observed
interval between litters and the ac-
tual gestation period should also be
considered. The results presented
here are thus intended merely to fur-
nish a Simple illustration Of the Fig. 9.— Coefficients of determination. Symbols
method. A more complete analysis as in figure 7'
of the relations among the factors which affect birth weight and later
growth will be presented in a later paper.
DETERMINATION IN TERMS OF CORRELATION
Having obtained a formula for correlation in terms of determination,
the question arises whether the converse is possible. For a special class
of cases such a formula is easily obtained.
Jan. 3, 1921
Correlation and Causation
57i
For a single cause and effect the required formula is merely d^-r2^
(fig. 10).
?4
Fig. 10.— Effect and one known cause.
The degree of determination by residual factors; that is, a\.0, is thus
1— r
If two causes are known, and the degree of correlation between them,
we have (fig. 11) —
,4
pK u.— Effect and two correlated known causes.
Br XA + Br XO~ X
\2
(i-^2xb)(i-»'2ab) I-^XB
r2xo = ^x-o =
I — y XA — r XB ' AB
i — r2
»'2xB-»'2ab+2»-xa'xb''ab.
Fig. 12.— Effect and three correlated known causes.
If three causes and their correlations are known (fig. 12), we have
cb'2xa + cb^xo = 1 . * r°m which
1 - Zr2XA + 2?rXArABr»x ~ zSriAWgcTCT + Sr2XAr2BC .
r2 = d =
' xo — ux-o
- y2AB - r\c - r\c + 2 rACrCBrv
572
Journal of Agricultural Research
Vol. XX, No. 7
In this expression 2r2XA means the sum of squares of the six known
correlations. 2rXArABrBX means the sum of the products of the groups of
three correlations, corresponding to the sides of triangles. There are four
of these triangles, XAC, XAB, XCB, ABC. 2rXArABrBC»''cx means the
sum of the three products of the groups of correlations which are
arranged in closed quadrilaterals, and 2r2XAr2BC means the sum of the
product of squared correlations in pairs which involve no common vari-
able (r2xxr2BC, r2xcr
:) (%. 13)-
The formula for four known causes is easily found by a continuation
of the methods used to find the others if attention is paid to the sym-
metry of the expressions. Since, how-
*^* - - .£"""* ever, this formula, as well as that just
given for the case of three causes, is some-
what cumbersome, it will be convenient
to use a more condensed notation.
<j>(XABC . . .) may be used for a func-
tion involving all possible correlations
among the variables (XABC . . .). In
the definitions 2r2 means the sum of the
Fig. 13.— Effect and four correlated known squares of all correlations ; 2rV2, the sum
of the product of all pairs of squared
correlations which involve no variables in common; Zrrr, Zrrrr, and
Xrrrrr are the sums of the products of all groups of correlations which,
represented by paths, form closed figures, triangles, quadrilaterals, and
pentagons, respectively. "St^rrr is the sum of the products made by
multiplying each triangle of correlations in the sense above by the sec-
ond power of those correlations which do not involve any of the vari-
ables in the triangle. The number of terms of each kind is given above
the brace, where it is more than one.
<t>(AB)
1 — r2 (2 terms).
<t>(ABC) = 1 - 2r*+ 22m- (5 terms).
<I>(ABCD) =1— 2r2 + 22m-— 22mr-t-2rV (17 terms).
10 10 15 12 15 10
<j>(ABCDE) = i— 'Lr2jr2Lrrr—2'Zrrrr-\-2Zrrrrr-\-'Lr2r2—2'Slr2rrr (73 terms).
jan. 3,1921 Correlation and Causation 573
The formulae for degree of determination by residual factors may be
written as follows :
dx-o = <i>(XA) in system XA.
4(XAB) . „,D
x-°= 4>(ab) m system XAB-
<t>(XABC) . vat,^
x-0 = 4>{ABC) m system XABC-
<t>(XABCD) . „ , D_n
x'° = <j>(ABCD) m s>"stem X^5CI?
The degree of determination by the known causes is now easily cal-
culated. When all causes of variation in X are constant except A,
variation of X is measured by o-'-cb^x arjd variation of A is meas-
ured by o-'-cb^aj writing the constant factors as subscripts to the left.
Assuming that the relation between A and X is linear, the deviation of
X determined by a unit deviation of A should be constant, whatever the
amount of variation in A . Thus :
. (TX=(TX-A = 0-'-CB<TX
aA °A 0"*CB°"a
In the case of the residual factor O, assumed to be independent of the
known factors A, B, C, etc., ...CBA(r0 = (r0,
and we have crx.0= ...cba^x
dv.n =
<t>(XABC...)_*\.0 ...CBA<72x
"x'° 4>(ABC.) <j\
Thus:
2 <j>(XABC.) 2
" -cba^x ^ABC.) ° x*
This should be the general formula for the squared standard deviation
with a number of constant factors.
Hence :
r2
4>{XBC...O) 2 /<f>(ABC...O)
'' 4>(BC...O) <7x/ <p(BC...O) (
<t>(XBC...O)
4>{ABC...O)1
U{XBC...O)
Px'A ^ 4>(ABC...O)
4>(XBC...O) 4>{XBC...)-dx.04>(BC...)
x'K~(j>{ABC...O) ~ 4>(ABC.)
17777°— 21 5
574
Journal of Agricultural Research
Vol. XX, No. 7
The general formula for partial correlation can easily be expressed in
the present terminology.
L^ X — DCB0^ Xl1
J
r2 =
DCB' XA
A. = i —
cf>(XABCD)ct>(BCD)
4>{ABCD)<t>{XBCD)
In some cases it may be of interest to find the degree of determination
when a number of factors not in the direct path between cause and effect
are assumed constant.
3°^X-A —
(o.-.rTS-.-CB0" xXuTsfr a)
(o".uts...Cb°'2a)(uts0' x)
J>(XBC...STU...O)4>(ASTU)
<j>(ABC...STU)<fi(XSTU)
RELATION TO MULTIPLE CORRELATION
The expressions denned as <f>(XABC...), etc., suggest the expansion of
determinants. It is in fact easy to show that <f>(XABC. ..N) =A.
Where
A =
The formula for Pearson's coefficient of multiple correlation has already
been given, ^x(ABC0) = -J i - ~L
where Axx is the minor made by
deleting row X, column X.
Evidently in this class of cases the coefficient of determination degen-
erates into a function of the coefficient of multiple correlation. For the
degree of determination by residual factors we have
_<f>(XABC.)
ix'°~ <t>(ABC.)
= i-R2
X(ABO")
in agreement with Pearson's results.
For the degree of determination by a known factor we have
0(XgC...O)_0(XgC...)-dx.o0(gC...) AAAAXX-AAAA„
flx'A 4>{ABC...O) 4>(ABC.) A2IX
Px-A
jan. 3,1921 Correlation and Causation 575
The last formula brings out the close relation between the path coeffi-
cients and multiple regression. As already noted, the most probable
deviation of X for known deviations of A, B, C, etc., is given by the
formula
X' AXA/1' AXBB' A/ £'
°x Axxo-A Axxo-B 0-A <7B
As already stated, Pearson's coefficients of multiple correlation and
regression were not devised especially for the analysis of causal relations.
The formula for multiple regression, for example, gives the most proba-
ble value of one of the variates for given values of the others regardless
of causal relations. In cases in which all the correlations are known
in a system including an effect and a number of causes the method can
be used to find the path coefficients and the degrees of determination
of the effect by each cause in the sense used in this paper. Such cases
in which the direct methods can be used are, however, relatively
uncommon. Where the system of paths of influence is at all com-
plex, involving perhaps hypothetical factors, the causal relations can
be analyzed only by the indirect method of expressing the known cor-
relations in terms of the unknown path coefficients, making the sums of
the degrees of determination unity and solving the simultaneous equations.
PART II. APPLICATION TO THE TRANSPIRATION OF PLANTS
A large body of experimental data on the factors which affect the rate
of transpiration in plants has been published by Briggs and Shantz (2).
These data are well adapted for use in illustrating the methods of analyz-
ing causal relations presented in part I of this paper.
The experiments which are used in this paper were conducted at
Akron, Colo. , in 1 91 4. A variety of crop plants were grown in sealed pots.
The total transpiration was measured each day. Among the environ-
mental factors studied were the total solar radiation during the day, the
wind velocity, the air temperature (in the shade), the rate of evaporation
from a shallow tank, and the wet-bulb depression (sheltered from sun but
not wind) . The correlations between the daily transpiration of each kind
of plant and the integrated values of the environmental factors were pub-
lished by Briggs and Shantz. In order to avoid the effect of seasonal
change in the plants, the logarithms of the ratios of the transpiration on
succeeding days were correlated with similar figures for the various factors.
The correlations between the various environmental factors for the 100
days from June 18 to September 25, 191 4, have been calculated by the
writer from the data presented by Briggs and Shantz. This period covers
all the crop periods but is longer than most of them. None of the corre-
lations appeared to depart much from linearity.
576
Journal of Agricultural Research
Vol. XX, No. 7
The daily averages, the standard deviations, and the correlations are
given in Table I.
Table I. — Daily averages, standard deviations, and correlations from experiments on
transpiration in crop plants made by Briggs and Shantz at Akron, Colo., IQI4
CORRELATIONS
Wind .
Radiation
Temperature
Wet-bulb depression
Evaporation
Small grains0
Rye
Sorghum, millet^
Sudan grass (in inclosure)
Sudan grass (in open). . . . .
Dent corn
Algerian corn
Cowpea, lupine c
Alfalfa d
Amaranthus
Wind.
-o. 01 ±0-07
- . 02 ± . 07
. 28 ± .06
.38 ± .06
.22 ± .04
. 19 ± .10
.2l8±
•52 ±
•32 ±
.28 ±
•33 ±
•335±
. 290 ±
. 04 ±
. 041
.07
.08
.08
.09
•°57
•03s
. 10
Radiation.
o. 01 ±0.07
570±
55 ±
52 ±
52 ±
62 ±
570±
430 ±
40 ±
•°5
.04
•03
.06
.030
.06
.07
.06
.06
. 042
.030
.09
Temperature.
-0.02 ±0.07
•47 ± -05
• 59 ± -OS
.56 ± .05
.71 ± .02
• 73 ± -°5
• 653± .026
.84 ± .03
.81 ± .03
.71 ± -°4
• 79 ± -04
•675±
•495±
•45 ±
.035
029
oS
Wet-bulb
depression.
28 ±0.06
48 ± -05
59 i -OS
83 ±
88 ±
94 ±
788 ±
83 ±
85 ±
81 ±
88 ±
785±
7oo±.
60 ±
. 02
• 025
019
Evapor-
ation.
o. 38 ±0. 06
.68 ± .04
.56 ± .05
-.83 ± .02
87 ± .02
91 ± .02
7I3± -°21
93 ± .01
82 ± .03
79 ± -°3
• 8S ±
• 775±
• 705 ±
• 56 ±
•03
.025
. 019
.06
Mean. a
Evaporation (shallow tank) (kilograms per square meter) 9. 70 2. 76
Integrated radiation (calories per square centimeter) 753 134
Air temperature, integrated mean (degrees Centigrade) 20. 10 3. 48
Integrated wet-bulb depression (hour degrees, Centigrade) 143 58
Wind velocity (miles per hour) 5-54 2. 24
a Averages x>i six similar correlations involving Kubanka and Galgalos wheat, Swedish Select and Burt
oats, Hannchen barley, and spring rye. The last, having on the whole the largest correlations, is also given
SCft Averages of four correlations, Minnesota Amber and Dakota Amber sorghum and Kursh and Siberian
Millet. These correlations were all very similar.
« Average of the similar correlations for cowpeas and lupine.
<* Average of four tests with alfalfa.
* Published as + 0.80, which seems too large. Recalculation gives + 0.52.
It will be interesting first to com-
pare the direct and indirect methods
of calculating path coefficients and
coefficients of determination. Let us
consider the relations of wet-bulb
depression (B) to temperature (T),
wind velocity (W), and radiation (R).
Since the direct methods are only
applicable in systems in which each
variable is connected with every
other variable, the diagram of rela-
tions is as shown in figure 14.
Outstanding factors, independent of
W, R, and T are represented by O.
*^M.
Fig. 14. — Relations between wet-bulb depression
(B), wind velocity (W), radiation (R), and tem-
perature (T) as assumed for direct analysis.
Jan. 3,i92i Correlation and Causation 577
INDIRECT METHOD
Six equations can be formed, expressing the six known correlations in
terms of the unknown path coefficients. A seventh equation represents
the complete determination of B by W, R, T, and O.
(i) rBw = o.2& = w + t(c+bs) + ub.
(2) ?-br= .4& = wb + ts + u.
(3) rm= .59 = w(c + bs) + t + us.
(4) rWR=- .01 = 6.
(5) Twt= — .02 = c+bs.
(6) rRT= 47 = s.
(7) o2 + w2 + f + u2 + 2wt(c + bs) + 2wub + 2Uts = I .
The values of b and s are given directly from equations (4) and (6) ,
and the value of c (=—0.0153) can then be obtained from (5). The
solution of (1), (2), and (3) gives w = 0.2921, t = 0.4735, and 14 = 0.2604.
Finally, from (7) we obtain o2 = 0.5138 as the degree of determination by
outst anding factors.
= 0.5138
pB.Vf=W =0.2921
pB.T=t = .4735
pB.R = u = .2604
dB.0 = o2
=
0.5138
dB.w = w2
=
•0853
"B-T= t"
=
.2242
dn.R = u2
=
.0678
dB.^=2wt(c+bs)
= -
•0055
dB.^ = 2wub
= —
.0015
dB.—=2uts
=
•"59
1. 0000
DIRECT METHODS
According to the formulae given in part I we have
${BWRT)
dR.0 =
dn.p =
(J>(WRT)
4>{BRT)-dB.MRT)
4>{WRT)
4>(BWT)-dB.0cj>(WT)
4>{WRT)
cj>(BRW)-dB.0ct>(RW)
where
B'T 4>{WRT)
4>{BWRT) = 1 - r2BW + 2rBVfrWRrRB- 2rByfryfRrKTrTB + r2BWr2RT
— r2BR+ 2rBwrWT^TB— 2rBwTwT»'TRJ'EB'+y2BR^2WT
— r2BT + 2rBRrUTrTB— 2rBRrRWrvrTrT:B+r2BTr2vrR
— r2WR+ 2ryrRrRTrTW
— r^wT
— r2RT
<t>(WRT) = 1 — r2WR — r2wT — ^rt + 2rWB.rHTrTw
<j>(BWR), etc., are analogous to 4>{WRT)
<t>(RT) -i-f8,, 4>(WT), etc., are analogous to 4>(RT).
578
Journal of Agricultural Research
Vol. XX, No. 7
By substitution of the correlations in these formulae the following
results are obtained:
4>(BWRT) = 0.4002
<t>{BWR) = .6884
4>{BWT) = .5665
4>(BRT) = .4668
cj>(WRT) = .7788
4>(BW) = 0.9216
<£(£#) = .7696
cj>(BT) = .6519
0(PFi?) = 0.9999
<j>(WT)= .9996
<K#T) = .7791
These give values of the coefficients of determination identical with
those given by the indirect method.
This method, as was shown in part I, is essentially the same as Pear-
son's method of calculating multiple regression.
Let A =
Let
I
*BR
fBT
fBW
fRB
I
fRT
to
fTB
fTR
I
^TW
rwB
fWR
r-wT
I
0.48
o.59
0.28
= 0.4002
48
1
•47
— .01
59
•47
1
— .02
28
— .01
— .02
1
ABB = A with column B, row B, deleted.
Abb588 0.7788, ABK = 0.2028, ABT = 0.3687, ABW = 0.2275
ABW - A
pB-vr=T— =0.2921
P =~
rB-R ^
-—=0.5139
0.2604
Abt
/'b-t = T- =0.4735.
-*bb
These values are identical with those obtained by the preceding
methods.
It will be seen that the first method, while apparently less direct than the
others, is really less laborious. The solution of three simultaneous equa-
tions requires merely the evaluation of a determinant of the third order
instead of one of the fourth order, as in the last method. The expression
<j>(BWRT) in the second method is, of course, merely an expansion of
the same determinant of the fourth order as that used in the last. The
indirect method, moreover, gives more insight into the processes followed
than the others in which there is a substitution in what appear to be
arbitrary formulae. In line with this last point, the indirect method is
more flexible in that it can be used to test out the consequences of any
assumed relation among the factors.
ANALYSIS OF CAUSAL RELATIONS
In attempting to interpret the present results in terms of causation,
we see at once that the scheme of relations chosen is not a very satis-
factory one. The wet-bulb depression was measured under shelter. Con-
sequently the coefficient of determination, dB<E = 0.0678, can not measure
Jan. 3, 1921
Correlation and Causation
579
the degree of direct determination by radiation, but determination by
some factor other than wind or temperature with which radiation is
correlated.
One should not attempt to apply in general a causal interpretation to
solutions by the direct methods. In these cases, determination can usu-
ally be used only in the sense in which it can be said that knowledge of
the effect determines the probable value of the cause. This is the sense
in which Pearson's formula for multiple regression must be interpreted.
If W, T', and R' are given deviations of wind, temperature, and radiation
from their mean values, the most probable value of the wet-bulb depres-
sion, B', is given by the following formula:
B' W . R' T
This formula can only be used for conditions which are similar to those
for which the values of the path coefficients were calculated. If path
coefficients were calculated in a sys-
tem which truly represented the
causal relations, the formula would
give the value of the wet-bulb de-
pression under any set of conditions
in so far as it is determined by the
factors considered.
The causal factors which actually
determine wet-bulb depression are
temperature, absolute humidity (H),
and wind velocity (fig. 15). Radia-
tion can be introduced into the scheme
as a factor correlated with these causal factors. Wind velocity is cor-
related to such a very slight extent with temperature and radiation that
its correlation with absolute humidity can probably be neglected without
serious error. The relations between radiation, temperature, and abso-
lute humidity are undoubtedly very complex. Radiation has a direct
positive influence on temperature. Both radiation and temperature have
positive effects on absolute humidity by increasing evaporation. Cor-
relation between absolute humidity and temperature would be expected,
because with reduced temperature the saturation point is reached at a
lower absolute humidity and the excess moisture is precipitated. In-
crease in humidity, on the other hand, tends to reduce the radiation
which reaches the earth, and directly or indirectly this has a negative
influence on all three of the correlations.
There are not enough data to estimate the importance of all of these
paths of influence. Even if we represent the complex of paths connecting
H, R, and T merely by three correlations, the diagram has eight paths to
solve. The six correlations between B, W , R, and T and the statement
FlG. 13. — Relations between factors of figure 14
and absolute humidity (II) expressing causal
relations better than figure 14 but adapted only
to indirect analysis.
580 Journal of Agricultural Research vol. xx.No. ?
in regard to complete determination of B by W, H3 and T furnish only
seven equations.
Fortunately, data are given in another paper by Briggs and Shantz (j)
from which an eighth equation can be derived. In this paper the average
value of each of the measured factors is given for each hour of the day.
The cycle of changes in wet-bulb depression follows very closely the
changes in temperature. In fact, there should be very little, if any,
regular hourly cycle of changes in absolute humidity, so that the wet-
bulb depression should be wholly determined by the temperature changes
except for some influence of wind velocity.
Let pB-T = t be the path coefficient which measures the relative influence
of temperature on wet-bulb depression in the variations from day to
day. Let pB-u = h, pB-w = iv> and let aT, aH, aw, and <rB be the standard
deviations of the daily differences in the various factors and in wet-bulb
depression. Let T' — T", etc., be the actual differences in temperature,
etc., at certain times. The difference to be expected in wet-bulb
depression, B' — B" , is as follows:
B'-B" T'-T" W'—W" , H'-H''
= 1-\ w-\ h.
While t, w, and h are assumed to measure the relative influence of tem-
perature, wind, and humidity in the variations from day to day, the
foregoing formula should apply under any conditions, if t, w, and h were
calculated from a system which represented truly causal relations.
The expression — t is shown in part I to give the change in wet-bulb
depression (B) directly caused by a unit change in temperature. The
relative importance of the various factors in determining the variations
from hour to hour is very different from that from day to day, but the
change in wet-bulb depression caused by unit changes in temperature,
wind velocity, or absolute humidity should always be the same so long
as the relations are substantially linear.
The greatest difference in temperature within an average day in the
data was between 5 a. m. and 3 p. m. This is given as 32. 70 F., or
18.1670 C. The difference in wet-bulb depression between these hours
was 21.80 F., or 12.1110 C. The difference in average wind velocity was
2.5 miles per hour. The standard deviations of the daily variations have
already been given. 0-1 = 3.48 day degrees C, <rB = 58 hour degrees C.
integrated for 24 hours. This means 2.4167 degrees C. 0-^ = 2.24 miles
per hour. We will assume that there is no difference in absolute humidity
(H' — H" = o). Substituting those values in the formula for wet-bulb
depression, we get
12. in 18.167 , 2.50
— = ^.f\ — ~w
2.4167 3.48 2.24
5.0114=5.2204/+ i.ii6iw.
jan. 3,i92i Correlation and Causation 581
We now have eight equations from which to find eight unknown path
coefficients.
(1) W= 0.28 = w + tc.
(2) *br — .48 — ts + bw + ah.
(3) >'bt = .59 = t + dh + wc
(4) ^= — .01 = 6.
(5) %i = — .02 = c.
(6) rRT = .47 = ^-
(7) w2 + h2 + t2 + 2wtc+2htd= 1.
(8) 5.01 14 = 5.2204.24- 1.1 i6iw.
Equations (4), (5), and (6) give b, c, and s directly. Solution of (1) and
(8) gives 2 = 0.8963, its— 0.2979.
From (2) ah= 0.0617
From (7) h2= .6570, fe= — 0.8105, a = — 0.0761
From (3) dh = — .3003, d= .3706
rBU = h + td= — 0.4784.
The coefficients of determination, the path coefficients, and the corre-
lations are thus as follows :
dB.T = 0.8034 ^B"T = °-^9^3 rBT = °-59°°
^b-h = -657° ^b-h= — -8105 rBn= — .4784
dB.^ = .0888 pB-w= -2979 *W= .2800
^B-ii= — .5384
^B-^= — -OI07 *HE= — .O761
I. OOOI
rHT= .37°6
rRT = .47OO.
It turns out that the differences between different days in wet-bulb
depressions are due to a somewhat greater extent to differences in tem-
perature (0.80) than to absolute humidity (0.66). The variation in wet-
bulb depression would be much greater were it not that these factors
vary together but act on wet-bulb depression in opposite directions and
so tend to balance each other (dB.^=— 0.54). Temperature shows a
rather strong positive correlation with absolute humidity (0.37) as well
as with radiation (0.47), but the various paths of influence between
radiation and absolute humidity almost balance each other (rHE = — 0.08).
These results can now be used in finding the relative importance of
the various factors which determine evaporation or transpiration. In
figure 16, X may represent either evaporation or the transpiration of
any plant. Radiation must be considered as a direct causal factor in
these cases.
582
Journal of Agricultural Research
Vol. XX, No. 7
The following four equations can be made with which to solve the
path coefficients from W, H, R, and T to X: s
rxw=w'
+ t'c
+ u'b
rxt =w'c
+ t'
+ u's
+ h'd
rXR =w'b
+ t's
+ u'
+ h'a
rXB =wfrjm + t'r6T + ufrm + h'rm.
Substituting the values already found for a, b, c, d, w, h, t, and rBH>
we have
yxw= + i.oow' — 0.022' — O.OIM'
rxT = — .02w'+ 1.00/'+ -47u' + 0.3706/?'
rXR = — .01 w' + -47^'+ loom'— .0761/1'
rXB= + .28w'+ .59*'+ .48M'— .4784/1'.
The solution is as follows :
w' = Px'yr= + 0.9971 rxw + o.oi43rXT—o.oo22rXB + 0.01 1 4rXB
t' = Px-T =— .2207rxw+ .8943rxT— .8175^ + .8228rXB
u' = Px-R = + .i488rxw— .3633^+1.4155^— .5067^
h' = px.u-+ .4607*^ + .7468^+ .4107^— 1.5772^.
It is merely necessary to substitute the values of the correlations of
evaporation or transpiration with wind velocity, temperature, radia-
tion, and wet-bulb depression, as
given in Table I, to find the four
path coefficients in each case. The
results are given in Table II. These
have all been checked by substitu-
tion in the fourth equation (rXB= +
o.28ie/ +0.59^' + 0.48M'— 0.4784/1').
Thecorrelationbetween evaporation
and the transpiration of any plant
can be deduced from the formula
rXE = wVbw + t'rET + mVer 4- feVuH.
The correlations of evaporation with
wind velocity, temperature, and
radiation have been given in Table I
as 0.38, 0.56, and 0.68, and that
with humidity can be calculated by the formula rEU = pE.K + apE.-R +
dpK.T=— 0.2651. Thus rXE= 0.38W' +0.56^' + o.68m' — 0.2651/1'. The
calculated results in column 6 of Table II are compared with actual
correlations between evaporation and transpiration in column 7. The
correlation of evaporation with itself comes out 0.839 by this for-
mula. There should, however, be an additional term (px-o^Eo) m the
formula to allow for correlation through other factors (O) than W, T,
R, and H. From Table III we find that evaporation is determined
Fig. 16. — Relations between evaporations or trans
piration (..V) and the system shown in figure 15.
Jan. 3, 1921
Correlation and Causation
583
to a considerable extent (dE.0 = o.i6i) by outstanding factors. The addi-
tional term in this case would have this value and when added to 0.839
gives 1, as it should. With one exception, the calculated correlation
between transpiration and evaporation is a little smaller than the actual
correlation. This means either that there is some additional factor
which should be allowed for or else that the path coefficients with W, T,
R, and H are not given quite their due weight, owing perhaps to lack of
complete linearity in the correlations.
Table II. — Table of calculated path coefficients
Wet-bulb depression
Evaporation (shallow tank)
Transpiration:
Small grains
Rye
Sorghum and millet ....
Sudan grass (inelosure) .
Sudan grass (open)
Dent corn
Algerian corn
Cowpea and lupine
Alfalfa
Amaranthus
Average transpiration
/>x.
o. 298
•395
238
2 00
234
539
339
297
349
35 J
3°3
052
-79
Tempera-
ture.
pn.T
0.896
•544
Radia-
tion.
/>X.R
.249
. 207
■ 203
• 130
•°59
■ 109
• 194
.214
■ 117
• 105
.181
Absolute
humidity.
-o. 811
" -437
Correlation with
evaporation.
Calcu-
lated.
o. 830
(■ 839)
826
852
741
Actual.
0.83
1. 00
87
91
713
93
82
79
85
77S
705
560
781
Table III. — Coefficients of determination
Wind
Tem-
pera-
ture.
rfx-i
Radi
ation
Abso-
lute
hu-
mid-
ity.
Joint determination.
</x.wt ds
.WR
rfx.TR
(fx.TH
— 0. on
O
0.
-O. 538
— . 009 —
OO3
O. 202
— . I76
— . 007 —
OOI
. 182
- -283
— . 007 —
OOI
. 166
- -369
— . 007 —
OOI
• 137
— . 224
— . 019 —
OOI
. I06
— . I40
- .013 -
000
.051
- .258
— . 010 —
OOI
.084
— . 244
— . 012 —
OOI
• 155
— .247
— . 010 —
002
• 143
- . 182
— . 007 —
OOI
. 067
— . I9O
— . 001 —
OOO
•°55
- .178
— .008 —
OOI
. 124
- .228
</x.i
Residual.
</x-c
Wet-bulb depression
Evaporation
Transpiration:
Small grain ,
Rye
Sorghum and millet
Sudan (inelosure) . . ,
Sudan (open)
Dent corn
Algerian corn
Cowpea and lupine. .
Alfalfa
Amaranthus ,
Average transpiration . .
o. 0S9
156
OS 7
044
055
290
"5
088
122
123
092
oo_<
07S
0.803
. 296
. 607
. 728
.516
■757
.861
.664
.724
• S°4
■364
•3M
•537
o. 156
. 062
•043
■ 041
■ 017
• 003
. 012
• 038
. 046
• 014
. on
•033
0.657
. 191
. 240
•34°
■ 177
•047
. 141
. 164
• 153
. 120
. 180
.183
. 176
OK)
01 8
013
004
003
007
Ol 2
on
OOS
007
(-
•125
.038
•293
. 062)
.O96
■237
.057
■ 247
•474
. 607
.277
The coefficients of determination are given in Table III. The differ-
ence between their sum and unity is given in the last column as dx.0>
the determination by outstanding factors. As suggested above, the
assumption that all the fundamental correlations are linear may involve
584 Journal of Agricultural Research vol. xx.No. 7
some error which would tend to underweight the coefficients of deter-
' mination between transpiration and the known factors and so over-
weight the apparent degree of determination by outstanding factors. In
certain cases, however, the residue is so small, in one case actually com-
ing out negative, that it is probable that this is not an important source
of error. The residual determination is greatest for the crops which
were cut twice during the season — namely alfalfa and amaranthus.
There were considerable periods following each cutting during which the
absolute value of the transpiration was small.
Wind velocity has about the same relative value as a factor in deter-
mining transpiration as it has in determining wet-bulb depression. Its
relative importance is a little greater for determining evaporation from
the shallow tank.
Temperature is somewhat more important than absolute humidity in
determining the variations in wet-bulb depression and rate of evapora-
tion from day to day. It is very much the most important factor in
determining the rate of transpiration in all the plants.
Radiation is an important factor in evaporation, coming out equal to
wind velocity and only slightly less important than absolute humidity.
In the plants, on the other hand, it is almost a negligible factor.
Comparing transpiration in the average plant with evaporation in the
sun from a shallow tank, we find that the former is influenced relatively
much more by temperature, to about the same degree by absolute
humidity, somewhat less by wind velocity, and very much less by radia-
tion. The four factors are much more nearly equal in importance in the
case of evaporation (g?e-t = o.3o, <fE.H = o.i9, dE.w=o.i6, dE.E = o.i6) than
in the case of transpiration (c/x.t = 0.55, dx.H = o.i8, g?x.w = 0.09, <ix.E = 0.04).
In comparing the importance of these factors it should be added that
radiation has an importance somewhat in excess of its direct influence,
in that its variations are correlated with those of temperature. Humidity
has reduced importance, since, though correlated with temperature, it
affects evaporation and transpiration in the opposite direction.
OTHER APPLICATIONS
The method of analysis presented here can readily be applied to the
problem of the relative importance of heredity and environment. An
application of this kind to the case of the piebald pattern of guinea pigs
has already been published (9), and one to the resistance of the same
animal to tuberculosis is in press.1 The method can be applied also to
such a problem as the determination of the effects of various systems
of mating, such as inbreeding, line breeding, and assortative mating on
the genetic composition of an originally random-bred stock.2
1 Wright, Sewall, and Lewis, Paul A. factors in the resistance of guinea pigs to tuberculosis
with special regard to inbreeding and heredity. In Amer. Nat., v. 55. 1921. In press.
'Wright, Sewall. systems of mating, i to v. In Genetics, v. 6. 1921. In press.
jan. 3,i92i Correlation and Causation 585
LITERATURE CITED
(1) Bravais, A.
1846. ANALYSE MATHEMATIQUE. SUR LES PROBABILITES DES ERREURS DE
situation d'un point. In Mem. Acad. Roy. Sci. Inst. France, Sci.
Math, et Phys., t. 9, p. 255-332.
(2) Briggs, Lyman J., and Shantz, H. L.
1916. DAILY TRANSPIRATION DURING THE NORMAL GROWTH PERIOD AND ITS
correlation with the weather. In Jour. Agr. Research, v. 7, no.
4, p. 155-212, 18 fig., 6 pi.
(3)
1916. hourly transpiration rate on clear days as determined by cyclic
environmental Factors. In Jour. Agr. Research, v. 5, no. 14, p.
583-649. 22 fig., pi. 53-55.
(4) G alton, Francis.
1888. co-relations and their measurement, chiefly from anthropo-
metric data. In Proc. Roy Soc. London, v. 45, no. 274, p. 135-145.
(5) ISSERLIS, L.
1914-15. on the partial correlation ratio, i-ii. In Biometrika, v. 10,
pt. 2/3, p. 391-411, 1914; v. 11, pt. 1/2. p. 50-66, 1915.
(6) Pearson, Karl.
1897. mathematical contributions to the theory of evolution.— hi.
regression, heredity, and panmixia. In Phil. Trans. Roy. Soc.
London, s. A., v. 187, 1896, p. 253-318.
(7)
1905. mathematical contributions to the theory of evolution.— XIV.
ON THE GENERAL THEORY OF SKEW CORRELATION AND NON-LINEAR
regression. Drapers' Co. Research Mem. Biom. Ser. 2, 54 p. 3 pi.
(8) Wright, Sewall.
1918. on the nature OF size factors. In Genetics, v. 3, no. 4, p. 367-374.
(9)
1920. THE RELATIVE IMPORTANCE OF HEREDITY AND ENVIRONMENT IN DE-
TERMINING THE PIEBALD PATTERN OF GUINEA PIGS. In ProC. Nat.
Acad. Sci., v. 6, no. 6, p. 320-332. 6 fig.
MEASUREMENT OF THE AMOUNT OF WATER THAT
SEEDS CAUSE TO BECOME UNFREE AND THEIR
WATER-SOLUBLE MATERIAL
By George J. Bouyoucos and M. M. McCool
Michigan Agricultural Experiment Station
INTRODUCTION
It has been shown that soils cause water to become inactive or unfree,
as is indicated by its refusal to freeze or to function as a solvent. The
magnitude of this unfree water has been measured by means of the
dilatometer method,1 which has proved most convenient, appropriate,
and unique for this purpose. The principle of this method is based
upon the fact that water expands upon freezing. If the amount of
expansion that a certain amount of water (i gm.) produces upon freezing
is known, then the quantity of water that freezes in the soil can be cal-
culated from the magnitude of expansion produced. On the basis of
this dilatometer method the water in the soil has been classified anew
as follows :
i. Gravitational water, unsuitable to plants.
2. Free water, readily available to plants.
I Capillary, adsorbed, very slightly available to plants.
| water of hydration 1 very unavailable to
1 water of solid solution J plants.
The free water is that which freezes very readily at the supercooling
of —1.50 C. ; the capillary, adsorbed water is that which freezes from
the temperature of — 1.50 to — 780; while the combined water is that
which does not freeze at all, even at the extreme temperature of — 780.
AMOUNT OF WATER THAT SEEDS CAUSE TO BECOME UNFREE
It is, of course, very well known that seeds absorb large quantities of
water and with a considerable force. Seeds like the lima bean, cowpea,
soybean, clover, and alfalfa absorb over 100 per cent of their dry weight
of water; while seeds like the wheat, rye, and corn absorb about 50
per cent of their dry weight of water. The great attraction that seeds
have for water is best realized by the fact that they will abstract the
moisture from the soils even down to the point of air-dryness. Whitney
1 Bouyoucos, George J. measurement of the inactive, or unfree, moisture in the sou, by
means of the dilatometer method. In Jour. Agr. Research, v. 8, no. 6, p. 195-217, 1 fig. 1917. Litera-
ture cited, p. 217.
classification and measurement of the different forms of water in the soil by means
of the dilatometer method. Mich. Agr. Exp. Sta. Tech. Bui. 36, 48 p., 5 fig. 1917-
and McCool, M. M. further studies on the freezing point lowering of soils. Mich. Agr.
Exp. Sta. Tech. Bui. 31, 51 p. 1916.
Journal of Agricultural Research, Vol. XX, No. 7
Washington, D. C Jan. 3, 1921
wi Key No. Mich.-i2
(387)
588 Journal of Agricultural Research vol. xx.No. 7
and Cameron 1 found, for instance, that when 50 gm. of seeds of cowpeas
were mixed with 50 gm. of soil containing 15 per cent of water, the
seeds had in 12 hours gained 12.1 per cent of water and had left in the
soil only 1.3 per cent — that is, the soil was reduced practically to air-dry
condition. It appears, therefore, that the power of seeds to absorb
water is very much greater than that of soils. Some attempts have
been made to measure the magnitude of the initial attraction that seeds
possess for water. Especially notable is the work in this direction of
Shull 2 who attempted to measure the attraction of seeds of Xanthium
for water, and then he used these seeds to measure in turn the moisture-
holding forces of soils. Shull found that the air-dry seeds of Xanthium
show an initial attraction for water of nearly 1 ,000 atmospheres.
Since it was found that soils cause water to become unfree, the extent
varying with the character of the soil, the question arose whether the
seeds also cause water to become unfree, and if so, to what extent. It
was reasoned and anticipated that since seeds possess a greater attraction
for water as evidenced by their power to abstract moisture from the
soil itself even down to the point of dryness, they ought to cause a
larger amount of water to become unfree.
In order to obtain information bearing upon these questions a general
investigation of the problem was undertaken. The type of dilatometer
used and the general procedure followed were the same as those used in
the study of soils. The procedure consisted in weighing out carefully
about 10 gm. of air-dry seeds and placing them in water to soak for
about two days. Then they were taken out, pressed between filter papers
in order to eliminate their excess of water, weighed again quickly, and
introduced into the dilatometer. The unoccupied space in the dilatom-
eter was then filled with ligroin, and care was taken to expel all the
air. The mouth of the dilatometer was then carefully stoppered, and the
contents were placed to cool in a temperature of — 30 C. When this
temperatuie was attained by the contents, as indicated by the column of
ligroin in the stem, which remained stationary, the water in the seeds was
caused to freeze. This was accomplished by taking hold of the dilatom-
eter by the stem and moving it gently in the cooling mixture until
solidification began, which was indicated by the rise of the ligroin in the
stem. The dilatometer was allowed to remain in the cooling mixture
with frequent movements until the rise of the ligroin in the stem ceased.
The total rise of the ligroin in the stem was taken to represent the total
amount of expansion due to the formation of ice.
In order to determine the effect of repeated freezing or of lower tempera-
ture upon the amount of water that seeds cause to become unfree, the
1 Whitney, Milton, and Cameron, F. K. investigations in soil fertility. U. S. Dept. Agr. Bur.
Soils Bui. 23, p. 30. 1904.
2 Shull, Charles Albert, measurement of the surface forces in soils. In Bot. Gaz. v. 62, no. 1,
P- 1-3 1. 5 fig- 1916. Literature cited, p. 29-31.
Jan. 3, 1921
Amount of Unfree Water Caused by Seeds
589
seeds in the dilatometer were thawed and refrozen either at the temperature
of — 30 or of — 200 C. In the latter case, the contents of the dilatometer
were allowed first to supercool at — 30 and to assume equilibrium at this
temperature; then they were put in the temperature of — 200, allowed to
remain there for about one hour, and were then placed back into the
temperature of — 30 and allowed to attain equilibrium.
In all, 14 different kinds of seeds were used. These were spring
wheat, winter wheat, barley, rye, white corn, yellow corn, broom corn,
alfalfa, alsike clover, mammoth clover, cowpeas, field peas, field white
beans, and black soybeans.
In Table I are presented part of the data obtained. They show the
amount of water the different kinds of seeds absorbed and the quantity
they caused to become unfree, as indicated by its refusal to freeze for the
first time at the temperature of — 30 C. The quantity of unfree water is
expressed both in cubic centimeters and in percentage based on the
weight of the air-dry seeds. The factor used for converting the volume
of expansion due to the ice formation into the corresponding weight of
water was that obtained experimentally and used in the study of the
soil — namely, 1 cc. of water expands approximately 0.1 cc. upon freezing.
Table I. — Amount of water that failed to freeze in seeds when they were supercooled and
frozen for the first time in a temperature of — j° C.
Kind of seeds.
Spring wheat. .. .
Winter wheat . . .
Barley
Rye
White corn
Yellow corn
Brown corn
Alfalfa
Alsike clover. . . .
Mammoth clover
Cowpeas
Field peas
Field white peas
Black soybeans. .
Weight of
air-dry
seeds.
Gm.
11. 2IO
xi. 250
10. 770
10. 230
12.07s
12. 215
10. 020
11. 3OO
II. 200
II. 200
9. 2IO
10. 070
10. 275
7. no
Weight of
water-
soaked
seeds.
Gm.
18. 290
18. 510
18. 080
18. 150
17. 640
17. 265
16. 230
25- 73°
24. 200
25.700
20. 790
20. 800
20. 320
16. 920
Absorbed water which
failed to freeze.
Cc.
2.880
3-36o
4-3xo
3.920
3-765
4.650
2. 510
8.430
7. 400
7. 100
6.680
7-73°
5-445
5-3io
Per cent.
25.70
30. IO
40. 02
40. 20
3I-I8
38.09
25-05
74. 60
66.08
63.40
72-54
76. 76
52.96
74.68
From the foregoing experimental data it is at once seen that the
amount of water which the seeds cause to become unfree is really very
great in nearly all the different kinds of seeds. It varies from about
25.05 per cent with broom corn to 76.76 per cent with black soybeans.
It appears that the alfalfa, clover, cowpeas, and bean seeds cause a
considerably larger amount of water to become unfree than the wheat,
rye, barley, and corn seeds. As has already been mentioned, this
17777°— 21 6
59o
Journal of Agricultural Research
Vol. XX, No. ?
percentage of unfree water is based only on the absorbed water; the hygro-
scopic moisture is not included in it. Hence the total amount of unfree
water in the seeds is still greater than is represented by these numerical
data.
In the foregoing investigation the seeds were supercooled and frozen
only once in the temperature of — 30 C. The investigations with soils
revealed the fact that repeated freezing and thawing and lower tem-
perature tended to reduce the amount of unfree water in soils, especially
in the fine-textured and colloidal soils. In order to ascertain whether
repeated freezing and thawing and lower temperature brought also a
diminution in the unfree water in the seeds, the latter were frozen and
thawed three times in a temperature of — 200. Finally they were super-
cooled to — 30, frozen in — 200 for one hour, and brought back again
to— 30, where the total expansion was measured. Table II contains
the results obtained from this investigation. For immediate and con-
venient comparison the results obtained at the first freezing are also
presented in this table.
Table II. — Effect of repeated freezing and thawing and low temperature on the amount
of water that fails to freeze in seeds
Kind of seeds.
Water
which
failed to
freeze,
(frozen
only
once).
Spring wheat 25. 70
Winter wheat 3°- J°
Barley 40. 02
Rye 40. 20
White corn 31. J8
Yellow corn 38-09
Broom corn 25. 05
Alfalfa 74- 60
Alsike clover 66. 08
Mammoth clover 63. 40
Cowpeas 72- 54
Field peas 76. 76
Field white peas 52. 96
Black soybeans 74-68
Water
which
failed to
freeze,
(frozen
and
thawed
four
times).
Per cent.
25.70
28.98
35-38
35-39
23.70
26. 62
13.08
40. 98
40. 18
41. 08
39-95
57-9°
26. 78
47.96
Difference
in favor
of seeds
frozen
only
once.
Per cent.
0. OO
1. 12
4. 64
4. 8l
7-48
II.47
II.97
33- 6a
25.90
22. 32
32-59
18.86
26.33
26. 72
It is readily seen that repeated freezing and thawing has a very marked
diminishing effect on the unfree water in the seeds, especially with
certain kinds of seeds. In those seeds which contained a tremendous
amount of unfree water at the first freezing, such as the alfalfa, clover,
peas, and beans, the diminution in the quantity of unfree water by
repeated freezing and thawing is very considerable, amounting in some
cases to over 33 per cent. On the other hand, in such seeds as the
Jan. 3. 1021 Amount of Unfree Water Caused by Seeds 591
wheat, corn, barley, and rye the process of repeated freezing and thaw-
ing had very little effect if any on the unfree water.
The process of repeated freezing and thawing, therefore, has practically
the same influence in seeds as it has in soils. In both cases it tends to
diminish the amount of unfree water in some seeds or soils more than in
others.
In explaining the decrease of the unfree water by repeated freezing
and thawing two hypotheses were presented. In the one it was sug-
gested that part of the water is held by the capillarities of the soil and
does not freeze. Upon repeated freezing and thawing these capillarities
are destroyed, and the water they held is liberated or becomes free and
freezes readily.
In the second hypothesis it was assumed that soils such as clays, clay
loams, silts, muck, and peats contained a considerable amount of colloidal
material which held water in such a manner that it does not freeze.
Upon repeated freezing and thawing, however, these colloids are coagu-
lated or destroyed, and the water they held is liberated or becomes free
and readily freezes.
These suggested explanations with few modifications may apply also
to seeds. There is no doubt that the living tissue as well as its capil-
laries and colloidal material are affected or destroyed by severe freezing.
It may be of interest to record here that when very old corn seed was
employed or corn seed that had been frozen in the field, no water was
caused to become unfree. Apparently long age or previous freezing
of the corn seed destroyed its power to cause water to become unfree.
This phenomenon, however, did not appear in the other seeds.
According to the classification of moisture in the soils based on the
dilatometer method, the water which freezes after the first freezing
may be classified as capillary-adsorbed water, while that which refuses
to freeze after the fourth freezing and at the low temperature may be
classified as combined, probably in the form of water of hydration and
water of solid solution.
However, the division of the unfree water into capillary adsorbed and
combined water is probably not so sharp in seeds as in soils, because in
the seeds there is a considerable quantity of water-soluble material
which causes a high freezing-point depression, and this in turn decreases
the amount of water that freezes at the degree of supercooling employed.
As is well known, there is always a tendency for an equilibrium to be estab-
lished between the liquid-solvent, solid-solvent, and the solute at any
temperature below freezing until the cryohydric temperature is reached.
Some of the water, therefore, which refused to freeze at — 200 C. or which
froze and melted again at — 30 may be due to the water-soluble material
of the seeds. It is believed, however, that the amount of water that
was prevented from freezing by the high freezing-point depression of the
seeds is probably not very great.
592
Journal of Agricultural Research
Vol. XX, No. 7
AMOUNT OF WATER-SOLUBLE MATERIAL IN SEEDS AS MEASURED
BY THE FREEZING-POINT METHOD
Recognizing the influence that high concentration of solution has
upon the quantity of water that refuses to freeze, the authors always
determined the freezing-point depression 1 of the seeds after they were
used for the dilatometer measurements. It was found that the magni-
tude of this depression was high for most of the seeds. Since the seeds,
however, used in the dilatometer measurements were allowed to stand
about two days in excess of water and were then subjected to alternate
freezing and thawing, it was thought that the depression values obtained
were the result of the biological and physical changes that the seeds
underwent. In order to ascertain, however, whether the seeds contained
water-soluble material in the dry condition they were ground very fine
and then portions of 10 gm. were mixed with 20 cc. of water in a
freezing-point tube. The mixture was allowed to stand for about 40
minutes, and then its freezing-point depression was determined in the
usual way. Table III contains the results obtained. The values of the
freezing-point depression have also been calculated into osmotic pressure
in atmospheres after the table of osmotic pressures worked out by
Harris and Gortner.2
Tabi.U III. — Freezing-point depression and osmotic pressure of dry seeds when 10 gm.
of powdered dry seeds were mixed with 20 cc. of water
Kind of seeds.
Freezing-
point
depression.
pressure.
0 C.
A tmospheres.
O. 280
3-375
•352
4- 243
.280
3-375
• 34o
4. 098
. 280
3-375
.580
6.988
. 610
7-349
.650
7-830
.650
7.830
•7i5
8.612
•55°
6.628
.685
8.251
.560
6.747
1. 180
J3- 336
1. 060
12. 760
Spring wheat
Rye
Buckwheat
White corn
Broom corn
Sorghum
Alfalfa
Alsike clover
Mammoth clover
Cowpeas
Field peas
Field white beans . .
Black soybeans
Speckled wax beans
Red kidney beans . .
The results in Table III are very surprising. They show most strik-
ingly that there is a tremendous amount of readily water-soluble material
in seeds, and in some seeds much more than in others. Thus the depression
varies from 0.2800 C. in wheat to 1.1800 in speckled wax beans. When
1 Bouyoucos, George J:, and McCool, M. M. op. cit.
2 Harris, J. Arthur, and Gortner, Ross Aiken, notes on the calculation of the osmotic pres-
sure OF EXPRESSED VEGETABLE SAPS FROM THE DEPRESSION OF THE FREEZING POINT, WITH A TABLE
FOR the values of p for A = o.ooi° To £ = 2.999°. In Amer. Jour. Bot., v. i, no. 2, p. 75-78. 1914.
Jan. 3,1921 Amount of Unfree Water Caused by Seeds 593
r
it is considered that this relatively large depression is obtained in a ratio
of 1 of seeds to 2 of water (10 gm. of seeds and 20 cc. of water), then it can
be imagined what the depression must be at a very low moisture content.
It really must be large. In the ratio given here it varies from 3.375 at-
mospheres in wheat to 13.336 in speckled wax beans. The great attrac-
tion that seeds possess for water and their ability to abstract it from soils
even down to the point of air-dryness must be due, therefore, partly, if
not largely, to their great osmotic pressure caused by their high content
of easily water-soluble material.
No experimental work was performed to prove definitely the nature of
the material in the seeds which went into solution to cause such great
depression. But it appears to be largely water-soluble proteins such as
albumins and probably also some of the mineral bases. It can not be
starch, which is the most abundant constituent in the seeds, because
that is very insoluble in water. A test showed, for instance, that 10
gm. of starch in the pure form in 20 cc. of water had a depression of only
0.025 ° C. Sugar, of course, which is soluble, is not supposed to be found
in dry seeds. Furthermore, to give the high depression obtained, there
has to be present a very large amount of sugar, because as it is well known
that this class of material does not dissociate. All evidences, therefore,
point to the proteins as the main class of constituents in the seeds which
produced such high depressions in the freezing point when dry seeds in
the powdered form were mixed with water.
SUMMARY
Seeds cause part of the water which they absorb to become unfree, as
is indicated by its refusal to freeze.
The dilatometer method is a convenient and appropriate method for
measuring the magnitude of this unfree water in seeds.
The amount of water that seeds cause to become unfree is very large,
varying from 25.05 per cent in broom corn to 76.76 per cent in black
soybeans, based on the air-dry weight of seeds. Repeated freezing and
thawing tends to diminish considerably the amount of unfree water, espe-
cially in some seeds.
Dry seeds contain a large amount of water-soluble material, as is evi-
denced by the high freezing-point depression. When 10-gm. portions of
seed flour are mixed with 20 cc. of water and the mixture is allowed to
stand for about 40 minutes or less, the freezing-point depression varies
from 0.2800 C. in wheat to i'.i8o° in speckled wax beans. At very low
moisture content the magnitude of this depression must be very great.
The magnitude of the osmotic pressure must also be correspondingly very
great.
The great power that seeds possess to absorb water and to abstract
it from the soil is partly if not largely due to their tremendous internal
osmotic pressure.
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Vol. XX JANUARY 15, 1921 No. 8
JOURNAL OP
AGRICULTURAL
RESEARCH
CONTENTS
Page
Inheritance of Syndactylism, Black, and Dilution in
Swine - - " - - -595
J. A. DETLEFSEN and W. J. CARMICHAEL
( Contribution from Illinois Agricultural Experiment Station )
Four Rhynchophora Attacking Corn in Storage - 605
RICHARD T. COTTON
( Contribution from Bureau ol Entomology)
Concentration of Potassium in Orthoclase Solutions Not a
Measure of Its Availability to Wheat Seedlings - - 615
J. F. BREAZEALE and LYMAN J. BRIGGS
(Contribution from Bureau of Plant Industry)
Composition of Tubers, Skins, and Sprouts of Three Varie-
ties of Potatoes -------- 623
F. C. COOK
(Contribution from Bureau of Chemistry )
Further Studies in the Deterioration of Sugars in Storage 637
NICHOLAS KOPELOFF, H. Z. E. PERKINS,
and C. J. WELCOME
( Contribution from Louisiana Agricultural Experiment Station )
Freezing of Fruit Buds ------- 655
FRANK L. WEST and N. E. EDLEFSEN
( Contribution from Utah Agricultural Experiment Station)
Effect of Various Crops Upon the Water Extract of a Typi-
cal Silty Clay Loam Soil ------ 663
G. R. STEWART and J. C. MARTIN
( Contribution from California Agricultural Experiment Station )
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE.
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WASHINGTON : GOVERNMENT PRINTINO OFPIOE : 1821
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS,
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
NEW
BUT a
JOBRNAL OF AGRICUITIIRAL RESEARCH
Vol. XX Washington, D. C, January 15, 1921 No. 8
INHERITANCE OF SYNDACTYLISM, BLACK, AND
DILUTION IN SWINE1
By J. A. DETLEFSEN, Professor and Chief in Genetics, and W. J. Carmichael,2 Asso-
ciate in Animal Husbandry, Illinois Agricultural Experi?nent Station
Our present point of view in animal and plant breeding is being shaped
to a large extent by experiments in the field of genetics. Probably the
plant breeders have profited more by these experiments than the animal
breeders; for there are relatively few precise observations on inheritance
in domestic mammals, for obvious reasons. While the animal breeder
can not afford to neglect the conclusions obtained with pedigreed cul-
tures of laboratory material, nevertheless the data accumulated directly
from domestic mammals will more quickly stimulate clear thinking and
intelligent practice. For these reasons, among others, the following
observations are presented and put on record.
The data in this study are derived from an original cross between a single
pure-bred registered mule-foot boar and a number of pure-bred Duroc-
Jersey sows, eligible to registration. Both boar and sows were owned
by Mr. J. H. Percival, of Champaign, 111. The results of the cross were
so striking and uniform that we were invited to examine the progeny
born in the fall litters of 1915 and in the spring litters of 1916. All
the Fx offspring, about 250 in number,3 were self-colored black and mule-
footed. Furthermore, the progeny resembled the mule-foot boar in
general conformation (in which, as a matter of fact, both the sire and
the Duroc- Jersey sows were much alike). The case is a good illustra-
tion of one type of prepotency, where the sire is homozygous in a number
of conspicuous dominant characters, such as black and mule-foot in this
particular instance. But the progeny inherited as much from their dams
as they did from the sire, as the next generation showed. The vigorous
hybrids were raised for the market and not for further breeding purposes,
as is the case generally with such hybrids. Since the material seemed
1 Paper No. 9 from the Laboratory of Genetics, Illinois Agricultural Experiment Station.
2 Resigned May 31. 1918, to become Extension Animal Husbandman, United States Department of Agri-
culture; at present Secretary of the National Swine Growers' Association. The writers are indebted to
Mr. J. B. Rice, Associate in Animal Husbandry, for much assistance after the resignation of the junior
writer.
3 The number of Fi young in this paper is conservatively estimated at about 250. The exact number
can not be given because the animals were kept in a large pasture, which made an exact count difficult.
There is no doubt, however, that all Fi individuals were black and mule-footed.
Journal of Agricultural Research, Vol. XX No. 8
Washington, D. C Jan. 15, 1921
wj Key No. Ill.-io
!Q (595)
596
Journal of Agricultural Research
Vol. XX, No. 8
promising for further genetic investigation, six Fx sows were purchased
in June, 1916.
The six sows, numbered 1 to 6 in Tables I and II, were bred back to a
Duroc- Jersey boar, since this type was recessive in a number of charac-
ters in the original cross. Each female, except 9 5, gave at least one
litter, and $ 3 gave two litters. A total of 42 F2 offspring by this back-
cross was thus obtained. The original mule-foot Px parent was without
doubt homozygous in mule-foot and black and probably had the genetic
formula BBMM, where B is a factor for black and M is a factor for mule-
foot. The Duroc-Jerseys had the genetic formula bbmm, where b
stands for red, and m for cloven-foot. The Fx hybrids were then heterozy-
gous in both black and mule-foot (BbMm) and, if the case is one of simple
Mendel ism, produced gametes BM + Bm + bM + bm with equal fre-
quency. Mating the Fx females to the Duroc-Jersey male should give in
Mendelian terms:
BM +
bm +
Bm +
bm
bM+ bm
BbMm 4- Bbmm + bbMm 4- bbmm
black black red red
mule-foot cloven mule-foot cloven
Fx gametes
Duroc-Jersey gametes
F2 zygotes
That is, the F2 classes would be of four equally frequent types. The
calculated and observed results agree, for there were produced 8 black
mule-foot, 11 black cloven-foot, 9 red mule-foot, and 14 red cloven-foot
where 10.5 of each kind is the calculated result. (See Pi. 70.) So far
as the evidence goes, the allelomorphic pair of factors for syndactylism
and cloven-foot is quite independent of the allelomorphic pair for black
and red. The ultimate recessive segregates, red cloven, bred true when
mated inter se and gave 30 red cloven in the F3 and F4 generations.
Table I. — Distribution of F2 segregates from mule-footX. Duroc- J 'ersey F, hybrids mated
back to Duroc-Jersey
Dam No.
Offspring.
Males.
Females.
Total.
Black
mule-
foot.
Black
cloven-
foot.
Red
mule-
foot.
Red
cloven-
foot.
Black
mule-
foot.
Black
cloven-
foot.
Red
mule-
foot.
Red
cloven-
foot.'
I
I
I
3
2
3
6
2
4
2
8
I
I
2
I
I
5
7
8
3
1
3
2
2
I
I
I
1
1
I
I
6
2
8
Total . . .
4
7
3
7
4
4
6
7
42
jan. 15, 1921 Inheritance of Syndactylism and Color in Swine 597
Table II. — Original data on the F2, F3, and F4 offspring from a cross of a mule-foot
boar on Duroc- Jersey sows
Fi
Dam No.
Duroc-Jersey
sire.
Off-
spring
No.
Sex.
Color.
Foot
charac-
ter.
Date of birth.
Remarks.
Good Colonel,
i a
?
Red...
Cloven . '
No. 4i5x7
ib
9
Yellow.
. . do . . . .
1 c
id
9
Cream .
..do....
Mule . . .
..do....
*Nov. 8, 1916
1 e
9
Lemon .
..do....
1 f
9
Yellow.
..do.....
2
do
2 a
2b
Black. .
..do....
Cloven .
..do....
2 e
r?
..do....
..do....
2d
r?
..do....
..do....
2 e
9
Yellow.
..do....
>Mar. 7, 1917
Saved for
breeding.
2 f
9
Lemon .
..do....
Do.
2 g
9
Red....
..do....
2h
3
Yellow.
Mule . . .
do
3 a
3b
9
9
Black. .
..do....
Cloven .
..do....
3 c
3
..do....
..do....
>Nov. 8, 1016
3d
(?
..do....
..do....
3 e
r?
..do....
Mule . . .
•?. ..... .
do
3 f
3 g
Yellow.
Black. .
Cloven .
Mule . . .
Saved for
breeding.
3"
cf
Yellow.
Cloven .
3 1
«?
..do....
..do....
Mar. 13, 1017
3 J
9
Black. .
..do....
3 k
9
Cream .
Mule . . .
Some roan.
3 1
9
Red...
Cloven .
4
do
4a
4b
9
9
Black. .
..do....
..do...
Mule.. .
4C
9
..do....
..do...
4d
9
Yellow.
. . do . . . .
•Jan. 19, 1917
4e
9
Cream .
Cloven .
Some roan .
4«
3
..do....
Mule. . .
4g
3
Red...
Cloven .
4h
3
Black. .
..do....
6
do
6a
6b
3
3
Yellow.
Cream .
..do....
. .do. .. .
Slightly
roan.
6c
3
Red....
..do....!
Much roan.
6d
6e
3
3
Black. .
..do....
Mule.. .1
..do....
-Apr. 8, 1917
6 f
9
Red....
. .do... .
6g
9
Black..
..do....
6h
9
..do
do....
[White spot
] on upper
1 lip.
F2
dam No.
F2 sire No.
Off-
spring
No.
Sex.
Color.
Foot
charac-
ter.
Date of birth.
Remarks.
2 e
jf
2e-a
2e-b
2e-c
2e-d
2e-e
3
3
3
3
9
Red....
..do
..do
..do
..do....
Cloven .
do....
do....
do... .
..do....
>Mar. I, 1918
Some doubt
as to de-
gree of
red in
this litter.
598
Journal of Agricultural Research
Vol. XX, No. 8
Table II. — Original data on the F2, F3, and Fi offspring from a cross of a mule-foot
boar on Duroc- Jersey sows — Continued.
F2
dam No.
F-2 sire No.
Off-
spring
No.
Sex.
Color.
Foot
charac-
ter.
Date of birth.
Remarks.
2f
■zi
21-a
2f-b
2f-C
2f-d
2i-e
2f-f
2t-g
2f-h
2f-i
2f-j
2f-k
2f-l
2f-m
9
9
9
9
c?
<?
c?
9
9
9
9
Cream.
..do....
..do....
..do
..do
Yellow.
..do
..do....
..do
..do
..do
..do....
..do
..do....
..do....
..do....
do....
do....
..do....
do....
..do....
do. . . .
do....
do....
..do....
do. . . .
Mar. 5, 1918
1
breeding.
Do.
Do.
F3
dam No.
F3 sire No.
Off-
spring
No.
Sex.
Color.
Foot
charac-
ter.
Date of birth.
Remarks.
2f-b ....
2f-a
2f-b-a
2f-b-b
2f-b-c
2f-b-d
2f-c-a
2f-c-b
2f-C-C
2f-C-d
2f-c-e
2f-C-f
2f-C-g
2f-C-h
c?
9
9
9
9
9
c?
c?
c?
9
9
Cream .
..do
..do
..do
Cream .
..do
..do....
Yellow.
Red
Yellow.
..do
Cream .
Cloven .
do....1
do...J
do...
..do...
do....
..do....
..do... .
do... .
..do....
do...
. . do . . . .
[Mar. 18, 1919
>Apr. 16, iqio
2f-C ....
2f-a
[ Lemon on
1 top of head
j and shoul-
1 ders.
("Few yellow-
hairs be-
t w e e n
[ ears.
Syndactylism has been recognized as an inherited character in man
by Lewis and Embleton (6),1 Lewis (5), and Pearson (7) ; in poultry by
Davenport (3); and in swine by Spillman (9). In man there is prob-
ably one main dominant factor allelomorphic to normal; and the case
shows simple Mendelism as we now understand it, although both Lewis
and Embleton and Pearson were not inclined to such a view. In poultry,
Davenport concluded that syndactylism was very imperfectly domi-
nant to its allelomorph, normal toes. Syndactylism versus cloven-
foot in swine has been cited as an illustration of monohybridism in a
number of textbooks, but no published data are available. Spillman
states :
It is interesting to note that in crosses between mule-foot hogs and ordinary breeds
the mule-foot character seems to be dominant.
1 Reference is made by number (italic) to " Literature cited," p. 604.
Jan. is, 1921 Inheritance of Syndactylism and Color in Swine 599
No statement is made regarding segregation. Kronacher (4) implied
that the character was transmitted pure after hybridization, for he says :
Der Zuchter v. Dtuiin-Kozicky liess im Jahre 1888 ein derartiges, gelegentlich
erhaltenes Einhuferschwein (polnisches Landschwein) von einem Yorkshireeber
decken und erhielt zur Halfte (5 von 9) solche Einhufernachzucht, die ihr charakter-
istisches Merkmal rein Eitervererbte.
It is difficult to know whether Kronacher really means that these
mule-foot hybrids gave no cloven-footed segregates or that the character
when transmitted showed no contamination after the cross and was
therefore "pure." Reference to the original source quoted by Kron-
acher leaves no doubt as to segregation, for von Dabrowa-Szremowicz
(1) states explicitly that, in attempting to fix the mule-foot character,
sporadic cases of cloven-foot crop out. He says :
Da bei den Schweinen es uberhaupt schwer ist, eine einheitliche und gleichmassige
Abart festzustellen, so treffen sich aueh noch bei den meinigen vereinzelte Falle mit
gespaltenen Hufen.
It is clear, then, that this case agrees with both Spillman's and our
own observations on dominance and with our observations on segrega-
tion.
The original mule-foot boar in these crosses was undoubtedly homozy-
gous (MM) in the factor for syndactylism, for every one of his offspring,
about 250, showed the mule-foot character. Six Fx sows (Mm) were
bred back to the cloven-foot Duroc- Jersey (mm), and each one gave
both mule-foot and cloven-foot segregates. The total F2 generation
thus produced was 17 mule-foot +25 cloven-foot, where theory calls
for 21 of each kind as the most probable result. The deviation, 4, is no
larger than one might reasonably expect as a fluctuation of sampling
/deviation 4 \
I v— r\ = = 1-83 J. It we add to these results those re-
\probable error 2.19 °J
corded by von Dabrowa-Szremowicz, we obtain 22 mule-foot+ 29
cloven, where 25.5 is the most probable value. In this total, the cal-
culated and observed results show even a closer agreement, for
deviation _ 3.5 _ ^
probable error 2.41
In experiments with the larger domestic mammals the usual apology
for small numbers must be made, for they often obscure the real facts.
In making our results a test against a monohybrid Mendelian hypothesis
we must not overlook the fact that our data might also admit of a dihybrid
interpretation with interaction of two factors to produce the mule-foot
character. For example, if mule-foot were due to the interaction of
X and Y, then the original mule-footed grandparent was XXYY and,
mated to xxyy females, gave XxYy in the Fj generation. Back-crossing
to xxyy would thus be supposed to give:
XxYy Xxyy + xxYy + xxyy
25 per cent mule-foot 75 per cent cloven-foot
600 Journal of Agricultural Research voi.xx.No. 8
We observed a ratio of 17 mule-foot to 25 cloven-foot in the F2 genera-
tion, while on this latter hypothesis the calculated results would be
10.5 to 31.5. The ^^ = iito=3'43' The odds aSainst Avia-
tions as wide or wider are about 45 to 1. But if we again add the
results of von Dabrowa-Szremowicz to ours, the observed ratio is 22 to
29, where 12.75 to 38.25 is the calculated ratio. In these combined
, ^ deviation 9.25 „, , , . , . .
results the = = 4.43. The odds against deviations as wide
error 2.09 ^ ^° &
or wider are now about 350 to 1 . In both cases the monohybrid explan-
ation is much more satisfactory. Furthermore, on a dihybrid hypoth-
esis we should sometimes obtain mule-footed when F2 cloven-footed
segregates are mated together. To test this, such matings were made.
Two of the three cloven-foot F2 daughters of 9 2 ( 9 2e and 9 2f in
Table II) were bred to a cloven-foot F2 son of 9 3 (<? 31", Table II).1
One F2 9 gave 5 F3 cloven-foot (4 c? <$ + 1 9 ) and the other F2 9
gave 13 F3 cloven-foot (5 d cf + 8 9 9 ). Therefore, a total of 18 F3
cloven-foot was obtained from F2 cloven-foot segregates bred inter se.
In the F3 generation two cloven-foot 9 9(9 2f-b and 9 2f-c) were mated
to their cloven-foot brother, tf 2f-a, and gave 4 and 8 cloven-foot respect-
ively. We may conclude that mule-foot and cloven-foot represent a
single allelomorphic pair, in which the syndactylous form is dominant and
the normal form is recessive, and that extracted recessives breed true.
As is common among mule-foot swine, the fused phalanges may sep-
arate along the line of fusion as the animal becomes older and heavier.
This splitting was infrequent in the front feet, but was occasionally seen
in the hind feet. There was never any difficulty in classifying the syn-
dactylous and normal at the time of birth or when the animals were young,
for syndactylism is a distinct discontinuous variation from normal.
There is, however, some variation in syndactylism itself. Fusion may vary
from complete, with no trace of separation on the hoof, to a less perfect
fusion with two deep parallel lines of demarcation. The former condition
is characteristic of the front feet, while the latter is the usual condition in
the hind feet. In an examination of 17 F2 mule-foot segregates, 14
showed complete fusion in the front feet, but 1 6 showed the deep lines
of demarcation on the hind feet. The factor for syndactylism acts differ-
ently on the front and hind feet. (See PI. 70.)
The relation of black to red in swine has never been quite clear. It is
well known that Poland China or Berkshire mated to Duroc- Jersey
usually produces a tortoise-shell type of red and black, but the amount
of each color varies markedly. Sandy, yellow, cream, or even white may
be substituted for red in such crosses, as shown by Severson (8). Wright
(10) advanced a suggestive hypothesis that such a tortoise-shell type of
sandy colored hog with black spots was selected in two directions to give
the characteristic color of the Berkshire or Poland China, on the one
1 The relationship of all animals recorded in this paper may be obtained from Table II, the original data.
Jan. is. 1921 Inheritance of Syndactylism and Color in Swine 601
hand, and Duroc-Jersey or Tamworth on the other. Selecting on the
basis of minor factors for the extension of black and for the dilution of
red to white gave the Berkshire color type, while selecting minor factors
for the restriction of black and for the intensity of red gave the Duroc-
Jersey type. In our crosses the self -black of the mule-foot does not act
like the black of the Berkshire with its peculiar pattern, but whether this
is due to a real difference in their genetic factors for black or is due to
variable spotting factors in the Berkshire as compared with the self of
the mule-foot remains to be shown. The six white points of the Berk-
shire may represent a highly selected spotting factor, or factors, with
numerous modifiers. By crossing such Berkshires to Duroc- Jerseys one
would expect to obtain a complex spotted hybrid. The mule-foot and
the Duroc-Jersey are both self-colored and, as our experiments indicate,
a cross between the two involves no striking spotting factors but shows
clear-cut segregation between self-red and self-black. We may, there-
fore, regard black (B) as a dominant allelomorph to red (b) in our crosses.
The original mule-foot boar (BB) was mated to Duroc -Jerseys (bb)
and gave about 250 Fx hybrids (Bb) which were self-black. The 6 Fx
sows mated to a Duroc-Jersey boar gave 19 blacks to 23 reds in the F2
generation, where 21 of each kind is the calculated ratio. The recessive
F2 red segregates gave 18 F3 reds. The F3 reds when mated inter se gave
12 F4 reds. Extracted recessive reds, therefore, breed true. The total
results indicate that black and red are allelomorphs in this cross in swine,
black being dominant to red. In all of the foregoing discussion the term
red includes red, yellow, lemon, and cream shades — that is, any form
showing red pigment but no black.
In any wide cross between two distinct varieties like the Duroc-Jersey
and mule-foot there are many factorial differences involved, and we are
not surprised to find numerous new variations in the F2 generation and
subsequent hybrids which were not seen in the parents. Thus, we
observed an occasional white spot on the feet or hoofs, white spot on the
upper lip, animals with varying amounts of roan, more variation in size,
and the like. Among the more striking variations seen in the F2 gen-
eration were the grades of intensity and dilution of red pigment. Although
red in the Duroc-Jersey varies somewhat, the red F2 segregates varied
much more than the original Duroc-Jersey parents. The black of the
Ft and F2 hybrids, on the contrary, did not vary perceptibly. This
seems to indicate that diluters of red may be carried by black swine but
that such diluters do not affect black. In this cross the original black
mule-foot sire evidently contributed diluters of red to the black Fx
hybrids; and such diluters segregated, giving more variability in red in
the F3 generation. We can hardly suppose that the Duroc- Jerseys con-
tributed the factors for this dilution, because Duroc-Jerseys mated inter se
do not show such dilute forms as we observed among our red segregates.
Samples of hair from each individual were saved. Comparing all F, reds
with each other, we classified these around four more or less arbitrary
602 Journal of Agricultural Research vol. xx.No. 8
modes — red, yellow, lemon, and cream, given in the order of most intense
to most dilute. Those classified as "cream" were, when adults, a very
light straw color, almost white. The 23 F2 individuals were distributed
as follows: 6 red, 9 yellow, 2 lemon, and 6 cream. It is not certain
that yellow and lemon belong to two genetically distinct classes. There
is little difference between them. If we group yellow and lemon together
as an intermediate shade, the ratio of 6 intense, 11 intermediates, and
6 dilutes suggests a 1:2:1 ratio; but this is probably a coincidence, and
we can not infer a single allelomorphic pair of factors for intensity and
dilution with incomplete dominance, as later experiments will show. We
do not know what the calculated ratio for the various shades of red in
such an F2 population should be, for we do not know the genetic consti-
tution of each Ft female or the Duroc-Jersey male with regard to these
diluters of red. This one fact, however, is clear — there was marked seg-
regation in shades of red in the F2 generation. Plate 70 shows some of
the variation in the intensity of red.
In order to test these dilute conditions, 9 2e, an F2 yellow, and 9 2f,
an F2 lemon, were mated to S 3f, an F2 yellow. It was thought that if
yellow and lemon were intermediate conditions between cream and red,
then these matings would give a range of forms from red to cream.
Female 2e gave 5 F3 offspring classified as red. They were discarded,
and unfortunately some doubt exists as to the exact shade of red. The
shade of red deepens as the animals grow older. We are quite sure they
were not cream, but they may have been either yellow or red. Some
were a deeper, more intense color than either F2 parent. Female 2f
gave 13 F3 young, of which 5 were cream and 8 were yellow. The
creams when born were absolutely white, and a microscopic examination
of their hair cleared in xylol and mounted in Canada balsam showed no
pigment. Later in life they acquired some yellow pigment in the medulla
of the hair but little or none in the cortex and gave the general appearance
of a very light straw-color. The presence of red, yellow, and cream
among the F3 offspring from yellow parents suggested that yellow might
after all be an intermediate condition and that a lighter shade like cream
is recessive. We did not know at that time whether a single pair of
factors with incomplete dominance was involved or whether there were
a number of independent factor pairs for yellow, the cumulative effect
of which gave the more intense shades
If a single alleomorphic pair with incomplete dominance were respon-
sible, then all the offspring from the F3 creams should have been cream.
Three F3 animals ( S 2f-a, 9 2f-b, 9 2f-e) classified as cream (white at
birth but very light straw-color when adults) were bred inter se to give the
F4 generation. However, the offspring from these creams were not all
cream, for 9 2f-b produced 4 creams, but 9 2f-c gave 4 creams, 3 yellows,
and 1 red. This hypothesis, therefore, becomes untenable. The dif-
ference between the creams and yellows or reds in this last litter, as
in all others, was a distinct one, and there can be no question as to the
Jan. i5, 1921 Inheritance of Syndactylism and Color in Swine 603
accuracy of classification. The fact that yellow may give red, yellow,
and cream and that the cream-colored may give red, yellow, and cream
leads us to believe that there is an interaction of factors producing
intensity of red and that similar somatic creams are not necessarily of the
same genetic constitution. The case appears much like the belt in the
Hampshire, where either belted X belted or nonbelted X nonbelted may
give both forms; or like purple and white aleurone in maize, where either
white X white or purple X purple may give both forms. We may add
that the adult creams can hardly be distinguished from Chester White
or Yorkshire color. Under the microscope these creams show little or no
pigment in the cortex of the hair but show yellow granules in the medulla.
Some white hairs from the Berkshire and Chester White may also show
yellow pigment. We have seen white hairs from the Berkshire which
show yellow pigment in the medulla exactly like our creams.
The fact that red hair may be so diluted as to be almost if not quite
indistinguishable from white hair suggests that the so-called white hair
in some breeds may really be a very dilute red. Severson's experiments
(8) show that Berkshire mated to Duroc-Jersey may give white and
black spotted rather than the usual red or yellow and black. If the
white hair of the Berkshire is really a dilute red, such a result would be
expected in occasional matings of Berkshire to Duroc-Jerseys carrying
recessive diluters; and there seems to be much evidence that Duroc-
Jerseys carry recessive dilution factors, for much lighter animals than the
standards require are known. Severson mated such a white and black
hybrid back to a Berkshire and obtained some red and black offspring.
Disregarding the black, this mating is like our matings of two creams
which gave reds, and it thus adds weight to our hypothesis that the
intenser shades, like red and yellow, are due to interaction of at least
two pairs of independent factors; but the more dilute shades, like cream
or white, are due to the absence of one or both interacting factors. That
is, zygotes with both interacting factors A and B would be red or yellow,
while zygotes with either A or B, or neither, would be cream or white.
The fact that creams or whites form one distinct grade and yellow and
red form another leads us to believe that the two groups are quite distinct.
The slight variations in red and yellow or in the creams may be due to
other minor factors. Summarizing, we may say that there are three
sources of evidence which indicate that cream or white may be dilute red,
that dilution and intensity are complex characters due to interaction of
independent factors, and that the so-called white hair in some breeds is
really a cream or very dilute red, as follows: (1) Yellow pigment was
found in the medulla of the hair of our creams and in the white hair of
Berkshires, (2) red offspring were derived from our creams mated inter se,
and (3) red and black spotted offspring were derived from Severson's
white and black spotted hybrid (from Duroc Jersey X Berkshire)
mated to Berkshire.
604 Journal of Agricultural Research vol. xx, No. s
SUMMARY
Syndactylism in swine is allelomorphic and dominant to normal
cloven-foot, and black is allelomorphic and dominant to red. The two
pairs of factors are evidently independent of each other.
The factor for syndactylism does not show quite the same effect on
the front feet as on the hind feet, for the fusion is usually less complete
in the latter.
The Duroc-Jersey and mule-foot are both self-colored in this cross
and transmit no distinct spotting factors. We have concluded tenta-
tively that the hybrids between Duroc-Jersey and Berkshire (or Poland
China) are spotted because the latter transmit highly selected dominant
spotting factors.
Intensity of red appears to be due to the interaction of independent
factors which do not affect black. Dilution of red or yellow to cream or
white takes place when either one or neither of the interacting factors
is present. The so-called white hair of some breeds like the Berkshire
and Poland China is really a very dilute red of genetic composition
similar to our cream segregates.
LITERATURE CITED
(i) Dabrowa-Szremowicz, S. v.
1905. eine neue abart vox schweinen. In Illus. Landw. Ztg., Jahrg.
25. No. 63, p. 564. 4%.
(2)
1905. Einhuferschweine. In Illus. Landw. Ztg., Jahrg. 25, No. 92, p.,
8io-8ii, 3 fig.
(3) Davenport, Charles B.
1909. INHERITANCE OF CHARACTERISTICS IN DOMESTIC FOWL. IOO p., 12 Col.
pi. Washington, D. C. (Carnegie Inst. Washington Pub. 121.)
Literature cited, p. 99-100.
(4) Kronacher, Carl.
1912. GRUNDZUGE DER ZUCHTUNGSBIOLOGIE . . . Xvi, 323 p., 95 fig., 9C0I. pi.
Berlin.
(5) Lewis, Thomas.
1908. ADDENDUM TO MEMOIR: " SPLIT-HAND AND SPLIT-FOOT DEFORMITIES."
In Biometrika, v. 6, pt. 1, p. 117-118.
(6) and Embleton, Dennis.
1908. SPLIT-HAND AND SPLIT-FOOT DEFORMITIES, THEIR TYPES, ORIGIN, AND
Transmission. In Biometrika, v. 6, pt. 1, p. 26-58, 3 fig., pi. 1-7.
Bibliography, p. 56-58.
(7) Pearson, Karl.
1908. ON THE INHERITANCE OF THE DEFORMITY KNOWN AS SPLIT-FOOT OR
lobster-claw. In Biometrika, v. 6. pt. 1, p. 69-79, pi- 8-16.
(8) Severson, B. O.
1917. color inheritance in swine. In Jour. Heredity, v. 8, no. 8, p.
379-381. 1 ng-
(9) Spillman, W. J.
1910. history and pecularities of The mule-Foot hog. In Amer. Breed-
ers' Mag., v. 1, no. 3, p. 178-182, illus.
(10) Wright, Sewall.
1918. color inheritance in mammals, viii. swine . . . In Jour. Heredity,
v. 9, no. 1, p. 33-38.
PLATE 70
The four types of F2 segregates from a cross between mule-foot boar and Duroc-Jersey
sows.
A. — Black mule-foot.
B. — Black cloven foot.
C. — Red mule-foot.
D. — Red cloven foot.
There is much variation in the intensity of red. The fusion in the hind feet is less
pronounced than in the front feet.
Inheritance of Syndactylism and Color in Swine
Plate 70
Q
Journal of Agricultural Research
Vol. XX, No. 8
FOUR RHYNCHOPHORA ATTACKING CORN IN
STORAGE
By Richard T. Cotton 1
Scientific Assistant, Stored-Product Insect Investigations, Bureau of Entomology,
United States Department of Agriculture
INTRODUCTION
Of the numerous insect enemies of stored corn there are four belonging
to the suborder Rhynchophora, or weevils, that are to a greater or less
extent of economic importance in the United States. Of these four, one
has received but little attention from economic entomologists, while of
the remaining three much has been published, but comparatively little
careful work has been done with the immature stages.
It is the purpose of this paper to present accurate drawings of the
immature stages of these weevils, together with carefully prepared
descriptions and keys, so that the various species may be readily dis-
tinguished in whatever stage they may be found.
The weevils under discussion represent two different families, Anthri-
bidae and Curculionidae, and three different genera, Araecerus, Caulo-
philus, and Sitophilus, two of the weevils belonging to the last genus.
KEY TO ADULTS
a. Beak short and broad.
b. Robust beetle, antennae inserted in small foveae upon the upper surface
of base of beak, last three segments of antennse forming a loose club.
Araecerus fasciculaius DeG.
bb. Slender, elongate beetle, antennae inserted at middle of beak, last few
joints forming a compact club Caulophilus latinasus Say.
aa. Beak elongate and slender.
c. Thorax with coarse, sparse, elongate punctures, wings lacking.
Sitophilus granarius L.
cc. Thorax with coarse, deep, very dense punctures, wings present.
Sitophilus oryza L-
KEY TO MATURE LARV^
a. Body slender, elongate, supplied with some or many long hairs, abdominal seg-
ments with hypopleurum not subdivided, mandibles armed dorsally with a
pair of bristles set close together.
b. Larger, 4.5 to 6 mm. in length, body profusely covered with long hairs.
Araecerus fasciculatus DeG.
bb. Smaller, 2 to 2.5 mm. in length, body sparsely provided with long
hairs Caulophilus latinasus Say.
1 The writer wishes to express his gratitude to Dr. Adam G. Boving, of the Bureau of Entomology, United
States Department of Agriculture, for his kindness in extending much valuable aid and advice in the study
of the larval forms and the preparation of the technical descriptions.
Journal of Agricultural Research, (605 ) Vo1- xx- No- 8
Washington, D. C Jan. 15. 1921
wk Key No. K-90
606 Journal of Agricultural Research vol. xx, No. 8
aa. Body short and stout, armed with but few small setae, abdominal segments with
hypopleurum subdivided into three lobes, mandibles armed dorsally with a
pair of bristles set far apart.
c. First three abdominal segments only, above divided into three distinct
areas, middle lobe of hypopleurum without seta. . . .Sitophilus oryza L.
cc. First four abdominal segments above divided into three distinct areas,
middle lobe of hypopleurum armed with a seta . Sitophilus granarius L.
KEY TO PUPAL STAGES
a. Antennae not geniculate, folded over on dorsum Araecerus fasciculatus DeG.
aa. Antennae geniculate.
b. Beak short and broad Caulophilus latinasus Say.
bb. Beak elongate and slender.
c. Inner wings rudimentary, almost completely concealed by
elytra Sitophilus granarius L.
cc. Inner wings well developed, extending well beyond tips of
elytra Sitophilus oryza L.
ARAECERUS FASCICULATUS !
SYNONYMY 2
Araecerus fasciculatus DeG.
"DeGeer. Ins. V, 1775. p. 276. t. 16. f. 2. — Wollast. Ann. Nat. Hist. V.
1870. p. 18. — Lucas. Ann. Fr. 1861. p. 399.
cacao Fabr. Syst. Ent. p. 64. — Oliv. Ent. IV. 80. p. 75. t. 2.f. 21. a-b.
jcapillicornis Say. Journ. Ac. Phil. V. 2. 1827. p. 249.
[mocstus Lee. Ann. Lye. I. p. 172.
cassiae Winthem. Dej. Cat. J. ed. p. 259.
coffeae Fabr. Syst. El. II. p. 411. — Gylh. Schh. Gen. Cure. I. p. 175 — Labr.
et Imh. Gen. Cure. I. nr. 55.
crassicornis Fabr. Ent. Syst. Suppl. p. 159; Syst. El. II. p. 399.
griseus Steph. III. Brit. IV. p. 211. t. 21. f. 2. (forte.)
japonicus Thunb. Nov. Act. Ups. VII, p. 122.
Perigrinus Herbst, Kaf. VII. p. 168. t. 106. f. 9.
saltatorius Falderm. in litt.
var. sambucinus Boisd. Voy. Astrol. II. p. 299 (forte.) — MacLeay, Dej. Cat.
3. ed. p. 259."
Araecerus fasciculatus (PI. 71) was described in 1775 by DeGeer from
Surinam. It is thought to have originated in India, but now it is
cosmopolitan in distribution. This beetle, commonly known as the
coffee-bean weevil, is robust, dark brown, and clothed with mottled
light and dark brown pubescence. The beak is short and wide.
ADULT
Ovate, convex. Dark brown to black or piceous, clothed with yellowish and dark
brown pubescence ; intervals of elytra alternately tessellate with brown and yellow-
ish; antennae, tibiae, and tarsi reddish brown, club fuscous; femora piceous at middle.
Thorax very finely and exceedingly densely punctate. Elytra with rows of fine,
close-set, feebly impressed punctures; intervals very finely and densely granulate-
punctate. Length 2.5 to 4.5 mm.3
1 Family Anthribidae, tribe Araecerini.
2Gemmlnger, M., and Harold, B. de. catalogus coleopterorum. v. 9, p. 2749. Monachii, 1872.
3 Blatchley, W. S., and Leng, C. W. rhynchophora or weevils of north eastern America, p.
42. Indianapolis, Ind. 1916.
Jan. i5l 1921 Four Rhynchoph or a Attacking Com in Storage 607
EGG
Egg shining, white, ovoid in shape; top broadly rounded, bottom slightly more
pointed. Length about 0.56 mm., width 0.35 mm.
LARVA
Mature larva 4.5 to 6 mm. in length; white, footless, fleshy grub with body curved,
wrinkled, and profusely covered with long hairs. Head very pale straw color; anterior
margin and mandibles slightly darker. Head longer than broad, somewhat oblong in
shape. Epicranial and frontal sutures faint and slightly lighter in color; there are also
two longitudinal, light stripes rising from the frontal sutures and running to base of
head. Frons almost triangular in shape; frons and epicranial lobes provided with
numerous long hairs. Antenna small, situated at anterior corner of frons. Mandibles
large, stout, triangular, with apex produced into an acute tooth; inner edge toward
apex provided with two acute subapical teeth and above protractor with a large molas
process or structure. Dorsal area of each mandible armed with a pair of stout bristle
set close together. Eye represented by a well-defined black spot beneath the exo-
skeleton. Clypeus and labrum present, both broader than long and about equal in
breadth. Labrum provided with four pairs of dorsal hairs and five pairs of short,
thickened, marginal setae; ventral surface of labrum with four small setae. Maxillae
elongate, terminated by a 2-jointed palpus and a single setose lobe. Maxilla armed
with numerous long hairs and a stout chitinized seta on palpifer just below the maxil-
lary lobe. Stipes labii fused with the basal joint of the 2-jointed palps and bearing
two setae on each side. Ligula and lingua fused and marked by a seta on each side.
Linguar region with numerous small asperities and a few setae. Behind linguar region
is a strong hypopharyngeal chitinization connected on each side with epicranium
with well-developed hypopharyngeal bracon. Chitinization anteriorly provided with
a cavity the bottom of which bears pointed processes. Posterior part of hypopharyn-
geal chitinization less heavily chitinized and limited by a chitinized frame which
gradually continues over into floor of oesophagus. Mentum and submentum sepa-
rated, men turn bearing two long hairs and submentum nine pairs of long hairs arranged
in four groups of four each and a median pair.
Pronotum simple and not divided. Mesothoracic and metathoracic segments are
above divided into three areas, representing praescutum, fused scuto-scutellar area,
and postscutellar area; below and adjacent to epipleurum is alar area. Below ventro-
lateral suture are hypopleurum, coxal lobe, and eusternum, all well-defined and
profusely provided with long hairs. Mesothoracic spiracle located on preepipleural
lobe of mesothorax near prothorax; larger than abdominal spiracles and differing
from them by being bifore whereas abdominal spiracles are monofore. Kidney-shaped
air tubes pointing dorsad. Ten abdominal segments: Ninth, small; tenth, reduced;
one to eight, each provided with monofore spiracles, that of eighth segment being
located slightly more dorsad and with air tube pointing cephalad instead of dorsad.
Praescutal and scutal areas of abdominal segments large and protuberant ; scutal area,
however, attenuating dorsad and not reaching the dorsal outline, scutellum and post-
scutellum flatter. Praescutum, scutum, and scutellum profusely armed with long
hairs. Epipleural lobes bulging and prominent, also well supplied with long hairs.
Measurements of larval stages
WIDTH OF
STAGE. LARVAL HEAD.
1 o. 22 mm.
2 34 mm.
3 < 58 mm.
4 78 mm.
5 90 mm.
608 Journal of Agricultural Research vol. xx, No. s
pupa
Pupa white when first formed, cast larval skin clinging tightly to last abdominal
segments. Length 3.75 to 4 mm.; width 2 mm. Tips of elytra pointed and termi-
nated with a long, chitinized hook nearly reaching seventh abdominal segment. Meta-
thoracic tarsi extending well beyond tips of elytra. Head rounded, beak short and
broad. Head profusely supplied with hairs. Antennae nongeniculate, folded over
on dorsum, tips nearly meeting on metanotum. Prothorax profusely supplied with
long hairs, femora apically armed with several hairs. Mesonotum and metanotum
each provided with two bunches or tufts of long hairs. Elytra armed with numerous
hairs. Each abdominal segment is armed with two rows of dorsal, and numerous
lateral, hairs. Seventh and eighth abdominal tergites apparently fused together;
the ninth segment bears two large bilobed fleshy processes armed with numerous
papilla?. The tenth segment is ventral to the ninth.
CAULOPHILUS LATIN ASUS >
SYNONYMY 2
Caulophilus latinasus Say.
" Rhyncholus latinasus, Say, Descr. N. Am. Cure. p. 30 (1831) Complete Writings,
1. p. 299 (nee Boheman).
Caulophilus latinasus, Lee. Proc. Am. Phil. Soc. xv. p. 340 (1876); Champ. Ent.
Monthly Mag. xlv. p. 121.
Caulophilus sculpturatus, Woll. Ins. Mader. p. 315, t. 6. figg. 4-4 a-c (1854).
Cossonus pinguis, Horn, Proc. Am. Phil. Soc. xiii. p. 442 (1873). Cossonus pici-
pennis, Sturm, in litt."
Caulophilus latinasus (PI. 72) was described from Florida in 1831 by-
Thomas Say. This weevil is now widespread over the State of Florida and
has been reported from South Carolina and Georgia. It is also known to
occur in Jamaica, Porto Rico, Mexico, Guatemala, and Madeira. It is
doubtless common throughout the islands of the West Indies and in the
countries of Central and South America.
It is commonly known as the "broad-nosed grain weevil," and is a
slender, elongate, reddish brown weevil with a short, broad beak.
Technical descriptions of the adult and immature stages follow.
ADULT
Elongate, rather robust. Reddish brown or piceous, feebly shining. Beak longer
than half the thorax, sparsely punctured, with a faint elongate fovea between the eyes.
Thorax as broad as long, moderately constricted near apex, sides strongly curved,
base slightly narrowed, feebly bisinuate; disk rather finely and evenly punctured,
with a broad, faint impression on basal third. Elytra subcylindrical, not wider than
middle of and more than twice as long as thorax, moderately convex; striae deep,
rather coarsely and closely punctured on basal half, more finely or obsoletely near
apex, the seventh and eighth united behind the humerus as in Allomimus: intervals
convex, indistinctly punctulate. Under surface sparsely punctured. Front tibiae
sinuate within.
Length 3 mm.3
1 Family Curculionidae, subfamily Cossoninae, tribe Cossonini.
2 Champion. G. C. rhynchophora. curculionidae. curcuuoninae (concluded) and calan-
drinae. In Biol. Centr.-Amer. insecta. coleoptera. v. 4, pt. 7, p. 40. 1909-1910.
" BlatchlEy, W. S., and Leng, C. W. op. cit.. p. 535.
Jan. 15, 1921 Four Rhynchophora Attacking Com in Storage 609
EGG
Egg opaque, shining white, bottom broadly rounded, top flattened and fitting into
a translucent cap. Length, without cap, 0.45 to 0.47 mm.; width 0.27 to 0.32 mm.
LARVA
Mature larva 2 to 2.5 mm. in length, a white, footless, fleshy grub, with body curved
and wrinkled. Head light brown or straw color, the anterior margin and mandibles
a darker brown. Head about as broad as long, almost circular in form. Epicranial
and frontal sutures distinct and light in color. There are also two oblique, longitudinal
light stripes rising from the frontal sutures and coalescing with the epicranial suture
near the base, of the head. Frons sub triangular, with a distinct dark median line run-
ning from posterior angle to middle, and indicating carina. Frons provided with
four pairs of large setae, sutural margins each bearing one seta. Epicranial lobes each
bearing the following setae: One close to posterior angle of frons and located in the
oblique, longitudinal stripe rising from the frontal suture, one small seta posterior to
this and near occiput, two anterior to it on disk of epicranium, two opposite middle
of frons, one opposite middle of mandible, one opposite hypostomal angle of mandible
and one on hypostoma near base of mandible. Epistoma represented by thickened
anterior margin of the front. Pleurostoma represented by somewhat darker, declivous
area surrounding the mandibular foramen. Mandibles stout, triangular, with the apex
produced into an acute apical tooth. Inner edge toward apex provided with a sub-
apical tooth and a small medial tooth, no molar structure. Dorsal area of each mandi-
ble armed with a pair of stout bristles set close together. Eye represented by a well-
defined black spot beneath exoskeleton. Clypeus broad at base, sides narrowing
toward apical angles; distinctly broader but not as long as labrum. Epistomal
margin provided with t\vo fine hairs on each side. Labrum about as broad as long,
rounded in front, provided with three pairs of large setae and five pairs of short, thick-
ened, marginal setae.
Maxillae terminated by a 2-jointed palpus and setose maxillary lobe. Maxillae each
provided with four setae as follows: One on first segment of palpus, two on vaginant
membrane between palpus and palpifer, and one stouter and larger one midway be-
tween palpus and cardo. The stipes labii enforced posteriorly by a median triangular
chitinization bear 2-jointed palpi and a single pair of setae. Ligula bearing four small
setae. Mentum and submentum fused and bearing three large setae on each side.
Pronotum simple and undivided; praescutal and scuto-scutellar areas roughly
indicated by rows of setae. Mesothoracic and metathoracic segments divided above-
into two areas representing praescutum and scuto-scutellum ; below and adjacent to epi-
pleurum is the alar area. Below ventro-lateral suture are a well-defined hypopleurum,
coxal lobe, and eusternum. The thoracic spiracle, located on the preepipleural
lobe of mesothorax, is bifore, with the fingerlike air tubes pointing dorsad, and is some-
what larger than the abdominal spiracles. Ten abdominal segments; ninth small,
tenth reduced. Each tergum of first eight abdominal segments divided above into
three distinct areas, praescutum, scutum, and scutellum. Below and adjacent to
epipleurum is the alar area. Abdominal segments provided with setae as follows:
Two on praescutum, five on scutellum, two on alar area, two on epipleurum, one on
coxal lobe, and two on eusternum. Each of the first eight abdominal segments bears
a bifore spiracle, that of the eighth being slightly larger than the rest.
Measurements of larval stages
WIDTH OF
STAGE. LARVAL HEAD.
1 o. 22 to o. 23 mm.
2 • 33 to .38 mm.
3 53to .57mm-
17776°— 21 2
610 Journal of Agricultural Research vol. xx. No. 8
PUPA
Pupa white when first formed. Length 2.8 to 3 mm.; width about 1.3 mm. Tips
of elytra attaining the sixth abdominal segment, tips of metathoracic tarsi not ex-
tending beyond wing tips. Head rounded, beak short and broad. Head provided
with two prominent spines towards vertex, two smaller ones on sides above eyes, a
spine on each side of front between eyes, two pairs on beak between frontal ones and
base of antenna, two pairs on beak between base of antenna and tip of beak, and four
pairs of small setae on tip of beak. Prothorax provided with two pairs of antero-
marginal setigerous tubercules, one pair of antero-lateral, two pairs of postero-lateral,
and four pairs of dorsal setigerous tubercules. Mesonotum and metanotum each pro-
vided with two pairs of spines. Abdomen with eight distinct dorsal tergites; dorsal
area of each armed with two pairs of large spines; lateral area of each tergite armed
with a spine at base of which is a small seta. Epipleural lobes each obscurely armed
with one or two minute setae. Ninth segment armed as usual with two prominent
pleural spines.
SITOPHILUS ORYZA >
SYNONYMY 2
Sitophilus oryza Linn. 1763.
" oryzae Linn. Amoen. Ac. VI. 1763. p. 395. — Oliv. Ent. V. 83. p. 97. t. 7.
/. 81. a-b. — Gylh. Schh. Gen. Cure. IV. p. 981. — Scriba. Stett . Zeit. 1857.
p. 377. — Kollar. Sitzgsb. Wien. Ac. 1848. V. p. 3.
frugilega Degeer. Mem. V. p. 273.
granaria Stroem. Dansk. Vid. Selsk. Skrift., II. p. §6.
quadriguttata Montrouz. Ann. Fr. i860, p. 910."
Var. zea-mais Motsch. Etudes Ent. IV, p. 77 (1855); Casey, Ann. N. Y. Acad. Sci. VI,
p. 686.
Sitophilus oryza (PI. 73) was described in 1763 by Linnaeus. 11 is
thought to have originated in India, but it is now cosmopolitan in dis-
tribution. It is the predominant species of the grain weevils in the
southern States of North America, where it is known as the "black or
rice weevil." It is easily the commonest and most destructive grain
weevil in the United States.
It closely resembles Sitophilus granarius in form but is readily distin-
guished by the presence of wings and the different punctuation of the
thorax. Technical descriptions of the adult and immature stages
follow.
ADULT
Reddish brown to piceous, opaque, elytra frequently with four rufous spots. Beak
slender, cylindrical, three-fourths as long as thorax, at base slightly dilated, above
with four rows of rather coarse punctures and with a slight fovea between the eyes.
Thorax longer than wide, constricted near apex, sides feebly curved, gradually diver-
gent to base; disc densely, deeply, and coarsely punctured. Elytra oblong, slightly
narrowed at tip, deeply striate, striae very coarsely and closely punctured; intervals
slightly convex, narrow, the sutural with a row of coarse punctures; each puncture,
both of thorax and elytra, bearing a very short yellowish seta. Beneath very densely
and coarsely punctured.
Length 2.1 to 2.8 mm.3
1 Family Curculionidae, subfamily Calandrinae.
2 Gemminger, M., and Harold, B. de. op. cit., v. 8, p. 2653. 1871.
3 Rlatchley, W. S., and Leng, C W. op. cit., p. 575.
Jan. 15, 1921 Four Rhynchophora Attacking Corn in Storage 61 1
EGG
Egg opaque, shining white, ovoid to pear-shaped in form, widest below middle,
bottom broadly rounded, neck narrowing sharply toward top which is somewhat flat
and bears a small rounded protuberance that fits into a cap or plug that cements the
egg into place. Length 0.65 to 0.70 mm., width 0.28 to 0.29 mm.
LARVA
Mature larva 2.5 to 3 mm. in length, a pearly white, fleshy grub; very thick-bodied,
ventral outline being approximately straight while dorsal outline is almost semicir-
cular. Head light brown in color, anterior margin and mandibles much darker.
Head longer than broad and somewhat wedge-shaped, sides broadly rounded from
middle to apex, which is slightly angular. Sides nearly "straight from middle to
anterior angles, lateral area with an oblique, longitudinal, lighter stripe or area. Epi-
cranial and frontal sutures distinct and light in color; also two oblique, longitudinal,
light stripes rising from the frontal sutures and coalescing with the epicranial suture
near base of head. Frons subtriangular with a distinct, dark median line indicating
carina, running from posterior angle to beyond middle. Sutural margins irregular
or sinuate. Frons provided with five pairs of large setae, sutural margins each bearing
a large seta. Each epicranial lobe with the following setae: One close to posterior
angle of frons and located within oblique longitudinal stripe rising from frontal suture,
one very small seta posterior to this and near occiput, two anterior to it on disk of epi-
cranium, two opposite middle of frons, one opposite middle of mandible, one opposite
hypostomal angle of mandible, and one on hypostoma near base of mandible. Epis-
toma represented by thickened anterior margin of front, distinctly darker in color,
with anterior margin declivous and slightly curving and lateral angles slightly pro-
duced and elevated where they support dorsal articulation of mandibles. Pleuro-
stoma represented by darker declivous area surrounding mandibular foramen. Man-
dibles stout, triangular, with apex produced into a broad apical tooth; inner edge
toward apex provided with a subapical tooth and a small medial tooth; no molar part.
Dorsal area of mandible provided with a pair of stout bristles set apart. Eye repre-
sented by a well-defined black spot beneath exoskeleton. Clypeus attached in front
of frons and broadly transverse; broad at base, sides narrowing toward apical angles,
slightly longer and broader than labrum, and bearing on epistomal margin two fine
setae on each side. Labrum distinctly broader than long with two small lateral and a
larger rounded median lobe. Labrum provided with six large setae behind middle,
two marginal, short, thickened setae on each of lateral lobes, and six similar marginal
setae on median lobe.
Maxilla with cardo present and distinct, stipes not divided into stipes proper,
subgalea, and palpifer, but one continuous piece with the anterior inner angle pro-
duced into a single setose lobe. Palpus 2-jointed, bearing a single seta near apex of first
segment. Three other setae found on maxilla, two located on vaginant membrane be-
tween palpus and palpifer and one stouter and longer midway between palpus and
cardo. No articulating maxillary area between maxilla and mental-submental region.
Labium with submentum and mentum fused and represented by a broad lobe bearing
three pairs of stout setae. Stipes labii posteriorly enforced by a median, triangular
chitinization, the anterior median section produced anteriorly between the palpi into
a small lobe-like ligula which is fused with the lingua. Each stipes labii bears a single
seta. Short, conical, 2-jointed palpi situated on anterior angles of stipites. Ligula
bearing four small setae. Prothorax not divided dorsally, but two areas, praescutal
and scuto-scutellar, roughly indicated by rows of setae. Mesothoracic and metatho-
racic segments divided above into two distinct areas, the anterior of which repre-
sents praescutum, and the posterior the scuto-scutellum and alar area. The thoracic
spiracle is located on a lobe pushed into prothorax from epipleurum of mesothorax.
612 Journal of Agricultural Research vol. xx, No. 8
It is bifore, elongate, larger than abdominal spiracles and placed with the fingerlike
air tubes pointing dorsad. Ten abdominal segments; ninth small, tenth reduced.
Each tergum of first three abdominal segments divided above into three distinct
areas, praescutum, scutum, and scutellum. Each tergum of fourth to eighth abdom-
inal segments divided above into only two areas, first containing praescutal and scutal
elements, second representing scutellum. Below these two areas and adjacent to the
epipleurum is the alar area. Abdominal spiracles placed anteriorly and in a small
separate corner piece, probably of alar area; spiracles bifore and found on abdominal
segments one to eight, that on the eighth being located slightly more dorsad than the
rest. Below a very indistinct and abrupt dorso-lateral suture and above a well-
defined ventro-lateral suture is a large, not subdivided epipleurum. The abdominal
epipleura are located considerably higher than the thoracic lobes. Below ventro-
lateral suture ishypopleurum subdivided into three lobes, one directly under the other.
Below hypopleurum is coxal lobe and below that sternum, consisting of eusternum
and a posterior triangular area representing parasternum or parasternum fused with
sternellum. Abdominal segments provided with setse as follows: One on praescutum,
a long and two short ones on scutellum, two on alar area located just above spiracle,
two on epipleurum, one on coxal lobe, and two on eusternum. One of the setae on
the scutellum is usually missing on abdominal segments five to nine.
Measurements of larval stages
WIDTH OF
STAGE. LARVAL HEAD.
i o. 22 mm.
2 32 mm.
3 48 mm.
4 64 mm.
PUPA
Pupa uniformly pearly white when first formed. Length 3.75 to 4 mm.; width
about 1.75 mm. Tips of wing pads attaining seventh abdominal segment, tips of
metathoracic tarsi extending beyond tips of inner wings. Head rounded, beak
elongate and slender. Head with two prominent spines toward vertex, a group of
two small spines and two spinules on each side above eyes, two pairs of small spines
near anterior margin and one on each side of front between eyes. Three pairs of
spines on beak between frontal ones and base of antenna, a pair of small ones on beak
midway between base of antenna and tip of beak, a pair on sides of beak between
latter pair and tip of beak, and two pairs of smaller ones on tip of beak. Prothorax
provided with one pair of antero-marginal setigerous tubercules, one pair of antero-
lateral, two pairs of medio-lateral, and four pairs of dorsal setigerous tubercules.
Mesonotum and metanotum each provided with three pairs of spines. Abdomen has
seven distinct dorsal tergites, the seventh being much larger than the rest, dorsal area
of each armed with a pair of large and a pair of smaller spines. Lateral area of each
tergite armed with a spine at base of which is a small seta. Epipleural lobes are
each armed with two minute spines. Ninth segment is as usual armed with two
prominent pleural spines.
Jan. iS> i9" Four Rhynchophor a Attacking Corn in Storage 613
SITOPHILUS GRANARIUS
SYNONYMY 1
Sitophilus granarius Linn. 1758.
granarius "Linn. Syst. Nat. Ed. X. p. 378. — Panz. Fn. Germ. 17. 11. —
Gylh. Schh. Gen. Cure. IV. p. gyj. — Jacq. Duv. Gen. Col. Cure. 1854. t.
2Q.f. 140. — Frisch. Besckr. All. Ins. 1720. II. p. 36. I. 8.
pulicaria Yam. ed. Voet. IV. p. 54. t. 37. f. 17. (forte.)
segetis Linn. /. c. p. 381.
unicolor Marsh. Ent. Brit. p. 275. — Steph. 77/. Brit. IV. p. g. "
Sitophilus granarius (PI. 74) was described in 1758 by Linnaeus.
It is thought to have originated in the regions cf the Mediterranean,
but is now widely distributed throughout the world. It occurs but
seldom in the southern States of North America, preferring the cooler
climate of the North.
It is a slender, cylindrical, chestnut-brown beetle with a slender,
elongate beak. Technical descriptions of the adult and immature
stages follow.
ADULT
Elongate-oblong, feebly convex. Chestnut brown to piceous, moderately shining.
Beak two-thirds as long as thorax, slender, cylindrical, finely and sparsely punctate.
Thorax sparsely punctate, punctures coarse and on the disc more or less fusiform.
Elytra deeply striate, striae punctured at bottom, not serrate; intervals smooth,
alternately wider and more elevated, especially towards the base; the sutural with a
row of elongate punctures. Pygidium coarsely cribate. Body beneath coarsely
and less densely punctured than in oryza. Length 3 to 4 mm.2
EGG
Egg opaque, shining white, ovoid to pear-shaped in form, widest below middle,
bottom broadly rounded, neck narrowing gradually toward top, which is somewhat
flattened and bears a small rounded protuberance that fits into a cap or plug that
cements the egg in place. Length 0.68 to 0.80 mm., width about 0.33 mm.
LARVA
Mature larva 2.5 to 2.75 mm. in length; a pearly white, footless grub, fleshy and
very thick-bodied, ventral outline being approximately straight while dorsal outline
is almost semicircular. Head and appendages of head similar in every respect to
those of Sitophilus oryza. Thoracic segments similar in external appearance to those
of 5. oryza. The abdominal segments are similar in form to those of 5. oryza with
the following exceptions which afford the best characters for distinguishing between
larvae of these two species: First four abdominal segments divided above into three
distinct areas, praescutum, scutum, and scutellum, whereas in the larva of S. oryza
the first three only of the abdominal segments are so divided. Middle lobe of the hypo-
pleurum of the abdominal segments of 5. granarius is provided with a seta. This
seta lacking in larva of 5. oryza.
1 Gemminger, M., and Harold, B. de. op. err., v 8. p. 2653. 1871.
s Blatchley, W. S., and I.eng, C W. op. err., p. 574.
614 Journal of Agricultural Research vol. xx, No.8
Measurements of larval stages
STAGE. WIDTH OP LARVAL HEAD.
i o. 25 to o. 26 mm.
2 36 to .37 mm.
3 47 to .48 mm.
4 61 to .65 mm.
PUPA
Uniformly white when first formed; length 3.75 to 4.25 mm., width 1.75 mm. Tips of
elytra attaining fifth abdominal segment, inner wings rudimentary and almost com-
pletely concealed by elytra. Tips of metathoracic tarsi extending beyond tips of
elytra. Head rounded, .beak elongate. Head has two prominent spines toward
vertex, a group of two small spines and two spinules on each side above eyes, two pairs
of small spines near anterior margin and one on each side of front between eyes, three
pairs of spines on beak between frontal ones and base of antenna, a pair of small ones
on beak midway between base of antenna and tip of beak, a pair on sides of beak
between latter pair and tip of beak, and two pairs of minute spines on tip of beak.
Prothorax provided with one pair of antero-marginal setigerous tubercules, one pair
of antero-lateral, two pairs of medio-lateral, and four pairs of dorsal setigerous tubercules;
also a pair of minute medio-lateral ventral spines. Mesonotum and metanotum
normally each provided with three pairs of spines; one or more pairs often missing.
