){T.07S V; AGRICULTURAL RESEARCH Volume XXIl OCTOBER I— DECEMBER 3, 192 1 PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINGTON, D. C. CONTENTS Page Occurrence of Quercetin in Emerson's Brown- Husked Type of Maize. Charles E. Sando and H. H. Bartleit i Biological Analysis of the Seed of the Georgia Velvet Bean, Stilzo- lobium deeringianum. BarnETT Sure and J. W. Read (15 text figures) 5 Effect of Soil Temperature upon the Development of Nodules on the Roots of Certain Legumes. FrEd reuel, Jones and W. B. TiSDALE (4 text figures; 3 plates) 17 Influence of the Period of Transplanting Western White Pine Seed- lings upon Their Behavior in Nursery and Plantation. E. C. Rogers (7 text figures) 33 A Dryrot Canker of Sugar Beets. B. L. Richards (6 plates) .... 47 Comparative Vigor of Fj Wheat Crosses and Their Parents. Fred Griffee 53 Temperature and Humidity Studies of Some Fusaria Rots of the Irish Potato. R. W. Goss (2 plates) 65 Blackleg Potato Tuber-Rot under Irrigation. M. Shapovalov and H. A. Edson (6 plates) 81 Microscopic Study of Bacteria in Cheese. G. J. HucKER (i plate) . 93 Further Studies on Relation of Sulphates to Plant Growth and Composition. Harry G. Mii^l,er loi Soybean Mosaic. Max W. Gardner and James B. Kendrick (2 plates) HI Influence of the Plane of Nutrition on the Maintenance Require- ment of Cattle. F. B. Mumford, A. G. Hogan, and W. D. Salmon 115 Turnip Mosaic. Max W. Gardner and James B. Kendrick (i plate) 123 Hydrocyanic Acid in Sudan Grass. C. O. Swanson 125 Nutrient Requirements of Growing Chicks: Nutritive Deficiencies of Corn. F. E. Mussehl, J. W. Calvin, D. L. HalberslEben and R. M. SandsuEdt (ii text figures) 139 Aecial Stage of the Orange Leafrust of Wheat, Puccinia triticina Eriks. H. S. Jackson and E. B. Mains (i plate) 151 A Transmissible Mosaic Disease of Chinese Cabbage, Mustard, and Turnip. E. S. SchulTz (4 plates) 173 Flora of Corn Meal. Charles Thom and Edwin LeFevre 179 , Hopkins Host-Selection Principle as Related to Certain Ceramby- u. cid Beatles. F. C. Craighead 189 in IV Journal of Agricultural Research voi. xxii Page Notes on the Organic Acids of Pyrus coronaria, Rhus glabra, and Acer saccharum. Charles E. Sando and H. H. BartlETT. . . 221 FertiHty in Shropshire Sheep. Elmer Roberts 231 Relation of Soil Temperature and Other Factors to Onion Smut Infection. J. C. Walker and L. R. Jones (2 text figures; 3 plates) 235 A Physiological Study of Grapefruit Ripening and Storage. Lon A. Hawkins (i text figure) 263 Absorption of Copper from the Soil by Potato Plants. F. C. Cook 281 Pale Western Cutworm (Porosagrotis orthogonia Morr.) J. R. Parker, A. L. Strand, and H. L. Seamans (i text figure; 4 plates) 289 Biology of Embaphion muricatum. J. S. Wade and Adam H. Boving (3 text figures; 2 plates) 323 Genetic Behavior of the Spelt Form in Crosses between Triticum spelta and Triticum sativum. Clyde E- LeighTy and Sarkis Boshnakian (3 text figm-es; i plate) 335 Plum Blotch, a Disease of the Japanese Plum Caused by Phyllo- sticta congesta Heald and Wolf. John W. Roberts (2 text figures; i plate) 365 A Comparison of the Pectinase Produced by Dififerent Species of Rhizopus. L. L. Harter and J. L. Weimer (2 text figures) .... 371 Hemotoxins from Parasitic Worms. Benjamin Schwartz 379 Ash Content of the Awn, Rachis, Palea, and Kernel of Barley during Growth and Maturation. Harry V. Harlan and Merritt N, Pope (5 text figures) 433 Temperature Relations of Stone Fruit Fungi. Charles Brooks and J. S. CoolEy (24 text figures) 45 1 Transportation Rots of Stone Fruits as Influenced by Orchard Spraying. CharlES Brooks and D. F. Fisher (6 text figures) . 467 Storage of Coniferous Tree Seed. C. R. Tillotson (2 text figures) . 479 Susceptibility of the Different Varieties of Sweet Potatoes to Decay by Rhizopus nigricans and Rhizopus tritici. L. L. Harter and J. L. Weimer 511 Index 517 Vol. XXII OCXOBER 1, 1921 No. 1 JOURNAI. OF AGRICULTURAL RESEARCH CONXENXS PagQ Occurrence of Quercetin in Emerson's Brown-Husked Type of Maize -------- i CHARLES E. SANDO and H. H. BARTLETT (Contribution from Bureau of Plant Industry and University ot Michigan) Biological Analysis of tlie Seed of the Georgia Velvet Bean, Stilzolobium deeringianum - - - - - - 5 BARNETT SURE and J. W. READ (Contribution from Arkansas Agricultural Experiment Station) Effect of Soil Temperature upon the Development of Nodules on the Roots of Certain Legumes - - - 17 FRED REUEL JONES and W. B. TISDALE (Contribution from Bureau of Plant Industry and University of Wisconsin) Influence of the Period of Transplanting Western White Pine Seedlings upon Their Behavior in Nursery and Plantation - -- - - - - - -33 E. C. ROGERS (Contribution from Forest Service) A Dryrot Canker of Sugar Beets ----- 47 B. L. RICHARDS (Contribution from Utab Agricultural Experiment Station) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINOa^ON, D. C. 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, Sfafe 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, Agricuh tural Experiment Station of the University of Minnesota R. L. WATTS, Dean, School of Agriculture, and Director; Agricultural Experiment Station, Tb4 Pennsylvania Staf* College All correspondence regarding articles from the Department of AgricultiU'e 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. Lipnaan, New Jersey Agricultural Experiment Station, New Bnxnswick, N. J. J01M£ OF AGRICiriAL RESEARCH Vol. XXII Washington, D. C, October i, 1921 No. i OCCURRENCE OF QUERCETIN IN EMERSON'S BROWN- HUSKED TYPE OF MAIZE By Charles E. Sando, Junior Chemist, Office 0/ Physiological and Fermantation Investi- gations, Bureau of Plant Industry, United States Department of Agriculture, and H. H. BartlETT, Department of Botany, University of Michigan, Collaborator, Office of Physiological and Fermentation Investigations, Bureau of Plant Industry, United States Department of Agriculture ' In connection witli the genetical studies of pigmentation in maize which have been carried on for several years at Cornell University by Prof. R. A. Emerson and his students, a plan was made for the writers to collaborate in parallel biochemical studies in the isolation and identifi- cation of the pigments from material of known genetical constitution, the latter to be furnished as a by-product of the Cornell experiments. As a beginning in the work it seemed desirable to undertake a study of the character pair purple versus brown. These are two of the general plant colors recognized in the Cornell experiments, the series running as follows: I, purple; II, sun-red; III, dilute purple; IV, dilute sun-red; V, brown; and VI, green. Full details of the genetic relations of these types are being published by Enierson,^ to whose memoir the reader should refer for full details. Here it may suffice to say that purple is a color type which is uncommon in cultivation and infrequent in experi- mental cultm-es. It is distinguished from other types by the fact that some purple color is developed even in seedlings grown in the dark. At maturity nearly all parts are more or less purple, including the culm, the brace roots, all leaf sheaths, the husks, even the inner ones, the cob, and the staminate inflorescence. In intensity of coloration purple stands at the head of the series of color types. Material of this type was furnished by Prof. Emerson to Dr. John W. Calvin, of the University of Nebraska, before we took over the general problem, and we have therefore confined our attention for the present to the brown type, awaiting a report on his study of the purple type from Dr. Calvin. • In connection with our work with maize we have received numerous favors from Prof. R. A. Emerson and Dr. E. G. Anderson, to whom we tender our best thanks. ^ ' Emerson, R. A. the genetic relations of generai, plant colors in maize. N. Y. Cornell Agr. . ^ Exp. Sta. Mem. 39, 156, p., n col. pi. 1921. or: _______ ■* "Journal of Agricultural Research, Vol. XXII, No. i j^ Washington, D. C. Oct. i, 1921 ^^ zl. Key No. G-244 i CD Journal of Agricultural Research Vol. XXII. No. I A few preliminary tests of the purple pigment convinced us that it was an anthocyanin of which the nonsugar portion was of the same general group as cyanidin, isolated by Willstatter ^ and his students from several sources, including the cornflower, Centaurea. His proof of the easy chemical transition from the anthocyanin to the flavonol series led us to look for a member of the latter series in the brovvTi maize. In accordance with expectations, we were able to isolate a glucosid of quercetin. This discovery makes it seem exceedingly likely that the anthocyanin of the purple type is a corresponding glucosid of cyanidin. The brown color type is a still more unusual one than the purple. It first appeared in Emerson's ^ cultures as a segregate in the second generation of the cross purple X green and is unkno%vn outside this series of cultures. Seedlings and young plants are wholly green. As the flowering period approaches, a brown color appears in the lower sheaths, and at flowering time the culm, sheaths, husks, and staminate inflores- cences are brown. Light is not essential to the development of the color. Our material of the brown type consisted of husks. ISOLATION AND IDENTIFICATION OF THE FREE QUERCETIN Ground husks were extracted in a large Soxhlet apparatus with redis- tilled 95 per cent alcohol for about 72 hours, and the alcohol was evapo- rated off in vacuo. The thin aqueous sirup was filtered from tarry matter and the filtrate boiled with animal charcoal. By shaking with ether it gave an etherial solution containing a free (nonglucosidal) fla- vone which proved to be quercetin. The ether was evaporated off, and the residue, after being dried in a desiccator, was extracted in a paper thimble, first with benzene, to remove tarry colloids, oils, etc., and finally, for a short time, with ether. The latter solvent dissolved part of the quercetin but left the bulk of it in the thimble. This portion was dried and acetylated for an hour with anhydrous sodium acetate and acetic anhydrid. After purification the acetyl derivative was quanti- tatively hydrolyzed with sulphuric acid in glacial acetic acid. The reac- tion mixture was diluted and the recovered quercetin was washed with cold water. The results are given in Table I. Table I. — Data on hydrolysis , by sulphuric acid in glacial acetic-acid solution, of the acetylated free flavone of brown maize husks Sample I. Samples. Samples Weight of acetyl quercetin (gtn.). . . . Weight of recovered quercetin (gm.) Percentage of recovered quercetin. .. 0.2521 .1484 58.86 0-3165 .1866 58.95 0.4908 .2902 59-13 1 Willstatter, Richard, and Everest, Arthur E. tjntersuchungen uber die anthocy.vne. i. user DEN FARBSTOFF DER KORNBLUME. /« Licbig's Ann. Chem., Bd. 401, Heft 2, p. 189-232, 4 fig. 1913. ' Emerson, R. A. op. ax. Oct. 1, 1921 Quercetin in Emerson s Brown-Husked Type of Maize 3 The mean of the three determinations is 58.98 per cent — in exact accord with theory. The entire yield of approximately i gm. of acetyl derivative was divided to make the above determinations. The quercetin obtained (0.6254 gin.) was again acetylated, yielding 0.8352 gm. of penta-acetyl- quercetin. The acetyl derivative melted at 190° to 192° C. The recov- ered flavone melted at about 305° to 306° with darkening. When mixed with quercetin from Bscholtzia (melting point approximately 305° to 310°) the mixture melted at 306° to 307°. In other charac- teristics the quercetin from maize was identical with a sample obtained by the writers ^ from rutin, a glucosid of quercetin found in Escholtzia petals. Combustions of the free quercetin and of its acetyl derivative were made, with the results shown in Table II. Table II. — Combustions of the free quercetin of brown maize husks and of its acetyl derivative Quercetin. Penta-acetylquercetin. Sample i. Sample 2 Weight of sample (gm.) Weight of carbon dioxid (gm.) Weight of water (gm.) Percentage of carbon Percentage of hydrogen 0-1353 .2951 .0404 59-47 3-34 o. 1126 .2430 .0403 58-85 4.00 0.2026 •4338 .0691 58.39 3.82 Theory requires: For quercetin, carbon 59.59 per cent, hydrogen 3.34 per cent; for penta-acetylquercetin, carbon 58.59 per cent, hydrogen 3.90 per cent. PREPARATION OF THE GLUCOSID After partition of the alcoholic extract of the brown husks between ether and water, the aqueous solution, containing as one of its chief con- stituents a quercetin glucosid, was treated with four successive portions of lead acetate. The first fraction of the lead precipitate was discarded. The second consisted largely of tarry matter and was therefore not used for the preparation of pure glucosid but yielded quercetin on hydrolysis after decomposition with hydrogen sulphid. The third and fourth frac- tions were combined, suspended in hot alcohol, decomposed with hydrogen sulphid, filtered, and evaporated to small bulk. A small quantit}' of impure glucosid separated out on standing, but the greater part was got by shaking the solution with ethyl acetate. The glucosid was purified only with great difficulty, by fractional solu- tion of the dry impure product in ethyl acetate and successive crystalli- zation of the purer fractions from water. The yield of pure glucosid ' Sando, Charles E., and Bartlett, H. H. rutin, the flavone pigment of escholtzia californica CHAM. /« Jour. Biol. Chem., V. 41, no. 4, p. 495-501, pi. 6-7. 1920. Journal of Agricultural Research voi. xxii, no. i obtained in this manner was insufficient for a thorough investigation, which must be deferred until a new lot of material is extracted. It was nearest in color to the "lemon yellow" of Ridgway's ^ color standards and melted to a cherrj^-red liquid at 220° to 222° C. A\Tien hydrolyzed it produced quercetin and apparently only one sugar, glucose, although the latter point is to be more thoroughly investigated. The osazone of the sugar melted at 204° to 206° and was evidently glucosazone. The quercetin obtained by hydrolysis was identified by its general properties and by combustions both of the free flavonol and of the acetyl deriva- tive. The latter melted at 191° to 193.5° and had the properties of penta-acetylquercetin. A sample weighing 0.4650 gm. gave 0.2735 &^- of quercetin, or 58.81 per cent by quantitative hydrolysis; theory requires 58.98 per cent. The results of combustions are given in Table III. Table III. — Combustions of the quercetin obtained by hydrolysis of the glucosid of brown maize husks and of its acetyl derivative Penta- acetyl- quercetin. Weight of sample (gm.) Weight of carbon dioxid (gm.). Weight of water (gm.) Percentage of carbon Percentage of hydrogen Quercetin. 0 1514 32«3 044S 59 13 3 29 0.1570 .3400 • 0537 59.06 3-83 Theory requires: For quercetin, carbon 59.59 per cent, hydrogen 3 .34 per cent; for penta-acetylquercetin, carbon 58.59 per cent, hydrogen 3.90 per cent. The glucosid is not one of the well-known ones but bears considerable similarity to one which Heyl ^ recently isolated from the pollen of rag- weed, probably Ambrosia artemisiijolia L., although he gives only the common name. SUMMARY In accord \vith the expectation that the broAvn-husked t5'pe of maize would be found to contain a flavonol, we have been able to isolate from brown husks both free quercetin and a quercetin glucosid of which a further investigation will be made. The two compounds in question are both lemon yellow in color. If they account for the truly brown color of the husks of this type, it must be through their tinctorial quality, probably through their adsorption on some colloid component of the brown tissues. It is very probable that the quercetin glucosid is the counterpart in the brown type of the anthocyanin of the purple type. The pigment of the latter will probably be found to be allied to cyanin. I RiDGWAY, Robert, color standards and color nomenclature. 43 p., 53 pi. (col.) Washington, D. C. 1912. ' Heyl, Frederick W. the yellow coloring substances of ragweed pollen. In Jour. Amer. Chem. Soc, v. 41, no. 8, p. 1285-1289. 1919. BIOLOGICAL ANALYSIS OF THE SEED OF THE GEORGIA VELVET BEAN, STIZOLOBIUM DEERINGIANUM By Barnett Sure; and J. W. Read, Laboratory of Agricultural Chemistry, University of A rkansas The velvet bean, Stizolohium deeringianum Bort.,is annually becoming more important in southern agriculture, and the acreage planted to this crop in the cotton belt is continually mcreasing. From 1915 to 1917 it is estimated that the area increased from less than 1,000,000 acres to more than 5,000,000 acres. The acreage in 191 7 was 1 19 per cent greater than in 19 1 6. It is the most vigorous growing annual legume in the United States ; and on account of its very rank growth and the common practice of cultivating it v/ith the corn crop it is chiefly used as a winter pasture for cattle and hogs, although much larger quantities of the beans are harvested from year to year and ground, either with or with- out the pods, for market purposes. Harvesting with the com crop for use as silage is also growing in favor. Because of the rapidly increasing interest in this crop as a feed and its very considerable promise in this respect, particularly to the South, it occurred to one of the authors^ that a biological analysis should give very fundamental information as to how the velvet bean might best be utilized for feeding purposes. Accordingly the Georgia Velvet Bean, commonly know as the Early Speckled, was chosen for our studies be- cause of its early maturity, general popularity, and adaptability to the more northern as well as to the other sections of the cotton area. The Georgia velvet bean seed has a very tough, hard hull which con- stitutes 12 per cent of the whole seed. In grinding the beans it was found impossible to grind the hulls in a satisfactory manner; conse- quently these were sifted out. The experiments reported in this paper were conducted with hulled seed. It was later found, however, that after the sifted hulls had been dried on a steam bath for from six to eight hours they could be ground ; and experiments were later inaugurated, introducing the hulls in the same proportions as they were found to exist in the seed, the results of which will be reported later, together with other data showing the supplementary relationships of the seed to the leaf and the biological value of the whole plant. The nutritive value of the seed and the whole plant in practical rations is also being studied at the present time. ' Credit for the inauguration and outline of the velvet-been studies as approved under the Adams Fund is due Prof. J. W. Read. Journal of Agriculural Research, Vol. XXII, No.i Washington, D. C. Oct. i, 1921 za Key No. Ark.-i (5) Journal of Agricultural Research voi. xxii. No. i The experiments reported in this paper were conducted ^vith albino rats, employing the standard technic adopted by the Department of Agricultural Chemistry of the University of Wisconsin. Preliminary experiments showed that young rats, 40 to 90 gm. in weight, will exist only from 7 to 12 days on a diet composed solely of Fig. I. — Gain in weight of lot 51 on ration of velvet beans, So per cent; butter fat, s per cent; No. 32 salts, 4 per cent; and dextrin, 1 1 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. At point x 9 per cent of the velvet beans was replaced by 9 per cent casein. the raw hull-less velvet bean seed and a liberal supply of distilled water. The food consumption could be increased 50 to 60 per cent, however, by feeding young animals the seed after it was autoclaved for one hour at 15 pounds pressure. Consequently, autoclaved velvet beans were Fio. 2. — G^a in weight of lot 52 on ration of velvet beans, 60 per cent; butter fat, s per cent; No. 32 salts, 4 per cent; casein, s per cent; and dextrin, 26 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embrvO, used in all- this work. In order to determine whether the water-soluble vitamin was destroyed in the process of autoclaving, controls were run with uncooked beans. Even when fed upon autoclaved beans, ammals, although consuming considerably more of the seed, existed only for a period of 17 to 21 Oct. 1,1921 Biological Analysis of Georgia Velvet Bean j days when the diet was composed solely of the seed. On diets com- posed of 80 per cent velvet bean with 20 per cent dextrin, and 60 per cent velvet bean with 40 per cent dextrin, eight animals, having an initial weight of 50 to 60 gm., maintained their weight for a period of eight weeks but made no growth. Likewise, when the velvet bean formed 60 to 40 per cent of the ration, respectively, as a source of protein, in the presence of all the other dietary factors, no growth resulted, but all the animals maintained their body weight for a period of six weeks, indicating that the proteins in the seed are deficient. Since these experiments lasted for periods ranging from six to eight weeks only, no charts were prepared illustrating the points mentioned. All the rest of our findings are illustrated in figures i to 15. When velvet beans formed 80 per cent as the source of protein in the ration (fig. i), very little growth resulted. Altliough at point xg per cent of the beans was replaced by 9 per cent casein, no appreciable change in the character of growth ensued, lack of response to the addi- tion of purified casein being due, as it will be noted from the following graphs, to the injurious effect of this high plane of velvet bean intake. Velvet beans fed at a 60 per cent level as a source of protein, supple- mented with 5 per cent casein, produced a fair amount of growth (fig. 2). When, however, 40 per cent velvet beans was the source of protein and the ration was further fortified with 9 per cent casein, the two females made normal growth for a period of four months and the two males grew at a rate even beyond the expectation curve (fig. 3). Rat 211 was unable to rear her young, although her litter was reduced from nine to four. Figure 4 shows that young animals are unable to make any growth on a ration composed of 80 per cent velvet beans as the source of salts. When 40 per cent velvet beans served as the source of salts, some little growth occurred during the first 10 weeks (fig. 5). It is evident, then, that at least part of the failure of lot 61 (fig. 4) must be ascribed to the harmful effect of the higher plane of velvet bean intake. A striking change in the character of growth is apparent when at point x 4 per cent of dextrin was replaced by 4 per cent of salt mixture No. 32.^ When I per cent sodium chlorid (NaCl) and 1.5 per cent calcium carbonate (CaCOg) replaced salt mixture No. 32 in the ration, very good growth was obtained for a period of three months (fig. 6) , indicating that the calcium, sodium, and chlorid ions furnish the necessary mineral supplements in the velvet bean seed. Figure 7 shows that when 1.5 per cent calcium carbonate alone replaces salt mixture No. 32 only a little growth results. * Steenbock, H., and Gross, E. G. pat soLUBtE vitamine. n. the fat-soi,ubi,e vitamine con- tent OP ROOTS together with some observations on their "WATER-SOtUBLB VITAMINE CONTENT. In Jour. Biol. Chem., v. 40, no. 2, p. 505. 1919. Journal of Agricultural Research voi. xxn. no. i 1 Figures i to 7 have indicated that velvet beans are detrimental to young experimental animals when fed at an 80 per cent level. The experiment on which figure 8 is based corroborates that fact. Although 60 Fig. 3. — Gain in weight of lot 53 on ration of velvet beans, 40 per cent; butter fat, s per cent; No. 3a salts, 4 per cent; casein, 9 per cent; and dextrin, 42 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. Y indicates point at which young were littered. when 80 per cent velvet beans served as a source of the fat-soluble vitamin, two animals made a fair amount of growth for a period of four SO 60 SO 60 Fig. 4.— Gain in weight of lot 61 on ration of velvet beans, 80 per cent; butter fat, 5 per cent; casein, s per cent ;and dextrin, 10 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. months; two rats died after six weeks. The failure of these two ani- mals can not be ascribed to the low concentration of the fat-soluble vitamin in the seed, since lot 70 (fig. 11) made normal growth for a period / ^ ^^ ^ ^? / %244 ,^ -*^ ^ -^ A24/ ,l>^ ^242 2 Oct. 1,1921 Biological Analysis of Georgia Velvet Bean of over five months when only 20 per cent velvet beans served as a source of that vitamin. 200 /80 /60 MO /20 /OO 80 60 40 Fig. 5. — Gain in weight of lot 69 oa ration of velvet beans, 40 per cent; butter fat, 5 per cent; casein, 9 per cent; and dextrin, 46 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. At point x 4 per cent dextrin was replaced by 4 per cent No. 32 salts. A considerable improvement in the character of growth is obtained when the plane of intake of velvet beans used to supply the fat-soluble vitamin is reduced from 80 to 60 per cent (fig. 9) . / 1 / / / / ) f / / i / / i / y / y' A / / / \^ /^ / y^ .>^ X 9 -^ B—H- 0274 *Z7S ^76 2 Fig. 6. — Gain in weight of lot 75 on ration of velvet beans, 40 per cent; butter fat, s per cent; sodium chlorid, i per cent; clacium carbonate, 1.5 per cent; casein, 9 per cent; and dextrin, 43.5 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. When the level of velvet bean intake was reduced to 40 per cent, normal growth was obtained. Rat 270 failed to rear her young, although her litter was reduced from eight to four (fig. 10). lO Journal of Agricultural Research Vol. XXII, No. I Even when the seed was reduced to as low a plane of intake as 20 per cent, it served as a very efficient carrier of tlie fat-soluble vitamin. It is also apparent that autoclaving for one hour at 15 pounds pressure had Fig. 7. — Gain in weight of lot 92 on ration of velvet beans, 40 per cent; butter fat, 5 per cent; calcium carbonate, 1.5 per cent; casein, 9 per cent; and dextrin, 44.5 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. no deleterious effect on this vitamin. Although excellent growth was obtained on this ration, mother rats No. 277 and 278 failed to rear their young in every case even when their litters ranging from 7 to 10 were reduced to only 4 (fig. 11). ^O Fig. 8. — Gain in weight of lot 63 on ration of velvet beans, 80 per cent; No. 32 salts, 4 per cent; casein, 5 per cent; and dextrin, 11 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted embryo. D indicates point at which rat died. Figure 12 shows that on reducing the level of velvet bean intake to 10 per cent as a source of fat-soluble vitamin the character of growth is considerably impaired. When 80 per cent velvet beans was used to supply the water-soluble vitamin very little growth resulted, one animal dying after three weeks on this ration (fig. 13). oct.i. I92I Biological Analysis of Georgia Velvet Bean II -^bl Fig. 9. — Gain in weight of lot 67 on ration of velvet beans, 60 per cent; No. 32 salts, 4 per cent; casein, 5 per cent; and dextrin, 31 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. At point x 4 per cent dextrin was replaced by 4 per cent additional casein. GM. 240 220 Fig. 10. — Gain in weight of lot 67 on ration of velvet beans, 40 per cent; No. 32 salts, 4 per cent; casein, 9 per cent; and dextrin, 47 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. Y indicates point at which young were littered. 12 Journal of Agricultural Research voi. xxn, no. Sixty per cent of velvet beans used to supply the water-soluble vitamin allowed only a very small amount of growth (fig. 14). G/^. Fig. II. — Gain in weight of lot 70 on ration of velvet beans, 30 per cent; No. 32 salts, 4 per cent; casein, 12 per cent; and dextrin, 64 per cent. The dextrin carried alcoholic extract of 10 gm. ether-extracted wheat embryo. Y indicates point at which young were littered. In order to determine whether the water-soluble vitamin was destroyed during the process of autoclaving, 40 per cent velvet beans was fed un- cooked. Very little growth resulted, nor was there any improvement SA7. 60 Fig. 12. — Gain in weight of lot 90 on ration of velvet beans, 10 per cent; No. 32 salts, 4 per cent; casein, 16 per cent; and dextrin, 70 per cent. The dextrin carried alcoholic extract of 15 gm. ether-extracted wheat embryo. in the character of growth when, at point x, lo per cent dextrin was replaced Avith lo per cent of an alcoholic extract of ether-extracted wheat embryo. It will be noted that after point x this ration is identical with Oct. 1,1921 Biological Analysis of Georgia Velvet Bean 13 that given to lot 53 (fig. 3) with the exception that lot 86 received the beans raw while lot 53 received the beans cooked. The striking dif- ference in the character of growth obtained in these two experiments must be attributed to the fact that the velevt bean seed uncooked is either toxic or indigestible at a concentration as low as 40 per cent. The /20 /OO 80 60 ^O Fig. 13. — Gain in weight of lot 62 on ration of velvet beans, 80 per cent; butter fat, s per cent; No. 32 salts, 4 per cent; casein, s per cent; and dextrin, 6 per cent. D indicates point at which rat died. cause of the deleterious effect of the raw seed is being studied and will be reported later. DISCUSSION The Georgia velvet bean, Early Speckled variety, has been found to be injurious when fed in the raw condition at as low a level as 40 per cent intake. This has been evidenced from an experiment where 40 per cent ^..^ * / / > t /^ ^ / ( Si^"' y / / 245 ^ i-..^ ^ ^ ^ --^ / WeEKS Fig. 14. — Gain in weight of lot 74 on ration of velvet beans, 60 per cent; butter fat, 5 per cent; No. 32 salts, 4 per cent; casein, 5 per cent; and dextrin, 26 per cent. At point x 4 per cent dextrin was replaced by 4 per cent additional casein. velvet beans uncooked formed the source of water-soluble vitamin (fig. 15). Growth was inhibited during the first six weeks of experi- mentation, after which time lo per cent dextrin was replaced by an alcoholic extract of lo gm. ether- extracted wheat embryo. This addi- tion of the water-soluble vitamin should have rendered the ration entirely satisfactory, judging by the character of growth obtained in a duplicate experiment where the beans were furnished cooked (fig. 3). H Journal of Agricultural Research Vol. XXII, No. I The nature of the possible toxicity of the velvet bean has been recently suggested by Miller * to be due to dihydroxypehnylalanine. Cooking the seed at 15 pounds pressure for one hour destroyed for the most part its harmful effects, but there was still some injury when fed cooked at as high a plane of intake as 80 per cent. When 80 per cent of the velvet bean served as a source of protein, little growth resulted, nor was there a response obtained after 9 per cent of the seed was replaced by 9 per cent of casein, although when only 40 per cent velvet bean was served as a source of protein, supplemented with the same amount of casein, excellent growth was obtained. Unpublished data in this labo- ratory show that the better growth on the lower level of seed intake is not to be attributed to the higher intake of dextrin. Additional evidence is apparent from the fat-soluble vitamin experiment that when cooked Gn /GO /20 /OO So 60 Fig. 15. — Gain in weight of lot 86 on ration of velvet beans (uncooked) 40 per cent; butter fat, 5 per cent; No. 32 salts, 4 per cent; casein, 9 per cent; and dextrid, 42 per cent. At point x 10 per cent dex- trin was replaced by 10 per cent of an alcoholic extract of ether-extracted wheat embryo. velvet beans are fed at an 80 per cent level some injury is still produced. Reduction of the plane of intake from 80 to 40 per cent results in con- siderable improvement in growth. That the velvet bean seed is very rich in the fat-soluble vitamin is evident from the fact that normal growth was obtained for a period of over five months when only 20 per cent of the seed served as the source of this syndrome. Reduction of the plane of velvet-bean intake to 10 per cent resulted in inferior growth. The fact that considerably inferior growth was obtained on lower levels of seed intake with larger amounts of casein and dextiin precludes, we believe, the possibility that our casein and dextrin might have furnished appreciable amounts of the fat-soluble vitamin at the higher levels of seed intake, where we had remarkable success. It is also apparent from these experiments that autoclaving the seed for one hour at 15 pounds pressure has no deleterious effect on the fat-soluble vitamin. ^ -s -^ X .^"^ rs< ^ ^ ^^ A X ^llr^ <^ ^ g^ '-3 0^ ^ ^ 5 2 meAS 1 Miller, Emerson R. DrHVDRoxYPHENYLALANiNe, a constituent of the velvet bean. Biol. Chem., v. 44, no. 2, p. 481-486. 1920. In Jour. Oct. 1,1921 Biological Analysis of Georgia Velvet Bean 15 The hull-less seed contained 27.5 per cent protein and therefore furnished 16.5 per cent protein when fed at a 60 per cent level ; however, this amount of protein was inadequate for growth even though all the other factors in the diet were rendered satisfactory by the addition of isolated purified food substances. Recently Johns and Waterman ^ have isolated two globulins and an albumin from the Georgia velvet bean and have reported analytical data on their composition, using the Van Slyke ^ method of protein analysis. Their results show that, with the exception of the albumin, which is low in histidin, the three proteins of the Georgia velvet bean are quite satis- factory for their diamino-acid content. However, since we have insuffi- cient chemical data on the amino-acid content of the Georgia velvet bean no correlation can be made at present between the chemical composition and the biological response of this seed. The nature of the amino-acid deficiencies is being investigated. The velvet-bean seed has also been found to be deficient for growth in the character of its salts ; however, sodium chlorid and calcium carbonate seemed to replace salt mixture No. 32 satisfactorily. The concentration of the water-soluble vitamin in the seed has been found to be low. Unpublished data show that the addition of the ground hulls in the same proportions as they occur in the whole seed does not improve the water-soluble vitamin content. It is not apparent from these experiments whether this vitamin was in any way destroyed during the process of autoclaving, since the seed was extremely injurious when fed uncooked. SUMMARY (i) The Georgia velvet bean seed, Early Speckled variety, when fed raw was found injurious to young rats even when constituting only 40 per cent of the total ration. (2) Autoclaving the seed for one hour at 15 pounds pressure destroys most of this injury, so that it is possible to include 60 per cent of the bean in a ration. A ration composed of 80 per cent velvet bean cooked still shows some harmful effects. (3) This seed, unlike most seeds so far studied biologically, is very abundant in the fat-soluble vitamin. The fat-soluble vitamin as it exists in this seed is quite stable after the seed is autoclaved for one hour at 15 pounds pressure. The water-soluble vitamin, however, is of low con- centration in the hulled seed. (4) Both the proteins and salts of the velvet bean have been found to be of deficient character for growth. 1 Johns, Carl O., and Waterman, Henry C. some proteins from the Georgia velvet bean, stizo- LOBIUM deeringianum. In Jour. Biol. Chem., v. 42, no. i, p. 59-69. 2 Van Slyke, Donald D. the analysis of proteins by determination of the chemical groups CHARACTERISTIC OF THE DIFFERENT AMINO-ACIDS. In Jour. Biol. Chem., V. 10, no. I, p. 15-SS, 2 fig. 1911. EFFECT OF SOIL TEMPERATURE UPON THE DEVELOP- MENT OF NODULES ON THE ROOTS OF CERTAIN LEGUMES By Fred Reuel Jones, Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant Industry, United States Department of Agriculture, and W. B. Tisdale, Instructor in Plant Pathology, University of Wisconsin INTRODUCTION During a search for the cause of a diseased condition of alfalfa under observation in 191 7 and 19 18 the senior writer was led by observations contributed by H. L. Westover, of the Office of Forage Crop Investiga- tions, to believe it likely that soil temperature, within the range which occurs in cultivated fields, affects the initiation and the development of nodules on the roots of alfalfa and perhaps all other legumes to such a degree that the assimilation of nitrogen by these plants is greatly modi- fied by this lactor during the summer. The probable importance of such an effect of temperature, should it be demonstrated, upon the develop- ment of alfalfa and other legumes and especially its possible relation to the disease in question seemed adequate reasons for making a beginning at the experimental determination of the facts. Experimental methods suitable for the performance of this work had already been highly devel- oped in the course of the study of soil-inhabiting plant parasites at the University of Wisconsin. Thus it came about that the collection of the following data was begun at Madison by the junior author in 191 7 and continued by both authors in 191 9 and 1920. A temporary suspension of the work is the immediate reason for the publication of this prelimi- nary report. In the beginning, interest was centered upon ascertaining to what extent soil temperature determined the number of nodules which any of a selected group of legumes might develop. Later the size and composi- tion of the nudoles appeared more significant than the number. Finally, it is seen that soil temperature probably affects profoundly the rate of nitrogen fixation within the nodules of the legumes studied and its assimilation by the plants. A complete demonstration of such an effect and a quantitative determination of its amount remains for the future. During the progress of the work certain striking effects of soil temperature upon the development of the plants quite apart from any relation to nodule formation have been noted. Journal of Agricultural Research, Vol. XXII, No. i Washington, D. C. Oct. i, 1921 zo Key No. G-24S 54817°— 21 2 (17) 1 8 Journal of Agricultural Research voi. xxii. No. x ENVIRONMENTAL FACTORS WHICH HAVE PREVIOUSLY BEEN FOUND TO MODIFY THE FORMATION AND DEVELOPMENT OF NODULES In the extensive studies which have been made of the conditions which may favor or hinder the development of nodules, no one, so far as the writers are aware, has concerned himself with the factor which is considered here — ^namely, the temperature of the soil. There are a number of factors, however, which are known to have very much influence upon nodule development, and two of these which may have become modified by our experimental methods for controlling soil tem- perature must be considered. These are soil moisture and the concen- tration of nitrates. With regard to soil moisture, there appears to be both observational and experimental data which indicate that high soil moisture tends to increase nodule formation. Gain * notes that peas grown in wet soil have far more nodules than those grown in drier soil close by. Wilson ^ in his experimental work reports that wet soil induces the formation of a greater number of nodules on soybeans. Fortunately, in experimen- tal work with controlled temperatures, it is comparatively easy to main- tain soil moisture at a predetermined point with very slight fluctuation. In the preliminary experiments, although no attempt was made to control soil moisture exactly, it is not believed to have fluctuated suffi- ciently to affect results appreciably. In the later work, soil moisture was maintained in each series at one-half the moisture-holding capacity of the soil used (14 per cent of the wet weight) by weighing the pots each day, if necessary, and restoring the water lost by evaporation and transpiration. It is believed that this method kept variation in soil moisture within such very narrow limits that this factor could not have produced appreciable variation in nodule formation. That the amount of nitrate present in the soil affects nodule develop- ment, completely inhibiting it when high concentrations have been reached, has been demonstrated by several investigators. Wilson ^ has added a considerable number of nitrates to soils in different amounts to determine the concentration at which nodule formation is inhibited by each of the compounds. Although complete inhibition is effected only at concenti"ations which are not likely to occur in normal soils, the marked effect of variations is so well attested that any differences arising unavoidably during an experimental series must be taken into account in the consideration of results. The control of the concentration of nitrates in the soil solution in a soil held at different temperatures offers difficulties which can be over- 1 Gain, Edmond. influence de i.'humidite sur le developpement des NODosixfes des l^gumineu- SES. In Compt. Rend. Acad. Sci. [Paris], t. ii6, no. 24, p. 1394-1397. 1S93. 2 Wilson, J. K. physiological studies of baollus radiocola of soy bean (soja max piper) and OF factors influencing nodule production. N. Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig. 80-94. 1917. Oct. 1. 1921 Effect of Soil Temperature on Development of Nodules 19 come only within certain limits. As was expected in advance, the rate of nitrification in soil differs greatly at the different temperatures, producing greatly different concentrations within a short time after a series of plants have been started. In addition, there is soon consid- erable difference in the size of the plants at the different temperatures and a consequent difference in ability to absorb nitrates. The varia- tions which arise from these causes can be limited somewhat by the use of soil low in total nitrogen, thus making impossible the accumulation of large amounts of nitrates in any case. Variations in the concentra- tion of nitrates which have been observed in the experimental work described here will be noted later, and their possible effect upon the results will be discussed. APPARATUS AND METHODS The apparatus used for the control of soil temperature in these experi- ments is that which has been used in the Laboratory of Plant Pathology at the University of Wisconsin for several years and needs no new description.^ In all cases plants were grown in metal cans 6 inches in diameter and 10 inches deep. The number of plants which could be grown in each can vnthout serious crowding of roots was 3 for soybeans, 5 for peas, and 10 for clover and alfalfa. The soil used was a sandy loam from a pasture which had never been cultivated. To this was added about an equal weight of sand in order that the total nitrate content should be kept low and that the mechanical condition of the soil should permit the easy removal of the roots. The temperatures noted in the different series were those at which the water was main- tained in the tanks in which the cans were set. Fluctuations of tempera- ture did not often exceed 1° C. from those given in the tables, and were of only a few hours' duration. Although record was made twice daily of the actual temperatures, it is not believed that a computation of the mean temperature from these figures would give a figure more significant than tlie convenient even numbers used here. It should also be noted that although the surface of the soil was insulated to some degree from loss of heat and moisture by the use of mineral wool, never- theless at the higher temperatures the surface soil to the depth of about I inch was usually cooler by i to i}4° than the water. However, it is believed that the larger part of the roots and nearly all the nodules were sufficiently deep in the soil below this cooler layer, so that error arising from this source is not considerable. Water was supplied through a glass tube which passed to the bottom of the metal can where it entered an inverted unglazed flower pot 3 inches in diameter, which acted as a reservoir. In the last series the > Jones, 1,. R. soil temperatures as a factor in phytopathology. In Plant World, v. 20, no. 8, p. 229-237, 2 fig. 1917. lyiterature cited, p. 236-237. 20 Journal of A gricultural Research \o\. xxii. No. i inverted pot was placed about 3 inches below the surface instead of at the bottom in the hope of maintaining a more uniform and rapid dis- tribution of moisture. This appears to have been an unfortunate change in method, since at the higher temperatures roots tended to col- lect around these pots, where they apparently developed more ex- tensively and produced more nodules than they had in the previous series. Inasmuch as no accumulation of roots took place at lower tem- peratures, it is not easy to explain this fact. Attention should here be drawn to the fact that two distinctly dif- ferent methods of securing data have been used. In the preliminary ex- periments, the plants were first grown in the cans at ordinary greenhouse temperature for about two weeks before inoculation was made, with the suitable strain of Bacillus radicicola Beyr., by pouring a water sus- pension of the organism around the base of the plants, and the cans were placed in the tanks adjusted at the predetermined temperatures. In the later series the plants were grown from seed in inoculated soil held at the required temperatures from the beginning. Several reasons led to the change of method. In the first place, one could not be certain that the bacteria poured around the plant in the first instance would become rapidly distributed through the soil at all temperatures. This inequality in rate of distribution might affect the number of infections and hence the number of nodules formed. At least it might tend to limit the formation of nodules to the roots near the surface of the ground where temperature is less exactly controlled. A second objection to this method appeared when the marked effect of temperature upon the morphology of the roots themselves was observed. The number of root hairs through which infection has been found to take place is much greater at lower tempera- tures than at higher. In view of the possible effect of this difference it appeared preferable to grow the plants from the beginning in inoculated soil at the designated temperatures, even though the plants thus produced would necessarily vary considerably in size. Data obtained by each of these methods will be presented. MEASUREMENT OF EFFECT OF SOIL TEMPERATURE UPON NODULE FORMATION When the experiments were begim it was assumed that different tem- peratures, if they are at all potent, would produce such a marked effect upon the number of nodules that count alone would give a significant expression of results. This expectation was fostered by the fact that Wilson 1 and nearly all previous investigators have used numbers to express similar experimental results. It will be seen from data given later that this hope was early disappointed. Different temperatures usually seemed to affect number not nearly so much as rate of develop- » Wilson, J. K. op cit. Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 2 1 ment and size. In fact, in some instances, volume of nodular tissue seemed in inverse ratio to number. With most legumes it is not easy to get an accurate dry-weight determination of small nodules, because these occur as swellings so closely attached to the root that it is hardly feasible to separate them from the true root tissue. The one species tried which gave least trouble from this source by reason of the distinct separation of its nodules from the root, even at early stages of development, was the soybean. For this reason, it alone was used in the final series recorded here. Of course it was soon realized during the progress of the work that vol- ume of production of nodules was only an easily observed index, significant chiefly in so far as it revealed important eff"ects of temperature upon the physiological processes which are dependent upon the nodular structures. It would be of greater interest, for example, to measure the amount of nitrogen fixed in these nodules produced at different temperatures and that portion which becomes available to the plant for use in its vital pro- cesses. The demonstration of an important limiting effect of temperature upon nitrogen fixation would be of no inconsiderable importance. Such an effect would probably be indicated by large differences in size of nodules, though it might occur without the appearance of such difference. In any case it seems easily possible to determine approximately the effi- ciency of nodules in the fixation of atmospheric nitrogen by growing parallel series of plants, inoculated and uninoculated, in the same kind of soil and at the same soil temperatures. If the amount of nitrogen in the inoculated plants (aside from that foimd in the nodules on those plants) is greater than in the uninoculated, the gain must be credited to the efficiency of the nodules. The gains thus found should be an accurate measure of the effect of soil temperature upon the fixation of available nitrogen in the legume used in the experiment, and a comparison of this gain with the weights of the nodules found on the inoculated plants should give an approximate idea of the relation existing between effici- ency of fixation of available nitrogen and volume of nodules. In the last series recorded an attempt was made to carry out this experiment with the soybean plant. Unfortunately some of the uninoculated plants in the series became inoculated during the experiment and developed a few nodules, thus making it necessary to discard the data so far as these controls are concerned. Thus an exact determination of the extent of the effect of soil temperature upon the fixation of nitrogen in the nodules of legumes remains to be made. For the present we can only ascertain the dry weights of the nodules themselves as they are found at the end of a period of time and determine the amount of nitrogen found within them. 22 Journal of Agricultural Research voi. xxii. no. i DISCUSSION OF THE LEGUMES USED AND THEIR BEHAVIOR UNDER THESE EXPERIMENTAL CONDITIONS Four legumes were selected for tlie soil temperature series requiring four different strains of Bacillus radicicola for their inoculation. One of these, the soybean, flourishes well at high soil temperatures; one, the Canada field pea, requires a low soil temperature for good growth; and red clover and alfalfa occupy intermediate positions. A few of the more striking reactions of the plants in these series will be noted. The Canada field pea does not flourish vigorously at a soil tem- perature as high as 30° C. (Table I) and is intolerant of temperatures above this point, maintaining roots only very close to the surface of the soil. It is perhaps misleading to infer that the lower surface temperature is alone responsible for the position of the roots, since in field plots in hot, exposed positions the death of deeper roots and the formation of surface roots has been noted in hot weather. Perhaps the more striking effect of the series of soil temperatures upon the soybean plants, aside from the fact of the wide range through which it grows vigorously, is the effect upon the color of the foliage. After the plants had become 5 or 6 inches tall, in both series the leaf color was much darker at the two ends of the series, especially at 30° C. and above, than at 2 1° and 24°. This difference persisted, tending rather to increase as long as the plants were grown. One striking difference in behavior between peas and soybeans on the one hand and clover and alfalfa on the other was noted in this series. The annuals formed a rather regular series of plants as judged by appear- ance (PI. 1) and also by dry weights (Table I). But with alfalfa and red clover the seedlings at the lower temperatures, 12° and 15° C, though little delayed in starting, remained small Alpine plants with thick dark green leaves and \vith much red color in the petioles; whereas at 18° the plants were more nearly what may be termed "normal" plants, larger, with fairly long petioles containing less red color. ^ EXPERIMENTAL DATA EFFECT OF SOIL TEMPERATURE UPON THE NUMBER OF NODULES FORMED As previously noted, in the fiirst two preliminary trials the seeds were planted in soil in the metal cans and grown for about 10 days at green- house temperature (about 22° to 23° C.) before they were inoculated with the suitable strains of Bacillus radicicola and placed in the tanks adjusted at the temperatures designated. When it was believed that sufficient time had elapsed for infection at all temperatiu-es, the tops were cut from the plants, dried, and weighed. The roots were carefully 1 Since this was written clover and alfalfa have been grown under similar conditions at controlled tem- peratures. The marked dwarfing of plants at 15° and 12° C. was found to disappear when the plants became older, and especially later in the spring when light intensity became greater. Oct. 1. 1921 Effect of Soil Temperature on Development of Nodules 23 washed from the soil and the nodules were counted. The count obtained is given in Table I. In the two later series the seeds were planted in soil which had already been placed in the tanks adjusted to the tem- peratures designated. The air temperature ranged from 14° to 18°. The counts obtained in these series are given in Table 11. Table I. — Average number of nodules produced on plants 26 days old grown at a soil temperature of about 20° C.for 10 days, after which mociilation was made and the tem- perature of the soil was maintained as indicated Temperature. "C. 10 to 12 15 20 25 30 35 40 Alfalfa. plants. IS 35 18 16 5 plants. Red clover. plants. 17 27 40 69 75 4 plants. 7 16 47 4 75 6 Soybeans. 6 plants. 46 61 37 35 6 plants. 14 25 33 28 Field peas. plants. 27 31 37 43 128 plants. 21 24 30 60 64 3 o The plants did not survive. Table II. — Average number of nodules produced on plants grown at the soil temperatures designated Temperature. Alfalfa. 20 plants 63 days old. Red clover. 10 plants 63 days old. Soybeans. 6 plants 63 days old. 9 plants 55 days old. a Field peas. S plants 32 days old. 5 plants 52 days old. 12 15 18 21 24 27 30 33 36 I. 19 1.6 16. 7 8.0 3-4 II. 6 10.7 3-5 1.8 5-0 12.5 24. 6 17.4 II- 5 8.9 5-3 o. o 6. I 5-9 4.0 II- 3 8.0 5-8 8.8 13.6 13.0 14.7 19.7 18.8 16.4 20.0 12.4 3-6 3-6 8.0 14. o 25.8 30.0 4.0 2.6 9.0 27.2 23.8 13.0 58.0 o- The larger number of nodules on the plants 55 days old as compared with those on plants 63 days old is believed to be due largely to the fact that this series of plants was grown in spring, when longer days promoted a far more vigorous growth than was produced by the other plants, which were grown in winter. Although it will be seen at once that the data in the two tables are not strictly comparable, nevertheless some temperature effects upon number appear. Most conspicuous of all is the greatly increased number upon peas near the upper thermal limit. But this increased number is accom- panied by a more than proportionate decease in size. No plant has been found to produce large nodules at 30° C. or above. Clover and alfalfa tend to produce their largest numbers of nodules in the middle portion of the range. Soybeans show no decided temperature effect at all, so 24 Journal of Agricultural Research voi. xxii. No. i far as number is concerned. However unsatisfactory these figures may be from several points of view,' nevertheless they establish one important fact beyond reasonable doubt : Modified soil temperature ^vithin the range which these plants can be expected to encounter in tlie field and even within which they can be grown \vith vigor under experimental conditions does not prevent the infection of roots by Bacillus radicicola and the formation of considerable numbers of nodules. In other words, B. radi- cicola, considered as a parasite, does not show the strongly marked inhi- bition of its ability to infect roots of plants that has been found in a number of fungus parasites.^ /Of- __A{oho^.€^.^ ' ... % /o\ \ /2 /■5 /8 30 33 36 2/ 24 27 ■PiG. I. — Comparison of dry weights of tops, roots, and nodules of soybeans given in Table HI, grcwn during November, December, and January. EFFECT OF SOIL TEMPERATURE UPON THE PRODUCTION OF NODULES AS MEASURED BY DRY WEIGHTS Although it is clear that soil temperature does not, in most cases, greatly modify the number of nodules produced, it was obvious from the very beginning that the size to which they developed was markedly and consistently affected. Such effect is shown graphically in Plate 2, where nodules from an equal number of plants are shown placed in rows and in Plate 3, where nodules from a larger number of plants are placed in tubes of equal diameter. Dry weights of the nodules shown are given in Table III. Data are given for the soybeans only for reasons already mentioned, but, judging by visual evidence obtained in studying all four legumes used, it is believed that all behaved in essentially the same manner. I Johnson. James, and Hartman. R. E., influence of soii, environment on the root-rot of TOBACCO. /» Jour. Agr. Research. V. 17, no. 2, p. 41-86, pi. 1-8. 1919. Literature cited, p. 84-86. TiSDALE, W. H. RELATION OF TEMPERATtree TO THE GROWTH AND INFECTING POWER OF FUASRIUM UNI. In Phytopathology, v. 7, no. s, p. 356-360, i fig., pi. u. 1917. Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 25 Now it would be expected, and it is clearly true, that plants grown in soils held at such widely different temperatures would show in the given time considerable difference in size and degree of maturity. The first question which will be asked regarding this difference in nodule develop- ment will be whether it does not correspond more or less approximately with corresponding differences in root or shoot development. Does it show a trend distinctly different from that of other portions of the plant ? When the figures given in this table are presented in graphs, the differences in trend become obvious. A comparison of the weights of the nodules with those of the roots (fig. i, 2) will show that in both /s /s 2/ 24 27 30 33 36 <^ Fig. 2. — Comparison of dry weights of tops, roots, and nodules of soybeans given in Table III, grown during April and May. series the maximum development of nodules occurs at 24° C, with very slight development at the extremes, 15° and 36°. Root develop- ment, on the other hand, rises much more rapidly at the lower temper- atures and is maintained at the the higher temperatures, reaching a maximum in the second series at a point 9° higher than that of the nodules. Root development is far more uniform at all temperatures than is nodule development. A comparison of nodule development with shoot development (fig.i, 2) shows that the effect of temperature upon the development of the two structures is quite different. As with root development, shoot develop- ment is relatively more vigorous at 15° and 18° C. than is nodule develop- ment, which increases greatly at 21° and reaches a maximum at 24°. "When at 27° the v/eight of nodules is beginning to diminish, that of shoots maintains its level or increases. Through the higher temperatures weight of nodules falls off rapidly, while that of shoots remains at the high level. 26 Journal of Agricultural Research voi. xxii, No. i Table III. — Dry weight per plant of shoot, roots, atid nodules prodticed in 6j days in the first series, grown in November, December, and January, and 55 days in the second series, grown in April and May Temperature. °C 12 15 18 21 24 27 30 33 36 First series. Shoot. Gm. o- '^33 . 410 • 432 .632 .875 .771 .818 .863 .996 Root. Nodules. Gm. O. 040 . IIO •135 . 140 •131 .108 .096 .116 . 108 Gm. O. 000 . 006 .008 •033 •043 .030 . 014 . 012 .005 Second series. Shoot. Gm. 0. 922 1.430 1. 710 1. 900 2. 620 2.540 2.440 2. 130 Root. Nodules. Gm. 3. 184 .318 . 269 . 296 •342 . 296 •437 . 422 Gm. O. 021 . 060 . 108 •145 .094 .089 . 076 . 042 OS 3S^ 5.<5.5 L T"— ' g N 5v !:c. y ^^ ^ ^ V / . y ^ f \\ ^ 0^ / 'V/ V , I f \ "*^^^^'- 0/ ^ by ^^ ^. -„«»■— "" ^\1^ .3 ^ /S /3 2/ 24 27 30 33 36 Pig. 3. — Ratios ot weight of nodules to weight (A tops and of roots. First experiment. In order to obtain a clearer view of the contrast between the effect of temperature upon weight of nodules and that of roots and shoots, the ratios of the weights of these parts of the plant have been determined and plotted (fig. 3, 4). If there is a direct relation between development of nodules and that of either the aerial or subterranean parts of the plants — if nodule development is conditioned by top or root development quite independently of the temperature factor which was varied in these experiments-^then the ratio should be approximately constant, or at any rate should be a straight line. A glance at the graphs shows that this is not the case. The ratios when plotted produce curves which are closely similar. No direct relation appears to exist between weight of nodules and that of either tops or roots under the conditions of these experiments. At 21° and 24° C. the weight of nodules is relatively larger than at temperatures above or below this region. The wide dif- Oct. I, I92I Effect of Soil Temperature on Development of Nodules 2 7 ference in the ratios and the consistent similarity of the curves can hardly be explained otherwise than as a temperature effect upon nodule development which is quite different from that upon development of root or shoot. EFFECT OF SOIL TEMPERATURE UPON THE COMPOSITION OF THE INOCULATED PLANTS In order that comparisons might be made of the amount of nitrogen found in inoculated and uninoculated plants, total nitrogen determina- tions were made of shoots, roots, and nodules of the plants grown in each series. Since the uninoculated plants did not remain free from nodules, the desired comparisons can not be made. Nevertheless the Od 07 \ %.03 y \ /^ \ y . ^ ^ \\ :5/ 4i ^ ^ =^cc^ s ^^y^ cPy \ '•x.^ /" \i / ^ -, X n^*^ "-. 1 1 sdQ. /O 12 /S /a 2/ 24 27 30 33 3$ 0£G7?£SS C£A^r/S7?^£>£ Fig. 4.— Ratios of weight of nodules to weight of tops and of roots. Second experiment. difference in nitrogen found were so marked in the series that the analyses of one series, the last that was grown, is given. Table IV. — Percentage of total nitrogen found in shoots, roots, and nodules of soybean plants grown at a series of soil temperatures in April and May Temperature °c. 15 18 21 24 27 30 33 36 Shoots. Roots. 2.42 2.89 2.86 3-40 4.27 2. 64 4. 22 2. 98 4-55 2.66 4.58 2. 62 3-98 2. 67 3-77 2.85 Nodules. 5-95 6.95 6.25 5-95 6.25 6.25 6. 00 5-70 The greatest differences in content of nitrogen are found in the shoots, the roots being very uniform and the nodules hardly less so. It will be seen that, generally speaking, the high nitrogen content of the top is 28 Journal of Agricultural Research voi. xxii, No. i correlated mth the best development of nodules, though the curve which would be produced by these figures when plotted in the manner of the preceding data would not have the same shape. The largest amount of notrogen is found at a higher temperature than the point at which the largest dry weight of nodules was found. A rather sudden increase in nitrogen at 21° C. as compared with 18° and a sudden fall at 33° as compared with 30° has characterized the series obtained thus far. The result of the analyses which have been made seems worth recording; but whether the low nitrogen content of the plants grown at both ends of the series is wholly due to the small nodules found on these plants, and whether the high nitrogen content of plants in the center of the series is due to large and presumably efficient nodules, likely as this connection appears, remains to be determined by more refined methods. DISCUSSION OF FACTORS THAT MAY HAVE HAD AN INFLUENCE UPON THE DATA GIVEN CONCENTRATION OF NITRATES IN THE SOII, In view of the fact already discussed, that large amounts of nitrates in the soil solution decrease nodule development and even inhibit it before the concentration becomes great enough to injure the plant directly, it is unfortunate that the soil used in these series should have had as much nitrate as was found (Table IV), even though the largest amounts are far below the inhibition point. There appears to be no data available in literature whereby we may know what is the maximum or the more usual amount of nodular structure formed on the roots of any of the legumes. Although the amount of nodular structure which peas may produce may be quite different from the amount which soy- beans may produce under the most favorable conditions, yet it may be worth while to record here that the writers have found in one instance a variety of wrinkled peas producing at the blossoming stage nodules whose dry weight was 2.2 times as great as that of the entire root system (average of 25 plants) ; and in individual plants the ratio of weight of nodules to roots was as high as 4.5 to i. However the ratio of weight of nodules to tops in these plants was 0.085 to i , a ratio not much different from that found under the best experimental conditions for soybeans recorded here (fig. 3, 4). However, the question of immediate interest here is whether or not the nitrate content of the soil used in tliese series was greatly changed at any of the temperatures at which it was held, and if there is any evidence that this change was of sufficient size and in the right direc- tion to indicate that it may have been responsible for the increased or decreased nodule formation at this temperature. In order to obtain information regarding the change which soil temperature maj'- have produced in the series, nitrate nitrogen determinations were made by Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 29 the colorometric method of a composite sample of the soil at the begin- ning of the experiment and of a sample from two cans of soil at each temperature when the plants were harvested. In addition, in order to get some clue to intervening changes, an unplanted can of soil was kept at each temperature from which a sample was taken at about the middle of the period. The results obtained are shown in Table IV. The effect of soil temperature does not appear to have been as definite and consistent upon the concentration of nitrate nitrogen as was anticipated. Until further data are available, it seems unwise to attempt to interpret the results. However, the very absence of large and consistent modifica- tion enables us to believe that this factor was not important in its effect upon nodule development. The only point at which nitrate accumula- tion became very large occurs in the second series where the unplanted soil shows at the end of 26 days a very high nitrate content at 21° and 24° C. If it is assumed that a similar concentration took place in the planted pot at an early stage in the development of the plants be- fore they were large enough to reduce it by absorption, it would be an- ticipated that a reduction in nodule production would be found here. In fact, however, this point of high nitrate formation is the point of highest nodule production, just as it is in the first series where no evi- dence of high nitrate content at any time was obtained. Table IV. — Nitrate nitrogen in the soil in which the soybeans grew and also in unplanted soil First series. Seconc series. Temperature. Planted soil. Unplanted soil. Planted soil. Unplanted soil. At start. At end. After 24 days. At end. At start. At end. After 26 days. After 61 days. °C. P. p. m. P. p. m. P. p. m. P. p. m. P. p. m. P. p. m. P. p. m. P. p. m. 12 20. s 20.5 8.6 16.3 II. 4 14.4 8-3 15 3-3 90 20.0 34 34 18 20.5 8.6 16.5 10. 6 90 14. 0 56 34 21 20.5 8.1 19-3 20.5 90 18.0 124 15 24 20. 5 10. 6 II. 8 14.4 90 8.5 150 17 27 20.5 10. 6 13-9 9.2 90 14. 0 88 30 30 20. 5 16.9 13-9 8-3 90 4.8 36 58 33 20.5 3-3 15-1 3-6 90 4.8 29 5° 36 20.5 9-7 21.8 3-6 90 30.0 22 30 MOISTURK CONTENT OF THE SOU. In view of the effect which high moisture content of the soil is reported by Wilson ^ and others to have in increasing nodule production, the moisture content of the soil in the later series was kept uniform at all 'Wilson, ]. K. physiologicai, studies of bacillus radiocola of soy bean(soja max piper) and OP FACTORS INFLUENCING NODULE PRODUCTION. N. Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig. 80-94. 1917- 30 Journal of Agricultural Research voi. xxii. No. i temperatures at one-half the moisture-holding capacity of the soil, previously determined to be 14 per cent of its dry weight. In order to get further evidence as to the extent of the effect of high moisture con- tent, a single can containing three plants was placed at each temperature in the last series with moisture content of 18 per cent of the dry weight — a distinctly wet soil. Accidents which befell several plants in the series produce irregularities in the figures which would require long explana- tion. Suffice it to say here that though the tops were increased in size there is no evidence that the nodules were increased either in number or size. Apparently moderate differences in moisture content of the soil were not large factors influencing results in the previous experimental work where exact control of soil moisture was not accomplished. HYDROGEN-ION CONCENTRATION OF THE SODv SOL,UTlON Inasmuch as it was considered possible that the extreme temperatures at which the soil was held might produce changes which would alter the hydrogen-ion concentration of the soil solution, and hence the formation and perhaps development of nodules, a determination of this environ- mental factor was made toward the close of the last series described. Samples of soil were taken from the unplanted pots at 15°, 24°, and 36° C. A determination of the hydrogen-ion concentration of the soil solution of the three samples by the colorometric method gave identical results, the Ph value being 6.3 in all three cases. Thus no evidence was obtained that temperature had altered this important factor in this series. SUMMARY (i) Preliminary studies have been made upon the effect of soil tem- perature on the development of four legumes, alfalfa, red clover, field peas, and soybeans, with special reference to its effect upon the infec- tion of these plants by Bacillus radicicola and the subsequent develop- ment of nodules. The larger part of the data were obtained by growing plants in soil held at a series of temperatures 3° apart from 12° to 36° C. The air temperature was uniform for all plants, ranging from 14° to 20°. (2) As was anticipated, the four plants differed in their ability to tolerate soil temperatures at the ends of the series. Peas were dwarfed at 30° C, clover developed poorly at 36°, while alfalfa and soybeans still grew very well at 36°. Soybean plants grown in the soils held at 12°, 15°, 33°, and 36*^ showed very dark green color of leaves, whereas those toward the center of the series became progressively lighter, those at 24° being lightest. (3) With regard to the number of nodules formed on plants grown in soil held at this series of temperatures, irregularities were found in each series; but no large consistent differences were discovered, except that at the extreme upper and lower limits at which a plant will survive the Oct. 1, 1921 Effect of Soil Temperature on Development of Nodules 31 number is reduced, and that peas usually produced greatly increased numbers at 30° C. All these species form nodules in soils at any tem- perature at which the plant can make a growth that is at all vigorous. (4) While variation in number was not consistent, size measured by the average dry weight per plant of all those formed on a number of plants was found to differ greatly and consistently within the series, at least so far as the soybean plant was concerned. The maximum weight attained on the soybean plant after a period of two months was found at a soil temperature of 24° C. Examination of nodules on the roots of the other legumes indicated that their maximum production occurred at about the same temperature. (5) Weight of nodules produced by soybeans was not found to be corre- lated with the weight of tops or of roots through the series of tempera- tures. Weight of tops was almost or quite as great at 30° to 36° as at 24° C, while weight of nodules declined rapidly at the higher tempera- tures. Weight of roots likewise showed no such diminution at the higher temperatures or even at the lower temperatures as did weight of nodules. With the soybean plant, and to a much less marked degree with the other plants, there was a correlation between weight of nodules and color of plant, the largest weight of nodules occurring on plants with the palest green color. (6) Generally speaking, plants with large nodules had a higher percent- age of total nitrogen in the tops, though this correlation is not exact. (7) Factors of soil environment that are regarded as having an influence upon nodule formation have been taken into account. Soil moisture has been controlled within narrow limits. Concentration of nitrates and the hydrogen-ion concentration of the soil solution have been recorded. It is not believed that variations in any of these factors are to be regarded as having produced the variations in nodule development recorded at the different temperatures in these series. PLATE I A. — Alfalfa plants grown 63 days in soil held at the temperatures indicated. B. — Red clover plants grown under exactly similar conditions with the alfalfa plants shown in A. Effect of Soil Tomporature on Development of Nodules Plate I Journal of Agricultural Researcli Vol. XXII, No. 1 Effect of Soil Temperature on Development of Nodulos Plate 2 ^-n .>^ r^' •* ^ 27"" 30° ^3° Journal ot Agricultural Research Vol. XXII, No. 1 PLATE 2 A. — Soybean plants grown 63 days in soil held at the temperatures indicated. B. — Nodules from 6 soybean plants (only 5 plants at 30° C.) grown 63 days at the temperattu-es indicated. One-half of the plants are shown in A. 54817°— 21 3 PLATE 3 A. — Soybean plants inoculated with Bacillus radicicola contrasted with uninoculated plants grown 55 days in soil held approximately at the temperatures indicated. The pots are grouped according to temperature, with the control on the left and the inocu- lated pot on the right in each set. B. — Nodules from 9 soybean plants grown 55 days in soil held at approximately the temperatures indicated. Effect of Gnil Temperature on Development of Nodules Plate 3 A 15' Journal of A_;ricultural Research Vol. XXII, No. 1 INFLUENCE OF THE PERIOD OF TRANSPLANTING WESTERN WHITE PINE SEEDLINGS UPON THEIR BEHAVIOR IN NURSERY AND PLANTATION By E. C. Rogers Forest Examiner, Forest Service, United States Department of Agriculture At forest nurseries in the northern part of the United States the work is customarily crowded into three or four weeks in spring immediately following the time when the soil can first be worked. Preferably, the stock is lifted, packed, shipped early, and spring sowing and transplanting are all usually crowded into this period. At the Savenac Nursery, Haugan, Mont., this spring congestion has been keenly felt; and the experiments outlined below have had for an object the determination of the safe limits of the transplanting season. The results may or may not apply beyond the local conditions prevailing in the region of western Montana and northern Idaho. FALL TRANSPLANTING Work was commenced upon this problem at the Savenac Nursery in the fall of 1913. By the use of the Mast trencher method, 600 i-year-old seedlings of western white pine {Pinus monticola Dougl.) were transplanted on each of the four following dates: August 15, September i, September 15, and October 10. In May, 19 14, these plants were examined and the overwinter losses were recorded. Loss by frost heaving, as indicated in the figures, includes not only plants completely thrown out but also those lying prostrate on the ground, even though they were quite firmly attached and still alive, because in that condition they would never recover sufficiently to be fit to plant. A few individuals showed the symptoms of winterkilling. These were about evenly distributed among the four units, in no case amounting to i per cent of the total. Figure I shows the loss by frost heaving. From one- third to one-half of the plants were heaved out during the cold nights of late October, before the coming of snovv^, and during the clear weather of late March and early April after the snow left. The loss was greatest in the October 10 unit. This may be explained by supposing that, because of the warmer soil temperature the individuals transplanted earlier had had time to make sufficient root growth to render them more resistant to the frost lifting, but that those transplanted latest were virtually heeled in. However, precise evidence on this point is lacking. As it had been suggested that possibly in the Mast V-shaped trench a pocket of loose soil was formed around the lower roots and that this Journal of Agricultural Research, Vol. XXII, No. i Washington, D. C. Oct. i, 1921 z p Key No. F-6 34 Journal of Agricultural Research voi. xxii. No. predisposed the plants to heaving, several rows of i-o western white pine seedlings, transplanted September 25, 1913, in open plowed trenches were likewise examined in May, 1914. From a total of about 12,000 trees the loss from frost heaving was 29.6 per cent and that from winter- killing 1.2 per cent. Here, again, nearly one- third of the plants were thrown out — a loss hardly 6 per cent less than that by the Mast trencher soo 4S.0 ^.o 3S.O 30.0 £S.O g 20.0 k ^ /s.o /o.o s.o o / / ^ / ^^ , /-OSS OP^/ia// /jrcZ/7S/0/c7/7/s ^ .__ /i^ay/7 Ja/7e2 Ji//7e/S /?uy./S S(sp^/ Se/:>A/S Oc/:? Fig. I. — I,oss by frost heaving of seedlings transplanted at different dates. method, which was used 10 days earlier. Apparently the method mat- tered little, the loss having been a necessary consequence of fall trans- planting. Lorey ^ found that fall transplants of Douglas fir and European larch led spring transplants in height growth in the transplant rows. More recently, Toumey ^ states that fall transplants lead in earliness of growth and in size, provided they escape winter injury. While no data with 1 Lorey, Tuisko. mitteilungen aus dem forstgarten und KtaTURBETRiEB. n. forstgarten INSBESONDERE. In AUg. Forst u. Jagd. Ztg., N. F., Jahrg. 70, p. 193-197. 1894. ' TouMEv. James W. seeding and planting: a manual for the guidance op forestry students . . . xxxvi, 4SS p., 140 fig. New York, 1916. Oct. 1, 1921 Infltience of Period of Transplanting 35 respect to those matters were collected, the heavy loss through heaving at Savenac Nursery more than balanced any possible gains of that kind. Fall transplanting is clearly so.unsafe that no further local experi- ments with it are necessary. SPRING TRANSPLANTING NURSERY TESTS Experiments in the spring of 19 13 had for their object the compari- son of three lots of 600 i-o western white pine each, transplanted on May 17, June 2, and June 16. The first summer's loss from drought increased with the lateness of the transplanting period, as is shown also by figure i. The June 16 lot looked less thrifty than the others at the end of the season, and it was concluded that^ in case of necessity, trans- planting could evidently be continued as late as June 15, though it is not desirable. As a control on the tests made in the spring of 19 13 it was arranged to transplant 1,000 i-o western white pine at Savenac Nursery every 10 days during the spring of 19 14. This was actually done on April 24, May I, May 9, May 20, May 30, June 12, June 19, June 30, and July 14. On each of these dates 100 more were removed from the seed- bed, of which the weights and measurements appear in Table I. Table I. — Weights and measurements of i-o western white pine on different dates of transplanting in the spring of IQ14 Date of Average length of stem. Average diameter of stem. Average weight. Per- centage Per- centage Per- centage Average new spring growth. trans- Lot No. of plants with buds closed. of plants with buds swelling. of plants plant- ing. Top. Root. with buds open. Needles. Rootlets. Inches. Mm. Gm. Gjn. Inches. Inches. Apr. 24 I 1-54 I. 17 0. 140 0. IIS 100 0 0 0. 0 COS May I 2 1-52 I 23 •i53 . 122 100 0 0 0 10 9 3 I. SO I 13 •143 . 090 90 10 0 0 20 20 4 1.90 I IQ .186 . 120 0 34 66 08 S4 30 S 1.99 I 17 .125 .oSi I 6 93 IS 62 June 12 6 3.30 I 28 .265 .150 0 I 99 27 I 07 19 7 2-43 I 22 .203 • 103 0 0 100 44 94 30 8 2. 30 I 46 •313 •139 0 0 100 73 (') July 14 9 2. 60 1-53 .274 .130 0 0 100 1. 02 0) 1 Not recorded. In Table I stem length is the distance from the ground line to the tip of the terminal growing point. The stem diameter was measured at the ground line. The average new growth of rootlets was based upon the longest rootlet noted in each plant examined and not upon all new rootlets. This figiure is only relatively correct, because some slight root breakage was unavoidable in taking up the plants from the seed bed. Root growth data were omitted in the two latest lots, since the older portions of the * Unpublished progress report. 36 Journal of Agricultural Research voi. xxii, no. i new roots were assuming a brown, mature appearance, and this made it difficult to determine the margin of growth. - Figure 2 shows graphically tlie condition of the seedlings as to length of stem and spring growth of needles and rootlets on the different dates of transplanting. Apr £4 May I May 9 Julijl-^ Maij£0 May 30 June 12 June IS June JO Time of 7~ran^ p/a nilng Fig. 2. — I^ength of stem and spring growth of needles and rootlets of seedlings transplanted at different dates. Table I brings out the following points : I. Root growth began prior to April 24 during the spring of 19 14, or over three weeks before visible stem gro^vth. Although not so indicated by the table, it was found that the earliest visible root elongation took place in the superficial soil layers, gradually progressing to deeper and deeper levels as the season advanced, presumably in response to changes in soil temperature. Oct. 1, 192 1 Influence of Period of Transplanting 37 2. The swelling of the buds began a little before May 9, and two-thirds of them were fully open by May 20. 3. Although individual variations existed — due largely, it is thought, to a lack of uniform density in the seed bed — stem length, stem diameter, and weight of top increased in general as the season advanced. 4. The proportion of the fresh weight of the plants contained in the root was greatest early in spring and decreased as the growth pushed ahead in May and June. Figure 3 brings out this point. Owing to the possibility of variation in the water content of top and root, dry weights would be of interest, but circumstances prevented their being obtained. so. Or o 4-50 =- 4-0.0 JSO 30.0 \ \ "~~\ Percsnta^s offrssh \A'eigh1r of plant- in root at different dates of transo/anting __- A\/eraffe fresh we/g/it ofp/ant ■~^~- ' ■ SO /\pri/2'}. Maj'l MayS May 20 May JO June/ 2 June/3 June SO Ju/y/4- 77/77G' o/' 7rt:?/7S/>/c?/7//no Fig. 3.— Proportion of fresh weight of roots of seedlings transplanted at different dates. Transplanting was done in adjacent parallel rows, and these were irrigated at intervals during the summer of 19 14. As early as August i there had come to be a marked differentiation in size and color. The April 24, May i , and May 9 units showed particular vigor and had a rich green color. The June 30 and July 14 lots showed much the same development as the earliest ones but had a yellow color, which gave the July 14 lot an almost sickly appearance. The remaining intermediate lots showed a healthier color than the later ones but lacked the size and development of the latter. This differentiation was increasingly marked at the end of the growing season. The loss from drought in these lots during the summer was very slight, the heaviest loss, 2.4 per cent, being suffered by the July 14 unit. 38 Journal of Agricultural Research voi. xxii. No. x On September ii, 1914, the season's growth of stem and needles was obtained by measuring every tenth plant in each lot, or 100 in each unit. The terminal buds of 500 plants in each lot were examined as to their maturity on this same date. Buds having a definite form, of a deep brown color, and covered with a protective coat of fine hairs were classed as mature. Plants without a single well-defined bud and those whose growing point had a tender green color, without the coat of hairs, were considered of immature development. For purposes of comparison, data similar to the foregoing were obtained from 2-year-old western white pine plants from a representative area of seed bed. These plants were of the same age and seed lot as the nine transplanted units, differing only in that they had been allowed to remain in the seed bed. These data are given in Table II. Table II. — Growth and development of western white pine during the first season in the transplant bed Date of transplanting. Lot No. Average seasonal stem growth. Average seasonal growth of needles. Percentage of plants with mature buds. Percentage of plants with immature buds. Apr. 24 May I 9 20 30 June 12 19 30 July 14 Not transplanted . Inches. 0-757 729 763 734 825 874 918 974 951 063 Inches. 0.934 952 897 424 566 570 611 715 943 131 80.0 75-7 75- 1 69. I 70.1 69.6 56.4 80.6 92. 6 93-5 20. o 24-3 24.9 30-3 29.9 32-4 43-6 19.4 7-4 6.5 By average growth of stem and needle is meant the growth for the entire season, regardless of whether that growth took place in the seed bed, in the transplant bed, or in both. Needle measurement was made in the middle of the sector of currently- grown stem. Table II brings out the following points : 1. The later the transplanting after the buds open, the higher the stem growth for the season. It appears that height growth practically ceases for a time after transplanting, the plant's energies being directed toward getting established in its new habitat. In other words, the height growth is roughly proportional to the length of time the plant is left in the seed bed. Hence, plants that were not transplanted made a higher stem growth than any of the transplanted lots. 2. The longest needle groAvth at the end of the season had been made by the first and last lots (Apr. 24 and July 14). The needle growth of the first lot had been made entirely in the transplant bed and was accom- panied by a deep green color, but that of the last lot had taken place in Oct. 1, I92I Influence of Period of Transplanting 3,9 the seed bed before transplanting and the needles of these looked much less vigorous. As figure 4 shows, the season's needle growth commences to fall with the May 9 lot, drops abruptly with the May 20 lot, then climbs gradually until the last lot equals the earliest. The stock that was not transplanted produced longer needles than any of the trans- planted units. 3. The difference in dates of transplanting had a pronounced effect upon the maturing of the fall buds. The earliest maturing lots were the two that were transplanted latest (June 30 and July 14). The less so k.O 0.0 %^^ c 2.0 1.6 10 06 Height growth erf in 1316. pric / planfj r +oJali/ 1. / / \ / / N \ / \ Growth \ of naedfe 3 during \ planti dif / fran^pi^ni^d at ^ s/ __^ --^^ ^v ^ : 1 AprZ^ Mayl Maij9 May20 MayJO Junel2 JunelS JuneJO Ju/(//4 Mf Time of Transpthntinq planted Tig. 4.— Increase in height and growth of needles of seedlings transplanted at different dates. favorable weather conditions appear to hasten preparations for winter by stock transplanted in summer. 4. Seedling stock of the same age and source (2-0) led transplants (i-i) in current stem growth and needle development no matter what the period of transplanting. The shock of the treatment, expressed quantitatively, resulted in a loss of 0.3 inch of stem growth and 0.2 inch of needle growth, even when the transplanting was done at the most favorable period. At the end of the season in which the transplanting was done it ap- peared that the plants lined out before the buds were open had suffered the least shock, and, judging from their unhealthy appearance, those transplanted in midsummer (July 14) seemed to have suffered most. No single item of weight or measurement appears to be a consistent indicator of the degree of severity of the shock. 40 Journal of Agricultural Research voi. xxii. No. On July I, 1916, 100 plants were washed out from each of these nine transplanted units by the aid of water under pressure. At this time, the stock could be considered to be in the i-2>^ age class. Data from these 900 plants are assembled in Table III. Table III. — Weights and measurements of 1-2% -western white pine Lot No. Average stem height growth (cur- reiit).a Average stem diameter. Average number of laterals. Date of trans- First order. Second order. Average total fresh weight of plant. Percent- age of weight in root. planting. 2 inches and up. 0.5-inch to 2 inches. 2 inches and up. 0.5-inch to 2 inches. Apr. 24 May I 9 20 30 June 12 19 30 July 14 I 2 3 4 5 6 7 8 9 Inches. 2-75 2-75 3.66 2.09 3-27 3-58 2.8s 2.74 1.82 Mm. 6.31 6.72 7.14 5-65 6.48 6.48 6.07 6.27 4-95 10.2 10.3 10.3 9.0 10. 5 8.1 9.1 10.2 2.6 5-4 6.7 6.1 6.1 4-5 5-2 5-3 5-1 5-3 3-4 6.3 4.1 3-2 2.9 3-6 3-7 3-8 2-5 15-3 27.4 17.2 13.0 13.0 II-3 10.5 12.4 9.9 Gm. 3-09 3.16 3-78 1.84 2.78 2.74 2.54 2.62 1. 41 43-4 45-6 36.2 45-7 41.4 36.9 39-4 42.7 48.2 o By current stem growth is meant the 1916 growth prior to July i. 73 7.0 6J GO § s.o J.S ao 2.6 ^^ ^ ^ / ^ ^ Diamett (me, Juli 1 r of sfern af ground /.ISI6, of stock trans- \ plan-f-e in the d at differ spring. of enr dates \ Apr. 2^ Maul Maij9 Maij20 Mat/JO JunelS Juna/S JuneJO Julijl^ 7~irrre of 7~ransp/aniing "Bic. 5. — Increase in diameter of stem of seedlings transplanted at different dates. Oct. 1,1921 Influence of Period of Transplanting 41 A study of the proportion of the fresh weight of plant in the root system shows that the relation between this proportion and the time of transplanting, which was so evident two years before, had entirely dis- appeared. All the plants were washed off, and the surfaces were allowed to dry in the air; but as the evaporating power of the air varied, it was not possible to compare directly the average fresh weights obtained at tlie time of transplanting with those obtained on July i, 19 16. All the weights obtained on the latter date are, however, comparable with one another. Figures 3, 4, and 5 illustrate further the current height growth of stem, the stem diameters, and the total fresh weight of the plants. A striking similarity will be noted in the curves in figures 3 (weight), 4, and 5. All agree in shovnng two minima, the first for the May 20 lot, and the second and still lower point for the July 14 transplanting. This bears out the conclusion reached in the fall of 19 14 that the shock of transplanting fell hardest upon the July 14 lot. But it now appears that the transplanting on May 20 was almost equally lasting in its unfavorable influence upon growth in the transplant bed. Between these two dates there appears to have been a period of about a month in which trans- planting worked less injury to the plant. As the roots of these plants were removed by washing, and with a minimum of breakage, it seemed worth while to compare the average numbers of laterals of the first and second orders. Figure 6 shows graphically the data obtained. In order to bring the curves close together, and thus facilitate comparison, the actual average number of lateral rootlets of the second order, between 0.5 and 2 inches in length, has in each case been divided by 2 in plotting the curves, and the average total number of lateral rootlets in the four classes has similarly been divided by 5. Because of the difficulty involved in recording them, and the limited time available, no records were obtained of the number of laterals of higher orders than the second, nor, in any case, of laterals less than 0.5 inch in length. The figures obtained are, however, considered indicative of the general nature of the root system. There is a close relation not only between the average number of laterals in each of the two length classes of the second order but also between these and the total number of rootlets of the recorded classes. On the other hand, the curves for the two classes of the first order run quite differently. Yet, so far as determining the total curve is concerned, these two classes could obviously have been neglected. There is a con- sistent decline in the fibrous development of the root system as the transplanting season advances. Transplanting in early summer, and even more so in late summer, holds back lateral root growth in the trans- plant bed, the effect being strikingly noticeable two years afterwards. 42 Journal of Agricultural Research voi. xxii. No. Although several seasons' observations had shown no noticeable loss through winter frost heaving of i-o western white pine transplanted in April and in early May, it was found in the spring of 1918 that of 2,922 i-i white pine transplanted June 15, 19 17, in connection with another experiment, 1,152, or 39.4 per cent, were heaved out during the late fall, winter, and early spring of 19 17-18. This is nearly as heavy a loss Ma(/9 May20 Mat/30 Junel2 JunelS JuneJO Jultjl^ Time of Transplanfina Apr 24 May I Fig. 6.— Number of lateral rootlets on seedlings transplanted at different dates. as had previously been recorded for fall transplanting. However, an unprecedented snowless period in December probably contributed to produce this result. A greater susceptibility to frost heaving on the part of late spring and summer transplants is the natural result of the poorer root development just referred to. The plant must rebuild its entire root system late in the season and so has a relatively poor anchor- age when the frost comes. Furthermore, Cannon * has found that the 1 Cannon, William Austin, root habits oP desert plants. 96 p., x7 fig., 23 pi. Washington, D. C„ igii. (Carnegie Inst. Wash. Pub. no. 131.) Oct. 1, 1921 Influence of Period of Transplanting 43 formation of an abundant lateral root requires a favorable water content in the soil and a sufficiently high soil temperature. Although summer soils are warm, yet, in spite of occasional irrigation, the greatest loss of transplants from drought at Savenac Nursery occurs during July and August, indicating that there is less available soil moisture during that period, or at least that there is a smaller balance for growth when the transpiration loss of the plant has been met. It seems, therefore, that in the foregoing series the plant organism was most deeply disturbed by being transplanted in midsummer. This appeared to be a consequence of the high evaporation and lack of moisture in the soil, along with the greater topheaviness of the plant. The May 20 transplants gave evidence of having been most severely set back, a result which must be attributed either to external conditions or to the internal state of the plant. The Savenac Nursery weather records show a precipitation of 2.16 inches in April, 1914, well distributed throughout the month, with only seven clear days. In May, previous to the twentieth, there fell 0.58 inch of rain, and 12 out of 19 days were cloudy or partly cloudy. On May 20, the soil was well stored with water and was favorable for the reception of plants. The maximum temperature on that day was 72° F., and it and the eight days following were partly cloudy. During the period from May 22 to 28, inclusive, 0.46 inch of rain fell, every day yielding at least a trace. The weather and soil conditions were, therefore, sufficiently favorable to convince the writer that the reason for the marked checking of the growth of the May 20 lot lay in the developmental stage of the plant itself. One-year-old western white pine seedlings, whose buds are just opening and whose tiny new needle fascicles are less than i/io inch long, show a particular sensitiveness toward removal and replanting. FIEI/D TESTS The influence of the season of transplanting upon the behavior of the tree in the plantation is of special interest to the forester. One hun- dred of the plants from each of the nine spring lots described above were planted October 6, 19 15, on the Wallace experimental area, near Wallace, Idaho. A northwest aspect — a typical white pine planting site — ^was selected. One row was devoted to each lot, and the rows were placed adjacent to each other and parallel, extending up and down the slope. The place where each tree was to be planted was previously marked by a cedar stake 16 inches long, whose top had been dipped in white paint to make it conspicuous among native cover plants. Each stake bore its lot number in black lumber crayon. The same man planted all the rows, using a uniform method. 44 Journal of Agricultural Research voi. xxii. No. On September 21, 19 16, and on November 7, 19 17, these plants were examined, their condition was noted, and the average height growth of stem was recorded, this average being based upon all vigorous living plants. Table IV gives the principal data secured. Table IV. — Date of transplanting, average current height growth, and percentage of trees surviving at the end of the igi6 and I gi J field seasons on the Wallace area Date of transplanting — 1914. Apr. 24. May I . 9- 20. 30 June 12. 19. 30- July 14. Average of all lots. Lot No. Average current height growth. 1916 Inches. 0-57 .91 •95 .82 .67 .96 .82 I. 00 ■65 Inches. O. 62 83 96 81 75 95 84 IS 84 Percentage of trees surviving. Fall 1916. Fall 1917 95-9 90.9 95-7 93-6 94.6 92-5 92.8 93-7 98.9 96. 2 94-3 The fact that the unusually dry summer of 191 7 caused almost negligible losses makes improbable any further changes of importance in the survival standing of the nine lots. The percentage of living trees of all lots in the fall of 191 7 was above 90. There is no superiority on the part of the early lots, the April 24 units standing lowest. The later lots have, on the whole, lived best. Both the May 20 and July 14 plants, while outclassed in the transplant bed, showed better than an average survival in November, 191 7. In fact, the time of transplanting had no apparent influence in the field. Figm-e 7 further illustrates the height growth of these plants. There is a marked similarity between the growth curves for 1916 and 191 7; hence each resembles the total growth curve for the entire two seasons. For instance, in each of the three curves the average point for the June 19 lot falls exactly upon the horizontal average line for that curve. To facilitate comparisons, the height growth curve from figure 4 is plotted in figure 7 also. This renders it possible to compare the growiJi made in the plantation the first year after planting (1916) Avith that made the same season prior to July i by individuals left in the transplant beds (curves A and D, respectively). There are certain points of resemblance between the curves of growth in transplant bed and in field. The May i and June 19 lots stand upon or very near the horizontal average line in both. The July 14 transplants stand low, and the May 9 and June 12 transplants stand high in both. But, on the other hand, the May 20 lot, which had a low growth rate in the Oct. 1, 1921 Influence of Period of Transplanting 45 transplant bed, lias an average rate in the field; but the May 30 and June 20 units reverse their positions with respect to the horizontal average lines. The planting out of the stock has evidently caused a somewhat Apr so May/. Maj'9 MaySO Ma/JO June I2 June/3 JuneJO July l4 T/mci of Transplanting Fig. 7. — Increase in height of seedlings transplanted at different dates. general rearrangement of growth rates in which the later lots tend to overtake the earlier ones, this rearrangement being accentuated after a second season in the field. The inferiority of the May 20 plants has 46 Journal of Agricultural Research voi. xxii, no. i disappeared; the July 14 stock slightly surpasses the April 24 lot; and there is nothing to indicate that summer transplants are not fully the equal of spring transplants, so far as growth after planting is concerned. CONCLUSIONS Transplanting western white pine seedlings at any time in the fall is not a safe practice at Savenac Nursery, because the frosts of the following late fall and early spring heave out the young plants. Results in the plantation thus far indicate that, where 1-2 stock is grown, the transplanting season may be extended from a date as early in spring as the ground can be worked until early July. The shock of removal from the seed bed is greatest when the transplanting is done in midsummer, on account of the high evaporating power of the air, the low water content of the soil, and the greater top-heaviness of the plant with its considerably increased transpiring surface. A particularly sev^e shock was also suffered when transplanting was done at the time of bud opening and before the rudimentary needle fascicles had reached ■^^g-inch in length. Stock transplanted at either of these critical periods lagged behind the other lots for at least two years in the transplant bed. However, when they were planted in the field as 1-2 stock, the plants survived as well as the others, with little, if any, inferiorit}'- in growth on account of the considerable rearrangement of growth rates following the planting. There are, nevertheless, other reasons which make transplanting at Savenac Nursery safer in spring than in summer. June and July trans- plants suffer more from drought the first season and, because of their poor root development at the end of the growing season, are more subject to frost heaving the following winter. The first disadvantage can be met by proper irrigation, but the second can not readily be prevented. The safest practice, therefore, is to confine this work as much as possible to April and early May. The foregoing conclusions apply to i -year-old seedlings which are to remain two years in the transplant rows, 1-2 stock being the only age class of white pine transplants at present grown at Savenac Nursery. Certain points brought out by this study may have an important bearing upon the season for field planting. It is probable, for instance, that subsequent lateral root development in the plantation may be decisively influenced by the time of planting. A DRYROT CANKER OF SUGAR BEETS By B. L. Richards Department of Plant Pathology, Utah Agricultural Experiment Station What appears to be an undescribed rootrot of the sugar beet was first called to my attention on August 5, 1920, by Mr. A. H. Bateman. Speci- mens of the diseased beets collected at this date at Cornish, Utah, ex- hibited numerous brown, circular lesions that varied from j^ inch to i inch in diameter (PI. 4 ; 8, D ; 9, A, B) . The outer surface of the root cov- ering these lesions, which in most cases remained entire, had so sunken as to give a definite undulating contour of alternating light and dark brown concentric areas or rings (Pi. 4). The removal of this outer layer of cells of the older lesions exposed deep cankers or pockets filled with hyaline- mycelium embedded in the dry remains of partially decayed host cells. This accompanying mycelium, when exposed to the atmosphere through the cracking open of the outer covering, appeared dark brown in color and immediately suggested the typical mycelium of the sterile, or "Rhizoctonia," stage of Corticium vagum B. and C. The general prev- alence of black sclerotial bodies on the outside of the diseased beets (PL 7, A), together with the microscopic examinations made at this time, confirmed this initial suggestion. An examination of the field from which these first diseased specimens were taken revealed the trouble to be of considerable economic impor- tance; at least 20 per cent of the beets in this field of 40 acres were diseased. The disease appeared to be confined to definite areas wherein every beet might be found infected. These diseased spots varied con- siderably in size and appeared to be widening most rapidly in the direc- tion parallel with rows. Three adjacent fields were found at this time to be seriously diseased, but none to the same degree as the field first visited. The progress of the disease in these fields appeared of such ominous character as to require immediate investigation. However, as the season was well advanced, little more than preliminary experiments were undertaken. The results to date, while definite, are not sufiiciently extensive to warrant final conclusions, and many of the important rela- tions of the disease remain obscm-e; nevertheless it is felt that the apparent economic importance of the trouble justifies a preliminary description at this time. The disease is first detected in the field by abnormal wilting of the leaves in the daytime with partial or complete recovery at night. Later Journal of Agricultural Research, Vol. XXII, No. i Washington, D. C. Oct. i, 1921 zm Key No. Utah-14 54817°— 21 4 (47) 48 Journal of Agricultural Research voi. xxii. no. i the older leaves fail to recover, turn brown, and die. This dying of the outer or older leaves continues with the progress of the disease in the root until all the leaves on the affected beets may succumb. Localized browning frequently occurs in the blade and petiole, but to date no suggestion of a parasitic relation has been found. Neither the petiole decay reported by Duggar (2) ^ nor the "western crownrot" described by Edson (j) have been found associated with thedryrot canker in the field. A peculiar type of crownrot, however, is found late in the season, usually well toward harvest time (PI. 6; 8, B). A study of a number of these crownrot specimens indicates definitely that the causal organism enters the beet below the stu-face of the soil and works upward in the tissues, eventually destroying the crown. The fungus has not been observed to attack the beet above the soil line. It is evident that the fungus is unable to destroy the outer corky cells of the beet root, but gains entrance to the inner tissue at a definite point and works tangentially just beneath this outer layer. As the fungus eats its way from the point of entrance the outer tissues, due to killing and subsequent drying out of the cells beneath, sink in such a manner as to produce the circular lesion with its very definite undulating contour of alternating raised and sunken concentric "rings" (PI. 4). The lesions appear first as a small, brown, sunken spot with a minute perforation in the center (PI. 8, D). The first definite concentric "ring" which is considerably sunken below the central area and usually dark brown in color is noted before the lesion reaches a diameter of >^ inch. With continued enlargement a second and somewhat broader "ring," less sunken and much lighter in color, results. Similar concentric areas are developed alternately until the fimgus reaches its limit of lateral spread. Individual lesions resulting from a single point of infection may obtain a size of from ^ to i inch in diameter and develop as many as eight alternate "rings" (PI. 4). When, however, adjacent lesions become confluent, as they frequently do (PI. 4; 8, D; larger lesions result which may in severe cases cover a large part of the root surface. In such cases large concentric rings are produced, which become common to a number of centers of original infection (PI. 4; 8, D; 9, A, B). Another characteristic feature of the disease results in cases where infection occurs at or near the apex of the root. The root in such an event is usually severed at the point of infection and the fungus advances upward, producing the typical dryrot with resultant concentric rings which may encircle the entire root (PI. 7, B). Again, cankers may occur with such frequency as to girdle completely the root (PI. 8, A). The distinctive feature of the contour, as shown in Plates 4 and 8, D, is obtained usually before the fungus penetrates deeply into the tissue of the beet and before a serious rupture of the outer layer occurs. With 1 Reference is made by number (italic) to "I,iterature cited," p. 52. oct.i.i93i A Dryrot Canker of Sugar Beets 49 the drying out and final cracking of this outer covering the fungus, possibly because of a better oxygen relation, eats radially into the beet, producing deep cankers (PI. 5 and 6). The decaying tissues rapidly dry out as the fungus advances inward, leaving the cavity partially filled with a dry, pithy residue. Frequently the content of the canker appears as a definite plug, which, upon wetting, may be removed intact from the cavity of the canker (PI. 9, D). Except for slight cracking, the outer layer of dead cells remains entire and furnishes a definite covering until the lesion has reached approxi- mately its limit of tangential spread. As the cells of this outer covering finally dry out the central perforation enlarges and ultimately gives rise to a definite crack which may extend the entire diameter of the lesion (PI. 4; 8, D; 5). Frequently adjacent cracks become confluent, resulting in large characteristic fissures, which in severe cases of the disease may obtain from 2>^ to 3 inches in length and from % to 1% inches in depth (PI. 5, 6). With numerous points of attack the beet by harvest time is converted into a dry, brittle shell filled with a pithy mass of host and fungous debris (PI. 6). During the season careful study was made of a large number of the beets taken from each of the different fields in which the dryrot had been found. In all cases the characteristic cankers exhibited the pres- ence of the sterile stage of Corticium vagum. This fungus, it was found, may be obtained regularly in a pure form from any part of the typical canker, provided the outer covering of the lesion is not previously de- stroyed. The brown layer separating the normal from the diseased tissue (PI. 8, A-C) has never failed to yield the fungus free from other organisms, and even from the open lesions cultures have been obtained with remarkable ease and regularity. The degree to which other organ- isms are found to be excluded is phenomenal. To determine the etiological relation of the fungus, inoculations were made September 3 on partially grown beets. In the process of inocula- tion the soil was removed to a depth of approximately 4 inches from 2 1 beets in each of five rows. Each of the 2 1 beets in the first row was punctured a number of times with a sterile needle, and the inoculum, consisting of the beet fungus, grown for several days on potato agar, was then scattered throughout the soil as the latter was replaced about the beet. Row 2 was inoculated exactly as row i except that in place of needle punctures slight incisions were made in the beet by the use of a sterile scalpel. The beets in rows 3 and 4 were wounded as in row i, and the soil was inoculated with two different "strains" of Corticium vagum,} Row 5 was left uninoculated, and the beets after wounding as in rows i and 2 were covered and grown as controls. All the wounded 1 These "strains" were obtained from the surface of a potato tuber in 1918 and have proved virulent on potato stems in both sterilized and unsterilized soil. 50 Journal of Agricultural Research voi. xxii. No. i beets in the control row healed normally. Infection occurred on but one beet in rows 3 and 4. The other beets in these two rows healed as perfectly as in row 5. The results of inoculation with the sugar-beet strain of the fungus in rows i and 2 are given in Table I. The types of lesions produced as a result of atrificial inoculation are shown in Plate 9, A. D. Table I. — Number of lesions on sugar beets inoculated with the sterile stage of Corticium vagum Beet No. Row I, needle puncture. Row 2, incision. I . 23 7 II 13 8 8 17 14 II 17 6 17 13 17 20 19 10 15 II 12 16 8 2 0 3- • 4. . \.A\^.^:^uu^^ 3 13 8 5 6 2 7 6 8 : 2 Q 6 10 .;. 4 II 4 6 12 I -J 7 8 14 IC 3 2 16 17 6 18 4 4 0 10 20 21 - - 0 Total 285 13-5 96 4-5 Average Instructions for the inoculating of sugar beets with the beet fungus without puncture or incisions were not followed. As a result the question as to the ability of Corticium vagum to attack the sugar beet independ- ently of other agents remains unsettled. It is quite conceivable that sugar-beet root aphis {Pemphigus betae Sloane) and other insects so prevalent in the soil may serve an important function in the initial en- trance of the fungus. Having once gained access to the lower tissue, however, it appears evident from the results that this particular "strain" of C. vagum is capable of producing the type of canker and dryrot with which it is so constantly associated in the field. The peculiar method of decay, together with the sharp line of demarca- tion between the diseased and the normal tissue (PI. 8, A-C; 9, C-F), pro- vide the most distinctive characteristics of the disease. A dark brown, watery layer invariably separates the dry, decayed mass occupying the cavity of the canker from the normal host tissue beneath. This layer oct.i.igzx A Dryrot Canker of Sugar Beets 51 when examined under the microscope is found to be composed of masses of hyaline, vigorously growing young hyphae ramifying through and between the rapidly decaying host cells. It is in this advance layer that the major portion of the tissue destruction occurs. The brown layer advances uniformly inward by additions from the normal host tissue, while the outer surface of the layer rapidly dries out and con- stantly contributes its substance to the pithy mass occupying the resulting cavity of the canker. The thickness of the layer is dependent largely upon the rate at which the moisture is lost from it outer surface as the fungus eats its way radially into the normal tissue. No evidence of direct penetration of tlie normal cells by the fungus has been found. On the other hand, it appears that dissolving enzyms precede considerably the advancing mass of young hyphae (PI. 8, C). This method of tissue destruction resembles in a very definite way that described by Ramsey (5), by which Rhizoctonia solani Kiihn attacks and produces a definite pitting of the mature potato tuber. A similar process of decay is described by Atkinson (r) for the "sore shin" of cotton. He states that — the fungus {Rhizoctonia solani) never seems to penetrate far into the living tissues, but kills as it goes, and the tissues become brown, depressed, and present the appearance of a plant having a deep and ugly ulcer at the surface of the ground. A type of decay most accurately resembling this particular beet rot is described by Richards (7) for the potato stem-canker caused by Corticium vagum. The early production of definite cankers by a slow corroding of the normal tissue, finally resulting in a complete dryrot of the beet, suggests a possible name "dryrot canker" for the disease here described. Various American workers. {2; 4, p. 243-^4; 3) have reported rootrots of the sugar beet which they attribute to the work of Rhizoctonia solani Kiihn. It appears difficult at this time, however, to determine the possible relation of these to the particular type of dryrot described in this article. The indefiniteness of the literature on the subject in fact does not justify any general statement as to the possible distribution of the disease. During September and October of 1920 a preliminary survey ^ was made of the beet-growing districts in four counties of Utah — Cache, Davis, Utah, and Salt Lake. The disease was found in 18 fields of the 51 visited in Cache County and in 3 fields of the 20 surveyed in Davis County. Very serious damage occurred in a number of these fields. No indication of the trouble v/as found in either Utah or Salt Lake Counties. The limited survey does not permit of an estimate of the loss to the total sugar-beet crop of the State; nevertheless, the general prevalence 1 This survey was conducted in cooperation with the Office of Plant Disease Survey, United States Department of Agriculture. The author wishes to express his indebtedness to Dr. G. R. Lyman for this support. 52 Journal of Agricultural Research voi. xxii. No. i of the trouble would indicate that under more favorable conditions the disease may become a serious factor in beet culture. It is not improbable that a thorough survey may discover the "dryrot canker" in every beet- growing district in this and surrounding States. Since the appearance of the author's abstract (6), Dr. George L. Peltier reports in a letter to the author that he noted during 1920 in Nebraska what appears to be the same trouble. Preserved specimens in the plant-disease herbarium of the Utah Agricultural College show that the disease was collected in Utah as early as 19 15. LITERATURE CITED (i) Atkinson, Leo. F. 1895. DAMPING OFF. N. Y. Cornell Agr. Exp. Sta. Bui. 94, p. 231-272, fig. 55. 6 pi. (2) DUGGAR, B. M. 1899. THREE IMPORTANT DISEASES OF THE SUGAR-BEET. N. Y. Cornell AgT. Exp. Sta. Bui. 163, p. 335-363, fig. 49-63. Some references to the lit- erature of beet diseases, p. 361-363. (3) Edson, H. a. i915. seedling diseases of sugar beets and their relation to root-rot AND CROWN-ROT. In Jour. Agr. Research, v. 4, no. 2, p. 135-168, pi. 16-26. Literature cited, p. 165-168. (4) Pammel, L. H. 1891. FUNGUS DISEASES OF SUGAR BEET. lowa Agr. Exp. Sta. Bui. 15, p. 234-254. 