^pWYOMBOTANICALGAWg, |9( JOURNAL OF AGRICULTURAL RESEARCH Volume XVI JANUARY 6— MARCH 31, 191 9 PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASHINGTON. D. C. d i ) XJ 1911 (Ji |-r|TJ'-VT <^r^^ I EDITORIAL COMMITTEE OF THE I UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS 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 Entomolgisl and Assistant Chief, Bureju of Entomology FOR THE ASSOCIATION H. P. ARMSBY Director, Institute of Animal Nutrttion, The Pennsylvania State College J. G. LIPMAN Director, New Jersey Agricultural Expert- ment Statum, Rutgers College W. A. RILEY Entomologist and Chief, Division of Ento- mology and Economic Zoology, Agricul- tural Experiment Station of the University of Minnesota 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa. (n) CONTENTS Page Determination of Acidity and Titrable Nitrogen in Wheat with the Hydrogen Electrode. C. O. Swanson and E. L. Tague. . i Ash Absorption by Spinach from Concentrated Soil Solutions. Rodney H. True, Otis F. Black, and James W. Kelly 15 Nitrates, Nitrification, and Bacterial Contents of Five Typical Acid Soils as Affected by Lime, Fertilizer, Crops, and Moisture. H, A. NoYEs and S. D. Conner 27 Effect of Certain Ecological Factors on the Morphology of the Urediniospores of Puccinia graminis. E. C. Stakman and M. N. Levine 43 Variations and ^Mode of Secretion of Milk Solids. John W. GowEN 79 New Biologic Forms of Puccinia graminis. E. C. Stakman, M. N. Levine, and J. G. Leach 103 Influence of Salts on the Nitric- Nitrogen Accumulation in the Soil. J. E. Greaves, E. G. Carter, and H. C. Goldthorpe. . 107 Physoderma Disease of Com. W. H. TisdalE 137 Injury to Casuarina Trees in Southern Florida by the Mangrove Borer. Thomas E. Snyder 155 Life-History Observations on Four Recently Described Parasites of Bruchophagus funebris. Theodore D. Urbahns 165 Cyanogenesis in Andropogon sorghum. C. T. DowELL 175 Effect of Certain Compounds of Barium and Strontium on the Growth of Plants. J. S. McHargue 183 Apple-Scald. Charles Brooks, J. S. Cooley, and D. F. Fisher. 195 Angular-Leafspot of Tobacco, An Undescribed Bacterial Disease. F. D. Fromme and T. J. Murray 219 Two Species of Pegomyia Mining the Leaves of Dock. S. W. Frost 229 Influence of Foreign Pollen on the Development of Vanilla Fruits. T. B. McClelland 245 A Blood-Destroying Substance in Ascaris lumbricoides. Ben- jamin Schwartz 253 Solubility of the Lime, Magnesia, and Potash in Such Minerals as Epidote, Chrysolite, and Muscovite, Especially in Regard to Soil Relationships. R. F. Gardiner 259 A Field Study of the Influence of Organic Matter upon the Water- Holding Capacity of a Silt-Loam Soil. Frederick J. Alway and Joseph R. Neller 263 (in) rv Journal of Agricultural Research voi.xvi Page Fusarium-Blight of Potatoes Under Irrigation. H. G. Mac- MiLLAN 279 Effect of Certain Grain Rations on the Growth of the White Leghorn Chick. G. Davis Buckner, E. H. Nollau, R. H. WiLKiNS, and Joseph H. Kastle 305 Ammonification of Manure in Soil. H. J. Conn and J. W. Bright 313 Index 351 ERRATA AND AUTHORS' EMENDATIONS Page 103, line 15, "C I 2879" should read "C I 5879." Page 104, line 19, "C I 4103" should read "C I 4013."' Page 161, line 31, "A. labena n. sp." should read " Labena a. sp." Page 168, the cuts of figures i and 2 should be transposed. Page 229-244, throughout the paper " Pegomyia affinis Stein." should read " Pegomyia vanduze Mallock 1919." Page 248, Table III, column 2, line 11, "4K" should read "3^-" Page 252, Plate 31, A, should be inverted. Page 260, Table I, the figures in column 2 should read, from top to bottom, "1.39, 1.94. 1.89, .24, .23, .ai, .17, .19, .21, .55"; the figures in coliunn 3 should read ".50, .10, .17, ,05, .10, .11, .10, Trace, .03, .10." Page 261. line 10, "0.27" should read "0.26" and "0.17" should read "0.08." Page 280, parag.aph 2, line 15, "41, B" should read "40, A." Page 285, paragraph 2, line 5, "38. D" should read "38, C." Page 286. paragraph 3, line 11, "tube" should read "tuber." ILLUSTRATIONS PLATES Nitrates, Nitrification, and Bacterial Contents op Five Typical Acid Soils as Afpected by Lime, FERTn.izER, Crops, and Moisture Plate i. Representative plates from i to 400,000 bacterial dilution of acid page yellow silty clay, cropped and held under optimum moisture conditions: A. — ^Aerobic plates, untreated. B. — Aerobic plates, treated with 2 tons of calcium carbonate. C. — Aerobic plates, treated with complete ferti- lizer. D. — Aerobic plates, treated with complete fertilizer and 2 tons of calcium carbonate. E. — Aerobic plates, treated with complete fertilizer and 6 tons of calcium carbonate 42 Plate 2. Representative plates from i to 400,000 bacterial dilution of acid whitish silt loam and acid brown silt loam cropped and held under opti- mum moisture conditions: A. — Aerobic plates, acid whitish silt loam, untreated. B. — Aerobic plates, acid whitish silt loam, treated with 3 tons of calcium carbonate. C. — Aerobic plates, acid whitish silt loam, treated with 500 pounds of acid phosphate. D. — Aerobic plates, acid brown silt loam, untreated. E. — Aerobic plates, acid brown silt loam, treated with 3 tons of calcium carbonate. F. — Aerobic plates, acid brown silt loam, treated with 500 pounds of acid phosphate. G. — Anaerobic plates, acid browTi silt loam, untreated. H.— Anaerobic plates, acid brown silt loam, treated with 3 tons of calcium carbonate. I. — Anaerobic plates, acid brown silt loam , treated with 500 pounds of acid phosphate 4a Plate 3. Representative plates from i to 400,000 bacterial dilution of acid black peaty sand, cropped and held under optimum moisture conditions: A. — Aerobic plates, untreated. B. — Aerobic plates, treated with 2 tons of calcium carbonate. C. — Aerobic plates, treated with complete ferti- lizer. D. — Aerobic plates, treated with complete fertilizer and 2 tons of calcium carbonate. E. — Aerobic plates, treated with complete fertilizer and 6 tons of calcium carbonate. F. — Anaerobic plates, treated with complete fertilizer. G.— Anaerobic plates, treated with complete ferti- lizer and 2 tons of calcium carbonate. H. — Anaerobic plates, treated with complete fertilizer and 6 tons of calcium carbonate 42 Plate 4. Representative plates from i to 400,000 bacterial dilution of acid dark-brown peat, cropped and held under optimum moisture conditions: A. — Aerobic plates, untreated. B. — Aerobic plates, treated with 2 tons of calcium carbonate. C. — Aerobic plates, treated with 20 tons of cal- cium carbonate. D. — Anaerobic plates, untreated. E. — Anaerobic plates, treated with 2 tons of calcium carbonate. F. — Anaerobic plates, treated with 20 tons of calcium carbonate 4a Plate 5. Representative plates from i to 40,000 bacterial dilution of acid yellow silty clay kept at different moistiu-e contents: A2. — Aerobic plates, from soil kept one-half saturated. Ho. — Anaerobic plates, from soil kept one-half saturated. Cj. — Aerobic plates of carbon-dioxid-siu-viving organ- isms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — Anaerobic plates from soil kept fully saturated. C3. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated 42 (V) VI Journal of Agricultural Research voi.xvi Page Plate 6. Representative plates from i to 40,000 bacterial dilution of acid whitish silt loam kept at different moisture contents: Aj. — Aerobic plates from soil kept one-fourth satiu-ated. H,. — Anaerobic plates from soil kept one-fourth saturated. Cj. — Aerobic plates of carbon-dioxid-surviv- ing organisms from soil kept one-fourth saturated. Aj. — Aerobic plates from soil kept one-half saturated. H2. — Anaerobic plates from soil kept one-half saturated. C2. — Aerobic plates of carbon-dioxid-stu-viving organ- isms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — Anaerobic plates from soil kept fully satu- rated. C3. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated 42 Plate 7. Representative plates from i to 40,000 bacterial dilution of acid brown silt loam kept at different moisture contents: Aj. — Aerobic plates from soil kept one-fovu-th saturated. Hi. — Anaerobic plates from soil kept one-fourth saturated. Cj. — Aerobic plates of carbon-dioxid-siu-viv- ing organisms from soil kept one-fourth saturated. Aj. — Aerobic plates from soil kept one-half saturated. Hj. — Anaerobic plates from soil kept one-half saturated. Cj. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — Aerobic plates from soil kept fully saturated. C3. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated 42 Plate 8. Representative plates from i to 40,000 bacterial dilution of acid black peat>' sand kept at different moisture contents: Aj. — Aerobic plates from soil kept one-fourth saturated. H,. — Anaerobic plates from soil kept one-fourth saturated. C,. — Aerobic plates of carbon-dioxid- surviving organisms from soil kept one-fourth satm-ated. Aj. — Aerobic plates from soil kept one-half saturated. Hj. — Anaerobic plates from soil kept one-half saturated. C2. — Aerobic plates of carbon-dioxid-sur- viving organisms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — Anaerobic plates from soil kept fully saturated. • C3. — Aerobic plates of carbon-dioxid-surviving organ- isms from soil kept fully saturated 42 Plate 9. Representative plates from i to 40,000 bacterial dilution of acid dark-brown peat kept at different moistiu-e contents: Aj. — Aerobic plates from soil kept one-fourth satxirated. H,. — Anaerobic plates from soil kept one-fourth saturated. C^ — Aerobic plates of carbon-dioxid- surviving organisms from soil kept one-fourth saturated. Aj. — Aerobic plates from soil kept one-half satmated. H2. — Anaerobic plates from soil kept one-half satiu-ated. C2. — Aerobic plates of carbon-dioxid-sur- viving organisms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully sattu-ated. H3. — Anaerobic plates from soil kept fully saturated. C3. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated 42 Physoderma Disease of Corn Plate A. Com leaf showing the effects of an attack by Physoderma zeae- maydis i54 Plate B. Sheath and culm of com plant showing the effects of Physoderma zeae-m^ydis ^ 54 Plate 10. Blade and sheath of com plant showing the effects of severe attack by Physoderma zeae-maydis. Jan. 6-Mar. 31, 1919 Illustrations vii Page Plate ii. Old sheaths and culms of corn showing effects of severe attacks by Physoderma zeae-maydis: A, B. — Badly diseased stalks broken over at weakened, infected lower nodes. C, E. — Portions of old attacked sheaths showing characteristic shredding. D. — Portion of an old infected stalk showing discoloration both on outside and in pith due to the attacks of the fungus 154 Plate 12. Blades and sheath of com showing the Physoderma disease pro- duced by inoculating plants in the greenhouse with a suspension of the sporangia of P. zeae-maydis in tap water 154 Plate 13. Physoderma zeae-maydis: Various stages in the germination of sporangia, formation of zoospore, and germination of zoospore, a. — Spor- angium, b, c, d. — Opening sporangia showing the earl)^ stages of zoospore formation, e. — Mature zoospores escaping through the ruptured apex of the endosporangium . /. — The collapsing endosporongium after the zoo- spores have escaped, g. — Zoospores, h. — Germinating zoospores 154 Plate 14. Physoderma zeae-m.aydis: a-f. — Zoospores germinating by fine threadlike hyphae which have penetrated the epidermal cell walls of a tender leaf of Indian com. In c, e, and/ the enlarged cells have begun to form in the epidermal cells of the host 154 Plate 15. Physoderma zeae-m,aydis : Mycelial stages within the host cells. a-d. — Drawings from ordinary' high-power magnifications showing the fibers and enlarged cells of the mycelium, e-g. — Drawings magnified with oil-immersion lens, b, d, g. — Notice the young sporongia at the ends of the short hyphae 154 Plate 16. Physoderm,a zeae-maydis: a-e, Mycelial fibers penetrating the cell walls of the host tissue. /, g, Different t^'pes of reproductive bodies 154 Plate 17. Physoderma zeae-maydis: Photomicrographs showing the different stages of the development in the host tissue (teosinte). A. — Notice the reproductive bodies connected by the very fine threadlike hyhae in the central cells of the figure. B. — Host cells filled with mature sporangia. . . 154 Injury to Casuarina Trees in Southern Florida by the Mangrove Borer Plate 18. A. — Casuarina trees planted along the water front, Belle Isle, Miami Beach, Fla., June, 1918. B. — Casuarina trees disfigured and killed by the mangrove borer {Chrysobathris tranquebarica) at Miami Beach, Fla. . 164 Plate 19. Chrysobothris tranquebarica: A. — Sex differences in the last abdominal segment. B. — Lateral and dorsal view of ovipositor. C. — Bark of red mangrove (Rhizophora mangle) showing how it is divided into plates 164 Plate 20. Chrysobothris tranquebarica: A. — Larval burrow in cambium of Australian pine (Casurina equiseii/olia), Miami Beach, Fla. B. — Larvae, ventral and dorsal views. C. — Pupa, dorsal and central views. D. — Female and male adult beetles 164 Plate 21. Chrysobothris tranquebarica: Adult male, dorsal view 164 Life-History Observations on Four Recently Described Parasites of Bruchophagus funebris Plate 22. A. — Liodontomerus perplexus: Adult female. B. — Eutelus bru- chophagi: Adult female 174 Plate 23. A. — Trim,eromicrus maculatus: Adult female. B. — Liodontomerus secundus: Adult female 174 VIII Journal of Agricultural Research voi. xvi Effect of Certain Compounds of Barium and Strontium on the Growth of Plants Page. Plate 24. A. — Effect of barium on the growth of cowpeas with and without calcium carbonate. B. — Stimulating effect of barium on root growth of cowpeas. C. — Effect of barium on the growth of soybeans 194 Angular-Leafspot of Tobacco, an Undescribed Bacterial Disease Plate 25. A. — A tobacco leaf showing an early stage of the angular-leafspot. B. — Angular leaf spots on a tobacco leaf 228 Plate 26. A. — Upper surface of a tobacco leaf affected with the angular- leafspot. B. — Lower siirface of a tobacco leaf affected with the angular- leafspot 228 Plate 27. A. — Angular leaf spots on a tobacco leaf as seen in transmitted light. B. — ^Atypical angular leaf spots on a narrow leaf of tobacco 228 Two Species of Pegomyla Mining the Leaves of Dock Plate 28. A. — Eggs of Pegomyia affinis. B. — Parasitized eggs of Pegomyia calyptrata. C. — Eggs of Pegomyia calyptrata. D. — A small mine on Rumex leaf, showing the original linear mine and the beginning of the blotch mine. E. — Atypical mine on Rumex oblusifolius produced by the larva of Pego- myia calyptrata. F. — A monstrosity, an adult P. calyptrata issuing feet first from its puparium. G. — A typical mine on Rumex crispus produced by the larva of Pegomyia calyptrata. H. — A mine on Rumex obtrusif alius, pro- duced by a nearly mature larva entering a new leaf to complete its develop- ment 244 Plate 29. A. — Fiske cages, used in studying the adult flies of Pegomyia spp., showing the arrangement in outdoor insectary. B. — Glass cylinder cage, used in studying habits of adult flies. C. — Fiske cage, used in studying habits of adult flies 244 Plate 30. A. — Ventral aspect of posterior segment of larva of Pegomyia affinis, showing lobes and anal opening. B. — Lateral aspect of posterior segment of larva of P. affinis, showing tubercles. C. — Lateral aspect of posterior seg- ment of larva of P. calyptrata. D. — Ventral aspect of posterior segment of larva of P. calyptrata, showing lobes and anal opening. E. — " Pseudo- cephalon" (Henneguy) of P. calyptrata: a, buttonlike areas referred to in text; 6, sensory papilla; c, antenna; d, mandibular sclerite; e, anterior spiracle. F. — Posterior spiracle of larva of P. calyptrata 244 Influence of Foreign Pollen on the Development of VANaLA Fruits Plate 31. A. — Left to right. Vanilla planifolia, first of each pair close-fertil- ized, second fertilized with- vanillon pollen. B. — Cross sections of same fruits 353 Pate 32. A. — Vanillon No. 13 fruits, close- and cross-fertilized. Lower row close-fertilized, upper fertilized with pollen of Vanilla planifolia. B. — Cross section of same fruits 252 Plate 33. A. — Vanillon No. 34: Fruits at right fertilized with pollen of Vanilla planifolia, left close-fertilized. B. — Cross sections of same fruits. 252 Plate 34. A. — Vanillon No. 43 fruits. Upper row fertilized with pollen of Vanilla planifolia, lower row close-fertilized. B. — Cross sections of same fruits 252 Jan. 6-Mar. 31. 1919 IllustrationS IX Pace- Plate 35. A. — Longitudinal sections of vanilla fruits. Left to right: Vanil- lon, pistillate, X vanillon, staminate; vanillon, pistillate, X Vanilla planifolia, staminate; Vanilla planifolia, pistillate, X vanillon, staminate; Vanilla planifolia, pistillate, X Vanilla planifolia, staminate. B. — Com- parative length of cleared columns and ovaries. Vanillon above. Vanilla planifolia below 252 A Field Study op the Influence of Orgaotc Matter upon the Water- Holding Capacity op a Silt-Loam Soil Plate 36. View of Field J., Minn. Agr. Experiment Station Farm, showing topography and surroimdings, looking from plot 6 to the bam which occu- pies part of plot 1 278 Fusarium-Blight of Potatoes Under Irrigation Plate 37. Effect of Fusarium-blight on seed pieces of potato: A. — Early- Ohio seed pieces: Control above; pieces inoculated with F. oxysporum below. B. — Early Ohio plant. See piece inoculated with F. oxysporum. C. — Early Ohio seed pieces: Control (left) and inoculated (right) seed pieces. D. — Seed piece well decayed, resulting from soil infection. E. — Seed-piece rot in field 304 Plate 38. A. — Inoculated and uninoculated stems of same potato plant. B. — Potato plant (control), showing method of inoculating with wedge of melilotus stem. C. — Seed-piece rot in field. D. — Potato plant naturally infected by F. oxysporum in the field 304 Plate 39. Potato stems showing seed-piece rot: A. — Stem split to show rot- ting due to organism entering through seed piece from soil. B. — Stem split to show slight discoloration at base where infection from soil-infected seed piece occiured. C. — Seed piece of potato plant shown in Plate 40, B. D. — Seed-piece rot in field 304 Plate 40. Potato plants affected by Fusarium blight: A. — Potato plant late in season with rolled leaves. Fusarium blight. B. — Top of potato plant consisting of three stems. C. — Potato plant of two stems; one at left showing Fusarium blight, or vnXt caused by seed-piece infection; one at right healthy. D. — Plant Adth rolled leaves gradually dying from Fusarium blight 304 Plate 41. A. — A field of potatoes showing the result of unfavorable cultural and soil conditions, by which seed-piece rot destroyed 60 per cent of the stand. B. — A field of potatoes planted with whole seed (rows to right) and cut seed (rows to left) at midseason 304 Effect op Certain Grain Rations on the Growth of the White Leghorn Chick Plate 42. A. — Lot i at the age of 3 months. B. — Lot 3 at the age of 3 months. C. — Lot 2 at the age of 3 months 31a TEXT FIGURES Determination of Acidity and Titrable Nitrogen in Wheat with the Hydrogen Electrode Fig. I. Graphs showing the hydrogen-ion concentration of wheat extract on titration to Ph 7 6 2. Graphs showing the hydrogen-ion concentration of wheat extract on titration Pa 8.3 6 106547°— 20 2 X Journal of Agricultural Research voi. xvi Page Fig. 3. Graphs showing the hydrogen-ion concentration of wheat extract on titration to Ph 9-3 6 4. Graphs showing the production of amino nitrogen at different temperatures 10 5. Graphs showing the total phosphorus in wheat extract at different periods of extraction and at different temperatures la 6. Graphs showing the inorganic phosphorus in wheat extract at different periods of extraction and at different temperatures 12 Ash Absorption by Spinach prom Concentrated Soil Solutions Fig. I. Graphs representing total ash and individual ash constituents found in spinach tops from plots receiving the substances indicated 20 2 . Graphs representing total ash and individual ash constituents in spinach roots from plots receiving the substances indicated 20 Nitrates, Nitrification, and Bacterial Contents of Five Typical Ac!id Soils Affected by Lime, Fertilizer, Crops, and Moisture Fig. I. Graphs showing the relation of aerobes and anaerobes to nitrification of five acid soils \\dth and without lime and fertilizer treatments 37 2. Graphs showing the relation of aerobes and anaerobes to nitrates in five acid soils kept at different moisture contents 38 Influence of Salts on the Nitric-Nitrogen Accumltlation in the Soil Fig. I . Graphs showing molecular concentrations at which the highest stimu- lation is noted 128 2. Graphs showing the percentage of stimulation at the above noted mole- cular concentrations (see fig. i), the untreated soil being counted as producing 100 per cent of nitric nitrogen 129 3. Graphs showing the molecular concentrations at which the various salts are toxic to nitrification ■. •. . 