Abdomen with seven distinct dorsal tergites, the seventh being much larger than
rest. Dorsal area of each armed with a pair of large spines and a pair of smaller ones.
Lateral area of each tergite armed with a spine, at base of which is a small seta. Epi-
pleural lobes each obscurely armed with two minute setae. Ninth segment armed as
usual with two prominent pleural spines.
PLATE 7*
A raecerus fasciculatus:
A. — Pupa, dorsal view.
B. — Pupa, front view.
C-Egg.
D.— Mandible.
E. — Mature larva.
F. — Ventral view of head.
G. — Labium and clypeus.
H. — Pupa, lateral view.
I. — Head, face view.
J. — Head, dorsal view.
K. — Head, lateral view.
Key to larval parts
al=alar area.
cox=coxal lobe.
dlsut=dorso-lateral suture.
ep=epipleurum.
eu=eusternum.
hvp=hypopleurum.
par = parasternum .
post=postscutellum.
pres = praescutum .
sc=scutum.
scut=scutellum.
vlsut=ventro-lateral suture.
Four Rhynchophora Attacking Corn in Storage
Plate 71
Journal of Agricultural Research
Vol. XX, No.
Four Rhynchophora Attacking Corn in Storage
Plate 72
Journal of Agricultural Research
Vol. XX, No. 8
PLATE 72
Caulophilus latinasus:
A. — Pupa, dorsal view.
B. — Pupa, front view.
C— Egg.
D. — Mandible.
E. — Mature larva.
F. — Ventral view of head.
al=alar area.
cox=coxal lobe.
dlsut=dorso-lateral suture.
ep=epipleurum.
eu=eusternum.
hyp=hypopleurum.
G. — Labium and clypeus.
H. — Pupa, lateral view.
I. — Head, face view.
J. — Head, dorsal view.
K. — Head, lateral view.
Key to larval parts
par=parasternum.
post = postscute 1 lum .
pres=praescutum.
sc= scutum.
scut=scutellum.
vlsut=ventro-lateral suture.
PLATE 73
Sitophilus oryza:
A. — Pupa, dorsal view.
B. — Pupa, front view.
C— Egg.
D. — Mandible.
E. — Mature larva.
F. — Ventral view of head.
al=alar area.
cox=coxal lobe.
dlsut=dorso-lateral suture.
ep=epipleurum.
eu=eusternum.
hyp=hypopleurum.
G. — Labium and clypeus.
H. — Pupa, lateral view.
I. — Head, face view.
J. — Head, dorsal view.
K. — Head, lateral view.
Key to larval parts
par=parasternum.
post=postseutellum.
pres=praescutum.
sc= scutum.
scut=scutellum.
vlsut=ventro-lateral suture.
Four Rhynchophora Attacking Corn in Storage
Plate 73
Journal of Agricultural Research
Vol. XX. Mo. 8
Four Rhynchophora Attacking Corn in Storage
Plate 74
Journal of Agricultural Research
Vol. XX, No. 8
PLATE 74
Sitophilus granarius:
A. — Pupa, dorsal view.
B. — Pupa, front view.
C— Egg.
D. — Mandible.
E. — Mature larva.
F. — Ventral view of head.
al=alar area.
cox=coxal lobe.
dlsut=dorso-lateral suture.
ep=epipleurum.
eu=eusternum.
hyp=hypopleurum.
G. — Labium and clypeus.
H. — Pupa, lateral view.
I. — Head, face view.
J. — Head, dorsal view.
K. — Head, lateral view.
Key to larval parts
par=parastemum.
post=postscutellum.
pres=praescutum.
sc= scutum.
scut=scutellum .
vlsut=ventro-lateral suture.
CONCENTRATION OF POTASSIUM IN ORTHOCLASE
SOLUTIONS NOT A MEASURE OF ITS AVAILABILITY
TO WHEAT SEEDLINGS
By J. F. BreazealE, Associate Biochemist, and Lyman J. Briggs, Physicist in Charge,
Office of Biophysical Investigations, Bureau of Plant Industry, United States Depart-
ment of Agriculturt
The object of the experiments described in this paper was to deter-
mine the availability of the potassium in solution of orthoclase by growing
wheat seedlings in aqueous orthoclase solutions, analyzing the seed-
lings for potassium, and comparing the results with those obtained from
suitable controls. The results show that the potassium present in solu-
tions of orthoclase is not appreciably absorbed by young wheat plants.
The conclusion is reached that potassium may be present in soil solu-
tions in such combination with other elements that it is not available to
plants.
The orthoclase used in our experiments was obtained near Riverside,
Calif., and contained a total of 12.5 per cent of potassium oxid (K20).
It was ground to pass a 60-mesh sieve. Different samples when brought
into equilibrium with water and analyzed1 contained from 2 to 9 parts
per million of soluble potassium, the saturation concentration not being
definite. There was, however, always some potassium present in the
aqueous solutions, the average concentration being about 4 parts of po-
tassium oxid per million of solvent.
The wheat was germinated on perforated aluminum disks floated on
water. When the plumules were about ]/2 inch long the seedlings were
transferred to other aluminum disks in the pans containing the culture
solutions. This early transfer prevents the young seedling plants from
absorbing the potash which exudes form unsprouted seeds.
The method of experimentation was, in general, to compare the potas-
sium content of wheat seedlings grown in orthoclase solutions with that
of similar seedlings grown in distilled water or other suitable control
solution free from potassium.
SOLUBLE POTASSIUM IN ORTHOCLASE NOT AVAILABLE TO WHEAT
SEEDLINGS
Wheat cultures were grown in orthoclase solution with and without
the addition of gypsum and were compared with cultures grown in dis-
tilled water alone and in distilled water to which gypsum had been added.
(See Table I, series a.) Although the orthoclase solutions were known
1 The J. Lawrence Smith method was used in the analysis.
Journal of Agricultural Research, Vol. XX, No. 8
Washington, D. C Jan, is, 1921
win Key No. G-216
(615)
616 Journal of Agricultural Research vol. xx, No. 8
from analyses to contain potassium, it will be noted that the wheat seed-
lings were unable to absorb any of it. This is of special interest, since
the avidity of wheat seedlings for potassium is very marked.
The culture solutions in series b, Table I, included a control of distilled
water (No. i), 40 gm. of finely ground orthoclase in 2,500 cc. of distilled
water (No. 3), and potassium chlorid solution containing 4 parts per million
of potassium oxid (No. 5). Culture solutions Nos. 2, 4, and 6 were similar
to No. 1, 3, and 5, respectively, except that gypsum was added to each
in excess, so that it would always be present in the solid phase. To each
of the six cultures were added also 50 parts per million of nitrate (N03) as
sodium nitrate and 50 parts per million of phosphoric acid (P2 05) as
sodium phosphate. Each solution, except those in which orthoclase
was present in the solid phase, was changed twice daily in order to insure
uniformity in concentration and freedom from bacterial disturbances.
The wheat seedlings were grown in these culture solutions for 10 days.
The analyses of the plants indicated, as before, that the wheat seedlings
were unable to remove potassium from the orthoclase solutions. This
was not due, however, to the diluteness of the solution, for in culture
solutions containing only 4 parts per million of potash as potassium
chlorid the plants were able to more than double their potash content in
10 days. The addition of nitrogen and phosphoric acid to the solutions
did not modify the nonavailability of the potassium in the orthoclase
solutions.
In series c the cultures were maintained for 17 days, all solutions being
changed daily. Nitrogen and phosphoric acid were added to one culture,
the sodium base being omitted. The results again showed no marked
absorption of potassium from the orthoclase solutions.
The plants in series d were grown for 15 days. The analyses, as in the
preceding experiments, showed no appreciable absorption of potassium
by plants grown in orthoclase solutions, but a marked absorption was
observed by plants grown in solutions of potassium chlorid. The presence
of gypsum or orthoclase in the potassium chlorid solutions did not mod-
ify the rate of absorption of potassium from these solutions by the
wheat seedlings.
The results in Table I, taken as a whole, show that the potassium in
orthoclase solutions is not absorbed in measurable quantity by the wheat
seedlings. On the other-hand, potassium in potassium-chlorid solutions
of equivalent concentration is readily absorbed by the plants.
Jan. is, 1921 Concentration of Potassium in Orthoclase Solutions 6 1 7
Table I. — Relative availability of potassium in orthoclase solutions and in potassium-
chlorid solutions
Culture
No.
ia
2a
3a
4a
ib
2b
3b
4b
5b
6b
ic
2C
3C
4C
5C
id
2d
3d
4d
5d
6d
yd
8d
9d
Culture solution.
Control (distilled water)
Control with CaSo,
Orthoclase (solid phase present)
Orthoclase with CaSo4 (solid
phases present)
Control
Control with CaSo4
Orthoclase (solid phase present)
Orthoclase with CaSo4 (solid
phases present)
KC1
KC1 with CaSo,
Control
Orthoclase (solid phase present)
Orthoclase with CaSo4 (solid
phases present)
Orthoclase with CaC03 (solid
phases present)
Orthoclase with 50 p. p. m.
N03 and 50 p. p. m. P,05
Control
Control with CaSo,
Orthoclase (solid phase present),
changed daily
Orthoclase and CaSo4 (solid
phases present), changed
daily
Orthoclase (solid phase) and 4
p. p.m. K,0 as KC1, changed
daily
KC1
KC1 with CaSo4
Orthoclase and KC1, changed
once
Orthoclase, not changed
KsOin
solution.
P. p. m.
O
O
2 tO 9
2 tO 9
to
to
to
to
to
Dry weight
of plants.
to «9
O
o
to 9
2 to 9
6 to 13
4
4
6 to 13
2 to 9
Gm.
i- Si
i-53
i-54
1.58
1.36
i-34
i-43
i-45
i-5°
1. 40
3.68
3-96
3.68
3-92
3-64
4.08
4.48
4-3°
4- 5°
4- 5°
4-45
4.85
4. 20
4. 10
K20in
100 plants.
Gm.
o. 0295
. 0281
. 0284
. 0272
•036S
.0368
. 0366
.0372
.0783
.0860
.0310
.0302
•0345
•0341
.0341
.0368
•0395
•0457
041 1
'. 0978
.0947
. IOIO
.0683
K2O in-
crease over
control.
Per cent.
O
- 4
- 3
i.S
+
+ 2
+ 114
+ 136
+ 11
+ 10
+ 10
o
+ 7
+ 33
+ 11
+ 166
+ 157
+ 175
+ 86
+ 5
AVAILABILITY OF POTASSIUM IN ORTHOCLASE SOLUTIONS NOT IN-
CREASED BY LIME OR GYPSUM
The application of lime and gypsum to orthoclase-bearing soils has
been considered by some workers as a means of increasing the availability
of the potassium in such soils. The authors 1 found in an earlier investi-
gation that the addition of lime or gypsum to orthoclase solutions con-
taining the solid phase did not increase the concentration of the potas-
sium in the solution. The data presented in Table I show that these
substances also had no effect on the availability of the potassium in the
orthoclase solution.
1 Briggs, Lyman J., and Breazeale, J. F. availability of potash in certain orthoclase-bearing
soils as affected by lime or gypsum. In Jour. Agr. Research, v. 8, no. i, p. 21-28. 1917.
6i8
Journal of Agricultural Research
Vol. XX, No. 8
AVAILABILITY OF THE POTASSIUM IN ORTHOCLASE SOLUTIONS NOT
INCREASED BY BOILING THE SOLUTION
The effect of boiling an orthoclase solution on the subsequent avail-
ability of the potassium is shown in Table II. In this experiment the
potassium content of the plants grown in the culture solution was com-
pared with that of the original seed. The analyses show that within the
errors of experiment the availability of the potassium was not modified
by boiling the orthoclase solutions.
TABLE II. — Effect of boiling orthoclase solutions on the availability of the soluble potas-
sium
Culture
No.
Material analyzed.
K2O in solu-
tion.
Dry weight
of plants.
K2O in 100
plants.
K2O increase
over control.
Original seed. . ,
P. p. m.
Gm.
Gm.
0. 0368
.0386
■°33°
Per cent.
O
2
3
Seedlings grown in orthoclase
solution (solid phase present).
Seedlings grown in orthoclase
solution (solid phase present),
boiled
2 to 9
2 tO 9
4. OO
4.28
+ 5
-8
AVAILABILITY OF POTASSIUM IN ORTHOCLASE SOLUTION NOT
INCREASED BY PRESENCE OF CARBON DIOXID
Carbon dioxid is universally present in the soil solution. It is con-
sequently desirable to determine whether the availability of the potassium
in orthoclase may be measurably increased by the addition of carbon
dioxid to the solution. A culture solution of orthoclase with the solid
phase present was accordingly prepared, and a portion of this solution
was saturated with carbon dioxid. Plants grown in the two solutions
showed no difference in their potash content (Table III). It conse-
quently appears that a weak acid, such as carbonic acid, in concentra-
tions equivalent to those found in soil solutions, does not increase the
availability of the potassium in orthoclase.
Table III. — Effect of carbon dioxid on availability of potassium in orthoclase
Culture
No.
Culture solution.
K2Oin
solution.
Dry weight
of plants.
K2Oin
100 plants.
K2O in-
crease over
control.
1
2
Orthoclase (solid phase present) .
Orthoclase (solid phase present)
saturated with CO-?
P. p. m.
2 to 9
2 to 9
Gm.
I. 92
I. 72
Gm.
O. 0284
. 0284
Per cent.
O
0
jan is, 1921 Concentration of Potassium in Orthoclase Solutions 619
SOLUBLE POTASSIUM IN ORTHOCLASE SOLUTIONS IS MADE AVAIL-
ABLE BY OXIDATION WITH ACIDS
To determine whether the soluble potassium in orthoclase could be
available by oxidation with acids, the following experiment was car-
ried out.
Finely ground orthoclase was added to about 100 liters of water, and
this mixture was shaken at intervals until equilibrium was established
and the maximum solubility of the potassium in the feldspar had been
obtained.
One-half of this solution was filtered through a padded folded paper
filter, and the clear solution, together with a few cubic centimeters of a
mixture of hydrochloric and nitric acids, was then evaporated to dryness
in Jena beakers. The excess of acids was driven off, and the solution
was brought back to volume with purified distilled water. A little cal-
cium carbonate (CaC03) was then added to insure alkalinity. Wheat
seedlings were grown in such cultures for 14 days, the solutions being
changed daily. The results are given in Table IV, series a.
Table IV. — Effect of oxidation of soluble potassium in orthoclase on its availability
Culture
No.
ia
2a
3a
4a
ib
2b
3b
4b
Culture solution.
Control
Orthoclase (solid phase present)
Orthoclase solution filtered and
evaporated with acids
KC1
Control
Orthoclase (solid phase present)
Orthoclase solution filtered and
evaporated with acids
KC1
K2Oin
solution.
Dry weight
of plants.
K2Oin
100 plants.
P. p. m.
Gm.
Gtn.
0
2. 42
0. 0326
2 to 9
2. 52
.0349
4
5
2.88
2.48
. 0722
. 0620
0
2 to 9
3-3°
2.66
.0203
. 0180
4
5
3-3°
3-36
•°3S7
.0815
K20 in-
crease over
control.
+ 7
+ 121
+90
o
+ 11
+ 76
The wheat seedlings grown in orthoclase solutions in which the potas-
sium compounds had been oxidized showed a total potash content at
the end of the experiment about twice that of the plants grown in dis-
tilled water. On the other hand, the plants grown in the untreated
orthoclase solution showed as before no gain in potash over the control.
A repetition of the experiment, Table IV, series 6, again showed a
marked increase in the potash content of the plants grown in the solu-
tions prepared from the oxidized solute. The orthoclase solution used
in this series of experiments had stood in contact with the powdered
mineral for about 2 months, being shaken at frequent intervals. The
experiment extended over 19 days, the culture solutions being changed
daily.
17776°— 21 3
620
Journal of Agricultural Research
Vol. XX, No. 8
It is of interest to note that in the first series of experiments the potas-
sium absorbed from the oxidized solute was equal to that absorbed from a
potassium-chlorid solution containing 5 parts per million of potassium
oxid. In the second series, the plants grown in the potassium-chlorid
solution showed relatively a marked increase in their potassium content.
INCREASED AVAILABILITY OF POTASH IN OXIDIZED ORTHOCLASE
SOLUTIONS NOT DUE TO ACTION OF ACIDS ON SUSPENDED
COLLOIDS
The orthoclase solutions used in the preceding experiments contained
some suspended colloidal material. It is therefore possible that the
observed increase in the availbility of the potassium may have resulted
from the direct action of the acids on the suspended colloids. To deter-
mine this point, a saturated solution of orthoclase was prepared and
filtered through a Pasteur-Chamberland tube. A part of this filtrate was
then treated with acids and evaporated to dryness, as described above,
and subsequently diluted to its original volume and used as a culture
solution. A portion of the original orthoclase solution which had not
received the acid treatment was used as a control. The results of two
experiments, made at different times, are given in Table V.
Table V. — Effect of freeing culture solutions from colloids
Culture
No.
Culture solution.
K2O in 100
plants.
K2O increase
over control.
ia. . . .
2a. . . .
ib
Gm.
O. 0272
•°S97
.0302
•055I
Per cent.
O
+ I20
O
2b
+ 83
The analyses of the plants show as before a marked gain in the potas-
sium content of the plants grown in the acid-treated solutions. The
colloids can not in this case be considered the source of the potash made
available by the acid treatment, since the colloidal material was removed
from the solution before the acids were added. We are consequently led
to conclude that the orthoclase solutions contain potassium in true solu-
tion (as distinguished from colloidal suspension) and that the potassium
is chemically combined in such a manner that it is not available to plants.
DISCUSSION
The failure of wheat seedlings to absorb the potassium found by
analysis in orthoclase solutions suggests that the potassium is combined
with other elements in a slightly soluble molecular complex. This is
supported by the fact that the potassium may be made available by
treatment with strong acids, which would result from the breaking
Jan. is, 1921 Concentration of Potassium in Orthoclase Solutions 621
down of the complex. We may also assume that the solute complex is
not dissociated, at least in such a way as to liberate potassium ions.
For we can say with some assurance that free potassium ions would be
absorbed by the wheat seedlings. We have evidence of this in the
selective absorption exercised by wheat seedlings on potassium-chlorid
solutions in which the potassium (either as K KOFH *s selectively
absorbed to such an extent that the culture solution becomes distinctly
acid.
The effect of the oxidation of the solute complex in orthoclase
solutions by hydrochloric and nitric acids is to reduce the potassium
in the complex to potassium chlorid or potassium nitrate (KN03), in
which form it dissociates and is readily absorbed.
The evidence presented in the case of orthoclase leads to the general
statement that the concentration of a specific plant food element in the
soil solution does not necessarily provide any measure of its availability.
The question of availability must be referred to the plant itself, except
perhaps in those cases in which the element in question is known to be
ionized.
The results of our experiments have an immediate bearing on various
investigations now in progress looking toward the utilization of ortho-
clase as a source of potash. It should be borne in mind that the appli-
cation of finely ground orthoclase, without other treatment, probably
does not contribute immediately to the available potash content of the
soil.
CONCLUSIONS
From the experimental data presented the following conclusions are
drawn, subject to the limitations imposed by the experimental error:
(1) The soluble potassium in aqueous solutions derived from finely
ground orthoclase is not absorbed by wheat seedlings to a measurable
degree.
(2) The availability of the potassium is not increased by the addition
of lime, gypsum, or carbon dioxid to the solutions or by boiling the
solutions.
(3) The soluble potassium in orthoclase solutions is made available by
oxidizing the solute with hydrochloric and nitric acids.
(4) The increase in the availability following oxidation is not due to
the action of the acids on suspended colloids, but is to be ascribed to
the breaking down of the complex solute molecule.
(5) The concentration of a specific plant food element in the soil
solution does not necessarily provide any measure of its availability.
The question of availability must be referred to the plant itself.
COMPOSITION OF TUBERS, SKINS, AND SPROUTS OF
THREE VARIETIES OF POTATOES
By F. C. Cook
Physiological Chemist, Miscellaneous Division, Bureau of Chemistry, United States
Department of Agriculture
PREVIOUS INVESTIGATIONS
The composition of the potato undoubtedly varies with the soil and
with the fertilizer used, as well as with other environmental and climatic
conditions. Since the sprouts depend for their growth on the tubers, the
composition of the tubers may influence that of the sprouts to no small
extent.
The composition of tubers from different varieties of potato plants has
not been investigated, nor has any extended study been made of the
composition and growth changes of sprouts from the same or different
varieties of tubers. Buckner (j),1 who has reported analyses of sprouts
skins, and tubers from one variety of potatoes for ash, phosphoric acid,
magnesium oxid, calcium oxid, and silica dioxid found a relatively high
percentage of ash in the sprouts.
The cause and regulation of rest periods in plants have been studied
for years, several investigations having been devoted to the effect of
various chemicals on tubers, with a view to shortening the rest period.
Experiments at the Arizona Agricultural Experiment Station (5) have
shown that ethyl bromid, carbon tetrachlorid, ammonia, gasoline,
ethyl chlorid, and bromin are effective in bringing dormant tubers into
activity — that is, in stimulating the buds. Seed tubers treated with
manganese chlorid and ethyl ether showed no differences i-n the growth
of foliage but exhibited a pronounced increase of tuber formation.
Miiller (6) claims to have shortened the rest period of tubers by storing
them for one month at o° C. Appleman (1) has found an increase of
both total and reducing sugar in tubers stored at o° C. According to
this investigator, the carbohydrate transformation during the rest period
depends entirely on the changing temperature. He has separated also
the nitrogenous and the phosphorus compounds of tubers stored for
various periods.
Schulze and Barbieri (9), in 1878, showed that potato sprouts con-
tained nonprotein nitrogen in addition to protein nitrogen and found
asparagin and solanin. It was shown that the potato contained 0.38
per cent nitrogen, practically one-half, or 0.18 per cent of which was in
1 Reference is made by number (italic) to "Literature cited, " p. 634-635.
Journal of Agricultural Research, Vol. XX, No. 8
Washington, D. C Jan. 15, 1921
wa Key No. E-15
(623)
624 Journal of Agricultural Research vol. xx, No. s
the form of protein nitrogen. Eighty-one per cent of the nitrogen in the
tubers proved to be soluble — that is, appeared in the pressed juice of
the potato. The sprouts contained 1.5 per cent of nitrogen on a dry
basis. In 1880 these same investigators (/o) isolated leucin and tyrosin
from an alcoholic extract of potato sprouts. Osborne and Campbell (7)
obtained a globulin called "tuberin," the properties of which they de-
scribe, and a small amount of another protein from potato. Sjollema
and Rinkes (11) have studied the hydrolysis of potato protein. They
precipitated the protein with a saturated sodium -chlorid solution, dis-
solved it in 10 per cent sodium chlorid, dialyzed it to remove the salt,
and finally reprecipitated it with alcohol. The nitrogen content of the
protein obtained was 14.9 per cent. Their investigation was divided as
follows : (1) Estimation of the various diamino acids (Van Slyke method) ;
(2) hydrolysis of protein by hydrofluoric acid; (3) estimation of different
diamino acids (Kossel and Patten method); (4) estimation of momo-
amino nitrogen by Fisher's esterfication method; and (5) estimation of
tyrosin. The result of their study of the hydrolysis of potato protein
showed that 100 gm. contained nitrogenous substances distributed as
follows :
Gm.
Ammonia 1.8
Histidin 2
Arginin 4
Lysin 3
Cystin 4
Glutaminic acid 4
Prolin 3
Gm.
Alanin 4. q
Leucin 12
Valin 1
Valin and alanin 8
Valin and leucin 1. 9
Phenylalanin 3.9
Tyrosin 4. 3
Ramsay and Robertson (8) have reported data on the rate of assimi
lation of food from the soil by the potato plant and the relative pro-
portion of each of the principal elements contained in the plants.
The fact that Bordeaux-sprayed potato plants in certain localities give
larger yields of tubers than unsprayed plants has been established by a
series of experiments extending over many seasons at the Vermont,
Maine, and New York Agricultural Experiment Stations. Stewart,
Eustace, and Sirrine (12), of the New York Experiment Station, reported
several years ago that one lot of Bordeaux-sprayed tubers was higher
in solids and starch than a corresponding lot of unsprayed tubers.
Charles D. Woods, of the Maine Experiment Station, has reported simi-
lar findings (13). The writer has analyzed several samples of Bordeaux-
sprayed and unsprayed tubers grown in Maine during the past three
seasons, generally finding a higher content of solids and nitrogen in the
sprayed than in the unsprayed tubers.
jan. is, 1921 Tubers, Skins, and Sprouts of Potatoes 625
OBJECT OF PRESENT INVESTIGATION
It was thought that some variation in the composition of sprouts of the
same or different varieties of tubers might be found. It was also believed
that the copper sprays used to control Phytophthora infestans, or late
blight of the potato, might influence the time of sprouting — that is,
increase or decrease the rest period compared with that of the unsprayed
tubers — or that these sprays might modify the composition of the sprouts
of the same varieties of tubers, just as copper sprays apparently influence
the composition of the tubers. An investigation, therefore, was under-
taken to determine, if possible, whether any of the changes just men-
tioned took place and to secure data on the chemical composition of
sprouts, skins, and tubers.
EXPERIMENTAL WORK
DESCRIPTION OF SAMPLES
In the course of some tests on the influence of copper sprays on the
control of Phytophthora infestans, or late blight of the potato plant, and
on the yield of the tubers, several samples of Maine and Connecticut
tubers dug in September, 1918, were stored in the laboratory at Wash-
ington, D. C, from October, 19 18, until they were analyzed in the spring
of 1919. Samples of Rural New Yorker (No. 12), Green Mountain
(No. 15), and Irish Cobbler tubers (No. 9) from Maine, from selected hills
where the vines were vigorous and healthy, as well as Green Mountain
tubers (No. 3 and 6) from Connecticut, taken from portions of the plots
which stayed green the longest were used for these tests. All the tubers
were held in a dark closet at laboratory temperature (average 700 F.)
from October, 1918, until February, April, or June, 1919. This rela-
tively high temperature may have affected the composition of both
tubers and sprouts. Several sprouts developed on each tuber, those on
the Rural New Yorker appearing later than those on the Green Mountain
and Irish Cobbler tubers. The sprouts of the Rural New Yorker tubers
were short and thick, while those of the Green Mountain and Irish Cob-
bler tubers were comparatively long and branching.
METHODS OF ANALYSIS
At the time of analysis the sprouts were removed from the tubers and
sifted to free them from adhering dirt. The tubers were washed and
dried and then pared as thin as possible, a difficult matter because of
their soft condition. The weights of the moist skins, tubers, and sprouts
were taken separately. The tubers and the skins were then ground
separately in a meat grinder, and each sample was well mixed and placed
in a Mason jar with rubber and top. The sprouts were placed in a stop-
pered bottle. The analyses were begun as soon as possible. Solids, ash,
phosphorus, and nitrogen determination were made on the moist samples,
626 Journal of Agricultural Research voi.xx,No. 8
the methods of the Association of Official Agricultural Chemists (2) being
used.
Water extracts of the sprouts, skins, and tubers were prepared by
macerating 50 gm. of the moist samples with a pestle in a mortar, then
rinsing the material into a graduated flask with water, adding 10 cc. of
toluene and making up to 500 cc. with water. The flasks were shaken
each minute for the first 5 minutes and then every 15 minutes for the
first hour, after which they stood overnight at room temperature. The
next morning the liquid was removed with a pipette and filtered through
glass wool and then through filter paper. The following determinations
were made on the water extracts: (1) Soluble nitrogen, employing
25 cc; (2) soluble phosphoric acid, employing 50 cc; (3) ammonia
nitrogen, employing 5 cc, by the aeration method of Folin (14) and
nesslerizing the volatile nitrogen; (4) separation of nitrogenous com-
pounds, employing 100 cc.
In making a separation of the nitrogenous compounds, 100 cc. of the
solution were acidified and heated to boiling. The coagulable protein
was removed first, then the remaining protein, by precipitation with
dilute lead acetate solution. The lead was removed from the filtrate
with hydrogen sulphid, the lead sulphid being filtered off and washed
with a dilute solution of hydrochloric acid through which hydrogen sul-
phid had been passed. The solution containing the amino acids, amids,
etc., was then made to volume, and the total nitrogen was determined
in an aliquot. The largest portion of the fdtrate was precipitated with
phosphotungstic acid according to the Hausman method, and the nitro-
gen in the filtrate (monoamino and amid nitrogen) was determined.
The nitrogen of the diamino acids and other bases was obtained by
difference.
Copper was determined in certain of the samples by the colorimetric
method, using potassium ferrocyanid and standard solutions of copper
sulphate. This method, which has been shown to yield identical results
with the electrolytic method, has the advantage of giving accurate
results when minute amounts of copper are present and of being appli-
cable when the electrolytic method is not.
RESULTS OF ANALYSIS
RELATIVE WEIGHTS OF SPROUTS AND TUBERS
The samples of sprouts, skins, and tubers numbered 1 to 3 and 4 to 6
(Table I) were of the Green Mountain variety. On February 1, 19 19,
the sprouts on these two sets of tubers constituted 4.6 per cent of the
total moist weight of sprouts, skins, and tubers. Samples 1,2, and 3 were
from vines sprayed with 5-5-50 Bordeaux spray, while samples 4, 5,
and 6 were from unsprayed vines. Samples 7,8, and 9 (sprouts, skins,
and tubers) were from Irish Cobbler plants. At the time of analysis,
Jan. 15, 1921 Tubers, Skins, and Sprouts of Potatoes 627
April, 19 19, the sprouts constituted 13.33 Per cent °f the total moist
weight of sprouts, skins, and tubers. These plants had been sprayed
with 5-5-50 Bordeaux.
The Rural New Yorker tubers (samples 10, n, and 12) and the Green
Mountain tubers (samples 13, 14, and 15) were grown at Foxcroft, Me.,
and had been sprayed with 5-5-50 Bordeaux spray. At the time of
analysis, April, 19 19, the sprouts of the Rural New Yorker tubers, a late
variety, constituted 3.5 per cent and the Green Mountain sprouts 7.2
per cent of the total moist weight. These two varieties of tubers, dug
from the same field late in September, 19 18, were stored in the laboratory
under identical conditions.
Samples of Green Mountain potatoes from Connecticut (samples 18
and 19), as well as the Irish Cobbler tubers grown in Maine (sample 21),
were held at laboratory temperature until June, 1919, when the sprouts
and tubers were analyzed separately. While the sprouts of the Irish
Cobblers were large and fresh, those of the two samples of Green Moun-
tain potatoes were partially dried and withered. No analyses of the
skins were made for these three samples analyzed in June because of
the difficulty of paring the soft tubers.
The variations in the percentage composition of sprouts obtained from
tubers stored under identical conditions can be explained only on the
basis of the presence in varying amounts of growth-promoting substances
in the different varieties of tubers.
COMPOSITION OF SPROUTS AND TUBERS
The analytical data in Table I include the distribution of nitrogen in
terms of total nitrogen. The total weight and percentage distribution of
the ash, phosphoric acid, and nitrogen compounds present in the sprouts
and tubers are given in Table II. Table III shows the ash, phosphoric
acid, and nitrogen results on a water-free basis.
628
Journal of Agricultural Research
Vol. XX, No. 8
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Tubers, Skins, and Sprouts of Potatoes
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630
Journal of Agricultural Research
Vol. XX, No. 8
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Jan. is, 1921 Tubers, Skins, and Sprouts of Potatoes 631
Solids. — The solids of the young sprouts of the Green Mountain and
Irish Cobbler varieties, samples 1, 4, 7, and 13, were exceedingly uniform,
notwithstanding variations in the water content of the tubers. The
moisture content of the sprouts seemed to be maintained at the expense
of the tubers. The Rural New Yorker sprouts, sample 10, contained
more solids than the other young sprouts. The older, partly dried
sprouts, samples 16, 17, and 20, were highest in solids. The moisture
content of the different varieties of tubers decreased with the period of
standing in the laboratory.
Ash. — The important feature of the ash analyses was the high per-
centage of ash in the sprouts as compared with that in the tubers, made
more evident on calculating the results to a moisture-free basis. The
skins showed a higher percentage of ash than the tubers. The sprouts
showed a selective action and withdrew the ash from the tubers in a
greater proportion* than it originally existed in them, so that the per-
centage of ash in the solids was nearly twice as high for the sprouts as
for the tubers. A higher percentage of ash was found in the old than in
the young sprouts and tubers.
Phosphoric acid (p2o5).— The phosphoric acid content of the sprouts
was greater than that of the skins or tubers. In the solids of the sprouts
it averaged 1.81 per cent and was less than 1 per cent for the skins and
tubers. In the ash of the sprouts it varied from 20 to 30 per cent, while
it was less than 20 per cent in the ash of the tubers and skins. From 60
to 76 per cent of the total phosphoric acid content of the young sprouts
was water-soluble as compared with but 50 to 60 per cent of the phos-
phoric acid content of the skins and tubers. Somewhat less phosphoric
acid was water-soluble in the older sprouts and tubers than in the younger
samples.
Nitrogen. — The nitrogen content of the sprouts was apparently
uniformly maintained. In the five samples of young sprouts examined
(No. 1, 4, 7, 10, and 13) approximately 0.75 per cent of nitrogen was
found. The older sprouts contained from 1.10 to 1.27 per cent nitrogen.
The different varieties of sprouts showed a uniform percentage of the
total nitrogen, both as protein nitrogen and as amid and monoamino
nitrogen. The amid and monoamino nitrogen formed about 40 per
cent and the diamino and other basic nitrogen formed less than 10 per
cent of the total nitrogen of the sprouts. A higher percentage of amid
and monoamino nitrogen was found in the older Green Mountain sprouts
(samples 16 and 17) than in the younger Green Mountain sprouts (sam-
ples 1 and 4). The sprouts contained a lower percentage of total nitrogen
in the form of coagulable protein but a higher percentage as total protein
than did the tubers. The younger sprouts also contained a lower per-
centage of the total nitrogen as amid and monoamino nitrogen and of
diamino and other base nitrogen than did the tubers. Based on the
632 Journal of Agricultural Research voi.xx.No.s
percentage of total nitrogen, the younger tubers showed a greater content
of water-soluble nitrogen than the older tubers. The samples of tubers
analyzed in June contained a larger amount of total nitrogen than those
analyzed earlier because of the added loss in water and the reduction in
sugar and starch of the tubers caused by respiration.
Ammonia. — The amount of free ammonia in the young sprouts was
constant. More ammonia was found in the skins than in the tubers or
sprouts. The older tubers apparently contained less ammonia than the
younger ones.
Copper. — All the samples tested showed copper, the sprouts contain-
ing somewhat more than the tubers or skin.
FACTORS WHICH MAY INFLUENCE THE COMPOSITION OF POTATO SPROUTS
Numerous factors may influence the composition of potato sprouts.
Excluding the various physiological and other diseases, a few of these
factors may be mentioned briefly.
Variety. — The analyses indicate that the composition of sprouts of
the same age from the three different varieties of tubers examined was
uniform. This was true in spite of the fact that the sprouts formed vary-
ing percentages of the total moist weight of tubers, skin, and sprouts and
contained varying percentages of the total nitrogen, phosphoric acid,
and ash.
Bordeaux spraying. — The results for solids and ash on the Green
Mountain sprouts, skins, and tubers from sprayed vines (samples 1,2,
and 3) were slightly higher than those on sprouts, skins, and tubers
from corresponding unsprayed vines (samples 4, 5, and 6). The distri-
bution of the nitrogenous substances showed the same general trend in
the two samples. The tubers from both the sprayed and unsprayed
plants formed sprouts with equal rapidity, judging by the percentage
weights of sprouts and tubers. The sprouts constituted 4.63 and 4.59
per cent of the total weight of sprouts, skins, and tubers of the two
samples at the time of analysis. The percentages of nitrogen, phos-
phoric acid, and a$h removed by the sprouts in the two cases were
remarkably uniform. While it is impossible to draw a definite conclu-
sion from the analyses of two samples only, the indication from these
and other samples is that the percentage of solids and nitrogen is higher
in the tubers from sprayed than in those from unsprayed potato vines.
Soil, climate, and fertilizer. — The potato is no exception to the
well-known fact that soil, climate, fertilizer, and other factors often in-
fluence the composition of the crop. Calculated to a water-free basis
(Table III), the Connecticut tubers and sprouts gave higher results for
ash, phosphoric acid, and nitrogen than the other samples, suggesting an
influence of soil and climate on the composition of the potato.
Age and growth. — The age of the sprout apparently influences its
composition. A higher percentage of solids and ash was found in the
Jan. 15, 192 1
Tubers, Skins, and Sprouts of Potatoes
633
older than in the younger sprouts. Many changes in the percentage of
water-soluble to total phosphorus and in the distribution of the nitro-
genous substances follow the growth of the sprouts. The principal
period of growth of the sprouts under the conditions of this test occurred
during the period up to March, or from 60 to.150 days after the tubers had
been dug. From 1 50 days until the end of June, or 270 days after digging,
the increase in weight of the sprouts was less. The sprouts of the Irish
Cobbler tubers analyzed in June (sample 20) constituted 17 per cent,
while those of the Green Mountain tubers (samples 16 and 17) constituted
5.5 per cent of the total weight of tubers and sprouts. The Cobbler is an
early potato and the Green Mountain a late one. Both varieties had
reached their limit of sprouting in June under the conditions of these
tests. Apparently the growth-promoting principle is much more active
or is present in larger amounts in the Irish Cobbler than in the Green
Mountain and Rural New Yorkers.
DISTRIBUTION OF NITROGEN, PHOSPHORIC ACID, AND ASH IN SPROUTS, SKINS, AND
TUBERS
The percentage distribution of nitrogen, phosphoric acid, and ash in
sprouts, skins, and tubers depends upon the relative weights of sprouts
and tubers. Although the sprouts of the Rural New Yorker tubers consti-
tuted 3.5 per cent of the total moist weight of tubers, skins, and sprouts,
they contained 6.32 per cent of the total nitrogen. The sprouts of the
Irish Cobbler on the same date constituted 13.33 Per cent of the total
moist weight and contained 14.81 per cent of the total nitrogen. Similar
ratios hold for the distribution of phosphoric acid and ash. This indi-
cates that the sprouts obtained the nitrogen, phosphoric acid, and ash in
certain proportions from the tubers, the tubers simply acting as reser-
voirs for the sprouts. The action of the sprouts was selective, as might
be expected in young, growing tissue. The solids of the sprouts con-
tained 4 per cent of nitrogen, while the solids of the tubers and skins con-
tained less than 2 per cent. In the Irish Cobbler the percentage of ash,
phosphoric acid, and nitrogen remaining in the tubers after sprouting
had ceased was less than 50 per cent of the total.
Buckner (3) found 17.77 Per cent of the total phosphoric acid in the
sprouts and 67. 1 3 per cent in the exhausted tubers. Because he found that
50 per cent or more of the mineral matter was left in the tubers, he thought
that a large amount of ash was necessary to bring about the katabolic
changes involved in sprouting. He obtained the following results :
Material examined.
New sprouts
Skins
Tubers
Ash in
solids.
Per cent.
9.91
8. 14
4-37
P2O5 in
ash.
Per cent.
12. 56
5-89
12. 4
CaO in
ash.
Per cent.
0. 90
1. 70
•75
MgO in
ash
Per cent.
2. 72
2.25
K2O in
ash.
Per cent.
40. 40
40.33
53- 52
Si02in
ash.
Per cent.
o-95
8.45
.60
634 Journal of Agricultural Research voi.xx.Mo. 8
Chlorin and other ash constituents in potato ash are not included in
Buckner's analyses.
SUMMARY
Analytical data for sprouts, skins, and tubers of three varieties of
Bordeaux-sprayed potatoes "stored at labratory temperature (average
700 F.) showed little variation in composition for the different varieties,
the age of the sprout apparently influencing the composition more than the
variety. " Data for Green Mountain sprouts, skins, and tubers from
Bordeaux-sprayed ~nd from unsprayed plants indicated that the spray
did not change the rate of growth or the composition of the sprouts.
Biological changes are taking place in the formation and growth of the
sprouts. The percentage distribution of the nitrogenous substances
showed the sprouts to contain more protein and less diamino and other
basic nitrogen than the skins and tubers. The sprouts showed a selec-
tive action in withdrawing from the tubers nitrogen, ash, phosphoric acid,
and water in larger proportion than was originally present.
The sprouts remained fresh and continued to grow as long as any water
was available in the tubers. The sprouts of the Irish Cobbler tubers
constituted 17 per cent of the total weight of the sprouts and tubers at
the time the tubers were exhausted, while the Green Mountain sprouts,
under the same conditions, constituted 5.5 per cent of the total weight.
An increased concentration or activity of the growth-promoting agent
or agents in Irish Cobbler tubers is suggested.
LITERATURE CITED
(1) Appleman, Charles O.
1914. BIOCHEMICAL AND PHYSIOLOGICAL STUDY OF THE REST PERIOD IN THE
tubers op solanum tuberosum. Md. Agr. Exp. Sta. Bul. 183, p.
181-226, 17 fig.
(2) Association of Official Agricultural Chemists.
1920. official and tentative methods of analysis. As compiled by the
committee on revision of methods. Revised to November 1, 1919.
417 p., 18 fig. Washington, D. C. Bibliographies at ends of chapters.
(3) BucknER, David.
1915. TRANSLOCATION OF MINERAL CONSTITUENTS OF SEEDS AND TUBERS OF
certain plants during growth, hi Jour. Agr. Research, v. 5, no. 11,
P- 449~458-
(4) Folin, Otto.
1910. NOTE ON THE DETERMINATION OF AMMONIA IN URINE. In Jour. Biol.
Chem, v. 8, no. 6, p. 497-498.
(5) McCallum, W. B.
1909. plant physiology and pathology. In Ariz. Agr. Exp. Sta. 20th Ann.
Rpt., [1908V09, p. 583-586.
(6) MullER, Hermann, Thurgau.
1885. BEITRAG ZUR ERKLARUNG DER RUHEPERIODEN DER PFLANZEN. In
Landw. Jahrb., Bd. 14, p. 851-907, 1 fig.
Jan. is. 192 1 Tubers, Skins, and Sprouts of Potatoes 635
(7) Osborne, Thomas B., and Campbell, George F.
1896. The proteids OF THE potato. In Jour. Amer. Chem. Soc, v. 18, no .7
p. 575-582.
(8) Ramsay, J. T., and Robertson, W. C.
1917. THE COMPOSITION OF THE POTATO PLANT AT VARIOUS STAGES OF DEVELOP-
MENT. In Jour. Dept. Agr. Victoria, v. 15, pt. 11, p. 641-655, illus.
(9) Schulze, E., and Barbieri, J.
1878. UEBER DEN GEHALT DER KARTOFFELKNOLLEN AN EIWEISSSTOFEN UND AN
amiden. In Landw. Vers. Sta., Bd. 21, p. 63-92.
(10)
1880. UEBER DAS VORKOMMEN VON LEUCIEN UND TYROSIN IN DEN KARTOFFEL-
KNOLLEN. In Landw. Vers. Sta., Bd. 24, p. 167-169.
(n) Sjollema, B., and Rinkes, I. J.
1912. die hydrolyse DES KARTOFFELEIWEISSES. In Ztschr. Physiol. Chem.,
Bd. 76, Heft 5/6, p. 369-384.
(12) Stewart, F. C, Eustace, H. J., and Sirrine, F. A.
1902. potato spraying experiments in 1902. N. Y. State Agr. Exp. Sta.
Bui. 221, p. 235-263.
(13) Woods, Chas. D.
1919. potato studies. Maine Agr. Exp. Sta. Bui. 277, p. 17-32.
17776°— 21 4
FURTHER STUDIES IN THE DETERIORATION OF
SUGARS IN STORAGE 1
By Nicholas KopELOFF, H. Z. E. Perkins, and C. J. Welcome, Louisiana Agri-
cultural Experiment Station
In a study of the deterioration of Cuban raw sugars stored under
normal conditions during the summer of 191 9 certain conclusions were
indicated concerning the correlation between chemical and bacteriolog-
ical analysis, with special reference to losses in sucrose content.2 It was
shown that the keeping quality of a sugar depends not only upon the
moisture ratio but likewise upon the content of microorganisms and
that any prediction concerning deterioration involves a concomitant
consideration of these two factors.3 In the present investigation of
sugars stored in 1920 the technic and procedure were identical with those
previously used, which have been described elsewhere;4 the only differ-
ence was that in 1920 the position of the bags in any single pile was re-
versed after four weeks' incubation to obtain uniformity of environment,
and the bags were placed on scantling 1 foot from the floor and were
protected by a covering of a single layer of sacks.
It was especially designed to have under observation as large a variety
of sugars as possible, and from the succeeding data it will be seen that
all extremes in polarization, moisture, and number of microorganisms
are to be found. This is not only true of the different marks chosen but
more significantly of the bags of each mark. As a rule 3 bags which
varied sufficiently to be considered representative of the mark were
chosen, and in some instances, where the variations in a lot were unusual,
6 bags were taken. It may be mentioned parenthetically that it was
planned to sample the bags monthly for six months, but because of the
postponed arrival of sugar it was necessary to delay the initial sampling
and thus curtail the number of analyses. In the succeeding tables the
names of the marks have been abbreviated to symbols, since there has
been no intention of subjecting any of the sugars to criticism. All the
sugars came from Cuba with the exception of 2 marks, M and A, from
Porto Rico. Seven of the 10 marks represent sugars transported by
vessel, the remaining 3 (Am, O, and Phil) having come by railroad via
1 Published by the courtesy of the American Chemical Society. Paper read at the meeting held in St.
Louis, April, 1920.
It is a privilege to acknowledge the invaluable assistance of Mr. J. McFetridge, whose interest made it
possible to carry out this investigation, and the efficient help of Mr. Salvant and his associates at Chalmette,
La.
2 Kopeloff, Nicholas, and Perkins, H. Z. E. the deterioration of cuban raw sugar in storage.
in Jour. Indus, and Engin. Chem., v. 12, no. 6, p. 555-558, 1920.
3 and Kopeloff, Lillian, the deterioration of cane sugar by fungi. La. Agr. Exp. Sta.
Bui. 166, 72 p., illus. 1919. Literature cited, p. 69-72.
1 — ■ — ■ Welcome, C. J., and Kopeloff, Lillian, the prevention of sugar deterioration. La.
Agr. Exp. Sta. Bui. 175, 58 p., 1 fig. 1920. Literature cited, p. 58.
Journal of Agricultural Research, Vol. XX, No. 8
Washington, D. C Jan. 15, 1921
wo Key No. La.~3
(637)
638
Journal of Agricultural Research
Vol. XX, No. 8
Key West. The following numbers of bags of each mark were received :
F, 2,303; Port, 4,694; Cun, 17,000; Agr, 13,500; Cab, 10,000; Am,
2,831; O, 1,406; Pil, 4,060; M, 11,300; Ag, 5,000 — totaling 23,070,080
pounds of sugar. The bags under observation were chosen from among
these.
In Table I are presented the chemical and bacteriological analyses of
the sugars under normal storage. The moisture ratio or factor of safety
Moisture
has been calculated according to the formula M. R. = _ , — -. — r- — >
a detailed discussion of which may be found in previous publications.1
The last column refers to the percentage of molds based on the total
number of microorganisms per gram.
Table I. — Chemical and bacteriological analyses of Cuban raw sugars in storage2
TRANSPORTED BY VESSEL
Tj
a
•g 6
ctf
u °
ri
3
*Q -
t
•3 S
a
a
a
at
0
a0
Mark
■g
j
0
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i- *>
No.
M
S
a!
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c
n
C3
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-
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•R ^
5
3
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u
a
c
0
a
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11
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2 5'5
C E 3
£ g g
g
PL,
G
Ph
J
s p
i
0
<
^
-
B
a
S
Per P
er
Per
Per
Per
cent. ce-
it.
cent.
cent.
cent.
[Mar.
8
93-2
2.90 1
83
0.30 0
43
+
35,000
+
1
[Middle.
Upr.
5
92
s
0. 7
2
80 I
99
0. 16
37
+
1.650,000
+
*
F 1...
J
(May
3
91
4
1.8
3
17 3
13
1.30
37
30,000
*
Mar.
8
94
2
2
31 1
30
•30
40
+
8,000
+
11
ISurface.
Upr.
S
92
7
1-5
2
34 2
10
.80
32
+
18,000
+
3
iMay
3
94
8
I
70 I
2ft
33
38.000
*
[Mar.
8
98
2
80
54
.28
44
+
9.000
+
2
[Middle
{Apr.
S
97
S
•7
96
S9
.05
38
+
1 70, 000
+
*
F a....
[May
3
96
9
1-3
1
00
77
•23
}-■
17,000
2
[Mar.
8
97
9
75
58
•27
ift
+
2.000
+
10
ISurface.
{Apr.
S
98
4
46
—
7,600
+
0
[May
[Mar.
3
8
97
96
8
4
1
7°
70
46
6}
31
47
'+'
2, 100
134,000
+
■33
0
[Middle.
\ Apr.
5
95
8
.6
1
62
96
■33
39
+
7,000,000
+
0
F3...
J
IMay
3
95
8
.6
I
67 1
SO
.87
40
10,000
*
[Mar.
8
97
0
I
20
66
•36
40
+
156,000
+
0
ISurface.
\ Apr.
5
96
6
•4
I
27
85
■ 19
37
+
5,500.000
+
0
(May
3
95
8
1. 2
I
50 I
37
■71
36
30.000
0
[Mar.
8
96
5
I
08 1
Of)
■39
31
+
240
+
3
[Middle.
< Apr.
(May
[Mar.
5
3
8
96
95
96
3
9
5
. 2
.6
I
I
1
00 1
30
10 1
05
93
OS
27
31
31
.„.
1.500
6,000
300
+
+
Port 1 .
•42
0
1 Surface.
\ Apr.
[May
5
3
96
96
5
3
0
. 2
1
T
02
10 I
98
29
—
800
+
0
(Mar.
8
96
0
I
18 I
16
•45
30
+
6,000
+
0
[Middle
Upr.
(May
5
3
95
95
9
8
. 1
. 2
I
I
10 1
28
09
97
27
—
98
—
Port 2 .
[Mar.
8
96
2
I
16 1
SO
.42
?o
+
1,150
+
0
ISurface.
{Apr.
(.May
(Mar.
S
3
8
96
95
95
0
8
5
. 2
•4
I
I
I
10 1
19 1
42 1
21
02
17
28
30
'+'
150
8,000
1,000
+
*
•5'
10
[Middle.
{Apr.
(May
Mar.
5
3
8
95
95
95
5
1
4
0
•4
I
I
I
25 1
42 1
42 1
09
04
04
28
30
31
'+'
2, 100
1,800
+
+
0
Port 3.
x
•55
0
[Surface.
{Apr.
5
95
5
I
10 I
0;
. 01
24
—
2 2 , 000
+
0
(May
3
95
4
0
I
37
98
?0
2, 150
0
1 Kopeloff, Nicholas, and Kopeloff, Lillian, factors determining the keeping quality of cane
sugar (with a chart for prediction). La. Agr. Exp. Sta. Bui. 170, 63 p., 1 fig. 1920. Literature
cited, p. 62-63.
— ■ Welcome, C J., and Kopeloff, Lillian, the prevention of sugar deterioration. La. Agr.
Exp. Sta. Bui. 175, 58 p., 1 fig. 1920. Literature cited, p. 58.
2 * Indicates negligible amount of mold.
Jan. 15, 1921
Deterioration of Sugars in Storage
639
Table I. — Chemical and bacteriological analyses of Cuban raw sugars in storage — Con.
transported by vessel — continued
Mark
No.
Cun 1.
Cun :
Cun 3.
Cun 4.
Cun 5..
Cun 6. .
Agr 1.
Agr 2.
Agr 3.
Cab 1..
Cab 2.
Middle.
Surface.
Middle.
Surlace.
Middle.
Surface.
Middle
Surface
Middle
Surface
Middle.
Surface
Middle.
Surface.
Middle.
Surface.
Middle
Surface.
Middle
Surface
Middle
Surface.
[Mar.
•Upr.
r
May 1 7
Mar. 22
Apr. 19
.May 17
{Mar. 22
Apr. 19
May 17
[Mar. 22
{Apr. 19
[May 17
fMar. 22
Apr. 19
May 17
[Mar. 22
{Apr. 19
[May 1 7
'Mar. 22
Apr. 19
May 1 7
Mar. 22
Apr. 19
..May 17
[Mar. 22
■(Apr. 19
[May 17
[Mar. 22
{Apr. 19
[May 1 7
[Mar. 22
SApr. 19
I May 17
'Mar. 22
Apr. 19
May 17
Mar. 30
Apr. 27
.May 2 1
(Mar. 30
{Apr. 27
(May 21
[Mar. 30
{Apr. 27
I May 21
[Mar. 30
Per
cent.
2. 00
1-75
1. 71
1.94
2. 10
2.15
1.68
2. 10
2. 21
1.50
1. 8S
2.00
1. 27
I- 52
1.66
132
1.80
2.08
1. 04
I- 51
1. 81
1. 20
1.30
1. 00
1. 16
1. 04
■94
1. 16
I. 14
1.30
1.50
1.62
I.60
1.28
1-45
1-54
1.40
Per
ceti I.
98
76
47
Per
cent.
■ 14
•67
•83
1. 62
i- 63
Per
cent.
1. 02
.96
.48
36
6g
3-~
400
85
I, IOO
1. 700
I IO, OOO
120.000
I.350
2,500
160,000
575
190
5,000
350
190
480
14.000
100,000
19,000
14,000
20,000
4, 100
600
2, 700
22, OOO
235
336
1,000
125
25O
3,000
475
575
560
67
1,000
3,100
125
35°
93°
120,000
115,000
145,000
18,000
7,500
215
190
1,800
I; ISO
283
300
220
300
55> 75°
225,000
250
2, 200
55,000
290
165
2,000
210
500
35°
sis
Per
c ent.
640
Journal of Agricultural Research
Vol. XX. No. 8
Table I. — Chemical and bacteriological analyses of Cuban raw sugars in storage — Con.
transported by vessel — continued
^
•d
3
■a 6
5
bi
3
a. a
M
1!
0
•0 s
O
Mark
No.
bi
bi
3
"a
e
S3
■4J
ni
N
to
w
3
to
3
0
a2
3 3
0 K
.H0
>-. O
Bo .
■SES
bi
3
'0
3
•0
.0
"3
"0
0
a!
a
a
a
0
3
a
V
3
0 c'5
•c a a
0 0 gf
■g
a
0
O
vJ;
3-S3
0
Ph
0
&
i-J
S
«
c
<
a
Q
£
P
§
Per
Per
Per
Per
Per
cent.
cent.
cent.
cent.
cent.
f
[Mar.
10
95.2
1.80
1.02
0. 61
0. ?s
1 10, 000
Middle.
{ Apr.
27
95-3
1.86
.80
Cab 3..
IMay
-• 1
95-8
•76
1-35
.40
■33
52,000
i) 260
(Mar
30
94.8
1.74
.69
1
{Apr.
27
95-4
1.30
.87
.28
[Surface.
(.May
21
95-4
1-33
.69
.29
8,000
TRANSPORTED BY RAILWAY
Am 2. .
Am 3.
Am 4.
Am 5.
Middle.
Surface
Middle.
Surface.
Middle.
Surface.
Middle.
Surface.
Middle.
Surface.
Middle.
Surface.
Middle.
Surface.
Middle.
Surface.
Middle.
Surface.
[Apr.
{Apr.
iMay
Apr.
Upr. .
(May 24
I Apr
■JApr. 29
|May 24
Apr.
{Apr.
IMay
Apr.
■jMay 29
I May 24
Apr
<Apr. 29
[May 24
Apr.
{Apr.
[May
Apr.
{Apr.
(May
IApr.
Apr.
May
Apr.
Apr.
May
I Apr.
{Apr.
IMay
Apr.
sApr. 29
[.May 24
i'Apr. s
May 3
May 26
Apr. 5
May 3
May 26
(Apr. s
^May 3
(May 26
Apr. 5
{May 3
IMay 26
(Apr. s
{May 3
[May 26
[Apr. 5
iMay 3
[May 26
96
96
96. 2
I.
2
5
I
3
S
I
2
2
4
I. I
• s
I. 0
•3
1.8
(. 00
5- 40
I.
2-
.21
}•
. 01
•SO
I.
. 00
2.
. 60
I.
■35
2.
.96
3-
•94
.90
■IS
I.
.92
•30
2.
.41
2.
.90
. 00
I.
•43
2-
.76
•47
I.
.80
2.
. 12
.14
■ 10
I.
•32
.16
I.
■33
2.
.90
•94
•85
. 00
I.
.09
I.
■32
2.
.24
.02
.80
80
.76
•85
■ S9
.86
•76
.68
• 70
.60
.60
.00
•93
•95
. 00
•93
.76
. 00
90
.
O. 40
I. IO
2.30
.48
.81
I.98
•36
.87
■33
•IS
.78
•36
I. 02
I' 31
■33
•35
1.38
•45
.88
1-38
.05
•45
■35
•36
1-23
I. 60
•39
.28
.08
■39
.67
I. 22
.40
. II
•54
.09
.09
•55
• OI
•57
• 02
•57
•63
• 02
•IS
•63
.08
. 12
>. 50
•37
•31
■25
•30
•27
•35
.28
•3°
•30
• 27
•43
.26
.26
.29
• 29
•29
•42
.26
.28
•3°
• IO
.26
.41
.24
.24
•32
• 27
.28
•44
•25
•23
.41
•36
•31
.24
—
. 21
—
.24
.19
—
•25
—
. 21
. 22
—
. 21
—
.19
. 20
—
.26
—
•27
.26
—
.24
—
.24
.18
—
•24
—
24
4,000
120,000
33.ooo
2,400
4.900
1.900
19,000
90, 000
20.000
850
14.000
1. 230
30, 000
65 , 000
2, 100
4.000
4.000
400
50. 000
3,000
750
1.500
280
540
7.000
115, 000
2,000
34°
40. 000
1. 420
300. 000
120. 000
16, 000
300
10.000
450
I, 700
43 5
+
+
415
33 5
200
+
+
350
550
*
140
—
2. 100
275
500
+
*
4.500
700
+
I, 500
+
1. 100
630
+
2 , 000
+
Jan. 15, 1921
Deterioration of Sugars in Storage
641
Table I. — Chemical and bacteriological analyses of Cuban raw sugars in storage-
Continued
transported by railway — continued
3
bt
•a
r, 0
a
a
ei
V 6
a
0
V,«
89
'•3 a
a
aH
|a
ftS
Mark
No.
W
a
.n
a
2
"o
0
S3
O
el
N
a
.2
"0
a
a
3
u
g
j
3
3
0
3
•a
u
_s
a
a
3
o5
11
0 c
11
a.
■8 ft
0-
0.2 ;
ill
111
■a
&
p
Ph
a
§ f
A
O
<
s
p
fc
p
§
Per F
er
Per
Per
Per
cent. ce
nl.
cent.
cent.
cent.
[Apr.
•i
96.0
1.36 0
52
0. 42 0
34
+
900
+
2
Middle.
{May
.3
95-9
. 1
1
20
60
.08
29
—
245,000
+
Pil 1
?6
96.6
[Apr.
5
95-7
1
10 1
oq
•45
26
—
340
+
4
Surface.
•J May
96.4
T
02
47
?8
—
240, 000
+
*
[May
26
96.7
81
?S
25
39.000
0
[Apr.
S
95-6
I
70
70
•51
39
+
4,250
+
0
Middle.
<May
3
95-2
• 4
I
53
7S
■05
32
+
80. 000
+
0
Pil 2
[May
26
95- 0
.6
I
20 1
26
•56
24
26. 000
0
[Apr
■>
95-8
I
30 I
10
•S4
31
+
550
*
1
25
25
[May
26
95-8
T
06
9'
1,850
+
[Apr.
s
94.4
I
88 1
?S
.42
34
+
210,000
+
0
Middle.
<May
3
94.4
I
60 1
16
.01
29
—
210,000
+
Pil 2
|May
26
94.0
■4
I
54 1
74
■39
26
5,500
0
[Apr.
{May
[May
S
96-S
I
00
Q4
.40
29
—
4.75°
+
0
Surface.
95-4
I. I
r
70
7«
37
+
26, 000
+
26
95-6
■9
1
35 1
3-
•38
31
1,900
9
TRANSPORTED BY RAILWAY AND VESSEL
M 2
M 3
Ag 1
Ag 2
Ag 3
Middle.
Surface.
Middle.
Surface.
Middle.
Surface,
Middle.
Surface,
Middle.
Surface
Middle.
Surface,
[Apr. 5
iMay 3
(May 26
Apr. 5
May 3
May 26
Apr. s
May 3
May 26
Apr. 5
May 3
May 26
Apr. 5
May 3
May 26
Apr. 5
May 3
May 26
Apr. 5
May 3
May 26
Apr. 5
May 3
May 26
[Apr. 5
{May 3
[May 26
[Apr. 5
{May 3
I May 26
[Apr. 5
{May 3
[May 26
[Apr. 5
<May 3
iMay 26
96
0. 7
.6
•9
1-7
•3
1.0
.6
•5
1. 1
•3
. 1
•5
. 2
•7
•4
1.0
.6
•5
•3
. 2
1. o
.6
30
• 17
1.
.70
• 29
• 36
1.
.86
1.
•15
1.
.02
1.
62
•23
•85
16
27
•33
1.
90
OS
03
00
1.
.16
1.
.04
•92
1.
.26
1.
•29
1.
75
. 20
•03
.70
.08
.88
•35
1.
•38
1.
■56
1.
.16
1.
■63
■43
1.
1.
0. 70
0. 16
•59
.66
.04
.62
.48
•50
.66
•35
1.05
•65
.08
.08
•63
.04
.60
•54
•13
. 10
•54
.80
. 22
• 19
•78
. 01
0
28
_
28
—
25
20
—
29
26
23
28
_
27
27
31
+
23
3 1
30
28
+
*
24
26
_
27
25
26
—
21
25
—
29
—
31
22
—
27
26
22
—
29
—
25
24
—
24
—
27
22
—
31
+
25
120,000
+
75,000
+
130,000
2,300
+
Lost
360
1,050
+
7°
—
400
260
+
370
+
225
35°
+
100.000
+
200, 000
800, 000
+
550
+
2 7 , 000
17s
—
250
+
650
310
+
2,600
+
540
325
+
4,000
+
4, SOO
200
+
4,500
+
150
117
—
200
+
2.750
230
+
450
+
1, 100
From the data given in Table I it will be seen that the sugars vary in
initial polarization from 92 to 98.2; in moisture from 0.75 to 2.90; in per-
centage of reducing sugars from 0.52 to 1.83; in percentage of ash from
642
Journal of Agricultural Research
Vol. XX, No. 8
0.28 to 1.02; in moisture ratio from 0.18 to 0.50; in number of micro-
organisms per gram from 67 to 134,000; and in percentage of molds from
oto94. It is apparent that certain generalizations may be drawn from
Table I — namely, that there is a reduction in polarization in practically
all sugars during storage, a fact already established. Furthermore, it
is apparent that a decrease in polarization is generally accompanied by
an increase in reducing sugars. As might be anticipated, when deterio-
ration sets in during the first four weeks of incubation, it continues through
the second four weeks, although it would be difficult to state whether the
deterioration is more active in the second period of four weeks than the
first. While it is not to be expected that the number of microorganisms
present can be correlated with polarization, nevertheless, in general, the
greatest number of microorganisms occurs where the moisture ratio is
highest, and as a corollary we have observed that the lighter colored
sugars having the higher moisture ratios deteriorate most rapidly.
The temperature and relative humidity in New Orleans during the
months of storage of these sugars are given in Table II. It may be said
that in 1920 these were somewhat lower than the average. Table III
graphically represents the differences between successive samplings
together with a comparison between the last sampling and the first.
There is a fairly close agreement to be found between the results for bags
of one mark ; therefore these bags have been summarized in Table IV.
Table II. — Temperature and relative humidity at New Orleans, La., during March,
April, and May, 1920
Month.
Relative
humidity.
Temperature.
Maximum.
Minimum.
Mean.
March
88
80
75
°F.
84
87
92
"F.
27
39
57
°F.
59-40
65-85
75-75
April
May
Average
81
88
41
67. 00
Table III.— Dijff
erences between successive samplings of
sugars in
normal storage1
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
Number
of
microor-
ganisms
per gram.
Molds.
F 1
("Middle .
[Surface .
(Apr. 15
^May. 13
[Mar. 18
Apr. 15
< May 13
l2Mar. 18
+
+
Per cent.
+
+
+
Per cent.
+
+
+
+
*
+
+
+
+
+
*
1 * signifies no change; + signifies increase; — signifies decrease.
2 Third sampling compared with first.
Jan. is, 1921
Deterioration of Sugars in Storage
643
Table III.— Differences between successive samplings of sugars in normal storage —
Continued
Number
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
of
microor-
ganisms
per gram.
Molds.
Per cent.
Per cent.
(Apr. 15
—
+
+
—
+
—
(Middle .
{May 13
—
+
+
—
—
+
F 2
l2Mar. 18
—
+
+
—
+
*
Apr. 15
+
—
—
—
+
—
[Surface .
May 13
—
+
—
+
—
*
[2Mar. 18
—
—
—
—
+
*
Apr. 15
—
—
+
—
+
*
(Middle .
May 13
#
+
+
+
—
+
F3
l2Mar. 18
—
—
+
—
—
+
Apr. 15
—
+
+
—
+
*
[Surface .
May 13
—
+
+
—
—
*
l2Mar. 18
—
+
+
—
—
*
Apr. 15
—
—
—
—
+
*
(Middle .
May 13
—
+
—
+
+
—
Port 1....
l2Mar. 18
—
+
—
*
+
—
Apr. 15
*
—
—
+
*
[Surface .
May 13
—
+
+
+
+
*
[2Mar. 18
—
*
—
—
+
*
Apr. 15
—
—
—
—
—
+
[Middle .
May 13
—
+
—
+
+
—
Port 2 . . . .
[2Mar. 18
—
+
—
*
—
*
Apr. 15
—
—
—
—
*
[Surface .
May 13
—
+
—
+
+
+
l2Mar. 18
—
+
—
#
+
+
Apr. 15
*
—
—
—
+
(Middle .
{May 13
—
+
—
+
—
+
Port 3....
l2Mar. 18
—
*
—
—
+
—
[Apr. 15
+
+
—
+
*
[Surface .
May 13
—
+
—
—
—
*
[2Mar. 18
*
—
—
—
—
#
Apr. 19
+
—
—
—
*
*
(Middle .
<May 17
+
—
—
+
+
*
Cun 1 . . . .
l2Mar. 22
+
—
—
+
+
*
Apr. 19
+
+
—
+
—
#
[Surface .
jMay 17
*
+
—
—
+
*
[2Mar. 22
+
+
—
+
+
#
[Apr. 19
—
+
+
+
+
—
(Middle .
May 17
—
+
+
—
+
*
Cun 2 . . . .
l2Mar. 22
—
+
+
+
+
—
Apr. 19
+
+
—
+
+
*
[Surface .
May 17
—
+ .
+
—
+
*
[2Mar. 22
—
+
+
—
+
*
'
[Apr. 19
*
+
—
+
—
— ■
Cun 3....
Middle .
{May 17
—
+
+
+
—
—
I2 Mar. 22
—
+
—
—
+
—
[Apr. 19
—
+
—
+
—
—
Surface .
May 17
—
—
—
—
+
—
,
[2 Mar. 22
—
+
—
+
+
—
[Apr. 19
—
+
+
—
+
*
Cun 4. . . .
Middle .
sMay 17
—
+
+
—
—
*
[2Mar. 22
—
+
+
—
+
+
[Apr. 19
—
+
+
+
+
*
Surface .
<May 17
—
+
+
—
—
*
.
I2 Mar. 22
—
+
+
—
—
*
2 Third sampling compared with first.
644
Journal of Agricultural Research
Vol. XX, No. 8
Table III. — Differences between successive samplings of sugars in normal storage-
Continued
Number
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
of
microor-
ganisms
Molds.
per gram.
Per cent.
Per cent.
'
(Apr. 19
+
+
—
+
+
—
Middle .
^May 17
+
+
—
+
+
—
Cun 5... <
I2 Mar. 22
+
+
—
+
+
—
[Apr. 19
+
—
—
—
+
*
Surface. •jMay 17
—
+
+
*
+
—
I2 Mar. 22
+
—
—
—
+
—
Apr. 19
—
+
+
+
+
+
Middle .{May 17
—
+
+
#
+
*
Cun 6 ....
[-' Mar. 22
—
+
+
+
+
+
Apr. 19
—
+
—
+
+
—
Surface .
<May 17
+
—
—
—
—
+
I2 Mar. 22
—
+
—
+
+
—
[Apr. 27
—
+
—
+
+
+
Middle .
4 May 21
—
+
+
*
+
—
Agr 1 <
I2 Mar. 30
—
+
—
+
+
+
[Apr. 27
—
+
+
+
+
+
Surface .
jMay 21
—
—
—
—
+
—
_
I2 Mar. 30
—
+
—
—
+
*
Apr. 27
—
+
+
+
—
*
Middle .
< May 2 1
—
—
+
—
+
+
Agr 2 s
[2 Mar. 30
—
+
+
—
+
+
(Apr. 27
+
—
—
*
—
+
Surf ace .
< May 2 1
—
*
+
—
—
—
I2 Mar. 30
—
—
—
—
—
*
Apr. 27
—
+
—
#
+
+
Middle .
i May 2 1
—
—
+
+
—
+
Agr 3
|2 Mar. 30
—
—
—
+
+
+
[Apr. 27
—
+
—
*
+
+
Surface .
I May 2 1
+
+
*
+
—
+
.
I2 Mar. 30
*
+
—
+
—
+
[Apr. 27
—
+
+
+
+
*
Middle .
< May 2 1
+
+
—
+
+
+
Cab 1 . . . .
[2 Mar. 30
+
+
—
+
+
+
Apr. 27
+
—
—
*
+
+
Surface .
< May 2 1
+
—
—
+
+
—
.
I2 Mar. 30
+
—
—
+
+
—
Apr. 27
*
+
—
+
—
*
Middle .
< May 2 1
+
+
—
+
+
*
(2 Mar. 30
+
+
—
+
+
*
[Apr. 27
—
—
*
—
+
+
Surface .
I May 2 1
—
+
#
+
—
+
[2 Mar. 30
—
+
*
#
+
+
(Apr. 27
+
+
—
+
*
*
Middle .
< May 2 1
+
—
—
*
—
*
Cab 3 ... .
!2 Mar. 30
+
—
—
+
—
*
[Apr. 27
+
—
—
+
+
—
Surface .
JMay 21
*
+
—
+
—
*
:
I2 Mar. 30
+
—
—
—
+
—
[Apr. 29
—
—
+
—
+
*
Middle .
i May 24
—
—
+
—
—
—
Am 1 . . .
<
I2 Apr. 1
—
—
+
—
+
—
[Apr. 29
—
+
+
+
+
—
Surface .
{ May 24
—
' +
+
—
—
*
1
I2 Apr. 1
—
1 +
+
+
—
—
2 Third sampling compared with first.
Jan. is, 1921
Deterioration of Sugars in Storage
645
Table III. — Differences between successive samplings of sugars in normal storage-
Continued
Number
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
of
microor-
ganisms
per gram.
Molds.
Per cent.
Per cent.
|Apr. 29
—
—
+
—
+
*
Middle .
| May 24
—
+
+
+
—
+
Am 2
I2 Apr. 1
—
+
+
—
+
+
1
[May 29
+
—
+
*
+
—
1 Surface .
j May 24
—
+
+
—
+
2 Apr. 1
—
+
+
—
+
+
[Apr. 29
—
—
+
—
+
+
Middle .
I May 24
—
+
+
*
—
+
I2 Apr. 1
—
—
+
—
—
+
[Apr. 29
—
+
+
*
*
—
Surface .
< May 24
—
+
+
*
—
—
)
I2 Apr. 1
—
+
+
*
—
—
[Apr. 29
—
—
+
—
—
*
Middle .
I May 24
—
+
+
+
—
—
Am 4
I2 Apr. 1
—
+
+
—
—
—
[Apr. 29
*
+
+
*
—
—
Surface .
JMay 24
—
—
+
—
+
+
I2 Apr. 1
— •
—
+
—
—
+
[Apr. 29
—
—
+
—
+
*
Middle .
I May 24
—
+
+
*
—
—
Am 5
I2 Apr. 1
—
+
+
—
—
—
[Apr. 29
—
+
+
—
+
—
Surface .
I May 24
+
—
—
+
—
+
,
I2 Apr. 1
—
—
+
+
—
[Apr. 29
+
—
+
—
—
*
Middle .
JMay 24
+
+
—
—
—
Am 6 , . . .
I2 Apr. 1
—
+
—
—
—
[Apr. 29
+
—
+
—
+
—
Surface .
I May 24
+
—
—
—
—
*
.
[2 Apr. 1
+
—
+
—
+
—
May 3
—
—
+
—
—
—
Middle .
< May 26
+
+
*
+
—
*
Oi
2 Apr. 5
—
—
+
*
—
—
May 3
—
+
+
+
—
+
Surface .
< May 26
+
—
—
—
+
—
.
I2 Apr. 5
*
+
*
+
+
+
[May 3
—
+
—
—
—
• —
Middle .
<May 26
+
—
+
—
+
*
O2
I2 Apr. 5
—
—
*
—
+
*
[May 3
—
+
—
+
+
—
Surface .
< May 26
+
—
+
+
+
*
.
I2 Apr. 5
—
+
—
+
+
—
[May 3
—
+
+
—
+
—
Middle .
{ May 26
+
—
—
*
' —
*
O3
<
I2 Apr. 5
—
—
+
—
+
—
[May 3
—
+
+
+
+
—
Surface .
{May 26
+
—
+
*
—
—
.
'Apr. 5
—
+
+
+
+
—
[May 3
—
—
+
—
+
—
Middle .
\ May 26
+
+
—
+
—
+
Pil 1
I2 Apr. 5
+
+
—
+
+
—
[May 3
+
—
—
+
+
—
Surface .
\ May 26
+
—
+
—
+
—
I2 Apr. 5
+
—
-
-
+
—
2 Third sampling compared with first.
646
Journal of Agricultural Research
Vol. XX, No. 8
TABLE III. — Differences between successive samplings of sugars in normal storage —
Continued
Number
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
of
microor-
ganisms
per gram.
Molds.
Per cent.
Per cent.
■
[May 3
—
—
+
—
+
*
Middle .
< May 26
—
—
+
—
—
*
P1I2
I2 Apr. 5
—
-
+
—
+
*
[May 3
+
Surface .
{ May 26
+
*
{- Apr. 5
*
:
[May 3
*
—
+
—
#
+
Middle . >!May 26
—
—
+
—
—
—
PU3
I2 Apr. 5
—
—
+
—
—
*
[May 3
—
+
—
+
+
+
Surface.^ May 26
+
—
+
—
—
+
2Apr. 5
+
+
—
+
—
+
f May 3
—
+
+
*
—
+
Middle . jMay 26
+
—
+
—
+
*
M 1
I2 Apr. 5
—
+
+
—
+
+
'[May 3
-f
+
+
Surface . < May 26
4-
+
I2 Apr. 5
+
+
+
.May 3
—
+
+
—
—
Middle . May 26
+
—
*
—
+
*
M2
2 Apr. 5
*
+
—
+
—
—
<
May 3
—
+
*
+
+
*
Surface. •{May 26
+
—
—
—
—
*
_
I2 Apr. 5
—
+
—
—
—
*
■
[May 3
—
+
+
—
+
+
Middle .
{May 26
—
+
+
—
+
*
M3
I2 Apr. 5
—
+
+
—
+
+
(May 3
—
+
+
+
—
+
vSurface .
< May 26
+
—
*
+
+
—
I2 Apr. 5
—
+
+
+
—
*
[May 3
—
+
+
+
+
*
Middle . { May 26
+
—
—
—
+
+
Ag 1
|l2Apr. 5
—
+
—
—
+
+
May 3
—
+
—
+
+
+
Surface. { May 26
+
+
+
+
+
—
I- Apr. 5
—
+
—
+
+
*
May 3
—
+
+
+
+
*
Middle .
< May 26
+
—
—
—
+
+
Ag 2
I- Apr. 5
—
+
+
+
+
+
May 3
—
+
—
+
+
—
Surface .
j May 26
+
—
+
—
—
—
,
I2 Apr. 5
—
+
—
+
—
—
[May 3
—
+
+
*
+
—
Middle .
< May 26
#
+
+
+
+
—
Ag 3
I2 Apr. 5
+
+
+
+
—
[May 3
—
+
+
+
+
—
Surface .
< May 26
+
—
—
—
+
+
•
I2 Apr. 5
—
+
—
+
+
—
2 Third sampling compared with first.
Jan. 15, 1921
Deterioration of Sugars in Storage
647
Table IV. — Summary of differences between successive samplings {average of bags of
same mark)1
Number
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
of
microor-
ganisms
per gram.
Molds.
Per cent.
Per cent.
Per cent.
I Apr. 15
—
—
+
—
+ •
—
Middle .
< May 13
—
+
+
*
—
+
F I to 3 . .
[2Mar. 18
Apr. 15
—
+
+
+
+
—
+
*
Surface .
May 13
—
+
—
+
—
*
.
l2Mar. 18
—
—
+
—
+
*
Apr. 15
—
—
—
—
+
*
Middle .
{ May 13
—
+
—
+
+
—
Port 1 to 3
l2Mar. 18
Apr. 15
*
+
+
+
+
*
Surface .
\ May 13
—
+
—
+
+
*
,
l2Mar. 18
—
*
—
—
+
*
I Apr. 9
—
+
*
+
+
—
Middle .
{ May 17
—
+
+
+
+
—
Cun 1 to 6
l2Mar. 22
Apr. 9
*
+
+
*
+
+
+
+
*
Surface .
{ May 17
—
+
*
—
+
*
.
l2Mar. 22
—
+
—
*
+
—
[ Apr. 27
—
+
—
+
+
+
Middle .
\ May 21
—
—
+
*
+
+
Agrito3.
l2Mar. 30
[ Apr. "9
—
+
+
+
*
+
+
+
Surface .
\ May 17
—
*
*
—
—
—
.
l2Mar. 22
—
+
—
—
—
*
[ Apr. 27
*
+
—
+
+
*
Middle .
{ May 21
+
+
—
+
+
+
Cab 1 to 3
l2Mar. 30
+
+
—
+
+
*
[ Apr. 27
+
—
—
*
+
*
Surface .
{ May 21
*
+
—
+
—
*
.
i2Mar. 30
+
—
—
*
+
—
[ Apr. 29
—
—
+
—
+
*
Middle .
{ May 24
—
+
+
*
—
—
Am 1 to 6.
I2 Apr. 1
f Apr. 29
—
*
+
+
+
—
+
Surface .
{ May 24
—
*
+
—
—
+
.
I2 Apr. 1
—
*
+
—
*
—
[ May 3
—
+
+
—
—
—
Middle .
{ May 26
+
—
*
*
—
*
O 1 to 3 . .
"Apr. 5
—
—
+
—
+
—
1
[ May 3
—
+
+
+
+
*
Surface .
| May 26
+
—
+
*
+
—
.
"Apr. 5
—
+
*
+
+
—
May 3
—
—
+
—
_U
*
Middle .
\ May 26
—
—
+
*
—
*
Pil 1 to 3 .
"Apr. 5
—
—
+
—
+
*
|
May 3
+
—
—
—
+
—
Surface .
\ May 26
+
—
+
*
*
—
:
'-'Apr. s
*
—
—
+
*
—
f May 3
—
+
+
*
—
+
Middle .
{ May 26
+
—
+
—
+
*
M 1 to 3 . .
"Apr. 5
—
+
+
—
+
+
May 3
—
+
+
+
*
+
Surface .
{ May 26
+
—
+
—
*
—
■
l2Apr. 5
-
+
+
+
—
*
1 *signifies no change; -fsignifies increase, —signifies 1
2 Third sampling compared with first.
648
Journal of Agricultural Research
Vol. XX, No. 8
Table IV.-
-Summary of differences between successive samplings {average of bags of
same mark) — Continued
Number
Mark No.
Part of
bag.
Date of
sampling.
Polari-
zation.
Moisture.
Reducing
sugar.
Moisture
ratio.
of
microor-
ganisms
per gram.
Molds.
Per cent.
Per cen I.
Per cent.
( May 3
—
+
+
+
+
*
Middle .
\ May 26
+
—
—
—
+
+
Ag i to 3 .
l2Apr. s
f May 3
+
+
+
+
+
+
+
—
Surface .
< May 26
+
—
+
—
+
—
1
l2Apr. 5
+
+
+
2 Third sampling compared with first.
It will be seen that in practically all instances there has been a reduc-
tion in polarization between successive samplings. With regard to
moisture content, however, there appears to be an increase in a majority
of instances. It is interesting in this connection to note that, with the
exception of the Cab sugars, an increase in polarization is accompanied
by a decrease in moisture content. Naturally, this means that there has
actually been a loss in weight of sugar. Furthermore, it will be seen that
the surface of each bag decreased in moisture content, or dried out, as
might be expected, much more rapidly than the middle of the same bag.
In the sugars which have deteriorated it will be observed that there has
been an increase in percentage of reducing sugars in successive samplings.
However, as a rule this increase is more noticeable in the middle of the
bag than at the surface where the deterioration does continue to progress
at the initial rate. The conditions of temperature and humidity were
such as to preclude the possibility of deterioration taking place more
rapidly from the surface of the bag than from the interior of the bag, as
occurs under average conditions which were noted in the previous experi-
ment.1 The moisture ratio was variable and does not permit of any
generalization.
In considering the number of microorganisms it will be seen that in
most instances there was an increase between successive samplings.
In general it was found in corroboration of the results previously set forth
that the increase in numbers of microorganisms was relatively more rapid
during the first month of incubation than subsequently. Likewise it is
to be noted that there is usually a greater number of microorganisms in
the middle of the bag than at the surface, where drying out occurs. It
will be shown in Table V, which is again corroborative of previous work,
that there is correlation between the number of microorganisms and
deterioration where the initial content is high or multiplication has been
rapid. The percentage of molds is variable, and a tendency to decrease
1 Kopeloff, Nicholas, and Perkins, H. Z. E. op. err., 1920.
Jan. is, 1921
Deterioration of Sugars in Storage
649
in the surface is to be noted during the first four weeks of incubation. It
is evident, therefore, that these results agree very closely with those pre-
viously obtained, and this is of added significance when it is remembered
that the range in variety of sugars is considerably greater.
Table V.
-Summary showing correlation between deterioration and number of micro-
organisms
Mark No.
Am
Cun 4.
Am 5.
Am 4.
Am 3 .
Am 3.
F 1...
Am 5.
Am 6.
Am 1 .
M3. .
Am 3.
Part of bag.
f Middle.
\ Surface.
J. . . do.
1 Middle.
...do.
. . . .do.
Surface .
Middle .
. . . do.
. . . .do.
. . . .do.
...do.
. . . .do.
....do.
Date of sampling.
Third. .
do.
do.
do.
do.
do.
do.
do.
do.
Second .
Third . .
Second .
Third . .
Second .
Loss in
polarization.
Gain m
reducing
sugar.
Per cent.
2-5
2.30
3-5
3-°
3-2
2. 0
1
I
I
1
93
63
58
60
2-3
1.8
1
I
38
38
1. 0
I
31
1.8
I
3°
1.6
I
23
1. 2
I
22
1. 2
I
10
1. 1
I
OS
•5
I
02
Number of
microorganisms
per gram.
120, OOO
4, 900
20, OOO
IOO, OOO
115, OOO
50, OOO
4, OOO
65, OOO
I, 650, OOO
70, OOO
120, OOO
4, 000
100, 000
30, 000
Table V consists of a summary arranged in such a manner as to bring
out clearly the correlation between the number of microorganisms and
deterioration. The order of bags is based upon the increase in reducing
sugars, since that represents the best criterion for determining deterio-
ration. In addition, it will be noted that the loss in polarization is pro-
portional to the gain in reducing sugars. Still more significant, however,
is the fact that deterioration occurs in the presence of the maximum
numbers of microorganisms. It may be mentioned that the number of
microorganisms set down opposite any figure for gain in reducing sugars
is the number occurring at the previous sampling, since that number was
responsible for the deterioration found at the time of analysis. With
three exceptions the greatest deterioration is to be found when there are
more than 20,000 microorganisms per gram, and the average deterioration
(represented by an increase of more than 1 per cent of reducing sugars)
is to be found where there are 174,000 per gram. It is interesting to com-
pare Table V with Table VI, which is a summary showing the maximum
numbers of microorganisms where no deterioration has occurred. It will
be seen at a glance that in only five instances has this number exceeded
8,000 per gram, the average being about 1 1 ,000 (unduly weighted because
of the Cab sugar which was especially heavily infected). Thus, a com-
parison between Tables V and VI reveals quite clearly that large numbers
of microorganisms are causally related to deterioration and that the con-
verse is likewise true.
650
Journal of Agricultural Research vol. xx, No. 8
TABLE VI. — Summary showing maximum numbers of microorganisms where no deteri-
oration occurs
Mark
No.
Part of bag.
Number of
microorgan-
isms per gram.
Hark No.
Part of bag.
Number of
microorgan-
isms per
gram.
("Middle
6, OOO
7, 000
6, 000
8, 000
2, 100
22, 000
8, 000
1, 100
5,000
14, 000
22, 000
1, 000
Agr 1
Agr 3
Cab 2
Cab 3
M 3
Ag 1
! Ag 2
Pil 1
Middle
3, IOO
P 1
\ Surface
("Middle
fMiddle
1 Surface
\ Surface
300
P 2
("Middle
("Middle . .
1 Surface
500
no, 000
P 3
\ Surface
do
fMiddle
JMiddle
\ Surface
I, 050
370
C 1
\ Surface
("Middle
do
C 3
\ Surface
fMiddle
do
4,500
24, 500
do
C 6
1 Surface
In Table VII the sugars analyzed have been ranked according to
deterioration as based upon the greatest loss of polarization during
normal storage. In compiling these data the analyses for all the bags
of each mark were averaged. It is evident that the deterioration in the
first six sugars mentioned was appreciable, the Am sugar being con-
siderably more deteriorated than any others. Inasmuch as this sugar
came by railroad as did the O and Pil sugars, it would be difficult to
regard the means of transportation as the sole limiting factor. Since
the former had a higher moisture ratio and considerably more micro-
organisms per gram, it is natural to suppose that it would deteriorate
more rapidly under any environmental conditions.
Table VII. — Sugars ranked according to greatest loss in polarization during normal
storage
Rank
Mark.
Am .
F...
Cun.
Agr.
M. ..
Ag..
Pil. .
O. . .
Port.
Cab.
Part of bag.
Middle
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
..do...
Average
loss in
polariza-
Rank.
tion per
bag.
I- 5
I
9
2
7
3
6
4
5
5
4
6
3
7
2
8
2
9
0
10
Am .
M. ..
Cun.
Ag..
F....
O. . .
Pil. .
Agr. .
Port.
Cab.
Part of bag.
Surface .
..do....
..do...
.do...
..do....
. do...,
..do....
..do....
..do....
..do...,
Average
loss in
polariza-
tion per
0.8
jan. i5> 1921 Deterioration of Sugars in Storage 651
It is interesting to note further in Table VII that in the majority of
cases the rank of sugars with regard to deterioration is the same for
the middle of the bag and for the surface. For example, the Am sugar
shows greatest deterioration both in the middle and at the surface,
while the Port and Cab sugars show least in both cases.
It has been shown that it is possible to predict the keeping quality of
a sugar (from the standpoint of mold infection) by the simultaneous
consideration of moisture ratio and number of organisms per gram.1
Evidence for a prediction based on the number of bacteria was likewise
advanced.1 In Table I the plus and minus signs in the columns labeled
"Deterioration predicted from moisture ratio" and "Deterioration
predicted from number of microorganisms per gram" represent the
prediction of deterioration based upon these factors considered inde-
pendently. In this case we have taken the critical moisture ratio and
the number of bacteria per gram which are required to produce deteriora-
tion in four weeks at this temperature and humidity of incubation as
30 and 200, respectively. (Table VIII.) Where these conditions were
higher, as in the experiment of 1919,2 less than half this number of micro-
organisms will produce similar effects. If attention is focused upon the
moisture ratio it will be seen that the factor of safety as worked out by
previous investigators holds true to a limited extent. In other words,
where the moisture ratio is above 0.30 to 0.33 deterioration usually sets
in, while sugars with lower moisture ratios usually resist deterioration.
However, there are any number of instances where this factor of safety
fails to function as an adequate criterion, and we may turn with some
confidence to the number of microorganisms per gram as a true index of
deterioration. In fact, a careful analysis of the data presented in Table
I shows that as a criterion for predicting deterioration the moisture ratio
or factor of safety proved to be in agreement with the analyses in 57
instances and failed in 86 instances; in other words, it was only 40 per
cent effective. On the other hand, the use of the number of micro-
organisms per gram as an index of deterioration resulted in 96 successful
predictions and 47 failures, or an efficiency of 67 per cent, which is 27
per cent better than the factor of safety. In the 65 cases where the
moisture ratio is in agreement with number of microorganisms for the
theoretical prediction of deterioration, there was practical confirmation
in the majority of instances.
1 Kopeloff, Nicholas, and Kopeloff, Lillian, op. cit., 1920.
2 Kopeloff, Nicholas, and Perkins, H. Z. E. op. cit., 1920.
17776°— 21 5
652
Journal of Agricultural Research
Vol. XX, No. 8
Table VIII. — Correlation of moisture ratio with number of microorganisms 1
MOISTURE RATIO
6
0
CO w
0 0
0
0
0
0
0
00
0
0
0
0
0
0
0
fa
0
0
10
0
0
0
00
0
0
0
0
O
+
O
i
+
*
+
*
*
+
*
*
*
+
*
+
-
*
*
+
+
+
-
+
~
+
*
*
*
+
-
*
+
•_■
+
*
+
-
—
+
+
*
+
-
+
+
*
+
+
+
-
-
+
+
+
+
+
+
+
+
+
*
+
-
+
+
+
+
+
+
*
+
+
+
+
1 + signifies deterioration; — signifies no deterioration; * signifies doubtful deterioration.
It will be seen from Table VIII, where there has been graphically illus-
trated the correlation between moisture ratio and number of microorgan-
isms per gram that while such a relationship is of necessity dependent
upon the environmental conditions at hand and it is hazardous in con-
sequence to derive any didactic conclusions, nevertheless certain gener-
alizations appear significant. For example, with more than 50,000
microorganisms per gram in practically all instances there was deteriora-
tion at every moitsure ratio employed. As the number of microorgan-
isms is increased beyond this point it is almost certain that deterioration
will occur at any moisture ratio generally occurring in Cuban raw sugar.
As the number of organisms per gram is decreased to about 500 we
have evidence of less deterioration at moisture ratios below 0.36. How-
ever, where the moisure ratio remains above 0.36 deterioration is effected
by more than this number. On the other hand, even where the moisture
ratio is reduced below 0.30, which is considered the critical point, there
is ample evidence to indicate that deterioration may be induced by more
than 200 microorganisms per gram. This corroborates the conclusions
arrived at in the investigations previously referred to x and emphasizes
again the necessity for reducing the mass infection in sugar. Thus, on
the basis of polarization, moisture content, and bacteriological analysis,
it is possible to predict the keeping quality of sugar and thereby intro-
duce considerable economy by immediately disposing of those sugars
which will deteriorate rapidly and storing only those proved to be capable
of storage without serious loss. As a matter of actual manufacture, it
should not be difficult to control the microorganisms to such an extent
1 Kopeloff, Nicholas, and Perkins, H. Z. E., op. cit.
and Kopeloff, Lillian, op. cit., 1919.
op. at., 1920.
Jan. is. 1921 Deterioration of Sugars in Storage 653
as to inhibit their detrimental activities. In this connection it may be
stated that recent experiments have enabled us to develop a method for
eliminating the microorganisms in sugar by the use of superheated steam
in the centrifugal which destroys over 90 per cent of the microorganisms.1
It is, therefore, evident that sugar deterioration depends upon the two
factors of moisture ratio and number of microorganisms per gram.
Furthermore, if the number of microorganisms is sufficiently reduced, and
if the moisture ratio is properly controlled, -sugar deterioration may be
satisfactorily prevented.
SUMMARY
(1) From the results presented a correlation has been established be-
tween deterioration and the number of microorganisms and between
deterioration and the moisture ratio. This makes it possible, as previ-
ously stated,2 to predict the keeping quality of sugar by a preliminary
bacteriological and chem ical analysis.
(2) From 3 to 6 bags of Cuban raw sugars, each of 10 different marks,
with moisture ratios varying from 0.18 to 0.50, were stored under normal
conditions in a large warehouse and were analyzed chemically and bac-
terio logically at the beginning and after four and eight weeks, respectively.
There was a loss in polarization in most of the sugars at the end of each
period, which was generally accompanied by a gain in reducing sugars
and moisture content.
(3) There was a decided increase in the number of microorganisms per
gram, especially during the first four weeks, which could be correlated,
within certain limitations, with deterioration. In general, there were
more microorganisms in the middle of the bag than at the surface. A
large initial infection or rapid multiplication of microorganisms was re-
sponsible for an increase in deterioration.
(4) It has been shown that the use of superheated steam in the cen-
trifugal will reduce the number of microorganisms more than 90 per
cent and consequently may eliminate deterioration if the moisture ratio
is likewise properly controlled.
1 Kopeloff, Nicholas, the prevention of sugar deterioration by the use of superheated steam
in centrifugals. In Jour. Indus, and Engin. Chem., v. 12, no. 9, p. 860-862, 1 fig. 1920.
2 Kopeloff, Lillian, op. cit., 1920.
FREEZING OF FRUIT BUDS
By Frank L. West, Physicist, and N. E. Edlefsen,1 Assistant Physicist, Utah Agri-
cultural Experiment Station
INTRODUCTION
Killing frosts occur in the late spring and early fall over large areas of
the United States, causing damage to the extent of several millions of
dollars annually. The commonest method of protection is to heat the
area by burning oil in pots distributed through the section that is endan-
gered. Heating is resorted to on a large scale in the citrus fruit sections
of California and less frequently elsewhere for the protection of such
fruits as apples, peaches, and cherries. The success of this practice de-
pends on the economical use of fuel and labor. If the predicted mini-
mum temperature is lower than the "critical temperature" by an amount
that exceeds the rise in temperature that the heaters will produce, or if
the minimum temperature is above the "critical temperature," then the
heaters should not be lighted. In order, therefore, to be able to tell
when to light the heaters, it should be known how hardy the buds are.
This paper gives the methods used and the results obtained from freezing
more than 24,000 fruit buds, most of them being apples and peaches,
and also the spring freezing temperatures and the yields of fruit in
orchards near Logan, Utah, from 191 3 to 1920.
THEORY OF INJURY DUE TO FREEZING
Pure water freezes at 32 ° F. Salts dissolved in water cause it to
freeze at a lower temperature than this, the amount of the depression of
the freezing point depending upon the nature of the salt dissolved and
also upon the concentration of the solution. Thus, a 5 per cent common
salt solution freezes at 270, while a 30 per cent sugar solution freezes at
only 290 F. W. H. Chandler2 found that the expressed sap from fruit
buds froze at 2 8° to 290 and in no case required a temperature below 2 8°.
The sap from Elberta peach twigs, extracted in March, froze at 28. 70,
while but two-thirds of the twigs of the same kind of fruit when subjected
in March to a temperature as low as io° froze. It is frequently found
that some of the buds withstand temperatures as low as 200 and mature.
The more concentrated the aqueous solution, the lower is its freezing
point, and in 'general the amount of the substance, especially if it is
organic, that will dissolve in water is but slightly affected by the sub-
stances that are already in solution. This allows the possibility of a
'Messrs. J. Z. Richardson, W. E. Goodspeed, and Scott Ewing rendered valuable assistance with the
laboratory and field work.
!Chandler, W. H. the killing of plant, tissue by low temperature. Mo. Agr. Exp. Sta. Re-
search Bui. 8, 309 p., 3 pi.: chart. 1913. Bibliography, p. 305-309.
Journal of Agricultural Research, Vol. XX, No. 8
Washington, D. C. Jan. 15, 1921
wp Key No. Utah-12
(655)
656
Journal of Agricultural Research
Vol. XX, No. 8
very concentrated solution, and each of these substances has its influence
in lowering the freezing point of the water largely independent of the
others. For these reasons, a rather low freezing point of a solution is
possible. A very concentrated juice, therefore, in the buds would be
expected to freeze at a fairly low temperature. In spite of this, however,
the unusual hardiness of some buds to freezing is really surprising. The
difference in sensitiveness to cold of different buds on the same branch
and of the same buds at different stages of development may be in part
due to the difference in quality and concentration of the cell sap.
Table I. — Classified list of the "danger points" for -various kinds of fruit as given by
different authors
Kind of fruit.
Petals closed
but showing
color.
In blossom.
Fruit set-
ting.
Authority.
Apples
°7
27
27
27
25
25
20
29
29
22
25
22
29
22
27
29
28
25
3°
3°
3°
22
3°
3°
22
1 3°
°F.
29
29
29
28
28
2S
3°
30
28
27
28
3°
28
29
29
29
28
31
30
31
28
31
31
28
31
31
°F.
3°
3°
3°
28
28
28
3°
3°
28
27
29
3°
28
29
29
29
28
3i
3i
3i
28
32
32
28
3i
3i
W. M. Wilson.1
P. J. O'Gara.2
Peaches
W. H. Hammon.
Paddock and Whipple.4
W. H. Chandler.5
W. M. Wilson.'
W. H. Hammon.3
Cherries
P. J. O'Gara.2
Paddock and Whipple.4
Garcia and Rigney.6
'W. M. Wilson.'
Pears
P. J. O'Gara.-
Paddock and Whipple.4
W. M. Wilson.'
P. J. O'Gara.2
W. H. Hammon.3
Paddock and Whipple.4
W. M. Wilson.'
P. J. O'Gara.2
W. H. Hammon.3 .
Paddock and Whipple.4
P. J. O'Gara.2
Plums
Apricots
Prunes
W. H. Hammon.3
Paddock and Whipple.4
P. J. O'Gara.2
W. H. Hammon.3
W. H. Chandler 7 reports minimum temperature and the resulting damage by natural frost. He also
reports his work on the artificial freezing of detached branches. Garcia and Rigney 8 placed self-registering
minimum thermometers in the orchard. After a freeze the percentage of frozen buds was determined, and
in the fall the yield of the orchard was obtained. Their work covered five years.
1 Wilson, Wilford M. frost. In Bailey, L. H., ed. Standard Cyclopedia of Horticulture, v. 3,
p. 1283. New York, 1915.
2 O'Gara, P. J. the protection of orchards in the paciftc northwest from spring frosts by
means OF fires and smudges. U. S. Dept. Agr. Farmers' Bui. 401, p. 20. 1910.
3 Garcia, Fabian, and Rigney, J. W. hardiness of fruit-buds and flowers to frost. N. Mex.
Agr. Exp. Sta. Bui. 89, p. 5. 1914.
4 Paddock, Wendell, and Whipple, Orville B. fruit-growing in arid regions . . . xx, 395 p.
illus. New York, 1910.
6 Chandler, W. H. op. err. , p. 146.
6 Garcia, Fabian, and Rigney, J. W. op. cit., p. 51.
'Chandler, W. H. op. cit. 1913.
8 Garcia, Fabian, and Rigney, J. W. op. cit.
jan. i5> 1921 Freezing vf Fruit Buds 657
When liquids are cooled to their freezing points, if there be none of
the solid material present, they rarely freeze. They may be cooled
several degrees further and kept for days without solidification taking
place. The introduction of as small an amount of solid as one-hundred-
thousandth part of a milligram is sufficient to cause freezing to begin.
The smaller the amount of liquid taken the easier it is to superfuse it,
and liquids contained in capillary tubes will remain for long periods of
time below their freezing point without solidification taking place. The
fact that the juice of the buds is confined in small capillary spaces will
help to explain in part the unusual hardiness of the buds and the great
difference in hardiness of buds that appear to be very similar. This
phenomenon explains why they may be cooled below their freezing points
and be warmed again without ice separating.
A classified list of the "danger points," as given by various investiga-
tors, is presented in Table I.
METHODS AND APPARATUS
NATURAL FREEZES
Each spring, for the last seven years, standard minimum thermometers
have been placed in especially prepared but simple shelters in fruit trees
of various orchards near Logan, Utah, and were read the day after a
minimum temperature of 32 ° F. or lower was experienced. A record
was made of the yield of fruit of the orchard for the season. The results
of this work are found in Table II.
ARTIFICIAL FREEZES
The first work consisted in freezing detached branches of fruit buds in
the laboratory by means of a specially designed thermostat, the air
surrounding the buds being cooled by means of common salt and ice and
warmed with an incandescent electric light, which was maintained con-
stant at an arbitrarily determined temperature in the usual way with a
relay. The extent of the injury was determined by cutting the buds
open and counting those that had been damaged and then calculating
the percentage that had been frozen.
Branches of trees were bent down into a vessel surrounded by a
second air chamber, the latter being surrounded by a mixture of ice and
salt. The minimum temperature was noted, the branch was tagged,
and the further development of the buds was observed and the yield of
fruit determined.
This method was modified by having the buds cooled by means of
evaporating liquid carbon dioxid instead of using ice and salt. A tank
of liquid carbon dioxid was connected to a metal coil that surrounded
the bud chamber. The very cold gaseous carbon dioxid cooled the bud
chamber, thereby cooling the buds to the desired temperature.
658 Journal of Agricultural Research vol. xx.No. 8
The fourth method consisted in freezing the whole tree by surrounding
and covering it with a two-walled metal vessel containing ice and salt.
The apparatus is shown in Plate 80.
The factors that determine the amount of damage done and that
need to be controlled in the experiment are:
1 . The kind of buds.
2. Their stage of development.
3. The minimum temperature.
4. The humidity.
5. The duration of the freeze.
6. The rate of thaw.
The first three are of most importance. By keeping the other factors
fairly constant and varying the fifth and sixth, little difference in the
results was noted. In almost every case in nature, as well as in our
experiments, the humidity just as freezing occurs is practically 100 per
cent. Transpiration into a closed vessel will ultimately give this result,
and the best desiccating agents will not keep the humidity down appre-
ciably. This holds true also in the orchard simply by the cooling irre-
spective of the transpiration, because even in such a dry section as
the arid West, with a humidity as low as 50 per cent and a cool spring
day of perhaps 450 F. noon temperature, the dew point would be 27. 50
F., which is about the temperature at which slight damage is caused.
Where the humidity is higher, as it is in most places east of the Rocky
Mountains and west of the Sierra Nevada Mountains, the dew would
collect and the humidity would be 100 per cent even before the buds had
cooled to the danger temperature. In all the work here reported the
humidity was practically 100 per cent.
While the whole tree was being frozen, several minimum thermometers
were suspended at different places in its branches, and the air was stirred
by an electric fan driven with storage batteries. The humidity was
determined by a continuous reading hygrometer, and the rate of thaw
and duration of freeze were recorded by means of a thermograph that
was placed in the branches.
The cost of the different methods is about the same for freezing the
same number of buds. Adjoining limbs and adjacent trees were thinned
to the extent that the branch or tree had been thinned by the frost,
and the yields in the fall were noted for comparison. A greater varia-
tion in the factors, and thus a greater number of different experiments,
can be secured for the same expenditure by freezing the branches on
the tree rather than the whole tree.
The results of the natural and artificial freezing experiments are
presented in Tables II to IV.
Jan. is, 1921
Freezing of Fruit Buds
659
Table II. — Temperatures produced and percentage of buds killed by artificial freezing
Kind of fruit.
Ben Davis apples.
Number of
buds.
Elberta peaches.
no
.935
, 172
101
8i3
,828
,490
28
127
, 217
12
29
49
40
33
48
55
58
. 715
,846
35
675
in
277
361
5i4
380
586
.195
189
372
349
38
22
42
62
35
061
42
355
749
194
37
27
507
Development.
80
16
70
49
78
Showing color .
....do
Full bloom
do
do
do
do
do
do
do
...do
Fruit setting. .
....do
do
....do
....do
....do
....do
Showing color.
....do
....do
....do
....do
....do
do
....do
....do
...do
....do
....do......
....do
Full bloom. .
....do
....do
....do
...do
....do
....do
....do
....do
...do
....do
....do
...do
...do
...do
...do
...do
Fruit setting.
....do
....do
....do
....do
....do
....do
....do
....do
Tempera-
Percentage
ture.
of damage.
°F.
22. s
88
25
45
24
81
24- 5
56
25
54
26
16
26
100
26. 5
36
27-5
54
28
0
28.5
0
25-5
93
26. 5
40
26. 5
23
27-5
21
27-5
59
27-5
62
28
461
i7- S
64
18
75
20
66
22. s
76 .
22.5
76
24
89
24
79
25
96
25
74
25
77
25
97
26
80
27-5
79
22
100
24
63
24
64
25
58
25
28
25
72
25
65
26
40
26
48
26
78
26
54
26
57
27
0
27
0
27
55
28
55
28
33
24- 5
3°
25
100
26
75
26. 5
48
27
75
27-5
56
27-5
48
28
43
29
33-3
66o
Journal of Agricultural Research vol. xx.no.s
Table III. — Temperatures produced and number of mature fruits harvested by artificial
freezing
Kind of fruit.
Number of
buds.
Ben Davis apples . .
Control limbs, un-
treated
Ben Davis apples. . .
30
38
18
60
19
39
36
12
19
37
119
32
149
64
55
44
45
35
45
7i
108
74
122
63
141
7i
52
87
64
88
i°5
106
69
68
69
Development.
Full bloom.. .
do
do
do
...do
do
do
do
...do
...do
....do
do
do
....do
...do
do
do
do
do
do
do
do
Full bloom. .
...do
...do
...do
...do
Fruit setting .
...do
...do
...do
....do
. ...d
.do.
.do.
.do.
.do.
.do.
Tempera-
ture.
20
20
20
20
23
23
23
23
24
2 5
2 5
2 5
2 5
25
28
2.S
28
28
88
20
25
25
25
25
25
28
28
28
28
28
Number
of fruits
harvested.
4
9
2
o
o
3
4
o
o
o
14
4
8
3
o
o
o
7
9
7
7
o
13
8
15
10
17
Table IV. — Result of natural freezes
Kind of fruit.
Showing color.
Full bloom.
Fruit
setting.
Percentage
killed.
28,25,27
27-5,27.5
26
24, 28
3°-5
28, 28. 5
31-5,3°
32
8
5
41
26
30
29
0
27
28
29
28
26,25
I 29,29,30
32
32
0
Jan. 15, 1921
Freezing of Fruit Buds
661
Table IV. Result of natural freezes — Continued
Kind of fruit.
Showing color.
Full bloom.
Fruit
setting.
Percentage
killed.
22
O
26
26
O
O
22
\\
26
26
32,3!
O
O
26,25
29,25,27
30
O
O
26
53
29, 29
30
3J»32
22
3°
0
25)3°
50
22
22
61
23
26
26
26
0
48
26
31) 23,32-
t 25,30
26
20
0
0
32
0
25
0
32
0
28
25
0
0
3°
3°
0
24
22
54
36
26
3°- 5
0
27.5,27-5
29,25
27, 3°
31-5,3°
56
0
20
26
22
32
55
SUMMARY
(1) Efficient orchard heating demands an economical use of labor and
fuel and requires knowledge of the temperatures that cause injury to the
buds.
(2) This paper contains the results of seven years' experiments in
freezing 24,000 apple, peach, cherry, and apricot buds, together with a
record of the natural freezes that occurred in the orchards near Logan,
Utah, during the same period.
(3) Ben Davis apple buds in full bloom have experienced temperatures
of 250, 260, and 270 F. without injury, but 280 usually kills about one-
fifth. Twenty-nine degrees or above are safe temperatures. Twenty-
five degrees kills about one-half and 220 about nine-tenths. On several
662 Journal of Agricultural Research vo1.xx.no. 8
occasions, however, apples matured on branches that experienced 200
when the buds were in full bloom.
(4) With Klberta peach buds in full bloom, 290 F. or above are the
safe temperatures, because even though occasionally 260, 270, and 280 do
no damage, yet on most occasions 280 will kill from one-fourth to one-
half. Twenty-six degrees kills about one-half of them and 22 ° about
nine-tenths. Temperatures as low as 180 have failed to kill all of them.
(5) With sweet cherry buds in full bloom, 300 F. is the safe temperature ;
250, 260, 270, 280 have done no damage; but 290 usually kills about one-
fifth. Twenty-five degrees usually kills about one-half, and when the
buds were showing color 220 killed only two-fifths of the buds.
(6) Sour cherries are hardier than the sweet varieties. When the buds
were showing color 230 F. did not harm them, and when they were in full
bloom 260 killed but one-fifth and 220 only two-fifths of them.