6 pi. (5) Ramsey, Glen B. 1917. A FORM OF POTATO DISEASE PRODUCED BY RHIZOCTONIA. In Jour. Agr. Research, v. 9, no. 12, p. 421-426, pi. 27-30. (6) Richards, B. L. 1921. a dry rot of the sugar-beet caused by corticium vagum. (Abstract.) In Phytopathology, v. 11, no. i, p. 48. (7)- 1921. THE POTATO STEM CANKER. Utah Agr. Exp. Sta. Bui. 178. PLATE 4 Sugar beet showing typical lesions as a result of natural field infection. Lesions as sho^v^ may become confluent and develop common concentric rings. Initial stages in fissiu-e formation are also evident. Photographed August 8, 1920. A Dryrot Canker of Su::ar Beets Plate 4 Journal of Agricultural Research Vol. XXII, No. 1 A Dryrot Canker of Surrar Beets Plate 5 Journal of Agricultural Research Vol. XXII, No. 1 PLATE 5 Sugar beet showing various stages in the rupture of the outer covering of the lesion resulting in the formation of deep fissures. The lesions shown on this particular beet have not reached the size normally attained before rupture occurs. PLATE 6 Late stage in the development of the disease, showing the beet as a dry shell par- tially filled with a pithy residue composed of mycelium and dead host tissue. The decay of tlie crown of this beet is a result of the fungus working upward from the point of infection below the surface of the soil. Remnants of the concentric rings of typical lesions are clearly visible. The cracking of the outer surface of the beet at this stage is shown to extend beyond the lesions. A Dryrot Canker of Sugar Beets Plate 6 Journal of Agricultural Research Voi. XXII, No. 1 A Dryrot Cankor of Su':ar Beets Plate 7 Journal of Agricultural Research Vol. XXII, No. 1 PLATE 7 A. — Portion of a sugar beet showing the typical sclerotial masses commonly found adhering to the beets in the infested areas. B. — Sugar beet showing the result of natiu-al infection near the apex of the beet, at which point the root has been completely severed. The dryrot advancing upward from the initial point of attack has produced the typical undulating contour so char- acteristic of the small lateral lesions. PLATE 8 Sections of diseased sugar beets, showing the abrupt drying out between the dis- eased and healthy tissue. The prominent "feeding surfaces" composed of recently killed cells and the young hypha is clearly evident. A. — Cross section, showing complete girdling of the beet by cankers resulting from separate points of infection. In such cases the continued penetration of the fungus may completely sever the root at the line of greatest infection. B. — Longtitudinal section of diseased beet, showing various stages of decay and the pulpy material partially filling the cankers. C. — Sugar-beet crown, showing the definite type of crownrot caused by the fungus worked upward from a point of infection below the soil surface (PI. 6). A small region of healthy tissue is shown to which a few sickly leaves were attached. D. — Section of beet stuface, showing progressive stages in the development of the lesions resulting from natural infection. The earliest visible stage is shown to exhibit a slight perforation of the outer surface at the center of the lesion. This small open- ing, present in all lesions, gradually enlarges with age and finally results in the large fissures (PI. 5). Various stages in the coalescence of lesions are especially evident. A Dryrot Canker of Su^ar Beets Plate 8 Journal of Asricultural Research Vi.1. XXII, No. 1 A Dryrot Canker of Sugar Beets Plate 9 Journal of Agricultural Research Vol. XXII, No. 1 PLATE 9 A, B. — Beets showing typical lesions produced by artificial inoculation. Needle punctures through which the fxmgus entered permitted of rapid drying out of the diseased tissue and of an early ruptiure of the outer layer of cells at the margin of the lesions. A number of the lesions, however, show the concentric rings so character- istic of the disease produced by natural infection. Cross sections of these lesions are shown in C and D. C, D. — Cross sections of the lesions in A and B. The lesions in C disclose the more advanced stage of the disease wherein the outer layers of cells are broken down. In D the outer layers of the lesions are more or less entire. E, F. — Cross sections of cankers resulting from natural infection in the field. A more advanced stage is shown than in C and D; otherwise the lesion produced by the natural and artificial method of inoculation appeared identical. ADDITIONAL COPIES or THIS PUBLICATION MAT BE PROCUBED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTINO OFFICE ■WASHINGTON, D. C. AT 15 CENTS PER COPY SUBSCRIPTION PRICE, $3 PER YEAR V Vol. XXII OCTOBER 8, 1921 No. 2 JOURNAL OF AGRICULTURAL RESEARCH CONTENTS Page Comparative Vigor of Fi Wheat Crosses and Their Parents - - -- - -- - - 53 FRED GRIFFEE (Contribution from Minnesota Agricultural Experiment Station) Temperature and Humidity Studies of Some Fusaria Rots of the Irish Potato - - - -- - -65 R. W. GOSS ( Contribution from Nebraska Agriculttual Kzperiment Station) Blackleg Potato Tuber-Rot under Irrigation - - - 81 M. SHAPOVALOV and H. A. EDSON (Contribution from Bureau of Plant Industry) Microscopic Study of Bacteria in Cheese - - - - 93 G. J. HUCKER (Contribution from New York Agricultural Experiment Station) Further Studies on Relation of Sulphates to Plant Growth and Composition - -- - - - - - 101 HARRY G. MILLER (Contribution from Oregon Agricultural Experiment Station) Soybean Mosaic - -- - - - - -111 MAX W. GARDNER and JAMES B. KENDRICK ( Contribution from Indiana Agricultural Experiment Staition) PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE' WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES ^W^ASHINQXON, D. C, EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCUTION OF LAND-GRANT COLLEGES FOR THE DEPARTMENT ElARL F. KELLERMAN, Chairman Physiologist and Associate Chief, Bureau of Plant Industry EDWIN W. ALLEN Chief, Office of Experiment Stations CHARLES L. MARLATT Entonwlogist and Assistant Chief, Bureau of Entomology FOR THE ASSOCIATION J. G. LIPMAN Dean, State College of Agriculture, and Director, New Jersey Agricultural Etptri- menl Station, Rutgers College W. A. RILEY Entomologist and Chief. Dtvisiott of Ento- mology and Economic Zoology, Agricul- tural Experiment Station of the University of Minnesota R. L. WATTS Dean, School of Agriculture, and Direetor. Agricultural Experiment Station. Th» Pennsyhania State College All correspondence regarding articles from the Department of Agricult^ire should be addressed lo Karl F. Kellennan, 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. JOIMALOFAGBiaiTllRALffiSEARCH Vol. XXII Washington, D. C, October 8, 192 1 No. 2 COMPARATIVE VIGOR OF F^ WHEAT CROSSES AND THEIR PARENTS ' By Fred Griffee ^ Instructor in Plant Breeding, Division of Agronomy and Farm Management, Depart- ment of Agriculture, University of Minnesota The comparative vigor of Fj crosses and their parents is a subject of much interest to the plant breeder. In crops where the technic of cross- ing is comparatively easy, the increase in vigor obtained in the F^ cross often more than pays for the additional trouble of producing the hybrid seed. In self-fertilized crops like the small grains where considerable labor is involved in making artificial crosses, it is apparent that F^ crosses can not be used commercially as a means of increasing crop yields. The suggestion, however, has been made by Anderson (ly that the added vigor of the heterozygous condition might be utilized in small grains by making a large number of crosses between strains which, when crossed, show a considerable increase in yield. Produce from Fj and F3 progeny could be used for seeding the general field, and the crosses could be repeated each year in order to keep up the supply of seed. Several theories have been advanced to explain the phenomenon of heterosis. The discovery of genetic linkage has led to the development of an adequate Mendelian explanation of the vigor so often obtained in Fi crosses. An excellent review of the development of this theory is given by East and Jones (^rot. Complete rot. Experiment 5, 19 15. — The results of earlier experiments having indicated that the influence of the relative humidity was nearly as great as that of temperature, it was decided to run a more complete test on the effect of the relative humidities at different temperatures. Since no apparatus was available by which the relative humidity and tempera- ture could be controlled at will, it was necessary to construct one. The principle employed in experiment 4 of using sulphuric-acid solu- tions of varying specific gravity in a closed chamber to obtain the different relative humidities was not used in this test. In preliminary experi- ments conducted in the same way as experiments 4 and 4A, the infection usually resulted in a softrot which gave good comparative results, but the type of rotting was not similar to that usually found in storage. Cultures from these softrots invariably yielded the Fusarium sp. used in the inoculation, and no bacteria were present in any case. Apparently I 74 Journal of Agricultural Research voi. xxii. no. 2 the absence of any aeration was the cause of this abnormal type of rot- ting, and the following method was devised to allow for aeration. The principle j[inally decided upon was that of passing a current of air, kept at a constant pressure, through sulphuric-acid towers and then over calcium chlorid and sodium hydrate. This gave a constant stream of dry, sterile air. The air was then passed over sterile water to bring it to a desired humidity. The amount of water necessary for a given humidity was determined by trials, and the air was then passed into the jars containing the tubers. An outlet was provided at the bottom of the jar. These jars were connected separately with the current of air and not in series. Relative humidities were obtained and used through- out the experiment as follows: i, 30, 70, and 100 per cent. These rela- tive humidities were used at three different temperatures — 9°, 12.5°, and 25° C. The set at 25° was placed in an incubator in the laboratory, the set at 12.5° was placed in a special low temperature incubator, and the set at 9° was placed in a well-insulated ice box. Each of these temperatures was maintained within a variation of 2° throughout the experiment. In this way four gradations of humidity at each of three temperatures were obtained. The method provided the tubers with suf- ficient aeration and secured sterile conditions throughout the experiment, since the jars containing the tubers were not moved or opened until the end of the period. The humidity readings were taken by the wet- and dry-bulb method, the thermometers being inserted into the stream of air at the entrance to the jar. The readings were found to vary, and at least 10 trial read- ings were taken for each jar after the preliminary determinations were made and the apparatus was set up. These readings ranged as follows : I to 10 per cent, 20 to 40 per cent, 60 to 80 per cent, and 90 to loo per cent. These were the greatest extremes found; and since a knowledge of the approximate relative humidity is all that is necessary in an experi- ment of this kind, these readings were taken to be sufficient, inasmuch as they showed a gradual gradation from approximate dryness to satura- tion. The ranges given above simply denote the possible error due to the method of taking the readings. The humidity necessarily remained constant, since the temperature, water surface, and air pressure were constant. It was found to be impossible to use the wet- and dry-bulb method to determine the relative humidity at the lower temperatures. The changes of temperature caused by opening the door to make the deter- minations were found to change the readings. Therefore the sets at 9° and 12.5° C. were installed temporarily at 25°, the preliminary deter- minations were made, and the readings were taken at that temperature and corrections made by the use of psychometric tables (13). The large battery jars were fitted with wire screen supports, and six tubers were used in each jar — four inoculated and two controls. The oct.8.i92i Temperature and Humidity Studies of Fusaria Rots 75 control tubers were separated from the inoculated ones by a thin layer of cotton. The entire apparatus was disinfected with formaldehyde gas before the experiment was set up. The tubers used were of the Up-to-Date variety. They had been kept over winter in a cool cellar, and a few sprouts which had started were removed. They were inoculated by wounding the epidermis and placing several drops of spore suspension in the wound. They were then placed in the jars which were closed with cork tops and paraffined. The inoculations were made with Fusarium oxysporum No. 8. The jars were opened up and the tubers examined after five weeks. Set I (9° C). At 10 and 30 per cent humidity the tubers were all healthy. At 70 per cent the tubers were sound with no penetration, although there was a slight growth of mycelium on the surface of the tuber at the point of inoculation. At 100 per cent the condition of tubers was the same as at 70 per cent, except that the external growth of mycelium was greater. All the control tubers of this set remained healthy, and both the controls and inoculated tubers had sprouted. Set 2 (12.5° C). At 10 per cent humidity the tubers were healthy. There was no invasion of the tissues. At 30 per cent, same as above with a slight external growth of mycelium at the point of inoculation. At 70 per cent the tubers were about the same as at 30 per cent. (PI. 10, A.) At 100 per cent invasion of the tissue had taken place for about 2 mm. beyond the wound, causing a slight browning of the tissue. On the surface there was a slight brown discoloration for several millimeters surrounding the point of inoculation and a slight growth of aerial myce- lium. All the control tubers in set 2 remained healthy, and both control and inoculated tubers were sprouting normally. (PI. 10, B.) Set 3 (25° C). The control tubers remained healthy and sprouted at 10 and 30 per cent relative humidity, while at 70 and 100 per cent there was a slight disorganization of the tissue around the eyes and the sprouts were all dead. No actual rotting was present or any fungus growth. With infected tubers at 10 per cent humidity all inoculations were successful and uniform. The fungus invaded the tissue for 2 cm. around the wound. Immediately below the surface at the point of inoculation there was in every case a cavity lined with a white mycelial growth. The tissue surrounding the cavity was of a granular appearance. Exam- ined under the microscope it appeared to be made up of a tangled mass of mycelium and starch grains. A sof trot extended out from this area, the tissue being light brown in color and completely invaded by mycelium (PI. 10, C). 76 Journal of Agricultural Research voi. xxii. No. 2 At 30 per cent humidity the rotting took place in the same manner as at 10 per cent, except that the cavity was larger and the rot extended through about 50 per cent of the tuber (PI. 11, B). At 100 per cent humidity there was a total rot of all tubers, most of the surface being covered with a white mycelial growth (PI. 11, C). The results of this experiment show very clearly that relative humidity plays a very important part in determining the amount of rot produced by Fusarium oxysporum. The only rot appearing at the low tempera- ture of 12.5° C. was in an atmosphere of 100 per cent humidity. Even at the high temperature of 25° complete rotting did not take place at the lower humidities. A gradual increase in the amount of rot corresponding to the increase in humidity was present in every case. The fungus can live and sporulate at the lower temperatures and lower humidities used in this experiment but apparently is not capable of penetrating the tubers under these conditions. It can be safely con- cluded that F. oxysporum under good storage conditions is not capable of producing a tuber-rot of great importance. CONCLUSIONS (i) Fusarium oxysporum, F. trichochecioides , and F. radicicola are all capable of producing a rot of the potato tuber. (2) In pure culture the amount of growth of all three species is nearly equal at 25° C, Fusarium oxysporum and F. radicicola increasing in growth up to 30°, where they produce their maximum growth. The growth of F. trichochecioides decreases above 25°, until at 30° very little or no growth takes place. It is more tolerant of the lower temperatures than the other two species. (3) Preliminary tests with different liquid media would indicate that the cardinal points for growth of these Fusaria vary to some extent with the medium used. (4) Experimental infection of tubers was produced with all three organisms under various conditions of temperature and relative humidit}'. (5) Preliminary tests with Fusariivm oxysporum indicated that the relative hmnidity plays a very important part in determining the amount of rotting. (6) In comparative tests with new and old tubers there is a distinct difference in the amount of rotting under the same conditions. The rotting was much more rapid and progressed much further in the old than in the new tubers. (7) Comparative tests with all three species at controlled relative humidities from i to 100 per cent and at controlled temperatures from 5° to 25° C. proved conclusively that — (a) A temperature of 25° C. is favorable for the production of a tuber rot by Fusarium oxysportim, F. radicicola, and F. trichothecioides. Oct.8.i92i Temperature and Humidity Studies of Fusaria Rots 77 (b) Fusarium oxysporum grows more rapidly and produces a more ex- tensive rotting of the tuber than the other two at a temperature of 16° C. and above. (c) Fusarium trichothecioides is capable of producing a rot at much lower temperatures than the others, in some cases causing rotting at 5°C. {d) The relative humidity plays a very important role in determining the progress of tuber rots and has the same influence on all three species. In every experiment it was noticeable that there was a gradual increase in the amount of rot corresponding to an increase in relative humidity. With a high humidity at a given temperature the rotting was always greater than at a temperature 5° to 10° C. higher but with a low humidity. The Fusaria used can all live and sporulate at the low temperature of 9°, and with low relative humidities, but they are not capable of producing a rot under these conditions. Inasmuch as the three species of Fusaria used in these experiments represent the common types causing storage-rots of potatoes, it is clear that considerable attention should be given to moisture as well as tem- peratures where incipient rot occurs in stored tubers. It is also en- tirely probable that a rotting of the tubers initiated at high temperatures and high relative humidities could be completely checked by submitting the tubers to lower temperatures and lower humidities. LITERATURE CITED (i) AppEt, Otto, and Wollenweber, H. W. I910. GRUNDLAGEN EINER MONOGRAPHIE DER GATTUNG FUSARIUM (LINK). Arb. K. Biol. Anst. Land. u. Forstw., Bd. 8, Heft i, 207 p., 10 fig., 3 pi. (i col.) Verzeichnis der wichtigsten benutzten Schriften, p, 196-198. (2) Bary, Anton de. 1861. DIE gegenwartig herrschende kartopfelkrankheit, ihre ursache UND ihre verhutung. eine pflanzenphysiologische unter- SUCHUNG . . . 75 p., I pi. I^ipzig. (3) BisBY, G. R. 1919. STUDIES ON FUSARIUM DISEASES OF POTATOES AND TRUCK CROPS IN MIN- NESOTA. TECHNICAL. Minn. Agr. Exp. Sta. Bui. 181, 58 p., 30 fig. Bibliography, p. 40-44. (4) Carpenter, C. W. 1915. SOME POTATO TUBER-ROTS CAUSED BY SPECIES OF FUSARIUM. In Joiir. Agr. Research, v. 5, no. 5, p. 183-210, pi. A-B (col.), 14-19. Litera- tiire cited, p. 208-209. (5) Edson, H. a., and Shapovalov, Michael. 1920. TEMPERATURE RELATIONS OF CERTAIN POTATO-ROT AND WILT-PRODUCING FUNGI. Ir„ Jour. AgT. Research, v. 18, no. 10, p. 511-524, 9 fig. (6) Frank, Albert Bernhard. 1898. UNTERSUCHUNGEN USER DIE VERSCHIEDENEN ERREGER DER KARTOFFEL- FAULE. In Ber. Deut. Bot. Gesell., Bd. 16, Heft 8, p. 273-289. jS Journal of Agricultural Research voi. xxn. No. a (7) Humphrey, H. B. i914. studies on the relation of certain species of fusarium to the to- MATO BLIGHT OF THE PACIFIC NORTHWEST. Wash. AgT. Exp. Sta. Bul. 115, 22 p., 5 pi. (8) Jamieson, C. O., and WollenwebER, H. W. I912. an external dry rot of potato TUBERS CAUSED BY FUSARIUM TRICHO" THEcioiDES, WOLLENW. In Jour. Wash. Acad. Sci., v. 2, no. 6, p. 146-152, I fig. (9) Link, George K. K. I916. A PHYSIOLOGICAL STUDY OP TWO STRAINS OP FUSARIUM IN THEIR CAUSAL RELATION TO TUBER ROT AND WILT OP POTATO. In Bot. Gaz., V. 62, no. 3, p. 169-209, 13 fig. Literatiire cited, p. 207-208. Reprinted as Nebr. Agr. Exp. Sta. Research Bul. 9. 1916. (10) Longman, Sibyl. 1909. THE DRY-ROT OF potatoes. In Jour. Linn. Soc. (London), Bot., v. 39, no. 270, p. 120-129, pi. 10. Literature referred to in the text, p. 129. (11) Manns, Thomas F. I91I. THE FUSARIUM BLIGHT (WILT) AND DRY ROT OF THE POTATO. PRELIM- INARY STUDIES AND FIELD EXPERIMENTS. Ohio Agr. Exp. Sta. Bul. 229, p. 229-336, 15 pi. (in text). (12) Martius, Carl Friedrich Philipp von. 1842. DIE KARTOFFEL-EPIDEMIE DER LETZTEN JAHRE ODER DIE STOCKFAULE UN RAUDE DER KARTOFFELN, GESCHILDERT UND IN IHREN URSACHLICHEN VERHALTNISSEN ERORTERT. 70 p., 3 col. pi. Miinchen. (13) Marvin, C. F. 1900. PSYCHROMETRIC tables FOR OBTAINING THE VAPOR PRESSURE, RELATIVE HUMIDITY, AND TEMPERATURE OF THE DEW-POINT ... U. S. Dept. Agr. Weather Bur. Bul. 235, 84 p., 2 fig. (14) Pethybridge, George H., and BowERS, E. H. 1908. DRY ROT OF THE POTATO TUBER. In Econ. Proc. Roy. Dublin Soc, v. i, pt. 14, p. 547-558, pi. 48- (15) PiZZIGONI, A. 1896. CANCRENA SECCA ET UMiDA dellE patate. In Nuovo Gior. Bot. Ital., n. s. V. 3, fasc. i, p. 50-53. (16) Pratt, O. A. I916. A WESTERN FIELDROT OF THE IRISH POTATO TUBER CAUSED BY FUSA- RIUM RADicicoLA. In Jour. Agr. Research, v. 6, no. 9, p. 297-310, Pl- 34-37- (17) 1916. CONTROL OF THE POWDERY DRYROT OF WESTERN POTATOES CAUSED BY FUSARIUM TRicHOTHEcioiDEs. In Jour. Agr. Research, v. 6, no. 21, p. 817-832, pi. 108. (18) Reinke, Johannes, and Bertholdt, Gottfried. 1879. DIE zersetzung der kartoffel durch pilze. 100 p., 9 pi. Berlin. (Untersuch. Bot. Lab. Univ. Gottingen. Heft i.) (19) Schacht, Hermann. 1856. bericht an das konigliche landes-oekonomie-collegium uber die KARTOFFELPFLANZE UND DEREN KRANKHEITEN ... 29, II p., 10 pi. (6, 8, 9 col.). Berlin. (20) Shereakofp, C. D. 1915. fusaria op potatoes. N. Y. Cornell Agr. Exp Sta. Mem. 6, p. 87-270, 51 fig., 7 pi. (col.). Literatiu-e cited, p. 269-270. (21) Schlechtendal, D. F. L. de. 1824. FLORA BEROLINENSIS, PARS SECUNDA. CRYPTOGAMIA. Berlini. Oct. 8, I92I Temperature and Humidity Studies of Fusaria Rots 79 (22) Smith, Erwin F., and Swingle, Deane B. 1904. THE DRY ROT OF POTATOES DUE TO FUSARIUM OXYSPORUM. U. S. Dept. Agr. Bur. Plant Indus. Bui. 55, 64 p., 2 fig., 8 pi. Literature, p. 61-62. (23) Stevens, Neil E. I916. A METHOD FOR STUDYING THE HUMIDITY RELATIONS OP FUNGI IN CUL- TURE- In Phytopathology, v. 6, no. 6, p. 428-432. Literature cited, P- 432- (24) Wehmer, C. 1896. UEBER DIB URSACHE DER SOGNENANNTEN "TROCKENFAULE" DER KAR- TOFFELKNOLLEN. hi Ber. Deut. Bot. Gesell., Bd. 14, Heft 3, p. loi- 107, 3 fig. (25) 1897. UNTERSUCHUNGEN iJBER KARTOFFELKRANKHEITEN. 2. ANSTECKUNGS- VERSUCHE MIT FUSARIUM SOLANI (DIE FUSARIUM-FAULE.) In Centbl. Bakt. [etc.], Abt. 2, Bd. 3, No. 25/26, p. 727-743, pi. lo-ii (i col.). (26) Wilcox, E. Mead, Link, George K. K., and Poole, Venus W. 1913. A DRY rot of THE IRISH POTATO TUBER. Nebr. Agr. Exp. Sta. Re- search Bui. I, 88 p., 15 fig., 28 pi. (i col.). Bibliography, p. 85-88. (27) WOLLENWEBER, H. W. 1913. STUDIES ON THE FUSARIUM PROBLEM. In Phjrtopathology, V. 3, no. i, p. 24-50, I fig., pi. 5. Literatiu-e cited, p. 46-48. PLATE lo Tubers inoculated with Ftcsariwrn oxyspomm and kept for five weeks at the fol- lowing temperatures and humidities: A. — 12.5° C, 70 per cent relative humidity. B. — 12.5° C, 100 per cent relative humidity. C. — 25° C, 10 per cent relative humidity. (80) Temperature and Humidity Studies of Fusaria Rots Plate 10 f B ^ Journal of Agricultural Research Vol. XXII, No. 2 Temperature and Humidity Studies of Fusaria Rots B Journal of Agricultural Research Vol. XXII, No. 2 PLATE II Tubers inoculated with Fusarium oxysporum and kept for five weeks at the fol- lowing temperatures and humidities : A. — 25° C, 30 per cent relative humidity. B. — -25° C, 70 per cent relative humidity. C. — 25° C, 100 per cent relative humidity. BLACKLEG POTATO TUBER-ROT UNDER IRRIGATION By M. Shapovalov and H. A. Edson, Pathologists, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant Industry, United States Department of Agriculture . OCCURRENCE AND GENERAL APPEARANCE ' '■ A bacterial field decay of the potato tuber, the real nature of which has not heretofore been adequately explained, prevails in certain irri- gated sections of the West. In early harvest, when the diseased tubers are apparently free from fungous invasion, the trouble has sometimes been assumed to be "sunscald"; during the winter months it has fre- quently been taken for a form of freezing injury. In other instances it has been confused with the so-called "jelly-end rot" and attributed either to Fusarium radicicola Wollenw. or to F. oxysporum Schlecht. It is probable, also, that on superficial examination some such material has been classed as "leak" {Pythium debar yanum Hesse), when conditions favored an extremely rapid progress of the decay, whether in the field or in transit. Specimens of this decay were received by the writers in 191 7 and 19 1 8 from Idaho, Nevada, and California. In material received in August the decay was soft and mushy (PI. 12, A-C). The aflfected tissues were in part brown to black, but mostly only slightly colored or colorless, though with a darker margin on the border line between the healthy and diseased portions. Disintegration, originating at one end of the tuber, was advancing irregularly over the surface. In some areas the decay was confined to the outer layer, just beneath the epi- dermis, while in others the deeper tissues also were involved. As a rule, the disease started at the stem end, but occasionally the eye end became infected first (PI. 12, B). Decaying material usually possessed a disagreeable odor. It is this soft type of the rot which some were inclined to regard as sunscald injury. Specimens received later in the season, during the months of Novem- ber and December, presented an entirely different appearance. The affected portions were not mushy, but more or less tough or dry and shrunken (PI. 12, D). The diseased area was dark brown in color, except when a fresh decay developed under favorable conditions deeper in the tissues. In the latter case it was practically of the same color as the normal flesh of the tuber, but soft and mushy in consistency. When such tubers were cut open and the cut surfaces exposed to the air, the diseased portions turned brown or even black. If the progress of the decay is completely anrested, the trouble may readily be mistaken for Journal of Agricultural Research. Vol" ^^^^ ^°- ' Washington. D.C. Key No'.'g-»46 (81) 82 Journal of Agricultural Research voi. xxii. no. 2 an inactive stage of jelly-end rot or for an after effect of freezing injury, particularly if the disease has made but little headway. The true nature of such obscure cases of the disease may be revealed with cer- tainty only by a series of cultural studies, coupled with experimental work and field observations. CAUSAL ORGANISM Isolations were made from every tuber of each of the four samples received from the West in 191 7-18. The results were surprising. In no case was Fusariu^n radicicola obtained; only one tuber yielded F. trichothecioides Wollenw. (from Nevada), two yielded Rhizoctonia (from Nevada), two F. oxysporum (from California), and a few gave miscellaneous, apparently saprophytic, fungi. Bacteria, on the other hand, were constantly present in the cultures, even when slightly acidu- lated potato agar was used. Carpenter (2)^ noted the presence of bacteria in jelly-end rot material, but he regarded these organisms as saprophytic, as they probably were. In the writers' cultures, however, the constant prevalence of one type of bacterial colony in the dilution plates was significant and warranted a detailed study of this organism. In the subsequent inoculation experiments with pure cultures it proved to be strongly pathogenic and produced a progressive decay of the tubers as well as a disease of the stems. A study of the cultural and biochemical features of the organism showed them to be fully in accord with the published description of the blackleg bacillus (5). MORPHOLOGY Short rod with rounded ends, also short chains; 0.5 to 0.9 Xi.c to 2.2 ;u, average 0.6 X i-S /x; flagella few, peritrichiate; no endospores and no capsules; stains well in aqueous gentian violet, aqueous methylene blue, aqueous fuchsin, anilin water gentian violet, alkaline methylene blue, and carbol fuchsin. CULTURAL FEATURES Agar stroke. — Growth moderate, filiform, flat to slightly raised, glistening, smooth, slightly opalescent; white, no odor; consistency slimy to butyrous; one strain distinctly viscid at first, but after a few replat- ings it lost its viscidity. Potato. — Growth moderate to abundant; filiform at first then spread- ing, slightly convex changing to flat, glistening, smooth to slightly ru- gose, yellowish white or dirty white; a decided odor of decayed potatoes on the third to fourth day at 22° to 25° C. ; consistency somewhat slimy; medium slightly grayed at first, changing later to either plainly gray, or purplish, or brown, or a combination of these shades. ' Reference is made by number (italic) to " Literature cited," p. 91-92. Oct. 8, 192 1 Blackleg Potato Tuber-Rot under Irrigation 83 Agar stab. — Growth somewhat best at top, abundant, spreading, filiform to slightly papillate. Gelatin stab. — Growth best at top, filiform along the line of punctm-e, liquefaction, beginning on the first day at 20° C, varying in shape from crateriform or funnel-shaped to saccate and broadly infundibuliform, complete in 7 to 12 days. Nutrient broth. — Usually slight ring and slight granular pellicle in young cultures, clouding moderate to strong, persistent; medium not discolored, odor absent; sediment compact, granular, somewhat dirty white; one strain decidedly viscid at first, but losing this character after a few replatings. j-mi/ibtia Jrff-io MiivK. — Coagulation and extrusion of whey at 25° C, beginning on the fourth day; coagulum not digested; one strain extremely viscid at first, but losing its viscidity in later replatings; medium not discolored. Acid production in milk. — A slight increase of acidity in milk cultiu-es was noticeable after 24 hours. Two series of tests were made at certain intervals within the period of 20 days, two to three cultures being used on each day for every strain. The average progress of the acidity of three western strains was as follows : AGE OP REACTION IN CULTURE. fuller's SCALE. 1 day ,, +12. 28 2 days .'. .: '. +13-49 3 days +22. 13 5 days +28. 00 10 days +32. 90 20 days .,;..„ . .^,. , i: K-. +41- 12 The average reaction of the control tubes was +11. 8 Fuller's scale. Two strains received from Dr. W. J. Morse, of the Maine Agricultural Experiment Station, were tested along with the western strains and gave similar reactions, one ("B. sol.") showing 37.0 and the other ("IIIA") 40.75 acidity on the twentieth day. The cultures were grown at 22° to 25° C. Litmus milk. — ^At 22° to 25° C. bleaching was complete at the end of three weeks; thorough reddening was accomplished in seven weeks. Gelatin colonies. — Growth rapid, form round, edge entire, lique- faction saucer-shaped. Agar colonies. — ^Surface colonies; growth rapid, usually round, but occasionally somewhat irregular, flat to slightly raised, entire to slightly undulate, finely granular with an internal ring surrounded by radiate striations; color pearly white, bluish opalescent by transmitted light; maximum diameter of colonies after 2 days 2 mm., after 3 days 4 mm., after 7 days 7 mm., after 14 days 9.5 mm. Buried colonies lens-shaped to nearly spherical, edge entire, color slightly yellow under hand lens. Fermi's solution. — Moderate clouding in 2 -day cultures; later growth becomes copious. ii^ mc*^": 54818*— 21 8 84 Journal of Agricultural Research voi. xxii, no. 2 Corn's SOLUTION. — No growth. Uschinsky's solution. — Growth was somewhat irregular in the ordi- nary Uschinsky's solution but was uniform and copious in the modified Uschinsky's solution, clouding being very strong on the fifth day. Sodium chlorid in bouillon. — Growth slightly inhibited by 3 per cent and more so by 4 per cent; no growth appeared in 5 per cent tubes until the third day, and only occasional tubes containing 6 per cent were clouded after 5 days. Morse reports no clouding for Bacillus atrosepti- cus Van Hall in concentrations higher than 5 per cent. At the end of two months, when conditions remained unchanged, transfers were made from 6 per cent and 7 per cent sodium-chlorid cultures of the western strains to sterile broth. In 48 hours all the transfers from 6 per cent solutions showed growth, and in three days clouding appeared in the majority of the transfers from the 7 per cent solutions, the remainder being dead. Growth in bouillon over chloroform. — Growth somewhat re- strained at first, but increasing gradually. On the fourth day there was a strong and uniform clouding in all cultures. Best medium for long-continued growth. — Morse considers that in the case of Bacillus atrosepticus neutral beef bouillon is best for this purpose. In the western strains the writers observed that the organisms can live even longer on the agar than on the broth when grown at ordinary laboratory temperature of 22° to 25° C. Their death on agar appears to be primarily associated with drying of the medium, while in broth it seems to be due to certain chemical changes in the substratum and takes place sometime before the liquid dries up completely. Six series of parallel broth and agar cultures were made and tested at diff"erent inter- vals, from 8 to 36 weeks, by making transfers to tubes of sterile broth. It was found that occasional broth cultures showed a somewhat weak- ened vitality, as demonstrated by retarded clouding, at the age of 16 weeks; some died after the expiration of 20 weeks, and none lived beyond 26 weeks. On the other hand, in no case was the agar culture dead before 26 weeks, and some remained alive even after 36 weeks. The experiment was carried on with 10 cc. of medium in each test tube. PHYSICAL and biochemical FEATURES Fermentation tubes. — Gas and acid production as well as growth in the closed arm was observed with dextrose, lactose, and saccharose. No acid and no gas with glycerin in cultures 1,3, and 5 days old. Ammonia production. — Feeble (tested by Folin's aspiration method). Nitrates in nitrate broth reduced to nitrites. Indol production. — Positive, but very feeble both in young and old cultures. Toleration of hydrochloric acid and sodium hydrate. — The writers' western organism grew in tubes having an initial reaction before Oct. 8, 1921 Blackleg Potato Tuber-Rot under Irrigation 85 final sterilization of +20 and —20, Fuller's scale, but not in those adjusted to +30 or to —30. Uninoculated tubes held as controls and titrated at the close of a 24-day incubation period showed marked changes from the original reaction, due doubtless in considerable measure to the absorption of gases with resulting chemical change. Tubes cal- culated for an initial reaction of -f 30 showed a final reaction of from +20 to +25; those originally +20 were about +15; those —30 were about — 10; and those —20 were about —6. Transfers from inoculated tubes calculated for an initial reaction of + 30 and above and — 30 and below made 24 days after inoculation developed growth in some cases in tubes from + 30, but not in those from more acid reactions nor from the alka- line broths. Vitality on culture media. — ^Long on bouillon, but still longer on agar. Temperature relations. — In freshly inoculated broth cultures exposed 10 minutes, occasional retardation of clouding began at 45° C. ; occasional growth was noted at various points between 46° and 50°; and in no case was growth present after heating above 50°. Optimum temperature for growth about 25°. Maximum temperature for growth between 33° and 35°. Minimum temperature for growth below 5°. Effect of sunlight. — Thinly sown agar plates exposed on ice for 30 minutes the latter part of March in Washington, D. C, resulted in 100 per cent killed. Cytase production. — Five-day-old 30-cc. broth cultures in 300-cc. Erlenmeyer flasks were precipitated by 160 cc. of 80 per cent alcohol, filtered, and the precipitate dried promptly in the air. The papers con- taining the dried precipitate were washed with 30 cc. of water, and the washings were received in a flask to which a few drops of toluene and three raw Irish potato disks 15 by 2 mm. were added. The disks gradually assumed a soft, cheesy consistency but did not entirely disintegrate. Microscopic examination showed the cells had lost coherence through softening of the middle lamella. The cellulose lamella and the starch content of the cells showed no evidence of change. Controls with uninoculated broth did not soften the disks. GROUP number 22 1. 1 II 3033 The last three points in this group number differ from those given by Morse (5) but coincide with the respective figures in Jennison's {3) revision, as reported by him at the fourth annual meeting of the Pacific Division of the American Phytopathological Society. The writers feel, however, that this may be largely a matter of interpretation of certain results and not necessarily an indication of actual difference in the organisms. Jennison studied 12 different strains of the blackleg bacillus, includingseveralof Morse's strains; but the results he obtained, apparently, 86 Journal of Agricultural Research voi. xxh. No. » were identical for all strains. The writers regard their western strains as nonchromogenic, although a certain yellow discoloration in cooked potato cultures might be taken as a suggestion of yellow pigment. They obtained no evidence of diastatic action on potato starch, nor of acid production with glycerin. It may, therefore, be concluded on the basis of the characters described that the pathogenic bacillus isolated by the writers from a peculiar soft decay of western potato tubers is essentially identical with the organism causing the blackleg disease of potatoes for which Appel's binomial Bacillus phytophthorus is regarded to be correct by Smith (7) . Morse (5) , who was unable to obtain an authentic culture of Appel's strain for his comparative studies of various blackleg organisms, believed that B. atrosepticus should be chosen in preference to other names he had under consideration, but stated that — There is nothing in the data here presented which bears on the relation between the organism originally described by Dr. Appel (/) as B. phytophthorits and the other strains of blackleg bacteria. EXPERIMENTAL WORK The pathogenicity of the bacterial organism described above was established by means of the following laboratory, greenhouse, and field experiments. PLANTING OF THE ORIGINAL MATERIAL Preliminary to the inoculation work some of the diseased western material was planted in the greenhouse as soon as the isolations were completed. Four tubers were selected and cut in halves so as to make eight seed pieces. Each piece was planted in a separate large pot filled with sterilized soil. Of these seed pieces one decayed completely in the soil before germination, six produced diseased plants, and one produced a plant considerably weakened though not clearly diseased. Some of the affected plants decayed while very young, others grew up to practi- cally normal size, developing blackening of the stem above ground and brown to black lesions on the underground portions. The lower leaves turned yellow, but the upper leaves wilted while green. In two cases the blackening of the stems was very intense (PI. 13, A) while in the remainder the appearance was less typical of the familiar field symptoms of the disease as it occurs in the eastern sections of the country. Black- ening of the pith of the stem developed to the very top in one case. When an affected plant was removed from the pot and the soil was care- fully washed off, it could be seen that the infection had spread from the seed piece to the stem (PI. 13, B). No tubers were produced in this experiment. Healthy sprouting Irish Cobbler tubers were replanted in these pots, but no infection was contracted by this new set of plants. Oct 8, 1941 Blackleg Potato Tuber-Rot under Irrigation 87 INOCUIrATlON OF HEALTHY TUBERS IN THE LABORATORY Over 60 tubers both new and old of the Netted Gem as well as of the Irish Cobbler varieties were inoculated in small lots at dififerent times with three strains of the western decay bacillus. When inoculations were made in wounds of any kind, whether on the side of the tuber or at either end of it, the results were invariably positive. The progress of the decay was much slower when uninjured potatoes were inoculated. In these latter cases the organism penetrated either through the eyes or through the young growing sprouts. If the infected potatoes are re- moved from the moist chamber after the decay has made considerable headway and are exposed to the dry air of the laboratory, the diseased tissues become shriveled and folded, resembling very closely the original specimens of natural infection (PI. 14, A, C). Ordinarily if the infected material is kept in moist chambers the decay is soft, mushy, spreading either equally throughout the tissue or sometimes more on the surface of the tubers, and is not confined to their piths as is usual in typical cases of blackleg. The color of the decaying areas ranges from that of the normal flesh to light or dark brown, often with blackish streaks or stripes in younger portions nearer to healthy tissues, but never black throughout. The margin is usually well defined, and there is no gradual transition from dead to sound tissues. This internal appearance changes considerably when tubers are taken from the moist chamber and are exposed to drying. The decay of the bark is then more or less arrested, and the disintegration centers mainly in the pith, so that a more or less sound shell surrounds the centers of the active decay. The diseased tis- sue is brown to black, the older regions becoming slimy (Pi. 14, B, D). In all cases the decay gives off a very strong putrefactive odor. PLANTING OF ARTIFICIALLY INOCULATED TUBERS IN THE GREENHOUSE Seven Irish Cobbler tubers inoculated with the western bacterial or- ganisms and partly decayed were planted in sterilized soil in pots. Four showed subsequently a stem decay and three remained apparently un- affected. One plant became girdled and died early. The disease ap- peared first on the remaining three plants in the form of black streaks in various positions on the stems, particularly at the leaf petioles. Later on in some instances the entire stalk became black at the base. Tuber- rot did not appear except on one tuber in one of the diseased pots. In this case it was a soft, watery decay, light in color, not typical for black- leg. The causal organism, identical with the original strains, was, how- ever, recovered from this area. Healthy sprouting Irish Cobbler tubers were immediately planted in the same pots in which these specimens were grown. The new plants were very vigorous, and none of them con- tracted the disease. 88 journal of Agricultural Research voi. xxn. No. a INOCULATION OF HEALTHY STEMS IN THE GREENHOUSE The stems of four young healthy potato plants were inoculated with 24-hour-old broth cultures of the three western strains of the bacillus injected by means of a hypodermic needle. A severe decay with an accompanying blackening resulted in all cases. FIELD EXPERIMENTS These experiments were conducted for two successive years at Arling- ton Farm, Va. In 1 9 19 Netted Gems and Irish Cobblers were used. The tubers were inoculated with the western strains of the blackleg organism a few days before planting. Six whole tubers and 20 halves of the first variety and 9 whole and 19 halves of the second variety were planted. In addition a number of uninoculated pieces of each variety were planted for controls. Planting was done on May 5. One half-tuber seed piece of each variety decayed in the ground. On July i one hill from the cut seed of Irish Cobblers was noted to show secondary symptoms character- istic of blackleg — ^namely, yellowing and rolling of the leaves. There was no blackening of the stem above ground. The underground portions, however, showed brown lesions and a brown rot of the stem at the point of attachment to the seed piece and somewhat above it. None of the remaining plants showed symptoms of the disease. At digging time, on September 15, no decay of the tubers was found, with the exception of one very small tuber of the Netted Gem variety which showed a soft bac- terial decay at the stem end. The progress of the decay, however, was checked, and the affected portion fell off, leaving only the sound part, so that the recovery of the causal organism was not possible. Since the hot weather after May 5 might have had something to do with the slight progress of the disease in 19 19, two sets of plantings were made on another piece of ground on the same farm in 1920 — one on April 8 and the other on May 6. Only Irish Cobblers were used this time. Twelve tubers were cut in halves through the inoculated wound so as to make 24 seed pieces for each of the two series. Inoculations were made a few days before planting. Eight tubers were inoculated with the three western strains of the blackleg organism and 4 with the "B. sol." strain received from Dr. Morse. Up to July 20 four hills out of 16 inoculated with the western strains in the earlier planting and 2 out of 8 inoculated with " B. sol." in the same series showed typical field symptoms of black- leg, including an intense blackening of the base of the stem. On the other hand, no hill of the series inoculated with the same organisms and on the same plan, but planted one month later, showed any signs of infection. At harvesting time, on July 20, a number of tubers in the planting of April 8 showed blackleg-rot, and in the later planting only 2 tubers were found showing the same decay. It appears, therefore, that the earlier planting, when the soil and the air temperatures were Oct. 8, 1921 Blackleg Potato Tuber-Rot und^r Irrigation 89 lower and the soil moisture was more abundant, greatly facilitated the development of blackleg. FIELD OBSERVATIONS The typical case of blackleg-rot on round varieties in the East has been figured in publications and charts issued by several agricultural institu- tions. As a rule, decay begins at the stolon end of the tuber with a comparatively small amount of rot visible on the outside or often only a small, black, circular opening. This opening leads to the interior of the tuber, where a progressive decay develops in the form of an irregular black, soft, or slimy hollow until nearly all of the tuber is consumed (PI. 15, D, B). However, the development of the disease may deviate from this type even in eastern and northern sections of the United States, when conditions are abnormal and favorable to the disease, such as those in moist places or in wet seasons. Morse stated (4) with refer- ence to blackleg in Maine that — When this disease occxirs on a field it doubtless is responsible for much of the soft rot of the tubers observed in wet seasons. It appears from certain observations made by the writers that under conditions of excessive soil moisture the bacteria in stems or seed tubers may be carried at least to the adjoining tubers of the same hill. The latter then become infected from the outside, or, if they are already infected through the stolons, the infection spreads in moist surround- ings more rapidly on the outside over the surface of the tuber, or evenly throughout the flesh. Specimens of this sort were observed on the Eastern Shore of Maryland and Virginia (PI. 15, G) and in Wisconsin, Minnesota, and Washington (PI. 15, F) on various round types of pota- toes. More accentuated symptoms of this order were found in irrigated sections of Colorado. The most peculiar manifestations of the blackleg tuber-rot were seen in the Snake River Valley of Idaho, where the Netted Gem variety is grown on a large scale. The following forms were observed there dm-ing a field survey arranged by the Office of Cotton, Truck, and Forage Crop Disease Investigations in 1920. I. The stem-end rot of pointed-end Netted Gems. The external appearance of this form is extremely misleading (PI. A, 1-4). It becomes prevalent in southeastern and eastern Idaho during the latter part of the season, shortly before the harvest. The relatively low temperature prevailing at this time of year is, no doubt, an important factor in the rapid progress of the disease. If the soil has plenty of moisture, freshly dug affected tubers show no shrinkage and preserve their natural shape (PL 15, A-C). In the course of two weeks the shrinkage is evident and the decay takes on an inward trend (PI. A, 4). By another two weeks, drying and folding of the decayed tissues become very pronounced, and the external appearance at this stage of the decay may well pass as an 90 Journal of Agricultural Research voi. xxn, ko. n illustration of any of a number of stem-end tuber-rots (PI. i6, A, B). When a Netted Gem tuber of pointed shape affected with this type of the disease is cut open longitudinally as soon as it is removed from the ground, four distinct regions of decay may, as the rule, be seen: (a) The extreme stem-end region is usually decayed throughout ; it is mushy or slimy in consistency and dark brown to black in color ; in the field this seldom extends deeper than the outer demarkation line of the decay, but in storage under favorable conditions the disintegration advances more rapidly in the inner tissues of the tuber, taking on a cup-like shape and leading ultimately to the formation of a slimy cavity (PL A, 4; 14, B, D; 16, B). (6) An area of fresh decay appears within the core just beneath the first region; it is practically colorless, though it occasion- ally contains dark or black streaks, and in the very early stages it has the consistency of hardened butter (PI. A, 4). (c) The cambium layer shows a brown discoloration extending sometimes close to the eye end; in advanced stages a portion of this region nearest to the stem end is more or less disintegrated and forms a channel attentuating toward the eye end until it gradually transforms into a mere browning of the vas- cular network which also gradually loses its intensity and finally disap- pears altogether; this condition is very distinct with some freshly dug tubers, but later on with the inward progress of the decay it becomes less pronounced (PI. A, i, 4; 16, B), {d) The decay of the outer layer develops in the bark region, is soft but not mushy in consistency and more or less dark-brown in color; it frequently extends over the tuber much farther than the pith decay, but not always as far as the cambium discoloration; its progress is checked after tubers are dug and exposed to drying (PI. A, i, 2; 16, B). 2. The shallow stem-end rot of round-shaped tubers. This type was observed mostly on the Idaho Rurals. Under conditions of abundant moisture the bacterial infection spreads from the stem end over the surface of the tuber and penetrates into the bark region, though not very deeply. When such tubers are taken out of the ground and exposed to the sun, as happens at digging, the infected areas dry up very promptly and form hard, black, shallow patches (PI. 16, E). The condition may easily be mistaken for the black fieldrot described by Pratt (6) and attributed to Fusarium radicicola. If, however, the tubers are again transferred to a moist place with a moderate temperature, a soft, mushy bacterial decay is likely to develop beneath these dry areas. On the contrary, under conditions unfavorable to the blackleg decay the status may either remain unchanged or become complicated by the entrance of various Fusaria and other rot-producing fungi. In the latter case it is impossible to determine the original cause of the disease. 3. Siderot of either round or long potatoes. It may penetrate inside of the tuber to a considerable depth, and when a freshly dug diseased oct.8.r«>4i Blackleg Potato Tuher-Rot under Irrigation 91 tuber is cut open it reveals a colorless buttery or mushy decay with black streaks, usually on the border line of the diseased and healthy tissues. If exposed to drying the decayed areas may become spongy and very much resemble the texture which is usually observed in Fusa- rium rots (PI. 16, D). In many instances, however, if the decayed region is sufficiently deep to prevent complete drying, sections through such tubers may show an inner layer of active bacterial decay. As is the case with the other forms of blackleg-rot this form, too, may become further invaded with various rot-producing or saprophytic fungi. The writers had under their observation a tuber of this type with a copious growth of Rhizoctonia on the outside all over the diseased area, while soft bacterial decay was still progressing within the tuber even in the dry laboratory atmosphere (PI. 16, C). SUMMARY (i) An organism isolated from western stem-end rotting potatoes is identical with Bacillus phytophthorus Appel in all the essential charac- ters commonly considered in the determination of bacterial species. (2) It is pathogenic to the potato, and inoculations of healthy stems or tubers with pure cultures produce, respectively, a rapid, soft decay of stems or a tuber-rot. (3) Blackleg tuber-rot under the field conditions in certain irrigated sections of the West, particularly in pointed-end Netted Gems, takes on a form atypical of the familiar manifestation of this disease in the East. (4) The external appearance becomes especially confusing when the affected areas dry up and shrivel in storage, but usually the trouble may be identified by cultural work or by planting diseased tubers under control conditions. LITERATURE CITED (i) Appel, Otto. 1903. UNTERSUCHUNGEN USER DIE SCHWARZBEINIGKEIT UNO DIE DURCH BAKTERIEN HERVORGERUFENE KNOLLENFAULE DER KARTOFFEt. In Arb. K. Biol. Anst. Land. u. Forstw., Bd. 3, Heft 4, p. 364-432, 15 fig., pL 8 (coL). (2) Carpenter, C. W. 1915. some potato tuber-rots caused by species op fusarium. in jout. Agr. Research, v. 5, no. 5, p. 183-210, pL A-B (col.), 14-19. Litera- ture cited, p. 208-209. (3) JENNISON, Harry Milliken. 192 1. BACILLUS ATROSEPTICUS VAN HALL, THE CAUSE OF THE BLACKLEG DIS- EASE OF IRISH POTATOES. (Abstract.) In Phytopathology, v. 11, no. 2, p. 104. (4) Morse, W. J. 1910. CERTAIN DISEASES OF MAINE POTATOES AND THEIR RELATION TO THE SEED TRADE. Maine Agr. Exp. Sta. [Misc. Publ.] 375, 12 p. 92 Journal of Agricultural Research voi. xxn. No. 2 (5) Morse, W. J. 19x7. STUDIES UPON THE BLACKLEG DISEASE OF THE POTATO, WITH SPECIAL REFERENCE TO THE RELATIONSHIP OF THE CAUSAL ORGANISMS. In Jour. Agr. Research, v. 8, no. 3, p. 79-126. Literature cited, p. 124-126. (6) Pratt, O. A. I916. A WESTERN FlELDROT OF THE IRISH POTATO TUBER CAUSED BY FUSARIUM radicicola. In Jour. Agr. Research, v. 6, no. 9, p. 297-310, pi. 34-37. (7) Smith, Erwin F. 1920. AN INTRODUCTION TO BACTERIAL DISEASES OF PLANTS. XXX, 688 p., 453 fig. [pi.] Philadelphia and London. Literature at end of most of the chapters. Blackleg Potato Tuber-Rot under Irrigation > Journal of Agricultural Research Vol. XXIi, N rivATE A Types of blackleg potato tuber-rot on pointed-end Netted Gem from Idaho, showing external as well as internal appearance. The photograph was taken two weeks after the tvibers were removed from the ground. The same tubers are shown in Plate 15, A, C, on the first day after digging. PLATE 13 Forms of blackleg tuber-rot in the West. A-C. — Specimens received in August, 1918, from Fresno, Calif. D- — Specimen received in December, 1917, from Fallon, Nev. I Blackleg Potato Tuber-Rot under Irrigation Plate 12 Journal of Agricultural Researcii Vol. XXII, No. 2 Blackleg Potato Tuber-Rot under Irrigation Plate 13 Journal of Agricultural Research Vol. XXII, No. 2 PLATE 13 Blackleg on stems resulting from planting the diseased western material. A. — Appearance of plant above the grotmd. B. — Spread of the infecion from the diseased seed piece to the new stem. 1 PLATE 14 Result of inoculation of healthy tubers with the bacterial organism isolated from the western diseased material. A, B. — Netted Gem variety. C, D. — Irish Cobbler variety. Blackleg Potato Tuber-Rot under Irrigation Plate 14 Journal of Agricultural Research Vol. XXII, No. 2 Blackleg Potato Tuber-Rot under Irrigation Plate 15 '^t *f \ \ Journal of Afrricultural Research Vol. XXII, No. 2 PLATE IS Different types of blackleg tuber-rot. A-C— Characteristic appearance on fresh specimens of the Netted Gem variety from Idaho. D, E. — T3rpical development on round varieties in the East. p^ G.— Other forms occurring on round varieties in various sections of the coimtry. Arrows in A, B, and C indicate the border line of decay. PLATE i6 Confusing forms of blackleg potato tuber-rot in the West (all specimens collected in Idaho). A, B. — ^Netted Gem variety one month after digging. Tuber B as it appeared on the first day after digging is shown in Plate 15, B. C. — Long Idaho Rural, showing secondary growth of Rhizoctonia on the outside and active bacterial decay in the inside. D. — Idaho Rural with deep side infection of blackleg decay which became dry and spongy on exposure to the sun. E. — Shallow surface infection which became dry and black when exposed to the sun after digging. Blackleg Potato Tuber-Rot under Irrigation Plate 16 Journal of Agricultural Researcli Vol. XXII, No. 2 MICROSCOPIC STUDY OF BACTERIA IN CHEESE By G. J. HucKBR Associate in Research, New York Agricultural Experiment Station INTRODUCTION Heretofore cultural methods have ordinarily been used in the study of cheese flora, but the usual routine technic has given only an inade- quate conception of the number and types of bacteria present. Although these cultural methods were employed in order to secure an idea of the number and varieties of organisms occurring in cheese, they have failed to establish the relative abundance of each type of microorganism in the cheese. This objection remains valid in the light of both quantita- tive and qualitative studies. The so-called "dilution technic," which involves the mass action of the organisms, has proved valuable in determining the type or group of organisms which predominates in a given sample ; but it does not furnish information regarding the general flora. This method often tends to give erroneous results, especially when the dilution medium used favors the growth of special groups. Under such conditions the results are influenced by the selective action of the medium. This has been true where milk was used as a dilution medium in examining cheese. The milk favored the growth of the lactic acid group; while the inert and nonlactose fermenting types or slow-growing cocci were overgrown, due in a large measure to the selective action of the medium. This procedure has well served its purpose in assisting to isolate the organisms for which it has a special adaptation. In general, cultural methods are preferable to a microscopic examina- tion because cultures can be isolated and studied independently — a feature which will always remain the outstanding advantage of these methods. Although subject to the same limitations as any microscopic method, the following method has been successfully used in this laboratory and has been employed in a routine way in determining the number of bac- teria in cheese. HISTORY Johan-Olsen {Sy, working with the molds which ripen "Gammelost" (a Norwegian cheese), mentions a sectioning method and implies that it resembles the usual histological technic but does not outline the procedure in detail. ' Reference is made by number (italic) to "Literature cited," p loo. Journal of Agricultural Research, Vol. XXII, No. i Washington, D. C Oct. 8, 1921 (93) 54818°— 21 4 Key No. N. Y. (Geneva) ,-6 94 Journal of Agricultural Research voi. xxii. no. 2 Troili- Peterson {11), in discussing tlie bacterial flora of Swedish "Giiterkase," mentions the microscopic examination of cheese as a control for the cultural procedure but does not give the technical details. She presents photomicrographs of cheese sections and states that some of the preparations were stained in methylene blue and that a few were examined unstained. Gorini (j), in studying the distribution of the bacteria in Grana cheese, presents the details of a method by which he prepared sections for microscopic examination. In his procedure he fixed and dehydrated samples of cheese by passing them through a series of alcohols of increasing concentrations until a strength of 95 per cent was reached. The usual histological methods of sectioning were followed, and the sections were stained in an aqueous solution of methylene blue. In the following year Rodella (9) reported a method used in his labo- ratory for preparing sections used in the direct examination of cheese samples. With his technic the samples were dehydrated and fixed by a method similar to that of Gorini and sectioned in the usual way. He found, however, that carbol-thionin gave better results as a stain than did methylene blue. Harrison (<5) outlined in detail a method for embedding and sectioning cheese which is similar to the common histological method, but like his predecessors he made no estimate of the number of bacteria present. During the year in which Rodella (9) presented his paper, Troili- Peterson {12) and Gorini (4) published notes discussing the question of priority raised by the practically simultaneous publication of their papers. It appears that the methods followed by Trioli- Peterson were similar to those of Gorini, but that she did not feel the necessity of pre- senting the technical details because of the universal knowledge of the common embedding methods. No results have been obtained in any of this work that permit a com- parison between counts made by the plate method, so commonly used in floral studies of cheese, and counts made by direct microscopic examina- tion. Following the method outlined below, comparatively accurate counts have been made by the direct method, and the number of the different types of bacteria have been determined as they actually exist in the cheese mass. TECHNIC EMBEDDING AND SECTIONING The samples of cheese were embedded by the usual histological technic and sectioned with a Minot rotary microtome. In sectioning, the micro- tome was so adjusted as to give sections 5 ^i thick. The sections were stained by the Gram method and with an aqueous solution of methylene blue. Oct. 8,1921 Microscopic Study of Bacteria in Cheese 95 In order to determine the effect of the embedding process upon the cheese, small measured cubes of cheese were subjected to the routine procedures. Only a slight shrinkage was found, indicating that the volume of embedded cheese when examined is approximately the same as that of the fresh sample. MICROSCOPIC EXAMINATION The preparations were examined with an oil immersion lens and a high power ocular, the most satisfactory combination being a 1.9-mm. fluorite objective with a numerical aperture of 1.32. Where a thick coverslip was used it was necessary to have a 3-mm. apochromatic objective with a numerical aperture of 1.4. Greater depth can be secured with compensating oculars than with the ordinary Huygenian oculars. The method, although at first used only for determining the types of organisms present in the samples and as a check on the usual plate method, was found useful as a means of determining the number of organisms present. In order to make such a computation the micro- scope was so standardized as to allow an estimate of the number of organisms per gram when only a small amount of the original section was examined. This computation is similar to that used in the direct method of counting bacteria in milk described by Breed and Brew (2). This was accomplished by measuring both the diameter of the micro- scopic field and the thickness of the section from which the amount of cheese actually seen in each field examined was determined. Knowing the volume and specific gravity of the cheese examined, the total number of organisms per gram can readily be computed. With the diameter of the field measuring 0.14 mm. (140 fx), the microtome so adjusted as to cut sections of a thickness of 0.005 nim. (5 ij.) , and a specific gravity of i, the amount of cheese examined per microscopic field would be 1/13,000,000 gm. — that is, each organism observed in a single microscopic field repre- sents 13,000,000 per gram. This factor may be computed by the following formula, in which any measure may be substituted : 1,000 , . , — -2 — 0 = factor per gram. In the above formula, r = the radius of the field examined in millimeters as determined by actual measurement. a = the thickness of the section in millimeters. b = the specific gravity of the cheese. The radius of the field, as has been stated, is determined by measure- ment with a stage micrometer and varies with the magnification and with the tyoe of ocular used. However, it was found advisable to adjust the 96 Journal of Agricultural Research voi. xxii. no. , draw tube of the microscope so that the field would be of the greatest possible diameter without losing definition, as the greater the diameter of the field the less the increment of error in the total counts. The thickness of the section is controlled by adjusting the microtome to cut sections of a desired and known thickness. If all the adjustments on the microtome are firm and a sharp knife is used, sections can be cut of uniform thickness with surprising accuracy. The thickness of the sections can also be remeasured with the fine adjustment screw on the microscope. Although not perfect, this method of measurement serves as a check upon the accuracy of the sectioning. The measurement is accomplished by focusing with the graduated fine adjustment screw on both the upper and lower surfaces of the section and noting the differ- ences in the readings between the two levels. The difference can be read in microns where graduations are given on the fine adjustment screw. To convert the per-cubic-centimeter counts into numbers per gram, the specific gravity of the cheese must be considered. As the specific gravity of all samples has been assumed to be approximately i, the counts are interchangeable. This assumption in regard to the specific gravity is arbitrary, but the variations in the specific gravity of cheddar cheese are so slight that the total count is not affected to any appreciable degree. Accurate determinations did not seem practicable, as the speci- fic gravity varies with the fat content and with the moisture and general consistency of the cheese. With the measurements and adjustments used in this laboratory the per-gram formula resolves itself into the following: r = 0.07 mm. (70 fx). a = .005 mm. (5 /x). 6 = 1.0. 1 ,000 7—- X I = approximately i-; ,000,000. 3.1416 X 0.0049 X 0.005 APPLICATION OF THE METHOD It is evident that this microscopic technic is subject to the limitations of any direct method of examination, many of which are unavoidable and are due to mechanical limitations or to the human error, which enters in when counts or estimates are made. QUALITATIVE EXAMINATION As previously stated, cheese has been examined microscopically by many investigators. The possibility of error is not as great when samples are examined to determine the types of organisms present as when total count is made, which is true of any microscopic work. Our present staining methods make possible a direct visualization of the microorgan- oct.8, igji Microscopic Study of Bacteria in Cheese 97 isms together with their morphological and other general characteristics, but an attempt to enumerate these types involves other difificulties. The direct examination of cheese in the different ripening stages is advantageous and important, since the different groups of organisms can be studied as they actually occur in the cheese mass, and their groupings and relative relationships noted. The grouping may be especially important when considered in relation to the number present. For example, an organism may be present in large numbers during the early stages of ripening, but appear in scattered and isolated groups containing only a few individuals. In some instances only single bac- teria were found through the mass. In such cases the total number of this group by the plate count may be large, but the grouping, as deter- mined by direct examination, may demonstrate that they are not ac- tively growing and playing a part in the ripening of the cheese. On the other hand, the presence of large clumps of organisms, with the size of the clumps increasing during ripening, indicates that such groups are developing in the cheese mass and are probably playing an important r61e in the changes involved. That this grouping of the organisms actually occurs can be seen in Plate 17, A. In this photomicrograph are shown the types and groupings of organisms found in a very green cheese, showing that the Streptococcus lactis-like organisms predominated and were scattered in pairs over the field. Any migration of these bacteria through the cheese mass appears to have been impossible, and one is impressed with the fact that growth and reproduction could not have been taking place rapidly or the number of individuals per group would have been larger. In Plate 17, B, which represents a section from a cheese 5 months older than that shown in Plate 17, A, the organisms are found in larger clumps with many of them so massed that accurate counting is impossible. From the examination of a series of sections from cheeses of varying ages, it has been found that the clumps increase in size as the cheese ripens, reaching a limit after seven to eight months. It is evident that the organisms in the clumps, mostly cocci and a few rods, are thriving and reproducing and must, therefore, change the surrounding medium as they utilize it for food. It is not within the scope of this paper to discuss the significance of this occurrence but only to point out that such variations are found when samples are examined directly. QUANTITATIVE EXAMINATION An objection often made to counting organisms in microscopic prepa- rations of dried liquids is the uneven thickness of the resultant dried film. This objection is eliminated when parafiin sections are used, as such sections are uniform in thickness and the organisms remain in their natural relationships. Boekhout and DeVries (z) at one time endeavored 98 Journal of Agricultural Research voi. xxii. no. 2 to show that the scattered organisms in cheese sections were due, in a large measure, to the breaking up and scattering of the clumps by the knife edge. This explanation will hardly appear plausible to anyone familiar j with the perfection of delicate histological sections prepared with a sharp knife. The grouping and clumping of the organisms often cause difficulty in accurately determining the number of organisms in the cheese sections. This is especially true in sections of old cheeses in which the bacteria tend to clump in large masses. The error can be overcome to a large degree by counting or estimating a large number of fields, the larger the number examined the smaller being the error in the final estimate. In a sample of green cheese where the organisms appear in large num- bers, but are evenly scattered, it is impracticable to count the entire field, and an ocular disk divided into quadrants may be inserted in order to facilitate accurate counting. In all cases 20 or more fields should be counted, and especially where the organisms are unevenly distributed. In such instances, typical fields which represent the general flora should be located by studying the entire section. COMPARISON OF DIRECT AND PLATE COUNTS Table I gives a few representative comparisons between direct micro- scopic and plate counts made from cheese samples in various stages of ripening. The plate counts average approximately one-twelfth the direct count, but no common ratio has been found to exist between the results obtained by the two methods. Wide variations in the ratios be- tween the counts were found, but in general the ratios from green cheese appeared to be larger than those from cheese more advanced in ripening. The above plate counts compare well with those found by other observ- ers who have examined cheddar cheese, Russell (10) found from 62 to 665 million per gram. Harrison and McConnell (7) found the count to be as high as 625 million per gram in the earlier stages of the ripening, while Harding and Prucha (5) observed from 37 to 177 million per gram. Several explanations may be offered to account for the apparent discrepancy between the results obtained by the two methods. The plate count is an estimate based on observations of the growth of organ- isms on some particular medium which, in cheese investigations, usually contains lactose. Lactose has been generally used because media con- taining this particular carbohydrate have been found to allow the develop- ment of a larger number of colonies than do sugar-free media. Investigators have based their cultural methods upon media giving the largest counts rather than upon media which might serve as an index to the relative number of types present. In comparing the microscopic counts with results obtained with the plate method, it may be noted that the types Oct. 8, 1921 Microscopic Study of Bacteria in Cheese 99 present in the cheese, as seen by direct examination, are not present in the same proportions on the plates, because those types which grow abundantly in the presence of lactose have outnumbered all groups which do not grow as readily on such media. Table I. — Relation between microscopic and plate counts obtained from cheese Approximate age of cheese. Miscroscopic count (millions per gram). Plate coimt (millions per gram). Cheese. Cocci. Short rods. Yeast. Strep- tococ- cus lactis. (Lister) Total. Strep- tococ- cus lactis. Miscel- laneous. Total. OOCIIXII 2.... 100 14,650 312 1,690 962 338 600 S, 200 780 260 26 26" 4.500 21,450 143 6,760 988 3.029 5,200 41,300 481 9.230 2,210 3.393 46 264 132 251 306 9 284 90 41 121 55 548 OOCI3 XII 2 . . . 6. 4,11 BCII 292 427 702 6. 26, II 5 months, 23 days. Unknown; a p - peared giecn. OO38I8 Results of plate counting may also be lower because of insufficient grinding and emulsifying of the cheese sample previous to plating. This appears to be especially significant in cases where investigators grind the sample with sterile quartz or sugar and suspend the ground mass in sterile water in preparation for plating. If the sample is not well ground, small particles of cheese remain in the emulsion, and the individual bacteria are not separated so as to allow them to grow into separate colonies on the artificial medium. Emulsions examined under the microscope often show comparatively large masses of cheese which have not been affected by the grinding process. CONCLUSION The microscopic examination of cheese embedded and sectioned by the usual histological method is a valuable and satisfactory method for studying the different stages of cheese ripening. Such a direct method of examination may be used to determine the number of organisms present in the sample. It also serves as an index to the types of organisms present and makes possible a study of the organisms as they actually exist in the cheese mass, allowing observations on the groupings and relationships during cheese ripening. The cultural methods do not yield as high a count as the microscopic method, due primarily to the selective action of the medium used and the difficulty of liberating the organisms from the cheese mass previous to plating. A combination of microscopic and cultural studies yields a far more complete picture of what takes place in cheese ripening than can be obtained by the use of either method alone. loo Journal of Agricultural Research voi. xxii, no. a LITERATURE CITED (i) BoEKHODT, F. W. J., and Ott de VriBS, J. J. 1899. UNTERSUCHUNGEN t)BER DEN RElFUNGSPROZESS DES EDAMER KASES. In Centbl. Bakt. [etc.], Abt. 2, Bd. 5, No. 9, p. 304-307. (2) Breed, Robert S., and Brew, James D. I916. COUNTING bacteria BY MEANS OP THE MICROSCOPE. N. Y. State AgT. Exp. Sta. Tech. Bui. 49, 31 p., 5 fig., i col. pi. Bibliographical footnotes. (3) Gorini, Costantino. 1904. SULLA DISTRIBUZIONE DEI BACTERl NEL FORMAGGIO DE GRANA. In R. 1st. Lombardo Sci. Let. Rend, ser, 2, v. 37, fasc. 2, p. 74-78, 2 fig. (on I pi.). (4) 1906. ZUR PRIORITAT DER METHODE DER KASEUNTERSUCHUNG DURCH MIKRO- SKOPISCHE SCHNITTPRAPARATE. In Centbl. Bakt. [etc.], Abt. 2, Bd. 16, No. 1/3, p. 66. (,5) Harding, H. A., and Prucha, M. J. 1908. THE BACTERIAL FLORA OF CHEDDAR CHEESE. N. Y. State Agf. Exp. sta. Tech. Bui. 8, p. 120-193. (6) Harrison, F. C. 1906. the distribution of lactic acid bacteria in curd and cheese of THE CHEDDAR TYPE. In Rev. G6n. Lait, v. 5, no. 18, p. 409-415, 9 fig. (on 4 pi. ) References, p. 413. (7) and CoNNELL, W. T. 1903. A COMPARISON OP THE BACTERIAL CONTENT OF CHEESE CURED AT DIF- FERENT TEMPERATURES. In Rev. Gen. Lait, v. 3, no. 4, p. 80-85; no. 5, p. 103-111; no. 6, p. 126-137, 1903 • "O- 7> P- i50~i55; iio- 8, p. 173-180, 1904. (8) Johan-OlsEn, Olav. 1898. DIE BEi kasEREifung wirksamen pilze. In Centbl. Bakt. [etc.], Abt. 2, Bd. 4, No. 5, p. 161-169, 17 fig. (on pi. 4-9, 4 and 5 col.). (9) RODELLA, A. 1905. EINIGES t)BER DIE BEDEUTUNG DER DIREKTEN MIKROSKOPISCHEN PRA- PARATE FtJR DAS STUDIUM DES KASEREIFUNGSPROZESSES. In Centbl. Bakt. [etc.], Abt. 2, Bd. 15, No. 4/5, p. 143-153- 5 ^g- (o" i pl-)- (10) Russell, H. L. 1896. THE RISE AND FALL OF BACTERIA IN CHEDDAR CHEESE. In Wis. Agr. Exp. Sta. 13th Ann. Rpt. i^g^jgb, p. 95-111, fig. 25-26 (26 on col. fold, pi.) (11) Troili-Peterson, Gerda. 1903. STUDIEN t;BER DIE MIKROORGANISMEN DES SCHWEDISCHEN GCTERKASES. In Centbl. Bakt. [etc.] Abt. 2, Bd. 11, No. 4/5, p. 120-143; No. 6/7, p. 207-2151, 5 fig. (on 3 pi.). (12) 1905. BEMERKUNGEN ZUR DER ARBEIT VON A. RODELLA " EINIGES t)BER DIE BEDEUTUNG DER DIREKTEN MIKROSKOPISCHEN PRAPARATE FCR DAS STUDIUM DES KASEREIFUNGSPROZESSES." In Centbl. Bakt. [etc.], Abt. 2, Bd. 15, No. 13/14, p. 430. PLATE 17. A. — Section of cheddar cheese i month old, stained with an aqueous solution of methylene blue, showing isolated pairs of Streptococcus lactis Lister throughout the field. X 500. B. — Section of cheddar cheese 6 months old, stained as in A. X 800. Microscopic Study of Bacteria in Clieese Plate 17 Journal of Agricultural Research Vol. XXII, No. 2 FURTHER STUDIES ON RELATION OF SULPHATES TO PLANT GROWTH AND COMPOSITION By Harry G. M1L1.ER Chemistry Department, Oregon Agricultural Experiment Station This is a continuation of an investigation, part of the results of which were reported in a former pubHcation {4}} As stated in the earher paper, the addition of the different forms of sulphur caused a marked increase in the dry weight of red clover, and beneficial results were obtained with oats and rape. One very noticeable result observed in the former work was the high nitrogen content of the clover grown on soils in the green- house receiving sulphur fertilizer, compared to that of clover receiving only the residual sulphur of the soil. This great increase in nitrogen assimilation by the clover where sul- phates were applied, and under the conditions described, led the writer to believe that the sulphates favorably influenced the activity of the legume bacteria. Especially did this appear true where the beaverdam soil was used. This soil contained o. 18 per cent sulphur with appreciable quantities of sulphate sulphur in the soil extract, and no beneficial result from sulphur fertilizer was expected. Oats did not respond to sulphur with this soil, although the sulphur content of oats (j) and the amount of sulphur removed by one crop of oats is as large as with a red clover crop. From present data, the responses of red clover so often obtained with gypsum compared to cereals can not be explained through a difference in sulphur requirement. With alfalfa the amount of sulphur removed is so large compared to the cereals and red clover that the addition of sulphates would apparently function directly as a plant food where in- creased growth results. An example of the latter would be the enormous increases in the yield of alfalfa obtained in southern Oregon (7) where sulphur fertilizers were applied to soils with a very low sulphur content. These authors, however, mention the favorable action that sulphur ferti- lizers had on the root development and nodule production of alfalfa. Duley (2) reports increase nodule production on red clover where sul- phiu- was added to soils. Pitz (5) observed increased nodule production and root development with red clover by applying gypsum to soil cultures. As far as the writer can ascertain, no correlation has been shown be- tween nodule production and nitrogen content of the plant, by influencing the development of the former, with ordinary sulphur fertilizer com- pounds. In this paper a study has been made of the effect of different ' Reference is made by number (italic) to "Literature cited," p. no. Journal of Agricultural Research. Vol. XXII, No. Washington, D. C. Oct. 8, 1921 zt Key Na Greg. 7 (lOI ) I02 Journal of Agricultural Research Vol. XXII, No. 2 concentration of sulphates on growth and nitrogen assimilation, and also the relation of total sulphur content of the plant as influenced by available nitrogen. The red clover and rape were used in this work. With clover, the initial concentration of legume bacteria has been varied by inocu- lating certain cultures, while others were not inoculated. Table I. — Analytical results with red clover on Medford loam soil Treatment. Weight of air- dried clover. N Total N. in- solu- ble in acet- ic- acid. Total S. Sul- phate S. Or- ganic S. P.cl. 2. 88 P.ct. 2- 13 P.ct. 0. 20 P.ct. 0.06 P.ct. 0. 14 ^■l'^ 2 10 20 .04 It) 2.8l 2 02 18 .07 II 3-31 2 10 16 . 02 14 3- 30 3-36 2 2 15 22 26 28 . 10 . 12 16 16 3-58 3-48 2 2 31 19 40 34 •25 • 14 IS 20 3-46 3-38 2 2 39 30 28 34 • 13 •IS 15 19 3-34 2 2 28 33 41 .27 •IS 14 18 Sin- S in soluble acetic- in acid acetic- solu- acid tion. I solu- tion. Weight of air- dried roots. Total S in roots. Total Nin roots. Ratio of tops to roots. 2. 64 2. 77 2. 74 2- 73 Control: Uninoculated . . Inoculated NaNOs: Uninoculated . . Inoculated NaNOa and HaSOt: Uninoculated . . Inoculated Na!S04: Uninoculated . . Inoculated CaSOi: Uninoculated . . Inoculated NaNOs and CaSOi: Uninoculated . . Inoculated Gm. 15-84 31. 24 17. 70 21- 29 18.82 23-35 15. 00 20. 31 16.49 16. 6s 14. 18 16.89 .08 .07 • 15 -17 09 Gm. 6. 00 7- 6s 6.45 7.80 7. 60 7. 10 8.60 8.20 6. 10 6. 20 P.ct. 34 2. 28 I. 23 I. 60 I 72 I 59 I 53 3 50 61 2 2 •55 I -55 2 2. 39 3. 92 In the first experiment Medford loam soil, designated as soil B in the previous publication (4), was used. This soil was heated in an electric oven where the temperature was gradually raised to 120° C. and main- tained for six hours. This was to destroy the legume bacteria present in the soil. Four kgm. of soil, after being mixed with the different fer- tilizers, were placed in paraffined clay pots and carefully seeded to red clover. One series was inoculated with Bacillus radicicola Bey. The solu- tion for inoculation was prepared by removing the growth of organisms from a culture and mixing with water. Each inoculated soil culture re- ceived a definite number of cubic centimeters of the bacterial solution, concentrated where the seeds were placed. The amount of different fer. tilizers added per pot was as follows : Sodium sulphate (NajSO^) , 3 gm. ; sodium nitrate (NaNOj), 2 gm. ; calcium sulphate (CaS04.2 H3O), 3.75 gm. ; and calcium carbonate (CaCOg) , 3 gm. The cultures were placed in the greenhouse on October 16 and harvested on March 18. Ten plants were allowed to grow in each pot. The cool temperature in the green- house did not permit rapid growth, and the plants were cut before ma- turity was reached. The treatment and analytical results are given in Table I. The cultures were weighed every other day, and the moisture was maintained at 20 per cent. In removing the roots the soil was shaken out of the pot and carefully loosened. The roots were then sepa- rated out and washed. After drying they were weighed, and the non- Oct. 8,1921 Relation of Sulphates to Plant Growth and Composition 103 volatile matter was determined by ashing a ground sample representing each cultm-e. This was done to correct for any excess weight due to adhering soil particles. The total sulphur was determined by the sodium-peroxid method. The sulphate sulphur was extracted by taking 2 gm. clover and 150 cc. of water and digesting on the steam bath for three hours. It was then slightly acidified with hydrochloric acid, and after standing for an hour the extract was filtered. Five cc. of 10 per cent barium-chlorid solution was used to precipitate the sulphate sulphur in the hot solution. After standing overnight the barium sulphate settled to the bottom of the beaker in all cases, and no particles of precipitate could be detected in the supernatant liquid. This liquid was carefully decanted off; and the white precipitate was washed on a Gooch crucible, dried, and weighed. Several of these precipitates were ignited, but since no appreciable loss in weight was detected this method appeared perfectly reliable for comparable results on sulphate sulphur in the different samples of plant material. For the determination of total nitrogen insoluble in acetic acid, i-gm. samples were digested with about 150 cc. of water on the water bath for two hours. The extract was then acidified with dilute acetic acid and filtered after standing about 30 minutes. The total nitrogen was determined on the precipitate by the Kjeldahl method. The filtrate was made alkaline with sodium carbonate, evaporated to a few cubic centimeters in volume, transferred to a nickel crucible, and total sulphur determined by the sodium-peroxid method. The dry weights of plant material produced show no increase in pro- duction that can be attributed to presence of sulphates. This result is different compared to the noticeable increase reported with the same soil before (4). As mentioned above, conditions were very unsatisfactory for growth, and the plants were cut before maturity five months after planting. In the former work reported, conditions permitted rapid growth, and the plants, though not mature, were harvested two months after seeding. As the soil used in this later work had been heated, there was perhaps some change in degree of solubility of soil minerals and in the biological flora. The concentration of added mineral salts was also greater in this work. Examination of the roots showed that all plants had become infected. In the uninoculated series, roots from cultures i and 2 contained very few nodules compared to the roots grown in the soil receiving sulphate fertilizer. This remarkable difference in nodule formation no doubt accounts for the low nitrogen content of the clover plants in pots i and 2. That these plants became infected without any artificial inocu- lation is not surprising. Wilson (8) found that — of fifteen legumes grown in Volusia silt loam soil, only one, Trifolium pratense, developed nodules without artificial inoculation. I04 Journal of Agricultural Research voi. xxii. No. a During the growth of the plants the inoculated series showed greater development, which is apparent upon examining the dry weights. Cultures i and 2 of the inoculated series show no effects from lack of sulphates, and all the sulphur-fertilized pots contained numerous well- developed nodules. The total nitrogen insoluble in acetic acid was no greater in some of the cultures receiving added sulphates than in the controls, so no statement can be made that sulphate addition caused this fraction to become larger. The nitrogen content of the fraction soluble in acetic acid is lower in i and 2 of the uninoculated series. The increase in percentage of sulphur caused by fertilizer treatment is accounted for generally by higher sulphate content. Although the organic sulphur is apparently higher in some, the results are not consistent with the total sulphur, to state that the former results from increased sulphur assimi- lation in this experiment. Total sulphur in the acetic-acid extracts runs parallel with sulphate sulphur results and is slightly higher, showing that there is some sulphur in the organic form not accounted for in the precipitate from acetic-acid solution. This was also found to be true with clover grown in other pots which was harvested while in blossom. The ratio in weight of tops to roots is greater in the inoculated sulphur- fertilized cultures than in the uninoculated sulphur-fertilized cultures. This difference does not appear to be due to inoculation alone, for this does not hold true in comparing i and 2 of both series, while the ratio of tops to roots in i and 2 of the uninoculated series is greater than the remaining four where there is heavy nodule growth. Amy and Thatcher (/) report a greater ratio in weight of tops to roots where inoculation was made with alfalfa and sweet clover. The sulphur content of the roots is larger than in the other portion of the plant, whereas the opposite is true in percentage of nitrogen. The second part of this work was carried on with beaverdam soil and red clover. Each pot contained 7 kgm. of soil, and the following amount of fertilizers were added as indicated in Table II: 12 gm. of calcium sulphate, 10 gm. of sodium sulphate, 2 gm. of sulphur, and 6 gm. of sodium nitrate. Two gm. of potassium chlorid and 10 gm. of calcium carbonate were added to all the soil cultures. Twenty red clover plants were allowed to grow in each pot, and the moisture content was kept at 40 per cent. The first crop grew at the same time as the clover on the Medford loam soil and was also cut before the blossoming stage. Three other crops were grown on these same cultures. The first was harvested on March 24, the second on May 20, the third on July 9, and the fourth on August 17. The last three crops were cut during the blossoming stage. The results are given in Table II. Oct. 8,1921 Relation of Sulphates to Plant Growth and Composition 105 TablS II. — Analytical results with red clover on beaverdam soil Treatmeat. Control CaSOi and NaNOs. CaS04 NaN03 NasSOi Na2S04 and NaNOs S SandNaNOs Crop I. Weight of air- dried clover. Gm. 46 46 Total N. P. a. 3-28 3- SO 3-41 3-25 3-47 3- 12 3-42 3-58 Total S. P.ct. Crop II. "Weight of air- dried clover. Gm. 102 91 80 83 Total N. P.ct. 2. 70 2. so 2. 40 2-53 2.49 2.47 2. 56 Total S. P.ct. Cropni. Weight of air- dried, clover. G.m 69 66 Total N. Total S. Sul- phate S. P.ct. o. 02 Or- ganic S. P.ct. O. 12 •15 . 12 •13 Treatment. Control CaS04and NaNOs.. CaS04 NaNOs Na2S04 Na2S04 and NaNOs. S. S and NaNOs. Crop IV. Weight of air- dried clover. Gm. Total N. Per cent. 3- 16 Total S. Per cent. 18 Sulphate S. Per cent. 03 15 12 06 Organic S. Per cent. IS IS IS 16 .16 16 14 .16 Bxammation of the foregoing data shows no result from sulphate application in the first two crops. There is no increase in dry weight in the pots receiving sulphur fertilizers, and in some crops the yield on the control is greater. In the third and fom'th crops, one distinctive difiference appears in the lower nitrogen content of the clover grown on the control soil cultures. The fact that the clover grown under condi- tions supplying more sulphate sulphur has a higher nitrogen content adds to the data already obtained pertaining to the favorable influence that sulphates have upon legume bacteria, the action of which results in a higher nitrogen content of the clover. According to experimental evidence, sulphates do not increase nodule production on all legumes. Wilson (8) reports that certain sulphates depressed nodule formation on the soybean. On the other hand, Prucha (6) mentions magnesium sulphate and calcium sulphate as exerting a beneficial influence on nodule development of the Canada field pea. The low nitrogen content did not appear to be due to the absence of sulphates in the plant tissue, thus limiting protein synthesis, as sulphates were present in all samples. However, the percentage of sulphate sulphur was lower in clover grown on the control pots. That the presence of available nitrogen or nitrogen assimilation by the plant tends to control or limit the total sulphur assimilation is illus- trated by data in Table III, io6 Journal of Agricultural Research Vol. XXII. No. a In this experiment sea sand was used which had been washed with dilute hydrochloric acid and large volumes of distilled water. The sand still contained sulphur compounds, but no sulphates soluble in dilute hydrochloric acid. Six kgm. of sand were mixed with lo gm. of calcium carbonate and placed in paraffined clay pots. Where elemental sulphur was used, 0.75 gm. was added at the same time. The other nutrients added were applied in solution form through a period of 70 days; the growing period was 80 days. The total amount of sodium sulphate which was added varied in the different cultures. Concentration i denotes 1.55 gm., concentration 2, 3.10 gm., etc. The same figures apply to calcium sulphate too. All cultures, with the exception of No. 3, 13, 20, and 21, received 3.9 gm. of sodium nitrate, and they each re- ceived 0.75 gm. Every culture received 2.6 gm. of potassium dihy- drogen phosphate and 1.3 gm. of magnesium chlorid. Twenty plants grew in each pot, and the moisture content varied fron 15 to 20 per cent in the different cultures. The weights recorded are the average of duplicates, and the analyses were made on a sample obtained by mixing the duplicates. The 22 cultures from 11 to 21, inclusive, were inocu- lated with legume bacteria and 5 gm. of beaverdam soil added to the same culture. Table III. — Analytical results of clover grown on sand receiving a nutrient solution Uninoculated series. Cul- ture No. Form of sulphur added. NasS04 ....do 3 ....do Na2S04 and CaSO^ . . NajSOi 2 Control S CaS04 ....do ....do NajSOi and CaSO* . . 2 Sul- phate con- cen- tra- tion. Weight of air- dry clover. Gm. 9.65 10. 7 3-95 8.7s 6.9 4- IS 6.62 7. 02 S-I Total S. Per ct. o. 26 .42 •30 •30 •34 Total N. Per ct. 3-76 3-77 2. 00 3- 50 3-69 3-90 3-75 3-8s 3- 80 Inoculated series. Cul- ture No. 013 14 tS j6 J7 18 19 a 20 a ji Weight of air- dry clover. Gm. 10. 00 7. 00 4.80 6.4s 6.8s 7-3 7- 77 7-3° 5.90 7-5° S-07 Total S. Perct. o. 27 30 Total N. Sul- phate S. Perct. 3.62 3-71 3.49 3- 70 3.60 3-90 3- 69 3- 70 3- 90 2. 42 2.46 . 12 .08 .18 ■ I."; .08 • 17 .18 .09 .14 Or- ganic S. Perct IS " Low nitrate. In the foregoing data the low sulphur contents occur in the clover grown in the pots receiving less nitrate nitrogen. In the inoculated series the sulphur content of the clover does not appear to increase by increasing the sulphate sulphur of the nutrient media. On the other hand, where there is a reduction in nitrate added, there is an appreciable reduction in the sulphur content of the clover. In the other series, where the concen- tration of legume organisms was not as great at the start, the percent- ages of sulphur generally run higher. In No. 3 the percentage of Oct. 8,1921 Relation of Sulphates to Plant Growth and Composition 107 sulphur is not lower than in some of the others, but here the total yield is small, and this often accounts for higher percentages of certain elements. The jdelds in No. 3 and 6 are about the same; but the sulphur content is higher in 6, although this culture depended only upon the sulphur in the sand. No. 2 shows response in sulphur con- tent to the higher concentration of sulphates in the media. The cor- responding culture 12 in the other series does not show higher sulphur content; and as the average sulphur content is lower in this uninoculated series, it appears that the legume organisms might have some effect on limiting the quantity of sulphur present in the clover hay. Table IV. — Data showing the sulphur-nitrogen relation in the portion insoluble in dilute acetic acid CaS04 and NaNOs. In soil treated with- NaNOa. Na2S04. NajSOi and NaNOs. Average. Ratio of Nto S in the insolu- ble por- tion. Aver- age ash con- tent. Crop I : Percentage of N insoluble in acetic acid Percentage of S soluble in acetic acid Percentage of S insoluble in acetic acid Crop II: Percentage of N insoluble in acetic acid Percentage of S soluble in acetic acid Percentage of S insoluble in acetic acid Crop III: Percentage of N insoluble in acetic acid Percentage of S soluble in acetic acid Percentage of S insoluble in acetic acid Crop IV: Percentage of N insoluble in acetic acid Percentage of S soluble in acetic acid Percentage of S insoluble in acetic acid Inoculated series, Medford loam: Percentage of N insoluble in acetic acid Percentage of S insoluble in acetic acid Uninoculated series, Medford loam : Percentage of N insoluble in acetic acid Percentage of S insoluble in acetic acid Per a. 2. 26 .08 •13 I. 70 . II .09 1.78 •13 .09 2.05 .19 . II Per ct. 2. 24 .07 . 12 1-57 .06 .08 I. 76 •05 .09 1.97 .09 •13 Per ct. 2.58 .09 I. 64 .07 .09 1.87 I. .16 . II Per ct. 2.15 .07 •15 1-73 . II .09 1. 85 . II .09 2. 12 .18 . II Per ct. 2.31 • 13- 1.66 m-1 1. 81 >i9. 4 •93 2.03 •"5 2. 20 ■125 I. 21 . II ni-s ► 17. 6 Per ct. 9.94 7-31 6-95 54818°— 21- io8 Journal of Agricultural Research Vol. XXII, No. 2 To say that percentage of sulphur will not increase regardless of sul- phate concentration in the nutrient media without increasing the avail- able nitrogen would not be in accordance with data already obtained. It does appear, though, that when the lack of nitrogen is sufficient to lower the nitrogen content compared to the normal nitrogen content of the clover there is a tendency toward decreased sulphur assimilation. It is interesting to compare the rape plant with the clover in this respect. The figures given in Table IV show that the clover cut before the blos- soming stage not only contains a higher percentage of total nitrogen but also a higher percentage of nitrogen insoluble in acetic acid. As the per- centage of nitrogen removed by this fraction varies, so also does the per- centage of sulphur. There appears to be a definite relationship between the sulphur and nitrogen content in this insoluble portion, thus adding more significance to this fraction in regard to quality and perhaps rep- resenting the true protein of the clover hay. No difference in ash con- tent caused by variation in fertilizer treatment was observed in the diflerent pots. The ash content of the different crops did vary however, as is sho\vn in Table IV. EXPERIMENTAL WORK WITH THE RAPE PLANT The Medford loam soil used in the first part of this work was used in this experiment. After the clover roots were removed the soil was re- turned to the pots and seeded to rape. Three gm., of sodium nitrate were added to those cultures which had received nitrate nitrogen in the clover experiment. The plants were harvested after a growing period of 50 days. At the end of this time there had been a cessation of growth, and the basal leaves dried up and fell off. The results appear in Table V. Table V. — Analytical results obtained with rape Treatment. Control , NaNOg NaNOg and Na, SO4. . Na2 SO4 NaNOj and CaSO^ CaS04 Number of plants. Weight. Total N. N insol- uble in acetic ac'd. Total S. Sulphate Org S. S anlc 5. Gm. Per cent. Per cent. Per cent. Per cent. Per cent. "7 12. 0 1-93 0. 90 0.35 0. 16 0 19 7 S-o 2.88 I 02 •39 . 12 27 7 12 2 2-35 93 . 22 •03 19 07 10. I 4. 14 95 . 16 . 02 14 S "•3 3-36 71 I. 46 I. 09 37 06 14. 0 2. 72 88 1.38 •97 41 7 2.4 2.57 91 3-^Z 2. 76 37 a 8 30 14-45 2-59 2.30 2,41 I. 26 2.05 ■83 3b 43 0 6 83 6 16.35 2.27 66 1-31 •93 38 07 2.4 2. 69 88 3.28 2.98 30 8 1.8 2.66 2. 70 2-37 33 Ash. Per cent. 13.6 19. IS- 3 9 o 18.3 17-7 22. o 19.4 14.7 16. 2 31. 9 21. 9 « Pots from the inoculated series. The presence of added sulphates appeared to have retarded growth of the rape, as there is a greater dry-weight yield on the controls. With oct.8,i92i Relation of Sulphates to Plant Growth and Composition 109 the cultures receiving nitrate fertilizer the addition of sulphate sulphur apparently caused increased growth or had just the opposite efifect. If the concentration of the sulphates was great enough to produce a toxic effect, the sodium nitrate may have counteracted this action. The wjiter has observed just the opposite effect with clover seedlings growing on agar agar-mineral salt nutritive media. One gm. of sodium nitrate per liter had a noticeably toxic effect, while the same concentra- tion of sodium sulphate produced no noticeably injurious effect. In the cultures containing both the same concentration of sodium nitrate and sodium sulphate there was an improvement in growth over the former sodium-nitrate cultures. Application of nitrates produced very good 5delds on a comparative low sulphur assimilation by the plants. The question naturally arises whether the rape does not absorb sulphur, if present, far in excess of that required for carrying on the synthesis of its organic compounds. This appears so noticeable in comparing the figures in Table V. Of course it is realized that the optimum concentration of nutrients for plant nutrition has always been a problem. The acetic acid-insoluble nitrogen is higher in the rape grown on the soil receiving nitrate fertilizer only, compared with that in the rape which received both nitrate and sulphate fertilizer. There seems to be a tendency of the sulphates to decrease this form of nitrogen. Sulphate application increased the organic sulphur and total sulphur content of the rape, while at the same time the presence of these sulphate compounds retarded growth where no nitrates were added. The extremely high sulphate content is very obvious in these samples of rape. This may account for the high ash content. The percentage of ash in the samples of rape varies considerably, depending upon the fertilizer treatment and magnitude of growth. Such a variation did not occur with the clover. SUMMARY Sodium sulphate and calcium sulphate had a beneficial effect on nodule development and nitrogen assimilation of the red clover grown on previously sterilized soil. On a similar series which was artificially inoculated with Bacillus radicicola at the time of seeding, sulphates caused no increase in nodule development. When a soil of high sulphur content was used, the nitrogen content in clover of the third and fourth crops was lower on the control pots than where either sulphur, calcium sulphate, or sodium sulphate was applied. As sulphate sulphur was present in all plants, the low nitrogen content could not be explained by a cessation in protein synthesis due to the absence of sulphates. This again shows the relation of sulphates to nitrogen assimilation and the favorable influence of sulphates on the legume bacteria or on some other agency controlling nitrogen assimilation. no Journal of Agricultural Research voi. xxii, no. 2 The ratio of nitrogen to sulphur in the portion of the clover plant insoluble in dilute acetic acid remains about the same, regardless of the stage in the development of the plant. This gives further support to the view that the nitrogen insoluble in acetic acid represents protein nitrogen. The total nitrogen and total nitrogen insoluble in acetic acid wias higher in those plants cut before the blossoming stage. With clover growing on sand cultures, it was possible, by reducing the available nitrate, not only to limit the growth and nitrogen content but also to decrease the sulphur assimilation. So, while sulphates appar- ently cause greater nitrogen assimilation through their beneficial effect on nodule development, the amount of sulphur taken up by the plant is limited by the total nitrogen absorbed. The rape plant assimilated a large amount of sulphur, although the presence of sulphates reduced the yield compared to the control soil cultures. Sulphate plus nitrate caused increased yields compared with those secured when nitrate was added alone. There does not appear to be any direct relation between nitrogen and sulphur assimilation in the rape plant. LITERATURE CITED (i) Arny, a. C, and Thatcher, R. W. 1915-17. THE EFFECT OF DH'FERENT METHODS OP INOCULATION ON THE YIELD AND PROTEIN CONTENT OP ALFALFA AND SWEET CLOVER. In JOUT. Amer. Soc. Agron., v. 7, no. 4, p. 172-185, 1915; v. 9, no. 3, p. 127-137. 1917. (2) DULEY, F. L. 1916. THE RELATION OF SULFUR TO SOIL PRODUCTIVITY, /n JoUT. Amer. Soc. Agron., V. 8, no. 3, p. 154-160. (3) Hart, E. B., and Peterson, W. H. 191 1. SULFUR REQUIREMENTS OF FARM CROPS IN RELATION TO THE SOIL AND AIR SUPPLY. Wis. Agr. Exp. Sta. Research Bui. 14, 21 p. (4) Miller, H. G. I9I9. RELATION OF SULFATES TO PLANT GROWTH AND COMPOSITION. In Jour. Agr. Research, v. 17, no. 3, p. 87-102, pi. 9-12. Literature cited, p. 100-102. (5) PiTz, Walter. 1916. EFFECT OF ELEMENTAL SULFUR AND OF CALCIUM SULFATE ON CERTAIN OF THE HIGHER AND LOWER FORMS OF PLANT LIFE. In JoiW. AgT. Research, v. 5, no. 16, p. 771-780, pi. 56. (6) Prucha, Martin J. 1915. PHYSIOLOGICAL STUDIES OP BACILLUS RADICICOLA OF CANADA FIELD PEA. N. Y. Cornell Agr. Exp. Sta. Mem. 5, 83 p. Bibliography, p. 79-83. (7) Reimer, F. C, and Tarter, H. V. I919. SULFUR AS A FERTILIZER FOR ALFALFA IN SOUTHERN OREGON. Oreg. Agr. Exp. Sta. Bui. 163, 40 p., 9 fig. Bibliography, p. 39-40. (8) Wn^ON, J. K. 1917. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF SOYBEAN (SOJA MAX piper) AND OF FACTORS INFLUENCING NODULE PRODUCTION. N. Y. Cornell Agr. Exp. Sta. Bui. 386, p. 363-413, fig. 80-94. Bibli- ography, p. 412-413- SOYBEAN MOSAIC ' By Max W. Gardner, Associate in Botany, and James B. Kendrick, Assistant in Botany, Purdue University Agricultural Experiment Station In a small field of Hollybrook soybeans in West La Fayette a typical mosaic disease was found August 25, 1920. A rather low percentage of the plants were affected, and the disease was more or less confined to one quarter of the field adjacent to which were several rows of garden beans affected with mosaic to a considerable degree. In another larger field of soybeans in the same locality no mosaic was found. Leafhoppers were very prevalent on the soybeans. The impression was gained that the disease might have spread from the garden beans to the soybeans, but as yet no evidence to support such a theory has been obtained. Clinton^ found soybean mosaic in 19 15 at Mount Carmel, Conn., and under the name of chlorosis or crinkling has given an excellent account of the leaf symptoms along with a good illustration. He found the disease on the varieties Medium Green, Wilson, Swan, Kentucky, Wing's Mikado, and Hollybrook, and states that the Hollybrook showed the most marked symptoms. He found the chlorosis without the crinkling on the varieties O'Kute, Ito San, and Manhattan. C. R. Orton ^ has reported the occurrence of mosaic in a field of Ito San soybeans at Girard, Pa., July 30, 1920. SYMPTOMS The mosaic symptoms on the soybeans were conspicuous and unmis- takable, resembling those characteristic of mosaic diseases in general. Affected plants were stunted, and petioles and intemodes were shortened to some extent. The leaflets were stunted, greatly misshapen, and puck- ered with dark-green puffy areas along the veins (PI. 18, A, C, D, B). Between these puffy areas the leaf tissue was etiolated. Affected leaflets tended to be asymmetrical, twisted, and curled downward about the margins (PI. 18, D, E). As in other mosaic diseases, the young, rapidly growing leaves showed the most severe effects, and in some cases whole leaflets or portions thereof were extremely stunted or killed outright by the disease (PI. 18, B). The mosaic symptoms were readily distinguish- able from a uniform crinkling of the leaflets which was rather common in this field and apparently attributable to insect injury. The pods on mosaic plants were stunted and flattened, less pubescent, and more acutely curved than those on normal plants (PI. 19, C, D). ' Contribution from the Botanical Department of Purdue University Agricultural Experiment Station, I^a Fayette, Ind. 2 Clinton, G. P. notes on plant diseases of Connecticut. In Conn. State Agr. Exp. Sta. Ann, Rpt., 1915, p. 446-447, pi. 23a. 1916. 3 Fromme, F. D. diseases of cereal and forage crops in the united states in 1920. In U. S. Dept. Agr. Bur. Plant Indus. Plant Disease Bui., Sup. is, p. 173- 1921- Mimeographed. Journal of Agricultural Research, Vol. XXII, No. a Washington, D. C. Oct. 8, 1921 zu Key No. Ind. -10 (III). 112 Journal of Agricultural Research voi. xxii, No. 2 Those borne at the upper nodes were more severely affected. The yield of seed was very materially reduced (PI. 19, A, B), since a considerable proportion of the pods contained no germinable seeds and the remainder as a rule not more than one or two seeds (PI. 19, D) . Even the germinable seeds were in general undersized. Observations made a month later showed that the mosaic plants were remaining green longer than the normal plants, so the disease evidently delayed maturity. FIELD INOCULATIONS In another field of soybeans in which no mosaic was present inocula- tions were made August 2 7 by rubbing the young intemodes with cotton soaked in the juice from crushed mosaic soybean leaves and then wound- ing these intemodes with a needle. One hundred and fourteen plants were thus inoculated, but no mosaic developed. Fifty- two plants were simi- larly inoculated, except that the juice of leaves from mosaic garden beans was used as inoculum, and none developed the disease. Forty-six garden bean plants were also inoculated in a similar manner with the virus from soybean mosaic, and none developed mosaic. SEED TRANSMISSION To determine whether or not the disease was seed-borne, a quantity of seed was saved from mosaic and healthy plants early in October for subsequent tests in the greenhouse. On October 25, 150 seeds from mosaic plants were planted in 25 pots of sterilized soil, 6 in each pot. By December 15, 124 plants had come up, and 18 showed unmistakable mosaic symptoms. None of the 148 controls grown from seed from normal plants showed mosaic. In a second trial about 180 seeds from mosaic plants were planted December 9 in 59 pots of sterilized soil. February 3, 192 1, 11 out of the 106 plants which were up showed mosaic. None of the 38 controls grown from seed from normal plants showed the disease. As a result of these two tests it is evident that about 13 per cent of the seedlings from seed produced on mosaic plants developed the disease. The mosaic seedlings were spindling (PI. 18, F, G), and the j&rst pair of true leaves were characterized by downward, longitudinal curling or rolling, a crinkling, and a faint etiolation or mottling. These leaves turned yellow prematurely. The leaves subsequently formed were greatly stunted and showed the mottling and crinkling more conspicu- ously than the first leaves. GREENHOUSE INOCULATIONS From these mosaic seedlings the disease was transmitted to healthy soybean seedlings. Several methods of inoculation proved successful. A number of inoculations made early in January yielded only negative results, but later better success was obtained. Oct. 8,1921 Soybean Mosaic 113 On January 26, twenty-five plants were inoculated by pricking with a needle at the nodes and rubbing the wounded areas with cotton soaked in the juice from crushed mosaic leaves. Eight plants used as controls were similarly treated, except that sterile water was substituted for the mosaic virus. Because of the unfavorable greenhouse conditions the plants made slow growth during the winter, so that the mosaic symptoms were very slow in developing. On March 5 two plants showed mosaic mottling on the young leaves. On March 25 two more showed mosaic, and on April 7 seven out of the 25 plants had developed the disease. The controls developed no mosaic. A number of inoculations were made March 2. In one series crushed mosaic tissue was inserted into slits made with a scalpel near the grow- ing points and on the petioles. On March 15 two of the seven plants thus inoculated showed mosaic symptoms on the young leaves, and on April 7 five had developed mosaic. In a second series of inoculations made the same date by cutting off one leaf at each node and smearing these wounded surfaces with crushed mosaic tissue, three out of eight plants showed mosaic symptoms on the new leaves March 15, or 13 days after inoculation, and on April 7 six plants had developed mosaic. In a third series five plants were inoculated by a combination of the two methods above described. Thirteen days later three showed mosaic mottling, and by April 7, or 37 days after inoculation, four had developed the disease. In a fourth series, five plants w^e inoculated by rubbing the under surfaces of the leaves with slightly crushed mosaic leaves forcibly enough to cause slight abrasions. On March 15, four of these plants showed the disease, and on April 7 all showed typical mosaic. None of the five control plants inoculated by one or the other of these methods without the application of mosaic tissue developed mosaic. At no time was there any spread of the disease in the greenhouse. In these inoculations the symptoms became evident only on the young leaflets. These in some cases developed distinct mottling, and in other cases they exhibited a slight degree of etiolation and the char- acteristic downward, longitudinal rolling. The incubation period under the conditions of this test was 13 days. Preliminary cross inoculations to garden beans and cowpeas have given negative results. Further tests are being made. Soybeans, therefore, are subject to a destructive mosaic disease which greatly reduces the yield of affected plants. The disease is transmis- sible from plant to plant and also is seed-borne. PLATE i8 A. — Typical mosaic leaf showing darker green puffy areas along the veins, B. — Mosaic leaf showing extreme sttmting of terminal leaflet. C. — Normal leaflet. D. — Mosaic leaflet showing longitudinal rolling. E. — ^Typical mosaic leaflet. F. — Mosaic seedlings from seed from a mosaic plant, showing stunting of the plant and longitudinal rolling of first leaves. G. — Normal seedlings from seed from a mosaic plant. Soybean Mosaic Plate If Journal of Agricultural Research Vol. XXII, No. 2 Soybean Mosaic Plate 19 D Journal of Agricultural Research Vol. XXII, No. 2 PLATE 19 A. — Upper nodes of a normal plant, showing yield of pods. B. — Upper nodes of a mosaic plant, showing effect of the disease on the yield. C. — Normal pod. D. — Type of pods produced by a mosaic plant. 54818° — 21 6 ADDITIONAL COPIES OF THIS PUBUCATION MAT BE PKOCUKED FEOM THE StTPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 15 CENTS PER COPY SuBSCEiPTiON Price, $3.00 Per Yeab A BBMBaHnBMBaai iP-r — Vol. XXII OCTOBER 15, 1921 No. 3 JOURNAL OP AGRICULTURAL RESEARCH CONXKNXS Page Influence of the Plane of Nutrition on the Maintenance Requirement of Cattle - - - - - - -115 F. B. MUMFORD, A. G. HOGAN, and W. D. SALMON ( Contribution from Missouri Agricultural Experiment Station ) Turnip Mosaic - - - - - - - - 123 MAX W. GARDNER and JAMES B. K^NDRICK ( Contribution from Indiana Agricultural Experiment Station ) Hydrocyanic Acid in Sudan Grass - - - - - 125 C. O. SWANSON ( Contribution from Kansas Agricultural Experiment Station ) Nutrient Requirements of Growing Chicks: Nutritive Deficiencies of Corn - - - - - - - 139 F. E. MUSSEHL, J. W. CALVIN, D. L. HALBERSLEBEN and R. M. SANDSUEDT ( Contribution from Nebraska Agricultural Experiment Station ) Aecial Stage of the Orange Leafrust of Wheat, Puccinia triticina Eriks. - - --- -- - 151 H. S. JACKSON and E. B. MAINS ( Contribution from Bureau of Plant Industry and Indiana Agricultural Experiment Station ) A Transmissible Mosaic Disease of Chinese Cabbage, Mustard, and Turnip ------- 173 E. S. SCHULTZ (Contribution from Bureau of Plant Industry) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICCLTDRE, WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINOTON, D. C. 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, ottd Director, New Jersey Agricultural Etperi- 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 Direct, 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 Agricultiu-al Experiment Station, New Brunswick, N. J. JOmAL OF ACRiaiTIIAL RESEARCH Voiv. XXII Washington, D. C, October 15, 192 1 No. 3 INFLUENCE OF THE PLANE OF NUTRITION ON THE MAINTENANCE REQUIREMENT OF CATTLE' By F. B. MuMPORD, Dean of the College of Agriculture, A. G. Hogan, of the Depart- ment of Animal Husbandry, and W. D. Salmon, Graduate Student in Animal Hus- bandry, College of Agriculture, University of Missouri. In 19 1 4 an investigation was begun at the University of Missouri to study some of the effects of underfeeding. Calves of beef-breeding stock were secured, and they were placed on three planes of nutrition. Group I was fed to grow rapidly, but not to become fat. Group II was placed on a lower nutritive plane and was fed to gain about }4 pound per day. Group III was placed on a still lower nutritive plane and fed to gain about V3' pound per day. At the present time three animals remain that were started on the investigation in 19 14. Seven others were added in 191 7. The older animals, therefore, have been under observation for seven years and the younger animals for four years. Under these cir- cumstances it seemed desirable to make a study of the maintenance requirement of steers at different ages and on different planes of nutrition.^ The ideal method of conducting an investigation of this kind would require a respiration calorimeter. Since that was impossible, the alter- native was to calculate the energy value of the feed consumed and cor- rect this for the estimated value of the gains or losses in body weight. The net energy of the feed consumed was calculated in accordance with procedures developed by Armsby.^ The energy value of the changes in body weight were calculated from the composition of steers that had been analyzed at this Station by the Department of Agricultural Chemis- try. So far as possible steers were selected as controls for this purpose that were of similar age, measurements, and weight and that had received similar treatment. ' The data for this paper were taken from the thesis of W. D. Salmon, presented at the University of Missouri, as partial fulfillment of the requirements for the degree of Master of Arts. The investigation was initiated by F. B. Mumford, Dean of the College of Agriculture, and by P. F. Trowbridge, at that time Chairman of the Department of Agricultural Chemistry. Since September 1918, E. A. Trowbridge, Chairman of the Department of Animal Husbandry, has had general supervision of the project. This article was prepared by A. G. Hogan, who has been in immediate charge since September, 1920. A large number of workers have contributed to the success of the experiment. ' The original data will be reproduced in detail in a subsequent publication. 8 Armsby, H. p., and Fries, J. A. net energy values for ruminants. Pa. Agr. Exp. Sta. Bui. 142, 20 p. 1916. Journal of Agricultural Research Vol. XXII, No. 3 Washington, D. C. Oct. 15, 1921 zv Mo. -J (115) ii6 Journal of Agricultural Research voi. xxn. no. 3 METHOD OF THE EXPERIMENT RATIONS The concentrate consisted of the following mixture: Com chop, 60 per cent; wheat bran, 30 per cent; linseed meal, 10 percent. The rough- age fed to the 3 old steers. No. 528, 589, and 585, from the beginning of the experiment until July 20, 191 7, was timothy. For the next 10 days a mixture of 5 parts timothy, 3 parts alfalfa, and 2 parts oat straw was fed. Following this the roughage consisted of a mixture of 60 per cent alfalfa amd 40 per cent oat straw. The animals were fed twice daily and had access to water at all times. Salt was accessible at feeding time. PERIODS The calculations are made for periods of 180 days, with the exception of the first period for the 3 older steers, which was as follows: No. 528, 130 days; No. 579, 142 days; No. 585, 150 days. The warm months of the year were selected for these periods to avoid a possible disturbing effect of low temperatures in the winter months. WEIGHTS The steers were weighed each morning after feeding but before water- ing. The weight given for the beginning of a period is the average of the 10 preceding days. The weight given at the end is an average of the last 10 days of the period ENERGY INTAKE The amount of dry matter consumed was calculated from the weight and composition of the feed consumed. The net energy was computed from this by the use of factors reported by Armsby and Fries. ^ For the concentrates the value 83.82 therms per 100 pounds dry matter was used. This is the factor given for Armsby's grain mixture No. 2,- which approxi- mates the grain mixture used in this experiment. For timothy hay the value 48.63 therms per 100 pounds dry matter was used. The factor for the roughage mixture used in the latter part of the experiment was cal- culated from the Armsby values, for alfalfa, 34.10 therms and for oat straw, 26.03 therms per 100 pounds dry matter. A mixture of 60 parts alfalfa and 40 parts oat straw would have a value of 30.87 therms per 100 pounds dry matter. The calculations of the energy value of the milk are based on factors published by Armsby.^ There are 29.01 therms per 100 pounds whole milk (4.4 per cent) and 14.31 therms per 100 pounds skim milk (0.2 per cent). From these values factors were computed for the different grades of milk used. 'Armsby, H. P., and Fries, J. A. op. cit. 2 Annsby's grain mixture No. 2, 60 per cent com meal, 30 per cent crushed oats, 10 per cent O. P. linseed meal. Our grain mixture, 60 per cent com meal, 30 per cent wheat bran, 10 per cent O. P. linseed meal. ^Armsby, Henry Prentiss, thb nutrition op farm animals, p. 719. New York, 1917. Oct. IS, 1921 ' Influence of Plane Nutrition on Maintenance 117 CHANGES IN BODY WEIGHT In order to obtain data concerning the maintenance requirement of these steers, it is necessary to calculate the energy gained or lost through changes in body weight. Our calculations are based on analyses previ- ously made by the Department of Agricultural Chemistry, University of Missouri.^ Control animals were selected from those on which analyses were available, on the basis of similar weights and measurements, and when possible of similar ages, daily gains, and daily consumption of dry- matter. In some cases suitable control animals were not available, and the composition of steers for those periods was estimated by interpola- tion, with the exception of the last period for steer No. 528. In this case a value published by Armsby^ was used. The average energy values of a pound gain as calculated by this method for steers in the three groups are given in Table I. For purposes of comparison the values given by Armsby are shown in the same table. Table I. — Energy values of a pound gain Approximate age (months). 6. 18 36 54 66 78 Group I. Therms. o- 95575 1. 0918 I. 7136 2. 1993 2. 50 3.00 Group II. Therms. o- 95575 I- 0583 I. 1608 I. 4104 I- 5352 I. 660 Group III. Therms. o- 8343 •9445 I. 0548 I. 1013 I. 4790 I. 6490 Annsby's values. Age. Months. I 2 to 3 5 to 6 II to 12 18 to 24 Energy. Therms. 1. 170 1-374 1.680 2. 292 3. 000 Armsby's values are consistently higher, as is to be expected. Our animals were thin and contained less than the usual amount of fat in the gain. .; „.j'f u.-A ..■...■.:■' f.,^.. vjorf In calculating the maintenance requirements per 1,000 pounds live weight, Moulton's ^ formula was used. He has shown that the surface areas of thin cattle are proportional to the ^ power of the live weight. The results of this calculation, on the basis of dry matter consumed, are given in Table II. The net energy required for maintenance was also calculated by another method,* based on the digestible organic matter of the feed. The following factors are given for the metabolizable energy of digesti- ble organic matter consumed: Roughage, 1.588 therms per pound; grains and similar feeds with less than 5 per cent digestible fat, 1.769 therms per pound. In the same publication the "Average energy expenditure by cattle per 100 pounds of dry matter eaten" is given. 1 These have not yet been published. * Armsby, Henry Prentiss, op. cit. ' MOULTON, C. R. THE AVAtLABILITY OF THE ENERGY OF FOOD FOR GROWTH. In Jour. Biol. Chem., V. 31, no. 2, p. 390. 1917. * Armsby, H. P., and Fries, J. A. op. cit. ii8 Journal of Agricultural Research voi. xxii. no. 3 Table II. — Average daily maintenance requirement as calculated from dry matter con- sumed Steer No. Number of periods averaged. Therms of net energy per i.ooo pounds, based on H power of live weight. Group I. Group II. Group III. 528 577 571 579 578 573 585 575 574 572 6 5. 870 5.280 5-730 3 3 e 4.920 3-830 4.409 1. •J c 4. 221 4.041 4.302 3-250 T. 7. •J Average of all animals for all periods 5-523 4-485 3-830 Table III. — Energy expenditure by cattle per loo pounds dry matter consumed Ration. Roughage : Timothy hay Alfalfa hay Oat straw Concentrate : Grain mixture No. 2 Energy ex- penditure. Therms. 35-47 53-03 46. 00 51-76 The coefficients of digestibility used in these calculations were derived from digestion trials conducted under similar conditions at this Station. These indicated that the digestibility of the ration varied with the rela- tive amounts of hay and grain fed. The factors used are given in Table IV. Table IV. — Digestion factors for organic matter Ratio of grain to hay. 1. 1 2-3 I. 3 1.3.4. or 5 1.6 or 7 1. 8,9, or 10 Hay only. Factor .6956 .6695 •6434 .6340 . 6229 .6030 0. 