130 4. Graphs showing the molecular concentrations which reduce the nitrifi- cation to three-fourths normal 131 5. Graphs showing the percentages of nitric nitrogen produced in 100 gm. of soil to which had been added 2 X lo-^ mole of the various salts, the untreated soil being counted as producing 100 per cent 132 Physoderma Disease of Corn Fig. I. Map showing the distribution of Physoderma zeac-maydis in the United States. Broken lines, P. zeae-maydis present: solid line, P. zeae- maydis causing damage 138 Injury to Casuarina Trees in Southern Florida by the Mangrove Borer Fig. I. Chrysoboihris tranqueharica: I,arva, dorsal, lateral, and ventral views.. . 159 2. Chrysoboihris tranqucbarica: a, Female pupa, ventral view; b, same, dorsal view 160 Jan. 6-Mar. 31, 1919 IllustratiOflS XI Life-History Observations on Four Recently Described Parasites OF Bruchophagus funebris Page Fig. I . Liodontomerus perplexus: Larva 168 2 . Liodontomerus perplexus: Pupa 168 3. Liodontomerus sccundus: Larva 170 4. Liodontomerus secundus: Pupa 170 5. Eutelus bruchophagi: Larva 171 6. Eutelus bruchophagi: Pupa 171 7. Trim,erom.icrus maculatus: Larva 173 8. Trimeromicrus m.aculaius: Pupa 173 Apple-Scald Fig. I. Graphs showing the effect of maturity upon susceptibility of Grimes apples to scald 197 2. Graphs showing the relative susceptibility to scald of eastern and western Grimes apples 198 3. Graphs showing the effects of temperature on apple-scald at the end of 4, 6, 7, 9, 13, and 16 weeks 199 4. Graphs showing the effects of temperature on apple-scald at the end of 2 , 3, 4, 5, 7, 12, and 16 weeks 200 5. Graphs showing the effects of temperature on apple-scald at the end of 2,3,4, 5, 9, and II weeks 20a 6. Graphs shomng the effect of temperature on apple-scald at the end of 5, 7,9, II, 13, 16, and 19 weeks 203 7. Graphs showing the effect of temperature on apple-scald at the end of 7 , 10, 12, and 18 weeks 204 8. Graphs showing the effect of temperature on apple-scald at the end of 12 , 14, 20, and 26 weeks 205 9. Graphs showing the effect of temperature on apple-scald at the end of 8, 12, 16, and 20 weeks 205 10. Graphs showing the effect of temperature on apple-scald at the end of 8, 12 , and 16 weeks 209 11. Graphs showing the relation of carbon dioxid to apple-scald production . 2 10 Two Species of Pegomyia Mining the Leaves of Dock Fig. 1. a, phar^'ngeal skeleton of first instar, Pegomyia calpytrata Zett; B, phar- yngeal skeleton of second instar, P. calyptrata; C, mandibular scle- rite of second instar, P. calyptrata; D, pharj'ngeal skeleton of third instar, P. calyptrata; E, phar}-ngeal skeleton of first instar, P. affinis Stein; F, mandibular sclerite of first instar, P. affinis; G, phary-ngeal skeleton of second instar, P . affinis; H, pharyngeal skeleton of third instar, P. affinis 233 A Field Study of the Influence of Organic Matter uton the Water- Holding Capacity of a Silt-Loam Soil Fig. I. Diagram showing arrangement of plots and crops on field J, University Farm, St. Paul, Minn. A is the original plan adhered to from 1893 to 1914, while B shows the cropping plan in 1915. C shows the arrange- ment of the samples taken for the two composites from a plot 265 2. Diagram showing the amount and distribution of the rainfall at Univer- sity Farm, St. Paul, Minn., during part of the season of 191 5. The dates of sampling are indicated by asterisks 269 .^iijBjjiMHHmiMKBMaeiiwvutM^ffniiiiiaiaim .III II— 11111111 Vol. XVI JAMUARY 6. 1919 No. 1 JOURNAL OF AGRICULTURAL RESEARCH CONXE^NXS Paga Determination of Acidity and Titrable Nitrogen in Wheat with the Hydrogen Electrode - - - - - 1 C. O. SWANSON and E. L, TAGOE (Contilbution from Kansas Acricultaral Experiment Station) Ash Absorption by Spinach from Concentrated Soil Solu- tions -- - - - -- - ~ "* 15 RODNEY H. TRUE, OTIS F. BLACK, AND JAMES W. KELLY ( ContributJon from Bureau oft»lant Industry ) PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE. WITH THE COOPERATION or THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WT^SHIKGTON, D. C. wkKuutmrnmimmii WASHiNQTON ! QOVERNMEWr.PmNTtNa OFFICE : 1810 ^ w*, EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chairman PhyiiUogist tend AssociaU Chief .Bureau of Plant Industry - EDWIN W. ALLEN Chief, Office of Experimeitt Stations CHARLES L. MARLATT Entomologist and Assistant Chief , Bureau of Entomology FOR THE ASSOCIATION H. P. ARMvSBY, Director, Institute ofAHimal Nutrition, The Pennsylvania Stale College E. M. FREEMAN Botanist, Plant Pathologisl and. Assistant Dean, Agricultural Experiment Station of the Unixersily of Minnesota J. G. LIPMAN Director , New Jersey Agricttliural Experi- ment Station, Rutgers 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 H. P. Armsby, Institute of Anim:^' ^'Mtritinn, State College, Pa. Vol. XVI Washington, D. C, January 6, 1919 No. i DETERMINATION OF ACIDITY AND TITRABLE NITRO- >^ew Y&r- GEN IN WHEAT WITH THE HYDROGEN ELECTRODE ■.--.> By C. O. SwANSON, Associate Chemist, and E. L. TaguE, Assistant Chemist, Department of Chem,isiry, Kansas Agricultural Experiment Station Acidity in wheat flour is usually determined by making a water extract of the flour at a definite temperature for a definite time and titrating this extract with a standard alkali, using phenolphthalein as an indicator. Since the quantity of alkali neutralized is governed to a considerable extent by the temperature and duration of extraction, a large number of workers extract at 40° C. for two hours. This method can also be used for wheat {Triticum aestivum), the grain being first finely ground. Because wheat contains the enzym phytase, as has been shown by several investigators,^ it is to be expected that the duration of extraction and the temperature used will influence the amount of standard alkali neutralized. Acidity in wheat or wheat flour is not due to the presence of free acids as that term is ordinarily understood. The varying amounts of alkali neutralized in different samples are supposed to be due to the presence of phosphates in less or greater amounts. This statement is supported by the fact that the greater the acidity the greater the amount of phosphor- us in the extract.^ Water extracts of wheat and wheat flour as ordinarily made are slightly turbid. This turbidity depends to some extent on the nature of the flour. Extracts from wheat and low-grade flour give clearer extracts than those from high-grade flour. This is probably due to the presence of greater amounts of electrolytes in the ground wheat and low- grade flour, as these would help to coagulate the turbid or colloidal matter. Because of the turbidity and the colloidal nature of wheat and flour extracts, absorption plays a part in the determinations of acidity when the colorometric method is used. ' Anderson, R. J. concerning the organic phosphorus compound of wheat bran and the HYDROLYSIS OFPHYTIN. N. Y. State Agr. Exp. Sta. Tech. Bui. 40, 31 p. 1915. ' Swansom, C. O. acidity in WHEAT flour; its relation to phosphorus and to other constitu- ents. Jn Jour. Indus, and Engin. Chem., v. 4, no. 4, p. 274-278. 1912. Journal of Agricultural Research. (l) Vol. XVI, No. i Washington, D. C. Jan. 6, 1919 qv Key No. Kans.-i8 2 Journal of Agricultural Research voi. xvi. No. i METHOD OF MAKING THE EXTRACT A good grade of Kansas hard wheat was used for this work. The wheat was finely ground, untempered in a burr mill. Fifty gms. of this ground material were weighed into a quart Mason jar and heated to the temperature used in making the extraction. Five hundred cc. of carbon- dioxide-free water, previously heated to the temperature employed, were then added, together with 5 cc. of toulene as a preventive of bac- terial action. The whole was thoroughly shaken and placed in a thermostat. The shaking was repeated every minute for the first 5 minutes, then every 15 minutes during the time of extraction. At the end of the extraction period the contents of the jar were poured into centrifuge cups and centrifuged for 5 minutes at 2,000 revolutions per minute. The supernatant liquid was then poured through a filter for the purpose of removing light floating particles. The filtrate was used for the following determinations: 1. The hydrogen-ion concentration or the Ph value of the extract. 2. The cubic centimeters of NI20 barium hydroxid used to titrate to the absolute neutral point of Ph 7. 3. The cubic centimeters of alkali used to titrate to the point of color change for phenolthalein or Ph8.3. 4. The cubic centimeters of alkali used to titrate to the point of color change for thymolphthalein or PhQ-S- 5. The amount of alkali necessary to reneutralize after the addition of neutral formaldehyde or the vSorensen method of determining amino nitrogen. 6. The total phosphorus in the extract. 7. The phosphorus in the extract precipitated by magnesia mixture. This may be considered phosphorus in the inorganic form. This method of determination is based on that used for the determination of inorganic phosphorus in animal tissues. APPARATUS USED IN DETERMINING HYDROGEN-ION CONCENTRA- TION Our apparatus contains the following pieces: One Kohlrausch slide wire bridge; one type B, No. 2500 Leeds and Northrup galvanometer; one Weston millivoltmeter and multiplier; Edison storage cells; and the hydrogen and the normal calomel electrodes made according to the directions of Hildebrand.^ Hydrogen made by the electrolytic process was used. As a precaution against impurities, the gas was washed in a train of alkaline permanganate and pyrogallic acid. 1 HlLDEBRAND, JOEL H. SOME APPLICATIONS OP THE KA'DROGEN ELECTRODE IN ANALYSIS, RESEARCH, AND TEACHING. In JouT. Amer. Chem. Soc, v. 35, no. 7, p. 847-871, 15 fig. 1913- Jan. 6. 19X9 Determination of Acidity and Nitrogen in Wheat 3 PERIODS OF TIME AND TEMPERATURES USED After some preliminary work the following periods were chosen: 5, 30, and 60 minutes, 2, 4, 8, 16, and 24 hours. Extractions were made at the following temperatures: 5°, 20°, 40°, and 50° C. Other temperatures and periods were tried in the preliminary work, but the results obtained were found of no added value. METHOD OF DETERMINING THE HYDROGEN-ION CONCENTRATION One hundred cc. of the solution prepared as above — namely, the extract of 10 gm. of ground wheat — were pipetted into the hydrogen-ion cell. This is closed with a rubber stopper through which the electrodes, as well as the tip of the burette, are inserted. In this way the carbon dioxid from the air is excluded. The hydrogen gas is bubbled through until equilibrium is reached. The reading of the voltmeter gives the figure for calculating the actual hydrogen-ion concentration of the solution. To facilitate this calculation, we have made a table giving the hydrogen- ion concentration corresponding to the volt readings from 0.281 to 1.090. It takes about an hour to obtain equilibrium, but this can be shortened by previously saturating the electrode with hydrogen. Barium hydroxid {N/20) was then added till the solution showed a volt reading of 0.686, indicating a hydrogen-ion concentration of Pg 7. This represents the absolute neutral point. The alkali was again added till the volt reading was 0.760, indicating a ?□ value of 8.3. This corresponds to the acidity as commonly determined by the use of phenolphthaiein as indicator. In several trials this indicator was added at this point, and gave the usual color change. The alkali was again added until the volt reading was 0.820, indicating a Pq value of 9.3. This is the point of color change of thymolphthalein, and this was actually determined by the use of the indicator. At this point 25 cc. of formaldehyde solution were added. This was made by mixing one part of 40 per cent formaldehyde with two parts of carbon-dioxid-free water and neutralizing to the hydro- gen-ion value Ph 9-3 before using. The hydrogen gas was bubbled through until equilibrium was reached, and the Pg value noted. Barium hydroxid {N/20) was then added until a Pg value of 9.3 was again reached. HYDROGEN-ION CONCENTRATION OF WHEAT EXTRACT The results obtained by these methods at temperatures varying from 5° to 50° C, and for periods varying from 5 minutes to 24 hours are given in Table I. Journal of Agricultural Research Vol. XVI, No. 1 Table I. — Hydrogen-ion concentration of ivheat extracts made at different temperatures and periods, together with the quantity of Njso barium hydroxid used in the titrating to change the hydrogen-ion concentration Tem- pera- ture. 5 S 5 5 20 20 20 20 20 20 20 20 40 40 40 40 40 40 40 40 50 50 50 50 50 Time. 5 minutes. . 30 minutes. 60 minutes. 24 hours. . . 5 minutes. . 30 minutes. 1 hour 2 hours .... 4 hours. . . . 8 hours. . . . 16 hours. . . 24 hours. . . 5 minutes. 30 minutes, 1 hour 2 hours. . . . 4 hours. . . . 8 hours. . . . 16 hours. . . 24 hours. . . 5 minutes. 30 minutes 1 hour 2 hours. . . . 4 hoiirs. . . . Volt reading. 663 661 666 661 662 ,662 ,667 ,663 ,661 ,661 ,661 ,656 .650 •655 ■ 654 • 643 • 654 •653 .651 ■ 654 .651 .652 •653 .648 ■655 Ph values. 6.60 6-57 6.65 6.57 6.58 6.58 6.67 6.60 6-57 6- 57 6-57 6.48 6.36 6.46 6.44 6.26 6.44 6.43 6-39 6.44 6-39 6. 41 6.43 6.34 6.46 Quantity of Ntzo barium hydroxid used to titrate to — 0.686 or Ph 7- Cc. 0-5 •5 • 4 1-5 1. I 1-3 1-5 2. 2 3-4 3-4 4-5 1.9 3-4 4.4 4.9 5-2 5-7 6.3 6.9 3-2 3-5 4.0 5-8 5-3 0.760 or Ph8.3- Cc. 1-3 1.6 5-5 2. I 3-1 14-3 16.8 3-9 7-4 II. I 14-3 17-3 17.9 18.0 18.2 6.5 10. 4 13-7 16.3 16. 4 0.820 or Ph9.3- Cc. 2.8 3-1 3-0 9.0 4.9 7-3 8.8 II. 8 13-5 19. o 22. 4 5-4 10. 7 IS- 7 19. I 22. 4 23.8 23 9 24. I 9.0 14.9 18.2 21. 4 21-5 Volt reading after adding formiil- dehyde. 0.820 or Ph9-3 after adding formal- dehyde. Cc. 0.778 •785 .790 .778 •763 .761 •763 .768 .770 . 762 .770 .770 .780 .771 .772 . 762 .772 .776 .776 .780 . 762 .770 •774 •774 . 780 Cc. 2.9 3-1 3-5 3-7 4-4 4-7 4-9 ^•7 2>-2> 3-7 4-3 4. I 4.0 4. I 4.2 3-5 3-6 3- I 3-2 3-0 The data in Table I show the following results : 1. The average hydrogen-ion concentration of the extracts obtained at 5° C. is Ph 6.60; at 20° C. it is Ph 6.59; at 40° C. it is Ph 6.43; and at 50° C. it is Ph 6.52. Thus, the higher temperature gives an extract of slightly higher hydrogen-ion concentration. 2. The hydrogen-ion concentration does not increase with the dura- tion of digestion. The average volt readings at 40° C. for 5 minutes, 30 minutes, and i hour is 0.653, corresponding to Ph 6.43. The average volt readings for 8, 16, and 24 hours are also 0.653, corresponding to Ph 6.43. The longer or shorter period of digestion does not increase nor decrease the hydrogen-ion concentration. The sHght variations obtained at 20° and 50° for the different periods are so small that they do not modify the above statement. Jan. 6, 1919 Determination of Acidity and Nitrogen in Wheat 3. A volt reading of 0.760 (Ph 8.3) corresponds to the point of color change of phenolphthalein. The reading 0.820 (Ph 9-3) corresponds to the point of color change for thymolphthalein. This was determined by adding these indicators at the points mentioned. 4. The amount of N/20 of barium hydroxid used to titrate to Ph 8.3 is greater than the amount used to titrate to Ph 7. and the amount used to titrate to Ph 9.3 is greater than the amount used to titrate to Ph 8.3, and these differences show a progressive increase as the time of digestion is increased from 5 minutes to 24 hours. 5. The values obtained at 20° C. are lower than those at 40° and at 50°. At 5° they are the lowest. x\t this temperature very little or no hydrolysis takes place. Hydrolysis, as well as proteolysis, is most active at 40°. 6. The amount of N/20 barium hydroxid required to titrate to Ph 7 at 40° C. increased but little after 16 hours. In titrating to Ph 8.3 or Ph 9.3 there is very little increase after 4 hours. For example, hydrolysis is slightly slower at 20° than at 40°, and also slower at 40° than at 50°. 7. At the end of 4 to 8 hours at 40° C, when hydrolysis is practically complete, three times the number of cubic centimeters are required to titrate to Ph 8.3 as are necessary to titrate to Ph 7. and four times as many are required to titrate to Ph 9-3- 8. The number of cubic centimeters used to determine the titrable nitrogen after the addition of formaldehyde shows a progressive increase with the duration of digestion, but the maximum is reached in about two hours, at 40° C. At 20° the increase is much slower and continues to the end of 24 hours. At 50° the final results obtained are lower than those at either 20° or 40°, but show no increase or decrease correspond- ing with the time. At 5° there is very little increase from 5 minutes to 24 hours. The number of cubic centimeters required to titrate to a definite point were only about one-third as many as those required to titrate to the same point at 40°. 9. The most outstanding result shown in Table I is the fact that, while the hydrogen-ion concentration shows no increase with the duration of digestion, the amount of N/20 barium hydroxid used to neutralize to a given hvdrogen-ion concentration increases in proportion to the duration of the time of digestion. A definite limit, however, is soon reached. This limit is reached soonest at the highest temperature. 10. The digestion of ground wheat in water produces a substance which is not ionized; yet it will neutralize definite amounts of standard hydroxid, and these quantities correspond to a certain extent with the duration and temperature of digestion. When the standard alkali is added, it is ionized and the quantity present can be determined by the method of titration. The results obtained with various concentrations at 5°, 20°, and 40° C. are graphically presented in figures i, 2, and 3. Journal of Agricultural Research Vol. XVI, No. I Figure i shows the results of titrating to Ph 7. At 20° the increase is slow and gradual. At 40° it is very rapid up to two hours, and then it is very gradual. Fig. 1.