(7) With apricots, 290 F. is the safe temperature; 260 and 270 killed
about one-fifth and 220 killed one-half. They are fairly hardy, but they
bloom so early that they are frozen oftener than any of the other fruits
studied in the experiments.
(8) The foregoing figures refer to the buds when in full bloom. Start-
ing from this stage, the earlier the stage of development the hardier the
buds are; and in general, when the fruit is setting the injury is from 5 to
10 per cent more than when they are in full bloom.
(9) Sour cherries are the hardiest, and then follow in order apples,
peaches, apricots, and sweet cherries.
(10) The fact that the same branch of buds will on one occasion ex-
perience 270 F. with 25 per cent injury and on another occasion take the
same temperature with no injury is no doubt due to the fact that the
juice is contained in capillary cells and supercooling results — that is, the
buds are cooled below the freezing point of the juice without the freez-
ing taking place. The great difficulty of killing all the buds even at
extremely low temperatures is due to the same cause together with the
fact that the cell sap may be very concentrated. Differences in the hardi-
ness of the different kinds of buds and also of the same buds at different
stages of development is due to differences in quality and concentration
of the cell sap.
PLATE 75
Apparatus for freezing entire tree.
Freezing of Fruit Buds
Plate 75
Journal of Agricultural Research
Vol. XX, No. 8
EFFECT OF VARIOUS CROPS UPON THE WATER EX-
TRACT OF A TYPICAL SILTY CLAY LOAM SOIL
By G. R. Stewart, Chemist, Hawaiian Sugar Planters' Association, and J. C. Martin,
Assistant Chemist, California Agricultural Experiment Station
The senior author has previously reported a series of investigations
carried on at the California Agricultural Experiment Station upon the
changes which took place in the water extracts from a group of selected
soils. These consisted of six silty clay loams and seven fine sandy loams.
All were typical soils brought from various places in California and repre-
sent a considerable range of past treatments and some variations in
known productive capacity. A large quantity of each soil was brought
to the Experiment Station at Berkeley, where it was sifted, mixed,
placed in two uniform containers, and afterwards kept under controlled
conditions. A crop of barley was raised upon all the soils during the
first year of the experiment in order to bring them into a somewhat
comparable state of tilth. During the second season one container of
each soil was cropped and the other was maintained as an uncropped
duplicate. Notable differences were found in the amounts of water-
extractable constituents from the cropped and the uncropped soils.
The water-soluble nitrates, calcium, potassium, and magnesium were
generally higher in the uncropped soils. Considerable differences were
also observed in the amounts of water-soluble constituents extracted
from the different uncropped soils. Further details of the experimental
methods and of the results obtained may be found in the original pub-
lication.1
The conclusion from our previous work, that barley reduces the nitrates
of soils to a low and fairly uniform magnitude independently of the soils'
crop-producing power and also tends to reduce the amounts of other
water-extractable constituents, seemed to require that the observations
be extended to include the effects of other crops. It was also deemed
desirable to study the effect of varying numbers of plants in accelerating
the changes observed and if possible to ascertain the rate of movement
of water-extractable substances through the soil.
The experimental work consists of two separate studies, one to cover
the specific effect of different types and numbers of plants, the other to
shed light on the movement of solutes through the soil.
1 Stewart, Guy R. effect of season and crop growth in modifying the son, extract. In
Jour. Agr. Research, v. 12, no. 6, p. 311-368, 24 fig., pi. 14. 191S. Literature cited, p. 364-368.
Journal of Agricultural Research, Vol. XX, No. 8
Waslnagton. D. C Jan. 15, 1921
vz Key No. Calif. -2 7
(663)
664
Journal of Agricultural Research
Vol. XX, No. 8
2,000
«3>,000
^OOO
Oj *,ooo
v: 6,000
^ 7000
6,000
S,ooo
/£?000
Tt/R>/V/R\S.
—aorsf beans.
RorAy oes.
■CORN.
UNCROPPED.
X
.* * 8
11
S I
EFFECT OF TYPE AND NUMBER OF PLANTS
A large portion of Yolo silty clay loam soil was sifted into a group of
eight containers. Each container was of the same size as those previously
used, 30 inches wide,
60 inches long, 18
inches deep, and held
approximately 1 ,800
pounds of soil.
One container was
planted to Golden
Ball turnips, one to
horse beans, one to
Early Golden Bantam
Fig. 1. — Decrease of water-soluble nutrients from the growth of various J
crops, as shown by increases in specific resistance. Crops were COrn, One to Burbank
planted May 13, and soil was sampled on dates given. n+atri A +h
to barley, the latter having, respectively, 24, 50, and 72 plants each.
In addition, one container was left uncropped as a control.
Water extractions were made at intervals of one to two weeks through-
out the major portion of the growing season. This period extended from
the middle of May to the end of September. All the crops except the
corn matured normally. The cool nights of the San Francisco Bay
region prevent corn planted in the spring from maturing till late in the
fall. The results with this crop, however, were of such a nature that
observations thereon became unnecessary after the maturation of the
other crops and were accordingly discontinued at that time.
The extractions were made in the proportion of 1 part of soil to 2 parts
of water. The mixture was triturated in a mortar for three minutes and
then filtered upon a
medium grade of semi-
quantitative paper in
an ordinary funnel.
The first portions were
poured back until
reasonably clear fil-
trates were obtained.
The conductivity of 3 8 « 2 S ;; $ « f; 8 <o
this solution was then I I I ^ S § ^ ^ *i i I §
determined by the Fig. 2.— Decrease of water-soluble nutrients from varying numbers of
Wheatstone b r i d PJ e barley plants, as shown by increase in specific resistance. Crops were
, . 1 - 1 planted May 13, and soil was sampled on dates given.
and is expressed m the
graphs as ohms of specific resistance. An increase of resistance, therefore,
represents a lowering of the concentration of electrolytes present. Work
performed in this laboratory on similar solutions has shown that this
method gives results which are comparable to those obtained by accurate
4000
3,000
4000
, 6,000
\ &000
0 7000
a, 000
e,ooo
BARLEY Z9-RLRNTS. .
- • — BARL EY 60 PLATA/ TS. \
BARLEY 72 PLAN TS.
—UNCROPPED.
-v-
Jan. is, 1921
Effect of Crops on Water Extract of Soil
665
determinations of total solids. The results of. these conductivity deter-
minations are plotted in figures 1 and 2.
Here we find that all the crops have reduced the concentration of the
water extracts during the middle of the growing season. It is interesting
to note in the cases of the barley crops that even the smallest number of
plants was sufficient to effect a substantial reduction of water-extractable
solutes by the time
the plants had be-
come well establish-
ed. The uncropped
soil, on the other
hand, maintained
a remarkably uni-
form concentration
throughout the per-
iod of observation.
Nitrates were de-
termined at a few
periods, and these
results are given in
graphs 3 and 4.
Here we see that
each crop at matur-
FiG. 3. — Decrease of water-soluble nitrates from the growth of various
crops. (Graphs=J£ NO3.) Crops were planted May 13, and soil
was sampled on dates given.
ity had depressed the soil's nitrate content to a minimum,
cropped soil constantly remained on a higher level.
MOVEMENT OF SOLUTES THROUGH THE SOU,
The un-
In this experiment two containers of the same soil were placed in the
greenhouse and buried in the ground, level with the floor for heat in-
sulation. Two* rows of sugar beets were planted across one end of one
container. These were spaced 6 inches apart in the row and 9 inches
between rows. The remainder of the container, some 40 inches in length,
was left bare. Two rows of barley were planted in one end of the other
container. The plants were spaced 6 inches apart and 6 inches between
rows. This left 40 inches of unoccupied ground.
The crops were started in December and were allowed to grow until the
following March. By that time the beets were about 2 inches in dia-
meter and the barley was fully headed.
Periodic observations of the concentration of the soil solution were
made by means of freezing-point determinations. Two samples were
always taken from each container, one from between the rows of beets
or barley and the other near the bare end of the tank. The freezing-
point depressions for both groups of samples are given in figure 5. The
last sample in April represents the condition we have previously observed
in soils when barley had made about the same growth.
17776°— 21 6
666
Journal of Agricultural Research
Vol. XX, No. 8
At this time a longitudinal section was cut in the soil, and the root
extension of both crops was studied. With the sugar beets it was found
that a thick, matted growth of fine rootlets extended from the second
row of beets to the
/ extreme end of the
container,. 41 inches
in all. Many of these
rootlets were branch-
es from the main
fleshy feeders. These
extended laterally
throughout the bare
end of the tank.
The main barley
roots were found to
extend 32 inches
from their plant
sources with the finer
rootlets extending 1
foot further toward
the bare end of the container. A portion of the roots also extended to
the bottom of the container and ran almost to the end wall.
The soil solution during the early stages of growth of both barley and
beets appeared to
Fig. 4.— Decrease of water-soluble nitrates from varying numbers of
barley plants. (Graphs=K NO3.) Crops were planted May 13, and
soil was sampled on dates given.
< JO°
^ .00s
Cj .090
Jo .oss
Jy .oso
(£ .075
Q) .070
<5 .065
k •«•«
£ .OSS
5 .050
> .0+0
Q) .035
5 .030
N .025
N.O20
HJ.O/5
HJ .0/0
fh .005
have a significantly
lower concentration
in the near neighbor-
hood of the plants
than at a distance
therefrom. It was
not until the early
part of April when
the plants had
reached a consider-
able size that the soil
solutions in the
cropped and un-
cropped ends of the
containers ap-
proached each other
in concentration.
Unfortunately for
the original objective of the experiment, the roots of the plants, in both
cases, appear to have penetrated the soil mass of the bare ends of the
containers about as rapidly as the concentration of the soil fell off.
BA&LEY BETPVEE// ROWS.
BB/H.EY/ITEA/0 OF TBMH.
BEETS BETtVEEH BO*VS.
BEETS /7TEA/0 OF TB/V/f.
8U 8w
U 11
Fig. 5. — Decrease in the concentration of soil solution shown by freez-
ing-point depression. Crops were planted December 3, and soil was
sampled on dates given.
jan. 15, 1921 Effect of Crops on Water Extract of Soil 667
There is, therefore, no proof here, either as to the rate of translocation
or the distance through which the soil solutes may move by diffusion.
But since the losses of concentration of the soil solution appear to be
somewhat proportional to root penetration, it would seem probable that
the rate of movement of solutes through the soil is less than the rate of
growth of the roots of normal barley and beets.
CONCLUSIONS
The gain in specific resistance and the decline in nitrate content of the
water extracts of soils planted to different crops warrant us in extending
the conclusions heretofore drawn from observations of the effects of bar-
ley. It is clear that the phenomena noted are not peculiar to the barley
plant but are characteristic of all the plants tested and probably apply
to all chlorophyll-bearing plants which root in the soil. The extent of
the reduction of concentration observed is variable with different crops.
We may not put too much stress upon the magnitudes of these differences,
however, because of the obvious differences in growth habits and life
history of the plants considered. It is interesting to note, however, that
corn which is commonly regarded as a "gross feeder" in ordinary fer-
tilizer practice has increased the specific resistance of the water extracts
more rapidly and completely than the other plants.
The second experiment sheds little light on the rate of movement of
solutes toward the plant roots. Inasmuch, however, as reductions in
concentration of water extracts of soil at a distance from growing plants
did not take place until that portion of the soil had become filled with
roots, it would seem that rapid and extensive movements of soil solutes
are probably not an incident of normal plant absorption.
SUMMARY
(1) The effect of crops of corn, horse beans, potatoes, turnips, and bar-
ley upon the water extract from a typical silty clay loam was studied
throughout the growing season.
(2) All the crops discussed in this paper reduced the concentration of
the water extract during the height of the growing season.
(3) The nitrate content of the soil was reduced to a very low figure
by all crops.
(4) An experiment in which the concentration of the soil solution was
studied by means of observations of freezing-point depressions in the
immediate vicinity and at a distance from the plants showed that con-
centrations are not significantly reduced until the portion of the soil
sampled is filled with plant roots.
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V
Vol. XX FEBRUARY 1, 1921 No. 9
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS
Page
Another Conidial Sclerospora of Philippine Maize - - 669
WILLIAM H. WESTON, Jr.
( Contribution from Bureau of Plant Industry )
Onion Smudge --------- 685
J. C. WALKER
(Contribution from Bureau of Plant Industry)
Variations in Colletotrichum gloeosporioides - 723
O. F. BURGER
(Contribution from California Agricultural Experiment Station )
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, D. C.
WMHINOTOH : QOVERKMENT PBINTIKO OFFtCB : t«!l
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and A ssociate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and A ssistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, Slate College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipraan, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
i«0
JOURNAL OP AGMCIILTHAL RESEARCH
Vol. XX Washington, D. C, February i, 1921 No. 9
ANOTHER CONIDIAL SCLEROSPORA OF PHILIPPINE
MAIZE
By William H. Weston, Jr.
Pathologist in Charge of Downy Mildew Investigations, Office of Cereal Investigations,
Bureau of Plant Industry, United States Department of Agriculture
Each year in the Philippine Islands the valuable maize crop suffers
very severe losses from the destructive activities of downy mildew (Scleros-
pora spp.). While the writer was studying this disease during the past
two years his attention was naturally directed to the question whether
the widespread destruction of maize throughout the thousand-mile extent
of these scattered islands was due in all cases to the same species of
fungus. A comparative study of material collected from many parts of
the provinces of Batangas, Laguna, and Rizal in the island of Luzon,
where the disease is most serious and where it was studied most inti-
mately, showed that in all cases the same causal fungus was involved.
This species of downy mildew was described in an earlier paper (12)1 as
Sclerospora philippinensis. It was only natural to suspect that some of
the abundant Philippine wild grasses related more or less closely to maize
would be found to harbor this or other Sclerosporas. As on the widely
distributed wild grass Saccharum spontaneum L. (PI. 77, A) the oogonial
stage of a Sclerospora had been very commonly encountered in great
abundance, this grass was obviously an object of suspicion. In Luzon,
however, despite extensive search, no conidial stage was seen on this host.
During a trip to the more southern Visayan Islands of Cebu, Bohol,
and Leyte, in which maize is a crop of very great importance, the writer
found that there, also, the maize plantings were suffering heavy losses
from downy mildew. As no microscope was carried, no study of the
causal organism was made at night during the period of conidium pro-
duction. However, inasmuch as the symptoms and the general effect of
the downy mildew were the same in these southern islands, the writer
inferred that the causal organism was that which he had found so widely
distributed on maize throughout the northern island of Luzon. Also the
wild grasses of these southern islands were carefully examined as possible
1 Reference is made by number (italic) to " Literature cited," p. 684.
Journal of Agricultural Research, Vol. XX, No. 9
Washington, D. C Feb. 1, 1921
wq Key No. G-217
(669)
670 Journal of Agricultural Research vol. xx, no. 9
hosts for downy mildew. After long search a clump of bugang grass
(Saccharum spontaneum) heavily infected by a conidial Sclerospora was
discovered by Mrs. Weston. Continued hunting brought the fungus to
light on the same host in two other places, all three cases being encoun-
tered in the rugged interior uplands of Cebu (PI. 76), which lie between
Carcar and Barili. In the island of Leyte, also, this Sclerospora was
again found on bugang grass on a hillside about three miles from Baybay.
No other cases of downy mildew either on this or on other hosts were seen.
Later, in a field of native sugar cane near Guadelupe cemetery outside
the town of Cebu, a single clump of cane was found infected with the
conidial stage of a Sclerospora.
The infected plants of Saccharum spontaneum and sugar cane were
transplanted to Los Banos, Luzon, for further study (PI. jj, B). There
a comparison of living material taken from these plants during the opti-
mum time of nocturnal conidiophore production showed that this downy
mildew from the southern islands was different from that previously
studied in Luzon. This discovery necessitated a revision of all available
material in order to determine whether or not other forms had been pre-
viously overlooked under the assumption that the collections were all
of the same form so commonly found in Luzon. Accordingly, living
material from maize, teosinte, and sorghum from the college plots and
from native fields in Batangas and Laguna provinces was compared with
the living material from the plants of Saccharum spontaneum and sugar
cane brought from Cebu. Dried, preserved, and mounted specimens
from maize collected in various parts of Luzon were compared with similar
specimens from maize obtained in various localities in Cebu, Bohol, and
Leyte. This survey showed clearly that all the material so far encoun-
tered fell into one or the other of two distinct species — one, the form
with shorter, broader conidia found on maize, etc., in Luzon and pre-
viously described as Sclerospora philippinensis, and the other, which will
be called Sclerospora spontanea, characterized by longer, narrower conidia,
and found on maize, bugang grass, and sugar cane in the Visayas. Once
this point had been established, a comprehensive study was made of the
two species to determine the resemblances and differences between them
in morphological and physiological characteristics.
COMPARATIVE STUDY OF SCLEROSPORA PHILIPPINENSIS AND
SCLEROSPORA SPONTANEA
FIELD CHARACTERISTICS
On maize, as observed in the field in the more southern islands and in
Luzon, the two species are apparently identical in their destructiveness
to the crop as a whole and also in their effect on the individual plants.
It is possible that quantitative studies of essentially similar fields infected
by the separate species would show some slight differences, but in general
appearance there is no distinction whatever between the two.
Feb. i,i92i Another Conidial Sclerospora of Philippine Maize 671
PHYSIOLOGICAL CHARACTERISTICS
Several varieties of maize grown in sterile soil and under controlled
conditions preventing contamination were infected with spores produced
on the living plants of bugang grass (Saccharum spontaneum) and sugar
cane brought from Cebu. Parallel inoculations were made also with
Sclerospora philippinensis . No difference was apparent either in symp-
toms or in the virulence of the resulting infection. Similar experiments
with seedlings of cultivated wheat, Setaria, Pennisetum, and several
species of wild grasses, including the very common aguingay (Rottboellia
exaltata L,.), anias (Andropogon sorghum var. halepense L.)> cogon (Impe-
rata cylindracea L.)> and tigbee (Coix lachryma-jobi L.), using the long,
narrow conidia of the southern species, were as uniformly unsuccessful
as they had been with Sclerospora philippinensis (12). Seedlings of
teosinte (Euchlaena luxurians Schrad.) and the wild grasses, Saccharum
spontaneum and Miscanthus japonicus (Thunb.) Anders., were success-
fully inoculated with both forms. No seeds of sugar cane were available
for planting. Had there been, there is little doubt in the mind of the
writer that infections in this case also could have been obtained. A
more detailed account of these inoculation experiments will be given in
a later paper. It should be said here, however, that the effect of the
Sclerosporas varied with the different hosts, being most destructive on
maize and least so on bugang grass; but the characteristic production of
conidiophores took place with uniform regularity at night on all
(PI. 78, B).
A comparative study of material of Sclerospora spontanea on these
different hosts showed that the distinguishing morphological character-
istics of the fungus had not been altered in any way. Moreover, even
after transition from one host to another through several generations,
the species remained constant and in no way approached S. philippinensis.
In like manner, after inoculating various hosts and passing through
several generations, S. philippinensis also was quite unchanged and
showed no tendency to approach the long-spored form.
The writer considers it quite possible that an exact statistical study of
large numbers of individuals infected by each of the two fungi would
reveal some slight quantitative difference in the area bearing conidia, or
in the rate of growth of hyphae through the host, or in some other aspect
not at once apparent to an ordinary comparative examination. It
should be noted here, however, that there is certainly no noticeable
physiologic difference between the two in virulence, range of hosts, or
general course of the resulting disease they produce.
MORPHOLOGICAL CHARACTERISTICS
Therefore, because the two forms differ morphologically rather than
physiologically, they were carefully compared in order to determine
whether the points of difference were sufficiently stable and well marked
672 Journal of Agricultural Research voi.xx,No. 9
to establish the long-spored form as a species distinct from Sclerospora
philippinensis .
Mycelium. — In morphological characteristics, extent, and relation to
the host tissue, the mycelium of the two fungi showed no distinctions
sufficiently marked or unvarying to warrant their use as a basis of
separation. However, the club-shaped hyphae (conidiophore initials)
which grow out through the stomata and develop into conidiophores are
different in the two forms, those of the long-spored Sclerospora being
markedly longer, more slender, and more irregular.
Conidiophores. — In general appearance the conidiophores of the two
Sclerosporas are noticeably dissimilar, those of the Visayan form being
markedly longer, more slender, and more spreadingly branched than
those of Sclerospora philippinensis. On analyzing this dissimilarity the
details of difference discussed in the following paragraphs are apparent.
The basal cell of the Visayan Sclerospora is very long (Pi. 79, A, D,
E, F, H), strikingly longer than that of Sclerospora philippinensis. The
length (140 to 260 yu) is greater not only actually but also relatively, for
even in the unusual cases when it is less conspicuously long (PI. 79, G)
the basal cell of the Visayan Sclerospora always exceeds or at least equals
in length that part of the main axis extending from the terminal septum
of the basal cell to the origin of the primary branches. In S. philippi-
nensis, the basal cell is always shorter than this part of the main axis.
Moreover, the basal cell of the Visayan Sclerospora is much more slender,
usually 5 to 8 /t at its narrowest diameter, and much less knobbed or
swollen at its base (PI 79, A, D, E, F, H) than is the basal cell of 5.
philippinensis.
The main axis of the Visayan Sclerospora expands more abruptly
above the basal cell and then constricts more distinctly (PI. 79, A, D)
just below the branches than in Sclerospora philippinensis. The greatest
diameter (22 to 32 /z), which usually slightly exceeds that of 5. philip-
pinensis, is thus placed, not just below the branches (as in 5. philip-
pinensis), but some distance lower (PI. 79, A, D, G, H).
The branches of the Visayan form generally are less constricted at
their point of origin, are of more uniform diameter, and are straighter,
less ascending, more spreading, and do not recurve, but stand out from
the main axis more stiffly. They are characteristically longer and more
slender, but, even if short and crowded, they stand out more stiffly than
in Sclerospora philippinensis. Although varying considerably in both
species, the number of conidia produced on conidiophores is approxi-
mately the same in 5. spontanea and in S. philippinensis. In the former,
32 to 48 are commonly borne, although as many as 88 or as few as 12
may less frequently occur.
The sterigmata also are straighter, less recurved, and stand out more
stiffly than in Sclerospora philippinensis, and, usually they are longer
(about 13 fx). It should be noted, however, that the length varies with
Feb. 1,1921 Another Conidial Sclerospora of Philippine Maize 673
the extent of the branch system, since in cases where this is reduced and
the primary branches or even the main axis give rise directly to sterig-
mata, these sterigmata are much larger (PI 79, B) than they are when
arising from quaternary or tertiary branches as the ultimate structures
of an elaborate system (PI. 79, A).
As a result of such differences, the conidiophore top of the Yisayan
Sclerospora has a more spreading, expanded appearance; and the long
axes of the branches, the sterigmata, and the conidia borne on them
stand out from the main axis like rays of a partly opened fan. In
Sclerospora philippinensis, on the contrary, the conidiophore top is more
compact and less spreading, the axes of branches, sterigmata, and
conidia being all approximately parallel to each other and to the main
axis.
These differences in the conidiophores of the two fungi are, on the
whole, relative rather than absolute and are influenced to some extent
by such environmental conditions as the depth and persistence of the
layer of dew in which they develop. Even these distinctions, however,
could be used as more absolute and less relative criteria if a very large
number of measurements of all parts of the conidiophores were made
and assembled to give an adequate quantitative impression. Even
from the qualitative rather than quantitative point of view, moreover,
these differences, although relative, are constant and distinct, and it
should be emphasized that they persist when the two fungi, developing
under exactly parallel circumstances on sister plants of the same age,
grown side by side under as nearly the same conditions of temperature,
soil, dew deposition, etc., as it was possible to obtain, were compared
by nightly examinations for several weeks.
Conidia. — Among the Peronosporaceae as a whole the characteristics
of the conidia have been found to be the most valuable basis for dis-
tinguishing species. This applies equally well to these two Sclerosporas,
since their conidia not only differ markedly and constantly in shape and
size but also remain relatively unaffected by changes in environment
and hosts.
In shape, the conidia of the Visayan Sclerospora are at once distin-
guished from those of Sclerospora philippinensis. They are not only
much more elongate but much more slender as well, the length being
frequently two or even three times the diameter. Consequently they
range from very elongate ovoid and obovoidal bodies to long narrow,
round-ended cylinders, but they are most commonly very elongately
ellipsoid in shape. A clearer idea of these variations may be gained
from Plate 79, I, J, K.
In such features as the rounded apex devoid of any papilla, the blunt
base with its apiculus of attachment, the hyaline, granular content,
and the thin wall, the conidia correspond to those of Sclerospora
674 Journal of Agricultural Research vol. xx, no. 9
philippinensis. As in the case of the latter species also, germination
is invariably by the protrusion of one or more germ tubes (PI. 79, I,
J, K).
In size, the conidia of the Visayan Sclerospora are very variable.
With respect to such widely varying bodies as the spores of this and
other genera of Peronosporaceae, recent investigations have shown that
it is no longer possible to delimit a species adequately by the extremes
or averages of a few measurements. Rather, there is required the
assembling and presentation in tables and graphs of a sufficiently large
number of representative measurements to give a quantitative as well
as a qualitative expression of the conidial characteristics of the species.
Accordingly, in order to obtain data adequate to identify the Visayan
form and to furnish a basis for comparing it with others, 700 conidial
measurements were made. These comprised measurement groups of
100 conidia from each of the two sugar-cane and the four Saccharum
spontaneum plants from Cebu, and from one maize plant inoculated
from the latter.
The conidia were taken from the leaves of the host at night during
the optimum period of conidia production — from 2 to 4 a. m. — mounted
in dew, and measured immediately.
Since, on examination, the seven measurement groups were found to
agree in all essential particulars, they were combined into the total of
700. For the purposes of comparison, 700 measurements of Sclerospora
philippinensis were secured in like manner.1 Of these, 300 were new
ones made of fresh conidia from teosinte and sorghum found infected in
the college plots and from Saccharum spontaneum seedlings artificially
inoculated from maize. All these groups were compared, found to agree,
and grouped into the total of 700.
In making these measurements, care was taken to include every
conidium in a marked area of the microscope field as the slide was moved
along by the mechanical stage. Only those conidia obviously injured or
those still attached to the conidiophores were excluded. The divisions
of the eyepiece equaled approximately 1.8 ll, and, with the magnification
used, it was possible to estimate with fair accuracy to one-third of a
division, or to about 0.6 fx. Consequently, the measurements are exact
to this extent — that is, the conidium recorded as 32 ll in length may as
well be 31 .4 ll or 32.6 ll instead of exactly 32 fx but not, in all probability,
3 1 or 33 fx. With a large number of spores such differences tend to equalize
themselves. As a result, the measurements presented here may be
considered as adequately representing the characteristics of the conidia
of the species involved.
1 The writer wishes to take this opportunity to call attention to an error in the tabulation of the previous
spore measures of Sclerospora philippinensis (12, p. no). In the table of length, the conidia measuring 41
to 42.9 m should be 23 in number instead of 24.
Feb. 1. 1921
Another Conidial Sclerospora 0} Philippine Maize 675
The measurements are summed up in Table I and are presented in
graphic form in figure 1. In addition, the biometric characteristics of
the two species are given in Table II. In making the calculations, the
directions and formulae of E. Davenport (j) and C. B. Davenport (2)
is /■? 16 ie eozz 29
Cl/WE T£ffor COMDr# IU
M/Cf?OHS
/&tr/o or length zoo/Meres?
FIG t -Comparison of the sizes of 700 conidia of Sclerospora spontanea with 700 conidia of S. phHippinensis;
A , variation of conidia in length; B, variation cf conidia in diameter; C, ratios of length to w.dth ol
conidia arranged in classes.
have been followed. The writer makes no pretense to a comprehensive
biometric study of the two Sclerosporas but has used this method solely
as a means to the end of presenting the accompanying data as a basis
of comparison between these and other species.
676
Journal of Agricultural Research
Vol. XX, No. 9
Table I. — Summarized measurements of conidia of Sclerospora spontanea and
Sclerospora philippinensis
Length.
Diameter.
Length over diameter.
Number of conidia
Number of conidia
Number of conidia
in 700.
in 700.
in 700.
Classes.
Classes.
Ratio classes.
S. spon-
S. philip-
S.spon-
S. philip-
S. spon-
S. philip-
tanea.
pinensis.
tanea.
pinensis.
tanea.
pinensis.
V-
1
1
2
5
17
It.
11 to 12.9. . .
13 to 14.9- • •
15 to 16.9. . .
17 to 18.9. . .
19 to 20.9. . .
11
175
39i
121
2
2
18
119
3"
199
2
5
41
154
211
\c\
t
■
25 to 26.9. . .
1
1.75 to 1.94- ■ •
7
27 to 28.9. . .
29 to 30.9. . .
31 to 32.9. . .
3
27
77
134
28
163
88
12
2.35 to 2.54. . .
102
24
33 to 34-9- •■
20
153
2.55 to 2.74. . .
154
8
3S to 36.9- • •
93
107
2.75 to 2.94. . .
161
4
37 to 38.9.. .
76
75
2.95 to 3.14. . .
88
39 to 40.9. . .
87
39
3.15 to 3.34. . .
37
41 to 42.9. . .
112
29
3-35 to 3.54. . .
22
43 to 44-9- ■ •
94
23
3-55 to 3.74. . .
5
45 to 46.9. . .
79
7
3-75 to 3.94. . .
3
47 to 48.9.
46
2
3.95 to 4.14. . .
0
49 to 50.9 .
22
0
4.15 to 4.34.. .
1
51 to 52.9- ■ •
IS
I
53 to 54-9- ••
12
55 to 56.9...
7
57 to 58.9. . .
5
59 to 60.9...
4
61 to 62.9. . .
0
63 to 64.9. . .
2
Table II. — Biometric constants of the conidia of Sclerospora spontanea and
Sclerospora philippinensis
Soecies.
Mean.
Median.
Mode
(approx-
imate).
Standard de-
viation.
Coefficient of
variability.
S. spontanea
S. philippinensis. .
42.o7±o. 145
34- S2± .113
41. 86±o. 142
34. I2± .142
41-43
33-32
5. 672±o. 102
4. 439 ± -080
i3-48±0. 247
I2.86± .235
DIAMETER
S. philippinensis. .
15- 79±o. 036
18. 40± .047
15. 84± 0.045
18. 36± .006
15- 93
18.36
i-395±o-025
i.834± .033
8. 83±o. 160
9-97± • 181
RATI
3 OF LENGTH TO
DIAMETEI
{
S. spontanea
S. philippinensis. .
2. 71 ±0.009
i-9i± .007
2. 7I±0. on
i.89± .008
2.71
I.85
0. 357 ±0. 006
. 266± . 005
13- 20±o- 242
I3-92± .256
An examination of the data shows clearly that the long-spored Visayan
form, Sclerospora spontanea, at least in regard to its conidia, is quite dis-
tinct from S. philippinensis. The location of the two frequency curves
shows that the great bulk of the conidia of 5. philippinensis fall between
the limits of 31 to 36.9 ju in length, and 17 to 18.9 n in width; while, on
the contrary, a like proportion of those of 5. spontanea are 37 to 46.9 n
in length and 15 to 16.9 n in width. The somewhat irregular character
Feb.1,1921 Another Conidial Sclerospora of Philippine Maize 677
of the length curve of the latter species does not, in the opinion of the
writer, indicate that it is bimodal, because, by using more inclusive
measurement classes of 4 fx or even 3 fx, the depression so noticeable with
the 2-ju classes smooths out and the curve becomes quite regular. More-
over, the difference between the modes as well as between the means and
the medians is still sufficiently great to emphasize strikingly the dissimi-
larity in size of the conidia of the two species.
It should be noted that, although the curves of frequency distribution
of the two species overlap slightly, size is none the less a valuable diag-
nostic criterion. In length, for instance, the curves overlap from 26 fx,
the lowest limit of the Visayan Sclerospora, to 52 ll, the highest limit
reached by Sclerospora philippinensis. As a result, it might be con-
tended that size is of no value in distinguishing between the two species
when applied at least to the conidia falling between these limits. While
this is true of any one conidium, experience shows that, if several are
measured, exceedingly few are to be found in this disputed region. For
practical purposes even 50 unselected conidia of each species are suffi-
cient to show the difference between them without any confusion due to
overlapping.
It is also worthy of note that the curves of the frequency distribution
of 700 conidia in both the Visayan species and Sclerospora philippinensis
differ in no essential particular from those of 500, 400, or even as few as
200 conidia.
Furthermore, in the ratios of length to width of their conidia, the two
species also show marked differences. The shorter, broader spores of
Sclerospora philippinensis most commonly show ratios of 1.55 to 2.14,
while in 5. spontanea the greater length as well as the lesser width of the
coniJia is expressed by the predominant ratios of 2.35 to 2.94.
In order to determine whether the differences between the biometric
characteristics of the two forms were indeed significant, the method
quoted by Rosenbaum (11) from Reitz and Smith was employed. This
method, which compares the difference between the mean or other con-
stants with the probable error of the difference, shows that in Sclerospora
philippinensis and 5. spontanea these differences without doubt are
significant and can not be the result of mere random sampling. This
significance is clearly brought out in Table III.
TABLE III. — Difference in means of Sclerospora spontanea and Sclerospora philip-
pinensis compared to the probable errors
Difference in means.
Difference in means divided by
probable error of difference.
Length.
Diameter.
Length over
diameter.
Length.
Diameter.
Length
over
diameter.
7. 55±o. 183
a. 61 ±0.058
0. 798±o- on
41.27
44.96
7o-39
678 Journal of Agricultural Research voi.xx, N0.9
The identity of the long-spored, Visayan Sclerospora, then, is clearly
established as quite distinct from Sclerospora philippinensis. Whether
this distinction is sufficient to entitle the former to specific rank depends
somewhat upon the judgment of the investigator. The matter could
be settled with greater finality if the two fungi were to be grown in pure
culture and compared in morphological and physiological details under
the controlled conditions of the laboratory, but unfortunately all attempts
to grow the two forms artificially have been unsuccessful. In view,
however, of such well-defined, although somewhat relative, morphological
differences in the conidiophores as the peculiarities of the basal cell and
the branch system, and the well-marked and easily measurable differ-
ences in size and shape between the conidia of the two fungi, and in view
of the constancy and persistence of these points of dissimilarity over a
wide range of hosts, through several generations of maize and during
three months' cultivation, the writer regards the Visayan form as worthy
of specific distinction from S. philippinensis. The species, therefore, is
described as new, and as it was first found occurring spontaneously on a
wild host, it is named 5". spontanea.
DIAGNOSIS
Sclerospora spontanea, n. sp.
Symptoms, effect on the individual host, and destructiveness to the maize crop
as a whole, as previously described by the writer for Sclerospora philippinensis (12).
Mycelial hyphae and haustoria as described for Sckrospora philippinensis; but the
clavate hyphae (conidiophore initials) which emerge from the stomata are longer,
more slender, and more irregular.
Conidiophores as in Sclerospora philippinensis, erect, single or grouped, develop-
ing only at night and in dew; comprising basal cell, main axis, more or less complex
dichotomous branching system, and terminal sterigmata; but differing in general
in greater total length (350 to 550 n) and more expanded top, and in particular as fol-
lows: Basal cell less knobbed and expanded at the base, more slender (least diameter
about 5 to 8 m). and longer (140 to 260 u), usually exceeding or at least equaling in
length the extent of the main axis from the septum to the primary branches. Main
axis usually expanding more abruptly above the septum to a greater width (22 to 32 /u)
and constricting noticeably (to about 20 u) below the branches. Branches longer,
more slender, less constricted at point of origin, less recurved and ascending, but
standing out more stiffly. Sterigmata longer (13 u), more slender, and straighten
Conidia resembling those of Sclerospora philippinensis in hyaline, finely granular
content, thin wall, rounded apex lacking papilla, and rounded base with apiculus of
attachment, and in invariable germination by tubes; but differing as follows: In shape,
longer and more slender, usually very elongately ellipsoid or cylindrical; in size,
showing greater length and less width, the majority being 39 to 45 /u long by 15 to 17 a
in diameter.
Oospores not yet encountered on maize, although an oogonial stage on Saccharum
spontaneum may prove to be connected.
Habitat. — Found in the Visayan group of the Philippine Islands principally on
cultivated maize (Zea mays L.), rarely on the wild grass bugang (Saccharum spon-
taneum L.), and once on cultivated sugar cane (Saccharum officinarum I,.). Inocu-
lated successfully upon the first two of these hosts and also upon teosinte (Euchlaena
luxurians Schrad.), and the wild grass Miscanthus japonicus (Thunb.) Anders.
Extremely destructive to maize, but much less so to the other hosts.
Feb. i,i92i Another Conidial Sclerospora of Philippine Maize 679
Material of the type will be found in the pathologic collections of
the Bureau of Plant Industry, Washington, D. C, and in the herbarium of
the Bureau of Science, Manila, P. I.
DISCUSSION
RELATIONSHIP
The two Sclerosporas, Sclerospora spontanea and 5. philippinensis, are
undoubtedly closely allied to each other. It is even possible that future
investigation will bring to light forms intermediate between them. Such
may be the downy mildew on maize seen by Prof. Reinking in the Cota-
bato Valley and by Gov. Coverston in Lanao Province, both of which
places are in the southern Island of Mindanao. On the other hand,
the Mindanao form may be as different from 5. spontanea and 5. philip-
pinensis as these have proved to be from each other. The writer feels
confident that on further search additional Sclerosporas will be encoun-
tered in the Philippines both on cultivated hosts and on wild grasses.
The relationship of the Philippine downy mildew Sclerospora to the
similar forms described on maize and related crops from other oriental
countries has been discussed in connection with Sclerospora philippi-
nensis (12). Unfortunately the matter can not be settled finally with
the data available. As the writer's discovery that suitable material
can be secured only at night is very recent, previous publications present
measurements and other data inadequate for comparison with living
material. In so far as one can judge, however, 5. spontanea, on account
of its longer, more slender spores, is even more sharply distinguished
than is 5. philippinensis from the Javan species, S. javanica Palm (io),
from the species of British India, 5. maydis (Rac.) Butl. (1), and from
the normal, short spored type of the Formosan species, i>. sacchari
Miyake (9). It is of interest to note, however, that in the greater length
of its conidia, the very character wherein it differs so distinctly from
these other oriental species, 5. spontanea tends to resemble the two
abnormally long-spored forms recorded by Japanese investigators. In
his account of 5. graminicola, Ideta (8, p. 143-145), in addition to conidia
of the size characteristic of the species, mentions a class of conidia having
the—
shape of a long ellipse, 38.4 to 57.6 n long by 19.2 to 24 /x wide.
Also, Miyake (9), in his account of 5. sacchari, describes conidia not only
of the usual shape and size, but also of an unusual type —
long ovate, 49 to 54 /x by 19 to 23 ju.
The descriptions and drawings of both these long types of conidia
remind one of the spores of 5. spontanea, even though the latter are
characteristically more slender. It is very probable that the occur-
rence of these long conidial types in Japan and in Formosa indicates the
680 Journal of Agricultural Research vol. xx, No. 9
existence there of strains or species of Sclerospora as yet unrecognized ;
but what their relationship and significance may be, future investigation
must determine.
The relationship of these two Philippine conidial forms to the oogonial
stage characteristic of the genus is as yet unknown. Whether Sclero-
spora philippinensis or Sclerospora spontanea is connected with the oogonial
stage which is so common on Saccharum spontaneum throughout the Philip-
pine Islands is yet to be established. The writer has attempted to germi-
nate the oogonia of the latter and to obtain inoculations with them, but so
far he has been unsuccessful. Until the precise connection is definitely
established, it is well to be cautious about assuming that the two types
of spores are with certainty different phases of the same species. It
may be worthy of note that the writer has found, in addition to the oogonia
on Saccharum spontaneum, similar spores on Miscanthus japonicus and
on cultivated sugar cane in the mountains of northern Luzon. On all
these hosts the oogonia are apparently the same species; and their
significance and importance will be discussed by the writer in a later
paper.
NONSPECIALIZATION
As the problem now stands, the Philippine maize-mildew presents an
interesting situation, since it involves two causal Sclerosporas quite distinct
morphologically but practically indistinguishable physiologically both in
their effect on, and in their virulence to, a range of hosts. The genus
Sclerospora seems, then, to present a marked contrast to the strong
specialization of the closely related genus Peronospora. In the latter,
the work of Gaumann (5, 6, 7) has shown that the species are strongly
specialized, being distinct on different hosts. This is true especially in
the Rubiaceae (7), but also to a marked degree in the Cruciferae (5) and
the Scrophulariaceae (6). The distinction holds both morphologically,
in the size and character of the conidiophores and conidia, and also
physiologically, in their inability to infect any host species but that from
which the spores were derived. Gaumann, therefore, regards it as highly
improbable that the same host species would be found to harbor more
than one species of Peronospora. In Sclerospora, however, we have
the two species, Sclerospora spontanea and 5. philippinensis, morpho-
logically distinct, yet both with equal ease inoculating the same series of
hosts, including members not only of the Maydeae but also of the Andro-
pogoneae.
SIGNIFICANCE OF OCCURRENCE
The finding of Sclerospora spontanea on a wild gramineous host is of
interest. Hitherto in spite of the attention which the destructive
oriental Sclerosporas have attracted, no conidial representative of the
genus has ever been reported as occurring naturally upon a wild host.
It is a question whether the occurrence of Sclerospora spontanea on wild
Feb. i, i92i Another Conidial Sclerospora of Philippine Maize 68 1
Saccharum in the Visayan Islands should be regarded as throwing light
on the problem of the origin of the Philippine downy mildews of maize.
In the opinion of the writer this and other facts indicate that the native
grasses of the Philippines were the original hosts from which the downy
mildews passed and are passing to such very susceptible introduced
crops as maize. On the other hand, one should not overlook the possi-
bility that the wild Saccharum clumps might have been infected with the
downy mildew from badly diseased maize growing near. In this con-
nection it should be noted that in two cases where Sclerospora spontanea
was found on wild bugang grass (Saccharum spontaneum) the infected
clumps were so far distant and so protected from any downy-mildewed
maize that there was little possibility of their having been infected thus.
In the other cases the infected bugang clumps were much older than the
mildewed maize adjacent; and, because inoculation experiments have
shown that bugang grass is susceptible only as comparatively young
seedlings, there is little doubt that the infection in the grass clump had
been carried over in the perennial rootstocks and had not been caught
from maize.
Moreover, it is worthy of note, also, that the wild Saccharum is very
resistant to the effect of the Sclerospora, while maize is exceedingly
unresistant. In contrast to the susceptibility to severe injury already
noted in maize, wild Saccharum, even though heavily infected, shows
only slight striping of the leaves (PL 78, B, C), remains undeformed, and
is not materially retarded in development. In spite of the downy
mildew the plants continue to grow vegetatively, to produce flowers
(PL 77, B), and to form, by tillering, dense clumps which by extensive
rootstocks persist from season to season, still supporting the active and
equally persistent parasite. Because, as a rule, it is the introduced host
which is most injured by a disease and the original, native host which is
relatively unaffected, the indications are that wild Saccharum and not
maize is the original host of Sclerospora spontanea.
The finding of Sclerospora spontanea on sugar cane is a second point of
interest. Because, in Formosa, the closely related species 5. sacchari
Miyake had proved indiscriminately destructive to both sugar cane and
maize, the writer, while in the Philippines, made especial effort to dis-
cover instances of the transmission of downy mildew from one to the other
of these hosts. The single case in Cebu, however, was the only one noted.
In this instance the single clump of sugar cane infected with 5. spontanea
was situated at the extreme edge of the field, separated only by a narrow
trail from a large planting of badly downy-mildewed maize. Although
the whole sugar-cane field was carefully inspected, no other cases of
Sclerospora were discovered. It is natural to infer that the sugar-cane
plant was infected from the neighboring maize, especially since the two
parasites proved to be the same. It is rather surprising, however, that
this lone cane plant, of all the thousands examined in scores of different
682 Journal of Agricultural Research vol. xx. No.9
fields adjacent to or even interplanted with infected maize, should be the
only one to succumb.
The matter is still further complicated by the fact that in Formosa
Miyake easily obtained the infection of sugar-cane plants grown from
cuttings, while in the Philippines the writer was not able to inoculate
cutting-grown plants of sugar cane, or even of Saccharum sponianeum,
although seedlings of this grass were readily infected (PI. 78, A). More-
over, in Formosa the effect of Sclerospora sacchari Miyake on sugar cane
is far more destructive than was the effect of Sclerospora spontanea on
this single cane plant. In the former the elongation and weakening of
the shoots and the conspicuous yellowish striping of the leaves are a dis-
tinct contrast to the stunting of the shoots and faint, pale green markings
of the leaves which characterized the Philippine specimens. Also,
although the latter died shortly after being transplanted, this was ap-
parently due to the severe treatment they had received rather than to
the destructive character of the Sclerospora. It is possible that Sclerospora
spontanea, in its essential individuality, is much less virulent to sugar
cane than Sclerospora sacchari, or it may be that some limiting factor is oper-
ative in the Philippines. The work of Fawcett (4) indicates that tempera-
ture differences may exercise an important limiting effect within a smaller
geographic range than from Cebu to Formosa. In any case, although
the matter is in need of further study, it can safely be said that in so far
as has been observed in the Philippines the production of sugar cane is
unaffected by Sclerospora spontanea or other conidial Sclerosporas.
SUMMARY
The downy mildew of maize which is extremely destructive in the
Philippine Islands has been found to be caused by the Peronosporaceous
genus Sclerospora. At first only one species was thought to be involved,
and this was described by the writer as Sclerospora philippinensis . More
recently the problem presented by the Philippine maize-mildew has been
still further complicated, since a second causal species of Sclerospora has
been found to be concerned also. The foregoing paper describes this
species as new {S. spontanea) and presents briefly its morphological and
physiological characteristics and its importance and relationship.
Sclerospora spontanea, the more recently discovered form, occurs in the
Islands of Cebu, Bohol, and Leyte, where it was found on the wild grass
Saccharum spontaneum L., on sugar cane (Saccharum ofjicinarum L,.),
and on maize (Zea mays L.). Sclerospora philippinensis, the species
first recognized, occurs in the Island of Luzon, where it was found on
maize, teosinte (Euchlaena luxurians Schrad.), and sorghum (Andropo-
gon sorghum [L.] Brot.).
Morphologically, Sclerospora spontanea is characterized by the rela-
tively much greater length and slenderness of its conidiophores in general
and of its basal cells and conidia in particular. In these respects it
differs markedly from 5". philippinensis, which has shorter, stockier
Feb. i,i92i Another Conidial Sclerospora of Philippine Maize 683
conidiophores, shorter, thicker basal cells, and shorter, broader conidia.
There are, moreover, some minor distinctions between the branch sys-
tems and between the sterigmata of the two species.
These differences remain constant for each species and are not influ-
enced by growth on different hosts even through several generations.
Both species have been artificially inoculated with equal ease from one
to another of the following hosts : Maize, teosinte, Miscanthus japonicus,
and Saccharum spontaneum. Attempts to inoculate sorghum artificially
were unsuccessful with both species. Because no seedlings of sugar
cane were available, no inoculation with either fungus was attempted.
Inoculations on sprouted sugar-cane cuttings were uniformly unsuccessful.
Since the size and shape of the conidia are the most useful criteria of
interspecies distinction, they are given in detail. Measurements of 700
conidia of each of the two species were combined into comparative tables
and graphs of frequency distribution in an attempt to present the dif-
ferences between them quantitatively as well as qualitatively.
Although morphologically the two species differ as has been described,
yet physiologically, in general effect in the field, in effect on the individual
plant, and in virulence to the same wide range of hosts no distinction
between them is apparent.
The discovery that two forms are involved complicates the problem
presented by the Philippine downy mildew of maize. Because two
forms morphologically different but practically indistinguishable in
physiologic effect are concerned in the same disease, there appears to
be a decided lack of that specialization which characterizes certain other
genera of the Peronosporaceae. It seems highly probable that still
other forms will be found to be concerned in similar diseases in the
Philippine Islands and throughout the Orient.
In addition to these two conidial species with a host range of maize,
teosinte, sorghum, sugar cane, Saccharum spontaneum, and Miscanthus
japonicus, the writer has encountered in the Philippines oogonial stages
of Sclerospora on Saccharum spontaneum, Saccharum ofjicinarum, and
M. japonicus. The oogonia on these three hosts are practically indis-
tinguishable. Whether these oogonial and conidial stages are quite
unrelated or are indeed only phases in the development of the same
organism remains to be determined.
Sclerospora spontanea, like 5. philippinensis, is closely related to the
other conspicuous conidial Sclerosporas of the Orient: 5. javanica Palm,
of Java; 5. maydis (Rac.) But., of India; and 5. sacchari T. Miyake, of
Formosa. All these forms are characterized by the predominance of
the conidial stage, the absence or great rarity of the oogonia, germina-
tion of the conidia by tubes, and the occurrence on maize, sugar cane,
and related hosts in the Orient. 5. spontanea, however, because of its
longer, more slender spores is as a species distinguished even more
sharply than 5. philippinensis from these other oriental representatives.
684 Journal of Agricultural Research voi.xx, No. 9
The discovery of Sclerospora spontanea on wild Saccharum spontaneum
is, in so far as the writer is aware, the first record of the occurrence of a
conidial Sclerospora on a wild host in the Orient. This occurrence, in
connection with other data, seems to the writer to indicate that the wild
grasses are the natural hosts of these oriental downy mildews from which
they have passed and are passing to susceptible introduced crops such
as maize.
LITERATURE CITED
(1) Butler, E. J.
1913. THE DOWNY MILDEW OF MAIZE (SCLEROSPORA MAYDIS (RAC.) BUTL.).
In Mem. Dept. Agr. India Bot. Ser., v. 5, no. 5, p. 275-280, pi. 8-9
(icol.).
(2) Davenport, C. B.
1904. STATISTICAL METHODS, WITH SPECIAL REFERENCE TO BIOLOGICAL VARIA-
TION. Ed. 2, rev. 223p.,diagrs. New York. Bibliography, p. 84-104.
(3) Davenport, Eugene.
[01907]. principles OF breeding. 727 p., illus. Boston, New York.
(4) Fawcett, Howard S.
191 7. preliminary note on the relation of temperature to the growth
OF certain parasitic fungi in cultures. In Johns Hopkins Univ.
Circ. 203 (n. s., 3), p. 193-194-
(5) Gaumann, Ernst.
1918. UBER DIE FORMEN DER PERONOSPORA PARASITICA (PERS.) FRIES. In
Beih. Bot. Centralbl., Bd.35, Abt. 1, Heft 3, p. 395-533, 47 fig- Zitierte
Literatur, p. 531-533.
(6)
1918. UBER DIE SPEZIALISATION DER PERONOSPORA AUF EINIGEN SCROPHULARIA-
CEEN. In Ann. My col., v. 16, no. 1/2, p. 189-199, 6 fig. Zitierte Litera-
tur, p. 199.
(7)
1918. UBER DIE SPEZIALISATION DER PERONOSPORA CALOTHECA DE BARY.
In Svensk Bot. Tidskr., Bd. 12, Hafte 4, p. 433-445, 2 fig. Literatur-
verzeichnis, p. 445.
(8) Ideta, Arata.
1914. handbuch der pflanzenkrankheiten japans. Ed. 4 enl., 936 p.,
illus., 24 pi. (8 col.). Tokyo. 1909-11. Text in Japanese; indexes
and bibliography (7 p.) in German, etc. Added title-pages in Japa-
nese, English, and French. A second Japanese t.-p., states that
this is ed. 6, 1914.
(9) Miyake, Tsutome.
191 1. ON A FUNGUS DISEASE OF SUGARCANE CAUSED BY NEW PARASITIC
fungus, sclerospora sacchari T. miy. In Rpt. Sugar Exp. Sta-
Govt. Formosa, Div. Path. Bui. 1, 61 p., 9 pi. In Japanese.
(10) Palm, Bj.
1918. onderzoekingen over de omo lijer van de mais. (With an English
summary.) In Meded. Lab. Plantenziekten [Batavia], no. 32, 78 p.,
8 pi.
(11) Rosenbaum.J.
1917. studies of the genus phytophthora. In Jour. Agr. Research, v. 8.
no. 7, p. 233-276, 13 fig., pi. 71-77. Literature cited, p. 273-276.
(12) Weston, William H., Jr.
1920. Philippine downy mildew OF maize. In Jour. Agr. Research, v. 19,
no. 3, p. 97-122, 3 fig., pi. A-B (col.), 16-25. Literature cited, p. 121-
25119°— 21 2
PLATE 76 »
Corner of a native-grown maize plot in the interior uplands of Cebu. At the edge
of this field, in which many maize plants were being killed by downy mildew, were
occasional clumps of the wild grass (Saccharum spontaneum L.) called "bugang" in
the Visayan Islands. One of these clumps, which was severely infected with Sclero-
spora spontanea, is shown at the left. The older, primary stalk of this clump, had died,
but although the remaining shoots were apparently uninjured, great numbers of
conidiophores were being produced on them, especially on the one held out for inspec-
tion. The base of this shoot was a few feet farther down the steep slope at the point
indicated by the arrow. Behind the central figure can be seen a maize plant notice-
ably discolored by the downy mildew.
1 Photographs by W. H. Weston.
Another Conidial Sclerospora of Philippine Maize
Plate 76
Journal of Agricultural Research
Vol. XX, No. 9
Another Conidial Sclerospora of Philippine Maize
Plate 77
Journal of Agricultural Research
Vol. XX, No. 9
PLATE 77
A. — Clump of Saccharum spontaneum, showing characteristic size and habit of
healthy plants under natural conditions. The measure is 2 meters tall.
B. — Clump of Saccharum spontaneum infected with Sclerospora spontanea. When
transplanted to this container in Cebu the infected plant comprised a single shoot
separated from the clump shown in the preceding plate. This shoot continued to
develop vigorously in spite of the downy mildew until after 5X months it had produced
the thriving clump shown. Conidiophores were still being produced in abundance,
especially by the younger stalks. Same measure as in A.
PLATE 78
A. — A young seedling (3 weeks old) of Saccharum spontaneum infected with
Sclerospora spontanea. On this seedling, which was artificially inoculated on the
second night after it emerged, conidium production began on the sixth night following
and recurred in increasing abundance on successive nights. In contrast to healthy
seedlings this plant betrays the effect of the Sclerospora in its pallor and in the presence
of a whitish " down" of conidiophores. These have collapsed on drying but can still
be seen on that part of the fourth leaf indicated by the pointer. X f£.
B. — Conidiophores on the leaf of Saccharum spontaneum. A portion of the upper
leaf surface of a downy-mildewed plant (PI. 77, B) showing remains of the whitish
"down" of innumerable conidiophores produced during the night. Although photo-
graphed as early as light would permit, the leaf surface has dried somewhat and the
fragile conidiophores have shrunk and matted together. X iK-
C. — Young shoots of Saccharum spontaneum arising after the primary stalk had been
cut, and like it severely infected with Sclerospora spontanea. The main plant, one of
the four downy-mildewed ones transplanted from Cebu, was cut off close to the ground.
All the subsequent shoots arising from the remaining base were, from the first leaf,
badly infected with Sclerospora and produced abundant conidiophores.
Another Conidial Sclerospora of Philippine Maize
Plate 78
Journal of Agricultural Research
Vol. XX, No. 9
Another Conidial Sclerospora of Philippine Maize
Plate 79
Journal of Agricultural Research
Vol. XX, No. 9
PLATE 79 »
A. — Typical conidiophore,2 showing characteristically long, slender, unknobbed
basal cell, relatively short main axis with its greatest diameter about midway to the
primary branches, and fairly well-developed branch system bearing long, slender
conidia. The number of conidia is somewhat less than that usually encountered.
From maize inoculated from Saccharum spontaneum. X 375-
B. — Upper portion of a conidiophore which has a poorly developed branch system
and hence bears few conidia on sterigmata which are relatively large. Several conidia
have been broken off in mounting. From maize. X 375-
C. — Portion of the branch system of a conidiophore, showing the conidia germinat-
ing while still attached to their sterigmata. From maize. X 375.
D. — Stalk portion of a typical conidiophore, showing long, slender, unknobbed
basal cell, and main axis which is slender above the septum, expands rapidly to its
greatest diameter about midway, and contracts again below the branches. From
Saccharum spontaneum. X 375-
E, F. — Typical basal cells of conidiophores. E from Saccharum spontaneum; F
from sugar cane. X 375-
G. — Stalk portion of a conidiophore with basal cell which, though unusually short,
nevertheless is longer than the extent of the main axis from septum to primary
branches. From Saccharum spontaneum. X 375.
H. — Typical stalk portion of a conidiophore from sugar cane. Compare with A
and D. X 375-
I, J, K» — Typical conidia showing variations in size and shape and method of ger-
mination by hyphae. I from maize, the lowest figure from material especially fixed
and stained to bring out the internal structure; J from Saccharum spontaneum; K
from sugar cane. X 375.
1 The drawings were made with the aid of a camera lueida. Figure A aad the ungerminated conidia of
figures I, J, and K are from fresh material. All the other drawings are from preserved specimens.
sIn comparing these drawings with the plates of Sclerospora phtlippinensis (12) it should be noted that
the latter give a somewhat misleading impression of the relative spreading of the branch system because
the conidiophores were flattened slightly in mounting.
ONION SMUDGE
By J. C. Walker
Assistant Professor of Plant Pathology, University of Wisconsin, and Pathologist, Office
of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant In-
dustry, United States Department of Agriculture1
INTRODUCTION
Smudge is a common disease of onions occurring both in the field and
in storage or transit. It is confined for the most part to the bulbs and
is characterized by dark green to black spots of variable size and shape
on the outer scales. The spots may be homogeneous in appearance or
may consist of numerous individual stromata scattered miscellaneously
or arranged in concentric rings. The disease is most common on the
white varieties of onions and damages materially the appearance and
market value of the crop. The causal fungus has heretofore generally
been known as Vermicularia circinans Berkeley, but as explained later
in this paper it should more properly be termed Colletotrichum circinans
(Berk.) Voglino.
The present investigations have been carried on with special reference
to the disease as it occurs in the districts of southeastern Wisconsin and
northeastern Illinois where onion sets are grown. The growing of white
onion "bottom sets" is an industry of considerable importance in these
sections, and the methods used in growing and handling the set crop are
often conducive to the excessive development of smudge during and
immediately following harvest. In this study attention has been given
primarily to the mycological and physiological aspects of the causal
organism, the relation of the parasite to the host tissue, the life history
of the fungus with relation to the production of disease, and the develop-
ment of remedial measures.
THE DISEASE
COMMON NAMES
A number of common names have been used in American and Eu-
ropean literature for this disease — namely, "onion Vermicularia" (j)2,
" Vermiculariose " (29), "black spot" (7, 50), "scab" (17, 21), "an-
thracnose" (7,36, 37, j^and "smudge" (26). The name " anthracnose "
1 This study was begun in the Department of Plant Pathology at the University of Wisconsin in 1914,
and the major portion was completed in 191 7. Since the writer entered the Office of Cotton, Truck, and
Forage Crop Disease Investigations in the latter year, observations have been extended to sections outside
of Wisconsin. Grateful acknowledgments are expressed to Dr. L. R. Jones, under whose immediate direc-
tion the work has been done, and to Drs. J. J. Davis and E. M. Gilbert, who have given valuable aid and
suggestions on the mycological phases of the problem.
2 Reference is made by number (italic) to " Literature cited, " p. 719-721.
Journal of Agricultural Research, Vol. XX, No. 9
Washington, D. C. Feb. 1, 1921
^r Key No. G-218
(685)
686 Journal of Agricultural Research voi.xx, N0.9
has been much used up to the present time. However, since the symp-
toms have little in common with those of the more common anthrac-
noses, and since it is believed that as simple and as descriptive a
name as possible should be chosen, the name "onion smudge" is used in
this paper to designate the disease, and this name is recommended
for general usage.
HOST PLANTS
White varieties of the onion (Allium cepa) are the chief ones affected
by smudge, but all varieties thoroughly tested have been found sus-
ceptible to at least a slight degree. The disease also occurs on shallots
(A . ascalonicum) and on leek (A . porrum) . It has never been found on
garlic (A. sativum).
HISTORY AND GEOGRAPHICAL DISTRIBUTION
Onion smudge was first described in 1851 by Berkeley (4) in England,
where it was found on the outer scales of a white variety. Subsequent
reports of its occurrence in Europe have been made by Massee (77) in
England, Bubak (8) in Bo1^ emia, and Voglino (35) and Allescher (1) in Italy.
The first collection of this disease in America, made by Michener, was
reported by Berkeley (5) in 1874. Since that time it has been re-
corded in literature as occurring in Rhode Island (3), Connecticut
(10, 19, 33), New York (20, 22), New Jersey (13, 25), Ohio (26), In-
diana (21, 34), Illinois (30), Wisconsin (23), and Alabama (2). Addi-
tional data furnished by the Plant Disease Survey show that it has
been present also in Massachusetts, Pennsylvania, Delaware, Mary-
land, Virginia, Georgia, Louisiana, Texas, Minnesota, and Iowa.
It is thus a disease of widespread occurrence; and, indeed, when one
considers the fact that thousands of bushels of infected "bottom "sets
are being shipped annually to all parts of the country and abroad, it is
reasonable to suppose that its distribution is even more general than
this summary indicates.
DESCRIPTION OP SMUDGE (PL. 80, 8l)
The disease is confined entirely to the scales and the lower portions of
the unthickened leaves which constitute the neck of the bulb. It first
becomes manifest upon the appearance of minute stromata which form
just beneath the cuticle of the host. These are dark green at first, be-
coming black with age. Depending on conditions of infection, the indi-
vidual stromata may be scattered miscellaneously over the surface of
the bulb, or, as is more commonly the case, they may be congregated in
smudgy spots around a few centers of infection. These spots are usually
roughly circular and variable in size. They often coalesce and occa-
sionally contain stromata arranged in concentric rings. Under moist
conditions the stromata bear acervuli which contain prominent setae
readily distinguished with a lens of low magnification. Cream-colored
spore masses frequently form on these fruiting bodies.
Feb. i, 192 1 Onion Smudge 687
Penetration of underlying dry scales by the fungus causes similar spots,
which are commonly surrounded by yellowish borders. On the fleshy
scales the disease first appears as minute, sunken, yellowish spots which
gradually enlarge and often coalesce. As the disease progresses, the
black stroma of the fungus usually appears; and, with the collapse of the
host cells, spots very similar to those on the dry outer scales result.
When the dark-colored stroma does not develop before the scale has
entirely dried down, the affected portions appear as slightly raised, yel-
lowish spots, giving to white onion sets an unnatural color which is
almost as detrimental to their market value as the black, smudgy spots.
The disease makes its appearance early in July under Wisconsin con-
ditions, the fungus living on the outer dead scales and increasing in
amount up to harvest time, when the outer two or three scales may be
affected. From this time on it penetrates farther into the bulbs, progress
depending upon environmental conditions. Badly diseased bulbs tend
to sprout prematurely in storage. In most severe cases the fungus pene-
trates the entire bulb and causes a complete collapse of the fleshy scales.
The foregoing description applies to the disease as it appears on white
onions. On colored varieties (red, yellow, and brown) the fungus is con-
fined, with rare exceptions, to the neck of the bulbs where there is
little or no pigment in the tissue, and the symptoms in these cases resem-
ble closely those on the corresponding parts of the white varieties.
On shallots the disease appears as smudgy spots very similar to those
on onion and is confined to the outer leaves or scales. On leeks similar
symptoms prevail.
OTHER DISEASES LIKELY TO BE CONFUSED WITH SMUDGE
Onion bulbs as they mature are subject to attack by a number of
fungi which develop saprophytically on the dead outer scales and pro-
duce symptoms which may easily be confused with those of smudge.
The most common of these are two species of Macrosporium (Macro-
sporium porri EH. and M. parasiticum Thum.) (33), and a species of
Phoma, probably Phoma alliicola Sacc. and Roum. (24). The Macro-
sporiums produce irregular, dark green spots which are due to ram-
ification of the mycelium through the dead scales, but which lack the
stromata and more or less regular outline of the smudge spot. In a
moist atmosphere the fungi fruit and develop a dark green mold due to
the production of conidia (PI. 81, F, G). In rare instances black peri-
thecia of M. parasiticum are found on the outer bulb scales. Phoma
produces small black pycnidia which are often difficult to distinguish
macroscopically from the stromata of the smudge fungus. It is com-
monly associated with M. porri (PI. 81, H). These two fungi commonly
attack both white and colored varieties, and in the latter case the pig-
ment in the outer scales is usually destroyed, giving a symptom which
is known in the trade as "onion blotch."
688 Journal of Agricultural Research voi.xx, No.9
Onion smut is sometimes confused with smudge, especially when the
former occurs on mature bulbs. In such instances, however, smut usu-
ally causes slightly raised, linear lesions which on colored varieties are
commonly accompanied by more or less destruction of pigment. The
exposure of the powdery spore mass upon breaking of the lesion estab-
lishes the identity of the smut fungus.
ECONOMIC IMPORTANCE
The importance of smudge as a detriment to the onion crop may
properly be considered from three standpoints — (i) that of reduction of
market value as a result of marred appearance, (2) that of actual shrink-
age of the bulbs in storage, due to fungus invasion, and (3) that of
increased sprouting of onion sets during storage. Thaxter (33) calls
attention to the reduction of market value caused by smudge, citing
an estimate by one grower of an actual loss of several thousand dol-
lars to his crop in one season on this account. There is little doubt
that marked spotting by this disease hampers greatly the disposal of white
onions, since they are usually grown at a greater expense than colored
varieties for a fancy trade which is prone to discriminate against dis-
figured stock. Under prolonged storage smudge causes a distinct shrink-
age of the bulbs and promotes premature sprouting. These last two
factors are not usually of material importance on large bulbs, but they
are of much significance with respect to onion sets. The latter are usu-
ally harvested in August and September and kept in storage until March.
The small bulbs are thus subjected to fungus invasion for several
months, and data presented later in this paper show that in badly dis-
eased sets the shrinkage may be doubled by smudge during this period.
Sets which sprout badly during storage are a total loss to the owner,
since they will not stand shipping and must be discarded. Much of the
sprouting of white sets in storage is due to severe attacks by smudge.
Experimental data in support of this statement are given later in this
paper.
It will be seen, therefore, that smudge is of greater importance than
would be suspected from casual observation. In the Chicago district
alone, where approximately 1 ,000,000 bushels of sets are grown annually,
the aggregate loss due to shrinkage in weight and sprouting probably
runs into many thousands of dollars.
CAUSAL ORGANISM
MORPHOLOGY
The morphology of the causal organism has previously been discussed
by Berkeley (4), Thaxter (33), Stoneman (32), Stevens and True (30),
and Kempton (16).
Mycelium. — The mycelium ranges from 2 to 8 microns in width, is
septate and branching, varying widely with age as to color and size. It
Feb. i, 1921
Onion Smudge
689
is at first hyaline with few septa, but later the walls thicken and take on a
dark green color, oil droplets become more numerous, and septation is
more frequent.
Stromata. — By close intertwining of the thick-walled mycelial threads,
dark green to black stromata, usually only a fraction of a millimeter in
diameter and few to several hundred microns thick, are formed beneath
the cuticle of the host (fig. 1). On nutrient media these stromata
commonly coalesce, forming a black stromateoid layer at the surface of
the substrate. This coalescence sometimes occurs on the host, but
more often the stromata remain distinct and are connected with one
another by threads of the dark-colored mycelium. During protracted
storage, or under poorly ventilated conditions, excessive stromatal
development may occur (Plate 83, B). Thaxter (33) describes large,
somewhat flattened sclerotia, "jet black externally and white within,"
Fig. i. — Conidia and appressoria of Colletotrichum, circinans. The f usoid conidia (C, D) germinate by one
ormore germ tubes, often becoming septate duringthe process(D). Dark-colored, thick-walled appres-
soria develop at the tip of the germ tubes, usually as the latter come in contact with the host cuticle (C,
D). Subsequent germination of appressoria commonly occurs (A , C). Terminal or intercalary appres-
soria-like cells, or chlamydospores, commonly develop within infected scales (B, E). Camera-lucida
sketch. X 750.
associated with the disease, though he does not definitely state
that they are connected with the causal organism. The writer has
never found bodies of this sort connected with the disease. On the other
hand, sclerotia of Botrytis spp., which cause decay of onion bulbs and
are commonly associated with smudge, compare favorably with his
description.
Appressoria or chlamydospores. — (Fig. 1). These bodies are vari-
able in size, dark brown in color, thick-walled, egg-shaped or roughly
circular, usually terminal but occasionally intercalary. In germination
drops on glass slides they form most abundantly where the germ tube
comes in contact with the slide and less commonly in the upper region
of the drop. Under such conditions they measure 6.5 to 8 microns by 4
to 5.5 microns. In Petri-dish cultures on various types of nutrient
agar they are almost invariably produced at the tips of hyphae which come
into contact with the glass surface. When "infection drops" containing
690
Journal of Agricultural Research
Vol. XX, No. 9
viable conidia are placed on the surface of onion bulbs, appressoria or
chlamydospores are formed in contact with the scale. Later they send
out germ tubes which penetrate the host. They are also commonly found
within the tissue of affected scales.
Acervuli. — The fruiting bodies are formed on the stromata which
develop beneath the cuticle of the host. Short, hyaline conidiophores
form in a palisade layer and rupture the cuticle of the host (fig. 2). One
to several acervuli form on a single stroma. In the study of the morphol-
ogy of the fruiting body the writer has found no evidence of a closed or
partially closed receptacle, as described originally by Berkeley (4). Its
true nature is more nearly in accord with the work of Stoneman (32),
who found not a pycnidium but an open fruiting body.
Fig. 2. — Acervulus of Collelotrichum circinans on artificially inoculated onion scale. Note the develop-
ment of the stroma in the subcuticular wall and the rupture of the cuticle by the formation of the
palisade layer of the sporiferous hyphae. Camera-lucida outline. X 265.
Setae. — Scattered throughout the acervulus are numerous setae
arising from the basal stroma. They are thick-walled, dark-colored,
o to 3 septate, upwardly attenuate, and 80 to 315 microns in length.
Conidia. — The conidia are borne acrogenously, being budded off one
at a time. They are fusiform, continuous, hyaline to slightly ochraceous,
somewhat curved, and obtuse at the very apex. Typically one prominent
vacuole is present in the center of the conidium, but under some conditions
the cytoplasm may contain many large vacuoles. As the spores are
budded off from the conidiophores they form a cream-colored, somewhat
mucilaginous mass on the top of the fruiting body. The spores vary
from 14 to 30 microns in length and from 3 to 6 microns in width. A large
majority, however, fall within the limits of 18 to 28 microns by 3 to 4
microns. They germinate usually by one, but occasionally by two or
Feb. i, i92 1 Onion Smudge 691
three germ tubes, which are pushed out at any point on the surface.
Septation of the spore commonly occurs during germination.
Perithecia^— Stevens and True (30) report the development of an
ascigerous form on onion sets heavily infected with Colletotrichum cir-
cinans and have referred the same to the new genus Cleistothecopsis.
The writer has never been able to prove C. circinans to be connected
with any ascigerous form found on onion. Stevens and True claim the
connection between the perithecia of Cleistothecopsis and C. (Volutella)
circinans on the following evidence:
(1) they occurred on sets badly infected with the Volutella; (2) no other fungi or
other types of mycelium were seen to be connected with them; (3) when studied in
various stages of development, the typical Volutella mycelium, which offers definite
characters for recognition, was seen in organic connection with them, as illustrated
in figure 18 (1), (4) the outgrowths from the perithecia are like those of the Volutella.
This evidence is hardly sufficient to prove that the two forms are stages
of the same fungus, especially since a large number of saprophytic or semi-
saprophytic forms very commonly occur on the dead outer scales of
onion bulbs and the differentiation of these from C. circinans on the basis
of the characters of the mycelium is sometimes very difficult. The writer
has, therefore, considered it advisable to use the binomial of the imper-
fect form until cultures from a single ascus or ascospore of the ascigerous
form are shown to be identical with C. circinans both as to morphological
characters and pathogenicity upon onion bulbs.
TAXONOMY
The taxonomic questions involved in this study concern first, the
proper position of the fungus in the present system of classification, and
second, the possible identity of the organism with other described species.
Berkeley (4) in the original description of the fungus refers to the
fruiting body as a perithecium and places it in the genus Vermicularia,
giving it the name Vermicularia circinans. Thaxter's (33) description
implies that the fungus has an open fruiting body, but he states that
in the early stages of its development a "sort of membrane" extends
over the basidia. Miss Stoneman (32) describes a thick basal stroma
bearing an open fruiting body. She also suggests that the characters
of the fungus resemble more closely those of the genera Colletotrichum
and Volutella than of Vermicularia. Voglino (33), believing the
fruiting body to be an acervulus, which would thus place the organism
in the order Melanconiales, transferred the species to the genus Colletotri-
chum. However, he gives no report of any study of the formation of
the fruiting body.
Stevens and True {30) in discussing the fungus describe a sporodochium
consisting —
of a pseudoparenchymatous inner tissue covered by a continuous surface layer. . . The
young sporodochium eventually ruptures its covering membrane.. .In all cases the
conidiophores are borne upon a raised superficial base which constitutes the sporodo-
692 Journal of Agricultural Research voi.xx, No. 9
chium, in contradistinction to the innate form of the acervnlus which has no such
base. The tubercular swelling, due to the massing of mycelium below and in the
epidermis, partakes of sporodochial character also, and while this subepidermal part
may not be regarded as constituting a true sporodochium it serves to emphasize the
tendency of the fungus to produce such structures. . .The structure is a tubercle with
a differentiated cortical outer layer. This outer layer ruptures and the tubercle
develops as a sporodochium.. .These facts exlcude the fungus from Vermicularia and
place it in the Tuberculariaceae under Volutella.
In the discussion later in this paper on the relation of the parasite to
the host it is shown that the development of the fungus commonly begins
in the outer wall of the epidermal layer of host cells. As the cellulose
becomes softened the hyphae multiply and a definite stroma forms with-
in this softened cell wall. Mycelium penetrates the epidermal and
underlying cells, and if humid conditions prevail the stroma will soon
occupy several layers of subepidermal cells. In good storage this process
is comparatively slow, but during a protracted period, especially if the
humidity rises considerably from time to time, the stroma commonly
does acquire a thickness of several hundred microns. An examination of
many sections has shown that regardless of the extent of its development
the stroma is always covered by the cuticle of the host. At the instant of
sporulation a palisade layer of hyaline hyphae interspersed with dark-
colored setae arises from the stroma, and in this process the cuticle is rup-
tured. This is shown to occur on stromata of widely different ages in
figure 1 and Plate 83, B. It is to be noted in the first illustration that the
stroma is of recent development, that it is confined to the outer wall of
the epidermal layer, and that the cuticle has been ruptured only by the
formation of the acervulus. In the second illustration, although the
stroma is much greater in extent, the host cuticle is still to be found in-
tact except where it has been ruptured by the two acervuli.
As pointed out by Saccardo (24, v. 3, p. 221-222, 233), certain species of
Vermicularia are characterized by imperfect or cup-shaped pycnidia, and
such forms approach the genus Colletotrichum. Obviously it is often dif-
ficult to determine the exact nature of the fruiting bodies, and as a result
many forms belonging in Colletotrichum have been placed in Vermicu-
laria. In the form under consideration there is no suggestion of pycnidial
development at any time during the development, of the fruiting body.
On the other hand, it does fall within the limits of the genus Colletotri-
chum. It is true that the basal stroma is much more highly developed
than in many of the better-known species of this genus. However, well-
developed stromata have been described in several species of this genus,
including Colletotrichum antirrhini by Stewart (31) and C. cereale by
Selby and Manns (27). In both cases the stroma develops beneath
the cuticle, which is ruptured only upon the formation of the acervulus.
It is quite possible that a critical study of the closely related species
classified at present in Vermicularia and Colletotrichum will lead to the
separation into another genus of those forms which develop acervuli above
Feb. i, 1921 Onion Smudge 693
thick basal stromata. This question, however, is not within the province
of the present paper. Those species of the Hyphales which are placed in
the family Tuberculariaceae are characterized by the grouping together
of the sporif erous hyphae in a superficial, conglutinate, sessile, or stipitate
mass, known as a sporodochium (24, v. 4, p. 635, 682). As already pointed
out, Stevens and True (jo) considered the fruiting body of the onion
smudge organism to be of this nature and on that basis have transferred
it to Volutella. In their description and figures, however, they seem to
have interpreted the host cuticle as part of the so-called tubercle and
thus as being of fungus origin. Were this true, the stroma would be super-
ficial, and the fungus would properly belong to the genus Volutella.
However, since the stroma is always subcuticular and the sporiferous
hyphae are subcuticular in origin, the form is more characteristic of Colle-
totrichum than of Volutella. Here again it is obvious that these two
genera need more critical study before their limits can be satisfactorily
denned. Meanwhile in the light of evidence just given, the writer con-
siders it more suitable to use the name Colletotrichum circinans (Berk.)
Voglino for the onion smudge organism.
The comparison of Colletotrichum circinans with other related species
has been very limited in this investigation. The list of species of this
genus which coincide closely with the one in question as to spore meas-
urements and general characters is large and extends over a wide host
range. Obviously the comparison of herbarium specimens is insufficient
basis for final conclusions under the circumstances. Critical comparison
has been confined to C. jructus (S. and H.) Sacc, described as causing a
fruit rot of apple. This species was originally described as a species of
Volutella (28), but it was later transferred to Colletotrichum by Saccardo
{24, v. 13, p. 1201)—
on account of the black setae and the acervulus being originally subcuticular.
Cross sections of apple fruits affected with C. jructus and with C. circinans
are compared in Plate 83, C, D. In both cases the development of the
stroma beneath the cuticle, which is ruptured only upon the formation
of the acervuli, is clearly shown. The former species was chosen for com-
parative study because the spore measurements and general characters
as previously described were closely similar to those of the onion smudge
organism and authentic cultures were available.
Cultures of the apple organism or diseased fruits were secured from
Prof. C. R. Orton, State College, Pa., Dr. L. R. Hesler, Ithaca, N. Y., Dr.
Charles Brooks, Washington, D. C, and Mr. G. A. Meckstroth, Columbus,
Ohio. Cross inoculation on apple and onion showed that Colletotrichum
circinans was able to produce a rot of apple fruit similar to that produced
by C. jructus (see Pi. 84, C). The formation of stromata and acervuli by
both species on apple is shown in Plate 83, C, D. The rate at which the
rot progressed, however, was uniformly slower in C. circinans. On onion,
694
Journal of Agricultural Research
Vol. XX, No. 9
C. fructus developed on the dead outer scale of the bulb, but no evidence
of further invasion as occurs with C. circinans was observed. Thus, the
two species are distinct as to pathogenicity.
Measurement of many hundreds of spores of several strains of both
species produced on several substrates including the natural ones —
namely, apple and onion — showed that the variations due to differences
between strains and substrates along with differences due possibly to
slight changes in environmental conditions precluded any distinction on
this basis. The slight difference in the shape of spores shown in figure 3
was quite uniform. The spores of Colletotrichum fructus have walls
nearly parallel throughout the middle half, and one end narrows much
more abruptly than the other.
A comparison of growth on potato agar gave further evidence as to
the distinction of the two species. The chief points of difference in
development on this medium are as follows: (1) Colletotrichum fructus
PlG. 3 —spores of Colletotrichum fructus (A) and C. circinans (£). Note the slight difference in shape.
In longitudinal section the walls of C. fructus are the more nearly parallel throughout the middle
half, while at one end they converge more abruptly. Camera-lucida sketch. X 750.
grows the more rapidly, (2) appressoria at the tips of hyphae coming in
contact with the glass surface in plate cultures are absent in C. fructus,
(3) the method of branching is quite distinct — that of C. circinans is
dichotomous while that of C. fructus tends to be monopodial in that
nearly straight threads of mycelium, which become dark-colored very
early and are greater in diameter, run out radially from the center of
the colony and send out hyaline side branches of less diameter. Stromata
develop at various points from these radial hyphae. This mode of
growth gives a somewhat stellate macroscopic appearance to the colony,
which differs from that of C. circinans, where distinctly radial hyphae
are absent and stromata are scattered. This macroscopic difference is
shown in Plate 84.
Thus, although the morphological characters are only slightly variant,
the two forms are considered distinct (1) because of difference in patho-
genicity, (2) because of difference in spore shape, and (3) because of
difference in type of colony on potato agar.
Feb. i, 1921 Onion Smudge 695
physiology
ISOLATION OF THE FUNGUS
Pure cultures of the causal organism are readily obtained by the
ordinary spore-dilution method. On potato-dextrose agar colonies
appear in three to five days. Single spore strains were isolated from
such cultures by means of the method described by Keitt (13). Isola-
tions thus made from many lots of diseased material collected in Wis-
consin, Illinois, Ohio, Connecticut, and Louisiana have yielded strains
which are closely similar in their behavior.
CULTURAL CHARACTERS
On potato agar (2 per cent dextrose) plates. — (See PI. 84,D, E.)
The conidium germinates within 6 to 8 hours, sending out one to three
hyaline germ tubes, which within 24 hours are many times the length
of the spore. Colonies become macroscopic in about 2 days. The
mycelium becomes somewhat thicker and denser in the center of the
colony, while the younger hyphae around the outer edge are thin-walled
and hyaline. Those branches of mycelium which come in contact with
glass plates usually produce dark-colored, thick-walled chlamydospores
or appressoria. Within 2 or 3 days stromata begin to form by abundant
branching from a definite point in the mycelium, which finally results
in a thick mass of hyphae. These hyphae assume an olivaceous color,
and by the fourth day the dark green stromata are macroscopic in size.
They form first at the center and later throughout the colony except at
the extreme outer edge. Occasionally they are arranged in such a
manner as to give the appearance of "fairy rings," but this is not a
constant characteristic. The appressoria and the stromata give the
young colony an olivaceous appearance. It becomes darker and almost
black with age as the stromata become denser and more numerous and
finally form an almost homogeneous stromateoid layer at the surface
of the substrate.
By the second day the colony shows a small amount of white aerial
mycelium. This increases somewhat with age and later takes on a
smoky gray appearance, masking the stromateoid layer to a certain
extent. In from three to five days fruiting bodies are formed on the
stromata at the center of the colony, and they continue to develop as
the colony grows. Conidia are produced in abundance in most strains,
accumulating in cream-colored or pinkish masses on the fruiting bodies.
The colony will continue to grow to an indefinite size if space and
nutrients are available. A diameter of about 25 mm. is reached in
seven days at room temperatures.
On potato agar (2 per cent dextrose) slants. — Growth is similar
in most respects to that on plates. Aerial mycelium tends to be more
abundant. Mycelium does not, as a rule, extend deeply into the agar to
form stromata. As the culture dries out the aerial mycelium forms a
25119°— 21 3
696 Journal of Agricultural Research voi.xx, No. 9
dense mat over the surface of the culture, its color usually becoming
slightly brownish with age. Spore masses often appear above this layer
of mycelium.
On other media. — The growth of the fungus was studied on 25
kinds of artificial media, including beef broth agar, corn meal agar, oat
agar, apple agar, synthetic agars, vegetable agars, cooked vegetables, and
fresh vegetable tissues. The character of growth on the various media
used was so uniform and so closely parallel to that on potato agar that a
separate description for each is unnecessary. The most noticeable dif-
ference was that correlated with the supply of sugar in the medium.
Where dextrose was omitted in the formula growth and sporulation were
very scanty, and the stromata were few in number and widely scattered.
On onion and apple agars made up without dextrose this difference was
less marked, probably on account of the presence of a considerable
amount of sugar in the plant tissues used. On synthetic agars * with
sugar added in the form of maltose, dextrose, lactose, and sucrose
copious growth took place with no evidence of preference for any one of the
carbohydrates used. Cooked bean pod, onion scale, carrot, potato, and
rice supported good development of the organism. On fresh onion and
apple, however, the growth was much retarded, and on fresh potato
and carrot it was very scanty. Stevens and True (30) report retarded
growth on onion broth agar made with red or yellow varieties. The
writer has found equally vigorous development on agar made from red,
yellow, and white types of onion.
RELATION OF TEMPERATURE TO GROWTH
Potato agar plates inoculated with mycelium or conidia of the fungus
were kept at temperatures ranging from i° to 35 ° C. The rate of growth
was determined by measuring the diameter of the resulting colonies or
thalli from day to day. In order to increase the accuracy of the results
Petri dishes of equal diameter containing equal amounts of agar were
used. In order to overcome the influence of variations in relative
humidity prevailing in different incubators the later experiments were
modified by placing the Petri dishes in moist chambers first and then
exposing them to the desired temperature. It was found after many
trials that the best comparative data could be secured at four to six
days. The growth was slight at i°, almost negligible at 20, but an
appreciable amount occurred at 8° to io° during a period of 10 to 14
days. Above this point the rate of growth increased rapidly, reaching
the optimum at about 260. At 310 to 320 little or no growth occurred
on potato agar. The growth at various temperatures on this medium
at the end of 6 days is represented graphically in figure 4.
1 Formula for synthetic agar used: Sugar, ioo gm.; peptone, 20 gm.; ammonium nitrate, 10 gm.; mag-
nesium sulphate, 2.5 gm.; potassium nitrate. 5 gm.; acid potassium phosphate, 2.3 gm.; calcium chlorid,
0.1 gm.; agar, 20 gm.; neutralized with normal sodium hydroxid.
Feb. i, 1921
Onion Smudge
697
A similar study of growth in tubes of onion decoction was made, with
essentially parallel results. The optimum on this medium appeared to
be slightly higher (270 to 290 C.) and slight growth occurred at 310.
SPORE GERMINATION
Relation OF medium. — For the studies upon spore germination a
few drops of the liquid medium to be used were placed in Van Tieghem
cells. A suspension of conidia in the same liquid was made, and a drop
of this was transferred to cover glasses, which were then inverted over
the cells and partially sealed with
S»
si
Vivo
s
Fig.
vaseline. The preparations were
placed in Petri dishes and exposed
to the desired conditions. For
some purposes open drops on glass
slides placed in Petri dishes lined
with moistened filter paper were
more suitable.
A comparative study of spore
germination in distilled water,
onion decoction,1 onion leaf ex-
tract,2 onion scale extract,3 soil extract (sterilized and unsterilized) ,4
and soil decoction 5 was made.
At room temperature germination in favorable liquid medium began
within 5 to 6 hours. At 24 hours practically all viable spores had germi-
nated. The percentage of germination in the drops was determined by
averaging the counts of several microscopic fields. The results of these
tests are summarized in Table I.
Table I. — Effect oj various media upon spore germination of Colletotrickum circinans
£ /O /S SO 3S OO
4. — Relation of temperature to growth of
Colletotrickum circinans on agar plates.
Medium.
Distilled water
Soil decoction
Soil extract, sterilized
Soil extract, unsterilized
Onion decoction
Onion leaf extract
Onion leaf extract, diluted with distilled water 1 to 10.
Onion scale extract
Onion scale extract, diluted with distilled water 1 to 10
Percent-
age of ger-
mination.
60
95
95
10
99
1 Onion decoction: ioo gm. onion scale in 500 cc. distilled water steamed one hour, filtered, and sterilized.
2 Onion leaf extract: Fresh onion leaves (green) crushed and the sap extracted by squeezing through
cheesecloth.
1 Onion scale extract: Fresh onion scale crushed and the sap extracted as in onion leaf extract.
4 Soil extract: 500 gm. black loam soil was supported in a glass funnel by excelsior and absorbent cotton:
500 cc. of tap water were poured over the soil; the filtrate was collected twice, and each time it was poured
over the soil. The third filtrate was divided into two parts; one part was left unsterilized and the other
part was sterilized in tubes at 15 pounds pressure for ]4 hour.
5 Soil decoction: soo gm. of black loam soil, to which had been added 500 cc. of distilled water, was steamed
at is pounds pressure for 14 hour. The liquid was filtered through filter paper and sterilized in tubes at
i; pounds pressure for yi hour.
698
Journal of Agricultural Research
Vol. XX, No. 9
The striking outcome of this comparison is the marked retardation in
unsterilized soil extract and the complete inhibition in. onion leaf and
onion scale extract. Even when the last two were diluted with 10 parts
of water no germination occurred. As pointed out in a previous note
by the writer (38), further experiments have shown the presence of at
least two distinct substances in onion tissue which are probably respon-
sible for inhibition of spore germination. A more detailed study of this
phase and its relation to the parasitism of the fungus will be included in
another paper. Cooked soil extract, soil decoction, and onion decoction
stimulate germination and promote rapid growth of the germ tubes.
It is evident that the cooking of the onion scale removes or destroys the
substances which are unfavorable for spore germination.
Relation of temperature. — Since conidia were found to germinate
well in distilled water, this medium was used for studies of the effect of
temperature on spore germination. A large number of tests were run at
a gradation of temperatures ranging from i° to 350 C. Spores were
found to germinate between the
limits of 40 and 320. Appressoria
developed in germination drops
throughout the same range of tem-
perature. At 350 to 370 slight swell-
ing of the spores took place, giving
them the appearance of ' ' involution
forms," but normal germination
did not occur. Figure 5 is a
graphic representation of the effect
of temperature as indicated by per-
centage of conidia germinating in
distilled water at 12 hours. Best germination occurred at about 200, but
good germination occurred between 130 and 250.
The temperature range for spore germination thus coincides closely
with that of fungous growth. The point of optimum development is
comparatively high, and this fact is significant in exolaining the occur-
rence of the disease in the field.
r
|
V
Fig.
so ss 30
— Relation of temperature to spore germina-
tion of Colletotrichum circinans.
EFFECT OF DESICCATION
In order to interpret more fully the development of the disease in the
field and the overwintering of the causal organism, the effect of desicca-
tion on conidia and stromata was studied in the laboratory.
On conidia. — Studies were made on conidia as they occur (1) in
masses on the fruiting body on the host, where they are embedded in
the mucilaginous material which surrounds them, (2) in similar masses
on potato agar, and (3) in water suspension, where the spores are sepa-
rated from one another, approximating to some extent conditions as
Feb. i, 192 1 Onion Smudge 699
they occur in nature when spores are disseminated by meteoric water.
Diseased onions bearing spore masses were brought in and allowed to
dry out gradually in the laboratory, and the viability of the spores was
tested from time to time. Ordinarily a large percentage lost their
vitality within 2 weeks, but in some cases good germination occurred
after 7 weeks. A small percentage of conidia from spore masses pro-
duced on potato agar and exposed to similar conditions germinated after
4 months. Spores in water suspension allowed to dry out on glass
slides were very sensitive to desiccation, little or no germination occurring
after 24 hours. It is evident, then, that the conidia are sensitive to
desiccation except when they remain in waxy masses on the host, in
which condition a small percentage will remain viable through extended
unfavorable periods. These results are in accord with the findings of
Hasselbring (14) for the somewhat closely related fungus Gloeosporium
fructigenum, causing the bitter-rot of apple.
On strom ata. — The stromata of the fungus are capable of withstand-
ing very long periods of desiccation. Test tube cultures of the fungus
on a large number of media were kept at room temperature for a period
of two years. Since the tubes were not plugged very tightly with cotton
the cultures dried out completely within four or five months. The
vitality of the fungus in this desiccated condition was tested by adding
sterile melted potato agar to the tube and slanting them until the fresh
medium hardened. Vigorous growth characteristic of the fungus re-
sulted from the cultures originally made on potato, beef broth, carrot,
corn meal, oatmeal, and onion agars, steamed rice and bean pods, and
fresh potato and onion plugs. The fungus was no longer viable on
synthetic agar, steamed potato, carrot, onion, and fresh carrot. Since
spores lose their vitality in such a long period of drying, it may be in-
ferred that the fungus lived through this extended period of desiccation
by means of the stromata which developed in the substrate. It is to be
expected from these results that the stromata which develop in the scales
of the host are capable of carrying the fungus over long periods of un-
favorable climatic conditions.
EFFECT OF FREEZING
On conidia. — Spores in water suspension exposed to freezing tem-
peratures are killed within a few hours. Fresh spore masses also are
very sensitive to low temperatures, but if they are allowed to dry out
before being exposed to freezing temperatures they will withstand such
temperatures for a month or more. In order to test the resistance of
conidia to the freezing weather of the entire winter period, infected
onion bulbs bearing spore masses were placed out of doors in a weather
instrument shelter at Madison, Wis., on December 7, 191 5. Germina-
tion tests showed a high percentage of these conidia to be viable at this
yoo Journal of Agricultural Research vol. xx,No.9
time. Tests made on January 22, 191 6, showed that by this date all
the spores had been killed. A similar experiment was carried out at
Madison in the winter of 1919-20. Infected bulbs bearing abundance
of spore masses were placed out of doors in October, 191 9, and protected
from rain and snow. A few viable spores were obtained on March 20,
1920. Thus, a few conidia may withstand Wisconsin winters if suffi-
ciently protected, but probably few, if any, live over under field condi-
tions.
On sTromata. — Agar cultures containing abundant stromateoid de-
velopment were kept out of doors during the winter months at Madison,
Wis., during which period there was much severely cold weather. In
all cases the cultures were found to be viable at the end of this time.
Stromata on onion scales have also been exposed in this region during
the winter period, and in every case they withstood the severe freezing
temperatures.
It is to be expected from the foregoing data that spore masses with-
stand short intervals of dry weather during the summer and furnish
ready inoculum upon the return of moist conditions. During extended
periods of unfavorable conditions, however, the stromata serve best to
perpetuate the fungus.
PATHOGENICITY
Inoculation experiments were performed on plants at various stages
of growth from young seedlings to mature bulbs.
Sterilized greenhouse loam soil was inoculated by spraying with a
water suspension of spores at the time of sowing onion seed. Three
hundred seeds of White Globe variety were planted in the inoculated
soil and the same number in uninoculated soil. Ten days later, as the
cotyledons were coming through the soil, the attack of the fungus became
evident by the rapid collapse of the succulent tissue at any point on the
young shoot. Acervuli of the fungus were present and continued to
develop on the diseased portions of the plants. Fifteen days after
sewing, 64 out of 123 plants in the inoculated pot were diseased, whereas
all of the 161 plants in the control pot were healthy. This experiment
was repeated several times, and in each case where sterilized soil was
inoculated a high percentage of the seedlings were killed. When un-
sterilized greenhouse soil was used the injury was greatly reduced,
the competition of other soil organisms evidently greatly limiting the
activity of the smudge fungus. Moreover, damping off of this sort due
to smudge has never been noted in old onion set fields, other factors,
such as low temperature at this early part of the season, probably limit-
ing the activity of the fungus.
Leaves of half -grown plants were sprayed with a spore suspension and
kept in a moist chamber for 24 to 48 hours. The fungus developed and
fruited on the lower leaves, which had reached a stage of "physiological
old age," but this never occurred on vigorously growing leaves.
Feb. i, 1921
Onion Smudge
701
The disease was produced many times by means of artificial inoculation
of healthy mature onion bulbs with suspensions of spores from pure
cultures, and the fungus was readily reisolated. A summary of these
inoculations is given in Table II. In certain cases when bulbs kept in a
closed chamber were thus inoculated, the experiment was unsuccessful.
It was found in such instances that although the spores were capable of
germination in water, they did not germinate in the drops on the bulbs.
The inhibitive effect of the volatile oil of onion on spore germination
was mentioned eailier by the writer (38). An accumulation cf this
substance when several onion bulbs are placed in the small space in a
moist chamber may possibly account for this lack of germination.
Further studies on this point will be described in a later paper.
More nearly uniform results were secured when sterilized soil was inocu-
lated by spraying with a spore suspension and healthy bulbs then inserted
in this medium for a week or 10 days. The outer scales usually became
uniformly infected in 7 or 8 days (see Pi. 81, C). When the bulbs were
removed and placed in storage, typical invasion of the underlying scales
occurred.
Table II. — Summary of inoculation and greenhouse experiments on onion bulbs
Type of inoculation.
Date of in-
oculation.
In soil
Dec. 3
Nov. 30
Dec. 16
Method of
inoculation.
Sprav
.do
.do
.do
.do
.do
.do
.do
.do
.do
Inoculated.
Num-
ber of
onions
used.
Percent-
age in-
fected.
IOO
IOO
IOO
80
o
IOO
IOO
IOO
IOO
IOO
IOO
IOO
Num-
ber of
days
before
first
note of
disease
Controls.
Num-
ber of
onions
used.
Per-
cent-
age in-
fected
In general, then, the fungus assumes the role of a weak parasite.
Actively growing portions of the plant are not attacked except in young
seedlings grown under certain conditions. In the field the fungus is
confined to the outer leaves or scales, the cells of which are dead or essen-
tially functionless. As the plant approaches maturity the dry outer
scales of the bulb are invaded, but the normal fleshy scales are not
affected at this time. A few cases have been noted where the fungus
702
Journal of Agricultural Research
Vol. XX, No. 9
attacked growing scales which were being parasitized by the smut
fungus, Urocystis cepulae, but apparently a weakening of the plant
is necessary before actual invasion of the growing parts occurs. Fol-
lowing harvest there is a gradual invasion of the dormant cells of the
fleshy scales of the bulb as previously described. The progress here
is usually slow, but in a moist, warm environment there may be a more
rapid invasion, resulting in decay of the resting central bud of the onion
set.
RELATION OF THE CAUSAL ORGANISM TO THE HOST TISSUE
METHODS
Onion bulbs from which the thin outer scales had been removed
were placed in moist chambers. Inoculum consisting of a suspension
of spores from pure culture in sterile distilled water was applied to the
uninjured surface of the exposed scales, either in drops by means of a
platinum loop or as a spray from an atomizer.
For the stud)' of penetration a razor section was cut tangentially
from the surface of the scale directly beneath the infection drop so as
Fig. 6. — Colletotrichum circinans: Stage of penetration of epidermal cell of onion scale at 66 hours after
inoculation. Camera-lucida sketch. Approximately X 430.
to contain the epidermis with a few layers of the immediately underlying
cells. This was examined directly in toto in a water mount, the absence
of chlorophyll in the host cells making clearing and staining unneces-
sary. For the study of the relation of the fungus to the host tissue
following penetration, pieces of inoculated scale as well as of naturally
infected fleshy scales were fixed in Fleming's medium fixative, washed,
dehydrated, embedded in paraffin, and sectioned according to standard
methods of procedure. In some material a satisfactory differentiation
of fungus and host was secured by omitting the bleaching of the micro-
tome sections (commonly done after using a fixative containing osmic
acid), which left the mycelium black, and then counterstaining the
host cell walls with orange G. In other cases the iron haematoxylin
and Delafield's haematoxylin stains gave satisfactory results.
PENETRATION
Under optimum conditions germination occurs within 10 hours and
appressoria are formed, either sessile or at the end of short germ tubes.
Usually the appressorium is flattened to some extent on the side adja-
Feb. i, 192 1
Onion Smudge
703
cent to the cuticle. The penetration tube is formed from the flattened
side of the appressorium and penetrates the cuticle directly (fig. 6, 7).
Blackman and Welsford (<5) have pointed out that solution of the host
cuticle by invading fungi has never been fully demonstrated; they
explain the invasion of bean leaf cuticle by Botrytis cinerea as mechani-
cal in nature. The mode of penetration in onion smudge was not
definitely ascertained, but it seems highly probable that the germ tube
from the adhering appressorium might pierce the thin cuticle by means
of mechanical pressure.
SUBSEQUENT DEVELOPMENT
The fungus hyphae, after penetration, develop first between the sub-
epidermal wall and the cuticle, which is rather elastic in nature and
can be raised considerably without being
ruptured. Figure 6 illustrates the extent of
invading germ tubes at 66 hours after inocu-
lation. The nature of the penetration tube
and the subsequent development beneath the
cuticle are shown in figure 7. In certain
other anthracnose fungi — namely, Colletotri-
chum lagenarium as reported by Gardner (12),
C. lindemuthianum by Dey (11), and Gloe-
osporium fructigcnum by Hasselbring (14) —
the penetration tube has been described as
invading the cell wall directly. This is also
the case in Botrytis cinerea on bean (6), al-
though the germ tube in this instance does
sometimes grow horizontally beneath the
cuticle. The softening of the subcuticular
wall in the case of onion smudge soon be-
comes apparent by its swelling and taking on a laminate appearance.
The hyphae grow through and between the laminae (fig. 8) and by rapid
development soon form the beginning of the stroma previously described.
The swelling of the outer wall eventually involves the entire lumen of
the epidermal cell . Although the greatest amount of fungus growth at
this stage takes place just beneath the cuticle, occasional hyphae pene-
trate underlying cells. As the hyphae attack these cell walls, softening
and lamination take place as in the subcuticular wall, while penetration
is seemingly accomplished partly by means of chemical action and
partly by mechanical pressure. The relation of mycelium to the
parenchyma cells just beneath the epidermal layer is also shown in
figure 8. In the case of bulbs inoculated in moist chambers the collapse
of invaded cells was not rapid, and there was no evidence noted of
injury to the cells in advance of the mycelium.
Fig. 7. — Cross section of epidermis,
showing early stage of penetration
by Colletotrichum circinans. Note
the empty appressoria with myce-
lium still wedged between the cuti-
cle and the subcuticular wall.
Material fixed 72 hours after inocu-
lation. Camera-lucida sketch.
X 700-
704 Journal of Agricultural Research voi.xx, N0.9
Under ordinary storage conditions, the progress of the fungus is
closely parallel to that just described, except that the progress is much
slower under this different environment. As described before, the first
macroscopic symptom of invasion from spots on the dry outer scale to
the underlying fleshy scale is a small, yellowish, slightly sunken area.
This usually increases in size very slowly in well-ventilated storage. A
cross section of one of these spots is illustrated in Plate 83, A, and a de-
tailed drawing from a similar section is shown in figure 9. The fungus
develops extensively at first just beneath the cuticle, and the softening
and lamination of the subcuticular wall is very slight. As invasion pro-
gresses, hyphae penetrate this wall directly, evidently by chemical solu-
tion rather than mechanical pressure, since the cavity is slightly larger
than the mycelium and there is no sign of bulging of the wall before
penetration is achieved. The collapse of cells beneath the epidermal
Fig. 8. — Cross section of epidermis (A ) and underlying parenchyma cells (S) of onion scale inoculated with
a suspension of Collelotrichum circinans spores and kept in a moist chamber at room temperature.
Note softening and lamination of cell walls by the invading hyphae. Material fixed five days after
inoculation. Camera-lucida sketch. A, X 308; B, X 350.
cell takes place before any appreciable invasion of hyphae occurs. In
the section shown in Plate 83, A, two layers beneath the epidermal layer
have collapsed, while only an occasional hypha is to be found beneath
the subcuticular wall. There is no evidence of softening of the cell wall.
Morever, in such lesions mycelium has never been found in the walls or
lumina of turgid living cells. This suggests that either the cells are
killed in advance of the hyphae or only slight invasion of the wall leads
to their collapse. This slow invasion, which prevails even after the cells
have become functionless, is surprising in view of what occurs when bulbs
are inoculated in moist chambers. Is it possible that the volatile oil
present in the onion scale is influential in checking the advance of the
fungus ?
Under moist conditions and optimum temperature the stroma develops
very rapidly in the subcuticular wall, and acervuli are formed in five to
Feb. i, 1921 Onion Smudge 705
six days after inoculation. This condition is shown in figure 2. In other
cases where sporulation is postponed through lack of proper environ-
ment the stroma continues its growth more slowly and eventually in-
volves a larger portion of the scale. The cuticle, however, remains
intact on the exterior and normally is not ruptured until the palisade
layer of conidiophores is formed. A cross section of a scale which had
been held in poorly ventilated storage several months is shown in Plate
83, B. Acervuli were produced upon exposure to proper conditions for
sporulation. Note that the cuticle is still present outside the extensive
stroma, except where it has been ruptured by the sporiferous hyphae.
FACTORS IN THE PRODUCTION AND PROGRESS OF THE DISEASE
OVERWINTERING OF THE CAUSAL ORGANISM
The experiments already reported on the effect of desiccation and
freezing upon conidia indicate only a remote possibility that the fungus
lives through the winter in this form under Wisconsin conditions. The
stromata, on the other hand, are capable of withstanding protracted
periods of drouth or freezing temperature. In order to confirm the
supposition that the fun-
gus actually overwinters W^ ^ — ^UST^^
and is widely disseminated ^Mfc^^^ggg^PWWBI^^
in this latter form, four lots * ■■■— id
rt -i • r -11 11 Fig. 9. — Cross section of onion scale naturally infected with
of heavily infected bulbs ^ ,, , t ■ u u n. r a 1 •
J Collelotrichum ctrcmans, showing the mycelium developing
Were placed OUt Of doors at first just beneath the cuticle and later penetrating the sub-
Madison, Wis., On Decem- cu^arwa»( Camera^lucida sketch. X 450. (Thisphase
' ' is illustrated further in PI. 83, A).
ber 7, 191 5.. One lot was
left in an instrument shelter near the surface of the ground, and the
remaining lots were buried in the soil at depths of 2, 4, and 6 inches,
respectively. Spore masses were present on this material at the begin-
ning of the experiment, and germination tests showed a high percentage
of the conidia to be viable at this time.
On January 22, 191 6, examination of spores from the bulbs placed
in the instrument shelter showed that they had completely lost viability
by that date. The four lots of bulbs were examined on April 12, 191 6.
Those which had been buried in soil readily produced conidia in abun-
dance upon exposure to humid conditions at room temperature. The
material kept in the instrument shelter had dried out considerably
during the winter and, though much slower to respond, eventually
proved to be viable by the production of spores. A similar experiment
conducted during the winter of 191 6-1 7 yielded confirmatory data.
It is to be expected that infected scales from the crop of the previous
season furnish a source of abundant inoculum for initial infection of
the growing crop. This, combined with the fact that in most onion-
growing sections it is the common practice to grow this crop successively
706 Journal of Agricultural Research voi.xx. No. 9
on the same field for many years, results in a heavy infection of a large
part of the white set crop annually. Examination of a large number of
fields in Wisconsin and Illinois has revealed the fact that "clean" white
sets are secured as a rule only from land growing its first crop of onions.
In a majority of cases the second crop of white sets is badly infected.
In all fields examined where the first crop of onions was being grown,
an occasional bulb infected with smudge was found. A satisfactory
explanation of these original infections has never been reached. Many
possible means of introduction of the fungus from neighboring infected
fields immediately suggest themselves, such as manure, farm imple-
ments, man and farm animals, drainage water, and wind, and undoubtedly
some of these often do play a part in the distribution of the disease.
The possibility of seed as a carrier is also to be considered in this con-
nection. Although smudge has never been found attacking the floral
parts of the plant, it is conceivable that those seed umbels which fall over
and come in contact with the soil before harvest might become infected
or be the means of introducing bits of infected scales to the seed. It
should be noted in this regard that the spores of onion smut, a disease
which is also confined to the bulb and leaves of the plant and in fact does
not attack onion seed plants, have previously been found on onion seed
samples (9, 18).
One experiment was performed on the relation of seed to the dis-
semination of the fungus. Samples of six varieties of seed were sown
in pots of sterilized soil in the greenhouse on December 5, 191 6. On
January 16, 191 7, all the seedlings were examined. Fruiting bodies of
Colletotrichum circinans were found on the outer scales of two seedlings
of the White Globe variety and of one seedling of the Queen variety.
No other signs of the disease were found. The identity of the fungus
was confirmed by isolation of pure cultures and comparison with authentic
strains. Two subsequent plantings of the same sample of White Globe
seed were made, but no further sign of the disease was found. The small
amount of the fungus occurring in this experiment is not surprising,
since only a very limited amount of infectious material can be expected
to be seed-borne. However, although the evidence at hand indicates
that the fungus is carried on seed to some extent, further data are
necessary before a final conclusion on this point can be made.
RELATION OF TEMPERATURE TO INFECTION AND TO DEVELOPMENT OF
THE DISEASE
Studies of the relation of temperature to the germination of conidia
and to their subsequent growth have shown the optimum to be about
200 C. for the former and 260 for the latter. The range in each case,
however, is wide. Accordingly a set of experiments was started for the
purpose of determining the range and optimum temperature for infection.
Sterilized loam soil in glass or glazed crock jars was inoculated with
a water suspension of spores. Healthy white onion sets were then
Feb. i, 1921
Onion Smudge 707
inserted in the soil; and the jars, each covered with a glass plate, were
placed in incubators running at temperatures ranging from 50 to 320.
In the first experiment 10 onions were placed in each of four jars
which were placed in incubators held at 50, 130 to 140, 230, and 280 to 310
C, respectively. The extent of the disease on the various lots at this
time is shown in Plate 82. It was apparent that infection took place
very slowly at 130 to 140, while that at 280 to 31 ° was slightly less ad-
vanced than at 230.