5832 Inasmuch as the thermal value of a pound of organic matter from grain differs from that of a similar weight of organic matter from rough- age, the Armsby factors ^ previously quoted in this paper could not be directly applied to the values obtained with the foregoing digestion coefficients. Those factors would not provide for the widely varying proportions of grain and hay. The following method, therefore, was 'Armsby, H. P., and Fries, J. A. op. cit. Oct. IS. 1921 Inf,uence of Plane Nutrition on Maintenance 119 used in computing tlie energy intake on the basis of digestible organic matter consumed. By use of the factors in Table IV, the weight in pounds of digestible organic matter in the mixed ration was determined for each period. This was multiplied by 1.588, the Armsby factor for metabolizable energy in a pound of digestible organic matter from hay. The thermal value of digestible organic matter from grain is 1.769, however, or 0.181 therms more. Therefore, each pound of digestible organic matter derived from grain was multiplied by 0.181, and the product was added to the result obtained by multiplying the total digestible organic matter by 1.588. This gave the total metabolizable energy in both the hay and grain. The digestibility of the organic matter of the grain was estimated by difference. This ranged closely around 80 per cent. The factors for energy expenditure are given in Table III. It seemed impracticable to calculate the net energy of the milk con- sumed on the basis of digestible organic matter, so the calculation was based on the quantity consumed, as previously described. Since the amount was small, however, the method of calculation would have little effect on the final result. [i;i> 0. The method used in correcting for changes in body weight has already been described, and the maintenance requirement as calculated on the basis of digestible organic matter consumed is given in Table V. Table v.- -Average daily maintenance requirement, as calculated front digestible organic matter consumed Steer No. Number of periods averaged. Therms of net energy per i.ooo pounds based on 5/8 power of live weight. Group I. Group II. Group III. 528 577 571 579 578 573 585 575 574 572 6 . . . . 6. 261 5-412 5-174 5. 260 4. 192 4-893 4.725 4-454 4-591 3-649 ■2 ... -2 . •2 . Average of all animals for all periods 5-777 4.869 4. 408 In determining the maintenance requirement on the basis of digestible organic matter, the calculations were based on digestion coefficients ob- tained at this Station under similar conditions. This method is probably more accurate than that of calculation on the basis of dry matter con- sumed, and for the animals concerned it gives a result about lo per cent higher. I 120 Journal of Agricultural Research Vol. XXII. No. 3 In calculating average results, obtained by both methods, four periods in which there were losses in live weight were omitted. The results for those periods were low, and we were uncertain as to whether the result was approximately correct or whether it was due to an incorrect assump- tion as to the energy value of the loss in weight. Most of the dry matter of the loss was probably fat, and if so, our calculation of its energy value was too low and so made our calculation of the maintenance requirement too low. One steer, No. 585, had a navel infection during the first period, accompanied by a very high maintenance requirement. This period also was discarded in calculating averages. INFLUENCE OF NUTRITIONAL PLANE There is a close parallel between the intake of net energy and the maintenance requirement of the animal. The record of steer 574 illus- trates that tendency. For the first period the average daily intake of net energy was 3.884 therms per 1,000 pounds, based on the 5/8 power of the live weight; and the maintenance requirement was 3.818 therms. For the second period the energy intake was increased to 5.783 therms, and the maintenance requirement increased to 5.1 19 therms. In the third period the energy intake was 5.253 therms, and the maintenance requirement was 4.836 therms. Table VI. — Dailymaintenance requirements of cattle — Net energy RESPIRATION EXPERIMENTS Num- ber of Investigator. Condition of ani- mal. Therms per i.ooo pounds live weight. ments. Maximum. Minimum. Average. 22 Armsby and Fries ^ . Medium do 7-430 6. 780 8.871 4-723 4-921 7-319 7 Kellner ^ 5-99^ do Fat 5- 742 7.946 LIVE-WEIGHT EXPERIMENTS Armsbv ' . . do'. ... Haecker * . . Eward ^ . . . Eckles^ .. Shirky 2. . . ... .do. . .. Our results . ....do. ... ....do. ... Thin do. . . Medium... do.... do.... do3.. Thin*.... Group I . . . Group II.. Group III. 7.044 6.039 5.676 7.850 7.079 5- OQ59 7.380 5- 724 5- 217 6. 136 4- 713 4. 662 6.450 5.841 4-953 4-915 3.809 3.276 50s 423 021 180 173 732 0245 777 5 5 4. 869 4. 408 ' Armsby, Henry Prentiss, op. cit., p. 291. 2 Shirkey, S. B. extent to which growth retarded during the early life of the beef animal CAN BE LATER REGAINED. Univ. of Mo. thesis, 1919. (Unpublished ) animal ' Corresponds to group I of this experiment. * Corresponds to group 11 of this experiment. Oct. 15. 1921 Influence of Plane Nutrition on Maintenance 121 In comparing the maintenance requirements of the three groups it should be kept in mind that group I does not represent a high plane of nutrition. The aim was to secure maximum growth with no considerable fattening. Their maintenance requirements as computed in this paper correspond closely to the average of 22 respiration experiments by Armsby and Fries ^ and of 7 by Kellner,^ on cattle in medium condition. A comparison of our results, and of those obtained by other investiga- tions, is given in Table VI. INFLUENCE OF AGE The ages represented in this experiment vary from 30 days for some of the calves at the beginning of the first period to more than 6 years at the close of the seventh period. Apparently there was no relation between the age and the maintenance requirement of these animals. Some of the steers showed a gradual decrease in the maintenance cost from the begin- ning to the end of the experiment. In such cases it was found that the energy intake per 1,000 pounds had also decreased. On the other hand, steers with an increasing energy intake showed an increased maintenance requirement. Maintenance trials on young animals usually give higher results than have been obtained with mature animals, but if age does influence the maintenance requirement the effect is too slight to be shown in a live- weight experiment of this kind. SUMMARY AND DISCUSSION There is a close relation between the amount of net energy consumed and the maintenance requirement. Periods of high energy intake were apparently periods of high maintenance cost, while periods of low energy intake were accompanied by a lowered maintenance requirement. The averages of the periods discussed show the following daily main- tenance requirements per 1,000 pounds live weight, calculated on the basis of digestible organic matter, and in terms of net energy: Group I, 5.777 therms; group II, 4.869 therms; and group III, 4.408 therms. If the maintenance requirement of group I is 100 per cent, that of group II is 84.4 per cent, and that of group III is 76.3 per cent. The calculations on the basis of dry matter consumed indicate even greater differences. The maintenance requirements as derived by this method may be compared as follows: Group I, 100 per cent; group II, 81 per cent; group III, 69.3 per cent. The estimated maintenance re- quirement of group I, as calculated by this method, is 30 per cent greater than the total net energy intake of group III. There is no apparent relation between the age of the animals and the amount of energy required for maintenance. ' Armsby, Henry Prentiss, op. cit. Turnip mosaic ^ By Max W. Gardner, Associate in Botany, and James B. Kendrick, Assistant in Botany, Purdue University Agricultural Experiment Station In one comer of a small field of turnips near South Bend, Ind., Octo- ber 12, 1920, a considerable percentage of the plants were found affected with an unmistakable mosaic disease. The symptoms were typical of mosaic diseases in general. The leaves were stunted, misshapen, and a lighter green with dark green blisters or puffy areas. Many of the leaves were extremely distorted by crinkling and folding (PI. 20, A). The dis- ease seemed to be confined to one area in the field, to some extent coin- cident with a heavy infestation of tarnished plant bugs. Several diseased plants were transplanted to pots in the greenhouse, where they continued to form new leaves during the winter. The mosaic symptoms exhibited by the new foliage formed under greenhouse con- ditions were not quite so extreme as had been noted in the field. One of these plants, with mottled and spindling leaves, is shown in Plate 20, B, as it appeared in December. Inoculation of a number of potted turnip and radish seedlings was made by breaking off a leaf and rubbing the wound with crushed leaf tissue from one of the mosaic plants. Out of 21 turnip seedlings inoculated early in January, 13 developed characteristic mosaic symptoms. The first symptoms were noted 26 days after inoculation. The turnips inocu- lated showed some varietal difference from the plants collected in the field in that the leaves were much less distinctly pinnatifid. Out of 46 radish seedlings, including both white and red varieties, similarly inocu- lated, none developed mosaic symptoms. A later series of inoculations was made January 26 by wounding the plants with a needle and rubbing the wounded areas with a piece of cotton soaked in the juice from mosaic leaves ground up in a mortar. Ten out of 14 turnip plants thus inoculated developed the mosaic disease. The first symptoms were noted 16 days after inoculation. No mosaic devel- oped among 13 control plants similarly treated except that sterile water was substituted for the mosaic virus. Twenty-two radish plants were also inoculated, and none of these developed the disease. Subsequent reinoculation of turnip plants from one of these radish plants produced no mosaic. The mosaic disease of turnips is therefore readily transmis- sible to turnips but not to radishes. ' Contribution from the Botanical Department of Purdue University Agricultural Experiment Station, I,aFayette, Ind. After this article was prepared it was learned that Eugene vS. Schultz, of the Bureau of Plant Industry, United States Department of Agriculture, was also working on this disease. Journal of Agricultural Research, Vol. XXII, No. 3 Washington, D. C. Oct. 15, 1921 zw Key No. Ind.- 1 1 (123) PLATE 20 A. — Leaves from mosaic turnip plants collected October 12, tqso. B. — Mosaic turnip plant transplanted to a pot in the greenhouse. Photographed December 20, 1920. (124) Turnip Mosaic Plate 20 Journal of Agricultural Research Vol. XXII, No. 3 HYDROCYANIC ACID IN SUDAN GRASS' By C. O. vSwANSON ^ Professor of Agricultural Chemistry, Kansas State Agricultural College In a previous paper ^ it was shown that hydrocyanic acid (HCN) is obtained from green Sudan grass by macerating, digesting in water, and distilling into a dilute solution of sodium or potassium hydroxid. Sev- eral experiments reported in that paper made it clear that this acid does not exist free in Sudan grass and is obtained only if the conditions of the determination are favorable to enzym action. It appears to be a common belief that hydrocyanic acid is developed by freezing. This merely bursts the green cells and thus performs the same function as maceration, with the result that the hydrocyanic acid is rapidly lost from frosted grass. It was also shown that while in some cases poisoning had been reported from pasturing Sudan grass, under normal conditions no poisoning took place either before or after the grass was frozen. It was suggested that when frozen the hydrocyanic acid had been liberated and then evap- orated as the grass dried. Because of the importance of the subject it was thought worth while to make further investigations. During the summer of 1920, material was obtained from a i/20-acre plot of Sudan grass grown by the Depart- ment of Agronomy of the Kansas State Agricultural College. The Sudan grass had been planted early in June in rows about 2 feet apart. On June 22, when the experiments were begun, the grass was about 6 inches high. These experiments were continued during the summer and early fall. METHOD OF DETERMINING HYDROCYANIC ACID At present there are no satisfactory quantitative methods for esti- mating hydrocyanic acid obtained from organic material. All are open to some objection. After considering several, the Prussian-blue method was adopted as best suited for the purposes of the present investigation. Because of simplicity in manipulation it is possible to run a large number of determinations at the same time. The amount of hydrocyanic acid obtained from the different samples was estimated colorimetrically, using standard solutions containing known amounts of potassium cyanid. One objection to the colorimetric measurements was the difficulty in ' Contribution No. 92 from the Department of Chemistry, Agricultural Experiment Station of Kansas State Agricultural College. * Credit is due Mr. Carl M. Conrad for efficient assistance in making the determinations reported in this paper. ' SWANSON, C. O. HYDROCYANIC ACID IN SUDAN GRASS AND ITS EFFECT ON CATTLE. Ill JoUr. Amer. Soc. Agron., v. 13, no. i, p. 33-36. 1921. Journal of Agricultural Research Vol. XXII, No. 3 Washington, D. C. Oct. 15, 1921 7x Key No. Kans.-J7 (125) 126 Journal of Agricultural Research voi. xxii. no. 3 obtaining a uniform blue color. Very often the precipitate was decidedly green. It was found that by warming and letting the precipitate stand for some time in loosely stoppered bottles a uniform blue color could be obtained. The use of nitric or sulphuric acid instead of hydro- chloric acid or the addition of potassium fluorid, all of which have been suggested by other workers, did not seem to eliminate, entirely, the green color. While the defects of the Prussian-blue method are fully realized, it compares favorably with other methods.' In no sense are the values reported in this paper to be regarded with the same degree of accuracy as a protein or even a crude-fiber determination. For this reason no conclusions should be drawn from the results unless the figures presented are uniformly consistent or the differences large. The calculations in this paper are based upon approximately 200 gm. of green material. When the grass was wilted or dry the weight of sample used was proportionately lessened. It is impracticable to secure green samples of uniform weights of dry matter, particularly if they are gathered during different hours of the day and throughout several weeks and months. Then, as will be shown in what follows, the hydrocyanic acid is localized in the plant, being present in the largest amounts in those portions of the plant possessing the greatest vegetative activity. For this reason leaves were separated from the stems whenever these were present. The amount of hydrocyanic acid obtained is small in proportion to the total weight of samples used. It was seldom more than 0.015 per cent. EFFECTS OF MACERATION The first sample was collected June 22, when the grass was about 6 inches high. This was cut into pieces about % inch long and digested for three hours in water at room temperature. Less than i mgm. hydrocy- anic acid was obtained. Another sample, taken the next day, was cut and thoroughly macerated by pounding in an iron mortar and was then digested in water. This sample gave 27 mgm. hydrocyanic acid. On June 28 a sample was secured and divided into two equal portions. One portion was cut and macerated as described above, and the other was cut and macerated with coarse, sharp sand. Both were digested in water for the same length of time. The portion macerated with sand gave 26 mgm. hydrocyanic acid, and the other gave 36 mgm. It ap- peared from this that maceration with sand was not necessary and might result in a loss. Subsequent experiments showed that as soon as the grass is macerated the hydrocyanic acid is liberated and for this reason may be lost. On August 18 a sample was divided into four portions and, after the preliminary treatment mentioned, was digested overnight, with the result given in Table I. * ViEHOVER, Arno, and Johns, Carl O. on the determination of small quantities op hydrocy- anic ACID. In Jour. Amer. Chem. Soc, v. 37, no. 3, p. 601-607. 1915. octis. I92I Hydrocyanic Acid in Sudan Grass 127 Table I. — Effect of maceration on liberation of hydrocyanic acid Sam- ple No. Treatment. HCN. No cutting or maceration Cut in feed cutter {}^ to ys inch) Cut and macerated slightly Cut and macerated thoroughly . . . Mgm. o 10 10 II From this it appeared that if the time of digestion is sufficiently long the amount of maceration is less important, provided, however, that the plant tissue is cut fairly fine. The smaller amount obtained from the grass on August 18, as compared with that obtained in June, is in accord with a general observation made during the summer, that as the season advanced smaller amounts were obtained from the 200-gm. portions. TIME REQUIRED FOR DIGESTION •tM,;;-n The time required for digestion in order to obtain the maximum amount of hydrocyanic acid was determened. A sample collected on June 28 was divided into three portions and similarly treated, except for the time allowed for digestion. The results obtained are given in Table II. Table II. — Effect of time of digestion on liberation of hydrocyanic acid Sample No. Treatment. HCN. I Digested 3 hours Mgm. 18 2 Digested 6 hours 32 32 3 Digested 24 hours This experiment seemed to show that digesting 3 hours was not long enough, while 6 hours was as effective as 24. On August 28 a similar experiment gave the results shown in Table III, the results in each case being an average of duplicate samples. Table III. — Effect of tim,e of digestion on liberation of hydrocyanic acid Sample No. Treatment. HCN. Digested two days. . . Digested three days . Digested four days. . . Digested seven days . Mgm. 10 10 10 O The portion digested seven days developed a very bad odor. Because of these results, the usual procedure with experiments reported in this paper was to macerate the sample and then digest at room temperature over- night. 128 Journal of Agricultural Research voi. xxn. No. 3 LOCALIZATION OF HYDROCYANIC ACID IN THE PLANT At three different times the grass was divided into leaves and stems. From 200-gm. portions the number of milligrams of hydrocyanic acid shown in Table IV were obtained. Table 1\ . — Hydrocyanic acid in leaves and stem s of Sudan grass Date collected. June 30 July 7.. July 21. stems. Mgm. Trace. The immaturity of the sample collected June 30 accounts for the moder- ate amount obtained from the stems. At a later date two tests were made on immature heads. No hydrocyanic acid was found. In the following tests reported in this paper leaves only were used unless otherwise stated. INFLUENCE OF STAGE OF GROWTH Since grass was cut almost every week throughout the summer there were afforded several opportunities to test the comparative amounts present in various stages of growth and development. The shorter grass was obtained from plants which had been cut once or several times. Table V. — Hydrocyanic acid in Sudan grass at different dates and stages of growth Date collected. Portion used. Average height. HCN. July 22. 26. Aug. 12 27. 30 Whole plant. ....do ....do Leaves ....do ....do Whole plant. do do Leaves do do do I Whole plant. Leaves do (Whole plant, do Leaves do fWhole plant. \Leaves (Whole plant, do Leaves do 6 inches 8 inches 12 inches 18 inches 24 inches 30 inches 4 inches 6 inches 12 inches Beginning to head Partly headed Fully headed Partly dead 4 inches 16 inches Headed 4 inches 10 inches 15 inches 24 inches 5 inches 20 inches 2 inches 12 inches Ready to head. .. . Blooming Mgm. 27 19 7 24 20 9 40 32 10 12 17 20 6 II 18 19 40 12 16 10 10 II 5 5 9 10 Oct. 15, I93I Hydrocyanic Acid in Sudan Grass 129 The results show that more hydrocyanic acid is found in the whole plant in the earlier stages of growth and less as the season advances. The difference is perhaps due to the large proportion of stems in the latter part of the season, since if leaves only are compared there is very little difference except where they are from mature plants. This indi- cates that most of the hydrocyanic acid is obtained from those parts of the plants where the vegetative activity is most pronounced. This agrees with the results obtained by Menaul and Dowell ^ at the Okla- homa Agricultural Experiment Station. These observations support the theory that hydrocyanic acid is an intermediate product between the nitrates and the amino acids. ^ DISAPPEARANCE FROM MACERATED MATERIAL As soon as the grass is macerated the hydrocyanic acid begins to pass off. This was demonstrated several times by suspending small pieces of sodium-picrate paper above some macerated grass in stoppered flasks. The paper very soon assumed a brown color. The quantitative deter- minations given in Table VI were made on samples macerated July 10 and treated as indicated. Table VI. — Disappearance of hydrocyanic acid in macerated grass _ ^X!'^ Treatment. | HCN. Digested in water two days Placed without added water in covered mason jar for two days then small amount of water added and distilled Placed in flask two days so that the hydrocyanic acid could escape only into the receiving flask, after which water was added and distilled 4 Left in open jar for two days, digested and distilled 5 Repeat of 3 but kept in flask overnight only Mgm. 25 26 Trace. 26 DISAPPEARANCE FROM GRASS AFTER CUTTING In a previous paper ^ it was stated that tests made on partially wilted grass may be worthless. In the experiments made at that time, the amount of sulphuric acid added was not carefully enough controlled. It will be shown in the following paragraphs that if acid is added beyond certain limits no hj^drocyanic acid will be obtained from either green or partially wilted grass. In each of the determinations given in Table VII the grass was macerated after the treatment stated and then di- gested in water overnight. 1 Menaul, Paul, and DowEi,!,, C. T. cyanogenesis in sudan grass: a modification of the franos- CONNELI, METHOD OF DETERMINING HYDROCYANIC ACID. In Jour. Agr. Research, v. i8, no. 8, p. 447-450. 1920. 2 Ravenna, C, and Zamorani, M. ntjove ricerche sulla fxjnzione fisologica dell, acido ciani- DRICO NEL SORGHUM VULGARE. In Atti R. Accad. Lincei, Rend. Cl. Sci. Fis., Mat. e Nat., v. 18, sem 2, no. 8, p. 283-287. 1909. Abstract in Chem. Abs., v. s, no. 6, p. 1113. 1911. ' SWANSON, C. O. HYDROCYANIC ACID IN SUDAN GRASS AND ITS EFFECT ON CATTLE In Jour. Amer. Soc. Agron. , v. 13, no. i, p. 33-36. 1921. I30 Journal of Agricultural Research voi. xxii. no. 3 TablR YII. — Disappearance of hydrocyanic acid from Sudan grass after cutting Date collected. Sample No. Treatment. HCN. June 29 July 7. Aug. 12 Sept. I. \Vilted in shade Green , control sample Wilted in sun for three hours Dried in sun from morning till evening, outdoors over night Dried in shade for same length of time as 2 Dried outdoors two days and nights Dried in the shade two days and nights Dried in shade tliree da)'s and nights Dried in the shade five days Dried in the shade two days Mgm. 28 36 15 24 IS 7 20 32 6 While these results are not uniform, they do show conclusively that hydrocyanic acid can be obtained from wilted grass. Because of this result an attempt was made to determine more accurately the amount of hydrocyanic acid that may be obtained from wilted and dried grass. EFFECTS OF KEEPING GREEN GRASS MOIST AFTER IT IS CUT A large sample of grass collected June 29 was placed stems down in a large bottle so that about one-fourth was immersed in water. At the end of different periods of time 200-gm. portions of the leaves were mac- erated and digested in water overnight. The amount of hydrocyanic acid obtained is given in Table VIII. Table VIII. — Effect of keeping grass moitt after cutting Sample No. Length of treatment. HCN. 6 hours Mgm. 32 20 2 22 hours ' 3 4 30 hours 8 48 hours 2 The results indicate that hydrocyanic acid slowly disappears from the grass after it is cut, but also that the grass may be kept for a while in the green condition without much loss of the hydrocyanic acid. Control samples taken at this time gave 30 mgm. hydrocyanic acid. When the grass was wholly covered with water or when the air was excluded the results were different. In each case in the experiment re- ported in Table IX, unless otherwise stated, the grass was macerated and digested at the end of the treatment given. This shows that the presence or absence of air has an intimate relation to the evolution of hydrocyanic acid. Experiments were performed in which the grass was kept in an atmosphere of carbon dioxid and also of Oct. IS, 1921 Hydrocyanic Acid in Sudan Grass 131 hydrogen. No hydrocyanic acid was obtained from the grass kept in an atmosphere of hydrogen, whereas from that kept in carbon dioxid con- siderable amounts were obtained. Some macerated grass was also placed in a desiccator from which the air was exhausted continuously. This did not seem to affect the amount of hydrocyanic acid obtained, but the experiment was not satisfactory. The effect of keeping the grass in different atmospheres needs further study. Table IX. — Effect of different treatments after cutting on hydrocyanic acid content Date collected. July 8. July 10. Treatment. 'Placed uncut in bottles and covered with water two days Distillate from this water Placed uncut in sealed mason jar with small amount of water two da^'s Placed uncut in sealed mason jar with small amount of chlo- roform two days Placed uncut in sealed mason jar for 2 days, no water Placed uncut in bottle 2 days, covered with water Obtained by distilling water from this Macerated and digested 2 daj^s in water amino substance — ^ protein substance. According to this theory the cells which con- tinue to be active use the hydrocyanic acid for the building of protein substance, and as more nitrates from the soil are not supplied for man- ufacture of more hydrocyanic acid, the potential amount present when the plant is cut is soon exhausted. When the grass was dried slowly in the shade the hydrocyanic acid disappeared more slowly than when it was dried in the sun, and the amount obtained from the hot-water treatment became approximately equal to that obtained from the longer digestion in cold water. This seems to mean that when the plant wilts the hydrocyanic acid is split off from glucocids and held in such loose combination that it can be set free by hot water and that practically all the hydrocyanic acid is in such combination, since additional amounts can not be obtained by further digestion. Splitting off begins as soon as the plant is cut. Determinations made on grass kept moist in the shade appear to show that after 24 hours all the hydrocyanic acid not otherwise used by the cells is in such a condition that it is soluble in water. In the test in which the grass was frosted the hydrocyanic acid dis- appeared very rapidly, though the results were not very consistent. EFFECT OF ACIDS On June 24 a sample of grass was placed in a flask after maceration, covered with water, and sulphuric acid was added to acid reaction. After it was digested and distilled as usual only a trace of hydrocyanic acid was obtained. From a sample of like material and similarly treated, except that no acid was added, 27 mgm. were obtained. On June 29 this experiment was repeated with the result that 8 mgm. were obtained when acid was used and 26 mgm. when it was not used. On June 30, i and 28 mgm. were obtained by these respective treat- ments. These experiments clearly indicate that the presence of acid has a very importance influence on the amount of hydrocyanic acid that may be obtained. To test the effect of the amount of acid used, four samples were prepared on July 14 and digested overnight in the fol- lowing: (i) water; (2) N/o.i sulphuric acid (HjSOJ; (3) N/0.2 sul- phuric acid; (4) N/i sulphuric acid. No hydrocyanic acid was ob- tained from any of the treatments with sulphuric acid, whereas the water digestion gave 30 mgm. On August 4 this experiment was repeated, using a weaker acid solution. Digestion in water gave 10 1 Ravenna, C., and Zamorani, M. nuove ricerche sdlla funzione fisiologica dell' acido cian- iDRico NEL SORGHUM VULGARE. In Atti R. Accad. Lincei, Rend. Cl. Sci. Fis., Mat e Nat., v i8, sem. 2, no. 8, p. 283-287. 1909. Abstract in Chem. Abs. v. 5, no. 6, p. 1123. 1911. 134 Journal of Agrictdtural Research Vol. XXI. No. 3 mgm. of hydrocyanic acid; N/o.oi sulphuric acid, 1 1 mgm. ; and in N/ 0.02 sulphuric acid, 4 mgm. On August 2 three samples were prepared and digested in N/i vSulphuric acid; in N/0.2 sulphuric acid; and in N/0.05 sulphuric acid. Just before distillation, sodium hydroxid was added to almost neutral reaction. From the N/0.05 sulphuric acid 18 mgm. of hydrocyanic acid were obtained; a trace was obtained from the N/0.2, and none from normal. The weakest of the acid solutions gave no more than water alone. The smaller amounts obtained from the water treatments at the later date is in accord with the general observation that as the season advanced less hydrocyanic acid was present. It was planned to determine the exact hydrogen-ion concentration at which the hydrocyanic acid is most easily split off, but time did not permit. It is hoped that this may be determined in the future. It was shown in connection with the hot-water treatment that when grass dries the hydrocyanic acid is changed into a free condition, so that simply adding hot water and distilling will drive off the hydrocyanic acid. To see if more would be driven off if acid was also present the following experiment was performed. Six samples of leaves were placed in the open in clear weather from 9 a. m. till 9 a. m. the next day. Then they were macerated and digested in water and in different concentrations of sulphuric acid. The results are given in Table XII. Table XII. — Effect of acid solutions in formation of hydrocyanic acid HiS04 added. Nil... NI0.2. NI0.05 HCN ob- tained. Mgm. H2S04 added. NI0.O2 NJO.OI Water. HCN ob tained. Mgm. Trace. 10 18 Thus, it appears that sulphuric acid is unfavorable to the liberation of the hydrocyanic acid even in the wilted material. To determine whether hot sulphuric acid would liberate the hydro- cyanic acid, hot water and sulphuric acid of varying normalities were added to green material immediately after maceration on July 16. The results are shown in Table XIII. Table XIII. — Effect of hot sulphuric acid on formation of hydrocyanic acid H2SO4 added. Nil.. NI0.5 NI0.2 NIo.i HON ob- tained. Mgm. Trace. Trace . H2SO4 added. NI0.05 NI0.02 NIo.oi. .. . Hot water HCN ob- tained. Mgm. O Trace. Trace. Oct. 15, 1921 Hydrocyanic Acid in Sudan Grass 135 This shows that the use of hot acid is similar to that of hot water and that acid has no power to spht off the hydrocyanic acid, at least in the concentration used. The traces obtained in some cases were no doubt due to liberation of hydrocyanic acid during maceration. It would, appear, however, that hot water was less destructive than hot acid. I/ike experiments with hydrochloric acid were performed with similar results. ,,^.. ^.r>{' .r..>!!;.-:i r..,^ On July 2 1 eight samples were prepared and digested at room tempera- ture in phosporic acid (Table XIV) . Table XIV. — Effect 0/ phosphoric acid on liberation of hydrocyanic acid H3PO4 added. Nji.. NI0.5 NI0.2 Njo.i HCN ob- tained. Mgm. H3PO4 added. NI0.05 N 1 0.02 Njo.oi Water. HCN ob- tained. Mgin, The results indicate that the inhibiting power of phosphoric acid (H3PO4) was somewhat less than that of hydrochloric (HCl) or sulphuric acid. This would be expected since the degree of ionization of phosphoric acid is less than that of hydrochloric or sulphuric acid. Experiments with tartaric acid gave similar results. EFFECT OF DIGESTING IN ALKALINE SOLUTION On July 20, 16 samples were prepared and digested in sodium-hydroxid (NaOH) and sodium-carbonate (Na2C03) solutions, respectively (Table XV). Table XV. — Effect of alkaline solution on formation of hydrocyanic acid NaOH added. Nji. .. NI0.5. NjO.2. NIo.i. Njo.05 N 1 0.02 Njo.oi Water. HCN obtained. Mgm. 19 Nas CO3 added. Nil... NI0.5. NI0.2. NIo.i . NI0.05 NI0.02 NIo.oi Water . HCN obtained. Mgm. o o o o Trace. 17 17 20 The results show the same general effect as that secured with acid solutions. 136 Journal of Agricultural Research voi. xxii. No. 3 EFFECT OF ACID OR ALKALI ON HYDROCYANIC ACID AFTER IT IS LIBERATED An experiment was performed to show what effect acid or alkaline solutions have on the hydrocyanic acid after it is liberated. The green, macerated material was digested overnight in measured amounts of water. Enough standardized acid or alkali was then added to give the normality desired, and distilled. The results are given in Table XVI. Table XVI. — Effect of acid and alkali on hydrocyanic acid after it is liberated Solution added. H2SO4 Hcl . . . H3PO4 NaOH HON obtained after treatment with solutions of- Nlr. Mgm. 9 16 Nlo.g. Mgm. N/o.i. Njo.os. Mgm. 16 20 2 Mgvi. 14 18 18 O Water. Mg7n. 20 20 No hydrocyanic acid passed over in the first distillate from the sodium- hydroxid solution. The mixture was acidified with sulphuric acid and then distilled with the results given in Table XVI. While the results obtained in this experiment are not very uniform, they do show that hydrocyanic acid can be obtained from acid and alkaline solutions if the hydrocyanic acid is in a free condition before the acids are added. The experiment also appears to show that the addition of acid or alkali resulted in diminishing the amount of hydrocyanic acid obtained. The experiment was also tried by digesting the grass in sulphuric acid and sodium hydroxid of the normalities N/i, N/0.2, N/o.i, and N/o.o^ and then neutralizing before distilling. In no case was any hydrocyanic acid obtained. INFLUENCE OF WEATHER From a sample taken June 23, when there had been no rain for three weeks, 27 mgm. of hydrocyanic acid were obtained. On June 30, after a heavy rain and a week of good growing weather, during which there was plenty of moisture, 30 mgm. were obtained. On July 24, when there had been a period of dry weather, the amount obtained was 7 mgm. The next day, following a rain during the night, the amount was 16 mgm. Several experiments indicated that the largest quantity was obtained when the plant was in the most vigorous growing condition. This is contrary to a common belief that stunting has some effect in increasing hydrocyanic acid. On the contrary, the potential amount may be lessened. Determinations were made on samples collected at sundown and also before sunrise. The data obtained were not conclusive in deter- mining the effect of light on the potential amount of hydrocyanic acid present. Oct IS. 1021 Hydrocyanic Acid in Svdan Grass 137 HYDROCYANIC ACID IN SUDAN HAY Two samples were taken from the outside of a stack of Sudan hay and two from the inside. No hydrocyanic acid was found. AMOUNT OF HYDROCYANIC ACID IN OTHER SORGHUMS On July 23 a sample of kafir was taken and separated into leaves and stems. From the leaves were obtained 16 mgm., and from the stems 10 mgm. of hydrocyanic acid. The kafir stems were very little developed. Sudan grass, tested the same day, gave 8 mgm. of the acid from the same weight of material. On July 26, just after a heavy rain, following a period of dry weather, a sample of kafir gave 72 mgm. and a sample of sorgo (cane) 42 mgm. of hydrocyanic acid. Sudan grass 6 inches high, tested on that date, gave 32 mgm. of hydrocyanic acid. On August 7 a sample of second-growth sorgo (cane) was received from LaHarpe, Kans. About one-fifth was quite dry, two-fifths were wilted and yellow, and two-fifths were green. The sample was some- what moldy. One portion digested in the usual manner gave 13 mgm. hydrocyanic acid. Another portion distilled at once from hot water gave 24 mgm., showing that the hydrocyanic acid was in free condition. Another sample of sorgo was sent in from Seneca, Kans. This was reported to have killed six cows. From the portion distilled from hot water 20 mgm. were obtained and from the portion digested in the usual way 36 mgm. On September 2 a quantity of Red Amber kafir was collected, and six portions were prepared, and treated with the results given in Table XVII. Table XVII. — Hydrocyanic acid in Red Atnber kafir Sample No. Treatment. Left in flask 15 minutes after maceration, after which hot water was added and distilled Digested overnight in water ^^,.^.,^,^. Digested overnight in N/o.^ H2SO4. ...-.'.?..'.. Digested overnight in Njo.i HjSO^ Digested overnight in N/o.oi H2SO4 Digested overnight in N/o.oi NaOH Mgm. 32 119 None. None. 40 36 Part of this experiment was repeated by putting macerated sorgo into boiling water at once. This gave 8 mgm. hydrocyanic acid, while that digested overnight gave 96 mgm. Another portion was divided into five portions. After maceration they were all digested in water overnight. Then to these portions standardized sulphtuic acid was added so as to make the normahties indicated. The results are given in Table XVIII. These determinations show without a doubt that sorgo and kafir con- tain much larger amounts of hydrocyanic acid than does Sudan grass, and also that the conditions for obtaining it are very similar. 138 Journal of Agricultural Research voi. xxii. No. 3 Table XVIII. — Effect of different treatments on liberation of hydrocyanic acid in sorgo Sample No. Treatment. HCN. Digested in water and distilled Digested in water and distilled Digested in water and distilled from Njo.^ H2SO4. . . Digested in water and distilled from NIo.i H2SO4. . . Digested in water and distilled from NI0.02 H2SO4. . Mgni. 72 80 64 72 89 EFFECT OF HYDROCYANIC ACID FROM GREEN SORGO ON A HORSE Ten-pound portions of green sorgo, testing the amount of hydrocyanic acid given in Table XVIII, were fed to a horse. No effect on respiration, pulse, or temperature could be observed by Dr. H. F. Lienhardt, of the Veterinary Division, who made the observations. Data presented in this paper show that such a degree of acidity as is found in the stomach of a horse would prevent liberation of hydrocyanic acid from the green material. Feeding wilted sorgo was not tried. SUMMARY (i) In this paper are presented data givmg the results of tests made on Sudan grass for hydrocyanic acid during the summer and early fall of 1920. (2) The maximum amount of hydrocyanic acid was obtained by mac- erating the material and digesting in water at room temperature for about six hours or overnight. (3) Practically all the hydrocyanic acid was found in the leaves. In well-developed stems none was found. (4) More hydrocyanic acid was found in younger plants than in those more mature. This is due mostly to stem development. If leaves only are used the dififerences are small, except when the plants approach maturity. More was found in the summer than in the fall. (5) Hydrocyanic acid does not exist as free HCN in the growing plant. It begins to be liberated as soon as the plant is macerated or undergoes wilting. (6) Liberation of hydrocyanic acid is intimately associated with enzym action. If this enzym action is inhibited by addition of hot water or acids, no hydrocyanic acid will be liberated. Hydrocyanic acid was obtained from wilted grass when hot water was added, because during the wilting process hydrocyanic acid was set free. (7) Hydrocyanic acid can not be set free from the green material by acids. (8) The action of strong alkali is similar to that of acids. (9) Most hydrocyanic acid is present when the plant is in a vigorous growing condition. (10) Sudan grass contains less hydrocyanic acid than sorgo or kafir. NUTRIENT REQUIREMENTS OF GROWING CHICKS: NUTRITIVE DEFICIENCIES OF CORN^ By F. E. MUSSEHL, Professor of Poultry Husbandry, J. W. Calvin, Associate Chemist, Nebraska Agricultural Experiment Station, with the cooperation of D. L. Halber- SLEBEN and R. M. Sandstedt Investigators in the field of nutrition have noted that chickens behave unlike rats and swine when limited to rations of com or wheat grains and their products. This fact has made necessary the planning and execu- tion of experimental work having for its object a determination of the values and deficiencies of our common feeding stuffs when used for poultry and egg production. The results of a series of experiments carried on at this Station with this objective are reported in this paper. From the experience of investigators ^ who have worked with other species, mainly rats and swine, it has seemed that systematic inquiry should be made into the (a) ash re- quirements, (b) protein requirements (quality and quantity), and (c) food accessory requirements. Earlier in- vestigational work with chicks by Osborne and Mendel ^ and Hart, Halpin, and Steenbock* indicates that another element, (d) the physical factor, is also of fundamental impor- tance and must be considered in any complete study of the nutritive values of a particular grain or ration. In our work lo-day-old vS ingle-Comb White Leghorn chicks were used, spe- cial care being taken to select for vigor, vitality, and uniformity in each lot. Nine chicks per lot were used for the first series of experiments. Chicks were weighed individually every seven days, and the growth curves selected are typical of each lot (fig. i-ii). They show the weight of the chicks at the beginning of the experiment and the change in weight thereafter. Records of the feed 1 Published with the approval of the Director of the Nebraska Agricultural Experiment Station. 'McCoLLUM, E. v., SiMMONDS, N. , and PiTz, W. the relation of the UNroENTiFiBD dietary fac- tors, THE fat-soluble a, AND WATER-SOLUBLE 6, OF THE DIET TO THE GROWTH-PROMOTING PROPERTIES OF MILK. In Jour. Biol. Chem., v. 27, no. i, p. 33-43, 6 charts (1-3, 6 in text). 1916. '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. ^Hart, E. B., Halpin, J. G., and Steenbock, H. use of synthetic diets in the growth of baby CHICKS. A study of LEG WEAKNESS IN CHICKENS. In Jour. Biol. Chem., v. 43, no. 2, p. 421-442, 2 pi. 1920. o 100 —- » — 1125 — * X /oo ■:HiaK 1 //oa I X ,3S ^2 Fig. I.— Graph showing unsatisfactory results from feeding ration of 100 parts yellow com and calcium carbonate grit ad libitum to chicks of lot II. The time at which chicks died is indicated by X. Journal of Agricultural Research, Washington, D. C. zy (139) Vol. XXII. No. 3 Oct. 15, 192 1 Key No. Nebr.-4 140 Journal of Agricultural Research voi. xxii. No. 3 (300 y y y / < y / f/ V 300 A H/ y Jj 700 \ \ 600 SOO a 4 y //^i '} / d y 300 J- X / X'' ^ -^ 0 ^ -^ Fio. a.— Graph showing slow but continuous growth of chick in lot 211, fed ration of 95 parts yellow com and 5 parts ash mixture Nutrient Requirements of Growing Chicks 141 /.soo A 200 /,/oo /.ooo 900 / / z' /^ / ^ / y / /f'^ / , ( i'^ / ii/i ^ / f i^ 3> .( )f^(i J^ ^ ^ i^ _,--- ^ ^' ^ 5*^ .-^ :^ r.K 2jj ^ ^ 0\ '■^rf lA ^ ^ ^J ^ ,a£^ u> 0 — I*""*' O 7/^2/ Ze 3S -^2 4& se 63 70 77 S4 S/ S8 /OS //2 //^ /Z6 Fig. 3. — Graph showing growth of chicks in lot 213, fed ration of 80 parts yellow com, 15 parts casein, and 5 parts ash mixture. 142 Journal of Agricultural Research voi. xxu,no.3 /,ooo 9CX) soo 700 600 t 500 10 ^oo 300 200 /OO / y / / / y / if?' ly / ki rjt^ 0^ A 1 j\ c^ I^ 403 J^ ■y ^ i^ /OO o CH/CK 2736 O 7 /4 2/ 2e 3S ^^2 ^& S6 63 70 77 S'^ S/ S3 Fig. 4. — Graph showing detrimental results of adding 5 parts butter fat to ration of 65 parts yellow com, 15 parts casein, 5 parts ash mixture, and 10 parts starch for chicks in lot 215. Oct. IS, 1921 Nutrient Requirements of Growing Chicks 143 /ooo soo TOO ^00 I ^00 ZOO too o /^ /■ / / y / .i(^^ f^ / .A t' ^ (?/?! ?i^ y 1/ )(^t ^ ^ *^^ J^ 'y O 7 /^ 2f 23 3,^ *Pe '^9 c5<5 G3 70 77 S^ S/ &3 Fig. 5.— Graph showing that the addition of 20 parts com gluten did not improve ration of 65 parts yellow com, 5 parts ash mixture, and 10 parts starch for chicks of lot 208. 144 Journal of Agricultural Reserach voi. xxn. no. 3 I 900 aoo 700 600 \soo \fOO 300 200 /oo o y y / / , ■y / w /^ '/ A ^ aI"^ ^ v> ^iQ ^ ^ ^^ ^ P •^ .*-' 300 ^ zoo O 7 /"f 2/ £3 3S ^2 -^9 S6 63 70 77 3^ 9/ 93 Fig. 6.— Graph showing that the addition of 5 parts butter fat (fat-soluble A) did not improve ration of s parts yellow corn, 20 parts corn gluten, 5 parts ash mixture, and 5 parts starch for chicks of lot 209. X- j/r K P7f j^.. — -*- 6/ rff< 27. '^z' Oct. IS, I93I Nutrient Requirements of Growing Chicks 145 'POO 200 /oo A I o %zoo _ -X "^ TWCK 27^ consumption of each lot were also obtained. Clean wood shavings were used for litter, and each lot was confined to a pen 2 by 8 feet in size until the chicks were 8 weeks old, when the near normal lots were given a yard 4 by 8 feet in size. Methods of feeding are known to have considerable influence on the efficiency of a ration; so a standard policy was established of dividing the ration into two parts — the scratch or coarse feed and the mash or fine feed. The rations were mixed so that equal quantities of mash and scratch feeds were provided. The mash feed con- tained all the supplemental ingre- dients, such as ash, butter fat, and purified casein. The casein used in the rations was purified by extracting repeatedly with distilled water slightly acid- ified with acetic acid . After extrac- tion the casein was drained and dried in an air oven at 70° to 100° C. and then ground. The butter fat was purified by melting at 40° in a water bath and was then centrifuged to remove ash, casein, and other material. The ash mixture^ used in our rations was composed of the following ingredients, parts by weight: Bone ash .jV^/:'^: 't';^;! . V'^'v'. .'. 50 Calcium carbonate Sodium chlorid Dipotassium phosphate Calcium lactate Magnesium sulphate Sulphur Iron sulphate ^i9 Fig. 7. — Graph showing that the addition of is parts soybean meal did not improve ration of 80 parts yellow corn and 5 parts ash mixture for chicks of lot 210. The time at which chicks died is indicated by A'. JXi.i . U. "■^^rA. 14 IS 10 5 3 2 I The results of our inquiries may briefly be summarized as follows: (i) Yellow com (maize) is deficient in several of the essential qualities necessary for the complete nutrition of growing chicks. A deficiency in the ash content of the yellow com kernel is no doubt responsible for the early failure of baby chicks when restricted to a ration of corn alone. Supplementing the corn kernel with 5 per cent of a complete ash mix- ture improved the ration so as to enable very slow but persistent growth. 1 Philips, A. G., Carr, R. H., and Kennard, D. C. meat scraps versus soy-bean proteins as a StJPPLEMENT TO CORN FOR GROWING CHICKS. In Jour Agf. Research, v. i8, no. 7, p. 391-398, i fig. , pi. 50. 1920 146 Journal of Agricultural Research Vol. XXII, No. 3 o JOO % ° S 100 o JOO •^ frH CHICK Vr\3 — X C»/Ch Z770 "■"^x CHICK zeas — X £>i9y.s Fig. 8.— Graph show- ing bad results from lack of roughage in ration of 65 parts yellow com, 15 parts gelatin, 5 parts but- ter fat, 5 parts ash mixture, and 10 parts starch for chicks of lot 217. The time at which chick died is indi- cated by X. (2) Yellow corn is deficient in quality and quantity of protein required for normal growth of chicks. The addition of more com protein by including corn gluten in the ration did not markedly improve the efficiency of the ration. Compare growth curves, lots 208 and 211. (3) The addition of 15 per cent purified casein to a basal ration of yellow corn and ash did improve the ration decidedly. Compare growth curves, lots 211 and 213. The amino acid deficiencies of the corn proteins are no doubt supplemented by the amino acid contribu- tions of the casein. (4) Supplementing the basal yellow com ration with certain other proteins, ^^g albumen, and gelatin, lowered rather than raised the efficiency of the ration. The poor results with rations 217 and 218 were probably due to a distinctly sticky physical quality which prevented normal nutrition. (5) The fat-soluble food accessory does not appear to be a limiting factor in a yellow com diet for baby chicks. The addition of butter fat to a yellow corn, casein, and ash ration (lot 215) did not improve but rather lowered the efficiency of the ration. A slight change in the physical condition of the ration may explain this lowered efficiency, though it is more probable that the butter fat addition temporarily stimulated growth so that the supply of some other essential accessory was exhausted earlier than would have been the case had the butter fat been omitted. It is apparent at least that the failure of chicks on ration 215 was not due to fat-soluble A starvation. (6) Green feeds make certain very valuable contributions to a ration for growdng chicks. The addition of wheat greens to a yellow com, casein, and ash ration effected a decided im- provement in the efficiency of the ration. An excess of the wheat greens was offered, and subsequent observations indicate that about 5 per cent (dry matter basis) of this kind of green food are consumed when offered regularly in excess. The helpful influ- ence of the wheat greens may have been due to, first, an improvement of the physical condition of the ration; second, a food accessory con- zoo /oo o 200 /oo I o ^200 /oo o /oo _„--' 5^' ^0 ^A//'^ ^x Thi^ $73? >v Thi^ 7^ V ^ Z73\ ^X o /«? 2/ za Fig. 9.— Graph showing that be- cause proper physical quality was lacking the addition of 15 parts egg albumen did not im- prove ration of 65 parts yellow com, 5 parts butter fat, s parts ash mixture, and 10 parts starch for chicks of lot 218. The time at which chicks died is indi- cated by X. Nutrient Requirements of Growing Chicks H7 /.■300 /.OOO 900 eoo y ndi if / A , 6 ^ ^LQ^ i^ y y" i> ^ ^ ^ soo 400 200 /OO eoo 600 .soo I Vi 300 400 ^ ^ ^- <:'.. H ^ * ♦;-^ / 'f.i fy y ^ff f' ^ -3 . o ^$'i. W'^ ^^ ii ^ €^ c> ^ ^ '0. l'^ i* d 0' c zoo /OO ~ O 7 /4 2/ 23 33 42 49 S6 63 TO 77 34 S/ S3 /OS //2 //S /ZS Fig. io. — Graph showing that the addition of excess of wheat greens improved ration of 8o parts yellow com, 15 parts casein, and s parts ash mixture for chicks of lot 227. 65508°— 21 3 148 Journal of Agricultural Research voi. xxu,no.3 /./oo /,ooo 900 800 700 (ft 600 \ 400 300 200 /CO / 4 / 4 7 4\ / ( J / jH t^ y t^ f ^ y y ^ ^' ^ 0 7 M 2/ Z8 3S f2 -^9 S6 63 70 77 S'^ 3/ 38 /OS //2 Fig. II.— Graph showing normal growth produced by ration of 65 parts wheat, 15 parts casein, s parts butter fat, 5 parts ash mixture, 10 parts starch, and excess of wheat greens for chicks of lot 205. Birds were kept on this ration until they produced eggs, at age of about 200 days. Octis, I92I Nutrient Requirements of Growing Chicks 149 tribution; third, an increased food consumption, due to a stimulatory effect on the appetite. A series of experiments to shed further hght on the specific contributions which green feeds make to the ration is in progress at this time. (7) It is possible to raise to normal maturity chicks confined to a small pen. Drummond ^ reports great difficulty in rearing chicks in confine- ment, and other investigators have noted some of the problems, especially leg weakness.' Our lot 205 grew to normal maturity, some of the pullets producing eggs when about 200 days old, though never having more range than was provided in a yard 4 by 8 feet in size. Ration 205, though not synthetic, is of interest because of its comparative simplicity. 1 Drummond, Jack Cecil, observations upon the growth of young chickens under laboratory CONDITIONS. In Biochem. Jour., v. lo, no. i, p. 77-88, i pi. 1916. 2 Hart, E- B., Hai,pin, J. G., and Steenbock, H. op. ai. AECIAL STAGE OF THE ORANGE LEAFRUST OF WHEAT, PUCCINIA TRITICINA ERIKS.^ By H. S. Jackson, Chief in Botany, and E. B. Mains, Associate Botanist, Purdue University Agricultural Experiment Station, and Agents, Office of Cereal Investiga' tions. Bureau of Plant Industry, United States Department of Agriculture ^ This paper presents, in part, the results of a study of the leafrusts of wheat, rye, barley, com, and related grasses which was begun in 191 8. One of the important phases of this investigation is the determination of the aecial relationships of the various races or species included in the collective species, Puccinia Clematidis (DC.) Lagerh. (P. Agropyri Ellis and Ev.), and other closely related forms. While a number of the rusts of this group which occur on wild grasses have been connected with aecia, their host limitations and interrelations are not well understood. This study is especially important in the case of the leafrust of wheat, P. triticina Eriks. So long as the aecial stage of this species was un- known, little progress could be made in developing our knowledge with reference to its origin, development, spread, and relation to other rusts. The results of the investigation of the aecial relationship of this rust are presented in the following pages. HISTORICAL REVIEW Three rusts are known to attack wheat : the black or stemrust, Puccinia graminis Pers. ; the stripe or yellow rust, P. glumarum (Schmidt) Eriks. and Henn. ; and the orange or leafrust, P. triticina. Of these the stem- rust is the only one for which the aecial stage has been determined. This rust was shown by De Bary to have its aecial stage on Berberis vulgaris L., and this relationship has since been demonstrated repeatedly by a number of workers in various parts of the world. The discovery of the place of Aecidium Berberidis Pers. in the life cycle of P. graminis caused De Bary (4, p. 207-211)^ to turn his attention to the study of other grass rusts having incomplete life cycles. This resulted in the discovery that P. rubigo-vera (DC.) Wint. (P. straminis Fckl.) on rye was connected with aecia on Anchusa officinalis and Anchusa arvensis. Sowings made with teliospores from rye resulted in the production of ' Published with the approval of the Director as a contribution from the Department of Botany, Purdue University Agricultural Experiment Station. Cooperative investigation between the Purdue University Agricultural Experiment Station and the Office of Cereal Investigations, Bureau of Plant Industry, United States Department of Agriculture. ' The writers wish to acknowledge their indebtedness to various pathologists throughout the country for aid in obtaining material for the cultural studies upon which this paper is based, and to Mr. Forest Fuller, temporary culture assistant during the spring of 1919, and Mr. Emile Mardfin and Miss Florence M. Smith, Scientific Assistants, Ofifice of Cereal Investigations, Bureau of Plant Industry, for assistance in carrying out the cultural investigations. ' Reference is made by number (italic) to "Literature cited," p. 170-171. Journal of Agricultural Research, Vol. XXII, No. 3 Washington, D. C Oct. 15, 1921 zz Key No. G-347 1^2 Journal of Agricultural Research v^oi. xxii. No. 3 aecia on Anchusa, and when sowings were made with aeciospores from Anchusa, uredinia on rye were developed. Sowings of basidiospores from rye upon Berheris vulgaris Hoi., Rhamnus Frangida, Rhamus cathartica, Ranunculus acris, Ranunculus bulbosus, Taraxacum officinalis, and Urtica dioica were without result. Nielsen {20, p. 37) 10 years later reported obtaining infection with aeciospores from Anchusa officinalis on both rye and wheat. Plowright {21, p. 168) states that in the fall of 1885 he obtained aecia upon Anchusa arvensis by placing wheat straw rusted with P. rubigo-vcra near that host. At the time this work was carried out the name Puccinia rubigo-vera was used for the leafrusts of wheat, rye, and barley, as well as for similar grass rusts having globoid urediniospores and long covered telia. Eriksson and Henning (11, p. 197-203, 2^7-2^9) separated this species into two — Puccinia glumarum, the stripe rust, and Puccinia dispersa Eriks., the brown rust. Under the latter they included the rust of wheat as well as that of rye. The rust of wheat, however, was considered as a forma specialis, Tritici, of Puccinia dispersa. As the leafrust of rye had been shown by De Bary (4) to be connected with aecia on Anchusa, Eriksson (jo, p. 254-2^7) sought for the same connection for the leafrust of wheat. His sowings of basidiospores from wheat upon Anchusa officinalis and A. arvensis, however, produced no infection, as was also the case when aeciospores from Anchusa were sown on wheat. No results were obtained when basidiospores were sown on Nonnea rosea, Myosotis arvensis, M. alpestris, Symphytum asperrimum and Pulmonaria officinalis, species of Boraginaceae related to Anchusa upon which unconnected aecia were known to occur. As a result of these cultures, Eriksson {10, p. 270) concluded that the orange leafrust of wheat was a distinct species and gave it the name, Puccinia trificina. Klebahn (17, p. 85-86; 18, p. 246) made rather extensive cultures in an endeavor to discover the aecial host of Puccinia triticina. Besides sowing aeciospores of Aecidium Anchusae Eriks. and Henn. on wheat he made sowings of basidiospores on Anchusa arvensis and Anchusa officinalis without result. Sowings of basidiospores also were made without success upon Triticum vulgare, Ranunculus acer, Ranunaihis asiaticiis, Ranun- culus auricomus. Ranunculus bulbosus, Ranunculus Ficaria, Ranunculus flammula, Rammculus lanuginosus , Ranunculus repens, Anemone ranun- culoidcs, Aconitum Lycoctonum, Aconitum Napellus, Berberis vulgaris, Nasturtium sp., Barbaraea vulgaris, Melandryum albutn, Coronaria fl.os- cuculi, Agrostetnma Githago, Rhamnus cathartica, Lythrum Salicaria, Ribes Grossularia, Aegopodium Podagraria, Pastinaca sativa, Valeriana dioica, Knautia arvensis, Tussilago Farfara, Taraxacum, officinale, Cen- taurea Cyanus, Achillea Ptarmica, Campamila rotundifolia, Ligustrum vulgare, Phillyrea sp., Echium vulgare, Lithospermum purpureo-coeruleum, Myosotis sp., Symphytum officinale, Glechoma hederacea, Prunella vulgaris, Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat 153 Rumex acetosa, and Urtica dioica. These results substantiate those obtained by Eriksson and indicate that the positive results reported by Nielsen {20) and Plowright {21) were probably due to a mixture of rusts or of hosts. The failure to obtain infection on Boraginaceous hosts has influenced other workers to turn their attention to other families in a search for the aecial hosts. Arthur (j, v. 9, p. 304), largely as a result of morphological studies, reached the conclusion that Puccinia triticina was best con- sidered a race of P. Agropyri, and upon this basis Arthur and Fromme (j. P' 333~337) have placed it in the collective species Dicaeoma Clerna- tidis (DC.) Arth. Several races of this collective species had been shown by workers in Europe and America to go to species of Clematis. Arthur thought that the aecial host might be either Clematis ftamm.ula or C. vitalha as these were the only common species of Clematis found in the wheat-growing regions of southern Europe, northern Africa, and western Asia, a region which at that time was considered as the probable home of the original wild wheat. His culture with wintered telia of the leaf- rust of wheat on C. flammula, however, was unsuccessful. According to Butler (d, p. 75) Cunningham and Prain (9) considered that there was considerable ground for believing that an Aecidium on Launaea asplenijolia, one of the Cichoriaceae, was the aecial stage of Puccinia triticina, as it was found throughout the greater part of the wheat-growing area of India. Butler, however, sowed aeciospores from this host upon wheat without obtaining infection. These unsuccessful attempts to demonstrate an aecial stage for Puc- cinia triticina have resulted in the development of the idea that the aecial stage of this rust has been lost and that it is able to maintain itself without one. In this connection a number of important facts have been established and a number of interesting hypotheses proposed. It has been shown by BoUey (5, p. 13-14), Hitchcock and Carleton (15, p. 1—2), Carleton {8, p. 21-22), and others that in certain regions, P. tri- ticina is able to overwinter by means of its uredinal mycelium and that no aecial host is necessary for the maintenance of this species. This does not appear, however, to be true for all regions where P. triticina is abundant {6, p. 11). A number of suggestions have been made to ex- plain the 5'early appearance of the rust in regions where the uredini- ospores or uredinial mycelium does not overwinter. It was considered possible that spores may be carried from other regions by the wind. The mycoplasm theory of seed transmissal has also been put forward as a possible explanation. Whatever may be the merits of these hypothe- ses, they have resulted in recent years in directing attention away from a search for the aecial host of this species. 154 Journal of Agricultural Research voi. xxii, no. 3 BASIS OF CULTURAL INVESTIGATIONS A study of Puccinia trUicina in comparison with other grass rusts with long covered teha shows that it can not be readily separated morpholog- ically from the leafrust of rye. The separation of this form as a species was made by Eriksson {10) because he obtained only slight infection on rye with urediniospores and was not able to obtain infection on Anchusa with basidiospores and because the teliospores germinated in the spring, while those of the rye rust germinated in the fall. The close morphological similarity, however, furnished considerable grounds for the assumption that the aecial host of leafrust of wheat was likely to be some species of Boraginaceae other than Anchusa, especially as another rust of this type, Puccinia hromina Eriks., has since been found to have its aecia on the Boraginaceous hosts Symphytum officinale and Pulmonaria montana, with very weak development of aecia on Anchusa {ig, p. 182-202). Unfin- ished investigations now being conducted in this laboratory strongly indicate that in America certain grass rusts having aecia on Boraginaceous hosts are very similar to the leafrust of wheat and rye. For these reasons it was considered desirable to test as many Boraginaceous hosts as were available, as possible aecial hosts for the leafrust of wheat. There is, however, still another group of grass rusts very similar to the orange leafrust of wheat to which Arthur (j, v. 9, p. 304) has called atten- tion. This group has aecia upon various Ranunculaceous hosts and in- cludes forms which have been separated from time to time, according to their aecial connection, together with slight morphological variation, into a number of species, including Puccinia persistens Plowr., P. perplexans Plowr., P. Agropyri, and P. alternans Arth, The writers felt from the beginning that the greatest possibility of success in the search for the aecial stage was to study thoroughly the genera of this family on which aecia were known to occur. The idea that Puccinia tritichia has lost its ability to develop an aecial . stage through long propagation by urediniospores, while admittedly pos- sible, was not considered to be fully substantiated. CULTURES MADE IN 1919 With these considerations in mind rather extensive sowings were made in the spring of 19 19 upon a considerable number of species of the fami- lies Ranunculaceae and Boraginaceae and the closely related family Hydrophyllaceae. For this purpose, 20 collections of telia of Puccinia tritici'na were obtained from various sections of the country during the summer and fall of 1918 and placed outdoors to winter. Early in March these began to germinate. Ten of the 20 collections gave good germina- tion and were sown upon various species of the above-named families and upon Ornithogalum umbellatum L., Impaiiens sp., and Catnassia esculenta (Ker.) Robins. (Quamasia hyacinthina). The results obtained are given in Table I. Oct. IS. 1921 Aecial Stage of the Orange Leaf rust of Wheat 155 Table l.—Data obtained in igig from sowing teliospores of Puccinia triticina, from 10 different localities, on various host plants, mostly of the families Ranunculaceae and Boraginaceae o- Host inoculated. No. ii8 (Okla.). No. 218 (Ala.). No. 418 (Tenn.). No. 618 (Ga.). No. 718 (Ga.). No. 818 (S.C). No. 918 (Ind.). No. 3518 (Wis.). No. 3818 (Wis.). No. 4Si8 (Wis.). - - _ - - - - - _ ■~ - - Anemone japonica Sieb. & - - - - _ Aquilegia canadensis L - ~ ■■■-'■' - - _ - _ Aquilegia glandulosa Fisch. . - = 1 Aquilegia vulgaris 1, - - ■:':;::;::::: _ Cimicifuga racemosa (L.) Nutt - - - _ - - - - - _ Clematis virginiana 1, ~ ■■'-"■ "" - - Delphinium "Belladonna." (Hort.).. - - " "— - "" - - - - _ Mertensia virginica (L-) Link - - — — _ Ornithogalum umbellatum. L - - - - — Camassia esculenta (Ker.) - - - _ - b "— b "— Ranunculus repens L - - - - _ Miihl - - — — i " — No infection. " Pycnia produced. ^ Two sowings were made, the other. Pycnia were produced from only one sowing, no result being obtained from The telial collections used in the cultures were all obtained from Triti- cum aestivum (T. vulgare) in the following localities : 118, from Stillwater, Okla., collected by J. D. Moore. 218, from Flint, Ala., collected by McClellan. 418, from Tennessee, collected by W. T. Evans. 618, from Carrolton, Ga., collected by R. O. Bums. 718, from Carrolton, Ga., collected by R. O. Burns. 1:^6 Journal of Agricultural Research , ,l Voi. xxii, No. 3 818, from Anderson, S. C, collected by R. O. Burns. 918, from La Fayette, Ind., collected by E. H. Toole. 3518, from Menah, Wis., collected by E. H. Toole. 3818, from Wisconsin, collected by E. H. Toole. 4518, from Superior, Wisconsin, collected by E. H. Toole. Negative results were obtained on all but two species of the hosts used. The collection from La Fayette, Ind. (No. 918), gave infection upon Thalictrum angustifolium and T. aquilegijolium, producing, however, only pycnia. It was impossible to carry this study further in 1919, as the above results were not obtained until late in the spring. The failure of aecia to develop from the two successful infections could be explained on either of two hypotheses. The conditions in the green- house may have been unfavorable, or the species of Thalictrum used may have been resistant. In either case, however, these results were inter- preted as indicating that the aecial host of the leafrust of wheat was some species of ThaHctrum. There was considerable basis for this assumption. All of the culture studies being carried on in this laboratory with the related rusts, occurring on wild grasses, and having aecia on members of the family Ranunculaceae, have indicated that while a given race may develop aecia on several species in one host genus with varying degrees of virulence it will not go to species of more than one genus. The rusts of this group show a very high degree of specialization. The two species of Thalictrum on which infection was obtained were foreign species, while the North American species, Thalictrum dioicum and T. polygarnum, were not infected. On this account it was thought that the susceptible aecial hosts for the leafrust of wheat probably were foreign species of Thalictrum. As the leafrust of wheat presumably is an introduced form, as explained in the following pages, this would be expected, and on that basis the species of Thalictrum should be western Asiatic or eastern European, corresponding to the region in which wheat is believed to have originated. CULTURES MADE IN 1920 In preparation for cultural studies for the spring of 1920 an effort was made during the summer and fall of 19 19 to obtain as many species of Thalictrum as possible. It was impossible to obtain material from for- eign botanical gardens in time to be of use, and the best that could be done was to secure such species of Thalictrum as were carried by nursery- men in this country, together with such native species as could be obtained through collectors in various parts of the United States. As a result 14 species were brought together. An appeal was also made to the plant pathologists in the various agricultural experiment stations tliroughout the country for aid in securing telial material of the leafrust of wheat. A very gratifying response to this appeal was made, and in this way 80 collections of telia were obtained and placed out to overwinter. Of these, 51 collections germinated in the spring of 1920 and were sown. The number of collections was so great that it was not possible to sow Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat 157 them on as large a number of species as was done in 19 19, and attention was devoted mainly to sowing upon species of Thalictrum. Of the 51 collections used 9 were sown upon as many Thalictrum species as possi- ble in order to determine the relative susceptibility of these species. The results are given in Table II. Tabl:^ II. — Data obtained in ig20 from sowing teliospores of Piiccinia triticina from nine different localities on -various species of ThalictrumO' Host inoculated. Labor- atory No. No. 5619 (Ga.). No. 6019 (N. C ). No. 7219 (N.C). No. 7819 (Tenn.). No. 8019 (Mich.). No. 8719 (Miss.). No. 11619 (Pa.). No. 12519 (Idaho). No. 15119 (Nev.) Thalictrum angustifo- 6 S 66 65 56 16 53 17 6s "5 114 19 98 55 61 0 I I 61 0 0 0 0 -(2) 0 I - 0 — 0 -(2) 61 0 0 0 0 I bi I I Thalictrum aquilegifo- i>i Thalictrum aquilegifo- - Thalictrum dasycarpum Fisch. and LaU Thalictrum De lavayi 01 I I I -(3) Thalictrum diocium X, Thalictrum flavum I/. . . . Thalictrum minus 1, I 0 - (2) 0 I I Thalictrum, minus adi- 0 Thalictrum occidentale Gray Thalictrum polycarpum S. Wats bi 0 I Thalictrum polygamum Muhl 0 - (2) I 01 I 0 I 0 -(2) I — a — Noinfection, ■ . v, u'.vr^'! •,>'.t>.-.i, T 0 Pycnia only produced. 1 Aecia following pycnia. A numeral in parenthesis following the sign indicates the number of times the results were obtained. ^ Although aecia were produced, the infection was weak. The following is a list of the sources of the telial material used in the cultures : 5619, from Athens, Ga., collected by C. A. Ludwig. 6019, from W. Raleigh, N. C, collected by Ludwig and Wolf. 7219, from Hickory, N. C, collected by C. A. Ludwig. 7819, from Tennessee, collected by C. A. Ludwig. 8019, from Coldwater, Mich., collected by B. W. Mains. 8719, from Canton, Miss., collected by C. A. Ludwig. 11619, from State College, Pa., collected by J. T. Adams. 12519, from Moscow, Idaho, collected by C. W. Hungerford. 15 1 19, from Reno, Nev., collected by G. R. Hoemer. In addition to the sowings indicated in Table II, culture 8719 was sown upon Aquilegia glandulosa F'isch., A. olympica Boiss., Clematis heraclaefoHa DC, C. paniculaia Thunb., C. recta L., and Ranunculus acris L-, all without infection. An examination of Table II shows that 12 out of the 14 species of Thalictrum were infected, Thalictrum occidentale and T. aquilegifolium apparantly being immune. The species on which infection occurred showed varying degrees of susceptibility. Thalictrum dasycarpum and 158 Journal of Agricultural Research voi. xxii. no. 3 T. polygamum gave mostly negative results or the occasional production of pycnia. T. angustifolium, T. aquilegijolium (5), T. minus, T. minus adiantifolium, and T. polycarpum showed occasionally a weak develop- ment of aecia, but usually only pycnia developed or no infection occurred. T. dioicum, in one case, showed a moderate development of aecia; in all other cases only pycnia developed, or no infection resulted. T. Delavayi and T. sp. (98) (PI. 21, A, B) showed fairly vigorous infection, accompanied in most cases by more or less hypertrophy and usually by well-developed aecia. T. flavum (PI. 21, C) and T. sp. (55) (Pi. 21, D) showed a very vigorous infection accompanied usually by pronounced hypertrophy of the infected leaf and petiole tissue and practically always with the production of well-developed aecia. An attempt has been made to check the determination of the species of Thalictrum used in these studies, but this has been difficult because a number of them have produced neither flowers nor fruit, and the leaf characters in this genus are in most cases extremely variable. Specimens of most of the species have been sent to Mr. S. F. Blake, of the Bureau of Plant Industry, Washington, D. C, who has kindly compared them with specimens in the United States National Herbarium and has given his opinion as to the identity of our material. The following list gives the species used above, their sources, and native distribution as accurately as they could be determined. The accession number of this laboratory follows the name of each species. , Thalictrum angustifolium L. (6) . Source : Seed from Brooklyn Botanic Garden. Distribution: Central Europe and Asia Minor. Thalictrum aquilegifolium L. (5 and 66) . Source : Bobbink and Atkins Nursery Co. Distribution: Europe, Middle and Northern Asia. (No. 66 was purchased for T. paniculatum.) Thalictrum dasycarpum Fisch. and Lall. (65). Source: Department of Botany, Michigan Agricultural College. Distribution: Northern and central United States and southern Canada. Thalictrum Delavayi Franchet (56). Source: Farr Nursery Co. Dis- tribution: Western China. Thalictrum dioicum L. (16). Source: LaFayette, Ind. Distribution: Eastern United States. Tlialictrum flavum L. (53). Source: Farr Nursery Co. Distribution: Europe, Western Asia, and Asia Minor. Thalictrum minus L. (17). Source: An American nursery. Distribu- tion: Europe, Asia, and eastern and southern Africa. Thalictrum m,inus adiantifolium (63). Source: Seed from Brooklyn Botanic Garden. Distribution: See T. minus. Thalictrum occidentale Gray (115). Source: Corvallis, Oreg. Distribu- tion: Mountains, California to British Columbia. Thalictrum, polycarpum S. Wats. (114). Source: Berkeley, Calif. Dis- tribution: California. Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat 159 Thalictrum polygamum Muhl. 19. Source: Ithaca, N. Y. Distribution: Eastern United States. Thalictrum sp. (98). Source: Palisade Nursery Co. Distribution: Exotic. Thalictrum sp. (55). Source: Farr Nursery Co. Distribution: Exotic. From the data presented above it is evident that a number of species of Thalictrum are susceptible hosts for Puccinia triticina. As far as the host determinations are at all certain, the evidence would indicate that the most susceptible hosts are from western Asia and eastern Europe, and doubtless in this region other species will be found of as great or greater susceptibility. The remaining collections of telia showing good germination were sown on one or more of the susceptible species of Thalictrum in order to determine how uniformly Puccinia triticina from the United States would go to Thalictrum. Table III gives the results of these cultures. Table III. — Data obtained in ig20 from sowing teliospores of Puccinia triticina, from many different localities, on four especially susceptible species of Thalictrum o- Nmnber and source of telia. 5119 Pa 11719 Pa.. . . 9819 W. Va. 6319 N. C. . 6419 N. C. . 7319 N. C. . 5819S. C. ... 5019 Ga. . . . 4119 Ala. . . . 4519 Ala. . . . 4419 Tenn.^. 17419 Tenn. 6519 Ky . . . . 4819 Ind. . . . 5319 Ind.... 10119 Ind. . . 10419 111 . . . 12419 Minn. . 3619 Iowa. . 9219 Mo. . . . 9319 Mo . . . . 9519 Mo 9619 Mo . . . . 3819 La 6719 Tex. .. . 7019 Tex.c. . 3219 Ariz. . . 61 19 Calif. . 122 19 Wash. 165 19 Wash. 199 19 Wash. I39i9 0reg. . T. sp. (ss). T Delavayi Sown also on T. angustifolium, producing pycnia, and on T. aquilegifolium without results. e Sown also on T. dasycarpum without results. i6o Journal of Agricultural Research voi. xxii.no. 3 Source of telial material used in cultures : 51 19, from York, Pa., collected by F. D. Kern. 1 1 719, from Bradford Count}^ Pa., collected by E- T. Nixon. 9819, from Morgantown, W. Va., collected by N. J. Giddings. 6319, from Statesville, N. C, collected by C. A. Ludwig. 6419, from Statesville, N. C, collected by C. A. Ludwig. 7319, from Biltmore, N. C, collected by C. A. Ludwig. 5819, from Clemson College, S. C, collected by C. A. Ludwig. 5019, from Tifton, Ga., collected by C. A. Ludwig. 41 19, from Bay Minette, Ala., collected by C. A. Ludwig. 4519, from Auburn, Ala., collected by C. A. Lud\vig. 4419, from Union City, Tenn., collected by Carl Kurtzweil. 1 7419, from Johnson City, Tenn., collected by C. A. Ludwig. 6519, from Lexington, Ky., collected by R. S. Kirby. 4819, from Mount Vernon, Ind., collected by E- B. Mains. 5319, from Washington County, Ind., collected by H. S. Jackson. 10119, from La Eayette, Ind., collected by E. B. Mains. 10419, from Bloomington, 111., collected by Koehler and Toole. 1 24 1 9, from Wasioja, Minn., collected by G. W. Martin. 36^9, from Ames, Iowa, collected by I. E. Melhus. 9319, from Columbia, Mo., collected by W. E. Maneval. 9419, from Columbia, Mo., collected by W. E. Maneval. 9519, from Columbia, Mo., collected by W. E. Maneval. 9619, from Columbia, Mo., collected by W. E. Maneval. 3819, from Baton Rouge, La., collected by Thiel and Lud\\4g. 6719, from Dallas, Tex., collected by W. H. Ballamy. 7019, from San Antonio, Tex., collected by R. S. Kirby. 3219, from Yuma, Ariz., collected by L. Y. Leonard. 61 19, from Chico, Calif., collected by R. M. Kelia. 122 19, from Dayton, Wash., collected by J. W. Hotson. 165 19, from Colton, Wash., collected by J. W. Hotson. 19919, from Puyallup, Wash., collected by G. R. Hoemer. 13919, from Oregon, collected by G. R. Hoemer. The data in this table, taken with those in Table II, show that Puccinia triticina from Pennsylvania, West Virginia, North Carolina, South Caro- lina, Georgia, Alabama, Mississippi, Tennessee, Kentucky, Indiana, Michigan, Illinois, Minnesota, Iowa, Missouri, Louisiana, Texas, Arizona, California, Washington, Oregon, Idaho, and Nevada gave positive results when sown upon Thalictrum. The following collections showed some germination but produced no infection when sown on Thalictrum : 11819, from Hopkinsville, Ky., collected by Carl Kurtzweil. 9419, from Columbia, Mo., collected by W. E. Maneval. 3919, from Fayetteville, Ark., collected by H. R. Rosen. 5419, from Memphis, Tenn., collected by A. F. Thiel, Oct IS, 1921 Aecial Stooge of the Orange Leaf rust of Wheat 161 7519, from Southampton, N. Y., collected by H. S. Jackson. 8319, from Rocky Ford, Colo., collected by J. G. Leach. 10019, from Buffalo, Minn., collected by G. W. Martin. 105 19, from Plainview, Nebr., collected by H. W. Thurston. 11019, from Vermillion, Minn., collected by G. W. Martin. 1 12 19, from Newark, Del., collected by T. F. Manns. Besides the above, the following collections were wintered, but no germinating teliospores were found, and in consequence they were not sown. 1419, from Santa Rosa, Calif., collected by H. S. Jackson. 3319, from Sonora, Mexico, near Yuma, Ariz., collected by L. Y. Leonard. 3419, from St. Louis, Mo., collected by E- B. Mains. 3719, from Jackson, Tenn., collected by Kurtzweil and Thiel. .5 4019, from Corvallis, Oreg., collected by G. R. Hoemer. ,i 5519, from St. Paul, Minn., collected by A. F. Thiel. .^ | 6619, from Hiawatha, Kans., collected by W. H. Ballamy.o\^ | 6819, from Marshall, Mo., collected by R. S. Kirby. ) n 6919, from Guthrie, Okla., collected by R. S. Kirby. 71 19, from Wellington, Mich., collected by G. H. Coons. 7919, from Nashville, Tenn., collected by C. A. Ludwig. 91 19, from Madison, Wis., collected by E. B. Mains. 108 1 9, from Manhattan, Kans., collected by L. E. Melchers. Ill 19, from Toledo, Iowa, collected by I. E- Melhus. 11919, from Fort Collins, Colo., collected by J. G. Leach. 12019, from East Lansing, Mich., collected by Acelia M. Leach. 12 1 19, from East Lansing, Mich., collected by Acelia M. Leach. 12319, from Pullman, Wash., collected by F. D. Heald. 1 38 1 9, from Fort Collins, Colo., collected by J. G. Leach. 21019, from Moscow, Idaho, collected by G. R. Hoemer. 25519, from Murfreesboro, Tenn., collected by Carl Kurtzweil. 25719, from Clarksville, Tenn., collected by Carl Kurtzweil. The accompanying map (fig. i) shows the source of collections used in the work together with the results obtained with them at La Fayette, Ind. This map shows that material from the States of Pennsylvania, West Virginia, Indiana, Illinois, North Carolina, South Carolina, Georgia, Alabama, Mississippi, Louisiana, and Texas gave germination uniformly and infected Thalictrum in all cases. A region represented by the States of Colorado, Oklahoma, Arkansas, Kansas, Nebraska, Minnesota, Iowa, and Missouri, with one arm running through Wisconsin into Michigan and another through Tennessee into Kentucky, gave material which usually did not germinate or, if germination was obtained, produced infection on Thalictrum in only a few cases. Whether this situation indicates the presence of another strain of the leafrust having different characteristics as regards its viability and power to infect Thalictrum, or whether it means that the season or climate was of such a nature that l62 Journal of Agricultural Research voi. xxii. No. 3 teliospores of a low vitality were produced, remains for future investiga- tion to decide. Telial material from the Pacific coast, while not viable in a number of cases, produced infection on Thalictrum in all cases where germination was obtained. The aecia produced from the following telial collections were sown back upon wheat: 4519, from Auburn, Ala. 4819, from Mount Vernon, Ind. 5019, from Tifton, Ga. 5619, from Athens, Ga. 5819, from Clemson College, S. C. 6019, from W. Raleigh, N. C. 6319, from Statesville, N. C. /u_ p/ \ ■ — j — I T^ L 1 L V \ / /^i- 2 0 i .+ 500~^ ?pr+^ ? V 1. +0 — < • \ \ \ /— ~— ^/ * 0° vXT r I XI A/ 0 A/OT SOi^A/. 0 + + V ,-> \ai4 Flo. I, — Map showing results obtained at LaFayettc, Ind., with leafrust material collected in different parts of the United States. 7219, from Hickory, N. C. 7819, from Tennessee. 8019, from Coldwater, Mich. 8719, from Canton, Miss. 9619, from Columbia, Mo. 9819, from Morgantown, W. Va. 10419, from Bloomington, 111. 11619, from State College, Pa. 1 24 1 9, from Wasioja, Minn. 125 1 9, from Moscow, Idaho. 151 19, from Reno, Nev. 165 19, from Colton, Wash. 1 7419, from Johnson City, Tenn. Oct. 15, 1921 Aecial Stage of the Orange Leaf rust of Wheat 163 These were each sown upon the variety of wheat known as Dawson Golden ChaflF, and in all cases positive infection was obtained followed by the development of uredinia which were typical of Puccinia triticina. Sowings of aeciospores also were made upon a number of grasses. Aecia which were produced from telia obtained from Hickory, N. C, Canton, Miss., and Moscow, Idaho, were used and Table IV gives the results. Table IV. — Results obtained in ig20 from sowing the aeciospores of Puccinia triticina produced from telia obtained in three different areas, on wheat and related grasses Host inoculated. Arrhenatherum elatius (L.) Mert & Koch. . . Agropyron caninum (L.) Beauv Agropyon cristatum Beauv Agropyron desertorum Schult Agropyron in£rme (Schribn. & Sm.) Rydb . Agropyron repens (L.) Beauv Agropyron tenerum Vasey Elymus atistralis Schribn. & Ball Elynius canadensis L Elymus glaucus Buckl Elym^us triticoides Buckl Elymus virginicus L Hordeum caespitosum Schribn Hordeum gu^soneanum Pari Hordeum jubatum L Hordeum pusillum Nutt Hordeum inurinum I, Hordeum vulgare L Hystrix Hystrix (L.) Millsp Notholcus lanatus (L.) Nash Secale cereale L Sitanion Hystrix (Nutt.) J. G. Sm. Triticum aegilops Beauv Triticum aestivum L Number and source of aecia. No. 7219 (N.C.). t Many. No. 8719 (Miss.). t One ure- dinium. t Many. No. 12519 (Idaho). t Few. t Many, — No infection. t Uredinia produced. Except for the one uredinium produced on Secale cereale, Triticum aestivum and T. aegilops were the only species infected DESCRIPTION OF AECIA The following description has been drawn from the aecia obtained in the cultures discussed above. Pycnia amphigenous, mostly epiphyllous, numerous, crowded upon more or less swollen reddish brown to yellowish areas 2 to 15 mm. in diameter, conspicuous, subepidermal, honey-yellow, globoid or flattened globoid, 80 to 145 /i broad by 80 to 130 fx high; ostiolar filaments 95 to 190 jj, long, agglutinated to form a prominent, broad column. 65508°— 21 4 V 164 Journal of Agricultural Research voi. xxii. no. 3 Aecia hypophyllous, crowded in more or less swollen, gall-like, reddish brown or yellowish areas 2 to 15 mm. in diameter, cupulate or short cylindric, 0.2 to 0.6 mm. in diameter, up to 0.5 mm. high; peridium white or yellowish, the margin erose or somewhat lacerate, recurved; peridial cells oblong or somewhat rhomboidal in longitudinal radial section, 14 to 19 by 18 to 29 IX, abutted or slightly overlapping, the outer wall 6 to 7 /x thick, transversely striate, the inner wall thinner 2 to 3 yLt, very coarsely verrucose; aeciospores angularly globoid or ellipsoid, 16 to 20 by 16 to 26 /x; wall colorless, thin, i /i or less, very closely and finely verrucose. The pycnia and aecia usually were produced in definite galls or swellings. These galls apparently were formed by the excessive enlargement of the cells of the infected areas, especially those of the mesophyll. When infection took place in the young, rapidly developing tissue of the petiole, galls developed (PI. 21, D) which were 10 or 15 times as large as the normal petiole. A very noticeable odor, resembling that of the hyacinth, was often detected as the pycnia reached maturity. GENERAL DISCUSSION OF RESULTS The discovery that species of Thalictrum are the aecial hosts for Puccinia triticina goes to support Arthur's contention {i, v. 9, p. 304) that the leafrust of wheat is closely related to grass rusts of the type of Puccinia Agropyri, having aecia on species of the family Ranunculaceae. A num- ber of cultures have been made with rusts of this type, connecting them with various species of Thalictrum. Plowright, in England {21, p. j8i), connected aecia on Thalictrum flavum with a rust on Agropyron repens. To this rust he gave the name Puccinia persistens Plowr. He considered Aecidium Ranunculacearum 7 Thalictri flavi DC, and Aecidium Thalictri fiavi (DC.) Winter as synonyms, and describes the aecia as occurring on thickened spots with aeciospores subglobose 17 to 20 by 20 to 30 /i. Fischer (12, p. 37-63), in Switzerland, cultured a rust from Poa nemoralis var. firmula on Thalictrmn minus, T. aquilegijolium and T. foetidum. On account of the morphological similarity, he concluded that his material belonged to Puccinia persistens, although he made no cultures on either Agropyron repens or T. flavum. He describes the aecia (ij, p. 347-349) as having peridial cells with the outer wall 4.5 to 13.5 ^ thick and the inner 2 to 6 fjL and aeciospores 10 to 21 /x broad and up to 28 /i long. An exam- ination of Sydow's Uredineen No. 725, issued as Puccinia persistens on T. aquilegifolium, shows the following measurements : Peridial cells, 18 to 23 by 21 to 26 /x; outer wall, 7 to g fx; inner, 3 to 5 /x; aeciospores, 16 to 19 by 19 to 26 n. Juel (j<5, p. 411), in Sweden, made cultures connecting aecia on Thalic- trum alpinum with a rust on Agrostis borealis and Anthoxanthum odor- atum. To this rust he gave the name Puccinia borealis Juel, and con- sidered Aecidium thalictri Grev. as a synonym. His description follows: Pycnia not present; aecia not causing hypertrophy of host tissue; aecio- Oct i^. 1921 Aecial Stage of the Orange Leaf rust of Wheat 165 spores about 13 ju in diameter. The Sydows {23, p. 718-719) give the measurement of the aeciospores as 13 to 18 /x in diameter or 13 to 16 by 18 to 20 M and note that no swelHngs are produced on the leaves of the host. An examination of Eriksson's Fungi Parasitici Scandinavici 432a, col- lected by Juel in Norway, gives the following measurements: Peridial cells, 16 to 19 by 19 to 29 /x; the outer wall, 10 /x; the inner, 3 to 4 ju; aeciospores 14 to 16 by 16 to 21 m- Rostrup (22, p. 269-273), in Denmark, obtained infection with aecio- spores from Thalictrum minus on Elymus arenarius and considered the rust to be Puccinia Elymi Westendorp. The writers have seen neither description nor material of these aecia. The Sydows (23, p. 827) mention that Lindroth in Finland connected an aecidium on Thalictrum m/ijus with a rust on Agropyron caninum. No description or material of this connection is available for study. In North America a number of connections have been established by the cultures of Arthur and of Fraser. Arthur (/, v, i, p. 248-249) reports culturing a rust found associated with aecia on Thalictrum sparsifiorum from Bromus Porteri to T. dioicum. To this he gave the name Puccinia alternans. He describes the aecia as having peridial cells 2 1 to 29 /x long with the outer wall 9 to 12 /x thick and the inner 5 to 7 /x and with aecio- spores 15 to 20 by 17 to 24 IX. A number of other species of Thalictrum are given as hosts. Arthur (j, v. 2, p. 226) also reports obtaining infection from telia on Agropyron resulting in aecia on Thalictrum alpinum but not on T. dioicum. This material he considered as belonging in Puccinia obliterata Arth., which he had previously shown as having aecia on Aquilegia. A study of the material obtained by this culture shows little or no hypertrophy of the host tissue. The peridial cells measure 16 to 21 by 24 to 32 /x, having the outer wall 7 to 9 ^u thick and the inner 3 to 5 /x. The aecio- spores measure 14 to 18 by 18 to 23 jx. Still another connection was obtained by Arthur (i, v. 8, p. 132-133) when he cultured a rust on Festuca Thurberi to Thalictrum dioicum, producing aecia. To this he later (2, p. 113) gave the name Puccinia Cockerelliana Bethel. He gives the peridial cells as 16 to 23 by 27 to 36 /x with the outer wall 6 to 8 /x and the inner 2 to 3 a« and aeciospores 18 to 24 by 20 to 29 IX with a wall 1.5 to 2.5 ix thick. The natural host for the aecia is given as T. Fendleri. Fraser {14, p. 131-133) reports sowing aeciospores from Thalictrum dasycarpwn on Elymus canadensis, E. virginicus, Agropyron tenerum, A. Richardsonii, Hordeum jubatum, Triticum vulgare, and Bromus ciliatus, obtaining infection on E. canadensis, E. virginicus, H. juba- tum, and B. ciliatus. When, however, the rust obtained upon B. cili- atus was sown on E. virginicus, A. tenerum., A. Smithii, A. repens, and H. jubatum no infection was obtained on these species. From these results Fraser concludes that two strains of Puccinia Agropyri KHis and E., 1 66 Journal of Agricultural Research voi. xxn. no. 3 were present in the aecial material on Thalictrum which he used for the culture. A study of the aecia used in these cultures shows the dimensions of the peridial cells to be 15 to 19 by 23 to 29 n with the outer wall 7 to 10 /x and the inner 3 fx thick and the aeciospores 14 to 19 by 19 to 23 p. in diameter. A comparison of the foregoing description of the aecia of Puccinia triticina with the measurements given for the various grass rust aecia on Thalictrum shows surprisingly little variation. The aecia of P. Cockerel- liana show the greatest difference, having larger peridial cells and some- what larger aeciospores with much thicker walls than the aecia of P. triti- cina. Slightly smaller aeciospores occur in P. horealis and P. obliterata, and the aecial infection causes little or no hypertrophy of the host. The remaining aecia differ mainly in slightly thicker walls of the peridial cells. It is evident that Puccinia triticina is closely related to P. persistens. Whether the name Aecidium Tlialictri-flavi (DC.) Wint. should apply to the aecial stage of the former is a question which can not be answered with the available information. De Candolle (7, p. giy) described A. Ranunculacearum for aecia occurring on the family Ranunculaceae and as a variety of this gives Thalictri-flavi without further description. Winter (24, p. 269) raises this variety to specific rank and gives a description which, however, could apply to the aecia of either rust. As both P. triticina and P. persist-ens are common rusts throughout Europe, there is no way of determining definitely to what aecia the name was applied beyond the fact that they were on Thalictrum flavum. As it has been shown that at least some aecia on that host in England belong to P. persistens the name A. Thalictri-flavi should be retained for the present as a synonym of that species, at least until aecia can be found in Europe upon T. flavum which will produce the leafrust of wheat. Upon their grass hosts these rusts present a somewhat greater varia- tion. They all have uredinia with globoid or ellipsoid urediniospores with a varying number of scattered pores, usually more than six, and with few or no paraphyses. The telia are long, covered by the epidermis, usually with more or less stroma present, and the teliospores are cylindric, more or less flattened at the apex, and with a very short pedicel. Puc- cinia Cockerelliana differs most noticeably from P. triticina in that the teliospores are much longer and the telia do not remain entirely covered by the epidermis at maturity. P. Elymi differs especially in the thicker and darker walls of the urediniospore and in the longer teliospores which are often many-celled. P. alternans, P. borealis, P. obliterata, and P. persistens differ but little, mostly in the tinting of the urediniospore wall and a slight variation in pore number. Although the morphological differences between Puccinia triticina and the related rusts discussed above are not great, their biologic specializa- Oct. IS, 1921 Aecial Stage of the Orange Leaf rust of Wheat 167 tion to their hosts is very pronounced. This appears to hold true for the aecial as well as the grass hosts. P. Cockerelliana and P. alternans go to Thalictrum dioicum, and P. Elymi to T. minus, as aecial hosts, neither of which is a favorable host for P. triticina. The rust of T. dasycarpum, used by Fraser {14) in his cultures, is on a host which was not infected by P. triticina. P. horealis and P. ohliterata on T. alpinum offer no com- parison, as P. triticina was not sown on that host. P. persistens, as cultured by Fischer on T. minus and T. aquilegifolium, is upon species of Thalictrum unfavorable for P. triticina, while P. persistens as originally cultured by Plowright upon T. flavum is on the most congenial host for the leafrust of wheat. It is very probable that Plowright and Fischer were working with two distinct biologic strains. Although T. flavum appears to be a favorable host for both P. persistens and P. triticina, and these two rusts are very similar in their morphology, the inability of the latter to infect Agropyron repens shows that it is biologically distinct from the former. A study is being made of the relationship of P. triticina to grass hosts other than wheat. From the data now at hand, it would appear that, in addition to the grasses listed in Table IV, species of Bromus, Festuca, Agrostis, Poa, and Anthoxanthum are immune from the leafrust of wheat. These results indicate that, as far as its telial host is concerned, P. triticina also is biologically distinct from other grass rusts having aecia on Thalictrum. A similar situation exists in the relationship of Puccinia triticina to rusts having aecia upon species of other genera of the Ranunculaceae. Slight morphological differences, such as urediniospore size, wall color, and pore number, exist among the different races producing aecia upon species of such genera as Actaea, Anemone, Clematis, Delphinium, etc. A similar, or perhaps greater, biologic specialization is also to be found among these races. The importance of these morphological characters and biological differences which occur among the members of this group can not be fully determined at present on account of our comparatively limited knowledge of but few races. Any final interpretation must await further study of a greater number of such races. On the basis of our present knowledge, the disposition of P. triticina must depend largely upon the species concept held. In Europe there is a tendency among certain students of the rusts to consider as species those rusts showing distinct biologic specialization regardless of the absence of morphological difference. In this country, on the other hand, the general tendency is to include in a single species all closely related forms having but little difference in their morphology. Forms limited to a definite host, or hosts, are considered as races of such species. On the former basis, P. triticina would be considered a distinct species comparable to P. Elymi, P. Agropyri, P. persistens, etc., while with the latter concept it would be united with all or part of these, each being considered a race of a 168 Journal of Agricultural Research Voi. xxii. no. 3 collective species to be designated, according to the limitations of the species concept held and the system of nomenclatm-e used, as P. Agropyri B. and E. (j, v. 9, p. 304), P. Clematidis (DC.) Lagerh., or Dicaeoma Clematidis (DC.) Arth. (j, p. 333-337). The close biological specialization of Puccinia triticina to wheat is of considerable significance with respect to the bearing it has upon the possible origin of this rust and of wheat itself. Since wheat is an intro- duced plant, it is logical to assume that a rust showing such close biolog- ical specialization to it is also introduced and of foreign origin. It is generally recognized among students of the rusts that a high de- gree of host specialization must have been acquired in certain groups of species at a very early stage in the evolutionary history of this group of fungi. It is also recognized that the host is the most important factor in the evolution of highly specialized pai-asitic fungi. As the higher plants have gradually developed during geological times, their rust parasites have developed with them. It therefore appears reasonable to assume that Puccinia triticina, which shows such a high degree of spe- cialization to wheat at the present time, had its origin as a distinct strain comparatively early in the development of the group of grasses from which our cultivated wheats have originated. The original distribution of the rust presumably would coincide with the distribution of the ancestral wheats. A study of the relative susceptibility of various species of Thalictrum to infection by this rust is of interest in this connection. The four most susceptible species of Thalictrum encountered in this investigation are all of foreign origin. The most susceptible of our native North American species, Thalictrum dioicum, does not compare in susceptibility with these four foreign species— T. flavum, T. Delavayi, T. sp. 55, and T. sp. 98 — but is comparable to the resistant foreign species such as T. minus. That these foreign susceptible species of Thalictrum are also to be con- sidered as indicating a foreign origin of the rust would appear to follow if the nature of aecial infection is considered. Heteroecious rusts in most cases infect their aecial hosts only for a comparatively short period of tune while the teliospores are germinating in the spring. The infection produced, not being able to propagate itself upon such hosts, causes little or no damage, and they are in most cases soon able to outgrow it. On this account it is hardly to be expected that a natural selection of resist- ant strains of aecial hosts takes place in nature comparable to that which occurs where the host is killed or prevented from producing seed. Should this occur in heteroecious rusts which are not able to survive adverse conditions in winter or summer by means of urediniospores, such a selec- tion would be fatal to the rust itself. For this reason the susceptibility of the aecial hosts of P. triticina may be taken as indicative of its origin. It is true that susceptibility of a host species does not necessarily indi- cate that such a species was a native host of the rust nor does resistance Oct. IS, i92t Aecial Stage of the Orange Leaf rust of Wheat 169 of some one species denote that the rust is not to be found in the habitat of such a resistant species, for susceptibility or resistance is not dependent upon the presence or absence of the rust but may develop with the species in any region. It is regarded as significant, however, that of the species of Thalictrum tested the most susceptible are exotic. This fact, taken with the foreign origin of wheat itself, is confirmative of the foreign origin of the rust. The native habitats of two of these species of Thalictrum are known with some degree of certainty. Thalictrum flavurn is found throughout Europe, western Asia, and Asia Minor. T. Delavayi is given by the Index Kewensis ^ as occurring in western China, probably indicating a distri- bution in the little-known mountainous regions of Tibet and Chinese Turkestan. These two species, taken together, would therefore indicate as the most probable original distribution a region in which the two Thalictrum species may border or overlap, such as that of southwestern Asia. Such an origin would indicate a like origin for wheat itself, which, we believe, would agree with the latest theories advanced as to the original home of wheat. Concerning the occurrence and distribution of the aecia of Puccinia triticina but little can be said with the data at hand. It is also probable that the aecial stage occurs, and probably assumes greater importance, in other regions than it may in either Europe or North America, where the rust is known to overwinter in its uredinial stage. Thus in such countries as India, where Butler has shown there is no oversummering of the rust, the Thalictrum species of the foothills may be of importance in starting the rust the next season. The question of the role which the aecia of the leafrust of wheat plays in its life history and distribution must, however, be left for future research to solve. Whether native species of Thalictrum serve as aecial hosts in North America and, if so, whether they serve as important factors in the development of the leafrust of wheat and whether there is more than one race of the leafrust, as indicated by the results obtained from the Great Plains area, or whether these results were due to other causes, such as climatic or seasonal effects weakening the vitality of the teliospores, are all questions on which further investi- gation is planned. Other species of Thalictrum from foreign botanic gardens also v/ill be studied in regard to their susceptibility to the orange leafrust of wheat. SUMMARY (i) The aecial stage of Puccinia triticina has been produced in green- house cultures upon several species of Thalictrum. (2) The various species of Thalictrum show varying degrees of sus- ceptibility to the rust. Thalictrum occidentale was apparently immune. Upon T. dasycarpum and T. polygamutn an occasional devlopment of > INDEX KEWENSIS PLANTARUM PHANEROGAMARUM. SUPPI.EMENTUM PRIMUM . . . CONFECERUNT THBOPHIlrUS DURAND ET B. DAYDON JACKSON. p. 42^. BniXCllis I9OI-06. lyo Journal of Agricultural Research voi. xxn. no. 3 pycnia took place. When T. angustijoliuvi, T. aqtiilegifolium, T. dioicum, T. minus, T. minus adiantijolium, and T. polycarpum were inoculated usually only pycnia resulted, with an occasional weak development of aecia, while in other cases no infection occurred. Two undetermined species of Thalictrum, as well as T. Delavayi and T. flainmi, when inocu- lated, showed a vigorous development of aecia, increasing in suscepti- bility in the order named. (3) Puccinia triticina is apparently limited to species of the genus Thahctrum, no infection being obtained upon species of Aconitum, Actaea, Anemone, Aquilegia, Cimicifuga, Clematis, Delphinium, Echium, Ilepatica, Hydrophyllum, Impatiens, Mertensia, Myosotis, Ornithogalum, Phacelia, Camassia, Ranunculus, or Trollius. (4) On account of the morphology and host relationships, Puccinia triticina is considered to be very closely related to P. persistens, P. borealis, P. alternans, P. ohliterata, P. Elymi, and P. Agropyri, but is separable from these rusts by its sharp biologic limitation to wheat. (5) Puccinia triticina is considered to be of foreign origin, because wheat, for which it shows close specialization, is an introduced host, and because the most susceptible species of Thalictrum which serve as aecial hosts also are exotic. LITERATURE CITED (i) Arthur, Joseph Charles. 1909-17. CtliTURES OF UREDINEAE IN 1908, I909, 1915, 1916 AND 1917. In Mycologia v. i, no. 6, p. 225-256; v. 2, no. 5, p. 213-240; v. 8, no. 3, p. 125-141; V. 9, no. 5, p. 294-312. (2) 1919. NEW SPECIES OF UREDINEAE — XI. In Bui. Torrey Bot. Club, v. 46, no. 4, p. 107-125. (3) and Fromme, Fred. Denton. 1920. DICAEOMA ON POACEAE. In North American Flora, v. 7, pt. 4-5, p. 269-341. (4) Bary, Anton de. 1866. NEUE UNTERSUCHUNGEN ubER urEdinEEn. In Monatsber. K. Preuss. Akad. Wiss. Berlin, 1866, p. 205-215, i pi. (5) BoLLEY, H. L. 1889. WHEAT rust. Ind. Agr. Exp. Sta. Bui. 26, 19 p., 9 fig. (6) Butler, E. J., and Hayman, J. M. 1906. INDIAN wheat rusts. In Mem. Dept. Agr. India, Bot. Ser., v. i, no. 2, p. 1-52, I fig., 5 pi. (1-4 col.). (7) CandollE, a. p. de. 1815. FLORE FRANfAisE. • • t. 5 (v. 6). Patis. (8) Carleton, Mark Alfred. 1899. CEREAL RUSTS OF THE UNITED STATES. A PHYSIOLOGICAL INVESTIGA- TION. U. S. Dept. Agr. Div. Veg. Physiol, and Path. Bui. 16, 74 p., I fig., 4 col. pi. Bibliography, p. 70-73. (9) Cunningham, D. D., and Prain, D. 1896. A note on INDIAN WHEAT-RUSTS. In Rec. Bot. Survey India, v. i, no. 7, p. 99-124. Oct. IS, 19" Aecial Stage of the Orange Leaf rust of Wheat 171 (10) Eriksson, Jakob. 1899. NOuvELLES ^TudES sur LA rouillE brunE dES c^r^alES. In Ann. Sci. Nat. Bot. ser. 8, t. 9, no. 2/4, p. 241-288, pi. 11-13 (col.). Lit- tdrature citee, p. 286-287. (11) and Henning, Ernst. i8g4. DIE hauptrESULTATE EinER nEUEN unTERSUCHUNG ubeR die GETREI- DEROSTE. In Ztschr. Pflanzenkrank., Bd. 4, p. 66-73, 140-142, 197- 203, 257-262. (12) Fischer, Eduard. 1898. ENTWICKLUNGSGESCHICHTLICHE UNTERSUCHUNGEN UBER ROSTPIUZE. . . X, 120 p., 16 fig., 2 pi. (Beitrage zur Kryptogamenflora der Schweiz, Bd. I, Heft. I.) (13) 1904. DIE UREDINEEN DER SCHWEIZ. xciv, 590 p., 342 fig. Bern. Literatur- verzeichniss, p. 558-576. (Beitrage zur Kryptogamenflora der vSchweiz Bd. 2, Heft. 2.) (14) Eraser, W. P. 1919. CULTURES OF hETEroecious RUSTS IN 1918. In Mycologia, v. 11, no. 3, p. 129-133. (15) Hitchcock, A. S., and Carleton, M. A. 1894. SECOND REPORT ON RUSTS OP GRAIN. Kans. Agr. Exp. Sta. Bui. 46, 9 p. (16) JuEL, H. O. 1894. MYKOLOGISCHE BEITRAGE I. ZUR KENNTNISS EINIGER UREDINEEN AUS DEN gebirgsgEgendEn skandinaviens. In Ofvers. K. Svenska Vetensk. Akad. Forhandl., Arg. 51, no. 8, p. 409-418. (17) Klebahn, Heinrich. 1900. beitrage zur kenntnis der getreideroste. II. In Ztschr. Pflanzen- krank., Bd. 10, Heft. 2, p. 70-96, I fig. (18) 1904. DIE wirtswechselnden rostpilze. . . 447 p. Berlin. Literatur, p. ix-xxxvii. (19) MtJLLER, Fritz. 1901. BEITRAGE ZUR KENNTNIS DER GRASROSTC. In Bot. Centbl., Beihefte. Bd. 10, Heft 4/5, p. 181-212, I fig.. (20) Nielsen, p. 1877. bemaerkninger om nogle rustarter, navnlig om en genetisk FORBINDELSE MELLEM AECIDtUM TUSSILAGINIS PERS. OG PUCCINIA POARUM N. SP. In Bot. Tidsskr., Raekke 3, Bd. 2, p. 26-42, 3 fig. (21) Plowright, Charles B. 1889. A monograph of the BRITISH UREDINEAE AND USTILAGINEAE . . . vii, 347 p., 13 fig., 8 pi. London. Authors quoted, p. 309-315. (22) ROSTRUP, E. 1898. ET NYT VAERTSKIFTE HOS UREDINACEERNE OG KONIDIER HOS THECAPHORA CONVOLVULI. In Overs. K. Danske Vidensk. Selsk. Fordhandl., 1898, No. 5, p. 269-276. (23) Sydow, p., and Sydow, H. 1904. MONOGRAPHiA uredinearum . . . V. I. Lipsiae. (24) Winter, Georg. 1884. die PILZE DEUTSCHLANDS, OESTERREICHS UND der SCHWEIZ. ABT. I. SCHIZOMYCETEN, SACCHAROMYCETEN UND BASIDIOMYCETEN. In Raben- horst, L. Kryptogamen-Flora von Deutschland, Oesterreich und der Schweiz. Aufl. 2. Bd. i, Abt. i. Leipzig. PLATE 21 A.— Infection produced upon Thalictrum sp. (98) inoculated with Puccinia triticina from Colton, Wash. (16519). B. — Infection produced upon Thalictrum sp. (98) inoculated with Puccinia triticina from Canton, Miss. (8719). ' C. — Infection produced upon Thalictrum flavjim (53) inoculated with Puccinia iriii- ana from Hickory, N. C. (7219). D. — Infection produced upon Thalictruin sp. (55) inoculated with Puccinia triticina from Hickory, N. C. (7219). (172) Aecial Stage of the Orange Leafrust of Wheat PLATE 21 Journal of Agricultural Research Vol. XXII, No. 3 A Transmissible mosaic disease oe Chinese CABBAGE, MUSTARD, AND TURNIP By E. S. ScHUivTz Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureati of Plant Industry, United States Department of Agriculture In the fall of 19 19, while the writer was selecting different kinds of plants for inoculation experiments with mosaic of Irish potatoes (Sola- num tuberosum Linn.), Dr. W. A. Orton called his attention to mottling in plants of Chinese cabbage (Brassica pekinensis (Lour.) Gagn.), mus- tard {B. japonica Coss.), and turnip {B. rapa, Linn.). The mottling resembled that of mosaic plants of other species, such as potato and tobacco. Diseased and healthy individuals were found in the same plot; the former appeared in groups in some parts of tlie field, suggesting an infectious character of this malady. Evidence bearing upon the nature of this disease, its symptoms, and means of transmission is presented in this paper. SYMPTOMS Mosaic of Chinese cabbage, mustard, and turnip produces a distinct mottling of the leaves, very similar to that of mosaic diseases of the Solanaceae. This mottling is produced by the appearance of irregular light green and dark green areas on the leaves (PI. B ; 22, D, E ; 24, A, B). These light green areas usually adjoin the veins, from which they may extend so as to include a considerable area of the leaf surface between the veins. Another very common macroscopic symptom of this disease is the characteristic ruffling and distorting of the leaf surface (PI. 24, A, B). On the raised areas the dark green patches appear. The leaf margins frequently are much more irregular than in healthy plants, causing some of the leaves to appear somewhat unsymmetrical. In addi- tion to these common abnormalities on the leaves the entire plant may be dwarfed, and the flower stalk and number of blossoms may be con- siderably reduced (PI. 22, B; 23, B). OCCURRENCE IN THE FIEED Since mosaic individuals appeared among Chinese cabbage, mustard, and turnip plants growing in adjoining plots, interspecific susceptibility was suggested. Furthermore, it was found that a large percentage of the plants were infested with aphids, Myzus persicae Sulz.,^ one of the 1 Identified by Dr. A. C. Baker, Entomologist, Bureau of Entomology, United States Department of Agriculture. Journal of Agricultural Research, Vol. XXII, No. 3 Washington, D. C. Oct. 15, 1921 aaa Key No. G-248 (173) 174 Journal of Agricultural Research voi. xxn. N0.3 casual agents in the transmission of mosaic and leafroll of Irish potato.^ In view of these field observations experiments on this disease were conducted in the greenhouse at Washington, D. C, during the winters of 1919-20 and 1920-21. TRANSMLSvSION WITH PLANT JUICE Chinese cabbage, mustard, and turnip plants showing mosaic mottling were taken from the field and planted in pots in the greenhouse. Only a small percentage of these mature and mosaic plants survived trans- planting, so that the supply of mosaic material for inoculations was thus considerably reduced, and therefore only a small number of healthy plants were inoculated at one time. Inoculations with juice were made by rubbing the leaves between the fingers so that considerable sections of the leaflets were crushed, apparently permitting the applied juice to be absorbed by such areas of the leaf as still remained free or partly free from mutilation. Such operations were performed chiefly upon the youngest leaves, the first applications being made when the plants had developed about five leaves. In Table I the results of these inoculations are presented. Table I. — Inoculations with juice from mosaic plants Variety and species inoculated. Time of inocu- lation. Source of juice. Southern turnip. Do. . Do. . Prize Mustard . Do. Do. Do. Pe-tsai or Chinese cabbage. Do Do Dec. 4, 1919 . do Dec. 6, 1919 . ...do /Mar. 8, 192 1 \Mar. 21, 192 1 fMar. 9, 192 1 \Mar. 21, 1921 Jan. 15,1921 Dec. 4, 1919 ....do Dec. 6, 1919 Mosaic Southern Prize tiu-nip. Healthy turnip. . . . Mosaic Green Moun- tain potato. do }....do [Mosaic mustard. . . . Mosaic pe-tsai or Chinese cabbage. do Healthy Mosaic Green Moun- tain potato. Number of plants inoculated. Number of plants mosaic." 9 6 9 5 0 0 5 0 6 0 8 5 5 3 8 6 5 4 0 0 Per cent- age mosaic. 67 63 60 1^ <» Date of last observation April 2, 1920 and 1921. The data indicated in Table I disclose the fact that mosaic mottling was obtained only when juice from a mosaic plant was introduced into I ScHULTz, E. S., FoLSOM, Donald, Hildebrandt, F. Merrill, and Hawkins, Lon A. investigations ON THE MOSAIC DISEASE OF THE IRISH POTATO. In Jour. AgT. Research, v. 17, no. 6, p. 247-274, pi. A-B (col.), 25-30. 1919. Literature cited, p. 272-273. ScHULTz, E. S., and Folsom, Donald, transmission of the mosaic disease of irish potatoes. In Jour Agr. Research, v. 19, no. 7, p. 3x5-338, pi. 49-56. 1920. ScHULTZ, E. S., and Folsom, Donald, leafroll, net-necrosis, and spindling-sprout of the irish POTATO. In Jour. Agr. Research, v. 21, no. i, p. 47-So, pi. 1-13 1921. Literature cited, p. 7S-80. Oct. IS, I92I Transmissible Mosaic Disease of Cabbage 175 a plant of the same or a closely related species.^ No mosaic mottling appeared on any of the cruciferous plants inoculated with juice from mosaic potato. With a more adequate supply ofcrucifer mosaic mate- rial and repeated applications it is probable that every plant treated would have developed mosaic mottling, such as has frequently been obtained with mosaic potato juice inoculations on the Irish potato.^ The first mosaic mottling was observed from 20 to 30 days after inoc- ulation, which also corresponds very closely with the incubation period for mosaic of Irish potato. The results in Table I also disclose successful inoculations on plants in different species of Brassica. Further evidence on this interspecific infection is presented in Table II on transmission by means of aphids. TRANSMISSION WITH APHIDS Since aphids were found on every mosaic plant examined in the field and on account of the fact that these insects have been found to transmit mosaic of tobacco,^ spinach blight/ and mosaic of potato,'' experiments were carried on with these insects. Aphids belonging to Myzus persicae Sulz. were used in this investigation. These insects were originally collected from the morning-glory and transferred to healthy turnip and mustard plants on which they were cultured while confined under cages until needed for inoculation. Neither the morning-glory nor the turnip or mustard plants on which these insects fed before being transferred to mosaic Chinese cabbage and turnip developed mosaic mottling. This indicates that the morning-glory apparently was free from mosaic, at least from the type which could infect the crucifers used in this experiment. When the healthy plants for inoculation had developed from five to eight leaves, aphids were transferred from the cultures to mosaic plants, where they were allowed to feed for a few days before they were intro- duced to the healthy plants. All inoculated plants also were confined in cages so as to prevent dispersal from one species to another. After the aphids had fed from 7 to 14 days on the inoculated plants they were killed by tobacco fumigation in a fumigation chamber. These plants were now allowed to grow without cages in a greenhouse where fumiga- tion was practiced at regular intervals for the control of aphids. Since mosaic mottling developed from 12 to 30 days after these insects were killed by fumigation, mosaic mottling can not be attributed simply to the mechanical injury produced by the aphids. This fact is further J Gardner, Max W., and Kendrick, James B. turnip mosaic. In Jour. Agr. Research, v. 22, no. 3, p. 123-124, I pi. 1921. - ScHXJLTz, E. S., Foi^oM, Donald, Hudebrandt, F. Merrill, and Hawkins, I,on A. op. ot. ' Allard, H. A. THE MOSAIC disease OF TOBACCO. U. S. Dept. Agf. Bul. 40, 33 p. , 7 pi. 1914. * McCuNTOCK, J. A., and Smith, Loren B. true nature of spinach-blight and relation of insects TO ITS transmission. In Jour. Agr. Research, v. 14, no. i, p. 1-60, pi. A (col), i-ii. 1918. 6 Schultz, E. S., Folsom, Donald, Hildebrandt, F. Merrill, and Hawkins, Lon. A. op. ciT. 176 Journal of Agricultural Research voi. xxn. no. 3 confirmed by the control plants which remained free from mosaic mottling after aphids taken from healthy plants had fed upon them. The results secured from inoculation with aphids are presented in Table II. Table II. — Transmission of mosaic of m,ustard, pe-tsai, and turnip by means of aphids Variety inoculated. Date of inocu- lation. Ap- proxi- mate num- ber of aphids trans- ferred. Source of aphids. Num- ber of plants inocu- lated. Date of first symptoms. Num- ber of plants mo- saic. « Per- centage mo- saic. Southern Prize tur- nip. Do Jan. 12,1920 Jan. 26, 1920 Jan. 12,1920 Jan. 22,1920 Feb. 24, 1920 Mar. 5, 1920 Mar. 10,1920 Jan. 12,1920 Jan. 19, 1920 do 5° SO 5° 12 100 100 50 50 100 100 50 so 50 25 50 2S 12 2S 25 SO Mosaic Southern Prize turnip. do 2 3 3 3 2 3 I I 3 I 3 I s 3 S 3 3 I 5 S Feb. 3 , 1920 Feb. 20, 1920 3 3 0 3 3 2 I I 3 0 3 0 5 3 3 2 3 I 3 0 100 Do Healthy turnip. . . Mosaic Southern Prize turnip. Mosaic mustard. . . do Purple Top turnip.. Seven Top or South- Prize turnip. Do Feb. 14, 1921 Mar. 20, 1930 Mar. 31,1920 Apr. 2,1920 Feb. 11,1920 Feb. 19, 1920 100 100 Do do do Do do Do Healthy mustard . Mosaic mustard . . . Healthy mustard. . Mosaic mustard . . . Mosaic Southern Prize turnip. do Do Jan. 26, 1920 . . .do Do Do Mar. 12,1921 Jan. 29,1921 Feb. 19, 1921 Feb. 8, 1921 Jan. 15, 1921 Jan. 29, 1921 Feb. 19,1921 Mar. 2,1921 Apr. 2,1921 Feb. 20, 192 1 Mar. 25,1921 Do Do 40 Do do Do Mosaic Chinese cabbage. do Feb. 14,1921 Feb. 20,1921 Mar. 38,1921 Do Do do 60 Do Mosaic Green Mountain potato. « Date of last observation, Apr. 2, 1920 and 1921. From the data indicated in Table II it is evident that aphids transmit mosiac of the crucifers in question between different species as well as among plants of the same species, as was suggested in Table I on juice inoculations by means of rubbing. It will also be noted that the period in which the first mosaic mottling appeared corresponds very closely to that obtaining with mosiac diseases of other plants. As with the plants inoculated by rubbing, the plants inoculated by means of aphids devel- oped the mosaic symptoms only on the younger leaves. Mosaic symp- toms on the inoculated plants were like those which were observed on mosaic lots taken from the field. Since turnips from mosaic plants taken from the field continued to produce mosaic foliage it is apparent that such plants become a source of infection if planted near susceptible varieties. Mustard seed from mosaic mustard plants apparently develop healthy seedlings. This was observed in loo seedlings, which were grown from seed from mosaic mustard in the fall of 1920; in this test every seedling was free from mosaic mottling. oct.is. I92I Transmissible Mosaic Disease of Cabbage 177 SUMMARY From these preliminary observations and experiments it appears that the crucifers here mentioned may be added to the Hst of plants suscepti- ble to mosaic, a disease whose cause has not been discovered but which can be transmitted from mosaic to healthy plants by direct transfer of juice as well as by means of aphids which apparently are very effective natural agents in the dissemination of this disease. PLATE B I. — Leaf from healthy turnip, control to mosaic turnip in figure 2. 2. — Leaf from mosaic turnip, mosaic induced by aphids transferred from mosaic turnip plant. 3. — Leaf from healthy mustard, control to mosaic mustard in figure 4. 4. — Leaf from mosaic mustard, mosaic produced by aphids from mosaic mustard. (178) A Transmissible Mosaic Disease of Chinese Cabbage Plate B Journal of Agricultural Research k Vol. XXII, No. 3 Transmissible Mosaic Disease of Cabbage Plate 22 Journal of Agricultural Researcli Vol. XXII, No. 3 PLATE 2 2 A. — Healthy turnip plant, control to B. Aphids from healthy turnip were allowed to feed on this plant. Planted the same time as B. B. — Mosaic on turnip plant, variety Seven Top or Southern. Mosaic mottling appeared 26 days after the introduction of aphids from a mosaic Southern turnip plant. C. — Leaf from A, healthy. D, E. — Two mosaic leaves from B. Mosaic mottling and ruflSing apparent on the diseased leaves. 65508°— 21 5 I PLATE 23 A. — Healthy mtistard plant, control to B. Planted the same time as B. B.^ — Mosaic on mustard plant produced by transferring aphids from mosaic mustard. Distinct mosaic mottling was noted 28 daj^s after introduction of aphids. Transmissible IVlosaic Disease of Cabbage Plate 23 Journal of Agricultural Research Vol. XXII, No. 3 Transmissible Mosaic Disease cf Cabbage Plate 24 Journal of Agricultural Research Vol. XXII, No. 3 PLATE 24 Leaves from plants shown in Plate 23, A, B. A, B. — Mosaic leaves showing mottling and ruffling. C— Healthy leaf. ADDITIONAL COPIES OF THIS PUBLICATION MAT BE PEOCUKED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 15 CENTS PER COPY Subscription Price, $3 Per Year V Vol. XXII OCTOBER 22, 1921 No. ^ JOURNAL OP AGRICULTURAL RESEARCH coN'rE>N'rs Page Flora of Corn Meal --------179 CHARLES THOM and EDWIN LeFEVRE ( Contribution from Bureau of Chemistry ) Hopkins Host-Selection Principle as Related to Certain Cerambycid Beetles ------- 189 F. C. CRAIGHEAD ( Contribution from Bureau of Entomology) Notes on the Organic Acids of Pyrus coronaria, Rhus glabra, and Acer saccharum - - - - - - 221 CHARLES E. SANDO and H. H. BARTLETT ( Contribution from Bureau of Plant Industry and University of Michigan ) Fertility in Shropshire Sheep - - - - - -231 ELMER ROBERTS ( Contribution from Illinois Agricultural Experiment Station ) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES ^W:A.SHlNaTON, D. c. 