-- Grapns showing the Ir/drogen-icn concentraUon of wheat extract ca titration to Pa 7. i -,_. ■ 1 , ■»h'i ■^ ^ " ^ I ^ / 2> '1 ,-- 1/ ^ ^ / ^ ^ / ^ / .-' ^ - J \^ ^ 4>': "" ^ 1^ i ^ \^ " .. ^ L> <§ <^ y 1 '^' 1 / / 1/ >J ^ " ! / — ■^ /! i ^ -"^^ ^ -— " - 1 ,1 1 1 1 yj> /JV L ..J ^ Vie. 2.— Graphs -.liowing the hydrogen-ion concentration of wheat extract on titration to Ph 8.3. 2S — — — \ i 1 ! =^ s>*c ,^ _i y ^ ^ J'^ ■~ A ^ ^ ^/ 7 i ^ _ fO f ^ ~ 7 ^ .^ n ^ <■ x' /7 X' _ ^ ».''■*' • > V" ~ ■ ~ t>^ "" ■ X / 7^ 1 / 1 ^- 'C^ s -y ~ \ / ^ J' 7 ' ~" ff rr ^ tu- ~ ^ 1 " :! i 1 1 1- £ ■■u / < 9 if < 9 V v / «" £ -^ Fig. 3.— Graphs showing the hydrogen-ion concentration of wheat extract on titration to Pm 9-. Jan. 6, 1919 Determination of Acidity and Nitrogen in Wheat 7 Figure 2 shows the results of titrating to Ph 8.3. At 5° the amount of standard alkaH required increases very slowly. At 20° the increase is very rapid up to four hours, followed by a gradual increase up to 24 hours. At 40° the increase is most rapid up to four hours. After that the increase is slight or practically none. The final results at 20° and 40° are not much different. Figure 3 shows the results of titrating to Ph 9.3. The general direc- tion of the curves are similar to those in figure 2, except that the values are higher. DETERMINATION OF NITROGEN IN AMINO FORM BY FORMALDEHYDE METHOD Some investigators neutralize to litmus ^ and others to phenolphthalein ^ before adding the formaldehyde. In Table I the results obtained by neutralizing to three points: Ph 7, Ph 8.3, and Ph 9.3, are given. That the latter two are the points of color change for phenolphthalein and thymolphthalein, respectively, in these extracts, was determined ex- perimentally. At Ph 9.3 the formaldehyde was added and then the titration repeated until the concentration was again Ph 9.3. The results of this last titration are given in the last column of Table I. The amounts given times 0.7 gives the milligrams of nitrogen in the amino form. If the formaldehyde had been added at Ph 8.3 and then the titration continued to the Ph 9.3, a larger amount of NI20 barium hydroxid would have been neutralized. Still larger amounts would have been neutralized if the formaldehyde had been added at the strictly neutral point, Ph 7, and then the titration continued to the Ph 9.3 point. These amounts are given in Table II, which has been calculated from Table I. Table II. — Quantity {in cubic centimeters) of NI20 barium hydroxid required had the neutralization been made to the points indicated before tlw formaldehyde was added Duration of extraction. 5 minutes. . 30 minutes . 1 hotir 2 hours 4 hours 8 hours 16 hours. . . . 24 hours. .. . First neutralization to Ph 8.3. 5° C. 20° C. 40° C. Cc. 3-0 2.9 5-7 Cc. 3-4 4-7 5-9 7.0 7.6 8.6 9.4 10.5 Cc. 3-2 6.6 8-3 9.1 9.2 9.9 10. o 10. I First neutralization to Ph 7. 5°C. 20° C. 40° C Cc. 3-8 4. I 4.0 9-7 Cc. 4-7 6.7 9. I 10.8 ^3-3 15-3 20. 3 22.8 Cc. 5-2 10. 6 15.0 18.5 21.3 22. I 23-9 21. 4 ■ Allen's Commerciai, Organic Analysis, v. 8, p. 479. Philadelphia, 1918. » AbDERHALDEN, E&III,, ED. HANDBUCH DER BIOCHEMISCHEN ARBEITSMETHODEN. Bd. 3, HiilftC I, p. 228. Berlin, Wien, 1910. 8 Journal of Agricultural Research Vol. XVI, No. I The figures given under first neutralization to Ph 8.3 are obtained as follows: The number of cubic centimeters of NJ20 barium hydroxid obtained by titrating to Ph 8.3 are subtracted from the number of cubic centimeters obtained by titrating to Ph 9.3. This difference is added to the figures in the last column of Table I. The figures so obtained are assumed to be the same as if the formaldehyde had been added at the concentration Ph 8.3 and then the titration resumed till the concentration Ph 9-3 was obtained. The figures under first neutralization to Ph 7 were calculated in the same way except that the differences between the number of cubic centimeters in the columns under Ph 7 and Ph 9.3 in Table I were used. The calculations made on these assumptions show that the "formol" or titratable nitrogen obtained by first titrating to Ph 8.3 and then adding the formaldehyde is over twice that obtained by titrating first to Ph 9.3 and then adding the formaldehyde. And further, if the for- maldehyde is added at Ph 7 and the titration is then continued to the concentration Ph 9.3 the amount is over four times as great. The results obtained by this method of calculation raises the question, To what point of concentration should the solution be titrated before the formaldehyde is added? To throw light on this point an extract from wheat digested at 40° C. for four hours was used. It was prepared as the other extracts used in this investigation. This was then titrated to the points Ph 7, Ph 8.3, and Ph 9.3, first without adding any formaldehyde, and second by adding the formaldehyde before starting the titration. Corrections were made for the differenpes in volume of these two. These corrections were o.i and 0.2 cc. The following results were obtained (Table III). Table III. — Quantity of NI20 sodium hydroxid required for neutralization with and without formaldehyde Treatment Quantity (in cubic centimeters) of N:'2o sodium hydroxid used to titrate to— Ph7 Ph8.3 Ph 9-3 Adding the formaldehyde before starting the titration . Titrating without formaldehyde 7.6 4.9 14-5 II. 2 18.0 14. 6 Increase due to formaldehyde 2-7 3- .3 3-4 One hundred-cc. portions of extract from the wheat prepared in the same way were then used to see what differences would be obtained if the formaldehyde was added after the titration had been made to the following concentrations: Ph 7, Ph 8.3, and Ph 9.3. Jan. 6. 1919 Determination of Acidity and Nitrogen in Wheat Adding Formaldehyde at Ph 7 Total quantity (cc.) of NI20 sodium hydroxid to neutralize to Ph 7 . 4. 9 Total quantity (cc.) of NJ20 sodium hydroxid to neutralize again to Ph 7 after adding formaldehyde 2. 7 Adding Formaldehyde at Ph 8.3 Total quantity (cc.) of NI20 sodium hydroxid to neutralize to Ph 8-3 • "-3 Total quantity (cc.) of NI20 sodium hydroxid to neutralize again to Ph 8.3 after adding formaldehyde 3.3 Adding Formaldehyde at Ph 9.3 Total quantity (cc.) of NI20 sodium hydroxid to neutralize to Ph 9-3 14-8 Total quantity (cc.) of NI20 sodium hydroxid to neutralize again to Ph 9.3 after adding formaldehyde 3.4 This shows that sHghtly higher results are obtained when the for- maldehyde is added at the higher concentrations and the solution is titrated again to the same concentrations. From data obtained in connection with some other work the follow- ing figures are added. The figures represent the number of cubic centi- meters of N/20 alkali needed to neutralize after the addition of for- maldehyde, the titrations ha\dng first been made to the concentrations shown. Concentration. Flour A . Flour B. Flour C. Ph 7 2.7 3-4 31 2-5 2.7 2.4 3-0 3-1 2.9 Ph8.^ Ph 9.^ One question not settled by the data given in this paper is to what point should the titration be carried after the addition of the formalde- hyde. This question is reserved for future work. On the basis of the above discussion we may say that the results in Table I show the following in regard to amino nitrogen : 1. At 5° C. there is practically no increase in the amount of amino nitrogen as the time of digestion is increased. 2. At 20° C. the amount of amino nitrogen reaches the maximum shortly after 8 hours. 3. At 40° C. the amount of amino nitrogen reaches the maximum at 2 hours. 4. The calculated results in Table II show a gradual increase in the amount of amino nitrogen to the end of the 24 hours with one minor exception at 40*^ C. The results on amino nitrogen are graphically pre- sented in figure 4. lO Journal of Agricultural Research voi. xvi, No. i PHOSPHORUS IN THE WHEAT EXTRACTS As previously mentioned, determinations were made of total phos- phorus and of phosphorus precipitated by magnesia mixture. From each extraction mixture two portions of 50 cc. each, represent- ing 5 gm- of ground wheat, were pipetted into beakers. Ten cc. of concentrated nitric acid were added and boiled until all the organic matter was destroyed, more nitric acid being added as needed. The residue was used for the determination of phosphorus in the usual way. To two other 50-cc. portions of the extraction mixture there were added 40 cc. of magnesia mixture, and after standing for 15 minutes, 25 cc. of concentrated ammonia. After a thorough stirring the beakers Fig. 4. — Graphs showing the production of amino nitrogen at different temperatures. - . j^ i^^^"" " - i ^ 1 — ± : + :± were allowed to stand overnight, and the contents were then filtered and the precipitate washed four times with 2 per cent ammonium hydroxid. The precipitate was then dissolved in 40 cc. of dilute (i 14) nitric acid and the filter washed with 100 cc. of hot water. The filtrate and vv^ash- ings were then boiled, in order to destroy organic matter, and the phos- phorus determination was completed in the usual way. For convenience, this will be called inorganic phosphorus. Whether or not that is the case may be questioned, and the merits of this method of determination are not discussed here. We simply used this method as one best suited to our purpose, and the results obtained (Table IV) are used for their comparative value. Jan. 6, 1919 Determination of Acidity and Nitrogen in Wheat ii Table IV. — Determination of phosphorus in wheat extract Tem- pera- ture. 5 5' 5 5 20 20 20 20 20 20 20 20 40 40 40 40 40 40 40 40 50 50 50 50 50 Duration r:{ extraction. 5 minutes . 30 minutes I hour .... 24 hours. .. 5 minutes. 30 minutes 1 hour 2 hours. . . . 4 hours. . . . 8 hours. . . . 16 hours. .. 24 hours. .. 5 minutes. 30 minutes 1 hour 2 hours. .. . 4 hours. .. . 8 hoiu's. .. . 16 hours. . . 24 hours. .. 5 minutes. 30 minutes 1 hour 2 hours. .. . 4 hours. . . . Total percent- age of phos- phorus in the extract. O. 018 . 029 . 027 .083 .044 .063 . 067 . 106 •159 •173 .208 . 190 .099 .183 . 210 . 240 •253 •254 •257 •259 . 162 •215 .236 • 254 .258 Percentage of inorganic phos- phorus in the extract. O. 019 . 020 . 020 . 070 .031 .031 .050 . 062 .083 . 092 .098 •113 .032 .116 . 146 .177 . 212 .223 •25 .24 . 109 •151 . 172 . 220 . 196 1. It will be noted that at 50° C. the total and inorganic phosphorus increases with the time of digestion. 2. At 20° C. the total and inorganic phosphorus increases with the time of digestion, but the total phosphorus does not show any increase after 16 hours. The ratio between the total and inorganic phosphorus varies considerably, but the latter averages a little more than one-half of the total. 3. At 40° C. the total and inorganic phosphorus increases with the time of digestion, but the maximum for both total and inorganic phos- phorus is reached at about 4 hours. The proportion of inorganic phos- phorus in relation to the total is much greater than at the lower tem- perature. After four hours the inorganic phosphorus is almost equal to the total. 4. At 50° C. the maximum of both total and inorganic phosphorus is reached in about two hours. The results are graphically presented in figures 5 and 6. Why does hydrogen-ion concentration remain the same or show no increase with the increased time of digestion, while the amount of N/20 barium hydroxid necessary to bring the solution to the concentration 12 Journal of Agricultural Research Vol. XVI, No. 1 Ph 7> Ph 8.3, or Ph 9.3 shows a constant increase corresponding with the time of digestion, and also an increase in the total and inorganic phosphorus ? The only explanation that we have to offer at this time is that the extract of wheat contains a definite amount of the hydrogen ion, O^S 1 ~ 4o' ,- '^ J?f ^ ■"" ~" ... r:. / ^.*w ' .^ir\ _ 1 — — <- _ j — -t- n $ / y 1 vz / \yc / / f L .5* r \06 1 ___ — — 1 _ — — ■^ ^ .1 Fig. s. — Graphs showing the total phosphorus in wheat extractat different periods of extraction and at different temperatures. and that this amount is not increased during the hydrolysis of the wheat because the phosphates which are produced during digestion are of such a nature that they undergo very small ionization in the water. When, however, the hydroxid is added, they undergo ionization. The per- centage of ionization in these phosphorus compounds must be very small. ajv ■ - r-' — 1 ""^ " ^ 2_ — — — ^ 1 n y ^ / / r __ _*j IS— K -^ — —I — — x- ^ - ^ J jt- f y , — ~~ \ Fig. 6. — Graphs showing the inorganic phosphorus in wheat extract at different periods of extraction and at dififerent temperatures. SUMMARY (i) This paper presents the results of a study in determining, by means of the hydrogen electrode, the different hydrogen-ion concentrations in the extract of ground wheat. The total phosphorus and the phosphorus precipitated with magnesia mixture were also determined in the extract. (2) Extractions were made at the following temperatures: 5°, 20°, 40°, and 50° C. ; and for the following periods: 5 and 30 minutes; i, 2, 4, 8, 16, and 24 hours. Jan. 6,1919 Determination of Acidity and Nitrogen in Wheat 13 (3) The temperature at which the extraction was made was found to have but little influence upon the hydrogen-ion concentration. The higher temperatures give an extract of but slightly higher concentration. (4) The duration of the digestion period did not influence the hydrogen- ion concentration. The average hydrogen-ion concentration when the extraction was made for 5 minutes, 30 minutes, and i hour was the same as when the extraction was made for 8, 16, and 24 hours. (5) But while the hydrogen-ion concentration of the extract shows no increase with the duration of the digestion, the quantity of NI20 barium hydroxid necessary to add in order to change the concentration to a definite point was greater in amount and within certain limits propor- tionate to the duration of the digestion. (6) The substances produced when v/heat is digested in water are not ionized until an alkali has been added. The amount of these substances produced bears a definite relation to the time and temperature used in digestion. A limit, however, is soon reached, and this limit is reached sooner at the highest temperature. (7) The amino nitrogen as determined by the Sorensen formaldehyde method is all extracted in two hours at 40° C. (8) At 20° C. the amount of phosphorus in the extract precipitated by magnesia mixture averages about half of the total. At 40° practically all of the total phosphorus is converted into forms that are precipitated by the magnesia mixture. (9) The hydrogen-ion concentration of the water extract of wheat is definite in amount. This concentration is not changed during the extraction in proportion to the time. The reason for this is that the conditions for ionization are not present until an alkali is added. When, however, this is added, ionization takes place and the amount of standard alkali necessary to add in order to lower the hydrogen-ion concentration to a given point bears a proportionate relation to the temperature and duration of the digestion period. ASH ABSORPTION BY SPINACH FROM CONCENTRATED SOIL SOLUTIONS By Rodney H. True, Physiologist in Charge, Otis F. Black, Chemical Biologist, and James W. Kelly, Laboratory Technician, Office of Drug-Plant, Poisonous- Plant, Physiological, and Fermentation Investigations, Bureau of Plant Industry, United States Department of Agricultiire INTRODUCTION In 1 91 5 when the authors were engaged in a study of the possible causes of spinach-blight, the theory was advanced that the disease was a form of "malnutrition" due, in a measure, to the accumulation of an excess of fertilizer salts in the soil solution. FERTILIZER SUBSTANCES USED In the hope of reproducing the symptoms seen, beds of spinach (Spi- nacia oleracea) grown on the grounds of the Virginia Truck Experi- ment Station,^ in cooperation with which this work was carried on, were given large applications of the commoner fertilizer salts, singly and in the usual mixtures. The so-called "acid mixture" was that generally employed by the truck farmers of the Norfolk region for use in their spinach fields. The "basic mixture" was made up of substances which would likely be neutral or basic in the soil. The constituents of each were approximately as follows : ACID MIXTURE. Pounds. Ammonium sulphate 340 Acid phosphate 830 Potassium muriate 170 Dried blood 260 Tankage 400 BASIC MIXTURE. Pounds. Sodium nitrate 450 Basic slag from Birmingham, Ala. 720 Potassium sulphate 170 Dried blood 260 Tankage 400 Single salts were supplied in two proportions : One was intended to be near the maximum; the other to cause clear injury. The substances applied are listed below in terms of pounds per acre : SXIBSTANCES. QUANTITY USED. Calcium carbonate 3 and 6 tons per acre. Magnesium carbonate i and 2 tons per acre. Potash 750 and 1,500 pounds per acre. Sodium nitrate Do. Sodium chlorid Do. Sodium sulphate Do. 1 The authors are indebted to the Director of the Virginia Station and to his assistants for help in many- ways. To Mr. J. A. McClintock, at that time Plant Pathologist of the Station, they owe an especial debt for careful notes made from time to time and for help rendered in other ways. Journal of Agricultural Research, Washington, D. C. qw (X5) Vol. XVI, No. I Jan. 6, 1919 Key No. G-171 1 6 Journal of Agricultural Research voi. xvi. no. i SUBSTANCES — continued. quantity used. Acid phosphate i,ooo and 2 ,000 pounds per acre. Complete mixture: Acid 2,000 and 4,000 pounds per acre. Basic Do. Stable maniu'e 20 and 40 tons per acre. Control plots alternated with those receiving treatment. The land used for planting had not been used for spinach for many years previously and was in good condition. It had received excellent treatment for several years and seemed to be very uniform in all respects. Each bed was 5 feet wide and 61 feet long, and received four rows of seed. The chemicals were applied on July 29, 191 5, and immediately hoed into the soil. The land vv^as kept free of v/eeds until September 11, when curled Savoy spinach seed was drilled in. In 10 days the stand could be reasonably well determined, and since the variation with the treatment was clearly seen, the result may be summarized here. PROGRESS OF PLANTS IN THE FIELD The " best" stand was seen in the beds receiving stable manure; "very good" in beds receiving magnesium carbonate; "good" in those with cal- cium carbonate, acid phosphate, sodium sulphate, and basic complete mixture; "scattering" only in those with potash, sodium nitrate, sodium chlorid, and complete acid mixture. The controls were in the class designated "Good," a few in "Very good." Owing to the unsatisfactory stand in some of the beds, all were re- seeded on September 23, and irrigated. On September 30 plants began to appear, a thick stand being seen by October 6. The usual cultiva- tion and thinning took place about a fortnight later. From notes taken on the beds as they appeared on October 27, cer- tain outstanding features may be developed. On taking completeness of stand, growth, and color as criteria, the treated plots were distributed in the following groups: Excellent : basic complete mixture. Very good (equal or better than the best control plots) : acid phos- phate, sodium sulphate (heavier treatment), magnesium carbonate, manure. Good (equal to the poorer control plots) : calcium carbonate, sodium sulphate (lighter treatment) . Poor (poorer than controls) : complete acid mixture, sodium chlorid, potash, sodium nitrate. The poor plots were marked by a yellowish -green color, poor growth, and death of some of the seedlings, giving a bad stand. The greatest injury was seen in those parts of the plots receiving the heavier treatment, Jan. 6, 1919 Ash Absorption by Spinach 17 Another review of the plots was made on December i, 191 5, with similar results. The "best" plot in the whole experiment was that receiving the basic complete mixture. Those having acid phosphate and sodium sulphate were "excellent." "Good" would be said of plots receiving magnesium carbonate; some- what less so, were those receiving calcium carbonate, which gave a very deep green color, and manure, especially the part of the bed receiving the lighter application. The four plots found to be "poor" were those having sodium chlorid, sodium nitrate, and acid complete mixture. Poorest of all was potash. All poor plots were alike in having a crusted soil surface, with a suggestion of moisture. At this stage samples were taken from several of the beds for ash analysis at Washington. ASH DETERMINATIONS The plants, after being divided into roots and tops, were ashed in an electric furnace at a low red heat of approximately 600° C. The total ash being determined, the chief constituents were worked out by the methods recommended by the Association of Official Agricultural Chem- ists.' The results are given in Table I. In the first section the lesults are calculated as percentages of the air-dry weight of the plant material, while in the second section the individual constituents are calculated as percentages of the total ash. Table I. — Ash constituents of spinach CALCULATED AS PERCENTAGES OF DRY MATERIAL Fertilizer. Sodium Sodium nitrate. chlorid. Sodium sulphate. Potassium chlorid. Calcium carbonate. Control. Tops. Roots.Tops. Roots. Tops. Roots. Tops. Roots. Tops. Roots. Tops. Roots. Total ash 19-94 8.87 '21.81 8.58 20. 