In the second experiment jars containing 10 onions each were held at
50 to 6°, 90 to io°, 140 to 15°, 170 to 180, 200 to 21. 50, 220 to 230, 260 to
270, and 300 to 320 C. They were allowed to remain for 17 days before
examination. At the end of this period, no infection had taken place
at 50 to 6°, a very slight infection at 90 to io°, and as the temperature
rose the amount of disease increased up to 2 6° to 270, at which point it
was greater than in any of the other jars. At 310 to 320 it was slightly
less than at 260 to 270. A third experiment confirmed the results of the
first two.
Infection takes place and the disease progresses, then, at or above io°
C, but it is quite evident that for very rapid development a temperature
of 200 or above is needed. Since the fungus develops in the soil prior to
infection, the range of soil temperature during the growing season is
undoubtedly an important factor in determining the severity of the
disease.
PRODUCTION AND DISSEMINATION OF CONIDIA
After the appearance of the first stromata on the bulbs, subsequent
spread of the disease is effected to a considerable extent by conidia
Sporulation does not take place except under fairly humid conditions-
In order to determine the range of temperature at which fructification
may occur, infected scales were placed in Petri dishes lined with moist-
ened filter paper and exposed in incubators running at a range of temper-
atures from 20 to 280 C. Abundant sporulation occurred within 36 hours
at 200 to 280. The process was much retarded at lower temperatures,
though a few spores were formed at 20 to 30 after several days.
Under optimum conditions for spore production the conidia accumu-
late on top of the acervuli, forming gelatinous masses which remain in-
tact among the setae. Exposure of portions of scales bearing fresh spore
masses over sterile agar plates has yielded no indication of spore dis-
charge. The mucilaginous material surrounding the spores appears to
dissolve partly when a spore mass is placed in water, and the conidia
thus become separated.
It is thus to be expected from the nature of the fungus that warm,
rainy weather is especially favorable for the development of smudge,
since high humidity promotes the production of spores, and meteoric
water, especially in the form of spattering rain drops, is important for
their dispersion and dissemination.
708
Journal of Agricultural Research
Vol. XX, No. 9
CORRELATION OF CLIMATIC CONDITIONS WITH THE DEVELOPMENT OF THE
DISEASE IN I 91 5-1 6
Plots of white onion sets were grown in 191 5 and 191 6 on land which
had previously produced many successive crops of onions and where
the smudge organism was known to be present in the soil. Soil tem-
perature records were taken at a depth of 1 to 2 inches during part of
the 191 5 season and most of the 191 6 growing season. The daily mean
soil temperatures and rainfall for these seasons are represented in figure
I
530
|«
K /o
§ S
VI I.O
!yao
— /9/s j Jj\rv
/ '' iyfj v
f
1 1 1
. 1 II
1 1 - ~- EE=^lliiEiii J P[i^EnFliii^j
j-*-1L1l^j1-.. .dfci L . .-aI^._^-_^-_._. 1. 1 _-
•367 // AS 19 23 27
fit/tr
6 /o /«»* ia zs 26
OUH£
a <s
10 & /& 22 26 30 1 5 9
JULY AUGUST
FlG. 10.— Chart from data collected at Racine, Wis., during 1915 and 1916, showing the daily mean soil
temperature at a depth of 1 to 2 inches, and the rainfall. The horizontal broken line represents the
optimum temperature for infection and development of the disease as indicated by controlled
experiments, the broken vertical line the date of first observation of the disease in 191s, and the heavy
vertical line the first appearance of the disease in 1916.
10. The rainfall records included here are compiled from data taken
at the Racine (Wis.) post office, approximately 3 miles from the onion
set plots. The progress of the disease between the time of its first sea-
sonal appearance and harvest is described for these two seasons, since
they represent distinctly different conditions which had varying effects
upon the progress of the disease.
IN 1915
On June 28 a very few dark green stromata were found, but no acer-
vuli or setae had developed. The soil temperature mean was now well
Feb. i, 1921 Onion Smudge 709
above 200 C. and remained between 200 and 270 for most of the time
until harvest. On July 2 a few scattered acervuli were found. A
slight precipitation was recorded on July 2, 2 inches on July 4, 0.02 inch
on July 5, and 1.17 inches on July 7. Following this rainy period there
was a marked increase in number of acervuli noted on July 10. A slow
rain fell during most of July 14 and part of July 15. On July 15 the
disease was prevalent above the bulbs on the unthickened portions of the
outer leaves which comprise the "neck." These infections were clearly
the result of spores spattered upon these portions from the bulb scales
by rain a few days previously. The rainy weather, which prevailed until
harvest, about August 10, resulted in continued spread and development
of the disease, so that the white sets were all badly spotted by the latter
date. Further observations showed that the development of the disease
in other fields followed closely that noted in the experimental plot. The
infection in practically all cases, however, was confined to one or two of
the outer dry scales, the fungus being unable to attack the fleshy scales
previous to harvest. On the yellow and red varieties the fungus was
very abundant on the uncolored portions of the leaves at the neck, but
the highly colored bulb scales remained entirely free from it. This has
been the usual observation with the colored types.
IN 1916
The month of July, 191 6, was extremely warm and dry as contrasted
with cool, moist weather of the same period in 191 5. The soil tempera-
ture mean passed 260 C. on July 2 and remained above that point for the
rest of the month. In fact, for a large portion of that period it was well
above 320, the maximum temperature for growth of the fungus on potato
agar. No signs of smudge were found until July 8. The extent of the
disease at this time was very meager, only a few acervuli being noted.
It is probable that the dry weather preceding this date checked the
fungus, in spite of the fact that the soil temperature was favorable.
Aside from 0.03 inch precipitation on July 8, 0.45 inch on July 20, and
0.14 inch on July 31, no rain fell during the rest of the month. More-
over, the soil temperature was well above the maximum for development
of the disease. On July 13 but very little smudge could be found.
On July 22 no further development was noted. The moisture from
the shower of July 20 disappeared very rapidly from the upper 2
inches of soil because of the extreme heat. A rainy period occurred
on August 3, 4, and 5, and following this Macrosporium porri
and Phoma alliicola developed rapidly. Smudge increased but very
slowly, however, probably because of the scarcity of viable spores..
Another heavy rain fell on August 9 and 10, and the weather then
remained clear until after harvest on August 23. At the latter date
the bulbs were examined carefully, and in general the sets were only
moderately infected. The disease was confined for the most part to the
portions of the bulbs below the surface of the soil, while the abundant
yio Journal of Agricultural Research voi.xx.No.o
infections on the necks which were so conspicuous in 191 5 were almost
entirely absent.
To summarize, the disease progressed most rapidly during the last
part of the growing season of 191 5, with the mean temperature range
between 200 and 300 C, accompanied by sufficient rainfall to promote
abundant spore production and dissemination as well as subsequent
infection. On the other hand, development was materially checked in
1 91 6 by extreme heat, together with lack of precipitation during July.
RELATION OF ENVIRONMENT DURING CURING TO THE DISEASE
The onion set crop is usually harvested in early August. The tops
are twisted or clipped and the small bulbs are placed in shallow crates 2
or 3 inches deep. These are stacked in the field in piles with temporary
roofs, where they are allowed to cure for several weeks. Usually the
fungus is well established upon the outer scales of the bulbs before they
are pulled, and thus further invasion is dependent largely upon the
environmental conditions which prevail during the curing and storage
periods.
The respiratory functions of the living cells in the bulbs continue after
the sets are pulled, and there is, in consequence, some accumulation of
moisture. This is counteracted in part by the use of shallow crates
which are exposed to natural air currents. In bright, windy weather
the bulbs cure rapidly, while rainy or humid weather retards the process
and favors the progress of the disease. A number of experiments were
conducted during 191 6, 191 7, and 191 8 to determine the effect of varied
amounts of external moisture during the curing period upon the develop-
ment of the disease.
Experiment 1. — On August 15, 191 6. a crate of white sets was taken
from the general run of the crop which had been harvested on August 9
at Racine, Wis. The outer scales were badly spotted with smudge, and
in some cases the second scale had been invaded. After removal to the
laboratory the bulbs were sprinkled with water while in the crates.
After two days a portion of this lot (5^ pounds) was dried for 24 hours at
450 to 520 C. and the remainder (14K pounds) was given no further
treatment. Both lots were placed under cover in a shallow crate, where
they were exposed to good conditions for further natural curing. They
were later placed in a well-ventilated onion warehouse held at about
350 to 400 F. On January 13, 191 7, both lots were examined. Most
of the outer dead scales present at harvest time had sloughed off during
storage, and in the dried sets the fungus had advanced very little from
these original infections. In the naturally cured sets, however, the
fungus, probably aided by the greater excess of moisture present, had
invaded several underlying scales, and these sets were badly spotted
even after the outer scales were removed. The sets in each lot were
then sorted into three classes — (1) free from disease, (2) slightly diseased,
Feb. x, i9ii
Onion Smudge
711
(3) badly diseased. The result of this classification is given in Table III,
and samples from the dried and the undried lots are shown in Plate 85,
A, B.
Table III. — Relation of artificial curing
to the development of onion
smudge
Condition at end of storage period.
Treatment.
Percentage
free from
disease.
Percentage
slightly-
diseased.
Percentage
badly
diseased.
Naturally cured
7
56
29
36
64
Artifically dried
8
Experiment 2. — On August 30, 191 7, several bushels of white onion
sets were secured from a field where the crop had been harvested on
August 16 and placed in stacks of shallow crates. The weather had
been clear during this intervening period, and good natural conditions
for curing had prevailed. Smudge was prevalent on the outer scales of
the sets at this time. In order to test the effect of exposure to moist
weather on the progress of the disease, a portion of this lot in the crates
was sprinkled with water daily for one week, approximating roughly what
often occurs when a rainy period comes during harvest. After one
week a part of the moistened lot was placed in a kiln drier, where the
temperature was held at ioo° to 1200 F., until the bulbs were thor-
oughly dried. The remainder of this lot was allowed to dry naturally
under cover. All the sets were then stored in a standard onion storage
house. Samples taken from a moistened and an unmoistened crate on
October 10 are shown in Plate 85, C, D. Marked increase in the amount
of smudge was very noticeable within a few days after moistening was
begun. On January 14, 191 7, the amount of smudge was estimated by
classifying several hundred bulbs from each of the three lots into either
of two classes, namely, (1) those free from smudge or only slightly
diseased and (2) those so badly diseased as to impair their market
quality. The results are given in Table IV.
Table IV. — Effect of varied conditions at harvest on the amount of smudge on stored onion
sets
Treatment.
Condition at end of
storage period.
Percent-
age free
from
smudge
or
slightly
diseased.
Percent-
age badly
diseased.
Best natural curing
Exposed to moist conditions after harvest
Artificially dried after exposure to moist conditions
25119°— 21 4
42
93
48
712
Journal of Agricultural Research
Vol. XX, No. 9
This experiment shows (i) that even under what may be considered
very good weather conditions for natural curing a considerable amount
of smudge will develop; (2) that exposure to moist weather for a week
after harvest practically doubled the amount of smudge; and (3) that
thorough artificial drying immediately after such exposure counteracts
the effect of excessive moisture.
Experiment 3. — The sets used in this experiment were from a late
sowing and consequently were not harvested until September 14, 1918.
Smudge was prevalent on the extreme outer scales of a large percentage
of the bulbs at this time. Five bushels were placed in shallow crates
in the kiln drier, in which the temperature was maintained at ioo° to
1200 F. One crate was removed at the end of one day, a second at the
end of two days, and the remaining three on the fifth day. Three
untreated crates used in the experiment were allowed to cure in a cov-
ered pile in the field with the remainder of the crop. On September 30
they were removed to a standard onion warehouse, where they were
stored during the winter with the artificially dried lots. On March 5,
1 91 9, when final notes were taken, a comparison of the artificially cured
and field-cured lots was secured by estimating the percentage showing
any signs of smudge after sets had been milled to remove the loose
scales.1 The results are given in Table V.
Table V. — Amount of smudge on artificially cured and field-cured onion sets at the end
of the storage period
Crate
No.
9
10
Nature of treatment.
Artificially dried.
do
do
do
do
Field-cured
do
do
Average of artificially dried crates.
Average of field-cured crates
Length of
treat-
ment.
Days.
I
3
5
5
5
16
16
16
Percent-
age
showing
any
signs of
smudge.
33
31
72
75
78
75
The foregoing experiments clearly establish the importance of moisture
as a factor in the advance of the disease during the curing and storage
periods. They also indicate that artificial curing immediately following
harvest greatly checks the progress of the disease as compared with
natural field-curing.
1 It is the common practice to run "bottom" sets through a fanning mill as they are taken from storage
in order to remove the loose outer scales.
Feb. i, 1921 Onion Smudge 713
RELATION OF STORAGE CONDITIONS TO THE DISEASE
The study of the disease in storage has been directed toward the
solution of three problems: (1) The importance of smudge as a cause
of premature sprouting of sets; (2) the extent of shrinkage, if any,
which can be brought about during the storage of onion sets; and (3)
the amount of new infection or actual spread from diseased to healthy
bulbs occurring during the holding period. While the data on these
points are by no means complete and the factors involved in the progress
of the disease during the storage period by no means fully studied, the
experiments here reported upon throw some light on the matter.
Observations on the first two questions were made in a standard onion
set warehouse at Morton Grove, 111. In practice, onion sets are stored
in crates about 4 inches deep with slatted bottoms, piled so as to allow
a 1- to 2-inch space between each two crates to facilitate circulation of
air. Sets are placed in storage during September and October. The
temperature is gradually lowered, following seasonal changes, until it
approaches o° C. (320 F.), an attempt then being made to hold it slightly
above this point. During extremely cold weather some artificial heat
in the house is necessary to prevent freezing, while ventilation is con-
stantly needed to remove excessive moisture.
The experiments were carried on during the winter of 191 8-1 9. The
extremely mild weather during this season prevented the temperature
of the house from being held as close to o° C. as is commonly the case,
while, on the other hand, ample opportunity for ventilation was afforded.
Continuous records of temperature and relative humidity were secured
by means of a Friez hygro-thermograph. The temperature gradually
lowered during October and November, the minimum temperature reach-
ing 0.50 C. (330 F.), on November 23, while the maximum temperature
commonly reached 12.70 C. (550 F.) during this period. During Decem-
ber, January, and February the temperature fluctuated between 0.50
and 7. 20 C. (330 and 45 ° F.). The relative humidity varied between 65
per cent and 85 per cent during October and November, while through-
out the remainder of the period it seldom went above 75 per cent and
not often below 60 per cent.
RELATION OP SMUDGE TO SPROUTING
Two lots of onions were used in these experiments, and, since they
differed somewhat as to time of maturity and method of handling, they
are here considered separately.
Experiment i. — Bulbs averaging about 1 inch in diameter were
selected from a lot of white sets harvested early in August and brought
into storage on August 22, 1918. Two groups were secured, one con-
sisting of 49 bulbs badly spotted with smudge and the other containing
47 perfectly healthy sets. The two lots had thus been grown and han-
dled alike and presumably differed only as to infection with smudge.
7i4
Journal of Agricultural Research
Vol. XX, No. 9
They were carried through storage and examined on February 18, 191 9.
The results are given in Table VI.
Table VI. — Relation of smudge to sprouting of onion sets in storage
EXPERIMENT 1
Condition of bulbs.
Total
number of
bulbs used.
Healthy.
Diseased .
47
49
Number
sprouted.
14
26
Percentage
sprouted.
29.7
53- o
Experiment 2. — The sets used in this experiment were sown late in
the spring and consequently were not harvested until about September
14, 1918. They were allowed to cure in the field in the normal manner
until September 30, when they were placed in storage. Three average
crates were selected at this time and kept under observation. At har-
vest time smudge was prevalent only on the dry outer scales of the sets,
but during the storage period it gradually penetrated the underlying
scales. When a final examination was made on March 5, 191 9, it was
clear that in nearly every case where the fungus had penetrated deeply
the bulb had sprouted and had thus become worthless. A typical
example of this condition is shown in Plate 81 , D. An estimate of the
amount of sprouting actually due to or intimately associated with smudge
was secured by counting 100 to 200 bulbs in each crate. The results
are given in Table VII.
Tabi.E VII.— Relation of smudge to sprouting of onion sets in storage
EXPERIMENT 2
Crate No.
Number of
bulbs
examined.
Total
percentage
infected by
smudge.
Total
percentage
sprouted .
Total
percentage
sprouted
and showing
advanced
stage of
smudge.
165
197
148
75
75
72
6.0
9.6
2. O
6.0
9.6
. 7
74
5-8
5-4
It is not to be construed from these data that smudge is always the
chief cause of premature sprouting of onion sets in storage, since un-
questionably other factors may often be entirely responsible. One of
these, the neckrot decay of the scales, commonly produces a similar effect.
It is apparent, however, that the invasion of the bulb scales by the smudge
fungus brings about some physiological change which promotes growth
of the previously dormant bud.
Feb. i, 1921
Onion Smudge
715
Economically this factor has considerable value, since bulbs which
sprout before the end of the storage period are usually a total loss.
RELATION OP SMUDGE TO SHRINKAGE OP SETS IN STORAGE
In order to secure bulbs as nearly comparable as possible except for
presence or absence of smudge, healthy and diseased sets averaging
about 1 inch in diameter were selected from a general lot of white sets
which had been harvested in early August, properly field-cured, and
placed in storage on August 22, 191 8. Four lots of 25 bulbs each were
secured which showed heavy smudge infection but no signs of any other
disease. Three lots of 25 each were selected which appeared to be per-
fectly healthy. All lots were weighed on October 15. Two diseased lots
and one healthy lot were kept in the warehouse throughout the experi-
ment under conditions previously described. In order to secure a high
relative humidity a special temporary chamber was made in the ware-
house and lined with moistened burlap. Thus, a relative humidity of
90 to 95 per cent was maintained at a temperature close to that of the
main warehouse. Two diseased and two healthy lots were placed in this
chamber for approximately four weeks and then removed to the main
warehouse room. The several lots were weighed on December 30, 191 8,
and on February 18, 191 9. The results of the experiment are given
in Table VIII. A constant increase in shrinkage of diseased sets over
healthy sets was to be noted. Before the end of the experiment sprouting
had occurred in most of the lots, and, as was to be expected, was more
prevalent in diseased than in healthy lots. Sprouting and the complica-
tion of contaminating parasites should be considered; but, since the
former is seemingly enhanced by the disease and the latter is not serious
in these cases, there is reason to believe that smudge is responsible in
large measure for the increase in shrinkage.
Table VIII. — Relation of smudge to shrinkage of onion sets in storage
Lot
No.
Condition of bulbs.
Environment.
Num
ber
of
bulbs
used
Origi-
nal
weight,
Oct. iS>
1918.
Percentage of
shrinkage.
Dee. 30,
1918.
Feb. 18
1919.
Condition at end
of experiment.
Diseased.
do...
Healthy .
Diseased.
.do.
Healthy
do
Average shrinkage of dis-
eased lots
Average shrinkage of
healthy lots
Ordinary storage.
.do.
do.
Exposure to high rela-
tive humidity for 4
weeks, followed by or-
dinary storage.
....do
do.
.do.
Gm.
291.8
277- S
319-3
324-3
284.5
6-S
7-4
2-5
8.9
19. o
II- 3
9. I
11.4
22. 4
12 sprouting; 1 in-
fected with neck-
rot.
is sprouting.
8 sprouting; 1 in-
fected with blue
mold.
16 sprouting; 3 in-
fected with neck-
rot.
7 sprouting.
5 sprouting.
7i6
Journal of Agricultural Research
Vol. XX, No 9-
SPREAD OF SMUDGE IN STORAGE
It has been claimed that smudge spreads from infected to healthy
bulbs in storage (17, 29). It is to be expected that under unusually
moist conditions this might occur. However, since considerable mois-
ture is necessary for sporulation and infection, the conditions which
prevail in good storage houses are not conducive to rapid spread of
the disease. Several experiments have been conducted during the course
of this investigation in which healthy bulbs have been marked and mixed
in lots of badly diseased sets. A summary of these experiments appears
in Table IX.
Table IX. — Spread of smudge in storage
Experi-
ment
No.
Storage conditions.
Standard onion warehouse. . . .
do
Cool cellar
....do
Moist chamber at room tem-
perature.
Length
of ex-
peri-
ment.
Days.
154
103
66
208
36
.Num-
ber of
healthy
bulbs
used.
34
40
20
30
20
Condition at end of experiment.
All healthy.
2 bulbs showed slight infection.
All healthy.
Do.
6 showed slight infection.
It was found that there was little or no spread of the disease under
ordinary storage conditions or in a cool cellar. In a saturated atmos-
phere some infection of healthy bulbs occurred. In practice, then, some
spread from diseased to healthy bulbs is to be expected where sets are
exposed to rain or very humid atmosphere such as might occur during
the curing period. However, with fairly dry sets kept in cool, well-
ventilated storage new infections are probably negligible.
CONTROL OF THE DISEASE
The control of this disease is obviously connected closely with the
handling of the crop at or immediately following harvest.
In 1 91 5 a spraying experiment was conducted on a plot of white sets
at Racine, Wis. The development of the disease in this plot has been
described on pages 708-709. Various schedules were used with 4-4-50
and 8-8-50 Bordeaux mixture plus soap, 4-50 copper sulphate, and 1-10,
1-16, and 1-32 lime sulphur. The sprays were applied upon the bulbs
and necks of the plants. Contact with the soil probably reduced the
disinfecting property of the chemicals, and their adhesiveness was limited
by the nature of the scales and leaves of the onion. No beneficial results
were secured even where the first application was made before the first
signs of the disease appeared and where the spraying was continued at
intervals of three to eight days until harvest. The complete failure of
Feb. i, 1921 Onion Smudge 717
this experiment was sufficient to show that sprays could not be used
successfully for the control of smudge.
Dusting of the sets in the crates at harvest time with lime or sulphur
has been suggested by Thaxter (33). In 1916 and 1918 dusting ex-
periments with lime, sulphur dust, and dry Bordeaux powder were con-
ducted without any positive results. This is to be expected, since, as
a rule, the outer scales of the bulbs became infected before harvest
and a disinfectant applied externally could hardly prevent further
invasion of underlying scales.
The importance of thorough curing and prevention of exposure to
humid conditions after harvest has been emphasized by Thaxter (33),
Clinton (10, p. 333) , Massee (17) , and Stevens and True (30) . The experi-
ments reported on the effect of drying of bulbs at harvest have shown
that rapid dehydration of the outer scales at this time checks further
invasion by the fungus to a large degree. Observations in the field by
the writer during the years 1914 to 1920 indicate that even the best
natural curing weather to be expected in the Middle West is not sufficient
to do more than partially check the disease on seriously infected fields.
Artificial curing offers a possible measure of control for smudge, and,
as already pointed out (37) , preliminary experiments indicate that neckrot
can also be checked by this treatment. Extensive control experiments
carried on in the Chicago district in 191 8 have shown that thorough dry-
ing very soon after harvest is necessary in order to check smudge materi-
ally. In the set-growing district a large portion of the crop is grown on
contract to be delivered at a central warehouse as soon as it has cured
sufficiently. The expense involved in this treatment would almost
necessitate that they be dried at a central point, preferably at the place
of storage. Therefore, in order to handle the large quantity received, a
fairly rapid process of drying would be essential.
Further experimental work is necessary before artificial drying can be
recommended as a general practice, and the results of control experi-
ments are reserved for later publication. In the meantime, the most
applicable remedial measures consist in prompt harvest and the best
use of natural climatic conditions in curing the white onion set crop,
including all possible protection from moist weather. This should be
followed by storage in a well-ventilated warehouse held as nearly as pos-
sible at 330 to 360 F.
SUMMARY
(1) Smudge is one of the most common diseases of white onion Gets in
Wisconsin and Illinois.
(2) It occurs also on shallot (Allium ascalonicum) and leek (A. porrum).
(3) The disease was first described by Berkeley in England in 1851 and
is now widely distributed in Europe and America.
yi8 Journal of Agricultural Research voi.xx.no.s
(4) Smudge is confined to the scales and neck of the bulb, where
it causes dark green to black spots. On fleshy scales it appears as sunken
yellowish spots which enlarge slowly, coincident with gradual shrinkage
of the scale. On colored varieties the disease is confined to unpig-
mented portions of the outer scales of the neck of the bulb.
(5) Spots on the outer scales of bulbs due to Macrosporium porri,
M. parasiticum, Phoma alliicola, and Urocystis cepulae may be confused
with smudge.
(6) Smudge becomes detrimental to the onion crop as a cause of (1)
the reduction of market value of white varieties, (2) shrinkage in storage,
and (3) premature sprouting of sets in storage.
(7) A detailed description of the morphology of the causal organism,
Colletotrichum circinans (Berk.) Voglino, is given. The ascigerous
form, Cleistothecopsis circinans, has been described by Stevens and True,
but complete proof of its connection with Colletotrichum circinans is
lacking.
(8) Inasmuch as the causal organism produces a subcuticular
stroma and a well-defined acervulus, the species is classified in the Melan-
coniales as Colletotrichum circinans (Berk.) Voglino. A comparative
study of the latter with C. fructus (S. and H.) Sacc. was made.
(9) The characteristic growth of the organism on culture media is
described.
(10) Growth on potato agar takes place between 20 and 320 C, while
the optimum is about 260.
(11) Spore germination is stimulated in soil decoction, onion decoc-
tion, and sterilized soil extract, as compared with that in distilled water,
while it is reduced in unsterilized soil extract and entirely inhibited in
onion leaf or scale extract.
(12) Spore germination occurs within the range of 40 and 320 C, while
the optimum temperature is from 200 to 260.
(13) Conidia are very sensitive to desiccation except when in spore
masses, in which condition a small percentage retain vitality for four
months or more. Stromata are very resistant to desiccation, retaining
vitality for two years or more.
(14) Conidia are sensitive to freezing temperatures, but dried spore
masses may withstand this environment for a month or more. Stromata
are capable of withstanding several months of freezing weather.
(15) The fungus is pathogenic upon the scales of mature bulbs, but
does not attack actively growing parts of the plant with the exception
of young seedlings, upon which it may cause "damping off" under certain
greenhouse conditions.
(16) Spores germinate and appressoria form within 10 to 12 hours.
The infection tube is pushed from the side of the appressorium adjacent
to the host cuticle directly through the latter. The mycelium then de-
velops for a time between the cuticle and the subcuticular wall, raising
Feb. i, i9« Onion Smudge 719
the former and eventually causing a softening of the latter. In bulbs
inoculated in moist chambers the fungus progresses fairly rapidly, caus-
ing softening and lamination of the walls and the gradual collapse of the
cell. The stroma involves the subcuticular wall at first and later the
underlying cells, but the cuticle remains unbroken until the acervulus is
formed. The process of invasion under storage conditions is essentially
the same but much slower.
(17) The fungus overwinters as stromata in infected scales.
(18) Infection occurs at or above io° C, but progress is very slow
below 200; the optimum is about 260.
(19) Conidia are produced abundantly under moist conditions and at
temperatures between 200 and 300 C. They are disseminated chiefly by
meteoric water, especially spattering rain.
(20) The disease develops most rapidly in the field when the mean soil
temperature range lies between 200 and 300 C. and is accompanied by
abundant rainfall. Extremely hot, dry weather in July checks progress.
Presence of moisture favors the progress of the disease during the curing
period, whereas artificial drying of sets immediately following harvest
checks it.
(21) Smudge tends to promote premature sprouting and increases
shrinkage of sets in storage. The disease may spread from bulb to bulb
in the crate under very moist conditions, but in proper storage this factor
is negligible.
(22) The important measures of control are protection of the har-
vested crop from rain, rapid and thorough curing, and provision of well-
ventilated storage at about 330 to 360 F.
LITERATURE CITED
(1) Allescher, Andreas.
1898-1901. fungi imperfecti . . . 1016 p. Leipzig. (Rabenhorst, L. Kryp-
togamen-Flora von Deutschland, Oesterreich und der Schweiz. Aufl.
2, Bd. i, Abt. 6.)
(2) Atkinson, G. F.
1897. some fungi from Alabama . . . Bui. Cornell Univ. (Sci.), v. 3, no. 1,
50 p. Bibliography, p. 2.
(3) Bennett, J. L.
1888. PLANTS OF RHODE ISLAND, BEING AN ENUMERATION OF PLANTS GROWING
WITHOUT CULTIVATION IN THE STATE OF RHODE ISLAND. 128 p.
Providence, R. I.
(4) Berkeley, M. J.
1851. [a new onion disease.] In Gard. Chron., 1851, no. 38, p. 595, 2 rig.
(5)
1874. notices OF north American fungi. In Grevillea, v. 3, no. 25, p. 1-17.
Continued article.
(6) Blackman, V. H., and Welsford, E. J.
1916. STUDIES IN THE PHYSIOLOGY OF PARASITISM. II. INFECTION BY BO-
trytis cinEREa. In Ann. Bot., v. 30, no. 119, p. 389-398, 2 fig., pi.
10. Literature cited, p. 397.
720 Journal of Agricultural Research vol. xx,No.9
(7) Britton, W. E., and Clinton, G. P.
[1918.] spray calendar. Conn. Agr. Exp. Sta. Bui. 199, p. 51-98, illus.
(8) BubAk, Fr.
1904. IN BOHMEN IM JAHRE 1902 AUFGETRETENE PFLANZENKRANKHEITEN.
In Ztschr. Landw. Versuchsw. Oesterr., Jahrg. 7, Heft io, p. 731-741.
(9) Chapman, George H.
1910. NOTES ON THE OCCURRENCE OF FUNGO JS SPORES ON ONION SEED. Mass.
Agr. Exp. Sta. 226. Ann. Rpt., 1909, p. 164-167.
(10) Clinton, G. P.
1904. DISEASES OF PLANTS CULTIVATED IN CONNECTICUT. Conil. Agr. Exp.
Sta. 27th Ann. Rpt., 1902/03, p. 279-370, pi. 9-28.
(11) Dey, P. K.
1919. STUDIES IN THE PHYSIOLOGY OF PARASITISM. V. INFECTION BY COL-
letotrichum lindemuthianum. In Ann. Bot., v. 33, no. 131, p.
305-312, pi. 21. References, p. 311.
(12) Gardner, M. W.
1918. anthracnose of cucurbits. U. S. Dept. Agr. Bui. 727, 68 p., 15 fig.,
8 pi. Literature cited, p. 65-68.
(13) Halsted, Byron D.
189 1. report of the botanical department. N. J. Agr. Exp. Sta. nth
Ann. Rpt., 1890, p. 323-453, illus.
(14) HassELBRING, Heinrich.
1906. THE APPRESSORIA OF THE ANTHRACNOSES. In Bot. Gaz., V. 42, no. 2,
p. 135-142, 7 ng-
(15) Keitt, G. W.
191 5. SIMPLE TECHNIQUE FOR ISOLATING SINGLE-SPORE STRAINS OF CERTAIN
types of fungi. In Phytopathology, v. 5, no. 5, p. 266-269, x nS-
16) Kempton, F. E.
1919. origin and development of the pycnidium. In Bot. Gaz., v. 68, no.
4, p. 233-261, pi. 17-22.
(17) Massee, George.
1903. A text-book of plant diseases caused by cryptogamic parasites.
ed. 2, 472 p., illus. London, New York.
(18) Munn, M. T.
1917. neck-rot disease of onions. New York State Agr. Exp. Sta. Bui.
437, p. 361-455. ll pl- Bibliography, p. 45°-455-
(19) OrTon, W. A.
1903. plant diseases in the united states in 1902. U. S. Dept. Agr. Year-
book, 1902, p. 714-719.
(20
(21
(22
(23
(24
1907. plant diseases in 1906. U. S. Dept. Agr. Yearbook, 1906, p. 499-508.
Osner, George A.
1917. additions to the list of plant diseases of economic importance in
Indiana. In Proc. Ind. Acad. Sci., 1916, p. 327-332.
Peck, Charles H.
1881. report of the botanist. In 34th Ann. Rpt. N. Y. State Mus. Nat.
Hist., p. 24-58, 4 pl.
Russell, H. L.
191 5. report of the director, plant disease survey. Wis. Agr. Exp.
Sta. Bui. 250 (Rpt. 1914), p. 33-39, fig. 14-17-
Saccardo, P.
1884-1913. SYLLOGE Fungorum . . . v. 3,1884; v. 4, 1886; v. 22, 1913. Patavii.
Feb. 1. 1921 Onion Smudge 721
(25) Schwarze, Carl A.
1917. THE PARASITIC FUNGI OF NEW JERSEY. N. J. Agr. Exp. Sta. Bui. 313,
226 p., 1056 fig.
(26) Selby, A. D.
I9IO. A BRIEF HANDBOOK OF THE DISEASES OF CULTIVATED PLANTS IN OHIO.
Ohio Agr. Exp. Sta. Bui. 214, p. 307-456, 106 fig. List of plant diseases
referred to in this publication, p. 1-7.
(27) and Manns, T. F.
1909. STUDIES IN DISEASES OF CEREALS AND GRASSES. Ohio AgT. Exp. Sta.
Bui. 203, p. 187-236, illus., 14 pi.
(28) Stevens, F. L., and Hall, J. G.
1907. an apple rot due To volutella. In Jour. Mycol., v. 13, no. 89, p.
94-99, 6 fig.
(29)
1910. diseases of economic plants, x, 513 p., illus. New York.
(30) and True, Esther Y.
1919. black spot of onion sets. 111. Agr. Exp. Sta. Bui. 220, p. 505-532,
19 fig.
(31) Stewart, F. C.
19OO. AN ANTHRACNOSE AND A STEM ROT OF THE CULTIVATED SNAPDRAGON.
N. Y. State Agr. Exp. Sta. Bui. 179, p. 105-110, 3 pi.
(32) Stoneman, Bertha.
1898. A COMPARATIVE STUDY OF THE DEVELOPMENT OF SOME ANTHRACNOSES.
In Bot. Gaz., v. 26, no. 2, p. 69-120, pi. 7-18. Bibliography, p. 114-117.
(33) Thaxter, R.
1890. report OF . . . mycologist. In Conn. Agr. Exp. Sta. Ann. Rpt., 1889,
p. 127-177, 3 pi.
(34) Van Hook, J. M.
1911. Indiana fungi. In Proc. Ind. Acad. Sci., 1910, p. 205-212.
(35) Voglino, P.
1907. I FUNGHI PARASSITI DELLE PIANTE OSSERVATI NELLA PROVINCIA DI TORINO
E REGiONi vicinE NEL 1906. In Ann. R. Accad. Agr. Torino, v. 49,
p. 175-202.
(36) Walker, J. C.
1917. studies upon the anthracnose of the onion. (Abstract.) In
Phytopathology, v. 7, no. 1, p. 59.
(37)
1918. control of neck rot and anthracnose of onion sets. (Abstract.)
In Phytopathology, v. 8, no. 2, p. 70.
(38)
1918. NOTES ON THE RESISTANCE OF ONIONS TO ANTHRACNOSE. (Abstract.)
In Phytopathology, v. 8, no. 2, p. 70-71.
PLATE 80
Onion smudge:
Onion sets (White Portugal variety) naturally infected with Colletotrichum cit'
cinans. Collected on August 27, 1919, several weeks after harvest, at Morton Grove,
111. Photographed September 23, 1919. Note in the three lower bulbs the small
sunken spots in the fleshy scales which mark the early stages of invasion of the living
tissue. Natural size.
(722)
Plate 80
Journal of Agricultural Research
Vol. XX, No. 9
Onion Smudge
Plate 81
Journal of Agricultural Research
Vol. XX, No. 9
PLATE 8 1
Onion smudge:
A, B, E, D. — Advanced stages of smudge after several months in storage. Note the
shrinkage of fleshy scales and the tendency to sprout.
C. — Bulb inoculated in a moist chamber with a suspension of Colletotrichum cir-
cinans conidia.
F, G. — Macrosporium sp. on outer scale of white onion sets.
H. — M. porri and Phoma alliicola on outer scale of white onion set. Natural
size.
PLATE 82
Relation of soil temperature to the development of smudge:
Onions kept in infected soil held at different temperature for nine days.
A.— 50 C.
B— i5°C.
C.-230 C.
D.— 320 C.
Slightly reduced.
Onion Smudge
Plate 82
Journal of Agricultural Research
Vol. XX, No. 9
Onion Smudge
Plate 83
n
f % v -*v
Journal of Agricultural Research
Vol. XX, No. 9
PLATE 83
Colletotrichum circinans and C.fructus:
A. — Photomicrograph of cross section of naturally infected onion scale. Note
that the fungus is confined largely between the cuticle and the subcuticular wall.
The epidermal cells and two layers of the parenchyma cells have collapsed, while the
uninvaded cells beneath the lesion are slightly enlarged and distended.
B. — Photomicrograph of cross section of an infected onion scale held for several
months in poorly ventilated storage. Note that the stroma is excessively developed
and that the cuticle is still intact except where ruptured by the acervuli.
C, D. — Photomicrographs of cross sections of C. circinans (C) and C.fructus (D) on
apple fruit. Note similarity between the two forms and the subcuticular origin of the
stromata in each case.
PLATE 84
Colletotrichum fructus and C. circinans:
A. — Dilution plate from spores of Colletotrichum fructus. Photographed on sixth
day. Note stellate character of colonies as compared with C. circinans in D. X 4/s-
B.— Individual colony of C. fructus on potato agar. Photographed on the fourth
day. Compare with C. circinans in E. X iH-
C. — Apple of Fameuse variety inoculated with mycelium from pure culture of
C. circinans. Photographed two months after inoculation.
D. — Dilution plate from spores of C. circinans. Photographed on sixth day. Com-
pare with C. fructus in A. X 4/s.
E. — Individual colony of C. circinans on potato agar. Photographed on fourth day.
Compare with.- C. fructus in B. X iK-
Onion Smudge
Plate 84
Journal of Agricultural Research
Vol. XX, No. 9
Onion Smudge
Plate 85
Journal of Agricultural Research
Vol. XX, No. 9
PLATE 85
Relation of curing conditions to the development of smudge:
A, B. — Comparison of onion sets artificially dried immediately after harvest with
those not dried. Photograph made at the end of the storage period after the two lots
had each been divided into three classes — namely, those free from disease, those
slightly diseased, and those badly diseased. (See experiment 1, p. 710-711.)
C, D. — Comparison of white onion sets cured in shallow crates in the field under the
best of natural conditions with part of the same lot after exposure to moist conditions
for one week. (See experiment 2, p. 711-712.)
25119°— 21 5
VARIATIONS IN COLLETOTRICHUM GLOEOSPORIOIDES1
ByO. F. Burger2
Instructor in Plant Pathology, Graduate School of Tropical Agriculture and Citrus
Experiment Station, University of California
The diseases of citrus trees and fruit known as wither-tip, leafspot,
anthracnose, and tearstain are all caused by the same fungus, Colleto-
Irichum gloeosporioides (Penz.) . These diseases have been found in Florida,
(4; 5; 9, p. 88), 3 California (j), West Indies, South America, Australia,
and Malta; and in practically all citrus-growing regions rather serious
outbreaks of some or all of these diseases have occurred from time to
time.
The smaller twigs of citrus trees are very frequently and severely
attacked by the fungus. It is quite common to see many of the small
twigs killed back 4 or 5 inches. These infected twigs soon turn to a light
brown color and sooner or later become dotted over with numerous
small black acervuli. After the rainy season begins, the spores, which
are imbedded in a gelatinous matrix, exude from the acervuli and are
washed down over the fruit and leaves, causing leafspot, tearstain, and
anthracnose of the fruit.
The spores must have an abundance of moisture in order to germinate.
Since the rainy season in California occurs during the winter and early
spring months, it is at this period that these diseases are most prevalent.
In Florida these diseases cause much damage to the citrus industry,
whereas in California they are considered of minor importance. This
difference in the amount of injury in the two States named is due, I
believe, to the difference in the amount of rainfall. During the dry
summer in California there is little evidence that Colletotrichum gloeos-
porioides is active. In Florida this fungus causes bloom drop and a
considerable amount of leaf spotting during the spring and summer
months, as well as anthracnose and tearstaining of the lipe fruit. Many
growers and agricultural workers believe that the fungus injury is
secondary. It has been stated repeatedly that the weak or injured tree
is more susceptible to an attack of C. gloeosporioides than the healthy
tree.
DESCRIPTION AND HISTORY OF THE FUNGUS
The fungus, Colletotrichum gloeosporioides (Penz.) was first described
by Penzig in 1882 as V ermicidaria gloeosporioides. In 1887 he placed
1 Paper No. 66, University of California, Graduate School of Tropical Agriculture and Citrus Experi-
ment Station, Riverside, Calif.
2 Resigned June i, 1918.
3 Reference is made by number (italic) to "Literature cited," p. 735-736.
Journal of Agricultural Research, Vo1- :xx> No- 9
Washington, D. C. Feb. 1. 192 1
ws Key No. Calif-28
723
724 Journal of Agricultural Research vol. xx, No. 9
it in the genus Colletotrichum. It was first collected in America in
1886 by Dr. Martin from Green Cove Springs, Fla., and was first reported
by L. M. Underwood (8) in 1891. However, the disease was not found
in California until some years later. It was reported by Essig (4) in
1909 from the Limoneira Ranch at Santa Paula, where it was causing
considerable damage to lemon trees.
In 1904, Prof. P. H. Rolfs (5) gave a very good description of the
fungus as it occurred on various citrus trees and fruits in Florida. He
says (5, p. 20) that the —
diseases . . . manifest themselves as wither- tip on orange, pomelo, and lemon twigs;
as leaf -spot on the leaves of the various citrous species; as anthracnose on lime blos-
soms, recently set limes, lime twigs, and lemon twigs; as lemon-spot on ripe lemons
and as canker of limes.
The following description is given by Prof. P. H. Rolfs:
Acervuli located on the surface of the leaf, twig or fruit; 90-270 n in diameter,
erumpent, superficial. Shape various, not uniform, occurring on either surface of
citrus leaves; disposed irregularly or in more or less concentric lines; pale to dark
colored. On tender lime twigs, tender lemon twigs, lemon fruits and lime fruits, pale
colored, dull red in masses, confluent. Epidermis breaks irregularly. Setae fuligi-
nous, ranging in length from 60-160 /x, frequently once or twice septate, disposed at
margin of acervuli. Frequently absent, and on tender lime twigs, tender lemon
twigs, lemon fruits and lime fruits usually absent.
Conidia broadly oval or oblong, 10-16 m by 5-7 n, hyaline; size variable in same
acervulus, usually with one or two oil drops. Developing from a well-defined stroma;
basidia, 3-18 m- In moist chambers the conidia stream from the break in the epi-
dermis. Intrabasidial setae, variable 8-30^ by 3-6/i, cylindrical or sometimes en-
larged at distal end; hyaline.
In 1 91 2 Clausen (/) described the fungus causing wither-tip of the lime,
Citrus medica, as Glocosporium limetticolum. He believes that Rolfs had
confused two forms and described them as one. Clausen uses the ab-
sence of setae as a distinguishing character from Colletotrichum
glocosporioides. It is the opinion of Stoneman (7), Edgerton (2), and
Shear and Wood (6) that the setae are variable as to presence or absence
and that they are not reliable morphological characters to use in separat-
ing genera. I have found them in some of my cultures of Colletotrichum
glocosporioides, while in other cultures they were absent. Another char-
acter he uses is the lack of a coarsely granular plasma filling the spores. I
have found several strains of this fungus which are considered to be
Colletotrichum gloeosporioides, whose spores are not filled with a coarse
granular plasma but appear at first to be homogeneous. Clausen also
uses growth characteristics as a means to identify the two strains.
Some of my strains had the same growth characteristics as the strain
which was obtained from Clausen — that is, a white mycelium and
abundant spore production.
Shear and Wood (6) in their bulletin on the genus Glomerella, have
brought together strains from various hosts and included them in one
Feb. i,i92i Variations in Colletotrichum gloeosporioides 725
species, Glomerella cingulata. To my knowledge, the perfect stage of
Clausen's fungus has not been found. Several of my strains produced
the perfect stage when first isolated, and the spores and asci were the
same as described for G. cingulata. It is, therefore, the opinion of the
writer, which will be presented in the following pages, that Colletotrichum
gloeosporioides as found in California is a polymorphic species, composed
of many strains.
STRAINS IN COLLETOTRICHUM GLOEOSPORIOIDES
In the fall of 19 16 when the writer began work at the Citrus Experi-
ment Station, the wish was expressed that he should study Colletotrichum
gloeosporioides. The different members of the Division of Plant Pathol-
ogy had isolated several cultures of this fungus from different citrus
hosts. Some of these differed from each other in their cultural character-
istics. It was suggested that these forms might have different regional
distribution, or that their differences might be due to the host. Other
isolations were made from the various citrus hosts; and these, together
with the cultures obtained from the different members of the Division of
Plant Pathology, were given laboratory numbers and were always spoken
of as strains. In all, 46 cultures were used in the study. Forty-two of
these represented all the important citrus districts of southern California,
and there was one each from Texas, Florida, Alabama and one kindly
furnished by Dr. C. L. Shear.
CULTURAL CHARACTERISTICS
The various strains were grown on five different media — corn meal
agar, green bean plugs, potato agar, lactose-beef agar and oatmeal
agar. Each strain was grown on these five different media for a period of
18 months. Transfers were made about every 5 weeks, and a record was
kept of the variations in growth occurring in each strain on the various
media. While most of the strains exhibited different cultural character-
istics on the various media, there were a few whose macroscopic charac-
teristics of the mycelium were much the same on all the media. Not
only did each strain vary in its growth characters on the different media but
some of the strains differed characteristically from each other. Therefore,
the variations exhibited by the various strains in their cultural character-
istics made it possible to classify them into the following five groups.
Group I: Mycelium white; spores abundant, salmon-colored in mass.
Group II : Mycelium grey to greenish black on the various media, very
little aerial growth on oat agar; spores abundant, salmon-colored or
yellowish in mass.
Group III: Mycelium gray to black on various media; no spore
masses on oat agar.
Group IV: Mycelium gray to black; spore production so abundant
on all media that the surface of the medium is nearly covered by a
bacteria-like mass of spores.
726
Journal of Agricultural Research
Vol. XX, No. 9
Group V: Mycelium gray to black, rather fluffy; no pink spore masses
on any medium; spore production scant and on some media no spores
produced.
Since the cultural characteristics of some strains changed, it became
necessary to reclassify the different strains on the following dates: Janu-
ary 27, 1917; April 16, 1917; September 13, 1917; and February 28,
19 18. Very few of the strains remained in the group in which they were
placed at the first classification. Under artificial cultivation the charac-
teristics of the various strains changed; therefore, they were placed in
different groups (see Table I). There were only three strains whose
characteristics remained constant in group I. In group II there was
only one strain which remained constant. It will be noticed that in
group IV cultures 296 and 299 remained constant until September 13,
19 17. At the next date of classification these two strains were placed
in group II. No strains were placed in group V until September 13,
19 17. This may be due to the fact that under artificial conditions these
strains lost their power to produce spores.
Table I. — Classification of strains of Colletotrichum gloeosporioides into groups
Group No.
Jan. 22, 1917.
Apr. 16, 1917.
Sept. 13, 1917.
Feb. 28, 1918.
I
a 295
0298
323
0429
a 295
0298
323
0 429
a 295
a 298
323
326
a429
496
502
901
934
955
0 295
a 298
a 429
496
561C
296
299
323
325
326
502
5IO
406
475
507
527A
II
326
459
496
502
5°7
5io
943
297
325
406
467
475
483
651
901
912
926
940
934
955
"990
326
459
475
483
502
5°7
536
297
325
406
467
560
56i
651
912
926
934
943
496
5IQ
910
940
a99<D
955
325
475
483
5IQ
536
612
615
507
560
561
651
a 990
527
536
536A
536B
561B
620
901
536C
560
&\
561A
527C
926
a990
Ill
912
943
912
943
955
612
IV
296
299
296
299
296
299
297
467
495
527
940
V
297
483
495
527B
651
934
940
<* Culture remained in its original class throughout the work.
VARIATIONS IN SPORE LENGTH
Since such great differences were found in cultural characteristics be-
tween the strains, the question arose whether differences could be found
in the spore length of the various strains. One hundred spores were
measured from each strain. The measurements were made in the fol-
Feb. i, 1921
Variations in Colletotrichum gloeosporioides
727
lowing manner: A dilute suspension of the spores taken from green
bean plugs was made in sterilized tap water, and a drop of the suspension
was placed on a microscope slide and covered with a cover glass. It was
necessary to make the measurements quickly, because the spores did not
remain quiet for any length of time. The image of the spore was thrown
on drawing paper by means of the camera lucida, and the length and
width were quickly marked with a pencil. The microscope was so ad-
justed that 1 micron on the micrometer scale in the eyepiece was equal
to 1 millimeter on the paper. Therefore, after the length and width
were indicated on the paper the spore size could be quickly ascertained
by means of a millimeter rule.
Table II.-
-Variation in spore length in the different strains of Colletotrichum gloeos-
porioides
Strain
Number of spores measuring (in microns) —
No.
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
26
296. . .
I
I
3
9
19
28
18
14
6
1
33
31
13
15
21
28
35
27
8
32
11
41
37
13
26
37
32
5
28
21
36
22
20
24
19
8
J9
22
20
22
31
26
3°
31
1
27
18
21
25
2
8
25
23
16
13
1
19
6
2
18
21
16
3
J3
3°
20
39
33
43
44
21
35
21
36
26
27
28
9
21
7
4
5
27
28
0
7
19
14
33
3
2
9
1
1
16
8
16
5
2
20
20
20
19
J3
18
28
27
25
27
26
17
21
2
6
10
1
2
11
J5
1
2
5
1
21
0
12
21
19
16
10
1
I
4
2
2
5
10
4
3
17
16
4
0
16
33
4
22
32
6
6
10
11
16
2
3
2
1
1
1
1
2
2
4
2
3
2
11
9
26
29
4
4
29
20
8
25
1
20
23
15
27
38
17
25
18
8
23
9
12
5
9
8
8
2
3
6
4
7
16
8
39
24
1
1
1
0
0
12
8
1
1
0
2
1
2
1
1
0
5
4
1
23
14
1
4
1
15
24
3
7
13
4
990...
I
O
0
3
2
12
1
6«;i.. •
1
4
3
■
6
7
1
1
0
11
2
0
4
3
12
4
10
11
8
8
22
12
I3
5
12
5
10
0
3
1
2
1
1
2
16
0
21
5
22
11
1
2
5
3
4
1
2
5
3
1
13
0
9
2
2
1
1
0
0
0
2
1
1
K1& . ■ .
c;6i.. .
514. . •
2
Kit . ■ ■
CI7. . .
1
cis. . .
3
0
1
1
1
2
2
047. . .
467 . . .
1
"?I2. . .
0
1
1
471?. . .
1
026. . .
483 .. .
2
7
5
1
1
0
1
12Z . . .
1
0
1
0
0
298. . .
It was soon determined that each strain had a certain range of vari-
ability in its spore length and width (see Table II). While there were
728
Journal of Agricultural Research
Vol. XX, No. 9
individual variations exhibited, yet it was soon determined that many
of the strains had the same mode for their spore lengths. Therefore, the
cultures were classified in regard to the modal length of the spores (see
Table III). The strains varied in their modal spore length from 12 to
20 /*. Most of the strains have their mode at 1 5 ju.
Table III.— Mode of the spore length of different cultures of Colletotrichum gloeosporioides
Spore No. measuring (in microns) —
12
296
527
13
14
15
16
17
18
19
20
323
325
502
295
297
298
299
429
459
5°7
5io
560
56i
926
934
940
943
990
475
483
512
5i3
5i4
5*7
524
536
326
406
467
5i5
651
912
901
955
|
|
1
Microns
6 7 & 9 10 II IS 13 14 16 16 17 18 19 20 21 22 23 24 25 26
#rz__z
zt%^zxt-v-Z^
XI I X V \ /
40
235
t
g30
o>
0
&20
Id
CD
Z 15
D
Fig. i.— Variability of strains of Colleloirkhum gloeosporioides in spore length.
It was soon observed that this classification could not be correlated
with the classification of the strains based on their cultural characteris-
tics. It was hoped that it would be possible to find morphological
differences correlated with the cultural characters, but this was not the
case.
In order to show the variability within the strain and the differences
between the strains, graphs were made representing the variability in
four strains (fig. i). Strain 296 has its modal spore length at 12 /jl, 507
Feb. i, 1921
Variations in Colletotrichum gloeosporioides
729
has its mode at 15 lx, 912 has its mode at 17 lx, and strain 901, which has
the largest spores of all the strains, has its mode at 20 /x.
There was also a certain range of variability in spore width. The
variability was not as great as in length. The widths ranged from 3 to
8.5 n; in most of the strains the mode was about 4 or 5 /x. In strain 901
the variability was from 5 to 8.5 /x with the mode at 6.5 /x.
In Table IV are given the calculated mean, standard deviation, and
probable error of each, for the spore length and width of eight different
strains. The measurements were made from spores taken from the
green bean plug medium.
Table IV.
-Table of calculated spore measurements for certain strains of Colletotri-
chum gloeosporioides
Strain No.
Mean length of
spore in microns.
a
0. 97 ±0
. 046
1. 7i±
.082
1. 4<3±
. 067
1. 42 ±
.068
1. i7±
.056
1. 64±
.078
2. 04±
•097
i.44±
. 069
Mean width of
spore in microns.
295
296
298
429
5°7
651
901
912
11. 54±o. 065
12. oi± . 115
14- 79±
14- 73 ±
14. i6±
17. 23±
20. 34±
5. 52 ±0.057
094
°95
079
no
x37
16. 99 ± .097
4- 2 ±
4. 68 ±
3. 26±
4. 91 ±
4. 52 ±
6.45±
4-7 ±
065
014
077
048
°35
132
no
0. 85 ±0. 041
• 97± -046
. 2I± . 010
i-i5± -OS5
• 7J± -°34
. 52± .025
1. 96 ± .093
1. 63 ± .078
This table shows that strains grown on the same medium under like
conditions vary greatly in respect to their spore sizes. We can, there-
fore, safely conclude that there exist individual differences in the various
strains in regard to certain morphological characters.
VARIATIONS IN THE DIFFERENT STRAINS INDUCED BY THE MEDIUM
The difference in growth characteristics occurring in the same strain
when transferred to the various media was very noticeable. The
various strains were grown on the five different media for a period of
one year. Transfers were then made from cultures growing on the vari-
ous media to different plates poured with the same medium. The plates
were kept at room temperature, and their growth characteristics were
noted. It was soon observed that some strains had been more affected
than others by their previous environment. While some of the variations
were slight, still it was impossible to account for this variation other than
as the effect of the medium.
On October 25, 1917, 20 cc. of potato agar were poured in sterilized
Petri dishes and allowed to harden. Transfers were then made from the
various strains as follows :
730 Journal of Agricultural Research Voi.xx.No.g
strain 429
Plates 1 to 4 were transfers from mycelium on corn meal agar.
Plates 5 to 8 were transfers from spores on corn meal agar.
Plates 9 to 12 were transfers from mycelium on green bean plugs.
Plates 13 to 16 were transfers from mycelium on glucose-potato agar.
Plates 17 to 20 were transfers from mycelium on lactose-beef agar.
Plates 21 to 24 were transfers from mycelium on oatmeal agar (spores).
Plates 25 to 30 were transfers from mycelium on oatmeal agar (mycelium).
On November 22 the final notes taken on the foregoing cultures were as follows:
Plates 1 to 4. White, woolly fungal growth covering the medium. Plate No. 4 was
distinctly zoned; spores in center of culture.
Plates 5 to 8. White, scanty fungal growth, which gave the culture a granular ap-
pearance; spores in center of culture.
Plates 9 to 12. White, cottony growth, not zoned, but in two plates there was con-
siderable dark mycelial growth; spores in center of culture.
Plates 13 to 16. Very scanty white mycelial growth; few spores.
Plates 17 to 20. White, cottony growth; no spores.
Plates 21 to 24. A membrane-like growth over the entire surface. Very little aerial
growth ; few spores.
Plates 25 to 30. White, scanty growth of a granular appearance; zoned.
STRAIN 561
Cultures made on glucose potato agar, December 18, 1917.
Plates 1 to 5 were transfers from corn meal agar.
Plates 6 to 10 were transfers from glucose-potato agar.
Plates 11 to 15 were transfers from oatmeal agar.
The final notes were taken on December 28, 191 7.
Plates 1 to 5. There is a gray, woolly aerial mycelium; growth in medium is dark
In plate 1 there is a white sector; no aerial growth but abundant spore production.
Plates 6 to 10. The growth is white, apprest, wet-looking; no spores.
Plates 11 to 15. No aerial mycelium, zoned, growth in medium white; good spore
production on surface.
STRAIN 560
Cultures were made on Petri dishes, poured with corn meal agar December 5, 1917.
Plates 1 to 3 transferred from corn meal agar tubes.
Plates 4 to 6 transferred from green bean plug.
Plates 7 to 9 transferred from glucose-potato agar.
Plates 10 to 12 transferred from lactose-beef agar.
Plates 13 to 15 transferred from oatmeal agar.
On December 17 the final notes taken on the foregoing cultures were as follows:
Plates 1 to 3. White growth in medium; good spore production.
Plates 7 to 9. White growth in medium; no aerial growth; no spores.
Plates 10 to 12. White, woolly aerial growth; no spores.
Plates 13 to 15. Growth in medium, dark; very scant aerial growth; no spores.
STRAIN 990
On October 16, 1917, corn meal agar plates were inoculated with strain 990, the
transfers being made from the various media.
Plates 1 to 4 transferred from corn meal agar tube.
Plates 5 to 8 transferred from green bean plug.
Plates 9 to 12 transferred from glucose-potato agar tube.
Feb. 1,1921 Variations in Colletotrichum gloeosporioides 731
Plates 13 to 16 transferred from lactose-beef agar tube.
Plates 17 to 20 transferred from lactose-beef agar tube.
Plates 21 to 24 transferred from oatmeal agar (mycelium).
Plates 25 to 28 transferred from oatmeal agar (spores).
The final notes were taken October 29, 191 7.
Plates 1 to 4. Gray, short mycelial growth.
Plates 5 to 8. Gray to black aerial mycelium, but in some spots there were no aerial
hyphae, growth confined to the medium; good spore production. The peculiar spots
were more or less in sector-like areas. Plate 6 showed definite sectors of black and
gray aerial mycelium, and in some sections the growth was confined in the medium.
Plates 9 to 12. Almost all the plates had a good growth of gray aerial mycelium,
while in others there appeared sectors where the mycelium was confined in the medium.
Plates 13 to 16. No aerial mycelium, but the growth was confined in the medium,
was light-colored, and was producing many spores.
Plates 17 to 20. The aerial growth is gray, woolly; some spores produced.
Plates 21 to 24. Gray felt-like growth covering the medium; no spore production.
Plates 25 to 28. These plates differed from plates 21, 22, 23, and 24 in that some of
the plates were zoned and produced more spores.
It is clear that there exist variations in a single strain which can not
be accounted for on any other ground than the effect of environment.
If, therefore, the differences in environment have caused these variations
in one year, there may be a possibility of certain environments causing
still greater variations which would be more or less permanent.
EFFECT OF THE MEDIUM ON SPORE SIZE
Spores were also measured from the different media to ascertain whether
the spore size had been affected. One hundred spores were measured
from five different media, and the mean length, mean breadth, standard
deviation, and probable error of the mean were calculated for five strains
(see Table V). It will be seen that the various media did affect the spore
size, but all strains were not affected alike by the same medium. While
it has been definitely shown that there exist different strains in Colleto-
trichum gloeosporioides, it also has been shown that these strains are
affected in growth characteristics and morphological characters by the
medium.
MUTATIONS
The variations which have been described in this paper occurring in
the various strains of Colletotrichum gloeosporioides have been shown to be
due to environmental factors. Not all the variations, however, which
occurred during the progress of the work are thought to be due to the en-
vironment. These variations which were thought to be induced by some
factor or factors other than the environment are in this paper called
mutations. These mutations have kept their peculiar characteristics
although grown under the same conditions as the cultures from which they
arose.
When the various strains were isolated in the fall of 19 16, they were
grown in plate cultures to study their growth characteristics. The
732
Journal of Agricultural Research
Vol. XX, No. 9
cultures in which the mutations occurred had greenish gray, fluffy, aerial
growth. None of the cultures showed any variation from the descrip-
tion given in the table. This seems to indicate that the cultures were all
pure.
Table V. — Differences in spore size of Colletotrichum gloeosporioides induced by various
media
STRAIN 295
Kind of medium.
Mean spore length
in microns.
a
Mean spore width
in microns.
a
Corn meal agar
Green bean plug
Potato agar
II. 54 ±0.065
14- 13 ± • XI4
13. 6 ± . 129
15-74 ± -319
13. 24 ± .071
0. 97 ± O. 046
i.69± .081
1. 92 ± . 092
4. 74± . 226
i.o6± .051
5. 52 ±0.057
4. 41 ± .11
4-9 ± -°55
4- 65± .044
5. 34 ± -018
O.85 ±0.041
I. 63 ± . 078
.835± .040
Lactose agar
Oat agar
.65 ± .031
. 27 ± .013
STRAIN 296
Corn meal agar . .
Green bean plug
Potato agar
Lactose agar
Oat agar
9-
14
±0
in
12.
01
±
"5
11.
87
±
118
13-
53
±
i°3
1 1.
98
±
078
I. 65 ±0. 079
I. 71 ± . 082
1. 75± .083
i-53± -°73
1. i6± .055
5. 48 ±0.052
4. 2 ± . 065
5-3 ± -°3
4v4^± -04
5. 23± .127
77 ±0.037
97 ± • 046
44 ± . 02 1
61 ± . 029
88 ± .090
Corn meal agar . .
Green bean plug
Potato agar
Lactose agar
Oat agar
11. 036±o
176
14-79 ±
094
12. 96 ±
144
13. 17 ±
126
12.98 ±
078
2
6i±o
124
1
40 ±
067
2
i4±
102
1
88 ±
090
1
i6±
°55
3. 34±o. 056
4. 68 ± . 014
4. 56± . 0188
4- 54± • 061
5. 56 ± .064
o. 836 ± o. 040
. 21 ± . 010
. 28 ± .013
.91 ± .043
• 95 ± -°45
STRAIN 429
Corn meal agar . .
Green bean plug
Potato agar
Lactose agar
Oat agar
13.03 ±0. 138
14-75 ± -°95
13.07 ± . 125
12. 75 ± . 101
13. 76 ± . 123
2. o5±o
.098
I-42±
.068
1. 87±
.089
I. 52±
. 072
i.8i±
.086
3-94±o. 036
3. 26± . 077
3. 99± . 122
3. 75 ± .047
4. 58± . 121
0. 53 ±0.025
1. 15 ± .055
1. 81 ± . 086
•7° ± -°33
1.80 ± .086
STRAIN 651
Corn meal agar . .
Green bean plug
Potato agar
Lactose agar
Oat agar
14.43 ±0.115
17. 23 ± . no
15. 11 ± .115
15. 67 ± . 121
15.06 ± . 113
1. 7i±o
.082
i.64±
.078
i-7 ±
.081
1.8 ±
.086
1. 67 ±
.080
4. 49 ±0.013
4- 52± -°35
5. I2± . OI7
4. 44± .042
5.38± .028
o. 19 ±0. 009
± .025
± . 012
± .030
± . 020
In the fall of 191 7, after the strains had been grown on aitificial media
for a year, they were again grown in plate cultures. In a few of the
strains there appeared some mycelial growth which differed in color from
Feb. i,i92i Variations in Colletotrichum gloeosporioides 733
the rest of the growth in that plate. These mutations usually appeared
as wedge-shaped or fanlike areas with the point of origin usually at the
center of the culture. Sometimes they occurred more toward the periph-
ery of the culture. (PI. 86, A, B.)
Mutations occurred in the following strains: 943, 297, 615, 495, 940,
510, 561, 536, 527, and 990. These mutations have remained true to
the characteristics manifested by the first culture. Figure 2 will serve
to illustrate the manner in which the mutations originated. Since these
strains were not progenies from a single spore, it was thought that there
might be a possibility of having a mixture of strains.
There are several types of these variations. One type had a white,
fluffy mycelium. A second type, where the mycelium was confined in
the medium, had abundant spore production on the surface. A third
type had varying shades of gray mycelium bearing spores. At first these
peculiarities in growth were regarded as modifications due to some envi-
ronmental factor. However, after these variations were transferred to
other culture tubes and the resulting cultures always exhibited the same
characteristics, they then were considered as mutations. Therefore,
single-spored cultures were made from one of the strains.
SINGLE-SPORED ISOLATIONS
On November 14, 191 7, single spores were isolated from culture 990.
The spores were taken from oatmeal agar, and a suspension was made in
sterilized distilled water. A platinum loop was used to transfer a drop of
the suspension to a cover glass. Each cover glass was examined with
the microscope, and when a drop contained only one spore the cover
glass was dropped into a test tube containing potato agar. Three cul-
tures were thus obtained and were designated as 990A, 990B, and 990C.
After the spores had germinated and had produced a mycelium, transfers
were made to the five media used in culturing the various strains. The
growth characteristics of cultures 990A, 990B, and 990C were identical
with those of the original culture 990.
On November 26, 1917, transfers were made from culture 990C to
potato agar plates. The resulting growth was composed of black and
white mycelia, with abundant production of spores in the center of the
culture (PI. 86, C). On December 12, 191 7, transfers were made from
the white and black mycelia to potato agar plates from the cultures
made November 26, 191 7. The plates made from the black mycelium
became black with some white mycelium. The plates made from the
white mycelium were white, but only slight traces of black growth could
be detected. All cultures produced abundant spores.
On January 9, 191 8, transfers were again made from the two kinds
of cultures obtained in transfers of December 12, 191 7, with results simi-
lar to the transfers of December 12, with the exception that there was
734
Journal of Agricultural Research
Vol. XX, No. 9
Fig. 2. — I, culture 510: A, greenish black mycelium; B, white mycelium. II, culture 943: A, black
mycelium; B, white mycelium; C, mycelium mostly in medium, growth zoned, abundant spore pro-
duction. Ill, culture 495: A, black mycelium; B, gray mycelium; C, white mycelium. IV, culture
527: A, gray mycelium; B, greenish black mycelium; C, white mycelium; D, black mycelium. V,
culture 940: A, greenish black mycelium; B, white mycelium, some greenish concentric circles; C, black
mycelium; D, white mycelium; E, white and black mixed.
Feb. 1,1921 Variations in Colletotrichum gloeosporioid.es 735
practically no black mycelium in the white cultures and but very little
white growth in the dark cultures.
Another set of transfers was made on January 24, 191 8, from the cul-
tures made January 9, with the result that the white cultures were pure
white but the black cultures still produced white hyphae. All plates
produced an abundance of spores.
Since the spores are asexual, I wished to determine if they would act
like parts of the mycelium when transferred. On January 29, 191 8, trans-
fers were made from the spores produced by the white mycelium, and
the resulting cultures were pure white, producing many spores. Also
spores were transferred from the black and white plates, and the resulting
cultures were black with some white hyphae, each culture producing many
spores.
The foregoing experiment seems to point to the fact that asexual spores
of Colletotrichum gloeosporioides act like mycelium when transferred.
The various types obtained by the mutations (fig. 2) are similar to the
strains I had in culture. Therefore, one might be led to conclude from
the foregoing data that Colletotrichum gloeosporioides is constantly giving
off new types under natural conditions, as well as in artificial cultures.
SUMMARY
(1) Colletotrichum gloeosporioides is a polymorphic species made up of
a number of strains.
(2) The various strains when grown on artificial media give distinct
cultural characteristics.
(3) Each strain is affected by its environment. The growth charac-
teristics as well as the spore size are varied by the medium on which
the strain is grown.
(4) This induced variation may be more or less permanent.
(5) There occur mutations in culture which iesemble the strains iso-
lated from the natural environment.
LITERATURE CITED
(1) Clausen, Roy E.
1912. A NEW FUNGUS CONCERNED IN WITHER TIP OF VARIETIES OF CITRUS
medica. In Phytopathology, v. 2, no. 6, p. 217-235, 1 fig., pi. 21-22.
Index to literature, p. 233-234.
(2) Edgerton, Claude Wilbur.
1908. THE PHYSIOLOGY AND DEVELOPMENT OF SOME ANTHRACNOSES. In Bot.
Gaz., v. 45, no. 6, p. 367-408, 17 fig., pi. 21. Literature cited, p. 405-
407.
(3) Essig, E. O.
191 1. WITHER-TIP OF CITRUS TREES (COLLETOTRICHUM GLOEOSPOROIDES Penzig).
In Pomona Col. Jour. Econ. Bot., v. 1, no. 1, p. 25-56, fig. 14-21.
(4) Fawcett, Howard S.
1915. CITRUS DISEASES OF FLORIDA AND CUBA COMPARED WITH THOSE OF CALI-
FORNIA. In Cal. Agr. Exp. Sa. Bui. 262, p. 149-211, 24 fig.
736 Journal of Agricultural Research vol. xx, No. 9
(5) Rolfs, P. H.
1904. WITHER-T1P, AND OTHER DISEASES OP CITRUS TREES AND FRUITS CAUSED
BY COLLETOTRICHUM GLOEOSPOROIDES. U. S. Dept. Agr. Bur. Plant
Indus. Bui. 52, 20 p., 6 pi.
(6) Shear, C. L,., and Wood, Anna K.
1913. STUDIES OF FUNGOUS PARASITES BELONGING TO THE GENUS GLOMERELLA.
U. S. Dept. Agr. Bui. 252, no p., illus., 18 pi. on 9 1. Literature
cited, p. 101-105.
(7) Stoneman, Bertha.
1898. A COMPARATIVE STUDY OF THE DEVELOPMENT OF SOME ANTHRACNOSES.
In Bot. Gaz., v. 26, no. 2, p. 69-120, pi. 7-18. Bibliography, p. 114-
117.
(8) Underwood, Lucien M.
1891. diseases OF THE orange in Florida. In Jour. Mycol., v. 7, no. 2, p.
27-36.
(9) U. S. Department of Agriculture. Bureau of Plant Industry.
1908. REPORT OF THE CHIEF OF THE BUREAU OF PLANT INDUSTRY, 1907. 93 p.
Washington, D. C.
Variations in Colletotrichum Gloeosporioides
Plate 86
Journal of Agricultural Research
Vol. XX, No. 9
PLATE 86
A, B. — Variation occurring in strain 900. The cultures were not made from a single
spore.
C. — Variation occurring in a culture of strain 990 which was made from a single spore.
25119°— 21 6
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Vol. XX FEBRUARY 15, 1921 No. 10
JOURNAL OP
AGRICULTURAL
RESEARCH
CONTENTS
Page
A Transmissible Mosaic Disease of Lettuce - - - 737
IVAN C. JAGGER
(Contribution from Bureau ot Plant Industry)
Leconte's Sawfly, an Enemy of Young Pines - 741
WILLIAM MIDDLETON
(Contribution from Bureau of Entomology)
Amylase of Rhizopus tritici, with a Consideration of Its
Secretion and Action ------- 7oi
L. L. HARTER
( Contribution from Bureau of Plant Industry)
A Comparative Study of the Composition of the Sunflower
and Corn Plants at Different Stages of Growth - - 787
R. H. SHAW and P. A. WRIGHT
(Contribution from Bureau of Animal Industry)
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE,
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
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WA8HINQTON : GOVERNMENT PRINTING OFFICE : I0St
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
CO
*****
for.
Ua«o*h
JOURNAL OF AG1TOCML RESEARCH
Vol. XX Washington, D. C, February 15, 1921 No. 10
A TRANSMISSIBLE MOSAIC DISEASE OF LETTUCE
By Ivan C. Jagger
Pathologist, Office 0/ Cotton, Truck, and Forage Crop Disease Investigations, Bureau
of Plant Industry, United States Department of Agriculture
During January, 1920, Romaine lettuce (variety Paris White Cos) in a
field of several acres at Sanford, Fla., developed a condition very sugges-
tive of a transmissible mosaic disease. The first symptom of disease was
a yellowish discoloration along the smaller veins of the younger expanding
leaves. This symptom was usually evident for only a few days, giving
way to a general yellowish, discolored appearance of the whole plant.
All gradations of discoloration occurred, from very marked to conditions
not distinguishable with certainty from normal. Close examination
usually revealed irregular blotches of an approximately normal green
color, which were usually located along the larger leaf veins. The
blotching varied from a few barely perceptible green areas on a yellowish
leaf to numerous pronounced green spots giving a marked mottled ap-
pearance to an occasional plant (Pi. 87, A). The leaves of diseased
plants generally seemed to be rather more wrinkled than those of normal
plants. Where plants became diseased only after reaching considerable
size, the older leaves, which were fully expanded on the first appearance
of disease symptoms, frequently continued to appear perfectly normal,
while all younger leaves developed the disease symptoms.
At the same time head lettuce (variety Big Boston) in a neighboring
field developed a similar diseased condition. The general yellowish,
discolored appearance of whole plants was frequently pronounced, but
in most cases the blotching was less marked than in the Romaine lettuce,
and a decided mottled appearance was never observed.
In general, diseased plants made a stunted growth. In severe cases
the plants were decidedly undersized, and occasionally the leaves formed
only a rosette, with no indications of a folding together of the tips to form
a head. Usually loose heads of poor quality were formed, although all
gradations of development, including occasional heads of practically
normal size and hardness, occurred. Often plants that showed marked
discoloration, mottling, and stunting soon after becoming diseased would
later seem to recover in part and to make a more or less normal growth
with only slight discoloration and mottling.
Journal of Agricultural Research, Vol. XX, No. xo
Washington, D. C Feb. 15, 1921
wt Key No. G-219
(737)
738 Journal of Agricultural Research voi.xx, No. 10
Attempts to islolate fungi or bacteria from the apparently healthy
plants were unsuccessful, at least in so far as it has not been possible to
isolate any organisms capable of producing the disease on reinoculation.
Furthermore, examination of the etiolated areas of the diseased plants
does not disclose the presence of any recognizable parasite.
Variations parallel in every respect to those described above have
been observed frequently by the writer in the mosaic disease of beans.
Approximately 75 per cent, or more, of the plants in these fields be-
came diseased. Frequent observations showed that aphids (Myzus
persicae Sulz.) were abundant on the lettuce during the time the disease
was developing. Similar conditions were observed in April, 19 19,
when the writer found what appeared to be the same disease in destruc-
tive amounts in several fields of head lettuce at Beaufort, S. C, which
were at that time nearly ready for harvest. Several growers stated that
aphids had been abundant in these fields a few weeks earlier. A disease
that seemed identical has also been observed every season for several
years in numerous localities in New York State, usually, however, affect-
ing only occasional plants and causing only minor losses. During four
seasons (1914-1917) it occurred in practically all fields of lettuce in the
vicinity of Rochester, N. Y., where aphids and other insects were usually
more or less abundant, while on the same farms lettuce grown during
the winter in the greenhouses, where aphids and other insects were held
at a minimum by fumigation, was usually entirely free from the disease.
In order to follow up experimentally these observations, which sug-
gested a relation between the mosaic disease and aphids, several insect
cages were constructed of cheesecloth, which were large enough to per-
mit the growing of several lettuce plants under each. Lettuce of both
the Big Boston and Paris White Cos varieties was grown from seed
under the cages in the field at Sanford, Fla., during the winter season of
1920, particular care being exercised to prevent any aphids from reaching
the plants except when intentionally placed on them. Myzus persicae
Sulz. was used in all the experiments.
On February 10 two aphids collected from several mosaic lettuce
plants were placed on each of 25 small healthy lettuce plants under an
insect cage. When these were examined, on March 8, there were 7
mosaic and 5 healthy Paris White Cos plants and 5 mosaic and 8 healthy
Big Boston plants. Twenty-five plants grown under an adjacent control
cage, under conditions comparable in every respect except that no aphids
had been placed on them, were all healthy, with the exception of one
mosaic plant. The plants were still small, having made slow growth
on account of cool weather. There were no aphids in the control cage.
In the aphid cage there were at least a few aphids on each plant, but they
were apparently not numerous enough to interfere materially with
normal growth.
Feb. is, 1921 A Transmissible Mosaic Disease of Lettuce 739
Six aphids from a colony on mosaic lettuce plants under a cage were
transferred on March 15 to each of 16 healthy, rapidly growing lettuce
plants under an insect cage. On March 27 several of these plants showed
the first symptom of the mosaic disease, as previously described, and
there were several aphids on each plant. On this date all aphids were
destroyed by drenching the plants with "Black Leaf 40" solution. On
March 3 1 there were 4 mosaic and 4 healthy Paris White Cos plants and
3 mosaic and 5 healthy Big Boston plants. Sixteen comparable control
plants under an adjacent cage were all healthy. On April 15 there
were 6 mosaic and 2 healthy Paris White Cos plants and 5 mosaic and 3
healthy Big Boston plants. All the 16 control plants were still healthy.
Both cages were free from aphids.
On March 22 three sets of comparable healthy, rapidly growing lettuce
plants under three insect cages were treated as follows: Ten aphids
obtained from the same colony on mosaic lettuce from which the aphids
in the preceding experiment were secured were placed on each plant
in cage No. 1. Ten aphids that had presumably never fed on lettuce
were collected from a potato field and placed on each plant in cage No. 2.
Cage No. 3 was left without aphids, as a control. The first symptom of
the mosaic disease was evident on 2 plants in cage No. 1 on March 30
(PI. 87, B). On April 14 all 4 Big Boston plants and 3 of the 5 Paris
White Cos plants in cage No. 1 showed the mosaic disease, while the 9
comparable plants in each of cages No. 2 and 3 were apparently healthy.
Aphids were abundant in cages No. 1 and 2 and were lacking in cage No. 3.
CONCLUSION
There occurs at Sanford, Fla., a serious infectious disease of lettuce,
apparently caused by a parasite not capable of isolation through ordinary
microbiological or bacteriological technic. The disease has been trans-
mitted experimentally from diseased plants to healthy plants by means
of aphids, particularly the species Myzus persicae Sulz. From the symp-
toms and general character of the disease, it should undoubtedly be
recognized as a true mosaic disease of lettuce.
PLATE 87
A. — Leaves of Romaine lettuce. Leaf in center from healthy plant; two others
from mosaic plants, one showing pronounced type of mottling and the other general
yellowish discoloration with few green blotches along larger veins.
B. — Young expanding leaves of head lettuce from experiment started March 22.
Leaf on left from healthy plant; two others from plant in early stage of the mosaic
disease, showing yellowish discoloration along smaller leaf veins.
(74o)
A Transmissible Mosaic Disease of Lettuce
Plate 87
Journal of Agricultural Research
Vol. XX, No. 10
LECONTE'S SAWFLY,1 AN ENEMY OF YOUNG PINES
By William Middleton
Scientific Assistant, Forest Insect Investigations, Bureau of Entomology, United States
Department of Agriculture
INTRODUCTION
The following paper on Leconte's sawfly, Neodiprion lecontei (Fitch),1
consists of a detailed description of the various phases of this insect and
summarizes the notes on the life and seasonal history. A few notes on
the economic importance and the means of control are added.2
In describing the larva special care has been taken, and such new terms
as have been introduced are carefully explained and illustrated. It is
believed that by the introduction of these terms it has been possible to
give a more nearly accurate description of the larva and that this termi-
nology will aid in the preparation of descriptions of larvae belonging to
allied groups. The terminology here used is the same as that applied
to Pteronidea ribesii (Scopoli), Neodiprion lecontei, and other sawfly
larvae in a paper ready for publication, and for the reasons therein con-
tained and to avoid possible confusion it seems advisable to continue the
use of the same letters to designate the same body areas.
Because of the feeding habits of the larva, Leconte's sawfly is an impor-
tant enemy to young pine trees in the eastern part of the United States.
It is especially injurious to nursery stock. While this paper deals briefly
with all of the phases of the insect, more detailed accounts of its life and
seasonal history, the damage done, and the means of control have been
reserved for future publications of a less technical nature.
DESCRIPTIONS
ADULTS
FEMALE (pl. 88, a)
Length of female 6 to 9.5 mm. Labrum narrowly rounded apically, the surface
shining and slightly concave; clypeus broadly subangulately emarginate, apical mar-
gin broadly depressed, the basal part convex, with small, poorly-defined punctures;
supraclypeal area flattened; antennal fovese large, shallow, connected with the deep
supraclypeal fovese; lateral foveae large, circular, deep; middle fovese and ocellar basin
shallow, poorly defined; postocellar area usually well defined, convex, wider
1 Order Hymenoptera, suborder Chalastogastra, family Tenthredinidae, subfamily Diprioninae.
2 All the rearing and experimental work on which this paper is based was carried on in the insectaries
and nurseries of the Eastern Field Station of Forest Insect Investigations, Bureau of Entomology, located
at East Falls Church, Va. The work has been done under the direction of Mr. S. A. Rohwer, specialist
in Forest Hymenoptera, and the author is indebted to him for the descriptions of the adults, helpful
suggestions, and many of the observations here recorded. Plate 88 was drawn by Miss Mary Carmody,
Plate 92 was photographed by H. B. Kirk, and Plates 89 to 91 were drawn by the author.
Journal of Agricultural Research, Vo1- :KX- No- IO
Washington, D. C. Feb- *. I921
I Key No. K-91
(74l)
742 Journal of Agricultural Research voi.xx.No.io
posteriorly, somewhat impressed medianly, about two and one-half times as wide as the
cephalo-caudad length; postocellar line distinctly shorter than the ocellocular line;
antennae robust, normally 19- jointed but varying from 18- to 21-jointed, apical joints
a little more than twice as wide as long, joints 3 and 4 subequal, the basal rami more
slender than the apical ones; pedicellum much wrider than long; head dulled, with
scattered shallow punctures; mesonotum shining, with separate distinct punctures,
anteriorly the punctures closer; scutellum with somewhat larger punctures; mesepis-
ternum punctato-reticulate ; first parapteron depressed anteriorly and ventrally omit-
ting the depressed area the outline forming an equilateral triangle; tergites, except
the ventral aspect, polished, impunctate; last sternite broadly, arcuately emarginate;
pad-like apical ventral portion of the sheath a little over four times as long as wide and
fitting close to the median ridge of sheath; venation normal. Head, prothorax, and
mesothorax rufo-ferruginous; mesosternum blackish to ferruginous; greater part of the
mesepisternum sometimes pale ferruginous; metathorax and abdomen black, ventral
aspect of tergites whitish, nates and sheath rufo-ferruginous, venter black or in part
ferruginous. Legs ferruginous, part of femora and bases of coxae blackish; bases of
tibiae and basitarsi whitish; occasionally the tibiae are all whitish. Wings vitreous,
subhyaline; venation dark brown. Antennae black.
male (pl. 88, b)
Length 5 to 6.5 mm. Labrum polished, the apical margin rather broadly rounded,
clypeus with the apical margin very gently arcuately emarginate, not depressed, the
surface sparsely punctured; lateral foveae practically wanting, other foveas as in
female; ocellar basin represented by a glabrous impression; postocellar area well-
defined , subconvex, not impressed , postocellar furrow arcuate ; postocellar line slightly
shorter than ocellocular line; head with large punctures, those on the front closer,
those on the vertex and occiput more widely separated; antennae 19-jointed; mesono-
tum with small separate punctures, those of the scutellum rather larger; mesepimeron
punctato-reticulate; hypopygidium broadly rounded apically, exceeding the geni-
talia. Black; labrum pallid; apices of mandibles piceous; legs below trochanters
and middle of venter reddish yellow. Wings hyaline, iridescent; venation pale
brown.
EGG
Egg 0.25 mm. long by 0.5 mm. broad; envelope very thin, whitish, smooth, shining,
translucent, and oval in outline.
LARVA (SIXTH INSTAR) 1
The following description is prepared from apparently full-grown
larvae from alcohol, approximating 21 mm. in length (Pl. 89, A).
1 In the description of sawfly larvae, both structurally and for color, it is necessary that particular areas
and regions of a segment or body wall be designated and that the designations adopted be applicable to
both the thorax and abdomen of the larva in all its stages. Further, the method, or system, should permit
by addition, elimination, change in shape, armature, and spotting of folds, areas, or regions, the comparison
with other larvae, and at the same time should avoid possible confusion of meaning. The following is a
suggestion for such a terminology and is the one used in the succeeding pages.
An intermediate (second to eighth, inclusive) abdominal segment of Neodiprion leccmtei (Pl. 91, B, E)
consists of tergum, pleurum, and sternum and begins with the transverse tergal fold immediately pre-
ceding that above the spiracle.
The tergum is composed of six transverse folds which are considered as representing four primary divisions
(A, B, C, D), with one, the third, twice subdivided (C'» 2> 3).
The pleurum is divided into three folds — the dorsal anterior one here called the preepipleurite, the poste-
rior one called the postepipleurite, and a ventral one called the hypopleurite — and two areas, one containing
the spiracle and the other, armed with a few spines, posterior to and adjoining that containing the spiracle.
The area containing the spiracle is at the lower extremity of fold B immediately above the preepipleurite
Feb. is, 1921 Leconte's Saw fly, an Enemy of Young Pines 743
HEAD (PL. 90, A-E)
Structural characters. — The dimensions of the head are 2.33 mm.
in height (dorsad-ventrad) by 1.75 mm. broad. The capsule (PI. 90, B, C)
is of thin chitin with two openings, the occipital foramen in the posterior
wall where the head joins the thorax and the buccal foramen in the
venter where the pharynx, mandibles, etc., are situated. The head con-
sists of the following sclerites, areas, and organs: Epicranium, eyes,
antennae, frons, adfrons, pleurostoma, hypostoma, clypeus, labrum,
and is termed the spiracular area, while the second area, that posterior to the above and armed with few
spines, is below folds C l> 2> 3 and is termed the postspiracular area.
The sternum consists of two transverse folds before the hypopleurites, one between and one behind
them. The hypopleurites bear the uropods.
These segmental divisions are all rather well defined externally by infoldings of the skin or body wall
(PI. 89, B; 91, D, E), which serve to bear the attachments of certain muscles. These muscles are of con-
siderable value in defining the folds but are not discussed here in detail, since they would require much
comparison of forms, bring matter irrelevant to the subject at hand into the paper, and can better be treated
fully in a separate paper after further study. It should be said, however, that the studies made thus far
seem to bear out the foregoing conclusions and to offer an excellent method by which to limit segments
and segment subdivisions and check up homology of the areas, abdomen to thorax, species to species, and
larva to adult.
The interpretation of the segmental composition and terminology outlined above is applied to the thorax
(PI. 91, A, D) in the following way: Each of the three thoracic segments (prothorax, mesothorax, and
metathorax) is 4-annulate tergally, and the ann illations when viewed with reference to ornamentation, shape,
position, and relation with one another homologize in order with the primary divisions (A, B,C, and D)
of the abdomen, the third, C, not being subdivided.
The pleurum is distinctly divided into four lobes, preepipleurite, postepipleurite, prehypopleurite, and
posthypopleurite, in all three segments; and the postspiracular area is present, in approximately its relative
abdominal position, in the mesothoracic and metathoracic segments, despite the absence or displacement
of the spiracle.
The sternum consists of three small, rather indistinct folds anterior to the leg's basal attachment to pre-
hypopleurite and posthypopleurite.
Further, the transverse circumference of the larva is divided into longitudinal areas of about equal width,
(PI. 91. F).
Tergum or Dorsum
Thetergum or dorsum in the present paper is intended to designate that portion of the larva which is
dorsad of the spiracular and postspiracular areas and which is divided into transverse folds or annulets
A, B, C, and D in the thorax, and A, B, C1'2-3 and D in the abdomen.
I<*.— Middorsal, a single longitudinal midtergal line.
I.— Dorsal, a pair of longitudinal tergal regions, one to either side of the middorsal line.
II.— Subdorsal, a pair of longitudinal regions, one to each side of the dorsal regions.
III.— Laterodorsal, longitudinal regions, laterad of subdorsal regions.
IV. — Supraspiraculai , longitudinal regions, laterad of latero-dorsal regions.
Pleurum or Latus
The pleurum or latus designates that portion of the larva between tergum and sternum.
V.— Spiracular, longitudinal regions, one to each side of the larva and ventrad of the supraspiracular
regions, with the abdominal spiracle situated therein in most sawfly larvae, including Neodiprion
lecontei.
VI. — Epipleural, longitudinal regions ventrad of spiracular.
VII. — Pleural, longitudinal regions ventrad of epipleural.
VIII.— Hypopleural or lateroventral, paired longitudinal regions, in which are situated the hypopleurites,
one to either side of the sternum and ventrad of the pleural regions.
Sternum or Venter
The sternum or venter designates that portion of the larva beneath the body between the uropods. The
ventrad projection of the uropods places them with reference to the position they occupy in relation to
other structures in the adventral longitudinal areas.
IX.— Adventral, paired longitudinal regions containing the uropods, one protruding from each hypo-
pleurite.
X. — Ventral, a pair of longitudinal sternal regions.
X« — Midventral, a single, midsternal, longitudinal line.
744 Journal of Agricultural Research vol. xx.No. w
epipharynx, tentorium (arms and bridge), hypopharynx, maxillae,
labium, and mandibles.
The epicranium is the largest area of the head, extending from the dor-
sal margins of thefrons and the lateral margins of the adfrons on the an-
terior wall of the head to the dorsal margin of the occipital foramen and
the lateral margins of the hypostomaon the posterior wall. The epicranium
is divided dorsally by a rather faint, median line, the epicranial suture
(PI. 90, G) , from the dorsal angle of the f rons to the occipital foramen,
and has a pair of slight, parallel seams beginning near the lateral extrem-
ities of the occipital foramen and extending a short distance dorsally.
It is moderately spined generally but has concentrations of spines in
the areas about the antennas, eyes, and pleurostomata. The eyes (Pi.
90, A, D) are a single simple pair, one occurring near each of the lateral
extremities of the head and slightly below a line drawn through the
dorsad -later ad angles of the frons. The antennae (Pi. 90, F) are paired
and occur one each about midway between each eye and the nearest
portion of the pleurostoma. They consist of an elongate projecting cone
anteriorly and two flat, floating pieces beyond, one of which is usually
faintly connected with a narrow band running forward around the cone.
Thefrons (Pi. 90, G) is an inverted, somewhat shield-shaped area and has
for its dorsal margin an angle projecting into the epicranium with its
apex at about the height of the head's greatest width. Its lateral mar-
gins are nearly parallel and about equal in length to the distance of
their separation, while the ventral margin is moderately concave. This
sclerite is spined according to a rather regular pattern, but the number
of spines and their position vary somewhat. The adfrons (Pi. 90, G)
consists of an elongate area of thick chitin situated laterad of the frons
and separating it from the epicranium. In outline each adfrons is some-
what triangular and supports the dorsal attachment of a tentorial arm
and the dorsal or anterior condyle for the mandible. The pleurostomata
(Pi. 90, B) are the thickened lateral margins of the epicranium which
extend in an arc around the base of each mandible and support
at their anterior and posterior extremities the points of articulation
of each mandible. The hypostoma (PI. 90, B) is a centrally narrowing
bridge with its dorsal margin formed by the somewhat angular lower
rim of the occipital foramen, its ventral margin formed by the
slightly curved posterior rim of the buccal foramen, and its lateral limits
defined by the slightly curved and thickened ridges running from the
lateral extremities of the occipital foramen to the ventral or posterior
fossae for the mandibles. The clypeus (Pi. 90, B) is a dorsally chitinous,
ventrally membranous area immediately below the frons and connect-
ing it with the labrum. It is armed with two pairs of spines arranged
to form a transverse row. These pairs are separated from each
other about two and a half times the distance between the individuals
constituting the pair. The labrum (Pi. 90, I) is slightly bilobed
Feb. 15, 1921 Leconte's Sawfly, an Enemy of Young Pines 745
or rounded laterally and subapically but has a median apical concavity
and is ornamented with a transverse row of two pairs of spines. These
two pairs are slightly farther apart than are the two spines compos-
ing each pair. The epipharynx (PI. 90, E, I) is a thin skin, armed to each
side apically, or under each lobe of the labrum, with a series of inwardly
diminishing, opposed setae or blades, lacking symmetry, which often vary
somewhat in number and arrangement. The tentorial arms (PI. 90,
B, C) are a pair of supports or struts diverging to the widely separated
pair of adfrontal triangles from the tentorial bridge (PI. 90, B, C), which
is a thickened central attachment of the hypostoma. The hypopharynx
(Pi. 90, E, J), or floor of the mouth, rests between and beyond the paired
maxillary laciniae and is a thin membrane, minutely ornamented. Each
maxilla (Pi. 90, J-N) is composed of cardo, stipes, palpifer, 4-jointed
palpus, galea, andlacinia. The labium (PI. 90, J, K, O) is composed of
submentum (or mentum and submentum fused), mentum (or labial
stipes), ligula, and, to each side of the latter and attached basally to the
mentum (or labial stipes), a palpiger surmounted by 2-jointed palpus.
The mandibles (Pi. 90, H) are 5-toothed.
Color. — The head capsule is orange-brown, excepting the spots sur-
rounding the eyes, which are black, and a part of the clypeus, which is
dark brown. The labrum is pale brown with its entire margin darkened,
the chitin of the maxillae and labium is brown to blackish, while the epi-
pharynx, hypopharynx, and ligula are pale white with their armatures
pale brown.
THORAX
Structural characters. — The prothorax (PI. 91, A) when examined
exteriorly and in its normal position appears to consist dorsally of but
two or three annulets, C and D always and B sometimes. This is due
to the constriction of the anterior circumference of the segment in its
connection with the head. An examination of the skin infoldings (PI.
91, D), however, will reveal all four of the primary divisions. On the
posterior margin of the segment, but caudad-ventrad of B, which is
always distinct supraspiracularly, there is a large, rather elongate area
in which the large thoracic spiracle is situated. Ventrad of B and an-
terior to this spiracular area is the preepipleurite ; below the preepipleu-
rite and the spiracular area is the postepipleurite ; and under the latter
comes the posthypopleurite, anterior to which, and rather strongly chiti-
nized, is the prehypopleurite. The prehypopleurite and posthypopleu-
rite support the 4-jointed legs. That part of the venter not occupied by
the prehypopleurite and posthypopleurite is divided by three transverse
folds into four annulations, the first annulation with a pair of latero-
ventral, chitinized areas, extending one from the base of each leg forward
to the occipital foramen, called neck plates. B supraspiracularly, C, pre-
epipleurite, postepipleurite, prehypopleurite, posthypopleurite, the leg
joints, and the second and third sternal folds are armed with spines.
746 Journal of Agricultural Research vol. xx, No. 10
The mesothorax (PI. 91, A, D) is not constricted in circumference
anteriorly and is readily seen to be composed of the four primary tergal
annulets, a small fold ventrad of A and anterior to the preepipleurite,
the postspiracular area, preepipleurite and postepipleurite, prehypo-
pleurite and posthypopleurite, 4-jointed legs, and four transverse
sternal folds. A, B, C, preepipleurite, postepipleurite, prehypopleurite,
posthypopleurite, leg joints, and third and fourth sternal folds are
armed with spines.
The metathorax (PI. 91, A, D) is similar to the mesothorax, except that
the small fold anterior to the preepipleurite and ventrad of A is larger and
bears hidden on its posterior surface an exceptionally small spiracle.
Color. — The prothorax is whitish with the iollowing exceptions: A
supraspiracular black spot on B; black prehypopleurite and leg joints;
and a pair of black sternal neck plates.
The mesothorax is whitish with the following exceptions: A subdorsal
black spot on A, B, and C; a spiracular and supraspiracular black spot
on B and C and the postspiracular area; a black preepipleural spot; and
black prehypopleurite and leg joints.
The metathorax is similar to the mesothorax.
ABDOMEN
Structural characters. — In an intermediate (second to eighth, in-
clusive) abdominal segment (PI. 89, B; PI. 91, B, E) the tergum consists
of six transverse folds (A, B, C '• 2' 3 and D). The pleurum is divided
into preepipleurite, postepipleurite, hypopleurite, spiracular area ven-
trad of B and bearing the spiracle, and postspiracular area posterior
to the spiracular area and below C1, 2l 3. The sternum is composed of
two transverse folds before the hypopleurite and one behind it. The
uropods project from the hypopleurites. Annulets A, B, and C2, post-
spiracular area, preepipleurite, and postepipleurite are armed with spines.
The first and ninth abdominal segments are similar (PI. 91 , C) but lack
a well-developed hypopleurite and uropod on venter and have four trans-
verse sternal folds.
The tenth abdominal, or anal segment (PI. 91, C) consists tergally of a
large undivided area termed the epiproct, or anal plate; pleurally, of a
somewhat triangular fold situated in the anterior portion of the segment
similar to the preepipleurite (the anal opening occurring transversely
across the apex of the segment) ; and sternally, of the postpedes, the
area from which they spring, and the postcallus below the anus. All
folds and areas, except the postpedes, are armed with spines. The
area around the base of the postpedes is, however, but slightly spined
or haired.
Color. — The intermediate (or second to eighth, inclusive) abdominal
segments are whitish, with the following exceptions: A subdorsal black
spot occurring and diminishing posteriorly on A, B, C1, and C2; a supra-
Feb. 15.1921 Leconte's Sawfly, an Enemy of Young Pines j/tf
spiracular black spot on B, C1, C2, and the dorsad extremity of post-
spiracular area; a black spot on preepipleurite ; and sometimes a small
blackish spot on postepipleurite. The first and ninth abdominal seg-
ments are similar but have the preepipleural spot smaller and the post-
epipleural spot almost always absent. The tenth abdominal, or anal,
segment is white but with the epiproct black.
LARVAL INSTARS
The larval life of sawflies of the group to which this species belongs is
divided into two distinct periods by a change of objective. The form
and color of the larvae differ considerably in these two periods. In the
first period the larvae are active and, as they devote most of their energy
to feeding, change rapidly in size. There are usually six molts. In the
second period the larva is more contracted, less active, and devotes its
energies to seeking a place for and constructing the cocoon. No feeding
is done in this second period and there is no molting. This second
period is generally termed the prepupal period, but other American
writers have referred to it as the ultimate stage.
These periods, stages, or instars are measured by the hatching of the
larvae from the egg and by the subsequent sheddings 01 moltings. The
larva molts after slightly varying passages of time, the extent of which
will be discussed later; and the molting, as a rule, is accomplished by the
longitudinal splitting of the prothoracic and mesothoracic skin mid-
dorsally, the breaking of the head capsule along the epicranial suture,
and the separating of the frons from the epicranium and the adfrontal
triangles. Through the opening thus formed the larva in its new skin
endeavors to extract itself from the old, and if successful begins feeding
anew, leaving the exuvia attached by the anal end to the needle.
The following descriptions of stages and approximate length of each
are the summary of notes from numerous rearings of larvae in quantities,
since it has been found that isolation of larvae not only tends to retard
development but often causes death. This method makes impracticable
an absolutely accurate account of the time spent by particular larvae in
each stage. The first appearance of shed skins and of what seemed to
be a new stage was, however, recorded and was utilized for description
and as an index for these approximations.
The larvae hatch from the eggs with slightly varying periods of incuba-
tion and develop at such different rates that following the first molt
there are always two and more often three or more stages present at one
time. From about the fifth stage a difference in size of the larva, depend-
ent upon sex, becomes noticeable, to confuse further an endeavor to de-
termine stages accurately.
All the stages are similar to the sixth stage, except as noted in the fol-
lowing desciiptions. A detailed description of the sixth instar has
already been given under the heading "Larva."
748 Journal of Agricultural Research voi.xx.No. 10
FIRST INSTAR
Structural, characters. — The larva increases in length from about
2 mm. at hatching to about 5 mm. at the beginning of the second stage.
In proportion the thorax is slightly large for the abdomen, whereas the
head is large for the thorax. The body spines are obsolete, and the spi-
racles are unusually large, having the appearance of being expanded.
Color. — The head is brownish with the eye spots, the labial and
maxillary chitin, and the apices of the mandibles blackish. The body
is unspotted and previous to feeding is entiiely yellowish gray, but upon
the filling of the alimentary canal it appears green or lead green. The
thoracic leg joints are blackish.
SECOND INSTAR
Structural characters. — The second stage develops in length from
about 5 mm. to 7.5 mm. The head is still large but the thorax and
abdomen are nearly normal in their relation to each other. The spiracles
are now about normal in their appearance, and the spines are becoming
fairly distinct.
Color. — The head is brownish with the eye spots and the labial and
maxillary chitin blackish. The body is unspotted and pale yellow-
green, with the spines appearing faintly grayish, and the thoracic legs are
blackish.
THIRD INSTAR
Structural characters. — The larva in the third instar grows in
length from 7.5 mm. to 10 mm. The head is still large, wider and higher
than the thorax, and the body spines are now prominent.
Color. — The head is brownish to brownish black, with the eye spots
black but not conspicuous. The lab rum, apices of mandibles, and chitin
of the labium and maxillae are brownish black. The thorax and abdo-
men are pale, usually unspotted, but in some larvae with very faint gray
supraspiracular spots and epiproct. The thoracic leg joints are black.
FOURTH INSTAR
Structural characters. — The larvae of the fourth stage lengthen from
10 mm. to 12.5 mm. The head is now about normal size in relation to
the body.
Color. — Approximately the same as in the sixth stage. The head
varies from brownish to orange and the body is normally spotted with
gray black.
FTFTH INSTAR
Structural characters. — The fifth stage increases in length from
14 mm. to 18 mm. Structure as in sixth stage.
Color. — Same as the sixth stage except that the head sometimes has
more brown and the body markings appear in some instances propor-
tionally larger and a deeper black than in the sixth stage.
Feb. 15, 1921 Leconte's Sawfly, an Enemy of Young Pines 749
SIXTH INSTAR
In this instar the larva grows from 18 mm. to about 22 mm. For
characters, see previous detailed descriptions of larva.
PREPUPA
The prepupa, or seventh larval instar, is the nonfeeding, cocoon-
spinning stage in which the larvae search out a suitable place to spend
their quiescent period. In size they usually measure from 10.5 mm. for
one which has spun a male or small type of cocoon, to 12 mm. for one
which has spun an average size large type or female cocoon.
HEAD
Structural characters. — The head is 2 mm. in height (dorsad-
ventrad) by 1.6 mm. broad, and except for being somewhat smaller is
similar to that in the preceding, or sixth, stage.
Color. — The head is pale whitish, usually grayish across the dorsum
above the eyes. The eyes are pale and are placed somewhat dorsad-
caudad of the center of the black oval spot surrounding them. The
antennal joints are inconspicuous, being yellowish white on a white
membrane. The frons, adfrons, clypeus, labrum, labium, and maxillae
are all pale, the heaviest chitin appearing only yellowish white while the
mandibles are pale excepting the teeth, which are brownish black.
thorax
Structural characters. — The thorax is similar to that of the sixth
stage larva.
Color. — The thorax is about the same as that of the sixth stage
except that the skin is pale white rather than yellowish white, with spots
grayish black rather than black; the mesothoracic and metathoracic
subdorsal spots are absent on fold C and very faint on B; and the legs
are entirely white.
ABDOMEN
Structural characters. — The abdomen is similar to that of the sixth
stage.
COCOON
The cocoon is a tough, single- walled, papery, red-brown cylinder with
hemispherical ends. The exterior, which is darker and less glossy than
the interior, shows some coarse threads and often has particles of sand
or other surroundings adhering to it. The cocoons vary in size for both
sex and individuals. In a number examined, the female, or larger
cocoons, varied from 9.5 to 11 mm. in length and from 4.5 to 5 mm. in
diameter, averaging 10.3 mm. long by 4.6 mm. in diameter. The male,
or smaller cocoons, vary from 7 to 7.8 mm. in length and from 3.2 to 3.5
mm. in diameter, averaging 7.5 mm. long by 3.4 mm. in diameter.
750 Journal of Agricultural Research voi.xx.No.io
pupa
Little is known of the pupa stage, but without doubt it is of short
duration, since pupae are rarely found when cocoons are cut open,
either shortly after being spun or up to the time they are a year old and
have practically all produced adults.
The following descriptions are prepared from a female pupa.
Structural characters. — The pupa is similar to, though some-
what larger and less hardened than the unemerged adults. The flagel-
lum of the antenna varies from 19 to 20 in the number of joints in the
specimens counted. The appendages are folded in or toward the venter
with the second pair of wings under the first pair which extend caudad-
ventrad. The shed prepupal skin is attached loosely to the apex of
the pupa's abdomen.
Color. — The pupa is entirely yellowish, the eyes, apices of the man-
dibles, and antennas being the first parts to darken with the develop-
ment of the adult.
UNEMERGED ADULT
The approach of the pupa toward the mature adult is accompanied
by a darkening, or coloration, and hardening of the body wall, which
before issuance becomes almost complete, and by the shedding or removal
of the pupal membrane or skin, by a reduction in size, and by an in-
crease in activity.
The following descriptions are prepared from an unemerged female
adult.
Structural characters. — The unemerged adult is similar to the
mature adult, and the shed pupal skin is attached loosely to the apex of
the abdomen.
Color. — The head is yellowish brown, with the eyes leaden, the anten-
nas brownish, the apices of the mandibles brown, and the labium and
maxillae yellowish white. The greater part of the thorax is yellow to
yellowish white, but some of the posterior sclerites (mesothorax in part
and all of the metathorax) are brownish. The wings are nearly com-
pletely developed with their veins brownish, and the legs, excepting
small portions, are yellowish white. The abdomen has the tergites (ex-
cept intersegmental skin) blackish with a broad, white, longitudinal
band along the spiracles; the pleural line white; the sternites white
medially, brownish near pleural line; and the reproductive parts mostly
yellowish.
LIFE AND SEASONAL HISTORY
The length of life of a colony, or the time between the depositing of
the first egg and emergence of the last adult, may be approximately
either 12 or 14 months — 12 months when the eggs are laid in the late
summer or early fall and 14 months when the eggs are laid in the later
spring or early summer. The length of life of a single colony has been
given the name "colony period."
Feb. is, 1921 Leconte's Sawfly, an Enemy of Young Pines
75i
From the cocoons of a single colony there are two periods of adult
emergence. The first period is termed "brood A," and the second
"brood B." When the colony period begins in late spring or early sum-
mer, brood A emerges in the late summer and early fall of the same year
and brood B emerges in the late summer and early fall of the following
year, making the length of the colony period 14 months. When the
colony period begins in the late summer, brood A emerges in the spring
and early summer of the following year and brood B emerges in the late
summer and early fall of the same year as brood A, making the length
of the colony period 12 months.
Thus (see year II in fig. 1) we may have adults of brood B of the first
colony period, brood B of the second colony period, and brood A of the
third colony period existing in the late summer of the same year. In
YEAR I
YEAR II
YEAR III
JUNE
JULY
AUG.
SEPT.
OCT.
f)PR.
M/tr
JUNE
JULY
AUG.
SEPT.
OCT.
apR.
MAY
JUNE
JULY
ffUG.
SEPT.
SGGS
LS/PME
COCOONS
ADULTS
EGGS
Lrt/H/ftE
COCOONS
rtOULTS
~
EGGS
LfiffME
COCOONS
ftDULTS
PlG. i.— Chart showing life and seasonal history of Neodiprion lecontei through the active period of three
years (November to March omitted, the insect being in the cocoon during this period).
the spring, however, it is possible only to have brood A, but these may be
from different colony periods (see year III in fig. 1).
The eggs are laid in a row of slits along one of the serrated edges of the
leaf (PI. 92, B) . These slits, the work of the female'ssaw, are about 1 .5 mm.
long and 0.8 to 0.9 mm. deep and have an interval between them about
equal to their length. They are somewhat shoe-shaped, the opening or
slit not entirely covering the pocket, and deepen slightly toward the
apex, or toe. These egg punctures are rather conspicuous, appearing
yellowish against the green of the undisturbed leaf tissue and becoming
brownish with age. Usually the leaves containing eggs die and become
noticeable some time after the hatching of the larvae.
In cage experiments the number of eggs laid by single females varied
from 25 to 178, with an average of 82. In six virgin females dissected
the number of eggs varied from 58 to 218, with an average of 139, so it is
752 Journal of Agricultural Research voi.xx.No.io
certain that in these experiments the maximum number of eggs was not
obtained. Available data indicate that approximately two-thirds of
all the eggs laid produce larvse.
As a result of there being two periods of adult emergence there are two
periods of oviposition and incubation during the year, coincident with
those of issuance, the first occurring in the late spring and early summer
(particularly May and June) and the second in the late summer and early
fall (late July, August, and early September).
The period of incubation as determined by the time elapsing between
the laying of the first egg and the hatching of the first larva varies from
13 to 21 days with an average, from six experiments, of 16 days.
For the first 5 or 6 days after oviposition very little change is noted
in the eggs, but beginning with the seventh or eighth -day a gradual
swelling is evident, so that by the ninth day there is a slight separation
of the sides of the mouth of the egg pocket. This separation increases
until it is 0.5 mm. in breadth shortly before the egg hatches and the larva
emerges.
The length of the larval feeding period, from the hatching of the eggs
to the appearance of the first prepupa, varies from 25 to 31 days, with
an average of 28 days.
During the whole of the feeding period the larvae are gregarious and
show little or no tendency to disperse. If disturbed while feeding they
throw back the head and thorax and remain motionless in that attitude,
attached to the needle only by the uropods.