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. ALIyEN Chief, Office of Experiment Stations CHARLES L. MARLATT Entomologist and Assistant Chief, Bureau of Entomology FOR THE ASSOCIATIOII J. G. UPMAN Dean, State College of Agriculfurei 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 Diredor: Agricultural Experiment Station; Tht Pennsylvania State College All correspondence regarding articles from the Department of Agriculture should be addressed to Karl F. Kellennan, Journal of Agricultural Research, Washington, D. C. All correspondence regarding articles from State Experiment Stations should be addressed to J. G. Lipman, New Jersey Agricultxiral Experiment Station, New Brunswick, N. J. joim OF AGrtrmL re Vol. XXII Washington, D. C, October 22, 1921 xM'^j^^ 4 FLORA OF CORN MEAL '^ >".:-.*. By Charles Thom, Mycologist in Charge, and Edwin LeFevre, Scientific Assistant, Microbiological Laboratory, Bureau of Chemistry, United States Department of Agri- culture INTRODUCTION Corn meal as it comes from the mill carries the mycelia of certain fungi which infect unground grain. In addition, numerous species of molds and bacteria, present in spore form as contaminations upon the surfaces of sound kernels or as saprophytes in partially spoiled grains, are recov- erable by routine cultural examination of the finished meal. Many experiments, extending over several years and including the work of vari- ous members of the Microbiological Laboratory, show that certain groups of organisms are practically always abundant in such cultures. Other species are usually present, but in smaller numbers, and many forms are obtained occasionally as accidental contaminations. In undertaking to study this complex flora, it may be possible to determine by routine cul- ture the species represented and something of their relative abundance in the sample, but the list so obtained gives little information as to the relative importance of the individual species as causes of spoilage in the product. The culture media commonly used in such routine examination of food- stuffs present conditions for the growth of microorganisms which differ greatly from those found in corn meal. The nutrients used in preparing such media are selected because they are readily assimilable to most organisms. These nutrients appear in solution or in jelly-like masses which contain high percentages of moisture. Corn meal, on the other hand, presents a range of composition, according to Winton and his associates {8),^ approximately as follows: Moisture, 10 to 18 per cent, but under usual commercial practices ranging from 12 to 15 per cent; protein, 5 to 10 per cent; fat, i to 5 per cent, according to the method of milling; nitrogen-free extract, including starch and sugar, 68 to 78 per cent. Of the nitrogen-free extract, sugars constitute perhaps 3 per cent, and gums and dextrin, some of which are readily fermentable,, perhaps an equal quantity. In dealing with this product as a substratum for organisms, the percentage of water found is an important limiting I Reference is made by number (italic) to " lyiterature cited," p. i88. Journal of Agricultural Research, Vol. XXII, No. 4 Washington, D. C. Oct. 22, 1921 aab Key No. E-16 (179) i8o Journal of Agricultural Research voi. xxii, no. 4 factor. Obviously this product, 'even at its maximum moisture content, presents a marked contrast to laboratory media as usually prepared. Nevertheless, corn meal has been so often found an unstable product that it is commonly milled only for consumption within a few weeks or by methods intended to eliminate the most readily fermentable portions of the grain. Under ordinary conditions of handling, spoilage in this product ap- pears in one of the following forms: Souring, rancidity, mustiness, the formation of clumps or balls, extensive concretions which may involve the solidification of an entire bag, or the formation of a hard, cylindrical outer mass with the center loose and mealy. Heating occurs only in the wettest samples. Much com meal, if held beyond a very short period, develops a musty, moldy, or sour odor and shows occasional balls or masses of meal held together by mold, which bring about losses in palatability and market quality in the product. Such changes as rancidity and the formation of extensive concretions into moldy masses are so obviously due to high moisture content and involve such losses that they have been almost eliminated from commercial practice. When losses occur the meal is found to carry more than a critical mois- ture percentage. This may be due either to milling com which is in- sufficiently dried or to the storage of the meal under conditions which will maintain a moisture content above the danger point. For the samples used in all series reported here this figure was approximately 13 per cent (2). CULTURAL EXAMINATION In routine cultural examination reported here, plain agar was used for bacterial counts, wort agar for mold counts, and dextrose-litmus shake agar to determine acid, gas, and anaerobic growth. The presence of particular organisms was determined by tlie use of special methods on special media. Experimentation covered a range wide enough to justify the restriction of routine cultures to the media already noted. After comparative study of many series of cultures. Table I is intro- duced as giving a group of cultural results fairly typical for commercial meal in sound, merchantable condition. The nine samples reported were purchased in different retail stores of Washington, D. C, during October and November, 1920. Four of them were yellow and fairly coarsely ground. The white meals were softer or more finely ground. All were bolted. All showed by microscopic examination traces of both bran and germ, although these portions were scanty in certain samples. The history of the samples was not obtained. These samples were sound in appearance and odor. There was no evidence of the multiplication of microorganisms. Among the bacterial colonies micrococci, members of the mesentericus and of the colon- aerogenes groups were characteristically present. Special tests in cab- Oct. 22, I92I Flora of Corn. Meal I8i bage juice showed, in four of the nine samples, the presence of lacto- bacilH with the morphology and cultural characters of the organism of pickle and sauerkraut fermentation. N>) bacterial colonies were obtained in plain agar from two of the samples. A duplicate of sample 9 proved equally negative. Mold colonies were obtained in all samples. These represented in varying proportions Aspergillus repens De Bary, A. niger Van Tieghem, A. fiavus hink, Fusarium, various mucors, and unidentified colonies. Table I. — Results of cultural examination of commercial corn meals Sample No. Bacteria per gram on plain agar. Molds per gram on wort agar. Bacteria per gram on dextrose- litmus agar. Acid colonies. I 10, 000 TO. 000 16, 000 Per cent. 50 5, 000 I, 000 t;=;, 000 T3. 000 •^ 42, 000 13, 000 10, 000 8, 000 60 60, 000 70, 000 5,000 10, 000 400, 000 20, 000 3,000 11, 000 5,000 50 60 6 7 8 10, 000 4, 000 3,000 0 30 A more extensive series of studies was conducted in cooperation with the Plant Chemical Laboratory of this bureau. The general results of this experiment are described elsewhere (2). In brief, during the spring of 1920, a series of bags of meal were prepared for this storage experiment from com bought by the mill in the regular course of business. This grain, while sold as No. 2, was obviously wet and barely passable as a fair product. Infected and even badly decomposed ears were not uncommon among the ears of corn received in bulk. Although the lots of meal included were milled at water contents varying from 12.7 to 16.18 per cent, the conditions of storage were such that no spoilage determinable by the senses took place. Cultures were made from the meal as freshly ground in April, then, beginning May 5, once each week until July. In all these cultures no evidence of multiplication of either mold or bacteria was found. It was, therefore, possible to follow the relative numbers of viable organisms in the various groups from the time of grinding through the four months of storage. In the freshly milled samples the average count of colonies of bacteria upon plain agar was about 1,000,000 per gram of meal, with variations from 600,000 to 1,600,000. Upon wort agar the count of mold colonies averaged about 100,000 per gram of meal, with variations in different samples from 70,000 to 160,000. Of the bacterial colonies observed about 60 per cent were acid producers. 1 82 Journal oj {Agricultural Research voi. xxn, no. 4 For comparison a special series of samples were prepared by adding 5 per cent of meal made from com markedly rotted with Diplodia and Fusarium. In the freshly grouted meal of this series the bacterial count upon plain agar was about 2,600,000. The count of mold colonies upon wort agar was about 1 10,000. About 70 per cent of the bacterial colonies were acid producers. After storage for approximately one month (May 20 and 21) samples from a particular lot of five bags of the regular meal showed an average count of 108,000 bacterial colonies and 15,000 molds. Samples from the same bags on June 30 showed an average count of 12,600 bacterial colonies, and 7,600 mold colonies. Without placing emphasis upon exact figures, these cultural results are fairly typical of the mass of figures obtained from cultures made weekly from representative samples involving the whole series of 88 bags of meal. These figures are readily comparable with those obtained from commercial samples (Table I). Discrepancies which occur may perhaps be accounted for by the fact that samples 3, 4, and 5 were evidently the product of local mills, sold fairly quickly after milling, while samples 2, 7, and 9 were clearly the product of special processes and handled under conditions involving much slower distribution. In this lot of meal, therefore, the conspicuous change due to storage was the drop in the number of viable organisms to about i per cent of the original number of bacteria and perhaps 10 per cent of the original number of molds. The larger part of this decrease occurred during the first six weeks, with a slow reduction throughout the succeeding periods. In connection with the study of these figures, data obtained by Thom and Stiles (unpublished) in examining Winton's {8) samples ^ in 19 14 were restudied and compared with the results here considered. Win- ton's com meal varied in initial moisture content from 19.27 to 10.79 per cent. In those lots of meal (A, B, and C) carrying moisture mark- edly above 13 per cent, the evidence of multiplication of molds and bacteria was clearly discernible. Musty odors and balls of meal held together by mold were present in every sample. In cultures, the count of colonies of molds and bacteria reached 13 million in the wettest lot. Of these several million were Aspergillus flavus. The predominant or- ganisms were molds rather than bacteria, but there was fairly clear evidence of some bacterial multiplication at the higher water percentages. In the roller-gi'ound samples of lots D, E, F, which did not spoil and whose water percentage was near to or less than 13, the total counts found by Stiles approached very nearly those already given in this paper. These examinations began too late in the storage period to show that part of the bacterial flora which dies off rapidly. The stored samples still showed some acid organisms, but micrococci and aerobic spore ' Samples of the meal studied were examined bacteriologically by G. W. Stiles, formerly of the Bureau of Chemistry, and for mold activity by Charles Thom, then in the Bureau of Animal Industry {8, p. zs)- Oct. 22, 1921 Flora of Corn Meal 1 83 formers of the mesentericus group formed the majority of the bacteria obtained. In the lots with moistm^e content decreasing toward 13 per cent there was progressive reduction in the number of active species of molds. Bxtensive experimentation showed clearly that Aspergillus repens was the agent which formed the balls of meal loosely held together with mold hyphae, which characterized meal containing barely enough water to start spoilage. In another series of experiments A. flavus began to be active only in samples containing about 16 per cent of water. Yeasts, mucors, and Penicillia were reported by Stiles only in the sample carrying about 19 per cent of water. During the examination of the preliminary samples in the 1920 ex- periment, an effort was made to identify the groups or actual species represented. As a matter of routine, inoculations were made from each flask prepared for diluting plates (consisting of 5 gm. of the meal to 45 cc. of sterile water) into the following media: Plain milk, gelatin, and litmus lactose broth. Smears were also made on Endo's agar in each instance. In every case there was prompt coagulation of the milk, with extrusion of whey, but no digestion of curd. Pink rings formed near the surface. Gelatin was liquefied in every instance, and acid and gas formed in all broth tubes. Growth in Endo's media indicated the presence of Bacterium aerogenes Escherich. Further cultural studies showed that Bad. aerogenes was the predominant bacterial species pres- ent in all these samples. This predominance was maintained throughout the series of examinations made. Microscopical examinations of smears made in each case, however, showed the presence of spore-bearing bac- teria, especially the mesentericus group, and micrococci of various kinds. Dextrose agar tubes often contained colonies growing deep in the media, indicating the presence of anaerobic bacteria. Yeasts were found in all samples, their growth being largely of the mycoderma type. The plates showed many mold colonies. Various mucors, species of Fusarium, Aspergillus flavus, A. niger, and occasional green Penicillia were ob- served. The species of molds present on the plates varied from period to period and with the sample. Molds were always more numerous on plates made from meal to which Fusarium and Diplodia had been added, but growth on these plates did not show dominance of these particular forms. Evidence of the effect of bolting upon the abundance of organisms was furnished in the 1920 experiment by the examination of samples of two series of five bags each, representing a single lot of meal, one-half of which was bolted and the other half unbolted. The bolting to which these samples were subjected removed a considerable part of the bran but little of the germ from the meal. After one month of storage, the bolted meal showed an average of 34,000 bacterial colonies and 20,000 mold colonies. The unbolted samples showed 108,000 bacterial colonies 184 Journal of Agricultural Research voi. xxii. no. 4 and 15,000 molds. This observation was confirmed by a restudy of Stiles's unpublished examination of Winton's (8) samples. Of every lot of com handled, part was ground in a stone mill without sifting or bolting and part was carefully " degerminated " and "roller" ground. In the bolting process all of the bran was taken out, and many of the samples consisted almost completely of homy endosperm. In that part of this series made up of meals in which no multiplication of micro- organisms occurred, bolting consistently reduced the cultural count of microorganisms below that of the stone-ground meal. Frequently the number found in the bolted meal was less than one-tenth of that in the stone-ground meal. By removing the bran, bolting takes away the largest area of contami- nation with saprophytic organisms. The tip of the kernel and the germinal area carry the majority of the infections found in com. Study of many samples of com over a period of years shows that invasion of the germinal area by molds is not uncommon in com which has not been fully matured or has not been promptly and thoroughly dried. Sam- ples have frequently shown the invasion of the germ in every kernel by Aspergillus repens. Recently samples representing a bulk shipment have shown nearly every grain to contain one or the other of two species of Penicillium. Meal therefore may be so milled and sifted or bolted as to remove the larger part of all contaminations, as well as those mold infections which do not involve general disintegration. The cleaning process before milling removes the grains thoroughly rotted by Fusarium and Diplodia. Corn has still been seen going into the rolls of a mill in which the low grade of the stock could not have been concealed if it had passed through a stone mill without being bolted. The product, however, was going into human food without showing tangible evidence of the low quality indicated by the unground grain. In other words, the fractional milling of low-grade grain makes possible such separation as turns the infected portions of the grain into oil stock or cattle feed and the solid or homy portions which are less obviously damaged into meal. The literature of maize deterioration is reviewed by Alsberg and Black up to 19 13 (7). The activity of Fusarium and Diplodia as causes of rotting in ear com was discussed by Burrill and Ban-ett (5) and that of Diplodia alone by Heald, Wilcox, and Pool (4). More recently McHargue (6) has studied the activities of certain fungi and their relation to commercial conditions in the handling of the product. Excessive moisture in the grain is regarded as the limiting factor in most cases of such spoilage. The factor of temperature must not be over- looked. The moisture content limit may be materially increased during the winter without evidence of the activity of microorganisms. The agents of spoilage in all the cases under review were primarily molds. The results already given in this paper harmonize in general with those Oct. 22, I92I Flora of Corn Meal 185 of McHargue. It has been possible, however, to go farther and indicate more clearly the groups of organisms regularly present and to record the conditions under which certain of them become active factors in spoilage. Routine mass or dilution cultures show that certain molds are recov- erable from practically all samples of meal. Among these are Rhizopus nigricans Ehrenberg and some of the mucors which frequently overgrow plate cultures within two days of incubation, although they probably are present only in spore form in the meal. Syncephalastrum, belonging to the same group, is not uncommon. Aspergillus flavus and A. niger are only occasionally visible factors in the infection of the unground grains, but they always appear as rapidly growing colonies in the mass or dilution cultures made. The brown masses of A. tamari Kita are commonly found with A. flavus. A. fumigatus Fres. and A. terreus Thorn. are frequently present but are quickly overgrown by the more active species already mentioned. A. repens, though practically always pres- ent, can be found only by careful search in the presence of these rapidly growing forms. Several strains of Penicillium are found in meal cultures. Peniciilia of the group with submerged orange mycelia and of the Citromyces group are probably most common. Penicillium expansum Link is reported by McHargue. P. oxalicum. Thom and Currie is found in many samples of meal, but rarely in miscellaneous cultural work. Strains related to P. luteum Zukal and P. purpurogenum O. Stoll are frequently present but usually indicate soil contamination rather than active growth in the corn or meal. One sample of corn rotted by a member of this series has been examined, but the conditions shown clearly indicated that the product had contained high percentages of moisture at the time the rotting occurred. Colonies of Fusarium develop from almost every sample of meal. Infections of this group are so abundant that conidia or grains of meal containing living hyphae are rarely absent. Cladosporium and Alter- naria are frequently found but represent spore contamination rather than infection. The other organisms observed in culture from time to time appear to represent excessive contaminations with spores due to unfavor- able conditions in the handling of the product, or, in certain species, to actual infection of the grain locally by the mold. The bacteria found in the fresh samples here considered were pre- dominantly Bacterium aerogenes. Certain other organisms have been regularly obtained in culture. When the necessary moisture is present, souring is so characteristic of the product that Round and Gore (7) found the addition of 3 per cent of fresh meal an adequate starter to insure the dominance of lactic acid fermentation in potato silage. Lacto-bacilli were present in four of the nine lots reported in Table I. According to unpublished records in the Microbiological Laboratory, Round found organisms of this group abundant also in fresh meal, but occasionally 1 86 Journal of Agricultural Research voi. xxn. no. 4 absent in old meal or meal made from old and thoroughly dried corn. Micrococci are constantly encountered in culture but have not been typed. Aerobic spore formers of the mesentericus group are always present, and in spore form they constitute the larger part of the living bacteria in some meals after long storage. This was clearly demonstrated by a series of experiments upon the possibility of producing a sterile meal with steam, dry heat, or both (unpublished cultural results of Ruth B. Edmondson). The spores of this group survived more heating than could be applied under practical working conditions to the product. Aside, however, from meal so wet as to be unmarketable, these experiments show no evidence of bacterial activity. One sample of apparently sound yellow meal showed the presence of Bacillus niger Migula in such extensive numbers that masses of meal placed upon culture media were promptly overgrown and with the agar turned bluish black with this species. The meal was contributed by Dr. S. S. Adams, of Washington, D. C, who reported the feces of a child apparently well to have been blue when fed this meal. When, however, com or meal is bottled and incubated at laboratory temperature (20° to 30° C), those species capable of developing under the conditions presented show active growth. In the authors' series such growth was not detected by physical appearance in products carry- ing less than 13 per cent of moisture. Certain stone-ground samples of Winton's series (8) showed some evidence of mold activity below that figure. Measurable changes in quality certainly occur in such meals during storage. Some experimental results have suggested the possibility that these changes in such meal are due to the distribution of infected material throughout the mass by the grinding of infected corn. This conflicts with the current trade belief that the natural enzyms of the germinal area are the chief causes of such deterioration, but reflects the findings of HofTer (5) and his coworkers that even selected seed com may be extensively infected. Examinations of commercial samples in the Microbiological Laboratory have shown extensive development of molds within the grain itself in com of other than the higher grades. In samples carrying 14 to 15 per cent of water the formation of balls and concretions in the meal begins to be evident. The principal agent in their formation appears to be Aspergillus repens, although many diffi- culties are encountered in fixing a minimum moisture percentage for the activity of this species. Changes involving the development of mold mycelium in the meal begin within the limit of 13 to 15 per cent of moisture. Incubation at 20° to 30° C. merely accelerates changes which would progress more slowly in colder places. Moist chamber experiments with meal inside this range of water content show the presence of active mycelia of more than a single species, but principally Aspergillus repens. When the percentage of moisture reaches 16, sev- eral species are clearly able to grow. Special studies with Aspergillus Oct. 22, I92I Flora of Corn Meal 187 flavus show that very little development of this species occurs below 16 per cent, but that from 16 per cent upward development of this species rapidly increases and the number of forms capable of growing rapidly rises. Among the characteristic saprophytic molds observed under these conditions, in about the order of their abundance under the con- ditions, are Aspergillus repens, Aspergillus flavus, Actinomyces sp., Penicillium sp. and Citromyces sp., Fusarium sp., Aspergillus candidus, Aspergillus ochraceous Wilhelm, Aspergillus iamari, and Aspergillus niger. Bacterial activity appears to be a concomitant of the disintegration due to mold action in such rotting processes as this. As indicated by Bailey and Thom (2, Table I), active disintegration by molds is accompanied by an increase in the water percentage of the sample. Bacteria follow rather than initiate the process in the samples studied, thus becoming a small factor in the merchantable product. Throughout this investigation a close correspondence has been observed between the flora of deterioration in unground com and the flora of the milled product. SUMMARY In seeking possible causes for the well-recognized instability of com meal, cultures show considerable numbers of molds and bacteria to be generally present. Among these the following species of molds were characteristic of many series of cultures: Fusarium sp., Aspergillus repens, A . flavus, A . tamari, A . niger, Citromyces (or Penicillium section Citromyces) sp., Penicillium oxalicum, P. luteum varieties, Mucor sp., Rhizopus nigricans, and Syncephalastrum sp., together with various yeasts and yeast-like fungi. Among bacterial groups, the colon- aerogenes group and lacto-bacilli were most abundant in fresh meal. Aerobic spore formers and micrococci were always present and persisted in the stored product. Within the range of composition found in merchantable meals, no bacterial activity was detected. Only one grade of unbolted meal showed signs of mold development below 13 per cent of moistiu-e. Above 13 per cent moisture, Aspergillus repens begins to be an active agent of spoilage somewhere between 13 and 15 per cent of moisture, varying with the form of milling practiced. Several other species of molds are active in meal containing 16 per cent moisture; and numerous forms, including some bacteria, develop when 18 to 20 per cent of moisture is found. Many samples of corn are found to carry extensive infections with Fusarium, Diplodia, Aspergillus repens, or Penicillium, especially in the germinal area and in the tip of the kernel. These sections of the kernel are removed in varying degrees by different milling systems. The bolted meals examined show a corresponding reduction in count of viable organisms as shown by culture. 1 88 Journal of Agricultural Research voi. xxu, no. 4 LITERATURE CITED (i) Alsberg, Carl L., and Black, Otis F. 19 13. CONTRIBUTIONS TO THE STUDY OF MAIZE DETERIORATION. BIOCHEMICAL AND TOXICOLOGICAL INVESTIGATIONS OF PENICILLIUM PUBERULUM ANI> PENiciLLiUM STOLONiFERUM. U. S. Dept. Agr. Bur. Plant Indus. Bui. 270, 48 p., I pi. Bibliographical footnotes. (2) Bailey, L. H., and Thom, C. 1920. SOME observations of corn meal in storage. In Operative Miller, V. 25, no. 12, p. 368-371, chart A-D. (3) BuRRiLL, Thomas J., and Barrett, James T. 1909. ear rots op corn. 111. Agr. Exp. Sta. Bui. 133, p. 63-109 incl. pi. i-ii, I col. pi. (4) Heald, F. D., Wilcox, E. M., and Pool, Venus W. 1909. THE life-history AND PARASITISM OF DIPLODIA ZEAE (SCHW.) LEV. Itl Nebr. Agr. Exp. Sta. 22nd Rept. [1908], p. 1-19 incl. 10 pi. Biblio- graphy, p. 7. (5) HoFFER, George N., and Holbert, J. R. 1918. SELECTION OF DISEASE-FREE SEED CORN. Ind. Agt. Exp. Sta. Bui. 224, 16 p., 20 fig. (6) McHargue, J. S. 1920. THE cause OF DETERIORATION AND SPOILING OF CORN AND CORN MEAL. In Jour. Indus, and Engin. Chem., v. 12, no. 3, p. 257-262. (7) Round, L. A., and Gore, H. C. 1916. A PRELIMINARY REPORT UPON THE MAKING OF POTATO SILAGE FOR CATTLE FOOD. In Proc. 3rd Ann. Meeting, Potato Assoc. America, p. 75-79. (8) WiNTON, A. L., Burnet, W. C, and Bornmann, J. H. I915. COMPOSITION OF CORN (mAIZE) MEAL MANUFACTURED BY DIFFERENT PRO- CESSES AND THE INFLUENCE OF COMPOSITION ON THE KEEPING QUALI- TIES. U. S. Dept. Agr. Bui. 215, 31 p. HOPKINS HOST-SELECTION PRINCIPLE AS RELATED TO CERTAIN CERAMBYCID BEETLES By F. C. Craighead Specialist in Forest Entomology, Bureau of Entomology, United States Department of Agriculture INTRODUCTION In connection with the reported dying of lodgepole pine (Pinus con- torta Loud.) over extensive areas in northeastern Oregon caused by the mountain pine beetle {Dendroctonus moniicolae Hopk.) and the threat- ened invasion by this beetle of the adjacent areas of yellow pine {Pinus ponderosa Laws.), detailed investigations were made by the Bureau of Entomology under the direction of Dr. A. D. Hopkins. Manuscript reports of these investigations, submitted in the summer of 19 lo, showed that the infestation by the beetle in the lodgepole pine was so extensive that there was no hope of controlling it, but that the comparatively small amount of infestation in the valuable stands of yellow pine was such as to warrant the undertaking of control, provided the beetle did not migrate from the lodgepole pine to the yellow pine. In a letter from Dr. A. D. Hopkins under date of July 30, 19 10, relating to a manuscript report of Mr. H. E. Burke, the following statement occurs which appears to be the first written reference to the host-selection principle : The more I consider the various features of the problem, the more I am convinced that it is entirely practicable to protect the yellow pine, even if we leave all but the immediately adjacent lodgepole pine to take care of itself. This is based on my belief that the majority of the broods of the beetles which have been breeding in the lodgepole will continue to confine their attack to that species, and gradually diminish with the reduced supply and their increased struggle to adapt themselves to the yellow pine. I may be wrong in this, but it is a matter worthy of careful considera- tion. Remember, that in all these years, there has been no marked or general migra- tion of beetles from lodgepole to the yellow pine. Therefore, it appears that the broods which are most dangerous to the yellow pine are those which have been breed- ing in it, and that these are the broods we will have to deal with mainly in our efforts to protect the best bodies of yellow pine. The control operations that were carried on during the following year, 191 1, were confined mainly to the yellow pine area. In manuscript reports by Messrs. W. D. Edmonston and George Hofer on a special examination of the yellow pine and lodgepole pine areas in the summer and fall of 1913, it is stated by Edmonston: In 1912 the examination of the areas on which insect control work was carried on during April, May, and June, 191 1, showed an average reduction of the infestation on the entire area, 76,430 acres, of close to 85 per cent. Journal of Agricultural Research Vol. XXII, No. 4 Washington, D. C. Oct. 22, 1921 aac (1S9) Key No. K-ioa 190 Journal of Agricultural Research voi.xxn.No. 4 Examinations made this season, 1913, show a still greater reduction in the infesta- tion; in fact, the infestation is so light tliat it is actually less on the treated areas than it is throughout any other area on this Forest. and — There was no reoccupation of the treated areas by broods from the lodgepole infested trees at higher elevations. and by Hofer — As we reached the summit near the North Powder Peaks we attained an altitude of 8,000 feet; the elevation at the Sheep Ranch is about 4,000 feet. From the summit of this divide for a distance of 10 miles north, 10 miles east, and 16 miles west we noted heavy infestation, both old deadings and the new work also, in both the lodge- pole pine and white bark pine, especially on both slopes of Antone Creek. No new infestation was found on the treated areas on Anthony Creek, Camp area. This seemed to furnish substantial evidence that the principle would hold. The principle as defined by Dr. Hopkins ^ is that an insect — species which breeds in two or more hosts will prefer to continue to breed in the host to which it has become adapted. In order to secure further evidence relating to this principle, the writer, after consulting with Dr. Hopkins, began a series of experiments in 19 14 with insects which infest two or more species of wood. The wood-boring Cerambycidae, or long-homed beetles, offered material which was very well adapted to the conduct of such experiments. Many species were easily available which exhibit great diversity in their selection of hosts in nature, as illustrated by those breeding exclusively in a single species of plant and those apparently attacking almost any wood. This varia- tion in host habits at once brought up the following questions : Will those species confined to a single host live in any other, and do the individuals coming from a certain plant of those species breeding in a variety of hosts select the same species of plant on which to oviposit ? Again, if such is the case, how do these host strains originate in nature? As these experiments progressed new problems came up demanding a broadening of the experiments from- year to year until, during the season of 1918, over 100 individual experiments were in progress. Fourteen species of insects and 21 species of plants were used, combining to form 45 host strains. It was thought desirable to conduct experiments on more species rather than more intensive experiments on a few species. It will be noted that certain experiments were not carried as far as others, due to the fact that time was not available or due to the absence of the writer at the critical time. At present several points remain to be con- clusively settled, and investigation of these will be continued another year or so. Nevertheless it is believed that sufiicient data have been accumulated to show definitely the extent to which the influence of the host applies to these insects. 1 Hopkins, A. D. economic investigations of the scox,ytid bark and timber beetles of north AMERICA. In U. S. Dept. Agr. Program of Work, 1917, p. 353. 1916. Oct. 22. I92I Hopkins Host-Selection Principle 191 HISTORICAL Very few references to the adaptation of insects to their host plants or the variation in their selection of host plants can be found. The most important paper dealing with the subject is that by Pictet.^ This author shows by several examples, (Ocneria) Porthelria dispar for one, that caterpillars of the second and third generations may be made to change their preferred food plants and that the adults reared from them exhibit changes in size and coloration. This paper is reviewed, and supplemented with reports of corresponding observations, by Schroder,^ who in a previous article ^ showed that even nidification (in Gracilaria stigmatella F.) and habits of feeding, combined with changes in reproduction (in the beetle Phratora vitellinoe L.). can be changed and that these acquired characters are transmitted spontane- ously from the third generation. In 1907 and 1908 Paul Marchal ^ succeeded in transferring numerous specimens of Lecanium corni Bouche from the peach (Amygdalus persica Linn.) to the black locust {Rohinia pseudacacia Linn.). Eggs hatched and larvae developed on the new host plant, spreading out over the leaves in large numbers, and in the fall migrating from the leaves to the wood for hibernation. In the summer of 1908 the insects com- pleted their development and had then the large size, deep coloration, and characteristic appearance of the insect described by Douglas as L. robiniarum, the attacks of which on the black locust had been severe in several European localities. This indicated that L. robiniarum was only a race of L. corni, resulting from individuals that had become transferred in some manner from the peach to the introduced American black locust. Dr. Marchal found great difficulty in reestablishing on the peach individuals of L. corni produced on the black locust. There are other records showing the acquired adaptation of certain species to new host plants, similar to those here cited. The practical application of such phenomena, however, has, so far as can be ascer- tained, first been recognized by Dr. A. D. Hopkins (referred to on p. 189 of this article) and presented by him in concrete form. In a paper prepared by M. Joseph Capus ^ on a nematode disease of peas in the Gironde and read by Paul Marchal at the session of July 10, 19 1 8, of the French Academy of Agriculture there is a record of injury to peas by a fungus (Fusarium vasinfectum var. pisi van Hall, considered as the conidial form of Necosmopora vasinfecta E. F. Smith, accompanied by 1 PicTET, Arnold, influence de l'alimentation et de l'humidite sur la variation des papillons. In Mem. Soc. Phys. et Hist. Nat. Genfeve, v. 35, fasc. i, p. 45-127, pi. 2-5. 1905. 2 Schroder, Chr. die literatur uber die FARBtmo der insekten des jahres 1905. In Ztschr. Wiss. Insektenbiol., Bd. 3, p. 162-164. i907- 3 i'BER experimentall erzielte instinktvariationen. In Verhandl. Deut. Zool. GeseU., Jahresversamml. 13, p. 158-166. 1903. * Marchal, Paul, le lecanium du rohinia. Compt. Rend. Soc. Biol. [Paris], t. 6s, p. 2-5. 1908. ^ Capus, Joseph, and Marchal, Paul, sur la maladie vermiculaire des pois dans la gironde. In Compt. Rend. Acad. Agr. France, t. 4, no. 25, p. 712-716. 1918. 192 Journal of Agricultural Research voi. xxii, no. 4 the nematode Heterodera schachti Schmidt). After pointing out the interdependence and relation of the two, M. Capus says: One might ask himself why this species, everywhere known for its injury to beets, does not establish itself on this plant in the Gironde and appears so abundantly on peas. Following M. Capus's explanations of this phenomenon. Dr. Marchal observed : Among the very interesting facts pointed out by M. Capus in his note I wish to call attention to the following: That injury to beets by Heterodera in the Gironde is not constant, is rare. We should recall in this connection the observations, already old but interesting, of the Dutch naturalist Ritzema-Bos. He has shown that when nematodes multiply in course of years without interruption on the same host, biologic races are formed adapted to this host which later pass to other vegetation with greatest difficulty, even when these are of those preferred by the species. It must be, there- fore, that, by virtue of the conditions of pea culture in the Gironde, a race of Heterodera schachti was formed especially adapted to peas and to the attack of which beets are resistant up to a certain point. There is no doubt that it will adapt itself to beets cultivated for a number of years in succession in the same soil infested with H. schachti. The experiments conducted at the Gipsy Moth Laboratory^ show that of the many plants tested a decided variation was found in regard to the susceptibility to attack by this insect. The plants were divided into four groups: I, favored species; II, favored food species after early stages; III, species on which a small proportion may develop; IV, species that are unfavored food. These results show that, although this insect has a wide variety of hosts on which it is capable of feeding, certain ones are selected in preference to others in the natural forests. As far as known, no observations have been reported showing whether or not several years' feeding on any particular host produces a strain which selects that in preference to others. In a recent paper Dr. C. T. Brues^ writes as follows (on p. 328-329): It has been claimed that the food habits may be modified experimentally, in that caterpillars reared on a strange plant (where they could be induced to select it) give rise to moths whose progeny more readily accept the new plant. It is very difficult to accept such evidence, at least as having any general application, without very clear and incontrovertible proof. If such transformations can occur so easily and become hereditary so quickly they should have entirely destroyed the coherent habits now existent, during the enormous period which has elapsed, for example, since the violet-feeding Argynnids were differentiated, since the holarctic and nearctic Vanes- sids have been separated, or while the world-wide Aristolochia-feeding Papilios were attaining their present distribution. That such a change has actually occurred in the caseof other groups seems equally evident, althoixgh, as has been shown, we can more easily believe that they may have arisen through mutations in maternal instinct not incompatible with larval tastes and then only in extremely rare cases and con- fined to certain groups. METHODS OF CONDUCTING EXPERIMENTS In connection with the experiments by the writer several types of cages, the particular type determined by the amount of material handled > MOSHER, F. H. FOOD PLANTS OF THE GIPSY MOTH IN AMERICA. U. S. Dept. Agr. Bui. 250, 39 p., 6 pi. 2 Brues, Charles T. the selection of food-plants by insects, with speoal reference to lepi- DOPTEROUS LARVAE. /» Amer. Nat., V. 54, no. 633, p. 3i2-33"2. 1920. Oct. 22, 1921 Hopkins Host-Selection Principle 1 93 and the exact conditions required, have been used in confining the colonies of beetles. It is essential to duplicate as closely as possible the conditions in which the insects are found in nature. For the larger logs and for experiments in which a large amount of material was used, an open wire insectary was constructed. This insectary is 40 feet long by 10 feet wide by 7 feet high. The foundation is of concrete, the side walls and top of i8-mesh galvanized wire screening, and over all a removable lattice- work roof was placed. This roof was adjusted to simulate shade conditions in the woods. It was removed in winter and replaced in summer. The floors were made of ashes to give good drainage. Cross partitions divided the insectary into seven compartments of different sizes. One room was entirely boarded in and roofed over. It was used for seasoning wood. Another was lined with cheesecloth, which was used for holding different cuts of wood under natural conditions until desired for use. In the other compartments were placed logs containing various species of insects. Where no danger is present of any infestation from the original host wood into cuts of different wood, it was possible to place several beetle species in the same compartment and continue their breeding in the same host from year to year. In this way forms such as Callidium in pine {Pinus spp.) , Neodytus capraea Say in ash {Fraxinus spp.), and Cyllene pictus Drury in hickory {Hicoria spp.) were placed together. The smaller insects, especially those in twigs and branches, were confined in glass museum cylinders of various sizes. The tops were kept in place so that a very constant degree of humidity could be maintained. This cage was found to give best results for the development of the larvae and, as no sand was needed, the adults were easily found in the cages. These jars were kept under a roof all the year in another insectary. Each of these insects has a particular preference for a certain condition or seasonal cut of wood. Also in some species the adults require food before ovipositing, consisting of green bark from twigs, leaves, or fungus s'pores. In the latter case the pustule of the chestnut blight {Endothea parasitica (Murr.) P. J. and H. W. Anderson) was used. Again, some require much moisture, others rather dry surroundings. The determi- nation of these factors sometimes delayed the successful continuance of a species for a year or more. When a new colony was collected from nature it was ascertained as nearly as possible when the tree died and the condition of the wood, also what degree of humidity was desirable. For instance, those insects naturally feeding in dead branches of a standing tree required drier conditions than those attacking branches fallen to the ground. In order to meet these conditions, wood of the various species used was cut every month or every other month of the year and stored under different conditions. Part was placed in the dry shed for dry seasoning, part hung or stood up in the open-air cage for normal air seasoning, and 194 Journal of Agricultural Research voi. xxii, no. 4 part laid on the ground in the wire cages for wet seasoning. The con- dition of the wood on which the insects were first found ehminated the use of certain of these periodic cuts and conditions of seasoning. How- ever, the first-year adults were usually caged with the choice of many of these cuts and the one infested most heavily was considered as the optimum condition and used afterwards for continuation of the succes- sive broods. The optimum cut could only be determined when sufficient material was given for the number of insects present, as an unfavorable cut may be attacked when the adults are confined on it without sufficient optimum material. Wood used a month or two after being cut is spoken of as green or freshly cut material. In many cases wood from several individual trees was used to avoid any possibility of offering an undesirable individual. To illustrate the variation in optimum conditions of wood, several examples are given: Callidium antennatum Newm. requires wood dry- seasoned over winter; Neoclytus capraea, wood cut during the late winter with the inner bark still sappy ; Liopus alpha Say in hickory, twigs cut in the early fall, air-seasoned for a while and then left on the ground over winter so that the inner bark sours somewhat. (This condition is brought about by the girdling habit of Oncideres cingulata Say.) Several terms which may need explanation are used in reference to the species of host wood: Primary host, or original host, refers to the wood in which the insect is found in nature and first caged in these experi- ments ; as secondary host is understood wood in which a colony has been successfully produced in the experiment, but it may or may not be recorded as a host in nature; an unfavorable host is one not recorded from nature and in which attempts to produce a colony have not been entirely successful. All experiments conducted are here given, although a few have been unsuccessful or have given no results. Occasionally failure to continue a colony is recorded. In all cases an explanation can not be given. It may be because of an improper cut of wood or of a peculiarity of the individual host. In one case partial failure was due to a nematode parasite causing sterility of the females; in another, the parent insects were entangled in spider webs and killed before ovipositing. Reference is made to larval transfers from one host to another. This is accomplished by making a smooth cell through the bark of the new host, partially filling it with frass from the larval mines of the original host, then placing the larva in this cell and finally tightly fixing a piece of bark over the cell. Such transfers do not injure the larva or affect its development. Many cases of transfer to the same host resulted in the survival of every larva. These experiments were conducted at tlie Eastern Field Station, East Falls Church, Va., and all flight dates of the adults and times of cutting of the wood refer to this locality unless otherwise stated. Oct. 22, I92I Hopkms Host- Selection Principle 195 OUTLINE OF EXPERIMENTS ON EACH SPECIES XYI^OTRECHUS COI,ONUS. EXPERIMENT I Xylotrechus colonus Fab. is found in nature in a wide variety of hosts. In fact, it feeds in nearly all hardwood deciduous trees of the eastern and central United States. It shows little or no preference for any exact condition of the wood, except that it will not attack perfectly seasoned material. The larvae can be found in dying standing trees or in logs felled in any month of the year provided they still contain a certain amount of moisture. The first flight of the year occurs in the last week of May or first week of June, reaching the maximum in about two weeks. A few adults emerge sporadically throughout the summer. From eggs deposited in June a few adults usually emerge in September, but the main brood remains as larvae until the next spring. These fall adults have never oviposited under confinement. The larvae feed entirely beneath the bark, or in the bark if it is thick. The pupal cell is made in the outer sapwood or in the bark. The wood of all species for this experiment was cut on April 15 unless otherwise stated. The colony was started by felling a red oak tree in March, 19 14. The wood was attacked during that June, caged soon afterwards, and the colony has since been maintained in red oak. From the original oak form colonies were secured in hickory (Hicoria), chestnut {Castanea dentata {M.QXsh..) Borkh.), locust {Robinia pseudacacia Linn.), red maple {Acer rubrum Linn.), and ash (Fraxinus sp.), in the following manner : QuERCUS. Experiment I. — During May, 1915, hickory logs were placed in the cage with oak intended to carry on the colony. Many adults were present, somewhat over 100, and the hickory as well as the oak was subsequently found infested. In June, 19 16, in the same cage stocked with oak for continuing the colony, chestnut and hickory wood was placed. There was again an overabundance of adults and all woods were infested. In June, 191 7, oak was placed in this cage to continue the colony and also ash, chestnut, locust, hickory, and red maple logs, all cut in February except the hickory, which was cut in April, 191 7; extra pieces of chestnut and maple, cut in November, 1916, and September, 1916, respectively, were also placed in the cage. There was an abundance of adults. In July these logs were examined and it was found that the oak was heavily infested; the chestnut and hickory were lightly infested; the ash, maple, and locust had no infestation. This same year, 191 7, adults were isolated on ash (I^), maple (I*), and locust (I^) with results as described in later paragraphs. 65583°— 21 2 196 Journal of Agricultural Reserach voi.xxn. no. 4 In June, 19 18, two pairs of adults from oak were caged on a collection of oak, hickory, ash, maple, and chestnut logs, all cut April 15, and of about equal size. Examination in July showed that the oak was heavily infested (over 50 larvae present), the chestnut contained 10 larvae, the hickory 7 larvae, and the ash and the maple none. At the same time a similar cage was prepared, and six pairs of adults were placed in it to test the influence of a greater number of beetles on the selection of hosts. The results showed the same relative propor- tion of infestation except that ash also was attacked. The maple was not infested. In June, 1919, this experiment was repeated with the same conditions except that the hickory sticks were accidentally omitted. They were examined in July and the infestation was as follows : The woods in the cage of two pairs of adults contained 28 larvae in oak, 22 in chestnut, and none in ash and maple; that of six pairs contained over 50 in oak, 19 in chest- nut, and none in ash or maple. HicoRiA. Experiment I ^ — May, 1915, hickory logs were placed (as before described) in the oak cage with the wood intended to carry on the colony. They were infested and in subsequent years kept isolated and continued as the hickory form. In June, 191 6, oak was placed in this cage, together with the hickory to continue the colony, and was subsequently found heavily infested. In June, 19 17, together with the hickory for reinfestation, chestnut and locust were placed in the cage. An examination in July showed that the chestnut contained a few larvae and the locust none. In June, 1918, two pairs of adults were isolated in a cage containing oak, hickory, ash, chestnut, and maple, all cut April 15, and of equal size. The results showed that the hickory was heavily infested by over 50 larvae, the oak contained 7 larvae, the chestnut i larva, and the maple and ash none. In June, 19 19, selection tests and selection quantity tests were carried out with this strain. The quantities and cuts of wood were the same as before, except that oak was accidentally omitted. In one cage two pairs of adults were isolated, the resulting infestation being hickory 18 larvae, chestnut 12 larvae, maple and ash none. In another cage six pairs of adults were isolated, the resulting infestation being hickory over 50, chestnut 40, and maple and ash none, Castanea. Experiment I ^. — In May, 1916, chestnut logs were placed (as before described) in the oak cage with the wood intended to carry on the colony. The wood was heavily infested, and these individuals have since been confined to chestnut. In May, 191 7, together with the chestnut, hickory was placed in this cage. The hickory was lightly infested. In June, 19 18, two pairs of adults were isolated in a cage containing oak, hickory, ash, chestnut, and maple, all cut April 15 and of equal Oct. 22, 1921 Hopkins Host-Selection Principle 197 size. An examination in July showed that the oak and chestnut were equally well infested, the hickory contained one larva, and the maple and ash none. RoBiNiA, Experiment I ^. — In June, 19 1 7, eight adults from oak were isolated on black locust cut in February, 191 7. The adults laid eggs, but all died later. In June, 19 18, the experiment was repeated with 15 adults and wood cut April 15. Many young larvae entered the bark, but by August 15 nearly all had died, and none lived to transform the next spring. The experiment was not repeated in 19 19. Acer rubrum. Experiment I*. — In June, 191 7, eight adults from oak were isolated on a piece of red maple cut February i, 19 17. A few larvae lived and three adults (two males and one female) emerged in 19 18. They were isolated in a cage containing oak, chestnut, hickory, ash, and maple, all cut April 15, but no infestation occurred in any wood. During August, 19 18, twenty larvae were transferred from oak to maple to con- tinue the species in this host. A few adults emerged in 1919, and were recaged on maple to develop a larger colony, which will be continued several years before testing the selection again. Fraxinus. Experiment I ^. — In June, 191 7, eight adults from oak were isolated on a piece of ash cut January i, 19 17. A fair infestation occurred, but the larvae developed slowly, and in May and June, 1918, only seven adults (both sexes represented) emerged, while many larvae were still in the wood. These adults were transferred to a cage contain- ing oak, chestnut, hickory, ash, and maple, all cut April 15, but no infesta- tion occurred in any wood. Larvae were again transferred to ash, and a few adults emerged in 19 19. These were recaged on ash, and several adults emerged in June, 1920, but failed to develop any larvae in the new wood. I, I\ I^, P. — During June, 1920, adults emerged from the oak, hickory, chestnut, and maple; strains and adults from all were recaged on the same wood and produced new colonies. No selection tests were made, and these strains will be continued for several years in the same wood before similar experiments are again attempted. CONCLUSIONS The original oak strain of Xylotrechus colonus shows a decided prefer- ence for a few woods, notably oak, chestnut, and hickory. Two years' trial failed to produce larvae capable of completing their development in locust, while the ash and maple colonies were maintained with difficulty. In nature these woods (ash, maple, and locust) have been found contain- ing thrifty colonies of this species. Originally the oak strain showed little preference as between oak, hick- ory, and chestnut; yet, after several years, strains were developed in each wood that showed a growing preference for the given wood. 198 Journal of Agricultural Research voi. xxn. no. 4 The number of insects present under identical conditions influences their selection of hosts. When few are present they concentrate on original or favored hosts; when more than can successfully oviposit on original hosts are present, less favored hosts are taken. CYIvI,ENE PICTUS, HICKORY HOST STRAIN. EXPERIMENT II The larvae of Cyllene pictus feed almost exclusively in hickory. A few specimens have been taken in wild grapevine (Vitis sp.), mulberry {Moriis rubra Linn.), osage orange (Toxylon pomiferum Raf.), and hackberry {Celtis occidentalis Linn.), but such instances are rare and of very local occurrence. In one locality near Harrisburg, Pa., all except one of these unusual food plants have been recorded. This borer is found generally distributed east of the Mississippi River. The optimum condition of wood is that cut during the winter, preferably in January, and left lying on the ground. November cuts are sometimes attacked, but no wood is suitable unless the inner bark is still sappy. Sticks cut at the time of emergence are too green for attack. The first emergence occurs about the middle of April and continues for three weeks. By September the larvae are full grown and have con- structed their pupal cells in the wood. They soon pupate, and in this stage they overwinter. The larvae feed about equally beneath the bark and in the wood. These experiments were started in April, 19 15, when adults were found ovipositing on a hickory log cut during the winter at Falls Church, Va. The strain has since been continued in January and February cuts of this wood, and other host strains have been attempted with varying suc- cess in grape {Vitis sp.), locust (Robinia pseudacacia) , ash {Fraxinus sp.), and mulberry {Morus rubra). Experiments were conducted as follows: ViTis. Experiment II ^ — In April, 1917, a piece of grape, cut in January, was placed in the same cage with much hickory used for the continuation of the hickory form. This grape was not infested. June 26, 19 1 7, sixteen larvae, about half grown, were transferred from hickory to grape cut in January. Nearly all these larvae lived, and the following spring 12 adults emerged. They were isolated in a cage con- taining several pieces of grape and one of hickory, both cut in February. Examination in June showed the grape to be heavily infested while the hickory contained no larvae. In April, 191 8, a large number of adults emerged from the grape. Two pairs were isolated in a cage containing one piece of grape 2 inches in diameter and 2 feet long, and one piece of hickory of the same size, both cut in January. Examination in July showed the grape to be very heavily infested, whereas the hickory contained only a few larvae. Robinia. Experiment II ^ — April 21, 191 7, three females and two males from hickory were caged on a piece of locust cut a month pre- viously. The females laid all their eggs on the locust and the young Oct. 22, 1921 Hopkins Host-Selection Principle 199 larvae bored through the bark, but by the middle of June all had died. June 15, twelve larvae 5 mm. long were transferred to locust and these all died by July 11, when three more, over half grown, were transferred. These lived to construct pupal cells and pupated, but all the pupae died during the winter. In April, 1918, five adults (three females and two males) from hickory were caged on locust cut during January, 19 18. The females laid all their eggs, but only a few larvae lived. These constructed pupal cells and pupated beneath the bark. In locust the larval mines are not normal, lying in almost all cases immediately beneath the bark instead of extending deep into the wood. About half the larvae made pupal cells in the outer sapwood while the others pupated beneath the bark instead of, as normally, deep in the wood. April 20, 19 19, a total of six adults had emerged and two pairs were caged on pieces of locust and hickory cut in January, 19 19. An exami- nation July 16 showed no infestation in either. Fraxinus. Experiment II ^. — April 24, 191 7, three females and two males were isolated on ash cut during January. The females laid all their eggs and the young larvae bored through the bark, but all died before June 15. At this time fourteen larvae 5 to 7 mm. long were trans- ferred to the same ash, and all died within a month. July 15, five more, over half grown, were transferred. They mined extensively beneath the bark, but all died before the end of September without pupating. July 24, 19 18, fifteen larvae, one-half to three-fourths matured, were transferred to ash cut in January. April 21, 19 19, a total of eight adults had emerged. One pair was caged on the January cut of ash, and two pairs were caged on January cuts of ash and hickory. July 16 the wood was examined, but in no case was it infested. MoRUS. Experiment II*. — April 29, 1918, three females and two males from hickory were caged on mulberry cut in January. The females laid eggs, and a very heavy infestation was secured. They developed normally and suffered little more than the normal rate of mortality experienced in hickory. April 21, 19 19, a total of 17 adults had emerged; two pairs were trans- ferred to a cage containing two pieces of mulberry and one piece of hickory cut during January, 19 19. In another cage containing the same quantity of wood four pairs of adults were transferred. In neither case was the quantity of mulberry sufficient to permit the development of all the larvae. Each piece was 2 inches in diameter and 14 inches long. July 16 the cages were examined, and that containing two pairs of adults was infested as follows: Hickory 6 larvae, mulberry over 30; that containing four pahs, hickory 13 larvae, mulberry very heavily in- fested, over 40. 200 Journal of Agricultural Research voi. xxn.No.4 Seasoned Hicoria. Experiment II*. — ^Attempts were made in April, 191 7, to develop a colony adapted to seasoned wood by caging two females and one male on wood cut in October and dry seasoned. Eggs were laid and larvse entered the bark but developed very slowly, never entering the wood. Only three lived to pupate, and these made their pupal cells between the bark and wood. All three pupae died during the winter. In May, 1918, the experiment was repeated with three females and three males and wood cut in November, 191 7. In the fall of 19 18 a number of larvse lived and pupated, but all were below normal size. Only a few adults emerged in April, 191 9, and these were below normal size. 1 1^. — Dr. A. D. Hopkins, in 19 16, recorded a dying hickory tree heavily infested by Cyllene picius with no evidence of primary injury from other causes. This suggested that a strain capable of attacking living trees might be produced, and attempts were made to secure a colony in such a tree. A small hickory 3 inches in diameter was selected and entirely stripped of leaves August 11, 1916. April 30, 191 7, it was again defoliated, and 80 adults were caged on it. The adults laid eggs and the young larvae entered the bark, causing sap to flow from the wounds. However, all died after growing to 3 mm. in length. In April, 19 18, the tree was again defoliated, and 156 adults were caged on it. The same results were observed. During both years the tree put out healthy foliage after artificial defo- liation, but it died in August, 19 17. In no case did the Cyllene larvae hve to mine more than % inch beneath the bark. QuERCUs. Experiment II^ — In transferring adults during the spring of 1918 to new hickory wood to continue a large colony, a piece of oak was unintentionally left in the cage. This cage contained six large hickory logs 4 to 6 inches in diameter and 5 feet long. The oak log was 3 inches in diameter and 4 feet long. During September, 1918, work of Cyllene was noticed on this piece of oak, and in the spring of 1919 it was separately caged. Five adults emerged in April — all very small, much below normal size. Two females and one male were transferred to a cage containing only oak; one pair to a cage of oak and hickory. These cages were examined July 16. Neither wood of the selection test was infested, but the oak wood on which two pairs were caged contained a few very small larvae. 11^^ — ^To test the influence of host selection on the condition of host. In April, 19 18, two males and two females from hickory were isolated in a cage containing a piece of grape and a piece of hickory of equal size — the grape of optimum cut, January, 19 18, the hickory less favorable, November, 191 7. Examination in July showed the grape to be heavily infested while the hickory contained very few larvae. Oct 22, 1921 Hopkins Host-Selection Principle 201 11^^. — To test the influence of an overabundance of adults and scarcity of the primary host on the host selection. Three pairs of adults from hickory were caged on a small piece of grape and a small piece of hickory (each 2 inches in diameter and i inch long), each cut during January, 19 1 8. Examination in July showed both woods to be infested, the grape containing a few more larvae than the hickory. In 1920 no adults emerged. II, 11^. — In 1920 only two strains were continued, those in hickory and those in mulberry. No attempt was made to reestablish the others that failed. CONCLUSIONS This species, although most commonly found in hickory, will readily adapt itself to several other plants, notably mulberry and grape, both of which are recorded as natural hosts. In some unfavorable hosts, or in an optimum host hi an unfavorable condition, the larvae may become established, but the mortality is high and the progeny seem to be sterile. After one year's feeding in a new host the larvae may select that host in preference to others. The selection of a host is influenced by the number of adults present and the quantity of the primary host, in that adults will prefer a sec- ondary host to overinfesting the original host. The selection of a host is influenced by the condition of the host, a favorable condition of secondary host being preferred to an unfavor- able condition of the original host. The optimum condition of any host capable of properly supporting growth of the larvae is of very restricted limits. CYI,I,ENE PICTUS, GRAPK HOST STRAIN. EXPERIMENT III This is the same species as previously discussed, having the same biological habits except that this host strain in grape (Vitis) was taken in nature at Hummelstown, Pa., in January, 1916, by J. N. Knull. Since then the colony has been continued at East Falls Church, Va., in grape cut in January or February. Other host strains have been pro- duced and experiments conducted as follows : HicoRiA. Experiment III ^ — When these adults emerged from grape (May, 19 16), three females and two males were isolated in a cage containing 10 pieces of grape and i piece of hickory cut in February, 19 16. No eggs were laid on the hickory. April 17, 191 7, two pairs of adults were isolated on hickory cut in January, 19 17. Eggs were laid and the larvae developed but not so rapidly as in the grape. June 15 they were under normal size. By September only two larvae were alive. One of these pupated but died during the winter. 202 Journal of Agricultural Research voi. xxn, N0.4 Many adults emerged from the grape in 19 18 and five pairs were caged on three small pieces of grape i}4 inches in diameter and 2 feet long and one piece of hickory about the same size, both cut in Januar}^ 1917- In July they were examined, and both hickory and grape were heavily in- fested. Quantity selection. Experiment III^. — To again test out the effects of host selection when an insufficient amount of wood is given than that required for the number of adults present, in April, 19 18, two pairs from grape were caged on grape and hickory cut in January, 191 8. The piece of grape was 2 inches in diameter and i foot long, the hickory 2 inches in diameter and 2 feet long. An examination in July showed both grape and hickory infested. This experiment was repeated in April, 1919, using two pieces of grape and one piece of hickory, all of optimum cut and equal size. In one case 1 pair of adults was isolated, in another case 3 pairs were used. The wood on which i pair was caged contained 5 larvae in hickory and 3 larvae in grape ; that on which three pairs were caged contained 4 larvae in hickory and over 25 in grape. The grape colony was not continued in 1920. CONCLUSIONS This host variety from nature had acquired a decided preference for grape. The selection of a host is influenced by the quantity of wood present for a given number of adults, in that the adults will select a new host in preference to overinfesting the original host. The tendency in this species in nature to confine itself to a certain host, either hickory or grape, is not as marked as in some other species. CYIvLENE PICTUS, HICKORY STRAIN II X GRAPE STRAIN III In order to determine whether crossing of these two host strains would influence the progeny in the selection of the host, males and females were isolated from their pupal cells in the spring of 191 7. April 17 three females from hickory and two males from grape were isolated in a cage containing hickory. Two females from grape and two males from hickory were isolated on grape. In neither case did mating occur as readily as when both sexes from the same host were paired. The sexes often ap- proached each other and moved away before finally copulating. Good infestations were secured in both cases. In April, 1918, one pair from hickory was caged on equal amounts of grape and hickory cut in January, 19 18. Only the hickory was infested. Two females and two males from grape were isolated on the same amount of grape and hickory cut in January, 19 1 8. Both woods were infested. These pieces were all 2 inches in diameter and 1 8 inches long. Oct. 22, 1921 Hopkins Host-Selection Principle 203 CONCIvUSIONS This crossing of the two host forms had no influence on the selection of hosts. The amount of wood and number of adults present influence the host selection, as shown when one female and two females were given the same amount of wood. CYI,I,SNE CRINICORNIS. BXPERIMENT XI,I Cyllene crinicornis Che v., found in the southwestern United States, is known to feed only in mesquite (Prosopis juliflora (Swartz) de C.) and occasionally on an allied legume, paloverde {Parkinsonia microphylla Torr.). In general its biology is similar to that of C. pictus, and it pre- fers the same conditions of wood. Adults begin emerging in the natu- ral range during late February, and part of this generation emerges in September. Mesquite infested with these larvae was sent to Falls Church, Va., by T. E. Snyder from San Antonio, Tex., April 27, 191 7. Adults emerged at Falls Church the following May and were caged on mesquite cut in March, 1918. A good infestation was secured and has since been con- tinued on mesquite. RoBiNiA. Experiment XLr. — In May, 19 18, two pairs were isolated on locust cut February, 19 18. May 31 the females were dead, and the abdomens were dissected and found to contain eggs. Probably no eggs were laid, and in July no evidence of larval work could be found. July 24, 19 1 8, seven nearly matured larvae were transferred from the mesquite to locust cut January, 1918. During May, 1919, five adults emerged. One pair was caged on locust cut in January, 1919, and one pair on both locust and mesquite. The mesquite was cut October, 19 18. July 16, 1919, these cages were examined, and the selection test showed that mesquite was not infested, whereas the locust contained several small larvae. The cage containing only locust was lightly infested. None of these larvae transformed in 1920. CONCLUSIONS The locust was such an unfavorable host that the adults would not oviposit on it, but larvae may live and transform for one or two genera- tions when forced to take it. CALUDIUM ANTENNATUM. EXPERIMENT IV Some confusion exists as to the taxonomy of the blue species of Calli- dium allied to Callidium antennatum. A number of species have been described of questionable validity. Two species have been experi- mented with — C. antennatum and C janthinum Lee. These two adults are easily separable, and their habits are also quite distinct. The 204 Journal of Agricultural Research voi. xxn, no. 4 former, so far as the Forest Insect records are concerned, feeds only in pine (Pinus) and spruce (Picea), and for this discussion will be confined to the form occurring in the northeastern United States. It shows a decided preference for a certain condition of the host, requiring wood that has been cut in the early fall or winter and is well seasoned. When the inner bark is still sappy the insects will not normally make their attack. Adults first appear about the middle of April, and the flight period continues about a month. One year is required to complete the life cycle. The larvae feed beneath the bark until half grown, then enter the wood to construct a long pupal excavation, at the end of which the pupal cell is chambered off. These experiments were started in December, 19 16, when infested Virginia pine was caged. In 19 16 and the following years the colony was continued in seasoned pine, and a form was also developed in spruce and freshly cut or green pine. Unsuccessful attempts were made to produce a juniper (Juniperus) strain. This insect has been reported as feeding in juniper (Juniperus) and maple (Acer). Juniperus. Experiment IV^ — In April, 19 16, juniper was placed in the cage together with pine; in addition, about 20 adults were isolated on a stick of juniper (both woods were cut in January, 19 16). In neither case was the juniper attacked, and the females isolated on juniper failed to oviposit. The same test was repeated in 191 7 with juniper cut in October, and the same results were obtained. During June and July, 20 larvae, from small to over half grown, were transferred to juniper. All finally died, some living a month. None increased in size before death. Picea. Experiment IV^. — April 12, 1916, a piece of seasoned spruce was placed in the cage, together with the pine, for reinfestation. An examination in June showed only one larva in the stick, and this was far below the normal size of those in pine. By July 11 this larva had died. It is possible that more eggs were laid on the spruce but the larvae died earlier. June 29, 1916, nine larvae about half grown were transferred to the same spruce wood. July 11, 191 6, three larvae were alive and 17 more were transferred. From these larval transfers 10 adults were secured in April, 191 7. They were caged on four small pieces of seasoned spruce and a piece of seasoned pine placed in the cage for one week, both cut in October, 19 16. Examination in July showed the spruce well infested, but only 4 larvae were found in the piece of pine. In 19 1 8 the same experiment was repeated, four females and three males from spruce being caged on four sticks of spruce and one of pine cut in September, 191 7. An examination in July showed that the spruce contained many larvae, but none were found in the pine. Oct. 22, 1931 Hopkins Host-Selection Principle 205 In 19 1 9 this was again repeated, but the results showed that pine was infested while the spruce contained no larvae. Both woods were of No- vember cut, but it is not likely that this would have such a decided in- fluence. However, six adults were isolated on spruce to continue the colony, and this wood was very lightly infested. No explanation can be offered as to the reason for this discrepancy from former results unless the spruce wood was in an unfavorable condition. During the same season another cage was prepared of pine cut during November, 1918, and green spruce cut in April, 1919. The pine was heavily infested ; the spruce contained no larvse. Experiment IV^. To test effects of condition of wood on selection. — April 23, 1920, one pair of adults from spruce was caged on a favorable cut of pine (November) and freshly cut spruce (April) . When the woods were examined on July 5, 1920, the pine contained many larvae while the spruce contained none. At the same time a pair was caged on November pine and November spruce, both optimum cuts. Several days later the female was found dead in the cage and had laid no eggs. Acer. Experiment IV^. — ^As maple has been reported as a host of this species, attempts were made in 19 16 to start a colony in this wood. On July 6, six half- grown larvae were transferred to a seasoned piece of wood, but by July 21 all but one had died and it was smaller than when transferred. This one died soon after. None of the larvae fed on the maple. Green pine. Experiment IV^. — In 1916 some of the wood used to carry this colony along was cut March i, and consequently little seasoned. It was very unfavorable for the ovipositing of the adults, but some eggs were laid on the pieces. The larvse developed slowly and at the time of pupation were below normal size. The adults secured in 1 9 1 7 averaged about one-half normal size. They were caged again on wood cut in March, 191 7. Adults were secured in 1918 and again caged on the same condition of wood, and a good infestation was secured. While the author was absent for a month from the field station in the summer of 19 18 these larvae were all killed by a fungus. The sticks were on the ground and so were caught in a period of rainy weather and were water soaked. Experiment IV, IV ^. — In 1920 the pine and spruce strains were continued in the same wood. CONCI^USIONS The pine form shows a decided preference for that host. It can live in spruce and then shows a decided preference for that host. It will not live in juniper or maple. In producing a new host strain a high mortality occurs in the young larvse. 2o6 Journal of Agricultural Research voi. xxn. No. 4 A colony can be produced in a host which is in an unfavorable condition, but the resulting adults are below normal size. Owing to the failure to continue the colony it can not be stated whether or not such a strain would show preference for the new condition of the host by selecting it voluntarily. CALLIDIUM JANTHINUM. EXPERIMENT V Under Callidium antennatum reference was made to C. janthinum Lee. It is distinguishable from the former by its smaller size, shining surface, and bluish green color of the adult, by the fact that the larva feeds only in juniper, and that the adults emerge about four weeks later in the spring. It requires wood which was cut during the late fall and which has not seasoned in contact with the ground. It will oviposit in greener wood than C. antennatum although the inner bark should not be sappy. The first flight occurs during the first to third week in May and con- tinues about two weeks. One year is required to complete the develop- ment. The larvae feed beneath the bark until half grown, then excavate long pupal chambers, the ends of which are plugged off for the trans- formation cell. These experiments were started with a lot of infested juniper branches from Hummelstown, Pa., collected by J. N. Knull in April, 1916. May 2, six adults emerged and were caged on juniper cut in April and rapidly dried in the house. Since then the colony has been continued each year in September and November cuts of juniper, which are preferred. PiNUS. Experiment V^ — May, 191 7, two pairs were isolated on pine cut in November, 19 16. A few eggs were laid from which larvse hatched and entered the bark. By July 10 all had died. The same test was repeated in 19 18 with similar results. CONCLUSIONS This species shows a decided preference for jumper and will not develop in pine from early stages. Larval transfers to pine with nearly matured larvae were not made. CAIvWDIUM ANTENNATUM AND C. JANTHINUM. VXIV; IV XV Because of the taxonomic confusion between these species (cited pre- viously) and with the idea that a crossing of these two forms might possi- bly influence the selection of a host, attempts were made to cross the species. VxIV. — In May, 19 17, four females of the juniper form were crossed with two males of the pine form and were caged on juniper. Both species had been previously isolated from the pupal cells to avoid all possibility of mating. These insects immediately mated, and the eggs were laid on the juniper from which a good infestation was secured. Oct 23, 1931 Hopkins Host-Selection Principle 207 April 17, 19 1 8, the first adult emerged from this cross. The second adult emerged April 25. May 3 and 4 eight adults emerged. All the 19 1 8 adults resembled the juniper form in color. These adults were all isolated in a cage containing juniper and pine cut in November, 19 17. An examination in July showed only the juniper to be infested, but by a very light brood. During the remainder of the summer all died except three larvae which constructed pupal cells. May 5, 19 19, two males and one female emerged and one pair was caged on juniper cut in November. A light infestation occurred. May 12, 1920, five adults emerged and were recaged on juniper. The selection of pine and juniper was not again tested. t;"- ,.,) hf>q^ 2 -2K 19 20 16 25 9 19 2K-3 II 00 10 19 2K-3 II 00 f ^^ 14 00 12 37 22 21 18 00 13 14 15 16 00 Uninoculated 00 00 25 45 00 i 17 00 Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 239 The foregoing experiment was repeated in a somewhat cooler house, in which the temperature remained close to 15° C. most of the time, rising to about 20° during the middle of the day. Under these conditions, the maximum length of the cotyledon was about 2)4 inches. The data from this experiment are reported in Table II. The plants became im- mune at approximately the same time as noted in the first experiment — between the nineteenth and twenty-fourth days after sowing, when the cotyledon had about attained its full growth and as the first leaf was emerging. It will be recalled that the basal portion of the cotyledon, as with each of the later leaves, forms a collar or sheath inclosing the lower parts of the younger leaves. The question arose as to whether or not immunity to smut infection is directly associated with maturity of the tissues. If so, it would seem that the explanation of this later escape of the onion plant from infection lies in the fact that the maturing basal sheath forms a thin but normally complete barrier of resistant tissue between the potentially infective soil and the deeper-lying embryonic tissues of the younger developing leaves. The removal of this mechan- ical barrier might, therefore, permit of infection at a later stage. In order to determine whether this is the case, the following experiments were undertaken. After the thirty-first day, when the onion seedlings had passed the so-called susceptible period, the cotyledons were carefully removed from the plants in one pot, and infected soil was placed around the base of the exposed first leaf. Sixty per cent of the plants thus treated became infected as shown in Table II, pot 9. This proves that the first leaves are susceptible even after the cotyledon becomes immune. On the fortieth day, a i-inch layer of infested soil was placed on top of pots 10 and 1 1 , so as to surround the first leaves in proximity to the axils. Pot 10 was left at the same temperature (15° to 20°) for 24 days and pot II was removed to a temperature of 25° to 28° for the same period. About 5 per cent of the plants in pot 10 showed infection of the first leaf as compared with 28 per cent in pot 11. The reason for the increased percentage of infection at the higher temperature has not been satisfac- torily explained. It may simply have been consequent upon the stimu- lated growth of the onion foliage. However this may be, it is evident that the basal portion of the first leaf remains susceptible to infection for a short time, at least, after it emerges from the cotyledon. From a summary of these results it appears that our own experimental data regarding the duration of tlie period of infection agree in the main with those of previous investigators. The conclusion seems justified that disease resistance is correlated with tissue maturity, and that the onion cotyledon becomes immune to smut infection at about the stage when growth ceases. The rate and nature of growth of the cotyledon will naturally vary with environmental conditions; hence variation in the actual length of the infection period is to be expected. The mature 240 Journal of Agricultural Research voi.xxii.no.s basal sheath of the cotyledon thus protects the embryonic region of the younger leaves from infection. That portion of the first leaf which emerges from the cotyledon is susceptible to infection for some little time after emergence, but since it ordinarily is not actually in contact with infested soil, this fact is probably not of practical significance. Table II. -Relation of stage of development of the option seedling to infection by Uro- cystis cepulae Inter- Inter- val be- Pot No. Method of exposure to inoculation. val be- tween sowing and in- ocula- tion. Length of cotyledon at time of inocula- tion. Condition of first leaf at time of inocu- lation. tween inocu- lation and final exami- nation. Total number of plants. Per- cent- age smut- ted. Days. Inches. Days. 1-3 4 Uninoculated lOO-f 24 Infected soil around base 13 I Not out 27 75 of cotyledon. 5 6 Do 16 2 to 2X 2 to 2X 2 to 2% do 24 24 24 24 34 41 19 36 35 8 Do 19 24 31 ... do 7 8 Do Just out. .. . K to iK inches Do above axis. 9 Cotyledons removed and infected soil placed around base of first leaf. 31 2 to 2^ . ..do 24 15 60 10 One-inch layer of infected soil placed on surface of old soil so as to cover lower inch of aerial por- tions of plants. 40 2 to 2K yi to 4 inch- es above axis. 24 36 6 II Same as No. 10, except that pot was transferred to a temperature of 25° to 28° C. 40 2 to 2>2 ...do 24 46 28 RELATION OF SOIL MOISTURE TO INFECTION Since there are these well-defined limits to the time of smut infection, the possibility becomes clearly evident that variable environmental factors during this limited period may exercise a controlling influence on the occurrence of the disease. As already stated, the problem seems to resolve itself primarily into the question of the relations of soil tempera- ture and soil moisture to infection. The results of several workers upon the grain smuts, as summarized by Jones (5), have shown that soil temperature may influence infection. Hungerford and Wade (4) have published evidence that high soil moisture, also, may favor infection of wheat by the smut fimgus Tilleiia tritici (Beij.) Wint. Variations in the moisture content of the surface layers of soil are likely to be wide, especially during the late summer planting season in the southern States when high temperatures and low humidity may cause rapid desiccation. Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 241 An experiment was therefore carried out in which onion seedlings were grown in smut-infested soil in pots which were held at different degrees of soil moisture. Galvanized iron pots 5 inches in diameter and 4 inches deep were used for these trials. Greenhouse sandy loam soil was used and its water-holding capacity was determined in advance by the two standard methods recommended by soil physicists ^ — that is, by means of the lo-inch cylinder and the i-cm. cup. The soils were brought to the desired low and medium water contents before they were placed in the culture pots ; and in those cases where the desired moisture approached the water-retaining capacity, the water content was finally adjusted after the soil was potted. Although these methods failed to secure exact uniformity in the physical compactness of the soils in the several series, they were considered satisfactory as to initial moisture conditions. The pots were weighed daily during the progress of the experiment, and water was added to replace the losses. Since it was realized that the surface layer of soil would change in moisture content through evaporation more rapidly than the lower layers, an effort was made to reduce this surface evaporation so far as practicable. To this end, tar paper covers were used until the seedlings came above ground, when glass covers were substituted for a few days, and finally mineral wool was packed on the surface about the seedlings to reduce evaporation. Absolutely uniform moisture throughout the pot could not be maintained even by this method, and the upper layers of soil unavoidably assumed a somewhat lower water content than the average for the pot. There- fore, at the end of the experiment, moisture determinations were made of the upper inch of soil, since this was the important part from the standpoint of smut infection. The soil was inoculated at the outset by the introduction of spores from diseased leaves and scales. The data from this experiment are given in Table III. Good germination took place within the range of 10 to 15 per cent moisture content (45 to 70 per cent of the moisture- holding capacity). A high percentage of infection also occurred within this range. Above 15 per cent there was some decrease in germination together with a gradual reduction in infection. At the extreme, how- ever, where germination of seed was practically eliminated (pot i), one of the two plants surviving became infected. It is evident from these data that a good percentage of infection may be expected at a soil mois- ture content up to the limit where good germination and growth of the host plant occur. There was a gradual reduction of infection below 5 per cent (23 per cent of the moisture-holding capacity), but this was not sufficient to insure good germination and support good growth. It may be concluded, therefore, that soil moisture does not function as a factor limiting infection with onion smut within the limits at either extreme where good germination and growth, of the host occur. 1 We are especially indebted to Prof. E. Truog, of the Department of Soils of the University of Wiscon- sin for advice in connection with this work. 242 journal of Agricultural Research voi. xxii, ^fo. s Table III. — Relation of soil -moisture to infection by Urocystis cepulae Pot No. 2. 3- 4- S- 6. 7- 8. 9- 10 II 12 13 14 Original moisture content (percentage of dry weight)." Moisture content of surface lay- er at end of experiment. 19.6 17. 1 16.3 18.2 20.0 15.0 15.0 10. o 10. o 10.7 5-0 8. I 7-5 6.6 27. 1 24.7 23-7 23-4 14.0 12.4 II. 6 9-3 9.0 2.4 2-5 1.4 3-4 1-3 Number of seeds planted. 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Total num- ber of plants. 52 48 50 60 66 66 71 66 32 66 63 37 Percentage of plants smutted. 50 O 42 71 90 97 85 94 59 50 19 35 « Water-holding capacity, as determined by lo-inch cylinders, was 22.3 per cent; as determined by the i-cm. cup, it was 27.8 per cent. The calculated wilting coefficient of the plants was 2.3 per cent. TEMPERATURE RELATIONS In calling attention to the importance of soil temperature as a factor in the development of certain plant diseases, Jones (5) points out that several investigators have stressed its bearing upon infection in the case of stinking smut of wheat, Tilletia triiici (Beij.) Wint., and of the oat smuts, Ustilago avenae (Pers.) Jens, and Ustilago Levis (K. and S.) Mag. Heald and Woolman (j) showed that the amount of infection with the stinking smut of wheat was reduced as the mean soil temperature rose above 65° F. (18.3° C.) or fell below 40° F. (4.4° C). Humphrey (4) states, for the same disease, that soil temperatues of 0° to 5° C. and above 22° C. are decidedly unfavorable to infection. In studying the relations of soil temperature to the developemnt of a parasitic disease, consideration must be given to the possible influence of such temperature upon the host and the parasite independently. This may enable one to analyze with more confidence the effects when host and parasite are subjected simultaneously to the experimental condition. This has been done as far as practicable in connection with the present work. Unfortunately, germination of the fungus spores under artificial con- ditions has been so scanty that the effect of temperature upon the fungus has been necessarily limited to inoculation experiments with infested soil. However, the disease is produced so readily and consistently by the latter method that a very accurate index to the development of the fungus can be secured by varying the condition of the infested soil dur- ing the germination and early growth of the onion seedling. The soil-temperature experiments were all carried out in the green- house at Madison, Wis., during the winter months. The apparatus in use Oct 29, 1921 Relation of Soil Temperature to Onion Smut 243 in the Department of Plant Pathology, University of Wisconsin, for the control of soil temperatures has been described by Jones (5) . Briefly, it consists of a series of water baths held at constant or nearly constant temperatures in which the glass or galvanized-iron culture pots are inserted.! s ^^-f?-' .'i.suuu-.. For these experiments galvanized-iron cylindrical pots 5 inches in di- ameter and 8 inches in depth were used. In order to overcome the in- fluence of the air temperature upon the upper layer of soil, the surface of the latter was kept at >2 to i inch below the level of the water. Tar- paper covers were placed over the pots until the seedlings came above ground; these covers were then removed, and a layer of mineral wool was placed on the surface of the soil. By this procedure the tempera- ture of the upper inch of soil was kept reasonably close to that of the deeper portions — that is, approximately that of the water in the tank. In order to follow any minor variations, readings were taken three times daily from thermometers inserted i inch below the surface of the soil. At the beginning of the experiments the moisture content of the soil was adjusted to two- thirds of the water-holding capacity. The pots were thereafter weighed at intervals of one to three days, depending upon the rate of water loss, and the moisture content was readjusted accordingly, either by adding water directly to the surface or by introducing it at the bottom of the pot through a glass tube. Obviously this method did not secure uniform distribution of moisture throughout the pot, and una- voidably the content of the upper layer of soil was somewhat lower than the average for the whole pot. It is believed that this variation had little if any influence, however, since other experiments, described earlier in this paper, showed that infection is quite uniform over a much wider range of soil moisture than here occurred. The seed was planted at a depth of I inch. Since the chlorophyll in the tops in some cases ob- scured the smut lesions, the plants were placed in alcohol acidified with acetic acid until thoroughly bleached before final examination for the disease was made. EFFECT OF SOIIv TEMPERATURE UPON THE DEVEI.OPMENT OF THE HOST Experimental studies to determine the relation of soil temperature to the rate and character of seed germination and seedling development were carried on in conjunction with those relating to infection, of which the results will be presented in the next section. It will be simpler, however, to discuss these two aspects of the problem separately, taking up first the relations of temperature to host development. Experiment I. — Seven pots of sterilized greenhouse loam soil were uniformly planted with 50 Red Globe onion seeds in each pot. One pot was then held at each of the following temperatures: 10° to 14°, 16.5° 1 since this description was published, numerous improvements have been made from time to time by members of the Department. As now in use these are termed the "Wisconsin soil temperature tanks." 244 Journal of Agricultural Research Vol. XXII, No. s to 1 8°, 19° to 22°, 24° to 26°, 27° to 29°, 30° to 31°, 35° C. The moisture content of the soil was held at two- thirds the water-holding capacity (22 per cent of dry weight). The air temperature of the greenhouse was kept at about 15° with a rise to 20° during the middle of the day. These conditions as to soil moisture and air temperature were such as had proved favorable for both host and parasite development in the earlier trials. The first seedlings to appear above ground were those at 27° to 29°, those at 24° to 26° came up shortly afterward, then those at 19° to 22°. Good growth took place at these three temperatures, but germination was very slow at lower temperatures. At the highest tem- perature, 35°, a few seeds germinated, but growth was very slight. The plants were all removed and the roots washed out on January 7, 1920, 29 days after the seed was sown. The data given in Table IV summarize the condition of tlie plants at this date. It will be seen that at this early stage in the development of the plants there was a tendency for best root development at about 21° or below, while the best develop- ment of tops took place at this point or above. Table IV. — Development of onion seedlings in sterilized greenhotise soil held at 22 per cent oj the dry weight or two-thirds the moisture-holding capacity, and at different soil temperatures. Data on January 7, IQ20, 2Q days after sowing Soil temperature. "C. 10 to 14 . . . 16.5 to 18. . 19 to 22 . . . 24 to 26. . . 27 to 29 . . . 30 to 31. . . Num- Total Per- Per- Per- centage with first leaf. Average Average ber of num- centage centage dry weight dry weight seeds ber of with 2 with 3 of tops per of roots per planted. plants. roots. roots. plant. plant. Gm. Gm. SO 41 22 0 17 0.00224 0 . 00046 50 30 17 3 70 .00293 . 00023 SO 32 Sb 25 100 .00396 . 00043 SO 28 32 0 82 •00307 .00028 50 0 14 SO 0 86 .00285 .00014 50 "8 13 0 63 .00212 Average total dry weight per plan I. . Gm. 0.0027 .0031 .0044 •0033 .0030 .002 1 » The reduced stand at 27° to 29° and 30° to 31° C. was due to damping-off fungi. Experiment II. — ^The experiment was repeated, with some modifica- tions, starting April lo, 1920. The Red Globe and Yellow Bermuda varieties were used. Two pots of each variety were kept at each of the following soil temperatures: 14°, 20°, 25°, 28°, 30° C. The air temper- ature ran sHghtly higher (20° to 30°) during the middle of the day and dropped to about 15° for the most of the night. Both the rate and the percentage of germination were noted, and the data are recorded in Table V. In both varieties the most rapid germination took place at 25°, although the rate was only slightly less at 20°, 28°, and 30°. At 14° the seedlings were distinctly slower in starting ofif. The plants from one pot of each series were removed on the thirteenth day. The dry weights of the tops and roots as given in Table V were so small at this age that com- parison on this basis does not have any great value. The tendency for rapid development of tops as compared with roots at 20° or above is, however, shown very strikingly in Plate 25. The plants in the remaining Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 245 pots were removed on the thirtieth day. The relative dry weights of roots and tops then secured are shown in Table V and those of the Red Globe are graphed in figure i. While the temperature relations of the two varieties were alike in their main features, there was an interesting minor difference, possibly indica- tive of the better adaptation of the Red Globe for northern culture and of the Yellow Bermuda for southern. In both cases with these onions, as indeed holds generally in our experiments with other plants, the best .0020 .008 I .00^4 \ I \.ooz \ .000 ^a^zr ^r^o^^,^. '^''■?'^rM>s\ I I .00/0 "1 I .000s \ Z'^" 20'' 2S'=' 23*^ 30'' .pooo Fig. I. — Relative developments of dry weight in tops and in roots of Red Globe onion as shown at end of 30 days' growth in a series of culture pots kept at the several soil temperatures indicated, with all other factors, including air temperature, alike for all. Note that the best root development occurs at the low temperatures (12° to 15° C.) whereas the tops are forced more strongly at higher temperatures (20° to 25°). root development occurs at relatively lower temperatures (12° to 20° C.) while best top development occurs at higher temperatures (20° to 25°). When the varieties are compared, it is seen that with the Globe both roots and tops grew relatively better at somewhat lower temperatures than did those of the Bermuda. Thus the data at 30 days show the maximum root development of the Globe at 14° while that of the Ber- muda was at 20° ; with tops the maximum was at 20° for the Globe and at 25° for the Bermuda. 246 Journal of Agricultural Research Voi.xxn. no. 5 0 •ia . t \ ■-H ' ^i2 , ■3^ 0^ 1- V- ^- 25^ ^ >- s 5 a •- S S S? S ?!•-"• " " ? S cS o*^^^^" ra T^ — •-t'O Ji-a '- -0 -d T) ^ ta S-d-a 9 . |8g8882 8^8S^s 4) 0 W 2 0 > "a <; 2 ^oo r 0\ ^ m b .Ot^ vOCOCOO C h Tj- M M r^so vo :> t^ .O>0 COI^O^MO 0\00> 1 s; -t r^ C NO vO t^ I V- M 0 (3 8 c OOOOO 000 ►^ fc- i<-r OOOOO 000 1 6 ... , ^ • 0 r^ Tf ov u^nO 00 M (»^ »o 1 n 0 2 \0 nC ■t .-o ■0 -a -a /q -a t3-OT3 a > 0 ^ < 0 a 2 2 -So « t> t^ 0^ 0 0 0 5^^ r*^ (N r^ ^ t 0 •d S 88 !0 8 8 0 888 000 9 -M . -0 >/■ 0 0 00 t^ 0 OWN 2-Sa ^!88 00 CO -0 rO « 88888 On r^oo 888 6 1 >*-. m 00 0 0 0 <^ ■* I' Ov N 0 fO r^ m-O 10 10 "J t fO ^ a >- g 1^^ ' J3 . <> fi Tf 0 OiO CO 10>0 M t- *j ► 10 f^ t^ I^ 0 1/^ 0 •0 »/io d 2gS 0 {h S-a •a CIS .g , t^ fO •^00 t^ M 0 wio On i r^ t^M3 0 M NO -0 >« 10 u Wfcx) "0 J3 . 0 "1 e< 0 \n 0 rr, ^ W500 0 to C9 .a& 0 t^ 0 NO 0 NO lO 10 ^■o 1 0 m rO D CO 0 t^ ** fO On ^•s>' NO i/i nr to lo ■>}• ■* (^ i-i m wg-S >t5 •a- 0 »nco 0 -Nt 0 1/100 0 ^ " " N M W5 « M M M CO V CS •*-! rt >. ■o 3 a rt t-i ►^^ 01 K" J2 0 5 PQ 0 "3 rt >< 1 Oct 29, 1921 Relation of Soil Temperature to Onion Smut 247 EFFECT OF soil. TEMPERATURE UPON INFECTION At present the chief interest in these data focuses upon the question of any possible bearing of the rate of development of the host plant at different temperattires upon predisposition to, or escape from, smut infection, recalling that such infection is practically limited to the seed- Hng stage before the maturity of the cotyledon. It has just been noted that the promptest seed germination and most rapid growth of tops during this early seedling stage occur at fairly high temperature, 20° to 25° C, with a rather pronounced drop in rate of aerial growth at tempera- tures below 20°. It is to be expected, therefore, that in the northern onion-growing sections where the seed is planted in early spring there will be a rather tardy germination and slow early development of tops, the growth energies during the seedling stage being directed under this climatic environment to a relatively stronger development of the root system. In the South where the seed is planted in the comparatively warm period of early autumn, we should expect a more rapid top growth at the outset, with correlated strength of root development coming later in the autumn as the soil becomes gradually cooler. The naturally infested soil was secured near Racine, Wis., from a badly diseased field of sandy loam rich in organic matter. The soil which was artifically inoculated consisted of a greenhouse mixture of loam and sand to which were added fresh spores from smutty onion leaves. In order to test the efficacy of this method of soil inoculation a preliminary planting of onion seed was made in advance of the final experiments. This gave a high percentage of smut infection, showing that the method of introduc- ing the inoculum was satisfactory. Several early trials indicated that below 25° C. soil temperature variations have little effect on the relative amount of infection. The results of two such experiments, nearly cover- ing the range of onion seed germination, are given in Table VI (experi- ments I and 2). It is evident from these figures that abundant infection occurred between 10° and 25°, both with naturally infested and with artificially inoculated soil. The number of pustules per plant as shown in Plate 25 proves that the fungus was very active even at low tempera- tures. Above 25° infection is reduced very rapidly, as indicated by both the percentage of infected plants and the number of pustules per plant. In order to determine more closely the point at which infection is inhibited four more serial experiments were conducted (experiments 3 to 6) in which the temperature was kept as constant as possible at 2- degree intervals between 25° and 31° C. The results given in Tables VII and VIII, and illustrated in Plates 26 and 27, show that abundant infection took place at 25° to 26°, while it was greatly reduced at 27° to 28° and completely inhibited at 29° or above. The infected seedlings from experiment 5 show the great reduction in the amount of disease per 248 Journal of Agricultural Research vo1.xxii,no.s plant at 27° to 28° as compared with 25° to 26°. It is interesting to note that infection was reduced more abruptly at 27° to 28° in the artificially- inoculated soil than in that naturally infested. This may be due to the age of the inoculum, a smaller percentage of the spores being functional in the former soil, or perhaps to the presence of a smaller amount of inoculum. Table VI. — Relation of soil temperature to infection of onion seedlings by Urocystis cepu lae Experiment i. Experiment 2. Naturally infested soil. Begim May 3, 1919; com- pleted May 30, 1919. Records not kept as to soil moisture nor as to air conditions. Artificially inoculated soil. Begim Dec. 10, 1919; completed Jan. 6, 1920. Soil moisture held at 22 per cent or two-thirds the water-liolding capac- ity. Air temperature 13° to 23° C, relative hu- midity 45 to 75 per cent. Soil temperature. Total ntmi- ber of plants. Smutted plants. Soil temperature. Total num- ber of plants. Smutted plants. "C. 10 to 13 25 5" 47 25 Per cent. 72 80 100 15 0 10 to 14 64 49 63 56 52 36 Per cent. 98 98 18 to 20 16 5 to 18 22 to 24 25 to 30 2 2 to 26 28 to 34 27 to 29 93 8 0 20 to •?! " stand reduced by damping-off fungi. Table VII. — Relation of soil temperature to infection of onion seedlings by Urocysti^ cepulae Experiment 3. Naturally infested soil pleted Jan. 10, 1920 Begun Dec. 20, 1919; com- Soil moisture held at 25 per cent or two-thirds the water-holding capacity. Air temperature, 13° to 2?° C: relative humiditv. Air temperature, 13 4S to 75 per cent Soil temperature. "C 19 to 22 23 to 26 26 to 27 27 to 28 Total num- ber of plants. 44 100 86 Smutted plants. Per cent. 93 96 S7 12 Experiment 4. Artificially inoculated soil. Begun Dec. 18, 1919; completed Jan. 12, 1920. Soil moisture held at 13 per cent or two-thirds the water-holding capacity. Air temperature, 13° to 25° C; relative humidity, AS to 75 per cent. Soil temperature. C. 23 to 26 26 to 28 29 to3i , Total num- ber of plants. 50 40 Smutted plants. Per cent. 98 12 O Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 249 Table VIII. — Relation of soil temperature to infection of onion seedlings by Urocystis cepulae Experiment 5. Experiment 6. Naturally infested soil. Begrun Jan. 16, 1920; com- pleted Feb. 10, 1920. Soil moisture held at 25 per cent or two-thirds the water-holding capacity. Air temperature, 13° to 25° C; relative humidity, 40 to 80 per cent.. Artificially inoculated soil. Begun Jan. 16, 1920; completed Feb. 10, 1920. Soil moisture held at 15 per cent or two-thirds the water-holding capac- ity. Air temperature, ij° to 25° C; relative hu- midity, 40 to 80 per cent. Soil temperatiu-e. Total num- ber of plants. Smutted plants. Soil temperature. Total num- ber of plants. Smutted plants. T. 25 to 26 89 103 77 43 Per cent. 100 47 0 0 °C. 25 to 26 106 98 47 30 Per cent. 27 to 28 27 to 28 29to3o.s 2Q to ^0. < 0 30 to •?2 0 It may be concluded from the foregoing experiments that a high per- centage of infection may be expected up to 25° C, above which there is a rather abrupt reduction, leading to complete inhibition at 29°. There appears to be no lower limit of temperature for infection within the range where onion seeds will germinate and normal growth occur. After it was clearly shown that no infection v/ould take place at 29° C, the question arose as to how long seedlings must grow at this temperature to become entirely immune. It has been shown that at moderate tem- peratures the plant becomes immune in about 20 days, or at about the time when the cotyledon has reached its maximum growth. To deter- mine whether or not this condition is altered when the plants are grown at 29°., several pots each of the naturally and the artificially inoculated soil were started off at this high soil temperature. Pots were then transferred from time to time to a lower temperature favorable for infection (15° to 20°) where they were held for about three weeks before they were examined for signs of the disease. The results of these experi- ments are summarized in Table IX. It is quite evident that the amount of infection was markedly reduced by an exposure of 15 to 18 days at 29° C. Complete inhibition of infection by even more protracted exposure to this high temperature was not attained. However, where infection did occur there was usually not more than one lesion per plant, which in the majority of cases was so situated that subsequent infection of newly forming leaves would be impossible. It is thus quite certain that where seedlings develop at about 29° for the first 20 days the amount of damage from smut will be negligible, especially in an area where the amount of inoculum is slight. 250 Journal of Agricultural Research voi.xxn.Ncs Table IX. — Effect oj different soil temperatures upon onion smut infection. In all cases except the fifth, tenth, and eleventh, the pots -were held for the stated period at 20° C, where infection was inhibited, then transferred to 15° to 20°, a temperature favor- able for infection. In the fifth and tenth, where the continuous temperature was high, note that practically no smut developed; in the eleventh, where the soil temperature was continuously low, note that practically all the plants were smutted; in the remaining series, where the plants were transferred from the higher temperature (29°) to the lower (75° to 20°) after varying periods, note that long exposures at the higher temperature tended to reduce the amount of infection. Extent of infection at end of experunent. Type of soil inocu- lation. Length of ex- posure t0 29''C Size of plants at time of removal to low temperature. Pot No. Total number of Per- centage dis- Extent of infection. plants. eased. Days. I 8 II Cotyledons i inch long. 16 94 63 per cent dis- eased first leaf. 2 15 Cotyledons 2 to 2>^ inches long. 17 41 12 per cent dis- eased first leaf. 3 ) ^T3 18 do 12 25 25 per cent dis- eased first leaf. .2^ 4 t-fl "^ 28 First leaves i-f inches high. 9 II II per cent dis- eased first leaf. 5 , 35 Continuous expo- sure at 29°. 6 0 6 1 12 Cotyledons iK to 2 inches long. 29 48 14 per cent dis- eased first leaf. 7 1 6 Coty le dons 2 ^to 40 13 3 per cent dis- Si 3K inches long. eased first leaf. 8 1 23 First leaves out in about one- 24 8 8 per cent dis- eased first leaf. half plants. 9 27 First leaves out in most plants. 16 13 6 per cent dis- eased first leaf. 10 38 Continuous at 34 3 0 per cent dis- ai 29°. eased first leaf. II 0 Continuous at 76 99 Most of these 15° to 20°. plants died in cotyledon stage. It is interesting to note also in this connection that continued expo- sure of onion roots to a temperature of 29° C. led to the gradual slowing up of groM^th. With the transfer of the pots to the lower temperatui-e (15° to 20°) both root and top development were greatly stimulated. In attempting to correlate these results one must keep in mind the fact that in nattire the temperature conditions under which the onions develop are much different from those in the experimental pots. Whereas in the pots the soil temperature is uniform throughout their depth, in the natural soil there is a gradual decrease in temperature at progressively greater depths. The temperature of the upper inch runs extremely high during the summer months because of direct exposure to the sun's rays, and this is the area critical for infection by onion smut. The young roots, on the other hand, as they develop progressively reach strata of lower temperature, which are more favorable for their growth. Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 251 EFFECT OF HIGH AIR TEMPERATURE UPON THE DEVEI^OPMENT OF SMUT The experiments reported above in which the soil temperature was varied were carried on at an air temperature of 15° to 20° C. The latter is considerably lower than the air temperature which prevails in southern onion sections at the time when young seedlings are starting off. This is shown for one section (Laredo, Tex.) in Table XIII, where the mean air temperature is about 30° during most of the onion-planting time. The question arose as to what efifect these high air temperatures might have upon the development of smut in the aerial portions of the plant. Five clay pots of onions in naturally infested soil were started off in a greenhouse running at about 25°. Previous observations had shown that the pustules become evident in the cotyledons on about the twefth day under these conditions. Accordingly the plants were allowed to grow at this temperature for nine days, at which time a few lesions were barely visible. In order to prove that good infection had aheady taken place, 10 plants were removed from infested soil and washed thoroughly in running water to remove any external inoculum, after which they were transplanted to clean soil. Within two days lesions were distinctly visible in these plants, and smut developed in 8 out of the 10. As a control on this method of removing the external inoculum 11 plants grown in clean soil were moistened and covered thoroughly with infested soil. They were then washed in running water and trans- planted to clean soil. No smut developed. On the ninth day after sow- ing, 4 of the 5 pots were removed to a greenhouse running at 30° to 33° C, one being allowed to remain at 25°. One pot was then transferred from the higher temperature back to 25° at the end of each the second, fourth, ninth, and fourteenth days. This exposure to the higher temperature resulted in a stimulation of host plant growth for a few days. When the plants were allowed to remain at this high temperature, however, for three weeks distinct stunting became evident, while more prolonged exposure resulted in death. The plants so transferred were allowed to continue growth at 25° for three weeks or more, when they were examined for the presence of smut. The final results are given in Table X. It was evident that the gradual elimination of smut which took place was proportional to the length of exposure to the higher temperature (29°). After 14 days of exposure only small lesions developed on 16 per cent of the onion plants, although presumably 80 per cent or more of these plants were originally infected while they were growing at the lower temperature. This experiment was repeated several times with prac- tically the same results, namely, that exposure of plants bearing in- cipient infections to a temperature of 30° to 33° for 12 to 15 days almost entirely checked further development of the parasite. 65768°— 21 2 252 Journal of Agricultural Research voi. xxii. No. 5 Table X. — Effect of high temperature, following infection, in inhibiting the further development of smut. The plants were from a series of pot cultures started at 25° C. and held there until incipient infection had occurred, then transferred for the period indicated to a high temperature, J0° to 33°, and finally brought back to the original 25°. Length of exposure to temperature of 30° to 33°. None (25° throughout) 2 days 4 days 9 days 14 days Percentage smutted. 94 45 37 33 17 It is to be noted that in the experiments just reviewed potted plants were used. The entire pot, thus including roots and tops of the experi- mental plants, was exposed to the stated temperature condition. The question then arose whether the results secured were due entirely to the effect of high air temperature upon the fungus or to an indirect effect of the changed conditions upon the metabolism of the host. In order to throw some light upon this point two experiments were con- ducted in which seedlings were grown in infected soil at three constant soil temperatures and each of two air temperatures, 25° and 30° to 33° C. The results (Table XI) at the lower air temperature (25°) coincided closely with those previously secured at air temperatures of 13° to 25°, inasmuch as abundant infection occmred when the soil temperature was 25°, while complete inhibition was attained at 30°. It is, however, significant to note, in comparison with the results in Table X, that, with the soil temperature held at 20° or 24°, the 30° to 33° air temperatm-e did not greatly check the development of the disease. It appears, then, that roots as well as tops must be exposed to the inhibitive higher tem- perature, 30° to 33°, in order fully to check the parasite after incipient infection has taken place. This suggests that the inhibitory effect may be due in part at least to the influence of the environmental conditions upon the metabolism of the host and not entirely to a direct effect upon the fungus itself. Table XI. — Effect of different combinations of soil and air temperature upon onion smut infection Air temperature. 2S 30 to 33 . Soil tempera- ture. 20 25 30 20 24 30 Experiment i. Number of plants. 23 68 42 Op 73 24 Percentage smutted. 100 97 86 o Experiment 2. Number of plants. 8S 49 22 41 .■50 30 Percentage smutted. 95 92 o 46 60 o 1 stand reduced by damping-o£f fimgi. Oct. 29, 1931 Relation of Soil Temperature to Onion Smut 253 ^FFBCT OF MODERATEI/Y HIGH TEMPERATURES UPON SYSTEMIC INVASION OF THE PI^ANT Thaxter (jo, p. 134) observed that in some instances the smut fungus may infect and develop in the cotyledon without invading the first leaf, with the result that the plant eventually outgrows the disease. Obser- vations lead us to believe that this may vary with different tempera- tures. It has been pointed out that at a temperature of about 25° C. the most rapid top growth of the onion seedling occurs, while at tem- peratures below 20° the top growth is much retarded. Two pots of infested soil were sown with onion seed and placed in greenhouses, one at 24° to 28°, with a maximum of about 36°, for one or two hours on sunny days, the other at 15° to 20°. A high percentage of cotyledon infection occurred in both pots. After 31 days 24 out of 29 plants at the high temperature were infected, but the pustules were all confined to the cotyledons and no infection of first leaves had developed, although the plants were now in the second leaf stage. At the low temperature, on the other hand, of approximately the same number of plants, only 9 had survived on the thirty-seventh day, and 8 of these showed infec- tion in the second leaves. It appears, then, that rapid growth of tops at about 25° may result in a large percentage of plants outgrowing the disease after the cotyledons become infected. The results of successive field plantings, discussed in the next paragraph, seem to confirm this judgment. The importance of the practical bearings of this matter are such as to justify further critical attention. EFFECT OF SUCCESSIVE PLANTINGS THROUGHOUT THE GROWING SEASON UPON INFECTION The laboratory experiments described early in this paper have shown that onion smut infection is greatly reduced where a constant soil tem- perature of 27.5° C. is maintained during the susceptible period of the plant's growth, while a temperature of 29° thus applied completely inhibits infection. Moreover, as explained in the last paragraph, when plants are growing in infested soil with temperature of air and soil held at about 25°, although a high percentage of cotyledon infection may occur, there is a greater tendency than at lower temperatures for the plants to outgrow the disease, owing to the rapid growth of tops. These results combined to justify the expectation that successive field plantings of onion seed throughout the growing season might show considerable variations in the percentage of smut infection. In the onion field the soil temperature usually varies widely during 24 hours, often reaching a maximum considerably above 29° during the day and descending to a minimum much below this at night. Under Wisconsin conditions the daily mean temperature gradually rises during the spring and early summer months and falls during the latter part of the growing 254 Journal of Agricultural Research Vol. XXII, No. s season. It seemed possible, therefore, that by making successive plant- ings a period might be found for this latitude when the mean soil tem- perature is sufficiently high to materially check or completely inhibit onion smut infection. In order to test this out, a series of plantings at intervals of from 8 to 14 days was begun on June 18, 1920, at Madison, Wis. Onion seed was sown in smut-free soil in an open trench about i inch deep and was then covered with naturally infested soil similar to that used in certain of the laboratory experiments. Two varieties. Red Globe and Yellow Bermuda, were used, one lo-foot row of each variety being put in at S/ 5 Fig. 2. — Graph showing the daily mean soil temperature at a depth of i to 2 indies as it occurred in the "successive planting" plots. Since the weather continued rather cool, one bed was covered with glass to insure a higher temperature. The temperature of the uncovered bed is shown by the solid line, the temperature of the glass-covered bed by the broken line. For further details see Table XII and and accompanying text. each planting. Temperatures of the soil at a depth of i to 2 inches were obtained by means of a self-recording thermograph. The hourly mean temperature for each day was then secured by adding temperatures as recorded for each hour and dividing the sum by 24. These computa- tions are represented graphically in figure 2. Since the weather in July was unusually cool, a higher mean soil temperature was secured for some of the plots by covering them with an ordinary glass cold frame. Inasmuch as the dry weather and high temperature would cause a rapid desiccation of the surface layer of soil, the plots were watered thoroughly on alternate days or oftener during the early growth of the plants. The Oct. 29. 