70 8.30 17.67 7.40 21.39 9.40 20.68 8.01 Silica (SiOs) Manganous o x i d (M113O4) 4-s6 • 035 I. II I- 13 5-22 3-50 •44 I- 03 .61 •13 1-77 .14 .29 •47 3- 25 .86 . 21 •92 •30 .07 3- 80 .02 I- 13 1.44 3-68 S-67 •47 1.22 •57 .09 1.46 .14 •45 •55 1.94 1.42 •23 1.16 .20 .06 4-72 .02 •83 I- 33 9. II .80 .76 1.29 .62 .08 1. 61 •045 •30 .41 2.91 1.28 •32 I- 13 ■24 .06 4- 30 •03 •94 1.68 6.56 1-53 •44 1-13 •49 .07 1.48 •OS •30 •S6 2.44 •54 • 15 .98 .18 •OS 7-97 •03 1.26 1. 14 7.20 1.70 •47 .98 •52 . II 3^i6 .07 •4S •49 2.97 •AS •23 I- 03 •45 .07 5^79 .025 1.14 1.38 8.60 •91 •52 1-33 •63 .09 1.48 Lime(CaO) Magnesia (MgO). . . Potash (K2O) Soda(Na20) Sulphur trioxid (SOs) •30 •34 3-44 ■33 Phosphorus pent- oxidCPzOs) Alumina ( AI2O3) . . . Ferric oxid (FejOa). .80 .26 .06 Total 17- 70s 8.28 18.09 7.61 19.56 8.305 17.17 6-73 21.38 9^37 20.4IS 7^40 1 1 Wiley, H. W., ED. oppiciai, and provisionai, methods of analysis, association op officlvl AGRICULTITRAL CHEMISTS, AS COMPILED BY THE COMMITTEE ON REVISION OF METHODS. U. S. Dept. Agr. Bur. Chem. Bui. 107 (rev.). 271 p., 13 fig. 1908, 92801°— 19 2 Journal of Agricultural Research voi. xvi, no. i Table I. — Ash constituents of spinach — Continued CALCULATED AS PERCENTAGES OF DRY MATERIAL — continued Fertilizer. Total ash Silica Manganous oxid. . . . Lime Magnesia Potash Soda Sulphur trioxid . . . . Phosphorus pent oxid Alvunina Ferric oxid Total Add phos- phate. Tops. Roots. .04 I- 13 1-33 6.40 2. IS •53 1.47 .70 20.98 •38 •49 I. 76 Complete add. Tops. Roots. S-40 •OS .96 1-33 8.69 .78 •58 1. 12 •6s 19.66 10.48 2.78 •OS •43 •S6 3-36 1.58 •35 I. 26 •33 .08 10.78 Complete basic. Tof)s. Roots. 18.60 4.01 •03 •97 1-34 8.70 .66 •53 1^32 •45 18. II 1.49 •32 .09 10.08 Manure. Tops. Roots. 4-47 .016 .84 I.S9 9.76 •45 .48 1.08 •51 19-306 Average. Tops. Roots 20.4s 1.36 .36 •IS S-2I .029 1-031 I^37 7-39 1.82 •53 I. 20 •S8 •097 11.46 19. 247 9-s8 2-39 .096 •38 •S3 3- 06 •8s • 28 1. 10 • 304 • 077 9.067 INDIVIDUAL CONSTITUENTS CALCULATED AS PERCENTAGES OF THE TOTAL ASH Sodium nitrate. Fertilizer. Tops. Roots. Sodium chlorid. Tops. Roots. Sodium sulphate. Tops. Roots. Potassium chlorid. Tor>s. Roots. Calcium carbonate. Tops. Roots. Control. Tops. Roots. Total ash Silica Manganous oxid. . . Lime Magnesia Potash Soda Sulphur trioxid . . . Phosphorus pent oxid Alumina Ferric oxid Total 19-94 8.87 21.81 8-58 20.70 8.30 17.67 7.40 9.40 8.01 22.57 .18 5-57 5-67 26.18 17-55 2. 21 S-17 3- 06 •6s 19.9s 1.58 3-27 5-30 36.64 9-70 2-37 10.37 3-38 .80 17.42 .09 5-18 6.60 16.87 26.00 2. II 17.02 1-63 5-24 6.41 22. 61 16. 55 2.68 13-52 2-33 -70 93-36 ,82. { 88.69 22. 80 . 10 4.01 6-43 44.01 3-82 3.62 6.23 19.40 •54 3- 61 4-94 35- 06 IS- 42 3-86 13-61 24.34 •17 S-32 9-51 37-13 8.66 2- 55 6.40 2-77 20.00 .68 4- OS 7-57 32-97 7-30 2.03 13-24 2-43 .68 37.26 .14 5-89 S-33 33-66 7-95 33-62 •74 4-89 5- 21 31-60 4.89 2-46 10.96 4.89 •74 38.00 . 12 S-SI 6.67 41.59 4.40 2.51 6-43 3- OS •44 18.48 •7S 3-75 4.24 42-95 4. 12 4.13 9-99 2.2s •75 90. 95 99. 6s 100. 00 98. 72 I 91.40 Fertilizer. Add phos- phate. Complete add. Complete basic. Manure. Average. Tops. Roots. Tops. Roots. Tops. Roots. Tops. Roots. Tops. Roots. Total ash 21.39 13.40 20.59 10.48 18.60 10.01 21-73 11.40 20.4s 9-s8 Silica 33-38 .19 5-28 6.22 29.92 10.05 2.48 6.87 3-27 .42 40-52 .67 2.84 3-66 13- 13 7-39 2.09 6.27 2-99 .60 26.23 -24 4-66 6.46 42. 20 3-79 2.82 5-14 3-16 -49 26.53 .48 4. 10 5-34 32.06 15.08 3-34 12.02 3-15 •76 21.56 .16 5-22 7.20 46.77 3- 55 2.8s 7.10 2.42 -54 23.08 .40 4.00 7-49 39-66 4.00 3-10 14-89 3-20 .90 20.57 .07 3-87 7-32 44-91 2.07 2. 21 4-97 2-35 -51 21.66 2.46 4-30 6-05 40.00 6. 14 3-51 11. 05 3-16 1-32 25.48 .14 5-04 6. 70 36. 14 8.90 2-54 5-87 2.84 •47 24-95 1. 00 3-97 S-S3 31-94 8.87 2.93 11.48 3-17 .80 Manganous oxid Potash Soda Sulphur trioxid Phosphorus pent- Alnmin^ Total 98.08 80.16 95-49 102. 86 97-37 100. 72 88.8s 99-65 94-12 94.63 Jan. 6,1919 ^^h Absorption by Spinach 19 In order to bring out more clearly the relationships involved in Table I, graphs have been prepared in which quantities are indicated on a uniform scale. In figure i appear the values found for the tops, and in figure 2 similar values for the roots. Each unit on the perpendicular axis represents o.i per cent of dry weight in every case. TOTAL ASH A casual inspection of these results reveals the fact that the total ash content of the tops calculated as percentage of dry weight, while showing considerable variation, is always greatly in excess of that of the roots, in obedience to the general rule.^ As regards the influence of specific substances on the total ash absorption certain coincidences may be noted. The total ash reached its minimum in both roots and stems in the plot treated with potash. It is depressed in the tops nearly as much in the presence of the basic mixture, to a less degree in the roots. In general, the quantity of ash constituents is kss for the roots in the plots treated with sodium salts and in the untreated control than in the other plots. Of the single salts calcium carbonate alone goes with a total ash in the roots, approaching that seen in the mixtures of several salts. This depressing action of the sodium salts is not seen in the ash content of the tops. Acid phosphate goes with the highest total ash seen in any sample of roots; sodium chlorid and manure, with the highest totals seen in the tops. INDIVIDUAL ASH COXSTITUENTS In examining the quantities of the different ash constituents seen in Table I it will be noted that in some cases there is a great variation among the different plots ; in others little difference is to be seen. Those showing great variation are silica, potash, and soda; those showing little change with the change in outside conditions are lime, magnesia, phosphorus pentoxid, sulphur trioxid, manganous oxid, alumina, and ferric oxid. It seems as though all plants were able to absorb these from the soil to a point of steady equilibrium without much regard to the substances offered. It appears that even when an excess of any of the ions present in this latter group of compounds is present no con- siderable increase in the absorption of these ions takes place. The quantity of ions absorbed from the group of variable salts seems much more subject to influence from the added salts. In some cases the ions present in excess are themselves absorbed in greater quantity. This seems to be the case with sodium-nitrate and sodium-chlorid plots, in which considerably greater quantities of soda appear in the ash of the tops than in any other plots. Sodium sulphate, however, gives no 1 PAiLADiN, W. PFLANZENPHYSiOLOGiE. Bearb. atifgrund der 6. russischen aufl. p. 88. BerUn, ig"- 20 Journal of Agricultural Research voi. xvi. No. i sao / \ N, / \ / 200 / \ \ "\ y \ / \ / /SO \ /so \ sic Fig. I.— Graphs representing total ash and individual ash constituents foimd in spinach tops from plots receiving the substances indicated. Jan. 6, 1919 Ash Absorption by Spinach 21 jwa /30 /^>o //o /oo s>o GO 70 eo so «^ ^o ^o yo FlO. 2. — Graphs representing total ash and individual ash constituents in spinach roots from plots receiving the substances indicated. 22 Journal of Agricultural Research Vol. XVI, No. I such result. The greater absorption of sulphur trioxid occurs in the only plot treated with great quantities of sulphates, but the increase is not great. In some cases a marked increase in a given constituent accompanies the presence in excess of some other ion. This is seen in the plot treated with sodium sulphate, in which potassium absorption in both root and top is high. A similar result is seen in the plots treated with stable manure and with both acid and basic complete mixtures. On the other hand, potassium absorption is decreased in the acid-phosphate plot in both tops and roots. Potassium chlorid in excess also accom- panies a reduction of potash in the ash of both parts of the plant. Silica is greatest in plots receiving calcium carbonate and acid phos- phate, lowest in those dosed with sodium salts, and in the control plot. It is interesting to note that, although the manganous oxid content when referred to dry weight is small in all cases, it is consistently higher in the roots than in the tops, acid complete mixture excepted, this being the only constituent which is not more abundant in the tops than in the roots. RATIOS BETWEEN PAIRS OF CONSTITUENTS In a number of cases there seemed to be some evidence that a roughly reciprocal relation exists between pairs of constituents. This tendency was most marked in the plots receiving mixtures of salts. Thus, in general, when silica was high, potash was low in both tops and roots. The silica-potash ratios, as worked out for the different plots, are summarized in Table II. Table II. — Silica-potash ratio in ash of spinach plants. Silica (5i6>2)=/ Potash (K2O). Fertilizer. Sodium nitrate. .. . Sodium sulphate . . Sodium chlorid ... Potassium chlorid . Calcium carbonate Acid phosphate . . . Complete acid Basic complete . . . . Manure Control It will be noted that, while both of these constituents belong to the group of the more variable ones, the ratio is less variable; silicia being unity, potash lies between i.oo and 2.00 in a majority of cases. In plots receiving heavy doses of calcium -containing fertilizers (calcium carbonate and acid phosphate) as well as in the tops from the sodium- chlorid plot, the silica exceeds the potash. Again, potash has high Jan. 6, 1919 Ash Absorption by Spinach 23 relative values, especially in the tops, in the plots to which the complete fertilizers were applied. The relation of potash and soda is of particular interest in view of the halophytic nature of the spinach plant. It is perhaps worth noting in this connection that the sodium-potassium ratio varies over a wider range, something to be expected perhaps in view of the greater variability in the quantities of these constituents present. In Table III these ratios are given, soda being unity, the values of potash appearing in the appropriate columns. Table III. — Soda-potash ratio in spinach ash. Soda {Na02)=i Fertilizer. Sodium nitrate. .. . Sodium sulphate . . Sodium chlorid . . . Potassium chlorid . Calcium carbonate Acid phosphate . . . Acid complete .... Basic complete . . . . Manure Control It will be seen that in a broad way, when the potash is high in the ash, soda is low, and vice versa. Potash, however, is always higher than soda, the excess being greater in the leaves than in the roots. High potash seen in the plot receiving sodium sulphate is accompanied by a very low soda content. The same is seen in the control as well as in both acid and basic complete mixtures and in stable manure. High soda is seen in the plots treated with sodium chlorid, in which potash reaches its minimum, and in that receiving sodium nitrate. The control culture and that receiving acid phosphate show the same less strikingly. Potash is in greatest relative excess in the plots receiving the complete fertilizers. These results suggest the possibility that sodium may be able to perform some functions in the plant which are usually performed by potassium. It seems likely that by giving proper mixtures of the alkalis it might be possible without detriment to the plant to get along with less of the expensive potassium constituent, thus protecting the potassium in the soil. In view of the fact that calcium is usually found in the ash of a great majority of plants in considerably greater quantity than magnesium* it is of interest to note the quantities found in these spinach plants. ' LoEw, Oscar. Liming of soils from a PBtYSiowKiiCAi, standpoint. In U. S. Dept. Agr. Bur. Plant Indus. Bui. I, p. 9-35. 1901. 24 Journal of Agricultural Research Vol. XVI, No. I It appears from data scattered through Wolff's ^ tables that in some cases the magnesium content exceeds that of calcium, especially in the beet family. In the leaves of these plants the calcium usually much exceeds magnesium, while the reverse holds for the roots. In the analysis given by Wolff ^ lime exceeds magnesia in spinach in an ap- proximately 2 to I ratio. In Table IV the calcium-magnesium ratio is shown for each experimental plot. Table IV. — Calciuin-magnesium ratio in ash of spinach plants. Lime {CaO)=i Fertilizer. Magnesia (MgO). Tops. Roots. Sodium nitrate I. 02 I. 60 1.27 1.79 .90 I. 18 1-39 I. 40 1.89 I. 21 I. 62 Sodium sulphate 1-37 1.23 1.87 I. 07 I. 29 1.30 1.87 I. 41 Sodium chlorid Potassium chlorid Calcium carbonate Acid phosphate Acid complete Basic complete Manure Control 1-13 It will be noted that magnesia exceeds lime in every case, regardless of the nature of the substances applied, except in that of the tops in the plot receiving lime, in which the lime exceeds the magnesia. Spinach seems to be even a more pronounced user of magnesium than the sugar beet. The field notes show that the plot receiving magnesium carbonate was somewhat better than that receiving calcium carbonate. Unfor- tunately no sample from the magnesium-carbonate plot was ashed. SUMMARY Spinach plants grown on the grounds of the Virginia Truck Experi- ment Station at Norfolk in beds given heavy treatments of fertilizer salts, singly and in mixtures, gave best results in plots receiving a com- plete mixture having a basic or neutral character in the soil (sodium nitrate, basic slag, and potassium sulphate) ; next best with acid phos- phate and with sodium sulphate ; poor in plots receiving heavy treatments of sodium chlorid, sodium nitrate, and acid complete mixture (i to 2 tons per acre); poorest with potassium chlorid. A study of the ash showed the highest total ash in the tops in plots with sodium chlorid, calcium carbonate, acid phosphate, and manure; lowest with potassium chlorid and basic complete mixture. The highest ash was in roots accompanied with acid phosphate and manure, the lowest with potassium chlorid and sodium salts. General excellent condition of ' Wolff, E. aschen-analysen. T. 2, p. 42-50. Berlin. 2 Wolff, E. op. ai, p. 128. jan.6. I9I9 A.sh Absorption by Spinach 25 the crops does not parallel high ash absorption, the best and poorest plots having plants with low ash. Ash constituents fall into two groups: (i) those present in quantities that show relatively little variation whatever be the chemicals added to the soil — lime, magnesia, phosphorous pentoxid, sulphur trioxid, manganous oxid, alumina, and ferric oxid; and (2) those which show great fluctuations in the quantity present — silica, potash, and soda. In the first group the plants seemed to be able to get the required quantity of constituents mentioned from the soil of all plots studied whatever was offered in excess, and reached an equihbrium that was little affected by the varying conditions. In the second group wide variations occur, sometimes with an increase of the ions offered in excess, as in sodium chlorid and sodium nitrate, sometimes by the absorption of something else, as increase in silica in plots receiving calcium carbonate and acid phosphate. Manganous oxid is the only constituent regularly present in greater proportion in the roots than in the tops. In some cases the high absorption of one constituent is accompanied by the low absorption of another, and vice versa. Such reciprocal pairs are silica and potash, soda and lime, and potash and magnesia. The silica-potash ratio is relatively steady. When silica equals i, potash varies between 1.16 and 2.18 in the tops and between 1.33 and 2.32 in the roots, except when the substance added to the soil is high in calcium, when the value of potash becomes less than unity in both tops and roots. The soda-potash ratio is much more variable, being always more than I in both tops and roots. When mixtures of salts are added to the soil, potash rises to very high relative values. There is a suggestion that sodium may perform some functions also performed by potassium, indicating the possibility that sodium might in part replace potassium in fertilizers. The calcium-magnesium ratio in spinach, both in leaves and in roots, is exceptional in having a value greater than unity. The only exception is seen in the tops of plants receiving a heavy treatment with calcium carbonate. This fact seems to suggest the practical importance of magnesium salts as fertilizers for spinach. ADDITIONAL COPIES OF THIS PITBUCATION MAY BE PEOCUKED FBOM THE SXJPEEINTENDENT OF DOCUMENTS GOVEENMENT FEINTING OFFICE ■WASHINGTON, D. C. AT 5 CENTS PER COPY StJBssEipnoN Peice, $3.00 Pee Yeab A Vol. XVI JANUARY 13, 1919 No. 2 JOURNAL OP AGRICULTURAL RESEARCH COISrXE^NTS Pt8« Nitrates, Nitrification, and Bacterial Contents of Five Typical Acid Soils as Affected by Lime, Fertilizer, Crops, and Moisture ___--- 27 H. A. NOYES and S. D. CONNER (.Contribution from Indiana Agricultural Experiment Station) Effect of Certain Ecological Factors on the Morphology of the Urediniospores of Puccinia graminis - - E. C. STAKMAN and M. N. LEVINE ( Contribtition from Minnesota Agricultural Experiment Station ) 43 PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASMINOXON, D. C. WASHIHQTON t OOVERMMENT PRINTINO OFFICE : I»I8 EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCL/VTION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chairman Physidogisl and Associate Chief, Bureau of Plant Industry EDWIN W. ALLEN Chief, Office of Experiment Stations CHARLES L. MARLATT Entomologist end Assi^nt Chief, Bureau of Entomology FOR THE ASSOCLATION H. P. ARMSBY Director, Institute of Anitnal Nttlrition, The Pennsylvania State College E. M. FREEMAN Botanist, Plant Pathologist, and Assistant Dean, Agrictdtural Experitnent Station of the University of Minnesota J. G. LIPMAN Director, New Jersey Agricultural Experi- ment Station, Rutgers College All eorrespondence 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa. A^. JOMALOFAGRIQlTimiSEARCH Vol.. XVI Washington, D. C, January 13, 1919 No. 2 NITRATES, NITRIFICATION, AND BACTERIAI. CONTENTS OF FIVE TYPICAL ACID SOILS AS AFFECTED BY LIME, FERTILIZER, CROPS, AND MOISTURE _ By H. A. NoYES, Research Associate in Horticultural Chemistry and Bacteriology, and ^^ ' -, S. D. Conner, Associate Chemist in Soils and Crops, Purdue University Agricul- ^^■TAt iural Experiment Station ''v INTRODUCTION The decay of organic matter and the transformation of nitrogen from one chemical combination to another were known and studied long be- fore bacteria were isolated. These phenomena were attributed to purely chemical agencies until the discovery of the function of soil bacteria proved them to be almost entirely due to microorganic life. Most in- vestigations in soil bacteriology have dealt with either the products of bacterial activities without reference to the number of organisms present or with only an enumeration of the bacteria present in the soil. This paper presents the results of an investigation taking into consideration both nitrates and bacterial numbers, as well as a correlation of the two, under certain specific conditions. HISTORICAL REVIEW NITRIFICATION The difficulties attendant upon keeping an adequate supply of avail- able nitrogen in the soil are so great that those bacterial activities which have to do w^ith nitrate formation are important and have been ex- tensively studied. As early as 1660 Digby (2)^ mentioned the value of nitrates in agriculture. He attributed the growth of plants to the "nu- tritional and attractional " powers of a "nitrous salt." Many agricul- tural writers of the early part of the nineteenth century followed the lead of Liebig, who claimed that nitrogen was not needed as a soil amend- ment. In 1856 Boussingault and Ville {8) independently published experimental results which proved that nitrates are markedly beneficial to plant growth, but it was not until 21 years later that Schloesing and Miintz {22) demonstrated that nitrification in the soil was due to .organ- ized ferments and does not take place in the absence of these ferments. 