The larvse for the first, second, and third stages eat only the epidermis
and the immediately adjoining tissue of the needles. The approximate
length of the first stage is 6 days, of the second 5 days, and of the third 5
days. Beginning with the fourth stage and continuing through the
sixth, the larvse eat the whole of the needle and occasionally portions of
the tender bark on the young twigs (PI. 92 , A) . Field observations on the
feeding upon the bark seem to indicate, however, that the species of the
tree may have more influence than the amount of foliage available. The
bark of the jack pine (Pinus banksiana) in Wisconsin and Virginia was
usually fed upon, even though there was plenty of foliage available.
The approximate length of the fourth stage is 5 days, of the fifth 4 days,
and of the sixth 4 days.
Following the larval feeding period comes the prepupal instar, a larval,
nonfeeding, cocoon-spinning, quiescent stage. The prepupae first seek a
suitable place and then spin their cocoons. In nature the cocoons have
only been found several inches under the surface of the ground under the
tree attacked.
After the cocoon is made the insect remains for a comparatively long
time as a prepupa, but shortly prior to the time of its emergence it trans-
forms to the pupa and then develops rapidly into the adult stage, which
FeD. 15, 1921 Leconte's Sawfly, an Enemy of Young Pines 753
cuts an end completely, or nearly so, from the cocoon, and issues. It is
in the cocoon that this insect passes the winter.
The length of the cocoon period, from its spinning until the issuance of
the adult, varies with the character of the colony. If the cocoons are
made by the larvae hatching from eggs laid in early summer (May or June)
there will be an emergence, called brood A, in the late summer or early
fall of the same year (late July, August, and early September) and a
second emergence from cocoons made by larvae of the same colony, called
brood B, in the late summer and early fall of the following year. In such
instances the length of the period between the first cococn and the first
adult of brood A varies from 13 to 23 days, averaging 18 days; and the
period between the first cocoon and the first adult of brood B varies from
364 to 379 days, averaging 371.
If, however, the cocoons are made by the larvae hatching from eggs
laid in the late summer or early fall the emergence of brood A will not
take place until late spring and early summer of the following year, while
brood B will emerge in the late summer and early fall of the same year as
brood A. In this instance the time elapsing between the making of the
first cocoon and the first emergence of brood A varies from 205 to 242
days, averaging 218 days, while that between the making of the first
cocoon and the first emergence of brood B varies from 292 to 342 days,
averaging 309 days.
The female adults seem to predominate throughout any period of
emergence and in a whole colony by the ratio of 3 to 1. Although the
females predominate for any given period of emergence or brood in the
sense in which it has been used in this paper, it is not unusual to find that
at either the beginning or end of the period males will emerge in the
majority.
EFFECT OF METEOROLOGICAL CONDITIONS
Eggs laid in late July and early August — that is, during the warmest
periods — hatch more quickly than those laid later or earlier in the year.
The particular period of the year, however, or the heat has not been
proved to be directly responsible for the speed of development, although
from temperature readings during the periods of incubation this would
seem to be a fact. For example, in June, 1917, when the mean tempera-
ture during the incubation period was 71.230 F., with a mean minimum
of 60. 140, eggs hatched in 21 days; in mid-August, when the mean tem-
perature during incubation was 74. 590, with a mean minimum of 63. 59°,
the eggs hatched in 18 days; and in late July and early August, 191 7,
when the mean temperature of the incubation period was 78.80, with a
mean minimum of 67. 690, eggs hatched in 13 days.
Further, the relation of humidity to development must be considered,
and it would seem from our records that high humidity tends to retard
incubation. For example, in June, 1917, when the average humidity
25150°— 21 2
754
Journal of Agricultural Research
Vol. XX, No. 10
during the incubation period was 74.30 per cent, eggs hatched in 21 days;
in mid-August, when the average humidity during incubation was 67.80
per cent, eggs hatched in 18 days; and in late July and early August,
1 91 7, when the average humidity of the incubation period was 65.57 Per
cent, eggs hatched in 1 3 days.
Table I. — Record of temperature and humidity during incubation period of Neodiprion
lecontei
Date.
Length of
incubation
period.
Mean maxi-
mum tem-
perature.
Mean tem-
perature.
Mean mini-
mum tem-
perature.
Mean rela-
tive humid-
ity.
1917.
Days.
21
13
18
°F.
82.33
89. 92
85-36
°F.
71.23
78.80
74-59
°F.
60. 14
67. 69
63-83
Per cent.
74-3°
July 30 to Aug. 11
65-57
Aug. 15 to Sept. 1
67.80
It is highly probable that normal development or acceleration is due
to the favorable combination or balance of both temperature and
humidity and that there are definite limits beyond which heat or moisture
would be either insufficient or excessive and result in retardation or
death.
Notes on the response of larvae of this species to meteorological influ-
ences are few and somewhat contradictory. The author has observed
a decided retardation of activity, feeding, and development, when damp,
cold, and cloudy weather occurs in the warm season, and a corresponding
acceleration on sunny days. Colonies were found feeding near Falls
Church, Va., on November 5, the day being bright but after a heavy
frost, while S. A. Rohwer records "nearly full-grown larvae feeding on the
sheltered side of a tree even though it was below freezing and snowing
hard," near Trout Lake, Boulder Junction, Wis., on September 21, 1913.
MATING AND COPULATION STUDIES
The females occasionally are, or seem to be, active in finding a mate,
but more frequently they appear to resist the attempts to mate offered
by the male, sometimes cutting off portions of his antennas and legs with
their mandibles. In those instances where copulation was observed
there were no preliminary attentions or courtship. Intercourse takes
place with the pair in positions in which their abdomens are opposed.
It was observed once that the male arrived in position by crawling over
the female from head to posterior end. When his abdomen had reached
the end of the female's he swung his under hers. During copulation the
wings are held flat against the body; the legs are spread rather far apart,
the forelegs projecting anteriorly, the middle legs slightly anteriorly, and
the hind legs posteriorly; and the antennae are <<Sually moved slowly,
up and down.
Feb. is. 1921 Leconte's Sawfly, an Enemy of Young Pines 755
Rohwer ' gives the following description :
Copulation lasts about 100 seconds and is accomplished by the two individuals
facing in opposite directions and the extreme end of the male abdomen being bent
at an obtuse angle because of the truncate abdomen of the female. The hypopygidium
of the male fits over the knob at the base of the sheath, the harpes grasp the sides
of the knob in the manner of a ball and socket joint, while the position occupied by
the parapenes, sagittae, volsellae, and penis valves, was not observed.
OVIPOSITION STUDIES2
After locating a suitable place for ovipositing, the female stands with
her legs grasping the needle, her abdomen bent ventrally so that its apex
comes in contact with the needle at a point between the mesothoracic
and meta thoracic tarsi. She seems to start the incision with the lance as
well as the lancets by pulling or sliding these away from her along the
^n
Fig. 2. — Position of end of abdomen of female when ovipositing, show-
ing the various parts and their position: i, lance; 2, apical part of
sheath; 3, basal part of sheath; 4, nates or ninth tergite; 5, eighth
sternite; 6, chitinized rods at base of lancet; 7, lancet.
needle in a fashion suggesting an attempt to catch a sharp point or tearing
edge in the tissue. After starting the incision she withdraws the lance
slightly and appears to use it to guide the lancets and to keep the
latter pressed against the front of the cut (fig. 2). After the insertion
of the lance and the lancets the female straightens or raises the ventrally
bent end of her abdomen, causing the ovipositor to form an abrupt angle
with it.
The chitinized basal rods of the lancets run along the chitinized ventral
side of the lance and turn into the abdomen towards the ninth tergite
Their up and down motion seems to be controlled by a somewhat side to
side movement of the nates, or ninth tergite. The lancets work opposite
each other except at withdrawal, when they are worked together up and
down arid back, following the lance through the arc of the cut they have
1 Rohwer, S. A. the mating habits of some sawfues. In Proc. Ent. Soc. Wash., v. 17, no. 4,
p. 195-198, fig. 1, pi. 22. 1915.
Page 196: Diprion lecontei.
2 Terminology used here is that adopted in a recent paper (still in proof) by S. A. Rohwer.
756 Journal of Agricultural Research vol. xx.No. 10
just completed. As the ovipositor is removed from the cut the female
squats over the freshly made opening and probably at this time deposits
the egg. The deposition of the egg could not be seen, but it is believed
that the egg does not descend through the ovipositor but that it is
dropped in place, leaving the body of the female through the spread bases
of the ovipositor, before the ovipositor is completely withdrawn.
The following is an account of the time spent by one female in each of
the different steps in the laying of an egg: In scratching the surface of
the needle endeavoring to start the incision, she spent 2 minutes and 13
seconds; in working the lance and lancets into the tissue, she spent
22 seconds; in beginning the pocket the female, with her abdomen bent
and close to the needle, worked for 27 seconds; and on the remainder of
the cutting of the pocket, with her abdomen raised, she worked 1 minute
and 49 seconds. The removal of the ovipositor and the deposition of the
egg were accomplished in 16 seconds.
PERIODIC APPEARANCE
Leconte's pine sawfly appears and disappears periodically. For several
years this species will be very abundant; then for a few years it will
become rare. The cause for this periodic disappearance has not been de-
termined, but it seems likely that some factors other than parasitism play
an important role, because we have no records which give a sufficiently
high percentage of parasitism to lead one to believe that this is entirely
responsible for a great reduction of the species. Investigation of certain
other means of natural control has thrown no light on the subject.
PARTHENOGENESIS
Experiments to determine if this species can reproduce parthenogeneti-
cally are inconclusive. In all these experiments only unfertilized females
of both emergence periods of brood A were used, and although all of them
weie failures the information acquired is inadequate to prove that the
adults of this brood can not reproduce parthenogenetically. Eight ex-
periments were performed, six of which produced eggs while two failed
entirely. In two experiments conducted under especially favorable con-
ditions the eggs hatched but the young larvae died without molting. It
is thus possible to state that females of brood A of this species can and
will lay eggs unfertilized and that these unfertilized eggs will hatch, but
in no experiments have these larvae produced adults.
HOSTS
This species appears to have three primary or preferred hosts and
a quantity of secondary or possible hosts. The primary hosts as de-
termined by observations in the field and the nursery are: Jack pine
(Pinus banksiana), which was subject to attack in Vilas and Oneida
Feb. is, 1921 Leconte's Sawfly, an Enemy of Young Pines 757
Counties in Wisconsin, at Kanawha Station, W. Va., and in the experi-
mental nursery at Bast Falls Church, Va. ; red pine (P. resinosa) , which
was commonly attacked in Vilas and Oneida Counties, Wis., and has
been recorded by a correspondent as being attacked at Hyde Park,
Dutchess County, N. Y., but which in experiments for oviposition by
adults and as food for larvae conducted in the nursery at East Falls
Church, Va., has always led to failures; and scrub pine (P. virginiana) ,
which is the native host of this insect through northern Virginia, Mary-
land, and Pennsylvania.
The secondary or possible hosts can not be ranked as complete hosts
capable of supporting the insect through all its various stages or as en-
tirely acceptable to females for cviposition. They have been deter-
mined by observation in the field and nursery, from correspondence and
literature, and through experimentation. They are white pine (Pinus
strobus) in Wisconsin and at Reading, Pa.; Scotch pine (P. sylvestris)
at Reading and Austin, Pa.; loblolly pine (P. taeda) Annandale, near
Falls Church, Va., and Clinton, La.; shore pine (P. contorta) at Kana-
wha Station, W. Va.; silver pine (P. monticola) in the nursery at the
Eastern Field Station; mugho pine (P. mughus), West Chester, Pa.; P.
eldarica, Yarrow, Md., chosen in the field and nursery; western yellow
pine (P. ponderosa) , used in experimentation (confining adults in a cage
upon the young tree); and longleaf pine (P. palustris),1 Austrian pien
(P. austriaca),2 and American larch (Larix americana)? mentioned in
literature and correspondence.
PARASITES
Neodiprion lecontei is subject to attacks by both parasitic insects and
a wilt. Four species of hymenopterous and four species of dipterous
adults have been reared from the cocoons of this species, but neither egg
parasites nor parasites which emerged from uncocooned larvae have
been obtained. The hymenopterous parasites were determined by
S. A. Rohwer as Exenterus diprioni Rohwer, Lagorotis diprioni Roh-
wer, L. virginiana Rohwer, and Perilampus hyalinus Say. Of these
parasites L. diprioni Rohwer is much the most abundant species, and
Perilampus hyalinus Say is probably a hyperparasite. The dipterous
parasites were determined by C. T. Greene as Phorocera claripennis
Macquart, Adomonita demylus Walker, Neopales maera Van der Wulp,
and Spathimeitenis spinigera Townsend.
The wilt of the larvae was probably a bacterial disease and was found
in Wisconsin by S. A. Rohwer, in 191 2. The larvae attacked were readily
distinguished by their lack of vigor and their white tracheal system,
1 Larvae sent in by a correspondent from Pinehurst, N. C, with the following note: "Eating the pine
needle of the longleaf pine in this vicinity."
2 RlLEY, C V. NINTH ANNUAL REPORT ON THE NOXIOUS, BENEFICIAL, AND OTHER INSECTS OF THE
state OF Missouri, p. 32-33. Jefferson City, Mo. 1877.
3 "When forced to, defoliate and girdle," in letter from W. D. Barnard, Boulder Junction, Wis.
758 Journal of Agricultural Research voi.xx, No. 10
which was conspicuous early in the disease when the larvae were yellow
and more noticeable later when the larvae became darkened. The wilt
was rather widespread in this locality of infestation, but though it killed
a considerable quantity of the larvae yet its success was limited.
From our notes and rearing records it would seem that none of the in-
sect parasites were abundant enough nor was the wilt sufficiently dis-
tributed and infectious to account for the periodic disappearance of this
species. It is certain that neither any nor all of these natural checks are
sufficiently numerous or effective to admit disregard of the artificial
control measures suggested below.
DISTRIBUTION
Neodiprion lecontei was described by Fitch from specimens collected
in New York, while Riley and Norton mentioned specimens coming
from Ridgewood, N. J. The localities represented in the United States
National Museum collection are Baltimore, Md., and Virginia (near the
District of Columbia), material collected by Theo. Pergande; and Long
Island, N. Y., material collected and reared by H. G. Dyar. The "Guide
to Insects of Connecticut" ' records the sawfly from Middletown,
Hampton, and Stamford, for that State. To these localities, through
collecting by members of the Bureau of Entomology and correspondents,
the following localities have been added (fig. 3) :2
Connecticut: Cheshire, Deep River, Ellington, New Haven, Norfolk.
District of Columbia: Throughout.
Louisiana: Clinton.
Maryland: Yarrow, Plummers Island.
Michigan: Remus.
Mississippi: Orange Grove.
New York: Hyde Park (Dutchess County).
North Carolina: Pinehurst.
Pennsylvania: Austin, Linglestown, Reading, West Chester.
Virginia: Falls Church and vicinity (generally throughout Arlington and Fairfax
Counties).
West Virginia: Kanawha Station.
Wisconsin: Generally throughout Oneida and Vilas Counties.
ECONOMIC IMPORTANCE
This species does considerable damage to both natural reproduction
and nursery stock by defoliating the trees. Complete or nearly com-
plete defoliation before late summer usually kills that part defoliated;
1 Viereck, Henry Lorenz, et al. guide to the insects of Connecticut, part hi. the hymen-
optera, or wasp-like insects, of Connecticut. Conn. State Geol. and Nat. Hist. Survey Bui. 22,
p. 44. 1916.
'Since this manuscript has been prepared this species has been received from the following additional
localities:
Connecticut: Hartford.
Florida: Orlando.
New Hampshire: Wonalancet.
Pennsylvania: Clearfield, New Germantown.
Feb. is, 1921 Leconte's Saw fly, an Enemy of Young Pines
759
and since this insect shows a very decided preference for young trees,
and the larvae often are numerous enough to strip the tree entirely of
leaves, many young pines are killed by this work alone. Trees not com-
pletely denuded often die because in their weakened condition they are
attacked by secondary insect enemies. When there is incomplete
defoliation and the tree recovers it is often stunted or misshapen and
is of little commercial or ornamental value.
FlG. 3. — Distribution of Neodiprion leconlei. The larger dots indicate places from which specimens have
actually been received. See also footnote 2, p. 758.
MEANS OF CONTROL
The control of this species depends largely on the extent and location of
the infestation. In large areas of either natural or artificial reproduction,
control because of its expense can not be generally practiced, but rangers
and lumbermen should make it a practice to destroy the colonies of these
larvae whenever they are found. The easiest way is to knock the larvae
from the trees and crush them with the foot.
In nurseries and in parks the control, in case of heavy infestation, can
best be attained before the larvae are full-grown and should consist of
thorough spraying. An arsenate of lead spray of 2 pounds of powder
to 50 gallons of water (or a ratio of 1 to 12) should be satisfactory. On
larvae which are discovered when young, less than >^-inch long, nicotine
760 Journal of Agricultural Research voi.xx.No.io
sulphate is a fairly satisfactory spray to use; however, because of the re-
sistance of conifers to arsenical sprays and because an arsenical treat-
ment gives more certain results, it is probable that the spray first
recommended should be used almost exclusively. In scattered infesta-
tions hand picking or knocking the larvae from the trees and crushing
them will be found to be much more economical and at least as effective.
Whenever these insects are observed in any locality and control
measures ate practiced against them, it is important that the territory
be carefully surveyed for the following 14 months, since it is possible
that some larvae may have escaped the treatment and have spun cocoons.
This possibility makes watchfulness necessary over the entire colony
period of the species in order that an emergence of adults from these
cocoons may not reestablish the infestation.
A. — Adult female.
B.— Adult male.
PLATE 88
Neodiprion lecontei:
Leconte's Sawfly, an Enemy of Young Pines
Plate 88
Journal of Agricultural Research
Vol. XX, No. 10
Leconte's Sawfly, an Enemy of Young Pines
Plate 89
Journal of Agricultural Research
Vol. XX, No. 10
PLATE 89 «
Neodiprion lecontei:
A. — Larva.
B. — Sixth -stage larva: The muscles of a single abdominal segment distributed over
several segments to show their numbers, position, and attachment.
PLATE 90
Neodiprion lecontei: Sixth-stage larva.
A. — Front view of head.
B. — Ventral (or apical) view of head capsule.
C. — Front view of head capsule.
D. — Lateral view of head. ,
E. — Sagittal section of head.
F. — Antenna.
G. — Frons, adfrons, and clypeus.
H. — Mandibles.
I. — Epipharynx and labium.
J. — Internal view of hypopharynx, maxillae, and labium.
K. — External view of maxillae and labium.
L. — External view of maxillae.
M. — Interior and apical view of maxilla.
N. — End view of maxilla.
O. — End view of labium.
EXPLANATION OF SYMBOLS
A, antenna.
AC, alimentary canal.
AdF, adfrons.
AE, attachment of extensor muscle.
AF, attachment of flexor muscle.
BF, buccal foramen.
C, clypeus.
Cd, cardo.
DC, dorsal or anterior condyle for mandible.
DF, dorsal or anterior fossa of mandible.
E, eye.
Epc, epicranium.
Ephx, epipharynx.
ES, epicranial suture.
F , frons.
G, galea.
Hypsim, hypostoma.
Hypx, hypopharynx.
Lac, lacinia.
Lbr, labrum.
Lig, ligula.
LP, labial palpi.
M, mandible.
MP, maxillary palpi.
Mtm, mentum.
OF, occipital foramen.
Pfr, palpifer.
Pgr, palpiger.
Plstm, pleurostoma.
PMBC, posterior margin of buccal cavity.
Sm, submentum.
St, stipes.
TA, tentorial arms.
TB, tentorial bridge.
VC, ventral or posterior condyle of mandible.
VF, ventral or posterior fossa for mandible.
Leconte's Sawfly, an Enemy of Youns; Pines
Plate 90
Journal of Agricultural Research
Vol. XX, No. 10
Leconte's Sawfly, an Enemy of Young Pines
Plate 91
^
%
vv§«
Journal of Agricultural Research
Vol. XX, No. 10
PLATE 91
Neodiprion lecontei: Sixth-stage larva.
A. — External view of the thorax.
B. — External view of the second and third abdominal segments.
C. — External view of the ninth and tenth abdominal segments.
D. — Internal view of thoracic skin.
E. — Internal view of the skin of the second and third abdominal segments.
F. — Diagrammatic cross section of the abdomen showing the longitudinal areas
of the body on its transverse circumference.
EXPLANATION OF SYMBOLS
Hypop, hypopleurite.
NP, neck plate.
Prep, preepipleurite.
Prhyp, prehypopleurite.
PSA, postspiracular area.
Psep, postepipleurite.
Pshyp, posthypopleurite.
SA , spiracular area.
Sp, spiracle.
Ia, middorsal; I, dorsal; II, subdorsal; III, laterodorsal ; IV, supraspiracular; V,
spiracular; VI, epipleural; VII, pleural; VIII, hypopleural or latero ventral ; IX,
ad ventral; X, ventral; and Xa, mid ventral.
PLATE 92
Neodiprion lecontei:
A. — Some defoliated twigs showing feeding on bark of stem.
B. — Eggs within needles of Pinus virginiana.
Leconte's Sawfly, an Enemy of Young Pines
Plate 92
Journal of Agricultural Research
Vol. XX, No. 10
AMYLASE OF RHIZOPUS TRITICI, WITH A CONSIDERA-
TION OF ITS SECRETION AND ACTION
By L. L. Harter
Pathologist, Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant
Industry, United States Department of Agriculture
INTRODUCTION
That certain mold fungi secrete amyclastic and other enzyms has
been known for a long time. However, much of the work in this direc-
tion has been centered around a few common forms, especially in the
genera Aspergillus and Penicillium. In fact, the same organism has
been selected by many investigators who studied the same or different
phases of enzymic production. The literature on the subject is already
very large and has been reviewed and listed in many of the publications
of recent years. For this reason the writer will refer only to such arti-
cles in the body of the paper as are germane to the particular subject
under discussion.
Rhizopus tritici was used for this investigation because it is responsible
for large losses of sweet potatoes and other vegetables under storage
and transportation conditions. Its parasitism has been proved repeat-
edly by inoculations into sweet potatoes, where it causes a rot identical
in appearance with that produced by R. nigricans. Preliminary ex-
periments were made with R. nigricans, which showed that it produces
amylase in abundance. No attempt has been made to duplicate with
R. nigricans the experiments carried out with R. tritici. So far as the
writer is aware these are the first experiments of the kind conducted with
R. tritici.
Some of the work of other investigators has been duplicated as far
as the method employed would permit, the purpose being to compare
Rhizopus tritici with some of the fungi hitherto studied. Some of the
results of previous investigators were corroborated, while others were
not, which indicates that no sweeping generalizations regarding all
fungi can be drawn from the study of a single organism.
METHOD OF EXPERIMENTATION
The investigations were carried out mostly with the powdered mycelium,
although the diffusion of the enzym into the culture solution was not
entirely disregarded. For certain phases of the work extracts of the
mycelium were used. The fungus was grown on a modified Czapek's
nutrient solution or on sweet potato bouillon for most of the comparative
Journal of Agricultural Research, Vol. XX, No. 10
Washington, D. C Feb. 15, 1921
wu Key No. G-220
(761)
762 Journal of Agricultural Research voi.xx.No. 10
studies. For some parts of the work Czapek's nutrient solution
was preferable, since it was then possible to cultivate the fungus in a
substrate of known composition. On the other hand, the fungus made
a luxuriant growth on sweet potato bouillon, and for experiments, such
as the influence of temperature on secretion, this medium was usually
employed.
The fungus was grown in 2-liter Brlenmeyer flasks containing about
750 cc. of the sterile solution, on which enough fungous felt was pro-
duced to carry out several comparative experiments.
Preliminary experiments showed that the fungus grew poorly on a
solution with sodium nitrate and cane sugar as a source of nitrogen and
carbon, respectively. Ammonium nitrate was therefore substituted for
sodium nitrate and glucose or potato starch, or both, for cane sugar in
Czapek's nutrient solution. The composition of the solution as finally
prepared is as follows:
Water j, 000. 00 cc.
Magnesium sulphate (crystals) . 5ogm.
Potassium acid phosphate 1. 00 gm.
Potassium chlorid . 50 gm.
Ferrous sulphate .01 gm.
Ammonium nitrate 5. 00 gm .
Glucose, starch paste, or both, in varying amounts to suit the require-
ments of the experiments as a source of carbon.
The sweet potato bouillon is prepared as follows: To the peeled
potatoes add double the weight of water; steam for one hour, then
squeeze out the liquid through gauze; steam a second time, filter into
flasks, and autoclave for 20 minutes at 13 pounds pressure. The sweet
potato bouillon always contains a considerable quantity of reducing
sugar and starch paste.
Rhizopus tritici grew well on both of these solutions and produced a
thick, heavy felt in from 7 to 10 days at a temperature of 250 to 35°C.
The better growth was made on the sweet potato bouillon. Contrary
to what might be expected, starch paste was more efficient as a source
of carbon in Czapek's modified nutrient solution than glucose. The
organism was grown in incubators, the temperatures of which did not
fluctuate more than 1 °.
At the end of the growth period the mycelium, which formed a thick
felt on the surface of the medium, was removed and washed in running
water for about 15 minutes. It was treated subsequently according to
Dox's (9) * modification of Albert and Buchner's " acetondauerhef e "
method. After washing, the mycelium was stirred constantly in an
excess of acetone for 10 minutes, squeezed as dry as possible, and treated
a second time for 2 minutes in a fresh supply. This acetone was removed
as in the former case, and the mycelium was treated with ether for 3
1 Reference is made by number (italic) to " Literature cited," p. 784-786.
Feb. is, 1921
Amylase of Rhizopus tritici
763
minutes. When air-dry the mycelium was put into small flasks and
held at a temperature of 90 C. until required for use. Experiments to
be discussed later will show that the mycelium can be held at 90 or even
higher for several months without any appreciable loss in its ability to
hydrolyze starch.
The hydrolysis by the mycelium or extract was carried out in 150-cc.
pyrex flasks. A weighed portion of the mycelium was ground in fine
quartz sand and transferred to the flasks, to which was added a measured
quantity of the starch paste solution made in distilled water. While
the percentage of starch is not material, a 0.5 per cent solution was used
for most of the work. After the addition of 2 cc. of toluol to each flask
as an antiseptic it was plugged by a cork with a small groove at the side
to allow for the escape of the expanded air when steamed at the close of
the experiment. Hydrolysis was carried out at different temperatures,
the results of which are shown elsewhere.
C. P. chemicals were used in the preparation of the culture media.
The Irish potato starch was obtained from Ehner and Amend. The
sweet potato starch was prepared by the writer. Preliminary experi-
ments showed that neither contained any reducing sugars. The sand
used for grinding the mycelium was purified by washing in distilled
water and then burning for an hour or more in a crucible. The water
in which sand so prepared was suspended did not reduce copper.
At the close of the digestion period the enzym was inactivated by
steaming the flasks in an Arnold steam sterilizer for about 15 minutes.
To avoid evaporation during the process of heating, oiled paper was
fastened with a rubber band over the cork and around the neck of the
flask. Before this method was finally adopted tests were made to deter-
mine the temperature reached in a given volume of solution in a given
length of time. Table I shows the results of these tests, made with tap
water in Erlenmeyer flasks, with an initial temperature of 140 to 150 C.
There was a small slit at the side of the cork to allow for expansion, and
a thermometer was run through it, with the bulb submerged in the water.
Table I. — Temperature reached by a certain volume of water when heated a given length
of time in an Arnold sterilizer {average of several tests)
Volume of
Capacity
Tempera-
water.
of flask.
ture.
Cc.
Cc.
°C.
Alinules.
5°
IOO
80. s
I
5°
IOO
93- 0
2
IOO
IOO
69. 0
I
IOO
IOO
89-S
2
IOO
IOO
96. 0
3
500
500
65.0
2
500
500
79-5
3
The loss of water by the use of the method described above was less
than 0.1 gm. in a flask of 150-cc. capacity containing 100 cc. of solution.
25120°— 21 3
764 Journal of Agricultural Research vol. xx,No.IO
After the flasks had been heated for 1 5 minutes the contents were filtered
through a fine quality of absorbent cotton to remove the mycelium and
sand. Filter paper was first tried but was finally rejected in favor of the
cotton for two reasons: (1) The solution filtered slowly, thereby intro-
ducing considerable error as a result of evaporation; (2) it removed
much of the nonhydrolyzed starch. After the filtrate cooled, the reducing
sugars were determined volumetrically, according to the method of
Clark (8). This is a quick and accurate method for the determination
of small amounts of reducing sugars by titrating the reduced copper
without removing it from the residual copper solution.
The results of starch hydrolysis set forth in the discussion of the
following experimental data are expressed in milligrams of reducing
sugars in a given volume of solution or in total reducing sugars formed.
The results are expressed mostly in milligrams per 10 cc, because 10 cc.
of solution are usually employed in making the titrations. If the quan-
tity of reducing sugars in 10 cc. of solution is known, the total reduc-
tion or that portion of the starch remaining nonhydrolyzed can be
calculated.
It is evident from the method employed that no account is taken of
products intermediate between the starch and reducing sugars. It is
likely that such products, for example dextrins, are formed in all cases,
but the determination of the reducing sugar meets the requirements of
the problem in hand, which has for its object mainly to show that a
vigorous starch-splitting enzym is formed by Rhizopus tritici, and also
some of the conditions upon which the production of this enzym -depends
and how certain environmental factors may influence its activity.
Various modifications of these methods were used in certain of the
experiments, but such changes in the methods required to meet the
needs of the experiments will be explained in sufficient detail when the
results of the experiments are presented and discussed.
It was shown by Dox (9) that a considerable autolysis of the fungus
mycelium actually takes place. In some enzym experiments where
hydrolysis is measured by the amount of reducing sugars formed, a
considerable error is likely to be introduced if a correction is not made
for the autolysis of the mycelium itself. A number of tests have shown
that the amount of autolysis produced from 0.25 gm. of mycelium sus-
pended in 50 cc. of distilled water varies from 1.20 to 7.39 mgm. per
10 cc, with an average of 6.38 mgm. Where a considerable amount
of reduction of the starch is involved, this amount would not introduce
a very considerable error. On the other hand, where the total hydroly-
sis is small a considerable error in the final results might be introduced.
In all experiments, except where the results would not be influenced
one way or the other, the autolysis of the mold was determined and
deducted from the total reducing sugars formed in the system.
Feb. is, 1921
Amylase of Rhizopus tritici
765
EXPERIMENTAL DATA
HYDROLYSIS OF RAW STARCH
Preliminary experiments showed that Rhizopus tritici produced an
enzym which hydrolyzed starch to reducing sugars. This fungus is com-
monly found as a cause of the decay of sweet potatoes in storage and
along with R. nigricans probably is responsible for the greater percentage
of decay attributed to the Mucoraceae. In just what form they utilize
carbohydrates when growing on the sweet potato is not known, but
that they are responsible for certain carbohydrate changes in the host
directly through their own activity or by stimulating the host to do so,
or both, will be shown by investigations now under way.
Most of the previous work with amylase secreted by fungi was carried
out with starch paste or soluble starch. This obviously is not the form
in which it occurs in the host, and although the enzym might digest
starch paste, it is not safe to conclude that it would act on raw starch, or
if at all, to the same degree.
Ward (24) concluded from the appearances of the starch grains of the
Irish potato that they were not acted on by Pythium, while Hawkins and
Harvey (14), on the other hand, found from a chemical determination of
the total starch present in the sound and rotted portions of the same
potato that the starch content was actually lower in the latter than
in the former. That all fungi do not behave the same as regards their
action on starch is evident from the fact that Hawkins (/j) found that
neither Fusarium oxysporum nor F. radicicola apparently alters the
starch content of Irish potato. It is evident from the results of the
authors just cited that no general conclusions can be drawn for all fungi
from the behavior of any one or more fungi. The first experiments,
therefore, were designed to test the comparative hydrolysis of raw starch
and starch paste. The results are given in Table II.
Table II. — Results of hydrolysis of raw starch expressed in terms of reducing sugar {aver-
age of several tests)
Mycelium.
Water.
Starch.
Time of
hydroly-
sis.
Tempera-
ture.
Hydroly-
sis in mil-
ligrams per
10 cc. of so-
lution.
Total
hydroly-
sis.
Source of starch.
Gm.
O.IO
.20
.20
.20
.20
.20
Cc.
5°
IOO
IOO
IOO
IOO
IOO
Per cent.
°-5
•S
•5
•5
•5
•5
Hours.
5-o
18.O
17- 5
17- S
17-5
i7-5
27- s
27-5
27-5
27. 5
27- s
27-5
I. 06
5-945
7-38
7.07
6.28
9. 12
Mgm.
5-3°
59- 45
73.80
70. 70
62.80
91. 20
Sweet potato.
Do.
Do.
Do.a
Irish potato.
Do.a
<* Starch macerated in sand before hydrolysis was started.
766 Journal of Agricultural Research voi.xx.No. 10
While an examination of Table II shows that both raw Irish and sweet
potato starch are hydrolyzed, no large amount of invert sugars are pro-
duced after hydrolyzation is carried on for 17.5 to 18 hours. Grinding
the starch in fine quartz sand does not seem to influence the amount of
hydrolysis appreciably.
That starch paste is more readily hydrolyzed than raw starch is evident
from the results of the following experiments. Two sets of flasks were pre-
pared to contain 0.2 gm. of powdered mycelium. To one set were added
100 cc. of sterile distilled water and 0.5 gm. of raw sweet potato starch,
and to the other 100 cc. of water containing 0.5 gm. of starch paste. A
third set contained 100 cc. of water and 0.2 gm. of mycelium but no starch.
Toluol was added as an antiseptic. Hydrolysis was carried on for 18
hours at 400 C. In the set with water and mycelium 1.98 mgm. of reduc-
ing sugar, representing autolysis of the fungus, were found per 10 cc. of
solution. This amount of reducing sugar was deducted from the results
obtained from the other two sets. Reducing sugars equivalent to an
average of 1.20 mgm. per 10 cc. of solution were obtained from the raw
starch, while 27.95 mgm. were obtained per 10 cc. from the starch paste
solution, or an amount more than 23 times as large.
INFLUENCE OF AGE OF MYCELIUM ON POWER OF HYDROLYSIS
To carry out any considerable number of comparative experiments at
different times the mycelium must be produced in quantity and kept for
some time. Before such material could be used for comparative studies
it was necessary to determine whether the mycelium lost its power of
digestion with age, and if so to what extent.
The mycelium was grown in large flasks on sweet potato bouillon.
At the end of 8 days' growth it was removed and prepared according to
the method already described. Hydrolysis was carried out at different
times at a temperature of 27. 50 C. for 19 hours by the use of 0.25 gm. of
powdered mycelium. A starch paste solution was prepared which con-
tained 53.4 mgm. of starch per 10 cc. of solution. This sterilized starch
solution was tightly stoppered to prevent evaporation and contamina-
tion and was stored at a temperature of 90. Two days after the mycelium
was collected the first experiment was conducted. Fifty cc. of the starch
paste and 0.25 gm. of the mycelium finely ground in sand were used in
150-cc. pyrex flasks, with 2 cc. toluol added as an antiseptic. Two flasks
with mycelium and starch paste and one control flask containing myce-
lium and 50 cc. of water were used in each test. The amount of autolysis
was deducted from the average of two closely agreeing samples. The
results appear in Table III.
There was a slight decrease in the amount of reducing sugars in the tests
of the last three months. From the results it seems safe to conclude that
the mycelium may be kept for several months without any appreciable
Feb. 15, 1921
Amylase of Rhizopus tritici
767
loss in reducing power. These results are in accord with those of Dox
(9), who found that mycelium may be kept almost indefinitely without
losing its activity.
Table III. — Amount of reducing sugars produced by the same samples of mycelium xised
at different times
Feb. 20.
Mar. s.
Mar. 18.
May 7.
June 10.
June 25.
Sept. 26.
Mgm.
216.337
Mgm.
225. 5
Mgm.
222. 65
Mgm.
233- 1
Mgm.
208. 55
Mgm.
205.4
Mgm.
204. 9
INFLUENCE OF DIFFERENT TEMPERATURES ON THE AMYLOCLASTIC
ACTIVITY OF THE MYCELIUM
Although it was shown by Table III that mycelium may be stored at
27.50 C. for a number of months without materially affecting the activity
of the enzym, it can not be concluded that it can be kept unimpaired at
any temperature. As a matter of fact, the following results show that
the hydrolytic power of the enzym is somewhat impaired when held for
a time at a high temperature.
The mycelium for these experiments was produced in six 2-liter flasks
containing about 750 cc. of sweet potato bouillon. At the end of the
growth period the mycelium was made into one composite sample and
held at a temperature of 90 C. for 18 hours. A sample was then removed
and its original hydrolytic power was determined. The remainder was
divided into three lots, one being stored at 90, one at 350, and one at 6o°.
To determine the original hydrolytic power of the mycelium two
0.25-gm. lots were weighed out and ground in fine quartz sand. To one
flask containing enzym powder were added 100 cc. of a 0.5 per cent
starch paste solution and to the other 100 cc. of sterile distilled water.
After the addition of toluol as an antiseptic both were digested for 18
hours at 400 C. In the former 2.2 mgm. and in the latter 33.46 mgm. of
reducing sugars were found in 10 cc. of solution, or a total of 22 mgm.
and 334.6 mgm. in 100 cc, respectively. These figures will serve as a
basis for comparison of future tests of the same lot of mycelium stored
at different temperatures. (Table IV.)
Table IV. — Amount of starch hydrolized by mycelium stored at different temperatures
for a given length of time
[Expressed in milligrams per 10 ec. of solution]
Tempera-
ture.
Original sam-
ple before
storage.
After 12 days'
storage.
After 39 days'
storage.
After 73 days'
storage.
°C.
9
35
60
33-46
29. 649
3 5- 148
22. 428
39- 429
37- 74o
21. 400
37-448
30. IOO
17. OOO
768 Journal of Agricultural Research voi.xx.No. 10
The results show that the hydrolytic power of the mycelium stored
at 6o° C. at the end of 73 days is somewhat more impaired than that of
mycelium stored at 350 and 90 for the same length of time. On the
other hand, the results indicate that the mycelium may be safely stored
for a considerable time at 90 and 350 without materially affecting the
enzym.
EFFECT OF TEMPERATURE ON THE HYDROLYTIC POWER OF THE ENZYM
It is generally understood that enzyms are more resistant to heat
when in the form of a powder than when in suspension. Kjeldahl (18)
found that the action of amylase at o° C. was very slow but increased
rapidly with the increase in temperature up to 6o° and at 700 became
insignificant. Similar results were obtained by Durandard (11), who
reports that the optimum temperature for the hydrolysis of rice starch
by an extract of Rhizopus nigricans to be 45 °. He obtained some
hydrolysis at io° and four times as much at 450 as at 300. It dimin-
ishes rapidly toward 55 °, becoming very feeble at 6o° and nothing at 700.
The writer found likewise the optimum temperature for the hydrolysis
of potato starch to be about 45 °, with a gradual decrease above that
temperature, becoming practically nothing at 6o°. Effront (12) con-
cludes also that the temperature has no other effect than to reduce the
diastatic power, and the nearer the temperature approaches 700 the
greater is the reduction. White (26) found that certain enzyms in dry
oats, among them diastase, were not injured on heating for 4K hours to
ioo°, but that an exposure for one hour at 1300 did destroy the ferments.
That the amylase contained in Rhizopus tritici is destroyed at a tem-
perature of 6o°C. is shown in the following experiments. Five-tenths gm.
of mycelium was extracted for 24 hours in each of two flasks containing
150 cc. of sterile distilled water at a temperature of 90. The contents of
the flasks were then filtered, and 100 cc. were pipetted into 250-cc. flasks.
Both flasks were exposed for an additional 100 hours, one at a tempera-
ture of 6o° and one at 90. The contents of each flask were then diluted
with 100 cc. of a 1 per cent starch paste and hydrolyzed for 18 hours
more at 400. At 6o° and 90 the reducing sugars formed per 10 cc. of
solution were on an average 1 .36 and 36.36 mgm. , respectively. Although
a little reducing sugar was formed, it is believed that it was derived by
autolysis of the mycelium during the period of extraction.
INFLUENCE OF GLUCOSE ON THE HYDROLYSIS OF STARCH
The stimulating and retarding effect of certain substances, especially
those identical with or similar to the products of hydrolysis, have been
subjects of investigations for a long time. Hill (75) found that glucose
interfered with the action of maltose, and Armstrong (1) pointed out a
number of cases where the reaction products inhibited the action of the
Feb. is, 1921
Amylase of Rhizopus tritici
769
enzyms. Kellerman (17) found that the alkalies without exception
seemed to be detrimental and the metals generally injurious to the action
of Taka diastase. From the results obtained by these and other investi-
gations it is evident that many substances influence the rate of action of
the enzym. The data shown here are the results of a single experiment.
Four flasks marked a,b,c,d were prepared, each to contain 0.25 gm. of
powdered mycelium. A second lot of flasks was prepared, and into flask
a were added 100 cc. of a 0.5 per cent starch paste solution; into flask b
125 cc. of a 0.5 per cent starch paste and 0.625 gm. glucose; into flask c
125 cc. of a 0.5 per cent starch paste and 2.5 gm. glucose; into flask d
125 cc of a 0.5 per cent starch paste and 6.25 gm. glucose. After thor-
ough mixing, 25 cc. were drawn from flasks b, c, and d, and the reducing
sugars were determined volumetrically. The contents of flasksb,c,andd
were then poured into the corresponding flasks containing mycelium
and digested for 18 hours at 400 C, with the results given in Table V.
Table V.— Amount of reducing sugars before and after hydrolysis
[Expressed in milligrams per 10 cc. of solution]
Sample.
Reducing
sugars original-
ly present.
Reducing
sugars present
at end of the di-
gestion period.
Increase in
reducing
sugars.
O
■53- °4Q
181. 580
438. 386
42. 476
86. 899
216. 080
472. 108
42. 476
33- 8S9
34- S°°
33. 722
It seems evident from the results of a single test that the presence of
glucose decreases the activity of the amylase, since the total reducing
sugars formed in sample a is considerably greater than in samples b, c,
and d On the other hand, the closely agreeing results of b, c, and d
indicate that the amount of glucose present at the strength used in this
experiment has no effect upon the hydrolysis of the starch.
RELATION OF QUANTITY OF STARCH PRESENT TO AMOUNT OF HYDROLYSIS
This subject naturally involves a consideration of the law of "mass
action " and in the literature on this subject there appears to be no
agreement of opinion on the question. The investigations show that so
far as enzyms are concerned so many factors influence the reaction that
no definite conclusion can be drawn. For example, Brown and Glen-
dinning (4) showed that when the concentration of the enzym relative
to the starch in the early stages is very small, the amount of starch
hydrolyzed per unit volume will be very large compared with the amount
of the combination of starch and enzym. If the concentration of he
unchanged substrate remains very large in relation to that of the
77©
Journal of Agricultural Research
Vol. XX, No. 10
combination, the latter will remain nearly constant in amount and equal
amounts of starch will be hydrolyzed in equal times, the curve being a
straight line. On trie other hand, when the concentration of the starch
has been greatly reduced, the amount of the combination and accordingly
the hydrolysis will follow more closely the law of "mass action." Similar
results were obtained by Armstrong working with lactose, maltose, and
emulsin. Other investigators have found various factors influencing the
reaction between the enzym concerned and the substrate. For a full
consideration of the theory involved in "mass action" the reader is
referred to a discussion of the subject by Bayliss (2).
The data submitted in Table VI are the results of a considerable number
of experiments which were varied to suit the requirements of the problem.
In the first series of experiments the amount of enzym power (0.25 gm.)
was constant and the volume of the starch paste solution was varied. The
time of hydrolysis was 19 hours at 320 C.
Table VI. — Total amount of reducing sugars and reducing sugars per 10 cc. of solution
in different volumes of a 0.5 per cent starch paste solution
Sample.
Volume of
solution.
Reducing sug-
ars per 10 cc.
Total reducing
sugars.
a
Cc.
5°
IOO
200
Mgm.
39. 984
25. 864
26. 600
16. 400
Mgm.
199. 92
258. 64
399.OO
328. OO
b
c
d
In sample a the reducing sugars per 10 cc. is considerably larger than in
sample d, while b and c are about the same. In total reducing sugars
found there is a progressive increase up to and including 150 cc, and then
a slight decrease. While in sample a some starch yet remained nonhydro-
lyzed, it is likely that on approaching the end point the rate of hydro-
lysis was slowed up. It is probable that a shorter period of hydrolysis
would have given a different curve and that the total reducing sugars
formed would have paralleled the reducing sugars per 10 cc.
Somewhat similar results were obtained when the total volume of solu-
tion (100 cc.) and the amount of enzym powder (0.25 gm.) were constant
but the quantity of starch paste was varied. A 1.5 per cent starch paste
solution was used in the dilutions, enough distilled water being added to
make a total volume of 100 cc.
The time of hydrolysis was 19 hours at 320 C. The average results of
parallel tests are shown in Table VII.
The results show an increase in reducing sugars with the increase in the
amount of starch present from sample a to sample c, inclusive, and then
a slight decrease. In sample a, although the end point had been more
closely approached than in any of the other samples, some starch still re-
mained unhydrolyzed . If it were not for the results obtained in samples d
Feb. is, 1921
Amylase of Rhizopus tritici
771
and e, it might be assumed that the accumulation of reducing sugars
acted as a paralyzer to further action of the enzym or, as has been sug-
gested by some investigators, the enzym entered into combination with
the products of the hydrolysis and consequently became inactive.
Table VII. — Amount of 1.5 per cent starch paste used, total reducing sugars, and reduc-
ing sugars per 10 cc.
Sample.
Total vol-
ume of solu-
tion.
Volume of
starch paste.
Reducing sug-
ars per 10 cc.
Total reducing
sugars.
a .-
O O Q O O O
OOOOOO
H H H M M M
Cc.
20
40
60
80
IOO
OO
Mgm.
21. 240
35-632
37. 842
33- 498
32-55I
5. 198
Mgm.
21. 24
356-32
378. 42
334- 98
325-5I
51.98
b
c
d
e
f
In the series of experiments reported in Table VIII different amounts of
a 1 per cent starch paste solution were used, and enough water was added
to make a total volume of 500 cc. One-fourth gm. of enzym powder was
added to each set of flasks. The time of hydrolysis was 1 8 hours at 400 C.
Table VIII. — Amount of 1 per cent starch paste used, total reducing sugars, and reducing
sugars per 10 cc.
Sample.
Total
volume of
solution.
Volume of
starch
paste.
a
Cc.
500
500
500
500
500
500
500
Cc.
20
SO
IOO
200
300
400
500
b
c
d
e
f
g
Reducing
sugars per 10
cc. of solution.
Mgm.
3- ^50
6. 3050
8- 9375
10. 4000
11. 9600
12. 1550
8- 1575
Total reducing
sugars.
Mgm.
159. 250
315- 250
446. 875
520. OOO
598. OOO
607. 750
407. 875
The amount of reducing sugars per 10 cc. increases with the increase
in the amount of starch from sample a to sample f and then decreases.
An approach toward the end point might here also account for the lesser
amount of hydrolysis in the more dilute solutions if the total reduction
in sample g, which contains the largest amount of starch, was not actually
less than in several of the other samples.
A final series of experiments was carried out in which the total volume
of 0.5 per cent starch paste was varied but the amount of enzym powder
(0.25 gm.) was constant. Hydrolysis was carried on for 18 hours at
400 C. (Table IX.)
There was a decrease in the reducing sugars per 10 cc. and an increase
in total sugars as the volume of the solution increased from sample a to
sample e, and then a reverse of the process.
772
Journal of Agricultural Research
Vol. XX, No. 10
Table IX. — Volume of 0.5 per cent starch paste solution used, total reducing sugars, and
reducing sugars per 10 cc.
Sample.
Volume of
starch
paste.
Reducing
sugars per
10 cc.
Total reducing
sugars.
a
Cc.
5°
100
200
300
400
500
Mgm.
45.16
37-64
28.28
19.74
14.42
9.80
Mgm.
225. 8
376-4
565-6
592.2
576.8
490. O
b
d
e
f
IS AN END POINT IN HYDROLYSIS REACHED?
Theoretically an end point should not be reached without shifting the
point of equilibrium of the solution. As a matter of fact, to settle the
question is difficult by any method, since there may be intermediate
products between starch and reducing sugars which are not revealed
by the iodin test and do not reduce copper. The experiments were made
with extracts of the mycelium. The mycelium (1.5 gm.) after powdering
was extracted in a pyrex flask for 24 hours at 90 C. in 300 cc. of distilled
water. The extract was then filtered. Two hundred fifty cc. of the
extract were then diluted with 250 cc. of a 2 per cent starch paste solu-
tion. After thorough mixing 20 cc. were drawn off, 2.5 cc. concentrated
hydrochloric acid were added and the mixture was hydrolyzed by boiling
for 2.5 hours. The solution was neutralized with sodium hydroxid
made up to 200 cc. with water, and the starch present was determined
as reducing sugars. A preliminary test showed that no reducing sugars
were present in the original starch paste solution. After hydrolysis
reducing sugars equivalent to 104 mgm. of starch per 10 cc. were found.
The solutions were mixed on May 22 and hydrolysis carried out at
450 C. Reducing sugars were determined approximately 24 hours apart
for several days thereafter with the results shown in the Table X.
Table X. — Amount of reducing sugars at different dates and equivalent in starch
[Expressed in milligrams per 10 cc]
Reducing
sugars.
Equivalent
in starch.
May 23
24, 9.30 a. m
24, 3.30 p. m
26
27
28
29
3°
31
June 9
56- 516
79. 236
84. 518
99. 968
103. 092
105. 364
108. 866
108. 866
108. 866
108. 889
52
73
78,
92.
95
97
101
101
101
101
560
689
602
970
87s
988
245
245
245
267
Feb. i5> 1921 Amylase of Rhizopus tntici 773
The results show that the amount of reducing sugars steadily increased
for the first 7 days but remained practically stationary thereafter. At
the end of 18 days a small amount of starch yet remained nonhydrolyzed.
To determine whether the addition of a small amount of starch would
stimulate further hydrolysis, 100 cc. of the solution described on page 772
were mixed with 100 cc. of an approximately 0.5 per cent starch paste
solution. A small amount (20 cc.) was drawn off, and the actual amount
of starch was determined. The remainder was hydrolyzed at 45 ° C.
After acid hydrolysis reducing sugars to the amount of 1,568 mgm.
were found in 200 cc. of the original solution. Of this amount 1,088.89
mgm. of reducing sugars and 27.33 mgm. of nonhydrolyzed starch
(equivalent to 29.38 mgm. reducing sugars) were brought over to the
solution when the dilution was made, making a total of 1,118.27 mgm.
reducing sugars. Deducting this amount from the amount originally
found (1,568 less 1,118.27 mgm.), the result gives the amount of reducing
sugars added in the form of starch, or 449.73 mgm. This is calculated
to be equivalent to 418.2489 mgm. of starch. To this amount should
be added 27.33 mgm., the quantity of nonhydrolyzed starch present
before the solutions were mixed, making a total of 445.58 mgm. starch
present in 200 cc. of the solution when hydrolysis was started. After
hydrolysis had gone on for 24 hours a sample was taken, and the reducing
sugars were determined, which gave in 200 cc. a total of iJ5i6.6 mgm.
There was no starch left in the solution according to the iodin test.
Since in the original solution there were 1,568 mgm. of reducing sugars
present, 51.4 mgm. (equivalent to 47.8 mgm. starch) remain unaccounted
for, except as intermediate products between starch and reducing sugars.
Parallel experiments, which will not be given in detail, gave similar
results.
The evidence brought out shows that an equilibrium is established
in the solution before quite all the starch is hydrolyzed. Also that if
more starch is added and the solution is diluted the starch finally dis-
appears so far as its presence is indicated by the iodin test.
So far as these and many other results go, an end point is reached if
the disappearance of the starch alone is considered. Viewed from the
standpoint of reducing sugars found, an end point is not reached. Many
experiments not designed primarily to demonstrate this point have
shown that no starch, as indicated by iodin, remains in the solution after
a definite length of time. On the other hand, starch is shown to be
present in some solutions by the same test after a considerable time.
It was also shown by experiments that if an end point was not reached
at a certain temperature, namely 45 ° C, the starch would completely
disappear in 24 hours by shifting the solution to a temperature of 350.
Perhaps an explanation of some of these facts may be found in the
results of other investigations. The results of the above experiments
show that all the starch was not accounted for as reducing sugars,
774 Journal of Agricultural Research vol. xx.No. 10
although in such solutions no starch was present, if judged by the iodin
test. This difference might be explained by the presence of dextrins as
intermediate products. Brown (j) claims that in the action of diastase
on starch the reaction ends when the composition of the product is 80.8
per cent maltose and 19.2 per cent dextrin. Maquenne and Roux (21),
however, suggest that the equilibrium of 80.8 per cent maltose and 19.2
per cent dextrin referred to above is due to insufficient activity of the
enzym and that if malt diastase is activated by acid in small amount
the whole of the starch is found to be converted into sugar, so no dextrins
remain. Bayliss (2) found that the amount of maltose produced in the
first stage was greater than the equilibrium position of Brown and
Heron because it was allowed to proceed for a longer time.
Although the writer did not use a temperature above 400 C, this
temperature might have had some bearing on the proportion of sugar to
dextrins, in accordance with the interesting results of Brown and
Heron (5).
These investigators found that the dextrinase is more injured by a
temperature of 68° C. than the amylase. According to this theory they
explain the fact that when starch paste is acted on by diastase which
has been exposed to a temperature of 68° there is less maltose and more
dextrin formed than when the enzym has not been so heated. This
raises the question as to just where the influence of temperature makes
itself felt. Furthermore, facts which might bear upon the question
were brought out by Tammann (2j), who reports that an increase of
hydrolysis was obtained in a stationary system by altering any of the
other conditions of the equilibrium, such as the addition of more amyg-
dalin, renewal of the products of the reaction, raising the temperature,
or increasing the dilution. In Tammann's work the retardation would
virtually be due to the accumulation of the products of the reaction.
GROWTH AND HYDROLYSIS IN A SOLUTION OF STARCH PASTE
The remarkable power of Rhizopus tritici to grow on almost any kind
of medium is evident when we consider that it can be isolated from a
great variety of decayed substances. Its ability to hydrolyze starch in
a solution poor in nutrient material was tested several times by inoculat-
ing a starch paste solution made with distilled water. While such a
solution would contain nutrient substances in addition to the carbo-
hydrates introduced in the form of starch, a considerable growth would
hardly be expected, but, nevertheless, a fair growth was made and
hydrolysis of the starch went on.
The experiments were made in Hrlenmeyer flasks containing 500
cc. of a 0.5 per cent starch paste solution. Some of the inocula-
tions were made with bits of mycelium and spores and some with spores
alone. Growth was slow at the outset, the colonies being submerged
Feb. is, 19*1 Amylase of Rhizopus tritici 775
at first, a felt forming later on the surface of the liquid. The solutions
were tested for reducing sugars at the beginning of the experiments,
but in no case were any found. The fungus must then of necessity have
either to utilize the starch directly or first have converted it into some
simpler form. From time to time some of the liquid was drawn off,
and the reducing sugars were determined. The results showed an
increasing amount of reducing sugars present with each subsequent
determination, from which it is evident that the fungus hydrolyzed the
starch in excess of its needs. If the growth continued long enough the
solution which was milky in color at first finally became clear, showing
that practically all the starch was hydrolyzed. Many experiments in
the course of these investigations likewise demonstrated clearly that the
fungus hydrolyzed the starch in the solution, although reducing sugars
were already present. Furthermore, the hydrolysis of the starch in a
solution of starch and glucose began very soon after inoculation, which
suggests that the enzym diffuses into the solution soon after the begin-
ning of growth. This subject will receive further consideration in the
discussion of an extracellular enzym.
EXTRACELLULAR ENZYM
The results in the following experiments show other interesting facts
in addition to the production of an extracellular amylase. Two nutrient
solutions a and b, differing in the source of nitrogen, were used. Solution
a had the following composition :
Water 1, 000. 00 cc.
Magnesium sulphate (crystallized) ^ .50 gm
Potassium acid phosphate 1. 00 gm
Potassium chlorid .50 gm
Ferrous sulphate .01 gm
Sodium nitrate 2. 00 gm
Starch 10. 00 gm
Solution b differed from a in that the sodium nitrate was replaced by
5 gm. of ammonium nitrate.
The chemicals were first dissolved in the water by steaming, after
which the starch was added and the entire mixture was sterilized by
autoclaving.
The growth in these two solutions was remarkably different. In a
the mycelium was mostly submerged, while in b a thick felt was formed
on the surface. Solution a produced in 16 days of growth a total dry
weight of 0.0298 gm. ; b, 0.7198 gm., or about 24 times as much. Both
solutions were inoculated on October 27. The reducing sugars and
starches were determined at stated intervals thereafter, as shown in
Table XI.
776
Journal of Agricultural Research
Vol. XX, No. jo
Table XI. — Amount of reducing sugars and starch present in solutions a and b at stated
intervals of time
[Expressed in milligrams per 10 ce. of solution]
Solution a.
Reducing
sugars.
Starch.
Solution b.
Reducing
sugars.
Starch.
Oct. 27
29
3i
Nov. 3
5
7
10
12
3 ('controls)
12 (controls)
o
5-3
21. 7
46. o
61. 4
°5-3
75- o
75-8
o
III. o
103.0
83.0
59- o
41. o
38.5
25.2
23-4
5-8
34-6
35-2
21. 6
14. o
7-7
6.0
106. o
96. o
47.0
24. o
25. o
23.8
22.8
21. 6
From Table XI it is seen that in two days reducing sugars in excess of
those used by the fungus were produced with a decrease in the amount of
starch. In the a solution the reducing sugars gradually accumulated to
the end of the experiment, while the amount of starch decreased, showing
that the fungus did not use a corresponding amount of the reducing sugars
formed. On the other hand, in solution b the reducing sugars increased
up to November 3 and then decreased to the close of the experiment,
while the starch decreased rapidly to November 3 and very little there-
after, which suggests that hydrolysis was slowed up as it approached
the end point and did not keep pace with the demands of the fungus
for reducing sugars. This condition is reflected in the amount of dry
matter formed, which is about twenty-four times greater in solution b
than in solution a. The amount of starch in the two solutions at the
close of the experiment was practically the same. It seems, then, that
an extracellular amylase was promptly secreted by the fungus and that
it hydrolyzed the starch in excess of the needs of the fungus in one case
(a) to the close of the experiment and in the other until November 3,
when the reducing sugars consumed exceeded those produced by the
hydrolysis of the starch.
Why the difference in the composition of the two solutions plays such a
fundamental role in the growth of the fungus can not be answered. As
previously stated, solution a derives its nitrogen from sodium nitrate
and solution b from ammonium nitrate. The growth in the latter case
was many times greater than in the former. Since solution a was vir-
tually Czapek's nutrient solution, it was tried at the outset for other
work of a similar nature and was later modified by the substitution of
ammonium nitrate for sodium nitrate. The solution so modified gave a
luxuriant growth of mycelium. Solution a, however, apparently had
no inhibitory action on the amylase, so that hydrolysis of the starch went
on unhindered.
Feb. xs, 1921
Amylase of Rhizopus tritici
111
REMOVAL OF AMYLASE BY FILTERING
The enzym powder was extracted for 24 hours in sterile distilled water.
The contents of one set of flasks was filtered through absorbent cotton,
which removed the fragments of mycelium, and the others were filtered
through four thicknesses of No. 1 Whatman chemically prepared filter
paper. A quantity of this filtered extract was then mixed with an equal
volume of a 1 per cent starch paste solution and hydrolyzed for 18 hours
at 400 C. At the close of the period of hydrolysis the reducing sugars
were determined in the usual way. The average of several parallel
experiments showed that when filtered through cotton, 172.51 mgm.
reducing sugars were formed in 100 cc. of solution but that only 129.32
mgm. were formed when filtered through filter paper.
INFLUENCE OF TEMPERATURE AT WHICH MYCELIUM IS GROWN ON ITS
POWER OF HYDROLYSIS
The investigations of the writer and others have shown that the
optimum temperature for the activity of amylase is about 45 ° C. and
that activity is reduced by higher and lower temperatures. Since these
results, however, were obtained from mycelium grown at one tempera-
ture, the question was naturally suggested whether the temperature at
which it was grown did not influence the amount of amylase produced.
The mycelium was grown on sweet potato bouillon in 2-liter Krlenmeyer
flasks. One set of flasks was incubated at 90, one at 290, and one at 400.
At the close of the incubation period (10 days) the mycelium was removed
from the flasks and treated with acetone and ether in the usual way.
The mycelium from the flasks held at the same temperature was made
into a compound sample and stored at 90 until used.
The hydrolytic power of the enzym was determined by the use of 0.25
gm. of powder in all tests but two. With the smaller amount of enzym
powder hydrolysis was carried out with 50 cc. of a 0.5 per cent starch
paste solution; with all others 100 cc. were used. The time of hydrolysis
was 18 hours at 400. At the close of the experiment, the enzym was inac-
tivated by steaming for 10 minutes. The results are given in Table XII.
Table XII. — Results of hydrolysis of starch by mycelium grown at different temperatures
Temperature.
Milligrams reduc-
ing sugars per
10 cc.
•c.
9
29
40
39- 7°o
26. 854
9-933
The results show a very striking influence of the temperature on the
production of amylase. A temperature of 400 C. represents about the
maximum temperature for growth and 90 the minimum, while a good
778 Journal of Agricultural Research vol. xx, No. 10
growth occurs at 290. At first thought one might suspect that at the
higher temperature the enzym diffuses out into the solution more readily
than at the two lower temperatures, and, indeed, one can not say such is
not the case. If the hydrolytic capacity of the enzym corresponded to
the growth of the fungus in the nutrient solution, as it does not, such a
theory might receive strong support. The poorest growth is at the
lowest temperature. At 90 the mycelium was mostly submerged, and
no fruiting had taken place. On the other hand, at 290 and 400 a thick
felt had formed, with some fruiting, though less at 400 than at 290.
QUANTITATIVE REGULATION OF AMYLASE
The results of many investigations have shown a quantitative regula-
tion of certain enzyms of various fungi. Brunton and MacFayden (7)
found that a bacterium produced diastase when cultivated on starch
paste but not when grown on meat broth. In the latter case a pep-
tonizing enzym was produced. Pfeffer (22) found that in several mold
fungi the secretion of diastase depended upon similar conditions, and
Brown and Morris (6) claim a similar regulatory action with barley, in
that when readily assimilable substances were supplied the secretion of
diastase did not take place, but when no such substances were available
diastase was formed at once. It was likewise found by Wortmann (27)
that certain molds had the power of excreting a starch-dissolving enzym
when starch grains were the only available food and that no secretion
took place if sugar or tartaric acid was offered to the organism along
with the starch. More recent workers have arrived at similar results
with different fungi. Went (25) showed that Monilia sitophila secreted a
number of enzyms, some of which were produced only when the particu-
lar substance on which they act was present in the culture solution.
Others were produced when substances chemically allied to the products
of hydrolysis were present. In general, however, he concluded that the
secretion of enzyms was not a hunger phenomenon, since those fungi
which were best nourished produced the most enzym. Dox (9), on the
other hand, demonstrated that for Penicillium camemberti, at least, the
enzyms were secreted regardless of the chemical nature of the substrate.
He found that by cultivating the fungus on a particular substratum the
quantity of the corresponding enzym may be increased, but that no
enzym not normally produced by the organism could be developed by
any special method of nutrition. Katz (16) in 1898 published the re-
sults of the regulating action of certain chemical substances in the
solution of the regulatory secretion of amylase by P. glaucum, Aspergillus
niger, and Bacillus megatherium and found that while the amylase secre-
tion was not prohibited by the presence of substances chemically allied
to starch, their effect was greatly to inhibit it. He found that the differ-
ent fungi did not respond exactly in the same way and cites as proof the
results with A. niger and P. glaucum. The presence of sugars ra the
Feb. 15, 1921
Amylase of Rhizopus tritici
779
solution had a much less inhibitory effect on the production of amylase
with A. niger than with P. glaucum. Similar conclusions were reached
by Duclaux (10) with P. glaucum and A. glaucus, though he considered
only the enzyms which diffused into the culture medium. The investi-
gations of Kylin (20) with P. glaucum, P. biforme, and A . niger corrob-
orate in a general way the results of other investigators. He found no
qualitative regulation of the enzyms studied by him (diastase, invertase,
and maltase), though a quantitative regulation was conclusively proved.
With P. glaucum the regulating secretion of diastase was greater than
with A. niger. Knudson (19), on the other hand, demonstrated a quali-
tative regulation of tannase with A. niger and P. sp. These fungi pro-
duced gallic acid by the fermentation of tannic acid when the latter
was added to a modified Czapek's nutrient solution, but if supplemented
with sucrose no tannase was formed. A number of other substances as
a source of carbon likewise failed to stimulate the secretion of tannase.
Young (28) studied the inulase formation by A . niger in a nutrient solu-
tion and found a well-marked quantitative regulation of the production
of the enzym. He showed that inulase was produced in greatest amount
in the mycelium (extracellular enzyms not studied) when inulin was
used as the source of carbon but was likewise produced when other car-
bohydrates were employed. The substances most closely allied to inulin
were most efficient in the production of the enzym.
The results of the writer's experiments which follow demonstrate also
a quantitative regulation of amylase in nutrient solutions. Sweet
potato bouillon and Czapek's modified nutrient solution (see p. 762)
with glucose and starch in combination or alone in varying amounts were
used as substrates.
In all these experiments the fungus was grown in 2 -liter flasks contain-
ing 1,000 cc. of solution. At the end of the growth period the mycelium
was removed and prepared in the usual way, according to the "aceton-
dauerhefe" method of Albert and Buchner, the mycelium from the flasks
of each series being mixed together to make a compound sample.
Experiment i . — The fungus was grown on Czapek's modified nutrient
solution with glucose or starch or both as a source of carbon. The
cultuies were incubated for 8 days at 32 ° C. Hydrolysis of starch was
carried out for 19 hours at 320 by using 0.25 gm. of enzym powder in 50
cc. of a 0.5 per cent starch paste solution. (Table XIII.)
Table XIII. — Source of carbon in Czapek's modified nutrient solution and amount of
hydrolysis by the enzym powder per 10 cc. of the substrate
Series.
Starch.
Glucose.
Reducing
sugars.
a
Gm.
5
0
5
Gm.
5
5
0
Mgm.
26. 19
34- IO
39-28
b
25120°— 21-
780
Journal of Agricultural Research
Vol. XX, No. 10
Experiment 2. — In this set of experiments sweet potato bouillon
was compared with Czapek's modified nutrient solution, the latter con-
taining different amounts of starch and glucose as a source of carbon.
The reducing sugars were determined in each series before inoculation
and after the fungous growth had been removed, the enzyms in the solu-
tions being inactivated at the end of the growth period by autoclaving
the solutions. The cultures were incubated for 10 days at 35 ° C. The
hydrolytic power of the enzym was determined by the use of 0.25 gm.
of powder in 100 cc. of a 0.5 per cent starch paste solution. The time
of hydrolysis was 1 8 hours at 400 C. (Table XIV.)
Table XIV. — Source of carbon in Czapek's modified nutrient solution, amount 0/
reducing sugars before and after the growth of the fungus, and the hydrolysis by the
enzym powder
[Expressed in milligrams per 10 cc. of the substrate]
Starch.
Reducing sugars.
Hydrolysis
by enzym
powder.
Series.
Before inoc-
ulation.
After re-
moval of fun-
gous growth.
Cm.
O
112. 186
112. 830
220. 570
6-599
40. 900
16. 882
92. 863
13.21
b
5.16
6.05
(J
Not determined a
24. 14
» Solutions a, b, and c were Czapek's nutrient solution; d was sweet potato bouillon. The reducing sugar
in b and c before inoculation was glucose.
The starch was not determined, but it was shown to be present in series
a, c, and d by iodin before the solutions were inoculated. When the
fungous growth was removed the starch had all disappeared in series a
and d.
From the results it is seen that the largest amount of hydrolysis took
place with mycelium grown on sweet potato bouillon (d), where reducing
sugars and starch both were originally present. On the other hand there
was considerably more hydrolysis with mycelium grown on starch alone
as a source of carbon (a) than where glucose was used alone (b) or in
combination with starch (c).
The reducing sugars in series b, c, and d were considerably less at the
end of the growth period than at the outset, showing that the fungus
made use of reducing sugars or had converted them into other sub-
stances.possibly alcohol, acids, etc. No starch remained in the solutions.
In series a the starch had entirely disappeared, but a small amount of
reducing sugar was present. In this case also the fungus had either used
a considerable amount of carbohydrate or had converted it into other
compounds.
The fungus made the best growth in series d, but it was good in all
and fruited abundantly in each of the solutions.
Feb. 15, 1921
Amylase of Rhizopus tritici
781
Experiment 3. — In the following experiments, Czapeck's nutrient
solution was used for series a, b, and c, and sweet potato bouillon was
used for d, the reducing sugars (glucose in b and c) and starch being
determined in the solutions before and after the growth of the fungus.
The digestion period was 12 days at 35 ° C.
In these experiments no account is taken of the amount used by the
fungus or that converted to other compounds by it.
The digestive power of the mycelium was determined by using 0.2
gm. enzym powder in 100 cc. of a 0.5 per cent starch paste solution,
which was hydrolyzed for 18 hours at 400 C. (Table XV.)
Table XV. — Amount of reducing sugars and starch in solutions before and after the
growth of the fungus; also the hydrolysis of starch by enzym powder
[Expressed in milligrams per 10 cc. of solution]
Before inoculation.
After removal of fungus.
Series.
Total reducing
sugars after
digestion of
starch.
Reducing
sugars before
digestion
of starch.
Starch
present as
reducing
sugars.
Total
reducing
sugars after
digestion
of starch.
Reducing
sugars before
digestion
of starch.
Starch
present as
reducing
sugars.
Hydroly-
sis of
starch by
enzym
powder.
a
b
43.62
No starch in
solution.
153- 60
449- 55
O
112. 19
112. 64
272. 56
43.62
O
40. 96
176. 99
29.47
IO. 69
36-56
33-97
144. 00
18.78
O
41. 18
38.25
9. IO
.78
•39
n- 54
c
d
75-15
182. 25
These results accord in general with those of the previous experiments,
series a and d having the greatest hydrolyzing power and b and c the
least.
Experiment 4. — The foregoing experiment was repeated, the solu-
tions being made to contain roughly the same amount of glucose and
starch. The hydrolysis of starch by the enzym powder was determined
by using 0.25 gm. enzym powder in 100 cc. of a 0.5 per cent starch paste
solution and hydrolyzing 18 hours at 400 C. (Table XVI.)
Table XVI. — Amount of reducing sugars and starch in the solutions before and after
the growth of the fungus; also the products of hydrolysis of starch by enzym powder
[Expressed in milligrams per 10 cc. of solution]
Before inoculation.
After removal of fungus.
Series.
Total reducing
sugars after
digestion of
starch.
Reducing
sugars be-
fore diges-
tion of
starch.
Starch as
reducing
sugars.
Total reduc-
ing sugars
after diges-
tion of
starch.
Reducing
sugars be-
fore diges-
tion of
starch.
Starch as
reducing
sugars.
Hydroly-
sis of
starch by
enzym
powder.
a
b
3L275
No starch.
144. 680
524. 000
No sugar
used.
105. 41
108. 68
300-37
3I.275
0
36. 000
223. 630
14.30
2.86
45-35
67. 60
107. 90
II.44
O
18.72
O
7-i5
1. 27
c
d
86.32
107. 90
1. 24
22.94
782
Journal of Agricultural Research
Vol. XX, No. io
Experiment 5. — This experiment was conducted in the same way as
experiments 3 and 4. The amyloclastic power of the enzym was de-
termined as in experiment 4. (Table XVII.)
Table XVII. — Amount of reducing sugars and starch in the solution before and after
the growth of the fungus; also the hydrolysis of starch by enzym powder
[Expressed in milligrams per io cc. of solution]
Before inoculation.
After removal of fungus.
Series.
Total reducing
sugars after
digestion of
starch.
Reducing
sugars be-
fore diges-
tion of
starch.
Starch as
reducing
sugars.
Total reduc-
ing sugars
after diges-
tion of
starch.
Reducing
sugars be-
fore diges-
tion of
starch.
Starch as
reducing
sugars.
Hydroly-
sis of
starch by
enzym
powder.
a
b
c
d
47-97
No starch
used.
213.98
508.56
O
122. 20
I20. 90
298. 48
47-97
0
93.08
210. 08
No starch
left.
No starch
used.
124. 80
217. 62
9- 23
33-93
44.72
144.30
O
O
80.08
73-32
26. 19
5-78
4.42
41. 08
An examination of the foregoing results shows a clear case of the
regulatory influence of the culture medium on the quantitative secretion
of amylase. In every case where starch (series a) alone was used as the
source of carbon the enzym powder hydrolyzed several times as much
starch in a corresponding length of time as when glucose alone (series b)
or in combination with starch (series c) was used. On the other hand,
the enzym powder from sweet potato bouillon (series d), which always
contained reducing sugars and starch and probably other carbohydrates,
hydrolyzed considerably more starch than the powder from the a series.
This exception is hard to explain, since it was obviously impossible to
determine the exact composition of sweet potato bouillon. That it
was a better medium for the growth of the fungus was quite evident.
The quantity of felt was always greater than in any of the other series.
The growth in the a series was likewise better than in either the b or c
series, starch alone appearing to be a better source of carbon than glucose
alone or in combination with starch.
These results seem to indicate that within the limits of these experi-
ments the solution which is best for the growth of the fungus is likewise
best for the secretion of amylase, regardless of the source of carbohy-
drates. It is probable that it is not so much the source of the carbohy-
drate which influences directly the quantitative production of the enzym
as the influence it has upon the growth of the fungus on which the secre-
tion of the enzym depends.
INFLUENCE OF THE AGE OF THE MYCELIUM WHEN REMOVED FROM THE
CULTURE ON THE PRODUCTION OF AMYLASE
It was shown by Dox, Young, and others that the greatest amount of
enzym is contained in the mycelium at about the beginning of the fruit-
Feb. is. 1921 Amylase of Rhizopus tritici 783
ing period. So far as the writer is aware, this fact has not been deter-
mined for Rhizopus tritici, and it was with the view of verifying it for
this fungus alone that comparative tests were made. In the experi-
ments carried out by the writer two different culture media were used —
namely, sweet potato bouillon and a modification of Czapek's nutrient
solution with a 0.5 per cent starch paste as a source of carbon. In the
former case the mycelium was removed from one set of flasks 3 days
after inoculation, when fruiting was just beginning. The mycelium was
removed from the other set of flasks 10 days after inoculation. The
difference in reducing power in this case was not large.
On the other hand, when the Czapek 's modified solution was employed,
the mycelium removed 5 days after inoculation (when just beginning to
fruit) hydrolyzed considerably more starch in a given length of time
than the mycelium removed 10 days later.
SUMMARY
(1) A vigorous starch-splitting enzym is secreted by Rhizopus tritici.
While some of the enzym is retained in the mycelium of the fungus, a
portion of it diffuses out into the substratum. The diffusion into the
culture medium begins soon after the substratum is inoculated, as was
shown by some of the experiments in which reducing sugars appeared
after 2 days in a nutrient solution with starch as the only source of car-
bon. The reducing sugars in such a medium accumulate in excess of the
needs of the fungus.
(2) The enzym is able to act on raw sweet potato and Irish potato
starch but much less energetically than on starch paste.
(3) The dried mycelium may be stored for several months at a tempera-
ture of from 90 to 35 ° C. without much deterioration, but at 6o° it grad-
ually becomes weaker.
(4) The optimum temperature for the digestion of starch is about
450 C. Above and below this temperature the amount of hydrolysis
becomes less, and at 6o° it is completely destroyed in 100 hours.
(5) If glucose is added to a system the hydrolysis of starch paste is
retarded. The quantity of glucose added does not seem to influence the
results. With a constant amount of enzym powder the total reducing
sugars formed in a solution of starch paste increases with the increase in
the volume of the solution up to a certain point and then decreases.
(6) An end point in the hydrolysis of the starch is not reached without
altering the equilibrium of the system. This was done by changing the
temperature and diluting the solution. If judged by the iodin test an
end point was obtained, but a quantitative determination of the reducing
sugars did not account for all the starch. It is probable that in this case
some of the products of the hydrolysis were dextrins which were not ac-
counted for as either starch or reducing sugars.
784 Journal of Agricultural Research voi.xx.No. 10
(7) When the enzym is in suspension some of it is removed by filtering
through Whatman chemically prepared filter paper.
(8) The temperature at which the fungus is grown has a marked in-
fluence on the production of intercellular amylase. With an equal
weight of enzym powder it was found that mycelium grown at 90 C.
hydrolyzed about four times as much starch in the same length of time
as mycelium grown at 400. The enzym powder of mycelium grown at 290
was intermediate between the other two. At these three temperatures
the best growth of the fungus was made at 290 and the poorest at 90.
(9) The results of these investigations show that there is a " quantita-
tive regulation" of the enzym. The hydrolyzing power of the mycelium
grown on Czapek's modified nutrient solution was much greater when
starch alone was used as a source of carbon than when glucose alone or
in combination with starch was employed. On the other hand, if grown
on sweet potato bouillon, which contains both starch and sugars, a unit
weight of the mycelium will hydrolyze more starch than when grown on
any of the other combinations. The vigor of growth of the fungus was
correlated with the hydrolytic power of the enzym powder. The results
seem to indicate that it is not so much the source of the carbohydrate
which influences the quantitative production of the enzym as it is the
influence which it has on the growth of the fungus on which the secretion
of the enzym depends.
(10) The enzym powder of young mycelium just beginning to fruit
was more active than the enzym from old mycelium.
LITERATURE CITED
(1) Armstrong, Henry E., and Armstrong, E. Frankland.
1907. STUDIES ON ENZYME ACTION. X. — THE NATURE OP ENZYMES. In
Proc. Roy. Soc. [London] s. B, v. 79, no. 533, p. 360-365.
(2) Bayliss, W. M.
1911. THE nature OP Enzyme action. Ed. 2, 137 p., 7 fig. London, New
York [etc.]. List of literature referred to, p. 121-132.
(3) Brown, Adrian J. ■ %
1904. LABORATORY STUDIES FOR BREWING STUDENTS. I93 p., 36 fig. London,
New York [etc.].
(4) Brown, Horace T., and Glendinning, T. A.
1902. THE VELOCITY OF HYDROLYSIS OF STARCH BY DIASTASE, WITH SOME RE
marks on enzyme action. In Jour. Chem Soc. [London], Trans,
v. 81, pt. 1, p. 388-400.
(5) and Heron, John.
1879. CONTRIBUTIONS TO THE HISTORY OF STARCH AND ITS TRANSFORMATIONS.
In Jour. Chem. Soc [London], v. 35, Trans., p. 596-654, 12 fold. tab.
(6) and Morris, G. H.
1890. RESEARCHES ON THE GERMINATION OF SOME OF THE GRAMINEAE. In
Jour. Chem. Soc. [London], v. 57, Trans., p. 458-528, 2 pi.
(7) Brunton, T. Lauder, and MacFadyen, A.
1890. the ferment-action of bacteria. In Proc Roy. Soc. [London], v.
46, 1889, p. 542-553.
Feb.is,i92i Amylase of Rhizopus tritici 785
(8) Clark, W. Blair.
1918. VOLUMETRIC DETERMINATION OP REDUCING SUGARS. A SIMPLIFICATION
OF SCALES' METHOD FOR TITRATING THE REDUCED COPPER WITHOUT
REMOVING IT FROM THE RESIDUAL COPPER SOLUTION. In JoUT. Amer.
Chem. Soc, v. 40, no. 12, p. 1759— 1772.
(9) Dox, A. W.
1910. THE INTRACELLULAR ENZYMES OF PENICILLIUM AND ASPERGILLUS, WITH
SPECIAL REFERENCE TO THOSE OF PENICILLIUM CAMEMBERTI. U. S.
Dept. Agr. Bur. Anim. Indus. Bui. 120, 70 p. Review of literature,
p. 16-35; bibliography, p. 66-70.
(10) Duclaux, E.
1899. traits de microbiologie. t. 2. Paris,
(n) Durandard, Maurice.
1913. l'amylase du rhizopus nigricans. In Compt. Rend. Acad. Sci.
[Paris], t. 157, no. 11, p. 471-474.
(12) Effront, Jean.
1902. enzymes and Their applications. Transl. by Samuel C. Prescott.
v.i. New York. Short bibliographies follow principal chapters.
(13) Hawkins, Lon A.
1916. EFFECT OF CERTAIN SPECIES OF FUSARIUM ON THE COMPOSITION OF THE
potato tuber. In Jour. Agr. Research, v. 6, no. 5, p. 183-196. Lit-
erature cited, p. 196.
(14) and Harvey, R. B.
1919. PHYSIOLOGICAL STUDY OF THE PARASITISM OF PYTHIUM DEBARYANUM
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p. 275-298, illus., pi. 35-37.
(15) Hill, Arthur Croft.
1898. reversible zymohydrolysis. In Jour. Chem. Soc. [London], Trans.,
v. 73, p. 634-658, 6 fig.
(16) Katz, J.
1898. DIE REGULATORISCHE BILDUNG VON DIASTASE DURCH PnVZE. In Jahrb.
Wiss. Bot. [Pringsheim], Bd. 31, p. 599-618.
(17) Kellerman, Karl F.
1903. THE EFFECTS OF VARIOUS CHEMICAL AGENTS UPON THE STARCH-CONVERT-
ING power OF taka diastase. In Bui. Torrey Bot. Club, v. 30, no. 1,
p. 56-70.
(18) KjELDAHL. J.
1879. undersogelser over sukkerdannende farmenter. In Meddel.
Carlsberg Lab., bd. 1, hefte 2, p. 107-184, illus. French resume,
p. 109-157 (separately paged).
(19) Knudson, Lewis.
1913. tannic acid fermentation. In Jour. Biol. Chem., v. 14, no. 3, p.
159-202, 2 fig. Bibliographical footnotes.
(20) Kylin, Harald.
1914. UBER ENZYMBILDUNG und enzymregulation bei einigen schimmel-
pn,zEN. In Jahrb. Wiss. Bot. [Pringsheim], Bd. 53, Heft. 4, p. 465-501.
Literatur-Verzeichnis, p. 500-501.
(21) MaquEnne, L. and Roux, Eug.
1906. influence de la reaction du milieu sur l'activitij de l'amylase ET
la composition des empois saccharifies. In Compt. Rend. Acad.
Sci. [Paris], t. 142, no. 3, p. 124-129.
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(22) Pfeffer, W.
1896. UEBER REGULATORISCHE BILDUNG VON DIASTASE AUF GRUND DER VON
HERRN DR. KATZ IM BOTANISCHEN INSTITUT AN GESTELLTEN UNTER-
suchungen. In Ber. Verhand. K. Sachs. Gesell. Wiss. Leipzig,
Math. Phys. CI., Bd. 48, p. 513-518.
(23) Tammann, G.
1889. uber die wirkung dER fermENTE. In Ztschr. Phys. Chem., Bd. 3,
Heft, i, p. 25-37, 4 fig.
(24) Ward, H. M.
1883. observations on the genus pythium (pringsh.). In Quart. Jour.
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(25) Went, F. A. F. C.
1 901. UEBER DEN EINFLUSS DER NAHRUNG AUF DIE ENZYMBILDUNG DURCH
monilia sitophila (monT.) sacc. In Jahrb. Wiss. Bot. [Pringsheim],
Bd. 36, Heft. 4, p. 611-664.
(26) White, Jean.
1909. THE FERMENTS AND LATENT LTFE OF RESTING SEEDS. In Proc. Roy. SoC
[London], s. B, v. 81, no. 550, p. 417-442. Special bibliography,
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(27) WORTMANN, Julius.
1882. UNTERSUCHUNGEN UBER DAS DIASTATISCHE FERMENTE DER BACTERIEN.
In Ztschr. Physiol. Chem., Bd. 6, Heft. 4/5, p. 287-329.
(28) Young, V. H.
1918. SOME FACTORS AFFECTING INULASE FORMATION IN ASPERGILLUS NIGER.
In Plant World, v. 21, no. 4, p. 75-87; no. 5, p. 114-133. Bibliography,
p. 132-133.
A COMPARATIVE STUDY OF THE COMPOSITION OF THE
SUNFLOWER AND CORN PLANTS AT DIFFERENT
STAGES OF GROWTH
By R. H. Shaw, Chemist, and P. A. Wright, Assistant Chemist, Dairy Division,
Bureau of Animal Industry, United States Department of Agriculture
INTRODUCTION
The sunflower plant is gaining recognition as a silage crop in certain
of the northwestern States where climatic or soil conditions are not
always favorable for the maturing of corn for silage purposes. In some
sections also there is a growing sentiment that sunflower silage offers a
more profitable feed than corn silage, because of the greater yield that
may be obtained per acre.
The Dairy Division is making an investigation of sunflower silage.
This paper, which is the first of a series, presents the results of a study of
the chemical composition of the sunflower plant at several different and
distinct stages of its growth as compared with that of corn grown under
similar conditions. The purpose of the study is to assist in selecting the
proper stage of maturity for ensiling.
The investigation of the corn plant was made partly as a basis on which
to study the sunflower plant and partly in connection with another in-
vestigation, the results of which will be published in a paper having to do
with the fermentation of corn in the silo.
HISTORICAL REVIEW
Numerous analyses of the sunflower plant have been published from
time to time. In some cases these have represented the whole plant,
but more often only the head or the seed. No record of any study of
the composition of the plant at different stages of growth has been found.
On the other hand, there have been several such studies, more or less
complete, made of the corn plant. Some of these will be briefly reviewed.
Roberts (5)1 selected periods of growth (1) when the plants were coming
into bloom, (2) when approaching roasting-ear condition, and (3) when
most of the ears were out of the milk . Basing his figures on the dry matter,
he found that the percentage of protein decreased from the first period
to the last, while the percentage of carbohydrates increased.
Ladd (j) concludes that the nitrogen steadily diminishes throughout
the period of growth, while the sugars rise and fall. The starch falls
slightly during the earlier stages and then rises rapidly until the plant
reaches maturity.
1 Reference is made by number (italic) to "Literature cited," p. 792-793-
Journal of Agricultural Research, Vol. XX, No. 10
Washington, D. C. Feb. 15, 1921
■wv Key No. A-56
(787)
788 Journal of Agricultural Research voi.xx.No.io
Morse (4) analyzed samples representing four stages of growth and
reached the same conclusions, with respect to the protein and carbo-
hydrates, as the other investigators.
Perhaps the most elaborate study of the subject was made by Jones
and Huston (2). Their study included the whole plant as well as the
stalks, leaves, and ears taken separately. Unfortunately their figures
for the whole plant are based upon yield per acre and so can not be com-
pared with those of the other investigators or with ours.
EXPERIMENTAL WORK
The crops for the experimental work were grown in a section of the
field at the Dairy Division Experiment Farm at Beltsville, Md., usually
devoted to silage corn. The preparation of the soil, the planting, and
cultivating were done under the supervision of T. E. Woodward, farm
superintendent.
The sunflower plants were of the variety known as Giant Russian, and
the corn was Boone County White. The sunflower plants thrived well in
this soil (Bibb silt loam), reaching a height in many cases of 10 and 12
feet.
In dividing the growing period of the corn plant into stages, more or
less arbitrary points must be taken. It is quite useless for the purpose
to select plants by their age or height, for it is easily possible to find at
any one time within a comparatively small area plants of the same height
and age at entirely different stages of maturity. Up to the time of tas-
seling, however, there are no easily recognized guides except height.
From that time until the plant is fully mature there are certain and
fairly distinct points that can be selected, based on the condition of the
silk and ears.
The task of selecting stages of growth of the sunflower plant offers
more difficulty, and it is quite impossible to divide it into anything like
as sharply defined stages as in the case of the corn plant. We endeavored
to differentiate the stages first by the height and later by the condition
of the flower and seed, but at best these points are very arbitrary.
The difficulties in selecting representative samples of whole plants for
chemical analysis are obvious. The plan we followed was to go through
a small area of the field and select from 6 to 20 plants of the proper
stage of growth and as nearly the same size and conformity as possible.
These were carefully wrapped in a specially prepared waterproof cloth
and taken immediately to the laboratory, where they were cut into
1 -inch lengths with a hand-power feed cutter.
A 1 -kilogram subsample was weighed out and dried in the steam
closet for the determination of starch. The remainder was ground to a
pulp in a power meat grinder, and a subsample was taken for moisture,
albuminoids, and total-protein determinations. A further subsample
Feb. is, 1921
Composition of Sunflower and Com Plants
789
was weighed out, from which the alcoholic extract of the pulp was pre-
pared according to the method described by Swanson and Tague (6).
Aliquot portions of the alcoholic extract were used to determine total
and reducing sugars according to the gravimetric cuprous-oxid method
of Walker and Munsen (7, p. 241).
Moisture was determined on a 5-gm. sample of the pulp by drying to
constant weight in a reduced pressure water- jacketed oven. The
subsample dried in the steam closet was ground to pass a 40-mesh sieve,
and starch was determined on the air-dry sample by the diastase method
with subsequent acid hydrolysis (1, p. no).
Tables I and II give the results of the chemical work on the whole
plants. The figures for total protein, albuminoid protein, reducing
sugars, nonreducing sugars, and starch are based on the dry matter.
Table 1.— Composition of sunflower plant at different stages of growth
Stage of maturity.
3 feet high
6 feet high
First flower
Rays ready to fall . . .
Rays dry and partly
fallen
Rays all fallen
Seeds hard and ma-
ture
Moisture
in fresh
material.
Per cent.
84.87
86.02
84. 09
83.90
75-58
74-37
69.68
Dry
matter.
Per cent.
15- 13
13.98
I5-9I
16. IO
24. 42
25-63
30-32
Moisture-free basis.
Total
protein.
Per cent.
8-59
8.01
7.04
9.44
6.80
7-°3
5-9°
Albumi-
noid
protein.
Per cent.
8.00
7-37
6-35
6. 22
6. 09
5-°4
Reducing
sugars.
Per cent.
12.36
18.95
15.96
J3-23
8.96
6.99
4- 15
Non- I
reducing Starch,
sugars. I
Per cent.
19. 08
15- 03
8-43
3.01
I. 40
.89
1.47
Per cent.
O.63
4. 6l
4-34
. 20
1.66
1. 90
Table II. — Composition of com plant at different stages of growth
Stage of maturity.
3 feet high
4K to 5 feet high. . .
Just tasseling
Just silking
Kernels forming
Milk stage
Silage stage (one-
half milk, one-half
glazed)
All glazed
Ready to shock
Moisture
in fresh
material.
Per cent.
84.21
85.14
81.65
81.56
8l. 20
77.60
68.69
64. 22
59-79
Dry
matter.
Per cent.
15- 79
14.86
18.35
18.44
18.80
22. 40
3i-3i
35-78
40. 21
Moisture-free basis.
Total
protein.
Per cent.
II. 14
9.42
9.90
8-95
8.99
8.97
7-3i
6.32
7.09
Albumi-
noid
protein.
Per cent.
IO. 26
8. 14
6-59
6-73
6.38
6.30
6.23
5.62
6. 14
Reducing
sugars
Per cent.
14. 69
16. 69
13- 13
18.23
20.37
J7-59
10.03
8. 50
7.71
Non-
reducing
sugars.
Per cent.
2-73
3-23
1-85
1.30
5-44
4-51
2.81
39
73
Starch.
Per cent.
1.52
1.66
1. 29
.86
3-45
2.87
24. 00
24. 78
21. 66
790 Journal of Agricultural Research vol. xx.No. io
DISCUSSION OF RESULTS
In studying the tables it should be borne in mind that the figures rep-
resent percentages based on the plants themselves and have no bearing
on the yield of the various constituents per unit of area. For example,
the proteids decline in percentage as the plant grows. This does not
mean, of course, that the amount of the proteids per given area decreases,
but rather that as the plant grows and increases in weight the proteids
do not increase in the same ratio.
Too much importance must not be placed on slight differences in com-
position from stage to stage of growth. Because of the difficulties in
sampling whole plants, small differences due to unavoidable errors are to
be expected, and conclusions are safest when drawn from the general
trend of the results rather than from particular figures.
Considering the sunflower plant first, it will be noted that the dry
matter steadily increases as the plant grows older. This, of course, is
what would be expected, but the fact is rather surprising that, even after
the rays had all fallen and the seeds had become dry and mature, the
plant still contained more moisture than the corn contained at the time
it was ready for the silo.
The proteids, both total and albuminoid, show a tendency to decline
as growth proceeds. This is somewhat contrary to what might be ex-
pected from the highly nitrogenous character of the seed.
The reducing sugars rise and then gradually decline. The nonreducing
sugars steadily and rapidly decline throughout the whole period of growth.
In the first stage there is one and one-half times as great a quantity of
nonreducing sugars present as reducing sugars. This relation, however,
is quickly changed, and in the last stage there is nearly three times as
much of reducing sugars present as nonreducing. The percentage of
starch is small, rising and falling with no apparent relation to the change
in percentage of the sugars.
Turning now to the corn plant, it will be noted, as would be expected,
that the dry matter steadily increases as the plant grows older. The
proteids, both total and albuminoid, decline slowly but quite regularly.
The sugars, both reducing and nonreducing, rise and fall but have an
upward trend until the kernels begin to mature, when there is a sharp
drop, accompanied by a sudden increase in the starch. This is at the
stage when the plant is storing starch in the kernels and is the stage usu-
ally selected for ensiling. The ratio of reducing and nonreducing sugars
changes, but within a somewhat narrow range. The reducing sugars
always greatly exceed the nonreducing. The starch rises and falls up to
the stage when the kernels begin to mature. Between the milk stage
and what may be called the silage stage the starch increased from 2.87
per cent to 24 per cent.
Comparing the sunflower and the corn plants, it will be noted that the
chief difference in the constituents studied lies in the amount and char-
Feb. is, 1921 Composition of Sunflower and Corn Plants 791
acter of the carbohydrates. Although no part of the present experiment,
silage was made of the sunflower plant at different stages of maturity,
and it was found that silage made from plants at the stage when the rays
were dry and partly fallen was excellent in quality. Comparing the plant
at this stage with the corn plant at the silage stage, it will be seen that
the starch and sugars combined constitute 1 1.2 per cent of the dry matter
in the former, of which only about one-fifteenth is starch, while the
combined starch and sugars in the dry matter of the latter constitute
nearly 37 per cent, two-thirds of which is starch.
There is no great difference in the percentage of proteids in the dry
matter of the two plants, but it is slightly in favor of the corn plant.
In selecting the best stage of maturity of a plant for ensiling, several
things must be taken into consideration. In general the stage must be
selected that promises the largest yield of food constituents in the silage.
This stage is not necessarily the one when the plant itself has the maxi-
mum amount of food constituents. The moisture content of a plant,
judging by the behavior of the corn plant when ensiled, plays an ex-
ceedingly important role. When silage is made from the corn plant
having a high moisture content there is a downward seepage of the
juice, carrying with it valuable food material. If the silo is tight this
juice waterlogs the bottom layer, rendering it unfit for feeding. If the
silo is not tight the juice leaks out and is lost altogether. Moreover,
high moisture in the plant is usually associated with high-acid silage.
On the other hand, a plant that has too low a moisture content is difficult
to pack closely enough to eliminate the air spaces that cause spoilage.
Silage produced from such plants is dry and lacks palatability.
Another point that should not be lost sight of is, of course, the yield
per acre. This point, aside from the high moisture content, would bar
out the three earlier stages of the sunflower plant. The fourth stage is
still too high in moisture. The last stage contains nearly 70 per cent
of moisture.
From the moisture content alone the sunflower plant at this stage
should make good silage, but here another factor must be taken into
consideration. The sunflower plant at this stage has lost some of its
leaves. The outer part of the stalk has become so hard and woody that
it would be difficult, if not impossible, to pack it closely enough to pre-
vent spoilage. This eliminates all but two stages, the one when the
rays are dry and partly fallen and the other when all the rays have
fallen. These stages are close together, and judging from the chemical
composition there is but little choice between the two.
There is but little difference in percentage between the total proteids
and albuminoid proteids in the sunflower plant at these stages and the
corn plant at the silage stage. The chief differences, as discussed in
another paragraph, lie in the sugars and starch.
792 Journal of Agricultural Research voi.xx.No.io
SUMMARY AND CONCLUSIONS
A study was made of the chemical composition of the sunflower and
corn plants at different stages of growth.
The dry matter in each increased gradually and consistently throughout
the entire period of growth.
There is no great difference in the percentage of proteids in the two
plants, but it is slightly in favor of the corn plant.
The reducing and nonreducing sugars in the sunflower declined some-
what irregularly but persistently during the growth of the plant. In
the first stage there was about one and one-half times as much nonreduc-
ing sugars present as reducing sugars. This relation was quickly changed,
and in the latter stages the reducing sugars greatly exceeded the non-
reducing.
The percentage of starch in the sunflower is small and rises and falls
irregularly throughout the growth of the plant.
The reducing and nonreducing sugars in the corn plant rise and fall
but with a marked upward trend during the growth of the plant until
the stage is reached where the kernels are maturing, when a sudden
drop occurs. The percentage of reducing sugars is always far in excess
of the nonreducing sugars.
The starch rises and falls until the kernels are maturing, when a
sudden rise occurs.
The chief difference between the two plants at the silage stage lies in
the amount and character of the carbohydrates.
From the results obtained in this study it would seem that the best
stage of maturity for ensiling the sunflower plant is when the rays of
the flower have become dry and are falling.
LITERATURE CITED
(i) Association of Official Agricultural Chemists.
19 1 6. REPORT OF THE COMMITTEE ON EDITING TENTATIVE AND OFFICIAL METHODS
OF analysis. 381 p., illus. Baltimore. From Jour. Assoc. Offic. Agr.
Chemists, v. i. no. 4, [pt. 2]; v. 2, no. 2 [pts. 1-2]; v. 2, no. 3 [pt. 2]. Biblio-
graphies at ends of chapters.
(2) Jones, W. J., Jr., and Huston, H. A.
1914. COMPOSITION OF MAIZE AT VARIOUS STAGES OF ITS GROWTH. EXPER-
IMENTS made . . . 1903. Ind. Agr. Exp. Sta. Bui. 175, p. 595-630, 10 fig.,
1 pi. (col.).
(3) Ladd, E. F.
1890. A study OF THE maize plant. In N. Y. Agr. Exp. Sta. 8th Ann. Rpt.
1889, p. 79-91.
(4) Morse, Fred W.
1902. silage studies. N. H. Agr. Exp. Sta. Bui. 92, p. 49-62, 2 fig.
(5) Roberts, I. P.
1888. growing corn for fodder and ensilage. In N. Y. Cornell Agr.
Exp. Sta. Bui. 4, p. 49-57, pi. 6.
Feb. IS, !92i Composition of Sunflower and Corn Plants 793
(6) Swanson, C. 0., and Tague, E. L.
1917. chemical studies in making alfalfa silage. In Jour. Agr. Re-
search, v. 10, no. 6, p. 275-292.
(7) Wiley, H. W., et al.
1908. OFFICIAL AND PROVISIONAL METHODS OF ANALYSIS. ASSOCIATION
OF official agricultural chemists. As compiled by the committee on
revision of methods. U. S. Dept. Agr. Bur. Chem. Bui. 107 (rev.), 272 p.,
13 fig.
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Vol. XX MARCH 1, 1921 No. 11
JOURNAL OP
AGRICULTURAL
RESEARCH
CONTENTS
Page
Evaluation of Climatic Temperature Efficiency for the
Ripening Processes in Sweetcorn - - 795
CHARLES O. APPLEMAN and S. V. EATON
( Contribution from Maryland Agricultural Experiment Station)
Some Lepidoptera Likely to Be Confused with the Pink
Bollworm --------- 807
CARL HEINRICH
( Contribution from Bureau of Entomology )
Biology of the Smartweed Borer, Pyrausta ainsliei Heinrich 837
GEORGE G. AINSLIE and W. B. CARTWRIGHT
( Contribution from Bureau of Entomology )
Effect of X-Rays on Trichinae - 845
BENJAMIN SCHWARTZ
( Contribution from Bureau of Animal Industry )
Relation of the Calcium Content of Some Kansas Soils
to the Soil Reaction as Determined by the Electro-
metric Titration -------- 855
C. O. SWANSON, W. L. LATSHAW, and E. L. TAGUE
(Contribution from Kansas Agricultural Experiment Station)
Green Feed versus Antiseptics as a -Preventive of Intes-
tinal Disorders of Growing Chicks - 869
A. G. PHILIPS, R. H. CARR, and D. C. KENNARD
( Contribution from Indiana Agricultural Experiment Station )
Comparative Utilization of the Mineral Constituents in
the Cotyledons of Bean Seedlings Grown in Soil and
in Distilled Water - - - - - - - 875
G. DAVIS BUCKNER
( Contribution from Kentucky Agricultural Experiment Station )
Sunflower Silage Digestion Experiment with Cattle and
Sheep - - - - - - - - - -881
RAY E. NEIDIG, C. W. HICKMAN, and ROBERT S. SNYDER
(Contribution from Idaho Agricultural Experiment Station)
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE.
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, E>. C.
WASHINGTON : GOVERNMENT PRINTING OFFICE : 1611
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
FOR THE ASSOCIATION
KARX F. KELLERMAN, Chairman J. G. LIPMAN
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES l. marlatt
Entomologist and Assistant Chief, Bureau
of Entomology
Dean, State College of Agriculture; and
Director, New Jersey Agricultural Expert'
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of EnUy
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director;
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
new v
SO;
JOURNAL OF AGRTOTOAL RESEARCH
Vol,. XX Washington, D. C, March i, 1921 No. 11
EVALUATION OF CLIMATIC TEMPERATURE EFFI-
CIENCY FOR THE RIPENING PROCESSES IN SWEET-
CORN
By Charles O. Appleman, Plant Physiologist, and S. V. Eaton, former Assistant
Plant Physiologist, Laboratory of Plant Physiology, Maryland Agricultural Experiment
Station
INTRODUCTION
Green sweetcorn for table use or packing into cans is picked while the
ripening processes are actively in progress. Since these processes greatly
change the chemical composition of the corn, it is obvious that the ears
must be picked as nearly as possible at the stage of ripening that will
furnish the most desirable quality. There is some difference of opinion
in regard to the chemical composition that gives the most desirable
quality to sweetcorn, especially for packing into cans. Attention
is usually focused upon sugar content, as sweetness is a desirable quality
of sweetcorn and, morever, the flavor appears to be associated with the
sugar content. This relationship may be merely a parallelism, but it
is certainly true that corn acquires a decided flat taste after the sugar
is reduced to low content either on the stalk or in storage. The fore-
going statement does not necessarily apply to naturally low sugar content
in certain varieties or to the same variety grown under different con-
ditions.
The percentages of starch and crude fiber are claimed by some to be of
equal if not of even greater importance than the sugar content. The
percentage of starch must be sufficiently high to give body to the corn,
while the amount of crude fiber must be kept as low as possible. Since
the starch and crude fiber increase at the expense of the sugar, the most
desirable stage for picking corn would seem to be a wise compromise
between sugar content and other constituents.
The present paper deals with the chemical changes in sweetcorn
during ripening and the effect of climatic temperature on rate of these
changes. An attempt has also been made to evaluate the climatic
temperature efficiency for these processes and to make the results of
some practical value as a guide for picking corn in different localities
and in different seasons in the same locality.
Journal of Agricultural Research, Vol. XX, No. xi
Washington, D. C Mar. i, 1921
yrx Key No. M6I.-3
(795)
Ql.
79^ Journal of Agricultural Research voi.xx.No. n
In this study a distinction has been made between the ripening and
the maturing processes. The corn is considered ripe when the growth
of the kernels ceases and the chemical changes in the corn have nearly
attained equilibrium positions — that is, it is ripe at the time after which
the ratios of the various constituents change very slowly and very little.
The maturing of corn consists essentially in the loss of water; therefore,
the rate at which corn matures depends largely upon the climatic condi-
tions which control evaporation.
CHANGES IN. CHEMICAL COMPOSITION OF SWEETCORN DURING
RIPENING
Stowell's evergreen corn grown from home-selected seed furnished the
material for this study. For each experiment 50 ears representing as
nearly as possible the same stage of ripening were carefully selected in the
center of the field. These ears were numbered consecutively and desig-
nated as being in the premilk stage. The husks were not yet firm, and
the silk was still green or red for about X inch beyond the tip of the
husks. The remainder of the silk was, as a rule, brown but not dry_
The kernels were inspected through a small longitudinal slit in the husks
which was afterwards carefully closed and tightly held with a rubber band.
The spikelets were still evident, the kernels small and spherical, and the
exudate was opalescent or cloudy but not milky. This is about the
earliest stage of ripening that will furnish sufficient kernel material from
a single ear for sampling.
Samples for analyses were taken at 10 o'clock a. m. every other day
during the ripening period. In order that the rate of change in chemical
composition during each succeeding 48-hour period might be determined
by comparing analyses from the same ear, as well as analyses from dif-
ferent ears, the following procedure was adopted : Samples of three rows
of kernels each were removed from ears 1 and 2. The husks were then
carefully brought back to place and held with rubber bands. After 48
hours a second pair of like samples was taken from the opposite sides of
ears 1 and 2. At the same time the first pair of samples was removed
from ears 3 and 4. At the end of the second 48-hour period the second
samples were removed from ears 3 and 4 and the first samples from ears
5 and 6. This overlapping method of sampling was continued through-
out the ripening period.
The treatment of the samples and the methods for the carbohydrate
determinations have been described in a previous paper.1 The methods
for fat, crude fiber, and total nitrogen were essentially those of the Official
Agricultural Chemists.2
1 Appleman, Charles O., and Arthur, John M. carbohydrate metabolism in green sweetcorn
during storage at different temperatures. In Jour. Agr. Research, v, 17, no. 4, p. 137-152. 1919.
2 Association of Official Agricultural Chemists, official and tentative methods of analy-
sis. As compiled by the Committee on Revision of Methods. Revised to Nov. 1, 1919. 417 p., 18 fig.
Washington, D. C 1920. Bibliographies at ends of chapters.
Mar.
Temperature Efficiency for Ripening of Sweetcorn 797
Table I shows the changes in chemical composition of the solids in the
corn during a typical ripening period. It was found that the rate of
ripening was fairly uniform in all the ears selected for the experiment.
Therefore the determinations from the four samples taken at each sam-
pling period were averaged instead of the first and second samples from
the same ears being compared, as was originally intended. Each percent-
age in the table, except the first set, represents an average of four deter-
minations. The averages for the first date include the determinations from
the first samples of ears 1 and 2. The averages for the succeeding dates
include the determinations from the first samples of two ears and the
second samples of the two ears that furnished the first pair of samples on
the previous date. The removal of the first sample from an ear does not
affect the rate of ripening in the kernels on the remaining half of the ear
if the husks are closed tightly and held in place.
Table I. — Changes in composition of sweetcorn during ripening
[Calculated as percentages of dry weight]
Date.
Starch.
Cane
sugar.
Reducing
sugars.
Fat.
Crude
fibre.
Total
nitrogen.
Protein
(total
NX6.25).
Aug. 3
5
7
9
11
13
15
17
19
18.36
25. 20
35-73
45-42
56.89
57-23
58.91
59- 15
60. 41
19-55
21.85
24-57
18.75
"•59
9-55
8.32
7.86
5-85
20. 07
13-93
9-45
5-43
3.01
2. 64
2. 24
1.97
1.77
2.97
4.04
3-99
4.44
4.81
5-25
5-°5
5.01
6. 01
7.92
6-37
4-63
2.58
2. 62
2.81
2-35
2-59
2.30
3-33
3.08
2-45
2. 09
2. 14
2. 01
2.03
2. 10
2. 20
20. 81
19.25
I5-3I
13. 06
13-37
12. 56
12. 70
13. 12
13-75
The chief changes in the percentage composition of the solids in the
corn during ripening consist in the depletion of sugars and the increase
in starch. In the very early stages the reducing sugars predominate but
very rapidly decrease as ripening proceeds. The percentage of cane sugar
increases until a maximum is reached and then decreases as the starch
increases. The reducing sugars predominate at the stage of highest
total sugar content; therefore this stage does not necessarily coincide
with the stage of greatest sweetness, as the reducing sugars are not nearly
as sweet as cane sugar. The highest content of the latter sugar is the stage
of greatest sweetness. The changes in the percentage of fat, crude fiber,
and total nitrogen occur during the very early stages of ripening. For the
remainder of the ripening period these percentages remain fairly constant.