1921 Relation of Soil Temperature to Onion Smut 255 data collected from this field plot are tabulated in Table XII. Obser- vations were made by pulling plants at several points in each plot, and examining for smut lesions after the chlorophyll had been removed by means of alcohol and acetic acid. The first observation was made on the twenty-first to the twenty-third day after planting. Subsequent observations were made as indicated in the table. TabIvE XII. — Development of onion smut in successive plantings in the field at Madison, Wis., ig20 Date of Variety. Treatment. First observa- tion, 21 to 23 days after planting. Second observa- tion, 29 to 31 days after planting. Third observation, 44 to 65 days after planting. planting, 1920. Ntim- ber of plants exam- ined. Per- cent- age smut- ted. Num- ber of plants exam- ined. Per- cent- age smut- ted. Time after plant- ing (days). Num- ber of plants exam- ined. Per- cent- age smut- ted.ff June 18 Red Globe... [Red Globe. . . Uncovered . ..do 108 62 45 41 20 52 40 35 46 26 S3 73 49 10 10 0 13 14 0 0 & SO 42 47 28 2>7> C40 «86 /;3 0 52 44 31 SI 3 39 June 26 Yellow Ber- muda. [Red Globe . . . ...do . . .do 65 65 65 53 53 53 53 61 24 9 135 39 91 45 July 10 Yellow Ber- muda. Red Globe . . . Red Globe... Yellow Ber- muda. Red Globe... Yellow Ber- muda. ...do Covered . . Uncovered ...do Covered « . Covered a . 0 0 II 13 9 24 July 19 " Covered for 15 days only. •6 Observation 39 days after planting. «^ Extent of infection: Systemic, 26 per cent; confined to dead cotyledon, 14 per cent. <* Extent of infection: Systemic, 10 per cent; confined to dead cotyledon, 66 per cent. « Extent of infection: Systemic, 5 per cent; confined to dead cotyledon, 81 per cent. / Extent of infection: All cotyledon infections. 0 Extent of infection: All systemic infections. An analysis of the data secured can be made by referring to Table XII and figure 2. It will be seen that the soil temperature mean gradually rose until July 23 to 29, after which there was a gradual drop. At no time did the mean in the uncov red plot reach the inhibiting temperature (29° C), but it closely approached this point during the warmest portion of the season. In the covered plot, however, the mean remained above 29° continuously until the cover was removed on August 3. The two important points to be considered in the respective plantings were (i) the amount of original infection which was determined three to four weeks after planting (see first and second observations in Table XII) and (2) the extent to which the disease either became systemic or was entirely outgrown by the plants during the following four or five weeks (see third observation in Table XII). 256 Journal of Agricultural Research voi. xxii. no. s Considering first the amount of original infection, it will be seen that a high percentage of disease resulted in all the plantings of June 18 and June 26. The somewhat lower infection in that of June 18 may be ex- plained in part at least by the fact that a smaller quantity of inoculum was used than in subsequent plantings. The next two plantings (July 10 and 19) were so made that the resulting seedlings were exposed during early growth to the maximum soil temperature of the season. By refer- ring to Table XII it will be seen that associated with this higher tem- perature there was a decided reduction in the amount of infection in even the uncovered plots, while in the covered plots, where the mean temperature remained continuously above 29° C, no infection whatever occurred. Considering, secondly, the extent to which the disease became sys- temic or was outgrown, it will be seen that in the planting of June 18 a majority of the infected plants showed systemic invasion at the second observation (thirty-ninth day). In the next planting (June 26), how- ever, by the time of the second observation most of the external signs of the disease were confined to the dead cotyledons. The amount of systemic infection increased somewhat, however, at the third observation (39 per cent). In the third planting (July 10) it is interesting to note first that the plants in the covered plot remained entirely free from infection. In the uncovered plot, although some cotyledon infection was noted at the sec- ond observation, no disease whatever was found at the third observa- tion. This indicates that the time when the temperature was at its highest point the infected plants succeeded best in outgrowing the disease. The field data secured in the foregoing experiments at Madison are thus in general accord with the experiments performed under controlled conditions. In such controlled experiments the amount of smut infec- tion falls as the soil temperature rises toward 29° C. and is totally inhib- ited above this temperature. Likewise in the field trials with successive plantings there was a gradual reduction in the amount of infection fol- lowing the rise in the mean soil temperature, with omplete inhibition of infection where the mean was kept above 29° for two or three weeks after planting. Complete freedom from infection under these Wisconsin field conditions was secured only by growing the plants under artificial condi- tions in which by covering the plants with glass the temperature was raised several degrees above the normal. It is, however, to be noted that the summer of 1920, when the foregoing results were secured, was some- what cooler than the average. The weather records of other years indi- cate that in a hot summer complete inhibition of smut infection would be secured by such summer plantings. Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 257 CORRELATION OF EXPERIMENTAL RESULTS WITH FIELD CONDI- TIONS OF THE SOUTHERN STATES These results obtained in both greenhouse and field experiments justify the question as to the part played by soil temperature in determining smut infection in the onion fields of the more southern States. As noted at the beginning of this article, a recent survey of southern onion sections indicates that smut is not prevalent in the southern fields — for example, in Texas — as it is in the northern onion sections. As was earlier explained, it is the practice in these southern fields to plant the onion seed in late summer or early autumn. It is thus quite possible that the mean temperature for the surface inch of soil in southern onion sections is considerably above the maximum for onion smut infection during and immediately following the sowing of seed. According to Mally (6), onion seed is sown in the Laredo district of southern Texas as early as August i , while most of the seed is planted about September 10 to 25. The mean air temperature as recorded at Laredo, Tex., by the United States Weather Bureau for August, September, and October, 1917, is given in Table XIIL Table XIII. — Mean air temperatures for August, September, and October, iQiy, at Laredo, Tex.''' Day of month. August. September. October. Day of month. August. September. October- "F. " F. " F. °F. "F. °F. I 89-5 85.5 71.0 17 90. 0 79.0 80.5 2 88.5 86.0 80.0 18 90-5 83.0 81. s 3 88. s 86.5 80.5 19 91. 0 7!?-5 76. 0 4 88.5 87.0 80.5 20 92. 0 79-5 67..-; S 89.0 88.0 80. s 21 90. 0 79-5 62. 0 6 88. <; 87-5 78.0 22 90. 0 80.0 63-5 7 89. i 88.0 81.0 23 90. 0 79-5 68. s 8 88.5 88.5 82.5 24 91. 0 79.0 61. 0 9 89.0 89-.=; 64-5 25 89-5 80. ■; 66.5 10 90-5 88.0 64-5 36 87.0 81. 5 76.5 II 90- .■; 83-5 70-5 27 89-5 81.5 74-5 12 88.0 85.0 73-.'; 28 86.5 71- S 78. s 13 89-5 86. =; 7S-0 29 89.5 71- 5 75- 0 14 88.5 88.5 77.0 30 84.5 71- S SO-."; I c 90- S 88.5 83-5 83-5 76. 0 80.0 31 86. 0 0 16 ° Obtained by averaging the daily maximum and minimum temperatures. Table XIII shows that the air temperature ranged very high during August and September, the onion-planting period. In this connection it is to be noted, moreover, that the records of Bouyoucos (i) in Michigan indicate that surface soil temperatures may considerably exceed air temperatures. Thus, his observations showed that the maximum tem- perature for the upper quarter inch of all the soils he studied was about 16° C. higher during hot, clear days than that of the air at an elevation 258 Journal of Agricultural Research Voi. xxu. No. s of 4 feet, while the minimum temperatm-e of all the soils used, except peat, was 0.5° to 1.0° C. higher, as a monthly average, than that of the air. Our own observations in Wisconsin are in general accord with these Michigan records. Assuming that the temperature of the surface layer of soil under Texas conditions likewise averages several degrees higher than the air, it is evident that the mean never went below the point where infection is entirely inhibited (29° C, or 84° F.) during August and seldom below it during September. Continuing up to October 8 there were only a few days when the air temperature fell below 27.5° C. (81.5° F.), the point at which our experiments have shown smut infection to be markedly reduced. It seems probable, therefore, that even if onion smut were introduced into this Laredo soil, it would stand small chance of infecting onion seedlings to the extent of establishing the disease as a permanent factor. The data available are not sufficient to justify the attempt at more detailed geographic correlation of onion smut occurrence with the temperature factor. We believe, however, that the conclusion is justified that soil temperature during the early seedling stage must be considered as a limiting factor in determining the occurrence of the disease in any locality. It must be left with local observers to make use of this fact in interpreting conditions as they occur in any particular region. SUMMARY Onion smut was first noted in the Connecticut River Valley in 1869. Since then it has successively appeared and become an economic factor in nearly all of the northern onion-growing sections from New York to Oregon. This has probably resulted from chance introduction of the organism with seed or bottom sets, followed by its accumulation in the soil where continuous cropping with onions is practiced. The disease has not appeared in the southern onion-growing sections of Texas and Louisiana, although they are exposed to similar chance introduction of the parasite and the continuous cropping method is common. These facts have raised the question as to wherein lies the explanation of the regional limitation of the disease. The southern method of cul- ture, characterized by special seed bed and transplantation of seedlings, does not offer sufficient explanation for the absence of smut. No differ- ence in susceptibility between northern and southern varieties has been found. Is regional limitation explained by differences in environ- mental factors in the North and the South at the time when the seedling is susceptible to infection, that is, during the first two or three weeks after germination? An analysis of certain of these factors in relation to infection has been the object of the present investigation. The cotyledon of the onion is susceptible to attack by the smut organ- ism up to the time it attains full growth, a period of about three weeks. Oct. 29, 1921 Relation of Soil Temperature to Onion Smut 259 varying somewhat with environment. Cotyledons remaining free from infection during this period become resistant and serve as a barrier to subsequent invasion of the embryonic region of the true leaves. Conse- quently, if infection is prevented by environing conditions during this period of susceptibility, the plant will remain free during the remainder of its growth. Experiments were conducted in which seedlings were grown on smut- infested soil held at various soil moisture contents. A high percentage of infected plants resulted over the entire range in which good germina- tion and growth of the host occurred. At either extreme, very high or very low moisture, there was some reduction in amount of infection, but with it occurred a corresponding decrease in seed germination and rate of growth of the plants. Soil moisture, therefore, does not appear as a serious limiting factor in onion smut infection. .^rj v/ot; The relation of soil temperature to the development of the host and the parasite was studied by growing plants in pots held experimentally at a series of constant soil temperatures in the special apparatus known as the "Wisconsin soil temperature tank." Seed germination and growth took place over a range of soil tempera- ture from 10° to 31° C. Most rapid seed germination and development of tops occurred at soil temperatures of 20° to 25°, while as a rule the best development of roots occurred below 20°. A high percentage of plants grown on smutted soil were infected at soil temperatures ranging from 10° to 25° C. A decided reduction in infection was noted at about 27°, and complete freedom from the disease resulted at 29°. In these experiments all plants were under uniform conditions of air temperature, which ranged from 15° to 20°. The relation of variations in air temperature to the development of the disease was then studied. Exposure of plants bearing incipient infections of the fungus in the aerial parts to an air and soil temperature of 30° to 33° C. so disturbed the relations between parasite and host as to preclude any further develop- ment of the disease. This was shown by growing plants at a tempera- ture favorable for infection (15° to 20°). Then, just as the pustules of the disease were beginning to appear (tenth to twelfth day), the plants were removed to a room held at 30° to 33°. This stimulated top growth for a few days, which was followed by a decided checking of the plants and death after three or four weeks. However, if after 12 to 15 days at the high temperature the plants were returned to the original tem- perature (15° to 20°), they grew normally, but the fungus in nearly all cases failed to produce spores, and the plants remained free from further invasion. Experiments were then performed in which seedlings were grown on infested soil held at 20°, 25°, and 30° C. with a uniform air temperature of 30° to 33°. A high percentage of infection resulted at soil temperatures 26o Journal of Agricultural Research voi. xxii, no. $ of 20° and 25°, but none at 30°, showing that high air temperature alone is insufficient to check the development of the disease. It appears probable that the failure of the fungus to complete its development in the case described above (where the plants after infection were exposed to an air and soil temperature of 30° to 33°) was brought about at least in part by some marked disturbance of the metabolism of the host and not simply by the direct effect of the high air temperature upon the fungus in the aerial parts of the seedling. Comparison between the development of the disease in plants grown at 15° to 20° and at 24° to 28° C. (air and soil) was made. A high per- centage of cotyledon infection occurred in both cases. At the lower temperature the disease proceeded as usual to the infection of the true leaves. At the higher temperature, however, the plants tended to out- grow the disease, this being associated with a more rapid rate of top development which apparently enabled the plants to slough off the smutted cotyledons before infection of the first true leaf occurred. The foregoing conclusions as to the dominant influence of soil tem- perature upon onion smut infection, while primarily based on greenhouse experiments, have been found to accord well with field developments. Successive out-of-door plantings at Madison, Wis., made in inoculated soil during the growing season, resulted in a gradual reduction of infection as the season advanced and the soil temperature rose. Complete freedom from smut was attained when the daily mean soil temperature at i to 2 inches depth remained at or slightly above 29° C. for two to three weeks. There was also a tendency, as the temperature rose, for the seedlings to outgrow the disease by the sloughing off of the diseased cotyledons before infection of the first leaf occurred. An examination of records from one of the southern onion sections (Laredo, Tex.) shows that during a good share of the critical period for onion smut infection (August and September) the mean air temperature is above that at which complete inhibition of infection was attained in our experiments (29° C. or about 84° F.). If we assume, as observed in northern sections, that the mean temperature for the upper layer of soil is several degrees higher than that of the air at this time of the year, it is reasonable to conclude that even though the smut organism were introduced into southern onion sections, its development would be pre- vented or greatly minimized, first, by the prevention of infection due to high temperatures, and, secondly, by the rapidly developing tops out- growing the disease, should occasional infections occur. In general we believe, therefore, that the regional distribution of onion smut in the United States is conditioned upon the soil temperature during the seedling stage of the plant's growth, the infection and develop- ment of smut being favored by the relatively low temperatures and inhibited by the high temperatures, with approximately 29° C. as the critical point. Oct. 29, 19" Relation of Soil Temperature to Onion Smut 261 It is hoped that the evidence here recorded may lead to the accumula- tion of further field data bearing upon this particular problem by inves- tigators in various places, especially in the southern States. It is also believed that these results illustrate well the importance of more per- sistent inquiry by the experimental method into the relation of environ- mental factors to the occurrence of disease of plants in general. (I (2 (3 (4 (S (6 (7 (8 (9 (10 LITERATURE CITED BouYOUCOS, George J. 1916. SOIL TEMPERATURES. Mich. Agr. Exp. Sta. Tech. Bui. 26, 133 p. Chapman, George H. I910. NOTES ON THE OCCURRENCE OF FUNGOUS SPORES ON ONION SEED. MaSS. Agr. Exp. Sta. 22 Ann. Rpt., 1909, pt. i, p. 164-167. Heald, Frederick D., and Woolman, H. M. 1915. BUNT OR STINKING SMUT OF WHEAT. Wash. Agr. Exp. Sta. Bui. 126, 24 p., 5 fig. (in text and on pi.). HuNGERFORD, Charles W., and Wade, A. E. 1920. RELATION BETWEEN SOIL MOISTURE AND BUNT INFECTION IN WHEAT. (Abstract.) In Phytopathology, v. 10, no. i, p. 53. Jones, L. R. 1917. SOIL TEMPERATURES AS A FACTOR IN PHYTOPATHOLOGY. In Plant World, V. 20, no. 8, p. 229-237, 2 fig. Literature cited, p. 236-237. Mally, F. W. 1915. THE BERMUDA ONION. Tex. Dept. Agr. Bui. 46, 56 p. MuNN, M. T. 1917. NECK-ROT DISEASE OF ONiONG. N. Y. State Agr. Exp. Sta. Bui. 437, p. 361-455, II pi. Bibliography, p. 4SO-455- SiRRiNE, F. A., and Stewart, F. C. 1900. EXPERIMENTS ON THE SULPHUR-LIME TREATMENT FOR ONION SMUT. N. Y. State Agr. Exp. Sta. Bui. 182, p. 145-172, i pi. Sturgis, William C. 1896. TRANSPLANTING, AS A PREVENTIVE OF SMUT UPON ONIONS. In Conn. Agr. Exp. Sta. 19th Ann. Rpt. 1895, p. 176-182, pi. i. Thaxter, Roland. 1890. the "smut" OF ONIONS, (UROCYSTis cEPUOrAE FROST). In Conn. Agr. Exp. Sta. Ann. Rpt. 1889, p. 129-154, pi. 1-2. Ware, Benjamin P. 1870. EXPERIENCE OF A PRACTICAL FARMER. In 17th Ann. Rpt. Mass. Bd. Agr. 1869, Appx., p. 1-16, PLATE 25 Relation of soil temperature to the development of onion seedlings. Upper row. — Seedlings of Red Globe variety 13 days old. Each cluster was the entire crop from one experimental culture pot. All were grown in like virgin soil and at the same air temperature (i5°to2o°C.) but with gradation in the soil temperature of the respective pots as follows (left to right): 12° to 14°, 20°, 25°, 28°, 30°. Note that there is a tendency for greater root development in relation to top growth at the lower temperatures. This was especially marked at the lowest temperature, 12° to 14°. For further details see Table V and the accompanying text. Lower row. — Seedlings of Yellow Bermuda variety grown imder same conditions as those in upper row. (262) Relation of Soil Temperature to Onion Smut Plate 25 L. Journal of Agricultural Research Vol. XXII, No. 5 Relation of Soil Temperature to Onion Smut Plate 26 Journal of Agricultural Researcli Vol. XXII, No. 5 PLATE 26 Relation of soil temperature to the infection of onion seedlings by the smut fungus, Urocystis cepulae. Representative seedlings taken from the experimental culture pots, showing the influence of soil temperature upon the amount of smut. All the pots alike contained smut-infested soil. The air temperature and other aerial factors were the same for all. Soil temperature was the only factor varied experimentally, the temperature grada- tions extending from about 10° to 30° C. Note the abundance of smut at 10° to 22°, as shown in the upper row. A slight re- duction occurred at 23° to 26°. At 27° to 29° the reduction is sharply marked. At 29° to 3 1 ° inhibition is complete. For the percentages of seedling infection and other details see Table VI and the accompanying text. PLATE 27 Relation of soil temperature to onion smut infection. This shows the results from a series of experiments in which the methods described for Plate 26 were repeated with the soil temperatures restricted to the critical limits between 25° and 29° C. and controlled more exactly. Note the marked reduction in infection at 27.5° and complete inhibition at 29°, thus establishing, but with more exactness, the conclusions illustrated in Plate 26. For the percentage of infected seedlings at these temperatures and other details, see Table VIII and the accompanying text. Relation of Soil Temperature to Onion Smut Plate 27 \ V ( \ ZS^'C 1 27.5°C Journal of AgriculturalResearch Vul. XXII. No. 5 A PHYSIOLOGICAL STUDY OF GRAPEFRUIT RIPENING AND STORAGE ' By IvON A. Hawkins Plant Physiologist, Office of Horticultural and Pomological Investigations, Bureau of Plant Industry, United States Department of Agriculture In an earlier investigation (7) ^ the changes in Florida-grown grape- fruit during storage were studied, particular attention being paid to the sugar and acid content of the pulp or edible portion of the fruit as influ- enced by some six different storage temperatures. It was found that the acid content decreased in cold storage while the total sugar content re- mained about the same. The percentage of cane sugar decreased and the reducing sugar content increased. At the higher temperatures, common storage (about 55° to 60°, 70°, and 86° F.) there was in some cases apparently an increase in acidity and a reduction in the amount of sugar, especially in fruit stored for long periods. The shrinkage, which was very marked in the ventilated packages at these high temperatures, made the obtaining of definite evidence on this point impossible. The investigation described in the present paper is concerned with the acid and sugar changes in the fruit on the tree as well as with the changes which take place, both in warm storage and in cold storage, in fruit picked at monthly intervals. The control of the pitting which occurs commonly on grapefruit in cold storage is given some attention. PLAN OF THE EXPERIMENTS " Common Florida " ^ fruit from two trees was picked at monthly inter- vals for four months, beginnmg July 27, making five different picks. At the last three pickings fruit was also harvested from two additional trees in the same grove. The fruit was expressed to Washington and sampled on arrival. Part of the fruit was placed in warm storage at about 70° F. and part in cold storage at 32°. Analyses were made of the fruit stored in the warm storage each month for two months and on fruit from the last three pickings stored in cold storage, after it had been in storage four months. By this plan it was possible to obtain data on the changes in the fruit on the tree from a month or so before the fruit was in con- dition to pick for market until the last of the season, and to compare the changes which took place in cold storage in fruit picked from the same trees at different times of the season. 1 This paper gives the results of a portion of the work carried on under the project " Factors affecting the storage life of fruits." 2 Reference is made by number (italic) to " Literature cited," p. 278-279. ' The writer's thanks are due Mr. W. J. Krome for the picking and shipping of all the " Common Florida" fruits used in these exijeriments. Joimial of Agricultural Research, Vol. XXII, No. s Washington, D. C. Oct. 29, 192 1 aag Key No. 0-251 (263) 264 Journal of Agricultural Research voi. xxii. no. s METHODS OF ANALYSES The fruit was prepared for sampling and sampled as in the previous work. Analyses were made for acids, sugars, both reducing sugars and total sugars, dry weight, shrinkage, thickness of peel, and percentage of peel. In addition the acidity and specific gravity of the expressed juice of the fruits were determined and the solids-acid ratio calculated after the usual method. The acidity determinations were carried out as in the previous work, as were practically all the other determinations with the exception of the extraction of the sugar from the pulp. In the sugar extraction a method was followed similar to that described in work on potatoes (6). The weighed pulp was placed in a liter volumetric flask which was then filled to volume with 85 per cent alcohol. It was allowed to stand with frequent shakings for about three weeks, the losses from evaporation, of course, being made up by adding alcohol. The alcoholic solution of sugar was then separated from the residue by filtration, and the sugars were determined in aliquots of the filtrate. The first lot of grapefruit was of small size, green in color, with very little juice in the pulp. No solids-acid determinations were made on this lot. They were, however, maintained in warm storage for two months. At the end of this period many of them had assumed the char- acteristic yellow color of the ripe grapefruit. The second pick, received August 29, was much further advanced, being about 50 per cent colored and of good size. The third and fourth picks, those of October 25 and November 28, respectively, were in fine condition for shipping and are what would be considered midseason fruit. The November 28 fruit was fair, possibly a little coarser than the two picks immediately preceding. No sprouted seeds were found in any of the fruits, however. The date of picking might be considered as in the latter part of the grapefruit season for this locality and for this variety. The analyses of grapefruit picked from trees i and 2, from warm storage at about 70° F. for one and two months, are shown in Tables I and II. In the analytical work the analyses were usually made in duplicate, and both analyses are given in the tables, as this furnishes evidence on the experimental error in the method of sampling. The tables are self-explanatory. An inspection of Tables I and II shows that in the first four pickings there is in all cases an increase in the acid content of the pulp, while in the last picking from both trees there is no decided increase. A com- parison of the acid content, as determined in the analyses of the pulp and the acid content of juice, shows a similar behavior. The acid content of the juice is, as a rule, markedly higher than that of the pulp, due, of course, to the fact that in the last-mentioned case the weight of fibrous material is taken into consideration in calculating the percentage Oct. 29, 1921 Physiological Study of Grapefruit Ripening 265 of acid. In the fifth, pick from both trees there is no decided variation in the acid content of the pulp during storage, and the percentage of acid in the juice does not change as much as in fruit from any of the other four picks. With the sugars, the percentage of reducing sugars and of total sugars is always greater at the end of two months in warm storage, except in the fifth pick. The reducing sugar increases most, due prob- ably to the inversion of some of the cane sugar which is less in all cases after two months in storage. It was brought out in the earlier publication on grapefruit storage that there was an indication that the acid content of the fruit was slightly increased during a long period of warm storage. It was pointed out also that definite evidence on this point was difficult to obtain because the structure of the fruit prevented accurate calculation of the shrinkage of the various portions. Further evidence, mostly of an indirect nature, may be derived from the data on sugar and acid content of the fruit, found in Tables I and II. As was mentioned above, there is in all cases an apparent increase in the acid content and the total sugar content of the pulp, due for the most part undoubtedly to loss of water during storage. In the tables it is noticeable that the solids-acid ratio is usually less after two months in storage. This indicates, of course, that the increase in soluble solids is not proportional to the increase in acidity and that some soluble substance or substances other than titratable acids decreased in the storage period. This occurs in five cases out of seven on which data were obtained. The other two cases, tree 2, third pick and fifth pick, show slight increases, 0.07 and o. 1 1 , respectively. These data are corroborated in the total sugars-acid ratios, which are calculated by dividing the percentage of total sugar as dextrose by the percentage of acid as citric. In the 10 cases the ratio of sugar to acid is less in 7, practically the same in 2, and greater in i. Indications are, then, that there is usually an increase in the ratio of sugar to acid under the conditions of the experiment. This could be brought about by either decreasing the sugar content of the fruit or by increasing the acid content or by a combination of these two factors. It is notice- able that in 6 cases out of 10 the acid-sugar ratio is greater after one month in storage than it is after two months at the same temperature. The acid and sugar in the fruit from warm storage will be considered later in comparison with the changes taking place in cold storage. There is in most cases not much variation in the percentage of dry matter during storage, though there seems to be a tendency, more marked in some cases than in others, toward an increase. This seems probable, as the shrinkage where determined is from 14.4 to 23.3 per cent for the full two months in storage. The percentage of peel always decreases during storage at this temperature, due to the loss of water and wilting. This is evident in the decrease in thickness of the peel, which is very marked, especially in the earlier picks. 65768°— 21 3 266 Journal of Agricultural Research voi. xxii, no. s CHANGES IN FRUIT ON TREES The analyses of fruit from the various pickings at the time it was placed in storage (Tables I and II) show marked differences in composition. PfRl con ri , \ X Mf, 10 fts 9 ^1 ^ .'^ ^-^ \^ :^ ■==r ^^'^ :^ ?^^ ^,_ >^ ■=^ ^^ 7 -^ — -| Z y /^ ^ — 6 55 5 y ^ y ^ •\ ^ o»&-- y- / / ■^o-V^^ ,o>-. •" 4 as 3 Z5 Z \3 1 y ' y / ^^ :^ ^i^ ■■—::. ^ ^<- Sucsc SE j^^,.^ "^^^ 2_S}i CPOSt. z^^ ,,/ y jjcSfiS- &• ^ ^ -^t^ "^^^ ^^^^^ Acid — .Tree 1 — .Tree 2 AU&i SEPT. \ ( XT 1 NOV. 1 DEC. 1 Fig. I. — Graphs showing changes in percentage of dry matter, acid, and sugars calculated as dextrose in fruit on two Common Florida trees from August to December. For convenience in comparison the data for sugars, acids, and dry matter are shown graphically (fig. i). In these curves the percentage is plotted on the ordinates and the time interval between pickings on the abscissae. Physiological Study of Grapefruit Ripening 267 t~. 00 r^ 0 0 00 t^ H C) M t^ a [ ■2 A B^ M ■* 1^ Oi OfOHtstooOOO tO-^OO M J^ 4 4 N N t^ t^vd d d »0 n' to M H 00 0 5 0 8^ TO (^ 10 o\vo 0 o> c^ t^ • H IT) 'S " " ^ to "1 (N 0\ to f^ . • to N •*00 ^ X sii^g M M 4 4 to to i^ t-*'d ■ • ■■)• tH M M 03 ^ W "i-> .— ,^ •— »—■*—.- _^_. : fe •d w .A ^ ro r^ 0 N 10 1; 0 CJ r^ 1/ 1 to tt to to N 10 M .^ ,. ) t4 s6 d d " 4 p^.s P _.>_ —^^.-^ -„-«^ --V— •i=i a 0 J 00 M O^t^O\O00f^^^0 -Ot^t^ 0 0 t~ lOOO lOvO HI t^vO -^ -^ ^00 4 4 N ci^O'vdtO d OC^-^j-MMt^ 0 a jH S a«, — ,^^-- ^w^ H M H M oS.H 0 CO t^ M M N Tf >0 « CO l^ fO 0 0 c< 0 ^ f 10 o\ t 3 0 H H 4 w ti vd d t^ 4 M M .i "^ t- 9, 0 M 0 t to N OvvO >o Tt M M u 0 1; '^ 0 0 0. 0 M ej 0 <^ " t 00 0 H «00 S.2 0 « r^ to to>d v3'>d d. M 10 M d 00 M •a 4 00 fO 00 0 CO oi-o a too tH to M to ai-sji ^ -T Tf 'J- o\oo 00 vo 00 o\ tooo CI H-aj^ *"* M lsa>^ M -0 W-O \0 00 M -^00 00 to M M 0 'a " " 0 00 00 »o a u^^O t^ M OiOO •a 0 rt;5 0 to N N N lnv^»}■o^0^c^to M 0 di 1 t^""^^ "^ U) ^ 0 00 M 00 H to to M t^ 01 to 00 tooo ?> 0 OJ M 0 0 lovo OS 0 lo 0\ tf-O to 10 Ct Tf to ^^«s H M c^ « « c^ lnir)4dd\t^i/S M d» t^ Ph.s 0 •2 , M'^ ■- a 0 1> ■*CC t^CO 00 >o 10 to to^O to »0 OS »o ts to 0 « too 0 tOvO 1^ 0 «io>o4didvts4 0 o\ 0 1 a g h-q| >_^" 1000 S §, S 0 00 0 0 CI 0 OS M ■* N ^J- rf to 0. t~ ti 00 c! w uS t^ d ,^ ri- M d -d m"" ^0 " " m •a il. CO fO r^ONOO CAt^O »oi?> tj- M VO H 0 (U . MMvooot^O»-^0 w T^ c* to Ah .2 m « •* m tt d\ cj\ 100 M cjs>d t3 I Mod •a cu— 0 00 t^ 01 Q 0 o\ 6 N M to M »n 0\ »o 10 r^ H tf tj- t^ M vd 10 •a 'a H w Qq -V---V __^_ _!!_" •a J, 5 a a-y 0 aa Q ro I^ 0 -0 0 f^ to 00 Tt- 0 o\ ov ao'^totoct ■(-> Wi 01 m Q 1" 1-t m "w --'— .--^ ^-— -— , "o i 2' w 0. C>'0 CO 00 t^ ^ >/^ 0 00 (U 0 D N >0 TT l^ 1 0 M -0 -0 to . Ci t^ to C< M « fosd ^.b"!, >_^^ ^-y— — V ■0 V •s 0 c« 0 1 i § •c ■3 "3 J •0 'S "3 3 J, J3 ^ S 1 ^ 3 S ?•«: -i: -3 -a c "o 7 '0 ■€ J It-? a ho 61 ' ^ 1 ^ |i|?l2 b 0 S S3fe-3-s? C8 Cl -U -1. ■0 a> 1 < p. Ai ft (- ft ft i" ft < <7) tfi 1 268 Journal of Agricultural Research voi. xxii, no. s J. So lo c^-O t >-» I^ M fO fO 0 N « O M CO t<; to o 00 ro ^^ O 0\ ^ I woo a 0 2^ ^ -so o rt g +; l-ll ) 0\ N O 00 vO ^OOOH M OCOW O t^O fD HI CO 04 ro fOO 'O ^ 0\ 0\ H -^ *ivO 00 O ^O fn Tj- fn ^ fO I I -^ 0\ O rt ■* 00 O Ov (^ 00 00 w) 't ^ 0\ CO VI ■ -^ f^ in M o\ t- lo W W W lO ■^ 4- o\ lO »o Tf M O CO "^ lOO iri ^ H O O O ^0 NvO lO 4 o\ -^ -^ vO 00 N M -tj- O O ■ t^ t^ lo w OS ^ rr f*j o» c> 6i*o *0 "(too lO -^ w ^O 00 ^ 01 "* Q O w H ro CO < a d .9 3 00 ro (O M fo t^ H 6 00 0\ Oi i 6 >o Forbes, R. H. certain effects under irrigation of copper compounds xtpon crops. Arlr. Agr. Exp. Sta. Bui. 80, p. 145-238. 16 fig., 4 pi. (i col.). 1916. Bibliography, p. 236-238. Part 3, Appendix: Methods of analysis, with the collaboration of E. E. Free and W. H. Ross, p. 229-233. Oct. 29, 1921 Absorption of Copper from the Soil by Potato Plants 285 Tabl^ II. — Copper found in soil SPRAYS APPLrED TO SOIL NEAR PLANT ROOTS Sample No. Date of sampling. Description of samples. Description of plots. Total copper found in soil. I July 26 Aug. 24 Sept. 3 Samples taken before any copper was added to the soils. Samples taken just before last application of copper to soil. Samples taken after the last application of copper to the soil. Control P. p.m. 2 2 3 4 5 6 7 8 9 10 Pickering "A" formula Bordeaux ^-^-c;o I 2 CUSO4 solution Control 2 S 211 256 250 2 Pickering "A" formula Bordeaux •?— •?— t;o CUSO4 solution Control Pickering "A" formula Bordeaux 3—3—50 225 243 449 CUSO4 solution SPRAYS APPLIED TO VINES IN COMMERCIAL PRACTICE July 16 Aug. 31 Samples taken before any sprayings were made in 1917. Samples taken after last sprayings were made in 1917. Control Pickering "C" formula. Bordeaux 5-5-50 Pickering "A" formula Control Pickering "C" formula Bordeaux 5-5-50 Pickering "A" formula DISCUSSION OF RESULTS VINES AND TUBBRS The leaves, stems, and roots of the plants from the soil receiving the Pickering spray showed an increased copper content with each successive analysis (Table I). The largest percentage of the copper was held by the leaves. The roots held an appreciable part of the copper, the amount increasing from o in the first sample to 0.0160 per cent in the sample taken on September 3. The tubers contained only minute amounts of copper. The plants from the Bordeaux treated soil showed irregularities, particularly with respect to the copper content of the roots and stems. The leaves and stems contained more copper than those of the plants from the Pickering treated soil, while the roots contained less copper than the roots of the plants from the Pickering treated soil. The amounts of copper found in the tubers were small. The vines grown in the soil treated with a solution of copper sulphate showed a marked progressive increase in copper content of the roots with each succeeding analysis. The leaves contained somewhat larger \ 286 Journal of Agricultural Research voi. xxn.No. s amounts of copper than the stems, but not as much as the roots. The leaves contained less copper than the leaves of the plants grown on the Bordeaux or Pickering treated soils. The tubers from the plot treated with copper sulphate solution were as low in copper as those from the other plots. The analyses of the various portions of the conti'ol plants showed the presence of copper, but in smaller amounts than in the plants grown on soil treated with the copper sprays. The results of the copper absorption experiments indicate that the potato plants, with the exception of those grown in the soil receiving the solution of copper sulphate where the roots were distinctly injured, distributed the largest part of the absorbed copper to the leaves, while the roots and stems contained appreciable amounts of copper. In all normally sprayed potato plants the largest proportion of the copper is said to be found in the leaves. The plants grown on the soil treated with a solution of copper sulphate were small and lacking in vigor. The roots had but few hairs, and showed other signs of injury. The large percentage of copper found in the roots, together with the small size of the roots, indicated some inter- ference with the normal metabolism of the vines. The toxic effect of the soluble copper salt was exerted primarily on the roots of the plants. It was apparent that the soluble copper sulphate had injured the potato plants, while the insoluble copper compounds had not. The vines from the Bordeaux plot contained a little more copper than the vines from the Pickering plot, indicating that the extra lime of the Bordeaux spray did not aid in preventing the absorption of copper by the plants. soil, The results of the analyses of the first and last samples of soil taken show that no water-soluble copper was found in any of the samples examined. The amount of copper in the first set of samples (Table II, No. 1, 2, 3, and 4) which were taken before any copper had been added to the soils, is practically the same in all cases. The sets of samples taken before and after the final treatment of the plots show the pres- ence of a large amount of copper in the samples receiving the copper treatments. This means that copper in an insoluble form may be pres- ent in the soil in marked amounts without exerting any apparent toxic action on the growth of potato plants. But little copper was found in the soil as a result of spraying with copper sprays according to commercial practice. On September 5, shortly after the last treatment of the soil, a few hills of potatoes were dug. The weights and number of the tubers, the per- centage of decayed tubers, as well as the notes taken on the size and appearance of the vines are given in Table III. Oct.29, I92I Absorption of Copper jrofyi the Soil hy Potato Plants 287 Table III. — Effect on potato tubers and vines of applications of sprays to soil Tubers." Spray used. Weight. stand and condition vines. Total. Average. Ounces. Ounces. Bordeaux (0.75 per 3 (i large and 2 8 2^ Normal. cent CUSO4). small) in ihill. Pickering (0.7 per 8 (5 large and 3 29 zH Do. cent CuSOJ. small) in 2 hills. Copper-sulphate solu- 17 (all small) in 4 23 iK Small and stunted. tion (0.75 per cent hills. CUSO4). Unsprayed (control) . 8 (3 large, 3 medium, and 2 rotten) in 2 hills. 24 3 Normal stand ; blight. a Rot found only on control tubers. These data show tliat the solution of copper sulphate had a very dis- astrous effect on the growth and yield of the tubers. The only decayed tubers found were obtained from the unsprayed plot. These results are so few that they can be considered only as suggestive. vSUMMARY Potato plants grown in soil treated with insoluble copper compounds contained more copper in the leaves than in the stems, while but little copper was found in the roots. The tubers showed only traces of copper. When the soil was treated with the copper sulphate solution, the roots were injured and the normal metabolism of the vines was disturbed. The tubers from these vines were small and the vines stunted. The roots of these plants held more copper than the leaves. The soluble copper sulphate added directly to the soil caused injury to the plants, while the insoluble copper compounds of the sprays did not. The excess lime of the Bordeaux spray did not reduce the amount of copper absorbed by the plants compared with the plants grown on the Pickering plot. Practically the same amounts of copper were found in all the soil samples tested. Samples of soil from sprayed potato fields showed but minute amounts of copper. ADDITIONAL COPIES OF THIS PUBLICATION MAY BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTrNG OFFICE WASHINGTON, D. C. AT 15 CENTS PER COPY Subscription Price, S3.00 Per Year V Vol, XXII NOVKIvIBE^R 5, 1921 No. 6 JOURNAL OF RE SEAJR.CH CONXKNXS Page Pale Western Cutworm (Porosagrotis orthogonia Morr.) - 289 J. R. PARKER, A. L. STRAND, and H. L. SEAMANS ( Contribution from Montana Agricultural Experiment Station) Biology of Embaphion muricatum - - - - - 323 J. S. WADE and ADAM H. BOVING ( Contribution from Bureau of Entomology ) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINGTON, D. C. GOVERNMENT PRINTING OFFICE 1921 EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF LAND-GRANT COLLEGES FOR THE DEPARTMENT KARL F. KEIyIi92i Pale Western Cutworm 291 DISTRIBUTION Published records of the occurrence of Porosagrotis orthogonia are as follows: Glencoe, Nebr., by Morrison (15, p. 2jg) ; Colorado, New Mexico, Arizona, and Utah by Smith {16, p. i2g) ; Rocky Mountains by Dyar (j. P- 139) ; Calgary, Alberta, (doubtfully) by Dod {2, 37, p. 53) ; Prairie, Alberta, by Hampson (9, p. 102) ; and southern Alberta by Hewitt {10, p. 177), Gibson (7) and Strickland {17). In Montana Porosagrotis orthogonia now occurs throughout the State east of the continental divide. It has been most abundant in the tier of counties which lies just east of the foothills of the main range of the Rocky Mountains and which extends from the Canadian border to within 100 miles of the southern border of the State. Mr. E. H. Strickland has kindly given the following information on the present distribution of Porosagrotis orthogonia in Canada : Our records indicate that it is practically confined to southern Alberta, extending as far north as latitude 51° and east to longitude 108°, although it has been recorded as far as Regina, Saskatchewan. The maximum intensity, however, is confined to an area that does not extend more than 100 miles east of the Rocky Mountains. Dr. William Barnes, of Decatur, 111., who has an extensive collection of western noctuids, has generously furnished the following records of Porosagrotis orthogonia specimens in his collections: Denver, Oak Creek Canyon, Lavetta, and Alamosa, Colo.; Deming and Fort Wingate, N. Mex. ; Provo, Vineyard, and Eureka, Utah; Yellowstone National Park, Wyo.; Reno, Nev. ; Redington, Ariz.; and Kern County, Calif. Mr. George M. List states that Porosagrotis orthogonia is fairly com- mon at Fort Collins, Colo., 430 moths having been taken at a trap dur- ing the season of 1920. )<;■ ;0 I; To Mr. C. N. Ainslie, of the Bureau of Entomology, United States Department of Agriculture, we are indebted for a record of 63 Porosagrotis orthogonia moths reared from a shipment of larvae from Dickinson, N. Dak., on June 10, 1920. From the foregoing records it is evident that Porosagrotis orthogonia occiu-s at least in scattering numbers throughout the southwestern and northwestern States with the possible exceptions of Oregon, Washing- ton, and Idaho, where as yet it has not been collected. Correspondence with entomologists throughout the territory where P. orthogonia has been recorded indicates that it has never been of economic importance outside of the heavily infested areas in Montana and Canada. METHODS OF STUDYING IN THE INSECTARY Larvae were reared in individual tin boxes 1.5 inches in diameter. The bottom of each box was covered with filter paper which was slightly moistened at each feeding. This prolonged the freshness of the wheat 292 Journal of Agricultural Research voi. xxn.No. 6 and dandelion which were used as food, and the cans could be easily cleaned by replacing the filter paper whenever it became soiled. Pupae were placed in moist, well-pulverized soil in individual glass vials I inch in diameter and 4 inches deep. Each vial was filled to a depth of 2 inches with well-firmed soil in which a round hole i inch deep was punched to receive the pupa, which was placed in it with the anterior end uppermost. A small twig was placed in each vial so that the moth upon emerging could hang from it and allow the wings to expand. The vials were closed by cheesecloth held in place by rubber bands. The best results were obtained by keeping the pupae in uniformly moist and mellow dirt. Extreme dryness or excessive moisture often resulted in the death of the pupse. Uniform moisture conditions were more easily obtained by allowing water to run slowly down the side of the tube instead of flooding it over the surface of the soil. After the moths emerged they were placed in wire s,creen covered tin cans 3.5 inches in diameter and 2.5 inches deep. An inch of moist soil was kept in the bottom of the cans, and alfalfa or clover blossoms were added each day for the moths to feed upon (PI. 30, A) and to hide under. Cutworm moths of all kinds seem very contented in these cans, and with Porosagrotis orthogonia no difficulty was encountered in getting the females to mate and lay eggs. Eggs were placed on filter paper in pint Mason jars with the caps lightly screwed down. A few drops of water were placed on the filter paper from time to time to provide the proper amount of humidity. IN THE FIELD A very good opportunity to watch the development of this insect under natural conditions was afforded in a heavily infested field at Wil- sall, Mont., in 19 19. This field was first examined on May i and was visited several times a month all summer. Many fields in other parts of the State were also visited, but that at Wilsall was the only one where Porosagrotis orthogonia was followed through all stages of its develop- ment. During the summer of 1920 a temporary field station was estab- lished at Willow Creek, Mont., in a district where thousands of acres of wheat had been destroyed during May and June. Moths appeared in large numbers during August and September and were under observa- tion at all hours, both day and night. Special attention was given to the egg-laying habits, and for this purpose two observation cages were set up. The cages were 2 -foot cubes with screen wire sides and solid metal tops. They were placed over sunflower plants, and the ground inside the cages was covered with soft dirt, stubble, clods, baked earth, and green plants, thus offering the moths nearly all the natural condi- tions of the neighborhood. Experiments with trap lights were also carried on at the Willow Creek field station. Nov. s. I92I Pale Western Cutworm 293 SEASONAL HISTORY AND HABITS DURATION OF EGG-IvAYING PERIOD During the season of 19 19 the eggs were found to be well developed in the ovaries when the female moth first emerged, and egg laying began and was completed within a short time under insectary conditions. The first eggs were obtained from reared moths on August 17, the aver- age period between emergence and the beginning of egg laying being four days. Moths collected in the field on August 26, which appeared to have just emerged, laid numerous eggs the following day and con- tinued to lay until September 9. In 1920 no records were available from reared moths, but a study of moths in the field seemed to indicate that the eggs that season were not fully developed when the moths first emerged. Thus out of 35 moths examined on August 24 only one had well-developed eggs in the ovaries. On September i many moths were found with the ovaries filled with well-developed eggs. These moths were mostly badly rubbed specimens, indicating that they had probably emerged some little time before. Our field observations show that the height of the egg-laying period is during the last week in August and the first week in September. Eggs in smaller numbers may be laid during the first three weeks in August and as late as October i . WHERE EGGS ARE LAID The first eggs obtained were from moths confined in tin rearing cans. When the soil in the cans was dry and light most of the eggs were placed from X iiich to i inch below the surface and could be found only by careful searching. When the soil was hard and lumpy eggs were scattered about on the surface and could be easily seen. In the rearing cans the eggs were not always laid in the soil. Many of them were placed on the stems, leaves, and flowers of alfalfa, and frequently scattering eggs were found on the sides of the cans or on the screen covers. Thus out of 243 eggs found in one can, 180 were found in the soil, 62 were found on the stems, leaves, and flowers of alfalfa, and i was found on the side of the can. Some were laid singly and others were in clumps of 2, 3, or 4, and sometimes as many as 40. In outdoor cages, where a variety of soil conditions and various kinds of vegetation were available, eggs were laid only in loose, dry dirt. Under field conditions the eggs are very difficult to find, and the only ones we have ever found were secured by carefully examining the soil at the exact spots where females were seen in the act of egg laying. Eggs are found most frequently in loose, mellow dirt from X to >^ inch below the surface. This is an important point, and will be discussed later in connection v^^ith the habits of the moths and from the standpoint of control methods. 294 Journal of Agricultural Research voi. xxii. No. 6 NUMBER OF EGGS The only females upon which we have complete recor ds are five moths reared and mated in the insectary. They averaged 315 eggs, the lowest number per moth being 248 and the highest 453. Ten moths brought in from the field averaged 132 eggs, but these had probably laid numerous eggs before they were caught. Under normal field conditions the aver- age number per female is probably between 300 and 400. The records of the individual moths are shown in Tables I and II. Table I. — Time between emergence and egg laying, length of egg-laying period, and number of eggs laid by reared specimens of Porosagrotis orthogonia Moth No. August, 19 19. Eggs in ovaries at death. Total 13 14 IS 16 17 iS 19 20 21 22 23 24 2S 26 27 28 29 30 31 ber eggs. E E 243 D E E E Ds 0 249 0 0 3 248 9 258 88 4S 96 66 30s S4 2SI 2 D 2S 12 283 26 75 D 12 453 E= emerged. D=dead. Average period between emergence and egg laying 4 days. Average period of egg laying 3 to 4 days. Average number of eggs i'^s- Table II. — Period of egg laying and number of eggs laid by Porosagrotis orthogonia moths brought in from the field and kept in rearing boxes Moth No. August. September. Eggs in ovaries at death. Total 26 27 28 29 30 31 I 2 3 4 5 6 7 ber eggs. Caught 63{ 1 D 100 D 23 45 2 6 IS 6 16 ] 4 91 0 88 103 30 I 0 0 152 167 do 116 do 22 2 74 5 43 3 I 1 D fD \ 3 m \ 3 m \i4 /D I 8 36 184 . .do 4 1 107 do f 112 6 do 4 35 26 56 IS 20 25 30 32 12 22 1 134 do 20 \ 127 8 .. ...do ( D 44 116 . ..do \26 1 106 do / D D=dead. Average period of egg laying 3 to 4 days. Average number of eggs 132. DURATION OF EGG STAGE The length of the egg stage is exceedingly variable, depending largely upon moisture conditions. It may be as short as 1 1 days or may extend over several months. Eggs laid in the insectary August 19 hatched August 3c. On September 30 several newly hatched larvae were found Nov. s, 1921 Pale Western Cutworm 295 at Wilsall. Hundreds of eggs were laid in the breeding cans during the last week in August and the first week in September, but with the ex- ception of three larvae which hatched August 30 no eggs hatched unless they were placed in a very humid atmosphere. The larvae mature within the eggs in from 10 to 20 days and may remain for months in this condition, waiting merely for proper moisture conditions to allow them to break through the eggshell. Examples of this may be of interest. Moth number 1427-G laid 160 eggs on August 24 and 25. These were placed on a piece of filter paper in a Mason jar. They were allowed to stand in the laboratory for 10 days and were then placed in an incubator and held at day temperature of 80° F. and night temperature of 60°. At the end of three weeks practically all the eggs had darkened, and the black heads of the young cutworms could be seen through the eggshells, but none had hatched. On October 4 a few drops of water were added to the filter paper, and when the jar was opened 24 hours later 70 larvae were found to have hatched. On October 1 1 water was again added to this jar, and 40 more larvae hatched out. On October 27 a few drops of water were added to another jar of 62 eggs which were laid August 29. In two hours 8 larvae had hatched, but no more hatched during the next six hours. Twenty-four hours later all of the eggs had hatched. On October 27 moisture was added to eggs that had been kept in the greenhouse since August 30, and in two hours many of them had hatched. On this same date one of these eggs was placed on a block of plaster of Paris, and water was slowly dropped upon it from a medicine dropper. At the second drop the larva began to move within the egg. Soon it began to move its mandibles and after several attempts the eggshell was punctured, and within 30 minutes after the first drop of water was added the larva was free from the shell and actively moving about. On November 20 eggs that were laid August 30 and had been kept for a month in a small tin can on a shelf directly over a radiator were examined, and living larvae were removed from them by carefully breaking the eggshells with fine needles. On November i twenty eggs which were laid on August 29 and had been kept indoors were placed in two small wooden boxes and buried in a pail of damp sand. The pail was set on the ground, outdoors, where it was covered with snow practically all winter. The eggs were exam- ined once a month, but none hatched until the second week in April when all the eggs were found to have hatched and the larvae were alive and vigorous. From our studies of the egg it would appear that if there is sufficient moisture and proper temperature condition the majority of the eggs will hatch in the fall, while if it is unusually dry or cold weather starts early the eggs will not hatch until the following spring. Strickland {18) found eggs on frozen ground December 3, which would indicate that in Canada some of the eggs at least do not hatch until spring. 296 Journal of Agricultural Research voi. xxii. no. 6 FIRST APPEARANCE OF I^ARV^E Larvae may appear in the fall. This is proved by the fact that three larvae were found at Wilsall on September 30 and that larvae hatched in the rearing boxes during October and November whenever sufficient moisture was added. Large numbers of larvae must have hatched at Wilsall during the fall of 1918, for as soon as the snow left the ground the following spring fourth- and fifth- instar larvse were found in large numbers. We have no records of injury to wheat during the fall months, but it seems quite probable that in years when there is considerable moisture and mild weather during October and November great damage may be done. The larvae begin to feed shortly after the wheat begins to grow in the spring. In 191 9 at Wilsall 80 acres of winter wheat were completely destroyed by May i, which indicates that the worms must have been active 10 days or 2 weeks previous to that date. In 192 1 at Willow Creek first-instar larvae were found on March 3. The weather had been warm for about a week, and winter wheat was starting to grow again. No larger larvae could be found, and it seemed as though the very small first-instar larvae must have just hatched. PERIOD OF LARVAIv FEEDING One of the reasons why Porosagrotis orthogonia is such a dangerous insect is the unusually long period of heavy larval feeding which extends until the middle of June or even to July 10 in the case of late-hatched specimens. Judging from the reports of injury received, the larvae attract most attention during the month of May and the first two weeks in June, differing decidedly in this respect from Chorizagrotis aiixiliaris, which generally has reached the height of its destructiveness by April 15 and has practically disappeared by May i. The length of the larval stage as determined for 20 larvae, 5 from each of 4 parent moths, under insectary conditions varied from 62 to 151 days and averaged 118 days, as shown in Table III. All of the larvae were kept under very similar conditions, and no reason has been found for the wide variation. They were always fed at the same time and were kept on one tray in individual rearing boxes, thus giving practically identical conditions of moisture, temperature, and food. In spite of this similarity of conditions we find that larva 2ie pupated 62 days after hatching from the egg, while larva 2 id from the same parent and from the same egg cluster took 124 days, or just twice as long, to reach the same stage of development. The number of instars was also found to vary. Thus the number of larval instars for the five larvae from each of the moths was as follows: Moth 21 — four had 7 instars and one had 8; moth D — all five passed through 8 instars; moth 42 — two had 7 instars and three had 8; moth 24 — one had 7 instars and Nov. 5, 192 1 Pale Western Cutworm 297 four had 8. According to the rearing records of other larvae than those shown in the table, several individuals passed through 9 larval instars and one passed through 10. Eight instars, however, is the usual number and the minimum is 7. Table III. — Duration of larval instars of Porosagroiis orthogonia under insectary conditions Larva record No. Number of days in each instar. IV. VII. VIII. Num- ber of days in larval stage. 2ia 2lb SIC 2 id 2ie Da Db Dc Dd De 42a 42b 42c 42d 426 Ma. Mb Mc Md Me Average 12 12 9 12 13 12 12 13 II 10 14 12 13 9 7 13 13 7 5 6 6 9 7 9 9 10 7 9 8 5 10 8 II 6 7 9 12 7 II 8 10 10 7 9 12 12 9 10 II 7 8 10 9 12 10 9 7 9 7 13 10 14 10 10 9 10 10 10 15 9 10 10 12 10 14 9 II II II 9 10 17 II 9 18 12 12 10 13 9 9 10 13 13 18 13 17 13 9 16 16 15 14 20 17 12 13 9 18 16 14 13 9.4 9.8 II- 3 14.4 44 29 32 16 14 14 14 21 16 19 25 41 34 14 20 18 18 26 45 37 48 63 45 51 32 2>Z 33 75 56 65 43 99 85 98 124 62 125 121 151 125 150 123 104 112 112 119 148 144 148 131 83 22. 6 29. 6 118 FEEDING HABITS OF LARVA The larva differs from that of most cutworms in its feeding habit in that it almost invariably attacks the plant below the surface of the ground. The most frequent type of injury is the eating away of the central stem and its surrounding sheaths from }i inch to i inch below the surface of the ground. In many instances the stem is severed and the lower part not eaten, and frequently it is only slightly chewed into before the worm moves on to another plant. Even slight injury to the under- ground portion of the central stem usually results in the death of the plant. A very small portion of each plant is actually eaten by this cutworm, and its capacity for destruction is thus greatly increased. The first indication of injury is the presence of wilting or dried plants which can be easily lifted out of the soil without bringing the roots. The destruction of grain that is just pushing through the ground is par- ticularly rapid, one worm being able to cut off plant after plant in quick succession. The worm usually moves along the drill row, taking each plant as it goes (PI. 30, D). Where one crop has been destroyed and 298 Journal of Agricultural Research voi. xxn.No. 6 the land has been reseeded the worms often attack sprouting grain and sometimes even gnaw into the kernels before they germinate. The larvae feed during both day and night. Freshly cut plants have been found repeatedly during the day and cutworms have been found with their heads inside the sheath of the plant in the act of feeding on the central stem. In the rearing boxes no difference could be noticed between the amount of day and of night feeding. According to observations made by Strickland and reported by Gibson (7) it is the habit of the larva — to travel over the surface of the soil and when a suitable plant for attack is found it immediately burrows and feeds just below the surface. In our observations we have never vdtnessed this habit. Considerable time has been spent during the late afternoon and evening in heavily infested fields, and except in rare instances we have never seen Porosa- grotis orthogonia larvse above ground. On the other hand, we have closely examined hundreds of newly attacked plants where there was no sign that the soil had been disturbed at the surface by the burrowing of the larva. In order to find out whether the worms would work from one plant to another without coming to the surface, wheat seedlings were started 3 inches apart in a flat in the greenhouse, and when the wheat was up about i inch 12 half-grown cutworms were placed in one end of the flat. At the same time a line of plaster of Paris was placed across the center of the flat so that any traveling of the worms over the surface at the center would be indicated by lines through the plaster of Paris. The seedlings at the end of the flat where the worms were intro- duced were immediately attacked, and within a week all of the seedlings in the flat had been cut off below the surface of the soil and without any indication that any of the larvae had crossed the plaster of Paris line. If it were the natural habit of Porosagrotis orthogonia to travel over the surface of the ground in getting from plant to plant, it would come in contact with poisoned bran mash scattered on the ground, and it seems as if there would be no difficulty in controlling it by the ordinary methods. The fact that poisoned bran mash is useless against Porosa- grotis orthogonia, together with our field and insectary observations, leads us to believe that it very rarely comes to the surface in getting from one plant to another but instead moves underground, generally along the drill row. We have received occasional reports from farmers stating that pale western cutworms had been seen feeding above ground during and im- mediately following rains, but only one instance of this kind has come under our own observation. This was at Willow Creek on June 27, 1920. A light thunder shower at 6.30 p. m. cooled the air and wet the ground to the depth of i inch. As soon as the storm was over, numer- ous cutworms were seen crawling over the surface in a wheat field that Nov. 5. 1921 Pale Western Cutworm 299 was known to be badly infested with Porosagrotis orthogonia. It was estimated that about 50 per cent of the total number of cutworms in the soil were on the surface at any one time. They appeared in greatest numbers in the spots where the grain had previously been cut off. At first they merely wandered about over the surface, but later on, as it became dark, they started feeding. Observations were continued until II p. m., at which time the worms were still moving about and feeding upon the leaves of wheat and grass. At 5 a. m. the next morning a few worms still remained on the surface, but all disappeared as the sun came up. The soil around stray wheat plants was noticeably stirred up where the worms had come up and gone down. That this habit of feeding above ground is not a common one is shown by the fact that only in rare instances have we ever found any injury to that part of the grain plant which is above the ground, and in such cases there was always some doubt as to whether the injury might not have been done by some other species. LARV^ DO NOT LEAVE FlElvDS AFTER GRAIN IS DESTROYED Another unusual habit of Porosagrotis orthogonia is that it seldom migrates even though its food supply becomes exhausted. If, in fol- lowing along the drill row, it fails to find a plant within a few feet, it simply remains where it is, perhaps for several weeks, without feeding or growing to any extent. In fields that are only partially infested the injury shows up as scattered bare spots, and in such places the larvae do concentrate along the edges of the standing grain, but we have never known them to migrate more than a few rods. This habit of remain- ing in the fields where grain has been destroyed has a very important bearing on farm practice, as will be shown by the following example: Eighty acres of winter wheat at Wilsall in 19 19 were completely de- stroyed by May i, the ground being left entirely bare. The field was reseeded to spring wheat the second week in May. On May 24 the grain was just coming through the ground and was being rapidly cut off by cutworms which had remained in the field since the winter wheat had been destroyed, some three weeks before. The worms continued to feed for several weeks and destroyed all the spring wheat. The ability to go for a long time without feeding was well shown by a half-grown larva which remained in a rearing can for 12 weeks with- out food and was then fed and reared to maturity. FOOD PLANTS In Montana this cutworm has been most commonly found feeding upon winter and spring wheat. Oats, barley, rye, flax, and alfalfa have also been attacked. In the insectary, larvae have fed readily and grown rapidly upon dandelion. In Canada, Gibson reports Porosagrotis 300 Journal of Agricultural Research voi. xxii no. e, orthogonia larvae as feeding upon fall and spring wheat, oats, barley, flax, beets, onions, cabbages and carrots. PERIOD OF INACTIVITY BEFORE PUPATION Although Porosagrotis orthogonia larvae are mature and have prac- tically ceased feeding by the middle of June they do not pupate until nearly a month later. During this period they occasionally feed slightly, but for the most part they remain in a semidormant condition, gradually turning whitish in color and shrinking in size just previous to pupation. This was noticed both in the field and under insectary conditions. Notes taken at Wilsall June 20, 1919, state that on that date cutworms were decreasing in numbers and were nearly all full grown. This field was visited again on July 4, when many whitish larvae were found, some of which had formed earthen cells, but no pupae were found in a two-hour search. Records kept on 75 larvae in the insectary showed an average period of 20 days of complete inactivity previous to pupation and a period of 26 days in which only very slight feeding took place. PUPAI^ PERIOD Pupation generally takes place about the middle of July. Out of 80 specimens collected as larvae at Wilsall in May, 1919, and reared to adults in the insectary, the average date of pupation was July 19, the earliest date July 2 and the latest August 1 1 . This checked out almost exactly with conditions in the field at Wilsall. About a month is spent in the pupal stage. The average length of pupal period of 80 specimens was 29^^ days, the shortest 2 1 days and the longest 40 days. The pupae are protected by a cemented earthen cell and are usually found at a depth of 3 to 4 inches in the soil beneath the plants where they last fed. SEASONAL ABUNDANCE OF ADULTS The earliest emergence of Porosagrotis orthogonia moths which we have on record is July 31, although Gibson {8, p. 30-ji) reports the emergence of a moth of this species on July 19, In general, the period of greatest abundance is during the last two weeks in August and the first week in September. The field at Wilsall where Porosagrotis orthogonia larvae destroyed two seedings of wheat during May and June, 19 19, was searched for moths on August 7, but none could be found. A trap light was run until mid- night on this date, and not a moth of this species was taken. On August 26 the field was again visited, and numerous moths were found during the day, and at night they came to trap lights by the thousands. The majority of the moths taken at this time were in prime condition and Nov. s, 1921 Pale Western Cutworm 301 looked as though they had just emerged. One week later the number of moths was greatly reduced ; the)'' were difficult to find during the day, and very few came to lights at night. On September 30 an entire day was spent in searching the same field, but not a moth could be found. The ovvTuer of the field had been disking and drilling throughout the month of September and during the first half of the month had frequently seen moths fly up as the ground was disturbed but had seen none after September 15. Seventy-five larvae collected in this field May i and reared in the insectary at Bozeman emerged as adults on the dates shown in Table IV. In 1920 at Willow Creek the first moth was caught on August 9, the heaviest flight was from August 19 to 24, and several moths were seen as late as October 8. Table IV. — Dates of emergence of Porosagrofis orthogonia moths in igiQ July 31. Aug. 4 5 6 7 8 9 10 12 13 14 IS 16 Number of moths. Aug. 17 18 19 20 22 23 24 25 26 27 28 31 Sept. I. Number of moths. 4 13 3 2 5 S 4 4 2 I I I 2 EGG-LAYING HABITS Egg laying was first witnessed at Willow Creek in 1920. Several gravid females were placed in outdoor observation cages in which a variety of soil conditions and vegetation was offered and were closely watched for several days. On the afternoon of September 4, at 4.45, one of these females was seen laying eggs. She crawled over clods, stubble, and plants, constantly feeling with the ovipositor the objects which she walked upon. On reaching a patch of soft earth she stopped and carefully worked the abdomen into the soil until the wings were flat on the ground. After remaining quiet for a short time she moved and repeated her actions in another spot. Three ovipositions were made in 15 minutes, and after each one the dirt was stirred as the abdomen was withdra-'vn and the hole left covered with dirt. The dirt around these holes was carefully removed with a teaspoon and eggs were found in clusters of 3 or 4 about % inch below the surface of the ground. A total of II eggs were recovered from the three ovipositions. 302 Journal of Agricultural Research voi. xxn, no. t Porosagrotis orthogonia moths were seen laying eggs in the open at Willow Creek on September 5, 1920. Just before dark moths were seen flying over a freshly worked, summer-fallowed field, being most abun- dant on the higher knolls and along the ridges where the soil was soft and loose. One moth was followed for some distance. She would fly a few feet, never getting over 10 inches above the ground, and would then crawl a short distance, continuously feeling the surface with her ovi- positor. On reaching soft dirt she stopped and laid eggs for six minutes, going through the same actions as the moth observed in the cage. When she left the ground she flew straight away for at least X ^^^ ^t a height of 20 to 30 feet above the ground and was finally lost to view. Five eggs were recovered from this oviposition. Other moths were seen flying to the ridges and knolls, but it was too dark for further observations on this date. A few days later another moth was observed laying eggs on a knoll in the same field. One oviposition was made which lasted 23 min- utes, during which time the moth was not in the least disturbed by any movements of the observer. When the ovipositor was finally with- drawn the moth swung around X iiich and started in again, this time remaining quiet for 1 7 minutes, after which she crawled under a clod to hide. This moth had oviposited for 40 minutes, and 12 eggs were re- covered from the two holes. Moths were seen ovipositing along the knolls and ridges in this field for several days. Moths in egg-laying show their preference for spots in the field where the soil is softest and also indicate a preference for freshly worked fields over those which have become caked and hard. Across the road from the freshly worked, summer-fallowed fields in which egg laying was observed was another summer-fallowed field which was spotted with Russian thistles and in which the soil was caked on the surface, due to a rain some two weeks earlier. Moths were continually observed flying into this field, but they usually flew on across it to the knolls in the freshly worked field, even though it was ]4 mile farther. Very few moths flew to similar knolls in the caked field, and those that alighted hid under the thistles or clods of dirt and made no attempt to lay eggs. Further evidence of this preference for mellow fields will be brought out later in this paper. ATTRACTION OF THE MOTHS TO LIGHTS Our first experiments in attracting the moths to lights were conducted at Wilsall on the evening of August 26, 19 19. A large Coleman gas lamp was placed on the ground in the field where the grain had been destroyed the previous spring. As soon as it grew dark Porosagrotis orthogonia moths began to come to the light at the rate of one every two or three minutes. The lamp was placed upon bare sandy soil and the ground was well lighted for several feet on all sides. The moths usually struck the Nov. 5, 1921 Pale Western Cutworm 303 ground from 2 to 15 feet from the light and then crawled toward it, where they could be easily picked up. As soon as it became totally dark the moths came to the light so rapidly that two men could not keep them picked up, and from 9 o'clock until midnight 282 females and 164 males of P. orthogonia were placed in rearing cans or killing bottles. This by no means represented the total number that came to the light, for hun- dreds escaped. Many different species of noctuids were attracted to the light, but fully 95 per cent were P. orthogonia. Ten of the females thus captured averaged 132 eggs. (Table II.) In another part of the same field a Duro moth trap was run through- out the nights of August 26 and 27, 19 19, and each morning the pan was well filled with Porosagrotis orthogonia moths. During the two nights 4,900 moths were caught, of which 4,200 were males. It is difficult to understand the preponderance of females caught at the larger light between 9 and 12 p. m. and the very small percentage of females caught at the smaller light during the entire night. Experiments with trap lights were conducted on a somewhat larger scale at Willow Creek. A trap was designed which was made up of utensils commonly found on every farm and which would serve other purposes when not in use as a trap light (PI. 30, B). It consists of a No. 2 galvanized-iron washtub and a No. 2 barn lantern. In addition, a galvanized-iron arch is made which fits across the tub and serves the dual purpose of deflecting the moths and holding the lantern. When the arch is wired firmly and the lantern swung in place the flame of the lantern is just above the edge of the tub. "W^Tien set in place, the tub is staked down to prevent its being blown over, and about 4 inches of water are poured into it. About }i to }{ inch of kerosene is floated on the water to kill the moths which fall into it. Eleven such traps were put out at Willow Creek, and observations were made during the flight period of Porosagrotis orthogonia. Two traps for catching moths alive were also used, and when these showed that P. orthogonia was beginning to fly the tub traps were put out on fields that had been heavily infested with worms. During the first few nights the catches were small and the moths were counted. The numbers increased nightly until the height of flight, which was from August 19 to 24, inclusive. The night flight gradually decreased after the latter date. When the numbers became too large to count they were estimated, and during the height of flight they were measured in pints. As a pint measure holds from 962 to 1,000 moths, the measuring of moths gave a fairly accurate count. During the heavy flight several of the traps ran as high as 4,000 moths in a single night. The entire season 's catch of Porosagrotis orthogonia moths in the 11 traps was 82,488. The catch on individual nights is shown in Table V. 304 Journal of Agricultural Research Vol. XXII, No. 6 Table V. — Number of Porosagrotis orthogonia moths caught at trap lights at Willow Creek, Mont., during the season oj igzo Aug. Sept. 13 14 IS 16 17 18 19 22 23 24 25 26 27 28 29 30 31 Weather conditions and remarks. Number of moths caught. 127 342 1,528 1,566 449 68 11,720 Rain in afternoon; night warm, cooler toward midnight Early part of night warmer than usual; warm after midnight. . . Warm all night Night moderately warm Cooler, especially so after 9.30 p. m. ; not as many moths flying . High wind in afternoon blew over traps; cold and windy after dark ; no moths out Windy; cold soon after dark, almost frost; moths flew only a few minutes Warm at 8 p. m.; moths flying heavily; windy and cooler after ir.30; fewer moths out Warm west wind most of night; heavy flight of moths 13 , 990 Same as night before; height of flight 9.15 p. m 14.950 Warm, light west wind 13 , 650 Warm, west wind; wind strong and cold after 12.30 a. m 16,250 Cold and cloudy after 9 p. m. ; southwest wind ! 7 , 490 Windy and cold ; moonlight; no moths flying ' Windy until 8 p. m.; clear, cold; full moon; no moths flying. Wind and heavy rain ; cold ; no moths Rain all morning; cold, windy night; traps not lighted ! Cool; little wind; few moths flying early; too cold after 9 p.m. . . 68 Clear, cool; bright moon; no moths flying Cloudy to 9.30; clear, cold; bright moon Moths flying in daylight after noon; few flying after dark No moths flying after dark; a few found feeding Few moths out after dark ; not attracted to lights Three Nociua c-nigrum caught; no others flying to light No moths flying to traps; traps taken up 119 171 Total. 82,488 In all observations made at Willow Creek no moth was ever seen to land on the ground on its way to a light trap as did the moths at Wilsall the previous year. This may have been due to the fact that the tub hid the light so it would not strike the ground, and in order to keep in the path of light the moth had to fly straight to the trap. This was usually the case, and for the most part moths flying to the traps came on a straight line from 4 to 15 feet above the ground. They either struck the arch or lantern or went straight on over the trap. On a still, dark, fairly warm night the moths would come to the traps in varying waves of abimdance for which there was no apparent reason. There would be a cloud of moths for a few minutes and then they would come in scattering two's or three's. If the wind was strong no moths were caught in the traps and no moths could be found moving about on the ground. No moths were caught during a rain or ever after a rain, as long as the ground and vegetation remained wet. When the moon was bright, moths were not caught nor were any seen flying, though they would start the minute the moon went behind a cloud. Practically no moths were caught after the temperature had dropped below 58° F. Nov. 5, 1921 Pale Western Cutworm 305 MOTHS BOTH NOCTURNAL/ AND DIURNAL During the last half of August when the nights were warm and night flying was at its height the moths remained inactive during the day, hiding under clods and weeds. As the nights grew colder, the moths flew only an hour or two after dark, and on September i they were seen flying during the day. On this date they began flying about 4.30 p. m. and were seen in abundance feeding upon sunflowers, golden rod, tumbling mustard, yellow greasewood and lamb's quarter. All but one of the moths seen at flowers at this time were males, a search under weeds and clods at the same time revealing only females. At 8 p. m. when the flower patches were visited moths were still feeding in large numbers, practically all of them being females. They paid no attention to lights, and none were caught in a trap light set close by. As the night grew colder all of the moths disappeared and could be found hiding under clods or weeds. On the following day moths were found feeding at flowers at i p. m., and at 3.30 p. m. a patch of yellow greasewood {Chrysanthus jrigidus), which seemed to be the favorite flower, had attracted dozens of Porosagrotis orthogonia, nearly all of which were males. At 5.30 p. m. the patch was again visited, and it was found that the males were then leaving and that females were flying to the flowers from a nearby summer-fallowed field. On September 3 moths of various species were found feeding during the morning, and at noon the flowers of the yellow greasewood were covered with moths, none of which were P. orthogonia. At 3 p. m. about 10 per cent of the moths present at flowers were P. orthogonia. These gradually increased in numbers until 6.15 p. m., at which time practically all other species had disappeared. After the moths had finished feeding they invariably flew toward the higher ridges and knolls in neighboring cultivated fields. Many of the moths in coming to the flowers were seen to fly from 200 to 500 yards directly against a stiff breeze. Moths were seen flying to flowers in large numbers until September 8, when a cold rain and wind occurred. The males were always found feeding earlier in the day and the females later, although neither was ever found before noon. ECONOMIC IMPORTANCE The record of this cutworm during the last 10 years has demonstrated its capacity for doing enormous damage to grain crops. When in 191 1 Porosagrotis orthogonia, then an obscure insect, suddenly increased in numbers and did considerable damage to grain in southern Alberta {10, p. iji) little importance was attached to it. In the following year, however, when 33 per cent of all the grain sown in the Lethbridge land district was destroyed and an accurate estimate by the superintendent of the experiment station at Lethbridge placed the actual loss from this insect at from 30,000 to 35,000 acres (jj, p. 506) it was looked upon as a pest of ; 65769°— 21 2 3o6 Journal of Agricultural Research voi. xxn, no. 6 major importance. During the last two years P. orthogonia has been re- sponsible for losses in central Canada amounting to several million dollars. In Montana the pale western cutworm has been on the increase since it was first noticed in 19 15 and is now the most destructive insect pest with which the grain grower has to contend. In 19 15 at Conrad 80 acres of wheat were destroyed and were reseeded to oats, which was also taken. Flax was then seeded, but this also was so badly injured that the owner plowed the field and summer fallowed it. This instance was typical of scores of losses in the district now composed of the counties of Chouteau, Teton, and Pondera. During the next two years, especially in 19 17, great losses were sus- tained throughout the north central portion of the State, due to cut- worms which worked entirely beneath the surface of the soil and which were doubtless no other than Porosagrotis orthogonia. In 1919 the pale western cutworm appeared in destructive numbers farther south and caused severe losses in Park and Jefferson Counties as well as in the previously infested area. A conservative estimate of the losses for the year based on the reports of county agents, hundreds of questionnaires returned by farmers, and the personal investigation of many fields in different parts of the State, is at least 200,000 acres. In 1920 the injury in Jefferson and Park Counties was more widely extended, and there was a decided increase in the damage done in many of the districts previously infested. The loss over the entire State for the year is placed at 250,000 acres, valued at $3,000,000. In the Willow Creek district in Jefferson County a careful survey conducted in 1920 showed that 29 per cent of the total seeded area had been destroyed by this cutworm, and a similar survey in several of the northern counties showed a loss of 35 per cent of the grain crops planted. To show perhaps a little more clearly what this cutworm has been doing it may be stated that 100 fields personally inspected during the summer showed a loss of 2,437 acres out of a total of 6,844 ^^ 1919, and in 1920 a loss of 3,382 acres out of 6,844, or 35.7 per cent in 1919 and 49.4 per cent in 1920. Mr. George O. Sanford, manager of the Sun River irrigation project, has stated to us that of the 15,300 acres seeded to crop on the Greenfield Bench in 1920, 7,345 acres was a total loss and that some damage was done to the remainder. Using the figures he has given for the average yields on the undestroyed acreage — wheat 11.5 bushels, oats 20.86 bushels, and flax 6.31 bushels — the average value of tlie prin- cipal farm crops of that section was at least $15 per acre. Accordingly, using that as a fair valuation per acre of the crops destroyed, the pale western cutworm inflicted a loss of $110,175 in this one comparatively small territory. Although these losses took place on irrigable land, no water was available until after the first of June. Were it not that irri- gation made it possible in some cases to reseed and grow a late crop on part of the originally destroyed area, the loss would have been 55 per cent instead of 48 per cent of the acreage in that district. Nov. 5. 1921 Pale Western Cutworm 307 EXPERIMENTS IN CONTROL ORDINARY CUTWORM CONTROI, METHODS NOT EFI^ECTIVE Early in our study of Porosagrotis orthogonia it became apparent that the ordinary method of scattering poisoned bran mash over an infested field was not effective in controlling this species. On May 2, 19 19, pois- oned bran mash was scattered over a heavily infested field in southern Montana at the rate of 20 pounds to the acre and was followed by three other applications on successive days. The field was exammed each day by the owner, who reported that he could not find a single dead worm. On May 7 the treated area was carefully examined by one of the writers but no dead worms could be found, nor could any decrease in the number of live worms be noted. On May 7, 19 19, poisoned bran mash was scat- tered over 2 acres of heavily infested wheat in northern Montana. During the next 10 days no results whatever were secured from this treatment. County agents and numerous farmers have reported that attempts to poison this species by the ordinary method of scattering poisoned bran mash over the surface have always resulted in failure. At Willow Creek in 1920 pale western cutworms were noticed crawling over the surface of the groimd in the evening after a rain, and an attempt was made to kill them by scattering poisoned bran mash during the night. The bait was scattered soon after dark over an area which in- cluded bare ground, scattering wheat, and a good stand of wheat, all heavily infested. Observations were made during the night by the aid of automobile headlights, and many of the worms were seen feeding upon the bait. Two days later a search was made for dead cutworms. In the area where there was no vegetation it was estimated that 60 per cent of the worms were killed; where there was a scattering of wheat the percentage of dead worms was 50; and where there was a good stand of wheat 43 per cent were killed. It is possible that several night appli- cations of poisoned bran mash during rainy weather might bring about a satisfactory control, but as yet we have not had the opportunity to try it. POISONED BRAN MASH HARROWED INTO THE SOU. Strickland reports {17) that poisoned bran mash harrowed into the soil gave gratifying results. This method was tried out at Wilsall in May, 19 19. Poisoned bran mash was scattered over 3^ acre of heavily infested wheat at the rate of 25 pounds to the acre. On several square rods where the worms were thickest the mash was worked well into the soil with a hand rake, and the remainder of the treated area was thor- oughly worked with a spike-toothed harrow. The plot was carefully examined three days after the poisoned bran mash was applied, and it was estimated that the treatment was not more than i per cent effective. Very few dead cutworms could be found, and eventually all of the wheat was destroyed. 3o8 Journal of Agricultural Research voi. xxn.No.6 POISONED BRAN MASH DRII,IvED INTO THE SOII# Since Porosagrotis orthogonia very rarely comes to the surface to feed, placing the poisoned bran mash beneath the soil was tried in the hope that the cutworms would thus come in contact with it and feed upon it. The most promising way of doing this seemed to be with a seed drill. This method was tried out in northern Montana at Havre and in southern Montana at Wilsall. TESTS AT HAVRE At Havre two formulae were used. FORMULA NO. I Shorts pounds . . 25 Paris green do ... . i Oranges 4 Molasses quarts . . 2 Water gallon. . i FORMULA NO. 3 Shorts pounds. . 25 Paris green do ... . i Molasses gallon . . i Water quarts . . 2 These mixtures after being prepared were spread out and allowed to dry for 24 hours. When dry, No. 2 was distinctly stronger smelling, although both had a good molasses odor. The reason for using the large amount of molasses in these formulae w^as to secm-e a distinct odor in the dried material which we hoped might attract cutworms in the soil for some little distance. The dried material was seeded into the ground with a Van Brunt drill at the rate of 16 pounds to the acre and at a depth of about 1.5 inches. Six acres were ti"eated. The drill was run at right angles to the rows of grain so that the worms in working from plant to plant would only move a few inches before coming in contact with the bran. The greatest difficulty encountered was in getting the bran to feed evenly through the drill. When it was sufficiently dr}^ to be well divided it was too light to force its way through and it was necessary to agitate the mixture continuously in the seeder box to get any-where near an even distribution. The field was examined two days after the poisoned bran mash was drilled in, and it was found that formula No. i had killed approximately 50 per cent of the worms while formula No. 2 gave slightly better results with a kill of about 55 per cent, which was not enough to prevent the destruction of the crop. Nov. 5. 1921 Pale Western Cutworm 309 TESTS AT WILSALt On June 8, 19 19, a similar test was conducted at Wilsall. The follow- ing formula was used : Shorts pounds. . 25 Paris green do .... ^ Salt :':v^...v;?..';:.:;v.r. : do % Molasses quarts . . 2 Water. gallon . . i After mixing, the mash was spread out to dry, which with a hot sun and a fair breeze was accomplished in half a day. The mixture was distributed over 25 acres of infested wheat at the rate of 12 pounds of the dry mash to the acre. Sixteen acres were sown with all the spouts of the drill working and 9 acres with every other one closed. The drill was run across the old grain rows. The greatest difficulty encountered was the same as in the test at Havre — the mixture was too light to feed evenly through the drill. This was overcome by using two men on the drill, one to drive and one to keep the bran shaken down where it would come in contact with the disks of the drill. This was done by frequently pounding the seeder box with a padded hammer and punch- ing out packed masses with a small stick. Dead and dying worms were found the second day after the poisoned bran was drilled in, and on the third day a careful examination was made and it was estimated that from 50 to 60 per cent of the worms had been killed. The field was examined two weeks later, and there was a very noticeable difference in the number of worms found in the treated and untreated areas, but this did not prevent total destruction of the crop. TEST AT WIL1.0W CREEK During May, 1920, poisoned bran mash was distributed with a grain drill over a very badly infested field at Willow Creek. Cutworms were uniformly scattered over a 40-acre field of spring wheat, and at the time the poisoned bran mash was applied had destroyed about half the plants. Conditions were ideal for a good test of control methods. The following mixtures were used : FORMUI,A NO. I Shorts pounds. . 25 Paris green do ... . i Molasses quarts.. 2 Salt pound. . i Water gallon . . i After mixing, this was thoroughly dried out and was then seeded 2 inches deep through a Van Brunt drill at the rate of 20 pounds to the acre. Two acres were sown. This mixture did not feed uniformly through the drill unless constantly agitated. 310 journal of Agricultural Research voi. xxu. no.6 FORMULA NO. 2 Shorts pounds . . 25 White arsenic ■ do ... . i>^ Molasses quarts. . 2 Salt pound . . i This was prepared and distributed in the same manner as formula No. I. Two acres were sown. FORMULA NO. 3 Shorts pounds . . 25 Paris green do .... i Salt do .... I This was mixed dry and seeded at the rate of 12^^ pounds to the acre. It ran through the drill about the same as the mixtures which were mixed wet and then dried. Two acres were sown. FORMULA NO. 4 Shorts pounds . . 25 White arsenic ; .* ! .'.'.. 1 do ... . i}4 Salt do ... . I This was prepared dry and then thoroughly mixed with an equal bulk of wheat. This combination ran through the drill very evenly, the wheat being heavy enough to carry the bran through the drill without clogging. Two acres were seeded at the rate oi \2}i pounds of bran to the acre. Three days after the poisoned bran was put out the field was examined and it was estimated that about 10 per cent of the worms in the treated areas had been killed. No difference could be seen in the effectiveness of the various formulae, and numerous living cutworms remained in all the plots. One week later the plots were again examined and the number of dead cutworms had not materially increased. A final examination of the field was made on June 14, three weeks after the poisoned bran was put out. Cutworms were found in abundance on all plots, and in plots 1,2, and 3 practically every spear of wheat had dis- appeared. In plot 4, which was seeded with a mixture of wheat and poisoned bran, the wheat was about 3 inches in height and was being rapidly cut off, 50 per cent of the new stand being already destroyed. From a practical standpoint the control on all plots was a complete failure and an absolute waste of materials. POISONED BAIT SPRAY FOR ADUI,TS The presence of large numbers of Porosagrotis orthogonia moths at flowers led us to try out the following poisoned bait spray: Water gallon . . i Molasses pint . . ^ White arsenic ounce . . }4 Amyl acetate do % Nov. 5, 1921 Pale Western Cutworm 311 This was scattered in coarse droplets over flowers and vegetation where moths were abundant. Many flies and bees were killed, but no moths were observed feeding upon the bait, and dead moths were never found in the vicinity of the sprayed vegetation. CUI^TURAI, METHODS AS A MEANS OF CONTROI, In our study of Porosagrotis orthogonia under field conditions we have repeatedly noticed instances where crops in one field were completely destroyed, while in an adjacent field the grain escaped unharmed. This suggested that the manner in which the ground was worked before the crop was put in might have been responsible for the great difference in the amount of damage done in the two fields, and in 1920 a survey was conducted with the object of determining the relation of cultural methods to cutworm abundance. This survey was conducted in two ways : (1) By an auto trip through the districts most heavily infested by means of which hundreds of farmers were personally interviewed and the histories of their fields obtained for the period 1919-20; (2) by questionnaires sent to all farm bureau members in counties where Porosagrotis orthogonia was known to be present. The percentage of cutworm losses under various cultural methods as shown by a study of fields, the owners of which were personally inter- viewed, is shown in Table VI. Table Vl. ^Percentage of Porosagrotis orthogonia injury in IQ20 under various methods of cultivation in preparation for seeding Cultivation between previous crop and 1920 crop. Number of Total Acres fields. acres. lost. 8 465 200 39 2,250 1,301 36 1.536 661 13 643 138 51 2,465 666 18 1.332 526 39 3. "4 267 Percentage lost. Fall double disked Spring double disked Spring single disked Fall-plowed ; disked or harrowed be 'ore seed- ing Spring-plowed" disked or harrowed before seeding Spring-harrowed Summer-fallowed 43- o 57- o 43- o 21. o 27. o 40. o 8.5 A study of the results shows a high percentage of cutworm injury in all cases where the stubble was only disked or harrowed before seeding. Fields which were plowed either in the fall or spring showed a somewhat lower percentage, while summer-fallowed fields showed only the very small loss of 8.5 per cent. While the average cutworm loss in summer-fallowed fields was low, yet several individual fields suffered severe losses. It was therefore decided to make a study of the histories of summer-fallowed fields during the two seasons of 19 19 and 1920. Since the moths were known to 312 Journal of Agricultural Research voi. xxii, no. 6 prefer loose mellow soil for egg-laying, it was thought that the condition of the ground in summer-fallowed fields during the egg-laying period might have considerable rafluence on the number of eggs deposited in the field and on the percentage of loss the following spring. Since egg laying does not begin until about August 15, fields which are not culti- vated or disturbed in any way after July 1 5 become more or less crusted and caked. Fields which are cultivated in any way during the last part of July or during August, on the other hand, are very likely to be soft and mellow during the egg-laying period, thus offering tlie very con- ditions which the moths are seeking. Forty-eight fields, for which we had data for both 19 19 and 1920, were therefore classified as crusted, if they were worked only before July 15, or as mellow, if they had been worked after that date. The percentage of loss for the variously worked fields is shown in Table VII. Table VII. — Percentage of Porosagrotis orthogonia injury during IQIQ and IQ20 in "crusted" and "mellow" sumtTier-f allowed fields. Data secured by personal interview with grower Condition of field and time of cultivation. Number of fields. Total acres. Number of fields infested. Acres lost. Percentage lost. "Crusted" — worked only before July 15 27 21 1,828 1,562 3 14 14 425 00. 7 27. 2 "Mellow" — worked after July 15. . Farmers were asked in questionnaires sent to farm bureau members in counties infested with Porosagrotis orthogonia whether they had noticed any relation between the condition of the soil in summer-fallowed fields during August and the amount of pale western cutworm injury the following spring. Sixty-eight grain growers answered this question. Fifty-three said that injury was most severe in fields where the surface soil was well pulverized, or, as one farmer stated it, "The more mulch the more worms." Seven reported that the greatest injury had oc- curred in fields that had been crusted during August, and five stated that they could see no relation between soil conditions and cutworm injury. The foregoing data, together with the fact that we have seen ovi- positing females show a distinct preference for mellow fields, leads us to the conclusion that the physical condition of the soil during the egg- laying period has a very important bearing upon the amount of Poro- sagrotis orthogonia injury that may occur the following spring. Accord- ing to the data at hand greatest injury may be expected in fields in which the surface soil is loose and well pulverized during the egg-laying period. This loose, mellow condition may have been brought about in summer-fallowed fields by tillage during late July and August or it may Nov. 5. I92I Pale Western Cutworm 313 be a natural condition such as is found on knolls and ridges where the soil is generally light and easily drifted. In fields where a crop is re- moved during July or August the surface crust may become broken and pulverized in numerous places by the disturbance of the soil in connec- tion with harvesting, thus offering the moths many desirable spots for egg laying. Injury may be least expected to occur in fields in which the surface soil is hard or crusted during the egg-laying period. In most instances this condition can be brought about by not disturbing the ground in any way between July 15 and September 15. If farmers in preparing their grain fields for seeding will be governed by these prin- ciples it is believed that Porosagroiis orthogonia injury can be greatly reduced. Fortunately this method of handling summer-fallowed fields does not interfere with approved farm practices, and in fact agrees very closely with the recommendations of the agronomists. NATURAL ENEMIES Unlike most of our common cutworms, Porosagroiis orthogonia suffers comparatively little from attack by natural enemies. Much difficulty has been encountered in rearing various other species taken in the field as larvae, particularly the army cutworm, Chorizagrotis auxiliaris, on account of the high percentage that developed disease or parasites. This has not been the case with the present species. Our records for 19 15 show that out of a large number of army cutworms reared indi- vidually only 35 per cent were brought through to the moth stage, parasites emerged from 24 per cent, 2 1 per cent died of disease, and the remaining 20 per cent died in the pupa stage, mostly on account of in- sect parasites. In 1919, 55 per cent of P. orthogonia larvse handled in the same way were reared to adults. Of the 45 per cent that died, very few seemed to die of any disease, and parasites emerged from only two larvae. In 1920, out of 960 Porosagroiis orthogonia larvae collected in the field, 13.7 per cent were parasitized, 12.2 per cent by Diptera and 1.5 per cent by Hymenoptera. The parasites which emerged were 14 Bon- netia compta Fall and i Peleteria robusta Wied. The common wild birds of the prairie are the most beneficial natural check that we have observed. The western grasshopper sparrow, Am- modramus savannarum bimaculatus Swainson, particularly, has been watched while digging out the larvae and carrying them away to its young. In many parts of the cutworm-infested regions it has been a common sight toward the last of June to see thousands of small excava- tions made by the western grasshopper sparrow, horned larks Otocoris alpestris leiicolaema Coues (PI. 30, C), and possibly other wild birds in their search for the larvae. Although the common ground squirrel, Citellus richardsoni Sabine, has at times been known to seek out and devour large numbers of cutworm 314 Journal of Agricultural Research voi. xxii, no. 6 larvae, we do not believe that ground squirrels are of much importance as a natural check. In some instances both larvae and adults of Calosoma teptdum Lee. have been observed to be especially numerous about cutworm-infested fields and are, we believe, one of the lesser important predators. DESCRIPTION OF STAGES EGG Spheroidal, flattened dorso-ventrally, glistening milk-white when first laid (PI. C, i), later becoming dull gray: i mm. in diameter, 8 mm. in height. Around the micropyle is the ■. . v.. .i. ..•,•■■•:••., .' usual rosette which lies in the cen- '/C ^:--^r'-^'''Vi'.'''f ■■(■''''■■.•' ter of a finely reticulated area about . •. .-• 0.3 mm. in diameter. The pattern of ■•, '• the reticulation is shown in figure i. From the edge of the reticulated area ]!...;.... about 30 slightly raised longitudinal ■/••.'.?■• ribs radiate toward the base, extend- fi. •' " ■ . ■.■."'^:;{.. ing approximately four-fifths the dis- '"'/""■■/•■■■■■■} •! V '■^..•"■•. tance from the apex to the base. "■•■■■'••■.••....• ■;'■"■ The ribs are sometimes irregularly branched or connected by cross ^^u'Z?'"'''''"^"''", "'■'A'^^'?!?'"-" ""'^^f'"'' ridges. The shallow channels be- about the micropyie. Highly magnified. " tween the ribs are transversely striated and lightly pitted. The chorion on the basal fifth of the egg is smooth and glistening without ridges or definite reticulation. I^ARVA ^ FIRST INSTAR Head width, 0.4 to 0.43 mm.; average 0.41 mm. Total length of body, 2.4 to 3 mm.; average 2.8 mm. The head (PI. 28, A) is a very dark glistening brown, almost black; clypeus and front same color as epicranium; adfrontals indistinct, but apparently extending almost to deep indentations at summit; mandibles, labrum, ring-shaped sclerite resting on mentum, and cardo dark brown; submentum, margins of stipes next the mentum, the antennae, and labial palpae brownish; ©cellar region blackish. The posterior portion of the thoracic shield dark brown; anterior margin very light brown. The thoracic legs are brownish with dark brown transverse lines immediately anterior and partially surrounding coxae. ■ In the description of the larva the naming of the various parts follows very largely the system of William T. M. Forbes U). Nov. 5, 1931 Pale Western Cutworm 315 Prolegs on segments 9, 10, and 13 (counting the head as the first seg- ment); crotchets brown; rudimentary prolegs slightly visible as small tubercles on segment 8. There is a reddish brown mottling over the lateral and dorsal regions, becoming more distinct along the posterior segments. Before the larva has taken food this coloration appears much darker. The general color of the newly hatched larva is brown. The spiracles dark brown, setae single and arising from minute brownish tubercles. SECOND INSTAR Head width, 0.47 to 0.576 mm.; average 0.53 mm. Total length of body, 2.8 to 3.77 mm.; average 3.3 mm. The head (PL 28, B) remains a dark, shiny brown; mandibles and labrum dark brown, mandibles at teeth and labrum at notch blackish; ring-shaped sclerite resting on mentum blackened ventrally and with its setae within the sclerite; cardo dark brown; submentum, margins of stipes next the mentum, antennae, and labial palpse brownish; ocellar region blackish. Thoracic shield dark brown posteriorly, but not as dark as head; anterior margin light brown. On each side of the light brown dorsal stripe is a dark spot on the anterior portion of the shield with several dark spots laterad. Prolegs on segments 8, 9, 10, and 13; crotchets on eighth segment con- sisting of only two or three hooks; rudimentary prolegs on segment 7 beginning to show; crotchets reddish brown. The dorsal stripe is gray, bordered with broken lines of brown; sub- dorsal and lateral stripes brownish; spiracles dark brown; setae single, tubercles brownish. THIRD INSTAR Head width, 0.68 to 0.786 mm.; average 0.75 mm. Total length of body, 4.19 to 5.5 mm.; average 4.29 mm. Head brown (PI. 28, C) with upper parts of lobes of epicranium dark brown; front brown but with its lower margin together with clypeus dark brown; mandibles reddish brown to black at teeth; labrum dark brown with blackened notch, ring-shaped sclerite resting on mentum blackened ventrally and with its setae within sclerite ; cardo dark brown ; submentum, margins of stipes next the mentum, antennae and labial palpae brownish; ocellar region blackish. Thoracic shield dark brown, almost black at posterior region; an- teriorly it is a lighter brownish gray with a small black spot on either side of the light dorsal stripe and with several dark spots laterad. Thoracic legs tinged with brown, claws and markings anterior to and partially surrounding basal joints reddish brown. 31 6 Journal of Agricultural Research voi. xxii, no. 6 The dorsal stripe along the body is made of broken gray which is bordered irregularly with brown. Subdorsal and lateral stripes brownish; general color same as dorsal stripe, or lighter, with a greenish tinge. Spiracles dark brown; setae single, tubercles greenish brown. FOtTRTH INSTAR Head width, 0.84 to 1.14 mm.; average 1.02 mm. Total length of body, 6.5 to 12.5 mm.; average 8.9 mm. Coloration of head slightly modified from preceding instar; front not as dark, clypeus a lighter brown, and cranial lobes considerably darker at top (PI. 28, D) ; mandibles are black at teeth and fade to reddish brown to dark brown near articulations; labrum dark brown with blackened notch; ring-shaped sclerite resting on mentum blackened ventrally and with its setae within the sclerite; cardo dark brown; submentum, margins of stipes next the mentum, antennae and labial palpae brownish; ocellar region blackish. Posterior part of thoracic shield dark brown; anteriorly it is lighter, and in this lighter area are three distinct dark spots on either side of the dorsal line and also larger dark spots toward the lateral ends of the shield. Thoracic legs tinged with brown, claws and markings anterior to and partially surrounding basal joints, reddish brown. Prolegs on segments 8, 9, 10, and 13 and rudimentary prolegs on segment 7; crotchets reddish brown. The dorsal line is a greenish gray partially broken and bordered with brown; subdorsal and lateral lines brownish; general color green to gray; spiracles dark brown; tubercles are greenish, setae single and ringed at base with a light color. FIFTH INSTAR Head width, 1.38 mm. to 1.98 mm.; average 1.83 mm. Total length of body, 11. 5 mm. to 18.0 mm.; average 16.1 mm. General color of head much lighter (PI. 28, E) ; ocellar region very dark brown; ocelli i, 2, and 6 colorless, other three dark; mandibles reddish brown to black at teeth; lower margin of labrum reddish, blackened at notch; cardo and submentum bro^vn with the margins of stipes next the mentum same color and with the stripe becoming wider about the base of the palpifer; sclerite resting on mentum blackened. The two bands of dark brown on the epicranium and bordering the adfrontals become prominent for the first time in this instar (PI. C, 2). The rest of the head seems to have lost color, leaving these two stripes which run from points even with the base of the clypeus to the second epicranial setae, above which they gradually fade out about the first epicranial setae. Nov. 5,1921 Pale Western Cutworm 317 The thoracic shield is brownish with a light dorsal stripe ; in the lighter area on the anterior margin of the shield and on either side of the dorsal stripe are distinct blackened spots, with other dark spots toward the lateral margins of the shield. Thoracic legs tinged with brown, claws and markings anterior to and partially surrounding basal joints reddish brown. Fully developed prolegs on segments 7, 8, 9, 10, and 13 are concolorous with body; crotchets brownish. The anal plate is marked with a trans- verse row of small brownish spots anterior to the setae. The dorsal line is a greenish gray and bordered with brown; the sub- dorsal and lateral stripes brownish; the general color is about the same or perhaps a trifle lighter than the dorsal stripe. SIXTH INSTAR Head width, 1.98 to 2.64 mm.; average 2.41 mm. Total length of body, 2.0 to 2.5 cm.; average 2.2 cm. Head light brown; ocellar region very dark brown; ocelli i, 2, and 6 transparent, other three brownish; mandibles reddish brown to black at teeth; lower margin of labrum reddish, blackened at notch; cardo and submentum brown with the margins of stipes next the mentum same color with the stripe broadening apically about the base of the palpifer. The two conspicuous bands of dark brown persist on the epicranium bordering the adfrontals (PI. 28, F); the stripes become a lighter color at top, ending near the first epicranial setae; a denser colored portion of each band follows the adfrontals almost to their apex. The thoracic shield is brownish with a light dorsal stripe ; in the lighter area on the anterior margin of the shield and on either side of the dorsal stripe are distinct blackened spots with other dark spots toward the lateral margins of the shield. The thoracic legs tinged with brown, claws and markings anterior to and partially surrounding basal joints reddish brown. Fully developed prolegs are found on segments 7, 8, 9, 10, and 13, con- colorous with body; crotchets brownish. The anal plate on the thirteenth segment, a pale green in color, possesses a transverse row of brownish spots anterior to the setae; a light-colored dorsal stripe runs through the plate. The dorsal stripe is a broken gray green bordered with light brown; the subdorsal is brownish, but the lateral has become lighter in color, brownish gray. Sets single, ringed at base with a light-colored area; tubercles greenish brown; spiracles black. SEVENTH INSTAR Head width, 2.70 to 3.18 mm.; average 2.93 mm. Total length of body, 2.9 to 3.2 cm.; average 3.02 cm. I Head light brown; ocellar region very dark brown, ocelli 1,2, and 6 transparent, other three brownish; mandibles reddish brown to black at 3i8 Journal of Agricultural Research voi. xxn.No. 6 teeth; lower margin of labrum reddish; ringed-shaped sclerite resting on mentum blackened ; cardo and submentum brown with margins of stipes, next the mentum, same color with the stripe broadening about the base of the palpifer. The bands of dark brown bordering the adfrontals have the same appearance as in the previous instar (PI. 29, A). The thoracic shield is dark brown with a distinct light-colored dorsal stripe on either side of which toward the anterior margin of the shield is a small blackened area with other dark spots toward the lateral margins of the shield. The thoracic legs are tinged slightly with brown toward the apical joints; claws reddish brown, and color markings remain the same about the basal joints. Prolegs on segments 7, 8, 9, 10, and 13 are concolorous, crotchets brownish; the anal plate is a gray green with dark spots in a transverse row anterior to the setae; a light-colored dorsal stripe runs through the plate. The dorsal stripe is gray-green, and the brown borders in the previous instars appear greenish in this one; the subdorsal and lateral stripes are greenish with a slight tinge of brown ; the general color is a greenish gray ; setae single and ringed at base with light area; spiracles black, tubercles green; the pulsating dorsal vessel can be easily seen through the epi- dermis. Eighth instar Head width, 3.18 to 3.42 mm.; average 3.36 mm. Total length of body, 3.1 to 3.6 cm.; average 3.34 cm. The general color of the head is a light brown with a slight yellowish tinge; ocellar region dark brown, ocelli i, 2, and 6 colorless, other three dark brown to black; the front is a trifle lighter in color than the clypeus; the adfrontals, which are made very distinct by a darker brown coloration following the frontal sutures and by the dark bands following the epi- cranial sutures, extend to the bottom of the deep indentation separating the epicranial lobes at the summit; the mandibles are black at teeth and at points of articulation, and between lies an area which is a very pale brown in color; submentum and cardo brown; chitinized brownish stripe on margin of stipes, next the mentum, broadened about base of palpifer; antennae, labial and maxillary palpse brownish. The two bands of dark brown which border the adfrontals and which are very conspicuous in the fifth, sixth, and seventh instars, are some- what reduced, especially the upper parts of the bands in the regions of the first and second setae, in the advanced stage of the last instar (PL 29, B) ; during the first days of the instar the bands extend from points even with the base of the clypeus to the second epicranial setae, running between the setae and the adfrontals; here they become slightly less dense in color and divided, parts of the stripes continuing along the Nov. s, 192 1 Pale Western Cutworm 319 adfrontals and the other, lighter but wider parts, extending back to the regions of the first epicranial setae. The thoracic shield is pale brown with a prominent whitish dorsal stripe; there are small spotted dark brown areas toward the lateral margins of the shield and several smaller brown spots on either side of the dorsal stripe. The thoracic legs are tinged with brown, especially laterad; about the base of the coxae and femora anteriorly are reddish brown stripes. The prolegs on segments 7, 8, 9, 10, and 13 are concolorous with body and possess reddish brown crotchets. In the first part of this instar (PI. C, 3) there are distinct dorsal and subdorsal stripes, the dorsal appearing as dark green and produced by the pulsating dorsal vessel beneath the epidermis, and subdorsal as brownish ; a broken whitish lateral stripe is quite distinct; in the advanced stage of the instar with the exception of the tubercles there are no mark- ings on the body, which becomes a bleached out yellowish color. The spiracles are black; setae, which are a reddish brown especially in the head region, are single; tubercles a greenish brown but imme- diately about the base of each seta there is a ring of lighter color; the anal plate is marked by a transverse row of dark brown spots anterior to the setae. PUPA Length 17.5 mm., width 5.7 mm. Typical noctuid pupa; labial palpa? exposed for entire length; maxillae, mesothoracic legs and antennae of practically same length and extend- ing almost to caudal margin of wings ; prothoracic femora exposed ; tips of metathoracic legs visible and mesothoracic legs not extending to eye pieces; dorsal cephalic margins of abdominal segments 5, 6, and 7 marked with many small chitinized circular pits which extend to ventral surfaces of segments but where they are fewer in number and less prominent. The slightly bifurcate, blackish, rough cremaster ends in two stout often incurved spines set far apart. The color of pupa varies from a light straw color to a dark brown, according to age (PI. C, 4). ADULT ' "Agrotis orthogonia" nov. sp. All the tibiae spinose. Antennae of the male strongly serrate. Middle of the second joint of palpi black, its outer edge and tip, as well as the third joint, light. Head and thorax gray. Anterior wings dark gray; all the markings well expressed; half-line followed by a white shade line; basal space lighter than the other portions of the wing; interior line forming a very long outward projection below the submedian vein, and another shorter one on the costa, the line is white and distinct, bordered with black on each side, between the submedian and subcostal veins it is straight, except one lobe below the median vein, to which the concolorous, black edged clavi- form spot is attached; subcostal median and submedian veins white, and contrasting 1 The description of the adtilt is quoted from Morrison (z^). 320 Journal of Agricultural Research voi. xxn. no. 6 (PI. C, 5); orbicular spot elliptical, with an outer black ring, within which appears a white annulus, inclosing the gray center; reniform spot large and of the usual shape, the portion of its black annulus, beneath the median vein, separated and very dis- tinct; exterior line rounded, formed of interspaced luniform marks, followed by a white shade line; subterminal space rather lighter than the median space, terminal space again dark; a series of partially effaced cuneiform marks, before the white subterminal line, which forms two short teeth on the second and third median branches. Pos- terior wings whitish at the base, with a black terminal band and contrasting white fringes. Beneath whitish, the center of the median space dark, and the neighbor- hood of the median vein, on the anterior wings, clothed with long soft hair. Expanse, 34 mm. Hab. Glencoe, Nebraska. Received from Mr. G. M. Dodge. (No. 66). The nearest ally of this fine species is the European Agrotis vestigiales Rett. LITERATURE CITED (1) COOLEY, R. A. 1919. SEVENTEENTH ANNUAL REPORT OF THE STATE ENTOMOLOGIST OF MONTANA. Mont. Agr. Exp. Sta. Bui. 133, 15 p. (2) DoD, F. H. Wolley. 1901-06, PRELIMINARY LIST OP THE MACRO-LEPIDOPTERA OF ALBERTA, N.-W. T. In Canad. Ent., v. 33, p. 40-42, 157-172, 1901; v. 36, p. 345-355. 1904; V. 37, p. 17-28, 49-60, 145-156, 173-184, 221-230, 241-252, 1905; V. 38, p. 45-54. 89-94, 263-267, 1906. (3) Dyar, Harrison G. 1902. A LIST OP NORTH AMERICAN LEPIDOPTERA... U. S. Nat. Mus. Bill. 52, xix, 723 p. List of works quoted, p. ix-xix. (4) Forbes, William T. M. 1910. A structural study op some caterpillars. In Ann. Ent. Soc. Amer- ica, V. 3, no. 2, p. 94-132, pi. 10-20. Bibliography, p. 125-127. (5) Gibson, Arthur. 1912. THE entomological RECORD, 1911. In 42d Ann. Rpt. Ent. Soc. On- tario, 1911, p. 89-112. (6) (7) 1912. cutworms and army-worms. Canada Dept. Agr. Div. Ent. Bui. 3 (70, Exp. Farm ser.), 29 p., 10 fig. 1914. A NEW destructive CUTWORM OP THE GENUS POROSAGROTIS, OCCURRING IN WESTERN CANADA. In Jour. Econ. Ent., v. 7, no. 2, p. 201-203. (8) 191 5. CUTWORMS AND THEIR CONTROL. Canada Dept. Agr. Div. Ent. Bui. 10. 31 p., 20 fig. (9) Hampson, G. F. 1908. notes on noctuidaE... In Canad. Ent., v. 40, no. 3, p. 102-107. (10) Hewitt, C. Gordon. 1912. report op the dominion entomologist, /w Canada Exp. Farms Rpts. [i9ii]/i2, p. 173-189, pi. 5. (11) 1914. report prom the division of entomology... [i9i2]/i3. In Canada Exp. Farms Rpts. [i9i2]/i3, p. 499-518, pi. iq. (12) 1915. REPORT FROM THE DIVISION OP ENTOMOLOGY... [i9i3]/i4. In Canada Exp. Farms Rpts. [i9i3]/i4, p. 851-876. Nov. s. 192 1 Pale Western Cutworm 321 (13) Canada Department op Agriculture. 1917. REPORT OF THE DOMINION ENTOMOLOGIST, [i9i5]/i6. 