1 Reference is made by number (italic) to "Literature cited, p. 41-42." Journal of Agricultural Research, Vol. XVI No. 1 Washington, D. C. Jan. 13, 1919 qx Key No. Ind.-s (27) 28 Journal of Agricultural Research voi. xvi. No. 2 Nitrification in soils is dependent upon several different factors, and chemists have not entirely agreed as to the conditions necessary for it to take place. It was early observed that calcareous material was nec- essary for the preparation of niter beds. Thouvenel (4) in 1787 found chalk and carbonate of lime to favor nitrification more than a number of earths and other chemicals. From the accumulated evidence that car- bonate of lime increased nitrate formation and the fact that acid forest soils often contained no nitrates the conclusion was reached by many in- vestigators that nitrification did not take place in an acid soil. In 1891 Warington (25, p. 51) said: A further condition of nitrification is the presence of a base with which the nitric acid when formed may combine. This condition is quite essential. Nitrification can only take place in a feebly alkaline medium. A little later in 1894 Deherain {8) (/>. 360) made the following statements : The nitric ferment does not act in an acid medium It is true that nitrifica- tion may go on in soil deficient in lime Moreover, the application of carbonate of lime to such soils is very beneficial and increases the production of nitrates. NITRATES IN ACID SOILS Twenty-two years before Warington {26) stated that nitrification could only "take place in a feebly alkaline medium" Houzeau {12), in 1872, reported nitrification in an acid soil. In 1908 Hall, Miller, and Gimingham (iz) found nitrates in an acid soil, but believing that nitrifi- cation could not take place in an acid medium, they atrributed the phe- nomena to the probable presence in the soil of small isolated particles of calcium carbonate. Since 1908 several workers have reported nitrifica- tion in acid soils. In 191 3 Petit {21) found pronounced evidence of such a condition, while the same year Abbott, Conner, and Smalley (/) re- ported the presence of large amounts of nitrates in an excessively acid soil. The water extract of the soil was acid in reaction and contained considerable aluminium. The next year Temple {23) reported nitrifica- tion in acid or nonbasic soils. White {26) in 191 5 from investigations on some unHmed and limed plots at the Pennsylvania Station found that nitrification was very active in many very acid areas. White remarks that— These results are entirely contrary to the general belief that nitrification ceases on very acid soils. Since nitrification is the result of oxidation reactions and due to bac- teria, it is affected by soil moisture and aeration. Schlosing {8), in 1868, found that rapid loss of nitrates occurred when a moist "humic soil" was kept in an atmosphere of nitrogen gas. Warington (25) in experi- ments at Rothamsted in 1880 found that saturating ordinary soil with water caused it to rapidly lose the nitrates it contained. Kellner {14) in 1891 and Kelley (zj) in 1914 found that flooded rice fields con- tained little or no nitrates. Jan. 13, 1919 Nitrates, Nitrification, and Bacteria of Acid Soils 29 BACTERIAL NUMBERS The conditions under which studies of the number of bacteria present in soils have been made have varied to such an extent that generaUza- tions rather than specific correlations have resulted. Chester (6) was the first to note that applications of lime increased the bacterial content of soils. He concluded that the favorable action was not — due to any direct action of the lime, but due to the more favorable reaction which the 4ime gave the soil. Later F'abricius and Feilitzen (jo), Engberding (9), and Brown (5) reported increased bacterial numbers as the result of liming, Engber- ding showed that in most cases a lack of lime accounted for low bac- terial counts. Kossowicz (16) summarizes the results of investigations by Houston, Th. Remy, Fabricius, and Feilitzen and C. Hoffman, as follows: Manuring brings about an increased bacterial content and betters the conditions for the development of those organisms already present in the soil. The time of the year and weather conditions influence the bacterial content of the soil. — Translation. Koch {15), Adametz {18) and others have shown that the majority of soil microflora consist principally of rod-shaped organisms. That anaerobic bacteria are present in great numbers has been shown by Ucke (24) , who found over 13,000,000 anaerobes present in a garden soil. Lohnis {18) states that the multiplication of soil organisms varies with different soil layers, and the number of bacteria present decreases with the depth, air and food being the first considerations. PRESENT INVESTIGATIONS Many* uncontrolled conditions, such as variations in temperature, moisture, and aeration, are constantly occurring in field practice. The data reported in this paper were obtained in order to ascertain the dif- ferences in bacterial numbers, nitrates, and nitrification of five var- iously treated typical acid soils, after these soils had been kept for 10 months under the same temperatures and controlled moisture conditions in pots where nitrates could not be lost by leaching. The soils used were all very acid and varied widely in organic matter. They were: (i) A yellow silty clay containing 0.7 per cent of humus, 0.07 per cent of nitrogen; (2) a whitish silt loam containing 1.3 per cent of humus, 0.12 per cent of nitrogen; (3) a brown silt loam containing 3.1 per cent of humus, 0.22 per cent of nitrogen; (4) a black peaty sand containing 5 per cent of humus, 0.4 per cent of nitrogen; and (5) a dark-brown peat containing 52 per cent of humus, 2. 04 per cent of nitrogen. More com- plete analyses of these soils and the changes in their acidities due to moisture changes are given by one of us in another paper (7). 20 Journal of Agricultural Research voi. xvi, no. 2 PREPARATION OF SOILS To obtain soils for the pot tests, quantities of field soil were taken from the surface 6 inches, sacked, transferred to the station where each soil was mixed over and over without drying, sieved, and potted. Equal weights of a soil were put in galvanized-iron paraffined culture pots 9.25 inches in diameter and 11 inches high. The soil was compacted to that of a good seed bed by dropping the pots a prescribed number of times onto the floor from a height of about 3 inches. The pots were, kept in the greenhouse and maintained at the desired moisture contents by weighing two to three times a week and replenishing the evaporated moisture with pure distilled water through an open tube extending from above the surface of the soil to an arch at the bottom of the pot. The surface of the soils of all pots except those kept fully saturated with water was cultivated from time to time to give a very thin dust mulch. The wheat stubble and growing clover were in the pots when sampled. The samples were taken to represent the entire depth of soil in the pot by the use of Noyes' bacteriologists' soil samplers (jp), and all determi- nations were made from these samples. ^ NITRATES AND NITRIFICATION WITH LIME AND FERTILIZER TREATMENTS The nitrates were determined by the phenoldisulphonic-acid method modified for the accurate determination of soil nitrates.^ The results are held to be equally accurate for all the soils, since the modified method takes into consideration the obtaining of a clear solution, the presence of soluble salts and interfering organic matter. The nitrification tests were made by the beaker method. One hundred gm. of each soil except the peat, of which 50 gm. were used, were placed in half -pint jelly glasses. Five cc. of a 2 per cent ammonium-sulphate solution were added and the soil was incubated for six weeks at 20° to 2 1 ° C. The moisture content at the end of the period of incubation was in every case within i per cent of what it was when the soils were sampled. Table I gives the acidity, crop yields, and nitrate data for each soil with the different lime and fer- tilizer treatments. The quantities of nitrates found in the untreated soils before incuba- tion showed that nitrification had taken place in every one of the acid soils. The amounts of nitrate present in the untreated soils when sam- pled were in proportion to their total nitrogen contents rather than in any relation to their acidities. The presence of growing clover in some of the pots lowered the ratio of the nitrates before incubation to those after incubation. Those pots which contained large growths of clover when sampled and which had received applications of lime alone con- tained less nitrates than the unlimed pots, which contained little or no 1 The pots used in this investigation were chosen from a series of different investigations on soil-acidity problems, and hence the lime and fertilizer treatments for each soil were not the same. 2 Noyes, H. A. the accurate determination of soil nitrates by the phenol disulphonic-acid METHOD. To be published iu Jour. Indus, and Engin. Chem. Jan. 13, 1919 Nitrates, Nitrification, and Bacteria of Acid Soils 31 clover. This shows that the nitrates present in the soils were greatly influenced by the growing crop. The limed pot in the brown silt-loam series was no exception to this, as the untreated soil on this series grew good clover. With each soil the amounts of nitrates found after incuba- tion were very much greater with lime than without lime, proving that calcium carbonate promotes nitrification in acid soils. As a rule, the less clover there was per pot the greater the ratio of nitrates before incubation to nitrates after incubation. Table I. -Effects of lime and fertilization on nitrates and nitrification of five typical acid soils Kind of soil and treatment per million pounds of soil. YELLOW SILTY CLAY. No treatment 2 tons of calcium carbonate . Nitrogen, phosphorus, po- tassium c Nitrogen, phosphorus, po- tassium , c 2 tons of calcium carbonate Nitrogen, phosphorus, po- tassium, c 6 tons of calcium carbonate WHITISH SILT LOAM. No treatment 3 tons of calcium carbonate . 500 pounds of acid phosphate BROWN SILT LOAM. No treatment 3 tons of calcium carbonate 500 pounds of acid phosphate BLACK PEATY SAND. No treatment 2 tons of calcium carbonate Nitrogen, phosphorus po- tassium c Nitrogen, phosphorus, po- tassium , c 2 tons of calcium carbonate Nitrogen, phosphorus, po- tassium, c 6 tons of calcium carbonate DARK-BROWN PEAT. No treatment 2 tons of calcium carbonate . 20 tons of calcium carbonate Acidity." Crop yields.6 Nitrates. Potas- sium nitrate. 2, 460 20 2, 800 1,360 20 1,380 460 20 460 I, 800 80 1,760 40 Calcium acetate. Wheat 4, 000 4, 125 750 3,000 500 3,000 3,750 750 3>750 6,750 3,500 6,750 3,000 750 2, 040 35, 000 I, 260 I27, 500 100 I 9, 250 Gni. 7 10 43 68 76 23 40 23 19 29 20 o. 5 52 o o. 5 48 Clover. Before incu- bation. Gm. P.p.ni 0 14 10 Tr. 2 Tr. 17 0 15 0 13 37 6 19 17 29 20 30 31 23 52 19 0 II 350 77 I 305 13 52 14 233 0 0 14 710 I, 216 154 After incu- bation. In- crease on incu- bation. P. p. m. P. p.m. 24 32 14 3.2 Tr. 0 184 184 873 873 38 879 48 19 862 19 92 852 119 69 800 1 100 340 585 1 — 10 508 473 168 913 861 I, 280 1,047 710 1,280 4,736 0 64 4,582 42 50 2 60 25 6 16 103 13 100 95 3 a Acidity determinations were made by Hopkins potassium-nitrate method and C. H. Jones calcium- acetate methods, and expressed in calcium-carbonate requirement per million. 6 Crop yields are given in grams per pot; average of two pots. c Chemically pure salts: 91 pounds of ammonium nitrate, 72 pounds of ammonium phosphate, and 100 pounds of potassium phosphate on yellow silty clay. No ammonium nitrate was used on black peaty sand. 2>^ Journal of Agricultural Research Vol. XVI, No. 2 The yellow silty clay containing 0.07 per cent of nitrogen and the black peaty sand containing 0.40 per cent of nitrogen received the same lime and fertilizer treatments, but gave quite different crop yields, ni- trates, and nitrification. These variations can not be entirely correlated with changes in soil acidity. On the yellow silty clay it took both lime and fertilizer to give a markedly increased nitrifying power, while on the black peaty sand, of higher initial nitrifying power, lime gave the large, increased nitrifying power. The whitish silt loam containing 0.12 per cent of nitrogen received the same lime and acid-phosphate treatment as the brown silt loam containing 0.22 per cent of nitrogen. Lime increased nitrification on both these soils more than acid phosphate did. SOIIv MOISTURE IN RELATION TO NITRATES AND NITRIFICATION In order to ascertain what effect keeping soils at different moisture contents without crop would have on the nitrates present in the soil and nitrification tests, samples were drawn from a series of pots where each of the five acid soils had been kept at different moisture contents. The nitrates present in the soils after standing 10 months with specified moisture contents are given in Table II. Table II. — Effects of variable moisture on nitrates and nitrification of five typical acid soils Acidity." Kind of soil and moisture treatments YELLOW SILTY CLAY. One-half 3, 07 Full \ I, 740 WHITISH SILT LOAM. One-fourth . One-half. . . Full BROWN SILT LOAM. One-fourth . One-half. . . Full BLACK PEATY SAND. One-fourth . One-half. . . Full DARK-BROWN PEAT. One-fourth One-half 1 2, 700 Full Potassium nitrate. Calcium acetate. Before in- cubation. P. p. m. 3.075 1,740 4. 500 3.125 24 0 1,550 1,860 888 3.125 4, 500 2,750 136 74 0 325 487 225 4,500 5. 000 2,500 265 319 0 I, 560 1,810 6, 500 6,250 140 315 925 4,750 0 2, 000 2, 700 1 3.360 1 31,750 32, 500 34, 750 178 618 0 After in- cubation. P. p. m. 19 82 128 100 122 Increase on incu- bation. 328 190 O 214 766 P. p. m. -5 o -165 -197 o -125 o 36 148 o Ratio be- fore and after in- cubation. 126 166 58 265 261 43 166 83 o Both methods are expressed in calcium-carbonate requirement per million. Jan. 13, 1919 Nitrates, Nitrification, and Bacteria of Acid Soils ^2> The results given in Table II show that the amount of water present in a soil is concerned with its nitrification, and further, that soils fully saturated with moisture do not contain nitrates either before or after incubation with ammonium sulphate. This table shows even more strono-ly than Table I that nitrification takes place in an acid soil, for the nitrates contained in the soils when sampled varied directly with the organic matter content of the different soils, but did not increase with lower soil acidities. The many instances where the nitrates in the soil when sampled were greater than those after incubation show that the nitrates present in these uncropped soils were near the maximum that could be present under the conditions of the experiment. METHOD OF OBTAINING COUNTS Field conditions are variable, and the results of these variations are apparent in the soil processes, due to bacterial agencies. It was believed that bacterial counts properly made would show some correlations among these different acid soils, the lime and fertilizer treatments, and the variable moisture contents they were kept under. Not only the nitrifying organisms but all classes of organisms had been given 10 months to respond to the different treatments, and an enumeration of both aerobes and anaerobes should show the types of bacteria predomi- nating under the different treatments. Plate counts were made from plates of high bacterial dilutions of each treatment according to the technic of Noyes and Voigt(2o). Unpub- lished work by one of us on aerobic and anaerobic soil bacteria has shown that the average of five plates of a bacterial dilution high enough so that all bacteria from i cc. of the dilution will have a chance to develop into colonies in 10 days, gives accordant results. The media used was Lipman and Brown(i7) modified synthetic agar, which exten- sive tests have proved to be satisfactory for the development of soil microorganisms. The carbon dioxid and hydrogen incubations were carried out in an atmosphere of flowing hydrogen or carbon-dioxid gas. AEROBIC AND ANAEROBIC COUNTS ON CROPPED, LIMED, AND FER- TILIZED SOILS The number of bacteria present under the different lime and fertilizer treatments are given in Table III. Table III shows that large increases in bacterial numbers result from the use of lime. These increases are largely in the aerobic organ- isms, although with the soils that contain considerable partially decom- posed organic matter the anaerobic count is also increased. Representative aerobic plates obtained from the yellow silty clay are shown in Plate i. The numbers of colonies per plate are small, allowing for maximum development;, yet no striking differences in kinds of microorganisms are apparent under the different treatments. Neither 34 Journal of Agricultural Research Vol. XVI. No. 2 lime nor complete fertilizer alone had any great influence on bacterial numbers, while complete fertilizer with 2 tons of lime more than doubled the bacterial index (sum of aerobes and anaerobes) of the soil. Six tons of lime with fertilizer did not increase the bacterial index as much as the 2 tons with fertilizer. Tabi,E III. — Effects of lime and fertilization on bacterial content of five typical acid soils Millions of bacteria per gram of dry soili Kind of soil and treatment per million pounds of soil. Air incu- bation. Hydrogen incubation. Bacterial index." Increase of bacterial index due to — Calcium carbonate. Fertilizer. YELLOW SILTY CLAY. No treatment 2 tons of calcium carbonate , Nitrogen, phosphorus, potassium « . . Nitrogen, phosphorus, potassium, c 2 tons of calcium carbonate Nitrogen, phosphorus, potassium, c 6 tons of calcium carbonate WHITISH SILT LOAM. No treatment 3 tons of calcium carbonate . . 