The formation and storage of starch is the chief process occurring in
the kernels during ripening. This is the resultant of a number of com-
plex processes in the plant, but it seems safe to conclude that the rate of
starch synthesis in the kernels is the controlling factor for several sup-
plementary processes in the ripening of the corn. For example, the rate
798
Journal of Agricultural Research
Vol. XX, No. ii
of movement of soluble carbohydrates from the stems and cob to the
kernels and the rate of hydrolysis of cane sugar in the kernels are both
controlled by the rate of starch formation. Most of the starch that is
stored in the kernels during ripening is formed from carbohydrates
already stored in the stem and cob when kernel formation begins. The
intensity of respiration does not change the ratios of the different car-
bohydrate constituents in the ripe corn. The carbohydrate transforma-
tions being reversible, their final equilibrium positions are maintained.
S /O
OJYS
Fig. i. — Comparison of early and late crops of sweetcorn in respect to changes in percentage composition
in equal lengths of time. Early crop (Aug. 3 to 18) indicated by solid lines. Late crop (Sept. 20 to Oct.
5) indicated by broken lines.
EFFECT OF SEASON ON THE RATE OF RIPENING
Two crops of corn from the same source of seed were planted so that
the first crop would ripen in August and the second in the cool autumn.
In order to compare the ripening rates of the early and late crops, it was
necessary to find a measure of the rate of ripening. The decrease in the
Mar. i,i92i Temperature Efficiency for Ripening of Sweetcorn 799
ratio of total sugar to starch was adopted for this purpose. Table II
and figure 1 show the changes in percentage of moisture, total sugar and
starch, and also the changing ratio of sugar to starch in equal times for
the two seasons, starting with the same stage of ripening in both cases.
By comparing these ratios it will be noted that the late crop required 15
days to reach the same stage of ripening as the early crop reached in 6
days. In other words, the rate of ripening was two and one-half times
faster in the early crop than in the late crop. During this period of
ripening the starch content in the early crop increased from about 2.5
per cent to 10.5 per cent, and in the late crop from about 2.7 per cent to
10 per cent. At the end of this ripening period the sugar to starch
ratios were 0.556 and 0.500, respectively, and the chemical composition
was such that it probably represented the best edible stage. By the nail
test the corn was in the typical milk stage, but a subsequent paper will
show that the chemical composition of the corn changes considerably
during the so-called milk stage.
Table II. — Comparison of early and late crops of sweetcorn in respect to changes in per-
centage composition in equal lengths of time
Time from first
examination.
Days
O
2
4
6
8
10
12
14
15
Early crop.
Moisture.
86-55
84.21
80.63
75- 89
72.05
70.47
67.78
65-5I
64.98
Total
sugars.
5-39
5-90
6.89
I 6.09
4.21
3- 75
3-5°
3-55
3.02
Starch.
2.47
3-98
6. 92
10.95
15.90
16.93
18.98
20. 42
20.94
Ratio of
sugar to
starch.
187
544
868
556
264
219
183
170
149
Late crop.
Total
sugars.
88.27
88.83
86.97
85-56
85.21
83.80
81.56
79. 26
77.69
6. 13
5-69
5-78
5-53
5-56
6.30
5. 62
5. 26
5.08
Starch.
Ratio of
sugar to
starch.
2. 72
2. 32
2.86
3-39
3-85
5-48
6. 90
8.71
10. 09
2.300
2-459
2. 168
1-747
1.448
1. 164
.879
• 673
0. 500
1 Same stage of ripening as late crop on fifteenth day.
b Same stage of ripening as early crop on sixth day.
EVALUATION OF CLIMATE TEMPERATURE EFFICIENCY FOR THE
RIPENING PROCESSES IN SWEETCORN
Since both the early and late crops of corn were grown from the same
source of seed and on the same type of soil, the great difference in the
rate of ripening must have been due to the different climatic conditions
which prevailed during the ripening periods. Of the climatic conditions,
temperature was the most important variable. The averages of the hourly
mean temperatures for the ripening periods of the early and late crops
were 830 and 650 F., respectively. The ripening processes being either
chemical or dependent upon chemical processes, the prevailing tem-
peratures for the two periods would be expected to have a very different
8oo Journal of Agricultural Research voi.xx.No.xi
influence on the rate of ripening. But these ordinary temperature
readings do not furnish a basis for a quantitative comparison of the tem-
perature efficiency in reference to these processes.
Various methods have been proposed for interpreting the observed
climatic temperatures in different localities and for different seasons in
the same locality, with reference to plant growth. Three of these
methods were applied to the fairly definite set of physico-chemical
processes involved in the ripening of sweetcorn. The first method
employed was one of direct temperature summation, similar to that
described by MacDougal.1
The integration was performed, with a planimeter, upon thermograph
records. The area between the 400 F. line and the pen tracing for each
day of the two ripening periods was first measured. Then the mean
temperature for each hour of a chosen day was computed from a thermo-
graph record, and 40 was subtracted from each hourly temperature.2
The sum of these results divided by the planimeter reading for the same
day gave a factor by which the planimeter reading for any 24-hour
period could be converted into hour-degree units of effective temperature.
The total number of hour-degree units was computed for the 6- and 15-day
ripening periods of the early and late crops, respectively. These units
express both the intensity and duration aspects of the temperature
factor. The adoption of the 400 as the starting point for the temperature
summations was based upon the facts that carbohydrate changes are
chiefly involved in ripening and that carbohydrate transformations in
green corn during storage are extremely slow below this temperature.
The results of the direct temperature summations given in Table III
show a slightly greater total number of hour-degree units of effective
temperature in favor of the late crop. Stevens and Higgins 3 have
shown that the temperature of green corn on the stalk in the shade is
nearly that of the air, while in the sun it is often above that of the air.
The period of ripening for the early crop here considered was character-
ized by high temperature and clear days, while the ripening period of
the late crop contained 2.5 times as many days, many of which were
cloudy. Since the temperature records from which the units of effective
temperature were computed were taken in an instrument shelter, the
sum of the hour-degree units for the early crop is probably a little less
than actually required.
Livingston and Livingston,4 realizing the need of some fundamental
principle of physiology upon which to base the value of temperature
1 MacDougal, D. T. the temperature of the soil. In Jour. N. Y. Bot. Garden, v. 3, no. 31, p. 125-
131, fig. 19-21. 1902.
2 The thermograph records were furnished by Dr. Earl S. Johnston of the Laboratory of Plant Pathology,
Maryland Agricultural Experiment Station.
3 Stevens, Neil E., and Higgins, C H. temperature in relation to quality of sweetcorn. In
Jour. Agr. Research, v. 17, no. 6, p. 275-284, 1 fig. 1919. Literature cited, p. 283-284.
4 Livingston, Burton Edward, and Livingston, Grace Johnson, temperature coefficients in
plant geography and climatology. In Bot. Gaz., v. 56, no. s, p. 349-375, 3 fig. 1913.
Mar.i,i9« Temperature Efficiency for Ripening of Sweetcorn 801
summations, were the first to apply velocity coefficients to the study of
effective climatic temperature conditions for plant growth. Upon the
basic assumption that the growth rate is unity at 400 F. and that it
doubles for each rise of io° C. (180 F.), they deduced temperature
efficiency values corresponding to temperatures, in whole numbers, from
400 to 990 F. These efficiency values are spoken of as exponential
indices. Since the rate of the carbohydrate changes in corn after it is
pulled has a temperature coefficient of about 2 for a range of tempera-
ture beyond the limits of the climatic temperature for either ripening
period, and since the chief process during ripening is the conversion of
sugar into starch, the exponential indices would be expected to furnish
the best criteria of the temperature efficiency for the ripening processes
in sweetcorn. In Table III are given the sums of the exponential indices
corresponding to the daily mean temperatures of each ripening period
under consideration, as well as the average daily index for each period.
The average daily index for the early season is 2.5 times greater than
that of the late season. If these indices furnish an approximate criterion
of the temperature efficiency for ripening of sweetcorn, the ripening
should have proceeded 2.5 times faster during the early ripening period
than during the late ripening period. The experimental data show that
this was actually the case; the late season required 15 days to carry
the corn to the same stage of ripening that required only 6 days in the
early season, a time ratio of 2.5.
More recently Livingston l has derived a new set of temperature
indices which he terms physiological indices, since they are based upon
Lehenbauer's actual measurements of the hourly rate of elongation of
the shoots of seedling maize plants. For the sake of comparison these
indices for the two ripening periods are also given in Table III, but it
will be seen at once that they do not furnish even an approximate cri-
terion of the temperature efficiency for the ripening processes in sweet-
corn. This may be at least partially explained by the fact that, for
the processes under consideration, the principle of Van't Hoff and
Arrhenius seems to hold for rather a wide range of temperature, while
in the elongation of maize shoots it holds only for a range of tempera-
ture from about 200 to 300 C.
1 Livingston, Burton Edward, physiological temperature indices for the study of plant
growth in relation To climate conditions. In Physiol. Researches, v. i, no. 8, p. 399-420, 4 fig.
1916. Literature cited, p. 420.
802
Journal of Agricultural Research voi.xx, no.
Table III. — Temperature indices in relation to ripening of sweetcorn
Crop.
Time.
between
premilk
and best
edible milk
stages.
Hour-
degree
units.
Exponential indices.
Physiological indices.
Sum.
Average.
Sum.
Average.
Early
Days.
6
6,42 5
7,393
31. Si
32. 22
5- 3°2°
2. 1458
640
319
107. O
21.3
Late
EXPONENTIAL INDICES AS A BASIS FOR AN APPROXIMATE PREDIC-
TION OF THE RATE OF RIPENING IN SWEETCORN
Since the rate of ripening appears to be inversely proportional to the
exponential indices, the proportions
6 : x : : y : 5. 3020
2 : x : : y : 5. 3020
furnish a basis for an approximate prediction of the number of days in
different localities and for different seasons in the same locality required
for corn to pass from the premilk stage to the best edible milk stage,
and also the maximum number of days that the corn may be expected
to remain in this condition. The first term of the first proportion is the
number of days actually required for an early crop to pass from the pre-
milk to the best edible stage, or from a starch content of about 2.4 per
cent to one of 11 per cent. The first term of the second proportion is
the maximum number of days that the corn of the early crop here con-
sidered remained in the best edible condition. The last term of the
proportions is the average of the exponential indices corresponding to
the daily mean temperatures for the 6-day period. By substituting for
y in these proportions the average of the exponential indices derived from
the normal daily mean temperatures for any season of any locality, the
value of x in the first proportion gives the approximate number of days
on the average that will be required for the corn to pass from the pre-
milk to the best edible condition. The value of x in the second propor-
tion gives the number of days that the corn may be expected to remain
in this condition.
Table IV gives the values of x for the usual ripening seasons of
four sweetcorn localities which show considerable variation in the normal
mean temperature for the ripening periods. In this calculation, the
normal mean temperatures calculated by Bigelow1 were employed.
1 Bigelow, F. H. the daily normal temperature and daily normal prectpitation op the united
states. U. S. Dept. Agr. Weather Bur. Bui. R, 186 p. 1908.
Mar.i,i92i Temperature Efficiency for Ripening of Sweetcorn 803
Table IV. — Comparison of the rates of sweetcorn ripening in different localities, based
upon the exponential indices corresponding to the normal mean temperatures of the
ripening seasons
Locality.
Ripening season.
Time be-
tween pre-
nnlk and
best edible
milk stage.
Length of
time in
best edible
stage.
Charleston, S. C...
Baltimore, Md. . . .
New Haven, Conn
Portland, Me
June 17 to 31.
July 1 to 15..,
Aug. 1 to 15..
Aug. 16 to 31.
Sept. 1 to 15..
Sept. 16 to 30
Oct. 1 to 15...
Aug. 1 to 15. .
Aug. 16 to 31.
Sept. 1 to 15.
Sept. 16 to 30
Days.
7.0
6-5
8.0
8-5
9-5
"•5
14. o
9-5
10. s
14. o
16. o
Days.
2-5
3-o
3-o
4.0
5-o
3-°
3-5
4-5
5-5
The results given in Table IV are simply the average expectations,
calculated for a 20- year period. If the mean temperature for a particular
season deviates to any considerable extent from the normal mean, the
rate of ripening for this season will be greater or less, depending upon the
direction of the deviation, than that calculated from the normal mean
temperature. In order to test the possible magnitude of deviation from
the average expectation, the ripening rates were calculated for the highest
and lowest mean August temperature at Baltimore from 1871-1918.
These results together with those calculated from the normal mean
August temperature for the same period are given in Table V. Data
were not available from which to derive the exponential indices cor-
responding to the daily mean temperatures for the month as was done
in calculating the data from normal mean temperatures given in Table
III. However, the results suffice to indicate that for the most extreme
seasons the number of days required for the two periods of ripening
under consideration -would not vary more than a day or two in either
direction from the calculated average. If the particular season in
question is unusually hot, one day would have to be subtracted from the
average prediction. If, on the other hand, the season is unusually cool,
one day would have to be added to the average expectation. This
applies particularly to Maryland conditions.
In making the foregoing predictions it was assumed that most of the
ears of a given crop will ripen at practically the same rate. This was
found to be true in the experimental crops grown from home-selected
seed. For canning purposes it is essential to use seed that will insure
the maximum uniformity in ripening.
804
Journal of Agricultural Research
Vol. XX, No. ix
Table V. — Rate of sweetcorn ripening during the month of August, calculated from
Baltimore temperatures
Temperature.
Exponen-
tial index.
Time
between
pre-milk
and best
edible stage.
Length of
time in
best edible
stage.
3. 8480
4. 6662
3- 4283
Days.
8-3
6.8
9-3
Days.
2.7
2-3
3- 1
Highest monthly mean, 1871—1918, 80. o° F
Lowest monthly mean, 187 1— 1918, 72. o° F
Stevens and Higgins state that the corn-picking season in Maryland
has a much higher average temperature than the corresponding season
in Maine, the difference being sufficient to cause considerably greater
deterioration in picked corn during a given period.1 They also derived
the exponential and physiological indices corresponding to the daily
normal temperatures for the corn-canning seasons of both localities.
The means of these two sets of indices were both greater for Baltimore,
Md., than for Portland, Me.; but they were unable to decide which
method furnishes the best criteria of the relative rates of deterioration of
picked corn in the two localities. The data presented in this paper and
in a previous paper by Appleman and Arthur 2 lend support to the
exponential indices as a good measure of the relative climatic tem-
perature efficiency for the deterioration of picked corn in different
localities.
The quality of canned corn may be influenced not only by the tem-
perature at which the corn is handled but also by the effect of tempera-
ture on the rate of ripening. A slow rate of ripening gives a greater
range in the number of days that the corn may be picked in good con-
dition. Corn that ripens in very warm seasons, for example in the
month of August in Maryland, requires very close attention lest the best
stage for picking be allowed to pass. The data presented in this paper
should furnish a more rational basis for picking green sweetcorn.
SUMMARY
Sweetcorn is considered ripe when the growth of the kernels ceases
and the chemical changes in the corn have nearly attained equilibrium
positions. The maturing of corn consists essentially in the loss of water.
The chief changes in percentage composition of corn during ripening
consists in the depletion of sugars and the increase of starch.
In the very early stages of ripening the reducing sugars predominate;
therefore the stage of highest total sugar content does not necessarily
coincide with the stage of greatest sweetness.
1 Stevens, Neil E., and Higgins, C H. op. cit.
2 Appleman, Charles O., and Arthur, John M. op. cit.
Mar. 1,1931 Temperature Efficiency for Ripening of Sweetcorn 805
Calculated as percentages of dry weight, the changes in fat, crude
fiber, and total nitrogen occur during the very early stages of ripening.
For the remainder of the ripening period these percentages remain
fairly constant.
The rate of starch synthesis in the kernels seems to be the controlling
factor for several supplementary processes. The rate at which the ratio
of total sugar to starch decreases is a good measure of the ripening rate
and was employed for that purpose.
Temperature is the controlling factor for the rate of ripening in sweet-
corn. Several temperature indices were employed to evaluate climatic
temperature efficiency for the ripening processes. The exponential
indices were found to furnish the best criteria of the temperature effi-
ciency for sweetcorn ripening.
A late crop of corn required 15 days for the same period of ripening
that required only 6 days for an early crop, a time ratio of 2.5. The
averages of the daily exponential indices for the two seasons were prac-
tically in the same ratio. Therefore, the rate of ripening in sweetcorn,
within a wide range of temperature, appears to adhere rather strictly
to the Van't Hoff-Arrhenius principle.
The rate of ripening being inversely proportional to the exponential
indices, a basis was furnished for an approximate prediction of the
number of days required in different localities and at different seasons
in the same locality for corn to pass from the beginning of kernel forma-
tion to the best edible stage, as well as the maximum number of days
that the corn may be expected to remain in this condition.
SOME LEPIDOPTERA LIKELY TO BE CONFUSED WITH
THE PINK BOLLWORM
By Carl Heinrich x
Specialist on Forest Lepidoptera, Bureau of Entomology, United States Department
of Agriculture
INTRODUCTION
The purpose of the present paper is to define the characters which will
distinguish the larva and pupa of the pink bollworm, Pectinophora
gossypiella Saunders, from those of other Lepidoptera attacking cotton
or related malvaceous plants and of still others feeding on plants
other than malvaceous but frequently found in the neighborhood of
cotton fields. A few (Dicymolomia julianalis Walker and Crocidosema
plebeiana Zeller, for example) so closely resemble the pink bollworm in their
habits and their larval stages that they are only to be distinguished by a
careful examination of their structure. It is hoped that the present
paper will make the differentiating characters clear and will enable
entomological workers to distinguish the forms treated.
The field work upon which this paper is based was conducted through-
out the area in southeastern Texas where the pink bollworm has been
found to occur, as well as in Cameron County, at the southern extremity
of the State. Special attention was devoted to discovering whether the
pink bollworm was attacking plants other than cotton. Thousands of
seed pods of okra and other malvaceous plants were examined. In one
case, at Smiths Point, in Chambers County, all the seed pods of a plant
related to cotton (Hibiscus lasiocarpus), growing in the immediate
vicinity of a field where a heavy infestation by the pink bollworm had
occurred during a previous year, were removed and given minute exami-
nation. Similar investigations were made with reference to other wild
and cultivated malvaceous plants growing in or about fields where the
1 This study was conceived and arranged by Dr. W. D. Hunter, in charge of the Pink Bollworm
Eradication, to aid the work of his inspectors. To the necessary preliminary field work the following
entomologists were detailed by Dr. Hunter: H. C Hanson, J. D. More, E. L. Diven, A. C. Johnson, and
Carl Heinrich. For a short period Mr. Herbert Barber was also associated with the work. The material
and notes on which the paper is based are all due to these workers. Especial mention should be made of
Emerson Liscum Diven, who had a major part in the investigations and who lost his life in an aeroplane
accident while scouting for cotton areas and who,* had he lived, would have worked up the results as
here given.
With the exception of Plate 107, all the drawings accompanying this paper were made under the writer's
supervision by Mr. H. B. Bradford, of the Bureau of Entomology. Plate 107 (also originally by Mr. Brad-
ford) is reproduced from Busck's article on the pink bollworm (In Jour. Agr. Research, vol. 9, no. 10, p.
343-37°, 1917)- The writer is especially indebted to Mr. Bradford for his painstaking and accurate drawings.
To Mr. Busck the writer is indebted for many helpful suggestions and both to him and to Dr. Dyar for
verification of some of the identifications.
Journal of Agricultural Research, (8°7) Vo1" XX? No-"
Washington, D. C. Mar- "> "921
wy
Key No. K.-92
808 Journal of Agricultural Research voi.xx.No.ii
pink bollworm had been found. In no instance was the pink bollworm
found in any plant other than cotton.
Thirty-eight species are considered here. Of these, six are described
as new, and four, already described, are recorded for the first time from
the United States. In each case the male genitalia of the type specimen
of the new species are figured. The essential larval and pupal characters
are referred to in the text as fully as possible, and purely descriptive
matter is reduced to a minimum.
FAMILY GELECHIIDAE
PECTINOPHORA GOSSYPIELLA (SAUNDERS), THE PINK BOLLWORM
(pl. ioi, a, b; 103, a; 105, c, E; 106, a; 107, a-d)
Depressaria gossypiella Saunders, 1843, in Trans. Ent. Soc. London, v. 3, pt.4,
p. 284-285.
Pectinophora gossypiella Busck, 1917, in Jour. Agr. Research, v. 9, no. 10,
P- 34.3-3 7°-
Inasmuch as the immature stages of the pink bollworm have been
already fully described in an earlier number of this journal 1 it will be
necessary here only to point out the structural characters which will
serve to identify its larva and pupa and distinguish them from those of
other Lepidoptera which, because of their habits, food plants, or general
appearance, might be mistaken for Pectinophora gossypiella. There is
no easy and ready-made method which will enable a layman to distinguish
an insect and be certain of its identity. This applies with particular force
to the pink bollworm. As Busck well states —
Definite and final determination of P. gossypiella in any stage can be made only by
the aid of a microscope
and he might have added, only by one reasonably experienced in insect
determination and familiar with the characters used in classifying
Lepidoptera. Nevertheless the pink bollworm has structural characters
by which it can be determined and its identity established beyond the
possibility of doubt. The specialist alone can pass upon these with certainty ;
but the average intelligent worker in the field can also use them, far
enough at least to say what larvae or pupa? commonly found in and about
cotton fields can not be P. gossypiella.
The combination of the following characters distinguishes the larva?
of the pink bollworm:
Three setae (III, IV, and V) triangularly grouped on the prespiracular shield of the
prothorax (Ti). (PI. 103, A.)
Setse IV and V closely approximate on the prol eg-bearing abdominal segments
(Am). (PI. 103, A.)
Setae III above (not directly before) the spiracle on the eighth abdominal segment
(Avm).
1 Busck, August, the pink bollworm, pectinophora gossypiella. In Jour. Agr. Research, v. o,
no. 10, p. 343-37°. 7 fig-, pl. 7-12. 1917. Literature cited, p. 366-370.
Mar. i, 1921 Lepidoptera Likely to be Confused with Pink Bollworm 809
On the ninth abdominal segment (Aix) the paired dorsal setae II not on a single
pinaculum (chitinized plate) and not appreciably closer together than the paired I
on the dorsum of the eighth abdominal segment; seta I no nearer to III than toll; VI
closely approximate to IV and V; group VII unisetose.
Prothoracic legs appreciably separated at their base. No anal fork on tenth abdom-
inal segment. Crochets of abdominal prolegs uniordinal and arranged in a circle broken
outwardly. (PI. 106, A.)
On each side of the thoracic shield near Seta Ib a small crescent or reniform spot (PI.
103, A) paler than the surrounding chitinized area.
On the epicranium the lateral seta (L1) behind the level of P1 and remote from A3
(that is, farther from A3 than A3 is from A2) and the anterior puncture (Aa) lying
between setae A1 and A2. (PI. 101, A.)
Each of these characters is possessed by other lepidopterous larvae,
but their combination is peculiar to Pectinophora gossypiella. No other
known larva that we have in this country possesses them all. I have not
seen caterpillars of (Gelechia) Pectinophora malvella Zeller,1 the only other
known species of the genus Pectinophora, or oi'Platyedra vilella Zeller,
which Meyrick considers congeneric with Pectinophora gossypiella.2
These may have most or all of the structural characters here given, but
as neither of them occurs outside of the Old World they do not concern
us at present.
The setal characters are fully illustrated on Plates 101, 103, and 105.
It will be noted that two slight changes have been made from the drawings
published in Busck's paper. The numbering of abdominal setae IV
and V has been reversed to correspond with our present conception of
the homologies of these setae; and the lateral puncture (La) of the
epicranium is shown directly posterior to rather than postero-ventrad of
seta L1. In Busck's figures3 the puncture is much too low.
The pupa (PI. 107, A-D) is evenly and densely clothed with a fine
pubescence; moderately stout, with a short, hooked cremaster surrounded
by 6 to 8 stout, hooked setae but without dorsal spines or other armature ;
labial palpi absent; maxillary palpi long, extending four-fifths of the
wing length; antennae long but not quite reaching to tips of wings;
vertex distinct but narrower than prothorax.
No other lepidopteron feeding on malvaceous planes in this country
has such a pupa. The fine pubescence and short, hooked cremaster are
easily discernible under a small hand lens and are enough to identify the
pupa which, when once seen, is not likely to be confused with that of
any other cotton-feeding species.
1 After this paper had gone to the printer we received from the Abb6 J. de Joannis of Paris a larva of
Pectinophora malvella. The structural characters are the same as those of Pectinophora gossypiella.
2 The Abbe Joannis also sent us a male moth of Platyedra vilella. A comparison of the genitalia of this
and Pectinophora gossypiella does not support Meyrick's contention.
3 Busck, August, op. err., 1917, p. 348, fig. 2, B.
810 Journal of Agricultural Research vol. xx, no. n
GELECHIA HIBISCELLA BUSCK
(PL- 93, c)
Gelechia hibiscella Busck, 1903, in Proc. U. S. Nat. Mus., v. 25, p. 869-871.
Gelechia hibiscella Busck, 1903, in Dyar, List North Amer. Lep., no. 5739.
This species was originally described from larvae collected on Hibiscus
moscheutos in the vicinity of Washington, D. C.
On the shores of Miller's Lake and Lake Charlotte in Chambers Co.,
Tex., we found the larvae fairly abundant in early September (191 8) on
both Hibiscus lasiocarpus and H. militaris and also occasionally on Kos-
telezkya spp. During October of the same year adults were reared from
these. The male genitalia compared with those of typical specimens
from the type locality agree in all details. A figure of the elaborate
and characteristic genitalia is given in Plate 93, C.
Gelechia hibiscella seems to be limited in food plant to Hibiscus and
one or two other closely allied Malvaceae. We have never found it on
cotton or okra, but there seems to be no reason why it should not thrive
on these. The feeding habits vary somewhat according to the charac-
ters of the plant on which the larvae feed. On the broader-leaved Hibis-
cus moscheutos around Washington and the similar //. lasiocarpus in Texas
the larvae feed chiefly on the leaves, rolling them up and partially biting
through the stems before pupation so that the folded leaf is easily shaken
to the ground by a slight wind. Within this roll they pupate. Occa-
sionally the larvae also attack the seed pods, but from the writer's obser-
vation this is rather rare in the broad-leaved species of Hibiscus. In
the narrow-leaved H. militaris and in Kostelezkya spp., on the other
hand, the habits are quite different. Here the larvae feed chiefly in the
flowers and seed pods, pupating in the withered flowers, and do not
attack or use the leaves at all.
There is no possibility of confusing this species with Pectinophora
gossypiella. The larvae as well as adults of the two are very different.
In Gelechia hibiscella the body of the larva from the beginning of the
metathoracic segment to the caudal end is white, longitudinally marked
with continuous, narrow, somewhat wavy, reddish brown stripes; one
pair on the dorsum, lying between the paired setae I; one subdorsal
stripe on each side, above seta III, and a lateral stripe in the spiracular
area. Except on the metathoracic and ninth abdominal segments none
of the body tubercles are touched by the longitudinal stripes but lie
between them on the white areas. The first two thoracic segments are
reddish brown with the anterior portion of the mesothorax white above.
The anal shield is yellow ; the thoracic legs and prothoracic shield are black.
The chitinizations about body tubercles moderate but conspicuous,
black or blackish brown, rounded or oval, and sharply defined; crochets
of prolegs uneven biordinal and in a complete circle, 32 to 36, brown;
anal fork present, rather stout, 6- to 8-pronged; head yellow-brown,
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 811
more or less suffused and mottled with black; ocellar pigment black,
continuous under all the ocelli. Full-grown larvae 22 to 23 mm. long.
The only caterpillar treated in this paper which could easily be con-
fused with this species is that of Gelechia neotr Ophelia Heinrich. The
latter, however, is at once distinguished by its 2-pronged anal fork and
the fusing of the middorsal stripes on most of the abdominal segments.
GELECHIA BOSQUELLA CHAMBERS
Gelechia bosquella Chambers, 1878, in Bull. U. S. Geol. Surv. Terr., v. 4, p. 87.
Gelechia basquella Busck, 1903, in Dyar, List North Amer. Lep., no. 5729.
A single moth of this species was reared September 23, 191 8, from
Cassia tora infested by larvae of Platynota rostrana Walker, collected at
Turtle Bayou, Tex. This species is not a malvaceous feeder and is only
mentioned here on account of the similarity of its larva to those of two
other species treated in this paper, Borkhausenia diveni Heinrich and
Noctuelia rufofascialis Stephens. It is very strikingly colored, the three
thoracic segments being a bright wine-red while the rest of the body is
green. The head, legs, thoracic shield, and body tubercles are black. The
red coloring of the thoracic segments, however, is not continuous as in
the two species just mentioned but is broken on the anterior portion of
the meathtorax by a broad encircling band of the greenish body color.
A detailed technical description of the larva is given by Dyar in Busck's
revision of the American Gelechiidae.1
GELECHIA NEOTROPHELLA, N. SP.
(PL. 94, c-g; 105, h)
Gelechia neotrophella, n. sp.
Antennae black. Palpi black, dusted with white. Face black, very slightly dusted
with white. Head and thorax black, heavily dusted with white. Fore wings black,
marked with overlaid white scales; the white dustings over the black forming an
oblique, basal grayish-white patch wider on dorsum than on costa, an obscure, rather
broad median fascia consisting of a narrow, oblique median streak clouded with grayish
before and behind, and a short white geminate costal dash at apical fourth ; cilia smoky
blackish fuscous. Hindwings and cilia pale smoky fuscous, somewhat shaded with
black toward apex. Legs black, dusted and annulated with white. Male genitalia
of type as figured (PI. 94, C-G). Alar expanse 12 to 13 mm.
Habitat. — Brownsville, Tex. (Diven and Heinrich).
Food plant. — Mimosa berlandieri. Larva a leaf-tier, spinning a tube of silk as it
feeds and so binding the leaves together.
Type. — Cat. No. 23739, United States National Museum.
Described from one male type and two male and six female paratypes.
Two generations were noted. From larvae collected February 3, 191 9,
moths issued March 5, and from larvae put in rearing early in May, 191 9,
adults emerged toward the end of the same month.
1 Busck, August, a revision of the American moths of the family gelechiidae, with descrip-
tions of new species. In Proc. U. S. Nat. Mus., v. 25, no. 1304 ,p. 864-865. 1903.
29666°— 21 2
812 Journal of Agricultural Research voi.xx,No. n
The larva is yellowish white, longitudinally striped with wine-red;
one rather broad middorsal stripe dividing into two thin parallel stripes
from the second abdominal segment forward; one moderately broad
subdorsal and one lateral stripe extending from hind margin of prothorax
and fusing on the ninth abdominal segment and forming on the tenth
a dark border around the outer edge of the anal shield; in the area of
seta VI a similar narrow sublateral stripe ; head and thoracic shield pale
yellow; crochets of prolegs 28 to 34, biordinal and arranged in a complete
circle; anal prolegs with a conspicuous blackish red chitinized spot on
caudal side; anal fork rather large, 2-pronged; full-grown larva 8 to
8.5 mm. long.
The species is close to and strikingly resembles Gelechia trophella
Busck, from which, however, it is easily distinguished by the male
genitalia. The structural differences are shown in Plate 93, A and B,
and in Plate 94, C-G.
The larva is not in any way to be confused with the pink bollworm,
from which it differs strikingly in superficial appearance. It resembles
somewhat the larva of Gelechia hibiscella Busck but is separable from
that species by food plant and structure. In G. neotrophella the anal
fork is 2-pronged, while in G. hibiscella it has from 6 to 8 distinct prongs.
In the latter, also, the dorsal stripes are nowhere fused.
TELPHUSA MARIONA, N. SP.
(PL. 94, a, b; 105, F; 109, g)
Telphusa mariona, n. sp.
Antennae black. Palpi cream-color, shading to white on upper side of second
joint; apical half of third joint and upper side of basal joint black. Face white.
Head and thorax cream-yellow. Forewiugs glossy black with two conspicuous
cream-colored spots; one, a short triangular dash on outer third of costa; the other,
an irregular spot of about the same size on dorsum just beyond middle; in some speci-
mens two or three minute and obscure patches of white or cream-colored scales along
termen; cilia blackish. Hindwings and cilia smoky fuscous. Legs black, ringed at
outer margins of the joints with cream -yellow or white. Male genitalia of type as
figured (PI. 94, A, B). Alar expanse 9 to 11 mm.
Habitat. — Brownsville, Tex. (J. D. More and H. C. Hanson).
Food plant. — Abutilon incanum. Larva a leaf-folder. Also taken on Abutilon
berlandkri, Malvastrum sp., Wissadula sp., and Sida sp.
Type. — Cat. No. 23740, United States National Museum.
Described from male type and 25 male and female paratypes reared
from larvae collected in late March and early April, 191 9, on Abutilon
incanum. Moths issued from middle of April to middle of May, 191 9.
Larva, full-grown, 6.5 to 7 mm. long; slender. Body yellowish white
with a subdorsal and a lateral longitudinal row of large red blotches and
a longitudinal row of smaller red spots on the level of seta VI and just
anterior to that seta on each segment; on the eighth abdominal segment
the paired subdorsal spots are fused and on abdominal segment 9 the
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 813
subdorsal and lateral spots are also fused; legs pale yellow; crochets
light brown, 18 to 20 in a complete circle, unevenly biordinal; thoracic
shield divided by a thin median longitudinal pale line, yellow with a
broad shading of fuscous on the lateral extremities and a smaller fuscous
patch at the center of the anterior dorsal margin; anal shield yellow
laterally shaded with fuscous; other chitinized areas smoky fuscous,
tubercles moderately chitinized; hairs moderately long, slender, yellowish.
Head light yellow with a narrow black shading at posterior lateral incision
of hind margin and a similar black dash on ventral margin of epicranium
adjacent to triangular pjate of hypostoma; ocellar pigment black, con-
tinuous under all the ocelli.
The larva is very similar in superficial appearance to the scavenger
worm (Pyroderces rileyi Wlsm.). It differs most strikingly in the ar-
rangement of the red markings, which are in spots or blotches rather
than in continuous bands, and in the possession of a well-developed
anal fork (PI. 105, F) entirely lacking in P. rileyi and the pink bollworm.
The pupa is easily distinguished from those of the other Lepidoptera
treated in this paper by the peculiarly scalloped and fringed posterior
margin of its eighth abdominal segment. (PI. 109, G.)
ISOPHRICTIS SIMIUEU-A (CHAMBERS) l
(PL. 95, A; 102, F)
Gelechia similiclla Chambers, 1872, in Canad. Ent., v. 4, p. 193.
Paltodora similiclla Busck, 1903, in Dyar, List North Amer. Lep., no. 5548.
In the dead flower heads of Rudbeckia sp. (commonly called "nigger
heads" in many parts of Texas) there are two species of lepidopterous
larvae which many nonentomologists have confused with Pectinophora
gossypiella. One of these when mature is about the same size as and
superficially like a full-grown pink bollworm. It is an olethreutid, how-
ever, and as such is easily distinguished by the setal arrangement of the
ninth segment which readily separates the two families Gelechiidae and
Olethreutidae. In the former the paired setae II on the dorsum of the
ninth segment are no closer together than the paired setae I on the dorsum
of abdominal segment 3 (Pi. 105, C) and I is as near II as it is III on the
ninth abdominal segment. In the Olethreutidae, on the other hand,
the paired II on the dorsum of the ninth abdominal segment are on a
single chitinization and closer together than the paired I on the eighth
abdominal segment. Also I and III are closely approximate (Pi. 105, B).
We have not succeeded in rearing the moth, so specific determination
can not be given. The family position of the larva, however, is certain.
1 The genus Isophrictis has been erected by Meyrick for those species formerly listed under the genus
Paltodora Meyrick having the second joint of the labial palpi clothed beneath with long rough spreading
hairs and having veins 7 and 8 of forewings out of 6. It replaces Paltodora for the North American species.
(Meyrick, E. on the genus paltodora. In Ent. Mo. Mag., v. 53.no. 636 [s. 3, v. 3, no. 29], p. 113. 1917-)
8 14 Journal of Agricultural Research voi.xx.No.ii
The other Rudbeckia feeder (Isophrictis similiella Chambers) belongs
to the same family as the pink bollworm and is much more abundant and
less local than the olethreutid. It feeds on the seeds of a number of
Compositae and is frequently found in sunflower heads. The larva when
mature often has a pinkish tinge and somewhat resembles an immature
pink bollworm except for its shape, which is distinctly spindle-like,
sharply tapering at both ends and decidedly stout for its length (1.5 to 2
mm. wide by 5 mm. long in full-grown specimens). The arrangement of
the setae of the anterior group on the epicranium is also characteristic;
A1, A2, and A3 are crowded very close together on the anterior dorsal part
of the head and L1, while remote from A3 as in most Gelechiidae, is well
forward near the ocelli. (Pi. 102, F.)
The pupa shows under the microscope a slight pubescence similar to
that of Pectinophora gossypiella but this is limited to the head alone.
Otherwise, except for the normal seta and a sharp, thorn-like, dor sally
projecting cremaster, the pupa is smooth. It is short and moderately
stout (1.5 mm. broad by 5.5 to 6 mm. long) with the wing cases reaching
nearly to and the metathoracic legs extending a trifle beyond the tip
of the abdomen.
Several moths of this species were reared from larvae collected at
various points in Chambers County and in the neighborhood 01 Galveston
and Houston. Larvae were collected in late August and early September,
1918, and adults issued from these from the middle to the end of Septem-
ber the same year. Other larvae, taken in October of 191 8, produced
moths the following May, passing the winter as pupae within the dried
flower heads.
The male genitalia of the moth are figured in Plate 95, A.
FAMILY OECOPHORIDAE
BORKHAUSENIA DIVENI, N. SP.
(PL. 96, C-F)
Borkhausenia diveni, n. sp.
Antennae white, faintly annulated with fuscous above. Palpi blackish fuscous,
broadly banded at base and apex of third joint with white; inner sides somewhat
dusted with white scales. Face white. Head white with a slight suffusion of fuscous
at vertex. Thorax white, heavily dusted with blackish fuscous; tegulae white, basal
half blackish fuscous. Forewings white, suffused and mottled with pale brown and
black scales, the brown suffusion obscuring most of the ground color at the base and
beyond the middle of the wing; an irregular black spot at base of costa; a similar black
spot on lower vein of cell close to base ; above and below it two smaller black spots;
at middle of wing a straight, rather broad, vertical fascia of blackish brown scales
inwardly margined by a distinct line of the white ground color; in the middle of this
fascia a round spot of distinctly paler brown scales with the black scales edging it
slightly raised ; on costa just beyond median fascia a poorly defined triangular patch
of brown and blackish scales; a small black dot at upper outer angle of cell and several
small obscure dark spots near tornus; cilia dirty white. Hindwingsand cilia grayish
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 815
fuscous. Legs fuscous on outer sides; banded with white on middle of tibiae and at ends
of joints; white on inner sides. Male genitalia of type figured (PI. 96, C-F). Alar
expanse 12 to 13 mm.
Habitat. — Brownsville, Tex. (E. L. Diven).
Food plant. — Lantana horrida. " Larvae making a narrow blotch mine at the edge
of the leaf and curling the edge near base, pupating within the mine" (Diven note).
Type. — Cat. No. 23741, United States National Museum.
Described from male type and one male and three female paratypes
reared from larvae collected April 22, 1919. Moths issued April 27 to
May 5, 1919- Named in honor of the late Emerson IJscum Diven.
The larva when full-grown is 7.5 to 9 mm. long; white, with the tho-
racic segments and the anterior portion of the first abdominal segment a
brilliant wine-red ; in fully fed specimens there is often a pinkish suffusion
on the dorsum of the abdominal segments; thoracic shield yellow,
posteriorly and laterally edged with dark brown; anal shield pale yellow;
other chitinized portions of thoracic segments dark brown; thoracic legs
blackish brown, paler on inner sides; body tubercles deep brown, minute;
setae pale, slender, moderately long; crochets of prolegs dark brown,
24 to 26, biordinal and in a circle broken outwardly ; spiracles pale yellow,
small, round, inconspicuous; no anal fork; head pale yellow with a dark
brown band on each side, extending from the ocelli to the lateral incision
of the hind margin; ocellar pigment black, continuous under the ocelli.
The pupa is rather stout and short, 1.5 to 2 mm. wide by 4.5 to 5 mm.
long; pale yellow-brown; smooth; caudal end rounded; cremaster ab-
sent ; wings and antennae extending to anterior margin of sixth abdominal
segment; labial palpi clearly defined but small, not extending to proximo-
lateral angles of maxillae; between genital and anal openings a divided,
blackish, chitinized rise, without spines, hairs, or other armature.
This species is easily distinguished from the other American forms in
the genus by the straight median fascia. I have placed it in Borkhausenia
advisedly, although strictly speaking it does not belong there. In any
further revision of the Oecophoridae, Borkhausenia divini with B.
conia Wlsm., B. fasciata Wlsm., B. episcia Wlsm., and probably B. orites
Wlsm., will have to be placed in a new genus. While agreeing with the
type of Borkhausenia (J5. minutella L.) on venational characters, they
differ markedly in genitalia. In B. minutella (Pi. 96, A, B) the harpes
are typically oecophorid and laterally placed, the uncus present though
small, the eighth abdominal segment simple, and the entire apparatus
symmetrical. In B. diveni and its allies, on the other hand (PI. 95-97),
the eighth abdominal segment is distinctly modified, the uncus is absent,
the harpes more ventrally placed, and the genital apparatus consistently
asymmetrical. The characters of their genitalia are those of the genus
Triclonella Busck, from which the species are separable on venation,
B. diveni and its allies having 5 of the hind wing distinctly separate at
base from the stalk of 3 and 4. The presence of a few raised scales on
8 1 6 Journal of Agricultural Research vol. xx, No. «
the forewing would seem to throw B. diveni into Mey rick's genus Erysip-
tila. The latter, however, is again distinct on characters of genitalia on
which it will have to be retained and recharacterized, as the raised scale
character does not seem to hold. It is possessed by B. diveni but not by
the other closely allied species (B. conia, B. fasciata, etc.). The genus
Erysiptila, while similar to these in some genitalic characters (for example,
the peculiar development of fused and armed soci and gnathus) and thus
separable from the genus Borkhausenia, has the organs symmetrical
throughout and the harpes laterally rather than ventrally placed. Of
the North American species now listed under the genus Borkhausenia
only three {B. pseudospretella Staint., B. haydendla Chambers, and B.
ascriptella Busck) agree with the type species on all characters. For the
present, however, B. diveni and its allies may be retained in that genus.
Until the entire family can be revised along lines suggested by the devel-
opment of genitalic structures there is nothing to be gained by erecting
a single genus on these characters.
FAMILY STENOMIDAE
AEDEMOSES HESSITANS WALSINGHAM 1
(pl. 95, b, c; 104, d)
Aedemoses hcesiians Walsingham, 1912, in Biol. Centr.-Amer., Lep. Heter.,
v. 4, p. 154.
Eighteen specimens (males and females) of a moth which Mr. Busck has
determined as this species were reared by Diven from larvae which he had
collected on "Mexican ebony" (Siderocarpus flexicaulis) at Brownsville,
Tex. The genus and species were described by Walsingham from a
unique female without hind legs, collected at Presidio Durango, Mexico,
and have not since been recorded. The present rearing, therefore, adds
another to our list of United States species. There can be no doubt of the
identification, as Busck has seen and is familiar with the Walsingham
type and our reared examples agree in all details with the description.
The larva is a leaf-tier, binding together several leaves and feeding
within the tie, eating first the epidermis and later all but the veins of the
leaves. It pupates within the tie, the pupa being naked and attached at
its caudal end by a strand of silk to one of the leaves.
The larva is a typical stenomid, slightly flattened and with seta III
antero-dorsad of and close to the spiracle on abdominal segments 1 to 7
(PI. 104, D); body white with four pale purplish brown longitudinal
stripes, one subdorsal pair just below the level of setae I and II, and a
dorso-lateral one just above the level of setae III; thoracic and anal
shields pale yellow; thoracic legs pale yellow, lightly shaded with brown;
1 Meyrick sunk the genus Aedemoses Walsingham as a synonym of the genus Stenoma Zeller, but on
insufficient grounds, as he disregards its very distinct venational structure in favor of general appearances.
(Meyrick, E. exotic microlepidopiera, v. i, pt. 13. p. 412. 1915.)
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 817
body tubercles inconspicuous, chitinized areas about them unpigmented
except around setae IP and IIb on mesothorax and metathorax where
they are pale brown; body hairs whitish yellow, rather long;*' abdominal
crochets yellow, 40 to 44, unevenly biordinal and in a complete circle;
anal fork absent; head pale yellow, the more heavily chitinized parts of
trophi lighter brown ; ocellar pigment black, continuous under the ocelli;
length, full grown, 7 to 7.5 mm.
The pupa is the typical short, squatty stenomid form; smooth, without
armature or processes of any kind except the very short, inconspicuous
primary setae and a pair of minute spines on the anal rise; seta III on
abdominal segments well forward of the spiracle; spiracles distinct and
rather large, very slightly produced ; wings, antennae, and metathoracic
legs extending to anterior margin of fifth abdominal segment; antero-
ventral margins of fifth abdominal segment curved around the edge of the
wing tips; labial palpi very small, not reaching to proximo-lateral angles
of maxillae; eighth, ninth, and tenth abdominal segments considerably
reduced and sharply tapering; prothorax broad, nearly one-third the
breadth of mesothorax; proleg scars distinct ; length 4 to 4.5 mm; width
1.5 to 2 mm.
Immature larvae were collected by Diven in late January, 191 9, and
feeding larvae as late as April 1, 191 9; from the latter, moths issued from
April 17 to 26 of the same year.
The male genitalia of the moth are figured in Plate 95, B, C.
FAMILY BLASTOBASIDAE
ZENODOCHIUM CITRICOLELLA (CHAMBERS)
(pl. 98, a-c; 102; 104, c; 105, I)
Blastobasis citricolella Chambers, 1880, in Rept. U. S. Dept. Agr. 1879, p.
206-207.
Blastobasis citriella Chambers, 1880, in Rept. U. S. Dept. Agr. 1879, p. 245.
Zenodochium citricolella Dietz, 1910, in Trans. Amer. Ent. Soc, v. 36, p. n-12.
Feeding in dry okra pods, in the seed pods of Hibiscus, and in old
or diseased cotton bolls we often found associated with Pyroderces rileyi
a dirty brownish larva with a glistening black head and thorax, spin-
ning a thin web in the seed pods within which it fed and pupated. A
number were collected at various places in Chambers County (Smith
Point, Point Bolivar, and South Bayou) and from these were reared a
number of adults agreeing in genitalic and other characters with authen-
tic reared specimens of Zenodochium citricolella Chambers in the United
States National Museum. The species is a scavenger and probably a
very general feeder, as it was originally recorded from dried oranges
and is to be found in almost any dry or diseased malvaceous seed pod.
Figures of the male genitalia of the moth are given in Plate 98, A-C.
Journal of Agricultural Research
Vol. XX. No. ii
The larva is easily distinguished from Pyroderces rileyi and the other
lepidopterous cotton feeders by the structural characters shown on
Plates 102, 104, and 105. The most striking features are the oval chiti-
nized plate on the submentum, the nearly complete fuscous circle sur-
rounding the chitinization of tubercle III on abdominal segments 1 to
7, and the typical blastobasid arrangement of the prothoracic legs (Pi.
105, I), set very close together with the coxal lobes touching each other.
The species probably has several generations a year. Larvse collected
in August, 1 91 8, produced moths in that month and throughout Sep-
tember. Others collected during November and December produced
moths the following April.
HOLCOCERA OCHROCEPHALA DIETZ
(PL. 98, D-F)
Holcocera ochrocephala Dietz, 1910, in Trans. Amer. Entomol. Soc., v. 36, p. 31-32.
A large series of moths were reared during February and March, 191 9,
from larvae collected December, 1918, in imperfectly opened and weevil-
infested cotton bolls at Brownsville, Tex. They agree with the descrip-
tion and the single female paratype of Dietz's species in the United
States National Museum, and I have no hesitation in so determining
them. The larval habits are the same as those of Zenodochium citrico-
lella. There probably has been some confusing of our material, as all
the larvae we have associated with the H. ochrocephala adults are iden-
tical with those of Z. citricolella. Probably, since the two species work
together in the same way and are superficially alike, the larvae of one
species was preserved and that of the other reared. It is extremely
unlikely that there should be two blastobasids in different genera with-
out a single structural difference in their larvae.
The male genitalia of the moth are figured in Plate 98, D-F.
HOLCOCERA CONFAMULELLA, N. SP.
(PL. 99, C)
Holcocera confamulella, n. sp.
Antennae deeply excised above basal joint and with truncate scale tuft; very
weakly ciliate. Palpi grayish ochreous, dusted with fuscous on outer sides. Face
grayish ochreous, vertically banded with fuscous. Head and thorax grayish white
mixed and suffused with fuscous scales. Forewings grayish white, suffused and
mottled with fuscous, the fuscous scaling giving the outer two-thirds of the wing a
distinctly gray-brown appearance, darkening into an ill-defined, outwardly angulate
antemedial fascia bordering a grayish basal patch and forming an irregular, broken,
and obscure vertical fascia beyond the middle; along the termen a few barely dis-
tinguishable fuscous spots; cilia grayish white. Hindwings very narrow, pale smoky
fuscous; cilia paler, tinged with ochreous. Legs whitish ochreous on inner sides;
the outer sides fuscous, spotted with white on tibiae and ringed with white or whitish
ochreous at ends of joints. Male genitalia of type figured (PI. 99, C). Alar expanse
14 to 15 mm.
Mar.i.i92i Lepidoptera Likely to Be Confused with Pink Bollworm 819
Habitat. — Brownsville, Tex. (More, Barber, Heinrich).
Food plant. — Fruits of Crataegus.
Type. — Cat. No. 23742, United States National Museum.
This species is very close to Holcocera modcstella Clemens, to which it
would run in Dietz's tables.1 It may eventually prove to be that species,
but in the absence of an authentic male of H. modestella from the type
locality it is better to risk a possible synonym than to make a doubtful
determination. I have seen no specimens of Clemens's species. The
male genitalia here figured fix the concept of H. confamulella and enable
its ready identification.
Five moths (male type and four male and female paratypes) were
reared April 10 to 21, 191 9, from fruits of Crataegus rather heavily
infested by larvae of Crocidosema plebeiana Zeller. The larvae of Holcocera
confamulella were not noted.
FAMILY ETHMIIDAE
ETHMIA DELLIELLA (FERNALD)
Psecadia delliella Fernald, 1891, in Canad. Ent., v. 23, p. 29.
Babaiaxa delliella Busck, 1903, in Dyar, List North Amer. Lep., no. 5935.
Ethtnia delliella Barnes and McDunnough, 1917, Check List Lep. Bor. Amer.,
no. 6645.
One moth reared April 30, 191 9, from Wissadida lozani heavily in-
fested by a stem -boring aegeriid (Zenodoxus palmi Neumoegen). Material
collected at Brownsville, Tex., by E. h. Diven, March 28, 191 9. Larva
and habits not noted.
ETHMIA BITTENELLA (BUSCK)
Tamarrha bittenella Busck, 1906, in Proc. U. S. Nat. Mus., v. 30, p. 730.
Ethmia bittenella Meyrick, 1914, Lep. Cat., pars. 19, p. 28.
Two pupae collected by Diven in galleries in stems of Wissadula lozani,
Brownsville, Tex., April 1, 191 9. Moth issued April 9, 191 9.
The larvae were not noted. The caterpillars of this family are, however,
to be distinguished from the others having three setae on the prespiracular
shield of prothorax and IV and V of abdomen approximate by the
presence of one or more secondary hairs on the body, usually on the
abdominal segments in the region of the prolegs. The prolegs them-
selves are long and slender as in the Pterophoridae. On abdominal
segment 9, seta I is higher than II.
1 Dietz, Win. G. revision of the blasiobasidae of xorih America. In Trans. Amer. Ent. Soc,
v. 36, no. i, p. 24-33. 1910-
820 Journal of Agricultural Research vol. xx, No. n
FAMILY COSMOPTERYGIDAE
PYRODERCES RILEYI (WALSINGHAM)
(PL. 102, a, b; 103, c; 105, d; 106, c; 107, E, F)
Batrachedra rileyi Walsingham , 1882, in Trans. Amer. Ent. Soc, v. io,
p. 198-199.
Batrachetra rileyi Dyar, 1903, List North Amer. Lep., no. 6059.
Pyroderces rileyi Busck, 1917, in Jottr. Agr. Res., v. 9, no. 10, p. 362-366, 370.
The larva of this common scavenger is frequently mistaken for the
pink bollworm. It is, however, very readily distinguished from it and
similar pink -banded larvae of the gelechioid and other groups.
Since a complete description of adult, larva, and pupa is given in
Busck's article on the pink bollworm,1 it will suffice here to call atten-
tion to the diagnostic characters of the immature stages.
For the larva these are :
Three setae (III, IV, and V) triangularly grouped on prespiracular
shield of prothorax; prothoracic IIa higher than P; IV and V on proleg-
bearing abdominal segments approximate; III on eighth abdominal
segment anterior to the spiracle; paired dorsal setae (II) on the ninth
abdominal segment not on a single chitinization, but closer together than
paired I on eighth abdominal segment (PI. 105, D) ; / and III approximate
on ninth abdominal segment (as in the Olethreutidae) ; IV and V approxi-
mate, with VI well separated from them on ninth abdominal segment;
crochets of prolegs uniordinal and in a complete circle ; anal fork absent ;
pink bandings on anterior and posterior margins (not in the middle)
of the segments.
The sum total of these characters is possessed by no other caterpillar
to be found on cotton.
The pupa (PI. 107, E, F) may be distinguished by the following
characters :
Pointed wing cases reaching to posterior margin of the sixth abdominal
segment; antennae reaching to tips of wings; maxillary palpi small and
not reaching proximo-lateral angles of maxillae; vertex wider than pro-
thorax; abdomen tapering, bluntly rounded, smooth except for primary
hairs and a cluster of strong hooked setae at posterior end and around
anal opening ; cremaster absent ; no labial palpi or exposed metathoracic
legs.
The drawings (PI. 102, 103, 105-107) show the distinguishing struc-
tural characters of larva and pupa. It will be noted that a correction has
been made in Busck's figure of the setal map of the ninth abdominal
segment of the larva which omitted one of the ventral setae. The setal
arrangement of the ninth abdominal segment with all setae in a row, I
approximate to III and VI well-separated from IV and V, can not be
1 Busck, August, op. err. 1917, p. 362-366.
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 821
considered a family character. It serves, however, to separate Pyroderces
rileyi from the gelechioid forms which it otherwise resembles.
FAMILY TORTRICIDAE
platynota rostrana (walker)
(Pl. 104, a; 105, a)
Teras rostrana Walker, 1863, in List Lep. Brit. Mus., pt. 28, p. 290.
Platynota rostrana Dyar, 1903, List North Amer. Lep.. no. 5383.
This species and the following two are rather general feeders and are
frequently found on cotton and other Malvaceae. We have reared
moths of Platynota rostrayxa from cotton, okra {Hibiscus esculentus),
Malvaviscus drummondii, Bastardia viscosa, Amaranthus spp., and Cassia
tora, collected at Brownsville and several localities in Chambers County.
The species is normally a leaf -feeder, tying the terminal leaves and pupat-
ing within the tie. We have, however, also found it occasionally feeding
on the flower buds of okra and on one occasion (Dec. 31, 191 8) Diven took
three larvae at Brownsville in dry cotton bolls, feeding on the lint. They
pupated in the loose lint, and moths issued February 7 and March 3, 191 9.
In the Chambers County localities larvae were collected during late August
and early September, 191 8, which produced moths late in September and
early in October of the same year. There are at least two and probably
three or more generations a year in Texas.
The larva is not likely to be confused with the pink bollworm. It is
easily separable on the setal characters figured on Plates 104 and 105.
The arrangement of the pared dorsal setae (II) on the ninth abdominal
segment (that is, on a single chitinization and considerably closer together
than any dorsal pair on the eighth abdominal segment) (PI. 105, A),
coupled with the normal micro characters of three setae on the pre-
spiracular shield of pro thorax, and a close approximation of IV and V
on the proleg-bearing abdominal segments, distinguishes the families of
the Tortricoidea. In Tortricidae proper (to which this and the two
following species belong) seta I on the ninth abdominal segment is much
as in the Gelechiidae (that is, rather well separated from III and often
as near to II as to III) (PI. 105, A). In the families Olethreutidae and
Phaloniidae, on the other hand, I and III are approximate and very
often on the same chitinization.
The pupa is typically tortricoid, with wings short and broad at the tip
(not tapering) and having the abdominal segments armed dorsally with
a double row of strong spines, those of the anterior rows larger and some-
what hooked (compare PI. 108, D). It is distinguished from that of the
common olethreutid malvaceous feeder (Crocidosema plebeiana Zell.) by
the presence of a well-developed, bluntly rounded cremaster entirely
lacking in the latter.
822 Journal of Agricultural Research voi.xx.No. h
PLATYNOTA FLAVEDANA CLEMENS
Platynota Jlavedana Clemens, 1861, in Proc. Acad. Nat. Sci. Phila., i860, p. 348.
Platynota flavedana Dyar, 1903, List North Amer. Lep., no. 5382.
One specimen reared by Diven (May 23, 191 9) from cotton leaves
collected at Brownsville, Tex., May 7, 191 9.
The larva was not noted.
The pupa is strikingly like that of Platynota rostrana Walker.
AMORBIA EMIGRATELLA BUSCK
(PL. 109, F)
Amorbia emigratclla Busck, 1910, in Proc. Ent. Soc. Washington, v. 11, p.
201-202.
Amorbia emigratella Walsingham, 1913, in Biol. Centr.-Amer., Lep. Heter., v. 4,
p. 219.
Two moths reared from cotton May 19 and 24, 1919 (E.'L. Diven) in
same material infested by Platynota flavedana, collected at Brownsville,
Tex., May 7, 191 9. The pupa has a conspicuous mid-dorsal, cuplike,
circular invagination near the anterior margins of the first seven ab-
dominal segments, the anterior dorsal margins themselves being strongly
chitinized and folded back into a projecting ridge; otherwise as in P.
rostrana.
The larva was not noted.
FAMILY OLETHREUTIDAE
CROCIDOSEMA PLEBEIANA ZELLER
(pl. 99, a; 102, c, d; 103, E; 105, g; 106, b; 108, a-d)
Crocidoscma plebeiana Zeller, 1847, in Isis von Oken, 1847, Heft 10, p. 721-722.
Eucosma plebeiana Walsingham, 1914, in Biol. Centr.-Amer., Lep. Heter., v.
4, p. 231-232.
Up to the present this almost cosmopolitan insect had not been re-
corded from the United States. Our collecting, however, showed it
well distributed and fairly abundant in Texas. In the United States
National Museum there are also several adults from California, so that
its known range may be said to correspond roughly with the distribution
of the Malvaceae. Adults were reared by us from the following plants:
Malvastrum spicatum (Brownsville, Tex., May, 191 9); hollyhock (Althaea
rosea) (Brownsville, Tex., May, 191 9); Malvaviscus drummondii (Smith
Point, Tex., November, December, 191 8; Anahuac, Tex., September,
1 91 8); okra {Hibiscus esculentus) (Double Bayou, Tex., November,
December, i9i8);and Kosteleyzkya spp. (Anahuac, Tex., November, 191 8).
Larvae were also collected in seed pods of H. militaris (Lake Charlotte, Tex.,
September, 191 8) and in flowers of H. rosa-sinensis (Smith Point, Tex.,
November, 191 8). They feed chiefly in the seed pods and on the seeds of
Mar. r, 1921 Lepidoptcra Likely to Be Confused with Pink Bollworm 823
the plants infested, but occasionally also on the pollen of the flowers. The
species is of special interest because its work and habits are almost identical
with those of the genus Pectinophora and also because the larva is fre-
quently pinkish and often has the outer crochets of the prolegs weakly de-
veloped or absent. It is easily mistaken for a half -grown pink bollworm.
It is readily distinguished, however, by the structural characters here fig-
ured (PI. 102, 103, 105, 106). The linear arrangement of setae III, IV, and
V on the prothorax, the position of III anterior to the spiracle on the eighth
abdominal segment, the well-developed anal fork (PI. 105, G), and the
olethreutid grouping of the setae on the ninth abdominal segment (PI.
103, E) separate it from all the larvae treated in his paper.
The characters of the pupal abdomen are shown on Plate 108, A-D.
Eucosma discretivana Heinrich and E. helianthana Riley exhibit
similar structures, but as neither of these species attacks Malvaceae
there is little or no likelihood of confusing them with Crocidosema,
We did not find C. plebeiana in cotton, but there appears to be no reason
why it should not attack that plant; and its possible presence and con-
fusion with the pink bollworm should be borne in mind in cotton in-
spection.
The male genitalia of the adult are shown in Plate 99, A.
EUCOSMA DISCRETIVANA, N. SP.
(PL. 99, B)
Eucosma discretivana, n. sp.
Antennae, palpi, face, and head dull, somewhat ashy fuscous. Thorax pale, dull
fuscous; tegulse fuscous with a very slight bronzy tint. Forewings dirty grayish
white marked with grayish fuscous; an outwardly angulate grayish fuscous basal patch
slightly wider on costa than dorsum; a somewhat paler, semioval patch on dorsum
before tornus and extending half way to costa; several narrow, obscure lines of fus-
cous scales extending outwardly from costa and faintly streaking the white areas; a
similar faint line extending from dorsum through middle of white area bordering basal
patch; entire termen narrowly margined by pale grayish fuscous: the wnitish areas
of the wing most pronounced just beyond basal patch and near tornus; cilia grayish;
costal fold deeply appressed and reaching nearly to middle of wing. Hindwings dull,
smoky fuscous, cilia grayish white with a dull fuscous band along their base. Abdo-
men grayish fuscous with silvery white scales along the sides and a few scattered silvery
scales beneath. Legs fuscous, shading to dirty gray-white on inner sides. Male
genitalia of type figured (PI. 99, B). Alar expanse 13 to 16 mm.
Habitat. — Sheldon, Tex. (A. C. Johnson).
Food plant. — " Wild myrtle. " Larva boring in the stem and forming a gall.
Type. — Cat. No. 23743, United States National Museum.
Described from male type and three male and five female paratypes
reared by A. C. Johnson, April 10 to 23, 1919, from larvae collected by
him March 14, 191 9.
It is very close to Eucosma obfuscana Riley, which it strikingly resem-
bles. The two species are, however, readily distinguishable on both
genitalia and slight but constant color differences. In E. obfuscana the
face, head, thorax, and base of antennae are inky blue-black, the dark
824 Journal of Agricultural Research voi.xx.xo. »
margin of termen of forewing pronounced and blue-black, extending
from the apex only a little over one-half the length of the termen, the
white scaling of the tornal area extending into the cilia of the anal angle
which are also white. In E. discretivana there is none of the blue-black
scaling so noticeable in E. obfuscana, and the entire termen is faintly-
dark margined. The cucullus of the harpes of the male genitalia is also
more narrowly elongate in E. obfuscana than in E. discretivana.
The larva is in general structure very like Crocidosema plebeiana, except
that setse I, III, IV, and V on the ninth abdominal segment are about
equally spaced and the anal fork is lacking. The body is cream-white
without markings; chitinized areas about body tubercles not pigmented;
hairs whitish yellow; thoracic and anal shields pale yellow, scarcely
pigmented; head light brown; crochets brown, 28 to 30, uniordinal and
in a complete circle; length, full-grown, 10 to 10.5 mm.
The pupa is similar to that of Crocidosema plebeiana but somewhat
larger, 8.5 to 9 mm. long by 2.5 mm. wide.
The two species are easily distinguished by their food plants and larval
habits.
EUCOSMA HELIANTHANA (RILEY)
Semasia helianthana Riley, 1881, in Trans. St. Louis Acad. Sci., v. 4, p. 319.
Thiodia helianthana Dyar, 1903, in List North Amer., Lep., no. 5186.
Eucosma helianthana Barnes and McDunnough, 1917, Check List Lep. Bor.
Amer., no. 7081.
We found a larva about the size of the pink bollworm and superficially
resembling it feeding in the flower heads and on the seeds of the large
garden sunflower. It was somewhat pinkish and had a pale kidney-
shaped spot on the thoracic shield similar to that of Pectinophora. It
had the characteristic olethreutid arrangement of setse on the ninth
abdominal segment and proved to be the caterpillar of Eucosma helian-
thana Riley, a species limited in food plant as far as I know to Helianthus.
As the pink bollworm does not attack sunflower and E. helianthana does
not attack cotton, there is no reason to confuse the two. The structural
differences are also easily seen under a binocular or a strong hand lens.
The pupa is similar to that of Crocidosema plebeiana but larger, about
the size of that of Eucosma discretivana.
Larvae were collected at Dickinson, Tex., September 28, 191 8, and
pupae also were found at Smith Point, August 30, 1918. From the latter
a moth was reared September 3 of the same year.
EASPEYRESIA TRISTRIGANA (CEEMENS)
Stigmonota tristrigana Clemens, 1865, in Proc. Ent. Soc. Phila., v. 5, p. 133.
Enarmonia tristrigana Dyar, 1903, List North Amer. Lep., no. 5275.
Laspeyresia tristrigana Barnes and McDunnough, 1917, Check List Lep. Bor.
Amer., no. 7220.
On the prairie lands and along the fences adjoining fields-that had been
planted in cotton the previous year (191 7) we frequently found a white
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 825
and pinkish larva feeding on the seeds of Baptisia spp. about the size
and with much the general appearance of the pink bollworm. Except
for the complete circle of crochets on the prolegs the superficial resem-
blance was rather striking. The structural characters are so obviously
different as to prevent confusion by a careful observer. The arrange-
ment of setae on the ninth abdominal segment is typically olethreutid
(Compare PI. 103, E; 105, B), and the grouping of the head setae is also
quite different from that of the pink bollworm; A1, A2, A3, and V lie in
almost a straight line, and the puncture Aa is well back of (almost directly
posterior to) A2 rather than between it and A1 as in Pectinophora gos-
sypiella.
The larva is most like that of Eucosma helianthana, from which it
differs in the size of the head, the color of the thoracic shield, and the
position of epicranial puncture Aa. In E. Jielianthana the puncture (Aa)
lies to the side directly dorsad of seta A2, between it and the adfrontal
suture, the head is smaller in the full-grown larva, and the thoracic
shield is brown with a more or less distinct hyaline kidney-shaped spot
on the side. In Laspeyresia tristrigana the shield is of the general body
color with a few small, irregular, scattered yellow spots. Neither species
has an anal fork.
The pupa is similar to that of Crocidosema plebeiana.
Several adults were reared during May, 191 9, from larvae collected in
August, 1 91 8 (Anahuac, Tex.) and in November, 19 18 (El Vista, Tex.).
FAMILY PHALONIIDAE
PHALONIA CEPHALANTHANA, N. SP.
(PL. IOO, A)
Phalonia cephalanthana, n. sp.
Antennae grayish black, palpi dull yellow, whitish above and on inner sides. Face
whitish. Head yellow. Thorax mahogany-red. Fore wings brownish overlaid with
mahogany-red mixed with a few blackish scales, the red scaling unevenly distributed,
forming an obscure but distinguishable outwardly angulate basal patch, a broad,
vertical, somewhat irregular median fascia, and a moderately broad, outwardly
oblique costal dash near apex, the latter extending from apical fifth of costa to below
middle of termen; other areas of wing brown, more or less streaked with reddish or
black scales; cilia mixed brown, red, and black. Hind-wings smoky gray; under-
side faintly mottled; cilia grayish white. Legs heavily dusted on outer sides with
grayish black; ends of joints and inner sides yellowish white. In general appearance
to the naked eye the insect is a rather pale wine-red, blotched with darker shading of
the same color. Male genitalia of type figured (PI. 100, A). Alar expanse 8 to 10 mm.
Habitat.— Shores of Lake Charlotte, Chambers County, Tex. (Heinrich).
Food plant. — Cephalanthas occidenialis.
Type. — Cat. No. 23744, United States National Museum.
Described from male type and 16 male and female paratypes reared
September 16 to 24, 1919, from larvae collected September 10, 1918; a
distinct and easily recognized species.
826 Journal of Agricultural Research voi.xx.No. h
The larva feeds in the seed pods. It is a dirty white with the chiti-
nized areas about the body tubercles conspicuous, moderately large, round
or oval, and a dull smoky fuscous, the chitinizations becoming heavier
and more extended toward the caudal end; on the eighth abdominal
segment paired setae I are on a single chitinization ; also paired II; on the
ninth abdominal segment paired II, I, and III are on a single shield; the
setal arrangement of the ninth abdominal segment is similar to that of
the Olethreutidae with I and III rather closely approximate; seta III on
eighth abdominal segment directly anterior to the spiracle; anal shield
brown; anal fork developed, 6-pronged; crochets of prolegs uniordinal
and arranged in a complete circle, 36 to 40; skin finely granulate; tho-
racic legs pale; thoracic shield the color of body except for a shading of
yellow along hind margins. Head yellow, shading to yellowish brown;
ocellar pigment slight, continuous but not filling the ocellar area; setae of
anterior and lateral group (A1, A2, A3, and L1) crowded well forward on
head ; A1, A2, and A3 forming a slightly acute angle ; L1 closely approximate
to A3. Full-grown larva 8 to 9 mm. long.
The pupa is similar to that of Crocidosema plebeiana except that the
caudal end is more rounded. There is no cremaster.
FAMILY AEGERIIDAE
ZENODOXUS PALMII (NEUMOEGEN)
Larunda palmii Neumoegen, 1891, in Ent. News, v. 2, p. 108.
Paranthrene palmii Beutenmiiller, 1901, in Mem. Amer. Mus. Nat. Hist., v. 1,
pt. 6, p. 316.
Paranthrene palmii Dyar, 1903, List North Amer. Lep., no. 4260.
Zenodoxus palmii Barnes and McDunnough, 1917, Check List Lep. Bor. Amer.,
no. 6735.
Several specimens of this species were reared during April and May,
1919, from larvae collected at Brownsville, Tex., January 23 and February
3, 1919, by H. C. Hanson and E. L. Diven. The caterpillars bore in the
stems of Wissadula lozani and are usually found well down in the stems
at the base of the plants near the roots. The adults agreed very well
with the description of Zenodoxus palmii Neum. I have since compared
them with the type in the Brooklyn Institute and have little hesitation
in determining them as that species, although they are a trifle small (alar
expanse 17.5 to 21 mm.).
The larvae of this family are not likely to be confused with those of the
pink bollworm and are easily identified by the peculiar arrangement of the
ocelli — that is, with ocelli I to IV grouped together forming a trapezoid
and V and VI well separated from the other four — and the crochets of the
prolegs. The latter are always uniordinal and in two transverse bands.
The setae on the ninth abdominal segment are much the same as in the
Olethreutidae.
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 827
The pupae have two rows of strong spines on the dorsum- of several
of the abdominal segments as in the Tortricidae, but the wings are
narrow and pointed, the maxillary palpi are large and conspicuous, and
the thoracic spiracle is normally well developed; thus they are dis-
tinguished readily enough from pupae of the latter group.
FAMILY PTEROPHORIDAE
EDEMATOPHORUS VENAPUNCTUS, N. SP., BARNES AND LINDSEY *
During April and May, 191 9, Mr. B. L,. Diven reared eight specimens
of a pterophorid moth from larvae feeding on the leaves of a composite
at Brownsville, Tex. These were referred to Mr. Lindsey, who determined
them as Oedematophorus venapunctus, an unpublished species, which he
and Dr. Barnes had recently described from collected material.
The species is not a malvaceous feeder and has no special interest here
apart from the rearing record and the structural peculiarities of the larva
and pupa which, while strikingly modified in this particular form, will
serve, nevertheless, to exemplify the family.
The pterophorid larvae have only two setae on the prespiracular shield
of the prothorax and setae IV and V approximate on the proleg-bearing
abdominal segments, as in the Pyralidae with which they are affiliated.
They have, however, in distinction from the Pyralidae proper, long stem-
like prolegs and a greater or less development of secondary setae. The
crochets are also peculiar, being uniordinal, few in number (6 to 8 in the
genus Oedemataphorus), and arranged in a quarter circle opening
outwardly. In O. venapunctus the secondary hairs are confined to a row
1 Inasmuch as the foregoing name was desired for this paper in advance of their proposed revision of the
Pterophoridae Drs. Wm. Barnes and A. W. Lindsey have kindly furnished the following description:
Oedematophorus venapunctus, n. sp., Barnes and Lindsey.
Head whitish ochreous between the antennae, elsewhere light brown. Antennae and palpi pale brown-
ish ochreous, almost white, the latter short, oblique or porrect. Thorax and legs of the same shade of pale
brownish ochreous, the fore and middle legs tinged with brown inside. Abdomen similar both above
and below, with a fine, browa, middorsal line.
Primaries concolorous with thorax, darker toward costa, especially in first lobe, though this shade is
scarcely evident in some specimens. Just before and below the base of the cleft is a small blackish brown
spot, isolated except in our darkest specimen, in which it is continued obliquely toward the costa by a
faint dark shade. In the outer margin of the second lobe there are four short, dark dashes on the tips
of the anal, cubital, and third median veins. These are very faint in some specimens. A similar but
heavier spot occurs on the inner margin of the first lobe a short distance before its apex at the tip of the
fifth radial. Two vague dots sometimes appear on the costal margin of this lobe, one just before the apex
and the other almost opposite the one on the inner margin. Fringes concolorous, slightly darker toward
the apex of the wing and with their bases slightly paler. Secondaries somewhat paler than primaries and
with a more grayish tinge. Fringes concolorous with slightly paler bases.
Expanse 15 to 18 mm.
Described from the following series: Holotype male, Brownsville, Tex., March; paratype male, same
locality; allotype and six paratypes females, San Benito, Tex., March and April. (Collection Barnes).
Paratype male, Brownsville, Tex., March, and paratype female, from San Benito, Tex., April, in United
States National Museum, type Cat. no. 23495.
This species appears to be allied to Oedematophorus paleaceus, O. stramineus, O. kellicctti, and related
species. It differs from the first two in the presence of the terminal dots and from the last two in that the
dot in the disc of the primaries is not contiguous to the base of the cleft. The form of the male genitalia
also differs from that of any related species known to us. We have been unable to place it as a described
Mexican or Central American species.
29666°— 21 3
828 Journal of Agricultural Research voi.xx.No. h
of 5 to 8 va the area normally occupied by seta VI. The body tubercles
are somewhat produced, especially on the pro thorax and tenth abdominal
segment, and the hairs themselves are swollen and bulbous. In addition
to the setae there are on all except the first thoracic and the last abdominal
segments several fingerlike projections from the skin. On the abdomen
these arise back of setse I, II, III, IV, and V from the base of their tuber-
cles and in the area back of the spiracle and seta group IV-V. The
prothorax is somewhat produced dorsally, and the head is capable of
retraction under the cover of this rooflike projection.
In the pupa the venter of the eighth, ninth, and tenth segments is
deeply concave with the lateral edges fringed by rather short flexible
setae. The ventral edge of the tenth segment and the anterior margins
of the concavity are also armed with clusters of slender, hooked hairs.
The caudal end is sharply pointed, but there is no distinct cremaster.
The larva is an external feeder, and the pupal period is very short.
Larvae collected by Diven from April 7 to 14, 1919, produced moths as
early as the ninteenth of the same month.
FAMILY PYRALIDAK
SUBFAMILY THYRIDINAE
MESKEA DYSPTERARIA GROTE
(PL. ioi, E, F; 104, B; 109, A-E)
Meskea dyspteraria Grote, 1877, in Canad. Ent., v. 9, p. 115.
Meskea dyspteraria Dyar, 1903, List North Amer. Lep., no. 4139.
This species was described by Grote from a single female collected in
Bastrop County, Tex. Up to the present it has been rare in collec-
tions, Grote's type and a male from the Riley collection being the only
representatives in the United States National Museum. Nothing was
known of its larval habits or life history. We succeeded in rearing a large
series of the moths and found their larvae rather abundant though locally
distributed. The larvae mine the stems of several malvaceous plants,
forming a conspicuous, elongate gall. The species seems to favor Malva-
viscus and Abutilon; but occasional larvae were found in galls on Kostel-
elzkyasp. (Anahuac, Tex., Aug. 13-14, 1918, More and Diven, collectors).
The species overwinters as larvae in the gallery, pupating in the spring and
producing moths during April and May. From larvae collected in Mal-
vaviscus drummondii at Wallisville, Tex., September 3, 191 8 (Hanson,
Diven, and Heinrich), October 28, 191 8 (Hunter, Busck, and Johnson),
and November 5, 191 8 (Barber, More, and Heinrich) moths were reared
during May 9 to 25, 191 9; in M. drummondii taken along the San Jacinto
River near Crosby, Tex. (Hanson), November 6, 191 8, moths issued May
4 to 10, 1 91 8. Larvae taken in Abutilon bcrlandieri, at Brownsville,
Tex., December 31, 1918, and in A. incanum at Barreta, Tex., January 5,
Mar. i, igii Lepidoptcra Likely to Be Confused with Pink Bollworm 829
1 91 9 (Hanson) pupated the latter part of March and produced moths
from April 5 to May 22, 1919. Neither larva nor work were found in cot-
ton or okra or on any of the various species of Hibiscus, though there
appears to be no reason why these plants should escape.
The full-grown larva is somewhat larger than a mature pink bollworm
(22-22.5 mm. long) and is easily distinguished from it by the pyralid ar-
rangement of the body setae (two setae only on prespiracular shield of pro-
thorax and IV and V approximate on proleg-bearing abdominal segments).
The structural characters of larva and pupa are fully illustrated in
Plates 101, 104, and 109. These and the larval habits will serve to
identify the species and distinguish it readily from any other lepidopteron
of similar food plant and habits.1
SUBFAMILY PYRAUSTINAE
NOCTUELIA RUFOFASCIAUS (STEPHENS)
Ennychia rufofascialis Stephens, 1834, Illus. Brit. Ent., Haust, v. 4, p. 33.
Botys (?) thalialis Walker, 1859, List Lep. Brit. Mus., pt. 18, p. 582.
Noctuelia thalialis Hampson, 1899, in Proc. Zool. Soc. London, pt. 1, p. 279, 1899.
Noctuelia thalialis Dyar, 1903, List North Amer. Lep., no. 4478.
Noctuelia rufofascialis Barnes and McDunnough, 1918, Contrib. Nat. Hist.
Lep. North Amer., v. 4, no. 2, p. 167.
The larva of this species is a seed-feeder in pods of Abutilon, Wissadula,
Malvastrum, Sida, and possibly other malvaceous or similar plants. It
feeds in much the same way as the pink bollworm and pupates in a thin
cocoon either in the empty seed pod or on the outside of the plant. Two
larvae were taken at Brownsville, Tex., April 11, 1919, by Diven feeding
in the young terminal shoots of cotton. This habit, however, is unusual.
When full-grown the larva is about the size of a full-fed pink bollworm
and seems ridiculously large for the small seed pods within which it must
accommodate itself. It is very strikingly and beautifully marked and
very similar to the caterpillers of Gelechia bosquella Chambers and Bork-
hausenia diveni, elsewhere mentioned in this paper. It is readily dis-
tinguished from them by the pyraloid setal arrangement of the pro-
thorax (two setae only in the prespiracular group). The general body
color is white with the thoracic segments and anterior half of the first
abdominal segment a deep wine-red. The remaining abdominal segments
are also partially encircled by a broad band of the same color. The head
is light yellow, and the thoracic and anal shields are yellow or brownish,
the legs smoky fuscous, and the crochets of the prolegs (7 to 10) uniordinal
and arranged in a circle broken outwardly as in the pink bollworm — a very
unusual structure in this subfamily.
1 It should be noted that puncture Aa on the epicranium is somewhat differently located on different
specimens, sometimes higher, sometimes lower, occasionally even lying between seta A3 and L1 and fre„
quently differently placed on opposite sides of the same head. Body seta IV on abdominal segment 9 is
also very often absent. When present it is always short and inconspicuous.
830 Journal of Agricultural Research voi.xx, no. »
Adults were reared during May, 191 9, from larvae collected in pods of
Abutilon and Malvastrum at Brownsville, Tex., December 27, 1918
(Hanson) , and April 12,1919 (Diven) . Other larvae were collected in seed
pods of Wissadula and vSida at Brownsville, but no adults were reared.
The species is not common and we found it only in the vicinity of
Brownsville.
PACHYZANCLA BIPUNCTALIS (FABRICIUS)
Phalaena bipunctalis Fabricius, 1794, Ent. Syst., c. 3, pars 2, p. 232.
Pachyzancla bipunctalis Dyar, 1903, List North Amer. Lep., no. 4344-
Several moths of this species were reared September 14 to 18, 191 8,
from larvae tying the terminal leaves and feeding on the seeds of the
common pigweed (A niaranthus hybridus) . Larvae were collected at Turtle
Bayou, Tex., September 4, 1918.
The caterpillars are typical Pyraustinae with the proleg crochets
triordinal and arranged in a penellipse.
All the Pyralidae are distinguished by having two seta* on the prespirac-
ular shield of the prothorax (IV and V) and IV and V approximate on the
proleg-bearing abdominal segments (compare PI. 103, B; 104, B). No
other group posesses this combination.
GLYPHODES PYLOALIS WALKER
Glyphodes pyloalis Walker, 1859, List Lep. Brit. Mus., pt. 19, p. 973-974.
Glyphodes pyloalis Hampson, 1899, in Proc. Zool. Soc. London, 1898, pt. 4, p. 746.
On a private estate near Alto Loma, Tex., the writer found a number
of pyralid larvae tying and feeding on the leaves of a mulberry tree.
A moth was reared from these which both Mr. Schaus and Dr. Dyar
have determined as Glyphodes pyloalis Walker. This record is of interest
because G. pyloalis Walker is a Chinese species which has not hitherto
been recorded from the United States. Unfortunately as the single
reared specimen is a female the genitalia could not be compared with
those of oriental specimens.
The larvae were collected September 27, 191 8. All died during the
winter except one which pupated about the middle of April, 191 9. The
moth issued April 19, 191 9.
SUBFAMILY CRAMBINAE
DICYMOLOMIA JULIANALIS (WALKER)
(PL. ioi, C, d; 103, b; 106, D; 108, E-H)
Cataclysta (?)julianalis Walker, 1859, List Lep. Brit. Mus., pt. 17, p. 438.
Dicymolomia julianalis Dyar, 1903, List N. Am. Lep., no. 4634.
The larva of this species is the caterpillar popularly known in the
cotton areas of Texas as the "white worm" and is the one most easily
and frequently confused with the pink bollworm. The two when full-
grown are about the same size, and both have the crochets on the
Mar. 1. 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 831
prolegs arranged in a circle broken outwardly. Dicymolomia julianalis
is also frequently found in cotton bolls. Its normal and favored food
plant is cattail (Typha sp.) in the spike of which it feeds and undergoes
its transformation. In some parts of Texas, however, we also found it
commonly in old and diseased cotton bolls, feeding upon the lint and in
some cases the cotton seeds. We did not, however, find it in any green
or healthy bolls. Larvae were collected in the region about Beaumont
during November, 191 8, and near Brownsville from December, 191 8,
until early April, 191 9. Adults issued from the latter part of March
until the middle of May. The species overwinters in the larval stage,
the caterpillars remaining in the fallen and rotting bolls and pupating
during February and early March.
While very similar in superficial appearance to the pink bollworm
and easily mistaken for it by one not familiar with larval characters,
the caterpillar of Dicymolomia julianalis is easily distinguished on struc-
ture. The position of the anterior puncture (Aa) of epicranium back
of seta A2 and the presence of only two setae on the small shield anterior
to the prothoracic spiracle at once separates it from Pectinophora.
The pupa is smooth except for the normal body seta and a half dozen
slender hooked spines on the cremaster and is not likely to be mistaken
for that of Pectinophora gossypiella.
The structural characters of both larva and pupa are fully figured in
Plates 101, 103, 106, and 108.
SUBFAMILY PHYCITINAE
MOODNA OSTRINELLA (CLEMENS)
(PL. 104, E)
Ephestia ostrinclla Clemens, 1861, in Proc. Acad. Sci. Phila., i860, p. 206.
Manhatta ostrinella Hulst, 1903, in Dyar, List North Amer. Lep., no. 4886.
Moodna ostrinella Barnes and McDunnough, 1917, Check List Lep. Bor.
Amer., no. 5795.
The larva of this species is a scavenger feeding in diseased cotton bolls
in company with and in much the same manner as Dicymolomia julianalis.
It is a smaller caterpillar (8 to 9.5 mm. long) when full-grown. The
heavy, ringlike chitinization about tubercles IIb of the mesothorax and
III of the eighth abdominal segment (PI. 104, E), which is so conspicuous
a feature on this and the following larva (Homoeosoma electellum), is a
character found upon most phycitine larvae but nowhere else, so far as I
know, outside of this subfamily.
The caterpillar of Moodna ostrinella is a nearly uniform dirty white;
thoracic shield smoky fuscous divided on dorsum by a wide median
whitish line; body tubercles dark brown; skin finely granulate; body
hairs moderately long, pale yellowish; legs whitish, ringed with smoky
fuscous; head pale yellowish brown; labrum and anterior margins of
epicranium blackish brown; ocellar pigment a black spot under each
832 Journal of Agricultural Research voi.xx,No. h
ocellus, not continuous; crochets evenly biordinal, alternating one long
and one very short hook, 40 to 44.
Larvae collected November 24, 191 8, at Kountz, Tex. Moth issued
April 7, 1 91 9.
HOMOEOSOMA EEECTELEUM (htJLST)
(PL. IOO, B)
Anerastia electella Hulst, 1887, in Entomologica Americana, v. 3, p. 137-13S.
Homoeosoma electelhim Hulst, 1903, in Dyar, List North Amer. Lep., no. 4865.
A large series of moths was reared April 23 to May 5, 1919, from larvae
collected at Brownsville, Tex., April 7, 1919, by E. L. Diven. The larvae
feed in the flower heads of a composite, making an untidy patch and
eating the bloom, stem, and seeds. The species appeared to be very
common.
The larva is pale smoky brown, longitudinally marked by two narrow
white dorsal stripes and a similar lateral stripe; spiracles black, thoracic
legs smoky fuscous; anal shield yellow, thoracic shield yellow, broadly
margined laterally and posteriorly with black ; head pale yellow, mottled
with yellowish brown and with a broad lateral black band and a blackish
shading toward anterior margins of epicranium; ocelli distinct; ocellar
pigment absent or confused in the lateral black of epicranium; general
structural characters as in Moodna ostrinella; width 6 to 7 mm.
The interesting and rather complicated genitalia of the male adult are
figured in Plate 100, B.
SUBFAMILY CHRYSAUGINAE
CLYDONOPTERON TECOMAE RILEY
Clydonopteron tecomae Riley, 1880, in Amer. Ent., v. 3, no. 12, p. 288.
Salobrana tecomae Dyar, 1903, List North Amer. Lep., no. 4526.
Clydonopteron tecomae Barnes and McDunnough, 1917, Check List Lep. Bor.
Amer., no. 5283.
The larva of this species feeds only in the seed pods of the trumpet-
flower vine (Tecoma radicans) . It is mentioned here only because its host
plant is often found in the neighborhood of the cotton fields and for that
reason it might be confused by the uncritical with the larva of Pectino-
phora gossypiella. It is easily distinguished, however. The spiracles
are rather large, oval, and black, the edges are heavily chitinized, and the
spiracle on the eighth abdominal segment is somewhat larger but no higher
on the body than the others; the proleg crochets are arranged as in the
Aegeriidae — that is, uniordinal and in two transverse bands — and the
prothorax has only two setae on the chitinization before the spiracle as in
other Pyralidae. It pupates in a cocoon within the seed pod.
Moths were reared by us August 30 to September 15, 191 8, from larvae
collected earlier in August (Anahuac, Tex.) the same year.
Mar. i, 1921 Lepidoptera Likely to Be Confused with Pink Bollworm 833
FAMILY NOCTUIDAE
Several species of this family feed upon cotton and malvaceous plants.
They are easily distinguished from the pink bollworm or larvae of any of
the other groups treated in this paper by the arrangement of the body
setae and the crochets of the prolegs. Like the Pyralidae they have only
two setae (IV and V) on the prespiracular shield of the prothorax, but
the position of IV and V on the proleg-bearing segments is quite different,
IV being remote from V and directly back of the spiracle (PL 103, D).
The crochets of the prolegs are also arranged in a mesoseries (PL 106, E).
The following species were reared.
SUBFAMILY AGROTINAE
HELIOTHIS (CHLORIDEA) OBSOLETA (FABRICIUS)
(pl. 103, d; 106, e)
Bombyx obsolcta Fabricius, 1793, Ent. Syst., t. 3, pars. 1, p. 456.
Heliothis armiger Dyar, 1903, List North Amer. Lep., no. 2300.
Chloridea obsolete Hampson, 1903, in Cat. Lep. Phal. Brit. Mus., v. 4, p
45- 657-
Heliothis obsoleta Barnes and McDunnough, 1917, Check List Lep. Bor. Amer.,
no. 1090.
This species is commonly known as the "corn earworm" or "cotton
bollworm." It feeds on a number of plants and often attacks cotton,
doing serious damage in some localities. The larva bores into the bolls,
making a large hole and destroying lint and seeds.
One moth was reared from a larva feeding on the leaves of Malvaviscus
drummondii at Brownsville, Tex. A larva was collected by E. L. Diven,
May 7, 1 91 9. The adult emerged May 29 of the same year.
HELIOTHIS (CHLORIDEA) VIRESCENS (FABRICIUS)
Noctua virescens Fabricius, 1781, Spec. Insect., t. 2, p. 216.
Chloridea virescens Dyar, 1903, List North Amer. Lep., no. 2296.
Chloridea virescens Hampson, 1903, in Cat. Lep. Phal. Brit. Mus., v. 4, p. 48.
Heliothis virescens Barnes and McDunnough, 1917, Check List Lep. Bor. Amer.,
no. 1091.
This species has very much the same habits as Heliothis obsoleta
Fabricius. Moths were reared September 8 and 17, 191 9, from larvae
taken feeding on seeds in okra pods August 19, 1918, at Double Bayou,
Tex. (E. L. Diven).
834 Journal of Agricultural Research voi.xx, No. n
SUBFAMILY ACRONYCTINAE
BAGISARA RECTIFASCIA (GROTE)
Schinia rectifascia Grote, 1874, in Proc. Boston Soc. Nat. Hist., v. 16, 1873/74,
p. 242.
Atethmia rectifascia Dyar, 1903, List North Amer. Lep., no. 2267.
Bagisara rectifascia Hampson, 1910, in Cat. Lep. Phal. Brit. Mus., v. 9, p. 156.
One moth was reared September 1 and one September 23, 191 8, from
larvae collected on Malvaviscus drummondii August 10, 191 8 (Anahuac,
Tex., J. D. More). Dr. Dyar, who determined the Noctuidae, informs
me that the larva of this species has not been described. Unfortunately
those preserved with the foregoing experiment are Catocalinae of some
kind and probably have no connection with the adults reared.
SUBFAMILY EREBINAE
ALABAMA ARGILLACEA (HUBNER)
Aletia argillacea Hiibner, 1820, Zutr. Samml. Exot. Schmett., fig. 399.
Alabama argillacea Dyar, 1903, List North Amer. Lep., no. 2555.
Several moths were reared from larvae feeding on the cotton leaves.
Larvae were taken September 25, 1918, at Dickinson, Tex., and moths
early in October of the same year. The species pupates within the
folded leaves on the plant.
ANOMIS EXACTA HUBNER
Anomis exacta Hiibner, 1810, Samml. Exot. Schmett., v. 2, pi. 411.
Anomis exacta Dyar, 1903, List North Amer. Lep., no. 2557.
One moth was reared September 1, 1918, from a larva collected on
Malvaviscus drummondii, Anahuac, Tex., August 14, 1918 (J. D. More).
The larva pupated August 2 1 , spinning a loose tie of several leaves.
ANOMIS EROSA HUBNER
Anomis erosa Hiibner, 1818, Zutr. Samml. Exot. Schmett., fig. 287.
Anomis erosa Dyar, 1903, List North Amer. Lep., no. 2556.
One moth from Brownsville, Tex., January 19, 1919, was reared from
a pupa in the tied leaves of Abutilon incanum (H. C. Hanson, collector).
FAMILY LYCAENIDAE
STRYMON MELINUS HUBNER
Strymon melinus Hiibner, 1818, Zutr. Exot. Schmett., fig. 121.
Uranotes melinus Dyar, 1903, List North Amer. Lep., no. 335.
Strymon melinus Barnes and McDunnough, 1917, Check List Lep. Bor. Amer.,
no. 352.
This caterpillar feeds on a great variety of plants, including practically
all the Malvaceae. On cotton it attacks the flowers and bolls, boring
into the latter and feeding upon lint and seeds and making, when half-
Mar. i, i93i Lepidoptera Likely to Be Confused with Pink B oil-worm 835
grown, a hole which reminds one very much of the exit hjle made by a
pink bollworm.
The larva itself looks nothing like -any of the others here treated.
It is spindle-shaped, sharply tapering at each end, broad in the middle
in proportion to its length, with a small head, the body covered with fine
stiff secondary hairs, and greenish yellow in color.
In addition to cotton we find it frequently on okra, Kosteletzkya spp.,
Malvaviscus drummondii, and Hibisctis spp. On these it fed on the
seeds, boring into the seed pods, or upon the blossoms.
The table of larval characters will serve to place the forms here treated.
The characters given are not to be understood as diagnostic in all cases.
In the Cosmopterygidae, for example, seta I is often as far from III
as it is from II as in the Gelechiidae or the Oecophoridae. There are
also a few exceptions to the gelechiid character (the remoteness of
epicranial seta V from A3). The characters hold, however, for all the
species here treated occurring on Malvaceae.
Characters of larva likely to be confused with the pink bollworm
1. Body depressed and spindle-shaped, covered with, secondary setae . . Lycaenidae.
Body otherwise 2
2. Setae IV and V on proleg-bearing abdominal segments closely approximate . . 3
Setae IV and V on proleg-bearing abdominal segments well-separated 13
3. Prespiracular shield of prothorax bearing two setae only 4
Prespiracular shield of prothorax bearing three setae 5
4. Prolegs long and slender; body of larvae normally with one or more second-
ary setae Pterophoridae.
Prolegs otherwise ; body with only primary' setae Pyralidae.
5. Body with one or more secondary setse Ethmiidae.
Body with only primary setae 6
6. Ocelli I to IV grouped together, forming a trapezoid; ocelli V and VI fairly
close together but well-separated from the other four Aegeriidae.
Ocelli otherwise 7
7. Paired dorsal setae II on ninth abdominal segment closer together than
paired I on dorsum of eighth abdominal segment; usually on a single
chitinization 8
Paired dorsal setae II on ninth abdominal segment at least as far apart as
paired I on eighth abdominal segment and not on a single chitinization . . 9
8. Setae I and III closely approximate on ninth abdominal segment 10
Setae I and III not closely approximate on ninth abdominal segment Tortricidae.
9 . Epicranial seta L,1 remote from A3 (farther from A3 than A3 is from A2) Gelechiidae.
Epicranial seta L1 approximate to A3, at least no farther from A3 than A3 is
from A2 11
10. Seta IP on prothorax higher than Ia Cosmopterygidae.
(in part : Pyroderces rileyi) .
Seta IIa on prothorax not higher than I a Olethreuttdae.
Phalonhdae.
11. Prothoracic legs very close together, coxae touching BlastobasidaE.
Prothoracic legs appreciably separated 12
836 Journal of Agricultural Research voi.xx, No. n
12. Setae III on abdominal segments I to VII antero-dorsad of and close to the
spiracle Stenomidae.
Setae III on abdominal segments I to VII dorsad of the spiracle; if occa-
sionally somewhat antero-dorsad not close to the spiracle OecophoridaE.
13. Seta IV directly behind the spiracle on proleg-bearing abdominal segments;
crochets of prolegs arranged in a mesoseries; two setae only on prespiracular
shield of prothorax; no secondary hair on body or head NoctuidaE.
(in part, as here represented).
PLATE 93
Male genitalia (Gelechiidae):
A. — Gelechia tropkella: Posterior part of tegumen, showing uncus and gnathos,
ventral view.
B. — G. trophclla: Lateral view of male genitalia with eighth abdominal segment
attached.
C. — G. hibiscella: Lateral view of male genitalia with eighth abdominal segment
attached.
Explanation of symbols applied to male genital organs on Plates 93-100.
Ae=aedoeagus (outer chitinous sheath of penis).
An=anellus (chitinous support of aedoeagus).
Ao=opening in tegumen through which anal tube passes.
Cl=clasper on harpe.
Cn=cornutus (cornuti) spine or spines on penis proper.
Cs=cucullus of harpe.
Gn=gnathos.
Hp=harpe.
Si=soci.
Tg= tegumen.
Ts=transtilla (a costal bridge, or sometimes elements thereof not united; con-
necting the harpes).
Vm = vinculum .
U=uncus.
A VIIIs=sternite of eighth abdominal segment.
A VIIIt=tergite of eighth abdominal segment.
Lepidoptera Likely to Be Confused with Pink Boliworm
Plate 93
Journal of Agricultural Research
Vol. XX, No. 11
Lepidoptera Likely to Be Confused with Pink Bollworm
Plate 94
Journal of Agricultural Research
Vol. XX, No. 11
PLATE 94
Male genitalia (Gelechiidae):
A —Telphusa mariona (type) : Lateral view of male genitalia.
B.-T. mariona (type): Posterior part of tegumen, showing uncus, ventral view.
C —Gelechia neotrophelia (type): Aedoeagus and penis.
D.-G. neotrophelia (type): Lateral view of male genitalia with aedoeagus and
eighth segment removed.
E.-G. neotrophelia (type): Posterior part of tegumen, showing uncus and gnathos,
ventral view.
p.— G. neotrophelia (type): Posterior half of harpes, ventral view.
G.-G. neotrophelia (type): Sternite and tergite of modified eighth abdominal
segment.
PLATE 95
Male genitalia (Gelechiidae, Stenomidae, and Oecophoridae) :
A. — Isophriclis similiella: Ventral view of male genitalia, spread.
B. — Aedemoses haesitans: Ventral view of male genitalia, spread.
C. — A. haesitans: Enlargement of typical split hair on cucullus.
D. — Borkhausenia fasciata: Ventro-lateral view of male genitalia, spread, showing
asymmetrical armlike projections from gnathos and costa of harpes.
Lepidoptera Likely to Be Confused with Pin
k Bollworm
Plate 95
Journal of Agricultural Research
Vol. XX, No. 11
Lepidopte
optera Likely to Be Confused with Pink Bollworm
Plate 96
E
Journal ol Agrl« ultural Research
Vol. XX, No. 11
PLATE 96
Male genitalia (Oecophoridae) :
A. — Borkhausenia miniitella: Aedoeagus.
B. — B. minutella: Ventral view of male genitalia, spread, aedoeagus omitted.
C. — B. diveni (type): Ventral view of male genitalia, spread.
D. — B. diveni (type): Dorsal view of an abdominal segment showing spinose con-
dition of abdomen.
E. — B. diveni (type): Modified tergite of eighth abdominal segment.
F. — B. diveni (type) : Modified sternite of eighth abdominal segment.
PLATE 97
Male genitalia (Oecophoridae):
A. — Borkhausenia conia: Portion of tergite of seventh abdominal segment, showing
spinose and chitinized character of caudal margin.
B . — B. conia: Ventral view of male genitalia, spread , aedoeagus omitted.
C. — B. conia: Aedoeagus.
D. — B. conia: Modified tergite of eighth abdominal segment.
E. — B. conia: Modified sternite of eighth abdominal segment.
Lepidoptera Likely to Be Confused with P,nk BoMworr
Plate 97
Journal of Agricultural Research
Vol. XX, No. 11
Lepidoptera Likely to Be Confused with Pink Bollworm
Plate 98
Journal of Agricultural Research
Vol. XX, No. 11
PLATE 98
Male genitalia (Blastobasidae):
A. — Zenodockium citricolella: Aedoeagus.
B. — Z. citricolella: Lateral view of male genitalia, right harpe and aedoeagus omitted.
C. — Z. citricolella: Right harpe.
D. — Holcocera ochrocephala: Ventral view of male genitalia, spread, aedoeagus
omitted.
E. — H. ochrocephala: Dorsum of an abdominal segment showing transverse row of
spines.
F. — H. ochrocephala: Aedoeagus and penis.
29666°— 21 4
PLATE 99
Male genitalia (Olethreutidae and Blastobasidae):
A. — Crocidosema plebeiana: Ventral view of male genitalia, spread.
B. — Eucosma discretivana (type): Ventral view of male genitalia, spread.
C. — Hokocera confamulella (type) : Ventral view of male genitalia, spread.
Lepidoptera Likely to Be Confused with Pink Bollworm
■ U , Si
r/L-' Ae
Plate 99
B
Journal of Agricultural Research
Vol. XX, No. 1 i
Lepidoptera Likely to Be Confused with Pink Bollwor
Plate 100
Journal of Agricultural Research
Vol. XX, No. 11
PLATE ioo
Male genitalia (Phaloniidae and Pyralidae):
A. — Phalonia cephalanthana (type): Ventral view of male genitalia, spread.
B. — Homoeosoma electellum: Ventral view of male genitalia, spread.
PLATE 101
Larval structures:
A. — Pectinophora gossypiella: Head capsule, dorsal view, showing arrangement of
setae.
B. — P. gossypiella: Head capsule, lateral view, showing arrangement of setae.
C. — Dicymolomia juUanalis: Head capsule, dorsal view, showing arrangement of
setae.
D. — D. juUanalis: Head capsule, lateral view, showing arrangement of setae.
B. — Meskea dyspteraria: Head capsule, dorsal view, showing arrangement of setae.
F. — M. dyspteraria: Head capsule, lateral view, showing arrangement of setae.
Explanation of symbols applied to larvae on Plates 101-106.
A1, A2, A3, Aa=anterior setae and puncture of epicranium.
Adf1, Adf2, Adfft=adfrontal setae and puncture of epicranium.
ADFR=adfrontal ridge of frons.
ADFS=adfrontal suture.
AF=anal fork.
E1, E2=epistomal setae.
F1, Fa=frontal seta and puncture.
FR= frons.
G1, Ga=genal seta and puncture of epicranium.
L\ La=lateral seta and puncture of epicranium.
LR=longitudinal ridge of frons.
0\ O2, O3, Oa=ocellar setae and puncture of epicranium.
P1, P2, Pa, Pb=posterior setae and punctures of epicranium.
SMp=platelike chitinization on submentum.
SO1, SO2, SO3, SOa=subocellar setae and puncture of epicranium.
X=Ultraposterior setae and punctures of epicranium.
Lepidoptera Likely to Be Confused with Pink Bollworm
Plate 101
Journal of Agricultural Research
Vol. XX, No. 11
Lepidoptera Likely to Be Confused with Pink Bollworm
Aors
Plate 102
so-'-" .■■'/■'/ Pr
sov /
y
''L'
so" ! ! ■
soJ oa oJ
c' aa
B
Journal of Agricultural Research
Vol. XX, No. 11
PLATE 102
Larval structures:
A. — Pyroderces rileyi: Head capsule, dorsal view, showing arrangement of setse.
B. — P. rileyi: Head capsule, lateral view, showing arrangement of setse.
C. — Crocidosema plebeiana: Head capsule, dorsal view, showing arrangement of setae.
D. — C. plebeiana: Head capsule, lateral view, showing arrangement of setse.
E. — Zenodochium citricolella: Labium and maxillse.
F. — Isophrictis similiella: Head capsule, dorsal view, showing arrangement of setse.
PLATE 103
Larval structures:
A. — Peclinophora gossypiella: Setal maps of first and second thoracic and third,
eighth, and ninth abdominal segments.
B. — Dicymolomia julianalis: Setal maps of first and second thoracic and third,
eighth, and ninth abdominal segments.
C. — Pyroderces rileyi: Setal maps of first thoracic and eighth and ninth abdominal
segments.
D. — Heliothis obsoleia: Setal maps of first thoracic and third abdominal segments.
E. — Crocidosema plebeiana: Setal maps of first and second thoracic and third,
eighth, and ninth abdominal segments.
Lepidoptera Likely to Be Confused with Pink Bollworm
.y
• /--ii
r.
£-
m
/?
■i
' /-'>
</£:::
■w
°//~ -
Y
■w
-w
r$~-
■m
Plate 103
-B
-1
--W
Tnkm
A
Am
Aye.
Ars
Ti Te £•
Journal of Agricultural Research
AID
Vol. XX, No. 11
Lepidoptera Likely to Be Confused with Pink Bollworm
Plate 104
Journal of Agricultural Research
Vol. XX, No. 11
PLATE 104
Larval structures:
A. — Platynota rostrana: Setal maps of first and second thoracic and third, eighth,
and ninth abdominal segments.
B. — Meskea dyspteraria: Setal maps of first and second thoracic and third, eighth,
and ninth abdominal segments.
C. — Zenodochium citricolella: Setal maps of first thoracic and third, eighth, and
ninth abdominal segments.
D. — Aedemoses haesitans: Setal map of third abdominal segment.
E. — Moodna ostrinella: Setal maps of second thoracic and eighth and ninth abdomi-
nal segments.
PLATE 105
Larval structures:
A. — Platynota rostrana: Setal maps of eighth and ninth abdominal segments,
dorsal view.
B. — Eucosma keliantkana : Setal maps of eighth and ninth abdominal segments,
dorsal view.
C. — Pectinophora gossypiella: Setal maps of eighth and ninth abdominal segments,
dorsal view.
D. — Pyroderces rileyi: Setal maps of eighth and ninth abdominal segments, dorsal
view.
E. — Pectinophora gossypiella: Prothorax, ventral view, showing position of legs.
F. — Telphusa mariona: Ventro-caudal view of tenth abdominal segment, showing
anal fork.
G. — Crocidosema plebeiana: Ventro-caudal view of tenth abdominal segment, showing
anal fork.
H. — Gelechia neoti -Ophelia: Ventro-caudal view of tenth abdominal segment, showing
anal fork.
I. — Zenodochium citricolella: Prothorax, ventral view, showing position of legs.
Lepidoptera Likely to Be Confused with Pink Bollworm
Plate 105
Journal of Agricultural Research
Vol, XX, No. 11
Lepidoptera Likely to Be Confused with Pink Bollworm
Plate 106
A
E
C
Journal of Agricultural Research
Vol. XX, No. 11
PLATE 1 06
Larval structures:
A. — Pectinophora gossypiella: Crochet arrangement of abdominal prolegs.
B. — Crocidosema plebeiana: Crochet arrangement of abdominal prolegs.
C. — Pyrodercet rileyi: Crochet arrangement of abdominal prolegs.
D. — Dicymolomia julianalis: Crochet arrangement of abdominal proleg.
E. — Heliothis obsoleta: Crochet arrangement of abdominal proleg.
PLATE 107
Pupal structures:
A. — Pectinophora gossypiella: Ventral view of pupa.
B. — Pectinophora gossypiella: Caudal end of pupa, lateral view.
C. — Pectinophora gossypiella: Mature pupa, ventral view, shaded to show eyes of
imago visible through pupal skin and characteristic pubescence of the pupa.
D. — Pectinophora gossypiella: Dorsal view of pupa.
E. — Pyroderces rileyi: Ventral view of pupa.
F. — Pyroderces rileyi, Dorsal view of pupa.
Explanation of symbols applied to pupae on Plates 107-109.
a = antenna.
a1 to a10=abdominal segments 1 to 10.
ao=anal opening.
c/=clypeus.
cr=cremaster.
/=front.
/1=femora of prothoracic leg.
/cr=fronto-clypeal suture.
<7=gena.
<7e=glazed eye.
go=genital opening.
/6=labrum.
/'^prothoracic leg.
/2=mesothoracic leg.
P=metathoracic leg.
//>=labial palpi.
rwc?=mandible.
m/>=maxillary palpus.
mx= maxilla.
/>/=pilifer.
se=sculptured eyepiece.
i1=prothorax.
i2=mesothorax.
i3 = metathorax .
v= vertex.
w1=mesothoracic wing.
Lepidoptera Likely to Be Confused with Pink Boliwor
Journai of Agricultural Res
Vol. XX, No. 11
Lepidoptera Likely to Be Confused with Pink Bollworr
Plate 108
Journal of Agricultural Research
Vol. XX, No. 11
PLATE 108
Pupal structures:
K.-Crocidosema piebeiana: Abdomen of female pupa, ventral view.
B __c. piebeiana: Abdomen of male pupa, ventral view.
C'_C piebeiana: Lateral view of an abdominal segment, showing arrangement and
character of dorsal spines; one spine greatly enlarged to show shape.
D __c. piebeiana: Abdomen of pupa, dorsal view.
■£—Dicymolomia julianalis: Dorsal view of pupa,
p _E>. julianalis: Caudal end of pupa, lateral view.
O.—D. julianalis: Caudal end of male pupa, ventral view.
H — D. julianalis: Ventral view of female pupa.