73 p., 4 pi. Ottawa. (14) Maxson, Asa C. 1920. PRINCIPAL INSECT ENEMIES OK THE SUGAR BEET IN THE TERRITORIES SERVED BY THE GREAT WESTERN SUGAR COMPANY. vii, 157 p., 30 fig. 9 col. pi. Denver, Colo. Bibliography, p. 147-148. (15) Morrison, H. K. 1877. descriptions OP NEW north AMERICAN NOCTUIDAE. In PfOC. BoStOH Soc. Nat. Hist., v. 18, 1875/76, p. 237-241. (16) Smith, John B. 1890. contribution toward a MONOGRAPH OF THE INSECTS OF THE LEPI- DOPTEROUS FAMILY NOCTUIDAE OF TEMPERATE NORTH AMERICA. RE- VISION OF THE SPECIES OF THE GENUS AGROTIS. U. S. Nat. MuS. Bul. 38, 237 p., 5 pi. (17) Strickland, E. H. 1915. POISONED BAIT FOR CUTWORMS. In Canad. Ent., V. 47, no. 7, p. 201-204. (18) 1916. THE CONTROL OF CUTWORMS IN THE PRAIRIE PROVINCES. Canada Dept. Agr. Ent. Branch Circ. 6, 8 p., 5 fig. 65769°— 21 3 PLATE C Porosagroiis orthogonia: —Eggs. — Cast head of fifth-instar larva. — Eighth-instar larva. — Pupa in earthen cell. — Adult, male. (322) Pale Western Cutworm Plate C 1 ^^i3^ Hit- i^i/^'.i Journal of Agriculturjil Researcli Vol. XXII, No. 6 BIOIvOGY OF EMBAPHION MURICATUM By J. S. Wade, Scientific Assistant, Cereal and Forage Insect Investigations, Bureau of Entomology; systematic description of the larva by Adam G. Boving, Bureau of Entomology, United States Department of Agriculture INTRODUCTION Considerable damage has been wrought during the past six or seven years by the larvae of Embaphion fmiricatum Say and related species of false wireworms to growing wheat and other field crops throughout the semiarid and middle western United States. The area of greatest injury embraces approximately the western half of Nebraska, Kansas, and Oklahoma, and the eastern third of Colorado and New Mexico, although losses of varying magnitude have been reported in various localities over the greater part of all these States. In view of the obscure character of the injury, it seems quite probable that much crop damage commonly charged to other causes in reality has been brought about by this pest. The steady transformation in recent years of grassy prairies into cultivated fields has been an important factor, because the removal of native food plants causes this and related species to feed more and more upon cultivated grains. The hardiness of the insect and the rapidity of its adaptation to changed conditions and to new host plants indicate that this species is potentially a serious menace to grain production within the infested region. EARLY RECORDS The species under discussion was originally described in 1824 as Akis muricata by Thomas Say (/) ,^ who stated that it — inhabits Arkansa at the Rocky Mountains and that — as it does not entirely agree witli any genus the characters of which Latreille has noted, it may be proper to remove it to the Blapsidae, under a separate genus, which may be named Embaphion. This description was reprinted in 1859 in the LeConte edition of Say's (x) works, with a brief supplementary editorial note indicating relationship of the genus Embaphion to the genus Eleodes. A single specimen from Texas was described as Eleodes contusum by LeConte (2) in 1853, who stated that it — resembles E. muricatum Say but is longer and narrower with the broad margin of the elytra more suddenly reflexed and almost perpendicular. Although so different in form, this genus is only distinguished from Eleodes by the inferior plane of the ' Reference is made by number (italic) to "I'ngeal bracon (PI. 32, A, G, H, hbr) is well developed as a chitinous rod in the buccal membrane between the ventral mandibular articulation and the hypo- pharyngeal region. Prothoracic legs (PI. 31, C, H, I; 32, B) considerably stronger than the mesothoracic and metathoracic ones and with coxae attached so closely together that they almost touch each other at base. Coxa of first pair about as long as wide ; many fine , scattered hairs on exterior and interior sm"f aces ; trochanter about as long as coxa, on the inner side (PI. 31, H) distally with two spinelike setae and also with a few other thin hairs; femur {fe) about as long and wide as trochanter, armed with five spinelike setae, also with many thin, scattered hairs; tibia {ti) about twice as long as thick, almost same length as femur but not fully as wide, armed with five spinelike setse and also with fine, scattered hairs; tarsus {ta) of almost same length as tibia, claw-shaped, strong, but rather slender, with backward-facing surface distally excavate and proximally carrying a round soft-skinned region around a short but strong seta; another and similar seta set close to it at the end of the excavation; otherwise no setae or hairs on tarsus. Second and third pairs of legs inserted farther apart than the first pair; the arrangement of their setae very similar to that of the first pair, but the proportional sizes between the articles somewhat different from those of the first pair. Prothoracic eusternum (PI. 32, B, eu) large, rhomboidal, anteriorly almost reaching the front margin of the segment, only separated from this margin by a small presternal area (y); the hypopletu-al chitinization {hi and h-^), and especially its prehypopleural part (A i), large and strong; prothoracic tergal shield siibquadrate, slightly wider than long, with anterior and posterior margins, as mentioned above, darker than the rest of the shield and finely longitudinally striated. Mesothorax and metathorax with transverse, sub triangular, narrow presternum {y), laterally adjacent to poststernellura (2) of the preceding segment; hypopleiiral chitinizations {h I and h n) well developed, but considerably smaller than those of prothorax; poststemellum of metathorax not present, preepipleurum of mesothorax {e i) subtriangular, carrying first thoracic spiracle; preepipleurum of metathorax not distinctly limited, carry'ing the rudimentary second thoracic spiracle; postepipleiuiim (e u) of both segments well developed, more or less fused with the corresponding preepipleura ; mesothoracic and metathoracic tergal shields transversal, subrectangu- lar, about twice as wide as long, right behind anterior margin with a dark transverse line; posterior margin darker than rest of segment, finely longitudinally striated. Typical abdominal segment (that is, one of the eight anterior abdominal segments) with fused sternal and hypopleural areas {ster), covered by a single, longitudinally rec- tangular shield, which posteriorly has a rather dark, transverse, longitudinally finely striated margin; one seta present near the anterior and one seta near the posterior margin; additionally the sternum of first abdominal segment is anteriorly densely Nov. 5, 1921 Biology of Embaphion muricatum 329 set with small, soft sets; similar outfit lacking on the other abdominal segments. Epipleural region narrow. Tergal region with a dark line above the spiracle. Tergal shield (ter) single, posteriorly with a dark, longitudinally striated margin. Anterior abdominal segments transverse, slightly wider than long; sixth, seventh, and eighth abdominal segments subquadrate. Ninth abdominal segment smaller than the preceding segment ; dorsal part or pygidium pointing upwards, subconically produced, above somewhat flattened, below broadly convex, apex obtuse, laterally with margin set with a single series of strong, short setse, whole siuface with scattered, fine setae; ventral part of ninth segment small, transverse, soft. Tenth abdominal segment (or "anal segment") small, with trilobate upper transverse anal lip, with a pair of conical and, except at the tip, setose ambulatory warts, laterally to anus a small triangular lower lip. Spiracles (Pi. 32, I) annular, shortly oval, transversely placed; opening at the bottom of cup-shaped peritrema, linear, unprotected by hairs. The number and development of setae on the first pair of legs vary according to species and do not offer any generic character. The same is the case with the setal arrange- ment of pygidium. Fig. I. — Pupa of Embaphion mu- ricatum, dorsal view. Fig. 2. — Pupa of Embaphion mw ricaium., ventral view. PUPA (FIG. I, 2) Length 11 mm. Width 5.6 mm. Free. Arcuate. Color pinkish white, with ferruginous tinge on pronotiun. Femora and tarsi fuscate, other appendages partly translucent. Pupa becomes more strongly colored immediately before issuance of adult. Head pressed to prostemum. Pronotum broad and projecting somewhat anterior to head, making the head nearly invisible from above. Frons impressed. Vertex prominent. Antennae placed backward near sides of prothorax. Mesonotum narrow. Legs not pressed against body. Tips of wing cases extending to the an- terior margin of metanotum. Second to fifth abdominal segments bearing on each side of tergites flat, lacerated protuberances, obtuse, pointed and directed posteriorly. Eighth segment ending in two slightly divergent, acute processes. 330 Journal of Agricultural Research voi. xxn.N>. e ADULT (fig. 3) * Oval to oblong-oval, brownish to piceous black, thoracic and elytral margins very- broad and foliaceous, strongly reflexed. Head small, less than twice as wide as long, plane, sides of the frons slightly promi- nent, punctate, punctures very feebly subasperate, fine, not dense, each with a small curved and short seta, frontal suture usually not visible. Antennce rather long, quite slender, outer four joints very slightly compressed and scarcely widened, tliird joint shorter than the next two taken together, fourth scarcely longer than the fiftli, the latter and sixth subequal, seventh shorter, eighth feebly shorter than the seventh and slightly triangular, ninth and tenth suborbicular, eleventh subovate. Pronotum with margins very broadly foliaceous, the margin more than one-half wider than the disc, the latter comparatively narrow, longer than wide at middle, very feebly convex, usually with irregular de- pressed areas; finely, more or less subas- perately and sparsely punctate; reflexed margins wider posteriorly and more or less concave, a little more distinctly punc- tate, punctures less sparse, each with a short curved seta; o/>ex deeply and feebly subquadrately emarginate, the emargina- tion about one-half wider than deep, sides almost parallel, and scarcely margined; sides evenly but not strongly arcuate, moderately converging from base to apex; base proper feebly arcuate, not margined and about equal to the length, 1^.' ,rally sinuate; apical angles rather narrowly rounded and formed by the advanced foliaceous margins and nearly as long as the head; basal angles are posteriorly prominent, subacute, and projecting back- ward over the basal angles of the elytra. Propleurcc smooth and impunctate; in- ferior surface of the foliaceous margins obsoletely punctate. Elytra oval to elongate oval; margins broad andreflexed, angles at humeri nearly rectangular and more or less truncate at base, posteriorly extending beyond the apex, the two meeting on a line with the suture above the true elytral apex, and defined from the same by a slight groove, borders evenly arcuate from base to apex or more or less parallel basally ; base evenly but not strongly emarginate; humeri proper broadly rounded and not prominent ; sides proper more or less evenly arcuate ; apex proper not produced and narrowly rounded; disc plane, feebly convex, at times slightly concave, the inflexed sides nearly straight and oblique, gradually and not strongly arcuately declivous behind; surface sculptured with approximate series of fine asperate punc- tures, which become more irregular and slightly denser laterally. Each puncture bears a short and ratlier robust cur\'ed seta. Epipleurce narrow, not attaining the humeral margin and not dilated, but gradually narrowing to apex, not defined from the inflexed sides of the elytra, and on the same plane; superior margin obsolete, except near apex; elsewhere represented by a line of punctures or a faint groove. Fig. 3. — Adult of Embapkion tnurkalum, dorsal view. ^Reprinted from F. E. Blaisdell (//, p. 473-476). Nov. 5, 1931 Biology of Embaphion muricatum 331 Sterna more or less dull, finely and not distinctly sculptured. Parapletirse smooth, rather sparsely but not very distinctly punctate. Abdomen horizontal, very finely and sparsely punctulate, obsoletely rugulose and quite evenly convex. Legs rather slender, moderate in length. Anterior femora mutic, pro tibial spurs similar in the sexes, the anterior slightly longer than the posterior. Protarsi simple. LIFE HISTORY AND DEVELOPMENT The principal observations on the hfe history and development of Embaphion muricatum, as given below, were made under laboratory and field conditions in south-central Kansas, at an altitude of approximately 1,200 feet. Under different conditions of latitude, altitude, and humidity there would doubtless be found more or less marked variations. The rec- ords are unfortunately based on incomplete studies for, owing to working conditions and to pressure of other duties, there was no opportunity to conduct a sufficiently extensive series of experiments to render all obser- vations conclusive. The eggs are deposited in loose, dry, or slightly moist soil at a depth of ^ to I inch, sometimes singly, but more often in clusters of two or three to a dozen or more eggs at one place. At temperatures ranging from 80° to 90° F. the average period of incubation is approximately 10 days, whereas at temperature's of 68° to 70° F. the egg stage is approxi- mately 13 days. Undoubtedly weather conditions and the time of year have a direct bearing on the duration of the egg period. During the later stages of development and shortly before hatching, the surface of the egg becomes light brown in color, and the shell appears to expand slightly and to become more flexible, while the movements of the young larva can be noted within. During the process of hatching, the struggles and the lifting pressure of the young larva burst the shell and the larva emerges by rather slow periodic movements, as its integu- ment is very soft and fragile. Though the young larva often remains for some time near the place of hatching, yet it is capable of locomotion soon after emergence. All normal eggs of the same egg cluster usually hatch within a short period, generally a few hom-s. While abnormal weather conditions may prolong the period of hatching, no injurious effects of such retardation are noted in the eggs. No infertile eggs were ever collected under field conditions. Soon after the emergence of the larvae the empty eggshells become more and more contracted and dried up, until eventually only tiny, shrivelled fragments remain. Upon hatching the larva averages 3.5 to 3.75 mm. in length and is yellowish white. The color changes sHghtly after each molt until at maturity the larva becomes a deep yellow. The length of the larva stage, according to an experiment consisting of 3 1 larvae hatched in June and kept in a cool cellar at an average tempera- ture of 68° F., averaged for the siu-vivors 79 days, while in an experiment consisting of 49 larvse, under similar conditions, it varied from 76 to 332 Journal of Agricultural Research voi. xxii, no. 6 96 days, though the average duration was 85 days. The larvae as hatched were placed in small tin salve boxes containing about X inch of slightly moist soil and split wheat grains. As the larA^se became large, whole wheat grains were used as food. Under field conditions many of the larvse appear to become nearly mature during late fall and overwinter in this condition. From about November i to March 15 in the latitude of southern Kansas they are exceedingly inactive and feed but little. The rapidity of growth of the larvffi undoubtedly depends to a large degree upon weather and seasonal conditions and the quantity and quality of food available. Shortly before the period of pupation the larva does not feed and assumes a semidormant stage of approximately 7 to 9 days' duration. The pupa stage, when rearings were conducted under laboratory conditions, comprised 18 to 20 days. The pupse are pinkish white im- mediately after transformation, and as development proceeds the color changes to light yellow. Shortly before the adults emerge the append- ages take on a yellowish brown tint. The newly issued adults are of a brighter color, and the chitinous portions of the body are soft. Within a few days, however, the color darkens and the integument hardens so that the newly emerged adults are not distinguishable. Under artificial conditions mating does not become general for a week or more after emergence. Oviposition and feeding appear to occur usually at night. The adults are crepuscular. They may be found abroad in greatest numbers on cloudy days or in early morning or late evening. On clear days, during the middle or warmer portion of the day, they remain under shelter. While usually inactive at such periods, if disturbed they will run with great rapidity. The insect may overwinter both in the adult and in the larva stages. In the latitude of southern Kansas, however, the mortality of such over- wintering adults is great. ENEMIES While the incomplete character of the life-history work performed with Embaphion muricahtm afforded comparatively little opportunity for obtaining parasites under artificial conditions, or for obtaining data on other enemies for possible use in control work, yet some noteworthy information was obtained. From adults of E. imiricaium collected by the MTiter from barley at Colby, Kans., on August 25 there were reared on October 23 adults of a parasite determined by A. B. Gahan of the Bureau of Entomology as Perilihis eleodis Viereck {13). No life-history work on these parasites was attempted. Considerable difficulty was experienced in rearing larvae owing to the presence in the cages of a fungus, Metarrhizium anisoplae Metsch. Soc. ; and though the apparatus and soil were sterilized, yet the mortality was sufficient at times to interfere to a marked extent with the rearing. A Nov. 5, 1921 Biology of Embaphion muricatum 333 number of the larvae in the cages were also attacked by an obscure bac- terial disease. This appeared to be identical with that described by Prof. Swenk {12). There would appear somewhere upon the body sutures small circular or irregularly shaped dark brown spots, and these, after a few days, would become larger, until in some instances they would cover one-third to one-half of the body surface. This disease usually caused the death of the larvae within varying periods of time. Larvas found under normal field conditions are sometimes found to be affected both with Metarrhizium and with the disease. CONTROL While no extended series of experiments relative to control of the insect thus far has been found possible, yet the information secured on the subject has been sufficient to assure the practical value of the measures here recommended in reducing or preventing damage. A systematic rotation of crops is one of the most effective procedures in cutting down dam.age. The maximum injury always may be found upon those areas where the ground has been cropped to wheat continu- ously for several years, whereas the minimum injury is found where com, kafir, milo, and other crops are grown which require some degree of cultivation during the growing season. An important factor in migra- tion and infestation lies in the fact that the beetles are wingless and therefore become dispersed much more slowly than do winged forms. A number of fields within infested areas, which were also infested by the corn earworm {Chloridea obsoleta Fab.) and other insects of somewhat similar habits, were plowed by farmers during late fall or early spring to destroy the pupae, and it was found that such measures were of consid- erable value in control of the false wireworms in the soil. The pupal cells were crushed and the pupae buried or thrown out upon the surface, where they were exposed to the elements and to predatory enemies. It is not only good farm practice but also advantageous as a control measure to destroy and remove from infested fields and adjacent fence rows all clumps of Russian thistles and other weeds or heavy growths of grasses likely to shelter these beetles. While adults in small numbers are known to feed upon poisoned bran mash and similar preparations, experiments in poisoning the larvae were not satisfactory. It appears doubtful that such poisoning will ever prove of practical value in dealing with this pest. Late sowing of wheat in the fall also has been tried as a possible control measure, but does not appear to be successful unless the season is a very dry one, and even then if the seed has to lie in the ground any appreciable length of time before rain and germination much injury is likely to result, for the larvae are most active in the dry, loose soil under such conditions. 65769°— 21 4 PLATE 31 Embaphion muricatum: A. — Epipharynx (eph) and anterior margin of labrum. B. — Head: cl., clypeus; fa, anterior angle of front; epi, epistoma; /, frons; epc, epicranium. C. — Lateral view of larva. D. — ^Mandibles and hypopharyngeal sclerite from below. Concavity of molar part of left mandible grinding against the sclerite. E. — Gula, labiiun, and right maxilla from ventral side: gu, gula; tp, tentorial pit; sm, submentum; me, mentum; stla, stipes labii; li, ligula; hyp, hypostoma; fm, fossa for mandible; fc, fossa for cardo; ar, maxillary articulating area; ca, cardo; sti, stipes maxillaris; &.y, basis of stipes; z>i and Uj' i^mer margin of stipes; jmo, mala maxil- laris (probably lacinia); pag, basal membrane of maxillary palp. F. — Dorsal side of right and left mandible, hypophar>Tigeal sclerite between them: a^ and a^, the bicuspidate mandibular apex; t, tooth of cutting edge; m, molar part; c, carinate edge on exterior side of cutting part of mandible ; s, soft-skinned, seta-bearing elevation below the carinate edge; e, margin of chitin framing the soft elevation. G. — Mandibles and hypopharyngeal sclerite from below; no grinding in this position. H. — Left anterior leg showing the anterior face of the leg hanging perpendicularly down from a horizontally placed larva. I. — Left anterior leg, exliibiting its posterior face: cox, coxa; ir., trochanter; /e, femur; ti, tibia; ia, claw-shaped tarsus, shortly but not correctly designated as "claw." Biology of Embapliion muricatum Plate 31 Journal of Agricultural Research Vol. XXII, No. 6 Biology of Embaphicn muricatum Plate 32 /^c^^^ Journal of Agricultural Research Vol. XXII, No. 6 PLATE 32 Embaphion muricatum: A. — Lateral view of buccal cavity with mouthparts removed: eph, epipharynx; hsc, hypopharyngeal sclerite; hbr, hypopharyngeal bracon;/jw, fossa of ventral con- dyle of mandible; oes, oesophagus (note the distance between sclerite and entrance to oesophagus). B. — Ventral view of head, the thoracic segments, and the anterior portion of first abdominal segment: y, presternum; eu, eustemum (Snodgrass) or basistemum (Crampton); hi, prehypopleurum ; hn, posthypopleurum ; 2, poststernellum (prester- num and poststernellum constitute together the ventral intersegmental region); ej, preepipleurum ; %, postepipleiunim ; te, tergite; ster, sternal shield of abdominal segments; ep, abdominal epipleimim ; ter, abdominal tergite. C. — Pygidium, lateral view. D. — Pygidium, dorsal view. E. — Pygidium, ventral view; IX, ninth abdominal ("pygidial") segment; X, tenth abdominal ("anal") segment. F. — Maxillse, ligula, labial palpi seen from the buccal cavity. (Hypopharyngeal region removed.) G. — Hypopharyngeal region, oesophagus, and hypopharyngeal bracon which all were removed from figure F: hsc, hypopharyngeal sclerite; hbr, hypopharyngeal bracon; fm, mandibular ventral xossa; oes, oesophagus. H. — Hypopharyngeal region, same piece as figure G, reversed: hsc, base from which hypopharyngeal sclerite originates; hbr, hypopharyngeal bracon; /w, mandibular ventral fossa; oes, oesophagus. I. — First thoracic spiracle. ADDITIONAL COPIES OF Xms PUBLICATION MAY BE PROCURED FEOM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFnCE WASHINGTON, D. C. AT 15 CENTS PER COPY Subscription Price, $3 Per Year iToL. XXII NOVEMBER 12, 1921 No. 7 JOURNAL OF AGRICULTURAL RESEARCH CONTENTS Page Genetic Behavior of the Spelt Form in Crosses between Triticum spelta and Triticum sativum- - - - 335 CLYDE E. LEIGHTY and SARKIS BOSHNAKIAN ( Contribution from Bureau of Plant Industry ) Plum Blotch, a Disease of the Japanese Plum Caused by Phyllosticta congesta Heald and Wolf - - - 365 JOHN W. ROBERTS ( Contribution from Bureau of Plant Industry ) A Comparison of the Pectinase Produced by Different Species of Rhizopus - - "" ~ " -371 L. L. HARTER and J. L. WEIMER (Contribution from Bureau of Plant Industry) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINGTON, D. C. GOVERNMENT PRINTING OFFICE 1921 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 ssisfant 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 EntoTnologist and Chief, Dnision 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. JOlNiaOFAGiaaiTiAllSEMCH Vol.. XXII Washington, D. C, November 12, 1921 ,^ No. 7 GENETIC BEHAVIOR OF THE SPELT FORM IN CROSSES BETWEEN TRITICUM SPELTA AND TRITICUM SATI- VUM ' By Clyde E. LEighTY, Agronomist, Office of Cereal Investigations, Bureau of Plant Industry, United States Department of Agriculture, and Sarkis Boshnakian, Depart- ment of Plant Breeding, College of Agriculture, Cornell University INTRODUCTION In connection with genetic studies of density in the wheat spike, it was noted that the appearance of dififerent specific forms in certain wheat crosses introduced marked irregularities in density curves of the second and following generations, and that the densities of Triticum sativum Lam., T. polonicum Linn., T. spelta Linn., etc., were affected in different degrees when a certain known density factor was introduced through hybridization. In some instances there were partial and sometimes total inhibitory effects in regard to density, depending upon the sub- species and also the kind of density factor involved in the cross. The occurrence of these irregularities which appeared to be caused by the spelt character in some crosses led to the study of the nature and genetics of the species T. spelta. Although studies have been made of the mode of inheritance of the spelt form in a large number of inter- specific crosses, only the different modes of inheritance in crosses where the parents are spelta and sativum are presented in this paper. In other crosses, such as turgidum Linn. X sativum, durum. X sativum, dicoccum Schr. X .yafwwm, etc., spelts invariably appear in the F2 generation. The mode of inheritance of these spelt forms is complex and variable, so their discussion here has been omitted. The plants on which these studies were made were grown, with a few exceptions, on Arlington Farm, near Washington, D. C, or on the Plant Introduction Station, Chico, Calif., both operated by the United States Department of Agriculture. The crosses were made at the former place in 1913. > The specific name T. sativum as used in this paper refers only to the forms T. vulgare Vill., T. compactum Host., and T. capitatum Schlz. These three forms are essentially the same species, their differences being merely a question of intemode length. The word wheat is frequently used as an English designation for these forms, and when so used does not include such other forms as T. durum Desf . , T. polonicum, etc. Journal of Agriailtural Research, Vol. XXII, No. 7 Washington, D. C. Nov. 12, 1921 aak Key No. G-isa 336 Journal of Agricultural Research voi. xxn, no. 7 SPECIFIC DIFFERENCES BETWEEN TRITICUM SATIVUM AND TRITICUM SPELTA The shape of the outer or sterile glume is an important character in the dififerentiation of wheat species. The glume of the true T. sativum form (fig. I , B) is, as a rule, soft, with a somewhat pointed apex. It is rarely and very weakly keeled along the entire length. About 0.5 to i mm. A B Fig. I. — Characteristic spikelets of Triticum spelta (A) and Triticum sativum (B). Note upright and tight position of the glumes of spelta and the loose, spreading habit of satiiiim. Spelta has a flat shoulder (a) with two or three indentations; the shoulder of sativum (d), though varying widely, is generally not so prominent. It is often rather tapering with no indentations. The base of the sterile glume of spelta (b) is broad, showing firm attachment to the rachis. The glumes can not be opened without breaking them at the base. In sativum the base (/) is narrow and is weakly attached to the rachis, and the glumes can be opened easily. There are one or more depressions («) at the base of the glume in sativum which are not present in spelta. The glume of spelta is more or less uniform in width. It is stiff and has prominent corrugations (c); that of jaijVwwj is narrow at base, widens, then tapers again gradually. It is very thin and soft, and the veins on the glumes are not so prominent. Sativum has a weak keel while spelta has a very strong one . The Spelta spikelet usually develops only two kernels; sativum, often develops three or more. above the point of attachment there is a wrinkle or depression. The base is rather narrow and is very weakly attached to the rachis. The glume characters of the typical spelt, on the other hand, are quite different (fig. i, A). The glume is stiff and thick, with a very blunt apex. It is strongly keeled and has no depression above the base, which is wide and firmly attached to the rachis. \ Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 337 Some wheats exhibit some of the spelt characters in a very weak form. For instance, some are more or less strongly keeled or have a flat shoulder at the distal part of the glume. While such wheats are classified as T. sativum, they are not true sativum in the sense used here. They do not represent the type. Aside from the glumes, these species have other distinguishing char- acters. Of these, the brittleness of the rachis and nonshattering qualities of the spikelet in spelt may be mentioned as contrasted with the sativum types, the latter being nonbrittle and shattering easily. Although the heads of the spelt varieties commonly grown at experi- ment stations in this country usually are lax, laxness of the head is not necessarily a characteristic of the spelt. Compactness may easily be introduced into spelts when crossed with T. compactum, which is really only a wheat carrying a genetic factor or factors for compactness. The characters differentiating these two species are recapitulated below. This list includes also some other minor characters. T. sativum. T. spclta. Outer glumes — Outer glumes — Weakly attached by narrow base. Firmly attached by wide base. Weakly keeled. Strongly keeled. Apex tapering. Apex blunt. Depression or wrinkle near point of at- No wrinkle near base. tachment. Veins not prominent. Prominent parallel longitudinal veins. Shoulder narrow to broad, with no Shoulder broad, with two or three dentation. dentations. Glume soft. Glume, lemma, and palea firm and thick. Spikelets — Spikelets — Kernel loosely held between glumes Kernel tightly held (nonshattering). (shattering). Spikelets spreading somewhat from Spikelets oppressed tightly against rachis. the rachis. Usually three or more kernels per Usually two and rarely three kernels spikelet. per spikelet. Rachis — Rachis — Tenacious. Fragile. There also are differences in the shape of the kernels of these two species (i).^ LINKAGE OF SPECIFIC CHARACTERS In order to understand the manner in which these two forms are here classified, it is necessary to refer to the linkage of their specific characters, without discussing the details. Most of the characters tabulated above show two limits of contrasts. When a wheat is crossed with a spelt, a gradation of forms naturally ' Reference is made by rmmber (italic) to " Literature cited," p. 363-364. 338 Journal of Agricultural Research voi. xxii.no. 7 appears in the Fj generation, ranging from the true spelt to the typical wheat form. In the segregates, a plant which has the glume form of the spelt invariably has a brittle rachis and a nonshattering habit. If the glume form is intermediate, the brittleness and shattering qualities also are intermediate These three characters do not segregate inde- pendently. A plant with sativum glumes, for instance, has not been found whose rachis is brittle (a characteristic of the spelt only), nor have we obtained a spelt-glumed plant which has as tenacious a rachis as that of T. sativum. These species may present other characters, such as pubescence, beardedness, glume color, etc. These are common to both and segregate independently irrespective of the species. The presence of correlation between some of these specific characters in sativum X spelta crosses also has been noted by von Tschermak (12). Unless the linkage is absolute it does not exclude the possibility of the occurrence of crossovers, but if crossing over ever occurs with respect to these characters it must be very rare. The linkage of specific characters of spelt is very similar to the linkage of a number of glume characters of the wild oats in crosses between wild and cultivated forms described by Surface (u) and by Love and Craig (5), It is absolutely necessary to bear in mind that the determination of the species is based only upon the presence, intensity of development, or absence of the specific characters (glume characters, brittleness of rachis, and seed-holding habit) , all of which are linked to a very large extent. These characters show no independent segregation. It is obvious, therefore, that they do not mendelize independently. The characters which do segregate independently, such as pubescence, beardedness, glume color, etc., are not taken into consideration.^ METHOD OF CLASSIFYING THE FORMS The second and subsequent filial generations of spelta X sativum crosses show numerous gradations between the two parental forms. Some of the F2 plants produced a progeny consisting of individuals which were decidedly spelts, while others produced a progeny which, while all spelts, yet resembled T. sativum to some extent and were distinctly different from the former group of plants where all the individuals were markedly spelt-like. Ten arbitrary classes were made in order to record the degree of in- heritance of spelt characters. The typical or intense spelt which ex- hibited all the specific spelt characters in their extreme forms was graded I. As the exhibition of the intensity of these characters diminished — that is, as they tended to approach those of the wheat — ^the heads were ' Even these independently segregating characters vary in intensity and quality in these two species when segregating in crosses. Nov. 13, 1921 Genetic Behavior of Spelt Form in Crosses 339 classed 2, 3, and so on to 9. Class 10 includes only true wheats, the forms which show absolutely no trace of the spelt characters. It must be emphasized that these 10 classes are purely arbitrary, based upon the appearances of the heads, and are not intended to represent any geno- typic classes. The types representing these 10 classes are presented in Plate 33, A. When the degree of inheritance of the spelt characters by a heterozy- gous population is recorded on two different occasions, a variation in the class frequencies naturally may be expected, but the general form of the curve remains practically the same. The error which may affect the conclusions rests in the decision as to whether a particular plant belongs to class 9 or 10. The degree of error depends upon the cross examined. Of the plants discussed herein, which were tested in the Fg generation, there were five cases where individuals belonging in class 9 had been placed in class 10 and only one case where a class 10 individual was erroneously recorded as belonging to class 9. In the tables these six corrections have been made. As the conclusions are based not merely on the F2 individuals but upon the progeny of these, it does not seem likely that this source of error could have affected the results to any extent. The spelt parents used for making the crosses graded from i to 4 ; the variety of wheat known as Gatineau ^ and herein considered as speltoid in form graded from 4 to 7; and all the T. sativum parents of course graded 10. As the discussions in this paper are confined only to the spelt character it does not seem necessary to describe further the agrono- mic and botanical characteristics of the plants used in making the crosses, as these have no direct bearing on the subject. FAMILIES SHOWING THE PRESENCE OF ONLY ONE FACTOR FOR SPELTING 2 The hybrids of the first filial generation (FJ of a spelta X sativum cross are slightly intermediate in form, resembling the spelt more than the sativum parent. They grade from 4 to 7, depending upon the cross. They possess all the characteristics of a spelt, but the spikelets may be somewhat more open and the grains may not be so firmly held within the glumes. The spelt characters are so nearly completely dominant that they inhibit all wheat characters. In the second generation a segregation is obtained where the indi- viduals vary, producing forms ranging from the typical wheat form to ' This variety originated as a cross of Red Fife 9 (T'. sativum) and Goose S {T. durum) (lo, p. 239). It is a striking illustration of the spelt-like segregates which appear when these two wheat species are crossed. The heads are sc much like spelt that at blooming time especially it is easily mistaken for spelt, but it thrashes free and is otherwise like the common wheats. ' For brevity, "spelting" is used throughout this paper in place of "inheritance of the spelt characters." 340 Journal of Agricultural Research vou xxii, no. ? the spelt. The curve of these gradations is not a binomial frequency curve, but, on the contrary, more individuals are found, as a rule, at the extremities of the range than near the center. This fact may be ob- served in Table I, which shows the forms of the Fj segregates. Table I. — Number of plants of the F^ generation falling into each of lo classes, based on presence or absence of spelt characters, with total number of spelts arid wheats actually obtained, calculated numbers on basis of j to i ratio, deviation, probable error, and ratio between deviation and probable error Series. Crosses. Number falling in class — To- tal. 13255a 13260a 13263a 3094a 3085a 13124a 13125a Spelt sel. i344oXDale Gloria set. 13401 Spelt sel. 13438 X Turkey (C. I. 3375) sel. 13389 Dale Gloria sel. i340iXspelt Black Bearded spelt X Early Red Chief Black Bearded spelt X Jones Long- berry Vulgare (C. I. 3338)Xspelt sel. 13437 Crimean (C. I. 3340) sel. 13351X spelt sel. 13437 63 25 81 50 76 5° Total of crosses . 418 Series. 13255a 13260a 13263a 3049a 3085a 13124a 13125a Crosses. Spelt sel. i344oXDale Gloria sel. 13401 Spelt sel. 13438 XTurkey (C.I- 3375) sel. 13389- Dale Gloria sel. 13401X spelt Black Bearded spelt X Early Red Chief Black Bearded spelt X Jones Longbcrry Vulgare (C. I. 3338) X spelt sel. 13437 Crimean (C. I. 3340) sel. i335iXspeltsel. 13437 Total of crosses . . . Number of plants. Obtained. Spelts. Wheats. 52 49 18 52 37 56 41 305 113 Calctilated. Spelts. Wheats. 54-8 47-3 18.8 60.8 37-5 57- o 37-5 313-5 IS- 7 6.2 20.3 12.5 19. o 12.5 104.5 Devia- tion. 8.5 Prob- able error. 2-5 2-3 1-4 2.6 2. I 2.5 2. I 6.0 Ratio Dev. P.B. •74 •57 3-38 .24 .40 I. 67 I. 42 As it is impossible to distinguish the homozygous spelts from the heterozygous forms, in determining the ratios all the spelt and speltoid forms (classes i to 9) have been grouped together and compared with Nov. 13, 1921 Genetic Behavior of Spelt Form in Crosses 341 the wheat forms (class 10), which show no trace of spelt characters. The proportions between spelta and sativum forms of each of the crosses taken separately and that of the totals of these two groups approximated the monohybrid ratio of 3 to i. The obtained ratio of the totals of the crosses was 305 speltoid and spelt forms to 113 wheats, the expections being 313.5 to 104.5, respectively, showing a deviation of 8.5 with a probable error of ± 6.0. Two of the Fj families, 13260a and 13255a, gave the results shown in Table II when the F3 generation was grown. Table II shows that the Fj population of the families tested consisted of individuals in the proportion of i homozygous spelt to 2 heterozygous forms and i homozygous wheat. Table II. — Number of F2 plants from series ij26oa and Ij2^ja which proved to be homo- zygous spelts, heterozygous forms, and homozygous wheats {i : 2 : i) when tested in the /• 3 generation 13260a, Spelt X Turkey. 132553, Dale Gloria X Turkey. Nature of data. Homozy- gous spelts. Heterozy- gous forms. Homozy- gous wheats. Total. Homozy- gous spelts. Heterozy- gous forms. Homozy- gous wheats. Total Obtained Calculated Deviation 8 7-5 •5 13 15.0 2. 0 9 7-5 •5 30 7 8.2 1.2 14 16.5 2-5 12 8.3 3-8 33 The ratios of the totals of the forms produced in the F3 generation by the heterozygous Fg individuals of these two families (Table III) seem to conform to the foregoing assumption, although the ratios of the forms produced by each of the F2 individuals sometimes are not so close to the 3 to i expectation. Of the total individuals produced by the Fj heterozygous plants of series 13260a (spelt X Turkey) , 212 were spelts and 71 wheats. These results were surprisingly close to the expectation, the deviation from the calculated numbers being but 0.3 with a probable error of ± 4.9. In series 13255a (Dale Gloria X spelt), the numbers obtained from the heterozygous individuals were 365 spelts and speltoids and 156 wheats; the deviation here was 25.8 with a probable error of ±6.7. This apparent dominance of the spelt character over that of the wheat and its segregation into the 3 to i ratio are in accord with the observations of Pitsch, as cited by von Tschermak {12, p. ijg) and of Kajanus (j). 342 Journal of Agricultural Research voi. xxii. no. Table III. — Numbers of spelts and wheats produced in the F3 generation from the F^ heterozygous plants, and comparison of these with theoretical expectations, calculated on the J to I basis Total F3 plants. Number of plants. Devia- tion. Probable error. Ratio Pedigree No. Obtained. Calculated. Dev. Spelts. Wheats. Spelts. Wheats. P.E. 132603-3 . . . . 6 7. ... 9. ... 10. . . 13- •• 14. .. 15. .. 19. .. 25. .. 28. .. 29. . . 30. .. 23 20 28 17 23 24 17 20 29 17 17 18 30 13 16 20 10 18 17 13 16 26 13 12 16 22 10 4 8 7 5 7 4 4 3 4 5 2 8 17-3 15.0 21. 0 12.8 17-3 18.0 12.8 15.0 21.8 12.8 12.8 13-5 22. 5 5-7 5-0 7.0 4. 2 5-7 6.0 4.2 5-0 7.2 4.2 4.2 4-5 7-5 4-3 I. 0 I. 0 2.8 •7 I. 0 . 2 I.O 4.2 . 2 .8 2-5 •5 1.4 1-3 I- 5 I. 2 1.4 1.4 1.2 1-3 1.6 I. 2 I. 2 1.2 1.6 3-1 .8 •7 2-3 •5 •7 . 2 .8 2.6 . 2 •7 2. 1 •3 Total.. 283 212 71 212.3 70.7 •3 4.9 . I i325Sa-7 •• • ■ 8. ... 10. . . II . . 15. .. 16. .. 17. .. 21 . . . 23 •• ■ 24. . . 25. . . 36 19 24 35 59 33 13 52 49 35 44 37 50 35 34 15 20 27 44 23 4 28 29 18 33 25 33 32 2 4 4 8 15 10 9 24 20 17 II 12 17 3 27. 0 14-3 18.0 26.3 44-3 24.8 9.8 39- 0 36.8 26.3 33- 0 27.8 37-5 26.3 9.0 4-7 6.0 8.7 14.7 8.2 3-2 13.0 12. 2 8.7 II. 0 9.2 12.5 8.7 7.0 •7 2. 0 •7 •3 1.8 5-8 II. 0 7.8 S.3 0 2.8 4-5 5-7 1-7 1-3 1.4 1-7 2. 2 1-7 1. 0 2. I 2. 0 1-7 4.1 •5 1.4 4.1 . I 1. 1 5-8 5-2 3-9 4.9 26. .. 28. .. 3°- •• 1.8 2. 1 1-7 1.6 2. I 3-4 Total.. 521 365 156 390.8 130. 2 25.8 6.7 3-9 FAMILIES SHOWING THE PRESENCE OF TWO SPELT FACTORS Of the crosses studied, two families, 13126a (Giant Squarehead X spelt) and 3019a (spelt X Salt Lake Club) produced a very low proportion of wheat types. Not much importance would have been attached to the irregular behavior of these families if an apparently similar behavior had not been observed in another sativum X spelta cross. The manner of segregation of the progeny of these two crosses is given in Table IV. Table IV. — Degree of spelling and proportions of spelts and wheats obtained in the F2 generations of spelta X sativum crosses which did not segregate in the 3 to T ratio Degree of spelting in class— Total. Niunber of plants. Devi- ation. Series. I 12 27 2 7 15 3 7 9 4 6 7 s 9 3 6 8 5 7 3 2 8 6 5 9 12 8 10 4 2 Obtained. Calculated. bable error. Spelt. Wheat. Spelt. Wheat. 13126a '. . . . 3019a ^ 74 83 70 81 4 2 69.4 77.8 4.6 5-2 0.6 3-2 I. 40 1.49 1 Series 13126a, Giant Squarehead (C. I. No. 3351, selection 13366) X spelt (selection 13437). ' Series 3019a, white spelt X Salt ijake Club. Nov. 12. 1921 Genetic Behavior of Spelt Form in Crosses 343 In series 13126a (Table IV), only 4 wheats were produced in a popula- tion of 74 F2 plants, while in series 3019a, 2 wheats were produced in an F2 population of 83 individuals. The deviation from the 3 to i ratio is so great that even by grouping class 9 with class 10 — that is, by making generous allowances for observational error — the proportion approached more nearly the 15 to i ratio. On the basis of the 15 to i ratio, the expectation in series 13126a, is 69.4 to 4.6, in series 3019a, 77.8 to 5.2. The deviations are 0.6 and 3.2, and the probable errors ±1.40 and ± 1.49, respectively. On examining the Fg generation produced from 27 plants of series 13126a, it was found that 12 of these had produced only spelts (Table V), two plants yielded only sativum types, and the remaining 13 F2 plants yielded progeny of mixed forms. Assuming that the spelt parent in this particular cross carried two spelt factors, Sj and Sj,^ the first two generations will consist of the following genotypic forms: Pi (Giant Squarehead) S1S1S2S2 X wibib2>-'2 (Winter spelt) T. sativum T. spelta Fi (Speltoid) F2 I SjSjS2S2 I ^1^1^2^2 4 W1S1O2S2 2 S1S1S2S2 2 Oj^Oj02S2 2 SiSiS2S2 I D]^OiS2S2 2 OjSj02W2 I SjSj^0202 Tot al I T. sativum. I q spelt. If these genotypes were carried through the F3 generation the theo. retical behavior of each of the Fg plants would be as follows: GROUPS. F2 GENOTYPES. PHENOTYPES OF THE Fs AND THEIR BEHAVIOR IN THE F3 GENERATION. A I SjSjSjSj I wheat will yield wheat only. B I SjSiS2S2 2 t3iOi02S2 1 S1S1S2S2 2 DiSib202 I SiSjv!)2v32 C 4 SiSiS2S2 4 Spelts will segregate 15:1 15 2 S1S1S2S2 1 ^ cr.«ife will oo^.-o„o+» ... \^ unstable forms. >7 spelts will yield spelts only. 4 SiSiS2S2 4 spelts will segregate 15:1 | 2SisaS }4 spelts will segregate 3:1 f Total 16 When the performances of the Fj plants were examined, a close approximation was found to the above-mentioned theoretical ratios. The numbers of constant wheats, constant spelts, and unstable spelt forms obtained are shown in Table V, together with the theoretical expectations. ' In the factorial explanations given in this paper the spelt factors are assumed to be Si and S2. Although the assumption of the factors si and sz to stand for the wheat (T. sativum) character will fully agree with the results obtained, so far as the ratios go, there is no evidence as yet to warrant the assumption that the wheat and spelt characters are allelomorphic to each other. In fact, results with other specific crosses show the possibility that these are caused by two sets of independent factors. The behavior of the sativum X spelta crosses may be compared with the behavior of a maize cross where one parent has yellow endosperm and purple aleurone color (YYCCPP), and the other differs from this by its lack of purple color (YYCCpp). In such a cross, where the F2 shows segregation into 3 purple to i yellow, the assumption that yellow and purple are allelomorphic may be used as a working hypothesis for crosses of this type, although it is not the correct explanation, as endosperm and aleurone color are two different characters altogether. 344 Journal of Agricultural Research voi. xxii.no. 7 Table V. — Frequencies of spelt and wheal classes in F3 progeny of F2 individuals of family Iji26a, Giant Squarehead X spelt FAMILIES CONSISTING OP WHEATS ONLY Class of F2 parent plant. Classes of spelt inheritance Fa — Number of plants. Pedigree numbers of Fs. I 2 3 4 5 6 7 8 9 10 4 10 Spelts and spel- toids. Wheats. Total. 7 10 10 4 10 4 10 2? FAMILIES CONSISTING OF SPELTS ONLY 2 5 3 9 7 3 6 4 5 6 6 5 9 4 3 I 2 3 6 4 2 2 I 2 I 3 4 4 I 6 I 2 3 3 I 2 2 I I I 2 I I 3 1 2 2 2 I I I I I I I I 3 I I I 13 2 3 2 I I 18 10 13 13 13 5 18 14 5 14 10 5 18 A, 10 c 13 13 13 5 18 7 8 12 IC 16 14 5 14 10 20 22 24. I 21; 5 Total 138 138 FAMILIES CONSISTING OF SPELTS AND WHEATS (heterozygous f 2 plants) \ I 5 6 8 9 8 8 7 5 8 I I 9 5 I 2 2 I I 2 2 3 2 I 3 5 2 3 5 I 5 I I 3 4 5 I 3 I I I 2 2 I I I I I I I I I I 2 I I I I I I I I 3 I 13 3 3 2 3 I I 2 2 I I I 4 I I I 4 2 I 16 9 5 3 7 14 7 14 16 5 18 4 5 I 2 2 I I 1 4 I I I 4 2 I 17 II 6 0 7 4 8 10 II i-j 15 II 14. 17 15 17 6 18 IQ 21 22 26 6 27 6 Total progeny of he- terozygous F2 plants. . Expectations 123 108. 75 22 36.25 145 SUMMARY AND GROUPING Types. Number of plants. Groups. Obtained. Calculated 7:8:1. Deviation. A Wheats producing wheats only 2 12 13 1-7 II. 8 13-5 0.3 . 2 B Spelts producing only spelts Cand D... Spelts producing both spelts and wheats. . •5 Nov. 12. 1921 Genetic Behavior of Spelt Form in Crosses 345 The agreement between the proportions expected and those obtained is very close indeed to the 7 to 8 to i ratio, and perhaps too close to be ordinarily expected from such a small population. The analysis may be carried a step further. As shown above, the plants which would show unstability in the F3 (groups C and D), were expected to be of two different genotypes. One of them, containing the S1S1S2S2 forms (group C), was expected to segregate in the 15 to i ratio, while the other (group D), containing the S1S1S2S2 and S1S1S2S2 genotypes, should segregate in the simple 3 to i monohybrid ratio. Apparently the individuals belonging to each of these two groups are those analyzed in Table VI. Table VI. — Analyses of unstable spelts of the F2 generation, series 12126a. Number of individuals of the F^ generation produced from Fn plants of groups C and D, compared with the theoretical expectation ■p2 INDIVIDUALS APPARENTLY SEGREGATING IN THE 1 5 TO I RATIO (GROUP C, 81818252) Total. Number of plants. Devia- tion. Probable error. Ratio Dev. P.E. Pedigree. F3 obtained. F3 calculated. Spelts. Wheats. Spelts. Wheats. I3i26a-i . . . . II . . . 13. . . 17. . . 18. . . 17 8 IS 15 17 16 7 14 14 16 IS- 9 7-S 14. I 14. I IS- 9 I. I ■ s -9 -9 I. I 0. I • s . I . I . I 0.68 .46 .62 .62 .68 0. I 1. I . I . I . I Total.. 72 67 5 67- S 4-5 • s 1.38 •3 F2 INDIVIDUALS APPARENTLY SEGREGATING IN THE 3 TO I RATIO (GROUP D, S1S1S2S2 AND S1S182S2) i3i26a-6. . . . II 9 2 8.3 2.7 0.7 0. 96 0.7 9 7 5 2 5-3 1-7 •3 •77 •4 4 II 3 7 I 3-0 8.3 I. 0 0 14. . . 4 2.7 1-3 .96 1.4 19. . . 6 5 I 4-S i-S •S .72 •7 21 . . . 22 18 4 16.5 s-s I- 5 1-37 . I 26. . . 6 4 2 4-S 1-5 •5 .72 •7 27. . . 6 S I 4-S I- 5 •S .72 •7 Total.. 73 56 17 S4-8 18.2 I. 2 2. 50 -5 Total Fa plants segregating in 15 to i ratio, obtained, 5. Total F2 plants segregating in 15 to i ratio, calculated, 6.7. Deviation, 1.7. Total F2 plants segregating in 3 to i ratio, obtained, 8. Total F2 plants segregating in 3 to i ratio, calculated, 6.7. Deviation, 1.3. The data in Table VI show that forms were obtained in the F2 some of which segregated in the 15 to i and others in the 3 to i ratio as ex- pected. The agreement to the theoretical numbers of the progeny of each F2 plant is as close as can be expected with such small numbers, even 34^ Journal of Agricultural Research voi. xxn, no. 7 though it is borne in mind that the values of the probable error are likely to be too high in data of this kind. Summing up the types of groups C and D, the former yielded 67 spelts to 5 wheats, and the latter group yielded 56 spelts to 17 wheats, the deviation being 0.5 and 1.2 and the probable errors ±1.38 and ±2.50, respectively. Of the 2 7 plants tested (Tables V and VI) , 5 (group C) showed an ap- proximation to the 15 to I ratio and 8 (group D) to the 3 to i ratio. The theoretical number of plants belonging to each of the two unstable groups was 6.7 — that is, one-fourth of the total Fj plants tested. Comparing the results obtained with those expected, it will be noted that there were in the F, generation two (1.7 actual) SjSiSaSg individuals less and one (1.3 actual) SjSiSjS, or s^SiSaS, more than expected (Table VI) . In considering the 15 spelt to i wheat segregation, it should be borne in mind that if the progeny of a heterozygous Fj plant is less than about 10 individuals, the chances are that the wheat form, which is expected to appear but once in a population of 16 individuals, will not be obtained. Such heterozygous plants producing only spelts and no wheats, on account of their small F3 population, would be classified under group B. Had Fj plants 13126a — 12, — 20, and — 25 produced more than 4 or 5 individuals, I or 2 of them might have produced a wheat form which would have placed them in group C. The experimental ratios then would almost coincide with the theoretical. Considering the closeness of agreement even in the details of the analysis, with such small numbers, there seems to be no question that we are dealing here with two spelt factors and that the ratio observed is the ratio of 15 to I. In the absence of more experimental evidence, the simplest hypotheses were given to account for the 3 to i and 15 to i segregations. Notwith- standing the surprisingly close agreements between the experimental and theoretical ratios, however, the real explanation concerning the produc- tion of the spelt character is still a matter of speculation. The same spelt parent plant was used in crosses 13124a, 13125a, and 13126a. The wheat parents were of different varieties. No satisfactory explanation can be offered as to why the same spelt parent should pro- duce a 15 to I ratio in cross 13126a and a ratio of 3 to i in crosses 13124a and 13125a. Three possibilities, however, may be mentioned. 1. The spelt plant used in the above-mentioned three crosses might have been heterozygous for one of the spelt factors. Such a heterozygous SjSiSjSj plant bears gametes producing 3 to i and 15 to i ratios in the F2 generation when crossed with a double recessive (SjSjSjSj) wheat. 2. The spelt parent may be assumed to have carried two spelt factors Sj and S2 and some of the wheats might have carried an inhibiting factor I. If the wheat carried the I factor the ratio of spelt to nonspelt would be about 3 to i and if it did not the ratio would be 15 to i. Nov. ij. I93I Genetic Behavior of Spelt Form in Crosses 347 3. The spelt might have carried a spelt factor Sj and in addition another factor 83, which would produce the spelt character if there were present its complementary factor C, which might have been supplied by the wheat parent. In this case if C were present the ratio would be about 8 spelts to i wheat; and if C were not present, it would be 3 to i. The ratios to be expected on the basis of this last possibility, however, are not in accord with the experimental results. The second explanation seems to be the most plausible of the three. The assumption that some wheats carry the factor for the inhibition of the spelt character is not a mere speculation but a fact, as will be seen later when the question of the production of synthetic spelts by crossing two wheats is taken up. As commercial strains of T. sativum are not purified with respect to inhibitors, there are undoubtedly some strains which contain individuals heterozygous for this factor. If such a plant is used, for instance, as the female parent and is crossed with a spelt carrying the factor for spelting, according to the hypothesis some of the seeds will produce Fj progeny where in some instances the ratio will sug- gest a 15 to I, and in others a 3 to i segregation. As will be seen later, other modifications of these ratios may also be expected to arise. PROGENY OF STABILIZED SPELTOID X SATIVUM CROSSES The speltoid form used in the crosses which will be considered now is commercially known as "Gatineau." The variety originated from a cross between T. durum and T. sativum. It grades usually from 4 to 7 in the classification used in this study for spike form and so resembles the commercial spelts in this respect. It does not have the brittle rachis of spelt, and the grain thrashes from the glumes more easily than the grain of spelt, being like some of the tight-glumed wheats. It is neither a typical spelta nor a typical sativum. The F^ plants of the crosses between Gatineau and T. sativum were almost like Gatineau. The Fj generation consisted of forms which were intermediate; typical spelts of classes i to 2 were not found. The classi- fication of the Fj individuals is reproduced in Table VII. Table VII. — Classes of F2 generation plants of two speltoid X wlieat crosses. Numbers of individuals obtained compared with the theoretical expectations Degree of spelting. Number of plants. De- via- tion. Series. I 2 3 2 I 4 2 0 5 4 5 6 9 9 7 10 7 8 II 9 9 15 15 10 15 17 Total. Obtained. Calculated. Prob- able Spel- toid. Wheat Spel- toid. Wheat 13228a ^ 13229a 2 0 0 0 0 68 63 Si 46 15 17 51.0 47.2 17.0 IS- 8 2. 0 I. 2 2.4 2-3 > Series 13228a Turkey (C. I. 3375. Sel. 13389) X Gatineau (C. I. 2959. Sel. 13403). 'Series 13339a Seneca Chief (C. I. 3372, Sel. 1338S) XGatineau(C. I. 3959, Sel. 13403). 348 Journal of Aqriculiural Research voi. xxn.No. 7 So far as the ratios of speltoid to wheat forms are concerned, these crosses segregated in the simple mendeUan fashion. In series 13228a (Turkey X Gatineau) there were 53 speltoids and 15 wheats, showing a deviation from the theoretical numbers of 2.0 with a probable error of ±2.4 In series 13229a (Seneca Chief X Gatineau) there were 46 speltoids and 17 wheats, the deviation here being 1.2 with a probable error of ±2.3. In both cases the approximations of the figures obtained to those expected are within the range of their probable erros. Therefore, it can safely be concluded that the ratio is 3 to i and that there very likely is but one spelt factor difference. The question of interest in the inheritance of this speltoid form (Gatineau) is not so much in its 3 to i ratio as in the way it differs in details from the spelt X wheat crosses first discussed, which segregated in the ratio of 3 to i. In order to compare the Fj curves of these two groups of spelt X wheat crosses, the Fj frequencies in Table VII (series 13228a and 13229a) and the first two series in Table I (13255a and 13260a) are represented graphi- cally (fig. 2). The comparative characteristics of the curves of these two sets of crosses are as follows : The curves of the true spelt X wheat crosses (13255a and 13260a) begin at class I , where they have their highest spelt frequencies. They gradu- ally drop until they reach classes 5 to 9, inclusive, where there seems to be an indefinite fluctuation of frequencies. Then the curves suddenly rise again at class 10, which contains the spelt-free populations. As to the curves of the wheat X speltoid crosses, the Fj spelt popula- tions begin at about class 3, where but a very few individuals are found. Beginning at class 5, the curves steadily rise until they reach their maxi- mum height at class 10. The curves produced by the true spelt crosses, it will be recalled, continuously dropped instead of ascending. When these two sets of crosses are compared it will be observed that, although there is but one spelt factor difference in each, the spelt factor present in crosses 13255a and 13260a is entirely different from the spelt factor present in crosses 13228a and 13229a. The wheat parent has had no influence is producing this variation in distribution, the same wheat parent plant, Turkey, C. I. No. 3375, selection 13389, having been used in crosses 13260a and 13228a. GENERAL DISCUSSION OF THE GROUPINGS WITHIN THE SPELT AND SPELTOID CLASSES Having discussed the question of ratios, let us turn our attention to the analyses of the details of the variations within the spelt and speltoid classes. The possibility of distinguishing the homozygous from the heterozyg- ous spelts of the Fj generation is of primary interest. Table VIII has Genetic Behavior of Spelt Form in Crosses 349 _ . 1 SER/ES /326ba ^P£^T/9/V^^ TUf^KEY SERI^ /325Sa fSPEET/^ND p/?L£ ^LORf/^ SERIES i3228a TURKEY /f/yPO^XIf^ ^^f- SERIES /3229 -V A ^ V --. .^^ r-' — k /" ^i O' ■i? ^ z V / ^ ^ «< <' ^ *^ (£* ^ >»: s le _ _ ~ — ^ is t3 ^- ^5" /9 7 A CLffSSES 0FSP£LTEDN£6a /O Fig. 3. — Comparison of Fj generation curves of the progeny of F2 spelts which yielded only spelts (series 13260a, 13255a, and 13126a). 70494°— 21 2 352 Journal of Agricultural Research voi. xxn. no. 7 Table IX. — Frequencies of spelt and wheat classes in F3 progeny of Fj individuals in family Ij2^§a, Dale Gloria X spelt FAMILIES CONSISTING OF SPELTS ONLY Pedigree number Fj. Class of F2 parent plant. Classes of spelt inheritance. Number of spelts and speltoids. Num- ber of wheats. Totals. I 2 3 4 s 6 7 8 9 10 I 2 2 I I 2 I 3 4 5 3 10 16 7 16 2 5 2 6 7 8 2 3 I I 7 I I I I I I I 3 14 17 6 21 43 15 25 2 I I 17 6 3 4 3 I I 21 6 43 25 14 19 FAMILIES CONSISTING OF WHEATS ONLY 5 9 12 10 10 10 10 10 10 10 10 10 10 10 10 II 25 13 17 21 19 38 23 29 20 33 14 II 25 13 17 21 19 38 23 29 20 33 14 13 18 20 22 27 29 31 32 33 FAMILIES CONSISTING OP WHEATS AND SPELTS 15- 16. 17- 21 . 23- 24. 25- 26. 28. 30. Expectations Probable error DcAnation Ratio between deviation and probable error. 3 3 20 9 2 3 I 3 4 3 2 2 2 2 .S 2 3 2 2 2 2 I 17 5 8 I I 5 8 7 5 6 4 2 4 3 4 7 5 H S 2 5 I I 3 I I 5 4 5 2 I I 2 2 6 7 9 3 6 4 2 2 3 2 5 I 2 I 9 7 I 4 2 4 2 24 II 2 2 2 I 3 4 6 10 I ^ S 2 rcrnlic I 2 2 I 6 prnrx 2 T? r 21 >1ftTlt« 34 15 20 27 44 23 4 28 29 9 33 25 33 32 356 384 ±6.61 18 2. 72 4 4 8 IS 10 9 24 20 17 II 12 17 3 156 128 Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 353 Table X. — Frequencies of spelt and wheat classes in F^ progeny of Fj individuals of series lj26oa, spelt X Turkey FAMILIES CONSISTING OP SPELTS ONLY Pedi^ee number F,. Class of F2 parent plant. Classes of spelt inheritance (F3). Number of spelts and speltoids. Num- ber of wheats. Total. I 2 3 5 6 7 8 9 10 8 2 2 4 I 2 2 3 3 5 10 12 4 4 2 10 9 3 I 5 8 6 I 8 6 3 12 II 20 13 12 3 20 16 20 21 2 I 22 13 12 23 24 27 16 I 3 20 I I 16 FAMILIES CONSISTING OF WHEATS ONLY I 10 10 10 10 10 10 10 10 10 17 14 15 15 30 20 30 12 24 17 14 15 15 30 20 30 12 24 17 a 4 5 II 15 IS 30 20 30 12 17 18 26 24 FAMILIES CONSISTING OP SPELTS AND WHEATS 3 6 9 I I 3 I 2 7 5 2 2 5 I 0 I I 5 6 10 4 8 7 5 7 4 4 ^ 5 2 8 13 16 20 10 18 17 13 16 26 13 12 16 22 10 4 8 7 5 7 4 4 3 4 S 2 8 23 II 13 5 10 10 5 6 3 5 3 7 I I 3 28 9 I 17 23 24 17 20 29 17 17 18 10 13 I 3 10 2 14 3 I IS 19 2 9 6 I 6 II 6 I 5 2 6 I 2 3 3 4 I I 25 28 29 I I 2 3 I I I 8 I 10 30 I 2 73 4S 20 30 ofhetet ous F2 ] Expectal Probable Deviatio] Ratio of ozyg- jlants. ions. . 6 7 S 5 19 32 71 212 212. 25 ±4- 91 ■25 •OS 71 70-75 283 error 1 deviation to crobable erro r The F3 populations of the series segregating in the 3 to i ratio (13255a and 13260a) have a general tendency to produce the maximum frequen- cies at class I , sometimes at class 2. In only 4 families out of 15 are there individuals in classes above the fourth, 10 individuals in 248 receiving the 354 Journal of Agricultural Research voi.xxii.no. higher classification. The few mdividuals found in these speltoid classes at present may be regarded as exceptions. Their significance will be considered later. The distributions of the families in the series segregating in the ratio of 15 to I (13 126a) (Table V) are entirely different. They do not take the general course described above. Some of them have very low frequencies at classes i and 2. Families 5, 7, and 12 have no individual in class i, the population of family 5 being composed of class 9 individuals only. Family 25 produced its spelts in classes 6 to 9, inclusive. Of the 12 families being considered 6 produced class 9 individuals, while among the families segregating in the 3 to i ratio there is but one instance (13255a, family i) where class 9 individuals have been produced. The explanation of the increased variability of the constant spelt- producing families of series 13126a, as compared with series 13255a and 13260a, will be found in the factorial explanations given for these two groups of crosses. Families 13255a and 13260a segregated in the simple monohybrid 3 to I ratio. By hypothesis, all the spelts producing only spelts are sup- posed to have the genotypic composition SS. As to the cross 13126a which segregated in the 15 to i ratio, it was shown that there were five constant spelt forms, namely : SjvSiSaSg SiSiSjSs S1S1S2S2 SiSjSzSg S1S1S2S2 Although these forms would keep on producing only spelts, they are not genotypically identical. The fact that in the families segregating in the 3 to i ratio there was only one genotypic spelt form and in the family segregating in the 15 to i ratio five such forms were present may account for the increased varia- bility among the pure-breeding spelts of the latter cross. The F3 progenies can not be separated into the five theoretical geno- typic groups just referred to because, among other things, there is positive evidence that modifiers also are concerned which have the tendency to shift the classes one way or the other. This phase of the subject will next be discussed. MODIFICATION OF THE DEGREE OF INHERITANCE OF SPELT CHAR- ACTERS, DUE TO THE PRESENCE OF MODIFYING FACTORS For the consideration of the subject of modifiers the analyses will be confined primarily to the spelt classes (i to 9, inclusive) of the progeny of the heterozygous F.^ individuals of series 13260a, shown in Table X. This family has been chosen because it represents a simple mode of segregation. Whatever is said about modifiers for this family will be found to apply just as well to the other families. It has been shown that only one spelt factor was concerned in the cross under consideration. All the Fj heterozygous plants had the formula Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 355 Ss and produced spelts and wheats in the 3 to i ratio. If these F3 heterozygous plants have all the same genotypic constitution with regard to the spelt factor we would expect to see them produce similar F3 spelt distributions. These distributions obtained experimentally are far from being uniform. For instance, plant i326oa-3 (Table X) pro- duced individuals mainly of classes 8 to 9, and plant i326oa-6 produced its spelts in classes i and 2 only. The progeny of i326oa-i5, on the other hand, showed no definite grouping, the curve spreading from one extreme to the other. It may be argued that (i) these variations are insignificant; (2) they may be due to variations in soil and other external conditions; or (3) they are merely nonheritable fluctuations. These arguments may be answered easily: 1 . In the first place, let us take the first two frequencies (classes i to 9, inclusive), namely, those of i326oa-3 and i326oa-6 (Table X). The means are 8.00 ±0.29 and 1.3 1 ±0.08. The difference between the two means is 6.69 ±0.30. This difference, it is seen, is very significant. Similar striking differences will be found when the means of the other distributions are compared. 2. With regard to variations due to soil conditions and external factors, it is only necessary to mention that these plants were grown on the same plot of the experimental field. The pedigree numbers following the family number represent the numbers of the rows in which the progeny of each of the Fj plants was grown. For instance, the progenies of plants i326oa-28, 29, 30 (at bottom of Table X) were grown in three adjacent rows, yet i326oa-28 was composed of individuals contributing to nearly all classes of spelting, i326oa-29 produced practically all typical spelts, and the progeny of i326oa-30 were nearly all speltoid forms approaching the wheat type. So this second objection may also be dismissed. 3. As to the nonheritability of these variations, the objection may be settled by comparing the Fj and F3 generations in terms of the coefficient of heredity. If these variations are nonheritable fluctuations, there should be no correlation between the Fj and F3. Putting the statement in the affirmative, if there is a significant correlation in the degree of spelting of parent and offspring of the F2 and F3, then it is a direct and indisputable proof that these variations are transmitted to the follow- ing generations — that is, they are heritable. The accompanying corre- lation table (Table XI) has been prepared with a view of determining the validity of this last objection. The x-axis represents the mean classes of the F3 and the y-axis represents the classes of the F2 individuals which produced these Fg forms. In this table are included all the progeny of the heterozygous forms of series 13255a and 13260a in order to have a sufficient number of individuals. The coefficient of heredity as calculated is 0.880 ±0.029. As this coefficient is over 0.5 and over 10 times its 356 Journal of Agricultural Research voi. xxu.no. 7 probable error it may be regarded as being very significant. As there is a significant correlation between these two generations, the variations under consideration are not fluctuations due to external conditions but are hereditary variations. Table XI. — Correlation table showing the classes of spelt inheritance o/Fj heterozygous plants, with the average degree of spelling of the F^ progeny of each F-^ plant (series Ij2j^a and 12260a) Classes of spelt inheritance (Fs) I 2 3 4 5 6 7 8 9 I 4 I 3 I I 2 I a 3 I I I K ^ a V 4 2 rt a h "S 5 2 2 M a Q 6 I K 7 I 2 I 8 9 I 2 CoeflBcient of heredity=o.88o±o.o29 If there were no interference due to modifiers, the curve of the spelt F3 progeny of the heterozygous Fj individuals would follow the spelt curve of the Fj generation, as both the spelts of the Fj curve and those of the F3 curves of heterozygous Fg individuals consist of SS and Ss spelt plants in the proportion of i to 2, respectively. An examination of the F3 spelts of heterozygous individuals in Table X shows that the curves of the 13 families vary considerably from the curve of the F2 generation (Table I, series 13260a), although the curve for the totals is much the same. Again, if there were no genetic interference, all the F3 progeny curves produced by heterozygous F2 plants would be expected to follow more or less the same course. The experimental results exhibit wide differences, as the comparison of the classes of individuals i326oa-3, -6, -7, etc., will readily show. Nov. li, 1921 Genetic Behavior of Spelt Form in Crosses 357 As the presence of multiple factors is entirely out of question, it being proved in this case that the parents of this cross differ in only one factor for spelting, the following explanation may be given to account for these variations. One or more sets of modifiers furnished by the spelt, by the wheat, or by both parents seem to be present where each set of modifiers was in a homozygous dominant condition in one parent and in the alternative condition or absent in the other parent. These modifiers in the presence of the S factor tend to intensify the spelt character. An example may be given to illustrate the effect which a modifier may produce in a spelt X wheat cross. The modifier which may cause dilu- tion of spelting may be represented by the factor D and may be assumed to be carried by the wheat parent. (If this factor were contributed by the spelt parent, the latter would have been a dilute spelt, which was not the case in these crosses.) The wheat parent will then be represented by ssDD and the spelt parent by SSdd. The genotypic forms of the successive generations will be as follows : Pi SSdd spelt. X SsDd ssDD wheat Fl semidilute 1 spelt. 1^2 I SSdd. 2 SSDd. I SSDD. I ssdd. 2 Ssdd. 4 SsDd. 2 SsDD. 2 ssDd. I ssDD. 3 typical 6 semidilute 3 dilute 4 nonspelts. spelts. spelts. spelts. 12 spelts. 4 wheats. This represents a ratio of 3 spelts of different grades to i wheat. If we assume that the nature of the modifier were to produce intensifi- cation of spelt inheritance in the presence of factor S, which in this case may have been carried either by the spelt or by the wheat, we will have, in the F, generation, 3 intense spelts, 6 semi-intense spelts, 3 normal spelts, and 4 wheats. Some of these spelts (intense, normal, or dilute) will breed true to those conditions ; others will produce some or all of these forms in different proportions as expected on the factorial hypothesis. If more than one set of modifiers are present the types and their proportions naturally become rather complex. If, in the crosses 13255a and 13260a, a diluting modifier has been intro- duced, we would occasionally expect among the spelts (homozygous or heterozygous) some which are grouped in the dilute speltoid classes. The F3 population of i3i26a-5 and -25 (Table V); i3255a-26 (Table IX) ; i326oa-3, and -14 (Table X) ; and a number of others represent such cases. The progeny of i326oa-2o, -6, -10 and others may represent spelts carrying some intensifying factor. 358 Journal of Agricultural Research Voi.xxn.No. 7 In conclusion, it may be said with certainty tJbat besides tlie S factor in series 13255a and 13260a and the Si and So factors in 13126a, modifiers are present which tend to dilute or intensify the spelt character. In statistical studies of density in wheat, the junior author has found two characters whose mode of inheritance is almost identical with that of the spelt character. When a dense wheat (T. compactiim) is crossed with a lax wheat (T. vulgare) a 3 to i segregation is found in the Fj gen- eration. The F2 density curve consists of two distinct curves. One of these is a skew curve in the dense classes which contains 75 per cent of the individuals. After a gap, the other curve, which is composed of the lax segregates containing the remaining 25 per cent of the F2 population, begins. Although the F3 progeny of these heterozygous dense plants of the F2 generation invariably produce bimodal curves similar to that of the F2 just described, their modes or the means of the dense and lax curves shift at times considerably toward the lax classes and sometimes toward the denser classes, much in the same manner as does spelting. A similar phenomenon has been observed by Nilsson-Ehle (8) among his dense X lax wheat crosses. The other parallel case is density of the type just mentioned, but in this case the modifier is known to be the spelt factor itself. The curves of the progeny of the heterozygous dense individuals of dense wheat X spelt crosses have the general bimodal form, but the populations which, in addition to density, carry the S factor always have their density curves shifted toward the lax classes. With some spelts, the S factor shifts the density curves so much toward the lax classes that this S has to be regarded also as an inhibiting factor for density. Hull-lessness in oats, according to Love and McRostie (7), is inherited in a similar manner. While this character segregates in the simple mendelian ratio of i hulled to 2 intermediates to i hull-less, the inter- mediate forms vary appreciably as regards the percentage of hulled kernels they produce. By correlating the percentage of hull-lessness of parent and offspring, they have shown that these variations within the I to 2 to I ratio are hereditary. The mode of inheritance of the spelt character as shown in Tables IX and X closely resembles also Castle's (2) case of hooded rats, which had for a time aroused considerable controversy for and against the question of inconstancy of unit characters. In numerous crosses between rats having the hooded pattern and the wild (totally pigmented) or the Irish (white belly) types the hooded pattern behaved as a mendelian recessive. The ratio of nonhooded to hooded F2 offspring was 3 to i , showing that the hooded condition is dependent upon a single factor difference. Among the hooded individuals a considerable degree of variation was observed with respect to the degree of the extension of this pattern. By making selections for many generations in plus and minus directions Nov. 12. 1921 Genetic Behavior of Spelt Form in Crosses 359 Castle was able to increase and decrease the pigmented area. His belief then was that the variations observed in the race of hooded rats were not mere fluctuations but were hereditary variations in the sense that the factor for the hooded condition had undergone alterations. The assumption of unit-factor inconstancy, which Castle applied to account for variation of pigmentation of his hooded rats of course can not be applied for the analogous variations in the groupings of the spelt individuals, for, if this were the case, variations in this same extent should have been present among the self -fertilized population from which the parental form was selected. The classes of spelt inheritance in the parental strain ranged from i to 4, the mode being between classes i and 2 . No departures nearly as great as those found in the homozygous extracted spelts of the F3 generation were observed among this parental population. The study of the F2 generation shows clearly that either some modifier or modifiers were introduced by the nonspelt parent or were carried by the spelt parent, but these modifiers were reduced to a recessive state as a result of crossing. PRODUCTION OF SYNTHETIC WHEATS BY CROSSING TWO SPELTS, AND SYNTHETIC SPELTS BY CROSSING TWO WHEATS The writers frequently have obtained synthetic spelts in interspecific crosses in wheat. No indication has been observed as to the possibilities of producing true wheats in crosses between two different nonwheat species. It is theoretically possible, however, that such forms eventually will be produced in crosses between certain kinds of spelts. This suppo- sition may be explained by taking as an example the results of one of the experiments discussed at length in this paper. In the case of cross 13126a it Avas shown in detail that two spelt factors Si and S2 were involved; that the F2 segregates which bred true to the spelt character were not all genetically identical; and that they were composed of five genotypic forms, namely: v3iOiv32^2 wiS202^2 W1D1V52S2 ^1^1^2'^2 Sj^Sj0202» As long as these forms are allowed to be selfed, as they are in nature, no wheats ever segregate; but, by hypothesis, in a number of crosses between these five forms, a certain proportion of wheats are expected to appear in the following manner : 1. Crosses producing no wheats: SiSiSaSaXany other genotype; SiSiSoSaXSiSiSgSa; S1S1S2S2XS1S1S2S2. 2. Cross where one out of every four F^ plants will produce 6^ per cent wheats : *-'l^l^2^2 X v5iSiv32W2 • 3. Crosses where half of the F^ plants will produce 6^ per cent wheats: ^l*-'l^2^2Xv3iSiv3202; Oiv3i02S2XSiSit52S2- 4. Cross where all F^ plants will produce 6^' per cent wheats: ^1^1^2^2XSiSiv32»32' 360 Journal of Agricultural Research voi. xxii. no. 7 If wheats segregate trom these crosses it will prove further the correct- ness of the two-factor hypothesis. It will also lead to the expectation that genotypic forms similar to the above, and other combinations as well, exist among the so-called pure commercial spelt forms and when the proper cross is made among these commercial spelts, a certain number of synthetic wheats may be produced in the Fj generation. It is easy to understand how the wheat character, being distinctly hypostatic, may be carried from generation to generation by the spelt type. But how can the spelt type segregate from a wheat X wheat cross? How can one conceive the spelt factor, which is so pronouncedly epistatic to the wheat character, as being carried by a wheat without being mani- fested phenotypically ? The explanation is simple. It was shown that modifiers are involved in these crosses. Common wheats occasionally carry modifiers which tend to dilute the spelt character. Some of these modifiers were shown to be so effective that they grouped all of the spelts in class 9. Most of the class 9 individuals, as recorded in the foregoing tables, resemble wheat so closely that no one would be likely to call them true spelts. If a certain diluting modifier can shift the spelts to class 9, a group of these may readily shift the spelt to classes between 9 and 10. If these diluting factors are reduced to a homozygous dominant condition, the dilute spelt which will be classified as 10 will breed true to type and be considered as wheat, although from a genetic standpoint such a form is a spelt. As long as such sorts are allowed to self-fertilize they will produce a so-called pure line consisting of a constant wheat type. Their spelt characteristics are exhibited only when crossed with a common wheat which carries the factor for dilution in a recessive state. In the second generation of this cross the segregates which carry the S factor with the factor for dilution in a recessive state; that is, SS dd, will be spelts. Fortunately, experimental evidence can be cited to support this statement. One of the writers has observed at the Kansas Agricultural Experiment Station over 20 spelts among Fj hybrid plants derived from a number of wheat X wheat crosses where one of the parents was a rust-resistant variety of winter wheat and the other was Preston, Marquis, or Haynes Bluestem, well-known varieties of spring wheats. These parental types and some of the spelt segregates are shown in Plate 33, B.^ None of the F^ plants in these crosses were spelts, or at least passed for spelts, although they might have shown some spelt characteristics in a weak form. In the Fj generation, however, depending upon the cross, the proportions of wheat to spelts varied roughly from 3 to i to over ' The authors are indebted to Professors John H. Parker and L. E. Melchers for allowing them to photo- graph these forms and use them in connection with this paper. Nov. ra, igar Gefietic BehavloY of spelt Form in Crosses 361 100 to I, with some sets of crosses producing no spelt at all. Besides the true spelts, a number of speltoid forms also segregated. The absence of the spelt type in the F^ generation shows that the absence of the spelt character in one of the parents was due to the pres- ence of an inhibitor in the parent plant which carried the S factor. If the appearance of the spelt form in the Fj generation was due to com- plementary factors furnished by both parents, the spelt should have appeared in the F^ generation. Such was not the case. The cultures consisted of over a thousand Fg plants. It is not now absolutely necessary to know how, or exactly in what proportion, these appeared. The purpose of citing these examples is to substantiate the views expressed above regarding the possibilities of producing synthetic spelts from wheat X wheat crosses, which might have been regarded as a mere speculation in the absence of this experimental evidence. The fact that there were no spelts grown near the P^ or F^ plants and that spelts appeared in more than one cross excludes the possibility of accidental or natural cross-fertilization. These observations show that common wheats may carry the spelt factor, but the latter can not express itself because one or more diluting or inhibiting factors are carried with it. Some of these diluting factors may be regarded as inhibiting factors which are not totally dominant but produce intermediacy in a heterozygous state. From the small percentage of spelts which appeared in the Fj genera- tion in some cases, it may be inferred that there is one and in some cases more diluting factors.^ The production of synthetic spelts in wheat X wheat crosses just con- sidered is similar to the synthetic production of T. dicoccum dicoccoides, the so-called "Wild Wheat" of Palestine, in the vulgar e X durum cross (6). The wild character, consisting of a number of interdependent specific characters, is strongly dominant over both the sativum and the durum types, as is the spelt type toward sativum. Yet in both instances the character showing strong dominance toward either of the parental forms was carried by one of the parents together with a factor inhibiting in one case the wild and in the other case the spelt characters. MODIFICATIONS OF MENDELIAN RATIOS The question of modifiers whose presence in the production of the spelt character was demonstrated in various ways brings us to the con- sideration of the modifications of mendelian ratios. As all spelts or all wheats are not alike with respect to their ability to intensify, repress, or inhibit the production of the spelt characters, it is natural to expect ' Nilsson-Ehle (.8, q) and Kajanus (j, 4) have also observed the occurrence of spelts in certain sativum X sativum crosses. They support the correctness of the foregoing observations and tend to preclude the assumption of the possibility of the occurrence of some accidental or natural crossing of one of the sativum patents with a spelt. 362 Journal of Agricultural Research voi. xxii. no. 7 certain modifications of the 3 to i ratio. For instance, in certain spelt X wheat crosses, depending upon the potency of the diluting factors, the proportion of wheats to spelts may increase in certain amounts. In some instances, the increase of wheats will be slight, so that the obtained deviation from the 3 to i ratio, which will be on the side of excess for the wheat class, will be considered within or near the limits of the probable error and the inheritance will be regarded as simple mendelian. Perhaps series 13255a, where an increased proportion of both constant breeding wheats and F3 wheat segregates of heterozygous Fj plants are obtained, represents such a case. It also is possible that the ratio may fall between 3 to i and i to i in case the diluting factor is very strong. Here, then, will be an excess in the homozygous wheat class at the expense of the homozygous dom- inant spelt class. If factors of both dilution and of intensification are introduced in the same cross, the experimental ratios will defy any attempt at simple factorial explanations. The writers have obtained a cross where the Fg generation suggested a possible 3 to i segregation, but on examining the F3 generation, which was composed of a fairly large population, neither the individual segregations nor the totals of .these approached in any way 3 to i or i to 2 to i expectations. It naturally follows from the foregoing discussion that in spelt-wheat crosses wide departures from simple ratios occasionally may be expected. SUMMARY T. spelta and T. sativum are differentiated by a number of linked specific characters, which are present in one species and absent in the other. These characters, so far as observed, are not inherited independ- ently but are transmitted as a group. In crosses between a spelt and common wheats the F^ hybrid shows dominance of the spelt, but this character appears in a somewhat diluted form. In the second generation all classes of spelt inheritance are ob- tained. In order to classify these forms, 10 arbitrary classes were erected, class i representing the true spelt and class 10 the total absence of this character. The intermediate classes represent diff'erent grades of spelt inheritance. In most of the material studied there was but one factor difference for spelt, but in two cases two spelt factors were present. Both 3 to i and 15 to I ratios were obtained. These ratios were verified after determin- ing the genotypic constitution of the Fj plants, which gave pure breeding spelts, inconstant spelts, and pure breeding wheats in the ratios of I to 2 to I and 7 to 8 to i , respectively. Of the constant spelts produced in crosses with this latter ratio, approximately half yielded (in the F3 generation) spelts and wheats in the ratio of 15 to i and the other half in the ratio of 3 to i, as expected on the two factor hypothesis. Nov. 12, 1921 Genetic Behavior of Spelt Form in Crosses 363 The speltoid form "Gatineau" when crossed with wheats gave also a 3 to I segregation of spelts and wheats, but the curves showing the classes of spelts produced by this cross were entirely different from the curves produced by the spelt-wheat crosses which segregated in the same ratio. Aside from the factor or factors for spelting, there is positive evidence showing the presence of intensifying and diluting modifiers which tend to affect the degree of spelt characters without affecting to any extent the ratios of spelts to wheat. Some of the diluting modifiers tend to act as inhibitors. The progeny of all heterozygous spelts of the Ss type do not produce a similar spelt curve. There are wide discrepancies in the spelt inheritance of the progeny of these forms. These variations within the spelt classes have been found to be hereditary and to be caused by modifiers. The theoretical possibility of producing synthetic wheats in crosses between certain pure-breeding spelts is shown. Experimental evidence also is presented showing that, in spite of the fact that the spelt character is dominant over the wheat form, the former may be synthetically produced by crossing certain wheats, provided one of the wheats carries a spelt factor together with an inhibitor and that the other wheat carries neither. It is shown that, if intensifying, inhibiting, and diluting modifiers are introduced in a cross, wide departures may be expected from the 3 to i and 15 to I ratios. LITERATURE CITED (i) BosHNAKiAN, Sarkis. 1918. THE MECHAmCAL FACTORS DETERMINING THE SHAPE OF THE WHEAT KERNEL. In Jour. Amer. Soc. Agron., v. lo, no. 5, p. 205-209, fig. 27. (2) Castle, W. E., and Phillips, John C. 1914. PIEBALD rats and SELECTION ... 56 p., 3 pi. Washington, D. C. (Carnegie Inst. Wash. Pub. 195). Bibliography, p. 31. (3) Kajanus, Birger. 1912. 0BER EINEN SPONTAN entstandenen weizenbastard. In Ztschf. Pflanzenziicht., Bd. i, Heft i, p. 13-24. (4) 1918. KREuzuNGSSTUDiEN AN winTERWEIzen. In Bot. Notisef, 1918, Haftet 5, p. 235-244. (5) Love, H. H., and Craig, W. T. 1918. the relation between color and other characters in certain AVENA crosses. In Amer. Nat., v. 52, no. 620/621, p. 369-383. (6) 1919. THE SYNTHETIC PRODUCTION oP WILD WHEAT FORMS. In Jonr. Heredity, V. 10, no. 2, p. 51-64, 10 fig. (7) and McRosTiE, G. P. 1919. THE INHERITANCE OF HULL-LESSNESS IN OAT HYBRIDS. In Amer. Nat., V. S3, no. 624, p. 5-32, 7 fig. 364 Journal of Agricultural Research voi. xxii, no. 7 (8) NitSSON-EHLE, H. 1911. KREuzuNGSUNTERSucHUNGEN AN hafer und weizen. II. In Lunds Univ. Arsskr., n. f., afd. 2, bd. 7, no. 6, 84 p. Literaturzerzeichnis zu den Einleitung, p. 20. (9) I917. UNTERSUCHUNGEN UBER SPELTOIDMUTATIONEN BEIM WEIZEN. In Bot. Notiser, 1917, Haftet 6, p. 305-329, i fig. (10) Saunders, Charles E. 1907. report of the cerEaust. In Canada Exp. Farms Rpts., 1906, p. 235- 256, I pi. (11) Surface, Frank M. I916. studies on oat breeding. III. ON THE INHERITANCE OF CERTAIN GLUME CHARACTERS IN THE CROSS AVENA FATUA X A. SATIVA VAR. KHERSON. In Genetics, v. i, ixO. 3, p. 252-286, pi. 2-3. Literattire cited, p. 285-286. (12) TscHERMAK, Erich von. 1910. WEIZEN (TRITICUM). KORRELATIONEN. BASTARDIERUNG. /n Fruwirth, C. Die Ziichtung der landwirtschaftlichen Kulturpflanzen, Aufl. 2, Bd. 4, p. 119-139, 164-187, fig. 11-12. Berlin. Bibliographical footnotes. PLATE 33 A. — Wheat spikes showing different degrees of spelting. i and 2 are intense spelts; 3 to 9 are intermediate forms, showing varying degrees of dilution of the spelt character; 10 is a pure-breeding wheat (sativum) form, showing no trace of spelting. The numbers represent approximately the types falling in the 10 classes of spelting. B. — Snythetic spelts produced in Fj generation in wheat X wheat crosses; a, b, c, and d are the sativum parent plants; b-i spelt form of the progeny of a X b; c-i to c-5 spelt forms of the progeny of a X c; d-i and d-2 spelt forms of the progeny a X d. Genetic Behavior nf the Spelt Form in Crosses Plate 33 ) \2 i<9 /^ Journal of Atjricultural Research Vol. XXII, No. 7 PLUM BLOTCH, A DISEASE OF THE JAPANESE PLUM, CAUSED BY PHYLLOSTICTA CONGESTA HEALD AND WOLF^ By John W. Roberts, Pathologist, Fruit Disease Investigations, Bureau of Plant Industry, United States Departinent of Agriculture INTRODUCTION In June, 1905, W. M. Scott of the Bureau of Plant Industry, United States Department of Agriculture, collected near Fort Valley, Ga., fruits of the Japanese plum (Prunus triflora Roxbg.) affected with a disease very closely resembling the apple blotch, due to Phyllosticta solitaria E. and E. In the diseased areas were spore-bearing pycnidia which were found also on the leaves in gray papery spots resembling those on apple leaves caused by Phyllosticta solitaria. On May 27, 1908, the disease was again observed by Scott on both fruit and foliage of the Burbank plum at Montezuma, Ga. It was found to be rather common in several orchards about Montezuma, in some cases causing enough damage to injure seriously the market value of the fruit. In one orchard a large part of the fruit was affected, and many specimens bore from 15 to 20 spots each. On May 29, 191 7, the writer collected near the same locality Japanese plum fruits and leaves affected with the same disease. In the single orchard in which the disease was found, most of the fruit was heavily nfected and rendered nearly worthless. Considerable difficulty was encountered in finding the disease again, as the Japanese plum industry in Georgia had about passed out. Lack of demand for the fruit coupled with the susceptibility of all parts of the tree to various diseases and insect pests had caused growers either to eradicate their trees or to let them die. At present there are almost no Japanese plum orchards remaining in Georgia, and all of the trees in which plum blotch was found have been eradicated. So far as the writer knows, then, the disease no longer exists, though it is to be looked for throughout the South as far west as Texas. Should the growing of Japanese varieties of the plum be revived in the South, blotch may prove to be one of its most serious diseases, as it is very destructive, and probably would be exceedingly difficult to control. The varieties found to be affected were Abundance, Burbank, and what was apparently an unnamed seedling. 1 A brief description of this disease was published as an abstract of a paper presented at the Ninth Annual Meetmg of the American Phytopathological Society. (Roberts, John W. plum blotch. (Abstract.) In Phytopathology, v. 8, no. 2, p. 74. 1918.) Journal of Agricultural Research, Vol XXII No Washington, D. C. Nov. i.. 1921 ^^ Key No. G-253 70494°— 21 3 (365) 366 Journal of Agricultural Research voi. xxu. No. 7 DESCRIPTION OF THE DISEASE The infected parts on the unripe fruit appear as dark-colored raised areas with fringed margins and are somewhat roughened by the presence of small blisters and depressions (PI. 34, B). As in the case of apple blotch, the skin often becomes ruptured as the fruit increases in size. On the ripe fruit the blotched parts appear as irregular browned areas 3 to 6 mm. in diameter and consist of an aggregation of from 4 to 20 sunken spots, each separate spot being i mm. or less in diameter. At this stage the spots have a peculiar light blue cast owing to the "bloom" of the ripe plum covering the browned epidermis. The diseased area is rather superficial, extending only slightly below the epidermis. The affected tissues become hardened and somewhat leathery and show no tendency to decay. Small, glistening pycnidia are produced in considerable numbers even in the younger spots. Quite commonly there are 25 to 30 scattered promiscuously about in each blotched area. Infection evidently takes place when the fruits are very young, since the spots found May 29 were well formed and bore pycnidia with mature spores. Judging from the writer's inoculation experiments, infection probably took place five to six weeks earlier, or about the middle of April. On account of its characteristic appearance on the fruit, the disease has been given the common name of "plum blotch." On the upper surface of the leaf blades (PI. 34, A), the spots are angu- lar, rather small (about 0.5 mm. across), brown when young, but later becoming gray or silvery in color. They may be numerous, as many as 200 sometimes appearing on a single leaf. Usually only a single pycnidium is present in each spot, except where two or more spots have coalesced to form a single large spot. Affected areas are also found on the petioles and on the veins of the lower surface, especially on the midrib. On these the diseased areas are much larger than on the upper surface of the blade and are black and sunken. Pycnidia, bearing spores, are present in great abundance. ,, Pycnidia, apparently identical with those found on the fruit and leaves, were found also in small light-colored, often slightly sunken areas on the twigs; but, as spores were lacking, positive identification could not be made. It is possible that these pycnidia had discharged their spores early in the spring and had brought about the early infections on the fruit. CAUSE OF PLUM BLOTCH By comparison with type specimens, the organism involved in the pro- duction of plum blotch has been found to be identical with the fungus described by Heald and Wolf ^ as Phyllosticta congesta. Heald and 1 Heaid, F. D., and Wolf, F. A. new spEaEs op texas fungi. /wMycologiav. 3, no. i, p. 8. 191 1. i Nov. 12, 1921 Plum Blotch 367 Wolf found the fungus on the leaves only of Prunus sp. in Texas Their description is as follows : Maculis minutis, .5-.8 mm diam., brunneis numerosis, venis limitatis; pycnidiis solitariis in quaque area, 50-125 ix diam.; sporulis globulosis vel leniter elongatis, hyalinis 6-9 m- On Prunus sp. Boeme (Texas) 1554 (Type). On the upper surface of the leaf are very numerous brown areolae bounded by the veins of the leaf. The lower surface may not be discolored. These minute spots fuse, and each contains at its center a single black pycnidium. The pycnidia contain globular or slightly oval, clear spores. Fig. I. — Section through a pycnidium of Phyllosticta congesta, showing spores. Natural infection on plum fruit, Georgia 1917. X 340. Heald and Wolf do not mention the fact that the older spots become gray or silvery, though the type specimens as well as those collected by Scott and the writer show this to be the case. The spots on these leaves and those on Georgia specimens collected by the writer show a marked resemblance, and the fungi found upon them are morphologically the same. The spots on the leaves collected by the writer have a greater tendency to fall out. ^iof-:;, The pycnidia (fig. i) are glistening, lens-shaped, erumpent, on the leaves 65 to 120 /x in diameter, on the fruit 60 to 120 /x in diameter. On the average, pycnidia on the fruit are somewhat larger than those on the leaves. Spores on the leaves measured 7 to 9 ju in diameter, on the fruit 8 to 9 /i. Spores from younger spots were invested with gelati- nous envelops which were sometimes lengthened into appendages (fig. 2). Spores from older spots do not show these envelops, and they are not to be found in the dried herbarium specimens. The young spores of Phyllosticta solitaria have sucli an envelop. In fact, P. solitaria and P. congesta resemble one another so closely that on purely morphological grounds they might be considered as identical. Since the ascogenous stage of neither fungus is known, the writer prefers to retain the name P. congesta as a matter of convenience, unless it is shown by cross inoculations that the fungus on the apple and that on the plum are identical in every way. Fig. 2. — Spores of Phyl- losticta congesta, with the gelatinous envelops which are sometimes present. From pycni- dia on plum fruit, Georgia 1917. X 680. 368 Journal of Agricultural Research voi. xxii. no. 7 Of course the final test of identity would lie in whether or not the as- cogenous stages of the two fungi, assuming them to exist, would prove to be identical. Specimens of Phyllostica congesia on fruit and foliage of Prunus triflora have been deposited in the Pathological Herbarium, Bureau of Plant Industry, United States Department of Agriculture. It is not known how the fungus is carried over from one season to another. If it occurs on the twigs, as the writer is inclined to think, there would be good reason for believing that production of spores from twig lesions in the spring would constitute an important infection source. It is also possible that the fungus survives the winter on leaves and fruit. On all the ordinary culture media the fungus shows about the same type of growth. On corn meal agar, beef agar, prune agar, potato agar, and oatmeal agar growth is very slow, and on all these media it presents the same appearance. There is a dense black mass of closely woven hyphae forming a raised and irregular aggregation of shining bead-like bodies which may be considered as sterile pycnidia, since they are more or less hollow bodies containing oil drops. The margin of the growth is often fringed; in fact on the above-named media the growth is almost as blotch-like as it is on the fruit of the plum. On sterihzed stems of Melilotus the growth resembles that on the agars, but spores are often formed though very scantily. On Japanese plum twigs growth is also very slow. Pycnidia and spores are formed in about two months. Pycnidia are formed on the bark and may also be formed at the cut end of the twig, in which case they are densely aggregated. Sterilized apple twigs proved to be the best medium for the production of spores, though two to three months must elapse before spore produc- tion begins. On this medium the only sign of growth by the fungus is the formation of a dense mass of hyphae and pycnidia, closely aggregated at the upper end or at an abraided place on the side of the twig. On all the media used the type of growth exhibited by Phyllosticta congesia differs somewhat from that of P. solitaria. On sterile apple twigs, for instance, the latter produces pycnidia which are scattered over the bark, whereas the pycnidia of the former are formed only at the cut ends of the twigs. In 191 7 the fungus was isolated from both fruit and leaves by the poured plate method, using spores, and by planting bits of the diseased tissues in plates. In the spring of 19 18, no spores had been obtained in cultures, but inoculations were made by spraying the young fruits and leaves of Abundance and Burbank plums with bits of hyphae and sterile pycnidia suspended in sterile distilled water. The results were negative in every case. Nov. la, I92I Plum Blotch 369 In 19 19, spores obtained from apple twig cultures and suspended in sterile distilled water were applied to fruit, foliage, and twig of Abundance plums on May 15. Where cultures originally obtained from plum fruits were used, two fruits were found with two typical blotches on each of them; three leaves were found with scattering spots, each spot typical of the disease and each bearing a single pycnidium with the character- istic spores of Phyllostida congesta. Like results were obtained by the use of cultures obtained from the leaves; one fruit showed three typical blotches with pycnidia and two others showed one; seven leaves were successfully infected. From all these artificially inoculated parts, the fungus was reisolated and proved to be P. congesta. No lesions were found on the twigs. Inoculations made upon Japanese plums with spores from pure cul- tures of Phyllostida solitaria gave negative results in 1918, 1919, and 1920, though the spores were applied to fruit, foliage, and twigs at frequent intervals throughout the spring. Though the inoculation experiments herein reported upon are suffi- cient to prove Phyllostida congesta the cause of plum blotch on leaves and fruit and show the fungus on the fruit to be identical with that on the leaves, they are not as complete as the writer should wish. All the inocu- lation work was done at Arlington, Va., under conditions probably un- favorable to the fungus, since it has been found naturally only in regions much farther south. It is planned to carry on further inoculation work with both the plum blotch and apple blotch Phyllostictas. The writer expects eventually to obtain successful inoculations on plum twigs using Phyllostida congesta as inoculum. CONTROL MEASURES No attempts to control plum blotch have been made. One would expect that control might be had by spraying with a strong fungicide at intervals beginning shortly after the petals have been shed as is the case with apple blotch. Dilute lime-sulphur solution and Bordeaux mixture injure Japanese varieties of the plum so severely as to preclude their use during the growing season. It is also doubtful whether or not dilute lime-sulphur solution would control severe cases of disease, since it will control only mild cases of apple blotch. Self-boiled lime-sulphur can be used with safety on the Japanese plum, but it is a fungicide which is even weaker than dilute lime-sulphur solution. It seems probable, therefore, that should this disease ever become an important one, its control will present a problem of considerable difficulty, though it is realized that the reasoning by analogy in which the writer has just indulged may easily lead to wrong conclusions. I 370 Journal of Agricultural Research voi. xxii. no. 7 SUMMARY Plum blotch, a hitherto unknown disease of the Japanese plum {Prunus iriflora), has been found in Georgia. In addition to the fruit, the leaves and possibly the twigs are affected. The lesions on fruit and leaves greatly resemble those of the apple caused by Phyllosiicta soliiaria E. and E. Varieties Abundance and Burbank were found to be susceptible. An unnamed seedling, probably also belonging to Prunus iriflora was found to be severely infected. From diseased fruits and leaves the fungus, Phyllosiicta congesta Heald and Wolf, was isolated and grown in pure culture. Spores obtained from cultures on sterile apple twigs when suspended in distilled water and sprayed on healthy fruits and leaves produced characteristic lesions of the disease. Phyllosiicta congesta Heald and Wolf is to be considered for the present as different from P. soliiaria E. and E-, though greatly resembling it. Inoculation experiments on plums using spores from pure cultures of P. soliiaria were negative. No attempts have been made to control plum blotch, but the possibili- ties of control are discussed. PLATE 34 A. — Plum leaves affected with Phyllosticta congesta, Georgia, 1917. B. — Plum fruits affected with Phyllosticta congesta, showing the characteristic ' blotches, " Georgia, 1917. I Plate 34 Journal of Agricultural Research Vol. XXII ,No.7 A COMPARISON OF THE PKCTINASE PRODUCED BY DIFFERENT SPECIES OF RHIZOPUS By ly. ly. Harter, Pathologist, and J. L. Weimer, Pathologist, Office of Cotton, Truck, atid Forage Crop Disease Investigations, Bureau of Plant Industry, United States Department of Agriculture INTRODUCTION Recent investigations by Harter and Weimer ^ showed that Rhizopus tritici Saito, an organism demonstrated to be parasitic on sweet potatoes, produces a powerful intracellular and extracellular pectinase which dissolves the middle lamella so that the cells readily separate without themselves undergoing any noticeable alteration. A suspension of 0.25 gm. of the enzym powder in 25 cc. of water was found to completely macerate sweet potato disks i mm. in thickness in two to five hours. Furthermore, the solution on which the fungus grew was even richer in pectinase, maceration of the sweet potato disks being completed in one to three hours. Since the foregoing results have been published Harter, Weimer, and Lauritzen ^ have concluded experiments which showed that out of 1 1 species of Rhizopus studied 9 were parasitic on the sweet potato. Furthermore, these investigators found that the species differed in degree of parasitism, both as regards the percentage of infection and the rapidity of decay. The present investigations, therefore, had for their object to determine (i) if pectinase is produced by all species of Rhizopus and, if so, to what extent and (2) if its production is any indication of the parasitism of the species. TECHNIC The methods employed in carrying out macerating experiments with the different species of Rhizopus were for the most part the same as those used in previous work to which reference ^ has already been made, although some slight modifications were necessary to meet certain phases of the problem. Three sets of experiments were carried out with each organism. All the species were included in a single experiment and the macerating action was determined for all at the same time, so that the results for each species are directly comparable for a single experiment. The culture medium was so prepared and in sufficient quantity as to make 1 Harter, L. I,., and Weimer, J. L. studies in the physiology of parasitism with speciai, refer- ence TO THE SECRETION OF PECTINASE BY RHIZOPUS TRITICI SAITO. In Jour. Agr. Research, v. 21, no. 9, p. 609-625. 1921. Literature cited, p. 624-625. * Harter, L. I,-, Weimer, J. L-, and Laitritzen, J. I. the susceptibility of the different varie- ties OF SWEET potatoes TO DECAY BY RHIZOPUS NIGRICANS AND RHIZOPUS TRITICI. In Phytopathology, V. II. 1921. In press. Journal of Agricultural Research, Vol. XXII, No. 7 Washington, D. C. Nov. 12, 1921 aam Key No. G-354 (371) 1 372 Journal of Agricultural Research voi. xxii. no. 7 it uniform in all the flasks for all organisms. Four flasks (2 liters), containing 300 cc. of the culture medium, were inoculated with each one of the species in each experiment, and the cultures were incubated for three days. The macerating action was determined for the following species of Rhizopus: chmensis Saito, nodosus Namysl, tritici Saito, maydis Bru- derl, delemar (Boid) Wehmer and Hanzawa, arrhizus Fischer, oryzae Went and Pr. Geerligs, nigricans Ehmb., reflexus Bainier, artocarpi Racib., and microsporus v. Tieg. It has been shown ^ that the different species of Rhizopus do not all have the same optimum temperture for growth. Some species thrive at high temperatures, some at relatively low temperatures, and others at a temperature intermediate between the two extremes. Therefore, the 1 1 species studied have been separated into three groups with respect to their temperature relations. In all the experiments connected with the present investigations the same grouping of the different species has been observed, thus subjecting each organism to as nearly the optimum temperature for its growth as possible. The cultures of chinensis were incubated at 40° C, those of nodosus, tritici, maydis, delemar, arrhizus, and oryzae at 30°, and those of nigri- cans, reflexus, artocarpi, and microsporus at 20°. Although so far as temperature is concerned the results are not strictly comparable, pre- liminary experiments showed that more reliable data could be obtained by growing the different organisms at temperatures suited to their growth than by subjecting them all to a uniform temperature. Some of the species, as for example nigricans, which requires a relatively low tem- perature, make no growth or only a feeble growth at 30° and none at 35°. On the other hand, chinensis, a high temperature form, makes a reduced growth at 30° and a feeble growth at 20°. | At the close of the incubation period (three days) the mycelial growth was lifted from the culture flask and the substrate was filtered through a fine grade of muslin. The mycelium was treated subsequently by acetone and ether according to the method previously described.^ The solutions from the different flasks in which the same species had grown were made into a compound sample thoroughly shaken, and 25-cc. portions were used for maceration experiments. Likewise all the fun- gous felts of the same organism grown in the different flasks were brought together and treated as one sample, a weighed portion of the dried mycelium being used for maceration of the raw disks. Two types of controls were run with each set of experiments, as follows: (i) Sweet- potato decoction on which the fungus had grown for three days, which after the removal of the mycelium was steamed for 15 minutes to inac- tivate the enzym; (2) decoction which was identical with that used for 1 Harter, X,. h; Weimer, J. L., and Lauritzen, J. I. op. cit. 2 Harter, I^. 1,., and Weimer, J. 1,. op cix Nov. 12, 192 1 Pectinase Produced by Different Species of Rhizopus 373 inoculation purposes but which had not supported a fungous growth. Maceration by the enzym in the solution and in the mycelium was carried out at a temperature of 40° C. for all the species. Before the addition of the raw sweet-potato disks the solutions and suspensions of the my- celium were held for one hour at 40° in order to bring them to the tem- perature at which maceration was to take place. A/o^o,sa OF AGRICULTURAL RESEARCH CONTENTS Pago Hemotoxins from Parasitic Worms - - - - - -379 BENJAMIN SCHWARTZ ( Contribution from Bureau of Animal Industry ) Ash Content of t^ie Awn, Rachis, Palea, and Kernel of Barley during Growth and Maturation - - - - - - 433 HARRY V. HARLAN and MERRITT N. POPE One cc. of a 5 per cent suspension of defibrinated blood washed three times in physiological salt solu- tion was used in experiments. c The extract used in these experiments was made by suspending lo gm. of fresh worm material in loo cc. of 0.85 per cent NaCl. d Two controls — 0.5 cc. and i cc. of salt solution containing 0.5 per cent phenol were tested on i cc. of the suspension of corpuscles. « Pooled blood from six rats. / Four samples of corpuscles were tested. 5. EXPERIMENTS WITH ASCARIS LUMBRICOIDES POWDER The hemolytic principle of Ascaris lumbricoides may be preserved by drying the parasites. Specimens collected at a local abattoir were washed in salt solution to remove adhering interstinal debris, dried superficially with filter paper, and then placed in vacuum over sul- phuric acid. When the specimens were sufficiently crisp they were powdered in a mortar and stored for future use. Ascaris lumbricoides powder when added to a suspension of washed blood cells of cattle, sheep, swine, etc., produces rapid hemolysis. As ip the case of extracts of the parasite, the hemolytic action is inhibited by normal serum. The hemolytic substance may be more easily obtained from dried than from fresh ascarids by extracting the worm material in physiological salt solution. This is no doubt due to the fact that the dried material can be readily crushed and the hemolytic substance which, as has already been indicated, is rather closely bound to the parasite, may be more readily liberated. The following experiments performed by the writer illustrate this point: Several swine ascarids were broken tip into small fragments but were not powdered in a mortar. A portion of this ma- terial was extracted in salt solution for a few hours and filtered. The filtrate was tested on washed sheep corpuscles with negative results. The remaining portion of dried worm material was thoroughly ground Nov. 19, 192 1 Hemotoxins from Parasitic Worms 399 in a mortar, extracted in physiological salt solution, filtered, and the filtrate tested on sheep corpuscles. The results in this case were positive. A number of experiments were made with salt-solution extracts of powdered Ascaris lumbricoides. Rabbit and sheep corpuscles were used in nearly all experiments with these extracts. The results of these experiments were positive when the extracts were made from thoroughly powdered material; otherwise the extracts were only slightly hemolytic. Extracts of powdered material of Ascaris hmihricoides were usually prepared as follows: A definite quantity of powder was added to a definite volume of physiological salt solution in a flask, the latter was shaken thoroughly, and the material was extracted for a few hours to two days in a refrigerator without the addition of any preservative, or extracted in an incubator, in which case a few drops of chloroform were added. The mixtures were then filtered, and in cases in which chloro- form had been added the filtrate was left in an open receptacle in order to get rid of the chloroform by evaporation. The salt-solution filtrates were then tested as to their hemolytic power. An example of results of these experiments is given in Table II, in which a number of tests are summarized. Table II. — Effects of salt-solution extracts of powdered Ascaris lumbricoides on red blood corpuscles « Experi- ment No. Kind of erythrocytes.'' I 2 Rabbit ...do •3. ...do 4 ...do C ...do 6 ...do 7 8 Sheep ...do 0 ... do 10 ... do II ...do 12 ...do Quantity of extract. 5 drops 8 drops 8 drops (boiled) 10 drops (boiled) 10 drops (heated at 60° C. 30 minutes) xo drops salt solution 8 drops 10 drops 10 drops salt solution 8 drops 10 drops 10 drops salt solution Results after 2 hours at 37° C. + -H + + + + + + + + + + + + Results after 20 hours.d + + + + + + + + + 1— indicates negative results. + indicates slight hemolysis. ++ indicates marked but incomplete hemolysis. + + + indicates complete hemolysis. ^ Five drops of a s per cent suspension of washed rabbit erythrocytes and a 3 per cent suspension of washed sheep erythrocytes were used in these experiments. c In experiments i to 8, inclusive, the following extract was used; 0.85 gm. of powder were suspended in 85 cc. of salt solution and extracted in an incubator for 24 hours. In experiments 10 to 12 the extraction was made as follows: i gm. of powder was extracted in 10 cc. of salt solution in a refrigerator. <* After remaining in an incubator for 2 hours the tubes containing the corpuscles and extracts were in some instances transferred to a refrigerator (8° C.) where they were kept for 18 hours longer before the final reading was taken. 