500 pounds of acid phosphate , BROWN SILT LOAM. No treatment 3 tons of calcium carbonate . . . 500 pounds of acid phosphate . BLACK PEATY SAND. No treatment 2 tons of calcium carbonate Nitrogen, phosphorus, potassium b Nitrogen, phosphorus, potassium, 2 tons of calcium carbonate Nitrogen, phosphorus, potassium, 6 tons of calcium carbonate DARK-BROWN PEAT. No treatment 2 tons of calcium carbonate . . 20 tons of calcium carbonate. Average . ^3. 010 3. 046 3.027 7.605 5- 244 5.021 14. 810 5-531 9. 904 23. 921 II. 164 3. 146 8.386 2.813 10. 583 16. 037 I- 554 3. 420 91. 846 12. 109 o. 100 .381 . 000 . 000 . 000 1-545 .898 . 000 2- 556 2.714 I- 154 1. 617 2. 907 .099 I. 760 ■997 I. 440 II. 752 1-585 3. no 3-427 3.027 7.605 5- 244 6.566 15. 708 5- 531 10. 093 26. 477 13- 878 4.300 10. 003 5.720 17- 797 2- 551 4. 860 103. 598 0-317 4-578 2. 217 4. 962 12. 077 13- 694 2.309 loi. 047 15-874 -0.083 4.178 9- 142 -1-035 16. 384 3-785 5-703 I. 420 .679 a Sum of air and hydrogen counts. • i .-, • i Average of five plates. No count indicates no colonies on plates from i:.4oo,ooo bacterial dilutions. <^ Chemically pure salts: 91 pounds of ammonium nitrate, 72 pounds of ammonium phosphate, and 100 pounds of potassium phosphate on yellow silty clay. No ammonium nitrate was used on black peaty sand . Lime more than doubled the bacterial indexes of the whitish silt and brown silt loams. Acid phosphate decreased the anaerobic counts of the whitish silt loam enough more than it increased the aerobic con- Jan. 13, 1919 Nitrates, Nitrification, and Bacteria of Acid Soils 35 tents so that the bacterial index was decreased. With the brown silt loam containing considerable undecayed organic matter the acid phos- phate increased both the aerobic and anaerobic counts somewhat. Plate 2 shows representative petri plates from each treatment for the two soils. This plate shows a marked similarity between the colonies on the aerobic plates from the limed pots of both soils. The similarity of the appearances of the plates from the limed and the phosphated pots of tl^e whitish silt loam, the similarity of all aerobic plates from the brown silt loam and the uniformity of colonies developing from the brown silt loam under anaerobic conditions are to be noted. The black peaty sand containing six times as much nitrogen as the yellow silty clay, received the same lime and fertilizer treatments as the yellow clay, but gives entirely different results. Ume and fertilizer both alone and in combination give increased bacterial indexes. While the aerobic organisms are increased by lime, the organic matter of the black peaty sand must be in an advanced stage of decay since the counts are lower than they should be if the organic matter was good food for bacteria. Plate 3 shows representative culture plates from this soil. These illustrations emphasize the effect of lime on bacterial numbers and the small proportion of the bacteria which are anaerobic. The dark-brown peat shows an increase of over 100,000,000 in bacterial index as the result of liming. Peats in situ are generally low in bacterial content. Working them over after drainage generally causes enormous increases in their bacterial content. This peat, even when aerated, had only iK times as many aerobic as anaerobic bacteria, but adequate liming increased the aerobes more than 60 times and the anaerobes over II times. The increase in anaerobes is believed to be associated with the large amount of organic matter present in the soil. Plate 4 shows representative petri plates of the colonies developing in air and hydrogen. Attention is called to the small variation in colony types on the anaerobic plates as compared to the aerobic. The aerobic culture plates from the heavily limed soil showed many chromogenic differences between colonies not observable in the photographs. SOIL MOISTURE IN RELATION TO BACTERIAL COUNTS In addition to the incubations in air and hydrogen another set of plates was incubated in an atmosphere of flowing carbon-dioxid gas for 10 days. No colonies developed on this set of plates while they were in carbon-dioxid gas. The counts given were computed from colonies developing in 10 days in air after the plates had been removed from the carbon dioxid.^ Table IV gives the counts under the different condi- tions of incubation and the various soil-moisture contents. * These organisms, as far as tested, have been found to be spore formers. 36 Journal oj Agricultural Research voi. xvi, no. Table IV. — Effects of variable moisture on bacterial content of five typical acid soils Millions of bacteria per gram of dry soil. Bacterial index. 6 liatios to bacterial in- dex as 100. Kind of soil and degree of moisture saturation. Air. Hydro- gen. Carbon- dioxid- surviv- ing.a Air. Hydro- gen. Carbon- dioxid- surviv- ing. YELLOW SILTY CLAY. One-half " I- SS6 . 184 2.792 3.688 3- 179 4.920 4-513 7-854 I. 641 2-363 3-316 2.425 1.796 2-257 0. 101 .032 •367 1. 171 ■353 1.879 I. 640 2.864 •453 I. 270 .129 1.988 I. 914 -735 0. 131 •075 .282 . 216 . 246 •533 .847 •575 .286 • 463 1. 071 I. lOI I. 204 ■499 1-657 . 216 3-159 4-859 3-532 6.799 6.153 10. 718 2.094 3-633 3-445 4-413 3.710 2. 992 94 85 88 76 90 72 73 73 78 65 96 55 48 75 6 15 12 24 10 28 27 27 22 35 4 45 52 25 8 Full 35 9 4 7 8 WHITISH SILT LOAM. One-fourth One-half Full BROWN SILT LO.\M. One-fourth One-half 14 5 14 13 31 25 33 17 Full BLACK PEATY SAND. One-fourth One-half Full DARK-BROWN PEAT. One-fourth One-half Full Averages 3-034 I. 064 -538 4. 098 74 26 13 o Incubated 10 days in carbon dioxid; then 10 days in air. * Sum of air and hydrogen incubation, c All figures were computed from 5 plates. The bacterial content, as well as the proportions of aerobes to anaerobes, was changed by the degree of saturation of the soil, but the nature of the soil had a greater effect than the moisture content on bacterial num- bers. The proportions of anaerobes to the aerobes which survived •carbon-dioxid incubation increased with soil organic matter when the soils were held under optimum moisture conditions. Plates 5 to 9 show representative petri plates from the i to 40,000 bacterial dilution of these soils. Figures A^, H^, and C^ in each plate show representative petri plates after air (A) , hydrogen (H) , and carbon- dioxid, then air (C) incubations of bacterial dilutions of samples from pots of soils kept one-fourth saturated with water. Figures Aj, H2, and C2 are from samples from pots of soils kept half saturated, while A3, H3, and C3 are from pots of soils kept fully saturated with water. Plates 5 to 9 show that the bacterial flora of each soil is different from that of every other soil. The soils kept one-fourth saturated with water Jan. 13, 1919 Nitrates, Nitrification, and Bacteria of Acid Soils 37 contained the largest numbers of microorganisms developing moldlike colonies, and the fully saturated soils gave culture plates containing the smallest numbers of spreading moldlike colonies. NiTRATEvS AND NITRIFICATION IN RELATION TO BACTERIAL COUNTS Lime increased both nitrification and bacterial counts. A study of Plates I to 4 shows that the increases in bacterial numbers can be asso- Fig. I.— Graphs showing the relation of aerobes and anaerobes to nitrification of five add Soils with and without lime and fertilizer treatments. ciated principally with the aerobic small, round, entire colonies on the petri plates. Figure i shows the relations between aerobic and anaerobic bacteria and the nitrates after incubation for the cropped, limed, and fertilized soils kept at optimum moisture content. The nitrates after incubation varied directly with the aerobic bacteria. The aerobic count and nitrates after incubation show that it is the increased number of aerobic organisms that are to be associated with increased nitrifi- cation. 38 Journal of Agricultural Research Vol. XVI, No. 2 Figure 2 gives soil nitrates, aerobic, and anaerobic bacterial numbers for the series of soils where moisture was the variable. These graphs shows that lack of aeration which changed the proportions of aerobes to anaerobes prevented a correlation between nitrates and aerobic counts. c? 1 / 7' / / 1 1 ^ jff 1 1 ^ ^^1 1 1 t / / \ \ y i J/ I / o> / ^^/p \ / '1 / \ 1 \ y •^ \ A / ~~^ \ / v/r/ 5-772 ^s- \ r \\ / \ \ V \ \ i / \ \ \ > \ ^/\/y' %\ r ' / / \\ ^^v^ \ ^ ^^ / ^<:^ ^^ / !i^ i^ / i^ (^ / y£^^. cy^y u^///r£- ^/zr s-zpcuffw zo^/^ /5zyf«r/r^.s>'j/vz> /3/PC^A^ ^^^s^r FiG. 2. — Graphs showing the relation of aerobes and anaerobes to nitrates in five acid soils kept at different moisture contents. GENERAL DISCUSSION After conducting bacteriological investigations on acid soils to ascer- tain "if it might be desirable to consider more carefully the possibilities of a system of acid agriculture," Bear (j) concluded that — the supply of nitrogen in acid soils may be maintained by growing acid-resistant legumes, of which the soy bean is one. Undoubtedly the use of acid phosphate aids Jan. 13, 1919 Nitrates, Nitrification, and Bacteria of Acid Soils 39 materially in the nitrogen-fixation processes of acid soils. Small applications of calcium carbonate are, as a rule, relatively more effective than large applications as a means of increasing the bacterial activities in acid soils. The problem of maintaining soil fertility resolves itself into maintain- ing and increasing the available supply of organic matter and nitrogen in the soil and the replenishing of the mineral elements. One system now generally recommended and used is to apply lime and phosphates, then to grow legumes, and to plow them under. This system of soil mainte- nance and impro^"ement is in accordance with the important role of soil bacteria in plant nutrition, and the results obtained in the controlled investigations reported here illustrate some good reasons for such a method of soil management. When the soil was limed, the aerobic bacteria concerned with oxida- tion reactions increased in numbers. This is illustrated by the increased bacterial numbers and nitrification wherever the soils were limed. Plenty of organic matter is necessary for high bacterial numbers, a condition which is well illustrated by the low bacterial content and nitrate results with the limed yellow silty clay (low in organic matter) compared with the high bacterial contents and nitrates on the limed brown silt loam and dark-brown peat (high in organic matter) . Mineral fertilizers serv'e as food for larger crops and larger crops in turn leave more residues in roots and stubble for bacterial food. The number of bacteria in an arable soil can be correlated with crop yield to about the same degree that soil moisture can be. Soil moisture is conceded to be the most vital single factor influencing crop yields ; yet because of so many other variable conditions it is not always possible to correlate soil moisture and crops any more than it is possible to always correlate bacterial numbers and crops. Below a certain minimum in moisture or bacterial numbers field soils will not produce crops; above that minimum, everything else being equal, crops may be in general correlated with bacterial numbers as well as with moisture. Changes in bacterial numbers, especially differences in the propor- tions of aerobes to anaerobes, are of prime importance in soil-biology studies. The results here reported under controlled conditions make it evident that soil-fertility investigations should include both chemical and biological examinations of the soil. SUMMARY (i) Controlled greenhouse investigations were conducted on five typical acid soils. In part of the experiments the soils were fertilized with calcium carbonate, acid phosphate, and complete fertilizer, cropped to wheat and clover, and kept at optimum moisture content, while in another series the soils were unfertilized, uncropped, and kept one- fourth, one-half, and fully saturated with water. (2) The results reported include crop yields, soil-acidity determina- tions, nitrates in the soil when sampled and after incubation with ammo- 40 Journal of Agricultural Research voi. x\i, no. 2 nium sulphate, and also the numbers of aerobic, anaerobic, and carbon- dioxid surviving microorganisms present in the soils. (3) All the untreated soils were quite acid and contained nitrates when sampled, showing that nitrification takes place in acid soils. (4) The amounts of nitrates present and the nitrifying power of the untreated acid soils varied with the organic matter and total nitrogen rather than with the soil acidity. (5) Calcium-carbonate additions markedly increased the nitrification of all five soils. (6) Fertilization tended to increase nitrification, but not so much as calcium carbonate did. (7) Regardless of treatments the presence of growing clover kept down nitrate contents of the soils. (8) The degree of saturation of the soils affected the nitrates present. As a rule, more nitrate were fotmd in soil kept one-half saturated with water than in soil kept one-fourth saturated. (9) The soils that had been kept fully saturated with water for the i o months contained no nitrates and formed no nitrates when incubated with ammonium sulphate. (10) The relation of nitrates present in the uncropped soils before incubation to the* nitrates present after incubation shows that the ni- trate contents of these acid soils tend to reach an equilibrium, above which no increase is obtained without additional treatment. (11) The bacterial flora of each soil was different from that of every other soil. (12) No bacteria developed into colonies visible to the eye as long as plates were incubated in an atmosphere of flowing carbon-dioxid gas. (13) Calcium-carbonate additions increased the bacterial contents of the soils. This increase was largely in the aerobic organisms. (14) Small increases in bacterial content resulted from the use of fertilizer. (15) The degree of saturation at which the soil was kept changed the proportions between the aerobic, anaerobic, and carbon-dioxid-surviving bacteria. (16) Cultures from samples that had been kept one-fourth saturated with water contained the largest proportions of organisms forming moldlike colonies. (17) Under optimum moisture conditions both without and with lime and fertilizer treatments the nitrates after incubation varied directly with the aerobic counts. (18) In general, the greater the aerobic bacterial content and the nitrifying power of the soil the larger the crop yields. (19) These investigations show many reasons why a system of soil improvement which includes the addition of lime, phosphate, and or- ganic matter is worth while. (20) It is evident that soil fertility investigations should include both chemical and biological examinations of the soil. Jan. 13. 1919 Nitrates, Nitrification, and Bacteria of Acid Soils 41 IvlTERATURE CITED (i) Abbott, J. B., Conner, S. D., and Smalley, H. R. 1913. THE RECI.AMATION OF AN UNPRODUCTIVE SOII, OF THE KANKAKEE MARSH REGION. SOIL ACIDITY NITRIFICATION, AND THE TOXICITY OF SOLUBLE SALTS OF ALUMINUM. Ind. Agr. Exp. Sta. Bui. 170, p. 327-374, 22 fig. (2) AlKMAN, C. M. 1894. MANURES AND THE PRINCIPLES OF MANUIilNG. 592 p. Edinburgh, London. Cites (p. 6-8) paper by Digby. (3) BEAR, Firman E. 1917. A CORRELATION BETWEEN BACTERIAL ACTIVITY AND LIME REQUIREMENT. In Soil Science, v. 4, no. 6, p. 433-462, 4 fig. References, p. 460-462. (4) Berthelot. 1871. SUR LA NITRIFICATION naturellE. In Ann. Chim. et Phys., s. 4, t. 22, p. 87-96. Abstract in Jour. Chem. Soc. [London], v. 24 (n. s. v. 9), p. 1000-1005. 1871. Cites (p. 88) paper by Thouvenel. (5) Brown, P. E. I912. BACTERIOLOGICAL STUDIES OF FIELD SOILS. I. THE EFFECT OF LIME. Iowa Agr. Exp. Sta. Research Bui. 5, p. 185-210. (6) Chester, Fred'k D. 1902. studies IN SOIL BACTERIOLOGY. In Del. Agr. Exp. Sta. 13th Ann. Rpt. [i90o]/oi, p. 50-73. (7) Conner, S. D. 1918. soil acidity as affected by moisture conditions of the soil. in Jour. Agr. Research, v. 15, no. 6, p. 321-329. Literature. Cited, p. 329. (8) Deh^rain, p. p. 1894. NITRIFICATION IN ARABLE SOILS. /«Exp. Sta. Rec, V. 6, no. 5, p. 353-366. Cites (p. 353) Boussingault and Ville; (p. 359) Schlosing. (9) Engberding, Diedrich. 1909. VERGLEICHENDE UNTERSUCHUNGEN UBER DIE BAKTERIENZAHL IM ACK- ERBODEN IN IHRER ABHANGIGKEIT VON AUSSEREN EINFLUSSEN. In Centbl. Bakt. [etc.], Abt. 2, Bd. 23, No. 21/25, P- 569-642. (10) Fabricius, Otto, and Feilitzen, Hjalmar von. 1905. ueber den gehalt an bakterien in jungfraulichem und kulti- VIERTEM HOCHMOORBODEN AUF DEM VERSUCHSFELDE DES SCHWEDI- SCHEN moorkulturvereins bei flahult. In Centbl. Bakt. [etc.] Abt. 2, Bd. 14, No. 6/7, p. 161-168. (11) Hall, A. D., Miller, N. H. J., and Gimingham, C. T. 1908. nitrification in acid SOILS. In Proc. Roy. Soc. [London], s. B, v. 80, no. 539, p. 196-212. (12) HouzEAU, Auguste. 1872. FAITS POUR SERVIR A l'hISTOIRE DE LA NITRIFICATION. COMPOSITION DES TERREAUX DE TANTAH (basse-6g\tte). In Ann. Chim. et Phy. s. 4, t. 25, p. 161-167. Abstract in Jour. Chem. Soc. [London], v. 25 (n. s. V. 10), p. 465-466, 1872. (13) Kelley, w. p. 1914. RICE SOILS OF HAWAII. Hawaii Agr. Exp. Sta. Bui. 31, 23 p. (14) Kellner, O. 1891. researches ON THE ACTION OF LIME AS A MANURE, WITH SPECIAL REGARD TO PADDY SOILS. In Imp. Univ. Col. Agr. [Tokyo] Bui. 9, p. 1-25. 42 Journal of Agricultural Research voi.xvi.no. 2 (15) Koch, Alfred. I91O. UBER LUFTSTICKSTOFFBINDUNG IM BODEN MIT HILFE VON ZELLULOSE ALS ENERGiEMATERiAL. In Centbl. Bakt. [etc.], Abt. 2, Bd. 27, No. 1/3, p. 1-7. (16) Kossowicz, Alexander. 1912. EINFUHRUNG IN DIE AGRIKULTURMYKOLOGIE. I. TEIL: BODENBAKTERIOLOGIE. 143 p., 47 fig. Berlin. Literatur, p. 105-130. (17) LiPMAN, Jacob G., and Brown, Percy B. 1909. NOTES ON METHODS AND CtTLTURE MEDIA. Ill N. J. Agr. Exp. Sta. 29tll Ann. Rpt. [1907] /08, p. 129-136. (18) LOHNIS, F. 1910. HANDBucH der landwirtschaftuchen bakteriologie. 907 p. Berlin. Cites (p. 510) paper by Adametz. (19) NOYES, H. A. 1915. soil sampling for bacteriological examination of soils. Jour. Amer. Soc. Agron., v. 7, no. 5, p. 239-249, fig. 13, pl> 4. (20) and VoiGT, Edwin. I917. A TECHNIC for the BACTERIOLOGICAL EXAMINATION OF SOILS. In PrOC. Ind. Acad. Sci. 1916, p. 272-301, illus. (21) Petit, A. 1913. DE la NITRIFICATION DANS LES TERRES HUMTF^RES acidES. In Ann. Sci. Agron., arm. 30 (s. 4, ann. 2), sem. 2, no. 4, p. 397-398. Abstract in Exp. Sta. Rec, v. 30, no. 5, p. 424. 1914. (22) Schloesing, T., and MOntz, A. 1877. SUR LA nitrification PAR LES FERMENTS ORGANISES. In Compt. Rend. Acad. Sci. [Paris], t. 84, no. 7, p. 301-303. (23) Temple, J. C. 1914. NITRIFICATION IN ACID OR NON-BASIC SOILS. Ga. AgT. Exp. Sta. Bul. 103, 15 P- (24) UcKE, Alexander. 1898. EiN BEiTRAG ZUR kennTnis dEr ANAfiROBEN. In Centbl. Bakt. [etc.], Abt. I, Bd. 23, NO. 23, p. 996-1001. (25) Warington, Robert. 