PLATE 109
Pupal structures:
A. — Meskea dyspteraria: Caudal end of female pupa, lateral view.
B. — M. dyspteraria: Abdomen of female pupa, ventral view.
C. — M. dyspteraria: Male pupa, dorsal view.
D. — M. dyspteraria: Caudal end of male pupa, lateral view.
E. — M. dyspteraria: Male pupa, ventral view.
F. — Amorbia emigratella: Abdomen of pupa, dorsal view.
G. — Telphusa mariona: Caudal end of pupa, ventral view, showing peculiarly
scalloped and fringed caudal margin of seventh abdominal segment.
Lepidoptera Likely to Be Confused with Pink Bollworm
PLATE 109
A1-
f i
■ - - q_
f 1
K_ J
>
.--a-
--a*
\ " 7"
-a*"
- a6
-a'
1 /
\ "1
--as
C
Journal of Agricultural Research
Vol. XX, No. 11
BIOLOGY OF THE SMARTWEED BORER, PYRAUSTA
AINSLIEI HEINRICH1
By George G. Ainslie, Entomological Assistant, and W. B. Cartwright, Scientific
Assistant, Cereal and Forage Insect Investigations, Bureau of Entomology, United
States Department of Agriculture
INTRODUCTION
The attention of the senior author was first called to the smartweed
borer in 191 2, when hibernating larvae were found in cornstalks at Frank-
lin, Tenn. The economic status of this insect was undetermined at that
time, but field and rearing records made in Tennessee and neighboring
States since then have indicated that it is of no importance as a pest. At
present, however, it is of considerable interest because of its similarity,
both in habits and appearance, to the European corn borer (Pyrausta
nubilalis Hiibner). Until recently, also, it has been confused with
another similar species, P. penitalis Grote, which feeds on lotus; and
the purpose, in part, of this paper is to rectify this error.
Although Dr. E. Mosher (7, p. 264) 2 recorded differences of structure
and the present authors found distinct variations in habit between the
insect under discussion and the true Pyrausta penitalis, the former was
first definitely recognized as an undescribed species by Mr. Carl Heinrich
(<5) of the Bureau of Entomology. Mr. Heinrich gives in detail the
morphological characters separating the species nubilalis, penitalis, and
ainsliei in all stages. Chittenden (1) has well summarized all the avail-
able records of the smartweed and lotus borers, although he was not
aware that two species were included.
DISTRIBUTION AND HOST PLANTS
The smartweed borer is known to occur in Massachusetts, New York,
Pennsylvania, Ohio, and Illinois ; and the writers have taken it at numer-
ous points in Tennessee and Kentucky and at Clemson College, S. C.
Polygonum pennsylvanicum, its principal food plant, occurs throughout
the eastern half of the United States, and it is likely that the distribution
of the borer is coextensive therewith.
The plants in which the larvae are found must be divided into two
groups, namely, food plants proper and shelter plants.
1 In recent papers by Flint and Malloch (j, 4), the name Pyrausta obumbralalis Lederer (misspelled
obumbratilis) is used for this species. While it is possible that ainsliei will prove to be a synonym of obum-
bratalis, it seems inadvisable at this time to use this latter name for this species, for, until Lederer's type
can be examined and its exact identity and relation to the other species under discussion determined, its
use will simply add confusion to a matter which seems in a fair way to be solved.
2 Reference is made by number (italic) to "Literature cited," p. 844.
Journal of Agricultural Research, Vol. XX, No. n
Washington, D. C Mar. 1, 1921
wz (837) Ke>' No- ^"93
838 Journal of Agricultural Research voi.xx, No. «
FOOD PLANTS
Riley (according to Chittenden, 1, p. 454), who first noted what was
probably this species, found larvae in stems of Polygonum incarnatum; and
Hart (5, p. 182) mentions that it has been reared from the same species
at Urbana, 111. Chittenden states that there is a moth in the National
Museum reared from stems of Polygonum hydropiperoides . The foregoing
references occur under the name of Pyrausta penitalis, but relate without
doubt to Pyrausta ainsliei. After investigating the matter in New York,
Dr. E. P. Felt writes that in his opinion —
Pyrausta ainsliei occurs very commonly in Polygonum pennsylvanicum in this section
[New York] and much more rarely in P. lapathifolium.
Mr. D. J. Caffrey writes that Pyrausta ainsliei has been reared from
Polygonum persicaria in Massachusetts.
The work of the present authors indicates very clearly that south of the
Ohio River, at least, Pyrausta ainsliei breeds only in Polygonum pennsyl-
vanicum. Despite the most careful and persistent search they have failed
to find either larvae or eggs, or any trace of them, on plants of any other
species even though growing in the immediate vicinity of Polygonum
pennsylvanicum and often in the same clump. The species of the genus
Polygonum are often confused, and determinations of plants for
entomological purposes are so often made carelessly or from insufficient
material that further work appears necessary in order that the occurrence
of this borer in species other than Polygonum pennsylvanicum may be
verified. As Polygonum incarnatum is now considered a synonym of
Polygonum lapathifolium the following are here listed as reported natural
food plants of Pyrausta ainsliei: Polygonum pennsylvanicum, Polygonum
lapathifolium, Polygonum hydropiperoides , and Polygonum persicaria.
It should be stated that although never found on them in the field,
larvae have been reared from eggs to full-size caterpillars on leaves of
curled dock (Rumex crispus) and buckwheat (Fagopyrum fagopyrum),
both of which are close relatives of Polygonum. Leaves of all com-
mon weeds and plants were offered to the larvae, but in every case except
the two mentioned above they were either refused or only slightly
gnawed. On leaves of lotus (Nelumbo lutea) the larvae in several experi-
ments starved to death after merely pitting the leaf surface. Mr.
Heinrich's statement (6, p. 175) that we have reared these larvae to
maturity on N. lutea is an error.
SHELTER PLANTS
The other group, shelter plants, includes all plants the stems of which
are entered by larvae seeking winter quarters. The list of such plants
will eventually contain practically all the pithy stemmed weeds and plants
the bark of which is not too dense to permit the entrance of the larvae.
Some of the larvae remain in the stems of smart weed, but for some
Mar. i, 1921 Biology of the Smartweed Borer 839
obscure reason many leave their food plant and seek entrance to anything
that will give them dry quarters through the winter. The plants in
which larvae have been found by the authors are as follows: Corn (Zea
mays), ragweeds (Ambrosia triftda and Ambrosia artemisiaefolia) , cockle-
bur (Xanthium communis), goldenrod (Solidago spp.), aster (Aster spp.),
timothy (Phleum pratense), cattail (Typha latifolia), beggartick (Bidens
bipinnata and B. jrondosa), and numerous other wild plant stems not in
condition for determination. Dr. Felt adds Brassica arvensis and Chit-
tenden (1) lists raspberry stems, to which the larvae gained entrance
through the cut ends. Eupatorium sp., in which larvae were found in
Missouri according to Chittenden, is also undoubtedly a shelter plant.
Aside from Polygonum spp. the foregoing plants are in no sense food
plants. The larvae burrow the stems enough to construct a cavity
sufficiently large to contain them ; and even in this process, as the authors
have observed, they do not swallow the plant tissue but eject it from the
mouth. It is this habit of seeking shelter wherever it may be found, es-
pecially in cornstalks, that seems likely to lead to some confusion, for
the larvae are so similar to those of Pyrausta nubilalis, the European corn
borer, that without careful laboratory study the two can not be differ-
entiated.
SEASONAL HISTORY AND HABITS
In Tennessee there are two generations of the smartweed borer each
year. Adults reared at Knoxville emerged from May 26 to October 30
with two well-defined periods of maximum abundance, the first from
June 20 to July 5 and the second from August 1 8 to 30. Moths emerging
in June at once oviposit, and the resulting larvae complete their growth
early in August and immediately pupate in their larval burnyws in the
smartweed stems. The moths emerge later in the same month and give
rise to the second generation of larvae, which reach full growth before
winter and without further feeding remain in the food or shelter plants
unchanged until they pupate in May and June of the following year.
Very few published data are available. Hart (5, p. 182) states that
moths (probably of this species, as there is no Nelumbo near Urbana) were
taken at light at Urbana, 111., from May 19 to August 6, and that a single
moth was reared July 1 . In Missouri moths issued from smartweed from
May 29 to June 6, and others are labeled October 9. Although scat-
tering data on this species are included in his paper, Chittenden's conclu-
sions do not agree with the actual life history as the authors have found it,
and his statements must be taken, in the main, to apply to Pyrausta
penitalis.
In a reared series of larvae from eggs hatching August 16 a number of
moths emerged October 13 and 15. This is difficult to explain except on
the ground of abnormal conditions, for it does not seem possible that in
nature moths emerging so late could produce another generation, and
840 Journal of Agricultural Research voi.xx.No. h
under natural conditions neither pupae nor moths have been found at
this time of the year.
HABITS OF THE MOTHS
The moths frequent low, moist situations where the food plants grow
normally. During the day they rest on or under the leaves and when
disturbed make low direct or circuitous flights within the bounds of their
haunts.
THE EGGS
Eggs have been taken many times in the field, but oviposition has not
been observed. It doubtless occurs at dusk or during the night, and
possibly on cloudy days, as the moths seem active only at such times.
The eggs are laid in small patches or often in rows, with the individual
eggs overlapping shingle fashion, on the underside of the leaves, more
often those near the tips of the branches, and either on the leaf blade
proper or close beside the midrib in the angle between it and the blade.
Near Union City, Tenn., on August 8, 191 9, the senior author found
an isolated clump of six plants of Polygonum pennsyhanicum. Thirty
egg masses were found on these plants, all but one or two close beside
the midrib on the under surface of the leaf. In three instances there were
more than one mass on a leaf, but the difference in the stage of develop-
ment clearly showed that they had been laid at different times. The num-
ber of eggs per mass varied from 4 to 1 6, the average being 9.3. In another
collection of 17 egg masses made at Knoxville, August 12, the number of
eggs varied from 7 to 14, with an average of 9.47 per mass.
As the egg has not heretofore been described, its description follows:
Egg. — Flat, thin, scalelike, laid in flat masses or rows of from 4 to 16, shingle
fashion, each egg about half overlapping its predecessor. The individual egg is broadly
elliptic, sometimes almost circular in outline, about 1.213 mm. long and 0.886 mm.
broad. Chorion evenly reticulated all over with a close network of very fine but
sharply elevated lines. Pale watery-greenish in color, nearly transparent when first
laid, soon becoming more opaque, after which the embryo takes shape as a darker
green, more transparent object in the center. No marked change then occurs until
just before hatching, when the eyes and the mandibles darken, the color spreading to
the whole head which becomes brown and plainly visible and appears detached
because of the paleness and practical invisibility of the larval body which lies bent
around the periphery.
The period of incubation in June and July is six days, in late August
five days.
HABITS OF THE LARVAE
Upon hatching, the young larvae at once enter the stem near the tip of
a branch, choosing the base of a petiole for their point of attack. That
they are somewhat gregarious at this stage is shown by the fact that all
the larvae hatching from one egg mass usually enter the stem at the same
point, which may be several inches from the egg mass. Thus in the first
Mar. i, 1921 Biology of the Smartweed Borer 841
and second instars burrows are often found containing a number of larvae.
Their work very quickly results in the wilting of the tender tips above the
point of attack, and these drooping tips soon become to the observer an
almost certain indication of the presence of the young larvae. As soon as
the food supply here is exhausted the larvae desert this portion of the stem
and scatter, each reentering at another point to make a burrow of its
own, and thereafter only one larva is found in a burrow, although it often
happens in a thickly infested stem that these burrows are practically
continuous. The stems of Polygonum pennsylvanicum are thick-walled
and succulent, with only a very small central cavity. The larvae cut
into this cavity, almost invariably entering at the swollen node just
below the base of the ocrea, and consume the succulent tissue, leaving
only the very thin, fibrous, outer bark. They do not hesitate to abandon
a burrow and seek another location whenever the food supply fails. The
larger stems are attacked first, but later the branches are utilized, often
those so small that the larvae can scarcely crowd into them. The bur-
rows are kept clean, all excrement being disposed of through the entrance,
which is left open, although with the growth of the plant it often partially
heals.
Larvae of the first generation make no effort to leave the smartweed
stems but pupate in them as soon as fully fed. Those of the second
generation attack the plants in the same way and feed as did their pro-
genitors until they are fully grown. This stage is reached about the last
of August, and thereupon many of the larvae abandon their host plant
and seek shelter elsewhere. Those entering cornstalks have been par-
ticularly noted. Neither thoroughly dry nor green stalks suit them as
well as those of intermediate condition. They enter preferably under a
leaf sheath or behind an ear. Their presence is indicated by the fluffy
white pith showered from the entrance hole upon the leaves below. The
entrance hole is perfectly round and clean-cut, and the burrow within is
of equal diameter, 3 to 3.5 mm., and is kept clean and free of all cuttings
and excrement. It turns downward from the entrance and is from 1
to 4 inches long. Early in October the larva closes the entrance with a
drum-tight sheet of silk, quite effectively camouflaged by the incorpora-
tion of a few brownish particles of the chewed bark.
As far as determined the larvae are not torpid during the period of
hibernation. Repeated collections of larvae in the field during the winter
show them always quick to respond when disturbed. There is no evi-
dence that they consume any food before pupation after leaving their
food plants in the fall. In making their winter burrows in the shelter
plants they do not swallow the tissue but discharge it from the mouth
in sawdust- like particles.
No very definite cocoon is constructed by either generation. In
smartweed the burrow is lightly plugged above and below the pupa
29666°— 21 5
842
Journal of Agricultural Research
Vol. XX, No. 11
with pith particles interwoven with silk, and sometimes in the larger cavities
a light cocoon is constructed, hardly more than a network of silk fibers.
The burrow formed in smartweed by the larvae of the first generation
runs upward from the entrance; and the pupal chamber, in which the
pupa lies head downward, is 1 or 2 inches above the exit hole. The
emerging moth breaks the partition and leaves the pupal envelope in the
chamber. In corn the cocoon is even less elaborate, and the most evident
difference is that the pupa lies head upward in the burrow.
REARING RECORDS
Eggs were easily obtained from moths collected in the field and con-
fined in lantern-chimney cages with a potted smartweed or in 1 -ounce
tin boxes containing a leaf of the same plant. The eggs hatched nor-
mally, and the young larvae were transferred singly to 1 -ounce tin boxes
for rearing. The larvae while young thrived on smartweed leaves, but
in later stages they preferred the stems.
Table I contains the condensed data obtained from a series of larvae
hatching from eggs laid July 21 and from miscellaneous rearings from
partly grown larvae taken in the field.
Table I. — Length, in days, of instars and stages of Pyrausta ainsliei
Stage.
Maximum.
Minimum.
Average.
Number
averaged.
Egg
Larva :
Instar I
II
Ill
IV
V
VI
Pupa
6
4
6
7
7
12
28
16
6
3
3
4
4
3
18
12
6
3-27
4.64
5.22
5- 55
7.66
23.67
13-33
J5
14
14
9
6
3
3
Total
69-34
Table II contains similar data obtained from a series of 60 larvae reared
individually from eggs hatching August 18.
Table II. — Length, in days, of instars and stages of Pyrausta ainsliei
Stage.
Maximum.
Minimum.
Average.
Number
averaged.
Egg
5
2
8
9
18
37
17
5
2
3
3
5
20
6
5
2
4.90
5-3°
10.31
24. 66
12. 20
Larva:
Instar I
II
Ill
IV
V
Pupa
60
5°
43
32
12
5
Total
64-37
Mar. i, 1921 Biology of the Smartweed Borer 843
It will be noted that in Table II only live instars are listed, but that
the fourth is nearly equal to the combined length of the fourth and fifth
of Table I. It is possible that an error has been made here, but the notes
are clear. The matter will be reviewed another year.
Chittenden mentions 11 and 17 days, respectively, as the lengths of
the pupal stage of two specimens reared by him from cornstalks from
Kansas.
NATURAL CONTROL
The smartweed borer varies greatly in abundance from year to year,
and this seems to be due, in Tennessee at least, to variation in the abun-
dance of its parasites. Here the most important of these appears to be
(Panzeria) Pyraustomyia penitalis Coq., as over 40 per cent of the larvae
taken in the field at Knoxville for rearing were killed by it. Chittenden
notes that this same species also killed more than 50 per cent of the
larvae taken by him in raspberry stems. The host grows normally and
reaches its final instar before the maggot emerges. In its last instar
the host becomes sickly and inactive, paler than normal, and finally
incloses itself in a loose webbing. The parasite maggot emerges and
pupates beside or partly within the remains of its host, often closely
crowded into the cavity with them. In the overwintering larvae the
parasite remains within its host's body until spring and about the middle
of May emerges and pupates in the normal manner. The pupal period
for the fly varies from 13 to 16 days, being more often the latter. The
flies that have been reared by the authors have emerged during two
distinct periods — May 30 to June 10 and August 18 to September 12 —
coinciding closely with the normal dates for the emergence of the moths.
This leads to the assumption that the flies must attack the host larvae
during their early instars.
Coquillett (2, p. 15, 17, 19, 27) records three other tachinid flies (two
of them quoted from Townsend (9, p. 467) ) as reared from " Pyrausta
penitalis" — namely, Exorista vulgaris Fall., Hypostena variabilis Coq., and
Phorocera comstocki Will., but the information given is not sufficient to
determine whether they are parasites of Pyrausta ainsliei or of the true
Pyrausta penitalis.
Cremastus facilis (Cresson) was reared by Chittenden.
Three apparently distinct hymenopterous parasites have been found
by the writers. One of these had* spun a white cocoon and attached it
to the remains of a host larva in its burrow. The second species was
represented by small grubs which filled a dead larva. These grubs later
made gray cocoons, only one of which developed. Two grubs of the
third species were found attached externally to a larva. One of the
grubs developed to an adult and was determined by Gahan as Micro-
bracon sp., a male, and not specifically determinable. The authors
have not received determinations of the other material.
844 Journal of Agricultural Research vol. xx,No. «
Aside from true parasites, a coleopterous larva found preying on a
larva of Pyrausta ainsliei was reared and determined by Schwarz as
Callida decora Fab. Larvae of Chauliognathus pennsylvanicus DeGeer
are often found in the burrows and doubtless make way with some of the
borers. In two instances they have been found feeding upon the con-
tents of the puparia in the stems. Forficulids have been found in the
burrows, but they probably act merely as scavengers.
LITERATURE CITED
(1) Chittenden, F. H.
1918. THE lotus borer. In Jour. Econ. Ent., v. 11, no. 6, p. 453~457. pi- 16.
(2) CoquillETT, D. W.
1897. REVISION OF THE TACHINID^E OF AMERICA NORTH OF MEXICO. U. S.
Dept. Agr. Div. Ent. Tech. Ser. no. 7, 164 p.
(3) Flint, W. P., and Malloch, J. R.
1920. the European corn-borer. 111. Div. Nat. Hist. Survey, Ent. Ser.
Circ. 6, 7 p., illus.
(4)
1920. THE EUROPEAN CORN-BORER AND SOME SIMILAR NATIVE INSECTS.
In 111. Div. Nat. Hist. Survey, v. 13, art. 10, p. 287-305, 44 fig.
(5) Hart, C. A.
1895. ON THE ENTOMOLOGY OF THE ILLINOIS RIVER AND ADJACENT WATERS.
In Bui. 111. State Lab. Nat. Hist., v. 4, art. 6, p. 149-273.
(6) Heinrich, Carl.
1919. NOTE ON THE EUROPEAN CORN BORER (PYRAUSTA NUB1LALIS HUBNER) '
AND ITS NEAREST AMERICAN ALLIES, WITH DESCRB?TION OF LARV^,
puPiE, and one new species. In Jour. Agr. Research, v. 18,
no. 3, p. 171-178, pi. 7-11.
(7) Mosher, Edna.
1919. notes on lepidopterous borers found in plants, with special
reference to the European corn borer. In Jour. Econ. Ent.,
v. 12, no. 3, p. 258-268, fig. 11-14.
(8)-
1919. NOTES ON THE PUP^E OF THE EUROPEAN CORN BORER, PYRAUSTA
NUBTLALIS, AND THE CLOSELY RELATED SPECIES P. PENITALIS.
In Jour. Econ. Ent., v. 12, no. 5, p. 387-389, fig. 18-19.
(9) Townsend, C. H. T.
1893. HOSTS OF NORTH AMERICAN TACHINID/E, ETC., I. In Psyche, V. 6,
no. 206, p. 466-476.
EFFECTS OF X-RAYS ON TRICHINA
By Benjamin Schwartz
Zoological Division, Bureau of Animal Industry, United States Department of Agriculture
INTRODUCTION
The object of the experiments that are described in this paper was to
determine whether X-rays exert deleterious influences on trichinae
(Trichinella spiralis), with a view to the practical application of X-ray
radiation to the destruction of trichinae in pork. These experiments
were performed with the cooperation of a commercial firm that was
operating X-ray machines in Florida. The experiments were planned
and the details arranged by B. H. Ransom, Chief of the Zoological
Division of the Bureau of Animal Industry, in consultation with the
roentgenologist of the firm in question. The former supervised the
tests made by the writer to determine the effects of the X-ray treatment
upon the trichinae, while the latter carried out the portions of the investi-
gations relating to the X-ray treatment, calculations of the X-ray dos-
ages used, etc.
The number of experiments that have been performed are insufficient
to warrant any definite conclusions concerning the feasibility of applying
X-ray radiation to the destruction of trichinae in pork in routine packing-
house procedure. Aside from the fact that the expense involved may
render that procedure impracticable, much more experimental work
than is presented in this paper would be required to demonstrate whether
X-ray treatment could be depended upon to destroy trichinae. The
experimental data at hand are of interest from a general scientific view-
point, however, and it is from that point of view that they are presented.
In a discussion of the effects of X-rays on the flour beetle (Tribolium
confusum), Davey, ! referring to his own work and the work of various
other investigators, states:
X-rays may act upon an organism (or on a single type of cell in that organism) in
one of three ways: (i) to produce a stimulation; (2) to produce a destructive effect
which takes place only after a certain latent interval; (3) to produce an instant de-
structive effect.
That the effects of X-rays on trichinae so far as they are injurious
become evident only after the parasites are subjected to influences that
stimulate them to growth and development, or, in other words, after they
reach the intestine of a host in which they normally attain sexual maturity,
1 Davey, Wheeler P. the effect of x-rays on the length of life of tribolium confusum. In
Jour. Exp. Zool., v. 22, No. 3, p. 575-576. 1917.
Journal of Agricultural Research, Vol. XX, No. 11
Washington, D. C Mar. 1, 192 1
ww (845/ Key No. A-57
846 Journal of Agricultural Research voi.xx.No. h
and accordingly, that X-rays act on trichinae in the second of the three
ways mentioned above, is indicated by the results of the experiments
recorded here.
METHODS OF EXPERIMENT
The trichinous meat used in these tests was obtained from hogs (series
I, II, III, and V) and guinea pigs (series IV). The animals were arti-
ficially infected by feeding them trichinous pork. The infested pork to be
exposed to X-rays was obtained from hogs that were killed several months
after artificial infection. Trichinous guinea-pig meat was obtained from
animals kept about a month after artificial infection.
Trichinous pork was packed in wooden or cardboard boxes in Wash-
ington, forwarded to Florida, where the exposure to X-rays was made,
and promptly returned to Washington, where it was fed to experimental
animals in order to determine the effects of the exposure. In a few
cases portions of the meat that had been exposed to X-rays were digested
in an acidified solution of scale pepsin, the decapsuled larvae were ex-
amined, and the results were compared with those of the feeding experi-
ments. Infested guinea pigs were shipped alive to Florida about 30
days after artificial infection. The animals were killed with chloroform
in Florida, the skins and viscera were removed, and the carcasses were
placed in boxes, exposed to X-rays, and returned to Washington.
The feeding experiments were performed in Washington. A quantity
of the treated meat was ground up in a meat chopper and fed to a number
of rats and, in some cases, mice. Unless they died as a result of infection
with trichinae the animals were killed at various intervals and examined
for evidence of infestation with trichinae as noted in connection with
each experiment. Controls on the meat from the same lots as those
exposed to X-rays showed that in all cases in which it was possible to
maintain controls the untreated meat contained viable trichinae capable
of normal development. In those cases in which the entire carcasses
of trichinous guinea pigs were exposed to X-ray treatment it was of
course not possible to maintain controls.
EXPERIMENTS
SERIES I
X-ray dosage. — The units of dosage used in this series of experiments
are described by the roentgenologist under date of January 20, 1917,
on which day the exposures to X-rays were probably made,1 as follows:
I adopted a purely arbitrary unit, 1,000 of which units are equivalent to a dosage
received at a distance of 5 inches from the focal spot of a Coolidge tube with a current
of 4.2 milliamperes and a pressure of 70 kilcvolts across the tube terminals. Treat-
ment continued for 42 minutes. In ordinary X-ray terms this is equivalent to 172
milliampere minutes with a 6% -inch gap and a 5-inch distance.
1 The meat was received in Washington on January 22, and feeding experiments were begun on January 23.
Mar. i, i92i Effects of X-Rays on Trichina? 847
Experiment i. — Strength of dosage, 2,899 units.
Twelve rats and two mice were fed in this experiment.
Rats 1,2, and 3 were fed on January 23. Rats 1 and 2 were chloro-
formed on January 26. No trichinae were found in the intestines.
Rat 3 died on February 23; diaphragm negative.
Rats 4 to 9, inclusive, were fed on January 25. Rat 4 was killed on
January 26. Trichinae were found in the intestine. The parasites
were about ready to molt. They were somewhat paler than normal.
Rat 5 was killed on January 27. No trichinae were found in the intestine.
No. 6 was killed on February 26; diaphragm negative. No. 7 was killed
on March 12 ; diaphragm negative. No. 8 and 9 were killed on March 15;
diaphragms negative.
Rats 10 to 12, inclusive, were fed on January 30. Rats 10 and 11
were killed on January 31. A few trichinae were found in the intestines
of each animal. The parasites showed evidences of growth. Most of
them were dead, however, having undergone granular degeneration.
Rat 12 was killed on February 1. A few trichinae were found attached
to the mucosa of the intestine. These showed evidence of growth.
Two mice were fed some of the treated meat on January 29. Mouse 1
was killed on January 30. No trichinae were found in the intestines.
Mouse 2 was killed on the same date. A few trichinae were found in the
intestines. The parasites were dead but showed evidence of growth.
No details of structure were made out because the parasites had under-
gone granular degeneration.
Experiment 2.— Strength of dosage, 966 units.
Nine rats were fed in this experiment. Rats 1 to 3 were fed on Janu-
ary 23. Rat 1 was killed on January 26. No trichinae were found in
the intestines. Rat 2 was killed on February 2. A few trichinae, appar-
ently fully grown, were found in the intestines. The parasites showed
rather striking malformations, which were especially pronounced in the
reproductive organs. The gonads were shrunken. The uterus of female
specimens contained eggs, but the latter were full of minute granules.
The receptaculum seminis, which in normal females is crowded with
spermatozoa, was empty.
Rat 3 was killed on February 6. No trichinae were found in the in-
testines.
Rats 4 to 9 were fed on January 25. Rat 4 was killed on January 27 ;
intestines negative. Rat 5 was killed on February 1 ; a few trichinae
were found in the intestines. The parasites showed marked evidence of
degeneration. The cuticle was wrinkled; internally numerous vacuoles
were seen; the sex cells appeared undeveloped; the worms showed very
feeble movements. No. 6 was killed on February 26; diaphragm nega-
tive. No. 8 and 9 were killed on March 15 ; diaphragms negative.
848 Journal of Agricultural Research voi.xx.No. »
Experiment 3. — Strength of dosage, 191 units.
Six rats were fed in this experiment. Three rats were fed on January
25. Rat 1 was killed on January 26. Trichinae were found in the in-
testine. The parasites appeared normal as to size and structure. Rat
2 was killed on January 29; trichinae in intestines normal; uterus of fe-
males packed with embryos. No. 3 was killed on February 2. Numer-
ous trichinae were found in the intestines; apparently normal.
Three rats were fed on January 26. One rat died on February 6.
Numerous larvae were found in the fluid expressed from the diaphragm.
Intestines showed numerous trichinae. The second rat was killed on
February 12. Numerous unencysted larvae were found in the diaphragm.
The third rat died on February 24. Numerous encysted trichinae were
found in the diaphragm.
Experiment 4. — Strength of dosage, 81 units.
Five rats were fed in this experiment. Rats were fed on January 23.
Rat 1 was killed on January 26; numerous live trichinae were found in the
intestines. Rat 2 was killed on January 29; intestines negative. Rat 3
was killed on February 3 ; numerous live trichinae in intestines. Rats 4
and 5 were killed on March 15 ; diaphragms heavily infested with trichinae.
Experiment 5. — Strength of dosage, 35 units.
Five rats were fed in this experiment. Rats 1 and 2 were fed on
January 23. Rat 1 was killed on January 26; numerous live trichinae
in intestines. Rat 2 was killed on January 27; results as in No. 1. Rats
3 to 5 were fed on January 29. Rat 3 died on February 5 ; numerous
live trichinae in intestines. Rat 4 died on February 24; diaphragm
heavily infested with trichinae. Rat 5 died March 2 ; results as in No. 4.
Experiment 6. — Dosage, 19 units.
Three rats were fed on January 23 with the meat treated in this ex-
periment. Rat 1 was killed on January 26; intestines contained many
live trichinae. Rat 2 died on February 12; diaphragm not infested.
Rat 3 died February 26; diaphragm heavily infested.
Artificial digestion tests in experiments i to 6. — Some of the
meat used in each experiment was digested in an artificial gastric juice
January 23. The trichinae thus freed from their capsules were examined
microscopically. They showed no visible evidence of injury, being
active under heat stimulation and remaining tightly coiled at room
temperature and thus behaving like normal trichinae.
Results of experiments of series I. — These experiments indicate
that trichinae are seriously injured by sufficiently high dosages of X-rays.
Although the trichinae in all six experiments when freed from their
cysts by artificial digestion showed no apparent evidence of having
been affected by the X-ray treatment, the parasites in the meat that
had been exposed to the heaviest dosage (experiments 1 and 2) failed
to complete their development when fed to experimental animals.
Instead of growing and developing in a normal manner, after the molt
Mar. i, i92! Effects of X-Rays on Trichina 849
that regularly occurs soon after the parasites reach the intestines, they
underwent degenerative changes, and even in those cases in which the
parasites developed to sexual maturity the reproductive processes
were seriously disturbed. That the reproductive organs are especially
susceptible to X-ray injury is clearly shown by the results of experiment
2. In this experiment the larvae succeeded in attaining maturity, but
the sex cells evidently failed to function.
It is also interesting to note that despite the fact that several rats
in experiment 3 were not fed until 6 days after the meat had been exposed
to X-rays, the animals developed an infection. Thus, in this experiment
there was evident neither an immediate nor a delayed effect of the X-ray
treatment upon the encysted parasites.
SERIES 11
Three experiments are included in this series. The units of dosage
used in this series have the same relative values as those in series I.
Under date of February 5, 19 17, the roentgenologist writes as follows:
The package marked "A" (experiment 7) was given 600 units, the package marked
" B " (experiment 8) 300 units, and the package marked " C" (experiment 9) 350 units.
The 300 units given to package " B " were given with low density and extra long time.
The packages marked "A" and " C" were given the 600 and 3 50 units, respectively, at
high tension — that is, close to the tube and with short time.
Experiment 7. — Strength of dosage, 600 units. The meat was
exposed 19 minutes.
Three rats were fed on February 8. Rat 1 died on March 1 ; diaphragm
negative. Rat 2 died on March 2; diaphragm showed a slight infesta-
tion with trichinae. Rat 3 died on March 6; diaphragm slightly infested
with trichinse.
Experiment 8. — Strength of dosage, 300 units. The meat was
exposed 46 minutes.
Three rats were fed on February 8. Rats 1 and 2 died February 12;
live trichinae were found in the intestines. Rat 3 died on February 26;
numerous larvae were found in the fluid expressed from the diaphragm.
Experiment 9. — Strength of dosage, 350 units. The meat was
exposed 10^2 minutes.
Four rats were fed on February 8. Rat 1 died on February 21;
numerous live trichinae in intestines. Rat 2 died on February 28;
diaphragm infested with encysted trichinae. Rat 3 died on March 1;
results as in No. 2. Rat 4 died on March 2 ; diaphragm heavily infested
with trichinae.
Results of experiments of series II. — The parasites in the meat
used in experiment 7 were evidently affected by the exposure. That
some of them, however, escaped the injurious influences of the exposure
to X-rays may be concluded from the results of the feeding experiments
which resulted in rather slight infections.
850 Journal of Agricultural Research voi.xx.No. n
SERIES III
In this series, which includes 12 experiments, the dosages used had
the same relative values as those of the preceding series. The time of
exposure and distance from the focal spot in the X-ray treatment of
th e various samples of meat in this series of experiments were not given
in concrete terms, but in experiments designated by the letter a the meat
was placed at twice the distance from the focal spot and held four times
as long as in experiments designated by the letter b.
Two rats were used in each feeding experiment. The rats were fed
on May 14.
Experiment ioa. — Dosage, 674 units.
Both rats were killed on June 15; diaphragms heavily infested with
trichinae.
Experiment iob. — Dosage, 674 units.
Rat 1 died on May 29; intestines negative; diaphragm negative.
Rat 2 died on June 12; diaphragm negative.
Experiment iia. — Dosage, 924 units.
Rat 1 died May 28; intestine negative; diaphragm negative. Rat 2
died on June 1 2 ; diaphragm negative.
Experiment iib. — Dosage, 924 units.
Both rats killed on June 15; diaphragms heavily infested.
Experiment 12 a. — Dosage, 1,363 units.
The rats were killed on June 15; diaphragms negative.
Experiment 12B. — Dosage, 1,363 units.
The rats died on June 17; diaphragms negative.
Experiment 13 a. — Dosage, 2,162 units.
The rats were killed on June 15; diaphragms negative.
Experiment 13B. — Dosage, 2,162 units.
The rats were killed June 15; diaphragms negative.
Experiment 14 a. — Dosage, 1,081 units.
Rat 1 dead June 5; one unencysted larva found in diaphragm. Rat 2
killed June 1 5 ; diaphragm heavily infested.
Experiment 14B. — Dosage, 1,081 units.
Rat 1 dead June 12; diaphragm heavily infested. Rat 2 killed June
15; results as in No. 1.
Experiment 15 a. — Dosage, 3,094 units.
Rats killed June 15; diaphragms negative.
Experiment 15B. — Dosage, 3,094 units.
Rats killed June 1 5 ; diaphragms negative.
Results op experiments of series III. — In this series of experiments
trichinous meat subjected to dosages up to 1,081 units proved to be
infective, whereas in experiment 2 (series I) a dosage of 966 units impaired
the vitality of the reproductive cells of the parasites. Whether this can
be accounted for on the basis of variation of trichinae to the effects of
X-rays or whether other factors were involved can not be stated.
Mar. i,i92i Effects of X-rays on Trichince 851
SERIES IV
Under date of June 28, the roentgenologist states that the meat used
in this series of experiments was —
exposed to the direct action of the rays at a distance of very nearly 2 5 cm . from the
focal spot of a Coolidge-type tube. The pressure across the tube terminals was 73
kilovolts, measured by standard sphere gap, and also by ratios. The current through
the tube varied during the time of treatment, which extended over a period of about
3 hours. The lowest reading was 4.2 milliamperes, the highest 4.9. This high
reading, however, was for only a short time after the tube was started. The current
gradually dropped during 10 minutes to 4.3 milliamperes, and during the rest of the
treatment fluctuated between 4.2 and 4.3 milliamperes.
The boxes were so placed that the rays from other tubes in the machine had very
little influence on the contents. By calculation it shows as negligible .
Box A was given an exposure of 42 minutes ; box B an exposure of 84 minutes ; box C
an exposure of 126 minutes; and box D an exposure of 168 minutes. Following the
system of measurement used by Davey,1 which has the merit of being a complete ex-
pression of X-ray quantity, these dosages would read:
MAM
Box A 1S0 — at 73 KV.
25- 'J
Box B 361 ^^at 73 KV.
13 n MAM f vxr
Box C 542 — — at 73 KV.
MAM
Box D 722 g- at 73 K\ .
The rats used in this series of experiments were fed on July 31 and
August 3. Five rats were fed in each experiment.
Experiment 16 (box a), 42 minutes. — Rats 1 and 2 died August 4.
A few trichinae were found in the intestines. The parasites showed evi-
dence of growth. The sex cells were strikingly disorganized. Other
organs also showed evidence of injury. Rat 3 was killed on August 20;
diaphragm moderately infested. Rat 4 died on August 29; diaphragm
moderately infested. Rat 5 died on September 16; diaphragm moder-
ately infested.
Experiment 17 (box b), 84 minutes. — Rat 1 died on August 6; intes-
tines negative. Rat 2 died on August 1 7 ; intestines and diaphragm neg-
ative. Rat 3 died on August 18; results same as in rat 2. Rat 4 died
on August 20; results same as in rat 2. Rat 5 was killed on September
10; diaphragm negative.
Experiment 18 (box c), 126 minutes. — Rats 1 and 2 were killed on
August 20; diaphragms negative. Rats 3 and 4 were killed on Septem-
ber 10; diaphragms negative. Rat 5 was killed on September 10; dia-
phragm slightly infested.
• Davey (op. cit., p. 586) states: " The voltage and distance are given directly and the product of the cur-
rentandtimeis given, thus, 'ioomilliampere-minutesat 25 cm. distance at 50 kilovolts.' This is usually con-
tracted to read ioo^1^-1 tt5okv. It will be noticed that distance is expressed in terras of its square.
This is because the intensity of X-rays varies inversely as the square of the distance.
&52 Journal of Agricultural Research vol. xx, No. n
Experiment 19 (box d), 168 minutes. — Rat 1 was killed on August 7;
intestine negative. Rat 2 was killed on August 20; intestine negative
and diaphragm negative. Rat 3 died on September 5 ; diaphragm nega-
tive. Rats 4 and 5 were killed on September 10; diaphragms negative.
Results of experiments of series IV. — The X-ray dosages used in
these experiments were clearly injurious to the trichinae. The smallest
dosage used (experiment 16) had some effect, though it did not destroy
the reproductive functions of all the parasites. In the three other experi-
ments in which considerably larger dosages were used only 1 infection
occurred among the 15 experimental animals on which the infectiousness
of the meat was tested, and that infection was slight.
SERIES v
This series included six experiments. The dosages used in these experi-
ments were not indicated, except that two samples were given similar
dosages and that the remaining samples received graded dosages.
Furthermore, the samples were mixed so that it is not known which sam-
ples received the larger or the smaller dosages. The samples were treated
on March 24. Experimental rats were fed in Washington on March 27.
Experiment 20. — Rat 1 died on April 5 ; no trichinae were found in the
intestines. Rat 2 was killed on April 9; intestines contained live tri-
chinae; female trichinae contained many embryos; diaphragm negative.
Rat 3 was killed April 16; intestines positive; diaphragm positive. Rat 4
was killed on April 24; diaphragm heavily infested.
Experiment 2 1 . — Rat 1 was killed on April 9; intestines contained live
trichinae, normal in appearance; female trichinae contained eggs and em-
bryos. Rat 2 was killed on April 16; intestines contained many live
trichinae. Rat 3 died on April 24; diaphragm heavily infested.
Experiment 22. — Rat 1 was killed on April 8; intestines negative;
diaphragm negative. Rat 2 was killed on April 16; diaphragm negative.
Rat 3 died on April 17; diaphragm negative. Rat 4 died on April 24;
diaphragm heavily infested.
Experiment 23. — Rat 1 was killed on April 9; live trichinae were
found in the intestines; sex cells were atrophied; no larvae were found
in the diaphragm. Rat 2 was killed on April 16; no trichinae were found
in the intestines; diaphragm negative. Rat 3 was killed on April 23;
diaphragm negative. Rat 4 was killed on April 23; one encysted larva
was found in the diaphragm.
Experiment 24. — Rat 1 was killed on April 2; intestines contained
numerous live and apparently normal trichinae. Rat 2 was killed on
April 8; live trichinae were found in the intestines; diaphragm negative.
Rat 3 was killed on April 16; intestines contained trichinae, apparently
dead; diaphragm negative. Rat 4 was killed on April 24; diaphragm
heavily infested. Rat 5 was killed on April 24; diaphragm negative.
Mar. i, 1921 Effects of X-Rays on Trichince 853
Experiment 25. — Rat 1 was killed on April 9; intestines contained
live trichina; sex cells of trichinae atrophied; diaphragm negative. Rat
2 was killed on April 16; diaphragm negative. Rat 3 died on April 19;
diaphragm negative. Rats 4 and 5 were killed on April 24; diaphragm
negative.
Results of experiments oe series V. — The results of these exper-
iments are in harmony with the results of the experiments recorded in
the preceding pages. Trichinae that showed sex-cell injuries (experiments
23 and 25) failed to produce a new generation. That a few larvae in
experiment 23 escaped injury is evident from the results of the feeding
experiment with rat 4. It is interesting to note, however, that despite the
fact that the parasites showed evidence of injury they were still alive on
the fourteenth day after artificial infection. This indicates that X-rays
exert a selective action on the sex cells of trichinae and that injuries to the
sex cells do not necessarily affect the other vital functions of the parasites.
DISCUSSION
The results of the experiments described in the foregoing pages show
that trichinae may be seriously injured by X-ray radiation. It is inter-
esting to note that in experiments 1 to 6 inclusive (series I), larvae isolated
from the treated meat by artificial digestion appeared to be unaffected.
These larvae were normal as to color and general appearance, as viewed
through the microscope and as indicated by their reactions to heat
stimulation. The examination was made three days after treatment.
The larvae from the meat treated in experiments 1 and 2 (series I) were
incapable, however, of attaining full sexual maturity in the intestines of
rats or mice. Those in experiment 1 and some of those in experiment
2 underwent granular degeneration, while others in the latter experiment
succeeded in attaining maturity without being capable of functioning
sexually. The fact that no spermatozoa were found in the receptaculum
seminis of the female indicates that successful copulation had not taken
place.
It is also of interest to observe that a considerable degree of variation
in resistance to X-rays is exhibited by trichinae, since certain dosages
proved to be destructive in some cases and not in others. This is pos-
sibly due, however, to other factors. It may be noted in this connec-
tion that trichinae exhibit considerable variation in their resistance to
cold * and in their resistance to heat.2
Assuming that a reliable and practically possible method of destroying
the vitality of the sex cells in trichinae by means of X-ray treatment of
infested meat can be perfected, which is quite uncertain, it is still ques-
tionable whether such a method would be acceptable as a prophylactic
1 Ransom, B. H. effects of refrigeration upon the larvae of trichinella spiralis. In Jour.
Agr. Research, v. 5, no. 18, p. 819-854. 1916. Literature cited, p. 853-854.
2 and Schwartz, Benjamin, effects of heat on trichin.e. In Jour. Agr. Research, v. 17,
no. 5, p. 201-221. 1919. Literature cited, p. 220-221.
854 Journal of Agricultural Research voi.xx, No. n
measure, inasmuch as trichinae are not inoffensive as intestinal parasites
apart from the damage done by their migrating larvae. Rats, for example,
commonly die from intestinal trichinosis prior to the migration of the
larvae, and human beings also often suffer seriously from the effects of
the intestinal stage of the parasites during the first few days after infec-
tion before the migrating larvae have been produced. Consequently,
unless the X-ray treatment has the effect of diminishing the injurious
action of the intestinal stage of trichinae upon the host as well as of
destroying their powers of reproduction, it can scarcely be considered a
satisfactory prophylactic measure. It is of interest to note in this
connection that Tyzzer and Honeij ' found that encysted trichinae that
had been subjected to radium radiation failed to develop in mice. These
investigators also determined that whereas radium radiation failed to
destroy sexually mature trichinae in live rats, trichinae in rats which
were radiated beginning with the second day after ingestion of trichinous
meat showed retardation in development. Radiation of the larvae in
rats before they have begun to develop proved fatal to them.
SUMMARY
(1) Encysted trichinae are injured by relatively heavy dosages of
X-rays. So far as has been determined the injuries are not visible in
the encysted or artificially decapsuled larvae as structural or functional
disturbances but become apparent only when the larvae reach a suitable
host animal in whose intestine they are normally capable of continuing
their development.
(2) Trichinae from meat that has been exposed to strong dosages of
X-rays undergo rapid granular degeneration in the intestines of suitable
hosts before they attain maturity.
(3) Encysted larvae that have been exposed to lower but still injurious
dosages of X-rays are able to continue development in the intestines of
suitable hosts. Such larvae, however, do not attain structural and
functional sex maturity. The sex cells appear to be atrophied, and no
evidence of successful copulation can be found. X-rays, therefore,
appear to exert a more or less selective action on the gonads of trichinae.
(4) Trichinae appear to exhibit considerable variation in their suscepti-
bility to X-rays, since certain dosages injured some parasites and failed
to injure others. Whether the apparent variation in susceptibility of
trichinae to X-rays is an expression of an actual physiological variation
or may be accounted for by other factors has not been determined.
(5) The experiments described in this paper do not warrant any
definite conclusions as to the feasibility of using X-ray radiation as a
practical means of destroying trichinae in pork.
1 Tyzzer, E. E., and Honeij, James A. the effects of radiation on the development of trichi-
NELLA SPIRALIS WITH RESPECT TO ITS APPLICATION TO THE TREATMENT OF OTHER PARASITIC DISEASES. In
Jour. Par., v. 3, no. 2, p. 43-56, 1 pi. 1916.
RELATION OF THE CALCIUM CONTENT OF SOME KAN-
SAS SOILS TO THE SOIL REACTION AS DETERMINED
BY THE ELECTROMETRIC TITRATION
By C. O. Swaxson, Associate Chemist, W . L. Latshaw, Analytical Chemist, and E. L.
TaguE, Protein Chemist, Department of Chemistry, Kansas Agricultural Experiment
Station
The importance of the soil reaction has led to the development of
numerous methods for testing the neutrality, acidity, or alkalinity of the
soil, and, if the soil is acid, for determining quantitatively the amount of
agricultural lime necessary to add to the soil in order that it may have the
reaction required for maximum crop production. No attempt will be
made to review the literature on this subject, and only a few citations will
be given.
Of the different tests designed simply to determine qualitatively the
reaction of the soil, the litmus paper test is one of the oldest, best known,
and probably most extensively used. This test has been subjected to
much criticism, but this is probably due more to bad paper and faulty
use than to intrinsic defects in the method. The official or Hopkins
method {15, p. 20)1 has been used for most of the acidity work done thus
far on Kansas soils. It was found, however, that in some cases the indi-
cated lime requirement appeared too low when studied in connection with
the known cropping conditions of the soil. The well-known Veitch
method (14) is probably the best quantitative measure of the lime require-
ments of the soil at the present time. There are several other methods
proposed to determine the lime requirements of the soil, and each has its
advocates. The strong advocate of any method is usually very free with
his objections to some other method. All methods are limited in their
application, and faults are often found with methods because the users
extend the application further than the originators intended.
One difficulty in determining the soil reaction is to obtain the soil solu-
tion in the same concentration as it exists around the soil grains. Various
methods have been proposed for securing this solution, but none have
received general acceptance. Another factor is the facility with which
the optimum reaction for best crop production is maintained in the soil.
The concentration of the soil solution is in a state of continuous change.
The film of water surrounding the soil grains in a soil of optimum water
content tends to become saturated with the salts present in the soil. The
1 Reference is made by number (italic) to ' ' Literature cited, ' ' p. 867-808.
Journal of Agricultural Research, Vol. XX, No. n
Washington, D. C Mar. 1, 1921
xa Key No. Kans. -23
(855)
856 Journal of Agricultural Research voi.xx, no. «
addition of rain or irrigation water temporarily reduces the concentration.
If some of the water is carried off in the drainage, it takes away a certain
amount of the dissolved salts. At the present time calcium is removed
from the soil more rapidly than any other base (5, p. 23). Lyon and
Bizzell (8) found in lysimeter experiments that the equivalent of 485
pounds of calcium carbonate per annum leached from some soils. The
continuous removal of calcium from the soil produces an unbalanced con-
dition known as lime deficiency or acid soil. When calcium carbonate
is added to the soil the balance is restored and the reaction is neutral or
slightly alkaline.
The studies presented in this paper are not designed to settle any differ-
ences of opinion relative to the meaning of soil acidity nor to decide
which is the best method of determining the lime requirement of the soil.
They are presented as a contribution to the partial solution of a very com-
plex as well as important problem. The electrometric titration has been
used by a number of investigators (6, 9, 10). Because of its intrinsic
value it was used in this study of the relation between the calcium con-
tent of some typical Kansas soil and the reaction.
MEANING OF SOIL ACIDITY
The following is usually taken as the meaning of acidity in soil: Total
acidity means the total quantity of hydrogen ions which may be pro-
duced when the equilibrium is continually shifted by the introduction
of hydroxyl ions. The quantity of hydrogen ions present at any one
moment is regarded as the intensity of acidity. This definition would be
inclusive and very convenient if it were not for the adsorptive power of
colloids in soil. It will be shown that this intensity of acidity may be very
small as related to the total acidity. Understood in this way, quantita-
tively, total acidity has the same meaning as potential acidity. Poten-
tial acidity may be due to undissolved substances, or to soluble com-
pounds only partly hydrolyzed or dissociated. It appears also to be due
to colloidal clay; but whatever it is due to, the conditions are such that
as soon as more hydroxyl ions are introduced the equilibrium is shifted
by the production of more hydrogen ions. The absolute neutral point
obtains when the number of hydrogen ions and the number of hydroxyl
ions are equal and each has a concentration of io-7 per liter.
SOILS USED IN THIS STUDY
Twelve counties in Kansas have been surveyed and mapped by the
Bureau of Soils, United States Department of Agriculture. Five of these
counties were worked in cooperation with the Kansas Agricultural Ex-
periment Station. These types have been sampled and analyzed by the
Department of Chemistry, Kansas Agricultural Experiment Station
(1-4, 11). Determinations have been made for nitrogen, phosphorus,
potassium, carbon, carbon dioxid, and calcium. On the basis of these data
Mar. i, 1921
Relation of Calcium Content to Soil Reaction
857
those soils which were thought most suitable were selected. In the
description of these soils, the type names given by the Bureau of Soils
are used. The soil numbers are those found in our soil series. The
samples had been taken usually in three strata — namely, surface o to
7 inches, subsurface 7 to 20 inches, and subsoil 20 to 40 inches. For
this work, surface soils mostly were selected, with a few accompanying
subsoils. The soil number has a whole figure and a decimal. A surface
soil is designated as 1083. 1, subsurface as 1083.2, and a subsoil as 1083.3.
Some of the samples were taken in only two strata.
The figures given for total calcium and carbon dioxid are taken from
the publications to which reference has been made. In addition the
authors have determined the calcium soluble in Nji hydrochloric acid
and in NJ5 hydrochloric acid and the reaction as determined by the
hydrogen electrode with accompanying titrations.
DETERMINATION OF ACID-SOLUBLE CALCIUM
Five gm. of soil were placed in 100 cc. Nji hydrochloric acid and
shaken for one hour on a shaking machine, then placed in a thermostat
at 400 C. and digested for 23 hours with occasional shakings. The
acid-soluble calcium was determined by the volumetric permanganate
method. The treatment with NI5 hydrochloric acid was similar ex-
cept that 10 gm. of soil and 200 cc. of the acid were used. The results
on the calcium determinations are given in Table I.
Table I. — Calcium and carbon dioxid in representative Kansas soils
GROUP I, SOILS WHOSE INITIAL REACTION WAS MORE ALKALINE THAN IS INDICATED BY PH 8.3
Soil
No.
1043
1227
1297
"99
I206
1 186
I"5
III9
1169
County.
Russell
Greenwood..
Montgomery
Jewell
do
Reno
Finney
do
Reno
Soil type.
Benton loam
Crawford clay
Oswego silt loams
Laurel very fine sandy loam.
Lincoln silty clay loam
Kirkland clay loam
Richfield silt loam
Pratt loamy fine sand
....do
Total
Ca.
Per
Ca. solu-
ble in
Nil HC1.
Per cent.
4. 01
Ca. solu-
ble in
NI5 HC1.
Per cent.
4. 19
3- 18
I- 03
■53
.69
•47
.28
.09
•°S
CO2.
Per cent.
37
Trace.
Do.
• 009
GROUP II, SOILS WHOSE INITIAL REACTION WAS BETWEEN PH 8.3 AND PH 7
1219
IO47
I039
1132
1268
I267
II2I
II3I
IO7O
1157
1120
I072
Il62
II60
Greenwood . .
Brown
Russell
Shawnee. . . .
Montgomery
do
Finney
Shawnee
Reno
do
Finney
Harper
Reno
do
Osage loam
Silt loam (bottom)
Summit silty clay loam
Osage silty clay loam
Osage clay (alfalla land)
Osage silty clay loam
Finney clay
Osage very fine sandy loam .
Pratt loam
Arkansas clay loam
Sandy loam
Brown loam
Pratt fine sandy loam
Arkansas fine sand
0.88 0
80 0
80
.80
66
57
• 83
62
54
.87
59
54
• 79
52
49
.76
49
3»
•78
31
30
•79
28
36
.42
20
14
• 7°
21
19
•44
iS
16
•31
18
13
•49
12
12
•55
08
07
.04
.16
Trace
None
Do.
Trace.
Do.
Do.
.003
Trace.
None.
.003
.002
29666c
858
Journal of Agricultural Research
Vol. XX, No. ii
Tablk I. — Calcium and carbon dioxid in representative Kansas soils — -Continued
GROUP m, SOILS WHOSE INITIAL REACTION WAS MORE ACID THAN IS INDICATED BY Pa 7
Soil
No.
1141
1136
1285
1287
io53
1284
1 190
1293
1191
"35
1271
"43
1256
1262
1233
1257
1265
1273
1275
1232
1277
1266
1239
1243
1280
1230
1279
1 148
1244
County.
Shawnee . . . .
do
Montgomery
do
Doniphan.. .
Montgomery
Jewell
Montgomery
Jewell
Shawnee
Montgomery
Shawnee
Greenwood..
Montgomery
Cherokee. . . .
Greenwood..
Montgomery
do
do
Cherokee. . . .
Montgomery
do
Cherokee. . . .
do
Montgomery
Cherokee. . . .
Montgomery
Reno
Cherokee
Soil type.
Total
Ca.
Ca. solu-
ble in
N/zUCl.
Summit silty clay loam ....
Oswego silt loam
Crawford loam
Summit silty clay loam. . . .
Brown silt loam
Crawford loam
Jewell silt loam
Bates stony loam
Colby silt loam
Crawford silty clay loam. . .
Bates loam
Boone fine sandy loam
do
Crawford loam
Oswego silty clay loam
Summit silty clay loam ....
Cherokee silt loam
Bates very fine sandy loam.
Oswego silt loam
Bates silt loam
Bates shale loam
Bates very fine loam
Summit silt loam
Cherokee silt loam
Bates loam
Neosho silt loam
Bates very fine sand
Dune sand
Bates fine sandy loam
Per cent.
42
Ca. solu-
ble in
NIs HC1.
Per cent.
o. 41
.18
.19
.18
•15
•13
.14
•13
. 10
.09
.09
.08
.07
Per cent.
o. 016
.013
None.
Do.
Trace.
None.
. 01
None.
. 01
. 01
None.
Trace.
Do.
None.
Trace.
Do.
None.
Do.
Do.
Trace.
None.
Do.
Trace.
Do.
None.
Do.
Do.
.00;
Trace.
DETERMINATION OF THE INITIAL REACTION AND THE ELECTRO-
METRIC TITRATION OF SOILS STUDIED
The apparatus used in these determinations was the same as that de-
scribed in previous papers (12, 13). Ten gm. of soil were weighed into
a 250-cc bottle which was used as the electrode vessel, and 100 cc of
carbon-dioxid free water were added. The bottle was closed with a large
rubber stopper through which were inserted the hydrogen electrode and
the capillary tube connecting with the calomel cell. The hydrogen after
bubbling through the soil suspension passed through a water trap, and
the tip of the burette used in the titration was inserted through a hole in
this stopper. In this way contamination from the carbon dioxid in the
air was prevented. These precautions are necessary, since these deter-
minations require a number of hours.
The distilled water used in this work was freed from carbon dioxid by
aeration. While water so treated is not as neutral as conductivity water,
the purity was sufficient for these determinations. The reaction of va-
rious samples of this water ranged from PH6 to PH6.6. One-tenth cc. of
N/io alkali would change the concentration from about PH 6 to PH 8.
The error due to the water is therefore small. After the apparatus was
adjusted, the hydrogen gas was bubbled through until equilibrium was
obtained. The time required for this depended somewhat on the char-
acter of the soil. During the entire time the electrode vessel was shaken
about 60 times per minute by a shaking device. As soon as the readings
on the millivoltmeter remained constant within a few millivolts for 15
Mar. i, 1921 Relation of Calcium Content to Soil Reaction
859
minutes, the soil suspension was considered to be at equilibrium. This
point was noted and taken as the initial reaction of the soil. A solution
of saturated calcium hydroxid is very near N/24. For the sake of facility
in making calculations this was made iV/25. Since the final end product
of calcium hydroxid or calcium oxid added to the soil is calcium carbonate,
this equivalent is used in making the calculations. One cc. of NJ25 cal-
cium hydroxid is equivalent to 0.002 gm. of calcium carbonate. One
acre of soil 7 inches deep is assumed to weigh 2,000,000 pounds. Since 10
gm. of soil were used in a determination, the ratio of the calcium car-
bonate equivalent of 1 cc. of the calcium hydroxid is 1 : 5,000. Accordingly,
each cubic centimeter of calcium hydroxid used to titrate is equivalent
to 400 pounds of calcium carbonate per acre.
When the voltmeter reading at the initial equilibrium point had been
obtained, the calcium hydroxid was added from the burette in small por-
tions at a time until the equilibrium was again obtained at voltmeter
reading equivalent to PH 7. The total number of cubic centimeters used
in the titration were recorded, and again small portions of calcium hy-
droxid were added till equilibrium was established at voltmeter reading
equivalent to PH 8.3. This is approximately the titration end point for
phenolphthalein. Again the calcium hydroxid was added until equili-
brium was established at reading equivalent to PH 10. The latter point
was somewhat arbitrarily chosen.
A few grams of special " K " calcium carbonate were suspended in water,
and after long shaking the reaction was found to be PH 9.23. This is a
little lower alkalinity than the value PH 9.5 obtained by Sharp and Hoag-
land (10). The reading PH 10 denotes a higher alkalinity than that
found in a normal soil.
The electrometric measurements then gave these data: The initial re-
action of the soil suspension stated as PH ; the total number of cubic cen-
temeters of calcium hydroxid (Af/25) required to change the reaction
to PH 7, PH 8.3, and PH 10, respectively. The results of these measure-
ments are recorded in Table II.
Table II. — Initial reaction of the soil and tlie number of cubic centimeters of NJ25 calcium
hydroxid used to change the reaction to the figures given °
GROUP I. SOILS WHOSE INITIAL REACTION WAS MORE ALKALINE THAN IS INDICATED BV Ph 8.3
Soil
No.
1 169
1227
1043
1221
I2Q7
1 199
III9
I206
1 186
County.
Reno
Greenwood
Russell . .
Greenwood
Montgomery
Jewell
Finney. .
Jewell. . .
Reno. . . .
Soil type.
Pratt loamy fine sand
Crawford clay
Benton loam
Oswego silt loam
do
Laurel very fine sandy loam .
Dune sand
Lincoln silty clay loam
Kirkland clay loam
Initial
Ph.
8.61
8.56
8.52
8.51
8.46
8.44
8.44
8.40
8-37
Cubic centimeter^ of Ca(OH)s
required to titrate to —
Ph 7.
Ph8.3.
0.7
5-4
3-7
9-3
2-5
12.3
4.4
4.0
° Figures arranged according to increasing hydrogen-ion concentrations.
86o
Journal of Agricultural Research
Vol. XX, No. ii
Table II.— Initial reaction of the soil and the number of cubic centimeters of N/25 calcium
hydro xid used to change the reaction to the figures given — Continued
GROUP II, SOILS WHOSE REACTION WAS BETWEEN Ph 8.3 AND Pa 7
Soil
No.
1039
1219
1120
1047
1132
1268
1267
1072
1160
"57
1070
1131
1121
1162
County.
Russell
Greenwood. .
Finney
Brown
Shawnee. . . .
Montgomery
...do
Harper
Reno
....do
....do
Shawnee. . . .
Finney
Reno
Soil type.
Summit silty clay loam. . . .
Osage loam
Sandy loam
Silt loam (bottom)
Osage silty clay loam
Osage clay (alfalfa land) . . .
Osage silt clay loam
Brown loam
Arkansas fine sand
Arkansas clay loam
Pratt loam
Osage very fine sandy loam
Finney clay
Pratt fine sandy loam
Initial
Ph.
8.03
7. Si
7-5°
7-49
7-45
7.40
7-36
7.28
7.26
7. 26
7- 2 2
7.19
Cubic centimeters of Ca(OH)«
required to titrate to —
Pu 7.
Ph 8.3.
Ph 10.
i-5
13-2
3-°
10. I
2.0
6.8
•S
1. 0
2.7
8.7
2.9
8.4
3-°
19- 1
3-3
8-3
1.4
3-5
3-8
10.4
5 3
13-0
2.0
4.6
1-7
5-1
6 9
16. 1
GROUP III, SOILS WHOSE INITIAL REACTION WAS MORE ACID THAN IS INDICATED BY Pu
1287
"43
1191
1 190
1285
1243
I23O
I0S8
II48
1266
I271
I284
II4I
"j5
1232
1136
1256
1279
1263
1277
1280
1273
1257
1293
1275
1233
1244
1239
Montgomery
Shawnce. . . .
Jewell
....do
Montgomery
Cherokee
....do
Doniphan. . .
Reno
Montgomery
....do
....do
Shawnee. . . .
....do
Cherokee. . . .
Shawnee. . . .
Greenwood . .
Montgomery
....do
....do
....do
....do
Greenwood. .
Montgomery
....do
Cherokee
....do
.. do
Summit silty clay loam. . . .
Boone fine sandy loam
Colby silt loam
Jewell silt loam
Crawford loam
Cherokee silt loam
Neosho silt loam
Brown silt loam
Dune sand
Bates very fine sandy loam
Bates loam
Crawford loam
Summit silty clay loom. . . .
Crawford silty clay loam . . .
Bates silt loam
Oswego silt loam
Boone fine sandy loam
Bates very fine sand
Cherokee silt loam
Bates shale loam
Bates loam
Bates very fine sandy loam
Summit silty clay loam. . .
Bates stony loam
Oswego silt loam
Oswego silty clay loam
Bates fine sandy loam .
Summit silt loam
6
77
2-5
5-3
6
7b
.8
9.0
6
72
1. 2
3-7
6
71
.8
3-6
6
65
1. 0
4.2
6
ss
1-3
6.2
6
S3
i-3
4.6
6
53
r-3
5-r
6
46
.6
2-3
6
36
2.6
5- 2
6
-<;
2.4
6-5
6
as
•5
i.S
6
15
6.7
'7-5
6
01
8.4
20. 0
5
92
4-3
9.4
s
S4
7-o
17.9
s
72
9.1
15-3
5
70
2.6
6.0
5
<6
6.7
10.3
5
56
6.8
15.0
5
54
5- I
6.0
S
53
6.1
11. 0
5
49
9.2
22. 0
S
49
3-1
4-5
S
35
8.2
10. s
4
99
8.2
M-3
4
99
7-r
12.4
4
68
9.8
19-3
13.0
7-9
12.5
14. I
8.9
10. 2
4-2
"•5
20. 2
4-7
31-4
35-2
16. I
30.6
29. 6
11. 7
23.2
29-3
13- S
24.1
35-3
9-1
18.8
25. 2
24.0
31-0
CLASSIFICATION ON THE BASIS OF REACTION
When the data obtained, both in the calcium determinations and the
electrometric measurements, were brought together it was found con-
venient to classify the soils into three groups. In group I were placed
those soils whose initial reaction was more alkaline than is indicated by
PH 8.3. In group II were placed those soils whose initial reaction were
less alkaline than is indicated by PH 8.3 but more alkaline than is indi-
cated by PH 7- In group III were placed those soils whose initial reac-
tion was more acid than is indicated by PH 7. In arranging the soils
within these groups the figures in Table I are given according to decreas-
ing amounts of calcium soluble in N/i hydrochloric acid. In Table II
the soils are arranged according to decreasing alkalinity, or increasing
acidity, as expressed by the PH values.
Mar. i,i92i Relation of Calcium Content to Soil Reaction 861
CALCIUM CONTENT OF SOILS STUDIED
The soils of highest calcium content are found in group I. The four
soils in group I which have as low calcium content as several of the soils
in group II, or lower, are from the drier portion of the State. The soils in
group III have an average lower calcium content than the soils in groups
I and II. In general, the soils of a high calcium content have a more
alkaline reaction than soils of low calcium content; yet because of the
exceptions, the calcium content alone can not serve as the basis of classi-
fication as acid or alkaline. Most of the soils in group I are from the sec-
tion of the State where acid soils are not usually found, whereas most of
the soils from group III are from the section of the State where acid soils
are more common. Sandstone-derived soils from the drier portions of the
State may have a comparatively small amount of calcium and yet have
an alkaline reaction.
In soils of high calcium content a larger percentage of the amount pres-
ent is soluble in acid than in soils of low calcium content. As the per-
centage of total calcium decreases, it is relatively less soluble. This is
true in comparing the groups and in comparing soils within groups. In
group I the proportion of acid-soluble calcium is greater than in group II,
and in group II it is greater than in group III.
The differences in the amounts of calcium in forms soluble in Nji hy-
drochloric acid and in A// 5 hydrochloric acid are small. For practical
purposes they are of equal value.
The pronounced differences between the amounts of calcium soluble in
hydrochloric acid and the total, especially in soils of low calcium content,
raises the question of the relative importance of determining the total
calcium in a soil or determining the amount soluble in cold dilute hydro-
chloric acid. These figures would indicate that the results obtained by
the acid digestion are more valuable. In soils of low calcium content, it
is present mostly in insoluble forms. While weathering gradually con-
verts this calcium into forms that are soluble, the amount of available
calcium obtained is insufficient for the needs of the soil. Such soils are
deficient in "agricultural lime."
The figures for percentage of carbon dioxid show that all the soils in
group I have some carbonates, that only 6 of the 14 soils in group II have
carbonates in larger amounts than traces, and that only 3 of the soils in
group III have carbonates in larger amounts than traces and in these the
amounts are very small.
RESULTS OF ELECTROMETRIC MEASUREMENTS
The results on the electrometric measurements found in Table II are
arranged according to decreasing alkalinity values or, which means the
same thing, increasing acidity values. The figures expressing cubic cen-
timeters of calcium hvdroxid under the different PH values in each case
862 Journal of Agricultural Research voi.xx.No. n
mean the total calcium hydroxid used to bring the reaction to that point.
In interpreting the results of these electrometric measurements the fol-
lowing factors must be considered: (i) Kind of soil with reference to the
amount of sand, clay, and organic matter; (2) influence of climatic condi-
tions; (3) amount of calcium present, particularly in the carbonate form.
The amount of sand, silt, clay, or organic matter present in a soil may
have a greater influence on the initial reaction than the amount of cal-
cium present. Pratt loamy fine sand from Reno County has the lowest
calcium content of the soils placed in group I, Table I, but it has the
highest alkalinity as shown in Table II. Benton loam No. 1043 and
Crawford clay No. 1227 are both high in calcium and both have a high
initial alkaline reaction. The clay soils and the silty clay soils as a rule
require more calcium hydroxid to change to a certain hydrogen-ion con-
centration than the sandy soils.
Soils placed in group III, Table II, are distinctly acid in reaction. As
the initial acidity increases, the amounts of calcium hydroxid needed to
change the reaction to neutral (indicated by PH 7) also increases, but not
uniformly. This is due to factors mentioned in the preceding paragraph.
The influence of clay is shown by the figures in Table III.
Subsoils as a rule contain a larger amount of calcium than the surface
soils, particularly calcium in the carbonate form. These same subsoils
usually contain a larger amount of clay but a smaller amount of organic
matter. The calcium was determined in a number of the subsoils cor-
responding to the surface soils mentioned in Tables I and II. The elec-
trometric measurements were also made. The results are found in
Table III. The figures for the surface soils are repeated from Tables I
and II for the sake of comparison. The results in Table III are arranged
within the groups with reference to the decreasing amounts of calcium in
the surface soils. The results show that, with few exceptions, the sub-
soils have a higher calcium content than the surface soil and that in the
majority of cases the subsoil requires a larger amount of calcium hydroxid
to change it to the same reaction as the surface soil.
The soils in which the calcium content is less in the subsoil than in
the surface soil are: 1297, Oswego silt loam; 1271, Bates loam; 1273,
Bates very fine sandy loam; and 1277, Bates shale loam. In the first
one of these soils the titration figure is larger for the subsoil than for the
surface soil. This would be expected from the larger clay content and
the smaller amount of calcium. The last two have sandy subsoils; and
while no mechanical analyses were made, observations recorded at the
time of taking the samples show that the subsoils have less clay than
the surface soils. Both these soils were acid, and the subsoil is more
acid than the surface soil. Yet the lesser amount of clay in the subsoil
was of more influence in determining the amount of calcium hydroxid
needed to bring to neutral reaction than the initial acidity.
Mar. i, 1921 Relation of Calcium Content to Soil Reaction
863
Table III. — Calcium content and electrotnetric measurements on subsoils in comparison
with surface soils
GROUP I . SOILS WHOSE INITIAL REACTION WAS MORE ALKALINE THAN IS INDICATED BY Ph 8.3
Count!-.
Soil type.
Calcium
soluble in
XI 1 HCl,
calculated
to equiva-
lent of
CaCOa
per acre
7 inches
deep.
Cubic centimeters of NI25
Ca(OH)j required to
titrate to —
Soil
No.
Ph.
Ph7-
Ph8.3.
Ph 10.
Pounds,
i 200, 500
\ 643 , 000
\ 165,000
\ 206, 000
/ 57.000
\ 42,000
/ 30,000
\ 40,000
/ 25,000
I 45-5oo
/ 4, 000
\ 5 , 000
8 "
3-7
1043. 1 .
1043-3 J
12271 >Greenwood..
8
8
8
8
9
8
9
8
8
8
8
56
56
75
46
03
44
°3
40
40
61
6r
5.0
1227.3 J
1297-1 JMontgom-
1297-3 J ery
"W1 \ Jewell
1 199- 3 J
Laurel very fine sandy loam. . .
Lincoln silty clay loam
:::::::::.::
4.8
1206. 3
1169. I
> do
VFinney
7.6
1169. 2
GROUP n, SOILS WHOSE INITIAL REACTION WAS BETWEEN Ph 8.3 AND Ph
1085. I
1085.3
IO47. I
1047-3
1039. 1
1039-3
1121. 1
1121.3
1070. 1
1070.3
H57- 1
H57-3
1 1 20. I
1 1 20. 3
1072. 1
1072.3
1162. 1
1162. 3
Riley...
Brown.
Russell.
VFinney.
Reno...
\....do..
>Finney.
>Harper.
JReno. . .
Laurel silt loam
Silt loam, bottom
Summit silty clay loam.
Finney clay
Pratt loam
Arkansas clay loam
Sandy loam
Brown loam
Pratt fine sandy loam . .
71.500
8.16
73 - 000
8.16
33.000
7-49
53.500
8.68
31,000
8.03
78, 500
8.28
15,000
7-17
23,000
8.44
10,000
7. 22
14,000
6.67
10, 500
7.26
76, 500
8-39
9. 000
7-5°
05, 500
8.92
9,000
7.28
26, 500
8.44
6,000
7- 05
6-95
12. 6
3-8
3-8
6.9
6.2
GROUP III, SOILS WHOSE INITIAL REACTION WAS MORE ACID THAN IS INDICATED BY Ph 7
7-9
7-9
17-7
5- 1
1.4
13.0
26. 7
10. 4
9.0
Richfield silt loam
Summit silty clay loam
Brown silt loam
Jewell silt loam
Colby silt loam
Crawford silty clay loam
Oswego silty clay loam
Summit silty clay loam
Cherokee silt loam
Oswego silt loam
Bates loam
Bates very fine sandy loam. . .
Bates silt loam
Bates shale loam
Cherokee silt loam
Neosho silt loam
Bates fine sandy loam
16,000
6-57
177, 000
8
34
14.000
6
77
17,000
6
13
1 3 , 000
1 4 , 000
6
6
53
32
1 2 , 000
6
71
6 7 , 000
8
30
11,500
6
72
38,500
Hi 500
38,000
8
6
6
44
01
So
8,500
4
99
1 1 , 500
8,500
23,000
8,000
4
5
7
5
42
49
90
56
12,500
7,500
12,000
5
5
5
20
3-
3-
10, 500
7,000
6
5
29
46
8, 000
5
53
6,000
5
44
7,000
5
92
8,000
7,000
5
5
56
4,000
4,500
9,500
4,000
7-500
2, 500
5
6
5
6
5
4
15
55
66
53
66
99
3>5°o
4
94
2-5
1-7
8.4
10.3
9.2
6.7
6.4
8.2
7-6
2.4
3-1
6.1
4.9
4-3
4.2
6.8
7-7
1-3
4.6
1-3
5-7
7-1
8.1
10. 4
5-3
3-8
5-1
15-3
3-6
3-7
20. o
22. o
2- 1
10.3
11. 9
10.5
14.8
6-5
6-5
11. o
9.6
9.4
10- 1
15- 1
13-4
6.2
8.7
4.6
II. I
12.4
15.2
864 Journal of Agricultural Research voi.xx, No.n
Of the 34 soils represented in Table III, 13 required less calcium
hydroxid for the titration of the subsoil than for the surface soil. Eight
of these 13 soils have over three times as much calcium in the subsoil
as in the surface soil. From the figures presented in Tables I and II,
it is shown that when a soil has large amounts of calcium, especially in
the carbonate form, the amount of calcium hydroxid used to bring to a
certain reaction was less than when the calcium content was smaller.
That is shown by comparisons of the groups. A large amount of calcium
has a greater influence than a larger amount of clay.
The result on soil No. 1273 can be explained by the lesser clay content
of the subsoil, as was pointed out in a preceding paragraph.
The following four soils have only slightly more calcium in the subsoil,
and yet they require less calcium hydroxid for the subsoil than for the
surface soil:
1 199.3, Laurel very fine sandy loam.
1 162.3, Pratt fine sandy loam.
1 12 1. 3, Finney clay.
1287.3, Summit silty clay loam.
The results on the first two may be explained by the lesser clay con-
tent of the subsoil. Finney clay 11 21.3 is an abnormal soil. The
sample was taken from the edge of a buffalo wallow. The probability
is that the surface soil had more colloidal clay than the subsoil.
Sample 1687 must be an exception; no explanation is apparant.
The foregoing presentation shows that most subsoils have a greater
calcium content than the surface soil and also that the subsoils require
a larger amount of calcium hydroxid to bring to the same reaction as the
surface soil. This must be due to the absorptive power of the colloidal
clay. It can not be due to a larger acid content or to a deficiency of
basic elements. The larger content of calcium should neutralize the
acidity, and since the calcium content is larger in the subsoil than in
the surface soil in can not very well be said than the subsoil is more de-
ficient in lime.
The initial reaction of a soil is not necessarily an indication of the
amount of calcium hydroxid required to titrate to a given hydroxyl-ion
concentration. In Table II the results are arranged according to the
decreasing hydroxyl-ion concentration of the soil before titration. If
these figures are studied in comparison with the figures in Table I it is
found that the amounts used to titrate do not correspond to the initial
reaction nor to the content of calcium except in the following general way.
The soils placed in group I have a larger calcium content than the soils in
group II, and those in group II have a larger calcium content than those in
group III. The quantities of calcium hydroxid used in titration are
greater for soils in group III than for soils in group II, and greater for
those in group II than for those in group I. But for individual soils
this comparison does not hold.
Mar. i, 1921 Relation of Calcium Content to Soil Reaction
865
The total acidity in the soil was mentioned in a preceding paragraph
as the total quantity of hydrogen ions which may be produced when
the equilibrium is continually shifted by the introduction of hydroxyl ions.
On such a basis it is possible to calculate the amount of lime required
to satisfy this acidity as measured by the electrometric titration. It was
also shown in a preceding paragraph that 1 cc. of N/25 calcium hydroxid
used in titrating 10 gm. of soil is equivalent to 400 pounds of calcium
carbonate per acre. Table IV has been prepared by using this factor
and the titration figures from Table II. In the last column of Table
IV are given the figures of the lime requirement of these soils as deter-
mined by the Hopkins method. It is at once seen that there is no close
agreement in the figures obtained by the two methods. This does not
necessarily argue for the greater practical value of the figures obtained
by the electrometric method nor against the Hopkins method. Similar
disagreements can be found if other well-known acidity methods are
compared. The figures presented in Table III make it appear that
some of the calcium hydroxid is taken up by colloidal clay. Just how
much this amounts to is not known, nor the manner. This forms part
of an investigation now going on at this laboratory.
Methods do not show any agreement as to the amount of calcium car-
bonate that should be added to an acid soil. Hopkins (7) states that 10
tons of limestone per acre on some soils is not too large a quantity.
The figures in Table IV show that the amounts of lime required to change
from a more acid reaction than denoted by PH 7 to neutral, or PH 7, is
not in general larger than the figures obtained by the Hopkins method,
though there is no agreement between individual samples. The amounts
required to change from the initial reaction to that denoted by PH 8.3
are not far from the amounts recommended for use on acid soils, and
the amounts required to bring the reaction to PH 10 are in all cases
less than 10 tons per acre.
Table IV. — Electrometric measurements in equivalents of CaC03 per acre in o to 7 inches
of the surf ace soil in comparison with amount of CaC03 required by the Hopkins method0
GROUP I, SOILS WHOSE ALKALINITY WAS ABOVE PH 8.3
Soil
No.
County.
Soil type.
Initial
Ph.
Pounds per acre of CaC03
equivalent to titration of —
Ph 7.
Ph3-
Ph 10.
8.61
8.56
8.52
8.46
8.44
8.44
8.40
8-37
280
2, 160
1.480
1,000
4,920
1,760
1,600
2,840
Pounds
per acre
of CaC03
required
by the
Hopkins
method.
1 169
1227
1043
1297
1 199
1119
1206
Reno
Greenwood . .
Russell
Montgomery
Jewell
Finney
Jewell
Reno
Pratt loamy fine sand
Crawford clay
Benton loam
Oswego silt loam
Laurel very fine sandy loam .
Dune sand
Lincoln silty clay loam
Kirkland clay
Alkali.
' Alkali'.
a It is assumed that i cc. of iV'25 Ca(OH)2 is equivalent to 400 pounds CaC03 per acre.
866
Journal of Agricultural Research
Vol. XX, No. ii
TablE IV. — Electrometric measurements in equivalents of CaC03 per acre in o to y inches
of the surface soil in comparison with amount of CaC03 required by the Hopkins method —
Continued
SOILS WHOSE REACTION WAS BETWEEN Ph 7 AND PH 8.3
Soil
No.
County.
Soil type.
Initial
Ph.
Pounds per acre of CaCOi
equivalent to titration of —
Ph7-
Ph3-
Pounds
per acre
of CaCOs
required
by the
Hopkins
method.
10S5
I°J9
1219
1120
1047
1132
1268
1267
1072
1160
"57
1070
"31
1121
1162
Riley
Russell
Greenwood..
Finney
Brown
Sho.wnee. . . .
Montgomery
do
Harper
Reno
do
.. ..do
Shawnee. . . .
Finney ....
Reno
Laurel silt loam
Summit silty clay loam
Osage loam
Sandy loam
Silty loam bottom
Osage silty clay loam
Osage clay (alfalfa land)
Osage silty clay loam
Brown loam
Arkansas fine sand
Arkansas clay loam
Pratt loam
Osage very fine sandy loam. .
Finney clay
Pratt fine sandy loam
8.16
8.03
7.81
600
1, 200
800
200
1,080
1,160
1, 200
1.400
560
1,520
2, 120
800
680
2. 760
3' 160
5,280
4.040
2, 720
400
3i48o
3-36o
7,640
3,320
I,4O0
4, 160
5,200
1.840
2.040
6,440
340
340
SOILS WHOSE ALKALINITY WAS BELOW Pn
1287
1 143
II9I
IIOO
1243
1230
1058
1 148
1266
1271
1284
1141
"35
1232
1136
1224
1256
1279
1265
1277
1280
1273
1257
1293
1275
1233
1244
1239
Montgomery
Shawnee ....
Jewell
....do
Cherokee
....do
Doniphan . .
Reno
Montgomery
....do
....do
Shawnee ....
....do
Cherokee
Shawnee ....
Greenwood..
....do
Montgomery
....do
....do
....do
do
Greenwood .
Montgomery
do
Cherokee. . . .
do
do
Summit silty clay loam ....
Boone fine sandy loam
Colby silt loam
Jewell silt loam
Cherokee silt loam
Neosho silt loam
Brown silt loam
Dune sand
Bates very fine sandy loam
Bates loam
Crawford loam
Summit silty clay loam ....
Crawford silty clay loam . .
Bates silt loam
Oswego silt loam
Summit stony loam
Boone fine sandy loam
Bates very fine sand
Cherokee silt loam
Bates shale loam
Bates loam
Bates very fine sandy loam
Summit silty clay loam. . . .
Bates stony loam
Oswego silt loam
Oswego silty clay loam. . . .
Bates fine sandy loam
Summit silt loam
6.77
1,000
2, 120
4, 560
6.76
320
3, 600
7,880
6. 72
480
1,480
5,200
6- 71
320
1,440
3, 160
6-55
520
2,480
5,640
6-53
520
1,840
3-56o
6- S3
520
2,040
4.080
6.46
240
920
1,680
6.36
1,040
2,080
4,600
6.29
960
2,600
8,080
6.25
200
720
1,880
6.15
2,680
7,000
1 2 , 560
6.01
3,36o
8,000
14,080
5-92
I, 720
3,76o
6,440
5-84
2,920
7. 160
12,240
5.82
2,400
5,240
9, 200
5-72
3.640
6, 120
11,840
5- TO
1,040
2,400
4,680
5-56
2,680
4, 120
9, 280
5-56
2, 720
6.040
II, 720
5-54
I, 240
2,400
5- 400
5-53
2,440
4,400
9,640
5-49
3,680
8,800
14. 120
5-49
1,240
1,800
3.640
5-35
3,280
4. 200
7, 520
4.99
3,280
5, 720
10. 0S0
4-99
2,840
4,960
9,600
4.68
3-920
7. 720
12.400
580
340
340
Alkali.
440
680
2,380
1, 700
,380
270
,020
40&
SUMMARY
(1) A number of soils from different parts of Kansas were analyzed
for total calcium, calcium in forms soluble in Nl$ hydrochloric acid,
and in N/i hydrochloric acid. The amount of carbon dioxid in these
soils was also determined.
(2) Ten-gm. samples of these soils were placed in 100 cc neutral
distilled water, and the initial reaction was determined by means of the
Mar. i, 1921 Relation of Calcium Content to Soil Reaction 867
hydrogen electrode. N/3 calcium hydroxid was added to change the
reaction to a higher alkalinity. The points determined were the num-
ber of cubic centimeters of Nj 23 calcium hydroxid needed to bring the
reaction (if lower) to PH 7, PH 8.3, and PH 10.
(3) In soils of a high calcium content, a larger percentage of the cal-
cium is in forms soluble in these dilute hydrochloric acid solutions than
in soils of a low calcium content.
(4) As a rule, soils of a high calcium content have a higher initial
hydroxyl-ion concentration than soils of low calcium content.
(5) The amount of N/23 calcium hydroxid required to change a soil
from a lower to a higher hydroxyl-ion concentration depends more upon
the amount of colloidal clay present than upon the calcium content.
(6) Subsoils, as a rule, have a higher calcium content than surface
soils. It required more calcium hydroxid to change these subsoils from
a lower to a higher hydroxyl-ion concentration than it did for the cor-
responding surface soils. This was true for most of the soils. The
exceptions were due either to a very high calcium content in the sub-
soil as compared with the surface soil, or to a larger amount of sand in
the subsoil, or to some unusual condition of the soil and subsoil.
(7) The amount of N/23 calcium hydroxid required to change the
acid soils to a reaction represented by PH 7, calculated in equivalent
pounds of calcium corbonate per acre, compares favorably with some
other current methods of determining the lime requirements of the soil.
(8) In some soils the amount of calcium hydroxid, calculated in
equivalents of pounds of calcium carbonate per acre, required to change
to a concentration represented by PH 8.3 is as great as the equivalent
amount of acid-soluble calcium present in the soil, or greater.
LITERATURE CITED
(1) Call, L. E., Throckmorton, R. I., and Swanson, C. O.
1914. soil survey op shawnee county, Kansas. Kans. Agr. Exp. Sta. Bui.
200, p. 717-749, map. (In cooperation with Bur. Soils, U. S. Dept.
Agr.)
(2)
1915. soil survey of rEno county, Kansas. Kans. Agr. Exp. Sta. Bui. 208,
48 p., map. (In cooperation with Bur. Soils, U. S. Dept. Agr.)
1915. soil survey of Cherokee county, Kansas. Kans. Agr. Exp. Sta.
Bui. 207, 46 p., map. (In cooperation with Bur. Soils, U. S. Dept.
Agr.)
(3)
(4)-
1916. soil survey of jewell county, Kansas. Kans. Agr. Exp. Sta. Bui.
2ii, 36 p., map. (In cooperation with Bur. Soils, U. S. Dept. Agr.)
(5) Htlgard, E. W.
1907. soils, xxvii, 593 p., 89 fig. New York, London.
(6) Hoagland, D. R., and Sharp, L. T.
1918. relation of carbon dioxid to soil reaction as measured by the
hydrogen electrode. In Jour. Agr. Research, v. 12, no. 3, p. 139-
148. Literature cited, p. 147-148.
868 Journal of Agricultural Research voi.xx,No.«
(7) Hopkins, Cyril G.
[iQIO.] SOIL FERTILITY AND PERMANENT AGRICULTURE. xxiii, 653 p., ilhlS.,
maps, ports. Boston, London, etc.
(8) Lyon, T. Lyttleton, and Bizzell, James A.
1018. lysimeter experiments. N. Y. Cornell Agr. Exp. Sta. Mem. 12, 115
p., 4 pi. Bibliography, p. 82-84.
(9) Plummer, J. K.
1918. studies in soil reaction as indicated by the hydrogen electrode.
In Jour. Agr. Research, v. 12, no. 1, p. 19-31. Literature cited, p.
(10) Sharp, L. T., and Hoagland, D. R.
1916. ACIDITY AND ABSORPTION IN SOILS AS MEASURED BY THE HYDROGEN
Electrode. In Jour. Agr. Research, v. 7, no. 3, p. 123-145, 1 fig.
Literature cited, p. 143-145.
(11) Swanson, C. O.
1914. chemical analyses of some Kansas soils. Kans. Agr. Exp. Sta. Bui.
199, p. 633-715.
(12) and Tague, E. L.
1918. chemistry of sweet-clover silage in comparison with alfalfa
silage. In Jour. Agr. Research, v. 15, no. 2, p. 1 13-132, 5 fig.
(13)
1919. DETERMINATION OF ACIDITY AND TITRABLE NITROGEN IN WHEAT WITH
THE hydrogen Electrode. In Jour. Agr. Research, v. 16, no. 1,
p. 1-13, 5 fig.
(14) Veitch, F. P.
1902. THE ESTIMATION OF SOIL ACIDITY AND THE LIME REQUIREMENTS OF SOILS.
In Jour. Amer. Chem. Soc, v. 24, no. 11, p. 1120-1128.
(15) Wiley, H. W.,etal.
1908. OFFICIAL AND PROVISIONAL METHODS OF ANALYSIS, ASSOCIATION OF
OFFICIAL AGRICULTURAL CHEMISTS. AS COMPILED BY THE COMMITTEE
on revision of methods. U. S. Dept. Agr. Bur. Chem. Bui. 107
(rev.), 272 p., 13 fig.
GREEN FEED VERSUS ANTISEPTICS AS A PREVENTIVE
OF INTESTINAL DISORDERS OF GROWING CHICKS
By A. G. Philips, Chief in Poultry Husbandry, R. H. Carr, Associate in Nutrition,
and D. C. Kennard, Assistant in Poultry Husbandry, Purdue University Agricul-
tural Experiment Station
The problem of raising chicks in confinement has engaged the attention
of many nutrition investigators for years. The difficulties encountered
have been attributed to various causes, such as lack of vitamines in the
feed, lack of exercise, and intestinal putrefaction. Whatever the causes
may be it is recognized that they have proved a serious handicap in
making use of the chick in nutrition work. The critical time in the life
of a chick is between the ages of 8 and 12 weeks. During this period
by far the greater mortality occurs when they are kept in confinement,
and this is a most serious objection to their use in nutrition investiga-
tion. Drummond 1 has made some study of the growth of chicks in
confinement and concludes that it is impossible to grow them success-
fully even when the feed is known to be suitable for growth. Osborne
and Mendel 2 also report difficulty in raising chicks in confinement and
have found the use of paper pulp to aid somewhat in lessening mortality.
Hart and his associates 3 report difficulty in growing young chicks in
confinement but have found no trouble in using birds weighing 3 or 4
pounds. The authors 4 have reported some success in raisdng chicks in
confinement, but at that time it was thought the fair growth obtained
was due to the green feed given in the ration. However, better results
have since been secured without any green feed in the ration. The green
feed was thougth to give the necessary succulence and add the vitamines
needed for growth; but later experience does not indicate this to be
true. The question now arises in the minds of the writers as to whether
greens are necessary in the ration of a young growing chick. In three
years' work with growing chicks in confinement there was no extra gain
in weight or decreased mortality where sprouted oats were fed, over that
of the control pens ; in fact the chicks receiving greens were less vigorous
than those in the other lots. It may be noted in this connection at Pur-
due University that in eight years of feeding 2 -year-old steers in pre-
paring them for the market there was no advantage gained, so far as the
1 Drummond, Jack Cecil, observations upon the growth of young chickens under laboratory
conditions. In Biochem. Jour., v. 10, no. i, p. 77-88, 1 pi. 1916.
2 Osborne, Thomas B., and Mendel, Lafayette B. the growth of chickens in confinement. In
Jour. Biol. Chem., v. 33, no. 3, p. 433-438, pi. 4-6. 1918.
3 Hart, E. B., Halpin, J. G., and McCollum, E. V. the behavior of chickens fed rations re-
stricted to the cereal grains. In Jour. Biol. Chem., v. 29, no. 1, p. 59- 1917-
4 Philips, A. G., Carr, R. H., and Kennard, D. C. meat scraps versus soybean proteins as a
supplement to corn for growing chicks. In Jour. Agr. Research, v. iS, no. 7, p. 391-398. pi. s°- 1920.
Journal of Agricultural Research, Vol. XX, No. n
Washington, D. C. Mar. 1, 1921
xc Key No. Ind.-8
(869)
870 Journal of Agricultural Research voi.xx, no. h
average daily gain or selling price was concerned, by those steers receiving
varying amounts of silage over those receiving only dry feed, except that
the gains were made in the former case at a slightly reduced cost as
compared with the latter, due largely to the fact that silage is cheaper
than clover hay.
OBJECT OF THIS INVESTIGATION
Since sprouted oats seemed to be inefficient in preventing chick mor-
tality, an attempt was made during the year 191 9 to find some means of
checking intestinal putrefaction, which postmortem examinations have
shown to be the principal cause of mortality. Accordingly it was de-
cided to try a series of different compounds which might be expected to
have an antiseptic effect or might serve to prevent impaction by reason
of their bulk.
THE EXPERIMENT
The stock used was 160 White Leghorn day-old chicks, which were
divided into 10 lots of 16 chicks each. Every precaution was exercised
to distribute the chicks so that they would be uniform in all lots. During
the first four days the chicks in all lots were given water and granulated
corn and had access to sand. Thereafter they were given their respective
rations. At this time each bird was leg-banded and its weight was re-
corded. They were weighed individually at the end of each 14 days
thereafter until the close of the experiment, at the end of 14 weeks. The
weight of feed consumed by each lot was recorded each time the chicks
were weighed.
The basal ration used was one which had proved most satisfactory dur-
ing the past two years of feeding trials, including two different experi-
ments— one with White Leghorns and the other with White Plymouth
Rock chicks. All lots received the basal ration consisting of 50 parts
cracked corn, 35 parts corn meal, 15 parts corn bran, 3 parts ash, 8.86
parts meat scrap, and 10.9 parts soybean meal (all parts by weight), and
were provided with 1 inch of sand on the floor. In addition to this, some
other factor was included in all lots, except in lot No. 1 which was the
control pen. Lot No. 2 was provided with oat straw litter to note what
effect the increased exercise or consumption of straw would have. Lot
No. 3 was fed like No. 2, except that it received green feed in the form of
tops of sprouted oats. The care of this lot represented the management
usually given brooder chicks, since it provided a well-balanced ration and
in addition supplied scratching litter and green feed. The exception to
the usual brooder practice was that the birds were kept in confinement.
Lots 3, 11, 13, and 14 are the lots reported in the tables as receiving green
feed.
The idea has been advanced by some that the benefit of the scratching
litter was derived not from the exercise it promoted but from the large
quantities of the litter that were consumed by the birds, providing an
Mar. i, 1921 Green Feed versus A ntiseptics for Growing Chicks 871
abundance of fiber which is considered so beneficial in the digestive tract.
In view of this possibility, lots 4 and 5 were fed straw pulp. The only
difference in the treatment of these two lots was that No. 4 received but
one-half as much of the straw pulp as did No. 5. This pulp was prepared
by taking strawboard (made of straw) and reducing it to a pulp with
water. This pulp, after most of the water was expelled, was mixed with
the dry mash. This was bulky, especially the mixture fed lot 5. The
actual dry-weight consumption of paper was approximately 2}4 and 5 per
cent of the ration for lots 4 and 5, respectively. This pulp was palatable
when mixed in the feed, and the chicks would eat it fairly well.
Lot 6 received the basal ration with hydrochloric acid added to the
drinking water at the rate of 1 part 36 per cent hydrochloric acid (HC1)
to 500 parts of drinking water. This is sometimes recommended as a
substitute for buttermilk for use as a preventive or corrective of black-
head in turkeys and of bacillary white diarrhea and coccidosis in chickens.
Tobacco dust, a by-product of tobacco manufacturing and a valuable
remedy against intestinal parasites, was given to lot No. 7 at the rate of
2 parts added to the basal ration. In like maner lot No. 8 received 2
parts of sulphur, and No. 9 received 6 parts of lactose. Lot No. 10 re-
ceived the basal ration with copper sulphate added to the drinking water
at the rate of 1 part copper sulphate crystals (CuS04) to 1 ,400 parts
of water.
The mortality records and weights for the different lots are given in
Table I.
Table I. — Weight and mortality of chicks
Lot
No.
Basal only
Basal -(-straw
Basal -j- straw and greens
Basal-j-2^2 per cent straw pulp
(No. 1)
Basal +5 per cent straw pulp
(No. 2)
Basal+HCl
Basal -j- tobacco
Basal -(-sulphur
Basal -(-lactose
Basal +CuS04
Basal -f greens a
Basal+greens &
Basal -j- no greens c
Basal+greens d
Age, 8 weeks.
Weight. Mortality.
257
252
257
247
252
305
223
305
295
306
243
186
225
20=<
Age, 14 weeks.
Weight. Mortality.
Gm.
644
475
524
630
638
639
535
605
617
653
384
360
486
458
a Experiment No. I (1918), White Leghorns, fed same ration as Lot No. 3.
b Experiment No. II (1919), White Plymouth Rocks, fed same ration as lot No. 3.
c Experiment No. II (1919), White Plymouth Rocks, no greens; basal ration containing 10 parts of pro-
tein from meat scraps only, instead of meat scraps and soybean meal,
d Experiment No. II (1919), White Plymouth Rocks fed same as lot No. 13 with addition of greens.
872
Journal of Agricultural Research
Vol. XX, No. ii
FECES NITROGEN
A study of the nitrogen of the feces was made to note if any increased
utilization or change in the nature of the nitrogen end products could be
obtained because of the added compounds. The data from composite
samples of the feces taken from the different lots are contained in
Table II.
Table II.- — Amount and distribution of feces nitrogen
Lot No.
Average
protein
consumed
per chick in
14 days.
Percentage
of total
nitrogen.
Percentage
of nitrogen
soluble in
AT/70 HC1."
Percentage
of nitrogen
insoluble in
Njw HC1.&
Percentage
of soluble
nitrogen in
total
nitrogen.
I
Gm.
58-5
56.78
68. 11
67.08
73-63
67.23
61.77
73-78
68. 17
68.32
2. 23
Lost.
2. 20
2. 02
1.77
I.83
i-35
2.36
2. 27
2. 18
O.94
I. 29
42. I
■2
.91
•94
.89
I- 15
•85
I.05
I. 02
I. IO
I. 20
I.08
.88
.68
•5°
1.08
41. 4
46.5
5°-3
62.8
c
6
7
63. O
8
44- 5
44- 9
5°- 5
" Urea, ammonia, and amino acid nitrogen.
b Uric acid and residual nitrogen.
DISCUSSION
Table I gives the results of the different rations outlined, including
such factors as green feed, antiseptics, fiber, exercise, and their effect
upon and mortality of the chicks. When the gain in weight and mor-
tality of the different lots are considered, a few points stand out promi-
nently and are suggestive as being worthy of further investigation.
The most important of these is the effectiveness of copper sulphate in
preventing mortality, probably because of its well-known antiseptic
properties. Since an antiseptic seems to be so effective, it adds addi-
tional evidence that one of the main causes of mortality of chicks grown
in confinement is the intestinal putrefaction so often noticed in the
autopsy of chicks. Sprouted oats is thought by some to be effective in
lessening mortality, especially when fed for a short time only and when
given as a supplement to a somewhat monotonous ration. It is possible
that under the conditions of the experiment no benefit was obtained from
its use with growing chicks when fed throughout the first 14 weeks of
the growing period. Lots No. 11, 12, 13, and 14 noted in Table I include
unpublished data obtained in previous experiments which are introduced
here as further evidence of the ineffectiveness of greens in preventing
chick mortality.
The sulphur received by lot 8 caused a continued looseness of bowels.
This did not seem to have any ill effect and may have been of some
advantage, since at 8 weeks of age this was one of the best lots. The
retarding effect of tobacco was pronounced and resulted in stunting the
growth during the first 8 weeks. There was a tendency for the chicks to
Mar. i, 1921 Green Feed versus Antiseptics for Growing Chicks 873
recover somewhat by the age of 14 weeks. The chicks in this lot always
seemed more wild and nervous than those of any of the other lots.
The use of hydrochloric acid in the drinking water of lot 6 seemed to
be of some benefit, inasmuch as the mortality was somewhat less than
the average and the growth was consistent throughout the experiment.
Strawboard pulp was supplied to the ration in lots 4 and 5 for the pur-
pose of adding bulk and thereby lessening the danger of impaction of the
contents of the small intestine and caeca common when feeding a grain
ration. It did not seem to aid in reducing mortality.
Lot 2 was given a litter of oat straw to encourage the chicks to exercise.
This did not prove successful in promoting growth, since this lot made
the smallest gain of all, nor did it tend to lessen mortality. Lot 1 , which
was the control lot, received only the basal ration. As shown in Table I,
this ration has proved its efficiency in promoting growth and has also
proved its inefficiency in checking mortality, especially during the time
between the eighth and fourteenth weeks.
It will be noted from Table II that in lot 6 and also in lot 7, which
received tobacco, the percentage of nitrogen in the feces was lower than
in most of the othei lots. Furthermore, it was found that the percentage
of nitrogen excreted as uric acid was less, indicating a somewhat greater
percentage of utilization of the nitrogen in the feed.
Lactose, which was added to the ration of lot 9 did not seem to aid in
lessening mortality or in promoting growth. This may be due to the
fact, as stated by Mendel and Mitchell,1 that birds, unlike mammals, have
no sugar-splitting enzyms in the small intestine; hence the sugar fed
was not converted into lactic acid to any considerable extent and thus
did not aid in checking intestinal putrefaction. This view is further
substantiated in the production of the usual amount of uric acid in the
feces, since otherwise nitrogen appearing as uric acid would probably have
appeared as a soluble ammonium salt, as noted in lot 6 in Table II, where
hydrochloric acid was used in the drinking water.
SUMMARY
(1) The tops of sprouted oats seem to be useless as a preventive of
digestive disorders or as an aid to the growth of chicks in confinement.
(2) The analysis of the feces indicated that chicks given hydrochloric
acid and tobacco powder produced less uric acid in their feces than did
the other lots.
(3) Tobacco powder added to the ration of growing chicks prevents
their normal growth and causes them to be wild and nervous.
(4) Hydrochloric acid, sulphur, and particularly copper sulphate offer
interesting possibilities of success in raising chicks in confinement.
1 Mendel, Lafayette B., and Mitchell, Philip H. chemical studies on growth- -I. the inverting
ENZYMES OF THE ALIMENTARY TRACT, ESPECIALLY IN THE EMBRYO, In Amer. Jour. Physiol., V. 20, no. I,
p. 81-96. 1907. Bibliography, p. 94-96.
29666°— 21 7
COMPARATIVE UTILIZATION OF THE MINERAL CON-
STITUENTS IN THE COTYLEDONS OF BEAN SEEDLINGS
GROWN IN SOIL AND IN DISTILLED WATER
By G. Davis Buckner l
Chemist, Kentucky Agricultural Experiment Station
The experiments of Schroder on the distribution of organic and mineral
constituents in seedlings of the kidney bean, Phaseolus vulgaris, pub-
lished in 1868 2 show that, in his fourth stage of germinating, when the
second and third joints with the trifoliate leaves have formed, the coty-
ledons, which have become much reduced in size and more or less shriv-
eled, still retain a considerable proportion of their mineral matter unused.
Schroder's analyses show that these shriveled cotyledons retain about
nine-tenths of their original calcium, whereas not more than one-fourth
of their phosphorus and about two-fifths of their potassium, sodium,
and magnesium remain. In regard to the calcium, however, Scroder
points out that his determinations appear to be too high and that this
result should be verified. In describing Schroder's experiments Pfeffer 3
remarks that —
complete removal of all of the essential elements is never possible, for even in a
starved plant, certain essential structural constituents can not be mobilized or
consumed.
In 1915, the author of this paper published some results 4 showing that
when the Kentucky Wonder garden bean was grown in distilled water,
approximately 86 per cent of the calcium, 50 per cent of the phosphorus,
and 40 per cent of the magnesium remained unused in the cotyledons as
compared with the amounts found in the normal cotyledons. In this
experiment the seedlings had been permitted to grow in distilled water
until they became etiolated and died from lack of food. These figures
approximate those given by Schroder.
The following experiment was undertaken with the view of comparing
the degree of utilization of the total ash and the elements calcium, mag-
nesium, and phosphorus in the cotyledons of bean seedlings grown in
distilled water and in garden soil.
In starting the experiment it seemed of primary importance to deter-
mine the distribution of the total ash and the elements calcium, magne-
sium, and phosphorus, which were to be studied, in the separate portions
1 The author gratefully acknowledges Dr. A. M. Peter's careful criticism of this manuscript.
2 Schroder, Julius, untersuchung Cber die vertheilung des stickstoffs und der mineral-
best andtheilE bei keimung der schmtnkbohne. In Landw. Vers. Stat., Bd. 10, p. 493-510. 1868.
3 Pfeffer, W. the physiology of plants . . . ed. 2, transl. and ed. by Alfred J. Ewart. v.
1, p. 584. Oxford, 1900.
4 Buckner, G. Davis, translocation of mineral constituents of seeds and tubers of certain
plants during growth. In Jour. Agr. Research, v. 5, no. n, p. 449-458. 1915.
Journal of Agricultural Research, Vol. XX, No. n
Washington, D. C Mar. 1, 1921
xd Key No. Ky.-io
(875)
876 Journal of Agricultural Research voi.xx,No. u
of the bean under consideration. Since Schroder used the kidney bean,
Phaseolus vulgaris, it was decided to use a kidney bean in this experiment,
in order to obtain more comparable results. The Kentucky Wonder
garden bean is a good example of this type, and, since it is well adapted
to this climate, it was chosen.
About 3,000 perfect beans were selected and, after thorough washing,
were allowed to soak in distilled water overnight, until the integuments
were softened. From 1,000 of these beans the integuments were care-
fully removed and saved as a separate portion. The cotyledons were
then carefully separated, and the embryos were dissected out. The 1,000
embryos and 200 of the cotyledons were separately analyzed, as were 400
integuments and 100 of the whole beans remaining. During these oper-
ations, care was taken that the separate portions did not become contam-
inated with dust or other foreign material. The materials were dried in
an electric oven at ioo° C. for 24 hours, after which they were weighed,
ashed, and the phosphorus was determined by the method of the Associ-
ation of the Official Agricultural Chemists,1 while calcium and magnesium
were determined according to the method of McCrudden.2 All the anal-
yses made during the progress of this experiment were similar in every
respect. The results are stated in Table I, calculated for 1 ,000 beans and
also as percentage of the moisture-free materials.
In determining the degree of utilization of the elements in question in
the cotyledons of beans grown under normal conditions in garden soil, 500
carefully selected beans \vere planted in a box of garden soil in a room
which received the proper amount of sunshine and ventilation. In this
room, also, the seedlings in distilled water were grown. Since the room
was used only for this purpose, the chance of contamination from dust
during the growth of the seedlings was very small. When the. bean seed-
lings had pushed the cotyledons well above the soil, the cotyledons were
carefully washed with distilled water and a camel's-hair brush to remove
any adhering soil. At all other times the watering was done from below,
so that no water touched the cotyledons. As growth advanced, the coty-
ledons became greatly shriveled and turned brown and finally dropped off
upon clean paper so placed as to keep them from falling on the soil. They
were then analyzed and calculated according to the method described.
The results will be found in Table I.
In that part of the experiment in which the seedlings were to be
grown in distilled water, 1,000 beans from a new lot of the same variety
(the first lot having been all used) were selected and sterilized by placing
them in an atmosphere of formaldehyde gas for four hours, after which
1 Wiley, H. W., et al. official and provisional methods of analysis, association of official
AGRICULTURAL CHEMISTS. AS COMPILED BY THE COMMITTEE ON REVISION OF METHODS. U. S. Dept. A^r.
Bur. Chem. Bui. 107 (rev.), p. 3. 1908.
2 McCrudden, F. H. the quantitative separation of calcium and magnesium in the presence
of phosphates and small amounts of iron devised especially for the analysis of foods, urine and
feces. In Jour. Biol. Chem., v. 7, no. 2, p. 83-100. 1910.
Mar. i, 1921 Utilization of Cotyledons of Bean Seedlings 877
they were washed with sterile, distilled water and germinated between
blotting papers which had been treated with hydrochloric acid and washed
free from chlorids with distilled water. The germinating dish was of
porcelain and was sterilized by heating at 1800 C. for two hours. The
beans were allowed to germinate until the radicles were 1 cm. in length,
when the integuments were removed and the radicles wrapped in sterile
absorbent cotton which had previously been treated with hydrochloric
acid and washed free from chlorids with distilled water. This cotton
gave practically no ash when incinerated. After the radicles had been
wrapped in the absorbent cotton, each bean thus prepared was placed
in the mouth of a test tube which had been thoroughly coated inside with
paraffin and was held there by applying a few drops of melted paraffin.
The test tubes were thoroughly washed with distilled water before the
distilled water in which the seedlings grew was placed in them. This
water was replaced as rapidly as it was removed by evaporation and by
transpiration. The bean seedlings were allowed to grow until they had
etiolated and wilted. The seedlings thus formed were uniform in size
and development, being about 7 inches in height, with a well-developed
root system and having two perfectly formed leaves which were somewhat
undersized. The etiolation of the leaves and cessation of growth was
taken as a point of maturity at which the cotyledons were removed, in
a brown and greatly shriveled condition. They were analyzed as already
described, and the results are presented in Table I. Inasmuch as a new
lot of Kentucky Wonder beans was used for this part of the experiment,
200 normal cotyledons from beans of this lot were analyzed and the results
included in the table for comparison.
878
Journal of Agricultural Research
Vol. XX, No. 11
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Utilization of Cotyledons of Bean Seedlings
879
In Table II will be seen the percentage distribution of phosphorus, cal-
cium, and magnesium in the ash of the separate parts of the beans anal-
yzed. Here we see that the percentages of phosphorus in the normal
cotyledons and those exhausted under the given conditions are fairly
constant, ranging from 32.62 in the exhausted cotyledons grown in soil
to 36.66 in the normal whole cotyledons of the same lot. The percentage
of phosphorus in the ash of the exhausted cotyledons of beans grown in
distilled water very closely approximates that in the ash of the normal
cotyledons of the same lot being 35.18 and 34.22, respectively.
Table II. — Analyses of the ash of beans and of the several parts
Material analyzed.