400 Journal of Agricultural Research voi. xxn. no. 8 6. EXPERIMENTS WITH EXTRACTS OF DIFFERENT ORGANS OF ASCARIS I^UMBRICOIDES It has already been stated that the body fluid of fresh specimens of Ascaris lumbricoides is not hemolytic and that this fluid acquires hemolytic properties as the parasites are kept in vitro. Extracts of entire worms, on the other hand, were found to contain a hemolytic substance which is apparently firmly bound to the tissues of the parasite. These facts appear to indicate that the hemolytic substance is liberated in rather small quantities and that it ultimately finds its way into the body fluid. That the liberation of hemolysin from the tissues and cells of the parasite is associated with metabolic processes of the worms is advanced as a plausible explanation of the facts. In the host animal the body fluid of the worm contains blood and blood products by which the hemolysin is apparently neutralized. In vitro, on the other hand, the blood elements disappear, as judged by the disappearance of oxyhemoglobin ; and meanwhile fresh hemolysin which has found its way into the fluid remains unbound. The question as to which morphological elements of Ascaris lumbri- coides secrete the hemolytic substance or substances is interesting. A number of specimens of the parasite were therefore dissected and the intestine, reproductive organs, and body wall were separated into different lots. Physiological salt-solution extracts from each lot were tested on hog blood, and in a few cases on sheep blood. In one series of experiments it was found that the extracts of the intestine were strongly hemolytic, whereas extracts of the body wall showed no hemolytic eftects. Extracts of the reproductive organs were moderately hemolytic. In a second series of experiments extracts of the intestine were found to be very markedly hemolytic, whereas extracts of the body wall and reproductive organs showed weak hemolytic power. In another series of experiments a number of worms were dissected, and the body wall, reproductive organs, and chyle intestine were sepa- rated into different lots. Each lot was washed in physiological salt solution and dried with filter paper. The material in each lot was then put in an incubator at 40° C. and allowed to remain there for 24 hours. Pulverized material from each lot was then suspended in physiological salt solution and tested on washed sheep corpuscles. Extract of the intestine produced rapid hemolysis at 37° (in about i hour), whereas extract of body wall of approximately the same strength as that of the intestine produced no hemolysis even after 3 hours at 37° followed by 18 hours in a refrigerator. Extract of the reproductive organs pro- duced no hemolysis after 3 hours at 37° but after an additional period of 18 hours at 8° a slight indication of hemolysis was observed. Nov. 19, 1921 Hemotoxins from Parasitic Worms 401 It may be concluded, therefore, that the hemolytic agent of Ascaris lumbricoides is primarily a secretory product of the intestine and that part of this substance finds its way into the body fluid where it is ap- parently neutralized by blood elements that are obtained from the host. 7. EXPERIMENTS WITH DIFFERENT CHEMICAL. FRACTIONS OF ASCARIS I^UMBRICOIDES In contrast to the comparatively slight solubility of the hemolytic substance of Ascaris lumbricoides in physiological salt solution is its ready solubility in lipoid solvents, especially in alcohol. Equal quanti- ties of powder were suspended in 5 cc. each of physiological salt solu- tion, 95 per cent alcohol, ether, and acetone for 48 hours. The filtrates were evaporated and redissolved in 5 cc. of physiological salt solution. These extracts were then tested on a 5 per cent suspension of washed rabbit red blood cells. The alcoholic extract was the most potent from the point of view of hemolysis. Acetone and ether extracts were about as potent as the physiological salt-solution extract. In a second series of experiments in which A. lumbricoides powder was extracted in the substances referred to above, the extracts were tested on sheep red blood cells. In those experiments the alcoholic extract was the most potent, while the physiological salt-solution extract and the ether extract were the least potent. Further experiments with different fractions of Ascaris lumbricoides were performed. Dried worm material was ground up in a mortar and extracted in four volumes of ether in a flask for 48 hours at 37° C. The ether was then removed from the worm material and saved and fresh ether was added to the flask. This was allowed to extract for 24 hours, the ether being removed at the end of that period and added to the first ether extract. To the worm material fresh ether was again added, and after 24 hours of extraction the mixture was filtered. The last ether filtrate was practically free from any extract, A portion of the ether extract was then evaporated and a brownish yellow fatty sub- stance left behind. This substance had the characteristic odor of A. lumbricoides. A small quantity of this substance was emulsified in physiological salt solution and tested on washed rabbit blood corpuscles, which it hemolyzed. A second portion of ether extract in solution was shaken with an equal quantity of distilled water and allowed to remain at room temperature for two hours. Two layers — ^namely, an ether layer (fraction i) and a water layer (fraction 2) — were separated. The ether layer (fraction i) was evaporated, and a fatty substance was left behind which was hemolytic to washed sheep corpuscles. A portion of this substance was redissolved in ether, and to this solution an equal quantity of a solution of sodium bicarbonate was added and the mix- ture was thoroughly shaken. The ether layer (fraction la) was removed 462 journal of Agricultural Research voi.xxn.TJo.ft and evaporated. A fatty substance free from the characteristic odor of A. lumbricoides was left after evaporating the ether. This substance had no hemolytic power. Inasmuch as sodium bicarbonate saponified the free fatty acids in the ether, it is evident that the hemolytic effect of the ether extract free from the water-soluble fraction is due to fatty acids. Flury {191 2) , in fact, came to the conclusion that the hemolytic power of ascarids is to be ascribed to free fatty acids of which the un- saturated fatty acids are of prime importance. Flury stated further- more that oleic acid is probably the most active principle of Ascaris hemolysin because of the known hemolytic powers of this substance. The watery layer (fraction 2) was opalescent and contained a thick suspension of a grayish substance which was found to be slightly hemo- lytic to sheep cells. The ether extract contains therefore two fractions, (i) a water-insoluble fraction which consists of neutral fats and fatty acids, and (2) a water- soluble fraction, both of which are hemolytic. The composition of the water-soluble substance was not definitely determined. This substance was tested and found to be soluble in 95 per cent alcohol and in hot and cold water. By acidifying a watery solution of the substance and shaking it with an equal volume of ether it was made to go into solution and was recovered in the ether layer. Another portion of the water-soluble sub- stance was salted out from water by adding a few drops of a strong solu- tion of sodium chlorid. It rose to the surface, where it formed a thick layer which was insoluble in salt solution. Bondouy {1908, 1910), who experimented with a similar chemical fraction of a species of Strongylus, identified it as consisting of soluble soaps, substances that are known to have hemolytic power. To recapitulate, an ether extract of Ascaris lumbricoides was divided into the following fractions : ( i ) An ether-soluble and water-insoluble frac- tion, and (2) a water-soluble fraction. Both fractions were hemolytic, the latter, however, only to a moderate degree. The fatty acid in the first fraction (fraction i) was saponified. The fatty acid-free fraction which was extracted in ether was not hemolytic. This fraction consists largely of neutral fats. The hemolytic potency of the ether extract of A. lumbricoides is therefore due largely to free fatty acid. That the water-soluble part of the ether fraction (fraction 2) is a mixture of soaps is probable. A portion of the remaining Ascaris lumbricoides powder (free from ether-soluble fraction) was extracted in distilled water for 48 hours in an incubator. The mixture was then filtered. The filtrate had a brownish color and a sweetish odor. Tests for proteins were positive. The residue was evaporated at 40° C. A portion of the residue was taken up in salt solution, to which it gave a yellowish coloration. Tested for its hemo- lytic power on sheep blood corpuscles, it produced rapid hemolysis. The remaining portion of the residue was extracted in 95 per cent alcohol for n Nov. 19, 1921 Hemotoxins from Parasitic Worms 403 24 hours. It was only partly soluble. After filtering off the alcohol, fresh alcohol was added and the extraction continued for 24 hours longer. The alcoholic extracts were evaporated and the residue was taken up with a small quantity of physiological salt solution. Tested for its hemolytic power, the results were strongly positive on sheep erythrocytes. The alcohol-insoluble fraction was not hemolytic even when large quantities were employed. These experiments are rather significant in view of the fact that they show quite conclusively that the hemolytic potency of Ascaris lum- hricoides extracts are due not to fatty acids alone but that another sub- stance or substances, soluble in alcohol and water, must be involved. The experiments described above were repeated several months later with similar results. Extracts of powdered ascarids in 95 per cent alcohol were made by adding about 6 volumes of alcohol to i volume of powder and removing the alcohol by filtration at intervals of two to three days and adding fresh alcohol. After evaporating the filtrates, which were all mixed together, a brownish residue was left behind which was only partly soluble in ether. The ether-soluble portion as well as the ether-insoluble portion was hemolytic. A portion of the powder, free from the alcohol-soluble frac- tion, was extracted in etlier, but when the latter was removed and evap- orated no residue was left behind. The remaining portion of the powder free from the alcohol-soluble portion was extracted in physiological salt solution, and this extract when tested on red blood cells was found to be nonhemolytic. These experiments show, therefore, that the hemolytic substances of Ascaris lumhricoides are all soluble in alcohol, and confirm the results of the earlier series of experiments with reference to the fact that the ether-soluble fraction of A . lumhricoides contains but a portion of the hemolytic substance. Part of the alcoholic extract was divided into two fractions — namely, an absolute alcohol-soluble fraction and an absolute alcohol-insoluble fraction. The latter was hemolytic, whereas the former showed no hemo- lytic power. An ether extract of Ascaris lumhricoides powder was redissolved in ether and divided into two fractions by adding acetone in excess, which resulted in the formation of a whitish precipitate. The precipitate was separated from the solution and found to be nonhemolytic. The acetone- ether solution was evaporated and taken up in salt solution. It was also found to be nonhemolytic, whereas prior to precipitation with acetone the ether extract was hemolytic. The precipitate was obtained in quan- tities insufficient to determine its nature. That it was probably largely lecithin ^ can hardly be doubted. As is known, lecithin in quantities in which it alone produces no hemolytic effect can activate other substances and cause them to produce hemolysis. That this actually occurs in the ' The presence of lecithin in ascarids was demonstrated by Flury Oiii^)- 404 Journal of Agricultural Research voi. xxii, no. 8 case of the ether-soluble hemolytic substance of A . lumhricoides appears probable from the experiments described above. It should also be stated that a 95 per cent alcohol extract of Ascaris lumhricoides developed a precipitate when kept in solution in 95 per cent alcohol at 8° C. This precipitate went into solution when the alcohol containing the extract was transferred to room temperature. The removal of this precipitate by filtering in a refrigerator yielded a whitish substance which had no hemolytic power, nor did the removal of this substance interfere with the hemolytic potency of the extract. 8. PROPERTIES OF* ASCARIS lyUMBRICOlDES HEMOI^YSIN At low temperatures ranging from 6° to 10° C. hemolytic extracts of Ascaris lumhricoides lose their potency. Mixtures of extracts and sus- ceptible corpuscles that showed complete hemolysis after 2 hours' incu- bation at 37° showed no trace of hemolysis after 24 hours at 8°. After being removed from the low temperatures and transferred to an incubator hemolysis occurred rapidly in such mixtures. In order to determine whether the hemolytic substance of Ascaris lumhricoides is absorbed by the red blood cells at low temperatures the following experiments were performed. Mixtures of washed red blood cells (rabbit and sheep) and hemolytic extracts were put in a refrigerator at 8° C. After 24 hours the super- natant fluid was removed from the corpuscles and the latter were washed three times in succession to free them from traces of extracts; to the washed corpuscles from which the supernatant fluid had been removed an equal quantity of salt solution was added, and the tubes were thor- oughly shaken and placed in the incubator. Hemolysis set in slowly. The supernatant fluid which was removed from the corpuscles was also tested as to its hemolytic potency, with inconstant results. In some cases it was found that it had lost its hemolytic potency completely, but in a number of cases it still retained its blood-destroying power. That the potency of the fluid that had been in contact with susceptible cor- puscles had been considerably reduced was evident, since it had but slight hemolytic power as compared with that of intact extract. Whether the hemolytic substance in contact with susceptible corpuscles at a low temperature becomes fixed to the cells or whether it is precipitated at a low temperature and escapes removal despite repeated washing has not been determined. Hemolytic extracts of Ascaris lumhricoides are highly resistant to heat. Heating at temperatures ranging from 56° to 60° C. for 30 minutes did not weaken the potency of the extracts. An exposure to 70° for two hours failed to destroy the hemolytic substance. Salt-solution extract as well as alcoholic extracts were heated to boiling, and after cooling they were tested on susceptible red blood cells. It was found that as a Nov. 19, 1921 Hemotoxins from Parasitic Worms 405 result of boiling the potency of the extracts was weakened but not destroyed. The hemolysin goes through the pores of Berkefeld, Chamberland, and diatomaceous filters. The filtrates are less potent, however, than nonfiltered solutions. V. EXPERIMENTS WITH AGGLUTINATING SUBSTANCES FROM ASCARIS LUMBRICOIDES In the course of experiments on hemolysis of red blood cells by ex- tracts of Ascaris luvibricoides it was observed that the cells frequently became agglutinated before hemolysis set in. The agglutinating effect of the extracts was especially marked on rabbit red blood cells and was observed only occasionally on sheep erythrocytes. Several experiments on hog erythrocytes showed them to be refractory to the agglutinating substance of the parasite. The agglutinating property of Ascaris lumhricoides with respect to rabbit-blood corpuscles was present almost invariably in physiological salt- solution extracts. Alcohol and ether extracts of entire worms were not entirely free from agglutinating properties, however. Unlike the hemo- lytic substances which are entirely removed from the worm material by alcohol and ether extraction, the agglutinating substance resists ex- traction in these solvents and may be recovered in the fraction of the worm material from which the alcohol-soluble and ether-soluble frac- tions have been removed. The salt-solution-soluble hemagglutinin does not appear as firmly bound to the cells of the parasites as the lipoidal hemolysin. The latter, as has already been stated elsewhere in this paper, is but slightly soluble in physiological salt solution unless the material is thoroughly triturated. Salt-solution extracts of coarsely powdered worm material that yield but a small quantity of hemolysin were found to contain a considerable quantity of agglutinating substance. In physiological salt-solution extracts of Ascaris lumhricoides that con- tain the hemolysin and the hemagglutinin the potency of the former may be suppressed by low temperatures {6° to 10° C), whereas that of the latter remains unaffected by those temperatures. The hemagglutinin from Ascaris lumhricoides is relatively thermo- stabile and differs in this respect from the hemagglutinin which Tallqvist (1907) isolated from Diphyllohothrium latum. The latter is injured by 30 minutes' heating at 55° C, whereas that of A. lumhricoides with- stands heating at temperatures ranging from 56° to 60° for 30 minutes. Hemagglutinating extract of A . lumhricoides was passed through a Cham- berland filter without injuring its potency. Summarizing, it may be stated that in contrast to the lipoidal hemoly- sin, which is inactive at 6° to 10° C. and which is but slightly soluble in physiological salt solution, the agglutinin of Ascaris lumhricoides is readily soluble in salt solution, slightly soluble in ether and alcohol, and 4o6 Journal of Agricultural Research voi.xxn.No. s active at low temperatures. It also differs from the hemolysin in its rela- tive specificity for certain species of erythrocytes. VI. THE EFFECT OF ASCARIS LUMBRICOIDES FLUID ON COAGULA- TION OF BLOOD As has already been stated, Weil and Boye {19 lo) found that as a re- sult of injecting the fluid of Ascaris equorum into rabbits the coagulation of the blood was retarded by 20 minutes. These investigators state, however, that they obtained negative results with rabbit blood in vitro. Leroy (19 10) likewise observed that the blood of dogs which had received injections of the body fluid of A. equorum exhibited a delayed coagula- tion time. Flury (1912) made observations on the coagulation of dog blood in contact with the fluid of ascarids in vitro and records a decided delay. His experiments with human blood were likewise positive. In view of the contention of Weil and Boye with reference to rabbit blood in contact with Ascaris equorum fluid in vitro, the writer tested freshly drawn rabbit blood to which various quantities of A . lumbricoides fluid were added, in order to determine if the coagulation power would be affected. The addition of 3 to 5 drops of the fluid to lo drops of blood delayed the coagulation time about 15 minutes as compared with that of normal blood. The addition of 8 drops of fluid to 10 drops of blood produced a 35-minute delay, whereas the addition of 10 drops of fluid to an equal quantity of blood resulted in a delay of 42 minutes. The body fluid of Ascaris lumbricoides retards the coagulation of blood in vitro as well as in vivo, but its power in this respect is rather limited. VII. EXPERIMENTS WITH HOOKWORM HEMOLYSIN (ANCYLOSTOMA CANINUM) The anemia which occurs in cases of infestation with hookworms has been ascribed to several different factors. The direct abstraction of blood by the parasites, the possible absorption of toxic substances from the digestive tract as a result of the ulceration of the mucosa, hemorrhages following the laceration of the mucosa by the worms (Loeb and his collab- orators), and the absorption by the host of hemolysins secreted by the parasites have been advanced as explanations. The last view was ac- cepted as a plausible explanation before any experimental evidence in favor of it had been advanced. That the data with reference to the pro- duction of hemolysins by hookworms appear to show that such absorp- tion probably occurs has already been pointed out elsewhere in this paper. Nov. 19, 19" Hematoxins from Parasitic Worms 407 I. EFFECTS OF SAI^T-SOI,UTlON EXTRACT OF FRESH WORMS ON RED BI,OOD CORPUSCI^ES In the following experiments the hemolysin was obtained from about 100 specimens of Ancylostoma caninutn collected from three dogs. The parasites were put into a bottle containing a physiological salt solution and kept in an ice box for about 24 hours after removal from the hosts, without any apparent loss of vitality. The extract designated as extract of fresh worms was prepared as follows: The parasites were ground up in a mortar containing a small quantity of a physiological salt solution, and the macerated material was then suspended in about 20 cc. of salt solution, shaken vigorously for a few minutes, and placed in a refrigerator overnight. The super- natant fluid was found to be hemolytic, as the following experiments will show. Experiment i. — ^To each of three tubes containing 0.5 cc. of a 2 per cent suspension of washed dog erythrocytes there were added, respec- tively, 5, 8, and 10 drops of the extract of fresh worms. As a control, to a fourth tube containing the same quantity of suspension of cor- puscles there were added 10 drops of a salt solution. The tubes were shaken thoroughly and placed in the incubator at a temperature of 37° C. At the end of 30 minutes the tube containing 10 drops of the extract showed complete hemolysis. The tube containing 8 drops of extract showed complete hemolysis 15 minutes later, while the tube containing 5 drops of extract showed partial hemolysis at the end of an hour. The control tube showed no hemolysis. The tubes were kept in a refrigerator overnight and no further change was noted. Experiment 2. — ^To three tubes each containing 10 drops of a 5 per cent suspension of washed sheep corpuscles there were added, respec- tively, 5, 8, and 10 drops of the extract of fresh worms. It was neces- sary to incubate the tubes at 37° C. for two hours before hemolysis was produced in any tube. The tube containing 10 drops of extract showed complete hemolysis; the tube containing 8 drops of extract showed partial hemolysis, while the tube containing 5 drops of extract showed no hemolysis. A fourth tube containing 10 drops of corpuscle suspen- sion and 10 drops of salt solution showed no hemolysis. These tubes were kept in a refrigerator overnight with practically no change in results except that hemolysis was complete in the tube containing 8 drops of extract and was faintly indicated in the tube containing 5 drops of extract. Experiment 3. — ^The extract of fresh worms was tested against washed rabbit corpuscles as in experiments i and 2. Ten drops of a 3 per cent suspension of washed corpuscles were completely hemolyzed by 5 drops of extract in 20 minutes at a temperature of 37° C. This experiment was controlled as usual. 4o8 Journal of Agricultural Research voi. xxn. no. s ExPBRiMEjNT 4. — ^Twelve drops of extract of fresh worms were heated for 30 minutes at a temperature ranging from 56° to 58° C. The addi- tion of 0.5 CO. of washed dog corpuscles from the same lot as that used in experiment i resulted in partial hemolysis after one hour of incuba- tion at 37°. The tube was kept in a refrigerator overnight and showed almost complete hemolysis the next day. Experiment 5. — ^A quantity of extract of fresh worms was heated at 60° to 65° C. for 50 minutes. To two tubes each containing 10 drops of extract that had been thus heated there were added 5 drops of rabbit and sheep corpuscles, respectively, of the same concentration as noted in experiments 2 and 3. No hemolysis was produced after two hours' incubation at 37°. The tubes were kept in a refrigerator overnight and showed slight hemolysis the following day. Experiment 6. — ^Twelve drops of extract of fresh worms were heated to boiling, and after cooling they were added to 0.5 cc. of suspension of dog corpuscles of the same concentration as in experiment i and were incubated for one hour, but no hemolysis was produced. After remaining in an ice box overnight the tube showed but a trace of hemo- lysis. Similar results were obtained when rabbit and sheep corpuscles were used. Control tubes showed no hemolysis. A second series of experiments with extracts of fresh worms was per- formed several weeks later. The details of these experiments follow. The extract referred to as extract II of fresh worms was prepared by macerating 29 live specimens of Ancylostoma caninum ^ obtained from five dogs shortly after the animals had been killed. The macerated material was suspended in 3 cc. of physiological salt solution, shaken vigorously, and allowed to extract at room temperature for about an hour before it was tested for its hemolytic power. Part of the extract was kept overnight in a refrigerator and was used the following day. The suspension of corpuscles and extract was incubated at 37° C. for periods shown in the table, the results were noted, and the tubes were then placed in a refrigerator for an additional period of 18 hours, when the final results were read. The data presented in Table III show that rabbit and dog corpuscles are more susceptible to hookworm hemolysin than the corpuscles of swine and cattle. Despite the fact that the latter were not hemolyzed by the extract used in these tests, they are not absolutely resistant to extracts of dog hookworms, as will be shown in another section of this paper. ' These specimens were washed several times in physiological salt solution. Nov. 19, 1921 Hemotoxins from Parasitic Worms 409 Table III gives a record of the experiments performed with this extract. Table III. — Effect of extract II of fresh worms (Ancylostoma caninum) on washed red blood corpuscles « Kind of corpuscles. & Rabbit Do Do Dog... Do Do Hog..., Do Do Cattle. . Do Do Quantity of extract. 5 drops. . 8 drops. . Control c . 5 drops. . 8 drops. . Control c. 5 drops. . . 8 drops. . , Control c. 5 drops. ., 8 drops. . , Control*:. Period of incubation. 1 hour. . . . ....do... ....do... 2 X hours. ....do... ....do... ....do... ....do... ....do... ....do... ....do... ....do... Results at end of in- cubation period. + + + + + + + + Results after 18 hours addi- tional in re- frigerator (S^C). + + + + + + + + + + + o ++ indicates marked though incomplete hetaolysis. -H-+ indicates complete hemolysis. — indi- cates absence of hemolysis. ''0.2 cc. of a 5 per cent suspension of washed blood corpuscles were used in all experiments summarized in this table. " Eight drops of physiological salt solution were added to the washed blood corpuscles in order to control the experiment. The sediment in the tube containing the extract of hookworms when shaken with 3 cc. of physiological salt solution yielded additional hemoly- sis, as the following experiments will show. Experiment 7.— After the supernatant fluid from the extract (extract II of fresh worms) had been removed the sediment was shaken up with about 3 cc. of physiological salt solution, v/hich was tested against a 5 per cent suspension of washed dog corpuscles from the same lot as that referred to in Table I. Three drops of corpuscles were completely hemolyzed by three drops of the extract after one hour's incubation at 37° C. This experiment was controlled as usual. Experiment 8. — Five drops of the same extract were boiled for about one minute. After cooling, three drops of dog erythrocytes from the same lot as that used in experiment 7 were added and the mixture incubated for 1% hours at 37° C. No hemolysis was produced. The tube was kept 18 hours in the refrigerator without any change. EXPERIMENTS WITH EXTRACTS OE DRIED WORMS The experiments recorded below were performed with the following extract : Fifty-eight mgm. of coarsely powdered worm material {Ancylostoma caninum) dried at 37° C. and kept in a small vial for about two years were extracted in 10 cc. of physiological salt solution for several hours. Unlike 4IO Journal of Agricultural Research voi. xxii. no. 8 the extract of fresh worms, which is opalescent, the extract of powdered material remained quite clear. Experiment 9. — To four tubes labeled from i to 4, each containing 5 drops of a 5 per cent suspension of washed rabbit corpuscles, there were added, respectively, 3, 5, 8, and 10 drops of the extract. To a fifth tube containing an equal quantity of corpuscles there were added 10 drops of physiological salt solution in order to control the results of the experi- ment. The tubes were incubated for i hour at 37° C, and kept for 18 hours longer in a refrigerator, after which the final results were read. Tube I showed no hemolysis, while tubes 2, 3, and 4 showed complete hemolysis. The control tube showed no hemolysis. Additional experiments with the same extract and the same corpuscles showed that the hemolytic action was very slow, since 10 drops of the extract in contact with 5 drops of the suspension of corpuscles failed to produce hemolysis after 2 hours' incubation at 37° C, but after an additional period of 18 hours in a refrigerator the tube showed complete hemolysis, whereas the control tube showed no trace of hemolysis. Experiment 10. — The extract of dried worms was tested on a 5 per cent suspension of washed corpuscles of cattle and swine as follows: To four tubes each containing 0.2 cc. of corpuscles there were added, respec- tively, I, 2, 3, and 5 drops of the extract, and the tubes were incubated for I hour. None of the tubes showed hemolysis. After remaining in a refrigerator overnight the following results were noted. Cattle corpuscles : The tubes containing i and 2 drops of the extract showed partial hemolysis, whereas the tubes containing 3 and 5 drops of extract showed complete hemolysis. Hog corpuscles: No hemolysis was observed in any tube. The foregoing experiments were controlled as usual. In the experiments described above the extract was not filtered but was added to the suspension of corpuscles together with some particles of worm material. In a second series of experiments performed several weeks later it was found that washed rabbit blood corpuscles were unaffected when placed in contact with an extract of dried hookworms, incubated for 3 hours, and then kept in a refrigerator for an additional period of 18 hours. While no record was made as regards the introduction of particles of worm material into the tubes containing the suspension of corpuscles, it is probable that the clear supernatant fluid alone was added. A repetition of the experiment on a later date yielded the following results. Experiment m — ^A small quantity of coarsely powdered worm mate- rial was extracted in physiological salt solution, filtered, and the filtrate tested on washed rabbit blood corpuscles. No hemolysis was produced. To the material which had thus been extracted a small quantity of physio- logical salt solution was added, the contents were thoroughly agitated, Nov. 19, 1921 Hemotoxins from Parasitic Worms 411 and a few drops containing worm particles were added to 0.5 cc. of a 5 per cent suspension of washed rabbit erythrocytes. After one hour's incubation hemolysis was complete. A tube containing corpuscle sus- pension alone showed no hemolysis. A repetition of this experiment yielded similar results. From the foregoing experiments it appears that the hookworm hemoly- sin is firmly bound to the cells of the parasite. In fresh worms a con- siderable quantity of free hemolysin is probably present in tlie tissues and fluids of the body, which is absorbed by the salt solution in the course of extraction. Since the sediment of extracts of fresh worms has been found to yield additional hemolysin after the first extraction, it is evident that salt solution does not absorb all the hemolysin present in the worms. The observation of Preti (1908) that tryptic digestion liberates the hemolysin is further evidence of a close union between the hemolysin and the cells of the worm. 3. EXPERIMENTS WITH EXTRACTS OF ALCOHOLIC SPECIMENS The experiments described below were performed with extracts obtained from specimens of Ancylostoma caninum which had been pre- served in alcohol for about three years. Unless otherwise stated the extracts were prepared as follows: The specimens were washed several times in distilled water, dried at room temperature, and powdered in a mortar; o. i gm. of the powder was suspended in 10 cc. of an 0.85 per cent solution of sodium chlorid and extracted in a refrigerator for about 24 hours. The supernatant fluid was then tested on the washed erythro- cytes of rabbit and sheep as follows. Experiment 12. — Five drops of a 5 per cent suspension of rabbit corpuscles plus 3 drops of extract showed complete hemolysis at a tem- perature of 37° C. in 2 hours. Equal parts of extract and corpuscle suspension showed complete hemolysis in 1^2 hours. This experiment was controlled as usual. Experiment 13. — Five drops of a 5 per cent suspension of washed sheep corpuscles were mixed with 10 drops of extract and incubated for 2 hours without producing any hemolysis. A similar experiment was performed a few months later with negative results, despite the fact that after incubating the mixtures of corpuscles and extract for 2 hours they were kept in a refrigerator for 18 hours longer. Experiment 14. — Five drops of a 5 per cent suspension of rabbit corpuscles were not hemolyzed by 5 drops of extract. Experiment 15. — A 5 per cent suspension of washed guinea-pig corpuscles resisted hemolysis after remaining in contact for 3 hours at a temperature of 37° C. with an extract of alcoholic specimens made by extracting 200 dried specimens in 6 cc. of physiological gait solution and mixing 3 drops of extract with 2 drops of the suspension of corpuscles. Fifteen drops of the extract in contact with 3 drops of the blood suspen- 70495°— 21 3 j^i2 Journal of Agricultural Research voi. xxii. no. s sion for 2 hours at 37° C. followed by 48 hours in a refrigerator resulted in partial hemolysis. Several controls in which the suspension of corpus- cles alone and equal quantities of the suspension of corpuscles and extract were employed showed complete absence of hemolysis. These experiments indicate that alcoholic specimens are much less potent in their hemolytic action than fresh specimens. This is doubt- less due to the loss of hemolytic substance to the alcohol. In confir- mation of this view the writer found that dried hookworms from the dog freed from their ether-soluble and alcohol-soluble fractions were not hemolytic to washed erythrocytes of rabbits. The ether-soluble fraction left rabbit corpuscles intact. The alcoholic extract was un- fortunately lost before it was tested for its hemolytic potency. 4. EFFECT OF NORMAL SERUM ON HOOKWORM HEMOLYSIN Experiment 16. — To each of four tubes containing 0.5 cc. of blood corpuscles from the same lot as that used in experiment i there were added 5 drops of fresh hookworm hemolysin described elsewhere in this paper, and i, 2, 3, and 5 drops of dog serum, respectively. The tubes were incubated for i hour at 37° C. No hemolysis was observed in any of the tubes. After the tubes had remained in an ice box overnight it was found that with the exception of the tube to which but i drop of serum was added and which showed a faint trace of hemolysis, inhibition of hemolysis was complete. Experiment 17. — Five drops of a 5 per cent suspension of washed rabbit corpuscles from a lot which was susceptible to extract of alcoholic specimens were only partially hemolyzed when 3 drops of normal rabbit serum were added. It was also found that as a result of heating the serum for 30 minutes at a temperature of 56° C. the antihemolytic prop- erty was neither destroyed nor impaired. Experiment 18. — ^Washed rabbit corpuscles, which were completely hemolyzed when equal parts of a 5 per cent suspension of cells and equal parts of fresh salt-solution extract were mixed and incubated for 20 minutes at37° C, were found to resist a double quantity of the hemolysm in the presence of various inactivated sera, as follows : In each of three tubes there were placed 5 drops of the suspension of corpuscles, 10 drops of the extract, and 2 drops of heated rabbit, horse, or dog serum (60° to 65° for 30 minutes). The mixtures were incubated for 2 hours without any resultant injury to the blood corpuscles. After having been kept in a refrigerator for 18 hours after incubation, the tubes con- taining dog and rabbit serum showed faint traces of hemolysis, while the tube containing horse serum showed no hemolysis. Experiment 19. — To each of two tubes containing 3 drops of unwashed rabbit blood there were added 7 drops of physiolo^cal salt solution. These mixtures were incubated for 2 hours with 5 and 10 drops of fresh ^tract, respectively, at 37" C. No hemolysis was produced. The Nov. 19, 193 1 Hemotoxins from Parasitic Worms 413 tubes were kept 18 hours longer in a refrigerator, with a resultant faint indication of hemolysis. Washed erythrocytes from the same rabbit were highly susceptible to the extract, since 10 drops of a 3 per cent sus- pension of corpuscles were completely hemolyzed by 5 drops of extract in about 20 minutes. Experiment 20. — ^To a series of tubes each containing 3 drops of a 5 per cent suspension of washed dog erythrocytes used in an earlier ex- periment and included in Table I there were added 5 drops of extract II of fresh worms and various blood sera diluted with an equal quan- tity of physiological salt solution and heated at 59° C. for 30 minutes. The data and results of these experiments, including the controls, are given in Table IV. Table IV.— Effects of various sera on hookworm hemolysinO' Tube N0.6 Kind and quantity of diluted sera. 3 drops (horse serum) 3 drops (dog serum) . . 3 drops (rabbit serum No serum Results after 3 hours' in- cubation at 37° C. + + + Results after 18 hours longer in re- frigerator. + + + + (I + -I- -f indicates complete hemolysis, -f indicates slight hemolysis. — indicates absence of hemolysis. b Three drops of a 2 per cent suspension of washed dog corpuscles and 5 drops of extract II of fresh worms were used in this series of experiments. 5. EFFECT OF COLD ON HOOKWORM HEMOLYSIN Experiment 21. — Dog corpuscles which were found to be highly susceptible to an extract of fresh worms at 37° C. remained intact after being kept for 5 hours on ice in contact with a quantity of extract suffi- cient to destroy the corpuscles at 37° in 30 minutes. The removal of the supernatant fluid following rapid centrifugation showed that it had completely lost its hemolytic potency, since it failed to hemolyze sus- ceptible dog corpuscles after remaining in contact with them for 2 hours at a temperature of 37° followed by 1 8 hours at a temperature of about 10° Experiment 22. — The foregoing experiment was repeated, substitut- ing susceptible rabbit corpuscles for dog corpuscles, with similar results. The loss of the hemolytic property of the extract in contact with sus- ceptible corpuscles at a low temperature can not be attributed to a possible injurious effect of cold, since it was found that the hemolytic potency of the extract was not injured after standing directly on the ice for 18 hours. Washed sheep corpuscles were readily hemolyzed by the refrigerated extract, whereas a control tube containing corpuscles alone showed no hemolysis. Experiment 23. — Six drops of dog blood corpuscles from the same lot as that described in experiment 7 were mixed with 10 drops of extract II 414 Journal of Agricultural Research voi. xxn. no. s of fresh worms and placed on ice for 3^^ hours. The mixture was centrifuged and the supernatant fluid was removed and to it there were added 2 drops of washed dog corpuscles from the same lot as used in the first part of the experiment. After 1 hour's incubation followed by 18 hours in a refrigerator the corpuscles remained intact. The corpuscles from which the supernatant fluid was originally removed were washed three times in salt solution and then incubated with a small quantity of salt solution for i hour. Complete hemolysis was produced. A control tube containing a similar quantity of corpuscles without any hemolytic extract showed no hemolysis when placed in an incubator. While this experiment appears to indicate that the hemolysin was fixed to corpuscles and was not removed by repeated washing, this conclusion must be accepted with caution, because the possibility remains that some frag- ments of worms which were introduced into the tube together with the hemolysin may have been responsible for the hemolysis of the corpuscles after the removal of the supernatant fluid. The fact that the latter had lost its hemolytic power affords, however, strong presumptive evidence of an absorption of the hemolysin by the blood corpuscles. 6. DISCUSSION The results of experiments with reference to the presence of a soluble hemolysin in hookworms (Necator and Ancylostoma) show quite con- clusively that when living specimens are macerated in physiological salt solution they yield a considerable quantity of hemolysin. The latter is characterized by relative thermolability, nonspecificity, and suscepti- bility to normal serum, in the presence of which it loses its potency. So far as its physiological properties are concerned, hookworm hemolysin resembles strep tocol5^sin, staphylolysin, tetanolysin, and other hemoly- sins of bacterial origin. It differs from the hemolytic substances of Diphyllohothrium latum in that it is destroyed by boiling. The con- clusion of Preti {igo8) that hookworm hemolysin is resistant to boiling is not sustained by Whipple {1909) and is also contradicted by the results of the present writer's experiments. Unfortunately, Preti has not published a full account of his experiments. His general conclusions are unsupported by details, and judging from the statements that he makes it does not appear that he controlled his experiments. The present writer's experiments indicate that the hookworm hemolysin is rather firmly bound to the tissues of the parasites, which probably accounts for the difficulty of obtaining strong hemolytic filtrates from salt solution extracts of powdered specimens. That the living worm secretes the hemolysin is evident, however, from experiments with ex- tracts of fresh worms. The unbound hemolysin from fresh specimens evidently disappears in the course of drying. This comparative insolu- bility of the hemolytic substance from dried specimens in physiological salt solution is perhaps the basis of the contention of Preti {1908) and of Nov. 19. 19" Hemotoxins from Parasitic Worms 415 Usami and Mano {191 8) concerning the insolubility in water of the hook- worm hemolysin. That Loeb and his collaborators used dried material has already been stated. The fact that normal blood serum has antilytic properties and inhibits the action of the hookworm hemolysin accounts for the negative results obtained by Loeb and Smith {1904) and for the weakly positive results obtained by Whipple {1909). In this connection it is important to recall the observations of Noc {1908) with reference to the presence of antihemolysins in the blood serum of normal persons and of those recover- ing from hookworm disease and from beriberi, and the absence of anti- hemolysins in patients suffering from these diseases. Noc's observa- tions are decidedly significant and do not bear out Whipple's view that the hookworm hemolysin probably bears no relation to the secondary anemia of ancylostomiasis. De Blasi's observations with reference to the presence of hemolysins in the blood serum of patients infected with hookworms and Noc's discovery that under certain conditions the anti- lytic action of the blood serum may become impaired appear to indicate that the hookworm hemolysin has potentialities of causing anemia and that in severe infections it probably plays an important role in the disease. Since cold (6° to 8° C.) inhibits the action of the hookworm hemolysin in vitro, and the supernatant fluid from tubes in which susceptible blood corpuscles and potent hookworm extract have been in contact for a num- ber of hours at a low temperature no longer has hemolytic properties, the view that the hemolysin is a complex organic substance, not unlike a toxin, in that it apparently consists of haptophore and toxophore groups, appears to be justified. By means of the haptophore group union between the hemolysins and blood corpuscles takes place, but the dis- solving or lytic action is produced by the toxophore group. Inasmuch as low temperatures do not appear to interfere with the absorption of the hemolysin by the corpuscles despite the fact that the latter remain undissolved, it is permissible to believe that the toxophore and hapto- phore groups of the hookworm hemolysin act independently of each other. This view is purely speculative, however, and further experi- mentation is required before it may be accepted without reservation. VIII. EXPERIMENTS WITH EXTRACTS OF CA.TTLE HOOKWORMS (BUSTOMUM PHLEBOTOMUM) Hookworms belonging to the genus Bustomum occur as parasites in the small intestine of ruminants. Bustomum phlebotomum is the species that infests cattle. According to observations of several investigators, cattle infested with hookworms show symptoms not unlike those of human beings that harbor species of Ancylostoma or Necator. Experiments with extracts of Bustomum phlebotomum similar to those performed with extracts of Ancylostoma caninum showed that the former, like the latter, contain a powerful hemolytic agent. The extracts 41 6 Journal of Agricultural Research voi. xxn. no. s referred to below were prepared as follows : Living worms were removed from the intestine of a calf, washed a number of times in physiological salt solution, and kept in a refrigerator at a temperature of 8° C. over- night. The following day the worms which were still alive were trans- ferred to fresh salt solution and crushed in a mortar. The crushed material was then suspended in about two volumes of physiological salt solution, shaken thoroughly, and centrifuged. The supernatant fluid which was opalescent was removed and tested as to its hemolytic power. Tested on a 3 per cent suspension of washed sheep blood corpuscles, it was found that 5 drops of the extract hemolyzed 5 drops of the suspen- sion of blood corpuscles in i hour at a temperature of 37°. Even i drop of extract hemolyzed 5 drops of the suspension of corpuscles after a few hours. Controls, that is, 5 drops of suspension of corpuscles plus 5 drops of salt solution, remained intact. It was observed that before hemolysis set in the contents of the tubes assumed a dark red hue. An extract from another lot of Bustomum phlehoiomum prepared as has already been described was tested on a 5 per cent suspension of washed rabbit cells. Five drops of extract produced hemolysis rather slowly upon 5 drops of suspension of corpuscles. In a third experiment an extract prepared from worms that had been kept in a refrigerator overnight was tested on four different tubes of cat- tle erythrocytes and on four different tubes of hog erythrocytes. The extract in question was prepared from living specimens as follows: Forty-five specimens were ground up in a mortar and suspended in 2 cc. of physiological salt solution. The suspension was centrifuged, and the opalescent fluid was removed and tested on a 5 per cent suspension of washed blood corpuscles at 37° C. Three drops of extract were added to 5 drops of corpuscle suspension. The experiments with each sample of corpuscle suspension were controlled by adding 3 drops of physiological salt solution to 5 drops of suspension of blood cells. The results of these experiments follow. Cattle blood corpuscles. — ^After i hour one tube of blood was par- tially hemolyzed and three tubes were intact. After 2% hours two tubes were completely hemolyzed and two were intact. After 3 hours three tubes were hemolyzed ; one was intact. The tubes containing the mixtures were placed in a refrigerator at 8° C. until the next day. When examined hemolysis was complete in all tubes. The controls showed no hemolysis. Hog blood corpuscles. — ^After i hour all tubes were intact. After 1^4 hours two tubes were partially hemolyzed and two intact. After 2}^ hours two tubes were partially hemolyzed and two completely hemolyzed. After 3 hours all tubes showed complete hemolysis. Controls were intact. Inasmuch as it was found that the hemolysin could be preserved by drying the worms, powdering the dried material, and storing it in a dark place, further experiments with Bustomum phlebotomum hemolysin were performed with dried material. The details of these experiments follow. Nov. 19. igai Hemoioxins from Parasitic Worms 417 To each of four tubes of defibrinated blood (3 drops of physiological salt solution plus 2 drops of blood) a small quantity of the powder was added, and the tubes were shaken thoroughly and placed in an incubator at 37° C. After 2 hours hemolysis was produced in all tubes. Two lots of cattle blood from different animals were collected in a 2 per cent solution of sodium citrate (about 2 volumes of blood to i volume of a 2 per cent sodium citrate). Tested against dry Bustomum phlebotomum powder the unwashed citrated blood became hemolyzed in about 2 hours at 37°. Small quantities of powder were also tested on each of fotur lots of washed cattle blood corpuscles with positive results. Hemolysis set in rapidly and was complete after i hour at 37° C. A few drops of a 3 per cent suspension of washed sheep corpuscles were hemolyzed by a small quantity of Bustomum phlebotomum powder. Similar results were obtained with washed rabbit erythrocytes. Bustomum phlebotomum powder extracted in physiological salt solution yields but a small quantity of hemolysin, as the following experiments will show. Eighty-five mgm. of powder were suspended in 5 cc. of physiological salt solution. A few drops of chloroform were added as a preservative. The mixture was kept at a temperature of 35° to 37° C. for 2 days and then filtered. The clear filtrate was tested on a 5 per cent suspension of washed rabbit cells. Equal parts of filtrate and suspension of cells yielded negative results. It was necessary to add 10 drops of filtrate to 3 drops of corpuscle suspension to produce hemolysis. Evidently the hemolysin is firmly bound to the parasite material and is but slightly soluble in salt solution. In fact, the powder which had been extracted was dried and retested on rabbit blood cells, which it hemolyzed rapidly. An alcoholic extract of fresh specimens of Bustomum phlebotomum was found to be decidedly hemolytic. The extract was prepared as follows : About 100 specimens were washed a number of times in physiological salt solution after they had been removed from the host. The specimens were then triturated in a mortar and extracted in about 2 volumes of 95 per cent alcohol for about a week at 37° C. The alcohol was sepa- rated from the worm material by filtration. The filtrate was evaporated and the residue was shaken with a small quantity of physiological salt solution, in which it dissolved, producing an opalescent solution. Tested on sheep red blood corpuscles this solution produced hemolysis. A quantity of the solution which hemolyzed 5 drops of a 5 per cent suspen- sion of washed sheep corpuscles in about 2 hours at 37° failed to produce hemolysis on an equal quantity of blood corpuscles in 20 hours in a refrigerator (8°), thus showing that low temperatures paralyze the action of the hemolysin. Likewise, normal horse serum (2 drops) inhib- ited hemolysis of 5 drops of washed sheep corpuscles to which sufficient hemolytic solution had been added to cause hemolysis in the absence of 41 8 Journal of Agricultural Research voi. xxn, no. 8 normal serum. The worm material from which the alcohol-soluble sub- stance had been removed was dried and pulverized. A portion of this powder was added to washed sheep corpuscles but failed to produce any hemolytic effect, showing that the hemolytic substances of Bustomum phlehotomum are completely soluble in alcohol. In a few experiments the effect of normal serum was tested with a view of determining when it contained bodies capable of inhibiting the action of Bustomum phlehotomum hemolysin. Washed rabbit corpuscles, belonging to a lot that were rapidly hemolyzed by a small quantity of the powder, resisted hemolysis in the presence of a few drops of normal rabbit serum. The effect of heat on the hemolysin was found to be the same as the effect of heat on Ancylostoma canimim hemolysin. A salt-solution extract of fresh worms was completely inactivated by heating it for 40 minutes at 60° C. IX. EXPERIMENTS ON THE POSSIBLE PRESENCE OF ANTICOAGUUNS IN HOOKWORMS A series of experiments was performed with a view of determining whether the two species of hookworms discussed in the foregoing pages (Ancylostoma caninum and Bustomum phlehotomum) secrete a substance that has the power of inhibiting the coagulation of rabbit blood. Salt- solution extracts of fresh and dried material from the two species were tested as follows. Into a series of tubes containing varying doses of extract, rabbit blood drawn directly from the marginal ear vein was allowed to drop. Bach experiment was controlled by allowing an equal quantity of blood to drop into tubes containing physiological salt solution. So far as the rapidity of coagulation of the blood was concerned, appreciable but not very marked differences were detected between the test and control tubes. These experiments were performed on the blood of several rabbits with uniformly negative results. Inasmuch as Loeb and his collaborators tested the anticoagulin from Ancylostoma caninum on dog blood and obtained positive results, it would appear that the writer's negative results may indicate that the anticoag- ulins in hookworms are either strictly specific for the blood of their host or that they are perhaps only relatively specific. Further data bearing on hookworm anticoagulin as well as anticoagulins from other nematodes are given in a separate paper (Schwartz, 1921). X. EXPERIMENTS WITH EXTRACTS OF HAEMONCHUS CONTORTUS Haemonchosis or stomach- worm disease is a disease of cattle and sheep due to the presence in the fourth stomach of a nematode parasite known as Haemonchus contortu^. Young animals are especially susceptible to Nov. 19, i92t Hemotoxins from Parasitic Worms 419 stomach-worm disease, and among other symptoms they show those of a rather severe anemia. As in hookworm disease, the direct abstraction of blood by the parasites undoubtedly plays a part in bringing about the train of morbid symptoms associated with loss of blood, but that other factors are involved — ^namely, a chronic intoxication of the host by toxic substances liberated by the parasites — appears probable. Furthermore, it is by no means unlikely that as the suceptible animals grow older they become more or less immune to the effects of the parasites, although they are by no means immune to infestation with the worms. Whether the immunity is developed as a result of a previous infestation or whether it is a natural immunity associated with maturity is not known. In fact the clinical phase of haemonchosis is still an almost unexplored field in parasitology. The following experiments were performed by the present writer with reference to the presence of a soluble hemotoxin in this parasite. A number of specimens of Haemonchus contortus (about 100) that had been removed from a calf shortly after death were washed a number of times in physiological salt solution and kept in a refrigerator overnight. The following day the specimens were still alive. They were ground up in a mortar with a small quantity of physiological salt solution. The crushed material was transferred to a test tube and allowed to remain at room temperature for about two hours. The supernatant fluid was then tested on a 5 per cent suspension of washed sheep corpuscles. After 2 hours at 37° C. a number of tubes containing graded quantities of extracts and 5 drops of washed red blood cells showed no trace of hemolysis. The tubes were then transferred to a refrigerator, where they remained 18 hours longer. A faint trace of hemolysis was present in the tube containing the largest quantity of extract. The control tube was intact. A second experiment of a similar nature was performed with another lot of fresh worms. In this case the extract was tested on washed sheep blood corpuscles, with negative results. Alcoholic specimens of Haemonchus contortus from sheep were washed in salt solution to re- move traces of the alcohol and then dried at 37° C. The dried mate- rial was pulverized, and part of it was extracted in salt solution and tested on washed sheep corpuscles, with negative results. The remain- ing portion of the dried material was extracted in 95 per cent alcohol and the extract suspended in salt solution. Tested on sheep corpuscles, this extract likewise yielded negative results. A number of fresh specimens of Haemonchus contortus were dried at 37° C. and pulverized in a mortar. Graded quantities of the powder were added to washed sheep blood corpuscles. After 2 hours at 37° followed by 18 hours in a refrigerator slight hemolysis was produced. 420 Journal of Agricultural Research voi. xxii. No. s To one tube a small quantity of carbolized^ horse blood serum was added. The serum inhibited hemolysis. Haemonchus contortus powder was also tested on four samples of washed cattle blood cells. The results were slightly positive after 2 hours at 37° C. followed by 18 hours in a refrigerator. Inasmuch as in the experiment described above washed red blood cells were used, a series of tests were performed in which unwashed defibrinated blood was used. In this series six samples of cattle blood were involved. The addition of various quantities of Haemonchus contortus powder yielded negative results after 3 hours at 37° C. Summarizing, salt-solution extracts of Haemonchus contortus are very slightly hemolytic to sheep and cattle erythrocytes. The faint hemo- lytic property is preserved by drying. The weakly positive results obtained by experiments in vitro do not favor very strongly the view which has been commonly accepted as regards the secretion of a hemo- toxin by H. contortus. It is quite possible, however, that the appar- ently weak hemolysin requires some activator which is supplied by the host blood. The fact that experiments in vitro were only slightly positive by no means precludes the possibility that an absorption by the host of the secretions of H. contortus is followed by a marked hemo- lysis. Another possibility, which has already been mentioned, is that only the blood of young animals is susceptible to the secretions of H. contortus. The subject requires further investigation. XI. EXPERIMENTS WITH TRICHURIS DEPRESSIUSCULA EXTRACT A small series of experiments with an extract of Trichuris depressi- uscula was performed as follows: About 60 specimens collected from several dogs were thoroughly washed in physiological salt solution and dried in an incubator. The dried specimens were then triturated and extracted in 3 cc. of salt solution overnight at 8° C. The clear filtrate was tested on rabbit and sheep erythrocytes. Five drops of a 5 per cent suspension of rabbit blood cells were hemolyzed by 3 drops of extract in about 2 hours at 37°. Equal mixtures of sheep erythroc}'tes and extract showed no hemolysis. The extract was boiled for about a minute, and after it had cooled it was tested on rabbit erythrocytes. It produced a faint indication of hemolysis, showing that boiling prac- tically destroyed the hemolysin. XII. EXPERIMENTS WITH CESTODE HEMOLYSINS It has already been stated that while an active hemolytic agent has been shown to occur in Diphyllohothrium latum, evidence that other species of tapeworms secrete hemolytic substances is rather incomplete. The presence of a hemolytic agent in D. latum is significant in view of the fact that this parasite is capable of producing a severe anemia under ' 0.25 per cent solution of carbolic acid in senun. Nov. 19, 1921 Hemotoxins from Parasitic Worms 421 certain conditions that are not yet understood. Inasmuch as ces- todes are not capable of causing anemia by direct abstraction of blood or by lacerating the mucosa, the etiological role of a hemotoxin, if such a substance can be demonstrated in forms that cause anemia, can hardly be denied. The discovery of Schaumann and Ta\lqvist{i8g8) and the subse- quent studies of Tallqvist (1907) and Faust and Tallqvist {1907) with reference to the D. latum hemolysin are of great significance and mark the beginning of the study of the pathogenicity of parasitic worms from the point of view of intoxication. Despite the fact that D. latum ap- pears to stand alone among cestodes capable of setting up a severe type of anemia, there is some evidence that other cestodes are also capable of bringing about anemia, perhaps not so intense as that produced by D. latum. Railliet {1895), Neveu-Lemaire {1912), Hutyra and Marek {19 1 3), and other writers on veterinary parasitology state that cattle and sheep that are parasitized by tapeworms show clinical symptoms of anemia. Adult cestodes parasitic in these ruminants belong to the genera Moniezia and Thysanosoma. Only one species of the latter genus is known in the United States, namely, Thysanosoma actinioides , whereas several species of Moniezia occur in this country. Investiga- tions by the present writer with reference to hemolysins in worms be- longing to the genera Moniezia and Thysanosoma have yielded the following results. A salt-solution extract of Thysanosoma actinioides powder made by adding the powder to salt solution and allowing the extract to remain at 8° C. for about 24 hours was found to be hemolytic to washed sheep blood cells and rabbit blood cells. In one experiment 150 mgm. of powder were extracted in 5 cc. of physiological salt solution overnight at a temperature of 8°. The supernatant fluid was filtered and the filtrate tested on washed rabbit blood cells. Equal parts of extract of suspension of corpuscles showed complete hemolysis after 2 hours at 37°. Further experiments with salt-solution extracts of dried material on washed sheep and rabbit blood corpuscles confirmed the presence of a soluble hemolysin in this parasite. Thus, an extract prepared by adding 0.2 gm. of powder to 2 cc. of salt solution was tested on rabbit and sheep blood corpuscles and yielded positive results. The action of the hemolysin was comparatively slow. To tubes each containing 5 drops of washed blood cells 5 and 10 drops, respectively, of the extract were added and incubated at 37° for 2 hours; hemolysis was not evident in the tubes. After an additional period of 18 hours during which the tubes were kept in a refrigerator hemolysis was complete in the tube to which 10 drops of extract had been added and marked but incomplete in the tubes to which only 5 drops of extract had been added. It should be stated in this connection that in several instances salt-solution extracts of dried T. actinioides were not destructive to red blood cells of sheep. Whether the red blood cells of certain animals are more resistant than 42 2 Journal of Agricultural Research voi. xxn.No. s others, or whether the different extracts used in these experiments varied in their hemolytic content, has not been determined. At any rate, salt-solution extracts of T. actinioides , so far as the experiments referred to above are concerned, are not strongly hemolytic. A quantity of Thysanosoma actinioides powder was extracted in four volumes of ether. The ether extract after it had been freed from all traces of ether was emulsified in physiological salt solution and tested on sheep blood corpuscles with positive results. After a second extraction of the powder in ether a quantity of powder free from the ether-soluble fraction was extracted in physiological salt solution, and this extract was found to be nonhemolytic. The remaining powder was extracted in 95 per cent alcohol. After filtration the alcohol was evaporated, and the residue, which had a waxy appearance and consistency, was dis- solved in physiological salt solution and tested on sheep red blood cells with positive results. Boiling did not destroy the hemolytic potency of this extract ; neither did cold inhibit its activity. Normal horse serum inhibited its action completely. After alcohol extraction the pow^der was extracted in physiological salt solution and tested on sheep red blood cells. It was only faintly hemolytic. Another lot of powdered Thysanosoma actinioides was extracted in 95 per cent alcohol three times in succession, each extraction lasting 48 hours. After the last extraction only a slight residue was left when the alcohol had completely evaporated. The residues were dissolved in physiological salt solution and tested on sheep and rabbit blood cells with positive results. Boiling did not destroy them and low tempera- tures had no inhibiting effect on them. The powder freed from the alcohol-soluble fraction was extracted in salt solution, and this extract was nonhemolytic. It may be concluded, therefore, that a hemolysin is present in Thysanosoma actinioides, soluble to some extent in physiological salt solution and completely soluble in alcohol. Ether extracts of T. actini- oides are hemolytic, but worm material freed from ether-soluble fractions still retain the hemolytic agent. That substances other than fatty acids are involved in the hemolytic effects of T. actinioides extracts is evident, since the ether extracts remove whatever fatty acids are present in the worms. The alcohol-soluble and ether-insoluble fraction of T. actini- oides resembles rather closely tissue lysins so far as the chemical and physiological properties of tissue lysins are known. Noguchi {1907) found that tissue tysins are soluble in 95 per cent alcohol, are not removed by ether extraction, and that they have the chemical properties of solu- ble soaps. In common with the latter they are destructive to red blood cells even at 0° C, are neutrahzed by normal serum, and are resistant to boiling. While the chemical nature of the ether-insoluble and alcohol- soluble fraction of T. actinioides has not been determined, its resem- Nov. 19. 1921 Hemotoxins from Parasitic Worms 423 blance to tissue lysins appears to be very close. The Ascaris lumhricoides hemolysin as well as the Bustomum phleboiomum and Ancylostoma caninum hemolysins are not active at low temperatures, as shown else- where in this paper. Experiments with a species of Moniezia similar to those performed with Thysanosoma actinioides have yielded negative results. The addi- tion of various quantities of powdered Moniezia material to suspension of washed red blood cells of rabbit and sheep produced no destructive action on the cells. A salt-solution extract of Moniezia powder was likewise nonhemolytic when tested on washed sheep blood cells. An ether extract was only slightly hemolytic, but after removing from the ether extract the acetone-insoluble fraction, presumably lecithin, its hemolytic potency was no longer manifest. The acetone-insoluble frac- tion had no destructive effect on sheep blood corpuscles. A quantity of Moniezia powder freed from the ether-soluble fraction by repeated ex- traction with ether was extracted for 72 hours in 95 per cent alcohol at 38° C. The alcohol was separated from the alcohol-insoluble powder by filtration and evaporated. The residue was taken up in physiolog- ical salt solution, in which it was only partly soluble, the insoluble por- tion forming a coarse suspension. This solution had a decidedly acid reaction. Tested on washed sheep red blood corpuscles, it produced no hemolytic effect. XIII. RESULTS OF EXPERIMENTS The data presented in the foregoing pages have already been sum- marized in connection with each separate topic. The discussion which follows is for the purpose of correlating, comparing, and differentiating the results obtained with various species of parasitic worms that have been referred to in this paper, and to consider the general bearings that the results have on the nature of parasitic infection. Hemotoxins present in parasitic worms contain one or more active principles. Of the latter, hemolysins stand out as of prime importance. Hemagglutinins and anticoagulins may be associated with hemolysins. Hemagglutinins have thus far been observed in Diphyllohothrium latum by Tallqvist (1907) and in Ascaris lumhricoides by the present writer. Tallqvist describes the hemagglutinin from D. latum as a water- soluble, alcohol- and ether-insoluble substance, decidedly thermostabile. The hemagglutinin observed by the present writer in extracts of Ascaris lumhricoides is resistant to heat and soluble in lipoid solvents, such as ether and alcohol, as well as in physiological salt solution. It is, there- fore, quite a different substance from the agglutinin of D. latum. Anti- coagulins have been found in species of Strongylus (Weinberg, 1908), in the larvae of Gastrophilus (Weinberg, 1908) , in species of Ascaris (Weil and Boye, Leroy, and the present writer), in Ancylostoma caninum (Loeb 424 Journal of Agricultural Research voi. xxn.No. s and his collaborators) , and in several other species by the present writer {tq2i.) The anticoagulin in Ancylosioma caninum is the most active of the anticoagulins observed in parasitic worms and is doubtless a factor in the anemia that is present in hookworm disease. The anticoagulin of Ascaris lumbricoides has but a feeble action, so far as available experi- mental data show. Hemolysins from parasitic worms, so far as they have been described in the literature, have certain properties in common with hemolysins of bacterial origin as well as with hemolysins that have been obtained from normal tissues by Korschum and Morgenroth, Noguchi, and others. These properties may be characterized as nonspecificity in action and relative simplicity of structure as compared with hemolysins that may be artificially produced in animals by immunization with red blood corpuscles. The experiments recorded in this paper do not in any case contradict these facts. Different species of blood corpuscles may show differences in resistance to hemolytic extracts of parasitic worms, but absolute resistance of any species of corpuscles has not been established. Similarly, extracts from different parasitic worms differ in their resist- ance to heat, but once their potency has been destroyed it can not be reactivated by normal serum. The only apparent contradiction to this statement is the result of a small series of experiments of Garin with Graphidium sirigosum, which, as has already been indicated, can not be accepted as conclusive in view of the small number of experiments. Hemolysins produced in an animal as a result of immunization with red blood corpuscles, are, as is well known, specific in their action, affecting only corpuscles against which the animal has been immunized, and complex in structure, since they act in combination with comple- ment and may be reactivated by normal serum after the complement has been destroyed. So far as their resistance to heat is concerned, hemolysins from para- sitic worms differ considerably. Heat-resisting hemolysins have been recorded by Tallqvist from Diphyllobothrium latum, by Weinberg from species of Strongylus, and by the present writer from Ascaris lumbricoides and Thysatiosoma actinioides. Hookworm hemolysins from worms of the genera Ancylostoma, Necator, Bustomum, and the hemolysin from Tri- churis depressiuscula are not as resistant to heat. The relatively thermo- labile hemolysins from these parasites resemble in this respect bacterial hemolysins, whereas the thermostabile hemolysins resemble in this respect tissue extracts. The solubility of hemolysins from parasitic worms in lipoid solvents, especially in alcohol, is another property that they have in common with tissue lysins. A property of the latter is also the nonimpairment of their activity at low temperatures, even at 0° C. So far as the results of experiments recorded in this paper are concerned, hemolysins from Nov. 19, 1921 Hemotoxins from Parasitic Worms 425 worms belonging to the genera Ascaris, Ancylostoma, and Bustomum are inhibited at 8°. The hemolytic effect of Thysanosoma actinioides extract is not inhibited at this temperature, however. This fact is important and clearly differentiates hemolysins of nematodes from the hemolysin of T. actinioides. In this respect, too, nematode hemolysins resemble bac- terial hemolysins. Finally, the action of hemolysins from parasitic worms is inhibited by normal serum. The antilytic property of the serum is thermostabile (Weinberg, igo8, and the experimental results obtained by the present writer). Tissue lysins, too, are inhibited by normal serum. Certain bacterial hemolysins are similarly susceptible to normal serum. On the basis of this discussion nematode hemolysins may be character- ized as relatively simple substances, thermolabile or thermostabile, depending on the species from which they are obtained, inactive at low temperatures (6° to 8° C), inactive in the presence of normal serum, nonspecific, soluble in alcohol and in physiological salt solution. Cestode hemolysins, so far as they have been investigated, are rela- tively simple bodies, thermostabile, active at low temperatures, inactive in the presence of normal serum, nonspecific, soluble in alcohol. The question naturally arises whether toxic products from parasitic worms are liberated from the bodies of the latter and get into the circula- tion of the host. Blanchard (1903), while accepting the evidence in favor of the view that parasitic worms elaborate toxic products, appears to doubt the etiological significance of these toxic substances because of the possibility that they are either not liberated by the worms or if liberated may be thrown out of the body before they can injuriously affect the host. The available evidence on this question appears to indi- cate that hosts harboring parasitic worms actually absorb the toxic products of the latter. The serological evidence in favor of this view has already been referred to. It may be added that the fact reported by Guerrini {1908) with reference to the presence of hemolysins in the blood serum of hosts harboring Fasciola hepatica and the findings of De Blasi that hemolysins occur in the blood serum of hosts harboring Ancylostoma duodenale tend to confirm the belief that parasites liberate their toxic secre- tions and that these secretions get into the circulation of the host. Wein- berg {1908) has made some interesting observations on the tissues of hosts harboring parasitic worms which argue directly in favor of the absorption by the host of toxic products liberated by the worms. Weinberg exam- ined histologically the organs of 32 horses infested with strongyles and obtained the following results: In the blood vessels he found a large number of mononuclear leucocytes containing iron granules. He also found similar granules in the spleen, liver, in the conjunctival tissue, in the Malpighian tubules and in the convoluted tubules of the kidneys, and in the canals of the right kidney. Histological examinations by the same investigator of organs from 30 monkeys infested with a species of 426 Journal of Agricultural Research voUxxn.No. s CEsophagostomum yielded similar results. In another paper Weinberg {1909) records that the injection of extracts of worms of the genus Strongylus into guinea pigs leads to a pigmentation of the spleen but seldom of the liver. From this, it appears that erythrocyte destruction takes place in animals that harbor hemotoxin-producing parasitic worms and that the disintegration products of the erythrocytes are ingested by leucocytes, arrested in certain organs, and eliminated through the excretory system. Whether the hemotoxic substances from parasitic worms are liberated during the normal life of the worms, or whether they are liberated only when worms sicken or degenerate, as appears to be the case in Diphyllo- bothrium latum, can not as yet be stated with certainty. In the case of D. latum the view that only certain specimens secrete the hemolysin has been advanced by a number of investigators, especially by Leichten- stern (1896). Tallqvist's experiments show that hemolysins are present in specimens of D. latum expelled from patients that show no symptoms of anemia as well as in specimens obtained from cases of severe anemia. Tallqvist's hypothesis that the hemolysin is eliminated when the worms disintegrate finds confirmation in numerous cases in which eggs of D. latum are present in the feces of patients, and anthelmintic medication fails to expel any worms and merely yields a mass of eggs. Another factor which may be of importance, and which, so far as the present writer is aware, has been entirely overlooked, is the fact that certain individuals may lack antilytic constituents in their blood and are thus susceptible to the toxin which other individuals are capable of neutralizing. That the antilytic properties of the blood may under certain conditions be absent is probable from the observations of Noc {1908) with reference to hookworm disease. Whether the observations with reference to D. latum are applicable to other parasitic worms, especially to nematodes, can not in the light of our present knowledge be stated with any degree of certainty. That parasites may die in the intestine or other location and disintegrate before they are eliminated from the body of the host is by no means improbable. Cultures of larvae of parasitic worms in vitro show that bacteria may kill the worms, and that the latter undergo degenerative changes,- such as complete internal disorganization, quite rapidly. That worms may be attacked by bacteria and other organisms in the body of the host is by no means improbable. Weinberg has in fact described what appears to be a disease in worms belonging to the genus Ascaris, which is characterized by the presence of certain pig- mented spots that are clearly visible through the cuticle. The present writer has observed this condition in specimens of Ascaris lumhricoides on several occasions. Whether parasitic worms liberate their toxic secretions during life or whether these substances partake of the nature of endotoxins and are not liberated from the bodies of the worms unless the latter disintegrate -T^ov. 19, 1921 Hemotoxins from Parasitic Worms 427 is still open to speculation, but the view that toxic substances from parasitic worms are of etiological significance in parasitic diseases i^ supported by convincing evidence. XIV. SUMMARY Extracts of A scar is lumbricoides contain active substances that affect blood deleteriously. The hemolysin which these extracts contain is a thermostabile, nonspecific, alcohol-soluble substance which appears to be rather firmly bound to the cells of the parasite, presumably to the cells of the intestine in which it is elaborated. The hemolytic potency of extracts of A. lumbricoides is not due solely to fatty acids, since chemical fractions of the worms from which the fatty acids have been removed by ether extraction are hemolytic. The hemolysin is neutralized by normal blood serum. The body fluid of A. lumbricoides shortly after removal from the host contains oxyhemoglobin and is nonhemolytic. It acquires hemolytic powers, however, as the worms are kept alive in vitro for a few days, and loses at the same time its oxyhemoglobin content. Body fluid from fresh specimens of Ascaris lumbricoides does not acti- vate a hemolytic system, and alcohol-soluble fractions of the worms from which ether-soluble substances have been removed does not act as complement in combination with inactivated normal guinea-pig serum. The hemagglutinin from Ascaris lumbricoides is a salt-solution-soluble substance and has special affinities for rabbit blood cells. Unlike the hemolysin, its action is not hindered by low temperatures (6° to 8° C). Ascaris lumbricoides secretes a substance that inhibits the coagulation of blood. This substance is present in the body fluid of the worm and iias but a comparatively slight potency. Ancylostom,a caninum secretes a nonspecific hemolysin, soluble in salt solution, relatively thermolabile and inactive at low temperatures. Normal blood serum inhibits the action of the hookworm hemolysin. Bustomum phlebotomum secretes a hemolysin having properties similar to that of Ancylostoma caninum. This hemolysin is completely soluble in alcohol. Salt-solution extracts of Haemonchus contortus have but a feeble hemolytic action. Salt-solution extracts of Ancylostoma caninum and of Bustomum phlebotomum do not inhibit the coagulation of rabbit blood to any marked degree. A weak hemolytic substance is present in extracts of Trichuris depressi- uscula. Thysanosoma actinioides contains an alcohol-soluble hemolysin. Alco- hol-soluble fractions of T. actinioides from which the ether-soluble fraction has been removed are hemolytic, showing that substances other than fatty acids are involved. The hemolysin from this cestode is active at 8° C. 70495°— 21 4 428 Journal of Agricultural Research voi. xxii. no. s and is neutralized by normal blood serum. Extracts of a species of Moniezia similar to those of Thysanosoma actinioides axe nonhemolytic. The view that hemolysins and other hemotoxic secretions of parasitic worms are of etiological importance in parasitic diseases appears to be well founded. LITERATURE CITED AlEssandrini, Giulio. 1904. SUlvLA PATOGENESI DELL'aNEMIA DA ANCHYLOSTOMA. In PoHclin., ScZ. Med., anno 11, fasc. 12, p. 541-549, fig. 1-2. Bibliografia, p. 548-549. 1913. SUL poterE battericida DEI vERMi INTESTINAL!. In In Onore del Angelo Celli nel 25° Anno di Insegnamento, p. 259-276. Torino. 1913. sulIv'azione patogena del giganthorhynchus gigas (goeze) E su ALCUNE SUE particolarita anatomiche. In In Onore d. Angelo Celli nel 25° Anno di Insegnamento, p. 349-371, fig. 1-14. Torino. Askanazy, M. 1896. DER PEITSCHENWURM EIN BLUTSAUGENDER PARASIT. In Deut. Arch. 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J. 1904. THE PRESENCE OF A SUBSTANCE INHIBITING THE COAGULATION OF THE BLOOD IN ANCHYLOSTOMA. In Proc. Path. Soc. Phila., n. s., v. 7, no. 6, p. 173-177. 1906. UEBER EINE DIE BLUTGERINNUNG HEMMENDE SUBSTANZ IN ANKYLOSTOMA CANINUM. In Centbl. Bakt. [etc.], Abt. i, Orig., Bd. 40, No. 5, p. 738-739- LussANA, Felice. 1890. contributo alla patogenesi dell 'anemia de anchilostomiasi. In Riv. Clin. Milano, anno 29, puntata 4, p. 759-776. Nov. 19. 1921 Hemotoxins from Parasitic Worms 43 1 Magath, Thomas Byrd. I919. CAMAtlvANUS AMERICANUS, NOV. SPEC, A MONAGRAPH ON A NEMATODE SPECIES. In Trans. Araer. Micros. Soc, v. 38, no. 2, p. 49-170, fig. A-Q, 1-134, pi. 7-16. NevEU-LEMairE, Maurice. I912. PARASITOLOGIE DES ANIMAUX DOMESTIQUES. MALADIES PARASITAIRES NON BACT^RiENNES. ii, 1257 p., 770 fig. Paris. Noc, F. 1908. 6TUDES SUR Iv'aNKYLOSTOMIASE ET LE b6rib6rI EN COCHINCHINE. In Ann. Inst. Pasteur, t. 22, no. ii, p. 896-916; no. 12, p. 956-981. NoGUCHi, Hideyo. 1907. 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DIE LEHRE VON DER ALLGEMEINEN UND ORTLICHEN " EOSINOPHIUE. " In Krgebn. AUg. Path. u. Path. Anat. [etc.], Bd. 17, Abt. i, p. 137-789. Literature, p. 138-235, 787-789. Schwartz, Benjamin. 1919. A BLOOD-DESTROYING SUBSTANCE IN ASCARIS LUMBRicoiDES. [Prelimi- nary paper.] In Jour. Agr. Research, v. 16, no. 9, p. 253-258. Litera- ture cited, p. 257-258. 192 1. EFFECTS OF SECRETIONS OF CERTAIN PARASITIC NEMATODES ON CO- AGULATION OF BLOOD. In Jour. Parasitology, v. 7, no. 3, p. 144-150. References cited, p. 150. SeydERHELM, K. R., and SeydERHELM, R. 1914. die URSACHE DER PERNIZIOSEN ANAMIE DER PFERDE. EIN BEITRAG ZUM PROBLEM DES ULTRA visiBLEN VIRUS. In Arch. Exper. Path. u. Phar- makol., Bd. 76, Heft 3/4, p. 149-201, chart i-io. Shimamura, Torai, and Fujii, Hajime. I917. UEBER DAS ASKARON, EINEN TOXISCHEN BESTANDTEIL DER HELMINTHEN BESONDERS DER ASKARIDEN UND SEINE BIOLOGISCHE WIRKUNG. (mitteilung I.) In Jour. Col. Agr. Imp. Univ. Tokyo, v. 3, pt. 4, p. 189-258, fig. 1-4. 1 432 Journal of Agricultural Research voi. xxn. no.s Tallqvist, T. W. 1907. ZUR PATHOGENESE DER PERNICIOSEN ANAMIE MIT BESONDERER BERUCK- SICHTIGUNG DER BOTHRiozEPHALUS ANAMiE. In Ztschr. Kiln. Med., Bd. 61, Heft 5/6, p. 427-532. Literatur, p. 528-532. Thaler, E. Jul. 1918. ZUR DIAGNOSE DER DARMBLUTUNGEN. In Miinchen. Med. Wchnschr., Jahrg 65, No. 17, p. 460. USAMi, K., and Mano, R. 1919. ON THE COMPLEMENT FIXATION TEST OF THE SERUM OF ANCINARIASIS. (Abstract.) In Nippon No Ikai (Japan Med. World), Oversea ed., no. 303, p. II. WEa, P. :6mile, and BoyI;, G. I9IO. ACTION DES EXTRAITS d'ASCARIS EQUORUM SUR LA COAGULATION DU SANG DE LAPIN. In Compt. Rend. Soc. Biol. [Paris], t. 69, no. 29, p. 284-285. Weinberg, M. 1907. SUR UNE H^MOTOXiNE d'origine vErmineuse. In Compt. Rend. Soc. Biol. [Paris], t. 63, no. 24, p. 13-15. 1907. ACTION DE l'EXTRAIT DE SCL^ROSTOMES SUR LE SANG DU CHEVAL. In Ann. Inst. Pasteur, t. 21, no. 10, p. 798-807. 1908. PASSAGE dans l'oRGANISME DES SUBSTANCES TOXIQUES s6CR6t6ES PAR LES HELMiNTHEs (scle;rostome, oesophagostome, ankylostome). In Compt. Rend. Soc. Biol. [Paris], t. 64, no. i, p. 25-27. 1908. SUBSTANCES h6M0T0XIQUES s6CR]6t6ES PAR LES LARVES d'oESTRES. In Compt. Rend. Soc. Biol. [Paris], t. 65, no. 25, p. 75-77. 1912. ToxiNES VERMINEUSES. In Bui. Inst. Pasteiu-, t. 10, no. 22, p. 969-977; no. 23, p. 1017-1026; no. 24, p. 1065-1072. and Julien. 19x1. SUBSTANCES TOXIQUES DE L'aSCARIS MEGALOCEPHALA. (rECHERCHES EXP^RiMENTALES SUR LE cheval). In Compt. Rend. Soc. Biol. [Paris], t. 70, no. 9, p. 337-339- and Keilin. 1912. UNE MALADiE DE l'ascaris MEGALOCEPHALA. In Compt. Rend. Soc. Biol. [Paris], t. 73, no. 28, p. 260-262, fig. 1-7. Wells, H. Gideon. I918. CHEMICAL PATHOLOGY, BEING A DISCUSSION OP GENERAL PATHOLOGY FROM THE STANDPOINT OF THE CHEMICAL PROCESSES INVOLVED. Ed. 3 rev., 707 p. Philadelphia and London. Whipple, G. H. 1909. the presence of a weak hemolysin in the hook worm and its rela- TION TO THE ANEMIA OF UNCINARIASIS. In Jour. Exper. Med., V. II, no. 2, p. 331-343- Yagi. S. 1910. UEBER DAS VORKOMMEN DER HAMOLYSIERENDEN SUBSTANZ IN SCHIS- TOSOMUM JAPONICUM, ERRGER EINER IN Jj\PAN EPIDEMISCH AUFTRET- ENDEN KRANKHEiT. In Arch. Exper. Path. u. Pharmakol., Bd. 62, Heft 2/3, p. 156-158. YosHiMURA, Yoshio. 1913. UEBER DIE HAMOLYTISCHE WIRKUNG DES SCHISTOSOMUM JAPONICUM MIT BESONDERER BERUCKsiCHTiGUNG DER ANAMIE. In Vcrhandl. Jap. Path. Gesell. Tokyo, 3. Tagung, p. 104. d ASH CONTENT OF THE AWN, RACHIS PAI.EA, AND KERNEL OF BARLEY DURING GROWTH AND MA- TURATION By Harry V. Harlan, Agronomist in Charge of Barley Investigations, and Merritt N. Pope, Assistant Agronomist in Barley Investigations, Office of Cereal Investiga- tions, Bureau of Plant Industry, United States Department of Agriculture INTRODUCTION The ash determinations here assembled were made with two primary objects in view. Earlier studies had shown that in the varieties under observation the awns possessed a physiological function. When they were removed the kernel development was retarded and the spike became brittle through the greater ash deposit in the rachis. In order to see if usable variations existed in the amount of ash deposited in the rachises and awns, a considerable number of varieties were studied. The previous experiments were not as complete as was desired. Mechanical difficulties had prevented the taking of samples to the point of absolute maturity. There thus existed a possible doubt as to the nature of the changes in the days immediately following the date when kernel sampling became impossible. The determination of ash in the awns and rachises was, therefore, continued for some time after maturity in one series of varieties at Chico, Calif. The results point a possible way to the securing of desirable non- shattering awnless and hooded varieties. They also throw some light on the ash content of the kernel during growth. MATERIAL USED Material for the study of ash in the barley spike was collected from several sources. Two series of samples originated at Aberdeen, Idaho. The awns, rachises, and paleas were obtained from the irrigation plots, the kernel studies of which were previously reported. In this series and the one from Minnesota the glumes were forcibly removed from the kernels. To eliminate the possible effect of imperfect separation when the glumes were thus removed, the kernels from a naked barley grown at Aberdeen were included for comparison. Two lots of samples were grown at Chico, Calif. The first of these consisted of a collection of varieties embracing a wide range of botanical characters. The second consisted of a lesser number of varieties, which were allowed to stand in the field for a time after ripening. Frequent samples were taken, and the change of ash after maturity was determined. Further use also was made of the data from an experiment carried on at St. Paul, Minn. Ash determinations were made on a number of varieties grown at Arlington, Va., the detailed results of which are not included. Journal of Agricultural Research. Vol. XXII, No. 8 Washington, D. C. Nov. 19, 1921 aao (433) KeyNo. G-2SS 434 Journal of Agricultural Research voi. xxn, no. a ASH OF THE AWNS The awns of barley contain a very high proportion of ash. One of the most finely divided carbons known has been secured from barley awns. This extremely fine division is probably caused by the high percentage of ash. The ash is deposited during the time the kernel is developing. At the time of their emergence the awns contain little ash and are very flexible. ,i< i^ V \ ^ ^ "*• / / / / ^ ( > 4 1. - / 1 < f 1 .*" »* \ \ao / f / ,»• / / ? . y /5 / 1 • ,0* / .'' / 1 / '•• ,' / J K PL 07 3 V ^m ^[ _^. / / / / .m. • —1 _ / ^ y / ^o f iO ./» /• ' / — ^ ^A / 5h 51 7h BU 92 02. Vf Zl '3Z 'n 51 6t 7Z ■Bi 9.3 03 1 1 i ?<3 ? ^ ^ t "it 7 / ' S FlQ. I.— Percentage of ash, by progressive three-daj' averages, in the awns of Hannchen barley grown on plots variously irrigated at Aberdeen, Idaho, in 191 7. The ash at flowering time and for a few days immediately following usually runs from 4 to 8 per cent of the dry matter. As may be seen in Table I, the awns of the Hannchen variety may sometimes contain a slightly higher percentage at flowering time. Varieties of this type, how- ever, contain more ash than do those of most other types of barley. The increase in the ash content after flowering is very regular. The daily Nov. 15, i94i Ash Content of Awn, Rachis, and Palea 435 increment is quite uniform. In figure i it will be seen that within the variety the rate of deposit has a direct relationship with the amount of moisture in the soil and probably with the amount of water transpired. On plot 8, which was not irrigated after flowering, the plants were suffer- ing from lack of water during most of the time the kernels were devel- oping. This lack of water is reflected in the percentage of ash in the awns. The awns on this plot never contained as high as i6 per cent of ash. The uniformity of the deposit was in no wise affected. The daily increment, however, was less than on plot i, which received ample irriga- tion. The normal ash content of tlie awn of Hannchen barley when grown in the western United States is over 30 per cent at maturity. As will be seen, the awns on the spikes from plot i reached this percentage several days before matturity. Plots 2 to 7 received the same treatment as plot 8 until the time of their final irrigation. Only one irrigation after flowering was given to any plot except plot i . The irrigation occurred on the day the first ash determination was reported. Following the application of water there was an acceleration in the rate of deposit of ash in plots 2 to 6, inclusive. The barley on plot 7 was maturing when the water was applied. The leaves had begun to wither and the awns were almost color-free at the time of the irrigation. The rate of deposit was not materially increased over the rate on plot 8. The maturation was, how- ever, delayed, and the final ash content of the awns was decidedly greater than in plot 8. Tabls I. — Percentage of ash in plots variously the awni irrigated, of Hannchen barley grown at Aberdeen, Idaho, in igij on eight different Date. Plot. I Plot 2. Plot 3. Plot 4. Plot 5. Plot 6. Plot 7. Plot 8. July 16 17 18 9.4 10.8 II. 6 13-9 14. 0 16.5 19. 6 20. 0 23.8 23-9 27-5 28.6 25-4 28.4 30.0 31.2 30.7 32-7 16. 2 12.5 14.2 16. I 14.7 16.5 17.7 19-3 20. 2 19.8 20. 6 21. 6 20. 4 20. 4 21-5 21. I 22. 4 21. 2 6.0 7 7 9 8 9 8 8.7 14. 0 15-7 18.4 18.8 19.8 21. 9 18. I 19.9 21. 9 21. 9 3 3 ? 19 7.8 7-9 9-7 15-1 16.3 15-0 18.7 5 23 24 25 26 27 28 29 30 31 Aug. I 2 ■2 13.8 13-4 16.5 13-4 17.2 17.7 17. 0 18.9 22. I 21. 7 19.9 21.5 22. I 24-5 25-4 24. 0 26. 2 22.7 9 12 10 13 II 10 13 II 12 13 13 15 15 13 13 4 7 2 8 S 0 10.3 13.2 16. 0 12. 2 15-4 18.4 18.3 21.5 23.2 18.0 21.7 22. 5 26. 2 22. 7 32.1 11. I 12. 6 13-9 13-5 13- I 13.2 13-8 16.6 ig. 0 21. 2 17.2 21.5 7 4 I 9 4 26. 5 24. 2 26.2 26.3 32-9 30.1 27.8 23. 2 32.3 20.5 23.1 23-9 22. 6 20. 2 20. 4 19.7 20. 0 25-7 4 5 6 28.2 33-^ 29. I 6 7 8 29. I 33-1 28.2 7 8 0 _ 436 Journal of Agricultural Research voi. xxn.No. 8 Table II. — Percentage of ash in awns, rachises, atid kernels of eight varieties of barley grown at Chico, Calif., and sampled on lo different dates PERCENTAGE OF ASH IN AWNS C.I. No. Variety. June 6. June June June i6. June Jime 2S. June 27. June 30. July 7- July 14. 531 234 257 916 261 19s 690 652 Hannchen Nepal ' . . Tennessee Winter. Odessa. .. Mariout. . Smyrna. . Coast,. Poda 36.7 16.6 29. I 34-3 34- I 32.0 31- 9 27-3 36. 34-4 31.8 35- 5 34- 6 32. I 35-6 34-5 35-5 26.6 28.4 33-2 34-6 36.7 29-5 32.8 31.0 34-4 34- 33-1 35- 35-2 37- 35- 0 29. 33- 0 30. 33- ^ *5; S. r"^ ' _ ..« 15. W7 /1 1 \ .^ 1*^ ^ P^ ^ W, \ -— ' :> ^ * > *% y ^ f»^ ■~~ ' I Z 3 9-^6 7 8 9 10 II 12 J3 /^ iS 16 17 18- 19 20 Zl Z2232^2S Fig. 4.— Graph showing percentage of ash on the basis of wet weight in the kernels of Hannchen barley on 2 plots differently irrigated, in 1917, and of Baku barley grown in another year at Aberdeen; Idaho. DISCUSvSION OF RESULTS The extremely heavy deposit of ash in the awns of barley indicates that the awn, or parts of the awn, are used as a depository for the excess ash absorbed by the roots. The fact that some varieties contain much more ash in the awns and rachises than others is due probably to two causes. There most probably is a difference between varieties in the amount of water transpired. As was shown in the irrigation plots at Aberdeen, this results in a marked variation of ash deposit. There may also be a difference in the selective functions of the roots of different varieties. Some varieties may absorb more ash from the soil than do others. This is strongly indicated in the ash content of the rachises. It is also of greatest importance in this connection. Varieties of the Coast type are characterized by a low ash content of the rachis. In most of the shatter- ing varieties the rachises are high in ash content. The hooded varieties have long been known to shatter badly. From results previously re- ported it would seem that much of this is due to the loss of the awn as an organ partially utilized for the elimination of ash. On the other hand. Nov. 19, 1921 Ash Content of Awn, Rachis, and Palea 447 v _/* ^fVA/ >^ "s V > — — — - ^- /'' ^^^ s^ 26 > K H 1 1 1 f- \ \ / f ;^ '*«• iH^ y ■ \ • 1 1 1 1 i 1 \ /6 / / > \ /?/9C///S /O \ '~' y' \ _,^^i»* <5 n / ^*»— n_^ .'' **•■ **—•*. O o t3 © Cb o o 3 P o B c+ O o m o B c+ o B 0) -J 1 CO o O Oi M- H- J—" pJ £3* to OQ CD m o CO CD o* CD »-t) O •-J CD e+ CD P o CD c+ tr O CD CD CD "d -1 P' > OQ -i O c I-" c+ P O o o 3 CfQ •-1 CD CO GQ •->• O 3 •d o o p »-l c+ B CD 3 c+ CO 3S G) -i CD -} CD .Q C O 3 W CD CO 03 CD P 3 O o 3 c+ M- 3 I 3 B CD 3 c+ O >-*} c+ 3* CD ►d P CO CO CD Ci c+ 3* CD O »-»• CD 3 O »-^ c+ 3* CD CO § 'O c+ << O O 3 *^ CD CD (0 00 CO tJ "" CD 3 O o ►d CO CD CD '^ 3 o a; O CD B o" CD -1 o pi nj O M- P 3 CO M- - 3 pi OQ M- p. CO ►d c: o* h-" ♦-»- o p c+ o 3 O o o P ♦t o CO ro P w o 3 O *^ o c: 3 P > O !-• <+ P w •d (D 3 o« o c Z H m o (A > H PI in o m > m z H 0 "n > O n o c r H c m "Vol. XXII NOVE^^BER 26, 1921 No. 9 JOURNAL OF AGRICULTURAIy RESEARCH CONXENXS Page Temperature Relations of .Stone Fruit Fungi - - - 4S1 CHARLES BROOKS and J. S. COOLEY (Contribution from Bureau of Plant Industry ) Transportation Rots of Stone Fruits as Influenced by Orchard Spraying - - --- - - " 467 CHARLES BROOKS and D. F. FISHER ( Contribution from Bureau of Plant Industry) Storage of Coniferous Tree Seed - - - - - 479 C. R. TILLOTSON (Contribution from Forest Service) Susceptibility of the Different Varieties of Sweet Potatoes to Decay by Rhizopus nigricans and Rhizopus tritici - 511 L. L. HARTER and J. L. WEIMER (Contribution froiti Bureau of Plant Industry) PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINGTON GOVERP Mr NT PRINTING OFFICE 1922 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- tnent Station, Rutgers College W. A. RILEY Entomologist and Chief, Division of Enlo- tnology and Economic Zoology, Agrtcul- iural 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, Ntv? Brunswick, N. J. DRNAL OF AGEICrailML ffiSEARi Vol.. XXII Washington, D. C, November 26, 1921 No. 9 TEMPERATURE RELATIONS OF STONE FRUIT FUNGI By Charles Brooks and J. S. CoolEY, Pathologists, Fruit Disease Investigations , Bureau of Plant Industry, United States Department of Agriculture The two fungi that cause the heaviest market losses on peaches and other stone fruits are Sclerotinia cinerea (Bon.) Wor. and Rhizopus nigricans Ehr. The former is often referred to under its conidial name of Monilia and is the cause of brown rot, while the latter is the cause of black mold rot. The present paper gives the results of investigations in regard to the temperature responses of these two fungi under various conditions of growth. In all the experiments except that reported in figure i the spores were inoculated into the fruit from pure cultures. Except where other- wise stated the cultures were obtained from the host into which the inoculations were made. The fruit was warm when inoculated but after inoculation was placed in moist chambers and stored at once at the temperature indicated. Five or more peaches or prunes were used at each temperature in each test. The fruit was at the proper maturity for picking and shipping and was carefully selected for quality and sound- ness. In dividing the fruit into lots for distribution at the various temperatures uniformity was secured by selecting seven peaches or prunes (or as many as there were temperatures) that were similar in size, color and degree of maturity and distributing these one each in seven moist chambers and repeating the process till the desired amount of fruit was obtained. Records were made of the diameters of the rots at intervals of one or two days, and the average of all the rots at a given temperature on a particular date was taken as a basis for plotting the curves in the accom- panying figures. The equipment used in securing the various tempera- tures has been described in an earlier publication.^ SWEET CHERRIES In 19 19 a temperature experiment was made on Governor Wood cherries. These had been shipped by express from Wallingford, Conn., to Washington, D. C, and arrived somewhat bruised and with considera- ble brown rot. The specked and rotten cherries were discarded, but the slightly bruised ones were included in the experiment. The cherries were divided into five equal lots and distributed without inoculation at ' Brooks, Charles, and Cooley, J. S. temperature relations of applb-ROT fungi. In Jour. Agr. Research, v. 8, no. 4, p. 139-164, as fig., pi- 1-3- 1917- Journal of Agriculture Research, Vol. XXII, No. 9 Washington, D. C. Nov. a6, 1921 aap Key No. 0-256 75308—22 1 (451) 452 Journal of Agricultural Research voi. xxii. No. 9 five different temperatures.^ After 10 days' storage notes were taken and results obtained as shown in figure i. All of the cherries at 15° and 20° C. were partially or entirely rotten and nearly all of those at 10°. At 5° sixty-six per cent were affected, and at 0° thirty-four per cent. The results show the great inhibiting effect of low temperatures but perhaps give greater emphasis to the extreme diffi- culty of controlling Monilia rot at any temperature when the fruit has already received bad treatment and an opportunity has been given for the rot to pass through its initial stages while the fruit was warm. PRUNES But one temperature test has been made on prunes. The fruit was from Wenatchee, Wash., and was shipped from that point in a pony refrigerator August 31, 1920, arriving in Washington, D. C, in good condition 13 days later. Inoculations were made with Monilia and Rhizopus, and the fruit was distributed at once to the various tempera- tures. Figure 2 shows the development of the rots 5 days after inocula- tion. PEACHES A large number of temperature experiments have been made with Monilia and Rhizopus on peaches. The Carman, Belle, and Elberta peaches used in the 191 8 experiments were purchased in the Washington market. The Belle and Elberta used in 19 19 were from Rockville, Md., and the experiment was started the day after they w^ere picked. The Carman and Belle peaches used in 1920 were from Vienna, Va., and were inoculated the day after they were picked. These peaches were slightly greener than those of the other experiments. The curves of the various figures show very great uniformity. The Rhizopus cultures from peaches gave results similar to the cultures from cherries and strawberries, both in temperature response and in rapidity of rotting. An interesting contrast is seen between the behavior of the fungi on peaches and on dextrose potato agar. A comparison of figiu-es 3, 4, 5, and 6 with figure 7 shows that Monilia has grown just as freely at the higher temperatures and much earlier and more rapidly at the lower temperatures when grown on peaches than when on agar. At 10° C. rots usually became evident on the fruit within 3 days, while on the agar there was practically no growth at the end of 7 days. At 5° the rots were well started in 6 days, while the agar colony had scarcely made an equivalent growth at the end of 14 days. At 2>^° the rots made a start in 8 to 12 days, but there was no evidence of growth on the agar at the end of 20 days. A comparison of figures 8, 10, 12, 14, 15, 16, and 17 with figures 9, 1 1 , and 1 3 show^s that the reverse condition holds with Rhizopus. This fungus made a more rapid growth and developed at lower temperatures on the culture media than it did on the fruit. On both food materials it had its most rapid growth at 30°. With the culture media tlie growth at 20° and 25° was but little slower than at 30°, but on the peaches the growth at 20° fell far behind that at 30°. At 15° and also at 10° the 1 Temperature equivalents: °C. °F. 'C. "F. 20 68 5 41 IS 59 o 33 10 50 Nov. 26,1921 Temperature Relations of Stone Fruit Fungi 453 /CO < 3 i5" /o /s £0 F:g. 1. — Xatural infections of brown rot on Governor Wood cherries. The base line shows the temperatures and the perpendicular the percentage of cherries affected with brown rot. % ^£:<^/?£-£>S C£/\/T/<3/?/9£>£r Vv~;'2. — Monilia (solid line) and Rhizopus (broken line) on Italian prunes. Temperature is indicated'on the base line and diameter of rot on the perpendicular. '^£>/9yS Z/P^^yiS Z.,ff sS Z6 /O /S zc Fig. 3,— Monilia on Elberta peaches. Experiment started August 2c, 1519. 1 454 Journal of Agricultural Research voi. xxii, No. 9 •?ii>/?K? ^0/9ys O Z.S ^ 7.5 JO /s ,a? Fig. 4. — Monilia on Belle peaches. Experiment started August 27, 1919. '4-P/?yS zo/?ys /O /S Fig. 5. —Monilia on Carman peaches. Experiment started August 5, 1923. <5 O 3/yfrs ZD/^ys zo Fig. 6.— Monilia on Belle peaches. Experiment started August aS, 1920 Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi 455 c 2. 'O ,'d' :so as so Fig. 7. — Peach Monilia on potato agar with 3 per cent dextrose added. In Petri plates. Experiment started November 22, 1918. Fig. 8. — Peach Rhiiopus on Eiberta peaches.! Experiment started Augiist 23, 1918. 456 Journal of Agricultural Research voi. xxii, No. 9 /oo % h \ J eo I % yoa^yis / yS£>l^y^ ^ '^ \ 1 f \ / 1 ■n \ jyatusK 7 / \ F^— < Y o ^.s -s /O /S ZO Z5 ,30 ^S Fig. 9. — Peach Rhizopus on potato agar with 2 per cent dextrose added. Experiment started November 32, 191S. 60 60 ?y& A ^f^a/fyis / / ( ^/ / / < u « ^^ Y 3ff/frs O Z.S ^ /O /S ZO »S JO Fig. 10. — Cherry Rhizopus on Elberta peaches. Experiment started August 23, 1918. Nov. 26.1921 Temperature Relations of Stone Fruit Fungi 457 /oo i \ Is: I j •tyo/fYS j )sa9y^ t \ t/'f/i9yJO /?£0/?££:<5 c£-Arr/<^/?/9^£: Fig. 12.— Strawberry Rhiropus on Elberta peaches. Experiment started August 23, 1918 458 Journal of Agricultural Research voi xxii. no. ? /CO O Z.5 S /O /S P.O <95- Fig. ij — Strawberry Rhizopus on potato agar with 2 per cent dextrose added. Experiment started November 33, 1918. O Z.5 S /O /6 20 Fig. 14 — Peach Rhizopus on Carman peaches. Experiment started August i, 1918. Nov. a6, I9SI Temperature Relations of Stofie Fruit Fungi 459 I N I — / 1 ^/ )^/^>i^ t 'i^ •3£/^YS Fig. 15.— Peach Rhizopus on Belle peaches. Experiment started August 37 1919. za/}y,S %% 'i.S ■S /C /^ 20 Fig. 16.— Peach Rhizopus on Carman peaches. Experiment started August s, 1920. 'SDi^YS ^5" 7.S /O /S ^c Fig 17.— Peach Rhizopus on Belle peaches. Experiment sUrted August 28, 1920. 460 Journal of Agricultural Research voi. xxii. no. 9 growth started earlier and proceeded more rapidly on the agar than on the peaches. The fungus finally made a slow growth on the agar at 5°, but on peaches it made no growth at this temperature and with the exception of a few overripe peaches none at 7K°- These contrasts in the behavior of the two fungi can probably be partly explained by the fact that Monilia is a parasite and adapted to growth on living material, while Rhizopus is a saprophyte and suited to growth on dead material, like the agar or inactive living material such as overripe fruit. It is interesting to note that with both fungi unfavorable food material and unfavorable temperatures work together in delaying growth, one unfavorable factor adding to the other in delaying or inhibit- ing activity. A study of figures 3, 4, 5, 6, 18, and 20 gives a detailed idea of what can be expected of Monilia rot at any transportation or storage tempera- ture. The results in the last two figures have been obtained by averag- ing those of the first four figures and therefore stand as a summary of the various experiments. With fruit that is infected with brown rot (Monilia) 3 days at 15° C. would result in heavy losses, 3 days at 10° would mean badly specked fruit that would go down rapidly at that temperature and that would be entirely destroyed by a day at "a higher temperature. Brown rot cannot get started in 3 days' time at 7K°. but by the end of the fourth day fruit at that temperature may be specked with rot. In 6 days the fruit at 5° may be spotted, in 9 to 12 days growth may be evident at 2}4°, and at the end of 3 weeks rots may have started at 0°. Brown rot does not develop rapidly at the lower temperatures even when well started, yet its later growth is inhibited far less than its initial stages. A study of figures 8, 10, 12, 14, 15, 16, 17, 19, and 21 shows that Rhizopus has more decided temperature limitations than those that have been pointed out for Monilia. The results in the last two figures have been obtained by averaging those of the first seven and therefore stand as a summary of the various Rhizopus experiments. At 15° and 20° C. the growth rate of Rhizopus rot is practically the same as that of Monilia rot, Rhizopus being a trifle more rapid at 20° and Monilia just a little more rapid at 15°. At 10° Monilia rot develops more than twice as fast as Rhizopus rot, and at 7^° Rhizopus is practically eliminated. Whether Rhizopus could make any start whatever at 7^° C. seemed to be determined mainly by the maturity of the fruit. The curves of figure 15 show that Rhizopus had not made a start at 7^2° in 11 days. The peaches at that temperature were still free from rot at the end of 14 days, were removed to a warm room at that time, and were entirely rotted with Rhizopus 2 days later. The results show that the fungus was held completely in check at j}4° but was alive and ready for rapid development when given a more favorable temperature. With the ex- periments reported in figure 17, Rhizopus had produced evident rotting at 7^° in 6 days. At that time only the ripest peaches were affected, but at the end of 12 days rots began to develop on the greener peaches. When once started at this temperature Rhizopus rot made a fairly rapid growth. The results as a whole show that with the usual number of days in transit for most peach shipments Rhizopus can produce little or no damage at 10° and none at 7/^°. Figures 3 to 21, inclusive, show the development of the rots when the fruit is stored at the given temperatures immediately after inoculation. Figures 22, 23, and 24 show the effect of i day's delay at a higher tem- Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi 461 perature than that at which the fruit was finally held. A study of the figures brings out the facts that with Monilia i day at 25° C. followed by I day at 10° results in as large spots as 5 days at 10°; i day at 20^ followed by i at 10° results in as large spots as 4 days at 10°; i day at 25° followed by i at 7^2°, in as large spots as 6 days at y}4°', i day at Fig. 18.— Monilia on peaches. A summary of the experiments on brown rot obtained by averaging the percentages of figures 3, 4, 5. and 6. 25° followed by i day at 5°, in as large spots as 10 days at 5°; i day at 25° followed by i day at 2X°, in as large spots as 12 days at 2^2°. It will also be seen that with Rhizopus i day at 25° followed by i day at 10°, or I day at 15° followed by 4 at 10°, results in larger spots than 7 days continuously at 10°. 80 I L /fy.s )ts/?/fys / i )&a^xs/ 7/ / // e < M. '//- / '?-0/9y,s \g£i9y,S £0 Fig. 19. — Rhizopus on peaches. A summary of the experiments on Rhizopus rot obtained by averaging the percentages of figures 8, lo, 13, 14, 15, i6, and 17. Peaches inoculated with Monilia and promptly cooled to 2^°, 5°, 7>^°, or even 10° C. have had but little or no rot at the end of 3 or 4 days, but similarly inoculated peaches delayed at 25° for i day» before storing at these lower temperatures have developed so much rot by the end of the fourth day after inoculation that they were commercially worthless. Peaches inoculated with Rhizopus and promptly cooled to 10° have been 462 Journal of Agricultural Research Vol. XXII, No. 9 3 DAYS 6 DAYS 12 DAYS 20 15 10 DEGREES 7.5 S CENTIGRADE Fio. 30. — Monilia on peaches. The drawings represent the average of the results from the various experl - ments. The shaded portions indicate the extent ot the decay. The upper series shows the size of the rots at the various temperatures after 3 days, the second series the size after 6 days, and the third the size after 12 days. 3 DAYS 6 DAYS DEGREES CENTIGRADE Fig. ai.— Rhizopus on peaches. The drawings represent the average of the results from the various experi- ments. The shaded portions indicate the extent of the decay. The upper series shows the size of the rots at the various temperatures after three days and the second series the size after six days. Nov. 26, 1921 Temperature Relations of Stone Fruit Fungi 463 60 J^Z.5' 6 8 /O /Z /?/9y^ /^^72£7P /A/0CaL/9T/0/Y I'?- /S Fig. 22. — Effect on Monilia rot of one day's delay at 25° C. The base line shows the number of days after inoculation and the perpendicular the diameter of the rots. The curves show the rate of development of the rots at the temperatures indicated at the end of the lines. The dotted lines give the results on the fruit placed at once at the given temperatures and the solid lines the results on similar fruit delayed one day at 25° before placing at these temperatures. Elberta peaches, August 20, 1919. Z 3 Wash., without to Wenatchee, ment after the j n ice and 5 days' Wash., under days' cool stor- storage with- ice and 5 days' age had been fol- Variety. Orchard treatment. out ice, iS° C. storage under lowed by 3 days' s '% "o. (64.4° F.). ice. warm storage. s a d a 1 .2 'S c ■(J IS .2 1 s 3 .2 1 3 •3 'i 3 f^ s pL Pi S A4 a S Pk (ti Italian... . Bordeaux 1.6 3- 2 4.6 0 0 9.6 0 0 2.1 As above but last omitted. . . Commercial lime-sulphur .6 12.5 2.1 •9 ■7 0 0 3-9 0.4 0 8.3 . I 71.8 1.3 1-3 17-5 .6 0-5 0 0 0 36.1 II. 0 .6 1-3 A£en Self-boiled lime-sulphur 1. 1 As above but last omitted. . , . 2 13-9 0 0 38. S 1.9 I. 2 c ! 7.6 I. I .8 •9 8.7 1.8 2.8 1.4 7.0 0 0 0 1-3 0 0 20. 3 34-6 .8 . 3 1.6 0 Untreated 1 0 ! 1 The prunes were harvested Sept. 15, shipped to Wenatchee, Wash., Sept. it, received Sept. 18, and notes taken Sept. 23. 2 No shipment. The contrast in the amount of brown rot (MoniHa) on the fruit from the different plots after shipment and storage is shown graphically in figure I. A study of the figure shows that the unsprayed fruit developed six to nine times as much brown rot under transportation and market si BO/?£>£/?UX SULPf-/U/? OUST SO-3e-/S — ^i so/?£>£:/9ax otASr b /o ;so .so '?c Fig. I. — Brown rot on Italian and Agen prunes after shipment and storage. 1919. conditions as the fruit from the plots receiving the best orchard treat- ment. The sulphur dust was as efficient as the sprays, but the Bordeaux dust was far less efficient. The results show that orchard spraying may have great value on the market even when the amount of disease in the orchard has been negli- gible. DISCUSSION OF RESULTS In order to obtain the composite results from the various prune and cherry experiments the data from the different spraying and shipping tests have been brought together and averaged. The results are shown in figures 2 to 6, inclusive. Nov. 26, 19:1 Transportation Rots of Stone Fruits 475 Figure 2 shows the comparative efficacy of spraying and dusting as determined by the average of the four years' results on prunes. Little contrast is shown between the two methods of treatment, both sulphur dust and self-boiled lime-sulphur having reduced the amount of brown rot at picking time from 4 per cent to approximately i per cent and i/,5T /(VT/frT£/? ^MPAfS/^ a£LJ^-Bi>/^£P L/M£-SULPHU^ S0-35-i5 SULPHUR OUST Fig. 2. — Comparative results from spraying and dusting in a four years' test on prunes. reduced the amount developed in shipments from 16 per cent to 2.5 per cent. It was pointed out earlier in the paper that the different spray appli- cations were probably not of equal value in the control of brown rot on the fruit. The great importance of the last application in this connec- tion is shown graphically in figures 3 and 4. A reference to these figures S^R/9y£D /'^r/^CSA/T/fGE OrMO/V/L//? /?OT o ^ /o /s ;so Fig. 3.— Brown-rot control of cherries as influenced by a late spray application (about three weeks before picking time). The average results obtained from 15 different shipping tests. shows that with the prunes approximately one-half and with the cherries approximately one-third the brown-rot control was due to this late spraying. The comparative results obtained with the different rots in the various shipping experiments are shown in figures 5 and 6. The term sprayed s^/?/9yEi? i/Ms^^t9y£ri>— -" — ^/¥?t9y££f • Sf^^y£ii/^Sr i^Pt/C/9T/CW 0/t?/TT£O i/MS/'^/fy£0 /'£^^£/Vr/?0£ OFAfOMl//? /rtTT O ^ /O /S Fig. 4. — Brown rot control of prunes as influenced by a late application of spray or dust (three to five weeks before picking time). The average results obtained from 7 orchard experiments and 11 shipping tests. as used in these two figures includes both dusting and spraying. As has already been pointed out, there was no year in which there was a serious epidemic of rot in the orchards under investigation. The amount of rot on the untreated cherries at picking time never ran as high as i per cent. The average amount of brown rot on the sprayed prunes at 476 Journal of A gricuUural Research voi. xxii. no 9 picking time, as shown in figure 6, was 1.6 per cent and the average amount on the untreated prunes was 4.6 per cent. The orchard loss from rot with either the prunes or the cherries would be considered of very minor importance in practical operations, scarcely justifying the expense of spraying; yet even under these conditions the orchard spray- ing has shown decided beneficial effects in the carrying quality of the fruit in transportation and storage. The good eft'ects, however, have been largely if not entirely confined to the control of Monilia rot. With both the prunes and cherries the unsprayed fruit has developed approxi- mately four times as much of Monilia rot as the sprayed fruit, but has shown .practically no greater susceptibility to Penicillium and Rhizopus rots. These contrasting results are in harmony with the nature of the different fungi. Monilia is a parasitic fungus and able to penetrate the sound skin of both ripe and green fruit; PenicilHum and Rhizopus are saprophytic fungi, able to attack only the harvested fruit and dependent upon bruises and skin cracks for first points of entrance. The Monilia spores come primarily from the orchard, but Penicillium and Rhizopus have an almost universal distribution. /?Or^L /9^/^ r ^ '^ ^^ /.^ ^ ZS- \sp/f/?yi^£> "^ =— ■ ■ P£N/C/LUU»? ^SP/?/?yS£> />A/D ffH/20PLis\y/vs/'/?/9rep i Fig. s. — A comparison of the average development of rot on sprayed and unsprayed cherries in i8 different shipping and storage experiments. Under such circumstances it would be expected that orchard spraying would furnish at least partial protection against brown rot in transpor- tation and storage, since it would both decrease the supply of spores and furnish a more or less complete protecting film on the fruit. On the other hand, orchard spraying could not be expected to have any appre- ciable effect upon the spore supply of fungi like PenicilHum and Rhizopus that are of general occurrence, and a film of spray on the skin could offer little protection against fungi that enter through breaks in the skin. While these contrasts in the different fungi are of importance in con- nection with the present studies, it should not be inferred from the fore- going statements that skin punctures have no effect upon the occurrence of Monilia rot, for it is well known that any abuse to the fruit is decidedly favorable to the development of the disease; ^ nor should it be inferred that Rhizopus and Penicillium are entirely unable to penetrate the sound skin, for when these fungi are once well established in a crate they may spread out from a center of infection without much regard to the sound- ness of the adjacent fruit. This is particularly true of Rhizopus, and especially where it is favored by a high temperature. Under such a condition it often spreads through a package of stone fruit in a most rapid and indiscriminate manner. 1 Ramsey, H. J. the h.\ndling and shipping op fresh cherries prom the wxi,i,.\mette valley U. S. Dept. Agr. Bui. 331, 28 p., ii fig., 1916. Nov. 26, 1921 Transportatio7i Rots of Stone Fruits 477 With all the rots temperature has been an extremely important factor. In the 1 9 19 experiments (Table VI) part of the fruit was stored at 15° C. and part at 5°, with striking contrasts in the results. Rhizopus was en- tirely eliminated at the lower temperature, and Penicillium and Mon- ilia were greatly reduced. Short shipments without refrigeration have resulted in heavy losses, while fresh fruit, both sprayed and unsprayed, has been shipped across the continent in pony refrigerators under ice with no decay upon arrival. Fruit that was free from rot after seven days in the refrigerators became badly decayed after standing one or two days in a warm room, the unsprayed fruit always developing the most rot but the sprayed fruit never remaining free from it. Refrigera- tion is always valuable; but it is evident that its importance increases with any decrease in orchard or packing-house care. It is evident that there is a widely distributed responsibility for the occurrence of stone fruit rots in transit and in storage. Orchard spraying may be one of the important factors in the control of Monilia rot on the /o /^ ieo -as- MmL//}fir/fciftA/G — ^ ■~^~~" ■~'^~~ % niHiHiinii Z-ZEEj — 1 - ^£:/^/c/iuuftt fsff^9y£P firrER SH//=W£AfT \t/f»aP/»TYEP ^W/'/HEA/T \UJVSP/I9r£p Fig. 6.— a comparison of the average development of rot on sprayed and misprayed Italian prunes in i: different shipping and storage experiments. harvested fruit, but as a protection against Penicillium and Rhizopus rots it has little or no value. SUMMARY (i) Orchard spraying has reduced the amount of Monilia or brown rot developed on sweet cherries in transportation and storage experiments from 24.3 to 6.4 per cent. All the cherries were from orchards where there was less than i per cent of rot on either the sprayed or unsprayed fruit at picking time. (2) In similar shipping and storage experiments with Italian prunes there has been an average of 28 per cent of brown rot on the untreated fruit and 7.1 per cent on the sprayed or dusted fruit. The amount of rot on the unsprayed fruit at picking time was 4.6 per cent and on the sprayed fruit 1.6 per cent. (3) About half the brown rot control secured in the shipping tests with prunes and about one-third of that secured with cherries was due to the spray application made three or four weeks before picking time. (4) There has been little contrast between the brown rot control secured with sulphur dust and that secured with the standard spray materials. (5) Spraying and dusting have had little or no effect upon the develop- ment of Penicillium and Rhizopus rots in transit and storage, their occurrence apparently being much more influenced by the prevalence of bruises and skin punctures. (6) The unsprayed fruit has shown a greater need of refrigeration than the sprayed, and the injured fruit a greater need than the sound. STORAGE OF CONIFEROUS TREE SEED By C. R. TiLLOTSor^ Forest Exartmier, Forest Service, United States Department of Agriculture During the period from 1909 to 19 13 the United States Forest Ser\'-ice was especially active in its reforestation program. There were large areas of deforested land on the national forests, and there was a sincere desire on the part of the organization to serve the public interest by bringing these lands into a productive state as soon as possible. The program involved the growing in nurseries and planting of many millions of young trees each year and also the sowing of seed directly on extensive areas of deforested land. To carry out the program large quantities of seed were needed. In the year 19 10 alone, 63,000 pounds of seed were collected. Foresters know that seed is not borne in the same abundance upon trees each year. A good seed crop in any region is often followed by one to several very poor or lean crops. It accordingly is desirable to collect during years of plenty seed in large enough quantities to last several years. The Forest Service did pursue this course and thereupon became confronted with the problem of how to store the seed so that it would not deteriorate greatly in germinative ability and energy before it could be used. This problem was not a new one. European foresters had been faced with it a good many years ago and had made substantial progress in its solution for some species. One of the most intensive sets of European experiments with coniferous seed was perhaps that of Dr. Adolf Cieslar ^ which was begun in the spring of 1886 and continued over a period of 1 1 years. Dr. Cieslar attempted to determine not only the efifect of air-tight storage on seeds of Norway spruce, black (Austrian) and white pine, but also the effect of heating these seeds before placing them in storage. He came to the conclusions that (i) storing under air-tight covering lengthens the life of these species of seed so that when stored in this way they often show a considerably higher germination percentage, especially in the later years of storing, than seeds of the same origin stored in the air, this difference in favor of air-tight storing of seed amounting to 33 per cent in the case of 6-year-old Norway spruce seed ; (2) storing seed away from the air also results in higher germinat- ing power; (3) tbe application of heat at 45° to 55° C. to seeds of white and black pine at the beginning of storing injures the viability of these seeds and also their germinating power to a considerable extent, but Nor- way spruce is injured less by this means, and its germinating power is even kept at a high point by strong heating; (4) heating at 30° to 40° C. for one hour has a not unfavorable effect on the seed of these three species. When stored away from the air, such seed maintains both viability and germinating energy at as high a point as that of unheated seed; in fact, the slightly heated seed shows in later years of storing, a tendency to germinate in a very rapidly rising curve. 1 Cieslar, Adolf, versuche uber aufbbw.