1892. NITRIFICATION. NITRIFICATION AND DENTIFRICATION. In U. S. Dept. Agr. Exp. Sta. Bul. 8, p. 42-76. (26) White, J. W. 1915. NITRIFICATION IN RELATION TO THE REACTION OF THE SOIL. In Penn. Agr. Exp. Sta. Ann. Rpt. 1913/14, p. 70-80, 4 pi. 92802°— 19 2 PLATE I Representative plates from i to 400,000 bacterial dilution of acid yellow silty clay, cropped and held under optimum moistiu-e conditions: A. — ^Aerobic plates, untreated. B. — Aerobic plates, treated with 2 tons of calcium carbonate. C. — Aerobic plates, treated with complete fertilizer. D. — ^Aerobic plates, treated with complete fertilizer and 2 tons of calcium carbonate. E. — Aerobic plates, treated with complete fertilizer and 6 tons of calcium carbonate. Nitrates, Nitrification, and Bacteria of Acid Soils Plate I Journal of Agricultural Research Vol. XVI, No. 2 Nitrates, Nitrification, and Bacteria of Acid Soils Plate 2 Journal of Agricultural Research Vol. XVI, No. 2 PLATE 2 Representative plates from i to 400,000 bacterial dilution of acid whitish silt loam and acid brown silt loam cropped and held under optimum moistiu-e conditions: A. — Aerobic plates, acid whitish silt loam, tmtreated. B. — Aerobic plates, acid whitish silt loam, treated with 3 tons of calcium carbonate. C. — Aerobic plates, acid whitish silt loam, treated with 500 pounds of acid phos- phate. D. — ^Aerobic plates, acid brown silt loam, untreated. E. — Aerobic plates, acid brown silt loam, treated with 3 tons of calcium carbonate. F. — Aerobic plates, acid brown silt loam, treated with 500 poimds of acid phosphate. G. — ^Anaerobic plates, acid brouTi silt loam, im treated. H. — Anaerobic plates, acid brown silt loam, treated with 3 tons of calcium carlxtnate. I. — Anaerobic plates, acid brown silt loam, treated with 500 poxmds of acid phos- phate. PLATE 3 Representative plates from i to 400,000 bacterial dilution of acid black peaty sand, cropped and held under optimum moisture conditions: A.— Aerobic plates, untreated. B. — ^Aerobic plates, treated with 2 tons of calcium carbonate. C. — ^Aerobic plates, treated with complete fertilizer. D. — ^Aerobic plates, treated with complete fertilizer and 2 tons of calcium carbonate. E. — ^Aerobic plates, treated with complete fertilizer and 6 tons of calcium carbonate. F. — ^Anaerobic plates, treated with complete fertilizer. G. — ^Anaerobic plates, treated with complete fertilizer and 2 tons of calcium car- bonate. H. — Anaerobic plates, treated with complete fertilizer and 6 tons of calcium car- bonate. Nitrates, Nitrification, and Bacteria of Acid Soils Plate 3 Journal of Agricultural Research Vol. XVI, No. 2 Nitrates, Nitrification, and Bacteria of Acid Soils Plate 4 Journal of Agricultural Research Vol. XVI, No. 2 PLATE 4 Representative plates from i to 400,000 bacterial dilution of acid dark-brown peat, cropped and held under optimum moisture conditions: A. — Aerobic plates, untreated. B. — Aerobic plates, treated with 2 tons of calcium carbonate. C. — Aerobic plates, treated with 20 tons of calcium carbonate. D. — Anaerobic plates, untreated. E. — Anaerobic plates, treated with 2 tons of calcium carbonate. F. — Anaerobic plates, treated with 20 tons of calcium carbonate. PLATE 5 Representative plates from i to 40,000 bacterial dilution of acid yellow silty clay kept at different moisture contents: Aj. — Aerobic plates, from soil kept one-half saturated. Hj. — Anaerobic plates, from soil kept one-half saturated. C2. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully satiu-ated. H3. — ^Anaerobic plates from soil kept fully saturated. C3. — Aerobic plates of carbon-dioxid-siu^iving organisms from soil kept fully satiu-ated. Nitrates, Nitrification, and Bacteria of Acid Soils Plate 5 Journal of Agricultural Research Vol. XVI, No. 2 Nitrates, Nitrification, and Bacteria of Acid Soils Plate 6 Journal of Agricultural Research -Vol. XVI, No. 2 PLATE 6 Representative plates from i to 40,000 bacterial dilution of acid whitish silt loam kept at different moisture contents: t Aj. — Aerobic plates from soil kept one-fourth saturated. Hi. — Anaerobic plates from soil kept one-fourth saturated. Ci. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept one-fourth satiu"ated. A2. — Aerobic plates from soil kept one-half saturated. Hj. — Anaerobic plates from soil kept one-half saturated. Cj. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — Anaerobic plates from soil kept fully saturate . C3. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated. PLATE 7 Representative plates from i to 40,000 bacterial dilution of acid brown silt loam , kept at different moisture contents: Aj.— Aerobic plates from soil kept one-fourth saturated. Hi.— Anaerobic plates from soil kept one-fourth saturated. Cj —Aerobic plates of carbon-dioxid-surviving organisms from soil kept one-fourth saturated. A2.— Aerobic plates from soil kept one-half saturated. H2.— Anaerobic plates from soil kept one-half saturated. C2.— Aerobic plates of carbon-dioxid-surviving organisms from soil kept half saturated. Aj.—Aerobic plates from soil kept fully saturated. H3.— Anaerobic plates from so^l kept fully saturated. C3.— Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated. Nitrates. Nitrification, and Bacteria of Acid Soils Plate 7 Journal of Agricultural Research Vol, XVI, No. 2 Nitrates, Nitrification, and Bacteria of Acid Soils Plate 8 Journal of Agricultural Research Vol. XVI, No. 2 PLATE 8 Representative plates from i to 40,000 bacterial dilution of acid black peaty sand kept at different moisture contents: A,.— Aerobic plates from soil kept one-fourth saturated. H,. — Anaerobic plates from soil kept one-fourth saturated. C,.— Aerobic plates of carbon-dioxid-surviving organisms from soil kept one-fourth saturated. A2. — Aerobic plates from soil kept one-half satiirated. Ho. — Anaerobic plates from soil kept one-half saturated. C2. — Aerobic plates of carbon-dioxid-siu-viving organisms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — ^Anaerobic plates from soil kept fully saturated. Cg. —Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated. PLATE 9 Representative plates from i to 40,000 bacterial dilution of acid dark-brown peat kept at different moisture contents: Ap — Aerobic plates from soil kept one-fourth saturated. Hi. — Anaerobic plates from soil kept one-fourth saturated. Ci. — Aerobic plates of carbon-dioxid-siu-viving organisms from soil kept one-fourth saturated. Aj. — Aerobic plates from soil kept one-half saturated. Hj. — Anaerobic plates from soil kept one-half saturated. Cj. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept one-half saturated. A3. — Aerobic plates from soil kept fully saturated. H3. — Anaerobic plates from soil kept fully saturated. C3. — Aerobic plates of carbon-dioxid-surviving organisms from soil kept fully saturated. itrates, Nitrification, and Bacteria of Acid Soils Plate 9 Journal of Agricultural Research Vol. XVI, No. 2 EFFECT OF CERTAIN ECOLOGICAL FACTORS ON THE MORPHOLOGY OF THE UREDINIOSPORES OF PUC- CINIA GRAMINIS ' By E. C. Stakman, Head of the Section of Plant Pathology, Division of Plant Pathology, and Botany, Department of Agriculture, University of Minnesota, and M. N. Levine, Field Assistant, Cereal Investigations, Bureau of Plant Industry, United States Department of Agriculture COOPERATIVE INVESTIGATIONS BETWEEN THE AGRICULTURAL EXPERIMENT STATION OF THE UNIVERSITY OF MINNESOTA AND THE BUREAU OF PLANT INDUS- TRY OF THE UNITED STATES DEPARTMENT OF AGRICULTURE INTRODUCTION Extensive studies have been made of biologic forms of Puccinia gratninis Pers., but these studies have been mainly on the physiological rather than on the morphological phase of the problem. The effect of host plants and other factors on the parasitic capabilities of biologic forms has been quite thoroughly investigated. Some work has also been done on the effect of host plants on the morphology of the fungus, but hardly as much as the importance of the problem warrants. The question whether biologic forms change readily in response to environmental conditions is important practically and scientifically. The measure of plasticity has usually been the parasitic performance of the rust. But if there is a tendency for biologic forms to change rather quickly, it is reasonable to expect that the morphology might change also. The object of this work, therefore, was to determine the effect of hosts and of physical factors such as heat, light, and humidity on the morphology or urediniospores. It would be desirable to include a study of teliospores and aeciospores also, but the difficulties are obvious. Since the effect of physical factors on the morphology of the uredini- ospores may be indirect — by affecting the vigor of the rust — the virulence of the rust under different conditions was also studied. Although it has been generally believed that the various biologic forms of P. graminis differ only functionally, yet as early as 1902, Ward {15, p. 2^6)^ suggested that each specialized form — is in coiirse of becoming a species and may during the lapse of time actually become a species of Puccinia, which will eventually show morphological differences in addition to the physiological ones it already shows. Freeman and Johnson (5, p. 14) expressed a similar opinion in 191 1. Stakman also (11) obtained some evidence that long association with * Published, with the approval of the Director, as Paper No. 143 of the Journal Series of the Minnesota Agricultural Experiment Station. 2 Reference is made by number (italic) to "Literature cited," p. 77. Journal of Agricultural Research, Vol. XVI. No. 2 Washington, D. C. ' Jan. 13. 1919 rb Key No. Minn.-as (43) 44 Journal of Agricultural Research voi. xvi, no.z a given host might change the urediniospore dimensions of a biologic form. Recently Long {8) working with Puccinia ellisiana Thuem. and P. andropogonis Schw., whose secial hosts are certain species of Viola and Pentstemon, respectively, found he could change the morphol- ogical characters of the urediniospores of these rusts by reversing their aecial hosts. Thus P. ellisiana after passing through Pentstemon as the aecial host acquired the morphological characteristics of the urediniospores of P. andropogonis ; likewise, P. andropogonis assumed the characters of P. ellisiana after passing through Viola sp. as the aecial host. Attention has been called several times to the fact that when a bio- logic form of stemrust develops weakly on a partly resistant host the urediniospores usually are appreciably smaller than they normally are. It seems, therefore, that physical factors might also influence the spore size by affecting the vigor of the rust. But, although a great deal of work has been done on the effect of environmental factors on the severity of rust attacks, the possible correlation between the degree of vigor of the rust fungus and the size of the spores has not been investigated thoroughly. Ward {15, p. 2J4) noted tnat differences of temperature, illumination, drouth, etc., affecting the transpiration, assimilation, and other proc- esses of the seedlings, also affect the period of germination, incubation and maturation of the rusts. Fromme {4, p. 50^-509) has tabulated a number of recorded observations of this nature. Johnson (7, p. 47) found the cardinal temperature of Pticcinia graminis on wheat, barley, and oats to vary from about 35 to 90° F. Butler and Hayman (i, p. 11) have not succeeded in producing rust arti- ficially on plants grown in the open in the hot weather, in India, and they doubted — whetlier the spores have power to infect when exposed to temperatxires exceeding 100° F. Christman (2, p. 106) — found by experiment that [in Wisconsin] in the cooler weather of spring the incu- bation period following inoculation with uredospores is usually lengthened to between three and four weeks. Although there was considerable evidence on the effect of these environmental factors on rust, their effect was investigated again, especially for the purpose of getting evidence of the effect on spore morphology. EXPERIMENTAL METHODS The methods employed in these experiments were (essentially the same as those described by Stakman and Piemeisel (14). But it was thought advisable to obtain additional data on the following points: Jan. 13. 1919 Morphology of Urediniospores of P. graminis 45 (i) Quantity of inoculum to be used; (2) optimum length of incu- bation; (3) condition of urediniospores necessary to insure uniform measurements; (4) number of measurements to be made of a given strain; and (5) method of computation to be employed. In order to determine the amount of inoculum to be used, eight sets of inoculations were made with very heavy, with moderate, and with exceedingly light applications of inoculum. The very heavily inocu- lated plants produced 132 successful infections out of 142 inoculations, or 93 per cent; 130 out of 142, or 91 per cent, of the leaves inoculated with a moderate amount became infected; and 104 out of 118, or 88 per cent, of those which had received a small amount of inoculum became infected. Whenever infection resulted, there was no perceptible differ- ence in the size of either the uredinia or the urediniospores, or in the virulence of attack in general. A liberal amount of inoculum was used whenever possible in all subse- quent experiments. Jaczewski (5, p. 330) found that the germination of urediniospores begins two or three hours after placing them in water or on the surface of the plant blades, after which it progresses very rapidly, provided the spores are fresh. Fromme (4, p. 513) points out that in order to obtain a successful infection on plants a saturated atmosphere is necessary. To determine the optimum length of the incubation period, wheat plants in 10 pots, each containing 10 wheat seedlings 6 days old, were inoculated with an equal and liberal amount of viable urediniospore material of P. graminis tritici and placed in two pans containing a small amount of water and then placed under glass bell jars. Equal amounts of water were put in both pans, and all other conditions were kept uniform. At the end of 12 hours two pots were removed from under the bell jars and set out on the bench; after 24 hours the second pair of pots were set out; and the rest were taken out from the pans in pairs every 24 hours following the second pair— that is, 48, 72, and 96 hours after inoculation. The first observation was made 80 hours after inoculation, and no signs of infection could be detected on the plants incubated for 12, 24, or 48 hours; but 6 plants of those which had been under for 72 hours and 12 of those that were still under the bell jar showed very indistinct, but apparently typical, rust flecks. These were later found not to be infection flecks, but the result of supersensibility, due to the long con- finement in the moist chambers. At 128 hours after inoculation clearly defined rust flecks appeared on all plants which had been incubated for 48, 72, and 96 hours. Of the 20 plants that had been under the bell jar for 24 hours, 18 plants were flecked, while only a single fleck showed on one plant of those that had been under only 12 hours. 46 Journal of Agricultural Research Vol. XVI, No. 2 The first uredinia began to burst through the epidermis 144 hours after inoculation, except on those plants which had been under the bell jar for 12 hours. On these plants the first and only uredinium appeared 10 days after inoculation. At this time the rust was well developed on all the plants that had been under for 48 hours, whereas those that were kept under 24, 72, and 96 hours showed the maximum infection only two days later. Although all the fully developed uredinia were in every case approxi- mately the same size, color, and shape, the virulence of the attack varied considerably. The plants kept under the bell jar for 48 hours produced the greatest number of uredinia per leaf; those which had been under 24 and 72 hours, respectively, somewhat fewer, and those which had been under 96 hours, still fewer; while only one uredinium appeared on the single infected leaf of the plants kept under for 12 hours. In the present work, therefore, all inoculated plants were kept for 48 hours in the moist chamber, then removed to their respective places on the greenhouse benches. It was found that the superficial layer of each uredinium contains larger spores, and when this layer is removed, the remaining spores are considerably smaller. But if the uredinium is allowed to produce a new crop of spores, those on the surface again attain the same dimensions as the original ones. For this reason precaution was taken to measure spores from uredinia in the same stage of development. Table 1.— Results of ineasuring varying numbers of uredinios pores of Puccinia graminis trilici and P. graminis avenae Experi- ment. No. Source of urediniospores. Triticum aestivum . do do do do Avena saliva do do do Num- ber of spores meas- ured. Spore dimensions. Range of length. 25 50 100 200 400 25 5° roo 200 26. 88-38. 72 26. 88-40. 32 26. 56-40. 32 25. 60-40. 32 23. 68-40. 32 23- 36-35- 20 23- 36-35- 20 23- 04-35- 20 21. 12-36. 48 Range of width. M 17. 92-22 17. 92-23 16. 96-23 16 32-23 16 32-23 17. 28-22 17. 28-22 16. 96-22 16 32-23 .08 .04 -36 -36 •36 .08 .08 .08 .04 Modes. 032. 00X19. 52 ^33. 60X19-84 32. 96X19. 84 32. 96X19. 84 32. 96X19. 84 O27. 52X19- 52 29. 12X19. 52 29. 12X19. 52 o Modes doubtful but showing tendency to form as indicated. b Mode of length doubtful but that of width definitely established. c Modes indeterminate. As to the number of spores to be measured from a given group, it was found that 100 gave equally as good results as 200 or 400, while when less than 100 were used the results were not always representative or conclusive. Table I gives the results of measuring different numbers of Jan. 13, 1919 Morphology of Urediniospores of P. graminis 47 spores from the same plant taken on the same day from uredinia on the same leaves. Stemrust of both wheat and oats was tried with similar results. As noted from Table I, the modes of a population of 100, 200, or 400 spores are the same, but the limits of variation are less in a popu- lation of 100 than in those of 200 or 400. It will also be seen from the table that at least 100 spores should be used. In the present investiga- tion 200 spore measurements were made for each experimental group until January i, 1916, which constituted about half of all measurements made. Beginning with this date 100 spore measurements for length and 100 for width were made, instead of 200 for each. As a comparative basis of dimensions in this work, the biometric mode is used in preference to the arithmetic mean. The mode represents the group containing the largest number of individuals of a certain size, thus indicating that this size is the prevailing one in a given spore population. Comparative calculations made show that, as a general rule, arithmetic means usually fluctuate around the biometric modes, as seen from Table II; and consequently there is, on the whole, but little difference between the two bases of recording. It will be seen that in most cases the figures are almost identical ; in two cases they are the same ; and only in one case is there a difference of 0.24 /t, which may be considered negli- gible, since two consecutive measurements of the same group of spores may give even greater variation. In this experiment 100 spores were measured in each case. Table 1 1 . — Correlation of bioinetric •modes with arithemeiic means of urediniospore dimen- sions of Pitccinia graminis tritici on wheat Number of generations rust was confined to wheat. ) Biometric modes. 