Normal cotyledons
Embryos
Integuments
Normal whole beans
Exhausted cotyledons (grown in soil). .. .
Exhausted cotyledons (grown in water)0.
Normal cotyledons"
Phosphorus
as PsOs.
Per cent.
36.66
44- 5°
3-44
28.33
32. 62
35-i8
34.22
Calcium as
CaO.
Per cent.
•47
•J7
•73
•32
•45
•45
■30
Magnesium
as MgO.
Per cent.
4.46
6.88
9.81
4-75
8.48
3.87
3- 27
a A different lot of Kentucky Wonder beans from those grown in soil.
In Table III will be seen the comparative amounts of dry matter, crude
ash, and the elements phosphorus, calcium, and magnesium used by the
seedlings grown in distilled water and those grown in garden soil. Here
we see that 92.3 per cent of the dry matter, 92 per cent of the total ash,
92.8 per cent of the phosphorus, 81.4 per cent of the calcium, and 84.9 per
cent of the magnesium of the cotyledons of beans grown in garden soil was
translocated to other parts of the plant before the cotyledons ceased to
function as a source of food supply. We see also that only 58.2 per cent
of the dry matter, 54.2 per cent of the total ash, 42.9 per cent of the phos-
phorus, 1 4. 1 per cent of the calcium, and 60 per cent of the magnesium in
the cotyledons was utilized by the seedlings grown in distilled water
cultures.
Table III.
-Comparison of percentages of material translocated from the cotyledons of
beans grown in distilled water and in soil
Material translocated.
Dry matter
Crude ash. .
Phosphorus
Calcium . . .
Magnesium
In soil
92.
3
92.
0
92.
8
8l.
4
84.
9
In distilled
water.
58.2
54-2
52-9
14. 1
60. o
880 Journal of Agricultural Research vol. xx, no. h
It is readily observed that considerably more of each of these elements
was utilized by the seedlings grown in garden soil than by those grown
in distilled water. This would seem to indicate either that the distilled
water is deleterious to the growth of seedlings grown in it or that some-
thing needed in the process of translocation was accessible when the
beans were grown in soil but not when they were grown in distilled water.
Distilled water even of the highest purity has been considered toxic
to seedlings grown in it, because of the difference between the osmotic
pressure within the root cells and that of the distilled water surrounding
them. The distilled water used in these experiments was obtained from
a Barnstead automatic water still and contained traces of copper and
calcium. In this case the toxic effect of the copper, if any could be
attributed to it, was counteracted by the calcium, as there was no
evidence of the characteristic poisonous effect of copper on the roots.
It is hoped that more light may be thrown on the subject of the utili-
zation of the mineral constituents in the cotyledons by the young plant
under varying conditions by experiments now in progress in this
laboratory.
SUMMARY
When beans were grown in soil, a notably larger amount of reserve
material was translocated from the cotyledons than when they were
grown in distilled water.
In both cases, a smaller proportion of calcium was translocated than
of phosphorus or magnesium.
SUNFLOWER SILAGE DIGESTION EXPERIMENT WITH
CATTLE AND SHEEP1
By Ray E. Neidig, Chemist, Robert S. Snyder, Associate Chemist, and C. W.
Hickman, Animal Husbandman, Idaho Agricultural Experiment Station
The object of the experiment reported in this article was to determine
the apparent digestibility 2 of silage made from sunflowers when fed to
cattle and sheep. Sunflowers have gained a wide reputation as a silage
crop in the Pacific Northwest, and much interest is being taken in their
growth on lands where corn can not be successfully grown. Sunflowers
are a hardier crop than corn, withstanding both drouth and frost to a
much greater degree. Another point in favor of sunflowers is the fact
that usually a greater tonnage can be secured in the semiarid regions.
Many claims are made concerning the high value of sunflower silage for
feeding purposes, but little is known at the present time as to its actual
value other than numerous practical feeding tests which indicate that
sunflowers are a very promising silage crop. Recently, however, the
Montana Agricultural Experiment Station has reported on the digestible
nutrients in sunflower silage made from a crop of sunflowers harvested
when the plants were approximately 5 per cent in bloom. While a full
report of the work has not been published, yet a summary of the digest-
ible nutrients found in 100 pounds of silage, together with the same
data on mature and immature corn, taken from Henry and Morrison's
"Feeds and Feeding" is given in Bulletin 131 as follows:
Digestible nutrients in 100 pounds of
sunflower silage
Digestible nutrients in 100 pounds of
mature corna
Digestible nutrients in 100 pounds silage
from immature corna
Total
dry sub-
stance.
Pounds.
21. 4
26.
Crude
protein.
Pounds.
I. 24
I. I
I. O
Crude
fiber and
nitrogen-
free
extract.
Pounds.
IO. 13
IS.OO
II. 40
Ether
extract.
Pounds.
o-37
.70
.40
Nutri-
tive
ratio.
9.8
IS- 1
12.3
a Henry, W. A., and Morrison, F. B.
1917.
FEEDS AND FEEDING
ed. 17, X, 691 p. Madison, Wis.,
From the digestible nutrients found in the sunflower silage and from
the practical feeding experiments carried on by the Montana Agricultural
Experiment Station, with dairy and beef cattle, ewes, and brood sows
they conclude that sunflowers are a valuable silage crop.
1 Published by the permission of Director E. J. Iddings, Idaho Agricultural Experiment Station, as
a joint project of the Department of Agricultural Chemistry and Animal Husbandry.
2 Throughout this article the coefficients of digestibility refer to the coefficients of apparent digestibil-
ity—that is, the difference in the weights of the nutrients of the silage fed and in the feces expressed in
percentages of the total nutrients eaten.
Journal of Agricultural Research,
Washington, D. C
xe
$1)
Vol. XX, No. 11
Mar. 1, 1921
Key No. Idaho-5
Journal of Agricultural Research vol. xx, No. n
During the past two years similar work has been carried out at the
Idaho Agricultural Experiment Station, a part of which is reported in
this article on the digestion experiments with cattle and sheep. The
silage used was made from a crop of sunflowers harvested when about
50 per cent of the sunflowers were in bloom, but when only a few seeds
were in the dough stage. The plan of the work and the data secured
follow.
PLAN OF EXPERIMENT
Three registered Shorthorn cows, No. 5, 6, and 7, were used in the
experiment. These were the only cows available at the time the experi-
ment was conducted. Their ages varied, cow No. 5 being 3 years old,
cow No. 6 being 10 years old, and cow No. 7 being 5 years old. These
cows were kept in specially prepared stalls, which were arranged so that
it was possible to obtain an exact record of all silage eaten, water con-
sumed, all silage rejected, and all feces voided. No record was kept of
the urine, either as to the amount voided or as to its chemical analysis.
Three yearling wethers, all pure-bred Shropshires, were placed in
specially constructed pens which facilitated the securing of records on
the amount of silage fed, silage eaten, water consumed, and feces voided.
The preliminary feeding period extended over a period of 10 days,
during which time the animals were given an opportunity to accustom
themselves to their surroundings, and also to ascertain the maximum
amount of silage that they would consume daily. It was found that 50
pounds was the proper amount to feed the cows, while 2 pounds were
sufficient for the daily sheep ration. The cows and sheep were fed one-
half the full ration both morning and evening. When the animals
appeared to be normal in every way a few days were allowed to elapse
and then the final digestion period of seven days' duration was begun.
During this period samples of the silage fed, silage rejected, and feces
voided were collected daily and composited. Daily records of the
amounts of silage fed, silage rejected, and feces voided were secured,
together with the daily weights of the animals. Chemical analyses were
made of all composite samples. The results are given on both the wet
and dry basis in Table I.
Table II contains the amount of silage fed to cows and sheep, the
water consumed, feces voided, silage rejected (called orts), and the daily
weight of each individual cow and sheep. Table III contains the total
weight of silage fed, the total nutrients contained in the silage eaten, and
the feces voided. The amount of nutrients and the percentage digested
are also given for each animal. In calculating the nutrients eaten, the
total nutrients contained in the silage rejected were subtracted from the
total nutrients contained in the silage fed. Hence the figures represent
the actual amount of dry substance and nutrients eaten. The results
are all expressed on the moisture-free basis.
Mar. i, 1921
Sun-flower Silage Digestion Experiment
883
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Vol. XX, No. ii
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Sunflower Silage Digestion Experiment
885
Table IV gives the apparent coefficients of digestibility for each
animal, together with the average coefficients for the three cows and
sheep, respectively.
Table V gives the pounds of digestible dry matter and pounds of
digestible nutrients in 100 pounds of sunflower silage. The individual
nutritive ratio for each animal is given, together with the average
nutritive ratio for the cows and sheep.
Table III. — Total weights of sunflower silage , feces , and water for the J-day period
[Results expressed in kilograms on moisture-free basis]
COW NO. 5
Dry
sub-
stance.
Crude
protein.
Crude
fiber.
Ether
extract.
Nitrogen-
free
extract.
Ash.
Silage fed minus orts
33- 096
16. 109
16. 987
51- 3°°
3-203
I.586
I. 617
5°- 5°o
9. 714
6. 293
3.421
35- 200
1.952
.472
I. 480
75. 800
14. 848
6. 206
8.642
58. 200
3-279
2.430
•849
25. 900
Feces voided
Amount digested
Percentage digested ....
COW NO. 6
Silage fed minus orts
Feces voided
Amount digested
Percentage digested
32- 731
3. 181
9-564
!-945
14. 799
15. 098
1. 660
5- 7oo
.482
7-511
17- 633
1. 521
3.864
1.463
7. 288
53- 900
47. 800
40. 400
75. 200
49. 200
3.242
2. 282
. 960
29. 600
COW NO. 7
Silage fed minus orts
Feces voided
Amount digested
Percentage digested
Silage fed minus orts
Feces voided
Amount digested
Percentage digested
Silage fed minus orts
Feces voided
Amount digested
Percentage digested
Silage fed minus orts
Feces voided
Amount digested
Percentage digested
33- 673
3. 229
10. 008
1. 960
15- l63
16. 893
1.776
6.342
.584
5.860
16. 780
1-453
3.666
1-376
9-303
49. 800
45. 000
36. 600
70. 200
61. 400
3-3^3
2. 511
.802
24. 200
SHEEP NO. 2
2.694
0.258
0. 801
o- 157
1-213
1. 144
. 122
•427
•036
•382
1-550
• 136
•374
. 121
.831
57- 500
52. 700
46. 700
77. 100
68. 500
o. 265
.177
.088
33- 200
SHEEP NO. 3
2.634
0-255
0. 772
0. 156
1. 190
1.286
. 127
•499
• 043
•439
1.348
. 128
•273
• "3
•75i
51. 200
50. 200
35- 4oo
72. 400
63. 100
SHEEP NO. 8
o. 261
.178
.083
31. 800
2. 664
O.256
O.787
0. 156
1. 202
.858
. 102
■331
. 027
.268
I.806
•154
.456
. 129
•934
67. 800
60. 200
57. 900
82. 700
77. 700
o. 263
• 130
• 133
50. 600
886
Journal of Agricultural Research
Vol. XX, No. ii
Table IV. — Coefficients of digestibility for cows and sheep
[Expressed in percentages]
Dry
sub-
stance.
Crude
protein.
Crude
fiber.
Ether
extract.
Nitrogen-
free
extract.
Ash.
Cow No. —
t
5x-3
53-9
49-8
5o. 5
47-8
45- 0
35-2
40. 4
36.6
75-8
75-2
70. 2
58. 2
49. 2
6l. 4
25- 9
29. 6
24. 2
6
7
Average for cows
5i-7
47-8
37-4
73-7
56- 3
26.6
Sheep No. —
2
57-5
67.8
52-7
50. 2
60. 2
46.7
35-4
57-9
77. 1
72.4
82. 7
68.5
63. 1
77-7
33-^
31-8
50.6
8
Average for sheep
58.8
54-4
46. 7
77-4
69.8
38.5
Table V. — Nutrients digested by cows and sheep in each 100 pounds sunflower silage
[Estimated on wet basis]
Dry
sub-
stance .
Crude
protein.
Crude
fiber.
Ether
extract.
Nitrogen-
free
extract.
Nutritive
ratio.
Cow No. —
c
Pounds.
IO. 9
"■45
10. 56
Pounds.
I.03
•97
.91
Pounds.
2. 22
2-55
2.31
Pounds.
o-93
•93
.86
Pounds.
5-56
4- 71
5-87
9.6
9.6
6
7
Average for cows
10.97
•97
2-34
.91
5-38
IO. I
Sheep No. —
2
12. 2
10.86
14.4
I. 07
1. 02
1. 22
2.94
2. 23
3-65
o-95
.89
1. 02
6-55
6.03
7-43
10. 9
7.
8
10. 9
Average for sheep
12.49
1. 1
2. 94
•95
6.68
10. 6
INDIVIDUALITY OF COWS AND SHEEP AS TO THE AMOUNT OF
SILAGE DIGESTED
An inspection of the tables shows that the three cows and three sheep
all varied considerably in the amount of dry substances digested. In
general, the same ratio of dry substance digested and nutrients absorbed
existed. The sheep showed a much larger variation in total dry matter
digested than was noted with the cows. The results of this one diges-
tion period indicate that there exists an individuality among animals
as to the thoroughness with which they digest their feed. This view is
supported by the recent work of Grindley l and his associates on diges-
1 Getndley, H. S.,Carmichaei., W. J., and Newlin, C. I. digestion experiments with pigs . . .
111. Agr. Exp. Sta. Bui. 200, p. 55-94, 4 fig. 1917-
Mar. ii, 1921
Sunflower Silage Digestion Experiment
887
tion experiments with pigs, in which they found individual differences
in pigs of the same age and species in the amount of feed digested which
prevailed throughout 40 digestion periods.
It is readily seen that to secure an average digestion coefficient with
any class of animals, a considerable number should be employed, which
would mitigate the factor of errors introduced by individuality of the
animals. If, however, a considerable number of animals are employed,
the work becomes very voluminous and necessitates a large number of
men to carry the experiment to completion. While these individual
differences are not very great, it is thought that a sufficiently close
digestive coefficient value can be obtained by using a smaller number
of animals. In this work it is believed that the average coefficient
obtained for the cows and sheep closely approximate the true digestive
coefficient. A comparison of the analysis of the sunflower silage fed at
this station and that fed at Montana, together with the digestible nutri-
ents contained in each silage, follows.
Table VI. — Comparison of sunflower silage fed at Idaho and Montana Agricultural
Experiment Stations
Dry
sub-
stance.
Crude
protein.
Crude
fiber.
Nitro-
gen-free
extract.
Ether
extract.
Ash.
Crude
fiber
and ni-
trogen-
free
extract.
Nutri-
tive
ratio.
Sunflower silage, Montana
Pounds.
21.4
21.21
21.4
21.21
21.21
Pounds.
2. 1
2.03
1.24
•97
1. 10
Pounds.
6.8
6.3
Pounds.
10.4
9-5°
Pounds.
o-5
I-2J
•37
.91
•95
Pounds.
1.6
2.09
Pounds.
Digestible nutrients in ioo
10.13
7.72
9.62
Digestible nutrients in ioo
Digestible nutrients in ioo
It is seen that a slight difference exists between the digestive nutrients
found by Montana and those obtained by us, but the difference is small.
No data are available as to the kind of animals used by Montana, hence
no comments can be made. The nutritive ratio found by Montana and
by Idaho is quite similar. Some of the difference is no doubt due to the
different stages of maturity of the sunflowers. Montana silage was made
from sunflowers cut when 5 per cent were in bloom, while Idaho silage
represents a crop cut when 50 per cent were in bloom.
Additional studies are needed to determine the best time to cut sun-
flowers in order to secure the maximum food value.
When the digestion coefficients of sunflower silage obtained from cattle
and sheep are compared with the coefficients of immature corn given in
the early part of this paper, it is seen that for protein the cows utilizep
practically the same amount from sunflower silage that they utilized
from immature corn. With sheep, there is slightly more digestible protein
888 Journal of Agricultural Research voi.xx. No. n
in immature corn silage. When sunflower silage is compared with
mature corn, it is seen that the cows utilize slightly less protein from
sunflowers than from corn silage, whereas sheep utilize similar amounts.
SUMMARY
(i) Analysis of sunflower silage fed at the Idaho Agricultural Experi-
ment Station indicated that it compared very favorably with corn silage.
(2) The digestible nutrients contained in sunflowers compare favorably
with the digestible nutrients in mature and immature corn.
(3) The nutritive ratio is somewhat narrower in sunflower silage than
in mature or immature corn silage.
(4) Sheep utilized slightly more nutrients in sunflower silage than did
cows under the conditions of this experiment.
(5) Where both corn and sunflowers can be grown, the selection of a
silage crop should depend upon comparative tonnage per acre and cost
of harvesting.
Vol. XX MARCH 15. 1921 No. 12
JOURNAL OF
AGRICULTURAL
RESEARCH
CONTENTS AND INDEX
OF VOLUME XX
PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE.
WITH THE COOPERATION OF THE ASSOCIATION OF
LAND-GRANT COLLEGES
WASHINGTON, E>. C.
WAtHIHOTON : GOVERNMENT PHINTINO OFPICS : l»SI
EDITORIAL COMMITTEE OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE AND
THE ASSOCIATION OF LAND-GRANT COLLEGES
FOR THE DEPARTMENT
KARL F. KELLERMAN, Chairman
Physiologist and Associate Chief, Bureau
of Plant Industry
EDWIN W. ALLEN
Chief, Office of Experiment Stations
CHARLES L. MARLATT
Entomologist and Assistant Chief, Bureau
of Entomology
FOR THE ASSOCIATION
J. G. LIPMAN
Dean, State College of Agriculture, and
Director, New Jersey Agricultural Experi-
ment Station, Rutgers College
W. A. RILEY
Entomologist and Chief, Division of Ento-
mology and Economic Zoology, Agricul-
tural Experiment Station of the University
of Minnesota
R. L. WATTS
Dean, School of Agriculture, and Director,
Agricultural Experiment Station, The
Pennsylvania State College
All correspondence regarding articles from the Department of Agriculture should be
addressed to Karl F. Kellerman, Journal of Agricultural Research, Washington, D. C.
All correspondence regarding articles from State Experiment Stations should be
addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New
Brunswick, N. J.
INDEX
Page
Abbella subflava, parasite of Eutetiix tenella.. . 250
Abies —
balsamea, hypertrophied lenticels 255-266
grandis, hypertrophied lenticels 25s
Abutilon —
berlandieri, food plant of —
Meskea ihyridinae 828-829
Telepkusa mariona 812
incanum, food plant of —
Meskea ihyridinae 828-829
Telepkusa mariona 812
Acer negundo, composition of green and albino
leaves 179
Acetic acid. See Acid, acetic.
Acid —
acetic, in sugar beet top silage 540-542
boric, value as disinfectant 86-110
butyric, in sugar beet top silage 541-542
carbolic, coefficients of chlorin antiseptics, ioc-102
citric —
availability of iron to rice plants in cal-
careous and noncalcareous soils 5°-54
in grapefruit 359-372
glutaminic, in potato protein 624
hydrochloric, effect on availability of po-
tassium 619-621
hypochlorous, value as disinfectant 86-110
lactic, in sugar beet top silage 540-542
nitric, effect on availability of potassium. 619-621
phosphoric —
effect on availability of potassium 616-617
in plants grown with ferrous sulphate and
gypsum 42
in plants grown with sodium bicarbonate
and sprayed with lime and iron salts. . 46
in potato tubers, skins, and sprouts. . . . 628-634
propionic, in sugar beet top silage 540-542
tartaric —
availability to rice plants in calcareous
and noncalcareous soils 50-54
effect on yield of volatile oil from Chinese
colza seed 130-131
valeric, in sugar beet top silage 541-542
Acid-base balance of poultry feed 141-149
Acidity of —
poultry feed mixtures 141-149
sap of normal and mottled orange leaves. 186-187
soil, relation of calcium content 855-868
sugar beet top silage 540-542
wheat, changes due to tempering 272-275
Acids —
diamine in potato sprouts 624
effect on availability of potassium 619-620
Acids-solids ratio in grapefruit 359-373
Acronyctinae, one species collected on Mal-
vaviscus drummondii 834
Adesmia spp. , hosts of Urophlyctis alfalfae in
Argentina 296
Page
Adomoniga demylus, parasite of Neodiprion
lecontei 757-758
Aedemoses haesitans, similarity to Pectino-
phora gossypiella 816-817
Aegeriidae, similarity of one species to Pecii-
nophora gossypiella 826-827
Aeration, relation to hypertrophied lenticels
on the roots of conifers 253-266
Aerobic bacilli in canned ripe olives 377-379
Age, effect on composition of potato tubers,
skins, and sprouts 632-634
Age of mycelium of Rhizopus iriiici, effect on
hydrolysis of starch 766-768
Agrotinae, similarity of certain species to
Pectinophora gossypiella 833
Aguingay. See Rottboellia exaltata.
Ainslie, George G., and Cartwright, W. B.
(paper): Biology of the Smartweed Borer,
Pyrausta ainslii Heinrich 837-844
Air, carbon -dioxid content in barns 405-408
Alabama argillacea, collected on cotton 834
Alanin in potato protein 624
Albumins, effect on freezing-point depression
of seeds 593
Alcohol, effect on —
tetanus 69
yield of volatile oil from Chinese colza seed . 130-13 1
Aletia argillacia. Syn. Alabama argillacea.
Alfalfa. See Medicago saliva.
Algae —
influence on effect of copper sulphate on
organisms in water 200-203
susceptibility to copper salts 197
Alkaline reaction, effect on chlorosis of plants . 42-47
Alkalinity —
influence on effect of copper on organisms
in water 200-203, 205
of poultry feed mixtures 141-149
of soil, relation of calcium content 855-868
Allium —
ascalonicum, host of Colletotrichum circi-
nans 686-722
cepa, host of Collelolrickum circinans . . . . 685-722
porrum, host of Colletotrichum circinans. . 686-722
sativum, host of Colletotrichum circinans. . 686-722
Allyl—
cyanid, formation during maceration of
Chinese colza seed 131
isothiocyanate, physical constants 127
thiocyanate, formation during maceration
of Chinese colza seed 131
thiourethane, formation during maceration
of Chinese colza seed 131
Almond, tropical. See Terminalia catappa.
Althaea rosea, food plant of Crocidosema ple-
beiana 822
Aluminum in normal and mottled citrus
leaves l67
889
890
Journal of Agricultural Research
Vol. xx
A maranthus — Pa ge
hybridus, food plant of Pachyzancla bipunc-
talis 830
spp., food plants of Plalynota rostrana 821
A mbrosia —
artemisiaefolia, shelter plant of Pyrausta
ainsliei 839
trifida, shelter plant of Pyrausta ainsliei 839
American larch. See Larix americana.
Amid and monoamino nitrogen in potato
tubers, skins, and sprouts 628-634
Ammonia —
effect in stimulating sprouting of potato
tubers 623
influence on efficiency of chlorin disinfect-
ants 102-109
in potato protein 624
in potato tubers, skins, and sprouts 632-634
Ammonium hydrate nitrogen in potato tub-
ers, skins, and sprouts 628-634
Atnorbia emigratella, similarity to Plalynota
rostrana 822
Amorpha nodule bacteria cultures, effect on
mi'k 550
Amphistoma, intestinal fluke in Tropics 194
Amylaseof Rhizopus tritici.with a Consider-
ation of Its Secretion and Action (paper) . 761-786
Anaerobic bacilli in canned ripe olives 377~379
Anagrus giraulti, parasite of Euteltix tenella. . 250
Ancylus caurinus, susceptibility to copper
salts i99
Andropogon sorghum. —
host of Sclcrospora Philip pinensis.
var. halepense, immunity to Sclcrospora
spontanea 671
Angelica, host of Gibberella saubinctii 16
Anias. See Andropogon sorghum var. hale-
pense.
Annelids, susceptibility to copper salts 198
A nomis exacta, collected on Mahaviscus drum-
mondii 834
Another Conidial Sclerospora of Philippine
Maize (paper) 669-684
Anthrax —
inefficacy of echinacea against 74~7S
spores, effect of chlorin disinfectants 94-98
" Anthracnose " of onions. See Collectotri-
chum circinans.
Antiseptics, comparison with green feed as
preventive of intestinal disorders in chicks 869-873
Apium graveolens, host of Sclerotinia minor 331-334
Aplastomorpha vandinei, parasite of Sitopki-
lus oryza 42I
Appleman, Charles O., and Eaton, S. V. (pa-
per): Evaluation of Climatic Temperature
Efficiency for the Ripening Processes in
Sweetcorn 795-805
A raecerus fasciculatus —
description 606-608
distinguishing characters 605-606
synonymy 606
A raucaria bidwellii, hypertrophied lenticels . 255-266
Arbutus, trailing. See Epigaea repens.
Arginin in potato protein 624
Artschwager, Ernst F. (paper): Pathological
Anatomy of Potato Blackleg 325-330
Ascospores of Giberella saubinetii n
Ash— page
carbon-free —
in plants grown with errous sulphate
and gypsum 42
in plants grown with sodium bicarbonate
and sprayed with lime and iron salts . . 46
in bean cotyledons 878
in Chinese colza seed 127
in potato tubers, skins, and sprouts .... 628-634
in sugar beet top silage 538-540
in sugars in storage 638-653
Asparagin in potato sprouts 623
Asparagus, host of Gibberella saubinetii 16
Aspergillus, attacking wheat treated with
formaldehyde 215
Aspergillus —
niger, enzymic action 778-779
terreus in canned ripe olives 377~379
A ster spp. .shelter plants of Pyrausta ainsliei . . 839
Atanasoff, Dimitr (paper): Fusarium-B light
(Scab) of Wheat and Other Cereals 1-32
Atelhmia reclifascia. Syn. Bagisara rectifascia.
Atriplex spp., hosts of Eutettix tenella 247
"Atypical" carbon dioxid in barn air 408
Austrian pine. See Pinus austriaca.
Axena saliva, host of Gibberella saubinetii 1-33
Avocado weevil. See Heilipus lauri.
Babaeaxa delliella. Syn. Ethmia delliella.
Bacillus —
aerogenes, relation of nodule bacteria to. . 543-556
alcaligenes, flagellation peritrichicor cephalo-
trichic 55a
anthracis —
effect of chlorin disinfectants 94-98
inefficacy of echinacea against 74-75
botulinus —
in canned ripe olives 375-379
inefficacy of echinacea against 71-72
bovisepticus, inefficacy of echinacea against. 72-74
cereus in canned ripe olives 377-379
cloacae in canned ripe olives 377-379
coli —
flagellation peritrichic or cephalotrichic. . 552
relation to Bacillus aerogenes 543
lactis viscosum, relation of nodule bacteria
to 543-SS6
megatherium, enzymic action 778
mesentericus in canned ripe olives 377-379
mycoides in canned ripe olives 377_379
pneumoniae, relation of nodule bacteria to. 543-556
pyocyaneus, effect of chlorin disinfectants
upon 88-110
radicicola —
comparison with cowpea-soybean bac-
teria 545-554
peritrichic flagellations 544
radiobacter —
comparison with cowpea-soybean bac-
teria 545-554
relation to Bacillus coli 543
relation of nodule bacteria to 543-556
solanacearum, not cause of Fusarium-wilt
of tobacco 515-536
tuberculosis, effect of chlorin disinfectants . . 98-100
typhosus, effect of chlorin disinfectants
upon 88-110
Oct. i, 1920-Mar. 15, 1921
Index
891
Bacteria — Page
in canned ripe olives 375-379
nodule, of leguminous plants 543-556
Bacteriological Study of Canned Ripe Olives,
A (paper) 375-379
Bacterium —
fluorescens liquefaciens in canned ripe
olives 377-?79
japonicum, possible name for cowpea-soy-
bean nodule bacteria 551
Bactrocera cucurbitae, host of O phis fletcheri . 423-438
Bagisara rectifascia, collected on Malvaviscus
drummondii 834
Barber, H. S., and Dietz, H. F. (paper): A
New Avocado Weevil from the Canal
Zone 111-116
Barley. See Hordeum spp.
Barn air, carbon-dioxid content 405-408
Bastardia viscosa, food plant of Platynota
rostrana 831
Batrachedra rileyi. Syn. Pyroderces rileyi.
Bean —
kidney. See Phaseolus vulgaris.
navy, nodule bacteria cultures, effect on
milk 550
seedlings, utilization of mineral constituents
in soil and in distilled water 875-880
Beans, horse. See Viciafaba.
Beet leafhopper. See Eutettix tenella.
Beet top silage 537-542
Beggar weed nodule bacteria cultures, effect
on milk 550
Beggartick. See Bidcns bipinnata.
Benzoate, ferric, availability to rice plants in
calcareous and noncalcareous soils 50-54
Beta, host of Gibber ella saubineiii 16
Bicarbonate, sodium —
effect on growth of rice 44~47
value as disinfectant 86-110
Bichlorid, mercuric, toxity to snails 196
Bidcns —
bipinnata, shelter plant of Pyrausta ainsliei. 839
frondosa, shelter plant of Pyrausta ainsliei. . 839
Biology of the Smartweed Borer, Pyrausta
ainsliei Heinrich (paper) 837-844
Bisulphid, carbon, formation during macera-
tion of Chinese colza seed 131
Black locust nodule bacteria cultures, effect
on milk 550
Blackleg of Solanum tuberosum 325-330
"Black spot" of onions. See Colletotrichum
circinans.
Blanfordia, intermediate host of Schistosoma
japonicum 198
Blastobasidae, similarity of certain species to
Pectinophora gossypiella 817-819
Blastobasis —
citricolella. Syn. Zenodochiumcitricolella.
citriella. Syn. Zenodochium citricolella.
Blood, dried, availability of iron to rice plants
in calcareous and noncalcareous soils 50-54
Blood flukes, control by destruction of inter-
mediate host 193-208
Blood serum, effect on efficacy of chlorin
disinfectants 89-110
Blueberry, highbush. See Vaccinium co-
rymbosum.
Boliworm, pink, similar lepidoptera 807-836
Bombyx obsolcta. Syn. Heliothis obsoleta. Page
Borate, calcium, value as disinfectant 86-110
Bordeaux spraying, effect on composition of
potato tubers, skins, and sprouts 632-634
Borer, smartweed. See Pyrausta ainsliei.
Boric acid. See Acid, boric.
Borkhausenia —
ascriptella, agreement with type species... 816
conia, similarity to Triclonella spp 815-816
drveni, similarity to Triclonella spp 815-816
episcia, similarity to Triclonella spp 815-816
fasciata, similarity to Triclonella spp 815-816
haydenella, agreement with type species. . . . 816
minulella, distinguishing characters 815-816
orites, similarity to Triclonella spp 815-816
pseudospretella, agreement with type
species 816
Botryosphaeria saubineiii. Syn. Gibberella
saubinetii.
Botrytis —
cinerea, growth of hyphae 703
spp. , sclerotia 689
Botulism —
caused by Bacillus botulinus 375-379
inefficacy of echinacea against 71-72
Botys(?) thalialis. Syn, Noctuelia rufofas-
cialis.
Bouyoucos, George (paper): Degree of Tem-
perature to Which Soils Can Be Cooled
without Freezing 267-269
Bouyoucos, George J., and McCool, M. M.
(paper): Measurement of the Amount of
Water That Seeds Cause to Become Unfree
and Their Water-Soluble Material 587-593
Brassica —
bcsseriana, seed 125-126
campeslris —
classification 118-122
chinensis —
classification 118-121
oleifera, n. f. See Brassica campestris
chinoleifera.
chinoleifera —
analysis of seeds 126-132
bactericidal action 134-135
botanical characteristics 122-126
classification 118-122
pharmacological action 133-134
substitute for mustard 117-140
volatile oil 127-132
pekinensis, classification 118-121
var. annua sativa chinensis, classification 120
var. chinensis, classification 118-122
var. sativa annua chinensis, classification 1 19-122
cernua —
Japanese mustard 117
leaf formation 124
juncea —
Chinese mustard "7
leaf formation 124
napiformis, leaf formation 124
nigra, leaf formation 124
oleracea bullata gemmifera, seed 125-126
orientalis, classification 1 18-122
pekinensis, classification "9
pe-tsai, classification "9
rapa —
classification "9
effect on water extract of soil 663-667
892
Journal of Agricultural Research
Vol. xx
Brauneria — Page
angustifolia —
habitat 64
medicinal properties 63-84
habitat 64
pallida, habitat 64
paradoxa, habitat 64
purpurea, habitat 64
Ereazeale, J. F., and Briggs, Lyman J. (pa-
per) : Concentration of Potassium in Ortho-
clase Solutions Not a Measure of Its Avail-
ability to Wheat Seedlings 615-621
Briggs, Lyman J., and Breazeale, J. F. (pa-
per) : Concentration of Potassium in Ortho-
clase Solutions Not a Measure of Its Avail-
ability to Wheat Seedlings 615-621
Bromid, ethyl, effect in stimulating sprout-
ing of potato tubers 623
Bromin, effect in stimulating sprouting of
potato tubers 623
Bromus sp., host of Gibberella saubinelii 16
Broom. See Sarolkamnus scoparius.
Buckner, G. Davis (paper): Comparative
Utilization of the Mineral Constituents in
the Cotyledons of Bean Seedlings Grown in
Soil and in Distilled Water 875-880
Buckwheat See Fagopyrumfagopyrum.
Buds, freezing 655-66?
Bugang grass. See Saccharum spontaneum.
Bullinus, intermediate host of Schistosoma
haematobium and f>. mansoni 198
Bunchberry. See Cornus canadensis.
Burger, O. F. (paper): Variations in Colle-
totrichum gloeosporioides 723-736
Butyric acid. See Acid, butyric.
Buxus, host of Gibberella saubinetii 16
Calandra —
frugilega. Syn. Sitophilus linearis,
tamarindi. Syn. Sitophilus linearis.
{Calandra) Sitophilus oryza. See Sitophilus
oryza.
Calcifugous plants, ecology 33-34
Calciphilous plants, ecology 33-34
Calcium —
borate, value as disinfectant 86-1 10
carbonate —
effect on growth of plants 40-44
in Kansas soils 864-866
hypochlorite, value as disinfectant 86-110
in bean cotyledons 878
in cropped and uncropped soils 663-667
in normal and mottled citrus leaves 166-190
in soil extract 387-394
in southern poultry feeds 143
oxid in potato tubers, skins, and sprouts . . 633
phosphate, effect on growth of plants 40-44
silicate, effect on growth of plants 40-44
relation to soil reaction 855-868
sulphate effect on —
availability of potassium 616-617
growth of plants 40-44
Callida decora, parasite of Pyrausta ainsliei. . . 844
Calluna vulgaris, growth on calcareous soil. . . 35
Calories, protein, in various poultry feeds 147
Cannabis, host of Gibberella saubinetii 16
Carbohydrates — Page
in sugar beet top silage 538-540
in sweetcorn 795-805
Carbolic-acid coefficients of chlorin antisep-
tics 100-102
Carbonate —
calcium —
cause of chlorosis in plants 36-49
effect on availability of iron in soil 47-49
effect on growth of plants 40-44
in Kansas soils 864-866
bisulphid, formation during maceration of
Chinese colza seed 131
dioxid —
effect on availability of potassium 618
Kansas soils 858
relation to hypertrophy of conifers 261
tetrachlorid, effect in stimulating sprouting
of potato tubers 623
Carbon-Dioxid Content of Bam Air (paper) 405-408
Carmichael, W. J., and Detlefsen, J. A. (pa-
per) Inheritance of Syndactylism, Black,
and Dilution in Swine 595-604
Carr, R. H. , et al. (paper) : Green Feed versus
Antiseptics as a Preventive of Intestinal
Disorders of Growing Chicks 869-873
Carrero, J. O., and Gile, P. L. (paper): Cause
of Lime-Induced Chlorosis and Availability
of Iron in the Soil 33-62
Cartwright, W. B., and Ainslie, George G.
(paper) : Biology of the Smartweed Borer,
Pyrausta ainsliei Heinrich 837-844
Carum —
carvi, host of Uropklyctis kriegeriana 313
incrassatum, host of Urophlyclis hemisphae-
rica 308
Cassia —
chamaecrista nodule bacteria cultures, effect
on milk 550
tora, food plant of Plalynota rostrana .... 811,821
Castanea vesca, calcifugous nature 34
Caulophilus latinasus —
description 608-610
distinguishing characters 605-606
synonymy 608
Causation and correlation 557-585
Cause of Lime-Induced Chlorosis and Avail-
ability of Iron in the Soil (paper) 33-6*
Cataclysta (?) julianalis. Syn. Dicymolomia
julianalis.
Calolaccus incertus, parasite of Sitophilus oryza 42a
Cattail. See Typha lati/olia.
Celery'- See Apium graveolens.
Cephalanthus occidentalis —
food plant of Phalonia cephalanthana 825
water lenticels 256
Ceratitis capitata, experimental host of Opius
fietcheri 423
Cercocephala elegans, parasite of Sitophilus
oryza 421
Chaetodacus cucurbitae, host of Opius fietcheri. 431
Chandler, Asa C. (paper): Control of Fluke
Diseasesby Destruction of the Intermediate
Host 193-208
Changes Taking Place in the Tempering of
Wheat (paper) 271-275
Oct. i, 1920-Mar. 15, 1921
Index
893
Chauliognathus pennsylvanicus, parasite of Page
Pyrausta ainsliei 844
Cheiw podium —
glaucum, host of Urophlyctis pulposa 313
murale, host of Euiettix tenella 247
spp. , hosts of —
Eutettiz tenella 247
Urophlyctis (Cladochytrium) pulposa 300
Chestnut. See Castanea vesca.
Chicks, green feed versus antiseptics as pre-
ventive of intestinal disorders 869-873
Chilling, influence in stimulating growth of
plants 151-160
Chinese colza. See Brassica campestris chi-
noleifera.
" Chloramin T," value as disinfectant 85-110
' ' Chlorazene. ' ' See ' ' Chloramin T. "
Chlorid—
copper, toxity to snails 196-197
ethyl, effect in stimulating sprouting of
potato tubers 623
ferric: —
availability to rice plants in calcareous
and noncalcareous soils 50-54
effect on growth of rice 42-44
in normal and mottled citrus leaves 166-190
manganese, effect on formation of potato
tubers 623
potassium —
absorption by plants 616-617
effect on concentration of soil solution. . . . 393
Ckloridea —
obsoleta. Syn. Heliothis obsoleta.
virescens. Syn. Heliothis virescens.
Chlorin —
disinfectants, germicidal value 85-110
in southern poultry feeds 143
Chlorinated lime, influence on effect of copper
sulphate in water 202
Chlorosis, caused by lime 33-62
Chrysauginae, similarity of one species to
Pectinophora gossypiella 832
Chrysophyllum oliviformae, host fruit of Cera-
titis capitata 423
Citrate, ferric —
availability to rice plants in calcareous and
noncalcareous soils 5°~S4
effect on growth ot rice 42-44
Citric acid. See Acid, citric.
Citrus —
aurantium, composition of parts of tree 162
aurantifolia, parent of limequat 469
decutnana, changes during storage 357~373
grandis —
composition of leaves 163
influence of —
humidity on development of Pseudo-
tnonas citri 494-497
temperature on development of Pseu-
domonas citri 471-488
temperature on growth 459-471
limonia, composition of parts of tree 162
medica, host of Gloeosporium limetticohim . . . 724
Citrus — Continued Page
mitts, influence of —
humidity on development of Pseudomo-
nas citri 494-497
temperature on development of Pseudo-
tnonas citri 471-488
temperature on growth 459-471
nobilis, var. deliciosa, composition of parts
of tree 162
sinensis, parent of hybrid Rusk cTErange. . . 459
Citrus-canker. See Pseudomonas citri.
Citrus leaves, normal and mottled, composi-
tion 161-191
Cladochylnum alfalfae. Syn. Urophlyctis
(Physoderma) leproidea.
Clawson, A. B., and Marsh, C. Dwight (pa-
per): Daubentonia longifolia (Coffee Bean),
a Poisonous Plant 507-514
Clematis, host of Gibberella saubinetii 16
Clevenger, Joseph F., et al. (paper): Studies
in Mustard Seeds and Substitutes: I. Chi-
nese Colza (Brassica campestris chinoleifera
Viehoever) 117-140
Climate —
effect on composition of potato tubers, skins,
and sprouts 632-634
relation to crown wart of alfalfa 319
Climatic temperature, effect on ripening proc-
esses in sweetcorn 795-805
Clonorchis —
human liver fluke 193-195
parasite of Melania 198
Clover —
Japan, nodule bacteria cultures, effect on
milk 550
red, nodule bacteria cultures, effect on milk. 550
sweet, nodule bacteria cultures, effect on
milk 550
See Trifolium spp.
Clydonopteron lecomae, similarity to Pectino-
phora gossypiella 832
Cocci in canned ripe olives 377~379
Cocklebur. See Xanthium communis.
Coffea arabica, host of Ceratitis capitata 423
Coffee bean. See Daubentonia longifolia.
Coffee. See Coffea arabica.
Cogon. See Imperata cylindracea.
Con —
lachryma, host of Sitophilus oryza 410
lachryma-jobi, immunity to Sclerospora
spontanea 671
Cold, influence in stimulating growth of
plants 151-160
Colletotrichum —
antirrhini, stromata 692
circinans, causal organism of onion smudge.
685-722
fructus, similarity to C. circinans 693-694
gloeosporioides, variations 723-73<>
lagenarium, growth of hyphae 703
lindemutkianum, growth of hyphae 703
sp —
cause of wilting of cereal plants 7
isolated from diseased potato vines. . . . 280-281
894
Journal of Agricultural Research
Vol. XX
Colloidal material, effect on freezing of water Page
in soil 591
Colloids, effect on oxidation of potassium. . . . 620
Colon bacilli in canned ripe olives 377~379
Color, inheritance in swine 595-604
Comparative Study of the Composition of the
Sunflower and Com Plants at Different
Stages of Growth, A (paper) 787-793
Comparative Utilization of the Mineral Con-
stituents in the Cotyledons of Bean Seed-
lings Grown in Soil and in Distilled Water
(paper) 875-880
Composition of Normal and Mottled Citrus
Leaves (paper) 161-191
Composition of Tubers, Skins, and Sprouts of
Three Varietiesof Potatoes (paper) 613-635
Concentration of Potassium in Orthoclase
Solutions Not a Measure of Its Availability
to Wheat Seedlings (paper) 615-621
Conidia of Gibberella saubinetii 9-1 1
Conifers, hypertrophied lenticels 253-266
Conium, host of Gibberella saubinetii 16
Control of Fluke Diseases by Destruction of
the Intermediate Host (paper) 193-208
Convolvulus, host of Gibberella saubinetii 16
Cook, F. C. (paper): Composition of Tubers,
Skins, and Sprouts of Three Varieties of
potatoes 623-635
Copper —
chlorid, toxicity to snails 196-197
in potato tubers, skins, and sprouts 629-634
nitrate, toxicity to snails 196
sprays, effect on potato sprouts, skins, and
tubers 625-634
sulphate, toxicity to snails 196-208
Corn borer, European. See Pyrausta nubila-
lis.
Corn —
comparison with sunflowers for silage 787-793
See Zea mays.
Cornus canadensis, influence of cold in stimu-
lating growth (PI. 29) 151-160
Coronilla, host of Gibberella saubinetii 16
Correlation and Causation (paper) 557-585
Correlations for crop yields in different years 337-356
Cosmopterygidae, similarity of one species to
Pectinophora gossypiella 820
Cossonus pinguis. Syn. Caulophilus laiina-
sus.
Cotton, Richard T. (paper)—
Four Rhynchophora Attacking Corn in
Storage 605-614
Rice Weevil, (Calandra) Sitophilusoryza. 409-422
Tamarind Pod-Borer, Sitophilus linearis
(Herbst) 439-446
Cotton. See Hibiscus lasiocarpus.
Cotyledons, utilization of mineral constitu-
ents in soil and in distilled water 875-880
Couch, James F., and Giltner, Leigh T.
(paper) : An Experimental Study of Echi-
nacea Therapy 63-84
Coville, Frederick V. (paper): The Influence
of Cold in Stimulating the Growth of
Plants 151-160
Cowpea nodule bacteria cultures, effect on
milk 55°
Cowpea-soybean bacteria, comparison with Page
Bacillus radicicola and B. radiobacter 545-554
Crab, wild. See Malus coronaria.
Crambinae, similarity of one species to Pecti-
nophora gossypiella 830-831
Cremastus facilis , parasite of Pyrausta sp 843
"Critical temperature" for fruit buds 655-662
Crocidosema plebiana —
difference from Platynota rostrana 821
similarity to Pectinophora gossipiella 807, 822
Crops, effect on water extract of soil 663-667
Crotonyl isothiocyanate —
in Chinese colza seed 127-133
physical constants 127
Crop growth, effect on physical state of soil. 397-404
Crownwart of Alfalfa Caused by Urophlyctis
alfalfae (paper) 295-324
Crude fiber —
in Chinese colza seed 127
in sugar beet top silage 538-540
Crustaceans, susceptibility to copper salts. . . 198
Cucurbita sp., host of Gibberella saubinetii 16
Cummins, A. B., and Kelley, W. P. (paper):
Composition of Normal and Mottled Citrus
Leaves 161-191
Cyanid, allyl, formation during maceration of
Chinese colza seed 131
Cystin in potato protein 624
Daubentonia longifolia (Coffee Bean), a
Poisonous Plant (paper) 507-514
Daubentonia longifolia —
lethal dose 51a
pathological effects 511
symptoms of poisoning 510
Decodon verticillalus, water lenticels 356
Degree of Temperature to Which Soils Can Be
Cooled without Freezing (paper) 267-269
Depressaria gossypiella. Syn. Pectinophora
gossypiella.
Desiccation, effect on Colletolrichum circi-
nans 698-699
Deterioration of sugars in storage 637-653
Detlefsen, J. A., and Carmichael, W. J.
(paper): Inheritance of Syndactylism,
Black, and Dilution in Swine 59S-604
Dextrose in grapefruit 359-372
Diachasma —
fullawayi, parasite of Ceratilis capitata 424
iryoni, parasite of Ceratitis capitata 424
Dialyzed iron, effect on growth of rice 42-44
Diaminci —
acids in potato sprouts 624
nitrogen in potato tubers, skins, and
sprouts 628-634
Dicymolomia julianalis, similarity to Pectino-
phora gossypiella 807, 830-831
Dietz, H. P., and Barber, H. S. (paper): A
New Avocado Weevil from the Canal
Zone in-116
Digitalis purpurea, growth on calcareous soil. 35
Dioxid, carbon —
effect on availability of potassium 618
in bam air 405-408
Diplococci in canned ripe olives 377-379
Disinfectants, chlorin 85-110
Dock, curled. See Rumez crispus.
Oct. i, 1920-Mar. 15, 192 1
Index
895
Page
Dourine, inefficacy of echinacea against So-82
Downy mildew of maize. See Sderospora spp.
Drechsler, Charles, and Jones, Fred Reuel
(paper): Crownwart of Alfalfa Caused by
Urophlyctis alfalfae 295-324
Drying, effect on formaldehyde injury to seed
wheat 231-242
Dryinidae, parasite of Eutettix tenella 231
Eaton, S. V., and Appleman, Charles O.
(paper): Evaluation of Climatic Tempera-
ture Efficiency for the Ripening Processes
in Sweetcorn 795-805
"Ebony, Mexican." See Siderocarpus Jlexi-
caulis.
Echinacea —
anguslofolia, medicinal properties 63-84
purpurea. Syn. Brauneria purpurea.
Echinacea therapy 63-84
Edlefsen, N. E., and West, Frank L. (paper):
Freezing of Fruit Buds 655-662
Edson, H. A. (paper): Vascular Discoloration
of Irish Potato Tubers 377-294
Effect of Season and Crop Growth on the
Physical State of the Soil (paper) 397-404
Effect of Various Crops upon the Water Ex-
tract of a Typical Silty Clay Loam Soil
(paper) 663-667
Effects of X-Rays on Trichinae (paper). ..845-854
Electrolytic hypochlorite solutions, effect of
ammonia i0}
Electrometric titration, indication of relation
of calcium content of soil to reaction 855-868
Emmer, host of Gibberella saubinelii 16
Empoasca sp. , host of A bbella sub/lava 250
Enarmonia tristrigana. Syn. Laspeyresia tris-
trigana.
Ennychia rufofascialis. Syn. Noctuelia rufo-
fascialis.
Enzym in Chinese colza seed destroyed by
tartaric acid 130
Enzym ic action —
in Pseudomonascitri cultures 450-455
of Rhizopus tritici on starch 761-786
Ephestia osirinella. Syn. Moodna ostrinella.
Epigaea repens, influence of cold in stimulat-
ing growth (PI. 30) 151-160
Erodiuin —
cicutariunt, host of Eutettix tenella 247
moschaium, host of Eutettix tenella 247
Erebinae, species collected on Hibiscus lasio-
carpus, Malvaviscus drummondii, and Abu-
tilon incanum 834
Ether —
ethyl, effect on formation of potato tubers . . 623
extract —
in Chinese colza seed 127
in sugar beet top silage 538-540
in sunflower and corn silage 881-888
Ethmia —
bittenella, similarity to Pectinophora gos-
sypiella 819
delliella, reared from Wissadula lozani 819
Ethmiidae, similarity of certain species to
Pectinophora gossypiella 819
Ethyl— Page
bromid, effect in stimulating sprouting of
potato tubers g23
chlorid, effect in stimulating sprouting of
potato tubers 623
ether, effect on formation of potato tubers . 623
Euchlaena luxurians, susceptibility to Sclero-
spora spontanea 671
Eucosma —
discretivana, n. sp 823-824
helianthana, similarity to Pectinophora gos-
sypiella 824
obfuscana, similarity of E. discretivana. . . 823-824
pkbeiana. Syn. Crocidosema plebeiana.
Eupatorium sp., shelter plants of Pyrausla
ainsliei 839
"Eusol," value as disinfectant 86-110
Eutettix tenella —
description 245-246
life history 247-248
natural enemies 250-251
seasonal history 248-249
Evaluation of Climatic Temperature Effi-
ciency for the Ripening processes in Sweet-
corn (paper) 795-805
Evaporation, effect on formaldehyde injury
to seed wheat 221-222
Ewing, Clare Olin, et al. (paper): Studies in
Mustard Seeds and Substitutes: I. Chinese
Colza (Brassica campestris chinoleifera
Viehoever) 1 17-140
Exenterus diprioni, parasite of Neodiprion
lecontei 757-758
Exorista vulgaris, parasite of Pyrausta sp 843
Experimental Study of Echinacea Therapy,
An (paper) 63-84
Exponential indices of ripening in sweetcorn . 802-
S04
Extract, soil, relation to soil solution 381-395
Fagopyrumfagopyrum, food plant of Pyrausta
ainsliei 838
Fasciola hepatica, liver fluke of cattle and
sheep 194, 198
Feed, poultry, potential acidity and alka-
linity 141-149
Ferric —
benzoate, availability to rice plants in
calcareous and noncalcareous soils 50-54
chlorid —
availability to rice plants in calcareous
and noncalcareous soils 50-54
effect on growth of rice 42-44
citrate —
availability to rice plants in calcareous
and noncalcareous soils 50-54
effect on growth of rice 42-44
oxalate, availability to rice plants in cal-
careous and noncalcareous soils 50-54
tannate, availability to rice plants in cal-
careous and noncalcareous soils 50-54
tartrate —
availability to rice plants in calcareous
and noncalcareous soi's 50-54
effect on growth of rice 43-44
896
Journal of Agricultural Research
Ferric — Continued Page
valerianate, availability to rice plants in
calcareous and noncalcareous soils 50-54
" Ferric humate," availability to rice plants
in calcareous and noncalcareous soils. . . . 50-54
"Ferric molasses," availability to rice plants
in calcareous and noncalcareous soils. . . 50-54
Ferrous sulphate —
availability to rice plants in calcareous
and noncalcareous soils 50-54
effect on action of gypsum 38-44
Fertilizer, effect on composition of potato
tubers, skins, and sprouts 632-634
Fiber, crude, in Chinese colza seed 127
Filaree. See Erodium cicularium.
Fluke diseases, control by destruction of inter-
mediate host 193-208
Fluminkola fusca, susceptibility to copper
salts 199
Fluminicola, member of family Amnicolidas. 198
Fluorid, potassium, effect on yield of volatile
oil from Chinese colza seed 130-131
Footrot of cereals caused by Gibberella saubi-
netii 6-7
Formaldehyde —
injury to seed wheat 209-244
physical properties 218-223
Fortunella japonica, parent of liraequat 469
Four Rhychophora Attacking Corn in Storage
(paper) 605-614
Foxglove. See Digitalis purpurea.
Fraxinus, host of Gibberella saubinetii 16
Freezing, effect on Collelotrichum circinans. 699-700
Freezing of Fruit Buds (paper) 655-662
Freezing-point depression —
of dry seeds 592~593
of sap of normal and mottled orange leaves. 186-187
soil, effect of moisture 390-391
Freezing point of soils 267-269
Fruit buds, freezing 655-662
Further Studies in the Deterioration of Sugars
in Storage (paper) 637-653
Fusarium —
arcuosporum, parasite on cereals 2, 21
arihrosporioides, parasite on cereals 2, 21
avenaceum —
causal organism of " snowmold " 20
parasite on cereals 2, 21
culmorum —
causal organism of "snowmold" 19
parasite on cereals 2, 21
similarity of conidia to those of Gibberella
saubinetii 16
var. leleius, parasite on cereals 2, 21
didymiuvi, casual organism of "snowmold". 20
discolor var. sulphureum, isolated from dis-
colored potato tubers 282
graminearum. Syn. Gibberella saubinetii.
herbarum —
causal organism of " snowmold " 20
parasite on cereals 2, 21
lolii, causal organism of "snowmold" 20
metachroutn, causal organism of "snow-
mold" 20
nivalc, cause of "snowmold" 19
Fusarium — Continued Page
oxysporum —
failure to alter starch of Irish potato 765
isolated from discolored potato tubers. 280-282
var. nicolianae, n. var., causal organism
of Fusarium-wilt of tobacco 521-536
radicicola, failure to alter starch of Irish
potato 765
redolens, parasite on cereals 2, 21
roseum. Syn. Gibberella saubinetii.
rostratum. Syn. Gibberella saubinetii.
rubiginosum, causal organism of "snow-
mold" 19
scirpi, parasite on cereals 2, 21
solani, parasite on cereals 2, 21
sublatum, causal organism of "snowmold". 20
spp., cause of vascular necrosis of potato
tubers 277
tabacivorum, causal organism of disease of
tobacco 517-518
tropicalis. Syn. Gibberella saubinetii.
Fusarium-Blight (Scab) of Wheat and Other
Cereals (paper) 1-32
Fusarium-Wilt of Tobacco (paper) 515-536
Garlic. See Allium sativum.
Gasoline, effect in stimulating sprouting of
potato tubers 623
Gastrodiscus, intestinal fluke in Tropics 194
Gelechia —
bosquella, similarity to Borkhausenia diveni
and Noctuelia rufofascialis 811
hibiscella —
similarity of G. neoirophella 812
similarity to Pectinophora gossypiella. . . 810-811
malvella. Syn. Pectinophora malvella.
neotrophella, n. sp 81 1-812
similiella. Syn. Isophrictis similiella.
trophclla, similarity of G. neotrophella 812
Gelcchiidae, similarity of certain species to
Pectinophora gossypiella 808-814
Germicidal value of chlorin disinfectants. . . . 85-110
Germination of wheat, effect of formalde-
hyde 211-244
Glyphodes pyloalis, leaf-tying pyralid 830
Gibbera pulicaris i. zeae maydis. Syn. Gibber-
ella saubinetii.
Gibberella —
saubinc/ii —
description 4-9, 15-19
dissemination of spores n-13
economic importance 3
hosts 1-9, 16
life history 9-11
parasite on cereals 1-32
tritici. Syn. G. saubinetii.
Gile, P. !_., and Carrero, J. O. (paper): Cause
of I,ime-Induced Chlorosis and Availa-
bility of Iron in the Soil 33-62
Giltner, Leigh T., and Couch, James F.
(paper): An Experimental Study of Echi-
nacea Therapy 63-84
Gingko bilboa, hypertrophied lenticels 255
Girdling, influence in stimulating growth of
plants 155
Gleditschia, host of Gibberella saubinetii 16
Oct. i, 1920-Mar. 15, 1921
Index
897
Page
Globulin in potato protein 624
G locos poriu m —
fruciigenum, growth of hyphae 703
limetticolum, parasite of Citrus medica 724
Glomerella cingulata, relation to Colle/otri-
chuin gloeosporioid.es 725
Glucose, effect on hydrolysis of starch by
Rhizopus triiici 768-769
Glutaminic acid. See Acid, glutaminic.
Glyceria aquatica, host of Gibberella saubinetii. 16
Goldenrod. See Solidago spp.
Gonatopus contortulus, host of Eutetiix tenella 251
Goniobasis, akin to Melania 198
Goniobasis plicifera, susceptibility to copper
salts 199
Gram-negative bacilli in canned ripeo lives 377-379
Gram -positive bacilla in canned ripe olives 377-379
Grapefruit. See Citrus decumana and C.
grandis.
Grass, bugang. See Saccharum spontaneum.
Grasses, hosts of Gibberella saubinetii 1-32
Green Feed versus Antiseptics as a Preven-
tive of Intestinal Disorders of Growing
Chicks (paper) 869-873
Grouseberry. See Viburnum americanum.
Growth, effect on composition of potato tu-
bers, skins, and sprouts 632-634
Growth of plants, influence of cold 151-160
Gyneria, host of Gibberella saubinetii 16
Gypsum, effect on —
availability of potassium 616-617
growth of rice 40-42
Hahn, Glenn G., Hartley, Carl, and Rhoads,
Arthur S. (paper): Hypertrophied Lenti-
cels on the Roots of Conifers and Their Re-
lation to Moisture and Aeration 253-266
Hansen, Roy, and Lohnis, P. (paper): Nod-
ule Bacteria of Leguminous Plants 543-556
Harris, J. Arthur, and Scofield, C. S. (paper):
Permanence of Differences in the Plots of
an Experimental Field 335-356
Harter, L. L. (paper) : Amylase of Rhizopus
tritici with a Consideration of Its Secretion
and Action 761-786
Hartley, Carl, et al. (paper): Hypertrophied
Lenticels on the Roots of Conifers and Their
Relation to Moisture and Aeration 253-266
Hawkins, Lon A.,andMagness, J. R. (paper):
Some Changes in Florida Grapefruit in
Storage 357-373
Heather. See Calluna vulgaris.
Hedera helix, composition of green and albino
leaves 179
Heilipus —
lauri, avocado weevil 111-116
persae, new avocado weevil 1 1 1-1 16
pittieri, avocado weevil 111-116
Heinrich, Carl (paper): Some Lepidoptera
Likely to Be Confused with the Pink
Bollworm 807-836
Heliotkis —
armiger. Syn. H. obsoleta.
(Chloridea) —
obsoleta, similarity to Pectinophora gos-
sypiella 833
virescens, similarity to H. obsoleta 833
Helix pomatia, copper content of body 200
Hendry.Mary F., and Johnson, Alice (paper):
Carbon-Dioxid Content of Barn Air 405-408
Heterogeneity in experimental plots 335-356
Hibiscus —
esculenlus, food plant of —
Crocidosema plebeiana 823
Platynota rostrana 821
lasiocarpus, food plant of —
Gelechia hibiscella 810-811
Pectinophora gossypiella 807-836
militaris, food plant of —
Crocidosema plebeiana 822
Gelechia hibiscella 810-811
rosa-sinensis, food plant of Crocidosema ple-
beiana 822
Hickman, C. W., et al. (paper): Sunflower
Digestion Experiment with Cattle and
Sheep 881-888
Highbush blueberry. See Vaccinium coryin-
bosum.
Histidin in potato protein 624
Hoagland, D. R., and Martin, J. C. (paper):
Effect of Season and Crop Growth on the
Physical State of the Soil 397-404
Hoagland, D. R., Martin, J. C, and Stewart,
G. R. (paper) : Relation of the Soil Solution
to the Soil Extract 381-395
Holcocera —
confamulella, n. so 818-819
mode stella, similarity of H. confamulella 819
ochrocephala, similarity to Pectinophora
gossypiella 818
Holly. See Ilex aquifolium.
Hollyhock. See Althaea rosea.
Homoeosoma electellum, similarity to Pecti-
nophora gossypiella 831-832
Hordeum spp. —
effect on water extract of soil 663-667
susceptibility to formaldehyde injury. . . 240-241
"Humate, ferric," availability to rice plants
in calcareous and noncalcareous soils 50-54
Humidity —
effect on —
formaldehyde injury to seed wheat. . . . 223-231
growth of Pseudomonas citri 447-506
relation to —
deterioration of sugars in storage 642-653
freezing of fruit buds 655-662
Hurd, Annie May (paper): Injury to Seed
Wheat Resulting from Drying after Disin-
fection with Formaldehyde 209-244
Hydrate nitrogen, ammonium, in potato
tubers, skins, and sprouts 628-634
Hydrochloric acid. See Acid, hydrochloric.
Hydrogen-ion concentration —
changes in tempering of wheat 272-275
of sap of normal and mottled orange
leaves 186-187
Hydrolysis of —
starch by Rhizopus triiici 765-783
sugar in ripening of sweetcom 795-805
Hydroxid, potassium, effect on yield of vol-
atile oil from Chinese colza seed 130-131
Hypertrophied Lenticels on the Roots of
Conifers and Their Relation to Moisture
and Aeration (paper) 253-266
898
Journal of Agricultural Research
Vol. XX
Hypochlorite — Page
calcium, value as disinfectant 86-110
sodium, value as disinfectant 86-110
solutions, electrolytic, effect of ammonia. . . 102
Hypochlorous acid. See Acid, hypochlorous.
Hypostena variabilis, parasite of Pyrausta sp. . 843
Ilex aquifolium, composition of green and
albino leaves 179
Imperata cylindracea, immunity toSclerospora
spontanea 671
Influence of Cold in Stimulating the Growth
of Plants, The (paper) 151-160
Influence of Temperature and Humidity on
the Growth of Pseudomonas citri and Its
Host Plants and on Infection and Develop-
ment of the Disease (paper) 447-506
Inheritance of Syndactylism, Black, and
Dilution in Swine (paper) 595-604
Injury to Seed Wheat Resulting from Drying
after Disinfection with Formaldehyde
(paper) 209-244
Inorganic iron compounds, availability to rice
plants in calcareous and noncalcareous soils 50-54
Intestinal disorders of chicks, green feed
versus antiseptics as preventive 869-873
Inula helenium, ingredient of ' 'Subculoyd
Inula and Echinacea" 65
Investigations of the Germicidal Value of
Some of the Chlorin Disinfectants (paper) . 85-110
Iodin number on ether extract of Chinese colza
seed 127
Ipomoea batatas, host of Gibberella saubinetii. . 16
Iron —
availability to rice plants 47-58
compounds, availability to rice plants in
calcareous and noncalcareous soils 50-54
dialyzed, effect on growth of rice 42-44
effect of—
carbonate of lime on availability in soil. . 47-49
soil water on availability 54-58
effect on —
chlorotic plants 38-39
growth of rice 41-47
in normal and mottled citrus leaves 166-190
in plants grown with ferrous sulphate and
gypsum 42
in plants grown with sodium bicarbonate
and sprayed with lime and iron salts. ... 46
in soil 33-62
in southern poultry feeds 143
Irish potato. See Solanum tuberosum.
Isophrictis similiella, similiarity to Peclino-
phora gossypiella 813-814
Isothiocyanate —
allyl, physical constants 127
crotonyl —
in Chinese colza seed 127-132
physical constants 127
para-oxybenzyl, difference from crotonyl
isothiocyanate 135
Ivey, J. E., and Kaupp, B. F. (paper): Study
of Some Poultry Feed Mixtures with
Reference to Their Potential Acidity and
Their Potential Alkalinity: 1 141-149
Ivy. See Hedera helix.
Jack pine. See Pinus banksiana.
Jagger, Ivan C. (paper) — Page
Sclerotinia minor, n. sp., the Cause of a
Decay of Lettuce, Celery, and Other
Crops 331-334
Transmissible Mosaic Disease of Lettuce,
A 737-740
Japan clover nodule bacteria cultures, effect
on milk 550
Jasmine, yellow bush. See Jasminum nudi-
florum.
Jasminum nudiflorum, influence of cold in
stimulating growth 158
Job's tears. See Coix lachryma.
Johnson, Alice, andHendry.Mary F. (paper):
Carbon-Dioxid Content of Barn Air 405-408
Johnson, James (paper): Fusarium-Wilt of
Tobacco 51S-S36
Jones, Fred Reuel, and Drechsler, Charles
(paper): Crownwart of Alfalfa Caused by
Urophlyctis alfalfae 295-324
Juglans, host of Gibberella saubinetii 16
Juniperus virginiana, immunity from hyper-
trophied lenticels 255
Kaupp, B. F., and Ivey, J. E. (paper): Study
of Some Poultry Feed Mixtures with Refer-
ence to Their Potential Acidity and Their
Potential Alkalinity: 1 141-149
Kclley, W. P., and Cummins, A. B. (paper):
Composition of Normal and Mottled Citrus
Leaves 161-191
Kennard, D. C, et al. (paper): Green Feed
versus Antiseptics as a Preventive of In-
testinal Disorders of Growing Chicks 869-873
Kopeloff, Nicholas, Perkins, H. Z. E., and
Welcome, C. J. (paper): Further Studies in
the Deterioration of Sugars in Storage. . . 637-653
Koser, Stewart A. (paper): A Bacteriological
Study of Canned Ripe Olives 375-379
Kosteleyzkya spp., food plants of —
Crocidosema plebiana 822
Gelechia hibiscella 810-81 1
Meskca thyridinae 828-829
Kundmannia sicula, host Urophlyctis alfalfae. 308
Lactic acid. See Acid, lactic.
Lagorotis —
diprioni, parasite of Neodiprion lecontei. . 757-758
virginiana, parasite of Neodiprion lecon-
tei 757-758
Lantana horrida, food plant of Borkkausenia
diveni 815
Larch, American. See Larix americana.
Larix —
americana, host of Neodiprion lecontei 757
laricina —
hypertrophied lenticels 255-266
influence of cold in stimulating growth
(PI. 21) 151-160
Larunda palmii. Syn. Zenodoxus palmii.
Larvae of aquatic insects, susceptibility to
copper salts 198
Laspeyresia tristrigana, similarity to Pectino-
phora gossypiella 824-825
Latshaw, W. L., et al. (paper): Relation of
the Calcium Content of Some Kansas Soils
to the Soil Reaction as Determined by the
Electrometric Titration 855-868
Oct. i, 1920-Mar. is, 1921
Index
899
Laiuca sativa — Page
host of Sclerotinia minor 33 1-334
transmissible mosaic disease 737-74°
Leafhopper, beet. See Euteitix tenella.
LeConte's Sawfly, an Enemy of Young Pines
(paper) 741-760
Leek. See A Ilium porrum.
Leguminous plants, nodule bacteria 543-556
Lemon leaves, composition 167-174
Lenticels, hypertrophied, on the roots of coni-
fers 253-266
Lepidoptera resembling pink bollworm. . . . 807-836
Lettuce. See Latuca saliva.
Leucin in potato sprouts 624
Life history and habits of the beet leafhop-
per 245-252
Ligustrum aurea, composition of green and
albino leaves 182
Lime —
carbonate, effect on availability of iron in
soil 47-49
cause of chlorosis in plants 33-62
chlorinated, influence on effect of copper
sulphate in water 202
effect on —
availability of potassium 617
growth of rice 44-47
in plants grown with ferrous sulphate and
gypsum 42
in plants grown with sodium bicarbonate
and sprayed with lime and iron salts 46
Limnaea —
bulimoides, susceptibility to copper salts. . 199-200
(Galba) bulimoides, susceptibility to various
salt solutions 196-208
proximo, rowelli, susceptibility to copper
salts 199
spp., intermediate hosts of flukes 193-208
Liver flukes, control by destruction of inter-
mediate host 193-208
Loblolly pine. See Pinus taeda.
Locust, black, nodule bacteria cultures, effect
on milk 550
Lohnis, F., and Hansen, Roy (paper): Nod-
ule Bacteria of Leguminous Plants 543-556
Longleaf pine. See Pinus palustris.
Lung flukes, control by destruction of inter-
mediate host 193-208
Lupine nodule bacteria cultures, effect on
milk SSo
Lupinus —
angustifolius, calcifugous 34
luleus, calcifugous nature 34
Lycaenidae, pests of Malvaceae 834
Lysin in potato protein 624
McCool, M. M., and Bouyoucos, George J.
(paper): Measurement of the Amount of
Water That Seeds Cause to Become Unfree
and Their Water-Soluble Material 587-593
Macrosporium —
parasiticum, parasite of Allium spp 6S7-68S
porri, parasite of Allium spp 687-688
Magnesia in plants grown with —
ferrous sulphate and gypsum 42
sodium bicarbonate and sprayed with lime
and iron salts 46
Magnesium — Page
in bean seedlings g*g
in cropped and uncropped soils 663-667
in normal and mottled citrus leaves 166-190
in soil extract 387-394
in southern poultry feeds I43
oxid in potato tubers, skins, and sprouts. . . 633
Magness, J. R., and Hawkins, Lon A. (paper):
Some Changes in Florida Grapefruit in
Storaee 357-373
Maize. See Zea mays.
Malus coronaria, influence of cold in stimu-
lating growth (PI. 22) 151-160
Mahastrum —
spicalum, food plant of Crocidosema ple-
beiana g22
sp., food plant of Telphusa mariona 81a
Malvaviscus drummondii, food plant of —
Bagisara reclifascia g,*
Crocidosema plebeiana g22
Heliothis obsoleta 8,,
Meskea thyridinae 828-829
Platynota roslrana g2I
Manganese —
chlorid, effect on formation of potato tubers1. 633
in normal and mottled citrus leaves 167
Mankatta ostrtnella. Syn. Moodna ostrinella.
Maple. See Acer negundo.
Maritime pine. See Pinus pinaster.
Marsh, C. Dwight, and Clawson, A. B. (pa-
per) : Daubentonia longifolia (Coffee Bean) ,
a Poisonous Plant 507-514
Martin, J. C, and Stewart, G. R. (paper):
Effect of Various Crops upon the Water
Extract of a Typical Silty Clay Loam
Soil 663-667
Martin, J. C, and Hoagland, D. R. (paper):
Effect of Season and Crop Growth on the
Physical State of the Soil 397-404
Martin, J. C, et al. (paper): Relation of the
Soil Solution to the Soil Extract 381-395
Measurement of the Amount of Water That
Seeds Cause to Become Unfree and Their
Water-Soluble Material (paper) 587-593
Medicago —
denticulata, host of Urophlyctis alfalfae in
Argentina 296
falcata, host of Urophlyctis alfalfae 296
sativa, host of —
Gibberella saubinetii 16
Urophlyctis alfalfae 295-324
Melania, intermediate host of Paragonimus,
Metagonimus, and Clonorchis 198
Melanotaenium alismatis. Syn. Physoderma
maculare.
Melilotus alba, growth of Gibberella saubinetii
cultures on 18
Melon fly. See Bactrocera cucurbitae.
Mentha aquatica, host of Physoderma menthae. 313
Meraporus —
calandrae, parasite of Sitophilus oryza 422
reguisitus, parasite of Sitophilus oryza 422
utibilis, parasite of Sitophilus oryza 422
Mercuric bichlorid, toxity to snails 196
Meskea dyspteraria, similarity to Pectinophora
gossypiclla 828-829
9<x>
Journal of Agricultural Research
Vol. XX
Page
Metagonimus, parasite of Melania 198
"Mexican ebony." See Siderocarpus flexi-
caulis.
Microbracon sp., parasite of Pyrausta sp 843
Microorganisms, relation to deterioration of
sugars in storage 637-653
Middleton, William (paper): LeConte's Saw-
fly, an Enemy of Young Pines 741-760
Mildew, downy, of maize. See Sclerospora
spp.
Mimosa berlandieri, food plant of Gelechia neo-
trophella: 811-812
Mimusops elengi, host fruit of Ceratitis capi-
tala 425
Mineral content of —
bean cotyledons, utilization in soil and in
distilled water 875-880
southern poultry feeds 143
Miscanthusjaponicus, susceptibility fo Sclero-
spora spontanea 671
Moisture —
effect on freezing-point depression of soil. 390-391
in Chinese colza seed 127
influence on formaldehyde injury to seed
wheat 238-240
in sugars in storage 638-653
in sunflower silage 883
in sweetcorn 799
relation to hypertrophied lenticels on the
roots of conifers 253-266
soil, effect on Fusarium -wilt of tobacco. . . . 529
Molasses, availability to rice plants in calcare-
ous and noncalcareous soils 50-54
"Molasses, ferric," availability to rice plants
in calcareous and noncalcareous soils S°~S4
Mold. See Aspergillus terreus.
Molds-
attacking wheat treated with formaldehyde 215
in sugars in storage 638-653
Momordica charantia, host of Chaelodacus cu-
curbitae 43 1
Monoamino nitrogen in potato tubers, skins,
and sprouts 624, 628-634
Monilia sitophila, enzymic action 778
Moodna ostrinella, similarity to Peclinopliora
aossypiella 83 1-83 2
Mosaic disease of lettuce 737-740
Mugho pine. See Pinus mughus.
Mustard substitutes 117-140
Myrosin, effect on yield of volatile oil from
Chinese colza seed 130-131
Myzus persicae, carrier of mosaic disease of
lettuce 738-739
Navy bean nodule bacteria cultures, effect on
milk 550
Nebulium sp., seeds host of Sitophilus oryza. . 410
Neclaria (later Colonectria) graminicola, con-
idia cause of " snowmold " 19
Neidig, Ray E. (paper): Sugar Beet Top Si-
lage 537-542
Neidig, Ray E., Snyder, Roberts., and Hick-
man, C. W. (paper): Sunflower Silage Di-
gestion Experiment with Cattle and
Sheep 881-888
Page
Nelumbo lutea, not food plant of Pyrausta
ainsliei 838
N eocatolaccus allsiraliensis, parasite of Sito-
philus oryza 422
Neodip rion lecontci —
control 759-760
description 741-750
distribution 758
economic importance 758-759
effect of weather 753_754
hosts 756
life history 75°-753
mating 7S4~755
oviposition 755-756
parasites 757-758
Neopales maera, parasite of Ncodiprion lecon-
lei 757-758
New Avocado Weevil from the Canal Zone,
A (paper) in-116
Nicotiana —
glauca, host of Fusarium oxysporum var.
nicotianae 524-525
rustica, host of Fusarium oxysporum var.
nicotianae 525
tabacum, host of Fusarium oxysporum var.
nicotianae, n. var 515-536
Nitrate —
copper, toxicity to snails 196
effect on availability of potassium 616-617
in soil extract 387-394
Nitrates in cropped and uncropped soils 663-667
Nitric acid. See Acid, nitric.
Nitrogen —
effect on availability of potassium 6i6-6r7
in allyl and crotonyl isothiocyanate 127
in feces of chicks 872-873
in normal and mottled citrus leaves 166-190
in plants grown with sodium bicarbonate
and sprayed with lime and iron salts .... 46
in potato tubers, skins, and sprouts. . 623, 628-634
in wheat, changes due to tempering 272-275
monoamino, in potato sprouts 624
Nitrogen-free extract in sunflower and com
silage 881-888
Noctua virescens. Syn. Helioihis virescens.
Noctuclia —
rufofascialis, similarity to Pectinophora
gossypiella 829-830
thalialis. Syn. .V. rufofascialis.
Noctuidae, similarity of certain species to
Pectinophora gossypiella 833
Nodule Bacteria of Leguminous Plants
(paper) 543"556
Nonadditive factors in correlation and causa-
tion 563-564
Nonlinear relations in correlation and causa-
tion 564-565
Nonprotein nitrogen in potato sprouts 623
Non-spore-forming bacilli in canned ripe
olives 377-379
Nutritive ratio of sunflower and corn silage . 881-888
Oats. See Avena saliva.
Odontites rubra, host of Uropklyctis magnus-
iana 313
Oct. i, 1920-Mar. 15, 1921
Index
901
Page
Oecophoridae, similarity of one species to
Pectinophora gossypiella 814-816
Oedemalophorus —
kellicotti, similarity of O. venapunctus 827
paleaceus, similarity of O. venapunctus 827
stramineus, similarity of O. venapunctus. . . . 827
venapunctus, n. sp 827-828
Oil, volatile, in Chinese colza seed 127-132
Okra. See Hibiscus esculentus.
Olethreutidae, similarity of certain species to
Pectinophora gossypiella 822
Olives, bacteria in cans 375~379
Olpidium viciae, cytological similarity to
Urophlyciis alfalfae 309
Onion Smudge (paper) 685-722
Opius —
flelcheri as a Parasite of the Melon Fly in
Hawaii (paper) 423-438
humilis, parasite of Ceratilis capitata 424
Organic —
iron compounds, availability to rice plants
in calcareous and noncalcareous soils 50-54
matter, effect on —
efficacy of chlorin disinfectants 89-110
Fusarium-wilt of tobacco 529
influence of copper sulphate in water. . 200-203
Orthoclase solutions, concentration of potas-
sium not a measure of availability to wheat
seedlings 615-621
Oryza saliva, growth on calcareous soil 38-58
Osmotic pressure —
in plants 156-157
of dry seeds 592-593
Overwintering of Gibberella saubinetii 14-15
Oxalate, ferric, avalability to rice plants in
calcareous and noncalcareous soils 50-54
Oxid—
calcium, in potato tubers, skins, and sprouts 633
magnesium, in potato tubers, skins, and
sprouts 633
potassium, absorption by plants 616-617
Oxidation of potassium, effect on availa-
bility 619-621
Oxygen, effect on hypertrophy of conifers. . 259-262
Pachyzancia bipunctalis, similarity to Pecti-
nophora gossypiella 830
Pak-choi. See Brassica campestris chinensis.
Paltodora similiella. Syn. Isophrictis simi-
liella.
(Panzeria) Pyrauslomyia penilalis, parasite of
Pyrausta ainsliei 843-844
Paraformaldehyde —
injury to seed wheat 211-244
physical properties 21S-223
Paragonimus —
lung fluke 193-195
parasite of Melania 198
Para-oxybenzyl isothiocyanate, difference
from crotonyl isothiocyanate 135
Paranthrene palmii. Syn. Zenodoxus palmii.
Pathological Anatomy of Potato Blackleg
(paper) 325-330
Peanut nodule bacteria cultures, effect on
milk 550
Pectinophora — Page
gossypiella, similar Depidoptera 807-836
malvella, similarity to P. gossypiella 809
Pediculoides ventricosus, parasite of Sito-
philus —
linearis 443
oryza 42i
Peltier, George L. (paper) : Influence of Tem-
perature and Humidity on the Growth of
Pseudomonas citri and Its Host Plants
and on Infection and Development of the
Disease 447-506
Penicillhim —
biforme, enzymic action 779
camemberti, enzymic action 778
glaucum, enzymic action 778-779
sp., attacking wheat treated with formalde-
hyde 215
Perilampus hyalinus, parasite of Neodiprion
lecontei 757-758
Perkins, H. Z. E., et al. (paper): Further
Studies in the Deterioration of Sugars in
Storage 637-653
Permanence of Differences in the Plots of an
Experimental Field (paper) 335-356
Pe-tsai. See Brassica campestris pekinsis.
Pkalaena bipunctalis. Syn. Pachyzancia bi-
punctalis.
Phalonia cephalanthana, n. sp 825-826
Phaloniidae, similarity of one species to Pec-
tinophora gossypiella 825-826
Phaseolus vulgaris, mineral content of cotyle-
dons 875-876
Phenylalanin in potato protein 624
Phenylthiourea in allyl and crotonyl isothio-
cyanate 127
Philips, A. G.t Carr, R. H, and Kennard,
D. C. (paper): Green Feed versus Anti-
septics as a Preventive of Intestinal Dis-
orders of Growing Chicks 869-873
Phelum pratense —
host of Gibberella saubinetii 16
shelter plant of Pyrausta ainsliei 839
Phoma alliicola —
parasite of A Ilium spp 687-688
similarity to Collctotrichum circinans 718
Phorocera —
claripennis, parasite of Neodiprion lecon~
te* 757-758
comstocki, parasite of Pyrausta sp 843
Phosphate —
calcium, effect on growth of plants 40-44
in normal and mottled citrus leaves 166-190
in soil extract 387-394
sodium, effect on availability of potas-
sium 616-617
Phosphoric acid. See Acid, phosphoric.
Phosphorus —
in bean cotyledons 878
in normal and mottled citrus leaves 166-190
in southern poultry feeds 143
water-soluble in wheat, changes due to
tempering 272-275
Phycitinae, similarity of certain species to
Pectinophora gossypiella 831-832
902
Journal of Agricultural Research
Vol. xx
Physa— Page
nuttalli, susceptibility to copper salts. . . . 199-200
occidentalis, susceptibility to copper salts. . 199
Physoderma —
agrosiidis, morphology Ji3~314
buimio, morphology 3;3
calami, morphology 314
(Cladochytrium) —
butomi, similarity to Vrophlyctis alfalfae. . 305
flammulae 3°5
maculate, similarity to Vrophlyctis alfalfae . 306
coinari, morphology 3 '3
eleochardis, morphology 313
gerhardti, morphology 313
graminis, morphology 3I3
hipurides, morphology 3 14
iridis, morphology 313
maculare, morphology 313
menthae, morphology 3 13
menyanthis, method of germination 314
(Protomyces) menyanthis, similarity to
Urophlyctis alfalfae 305
schroeteri, morphology 3I3
spargani, morphology 3r4
speciosum, morphology 314
vagans, morphology 3 '3
zeae-maydis, morphology 313
Physopsis, intermediate host of Schistosoma
haematobium and S. mansoni 198
Phytolacca, host of Gibberella saubinctii 16
Picea —
canadensis, hypertrophied lenticels 255-266
mariana, hypertrophied lenticels 255-266
pungens, hypertrophied lenticels 255-266
rubens, hypertrophied lenticels 255-266
Pigweed. See Amaranlhus hybridus.
Pimpinella nigra, host of Urophlyctis kricge-
riana 313
Piuc —
Austrian. See Pinus austriaca.
jack. See Pinus banksiana.
loblolly. See Pinus taeda.
longleaf. See Pinus palustris.
maritime. See Pinus pinaster.
mugho. See Pinus mughus.
red. See Pinus resinosa.
Scotch. See Pinus sylvestris.
scrub. See Pinus virginiana.
shore. See Pinus conlorta.
silver. See Pinus monticola.
western yellow. See Pinus pondcrosa.
white. See Pinus slrobus.
yellow. See Pinus ponderosa.
Pinkbollworm, similar lepidoptera 807-836
Pinus —
austriaca, host of Neodiprion leconiei 757
banksiana —
host of Neodiprion lecontei 756-757
hypertrophied lenticels 255-266
. caribaea, hypertrophied lenticels 255-266
conlorta, host of Neodiprion lecontei 757
coulteri, hypertrophied lenticels 255-266
eldarica, host of Neodiprion lecontei 757
excelsa, hypertrophied lenticels 255-266
marilima, hypertrophied lenticels 255
Pinus — Continued Page
monticola —
host of Neodiprion lecontei 757
hypertrophied lenticels 255-266
mughus, host of Neodiprion lecontei 757
palustris, host of Neodiprion lecontei 757
pinaster, calcifugous nature 34
ponderosa —
host of Neodiprion lecontei 757
hypertrophied lenticels 253-266
var. scopulorum, hypertrophied len-
ticels 253-266
resinosa —
host of Neodiprion lecontei 757
hypertrophied lenticels 255-266
rigida, hypertrophied lenticels 255-266
strobus —
host of Neodiprion lecontei 757
hypertrophied lenticels 255-266
sylvestris —
host of Neodiprion lecontei 757
hypertrophied lenticels 255-266
taeda, host of Neodiprion lecontei 757
virginiana —
host of Neodiprion lecontei 757
hypertrophied lenticels 255-266
Pipunculus —
industrius, parasite of Eulettix tenella 250-251
vagabundus, parasite of Euteltix tenella. . . 250-251
Planorbis calliogly plus —
intermediate host of Schistosoma haemato-
bium and .S". mansoni 198
susceptibility to copper salts 199
Plant growth, influence of cold 151-160
Plants.transpiration, correlation and causa-
tion 57S-585
Platyedra vilclla, similarity to P. gossypiella. . 809
Plalynota —
ftavedana, similarity to P. roslrana 82a
roslrana, similarity to Pectinophora gossy-
piella 821-823
Pod-borer, tamarind. See SUophilus linearis.
Polarization of sugars in storage 638-653
Polygonum —
hydropiperoides, food plant of Pyrausta
ainsliei 838
incarnalum. Syn. P. lapalhifolium.
lapathifolium, food plant of Pyrausta ains-
liei 838
pennsylvanicum, food plant of Pyrausta
ainsliei 837-844
persicaria, food plant of Pyrausta ainsliei. . . 838
Polynema eutetlixi, parasite of Euteltix tenella. 250
Poncirus trifoliata, influence of temperature
on —
development of Pseudomonas citri 483-488
growth 459~47i
rest period 459
Potash—
in plants grown with —
ferrous sulphate and gypsum 42
sodium bicarbonate and sprayed with
lime and iron salts 46
Sec Potassium.
Oct. i, 1920-Mar. is, 1921
Index
903
Potassium — Page
chlorid —
adsorption by plants 616-61 7
effect on concentration of soil solution 393
concentration in orthoclase solutions not a
measure of its availability to wheat seed-
lings 615-621
fluorid, effect on yield of volatile oil from
Chinese colza seed 130-131
hydroxid, effect on yield of volatile oil from
Chinese colza seed 130-131
in cropped and uncropped soils 663-667
in normal and mottled citrus leaves 1 66- 1 90
in soil extract 387-394
in southern poultry feeds 143
oxid, absorption by plants 616-617
sulphate, effect on concentration of soil
extract 38S-389
Potato. See Solatium tuberosum.
Poultry feed mixtures, potential acidity and
alkalinity 141-149
Privet. See Ligustrum aurea.
Prolin in potato protein 624
Propionic acid. See Acid, propionic.
Protein —
calories in various poultry feeds 147
crystals due to potato blackleg 326-330
in Chinese colza seed 127
in sugar beet top silage 538-540
in sunflower and com silage 881-8S8
Proteins, effect on freezing-point depression
of seeds 593
Proteus bacilli in canned ripe olives 377-379
Pruning —
influence in stimulating growth of plants. . 155
top, effect on hypertrophy of conifers 258
Psecadia delliella. Syn. Ethmia delliella.
Pseitdomon as —
citri, influence of temperature and hu-
midity 447-506
japonica, possible name for cowpea-soybean
nodule bacteria 551
radkicola. Syn. Bacillus radicicola.
tumefaciens, causal organism of bacterial
crowngall 295
Pterophoridac, similarity of one species to
Peclinophora gossypiella 827-828
Pyralidae, similarity of certain species to
Pectinophora gossypiella 828-834
Pyrausta —
ainsliei —
control 843-844
hosts 837
seasonal history 839-840
mibilalis, similarity of P. ainsliei 837
obumbratalis. Syn. P. ainsliei.
penitalis, similarity of P. ainsliei 837
Pyraustinae, similarity of certain species to
Pectinophora gossypiella 829-S30
Pyraustomyia penitalis, parasite of Pyrausta
ainsliei 843-844
Pyroderces rileyi, similarity to —
Peclinophora gossypiella 820
Telphusa mariona 813
Zenodochium citricolella 818-819
Ragweed. See Ambrosia trifida and A. arte-
misiaefolia.
Ram, influence in dissemination of spores of Page
Gibber ella saubinetii 12-13
Rattlesnake venin, inefficacy of echinacea
against 75-77
Reaction, soil, effect on Fusarium-wilt of to-
bacco 528-529
Red clover nodule bacteria cultures, effect on
mi]k 550
Red pine. See Pinus resinosa.
Reducing sugars —
in grapefruit 359-373
in sugars in storage 638-653
Relation of the Calcium Content of Some Kan-
sas Soils to the Soil Reaction as Determined
by the Electrometric Titration (paper). 855-868
Relation of the Soil Solution to the Soil Ex-
tract (paper) 381-395
"Resting," relation to chilling of plants 159
Rhizobaclerium japonicum, name given by
Kirchner to soybean nodule bacterium 551
Rhizopus —
nigricans, similarity to R. trilici 761
sp., attacking wheat treated with formalde-
hyde 215
tritici, secretion and action of amylase. . . 761-789
Rhoads, Arthur S., et al. (paper): Hypertro-
phied Lenticels on the Roots of Conifers and
Their Relation to Moisture and Aeration 253-266
Rhus copallina, water lenticels 256
Rhyncholus lalinasus. Syn. Caulophilus lat-
inasus.
Rhynchophora attacking corn in storage. . 605-614
Rhynchophorus linearis. Syn. Sitophilus line-
aris.
Rice, growth on calcareous soil 38-58
Rice Weevil, (Calandra) Sitophilus oryza (pa-
per) 409-422
Ripening of sweetcorn, effect of climatic tem-
perature 795-805
Robinia, host of Gibberella saubinetii 16
Rootrot of cereals caused by Fusarium spp.
and Gibberella saubinetii 2
Rottboellia exaltata, immunity to Sclerospora
spontanea 671
Rubus, host of Gibberella saubinetii 16
Rudbeckia —
pallida. Syn. Brauneria atrorubens.
purpurea. Syn. Brauneria purpurea.
Rumex —
britannica, host of Urophlyctis major 313
crispus, food plant of Pyrausta ainsliei 838
scutatus, host of Urophlyctis rubsaameni 313
Rusk citrange, influence of —
humidity on development of Pseudomonas
citri 494-497
temperature on development of Pseudo-
monas citri 471-488
temperature on growth 459-471
Russian thistle. See Salsola kali var. tenui-
folia.
Rye. See Secale cereale.
Saccharum spontaneum, host of Sclerospora
spp 669-684
Salobrana tecomae. Syn. Clydonopteron teco-
mae.
904
Journal of Agricultural Research
Vol. xx
Salsola kali var. tenuifolia, host of Eutetlix Page
tenella 247
Sambucus canadensis, water lenticels 256
Sanicula —
menziesii, host of Urophlyctis pluriannu-
latus 312
spp., blisterlike galls on 299
Sap-
composition in orange leaves 182-187
pressure, relation to hypertrophy 260
Sarothamnus scoparius, growth in calcareous
soil 35
Sawfly, LeConte's. See Neodiprion lecontei.
"Scab" of onions. See Colleiotrichum circi-
nans.
Scavenger worm. See Pyroderces rileyi.
Schinia rectifascia. Syn. Bagisara rectifascia.
Schistosoma —
haematobium, parasite of Bullinus, Planor-
bis, and Physopsis 198
japonicum —
cause of schistosomiasis 193, 198
parasite of Blanfordia 198
mansoni, parasite of Bullinus, Planorbis,
and Physopsis 198
Schwartz, Benjamin (paper): Effects of X-
Rays on Trichina 845-854
Schistosomiasis, caused by blood flukes 193
Scirpus, host of Gibberella saubinctii 16
Sclerospora —
graminicola, conidia 679
javanica, difference from Sclerospora philip-
pinensis 679
maydis, difference from Sclerospora philip-
pinensis 679
philippinensis, causal organism of downy
mildew of maize 669-684
sacchari, conidia 679
spontanea, n. sp 669-684
Sclerotinia minor, n. sp., the Cause of a Decay
of Lettuce, Celery, and Other Crops
(paper) 331-334
Scofield, C. S., and Harris, J. Arthur (paper):
Permanence of Differences in the Plots of
an Experimental Field 335-356
Scotch pine. See Pinus sylirstris.
Scrub pine. See Pinus virginiana.
Season, effect on —
physical state of soil 397-404
ripening of sweetcorn 798-799
Secale cereale, host of Gibberella saubinelii . . , . 1-32
Seed wheat, injury from formaldehyde. . . . 209-244
Seeds, mustard, substitutes 117-140
Seedling-blight of cereals caused by Gibberella
saubinetii 5
Seedlings, wheat, availability of potassium in
orthoclase solutions 615-621
Septicemia, inefficacy of echinacea against.. 72-74
Sequoia spp., reported immunity from hyper-
trophied lenticels 255
Serum, blood, effect on efficacy of chlorin
disinfectants 89-110
Sesbania cavanillesii. Syn. Daubenlcmia longi-
folia.
Shallots. See Allium ascalonicum.
Shaw, R. H., and Wright, P. A. (paper): A Page
Comparative Study of the Composition of
the Sunflower and Corn Plants at Differ-
ent Stages of Growth 787-793
Shore pine. See Pinus conlorla.
Shrinkage of grapefruit in storage 360-372
Sida sp., food plant of Telphusa mariona 812
Siderocarpusjlexicaulis, food plant of Aedemo-
ses haesitans 816
Silage —
sugar beet top 537-542
sunflower, digestion 881-888
Silicate, calcium, effect on growth of plants . . . 40-44
Silica —
in potato tubers, skins, and sprouts 633
in normal and mottled citrus leaves 166-190
in plants grown with ferrous sulphate and
gypsum 42
in plants grown with sodium bicarbonate
and sprayed with lime and iron salts 46
in soil extract 387-394
Silver pine. See Pinus monlicola.
Sinapis —
alba, white mustard 117, 123, 125-126
brassicata, classification 119
chinensis, classification 119
juncea var. napiformis. Syn. Brassica
napiformis.
pekinensis, classification 1 19
Silophilus —
granarius —
allied to S. oryza 409
description 613-614
distinguishing characters 605-606
synonymy 613
linearis —
life history 440-443
parasite of Tamarindus indicus 439-446
parasites 443
oryza —
control 423
description 610-612
distinguishing characters 605-606
food 410-41 1
life history 41 1-421
parasites 421-422
synonymy 610
Skins, Irish potato, composition 623-635
Smartweed borer. See Pyrausta ainsliei.
Smudge, onion. See Colleiotrichum. circinans,
' 'Snowmold," caused by Fusarium spp 19-20
Snyder, Roberts., et al. (paper): Sunflower
Digestion Experiment with Cattle and
Sheep 881-888
Soda in plants grown with —
ferrous sulphate and gypsum 42
sodium bicarbonate and sprayed with lime
and iron salts 46
Sodium —
bicarbonate —
effect on growth of rice 44-47
value as disinfectant 86-110
hypochlorite, value as disinfectant 86-110
in normal and mottled citrus leaves 16G-190
in soil extract 3S7-394
Oct. i, 1920-Mar. is, 1921
Index
905
Sodium — Continued. Page
phosphate, effect on availability of potas-
sium 616-617
in southern poultry feeds 143
Sodium-toluene-sulphon-chloramid. See
"Chloramin T."
Soil-
availability of iron 33-62
effect of —
carbonate of lime on availability of iron. . 47-49
season and crop growth on physical
state 397-404
various crops on water extract 663-667
effect on composition of potato tubers,
skins, and sprouts 632-634
moisture, effect on Fusarium-wilt of tobacco 529
reaction, effect on Fusarium-wilt of to-
bacco 528-529
relation of calcium content to reaction. . . 855-868
solution, relation to soil extract 381-395
temperature, effect on Fusarium-wilt of
tobacco 527-528
Solanin in potato sprouts 623
Solatium tuberosum —
attacked by blackleg 3zS"33°
berries host of Gibber ella saubinetii 16
composition of tubers, skins, and sprouts. . 623-635
effect on water extract of soil 663-667
vascular discoloration of tubers 277-294
Solidago spp., shelter plants of Pyrausta
ainsliei 839
Solids, soluble, in grapefruit 359-372
Soluble solids in grapefruit 359-372
Solution, soil, relation to soil extract 381-395
Somasia helianthana. Syn. Eucosma helian-
thana.
Some Changes in Florida Grapefruit in Stor-
age (paper) 357~373
Some Lepidoptera Likely to Be Confused
with the Pink Bollworm (paper) 807-836
Sorghum —
susceptibility to formaldehyde injury 241
See Andropogon sorghum.
Soybean-cowpea bacteria, comparison with
Bacillus radicicola and B. radiobacter 545-554
Soybean nodule bacteria cultures, effect on
milk 550
Spalangionorpha fasciatipennis, parasite of
Sitophilus oryza 422
Spatkimeitenis spinigcra, parasite of Neodi-
prion lecontei 757-758
Spelt. See Triticum spelta.
Species, new 114, 333, 431, 678, 811, 812-813,
814-816, 818-819, 823-824, 825-826, 827-828
Spore-forming bacilli in canned ripe olives. 377-379
Sprouts, Irish potato, composition 623-635
Stahl, C. F. (paper): Studies on the Life
History and Habits of the Beet Leafhop-
per 245-252
Staphylococci in canned ripe olives 377~379
Staphylococcus aureus, effect of chlorin dis-
infectants upon 88-110
Starch-
hydrolysis by Rhizopus tritici 765-783
in Chinese colza seed 127
Starch — Continued. Page
in plants, influence of cold in transforming
to sugar 153-154
in sweetcorn .• 795-805
Stenomidae, similarity of one species to
Pectinophora gossypiella 816-81 7
Stewart, G. R., and Martin, J. C. (paper):
Effect of Various Crops upon the Water
Extract of a Typical Silty Clay Loam
Soil 663-667
Stewart G. R.,etal (paper): Relation of the
Soil Solution to the Soil Extract 381-395
Stigmonota tristrigana. Syn. Laspeyresia
tristrigana.
Stipa, host of Gibberella saubinetii 16
Stizolobium vines, availability of iron to rice
plants in calcareous and noncalcareous soils 50-54
Storage, effect on —
Colletotrichum circinans 713-716
deterioration of sugars 637-653
grapefruit 357"373
Strophostyles nodule bacteria cultures, effect
on milk 550
Strymon melinus, pest of Malvaceae.' 834
Studies in Mustard Seeds and Substitutes: I.
Chinese Colza (Brassicacampestris chinolei-
fera Viehoever) (paper) 117-140
Studies on the Life History and Habits of the
Beet Leafhopper (paper) 245-252
Study of Some Poultry Feed Mixtures with
Reference to Their Potential Acidity and
Their Potential Alkalinity: I. (paper)... 141-149
"Subculoyd Inula and Echinacea," medici-
nal properties 65-84
Sucrose in grapefruit 359-372
Sugar Beet Top Silage (paper) 537-542
Sugar —
in grapefruit 359-372
in plants, influence of cold in transforming
from starch 153-154
in sweetcorn 795-805
Sugars, deterioration in storage 637-653
Sulphate —
calcium, effect on —
availability of potassium 616-617
growth of plants 40-44
copper, toxity to snails 196-208
ferrous —
and molasses, availability to rice plants
in calcareous and noncalcareous soils. . 50-54
availability to rice plants in calcareous
and noncalcareous plants 50-54
effect on action of gypsum 38-44
in normal and mottled citrus leaves 166-190
in soil extract 387-394
potassium, effect on concentration of soil
extract 388-389
Sulphur —
in normal and mottled citrus leaves 166-190
in plants grown with ferrous sulphate and
gypsum 42
in southern poultry feeds 143
Sunflower plants, comparison with corn for
silage 787-793
oo6
Journal of Agricultural Research
Vol. XX
Sunflower Silage Digestion Experiment with Page
Cattle and Sheep (paper) 881-888
Swanson, C. O., Latshaw, W. L., and Tague,
E. L. (paper): Relation of the Calcium
Content of Some Kansas Soils to the Soil
Reaction as Determined by the Electro-
metric Titration 835-868
Sweet clover nodule bacteria cultures, effect
on milk 55°
Sweetcorn, effect of climatic temperature on
ripening processes 795-805
Swine, inheritance of syndactylism 595-604
Syndactylism, black, and dilution in swine,
inheritance of 595-604
Tague, E. L. (paper): Changes Taking Place
in the Tempering of Wheat 271-275
Tague, E. L-, et al. (paper): Relation of the
Calcium Content of Some Kansas Soils to
the Soil Exaction as Determined by the
Electrometric Titration 855-868
Tamarack. See Larix laricina.
Tamarind Pod-Borer, Sitophilus linearis
(Herbst) (paper) 439-446
Tamarind. See Tamarindus indicus.
Tamarindus indicus, host of Sitophilus
linearis 439-446
Tamarrha bittenella. Syn. Ethmia bitlenella.
"Tan disease" of fruit trees 263
Tannate, ferric, availability to rice plants in
calcareous and noncalcareous soils 50-54
Tartaric acid. See Acid, tartaric.
Tartrate, ferric —
availability to rice plants in calcareous and
noncalcareous soils 50-54
effect on growth of rice 42-44
Taius —
brevi folia, hypertrophied lcnticels 255-266
cuspidata, hypertrophied lenticels 255-266
spp., reported immunity from hypertro-
phied lenticels 255
Tecoma radicans, food plant of Clydonopteron
tecomae 832
Telephusa mariona, n. sp 812-813
Temperature —
at which fruit buds freeze 655-662
climatic, effect on ripening processes in
sweetcorn 795-805
effect on —
carbohydrate transformation in resting
potato tubers 623
formaldehyde injury to seed wheat. . . . 236-240
growth of Collelolrichum circinans 696-697
growth of Pseudomonas cilri 447-506
hydrolysis of starch by Rhizopus tritici. . . 767-
768,777-778
tempering of wheat 272-275
influence of copper sulphate on organisms
in water 200-201
relation to deterioration of sugars in
storage 642-653
to which soils can be cooled without freez-
ing 267-269
soil, effect on Fusarium-wilt of tobacco. . 527-528
Tempering of wheat 271-275
Tenebroides maurilanicus, parasite of Sitophi-
lus oryza 422
Teosinte. See Euchlaena luzurians. Page
Teras tostrana. Syn. Platynoia rostrana.
Terminalia catappa, host of Ceratiiis capitata. 423
Tetanus, inefficacy of echinacea against 67-70
Tetrachlorid , carbon, effect in stimulating
sprouting of potato tubers 623
Thiocyanate, allyl, formation during macera-
tion of Chinese colza seed 131
Thiodia hclianthana. Syn. Eucosma helian-
thana.
Thiourea in allyl and crotonyl isothiocyanate . 127
Thiourethane, allyl, formation during mac-
eration of Chinese colza 131
Thistle, Russian. Sec Salsola kaliv&r. tenui-
folia.
Thuja spp., reported immunity from hyper-
trophied lenticels 355
Tigbee. See Coix lacltryma-jobi.
Tilley, F. W. (paper): Investigations of the
Germicidal Value of Some of the Chlorin
Disinfectants 85-110
Timothy. See Phleum pratense.
Titration, electrometric, indication of relation
of calcium content of soil to reaction 855-868
Tobacco stems, availability of iron to rice
plants in calcareous and noncalcareous soils. 50-54
Top pruning, effect on hypertrophy of coni-
fers 258
Tobacco. See Nicoliana iabacum.
Tortricidae, similarity of cetaain species to
Peclinophora gossypiella 821-822
Trailing arbutas. See Epigaea repens.
Transmissible Mosaic Disease of Lettuce, A
(paper) 737~740
Transpiration —
effect on hypertrophy of conifers 259-262
of plants, correlation and causation 575-585
Trichinae, effect of X-rays 845-854
Trichinella spiralis, effect of X-rays 845-854
Trifolium —
monlanum, host of Urophlyciis bohemica . . . 313
spp., hosts of Gibberella saubinetii 16
Triticum —
repens, host of Gibberella saubinetii 16
spelta, host of Gibberella saubinetii 1-32
spp.—
hosts of Giberella saubinetii 1-32
seed, injury from formaldehyde 209-244
seedlings, availability of potassium in
orthoclase solutions 615-621
Tropical almond. See Terminalia catappa.
Trumpet flower vine. See Tecoma radicans.
Trypanosoma equipcrdum, inefficacy of echi-
nacea against 80-82
Trypanosomiasis, inefficacy of echinacea
against 80-82
Tsuga canadensis, hypertrophied lenticels. 255-266
Tubercle bacillus, effect of chlorin disinfect-
ants 98-100
Tuberculosis, inefficacy of echinacea against. 77-79
"Tuberin" in potato sprouts 624
Tubers, Irish potato, composition 623-635
Turnips. See Brassica rapa.
Thyridinae, similarity of one species to Pecli-
nophora gossypiella 828-S29
Oct. i, 1920-Mar. 15, 1921
Index
907
Typha— Page
latifolia, shelter plant of Pyrausla ainsliei. . S39
sp., food plant of Dicymotomia julianalis. 831
Tyrosin in potato sprouts 624
Ulmus, host of Gibberella saubinetii 16
Uranolcs melinus. Syn. Strymon melinus.
Urocyslis cepulae, similarity to Collelotrichum
circinans 718
Urophlyctis —
alfalfae, parasite of alfalfa 293-324
bohemica, haustoria 313
(Cladochytrium) pulposa, parasite on Chen-
opodium spp 300
hemisphaerica, similarity of U. alfalfae in
growth 303
kriegeriana —
haustoria 313
Syn. U. hemisphaerica.
leproidea. similarity of U. alfalfae in growth . 303
magnusiana, haustoria 313
major, haustoria 313
{Physoderma) leproidea, parasite of beets. . 300
pluriannulatus , similarity to U. alfalfae. . . . 312
pulposa —
apical apparatus on vegetative cells 306
haustoria 3 13
rubsaameni —
haustoria 313
nuclear behavior 308
Vaccinium —
corymbosum, influence of cold in stimulat-
ing growth 156-160
sp., calcifugous nature 34
Valerianate, ferric, availability to rice plants
in calcareous and noncalcareous soils 50-54
Valeric acid. See Acid, valeric.
Valin in potato protein 624
Variations in Colletotrichum gloeosporioides
(paper) 723-736
Varietal resistance to Fusarium-wilt of
tobacco S3°SS3
Variety, new 118-140,521,525-536
Vascular Discoloration of Irish Potato Tubers
(paper) 277-294
Ventilation in barns 405-408
Vermicularia —
circinans. Syn. Collelotrichum circinans.
gloeosporioides. Syn. Colletotrichum gloeo-
sporioides.
" Venniculariose." See Colletotrichum cir-
cinans.
Veriicillium albo-alrum, cause of vascular
necrosis of potato tubers 277
Vetch nodule bacteria cultures, effect on milk. 550
Viburnum americanum, influence of cold in
stimulating growth (PI. 21) 151-160
Vicia faba —
composition of green and albino leaves. . . . 182
effect on water extract of soil 663-667
Viehoever, Amo, Clevenger, Joseph F., and
Ewing, Clare Olin (paper): Studies in
Mustard Seeds and Substitutes: I. Chinese
Colza (Brassica campestris chinoleifera
Viehoever) 117-140
Page
Volatile oil in Chinese colza seed 127-132
Volutella circinans. Syn. Colletotrichum
circinans.
Walker, J. C. (paper): Onion Smudge 685-722
Water-
caused to become unfree by seeds 587-593
effect on —
availability of iron in soil 54-58
hypertrophy of conifers 258-262
tempering of wheat 272-275
extract of soil, effect of various crops .... 663-667
Water-soluble —
nitrates in cropped and uncropped soils . . 663-667
phosphorus in wheat, changes due to tem-
pering 272-275
Weed, beggar, nodule bacteria cultures,
effect on milk 550
Weevil —
avocado. See Heilipus lauri.
rice. SeeSilophilus oryza.
Welcome, C. J., etal. (paper): Further Studies
in the Deterioration of Sugars in Storage. 637-653
West, Frank L., and Edlefsen, N. E. (paper):
Freezing of Fruit Buds 655-662
Western yellow pine. See Pinus ponderosa.
Weston, William H. (paper): Another Coni-
dial Sclerospora of PhilippineMaize 669-684
Wheat-
tempering 271-275
See Triticum spp.
White pine. See Pinus strobus.
Wild crab. See Malus coronaria.
Willard, H. F. (paper): Opius fletcheri as a
Parasite of the Melon Fly in Hawaii 423-438
Wind, influence in dissemination of spores of
Gibberella saubinetii n-12
Wissadula —
lozani, food plant of Zenodoxus palmii 826
sp., food plant of Telphusa mariona 812
Worm, scavenger. See Pyroderces rileyi.
Wright, P. A., and Shaw, R. H. (paper): A
Comparative Study of the Composition of
the Sunflower and Com Plants at Different
Stages of Growth 787-793
Wright, Sewall (paper): Correlation and
Causation 557-585
Xanthium communis, shelter plant of Py-
rausta ainsliei 839
X-rays, effect on trichinae 845-854
Yeasts in canned ripe olives 377"379
Yellow pine. See Pinus ponderosa.
Zea mays —
effect on water extract of soils 663-667
host of Physoderma zeac-maydis 3 T3
host of Sclerospora spontaneum 669-684
in storage, attacked by Rhynehophora . . 605-614
shelter plant of Pyrausla ainsliei 839
Zenodochium citricolella, similarity to Pec-
tinophora gossypiella 817-81S
Zenodoxus palmii, similarity to Peciino-
phora gossypiella 826-827
o
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