\hruno vo^f NADELHOLWAME>f unter luftdichtem VBRSCHLussE. /» Centlbl. Gesam. Forstw., Bd. 23, Heft 4, p. 167-174- i^97- Journal of Agricultural Research, Vol. XXII, No. 9 Washington, D. C. Nov. 26, 1921 aar Key No. F-7 (479) ^.So Journal of Agricultural Research voi. xxii, no. 9 Experiments conducted by the German Chief Forester Haack ^ at the Eberswald Forest Academy from 1906 to 1909 confirm some of the results of Dr. Cieslar and give additional information on the subject of seed storage. His experiments were confined to seed of Scotch pine. He found that exclusion of air can not wholly prevent a loss of germinating power with increasing age of the seed. This is manifested less by a final decrease of germinating percentage than by a falling off in germinating energy. But compared with that of seed stored in the air, this decrease of germinating power is extremely slight. After three years the air- tight seed had a germinating power of nearly 90 per cent as against 22 to 70 per cent for seed stored in the open air in the same room. Another conclusion of Haack's was that in no case should seed that has been shut up in air-tight containers without previous thorough drying be placed in a storeroom in which the temperature is likely to increase, even if only occasionally (for instance, in attics). This con- clusion was based on two experiments. In one, two air-tight bottles were placed on thermostats heated to 36° C. and left for eight weeks. One of the bottles contained air-dried seed, the other seed from which 5 per cent of its weight in moisture had been removed in an exsiccator. In the test, the former germinated only a little over i per cent, the latter 96 per cent. In a similar experiment at a temperature of 30° C, the germination test at the end of four months gave 40 per cent and 9^ per cent, respectively. It might have been just as well for Chief Forester Haack to emphasize the necessity of thorough but not excessive drying of the seed before placing them in air-tight containers rather than stress the point of storing in a fairly cool room not subject to a rise in tem- perature. Such rooms are to be had only by some special arrangement. Later in the same article, Haack does state that in storing pine seed air-tight, care should be taken that the seed is neither moist nor over- dr)^ He decided that the degree of dryness which the seed has when it comes from the kiln or when spread out in a well-heated room or dried in the sun would probably be best — about i to 2 per cent lighter than its average weight in the ordinary seed bin. His experiments showed that long-continued drying is harmful to pine seed. The ger- minating power of Scotch pine seed left in the exsiccator for four years fell to 16 per cent at the end of that time, while the same seed not dried in this way still show-ed a germination of 80 per cent. Haack conducted one experiment to determine the eft'ect of different temperatures of the storeroom upon seed in air-tight containers. Scotch pine was stored for three years in a room heated to a temperature oi 20° to 25° C, in an unheated room, and in a cellar i meter in depth Both cellar and unheated room were free from frost in winter; in sum- mer the unheated room was somewhat warmer than the cellar. The results indicated that a temperature of 20° to 25° C. in the heated room was injurious to the seed. The germination percentage dropped from 96 to 82 in the three years. With strong seed there was very little decrease in germination of seed stored either in the basement or in the unheated room. With weak seed, however, not thoroughly dried before being placed in the container, there was, after three years, a difference in germination of 20 per cent in favor of the seed stored in the basement. These experiments of Dr. Cieslar and Chief Forester Haack, while thorough in themselves, w^ere confined to only four species, three of 1 Haack [Otto H. A.], der kiefersamen. vErhaltnis zwischen keimprozent und praktischem WERT, mehrjahrige /ufbewahrung ohne verminderung des keimprozents. In Ztschr. Forst. u. Jagdw., Jahrg. 41, Hett 6, p. 353-381, i fig. 1909. Nov. ; o, 1921 Storage of Coniferous Tree Seed 48 1 them European. In order to meet an immediate need for information, it seemed desirable to extend and expand upon them somewhat to those American species most used in reforestation operations on the national forests. These species were western yellow pine {Pinus pondc- rosa Law.), western white pine {Pinus monticola Dougl.), white pine (Pinus sirobus Linn.), Engelmann spruce (Picea engelmanni Engelm.), Douglas fir {Pseudotsuga taxifolia (Law.) Britton), and lodgepole pine (Pinus contorta Loud). The study should now be followed up with those more sensitive coniferous seeds, the true firs, the cedars, arbor- vitas, redwoods, and the numerous species of American hardwoods of which so little is known. The study brings a realization of the fact that it is a mistake to deal with so many variables in an intensive project of this nature. It is difficult if not impossible to be sure of the cause or causes for any particu- lar result. There is now a need for further investigations of slightly smaller scope, in which the variables will be reduced to a minimum and in which by laboratory methods, the exact physiological, chemical, and any other changes which the seeds undergo can be followed closely. These changes almost surely will throw light upon the behavior of seed in storage. FACTORS AFFECTING EXPERIMENTS Some of the conditions under which this study was carried out should be stated. Because of a shifting in personnel the study in various stages has come under the direction of several men. This has not been con- ducive to the best development of the project, and it may be that the analyses of the results are not so thorough as though made by the one who conceived the study. Apparently through oversight, no tests of the seed were made before they were put in storage. It is not known, accordingly, to what extent deterioration progressed in the seed during its first year of storage. That there was deterioration in the case of most containers is shown by the very general superiority after one year of the seed stored in air-tight bottles. Because of this lack of an original test, it has, in making analyses of the results, been necessary to use as the basis of comparison, the germination of seed after storage for one year in the air-tight bottles. On account of the large number of variable factors involved (6 species of seed, 5 kinds of containers, 13 storage points, and 3 temperature con- ditions at each of these points), the general conclusions are by no means fully supported by the results in every individual test. It is thought, however, that the average results are a safe criterion of what may in general be expected of these coniferous seed in storage. Fresh seed, with the wings removed, of the species previously men- tioned was obtained during the fall and winter of 1908-9 in the amounts and from the sources indicated below: Picea engelmanni, lo pounds, San Isabel National Forest, Colorado. Pinus monticola, 55 pounds, Coeur d'Alene National Forest, Idaho. Pinus contorta, 12 pounds, Deerlodge National Forest, Montana. Pinus ponderosa, 70 pounds, Boise National Forest, Idaho. Pinus strobus, 30 pounds, New York State. Pseudotsuga taxifolia, 25 pounds, San Isabel National Forest, Colorado. "When the seed was all brought together at Washington, D. C, it was spread out thinly on a floor and fanned steadily for two days by means of an electric fan. The object was to dry the seed coats thoroughly. 482 Journal of Agricultural Research voi. xxii.no. 9 Each lot of seed was then divided roughly into portions of about 600 to 800 seeds each, and these were distributed equally among the following containers : 1. Ordinary manila paper coin envelopes. 2. Similar envelopes soaked in melted paraffin. 3. Cotton cloth bags. 4. Similar bags soaked in boiled linseed oil and dried. 5. Glass bottles which after filling were sealed air-tight with paraffin. Seed of all six species stored in each of the five containers constituted one test set of samples. For convenience in handling, shipping, and storing, each test set was placed in a small wooden box lined with a wire mesh to prevent the access of rodents. POINTS OF STORAGE It was one purpose of this study to determine whether seed deteriorated in storage to a greater extent in one geographical region than in another. AJjV-~ ^7r~ _____^^ V\\ 7 \ r ""^--^--^ ^.. i^/ r f U'^vV^ ^ ©'■^"^^ ^^^ 1 /^"/jt^.i ^- A ^ ""%S v. \^ iV- 1 ^f^"^"'-' ^""^ -J v^ V Y ' % ^ r l^ , — ^ — ^. Fig. I. — Map showing points at which coniferous seeds were stored to test effect of geographical locatioa Thirteen points of storage, accordingly, as indicated below and on the map (fig. i), were selected. These, it will be noted, are rather widely scattered over the United States and afford a fair basis for arriving at some conclusions concerning this particular aspect of seed storage. POINT OP STOR.\Ge ' APPROXIMATE ALTITUD3 I. Ann Arbor, Mich 875 feet a . Dundee, 111 700 feet 3. Fort Bayard, N. Mex 6, 500 feet 4. Halsey, Nebr 2, 700 feet 5. Ithaca, N. Y 800 feet 6. Lake Clear Junction, N. Y i, 600 feet 7. Lawrence, Kans 800 feet 8. New Haven, Conn 30 feet 9. Pikes Peak, Colo 9, 000 feet 10. Pocatello, Idaho 4, 500 feet 11. State College, Pa i, 150 feet 12. Warsaw, Ky 400 feet 13. Waukegan, 111 600 feet Nov. 26, 1921 Storage of Coniferous Tree Seed 483 CONDITIONS OF STORAGE Another point on which it was hoped this study would throw some light was the eflfect of several conditions of temperature on seed in stor- age. At each of the geographical points mentioned, accordingly, the cooperators in the study were requested to store the seed where each of the following conditions of temperature would prevail : 1. Ordinary indoor temperature, such as an office shelf where the temperature would always be above the freezing point. 2. Fluctuating temperature, as in an outbuilding or unheated garret where the temperature would follow rather closely the actual outdoor variations. Proximity to a stable was to be avoided. 3. Fairly uniform low temperature, such as prevails in an unheated basement or cellar. To just what extent these conditions obtained and were entirely comparable at all points of storage, it is not possible to say. It would not be surprising if there were considerable differences, particularly in the low-temperature conditions. It is believed, however, that they were similar enough to warrant the drawing of general conclusions from the tests. PERIOD COVERED BY STUDY The study was planned to cover a period of approximately five years. The seed was sent to the 13 points of storage during March, 1909. In January, 19 10, and again in January, 191 1, 19 12, and 19 14, three test sets (one stored at each of the three temperature conditions) were for- warded by express from each of the storage points to Washington, D. C, for testing. It is thus seen that tests were carried on after the seed had been in storage for periods of approximately one, two, three, and five years. There was no test of seed in storage for four years. From a practical standpoint, at least, it seemed that tests covering a period of five years would be sufficient. It is unhkely that seed in commercial quantities at least will be stored for a longer time. As a matter of interest, how- ever, a few of the seeds which had been stored in bottles were carried over for another five years and tested during the year 19 19. This phase of the study will be taken up in more detail later (p. 510). SEED-TESTING OPERATION The seed-testing operation was a simple but rather large undertaking. There were carried on during each of the four years 195 tests for each of the six species. Two hundred seeds were used in each test. Ordi- nary greenhouse wooden flats about 14 by 18 by 4 inches in depth were nearly filled with fresh sand, which was compacted and smoothed off; the seed for each test was scattered uniformly over the surface, pressed into the sand by means of a board, and then covered with yi inch to % inch of sand. The flats were then set on greenhouse benches where the sand was kept moist during the course of germination by sprinkling it with an ordinary watering pot equipped with a fine rose or spraying nozzle. The seeds were protected from mice by covering the flats with frames made of fly screen. Ants were troublesome at first, but they were successfully combatted by scattering napthalene flakes on the benches. During the winter and spring months when artificial heat was employed in the greenhouse, the temperature sought was about 70° F. 75308—22 3 484 Journal of Agricultural Research voi. xxn. No. 9 during the daytime and about 50° at night. There were, of course, some variations in this, particularly as summer approached. On bright, sun- shiny days in late spring or early summer, the temperature in the green- house sometimes approached 100° during the middle of the day. By that time, however, the germination tests were practically completed for all except the slow-germinating eastern and western white pines. A careful day by day record was kept of the germination. As the seeds sprouted and developed a short radicle they were plucked out of the sand and discarded. While this method of conducting germination tests for all kinds of seed, particularly those which germinate very slowly like the white pines, is not considered ideal, the results secured for the several years are at least comparative. CONCLUSIONS In noting the conclusions, the reader should keep in mind that they have reference to coniferous seed only, and that they are based upon the results of one series of tests with only six species of coniferous seed and may not accordingly be applicable to all coniferous seed, or even to the same kinds of seed from other sources. It should also be remem- bered that the seed used in this experiment ^vas thoroughly air-dried before it was placed in air-tight storage. (i) Storage of coniferous seed in the air-tight bottle is far superior in every respect to storage in any other container. The average germina- tion for the 5 -year period of seed stored in bottles over that stored in the next best container was 22 per cent. (2) Thoroughly air-dried coniferous seed stored in air-tight bottles is little if at all affected by such differences in temperatures as exist between a location where the temperature follows the natural fluctua- tions, a location indoors where the temperature never falls below freez- ing, and a location in an ordinary cellar or basement. (3) Coniferous seed stored in air-tight bottles is little if at all affected by the geographic location of the storage point. (4) The quality of coniferous seed, by which is meant its value in terms of both germinative energy and germinative ability, is much superior in the case of seed stored in an air-tight bottle to that stored in any other receptacle. This is manifest even at the end of one year of storage. (5) Following the air-tight bottle, the various containers, in the order of their merit, fall into the following sequence: paper bag paraffined, paper bag, cloth bag, and oiled cloth bag. It should be noted that an ordinary paper bag closed at the top is superior to a cloth bag for seed storage. The oiled cloth bag is practically worthless as a container. (6) The use of any of the containers except the air-tight bottle results in such rapid deterioration after one or t\vo years of storage under the temperature conditions of this experiment as to render the seed, partic- ularly of Engelmann spruce, Douglas fir, and white pine, of very little worth. (7) Storage at the indoor temperature is superior to that at the fluctuating or low. Storage at the low temperature shows the poorest results. This low temperature has reference not to a low uniform tem- perature of freezing or less but to that of an ordinary cellar or basement. The difference in germination percentage is not great under these three conditions but is sufficient to make indoor storage preferable to the other two conditions. Nov. 26, 1921 Storage of Coniferous Tree Seed 485 (8) Some geographic locations are more favorable for seed storage than others. Fort Bayard, Pikes Peak, Pocatello, and Lake Clear Junction — all points of relatively high altitudes and, with the possible exception of Lake Clear Junction, of low relative humidities — stand out as exceptionally favorable localities. Four middle-western points, Waukegan, Dundee, La\\Tence, and Warsaw, and one Atlantic sea- board point. New Haven, stand out as unfavorable localities for seed storage. Such points should apparently be avoided where ordinary methods of storage are followed. No one of the geographic locations shows marked superiority over another when the seeds are stored in air-tight bottles. (9). In respect to sustained vitality, the seeds employed in this study range themselves in the following sequence, Avith the strongest first: western yellow pine, lodgepole pine, western white pine, white pine, Engelmann spruce, and Douglas fir. WHAT THE STUDY SHOWS The points brought out by the study can be shown better, it is thought, by the accompanying tables and curves with a few comments than by lengthy discourse. EFFECT OF CONTAIN'ER Table I, together with the curves (fig. 2), brings out what was very evident during the progress of the study, the striking superiority of the seeds stored in the air-tight bottles over those stored in any other con- tainer. This is particularly true when the storage period extends beyond one year and is more striking in the case of Engelmann spruce, Douglas fir, and white pine than in that of lodgepole, western yellow, and western white pines. The seeds of the former three species are apparently more likely to deteriorate than those of the latter three and after two years of storage are of little worth. It seems safe to assume (barring any hypothesis of post-ripening of the seed during storage) that the germination of the seed before it was put in storage was at least equal to that of the seed stored in bottles at the end of one year. Based on this assumption. Table I shows that the average deterioration for all species has at the end of five years been for seed stored in a paper bag 45 per cent; in a paper bag paraffined, 42.3 per cent; in a cloth bag, 47.8 per cent; in a cloth bag oiled, 51.4 per cent; and in the air-tight bottle, 10.8 per cent. In this connection it should be noted that Pinus ponderosa stored four years, Picea engelmanni and Pseudotsuga taxifolia three years, and Pinus contorta two j^ears show little if any decrease in the total germination of bottle-stored seed. In fact, germination at the end of two and three years has in some cases been greater than at the end of one year. The behavior of Pinus strobus and Pinus monticola seed is a puzzle. It will be noted that the germina- tion percentage of bottle-stored seed decreases through the second and third years, but at the end of the fifth year (19 14) it equals or betters that of the second year (191 1). The 1914 germination of these two species with seed stored in the other four containers is practically equal to the 1912 germination. Possibly the conditions for germination in 1914 were somewhat superior to those in 1912, or there may have been some physiological development to account for it. 486 Journal of Agricultural Research Vol. XXII, No. 9 80 70 k \40 \ 20 JO 0 50 S 40 b 20 \ P//VC r^ wp£r/ fOSf '45) py f--4?> '\ ■■. \ (4-L- \ \ N n~:r^ ■ . ^• -^H^ 2. 3 5 YE/IRS /N STOR/tOE do 70 k ^60 ^40 k /O 0 P/A/i /J- a wra, ?r/f Jfil- (2) \ '\ -ci{)\^ ■• \ {3h^- A \ ^ \\^ \ '"''-■ -., ^^-^ ~-. ^ (41.- ■^_ .. __ 30 70 ^60 Z 3 5 YE/19S IN3T0/?/i6E 20 10 0 P/CC r £^)b£:lm)^nn/\ / \ '(5j \ \ \ *»^ >J nN. ^-J w, {^ ^ '\ "^^ :^^^ \ \ ==^5--, s\ ^ ---., 1 2. 3 5 yej^rs w sroP^G£ . PI ^/US V STPCPi 3US i5) \ \(2.) \ K ^ ^ *\ ,,. ^, ,,--■ '' m-^\ \ ' \ ■v . = -^^"H I 3. 3 5 YE/fRS /A/ srOPt^G£ ~^5 Ye/fPs M sroP/^6c 3 ITdi Z 3 U, d) 5 YE/fPS /N STOP/^S£ f¥IPER B/fffS A. CLOTH a/fO.C?/ LEO P/JPER B^6, P/?/9/tFF/NED S BOTTLE F!/P-T/CHT J CLOT// B/f6 Fig. 2. — Graphs showiug value of different kinds of containers for the storage of coniferous tree seeds Nov. 26, 1921 Storage of Coniferous Tree Seed 487 Table I. — Germination percentages of seed stored in different containers and under different temperature conditions Seed tested for— Seed tested for— 30 days. 33 days. 3i days. 34 days. 95 1 80 93 days. days. days. 96 days. Temperature and container. Picea engelmanni. Pinus contorta. 1910 1911 1912 1914 Aver- age for all 4 years. 1910 1911 1912 1914 Aver- age for all 4 years. Fluctuating tem- perature; Paper bag Paper bag, par- affined Cloth bag Cloth bag. oiled. Bottle, air-tight. 47- 0 40.4 37-7 26. 7 67-1 38.8 43-2 27-5 20. 9 79-7 20.3 25.0 iS-o II. 6 70. 0 10.3 11. I 6.7 8.3 54-4 29. I 29.9 21. 7 16. 9 07-8 66.0 74.0 55- 0 18.2 7S-0 55- I 62.3 47-3 13-5 73-2 40. 8 45-2 32-2 15.0 60. 0 34-6 44.0 36. 9 13-5 59-8 49.1 56.4 40.4 iS-o 67.0 Average for all containers. . . Average for all containers for all 4 years 43-8 42. 0 28.4 18.2 33-1 57-6 50-3 38-6 35-8 45-6 Indoor temperature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled . Bottle, air-tight. Average for all containers. . . Average for all containers for all 4 years 52. I 55- I 51- 3 38.7 67.8 45- 7 51-5 39-5 31-8 74- 7 31- I 30. I 17-3 15-3 73-6 7-4 9. 2 7-9 4-5 61. 9 34. II 68. 8 1 bi. 2 36-5 77-2 57-4 29. 0 42. I i;i- 3 22. 6 16. 8 20. 8 69. si 72-1 75- 8 44-6 49-9 34-6 16.2 58- 4 36-9 45-9 21. 2 9-5 54-9 52-9 57-6 37-3 15-8 65-3 53. 0 48- 6 33- 5 18.2 38-3 55-4 53-3 40. 7 33- 7 45-8 I.,ow temperature; Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled. Bottle, air-tight. 30. 2 ; 19-2 40. 8 23-6 24-3 15- 9 16. I i 8. 3 67- 5 ] 77-2 10.5 "•3 6-3 ■3 76.0 4-7 1.8 I. 2 57-8 1 IS- 7 ; 63. 9 1 2C. I : 70-7 12. I 49. 2 6. 5 ' 12. 7 (19. 6 : 72. 9 49.6 49-6 46.4 16.6 30- 7 34- 0 31-6 14-5 64. 2 29. 0 37-0 29. I 13-5 61.8 43-3 47-8 39-1 14-3 69. 0 Average for all containers. . . Average for all containers for all 4 years 35.8 28.8 2C. 9 13.6 ' 53-9 i 1 24.8 47-9 35- 0 34- I 42- 7 Average for all 3 temperatures: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled Bottle, air-tight 43- I 45-4 37-8 27. a 67-5 34-6 39-4 27. 6 20.3 77-2 20. 6 22. I 12.9 9.1 73- 2 e.8 8.3 5-S 4- 7 58.0 i 26. 3 ! 66. 2 28. 8 74-0 21. 0 48. 8 15-3 IS- 6 69.0 1 73-3 55- 3 56. 4 48-3 17.0 75-4 38- 7 43-0 32-8 15-2 60. 9 33-5 42-3 25- 7 12. 2 58.8 48-4 53-9 38-9 15- S 66.9 Average for all containers and tem- I)eratures 44.2 39-8 27.6 16.7 55- 6 50- 5 38.1 34-5 488 Journal of Agricultural Research Vol. xxir. No. 9 Table I. — -Germination percentages of seed stored in different containers and under different temperature conditions — Continued Seed tested for— Seed tested for— days. days. 34 days. ,34 days. 103 117 days. 1 days. 1 117 days. 113 days. Temperature and container. Pseudotsuga taxifolia. Pinus ponder osa. 1910 1911 1913 1914 Aver- age for all 4 years. 1910 1911 1912 19 '4 Aver- age for all 4 years. Fluctuating tem- perature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled. Bottle, air-tight. 22.7 25-7 14.3 3-9 44-3 14-7 ■ 30.3 ii-S 6.6 Si-S 6.9 6.6 6-3 1.8 4S-S 0. 5 1.4 . I . I 30- 4 II. 2 13-5 S.o 31 43.9 76.0 75-6 75-2 71.6 78.3 63- 9 61. I 63- 7 60.6 72.2 55- S 38.1 . 34-8 43.7 74-4 43-3 30.2 42.4 32.6 72.8 63.3 61.3 39.0 53-4 74- 4 Average for all containers... Average for all containers for all 4 33. 3 20- 9 13-4 6-3 IS- 8 7S-3 64- 3 s3-3 4S.7 61.6 Indoor temperature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled. Bottle, air-tight. 28.7 34-5 34.7 II. 3 49.1 14-3 17-7 12-3 3-3 48.6 4-3 6.0 3- 7 •9 45-8 •4 •9 •S •7 33.6 13. 0 14.8 10.3 4.1 43- 0 7S-a 77-1 77.3 74-4 76. I 67.7 67-8 65.8 61.8 72.1 62.6 63-6 62.6 S6.4 7S-3 SI- 5 S8.i 44.6 38-3 76.6 64-3 66.7 62.6 S7-7 75- 0 Average for all containers.. . Average for all containers for all 4 years 39.7 19- 3 13. I 6.2 16.8 76.0 67.0 64. t 53-8 65.3 Low temperature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled . Bottle, air-tight . 13- S 19. 6 8-S 6-5 49.8 7-7 9.8 6.0 6.9 50.8 •7 2.6 •3 47-7 1-3 1.4 1.6 ■ I 33-9 5-8 8.4 4. I 3-4 45-6 73.1 71.8 73-9 68.3 78. S 48.1 S3- 4 49.2 SO-S 67-3 42.6 30- S 38.9 39-4 7S-9 32- S 32.6 29-9 37.6 75.0 48.8 S2-I 48.0 49.0 74-2 Average for all containers Average for all containers for all 4 years 19. 6 16.2 10.3 7-7 13- S 72.9 S3- 7 49- S 41-5 54- 4 Average for all 3 temperatures: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled . Bottle, air-tight. 21. 6 36.6 iS.S 7-3 47- 7 "■3 IS- 9 9.9 S-6 so. 3 40 S-I 3-4 •9 46- 3 •7 I. 3 • 7 •3 3I-0 9-7 13. 3 7-5 3-S 43-8 74-4 74-8 75-4 71-4 77-6 S9-9 S3- 6 60.8 S7-4 S9- 6 S2- I S7- 6 48- 2 70. S 75- 2 43-1 47.0 39-0 36-2 74-8 S7-8 60. 0 56.5 47-9 74- S Average for all containers and tem- peratures.... 2.vS i8.8 11.9 6.8 74-7 6r-7 S7-3 48.0 Nov. 26, 1921 Storage of Coniferous Tree Seed 489 Table I.— Germination percentages of seed stored in different containers and under different temperature conditions — Continued Seed tested for— Seed tested for— days. 126 days. 129 126 days. days. days. 126 days. 129 126 days. days. Temperature and container. Pinus strobus. Pinus monticola. 1910 1911 1912 1914 Aver- age for all 4 years. 1910 1911 1912 1 Aver- years. Fluctuating tem- perature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled . Bottle, air-tight . 40. 6 44.0 30-5 24-5 56.0 27-3 29-9 18.4 7-9 46. S 8.8 9.9 8.3 5-0 30-7 9.8 8.9 8.6 2. 2 49.2 21.7 23.2 16. s 9-9 45- 6 36.0 36. 7 30-3 18.7 41. 1 24.7 27. 0 II- 3 10.4 27-7 6.2 5-7 30 4-7 7-8 6.0 1 7.S 4-4 3-0 25-9 18.2 19.2 12.3 9 2 25.6 Average for all containers... Average for all containers for all 4 years 39- I 26. 0 12- S IS- 7 23-3 32.6 20. 2 s-s 9-4 16. 9 Indoor temperature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled. Bottle, air-tight. 46.6 49-9 3SO 23. 2 58. 5 28.7 32.9 13-4 II. 3 SO. 6 9.6 12.8 S.8 5- I 33.6 4-4 4.0 3-2 1-7 SO- 7 22.3 24.9 14.4 10.3 48.4 36-2 39-2 32-1 22. 0 39-7 26.3 24.2 is- 1 13-0 24. s 7-0 7-3 3-8 3-6 9-2 6.2 9-9 4-3 3-6 33-9 18.9 2a 2 13.8 10.6 26.3 Average for all containers.. . Average for all containers for all 4 years 42.6 27- 4 13-4 12.3 24.1 33-8 20.6 6.2 II. 6 18. 1 Low temperature: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled . Bottle, air-tight. 31-7 37-8 27-3 16. 2 58.9 23-3 27.9 12. 2 6.8 48-9 4.9 9.0 I. 2 • 7 27. 0 S-o 4.6 2. 2 .8 46. 2 16.2 19-8 10.7 6.1 4S-3 33.6 34-7 26.7 19-3 44.8 17-9 22. 2 10.7 6.2 22.3 6.0 6.2 3-7 .9 7-7 3-3 S-4 2. 2 1-3 28.8 IS- J 17. I 10,8 6.9 26. 0 Average for all containers.. . Average for all containers for all 4 years 34-4 23.8 8.6 II. 8 19. 6 31-8 16. 0 4.9 8.2 IS-' Average for all 3 temperatures: Paper bag Paper bag, par- affined Cloth bag Cloth bag, oiled Bottle, air-tight 39-6 43-9 30-9 21-3 37.8 26.5 30.2 14. 7 8.6 48.7 7. 8 1 6. 4 10. 6 ' 5-8 S- I 4-7 3.6^ 1.6 30. 4 ' 48. 7 20. I 22.6 13- 9 8.8 46.4 35-3 36.9 29-7 20. 0 41.9 23. 0 24-5 12.4 9-9 25.0 6.4 6.4 3-5 3-1 8.2 5-2 7-6 3-6 2.6 29-5 17- S 18.9 12.3 8.9 26.2 Average for all containers and tem- peratures . . . 38.7 25- 7 II. 5 13-4 32.7 1 18.9 S-5 9-7 490 Journal of Agricultural Research Vol. XXII. No. 9 Table I. — Germination percentages of seed stored in different containers and under different temperature conditions — Continued Temperature and containers. Fluctuating temperature: Paper bag Paper bag, parafifined Cloth bag Cloth bag, oiled Bottle, air-tight Average for all containers Indoor temperature: Paper bag Paper bag, paraffined Cloth bag Cloth bag, oiled Bottle, air-tight Average for all containers Low temperature: Paper bag Paper bag, parafifined Cloth bag Cloth bag, oiled Bottle, air-tight Average for all containers Average for all 3 temperatures: Paper bag Paper bag, parafifined Cloth bag Cloth bag, oiled Bottle, air-tight Average for all species. 51-3 55. 5 43- 7 31- I 60.6 40.8 45.9 23. 2 62. I 46. 7 39. 7 37-3 37- 5 40. 6 3C. o 20. o 58.5 37-3 23- I 25. I 19.9 14.5 SI. I 26. 56-5 28,3 21-3 16.3 49-3 28.3 27.6 31. I 18.3 13-4 48. 12-3 14.3 II. I 9. I 50. 6 16. o 18.7 13-3 9.6 50. 2 Average for all 4 years for all species. 31.6 33.9 26. 3 18.0 54.6 32. 9 34. I 36.8 '7-9 30. I 54. 7 34.7 27. 6 20. 8 28.4 Possibly the germination test was not continued long enough to deter- mine the comparative germinative ability of the seed. WTien the test of bottle-stored seed was continued in 191 2 for 270 days, an average germi- nation of 58 per cent for Pinus monticola and 76 per cent for Pinus strobus resulted. This is in contrast to germination percentages of 8.2 and 30.4 for the 129-day period shown by Table I. Again, in 1914 when the test was continued for 155 days, the average germination percentages were 42 and 58.5, as contrasted with 26.2 and 48.7 for the 126-day period, also shown in Table I. When the germination figures are curved, how- ever, they indicate that had the tests been continued longer during the years 1910 and 191 1, there would have been a considerable increase in the germination percentage shown by the tests of those years. It is believed, accordingly, that the figures in the table are a very fair cri- terion of the comparative value or condition of the seed for the years in question. This does not explain the reason for the sudden jump in ger- mination of bottle-stored seed in 19 14. Frankly, the writer is not able to assign any definite reason. The delayed germination in nurseries of these two seeds, particularly Pinus monticola, always is a source of trouble, and it is not surprising that in these tests they have not followed a course similar to the other seed. There is much to be learned about these two species. In addition to pointing out the very evident superiority of air-tight storage. Table I shows that the other containers should in respect to their merit be placed in the following sequence : paper bag paraffined, paper bag, Nov. 26. 192 1 Storage of Coniferous Tree Seed 491 cloth bag, cloth bag oiled. This is somewhat interesting. Small lots of seed of a few pounds are perhaps stored more often in a cloth bag than in any other container. This study indicates that on the average an ordinary heavy manila type of paper bag would, if tied at the top, be superior to the cloth bag. If the paper bag can be treated with a coat of paraffin, it will be still better. In fact the germination percentage of Pinus contorta in the paraffined paper bag was slightly greater than, and of P. ponderosa nearly equal to, that of the sealed bottle after one year's storage. The oiled cloth bag is decidedly inferior. This is much less pronounced with P. ponderosa than with the other species. Whether the general inferiority of this container is due to penetration of the seed coat by the oil and consequent injury of the embryo, to the prevention of access of water to the embryo when the seed was sown, or to some other cause is unknown. Regardless of the reason, the truth is evident that such a container should be avoided in storing any of these seeds. The superiority of the bottled seeds over those in other containers is in all probability due to the almost complete suspension of physiological activity by the seed thus stored. (This was not verified by any experi- ruents undertaken in connection with this study.) The two conditions essential for such activity are warmth and moisture. The seed in the bottles was of course exposed to high enough temperature to induce respiration, but the necessary amount of moisture for any great degree of activity was not present. Such moisture as was present in the seed or bottled air could not be increased by additions from without. On the other hand, the seed in the other containers was intermittently sub- jected to both temperature and atmospheric moisture conditions sufficient at times to induce rather active respiration. Such respiration can be carried on only by using up food material stored in the seed itself, with the consequent gradual weakening of its germinative ability. The seed stored in the paraffined paper bag and the plain paper bag were, it is believed, less subject to changes of atmospheric moisture than that stored in the ordinary cloth bag. The rate of deterioration was in consequence less rapid. It is quite generally held by forest tree seed investigators that the true criterion of the quality of seed is not alone its germinative ability or viability (germination percentage) but rather this germinative ability in conjunction with germinative power or energy. Rapidity of germina- tion (the germination percentage at the end of a certain period of time) is the measure of this germinative energy. This period is measured from the date of sowing the seed through the time that germination is pro- ceeding steadily and rapidly and at the end of which it starts to fall off rather abruptly. If the progress of germination is plotted and curved, the point at which the curve begins to fall off rather abruptly and flatten out will represent the number of days which should be selected for that species. Now, the quality of the seed will be determined by the mean of the germination at the end of this period and the final germination percentage. By final genninative percentage is not meant here the absolute final but rather that at the end of a reasonable period. Accepting this hypoth- esis, Table II was prepared, and the curves in figure i were drawn from it. They represent the quality of each species of seed stored in each container at the end of one, two, three, and five years. The periods 492 Journal of Agricultural Research Vol. XXII, No. 4 selected for the measure of germinative energy and final germination were as follows: Engelmann spruce . . Dou^^las fir Lodgepole pine Western yellow pine. White pine Western white pine.. Period of Period of «emunati%-e final ger- energy. mination . i; 3^ IS 30 55 80 45 105 35 115 45 "5 The germinative energy periods were selected through the use of the germination curves as the guide. Other investigators may not agree with these periods, and the writer will admit that other lots of seeds might indicate periods of different length. The period of final germi- nation is the same as that shown in Table I for the year 1910. It was necessary for comparative results to select a period not longer than that over which the tests were conducted in any one of the four years. The tests were discontinued sooner in 19 10 than in any other year, and this led to the selection of the test period during that year. Table II. — Quality of seed after storage for i, 2, j, and 5 years Species. Picea engelmanni . Pinus ponderosa. Pin us contorta. Paper bag Paper bag paraffined . Cloth bag Cloth bag oiled Bottle air-tight Paper bag Paper bag paraffined . Cloth bag Cloth bag oiled Bottle air-tight Paper bag Paper bag paraffined . Cloth bag Cloth bag oiled Bottle air-tight Paper bag Paper bag paraffined . Cloth bag Cloth bag oiled j Bottle air-tight Pinus strobus , Paper bag Paper bag paraffined . I Cloth bag j Cloth bag oiled I Bottle air-tight Pinus monticola Paper bag Paper bag paraffined . Cloth bag i Cloth bag oiled • Bottle air-tight Pseudotsuga iaxifolia. Container. Quality expressed in percentage after stor- age for — I year. 42. O 44.2 35-6 26. o 66.6 52.2 53- 8 .54-9 36-4 64.8 59- o 66.4 44-7 15. o 70.4 20.8 26.0 15.0 6.8 46.3 29.9 32-9 24. o 17.0 49.6 ] years. 19-3 33.8 38.6 26.8 19-3 76.6 39-8 41. o 39-6 39-7 58.0 .54-3 55-2 47-4 17.8 73-9 II. 2 14. 6 8.8 5-2 47. 2 13-2 13.6 7.8 5-8 26.6 17.8 18.2 9.4 7.8 16.6 3 years. 11. 7 12. 4 5 55 27' 39 38, 26, 46, 33-^ 37-6 27. 7 13-3 56.3 2.8 3-3 2. 2 .6 32.4 3-0 4.2 14.8 3-2 2.4 1.6 1-4 2.9 S years 5-4 6 6 4 4 3-6 35- o 33-4 35.8 21- 5 20. I 47-5 36. I 33.8 ig. 3 9-9 55. 3 •5 •9 •5 . 3 27. 6 30 2-5 3. 2 .8 22. 6 3- r 4-4 3. 2 I- 5 17.3 Nov. a6, t9it Storage of Coniferous Tree Seed 493 These curves are interesting. They emphasize more than ever the superiority of air-tight storage over any of the other methods. It will be recalled that, judged by final germination percentage only (Table I), the quality of lodgepole and western yellow pine seed stored in paraffined paper bags was at the end of one year practically equal to that of seed stored in the bottles. The curves adequately dispel this idea of equality. The bottle-stored seed of western yellow pine and lodgepole pine excels that in paraffined paper bags by 1 1 and 4 per cent, respectively. Further- more, at the end of five years the bottle-stored seed of all species except western yellow pine is practically equal or superior to that stored for only one year in cloth bags, and the bottle-stored seed of western yellow pine is superior to that stored for two years in any of the other containers. Douglas fir, Engelmann spruce, and lodgepole pine seed stored in bottles, western yellow pine in oiled cloth bags, lodgepole pine in cloth and oiled cloth bags, and western white pine in paper and paraffined paper bags show some appreciation in quality at the end of the second year over that at the end of the first ; there is in general a marked and fairly uniform de- terioration of seed for a 3-year period, after which it is less rapid ; the pre- viously expressed relative merits of the various containers is confirmed — that is, in the order of their merit they should be ranged in the sequence of air-tight bottle, paper bag paraffined, paper bag, cloth bag, and oiled cloth bag. The oiled cloth bag is so inferior that it should receive no consideration at all for seed storage purposes. EFFECT OF TEMPERATURE Of the three conditions of temperature under which the seed was stored. Table I clearly indicates that the highest average germination percentages were secured with that stored at the indoor temperature, followed in order by the fluctuating and low temperatures. The differ- ences in the average germination percentage for the indoor and fluctuating temperatures is only 1.8 per cent, but the superiority of indoor tempera- ture conditions over those in an ordinary basement or cellar is indicated by an average excess germination of 6 per cent. This general superiority, it will be noted from the table, is consistent with all of the species in- volved. Here again, however, the superiority of air-tight storage is evi- denced by the fact that the seed stored in bottles at the low temperature shows no inferiority but in fact a slight superiority (0.3 and 0.4 per cent) over that stored under indoor and fluctuating temperature conditions. Leaving the general averages in Table I and analyzing the results in Tables IV to IX, it will be noted that the superiority of seed stored at the indoor over that stored at the fluctuating and low temperatures seems to vary with the species about as follows: Engelmann spruce, Douglas fir, western yellow pine, western white pine, eastern white pine, and lodgepole pine. It will be noted that the two species, Engelmann spruce and Douglas fir, most susceptible to deterioration in storage were the most favorably affected by storage at the indoor temperature. The better results from storing at the indoor temperature was most marked at Halsey, New Haven, Lawrence, and Ann Arbor. Poor germination resulting from storage at low temperature was most pronounced at New Haven, Pikes Peak, Pocatello, Waukegan, Halsey, and State College. 494 Journal of Agricultural Research Vol. XXII, No. 9 Table III. — Germination percentages of seed at the several storage points Location. Ann Arbor Dundee Fort Bayard Halsey Ithaca Lake Clear Junction Lawrence New Haven Pikes Peak Pocatello State College Warsaw Waukegan Picea engelmanni. I9I0 1911 191 2 1914 53-7 44-9 28.3 13-2 25-7 18.5 IS- 9 12-9 1 6s-7 66.5 56.8 30- s ! 45-6 44- 7 22- S II- 4 43-5 41.9 24.9 14-4 SI- 3 56.7 3S-9 16.3 22. I 18.4 12.4 3-4 42.7 29- 3 19. I 6.1 ! 59-6 53-8 S4-2 43- 0 49-5 61.6 47- S 2S-0 46.4 32-5 19. 6 14. I 30- 0 15-6 II. 6 12.3 ! 40.5 33-4 20. 9 14.0 1 Aver- age. Pseudotsuga laxifolia. 35- o 18.2 54-9 31- I 31-2 40. I 14. I 24-3 52-7 45-9 28- 2 17.4 27. 2 191 1 191 2 16.7 10. 2 13.8 9.9 34-4 18.8 15-9 8.9 17-3 II- 7 31- I 15- I 11.9 9-7 II. 0 9-3 28.0 21. 6 25-6 14-7 12-5 9-3 II. 0 8-3 IS- 5 10.4 8.; 9' 13- ■ Location. Pinus contorta. Pinus ponderosa. 1910 1911 1912 1914 MV: 1 -- 191 1 1912 -- ^agr Ann Arbor 56.0 36.8 44-2 36.9 46-5 39-9 39-7 40- S 30.8 38.3 50. 2 41.8 32- I 24-3 33-3 22. 9 28.2 SI- 9 32-4 30.1 40. 6 22.3 29. 0 S5-6 44- I 32-5 24-3 33-3 40. 0 42.8 55-6 77.6 75-2 78.1 73.8 59-3 69.9 75-1 72-5 67- 4 66.9 58.4 45-8 49-2 61. I 52- 7 52-3 69.8 68. e Dundee 52 69 61 64 70 44 38 56 64 66 35 68 8 8 6 8 I 8 6 6 5 2 5 Fort Bayard Halsey 63. 2 j 60. 7 so- 9 i 57- I S3-S S3-0 52-9 SO-S S8-S 1 43-8 56. 1 44. 8 66. 1 cs: T 72- 7 73- 9 Ithaca 44-1 I 7S-S 46. I 1 7S- 8 38.9 68.8 42. I . 74- 4 57-5 ' ■ 7S-7 51.0 1 75-8 40. 9 ! 71. 8 34- 7 76- I 63. 5 67. 8 65. 2 , 69. 5 Lake Clear Junction. Lawrence New Haven 28. 2 46. 8 Pikes Peak 57- 4 59-7 64. 9 66. 6 Pocatello 63.8 48.7 so. 3 48. s 54-4 SO- 2 39-9 SS-8 State College 49. I 60. 0 Waukegan ... . 47-3 65-4 ' Location. Pinus strobiis. Pinus monticola. 1910 1911 191 2 1914 Aver- age. I9I0 1911 1912 1914 Aver- age. Ann Arbor 36-4 29-3 51-8 40-3 39-1 48-9 20. 7 35- 2 49-9 54-3 36.9 23-4 35-6 26.3 21.8 44-9 24.9 26. 9 36.0 10.0 15- 5 35-0 40-5 23-5 9-9 19-5 10.5 8.2 29-3 10. I 8.7 13-5 7-9 6.8 19.9 19. 0 9-4 5-6 7.6 10. 2 10.9 16. 7 10. I 10. 2 14. 2 II. 0 20. 9 17.6 35-7 21.4 21. 2 28.2 12.4 t6. A 32.8 30.8 42.6 33-9 31.0 31- I 23-1 35-3 38.2 35-9 33-9 27-6 29-3 II. 0 19.9 52. I 37-9 28.7 19.8 8-5 11. 6 10. I 14.0 14.0 5-3 Ij- 7 6.4 5-6 6-5 6-1 5-1 5-8 4-6 1.6 7-1 10. 3 6.9 2. I 4-6 11.9 9-3 27-9 S-9 7.0 12. 7 7-3 5-4 16. 0 9.1 4-3 4-7 4-3 15. 5 Dundee 16.4 Fort Bayard Halsey 32.3 21. 0 Ithaca 18.0 Lake Clear Junction. Lawrence New Haven 17-4 10.9 Pikes Peak 30- 6 1 33.9 20.0 t 33.5 9.4 I 19.8 17.9 State College Warsaw 14-8 10. 0 Waukegan 9.8 18. 1 12-9 Location. Ann Arbor Dundee Fort Bayard Halsey Ithaca Lake Clear Junction Lawrence New Haven Pikes Peak Pocatello State College Warsaw Waukegan Average of all species. 46. 1 38.2 55-8 44-8 45- I 49.6 34-5 43- 7 53-3 36.8 26.3 39- o 34-6 28.0 21.6 38.0 24.8 25-8 30. 2 18.4 19-0 34-9 33-0 24- o 14-5 Average ofaU species for all 4 years. 21-4 IS- 6 35-5 20.8 21-5 2S-8 II. 8 13-8 35-3 28.3 19.0 13- I 19 I 32.0 27.0 46. 1 33-2 32.9 37-3 22- 5 35- 7 40- 1 39- c 29-3 21. 7 29-9 Nov. 26, 1921 Storage of Coniferous Tree Seed 495 Average for each container at all 3 temperatures for all 4 years. •jqSij-jiB aniog 1 ■w 4 \ 6 t- •paito 3Bq qjOQ 1 Si ° " ^ 1 t 0 0 f •3Bq qjoio 1 cj « ;^ oc - ■pang -jBiBd Heq jadt;,! i « 00 >/l 0 i^ d, r^ 1/ •Scq jadBj : Tf n CI 0 i "^ "" S, S' •SIB3A 1' i[e JO} sJsnrejnoD \\ti ioj aSBi^Ay 0 "io " 6 fo f*^ Tf ro 1 0 li 0 0 n 1 >^ li 06 d* !-• » 1 CO •» t", 3s3 m 4 1 T'? : 4-> a •0 •sjaniB^noD IJB JOJ 'aSBjaAV 0 « ^ i « « w ov •nj| mor^ltil « »ndind|| sd-^fOMi •V\Svx-iye ajjioa 000 'i r^ r^ II 1/1 U1 0 d « w 1 r^ t^ 1/1 r-ll oovn ti mintn in|| ■4 doovn w cJint^ >nj ■o II -^so r- 0 /i 0 0 1 d M 4 >n 1 0 0_^ 0 II m 0 >n 1 W5 II r^ 1 00 t^'^^^ r^ 11 \0 t>. r.. 1 t-» || ■paiio 3Bq qiojo 0 0 • 0 1 : 1 ° °^ 1 J^sS^ o 4 W M ■ o 1 •3Bq q^op 0 /i M w d 0 :i ;M 0 0,^ 0 •* t'^ 2 TOO Tj- -i- " -J- to •a •sjaniBinoo IIB JOJ aSBjaAV 0 00 a. i>>o6 d t ^ ^ ? |i ^0 0 m 0> wi M m 06 d f^ m 0 W M O 1 |s» |] sd A M 4 1 •*0 N 1 t 11 •;q3r|-jre ap^oa 000 inoo >>. 00 lo r~ 0 10 0 06 t^ 't t^OO 00 N 000 1^ inoO|N|| ^ dti'* -d «Me;rill 00 t^r^t^ r^ oooooolooll •paiio 3Bq qjop 0 0 >" c; 0 m m en 0 m o in dv ■* >d 4 " M M so m t Mm 111 •SBq qiop 0 d t5-fl-d « modi ■«• 00 r^so t^ T^ fs- M Tj- II • 0 1/1 >n 3BqjadBd^ i ^^ ^ 0 0 ui >n O OmO m| OOmlmj ^ sS-tiR s^ 1 ts^'' 1%! 1910, 28-day test. •sjaniBjnoa IjB JOJ 'a3BjaAV M M 0 i>io d t^ t CO Tf ci 06 MD r^ 1 00 OS w r^ c? so so >0 so sd ds M m ■* m fn 1 rr li •iq3ri-jiB apjoa vn 0 0 d vd -i %0 so 0 S r^ t^ t^ vO 0 t~ 00 OOOlr^ Omo wl d r^dsdiltH >-* o f^ ool r^ r^vosojr^ sor-t^ sol •paito 3Bq qiop >/-! 0 "1 di " i i « moo •4 M fn so t^sO n o O m N so ■* m M d O w t « ■3Bq qjop in 0 0 dvo 06 n r- -^t 4 1 • .no 1 •:«" « ^-v "1 o so so tn 1 mmo m CO so o sd sd o 1 t m m 1 •pang -jBJBd Ssq jadBd 0 0 •0 c 3 a.' > C .- tJ S; 3 3 0 § •a 0 4; bs a > < 'i 8 s 3 a > < be ■: e • 'l' •1 I-. ^^■§ 3 a > < 496 Journal of Agricultural Research Vol. XXII, No. 9 &i J •5r° •qilSll-JlE 51110a 0 0 0 in iff ^ « K •paiio Scq 1110I3 | "^- ^ '^' <^ S « ? C »« o 1 V) o> -V O •2Eq moQ 00 . ^ - -^ 1 ~ r^ ■ M £ B E '^ ■pany i u-. -^ r- « -jBiBd Scq aadc J | S" 4 "^ IS > 8S M < 1 Oi o » r. •3Bqj3dEd 1 ^ .:. .o ^ •SJB3-^ t" l]E JOJ saantB^uoo hb joj aSEJSAV «0 M 00 •0 roOO « r^ rn •« 4 ^■^■^^ ■* M 06 t^ ■a- IIB JOJ aSBjaAv 1 J j-^ in ■^ 1^1 HI M ■ 0.6, : 0 3 S ■o 0^ 1 "> o,^ i 1^ t^^ — n in 0 •« 1 f- 1 t~ >noo <^ 1 n 0 >n II ^1 4S:K; 4ll 0 m • t<5 i v6 1 •?Bq jadBd 0 0 ■ 00 1 in m 0 r- 1| « 1 .H f*3 ^ (*3 ,i ° : 1 "^ ; 0 ; '■ &. '■ 1 rsjamtinoo 1 ^. ° ° IJE joj 'aSBjaAV 1 5- ^ « W -^ ■* H \0 i^ 0\ 01 ■y, r- 1 S ■pan© SBq qiop j l°y my 0 0 "-. oc 11 :| : c. OC V •SBq ipoo 1 .; ^ 4 M II 0 ■*« 1 in II 0 m ■ 1^ S. -}BJEd Seq jadEj | ?o w 0 0 in 0 00 II M 06 >n -j 11 I-. inso 0 II 0 • 0 m 0 • r-o6 • 00 i 0 <«o •SBqjadBd: j-An «| o»n»n|i^|[ »no-joo -0 A woo i 1 ■■*•■«• rn r 0 ct Tt- 1 ^ II « ■ 1 0 m ■ is : 00 1 1 lie JOJ aSEjaA-^r | IJJ ^"S •* ■<)• ■»-© 1 "1 II M S, « 1 N n 0 0 1^ •* 1 i 3 1 0 0 m •jqSiVJiB aiHOg 1 ^5 .^ « ! vO vO -o ir, mmo 0|| OOOItn vO 1 r^io t^ vO !! vo -O ^C 1 *0 000 i >v 1 0 w 10 •pajio SBq qioio , 0, ^ » "S in m 0 1 0 1 mo- « 0 1 n 1 0 0 • t^ i op ■SBq qiojo 1 "2 7^ Tf -0 m b-. 1 m II M • mom 5-0 " 5> •pang ° ° ° -jBJEd 3Bq jadBj 5 S5 3j n o>nin|r~|| OOm oo 5 « 06 « 1 >j j .*o « ly 1 ?•?":• 1 f^O w 0 ■<»■ 1 1 lo 0 ■SBq jadBnO|0O|| mo- >n 1 moo 1 00 « 6 00 0 n 0 u 5 ■^ E i 13 C n 0 U T! 0 .. c 2:1 CO 1: 0 C £ t >4 1 •c c ^ c ^ < c r ►4 1- C c T 1: 0 0 < c : : : c ■ • • c3| : ; ils : a <^ 0 > T ^-c 5 u < Nov. 26, 192 1 Storage of Coniferous Tree Seed 497 00 00 1^ 00 ■ 00 'T 0 5 0 r^ "- 2 ^. ir, 0 ■» N ^. 0 «' :: ■^ 0 00 N t* 10 Ov ■» a ■» 0 0 t- >d m* w -0 06 «^ M 0 00 « d d t^ 1^00 ■* 01 10 M m 11 0 to in .«• w m tf 1 •v M M II N IH too 1 M « di-o 4 M M M M c^ Oi «^ 0 m H ■^ 'T 0 0 woo 00 1 m 10 i/i d. 4 A 10 ■O 1 -0 lo r- 00 1 000 in d t^ ^ « 1 00 too d 0 m m n 1 0 1 mom i^ •^ 6^ fh d t~ m t~ vO 0 0 w 6> " 6 d ■* ■ j *? " 4 0 >o « m 0 ; m dm- 06 1 ■ vC 0 0 06 v6 n" d> ■ 1 -^ • 06 0 0 0 ] IT) U~j »A ro 1 -T . m " ■ id M 00 1^ 4 vO m n ■t " o_^ 1 lO >d cj^ d m ov ■* 0 t^ a 0 4d« 1 o\ 0^ 1 ^ N I/-, 10 0 fl in 0 "1 1 t^ 0 m^ 00 m£. ^0 m m 0 0 mom 0 1 r^ 00 ^O 00 CO 1 r-^ 0 0 • 0 m 0 ; in l^ ■ vO in . 00 d '■ •£ ■ ■ ■ di 0 0 o 0 «- m. 0 0 m II 0 .n^ 4 1 1 00 vd M m f 000 1^ r» 0 0 0 m 0 If'^.'S 1 ^ 0 0^ 0 0 m, m r- 0 d *^ t^ 0 so w 0 -0 m in vO 1 00 " d 5 =; 00' <~00 6 1 0 0 m 1 r^o 0 >o 0 0 m 1 CO d 0 0 • is '■■ ,., m m, .0 m 00 9 E^-^k ^^ t^ ! IT, V, ■ 0 tr, 0 'n 7 4 m m • " 0 0 m m. II ^ CO It i^ 10 1 l^ 0 >o 0 a i^ j^ 01 0 m 0 j m 1] 10 lo 0 m m 1 r^o po 1 m II roo co ■T ■«■ ^ i^ 4mm ill i ir. 0 >^. t- 1 i^. 0 0 <^ 1 mom " 000 0 <. 4 -t r^ r^ N 6 0^ moo di « t^ 1 TTO m '^ 000 vo t- in 0 vO m. 0 m mo -0 0 m m, 0 m M C^CO M D- c^ tn \r^ vO 0 000 ^1 m m 0 M ^ 11 0 m 0 5- 1 "^o •;' *? 0 m, 0 c< 1 « 0 >^- 0 fO M 6 vO 0 1- :; f~-a5 d 1 rn r^ w 1 m 0 m 0 CO m •* ci I rf N 0 M t^ 1 0 0 m I 0 m, 0 °° jl °^ H 00 w -d 11 t^ ■^. 10 0 ■k r^ &• ^o 1 omm||r--| mom 1 n S"?! !| !* i 9 mS 0 1 °":'° 1 '^^■^ CO 0 m m 0 1 .c io 9 « ?o 1 -c- 000 m 0 m II 0 11 0 m m f~ ji 0 0 0 s 1 ^1 KS 8 'i '5 0 0 V ' t s u 1^ CO -^ St;! tj I' 4) 0! ..E N 5 £ ' oi 0 ! C 0 1 ; s-d !»" '-^ O S- in>2..i:i-t 498 Journal of Agricultural Research Vol. XXII, No. 9 Average for each con- tainer at all 3 tem- peratures for all 4 years. •jqaij-jie apjoa 1 "^ 1 ^ 3 °4 u 1? •paijo acq q^op 1 1 o « 1 « 1 o 1 ^ •3Bqq?op 1 ■» in \ n 1 o 1 M •psay -jBJEd 3Bq jadBj s " It u 1* •3Bq jadBj 1 |i n Ol 1 I- 1 (7. M 00 1 6 JOJ sistiTB^noo \\e joj aSBjaAy 2 2 2 « •^ 0 m d\o6 d -o m rt -.r to •* O n 1 TT Osdo m jn •4-> >. •a •sjauiBjnoo [JB JOJ aSBJaAV looOMiio oot^l^i MOlOliml COMOJfO 00 V),^.^ t^ [1 » AoinOoo Oc»(i|6.i o>Ao|^| tiinin* WM^^ toi i^ •jqSij-jiB 3n;oa ui o O 1 c« 1 OOOIOI >nio>oioO|| \n\n Q t^ 1 m ^ ^l ^1 c*5 ^ xrt\ ^11 M CI « « 0 "",-^1 <^ 1 o a t^S, 00 d> •paiio 3Bq q;oi3 ;l i • o 0 r~ . 1 •* M « ■I : S • *^ •3Bq qjoio :| : 1 1 : ° jI^ 1 : £| ; •pang -jBJBd 3Bq jsdBj *? « m ■ m| o d> ; jl 00 : J •3Bq JadBj • : d|" 1 "^"^l 2 : 1 : i 5) i ■V •SJamBjnoo JIB JO} aSBjaAV "'OOOltH -00\N oil -O Ov,^l « d.M-do6 dMM|M d. •;q3tvJiB ap^oa uiioiolooj omOiml m w^^ \r, uiioiolM omwjfo o 6»-*fo»o ood^wd* d M.2^ "d *o M M fo d »^oo wi -d •pane 3Bq q^op :| : 1 1: 1 O -.-^i m • • M : " ■ '■ '■ •M •3Bq q^op o • 1 o 1 1 ; 1 " ^£ ^ 1 : " " m •patrg -JBJBd 3Bq jadB J m o vol o 1 m m,^| o mo 2 ^^\ ^ ' *^ *? o "■ tiiv • 00 o •8Bq jadBj « lo o 1 O d ■ n 1 M 1 : m o,^ n • *? 00 M ir *^ 4-) •sjamBinoD IJB JOJ aSBjaAV t^ Oi >o t^ <^ 1^00 O 1 n M o 1 00 1 OnOltJI oomm ov ddi-*'* -i ^ w oo6 dv d ■«■ 5: ^o d d •* M CO m m m| 00 'd •paijo SBq q^ojo : :| : ? ■ m 11 o m o 1 00 1 • • ■ o m -^QO MJ : : 1 : :l i : •3Bq q^op O >o| 00 O m m to d>« i m 1 0 m 1 °° m o « 6 • 1 00 i 1 "^ 0 •pang - JBJBd 3Bq jadBj[ 0 0 lO] 00 o o 1 0 O 0 ml m o d o «^ •* -a- N 1 CO m 0 O 1 « tooo m t^ t^ M m 00 t^ •3Eq jadBj Omo «| 0 ■ •I'^l >^'00|0 wioioooil oo dvvo^vd «••* lo vioo (o 00 00 * VI « t^ 0 t^ c c ? >. T3 6 o" •sjaaiB^noa IjB JOJ agBJaAv o I-t ^1 w 1 1^ r*oo 1 w 1 00 00 w « II 00 ^vo o | m oo 't « || :• d«^r^|d d^noO|00| ioo>o|0| 10U10 dvi^oo-^ vdMd\d( loto'd n ommioi OOmlm « Awmdv dd*M ■c •pajio 3Bq qiojo : ° ■ 00 . M • VO 1 i i III \li\^\ n : l\ I" :|" 0 •3Bq q:>op O loiOj 0 1 wo |io| .omjOO moo «| OmO|m| u .p9r>.Md ^om i^ oOMwd ( 1 t .^(OlN MCO Ml CiOMlCll T •pang -jBJBdSBqjadBj O O O 1 fO lo •*'d 00 Oiomltol mmo >^ 000]to| mooim u todit^pd t^dtipd ^ •rftowltol wmtolto ■! •3Bq jadB(i •noOjUih moo mi omoooj momlf^l oo>o|«| u ■* + dds «oti« d\N-*M MOv^od'd t^oo d >• Hciwlwll 1 W(*3fO'*>l fofow'wl c»(^«'(*;l ^ ,2 •5 a o 3 « V a. § a rt 9 'S a (U 00 a o m 1- a < O •§ 4; : 2 3 o o •a c 5 3 2 > < S : : : 1 1 I > < 1 a S : 11 2 > < oi '. ■ a : -3-0 CQ -> ; si ►4 > u St jl :ii 01 -I Nov. 26, 1921 Storage of Coniferous Tree Seed 499 ■* ^ 2 00 5 ■"t Oi f 0 ~^ ° 11 "^ ; ° 00 « ^" „mO>0 0 r- t^ i^ 0 0 f' ^ d 01 .«■ CO .» 0 '^ ^ d JO ■* t-0 0> HI 06 d> 0 •* m 0 ~ 6 -k 0^ CI [I r-- » -t «-^ w 11 0 d> 0 " 00 0 t^ 06 d\ " 0 d 0 -^ 0 1 d c 1 0 00 dv d> )"oT 0" n r^ 0 to "t M C - 0 0 1 0 1 1^ « -o : M ^ -O 1 0 ^ .1- Ov 1 lO-O Ov "1 II t~ 0 fO w oocir^ in o6f^»n di o^dr^ f.. 1 On 01 m 0 0 (O ■* 1 00 u^« 4 06 ^ 1 ^ 01 r^l M 1 r- « vd d 0 0 0 m dv w 0 1 00 V. 1 M 1 000 d « i^ r- Ommjr-l 00 m OS Hdd\f^ oodo m mom H 40 ci to to H C« 1 m 1 i/i 0 0 1 00 I « 1 ^ W M 1 W 00m m II r- t^H to d|| 0 H " 1 1 . 0 m' ^ 1 II 'Tl 1 ' "^ T " !l 1 \ ^^ M "? i 1 1 0 0 1 ^s r^ 0 0 1 1 : 1 C^ 1 0 0 -* m 1 ° T "1 1 : 1 ^ oc < 1, M 1 ■♦00 0 1 t^ II vo r< c^ ro 1 r^ to t~ 1 « * 1 ?)S"° 1 ^ m 0 t~ •» d d\ -5- d oo5^ a 11 r^oo 0 00 tooo 0 1 "t 0 dodo ti »n»o 1^ 000 r^[ »/iOO loti ■«- n-^d 00 r^io- N| OOm|a)| 000 0 d»o ro cs m moo • < ■a 0 0 1 0 0 0 06 « -d •y 1 : 1 ^ CO >n 0 oi . 1 cm 1 ^ 0 0 d to to 0 "? 1 : •no 00 (^ <> 000 fo »o (n 0 0 0 m >o >t ■* 00 com 00 II omm §vS^ Kill aij CO mom tH tOOO t^ m rf to m to oomoo ommr..jt>. 0 M 0 r^ ^ c^ fooo di i-i 1^ mmmm tttm-J-lm m 0 06 T " *? " 0 m 1 ^ 0 m . 06 ■ • dv : : ■ l'^ 1 ; t 0 m M II d M r^ • 1 cm '■ \ ^ -k ■a- m 0 1 moo ci do " m m 0 . ci ■ kO • 1^ 1 0 0 m| N c m to ■ 0 c 0 0 !^ t^ 00 mo 0 moo ci 0 d vd -i- dv • 0 06 1 i d d • to m 0 0 r^ •o 0 •l d\ M mo 00 to m m 0 ci 9 c^ 06 M 0 M : 0 mp m m ■ 1 0 m d to • 06 ci « 10 -0 ■*vd d 0 L t Ov fO m vO 0 f^ 1 -^t II fooo 0 06 d(ot~-d d-S"* 0 1 m too to ci m d [00 N 0 00 1 C^ "t o>oo 1 -3- 00 lO 0 I- 1 \n 0 V 9,y 5^^ m m 0 M m -t 0 1 m m 0 f \ * ^ m M d ci m . 00 1 m 0 m| 1-- 1 (^ w ci ti t^ 1 m 1 ^ m to 1 t mom t^ 00 t>. 00 t^ 1 00 C^ 0 . 0 • 0 ■ tr, m to II 00m ' to -^ d\ 1^ d^ ' 00 mom 06 oh' t^ Ml H : : m^ ■ m 0 06 1 mm to mom t^i omm d t^dm t^ MOON ^- -i-o 0 1 ti t/1 0 00 m 0 00 d 00 0 a _■< 0 i 00m vd d 00 06 10 >o 0 N d>-d M to il mom to 1 00m d\ mvovd 6\ Ommifoll ommjr^l ooo|t'||o toAcioo ommm d\d.-t|do ■* -* m 0 m 1 mom 00 t^ t^ ci r^ moo GOI OQio in 0 N 06 CO 2-0 VI N| moO|00| moO|m 06 oot^oot^l oocimoo tood ^ M ti 1- c 3 1 t- 1. > < 1 la 1 c c ■c c 1 n a u > < 1 a V 0 0 ■a c c a > < II 0 C I £ > < 0 0 0 ■0 c & 3 0 > ^5, Sit; a § •0 3 > < it 01 5 8 > d.a 1— t 4-t t-t 8 •a & 3 at < 1 2 £ 0 v 2 > < 7.5.308—22- 500 Journal of Agricultural Research voi xxii, No. 9 &i "O •jqSiVJiE amoa 1 0 **5 vO ><> 4 1^ •paiio 3Bq mojo t~ 0 « 0 "o M <5 «• 00 jj >0 ■* r- >o -0 Average for < tainer at a peratures years. •3Bq qiojo I S ^ s •pang -}B4BdaBqi3dB, t^ 1 -^ •«00 M rid - vO l^vO vO 00 vO m *i d m ■O J~vO .0 00 0 •sjatriB^uoo j|B JOJ aSejaAV 1^ « 0 "1 000. 0 -0 ci fO c- d mpj Pi r- r^ r^ r* 11 <> ■^ -* 1 « fi 1 »0 ri r^ d r^ 1! 100 vol 0 vO vO" -0 1 ■jqSiVJiB smog 000 «- 00m vO cc e^ vj 1/1 0 ^ ^ rn v/) M •^ 00 r^oo 0 d m m 0 1 r/-. tivd m vd r- rv. f* I r- m 0,^ 4odS, vd •paiio 3Bq mo|3 0 ^ 1 0 0 m n 000 0 t-- «^ 0 0 0 *^ »o & diS. TT m ill •3Bq moiD 000 0 1/1 vn 1^ 00 £> " « f^ 10 « 00 0 m 0 moo 00 m t^vo ti W « M 06 ^1 « t^ Tf( m 0 °.-^ ti-dS. vO vO 0 4 vO •pang -JBJBCJ 3Bq JadB J moo m 000 uiod N oc d oe 0 0 xnO ^ 06 w NO C> t^ 11 0 0 0 1 r^ r-. 1! 0 "O o 1 ^o m. 0^ ci odS, ■0 m III •3Bq jadBj 0 VI 0 00 "O vj 0 M d« d vd «^ »^ d T r-00 -0 r^ 0 m m t- t^ t^ 0 0 m| 00 ri 4 d fO vO vO vO 1 0 1 "^ 0^ d mO, so VO 00 1 •sjauiB^noD IjB JOJ 'a3BJaAV f^ TT 0 ui w -S-00 00 Rv^^ 00 1 •-• 0 00 1 0 « 0- m m d 00 -0 t~ m 3! •jqSiVJtB aiwoa >« >« 0 00 i« >/l 0 dod d >>i ^ d pi r.. 1^ r~ r^ r^oo oo 0 0 O— , 1^ r-^^ t^ 1 0 m mj (^ 4od vd vd 1 m t^-o 1 vo 0 m m t^ t^oo «^ !1 •pajio Seq q^oj^ 000 1^ ^ t^ r^ N 0 *0 "1 nC U-, m 0 mnod r^, -^ -^ ■^ m 0^ W5 0 m mi 0 4d ^ vb ! \0 r* «^i VO 0 l«od IvO ■» 1 0 0 *ii ■3Bq qioo t^ tA 1^, 00 M M d in 1- t- vo •* >n r<) ^ vd 42, vO vO 0 »0 0 0 1 QO 0 "O r* li^l nO :§ 00 vO ■pang -jBJBd 3Bq ladBtj 000 0 1 0 m o d M r* 0 "— , ^dO, 00 0 0 0 f^ 06 od OH p' -0 so 0 ^0^0 "-1 0 vc c vg 1 r>od t^ m t^ II vO 11 •3Bq jadBj 000 "1 »0 ^ vO -O moo c>o6 M 1 1 "J- 4 m 0^ mtiO, ■O vO 2 0 0 0 1 r- sO 0 0 1 -O 0 0 N M 4 t^ m. r^ 1 ■* vO 1 ■sjamBinoa \\c JOJ aSBjaA-y 0.0 •* f- d ^. ;j d. 1 r- a M ^•6 6 1 r^«o t^ 0. M m,x mod " r^ r^ t^ 1 0 00 d "^00 t^ «^ m. ^ •iqSiVJiB ainoa 000 t^ >A,od ^0 06 <0 0 t^ vO mom 0 moo ti d 4 t^oo vO r>- Il r- r^oo 1 r^ «>d* r* >< T3 •pajio 3Bq q^ojo in vr> 0 0 3 <>. d, li 1 moo 1 r- mvO •0 vO 0 0 m rv^i m f^ r* f^ 1^ r^ 1 10 0 voi r- 000 Sv^S, ^0 1 c ■3Bq q^ojo trtv-.O m ro M 0 d mom posd vd 1^ 0 0 m 00 rr, 6 ^ 0 c-ai t~ M 0 0 0 1 I- 0 m m M M 00 t^ t^ m 111 Ov •pang -JBJBd 3Bq jadBj 0 >o >n 0 « d d >^ M d ti 00 000 a ti - m r- 00 vi^ioo mom r* t-.->o Sll •3Bq jadBj \nir,v. m >n 0 0 d. d fi t^ r^ \n ci ^«0 -o 10 r^ t^vo "1 0 -"O d md d t^ 1 t^ r^ I^ 1 CO 0 0 v^i 00 I ^-0 0 vA M d 0 0 m 6 fh fh t^ r^ m. •o II •sjaniBjnoo IJB JOJ aSBJaAV t^ d.\o r^ ^ ti f^ e^ 1^ t^ t^ l^ r^ i^ M 1 ^0 « m 0 M vd r>. 1 r^oo «^ 00 ci vd « 1*; 1 r^ r- r- r* 1 r>- 00 ■>)■ M »o f^ dv t~ r^ 1^ ^- 1 w •;q3iVJ]B a|«oa 0 >n m 0 vd d r^ 06 f^OO t^ t^ 1 mom ! t^oo t^ r^ 00 00 t^ « 1 0 0 0 j 0 •^ 4^0 CO ^d 00 I r- r^ r>. t r^ 4od d (^ vo r» t^ 1 •paiJO 3Bq qjoio moo m d w d d 00 00 CO 00 0 0 0 (^ t^ t^ t^. 0 m 0 4 00 M vd t^ moo i^ 00 0 0 »n 00 M r^ d od M t^ %o 00 "O r^ 0 0 m vd M ri I--00 r^ 00 2 •3Bq qiO|3 m V. 0 "n II 0 0 m r^oo »^ t^ \\ I-** 1^ r^ 00 1 moo dv dv d 1 VO 1 0 0 0 1 r- <> M dvvd 1 v^i 44-^ t^ 1^00 t 0 0- •pang -JBJBd SBqjadBj m m 0 0 1 U-, m 0 ^ f^ 0 00 r^ (>. fi 00 1^ r* r* 1 i^ 1-^ r^ t^ 1, 0 m m ml 6 ri <> r* 1 00 00 vO 1 f^ 1 00010 1 r* 1 t^ t^O 1 r^ m m 0 od d. d m •3Bq jadBj "-.00 n moo m Omo ^ oc 4 vd Mod r^ -^ ^ -^sd CO I t* r* r* 1 r* m m 0 «^vO 00 a 0 •5 g V 3 (8 t s ■0 a « a a 1 tn J3 il n < 1 0 » 0 i < S3 S 0 8 C &' 3 si 2 > si c 8 •0 c 0 a > < 8 •0 z 3 > 6 • • C 8 •0 c c CO > < Storage of Coniferous Tree Seed 501 O r^ O 00 O ^ aioO j ir r^ -(i- lA I -^ -"t 4 <\ 00 d "-^ U-) ir. O 1 t^ r^CO OC I t^ f- -^ O CO a> rol r^ O r- M _t>-)_'d-__ O OsO 00 f:!' ■O ^ nO O O v^. O O ID O O I r^ 6 ^ •>-. O O I in & .'•0__ o o ] ooii-o aw I olio in O O I M I 000 r-O O I O O O ' "-. 11 "J O O 1 w- 1 O M 00 O O r^ ^ UC 00 o o 00 tjO <*; 00 O 00 t ^C O O VO M r- II r-00 00 000 0001 : S ^ o be o.g > ji o >-3 r^ r-O 1 O r- t- M rl- •2^ ■SfcJ23 fig ni bt c« > "o tj o > -* S.H SE«3 ^ 0 (^ i -SBq qioj3 1 T ' 1^ w oi r* Oi '.n lO » 0 •pang | . -JEJBd 8Bq J3dE •aeqiadEj 00 0 ►- 'n »o \6 06 06 M ■SJB3A «- IIB °? T'^ 0 1 "'^T 1 5^? 00 0 - « 0 ij TO fO c« ^ 6 >o VI 1 TOC M T vio VI V) 11 T T T 'i 000 0 T T 1 1 a ■? o a -4- IIB JOJ 'aSBJSAV 1 v2 "2 4 ^ ll « N « °S ' >§'S-T " «o6o|tH| « 1^2/ 0 j vn O n •iq3lVJlB3l«oa 1 4"0 ^ r; - ,:. 1 WWVO '-^i'vOOO r- 1 V, 0 0 0 ! ? So ■1 •. 0 II 0 0 0 1 0 1 • 0 1 .0 •»! 0 ""--vl « ! •paito Seq qjoi^ ; jJ! ^ ^ | : "^ : M 000 VJ (n T 0 1 0 « A T 1 0 1 V, 0,^ 00 V. 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VI 0 1^ •' OO Oi m 000|0| -oviinll •SBqqjOQ 1 ^^% «i II >o VI 0 0 1 0 0 V, 06 w fi CO T 1 0 t^ VI 00 1 0 0 vi| VI 0 VI Vlj ro II ■pang 1 o >n o ti m « o -jBJEdSBqjadEj 1 ^ S S" S 9 i,^ 0|| ..v,.. r,|| 0 v,o ^ TO-I ?|f m\\ N 0 f^ 00 00 i^ i» ■» oil Ot^T vi) viTvi VI fi 1 .«u,or~ ./../>0 •3BqjadBa| .a;o o 53;. cTi 1 0 V, V, r<5 V, 0v>|0 VIVIQ t^l fh 6 TOO w **1 d A T 0 01-.tIo Oin ■* vil •sjaniBinoa 1 co 0 i^ 0 01 r^ - IjB JOJ "aSBjaAv 1 ilj, ? ■S, S S S 0> II VI 0 « VI II 0 0 VI oil vivitIviI vivim ml test. •,q3!Vi!«3moa| |n 1 1^1 T 1 r^ !>• V) r- II t^o 00 00 II vivivi|n| v>vivi|00| 0 Anmoo MviwA <^ll ooovilol oooot^lr-l day •paito 3Bq qiop | « A - ^ ^°° "' 00 1 V, VI V) vi|vioOv> -OOOj 06 Tl-'W T -MOO fO » 1 0 .00 ^ 000 •3eq qjop | ^^^ 0 | ^ ^ * <-l II 0 V, 0 01 660 T II 00 r~o ci| 0>/iO|V. 1 OOviwj n \ o6r.-to MTM A o 'paug m 0 »/> r- 1 1/1 1/1 m -jBJEd3BqjadBj g,*-^ f: || 0^^ V, 1 000 01 T M 06 r* 1 00 00 m n v>v>vi|oo| vioO|«j " onmo d"dr^| ooll oot^r^lr-j t^ooooit'-l •SBq JadBjj 00010 11 >no vi| f/) 1 t^ \\ '^. « d 06 06 T ci 06 0 II i-vivij V) 1 1^ r- vil 0 li d 0 •5 a 3 a ■a □ □ O 00 - 4 -r X '■ 0 b» ■ '" B '■ sis c 3 J ^ ..-1 2 — ts ^ -3S 3 > < f - c 5 8 0 a. r > < "a ?. 11 , r 0 0 -J c B d > < a 0 8 -a c 0 en 0 V > Nov. 26, 1921 Storage of Coniferous Tree Seed 503 >c ■o -6 p^ § 0 t^ . CO 0 M "•. ir, rr, 0 00-OOCX>%0'.« -O ^ rr -^ 0 ■0 0 c-dii 1 EEi ■* 1 5o^)K 1 •* -^o 0 jj r- ■ t lo-eTTj- m \l ^ ■^ r^. 1 H ^ 0, jl ^ ►, .-.-1 r- |] K. 1-00 \ ^ \l \ 6 11 -0- -^ 1^1 -^ 1 «n ..a- t^ 11 w 1 tr 1 0^0. 101 Or^'a-l'O (O^OltHii r^^t>/. 1 Cfl i U-3SO .^1 Ul vO P4 ttI .^11 f*^ M r^ 10 1 rto O0|'^l| ->Piin|r^|br, M 0 in 0 ^ j cl 06 a jj "t 0 .^ "1 0 ^ >^- 0 ^ ii^i Isc 1 ^o-o^oi<^ -ovooolf^ll o'^t^ 0 II >o 0 0 1 " 11 >^. m 0 1 "^ || f» -o II t~.o .0 1 .C II t^ t- i-~ 1 .c II 1/^ -fo 0 ■ 0 ■ 0 1 0 ; : r- O.Otnl"-. 0>n>r-^| U-, 00 ;| :° ]\'^-\\ :' ::: 7\: .r. 0 0 N ./■. 0 0 00 0 0 ; li-; 1 ./I 10 ■/-, 1 ir. II 000 <- 1 « ^.00 0 .own 10 0 0 ; 1". 1 vr>0>/.|l^ OOOl'^ll OO"-: I")! OvCwilio -Om^|.»il lO-Jfro lo. 11 0 0 0 1 f^ II 0 "1 "-; 1 r~ 1 10 i>. M &0C No 1 i^vc -a- n \ N 000 0 1 ^0 ^ « t^ M • 1 -^ >/-. 0 0 « vn vo 0 1 t~ li I/-. >/1 "-. »o ; 0 ^. 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Ij lOOO \o cooo OOco -^Oco Ol ^d-^ oo o 0 0 1 r- s, 24s>k •pang -jEJBd8BqjadB^ roll OOO w t^ -^ di »-■ •«■ ■* m col •>)■ •a^q J9dB j 00"l 00 II 0>oO <«|j OOO 0|| iriuiio d 30 0 >o rf 1 ■* S : : o : g : 2 .' > ■> < •: c a 53 »; •^s bt K- -S 2 J^S s fe- ll & s ^ Nov. 26, 1921 Storage of Coniferous Tree Seed 507 •* 0 »*:) ■^ 6 ■d- ■a 10 * O" " "/ T "^ ■ ° '°- 0 ..... . r^OTToooovOM " ■» 0 i n N 00 M -^ M ■* ■^ M W -0 ^ n CT. -* w w w 1 ~D 0 0 n d 6 M d ^S2 « " iVi <2 in n t~ 0, W H ■9 \ vd " -t 1 ° 00 0 0»j a II 0 r^vO 1 0 II 00 00 r^ 06 jj w t- 1 6 0 0 1 6 "'^ 2 M 1 0 00 Ov w d d d\o6 1 ■» tooo "1^1 »^ W P) d M r5 M d d d 00 d i 0 0 >o W5 <^ •«■ w 1 oow 06 r; M <5 m 1 rO'O t^ |M| OOw|W| 000 0 oow 1 w 1 -^-o M 1 t-2 1 -a- W W W wvo •* 1 00 0 w w w d, dv N 1 w t -t ->}■ 1 0 Ii W 0 W; 1 0 II WWO t^ M d d ^ to i^\d 1 Tt 1] w wvo 1 w II w -a- ■* 0 1 1 li 1 ^ 0 1 ^ 1 \ ^ ■ 0 1 -o 1 j wo ! 5 ^ u ^ 1 ^^ \ 1 1 1 "'^ 000 •-I O-^OS « MM M 0 0 M t^ •a- r^ 1 0 1 0 -d d 1 r« 1 to 0 1 ° |. 1 I ^ ./I wo -^ 1 1 0 0 1 w -a- Ii i 0 0 \ <5 ■ r~^ r. w M o>r~ Ov 1 >ri 0 n 00 «i 'O 0 •*■ -0 •ri 0\\0 cS M ON w 1 0 W D^ d M i^^ •a- 1 (N 00 0\| 0 [ d ^c -* w| w 1 to w a doo 4 -0 1^ 0 0 0 w 00 0 WW 1 10 1 w w 0 r~ <>. 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" M 3 0. 06 w«0|w|| wwo to 0 w W w 06 w ■* 0 0 ; 0 t- WOO t^ OV d oow « II woo 06 5 *" •* W OOW|W oww|t^ ■5- «6no6 voni^o owo W^O 5 ,, , wow 01 w 0 0 00 tK w M -a- vd ■* ) to Tl- to 1 to OWO ? 5 www W M WOO Ksi all -^2 w woO|N|| wwo n wow W W 06 M t^ ^0 6^'0 t^ 06 6 t^ Ml fO-^T-llr-lll WWM ^ ^O^Oc*) to j woo w vd d « Pi 0 0 w w to 1 ^6 w • d w 5- lo in. 0 .0 ->« 0 = 1 • h a '^ > ■< 1 h h5 1 0 9. d at; l| to 2 1 S 3 0. 2 > < ^1 Pi B 1 •3 > b 61 0 0 0 ►4 I > < 0 0 -3 r 1 h4 as > 0 0 •0 c &' 3 > < to § c 1 s 1 -*- c g 2 c 01 bi n 5o8 Journal of Agricultural Research Vol. XXII. No. 9 Various experiments with tree seed go to prove that storage at a uniformly low temperature (o° to 32° F.) is preferable to that at higher temperatures. It is easy to understand that at such low temperatures there would be little if any physiological activity of the seed and con- sequently little wasting of stored-up food and energy. Had the seed in this study been stored at such low temperatures it is very probable that it would have excelled in germination that stored at the other two temperatures. In this study, however, the seed was stored in basemient or cellar where the summer temperature was doubtless fairly high. Moreover, as is usually the case in such locations, the atmospheric humidity was also doubtless high. The conditions, in fact, during summer were such as to be more conducive to physiological activity than under the fluctuating or indoor conditions of storage. Hence, it is reasonabe to suppose that there was more of such activity with the consequent deterioration of the seed. The lesson to be learned from this particular phase of the experiment is to avoid ordinary basements and cellars for storing seed in unsealed containers, and that in northern temperate climates storage indoors where the temperature never goes below freezing is preferable to storage where the temperature follows the natural variations. EFFECT OF GEOGRAPHICAL LOCATION Ordinarily, the seed dealer will perhaps have to store any seed which he possesses at his own establishment regardless of its location. It v^nll be a matter of interest, however, to learn that some geographic locations seem much more suited for the purpose than others. For seed in con- tainers other than the sealed bottle, there was a very consistent superiority shown in the germination of that stored in some locations and a very consistent inferiority of that stored in others. Table III brings this out. Note, for instance, the consistently high relative germination of seed stored at Fort Bayard, Pikes Peak, Pocatello, and Lake Clear Junction; on the other hand, note the relatively low germination of seed stored at Dundee, Lawrence, New Haven, Warsaw, and Waukegan. These are averages for all containers. Knowing, now, that seed in air-tight bottles is not so much affected by adverse conditions as that in other containers, it is believed, accordingly, that it should not be considered in determin- ing the effect of the geographic location upon stored seed. The effect is better shown by the behavior of seed stored in the cloth bags. Using the averages shown in Tables IV to IX, it is found that the towns range themselves in the following order: Town. Fort Bayard Pikes Peak Pocatello Lake Clear Junction Ithaca Halsey Ann Arbor State College Waukegan. .. New Haven . Dundee .... Lawrence . . . Warsaw. . . . Average gennina- tion per- centage for all 4 years of seed stored in cloth bags. 23.0 20. 6 18.3 16. o 13.2 9-7 Nov. 26, 1921 Storage of Coniferous Tree Seed 509 Fort Bayard stands out as an exceptionally favorable storage point, and Pikes Peak, Pocatello, and Lake Clear Junction follow it rather closely. On the other hand, New Haven, Dundee, Lawrence, Warsaw, and Waukegan seem especially unsuited as locations for seed storage in ordinary containers. There was not enough information collected in con- nection with this study to show the reason why some points give so much more favorable results than others. It seems probable that it is due to some climatic factor or factors which in turn have their effect upon the physiological activities of the seed. Fort Bayard, Pikes Peak, Pocatello, and Lake Clear Junction are in high altitudes, and the first three places at least experience conditions of low relative atmospheric humidity. Waukegan, Dundee, Lawrence, and Warsaw are middle western loca- tions of moderate altitude which experience severe temperature fluctua- tions and higher relative humidity than the first three locations men- tioned. During the summer when such changes would most affect seed in storage. New Haven experiences higher relative hum.idities than the middle western locations and severe climatic fluctuations are somewhat less marked there. Regardless of the reason, the study indicates that middle western points and perhaps Atlantic coast points should if possible be avoided as locations for storing coniferous tree seed in ordinary containers, especially for periods of several years. This conclusion for Atlantic coast points may not be entirely warranted as it is based on results at only one station. There is, however, comfort for those wishing to store seed in such locations when the germination of bottle-stored seed is examined. Based on the average germination percentage of such seed for all species and all years, the towns line up in the following sequence : Towa. Dundee Fort Bayard Waukegan . . Pikes Peak. Ithaca Pocatello . . . Halsey Average germina- tion per- centage for all 4 years of seed stored in air-tight bottles. Town. Lawrence Lake Clear Junction Ann Arbor New Haven State College Warsaw Average germina- tion per- centage for all 4 years of seed stored in air-tight bottles. This tabulation indicates, and it can be corroborated by inspection of Tables IV to IX, that the bottle-stored seed in this study was not affected by climatic conditions at the points of storage. Two of the points, Dundee and Waukegan, which the study indicates were very unfavorable storage points when ordinary methods of storage are followed, appear in the case of bottle-stored seed to be among the most favorable locations. Here again the lesson is obviously to use air-tight methods of seed storage. The geographic location of the storage point will then be of little or no consequence. 5IO Journal of Agricultural Research voi.xxii. N09 RESULTS OE STORAGE AT END OF TEN YEARS Reference was made (p. 483) to the fact that some of the bottle-stored seed was carried over for another 5 years and then tested again. The seed so carried over was that of each of the six species which had previous- ly been stored at the indoor temperature at Dundee, 111. P'ollowing the test during the winter of 1914, the bottles were resealed air-tight and then stored until January, 19 19, on a shelf in the Forest Service office building at Washington, D. C. The 1914 test of these seed showed a germina- tion percentage of 71.5 for Engelmann spruce, 43 for Douglas fir, 61.5 for lodgepole pine, 82 for western yellow pine, 74 for western white pine, and 56.5 for white pine. The test in 19 19 was carried on for 167 days under the same conditions as in previous years. Engelmann spruce, Douglas fir, and white pine germinated not at all, while lodgepole pine germinated to the extent of 9 per cent, western yellow pine 22 per cent, and western white pine 6.5 per cent. Although the test was continued for 167 days, the lodgepole pine had completed its germination in 90 days, western yellow pine in 75 days, and western white pine in 130 days. Due to the fact that these seeds were exposed to the air at the end of 5 years, although the bottles were afterwards resealed, this particular part of the experiment does not truly indicate whether seed can be successfully stored for 10 years without great deterioration. It does give an idea of the relative sustained vitality of the species concerned. It has been rather apparent all through the study that as respects general sustained vitality, the species will rank in the following sequence, the more vital species being placed first: Western yellow pine, lodgepole pine, western white pine, white pine, Engelmann spruce, and Douglas fir. If the seeds were se- cured from different sources than those of this study, the sequence might be altered. SUvSCKPTlBILlTY OF THE DIFFERENT Vx\RIETIES OF SWEET POTATOES TO DECAY BY RHIZOPUS NIGRI- CANS AND RHIZOPUS TRITICI By L. L. Harter. Pathologist, and J. I,. Weimer, Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant Industry, 'United States Departinent of Agriculture INTRODUCTION Although Rhizopus yiigricans Ehrb. has generally been regarded as the cause of the softrot of the sweet potato (Ipomoea batatas Lam.), Harter, Weimer, and Lauritzen^ have shown by recent experiments that a similar decay may be caused by the following additional species of the genus: tritici Saito, nodosus Namysl, reflexns Bainier, delemar (Boid), Wehmer and Hanzawa, oryzae, Went and Pr. Geerligs, maydis Bruderl., arrhizus Fischer, and artocarpi Racib. Their investigations showed that in order to obtain infection with the dififerent species it was necessary to incubate the potatoes at a temperature suited to the growth of the par- ticular species of Rhizopus with which they were inoculated. They group the different species into high, intermediate, and low temperature forms, R. nigricans Ehrb. and R. tritici, the two species concerned in the present investigations, belonging to the low and intermediate forms, respectively. The parasitism of the dififerent species of Rhizopus was determined by them for the Yellow Jersey variety of sweet potatoes only. Although softrot occurs on all the varieties, it is the general belief that some of them are more resistant to decay than others. The Jersey type of sweet potatoes, for example, is recognized as a poor keeper, while some of the varieties grown in the South are thought to keep well in storage. The present investigations were conducted in order to throw more light on the relative susceptibility of a number of the commercial varieties to infection and decay by R. nigricans and R. tritici. R. nigricans was employed for the major portion of the inoculations because it is the species commonly found under storage-house conditions and seems to be responsible for most of the loss. R. tritici although not so common as R. nigricans is a very parasitic species, especially under artificial conditions. A comparison of these two species gives some idea of the results that may be expected from them, one requiring relatively low and the other one an intermediate temperature, or at least a tem- perature considerably above that recommended for the storage of sweet potatoes. METHODS OF EXPERIMENTATION The susceptibility to infection and decay by R. nigricans was deter- mined for the following varieties of sweet potatoes: Big Stem Jersey, Little Stem Jersey, Southern Queen, Porto Rico, Dooley, Georgia, Pierson, Dahomey, Triumph, Gold Skin, Haiti, Nancy Hall, Early CaroHna, Yellow 1 Harter. L. L., Weimer, J. L., and Lauritzen, J. I. the decay of sweet potatoes (ipomoea batatas) produced by different species of rhizopus. In Phytopathology, v. ii, no. 7, p. 279-284. Literature cited, p. 2S4. 1921. Journal of Agricultural Research. Vol. XXII, No. 9 Washington, D. C. Nov. 26, igar aas Key No. 0-258 (511'; 512 Journal of Agricultural Research Vol. XXII, No. 9 Belmont, and Red Brazil. The parasitism of R. tritici was determined for all the above-named varieties except the last four. The mechanical operations involved in carrying out these experiments are identical with those previously employed ' and will not be discussed here. The potatoes inoculated with R. nigricans and R. tritici were incubated at temperatures of from 20° to 22° and 30° C, respectively, these temperatures having been found in previous experiments to be favorable for the growth of these two organisms. Records of the per- centage of infection and of the progress of decay were made at the end of 48 hours after inoculation and every day thereafter to the close of the experiment. The experiments were allowed to run for from four to six days. EXPERIMENTAL DATA The results of the inoculations of the different varieties of sweet pota- toes with R. nigricans are shown in the following table. The figures in columns i and 2 were obtained by taking an average of the results of the several experiments. The data presented in the second column are based on an estimation of the percentage of the total amount of decay when the experiments were terminated. Table I. — Percentage of sweet potatoes infected and the estimated percentage of decay at the end of the experimeii t Variety, Porto Rico Big Stem Jersey. .. Triumph Pierson Gold Skin Little Stem Jersey Georgia Early Carolina. .. . Percent- Per- age of cent- decay age at the of end of infec- the tion. experi- ment. 07 7=^ 100 88 97 60 97 73 1 100 100 100 gS 00 93 100 95 Variety. Nancy Hall Florida Red Brazil Haiti Dahomey Southern Queen Yellow Belmont Dooley. Per- cent- age of infec- Percent- age of decay at the end of the tion. experi- ment. 90 28 100 40 100 95 100 93 97 87 53 23 100 100 93 98 The results show that a large percentage of the potatoes became infected by the method employed. There were cases, especially among the more resistant varieties, where only a very small amount of the tissue about the well decayed. In view of this fact it was frequently difficult to decide whether infection had actually taken place or whether the small amount of decay was due to an enzym in the inoculum at the time the inocula- tions were made. The writers finally decided to regard as infected all potatoes which had been softened for i cm. or more beyond the margin of the well. It is not unlikely that the percentage of infection of some of the varieties, especially those which resisted further decay, was actu- ally placed too high by this method. ' H.^RTER, L. L., Weimer, J. L,., and Adams, J. M. R. sweet-pot.ito storage-rots. In Jour. Agr. Research, v. 15, no. 6, p. 337-368, pi. 21-27. 1918. Literature cited, p. 366-368. Barter, I,, h., Weimer. L., and Lauritzen ,J. I. op. cit. Nov. 26, J92I Susceptibility oj Sweet Potatoes to Rhizopus 513 Although it was easy to estimate the relative amount of decay between very susceptible and very resistant varieties, it was more difiBcult to determine the percentage of total decay at the end of a given time. The percentage of the entire potato that would be decayed at the end of a certain number of days depended to a considerable extent on the size and shape of the potatoes. Naturally a larger percentage of a small sweet potato would be decayed in a given time than of a large one; also a short to nearly spherical potato would be completely decayed sooner than a long, cylindrical one. As to shape, the potatoes differed greatly. The potatoes of some varieties were short and chunky, while others were long. In view of these facts the writers wish to emphasize the danger of putting too much reliance on small differences. The results, however, show some wide differences which the authors believe to be a fair estimate of the relative susceptibility of the different varieties. With respect to their susceptibility to decay by R. nigricans, the dif- ferent varieties of sweet potatoes can be divided roughly into three groups as follows: i, very susceptible; 2, quite resistant; and 3, inter- mediate. To the first belong Gold Skin, lyittle Stem Jersey, Georgia, Early Carolina, Red Brazil, Haiti, Yellow Belmont, and Dooley; to the third group belong Porto Rico, Big Stem Jersey, Triumph, Pierson, Florida, and Dahomey; and to the second, Nancy Hall and Southern Queen. As might be expected, a considerable variation exists between the different varieties of a single group. Furthermore, there is no sharp line of separation between the more resistant and the more susceptible varieties of two contiguous groups, the differences frequently being no greater than that which exists between varieties of the same group. The Gold Skin is by far the most susceptible variety studied, decay be- ing completed considerably in advance of that of any of the other vari- eties. The Little Stem Jersey is likewise very susceptible and with the iGold Skin stands out conspicuously as regards the rapidity with which t decays. The Big Stem Jersey, a variety grown extensively in the northern range of the sweet-potato belt, decays fairly rapidly and completely, and must be ranked high as a susceptible variety in the intermediate group. Nancy Hall and Southern Queen are the only representatives of the resistant group. Although the table shows a high percentage of infection, the amount of decay was always small, and within the limits of these experiments they must be regarded as much more resistant than any of the other varieties. The Florida is the only other variety that approximates these two in resisting decay by R. nigricans. Although the object of these experiments was primarily to determine the susceptibility of the different commercial varieties of sweet potatoes to decay by R. nigricans, one single set of inoculations was made with R. tritici, using the same varieties with the exception of the four already mentioned. R. tritici was found to be parasitic on all the varieties. One outstanding fact as a result of these experiments seems to be that the Nancy Hall and Southern Queen, varieties which are especially re- sistant to R. nigricans, are rather susceptible to decay by R. tritici. A further comparison, therefore, was made of the relative parasitism of these two species on Nancy Hall and Southern Queen, using the Little Stem Jersey, a very susceptible variety, as a control. After inoculation of the three varieties in the usual way with R. tritici and R. nigricans they were divided into two lots. One lot was in- 514 Journal of Agricultural Research Vol. XXII, No. 9 cubated at 30° C, a temperature favorable for R. tritici, and the other lot at 20° to 22°, a temperature favorable for the growth of R. nigricans. None of the potatoes inoculated with R. nigricans and incubated at 30"^ became infected, showing that this temperature is unfavorable to in- fection by this fungus. On the other hand, all those inoculated with R. tritici became infected and were completely decayed in three or four days. A direct comparison of these two organisms at 30° was therefore impossible. The percentage of infection as well as the total amount of decay of all three of these varieties by both organisms when incubated at a temperature of 20° to 22° for seven days is shown in Table II. Table II. — Percenlaije of infection and estimated percentage of decay of three -varieties of sweet potatoes by R. nigricans and R. tritici after seven days at 20° to 22° C. Variety, Organism. Percentage of infection. Percentage of decay. Southern Oueen R. nigricans R. tritici R. nigricans 70 100 80 100 100 15 Nancy Hall 85 40 Little Stem Jersey R. tritici R. nigricans R. tritici 100 80 100 An examination of this table shows that at this temperature the Little Stem Jersey variety was decayed much more readily than either Nancy Hall or Southern Queen, by both organisms. However, decay was much more rapid by R. tritici than by R. nigricans, being completed by the former in three to four days and not quite completed by the latter in seven days. The potatoes of the Nancy Hall and Southern Queen varie- ties were 100 and 40 per cent and 85 and 15 per cent decayed by R. tritici and R. nigricans, respectively, at the end of seven days. A comparison of the three varieties shows that the estimated amount of decay caused by R. nigricans at the end of the experiment was about 15, 40, and 80 per cent for Southern Queen, Nancy Hall, and Little Stem Jersey, respec- tively. The total amount of decay caused by R. tritici, on the other hand, was 85, 100, and 100 per cent, respectively. The results therefore show that the Little Stem Jersey is quite susceptible to decay by both species, while Nancy Hall and Southern Queen are very resistant to decay by R. nigricans only. The percentage of infection of the Nancy Hall potatoes was 95 and 80 by R. tritici and R. nigricans, respectively, while the Southern Queen potatoes were 100 and 70 per cent infected by the same organisms. On the other hand, Little vStem Jersey potatoes were 100 per cent infected by both species. These results indicate what occurs at temperatures of 20° and 30° C. under the conditions existing in these particular experiments. A tem- perature of 30° proved to be too high for R. nigricans and 20° to 22°, while not optimum for either organism, is probably more favorable for decay by R. tritici than by R. nigricans. On the other hand, if a much lower temperature had been employed it would have been more favorable to R. nigricans but less favorable to R. tritici. The results, however, do show a considerable difference in the three varieties with respect to their relative susceptibility to decay by the two species of Rhizopus at the temperatures tried. Nov. 26, 1921 Susceptibility of Sweet Potatoes to Rhizopus 515 SUMMARY (i) R. nigricans is parasitic on the following varieties of sweet potatoes : Porto Rico, Big Stem Jersey, Triumph, Pierson, Gold Skin, Little Stem Jersey, Georgia, Early Carolina, Nancy Hall, Florida, Red Brazil, Haiti, Dahomey, Southern Queen, Yellow Belmont, and Dooley. R. tritici was found to be parasitic on all the above-named varieties except Early Caro- lina, Florida, Red Brazil, and Yellow Belmont, on which it was not tried. (2) With respect to their susceptibility to decay by these two species of Rhizopus, the varieties can be separated roughly into three groups- — first, those that are very susceptible ; second, those that are very resistant ; and, third, those that are intermediate between the first and second. To the first group belong the Gold Skin, Little Stem Jersey, Georgia, Early Caro- hna. Red Brazil, Haiti, Yellow Belmont, and Dooley; to the second, Southern Queen and Nancy Hall ; and to the third, Porto Rico, Big Stem Jersey, Triumph, Pierson, Florida, and Dahomey. (3) Nancy Hall and Southern Queen, the two most resistant varieties, were more susceptible to decay by R. tritici than by R. nigricans at a temperature of from 20° to 22° C. Neither of these varieties, however, deca5'-ed as readily as the Little Stem Jersey under similar conditions and used as a control. (4) The results of these experiments show that all the varieties tried (16) are more or less susceptible to decay by R. nigricans but that there are some varietal differences. Likewise, the results show that those varieties which decayed most readily under the conditions of these experiments are the ones which have been observed to decay most readily under commercial storage-house conditions. 75308—22 5 Vol. XXII DECEMBER 3, 1921 No. 10 JOURNAL OF AGRICULTURAL RESEARCH CONTENTS AND INDEX OF VOLUME XXII PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE WITH THE COOPERATION OF THE ASSOCIATION OF LAND-GRANT COLLEGES WASHINGTON, D. C. GOVERNMENT PRINTING OFFICE 1923 EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF LAND-GRANT COLLEGES FOR THE DEPARTMBNT K. F. KELLERMAN, Chairman Phytiologist and Associate Chkf, Bureau of Plant Industry B. W. ALLEN CUrf. Office of Bxptriment Stations C. L. MARLATT Entomologist and Associat* Chief, Bureau of Entomolocy FOR THB ASSOCIATION J. G. LIPMAN Dean, Stale College of Agriculture, mtd Director, New Jersey Agricultural Experi- ment Station, Rutgers College W. A. RILEY Entomologist and Chief, Ditision of Enkh mology and Economic Zoology, Agricnt- tural Experiment Station of the Untsersity of Minnesota R. L. WATTS Dean. School of Agriculture, and Dirtttof, Agricultural Experiment Station, The Pennsylvania State College All correspondence regarding articles from the Department of Agriculture should be addressed to K. 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 Brtmswick, N, J. ERRATA AND AUTHORS' EMENDATIONS Page 54, line i. should read "Fj or Fi" instead of "Fa or F." Page s8. Table V, under last column the first number should read 33 instead of 23. Page 68, line 24, should read "with a nutrient solution" rather than "with an nutrient solution." Page 69, lines 9 and 11, should read "O. A. Pratt" instead of "A. C. Pratt." Page 76, line 3, should read "Plate 11, A" instead of "Plate 11, B." Page 80 and following, in Plates 10 and 11, fis^res A and C of Plate 10 should be interchanged with figures A and C of Plate 11. Page 174, line i, should read "causal" not "casual." Pages 175 and 177, and Plates 2a and 24, running head should read, "Transmissible Mosaic Disease of Chinese Cabbage " instead of " Transmissible Mosaic Disease of Cabbage." Page 178, colored plate is by J. Marion Shull. Page 242, line 8, should read "Humphrey, as reported by Jones (5)," not "Humphrey (4) states . . . ." Page 282, line 32, should read "at each treatment" instead of "to each treatment." Page 283, line 17, should read "nitric acid," not ''nitri acid." INDEX Page ABSORPTION OF COPPER FROM THE SOIL BY POTATO PLANTS 281-387 Acer — nibrum, host of Xylotrechus colonus 195-198 saccharum, organic acids 221-229 Achillea ptarmica, susceptibility to Puc- cinia triticlna 152-172 Acid — amino, in G€orgia velvet beans 15 citric, in grapefruit 263-279 diainiao, in Georgia velvet bean 15 hydrocyanic, in Sudan grass 125-138 :iialic, in Pyrui coronaria 221-229 phosphoric, effect on hydrocyanic acid in Sudan grass 135 succinic, in Pyrus coronaria 22 1-229 sulphuric, effect on hydrocyanic acid in Sudan grass. 133-135 Acid producers in corn meal 181-188 Acid-sugar ratio in grapefruit 263-279 Acids— effect on hydrocyanic acid in Sudangrass. 133-135 organic, of Pyrus coronaria , Rhus glabra, and Acer sacckarutn 221-229 Aconitum— Fischeri, resistance to teliosporesof Pticcinia triticiiia 155-172 Lycoctonutn, susceptibility to Puccinia triticiiia 152-172 Napellus, susceptibility to Puccinia triti- cina 152-172 Actaea spicaia, resistance to teliospores of Puccinia tritichia 155-172 Actinotnyces sp. in com meal 187-188 AECIAL STAGE OF THE ORANGE LEAFRUST OF WHEAT, PUCCINIA TRITICINA ERIKS 151-172 Aecidiuin — Anchiisae, pathogenicity on wheat 152 Berberidis, in life cycle of Puccinia grami- nis 151-172 Ranunculacearum 7 Thalictri Havi. Syn. Aeculiiim Thalictri Jiavi. Thalictri flavi, rust on Agropyron repens. . . 164 Aegopodiuin Podagraria, susceptibility to Puccinia triticina 152-172 A gropyron — caninum, resistance to aeciospores of Puc- cinia triticina 163-172 cristatum, resistance to aeciospores of Puc- cinia triticina 163-172 desertorutn, resistance to aeciospores of Puccin'a triticina 163-172 inerme, resistance to aeciospores of Puccinia triticina 163-172 Page ^4 gropyron — Coniinued. repens, resistance to aeciospores of Puccinia triticina 163-172 Rickardsonii, host of Puccinia Agropyri. . . . 165 Smithii, resistance to aeciospores of Puc- cinia A gropyri 165 t&nerutn, resistance to aeciospores of Puc- cinia triticini 163-172 Agrostemma Githago, susceptibility to Puc- cinia triticina 152-172 Agrostis — borealis, host of Puccinia triticina 164-172 orthogonia. Syn. Porosagrotis orthogonia. Akis muricota. Syn. Embaphton muricatum . Albumen, egg, effect on growing chicks 146 Albumin in Georgia velvet bean 15 Alfalfa, effect of soil temperature on develop- ment of nodules 17-31 Alkaline solution, efl'ect on hydrocyanic acid in Sudan grass 135-136 Alternaria sp. on pitted grapefruit 277 Atnbrosia artemisiifolia, glucosid 4 Amino acid. See Acid, amino. Ammodrainus savannaruni bim avtilatu s , enemy of Porosagrotis orthogonia 313 A mygdalus persica— effect of temperature on damage from rots 452-465 host of Lecaniu m corni 191 Aiichusa — arvensis, host of aecial stage of Puccinia rubigo-vera 151-173 ofvcinaUs, host of aecial stage of Puccinia rubigo-vera 151-172 Ancylostoma— caninum, nonhemolytic effect 383 duodenak, hemotoxins 382-432 Anemone — canadensis, resistance to teliospores of Puccinia triticina 155-173 cylindrica, resistance to teliospores Puccinia triticina 155-172 japonica, resistance to Puccinia triticina. . 155-173 ranunculoides, susceptibility to Puccinia triticina 152-173 A noploccphala — perfoliata, hemotoxins not found 383 pHcata, hemotoxips not found 382 Anthocyanin in maize 2-31 Anthoxanthum odoratum, host of Puccinia triticina 164-172 Anticoagulins in hookworms 418-420 Aphids, carriers of mosaic of Chinese cab- bage 173-178 Aphis, sugar-beet root. See Pemphigus betae. Apple blotch. See Phyllosticia solitaria. 5i8 Journal of Agricultural Research Vol. xxir Page Apple, crab. See Pyrus coronaria. Aqiiilegia — alpina, resistance lo teliospores of Puccmia trilicina iSS"'?* canadensis, resistance to teliosi>ores of Puccinia trilicina 155-173 chrysantha, resistance to teliosjwres of Puccinia trilicina 155-172 glandulosa, resistance to teliospores of Puccinia trilicina 155-172 olympica, resistance to teliospores of Puccinia trilicina 157-172 Skinneri, resistance to teliospores of Puccinia trilicina 155-1 72 xnilgaris, resistance to teliospores of Pucciniii trilicina 155-172 Army cutworm. See Cliorizagrotis auxiliaris. Arrhenatiierum clalis, resistance lo aeciospores of Puccinia trilicina 163-172 Ascaris — conocepkala, cause of anemia 385-432 luinbricoides, hemotoxins 385-432 Ash, bone, effect on growing chicks 145-149 Asb. See Fraxinus sp. ASH CONTENT OF THE AWN. RACHIS. PALEA, AND KERNEL OF BARLEY DURING GROWTH AND MATURATION 433-449 A spergillus — candidus in corn meal 187-188 Jlavus in corn meal i8i-i88 futnigalus in corn meal 185-188 niger in corn meal 181-188 ockraceous in com meal 187-188 re pens in com meal 181-188 tamari in corn meal 185-188 terreus in com meal 185-188 Awn of barley, ash content 433-449 Bacillus — coli communis, effect on aspartic acid 224 nigcr in corn meal 186-188 phytophlhorus, cause of balckleg potato tuber-rot 81-92 radicola — effect of sulphur 102-110 used in tests of effect of soil temperature on nodule development 20-31 tetanus, blood toxin 381 Bacteria — in cheese 93, 100 in corn meal 181-188 Bacterium aerogenes in com meal 183-188 Barbaraea vulgaris, susceptibility to Puccinia trilicina 152-172 Barley, ash content 433-449 Bartlett, H. H., and Sando, Charles E.: NOTES ON THE ORGAJ^IC ACIDS OF PYRUS CORONARIA, RHUS GLABRA, AND ACER SACCHA- RUM 221-229 OCCURRENCE OF QUERCETIN IN EMERSON'S BROWN-HUSKED TYPE OF MAIZE 1-31 Bfiin, velv-jt. See Stizolobium deeringianum. Beetle, pine. See Dendroct&nus inonticolae. Page Beetles, cerambycid, host-selection princi- ple 189-220 Beets, sugar, dryrot canker 47-52 Bcrberis vulgaris — failure to become infected from basidio- spores of Puccinia rubigo-vera 152 -i 72 susceinibility to Puccinia trilicina 152-172 BIOLOGICAL ANALYSIS OF THE SEED OF THE GEORGIA VELVET BEAN, STIZOLOBIUM DEERING- IANUM 5-is BIOLOGY OF EMBAPHION MURICA- TUM 333-334 Black locust. See Robinia pseudacacia. Black mold rot, caused by Rhi2o pus nigri- cans. 451-465 BLACKLEG POTATO TUBER-ROT UNDER IRRIGATION 81-92 Blight, chestnut. See Endothea parasitica. Blotch- apple. See Phyllosticla solitaria. plum. See Phyllosticla congesta. Blue mold rot. See Penicillium cxpansum. Bone ash, effect on growing chicks 145-149 Bonnetia compla, parasite of Porosagrotis orthogonia 313 Boshnakian, Sarkis, and Leighty. Clyde E. : GENETIC BEHAVIOR OF THE SPELT FORM IN CROSSES BE- TWEEN TRITICUM SPELTA AND TRITICUM SATIVUM 335-364 Boving, Adam G., and Wade, J. S.: BI- OLOGY OF EMBAPHION MURICA- TUM 323-334 Brassica — japonica, mosaic disease 173-178 pekincnsis, mosaic disease 173-178 rapa, mosaic disease 173-17S Bromus — ciliatus, host of Puccinia Agropyri 165 Porter i, host of Puccinia allernans 165 Brooks, Charles, and Cooley, J. S.: TEMPERATURE RELATIONS OF STONE FRUIT FUNGI 451-465 TRANSPORTATION ROTS OF STONE FRUITS AS INFLUENCED BY OR- CHARD SPRAYING 467-477 Brown-huskcd maize, quercetin 1-31 Brovm rot. See Sclcrolinia cinerea. Bustomum phlebnlomum, heinptoxins 415-432 Cabbage, Chinese. See Brassica pekinensis. Calcium- carbonate, effect on- growing chicks 139-149 plant growth 102-110 toxicity of copper sulphate 281 lactate, effect on growing chicks 145-149 sulphate, effect on plant growth ■ 102-110 Callidium — antennatum, host selection 194-220 janthinum, host selection 203-220 Camassia esculenta, resistance to teliospores of Puccinia trilicina 155-172 Campanula rolundifolta. susceptibility to Piiccinia trilicina 152-17; Oct. i-Dec. 3, 1921 Index 519 Page Canker of sugar beets 47-52 Calvin, J. W., et al.: NUTRIENT RE- QUIREMENTS OF GROWING CHICKS: NUTRITIVE DEFICIEN- CIES OF CORN 139-149 Camallanus ainrricanus, oxyhemoglobin in body fluid 392-432 Carbonate — calcium, effect on — growing chicks 139-149 plant growth 102-110 toxicity of copper sulphate 281-287 sodium, efiect on hydrocyanic acid in Sudan grass 135-136 Casein, effect on growing chicks 139-149 Caslanca dentala, host of — Hyperplalys maciilaius 217-219 Xylotrechus cuhnus 195-198 Cattle hookworm. See Bustomum phkboto- muni. Cattle, maintenance requirement 115-121 Celtis occidentalis, host of — Cyllene picius 198-203 Molorchus bimaculatus 212-213 Centaurea Cyanus, susceptibility to Puccinia triticina 152-172 Cerambycid beetles, host -selection principle. 189-220 Cfrcis canadensis, imrrmnity to Molnrchus bimaculatus 212-213 Cestode hemolysins 420-432 Cheese, bacteria 93-100 Cherries, effect of — orchard spraying on transportation rots. . 467-477 temperature on damage from rots 441-465 Chestnut. See Castanea dentata. Chestnut blight. See Endothea parasitica. Chinese cabbage See Brassica pekinensis. Chlorid, sodium, effect on growing chicks. . 145-149 Chorizagrotis auxiliaris, comparative des- tructiveness of Porosagrotis orthogonia 289 Cimicifuga racemosa, resistance to teliospores of Puccinia triticina 155-172 Citelhis richardsoni, enemy of Porosagrotis orthogonia 313 Citric acid. See Acid, citric. Citromyces sp. in corn meal 187-188 Cladosporium sp. on pitted grapefmit 277 Clematis — Douglasii, resistance to teliospores of Puc- cinia triticina 155-172 fiatninula, probable aecial host of Puccinia triticina 153-172 Fremontii, resistance to teliospores of Puc- cinia triticina 155-172 heraclaefolia, resistance to teliosirares Puc- cinia triticina 155-172 ligustici folia, resistance to teliospores of Puccinia triticina 155-172 orientnlis, resistance to teliospores of Puc- cinia triticina 155-172 paniculata, resistance to teliospores of Puc- cinia triticina 157-172 recta, resistance to teliospores of Puccinia triticina 155-172 Page Clematis — Continued. virginiana, resistance to teliospores of Puc- cinia triticina 155-171 ■citalba, probable aecial host of Puccinia triticina 153-171 Clonorchis sinensis, hemotoxins 380-432 Colletotrichum sp. on pitted grapefruit 277 COMPARATIVE VIGOR OF Fi WHEAT CROSSES AND THEIR PARENTS.... 53-63 COMPARISON OF THE PECTINASE PRODUCED BY DIFFERENT SPEC SPECIES OF RmZOPUS, A 371-377 Coniferous tree seed, storage 479-510 Cook. F. C: ABSORPTION OF COPPER FROM THE soil, BY POTATO PLANTS 281-287 Cooley, J. S., and Brooks, Charles: TEM- PERATURE RELATIONS OF STONE FRUIT FUNGI 451-465 Copper, absorption from soil by imtato plants 281-287 Copper sulphate, toxicity to potato plants. . 281-287 Com, nutritive defeciencies 139-149 Com meal, flora 179-188 Cornus florida, host of — Hyperplalys maculatus 217-219 Molorchus bimaculatus 212-213 Coronaria floscuculi, susceptibility to Puc- cinia triticina 152-172 Corticium vagum, cause of dryrot canker of sugar beets 47-52 Crab apple. See Pyrus coronaria. Craighead, F. C: HOPKINS HOST- SELECTION PRINCIPLE AS RE- LATED TO CERTAIN CERMBYCID BEETLES 189-220 Cutworm — army. Chorizagrotis auxiliaris. pale western. See Porosagrotis orthogonia 289-322 Cyanidin, glucosid, in maize 2-31 Cyllene — crinicoriiis, host selection 203 picius, host selection 193-220 Delphinium — ajacis, resistance to teliospores of Puccinia triticina 155-172 "Belladonna," resistance to teliospores of Puccinia triticina 155-172 consolida, resistance to teliospores of Puc- cinia triticina 155-172 geyeri, resistance to teliospores of Puccinia triticina 155-172 Dendroctonus monticolae, parasite on Pinus contorta 189-220 Dextrose in grapefruit 263-279 Diamino acid. See Acid, diamine. Digestion, liberation of hydrocyanic acid. ... 127 Diphyllobothrium latum, hemotoxins 381-432 Dipotassium phosphate, effect on growing chicks 145-149 Dipylidium caninum, hemotoxins 382-432 Dogwood. See Cornus florida. DRYROT CANKER OF SUGAR BEETS, A 47-5« k 520 Journal of AgrictUtural Research Vol. XXII Page Echium vulgare, susceptibility to Puccinia triticina 152-172 Edson , H . A . , and Shapovalov , M . : B LACK- LEG POTATO TUBER-ROT UNDER IRRIGATION 81-92 EFFECT OF SOIL TEMPERATURE UP- ON THE DEVELOPMENT OF NODU- LES ON THE ROOTS OF CERTAIN LEGUMES 17-31 Egg albumen, effect on growing chicks 146 Eleodes contiisum, resemblance to Embapkion iuuricalum 323 Elymu.1 — ausiralis, resistance to aeciospores Puccmia triticina 163-172 canadensis, resistance to aeciospores of Puc- cinia triticina 163-1 72 glauctis, resistance to aeciospores of Puc- cinia triticina 163-172 triticoides, resistance to aeciospores of Puc- cinia triticina 163-172 virginicus, resistance to aeciospores of Puc- cinia triticina 163-172 Embapkion — concavum, resemblance to Embapkion muri- catutn 314 muricatum — biology 323-334 control 333 description 326-332 distribution 324-325 enemies 332-333 Endotkea parasitica, food of Neoclytus capraea . 193 Euxoa ochragaster, connection with Porosagro- tis orthogonia 290 Fasciola hepatica, hemotoxins 380-432 FERTILITY IN SHROPSHIRE SHEEP. 231-234 Festuca Thicrberi, host of Puccinia Cockerel- liana 16s Fisher, D. F.. and Brooks, Charles: TRANS- PORTATION ROTS OF STONE FRUITS AS INFLUENCED BY OR- CHARD SPRAYING 467-477 Flavone in maize 3-31 FLORA OF CORN MEAL 179-188 Fraxinus sp., host of — Neoclytus capraea 210-211 Xylotrechus colonus 195-198 Fruit fungi, temperature relations 451-465 Fruits, stone, influence of orchard spraying on rots 467-477 Fungi, of stone fruits, temperature relations. 451-465 FURTHER STUDIES ON RELATION OF SULPHATES TO PLANT GROWTH AND COMPOSITION loi-iio Fusarium — eumartii, effect of temperature 66-80 hyperocysporum, effect of humidity 71-80 oxysporum — cause of rot of Irish potatoes 65-80 confused with causal organism of black- l^e potato tuber-rot 81-92 fadicicola — confused with causal organism of black- leg potato tuber-rot 81-92 effect of temperature 66-80 Page Fusarium — Continued. trichothecioides, effect of temperature 66-So sp.— in corn meal 187-188 on pitted grapefruit 277 trichothecioides, isolated from potato black- leg tubers 82 lubirivorum, effect of temperature 66-80 vasinfectum var. pisi, host selection 191-220 Gardner, Max W.. and Kendrick. James B.t TURNIP MOSAIC 123-124 SOYBEAN MOSAIC 111-114 GENETIC BEHAVIOR OF THE SPELT FORM IN CROSSES BETWEEN TRI- TICUM SPELTA AND TRITICUM SATIVUM 335-364 Geographical location, effect on coniferous tree seed in storage 508-510 Georgia velvet bean. See Stizolobium deering- ianum. Glechov'.a hederacea, susceptibility to Puccinia triticina i5J-i7» Globulins in Georgia velvet bean 15 Glucosid of quercetin in maize 2-31 Gluten, effect on growing chicks 144 Goss, R. W.: TEMPERATURE AND HUMIDITY STUDIES OF SOME FU- SARIA ROTS OF THE IRISH POTATO 65-80 Gracilaria stigmatella, change in nidification. . , 191 Grape — See Vitis vinifera. wild. See Vilis sp. Grapefruit, ripening and storage ;63-279 Graphidium strigosuni, heruoto.xins 3(57-432 Grass, Sudan, hydrocyanic acid 1^5-138 Griffee, Fred: COMPARATIVE VIGOR OF Fi WHEAT CROSSES AND THEIR PARENTS 53-63 Ground squirrel. See Citellus richardsoni. Growth of plants, effect of sulphur loi -no Kackberry. See Celtis occidcntalis. Haemoncltus contortus, hemotoxins 387-431 Halbersleben, D. L., et al.: NUTRIENT REQUIREMENTS OF GROWING CHICKS: NUTRITIVE DEFICIEN- CIES OF CORN 139-149 Harlan, Harry V., and Pope, Merritt N.: ASH CONTENT OF THE AWN. RACHIS, PALEA, AND KERNEL OF BARLEY DURING GROWTH AND MATURATION 433-449 Harter, L. L., and Weimer, J. L.: A COMPARISON OF THE PECTINASE PRODUCED BY DIFFERENT SPE- CIES OF RHIZOPUS 371-377 SUSCEPTIBILITY OF THE DIFFER- ENT VARIETIES OF SWEET POTA- TOES TO DECAY BY RHIZOPUS NIGRICANS AND RHIZOPUS TRI- TICI S"-SiS Hawkins, Lon A.: A PHYSIOLOGICAL STUDY OF GRAPEFRUIT RIPENING AND STORAGE 263-279 HEMOTOXINS FROM PARASITIC WORMS 379-43» Oct. i-Dec. 3, 1921 Index 521 PilgC He polka sp., resistance to leliospores of Puc- cinia triticina 155-172 Heterodera schachti, host selection 192 Histidin in Georgia velvet bean 15 Hogan, A. G., et al.: INFLUENCE OF THE PLANE OF NUTRITION ON THE MAINTENANCE REQUIRE- ilENT OF CATTLE 115-121 Hoowkorm — of cattle. See Bustomum phlebotomum. of dog. See Ancylosloma cannium. of man. See Necator americanus. of man, Old World. See Ancylostinna duodenale. HOPKINS HOST-SELECTION PRINCI- PLE AS RELATED TO CERTAIN CERAMBYCIDi: BEETLES 189-220 Hordeum — caespitosuin, resistance to aeciospores of Puccinia triticina 163-172 gussoneanum, resistance toXaeciospores of Puccinia triticina 163-172 jubatum — host oilPuccinia Agropyri i6s resistance to aeciospores oilPuccinia triti- cina 163-172 murinuiii, resistance to aeciospores of Puc- cinia triticina 163-172 pusilluut, resistance to aeciospores of Puc- cinia triticina 163-1 72 vuhare, resistance to' 'aeciospores of Puc- cinia triticina 163-172 Homed lark. See Otocoris alpestris leuco- laeina. Host-selection principle 189-220 Hucker, G. J.: MICROSCOPIC STUDY OF BACTERIA IN CHEESE 93-100 Humidity, effect on Fusaria rots of Irish po- tatoes 65-80 HYDROCYANIC ACID IN SUDAN GRASS 125-138 Hydrogen-ion concentration, effect on devel- opment of nodules ou legiunes 30 Hydrophyllum appendiculatuni, resistance to teliospores of Puccinia triticina 155-172 Hydroxid, sodimn, effect on hydrocyanic acid in Sudan grass 135-136 Hylolrupes ligneus, host selection 207-210 Hyperplatys — aspersus, host selection 217-219 maculatus, host selection 2 1 7-219 Hystrix Hyslrix, resistance to aeciospores of Puccinia triticina 163-172 hnpatiens sp., resistance to teliospores of Puc- cinia triticina 155-172 INFLUENCE OF THE PERIOD OF TRANSPLANTING WESTERN WHITE PINE SEEDLINGS UPON THEIR BEHAVIOR IN NURSERY AND PLANTATION 33-46 INFLUENCE OF THE PLANE OF NU- TRITION ON THE MAINTENANCE REQUIREMENT OF CATTLE 115-121 Ipomoea batata:, susceptibility of different varieties to Rhizopus spp 511-515 Irish potato. See Solatium tuberosum. Page Iron sulphate, effect on growing chicks. . . . 145-149 .Tackson, H. S., and Mains, E. B.: AECIAL STAGE OF THE ORANGE LEAF- RUST OF WHEAT, PUCCINIA TRI- TICINA ERIKS I5I-I7J Jones, Fred Rcuel, and Tisdalc, W. B.: EF- FECT OF SOIL TEMPERATURE UPON THE DEVELOPMENT OF NODULES ON THE ROOTS OF CER- TAIN LEGUMES 17-31 Jones, L. R., and Walker, J. C: RELA- TION OF SOIL TEMPERATURE AND OTHER FACTORS TO ONION SMUT INFECTION 235-263 Kafir, hydrocyanic acid 137 Kendrick, James B., and Gardner, Max W.: SOYBEAN MOSAIC 111-114 TURNIP MOSAIC 123-124 Kernel of barley, ash content 433-449 Knautia arvensis, susceptibility to Puccinia triticina 152-172 Lactate.'calcium, effect on growing chicks. 145-149 Lark; horned. See Otocoris alpestris leuco- laema. Lnunaca';asplenifolia, suspected accial host of Puccinia triticina 153-172 Leafiusl, orange.£ See Puccinia triticina. "Leak," confused with blackeg potato tuber-rot 81-92 Lecanium — corni, host selection 191 robiniarum, host selection 191-220 LeFevre, Edwin, and Thom, Charles: FLORA OF CORN MEAL 179-188 Legumes, effect of soil temperature on devel- opment of nodules 17-31 Lcighty, Clyde E., and Boshnakian, Sarkis: GENETIC BEHAVIOR OF THE SPELT FORM IN CROSSES BE- TWEEN TRITICUM SPELTA AND TRITICUM SATIVUM 335-364 Ligustrum vulgare, susceptibility to Puccinia triticina 152-172 Liopus alpha, host selection 194-220 Liriodendron tulipifera — host of Hyperplatys maculatus 217-219 immunity to Neoclytus erythrocephalus . . . 213-315 Lithospermum purPureo-coeruleum, suscepti- bility to Puccinia triticina 152-172 Locust. See Robinia pseudacacia. Lodgepole pine. See Pinus contorta. Lythrum Salicaria, susceptibility to Puccinia triticina 152-172 Macracanthorhynchus hirudinaceus, hemo- toxins 387-432 Magnesimn sulphate, effect on growing chicks 145-149 Mains, E. B., and Jackson, H. S.: AECIAL STAGE OF THE ORANGE LEAF- RUST OF WHEAT, PUCCINIA TRI- TICINA ERIKS 151-172 Maize — nutritive deficiencies 139-149 quercetin in brown-husked 1-31 Malic acid. See Acid, malic. Maple, red. See Acer rubrum. 522 Journal of Agricultural Research Vol. XXII Pase Maple, sugar. See Acer saccharuni. Meal, corn, flora 179-188 Melandryum album, susceptibility to Puc- cinia triticina 152-172 Mertensia virginica, resistance to teliospores of Puccmia triticina 155-172 Mesquite. See Prosopis juliflora. Metarrkizium anisopkae, parasite of Emba- phion muricatum 332 MICROSCOPIC STUDY OF BACTERIA IN CHEESE 93-100 Miller, Harry G.: FURTHER STUDIES ON RELATION" OF SULPHATES TO PLANT GROWTH AND COMPOSI- TION lOI-IIO Mold, blue. See Penicillium expansuvi. Mold rot, black, caused by Rhizoptts nigri- cans 451-465 Molds in com meal i8i-i88 Molorchus bimaculatus, host selection 212-213 Monilia. See Sclerotinia cinerea. Moths rubra, host of Cyllene pictus 198-203 Mosaic — of Chinese cabbage 173-178 of soybean 111-114 of turnip 123-124 Mulberry. See Morus rubra. Mulliceps midticeps, hemotoxins 382-432 Mumford, F. B., Hogan, A. G., and Salmon, W. D.: INFLUENCE OF THE PLANE OF NUTRITION ON THE MAIN- TENANCE REQUIREMENT OF CATTLE 115-121 Mussehl, F. E., Calvin, J. W., Halbersleben, D. L., and Sandstedt, R. M.: NUTRIENT REQUIREMENTS OF GROWING CHICKS: NUTRITIVE DEFICIENCIES OF CORN 139-149 Mustard. See Brassica japonica. Myosotis — alpestris, failure to become infected from basidiospores of Puccinia rubigo-vera . . . 152-172 arvensis, failure to become infected from basidiospores of Puccinia rubigo-vera. . . 152-172 palustris, resistance to teliospores of Puc- cinia triticina 155-172 scorpoides, resistance to teliospores of Puc- cinia triticina 155-172 sp., susceptibility to Puccinia triticina. . . 152-172 Myzus persicae, carriers of mosaic of Chinese cabbage 173-178 Nasturtium sp., susceptibility to Puccinia triticina 152-172 Necator americanus, hemotoxins 383-432 Necosmopora vasinfecta, host selection 191-220 Neoclytus — capraea, host selection 193-220 erythrocephalus , host selection 213-215 Nitrate, sodium, effect on plant growth. . . . 102-110 Nitrates, effect on development 011 nodules on legumes 18 Nitrogen, effect of soil temperature on assim- ilation by legumes 17-31 Nodules, effect of soil temperature on devel- opment 17-31 Page Nonnea rosea, failure to become infected from basidiospores of Puccinia rubigo-vera 152-17* NOTES ON ORGANIC ACIDS OF PY- RUS CORONARIA, RHUS GLABRA, AND ACER SACCHARUM 221-220 Notholcus lanatus, resistance to aeciospores of Puccinia triticina 163-172 NUTRIENT REQUIREMENTS OF GROWING CHICKS: NUTRITIVE DEFICIENCIES OF CORN 139-149 Oak, white. See Quercus alba. OCCURRENCE OF QUERCETIN IN EMERSON'S BROWN-HUSKED TYPE OF MAIZE 1-31 (Ocneria) Porthetria dispar, host selection 191 Oneideres cingulata, host selection 194-220 Onion smut. See Urocystis cepulae. Orange leaf rust. See Puccinia triticina. Orange, osage. See Toxylon pomiferunu Ornithogalum umbellatum, resistance to telio- spores of Puccinia triticina 155-172 Osage orange. See Toxylon pomiferum. Otocoris alpestris Icucolaema, enemy oi Poroses gratis orthogonia 313 Palea of barley, ash content 433-449 PALE WESTERN CUTWORM (POROS- AGROTIS ORTHOGONIS MORR.) . . . 289-322 Paloverde. See Parkhisonia microphylla. Parker, J. R., Strand, A. L., and Seamans, H. L.: PALE WESTERN CUTWORM (POROSAGROTIS ORTHOGONIA MORR.) 289-322 Parkinsonia microphylla, host of Cyllene crmi- cornis 203 Pastinaca sativa, susceptibility to Puccinia triticina iS2-i7« Peach. See Amygdalus persica. Pectinase produced by species of Rhizopus. 371-377 Peleteria robusta, parasite of Porosogrolis or- thogonia 3 13 Pemphigus betae, carrier of cortichim. vagii m.. . 50-52 Penicillin m — expansu m in com meal 185-188 lutcu m in com meal 185-188 oxalicu m in corn meal 185-188 purpurogenum 185-188 sp. on pitted grapefruit 277 Penta-acetyl-quercetin in maize 3-31 Perilitus eleodis, parasite of Embaphion muri- catum 332 Phacelia — Purshii, resistance to teliospores of Puccinia triticina 155-172 tanaceti folia, resistance to teliospores of Puc- cinia triticina 155-172 Phillyrea sp., susceptibility to Puccinia triti- cina 152-172 Phosphate, dipotassiiim, effect on growing chicks 145-149 Phosphoric acid. See Acid, phosphoric. Phratora vitellinoe, inherited changes in habits 191 Phyllosticta — congesta, cause of plum blotch 365-370 solitaria, cause of apple blotch 365 Oct. i-Dec. 3, 1931 Index 523 Page PHYSIOLOGICAL STUDY OF GRAPE- FRUIT RIPENING AND STORAGE, A 263-279 Picea engelmanni, storage of seed 481-510 Pigmentation of maize 1-31 Pine— lodgepole. See Pinus contorta. western white. See Pinus tiionticola. yellow. Pinus pandetosa. Pine beetle. See Dendroctonus inonticolac. Pinus — contorta— host of Dendroctonus tnonticolae 189-220 storage of seed 481-510 monticola — influence of period of transplanting on growth of seedlings 33-46 storage of seed 481-510 ponderosa — threatened by Dendroctonus Tnonticolae. 189-220 storage of seed 481-510 sirobus, storage of seed 481-510 PLUM BLOTCH, A DISEASE OF THE JAPANESE PLUM, CAUSED BY PHYLLOSTICTA CONGESTA HEALD AND WOLF 365-370 Plum, Japanese. See Prunus triflora. Poa nemoralis var. firmula, host of Puccinia triiicina 164-172 Poplar, yellow. See Liriodendron tulipifera. Porosagrotis — delorata. Syn. Porosagrotis orthogonia. orthogonia — control 307-313 distribution 291 economic importance 305-306 history 289-290 life history 293-305, 314-320 natural enemies 313-314 Porthetria dispar, host selection 191-220 Pope, Merritt N., and Harlan, Harry V.: ASH CONTENT OF THE AWN, RA- CHIS, PALEA. AND KERNEL OF BARLEY DURING GROWTH AND MATURATION 433-449 Potato- Irish. See Solanum tuberosum. sweet. See Ipomoea batatas. Potato tuber-rot, blackleg 81-92 Prosopis juUfiora, host of Cyllene crinicornis. . 203 Prunella vulgaris, susceptibility to Puccinia triticina 152-172 Prunes, effect of — orchard spraying on transportation rots. . 471-477 temperature on damage from rots 452-465 Prunus triflora, host of Phullosticta congesta . 365-370 Pseudotsuga taxifolia, storage of seed 481-510 Puccinia — Agropyri — ascia on Ranunculaceous hosts 154 rust on Thalictnim dasycarpum 165 alternans^ aecia on Ranunculaceous hosts 154 rust on Bromi's Porleri 165 borealis, rust on A grostis borealis 164 Page Pwccmja- Continued. bromina, pathogenicity to Symphytum officinale 154-172 Clematidis, aecia 151-172 Cockerelliana, rust on Festuca Thurberi 165 dispersa, brown rust of wheat 152-172 glumarum, on wheat 151-172 graniinis, on wheat 151-172 obliterata, rust on Thalictrum alpinum 165 perplexans, aecia on Ranunculaceous hosts. . 154 Persisiens, aecia on Ranunculaceous hosts. . 154 rubigo-vera, on rye 151-172 straminis. Syn. Puccinia rubigo-vera. triticina, aecial stage 151-172 Pulmonaria — inontana, host of Puccinia bromina 154-172 officinalis, failure to become infected from basidiosporesof Puccinia rubigo-vera 152-172 Pyrus coronaria, organic acids 221-229 Pythium debaryanum, confused with causal organism of blackleg potato tuber-rot 81-92 Quamasia hyacinthina. Syn. Camassia esculenia. Quercetin, in brown-husked maize 1-3 1 Quercus alba, host of Neoclytus capraea 210-211 Rachis of barley, ash content 433-449 Ragweed. See Ambrosia artemisiifolia. Ram, relation of age to fertility 232-233 Ranunculus— susceptibility to Puccinia triticina 152-172 aconitifolius, resistance to teliospores of Puccinia triticina 155-172 acris — failure to become infected from basidios- poresof Puccinia rubigo-vera 152-172 resistance to teliospores of Puccinia tri- ticina 155-172 asiaticus, susceptibility to Puccinia triti- cina 152-172 auricomus, susceptibility to Puccinia triti- cina 152-172 bulbosus, failure to become infected from basidiosporesof Puccinia rubigo-vera. . . . 152-172 Ficaria, susceptibility to Puccinia triti- cina 152-172 flammula, susceptibility to Puccinia triti- cina 152-172 lanuginosus, susceptibility to Puccinia triticina 152-172 repens, susceptibility to Puccinia triticina. 152-172 Read, J. W., and Sure, Bamett.: BIOLOG- ICAL ANALYSIS OF THE SEED OF THE GEORGIA VELVET BEAN, STIZOLOBIUM DEERINGIANUM .... 5-15 Redbud. See Cercis canadensis. Red maple. See Acer rubrum. RELATION OF SOIL TEMPERATURE AND OTHER FACTORS TO ONION SMUT INFECTION 235-262 Rhanius — cathartica, failure to become infected from basidiospores of Puccinia rubigo-vera .... 152-172 Frangula, failure to become infected from basidiosporesof Puccinia rubigo-vera. . . . 152-172 Rhizoctonia solani, similarity to Corticium vagum 51 524 Journal oj Agricultural Research Page Rhizopus — arrhizus — pathogenicity on sweet potato S'l production of pectinase 372-377 artocarpi — production on pectinase 372-377 pathogenicity on sweet potato 511 chinensis, production of pectinase 372-377 deUmar — pathogenicity on sweet potato 511 production of pectinase 372-377 maydis — pathogenicit y on sweet potato 511 production of pectinase 372-377 microspoTus, production of pectinase 372-377 nigricans — in com meal i8s-iS8 pathogenicity on different vurieties of sweet potatoes sn-sis production of pectinase 372-377 temperature relations 451-465 nodosiis— pathogenicity on sweet potato 511 production of pectinase 372-377 oryzae — pathogenicity on sweet potato 511 production of pectinase 372-377 reftexus — pathogenicity on sweet potato 511 production of pectinase 372-377 spp., production of pectinase 371-377 irHici — pathogenicity on sweet potato 511 production of pectinase 371-377 Rhus glabra, organic acids 221-229 Richards. B. L.: A DRYROT CANKER OF SUGAR BEETS 17-52 Ribes Grossularia, susceptibility to Puccinia triticina 152-172 Roberts, Elmer: FERTILITY IN SHROP- SHIRE SHEEP 231-234 Roberts. John VV.: PLUM BLOTCH, A DISEASE OF THE JAPANESE PLUM, CAUSED BY PHYLLOSTICTA CON- GESTA HEALD AND WOLF 365-37° Robinia pscudacacia, host of — Lecanium corni 191-220 'Xyloirechus colonus 195-198 Rogers, E. C: INFLUENCE OF THE PERIOD OF TRANSPLANTING WESTERN WHITE PINE SEED- LINGS UPON THEIR BEHAVIOR IN NURSERY AND PLANTATION 33-46 Rot- black mold, caused by Khizoptis nigricans 451-465 blue mold. See Penicillrum expansum. brown, caused hy ScUrotinia cinerea 451-465 Rots, transportation, influence of orchard spraying 4*7-477 Roughage requirements of chicks 146 Rum^z acelosa, susceptibility to Puccinia triiicina 152-172 Salmon. W. D., et al.: INFLUENCE OF THE PLANE OF NUTRITION ON THE MAINTENANCE REQL^RE- MENT OF CATTLE 115-1M Page Sando. Charles E., and Barllett, H. H.: NOTES ON TNE ORGANIC ACIDS OF PYRUS CORONARIA. RHUS GLA- BRA. AND ACER SACCHARUM.. 221-229 OCCURRENCE OF QUERCETIN IN liMERSON'S BROWN- HUSKED TYPE OF MAIZE i-ji Sandstedt, R. M.. et al.: NUTRIENT RE- QUIREMENTS OF GROWING CHICKS: NUTRITIVE DEFICIEN- CIES OF CORN 139-149 Schistosoma japonicum, hemotoxins 387-432 Schultz, E. S.: A TRANSMISSIBLE MOSAIC DISEASE OF CHINESE CABBAGE, MUSTARD, AND TUR- NIP 173-17S Schwartz. Benjamin: HEMOTOXINS FROM PARASITIC WORMS 379^432 Sclerotinia cinerea, temperature relations. . . 451-465 Seamans. H. L., et al.: PALE WESTERN CUTWORM (POROSAGROTIS OR- THOGONIA MORR.) 289-322 Secale cerealc, susceptibility to aeciospores of Puccinia triticina 163-172 Seed of coniferous trees, storage 479-510 Sequoia sempenirens, host of HyhtruPes lig- neus 209-210 Sermn, efifect on hookworm hemolysin 41 2-413 Shapovalov, M., and Edson, H. A.: BLACK- LEG POTATO TUBER-ROT UNDER II^TUOATION 8i-9» Sheep, Shropshire, fertility 231-234 Shropshire sheep, fertility 231-234 Sitanion Hystrix, susceptibility to aeciospores of Puccinia triticina 163-172 Smut— of oats. See Ustilago avenae. onion. See Urocystis cepulae. stinking, of wheat. See TiUelia tritici. Sodium — carbonate, efitect on hydrocyanic acid in Sudan grass 135-136 chlorid, effect on growing chicks 145-149 hydroxid, effect on hydrocyanic acid in Sudan grass 135-136 nitrate, effect on plant growth 102-110 sulphate, effect on plant growth 102-1 10 Soil temperature, effect on — nodules 17-31 onion smut 235-262 Solanum tuberosum — absorption of copper from soil 281-287 Fusaria rots 65-80 Sorghums, hydrocyanic acid 125-138 Soybean meal, effect on growing chicks 145 SOYBEAN MOvSAIC 111-114 Soybeans, effect of soil temperature on nodule development 23 Sparrow, western grasshopper. See Ammo- dranius s^vannarum bimaculatus. Spelt fonn, behavior in wheat crosses 335-364 Spraying, influence on transportation rots of stone fruits 467-477 Squirrel, ground. Citellus richardsoni. Sterility in wheat crosses 58-61 Oct. i-Dec. 3, 1921 Index 525 Page 145-14Q 139-149 102-110 lOI-IIO 145-149 Stomach worm of sheep. Sec Hacmcnckits contortus. Stone fruit fungi, temperature relations. . . . 4^1-465 Stone fruits, influence of orchard spraying on rots , 407-477 STORAGE OF CONIFEROUS TREE SEED „, , . 479-.S10 Strand, A. L., et al.: PALE WESTERN CUTWORM (POROSAGROTIS OR- THOGONIA MORR.) 289-322 Stinking smut of wheat. See Tilktia tritici. Succinic acid. See Acid, succinic. Sudan grass. See Grass, Sudan. Sugar-beet root aphis. See Pemphigus hUae. Sugar beets, dry-rot canker 4.-,, Sugar maple. See Acer sacckarum. Sugars in grapefruit 26^-70 Sulphate- calcium, effect on plant growth 102-110 copper, toxicity to potato plants 281-287 iron, effect on growing chicks magnesimu, effect on growing chicks sodium, effect on plant growth Sulphates, relation to plant growth Sulphur, effect on growing chicks Sulphuric acid. See Acid, sulphuric. Sumac. See Rkus glabra. Sure, Bamett, and Read, J. W.: BIOLOGI- CAL ANALYSIS OF THE SEED OF THE GEORGIA VELVET BEAN, STIZOLOBIUM DEERINGIANUM SUSCEPTIBILITY OF THE DIFFER- ENT VARIETIES OF SWEET POTA- TOES TO DECAY BY RHIZOPUS NI- CRICANS AND RHIZOPUS TRITICI. 511 Swanson, C. O.: HYDROCYANIC ACID IN SUDAN GRASS Sweet potatoes. See Ipomoea batatas. Symphytum — asperrimum, failure to become infected from basidiospores of Puccinia rubigo-vera 152-172 officinale — host of Puccinia bromina susceptibUity to Puccinia trilieina.. Taenia — saginata, hemotoxins iSo- ,-,-, ,. , , 300^433 sclruin, hemotoxms ,go .-^ Tapeworm. Sec Diphyllobothriunt laiunt. Taraxacum officinalis, failure to become in- fected from basidiospores of Puccinia rubigo- vera TEMPERATURE AND HUmidity^^ '^' STUDIES OF SOME FUSARIA ROTS OF THE IRISH POTATO 65-80 Temperature — effect on hookworm hemolysin 413-414 soil, effect on— nodules j^,^ ^ onionsmut 235-262 TEMPERATURE RELATIONS OP STONE FRUIT FUNGI 4S1-465 Thalictrum — angustifolium, susceptibility to teliospores of Puccinia triticina j^5_j ^ j 125-138 154-172 152-172 157-173 157-172 • 157-172 of • 155-172 . . . 165 240 24a _,, , Page 1 n-dltctrum — Continued. aquilegifolium, susceptibility to teliospores of Puccinia triticina 155-172 dasycarPuvi, susceptibility to teliospores of Puccinia triticina Dclavayi, susceptibility to teliospores of" Puccinia triticina dioicum. resistance to teliospores of Puccinia triticina Fendleri. host of Puccinia CockeTelliana .... 165 foetidum. host of Puccinia triticina 164-173 viinus — resistance to teliospores of Puccinia trit- icina ,. ... 155-173 adtanttfolxum. susceptibility to teliospores of Puccinia triticina 157-172 polycarPum, susceptibility to teliopores of Puccinia triticina polygamum. resistance to teliospore; Puccinia triticina sparsiflorum. host of Puccinia alfernans Thorn, Charles, and LeFevre, Edwm- FLORA OF CORN MEAL jjg-.ss Thysanosoma adinioides. hemotoxins 421-432 Tilletia tritici, effect of — soil moisture soil temperature Tillotson,C. R.: STORAGE OF CONIFER- ^Uf^^^ESEED ,„.3.„ Tisdale. W. B., and Jones, Fred Rcuel- EFFECT OF SOIL TEMPERATURE UPON THE DEVELOPMENT OF NODULES ON THE ROOTS OF CER- TAIN LEGUMES Toxylon Pomiferum, host of Cyllene pictus. ^98-203 TRANSMISSIBLE MOSAIC DISEASE OF CHINESE CABBAGE. MUSTARD AND TURNIP.A 173-178 TRANSPORTATION ROTS OP STONE FRUITS AS INFLUENCED BY OR- CHARD SPRAYING 467_4„ Trichostrongylus retortaeformis, hemotoxins . 387-432 Trichuris — depressiuscula, hemotoxins triciura, hemotoxins Triticum — aegilops, susceptibility to aeciospores of Puc- cinia triticina ,63-172 aesttium, host of Puccinia triticina 151-172 capilatum. behavior of the spelt form in crosses , ^ ^ , 335-364 com Pactum — behavior ot the spelt form in crosses . vigor of Fi of crosses dicoccum— behavior of spelt form in crosses ^^^ vigor of Fi of crosses " , ^^^^ dicoccoides, behavior of spelt form in crosses 361 durum — behavior of spelt form in crosses vigor of F 1 of crosses polonicum, effect of crossing 335-364 satixum. behavior of spelt form in crosses with T. spella 17-31 420 384-432 • 335-364 , ... 54-63 335 54-63 335-346 526 Journal of Agricultural Research Vol. XXII Page Trilintm— Continued, spelt a, behavior of spelt form in c rosses with T. sativum 33S~364 turgidum, behavior of spelt form in crosses. 335 vulgare — behavior of spelt form in crosses 335-36^ vigor of Ki of crosses. S4-63 Syu. Tritkum aestivum. TroUius europeiis, resistance to teliospores of ^ Puccinia trilicina 1 5S~i72 Tuber-rot, blackleg 81-9 Tulip tree. See Liriodendron tulipifera. Turnip. See Brassica rapa. " ^ TURNIP MOSAIC 123-12 Tussilago Farfara, susceptibility to Puccinia triticina 152-1 7a Urocystis cepulae, effect of soil temperature 235-26* Urlica dioica — failure to become infected from basidiospores of Puccinia ritbigo-vera 152-172 susceptibility to Puccinia triticina 152-172 Ustilago — avenae, effect of soil temperature 242 levis, effect of soil temperature 242 Valeriana dioica, susceptibility to Puccinia triticina 152-172 Velvet bean. See Stizolobium deeringianum. Vitis— vinifera, succinic acid 224 sp., host of Cyllene pictus 198-203 Page Wade, J. S., and Boving, Adam G.: BIOL- OI.OGY OF EMBAPHION MURI- CATUM 323-334 Walker, J. C! , and Jones, L. R. : RELATION OF SOIL TEMPERATURE AND OTH- ER FACTORS TO ONION SMUT IN- FECTION 235-262 Water, hot, effect on hydrocyanic acid in Su- dan grass 131-133 Weather, effect on hydrocyanic acid in Sudan grass 136 Weimer, J. L., and Harter, L. L.: A COMPARISON OF THE PECTINASE PRODUCED BY DIFFERENT SPE- CIES OF RHIZOPUS 371-377 SUSCEPTIBILITY OF THE DIFFER- ENT VARIETIES OF SWEET POTA- TOES TO DECAY BY RHIZOPUS NIGRICANS AND RHIZOPUS TRIT- ICI s"-sts Western white pine. See Pinus monticola. Wheat crosses, vigor of Fi seedlings 53-63 Wheat, orange leafrust 151-172 Whipworm. See Trichuris trichiura. White oak. See Quercus alba. White pine, western. See Pinus monticola. Worms, parasitic, homotoxins 379-43» Xylotrcchus colonus, host selection 195-220 Yellow pine. See Pinus ponderosa. Yellow poplar. See Liriodendron tulipifera. c New York Botanical Garden Librar 3 5185 00263 3756