33.28X19-84 7 1 32. 00X19. 84 10 32. 64X20. 16 Arithmetic means. M 33.36X19. 69 31.58X19-84 32. 64X20. 02 The apparatus used for meteorological observations is fully described in the discussions of the particular experiments performed. General notes on the behavior of the various cultures were taken at the close of each urediniospore generation before transfers were made to new plants, on the average every two or three weeks. The preliminary spore meas- urements were made of the original rusts found on the grasses in the field, the subsequent measurements were made on the first following generation and once or twice more during the period the rust was kept in culture. For color determination Ridgway's * chromotaxia was used. The Zeiss screw micrometer was used for measuring the urediniospores. 1 Rtogway, Robert, color standards and color nomenclature. 43 p., 53 col. pi. Washing- ton, D. C. 48 Journal of Agricultural Research voi. xvi.no.s MORPHOLOGY OF BIOLOGIC FORMS STUDIED The following biologic forms were investigated: Puccinia graminis tritici Erikss. and Henn., P. graminis tritici-compacti Stak. and Piem,, P. graminis secalis Erikss. and Henn., P. graminis avenae Erikss. and Henn., P. graminis pMeipratensis (Erikss. and Henn.) Stak. and Piem. and P. graminis agrostis Erikss. It has been stated by Stakman and Piemeisel {14, p. 484) that — In general, the size and shape of urediniospores of different biologic forms of Puccinia graminis are similar. If, however, large numbers of spores are measured and the arithmetical mean or biometrical mode is determined, it becomes quite apparent that there are appreciable and fairly constant differences, provided the spores measured be taken from congenial hosts. This was substantiated by the writers by many thousands of spore measurements and careful computations. It is necessary, however, to maintain uniform cultural conditions, since the range of variability in size of urediniospores under different conditions is sufficiently great to cause overlapping in some cases. A summary of the outstanding morphological features of the urediniospores of the biologic forms studied is given below. P. graminis tritici. — ^The urediniospores are quite constant in size, shape, and color. They are the longest of all the biologic forms of P. graminis, but in width they exceed only slightly those of P. graminis avenae. Their shape is elliptic to ovoid, color light cadmium-yellow. P. graminis tritici-compacti. — The urediniospores are very similar to those of P. graminis tritici, but are slightly shorter, and consequently are inclined to be ellipsoid and oval. In color they are somewhat duller. P. graminis secalis. — The spores are uniform in size, color, and shape. The color is dull, ashy yellowish to grayish; in length they are somewhat shorter than those of P. graminis on oats, width approaching that of spores of P. graminis phleipratensis; in shape cylindric-elliptic. P. graminis avenae. — ^The size and shape of the urediniospores are very variable. The shape ranges from ellipsoid to ovoid to pyrifonn to sub- plobose, even when grown on its type host, Avena sativa. Their color is similar to that of spores of P. graminis tritici. P. graminis phleipratensis. — The spore shape is predominantly pyrif orm ; they are very short and fairly uniform; their color is even duller and more grayish than that of spores of P. graminis secalis. P. graminis agrostis. — ^The spores are remarkably constant in size, but are smaller than those of any other form. In color and shape they resemble spores of P. graminis phleipratensis, but possibly are not quite so pyriform. The spore dimensions for the above biologic forms are given in Table III, in order to faciUtate ready comparison. The "size limits" in this table show the extreme variations of all of the urediniospore dimensions Jan. 13, 1919 Morphology of Urediniospores of P. graminis 49 of a given form studied. The "mode averages" were obtained by find- ing the arithmetic mean of all modes of a given biologic form cultured on various congenial hosts. Table III. — Comparative sizes of urediniospores of biologic forms of Puccinia graminis Biologic form. Size limits. Mode averages. P . graminis tritici M 23. 04-41. 92X15. 04-24. 96 23. 68-40. 00X14. 40-25. 28 17. 92-38. 72X13. 44-21. 44 19. 20-37. 12X13. 76-25. 60 16. 00-32. 00X11. 84-21. 12 15. 04-31. 68X12. 16-20. 48 32. 36X19. 82 31. 72X19.48 27. 14X17. 26 28.48X19.46 23.04X17. 24 22.48X15.95 P. graminis tritici-compacti P . graminis secalis P. graminis avenae P . graininis phleihratensis P . graminis agrostis Table III shows distinctly the considerable variation in the size of spores of the different biologic forms. The uredinospores of P. graminis tritici are the largest of all, while those of P. graminis tritici-compacti are less than i/x shorter and only a fraction of a micron narrower. The other forms vary more perceptibly. The spores of P. graminis secalis approach those of P. graminis avenae in length, the latter resembling those of P. graminis tritici in width. The spores of P. graminis phlei- pratensis are similar in width to those of P. graminis secalis, but con- siderably shorter; while P. graminis agrostis has smaller uredinospores than any other biologic form of P . graminis studied. Relative to shape, the six biologic forms discussed in this paper could be classified in two principal groups; the ellipsoid-cylindric group, con- sisting of P. graminis tritici, P. graminis tritici-compacti, and P. graminis secalis; and the ovoid-subglobose group, including P. graminis avenae, P. graminis phlcipratensis, and P. graminis agrostis. Stakman and Piemeisel {14) made an identical classification of these forms on the basis of their parasitism. It is interesting to note that the morphological differences between the individual biologic forms of Puccinia graminis are fully as great and distinct as those between many generally recognized species of fungi. Because of similar morphological variation in certain biologic forms of Erysiphe graminis, expressed by distinctive characteristics in the color of the conidia and in some cases also in their size, Salmon (9) concluded that those forms were "incipient morphological species." The same may be true of the biologic forms of Puccinia graminis. INFI.UENCE OF HOST If biologic forms of Puccinia graminis are incipient species, they are probably evolving gradually. If the change is sudden and accidental, finding the evidence may be merely a matter of chance. If, however, 92802°— 19 3 50 • Journal of Agricultural Research voi.xvi.no.z the change is a gradual one, it is reasonable to hope that some evidence of this change may be obtained by the methods used in the present work. Tv/o lines of work were pursued: (i) Attempts were made to develop a number of morphological strains of a given biologic form by culturing it for fairly long periods of time on several different hosts, and (2) attempts were made to unify spore sizes of different biologic forms by growing them on the same hosts. For instance, P. graminis tritici develops well on common wheat, barley, and on various species of Agropyron, Hordeum, and Elymus. The writers tried to ascertain whether these hosts exerted an appreciable effect on the rust when it had been confined to them for considerable periods of time. Again the secalis, tritici, and triiici-compacti forms grow about equally well on barley. Theoretically, therefore, it could be assumed that they ought to become morphologically similar if grown on barley long enough. In fact, all of the biologic forms discussed in this paper develop at least weakly on barley. It could be assumed that if they could all be grown on barley long enough, they would eventually become similar morpho- logically. The results of the effect of hosts are given on Tables IV to XII. KEY TO TABLES IV TO XII In Tables IV to XII the host from which the rust was originally cultured is given in the second column. Intermediate hosts refer to the hosts on which the rust had been grown up to the time the plant was inoculated. The term "intermediate host" is not used here in the sense of bridging. W, O, B, and R refer to wheat, oats, barley, and rye, respectively. Other symbols are explained when used. The number of " urediniospore generations" (successive transfers) on the host is indicated by the figure immediately following the symbol for that host. Thus, R2B4R1W2B5 indicates that the rust was transferred to rye twice, then to barley four times, followed by one transfer to rye, two to wheat, and five to barley. The degree of infection is self- explanatory. The result of inoculation is given in the usual manner in the form of a fraction, the denominator showing the number of plants inoculated and the numerator the number which became infected. Attempts to Develop Morphologic Strains of Biologic Forms by Culturing ON Different Hosts To determine whether or not a given biologic form of P. graminis has a tendency to split up into a number of different morphological strains on account of confinement for fairly long periods of time to a number of different hosts, a series of experiments was conducted with the six biologic forms indicated above. The host plants employed were very frequently of distant taxonomic relationship, but, unless they were equally congenial to the parasitic attack of the fungus, their effect was not considered when the final conclusions were drawn. The result of this phase of the work, which extended over a period of two years, is given in Tables IV to IX. Jan. 13,1919 Morphology of Urediniospores of P. graminis 51 Many inoculations were made with P. graminis tritici on wheat, barley, rye, and Hordeum jubatum (Table IV). All except rye are very sus- ceptible. On congenial . hosts the spores remained quite constant in shape, size, and color, irrespective of their origin and subsequent history. On rye, however, an uncongenial host, both the uredinia and the uredini- ospores became appreciably smaller, especially in length. These results are not in accordance with those of Freeman and John- son (j, p. 28), who say: The host-plant exercises a strong influence, not only on the physiological and bio- logical relationship, but in some cases even on the morphology of the uredospores. The difference in results might possibly be explained by supposing that Freeman and Johnson worked with a mixed strain, or that they did not measure enough spores. Their rust, however, may actually have changed. It will be readily seen from Table IV that the writers were not able to change the dimensions more than about i n, which is within the range of experimental error. The color of the urediniospores of P. graminis triciti is pale cadmium- yellow; their shape predominantly elliptic to ovoid; size limits 23 to 42 by 15 to 25 n, and average modes 32.36 by 19.82 n. Results obtained by Stakmanand Piemeisel {14) showed that the biologic form of rust, P. graminis tritici-compacti, discovered west of the Rocky Mountains on several different grasses and on club wheat, varied para- sitically from P. graminis tritici, found east of the Rockies. Many com- mon aestivum wheats, such as Haynes Bluestem and Fife, are resistant to this biologic form, while barley is fairly tolerant and the club wheats inoculated and Pacific Bluestem are very susceptible. Spore measure- ments of over a dozen strains (Table V) indicate that, whereas the spore sizes on the susceptible hosts vary but little (less than i /x) from those of P. gratninis tritici, yet they are on the average nearly 2 ix shorter on the tolerant hosts and almost 4 /x shorter on the resistant ones. The width of the spores does not seem to be influenced by the host. Identical results were obtained with P. graminis tritici-compacti, found in the summer of 1 917 in Louisiana and Alabama, on several soft wheats. The constancy of size is remarkable, and, like the western strain, the southern strain, too, exhibits a special affinity for club wheats, while the most of the hard wheats are resistant. The color of the urediniospores of P. graminis tritici-compacti is prac- tically the same as that of P. graminis tritici. The spores are slightly shorter, and ovoid to ellipsoid in shape. 52 Journal of Agricultural Research Vol. XVI. No. 2 ^ I 42 .9 'T^ tiT > o «'l fi3 X X XXX xxxxxxxx H p3 pj fj N M ■O r^ ro ro r^ fO i ■* O ^ O VO :^ o ■* ro t^ ro 5j- ro N ro W t^ -1 W cy a CI 0 ^ ^ vA M 3 O O Ov o fO rj M cj C) c< o5 ci ci ro CO CO CO CO CO CO c> O 4 N 00 VO 3" 4 VO 0\ o l^ VO CO o o CO t CO CI CO CO CO CO CO o o CI N N CI M ^ .^ 1 o it 1 CI V it 4 *^ fo CO VO CO VO VO o X X X X X X X X X X X X CI N =§ >o 00 M CO CO Ov CO O o 00 d Ov ri '^ ^ CO •^ CO 'd- it 1 1 Tt k u /, o o o VO VO VO X X •* o o 00 00 d o 00 d 00 •ft f' T^ -i- -* "P Tt CO 1 it h h 1 h <^ <^ Ov N VO o CO VO M VO Cor~0 ,v,|^ COlCO CDItr~- 0M05 COI-^ f— iIf— 1 "^MCO eOiCO C X X X X X X X X X X X X X =iXXXXXXX X X X X X X ~^ 00 N >0 r 7" M N 4 o O I (M Ico iM ICO »ni iic '^ li-i m Ph w cJ p? pf ^ p: d" « « ^ pq m 1 :< ^ Pi ^ ."2 5 S ffl P5 W Ph Si w S 8 S I -3 I S: 00 ^, S 5 I 8 « I h. E-. Jan. 13. 1919 Morphology of Uredinios pores of P. graminis 55 T^ f) c > 00 10 w cS c> c> H M M XXX 00 0 ^ rf 00 M 00 00 00 M N M M Tf 0 r- 0 Tl- M ro M NOP) 1 i 1 00 M N N rO rO t-^ >o ^ H M M XXX 0 ^ ^ vO cs >-i ro 4 ^ CO rp rp 1 I 1 ^ SO rt 0 CO H CO ro H N M N o^p ^K -^1^ c C c "C X 1: 'c OC S f c ^ ^^ 1 S 1 > -^ 0. ^. -S ^ ^ .•* "p S ^ ^ h to «. d \ c § w d i ^ ^ ^ S J -§ S 1 1 A, "s ^ t. CJ '-0 - ~ 9 a ii-S! o kj3 ■ij II •^ •v w Q f^ c% ;^ •^ s •> * 1 f U] 1 u HH W > s tt s ►4 n 8 <: -Si H • wEi^ t^ t^ r^ M3 t-^ r^ ri M M M M M M X X X X X X X vO o 00 O \o o M " >o 00 Tt ro 2" "^ *^ W M M '"' M M X X X X X X i r 6 1) 2 -I-' ^ ^ ,« ^ 14 0) cfl T) cfl L. « cfl Tl (U O rr pq 03 ; w ^" i'' w n w pq « i pp pT pS^-: Pi pq cq ^ PiW pq P^^ ^ tt: t& 58 Journal of Agricultural Research Vol. XVI, No. 2 ■^ § * -3 ^ n ^ 1 «, a 1" q 1-4 »-( > Q xs ir •J K ,3 1 H ^ s 1 >« a, n -* ^C 0 « ^O Ov 0 O^ Qs O^ O^ CS O^ OC OC) H H H 0 H H H H 73 ^XXXXXXX XXXX XX =i ■* OC OC f« « T 0 to to ro ro cs PJ CN M M cs N Pf P) PI PI C-i 1 0 4 0 ri- \Q CJ 00 0 VO 0 00 Pi N S a vO c "S- 0 CO t^ O 0 po •* 0 i-i '-' lO CO C4 ro fO w o VO VO to vc ^c VO vO W H w M w w M >-* ►-' =^XXXXXXX XXXX XX cs CN ei H CT ff IH H CS H M 1 £:3K'*'lSSISS^?5i?3-i'o§^l?3 SISSISSISS^ SIS'^P Pi i -, , / ^ c 0 0 0 v: 0 0 vO •+ M M (^ CO 6 ro ro fO T> ^P 1 h Jt 1 C-l ^ H OC 00 10 IN 0 0 c^ C^ Ov « « M " w «=!§ -'IS SiS ^la (U ^ u J^ c. c 73 a t: •c V 0 > 8 y S ^ i g 1 1 i 1- 3 »2» •g .s* 0 Q, Jt '^ ^ &: tJ- tn o t^ 00 6o Journal of Agricultural Research Vol. XVI, No. 2 5^ 8 5^ s -S M Si: ja 8 ■p ^ a ■^ ^ Itf fi3 ,X X X X X X X X X X X X Ht M 00 00 00 M ■* M o On 00 Ov Ov o (N N H Ht M M 1 I, /> A J> ^ 1 ■ X X X X p ^^ o X X O 64 Journal of Agricultural Research Vol. XVI, No. 2 =iX X X X X X X X X X X X CI CO .? 4 MO 4 0 •^ xxxxxxxxx X X X X 8 1 s ;■§ I s s OS D CO 00 d o co' o 00 M * ro ■P ^ ^ 'P t 1^ ^ . Ir }. ^ 1 h 1 00 h oJ, 7v N ^ o ro o N o « b 2 (U o PQ w m pT eq PQ pT e^ n ^ '^ w i-i >ih S.o W ag' Jan. 13, 1919 Morphology of Urediniospores of P. graminis 65 00 <3 00 0 vO N 0 M 0 On t>- vO t^ r^ VO H M M M M X X X X X 0 p) ■* () 0 « >o 00 w t^ t-- t^ t^ 00 M M « c< N M X X X X X -t fC N P) 10 CO ro ^ ro "^ A vA 1 M ^ 4 « t^ t^ r^ ^ 0 ^j N M M M C-l M P) CI CO 100 »0 ILO '-O li— 1 CO it^ 10 ICO l-HlrH CmIiMi-hIiM rH li-l r-lli-i ^ ;! Pl p? e{ pT p? pT p^' pT 00000 •o -a T3 "O "d T^ to VO «^ 00 93802°— 19- 66 Journal of Agricultural Research voi.xvi.no. 2 Johnson (<5, p. 8) gives the urediniospore dimensions of timothy-rust as 18 to 27 ju in length and 15 to 19 m in width. Stakman and Jensen {12, p. 214) found that on timothy the spores ranged from 17 to 31 /i in length and from 14.5 to 23 n in width, the modes falling at about 26 and 18 /!• On barley they found them to be smaller than those produced on any other host, ranging from 18.5 to 28.3 /x in length and from 13 to 20 ju in width, with modes at about 23 and 17 m- In the present work it was found that all the modes, except those of spores cultured on barley, fluctuated about those of a strain of P. graminis phleipratensis , which had been confined to timothy for more than a year. The modes fluc- tuated about 24 and 17 fi, varying perceptibly with the existing climatic and edaphic conditions. That barley is not a congenial host for timothy-rust is shown by the slight virulence of infection (Table VIII), very small uredinia, and con- siderably decreased size of the urediniospores, with average mode of 21.28 by 14.24 ju. The results of inoculation with P. graminis phleipra- tensis, obtained from two different sources — timothy {Phleum praiense) and orchard-grass {Dactylis glomerata) — and grown on three different hosts (timothy, orchard-grass, and oats), show (Table VIII) that the urediniospores retain their characteristic size, except for small negligible variations, whether parasitizing very congenial or merely tolerant hosts. The spore shape is predominantly pyriform, and the color is dull, dirty yellow to grayish. The infection capabilities of P. graminis agrostis are similar to those of P. graminis phleipratensis and P. graminis avenae The uredinio- spores of this rust are the smallest of all the biologic forms of P. graminis, especially in width. In shape and color they resemble very closely timothy-rust spores although not quite so dominantly pyriform. The spore dimensions are given in Table IX, The results given in Table IX show clearly that the influence of host on the size of the spores was negligible. Attempts to Unify Spore Sizes of Different Bioi,ogic Forms by Culturing Them on the Same Host P. graminis tritici has a number of hosts in common with P. graminis secalis. P. graminis avenae and P. graminis phleipratensis also have several hosts in common. The work was directed toward an attempt to determine whether the spore morphology of these biologic forms could be made identical by the use of common hosts. Table X gives the results of using Hordeum, vulgare as a common host to unify P. graminis tritici and P. graminis secalis, and in Table XI are given the results of using Avena sativa as a common host for P. graminis avenae and P. graminis phleipratensis. A condensed tabulated summary of these results is given in Table XII. Jan. 13, 1919 Morphology of Urediniospores of P. grammis 67 •2 •i o S s " '??>.'' g •Si s 5^ <>■ •0 Q » ? •~-s 3 '^i ■«'■«, a, 1 s «, s a, s 1^ X o3 «iii aX X X X X X X X X X 0 0 't- 0 rr' VO r^ 1^1 rri (v* IN N 0) ^ 1 h 1 h 0 r^ 0 ro 0 6 6 d M 6 N f) 7 cs (N N VD '4- -1- M -^ r^ 0 00 «~- X X X 0 00 M 00 10 ri- ro <^ ^ J, P) 0 X X X X X X X 6 6 M fO ^ 0 N 00 M 00 ro rO 01 rO n ^ ^ 1 /> /> 0 0 0 vO MO i^R^" W S W 0000 00000 O PL, O4 Oi P< flH f^ Ph ■24 S-38 Heavy' . . . ....do.... ....do..., ....do..., Moderate ....do.... 22. 72-43. 84X16.96-22.40. 26. 24-40. 96X17.60-22. 72. 25. 28-40.32X16.96-23.04. 25.92-40.00X16.96-23. 36. 24-96-38. 72X16.32-23.36. 22.40-38.08X16.64-21. 76. 33.28X19-84 33. 28X20. 16 32.64X19.84 32.64X20. 16 32.32X19.84 30.40X19. 20 Effect op Illumination In testing the effect of illumination two series of plants were em- ployed, one of which was kept beneath a double-layer muslin cage, while the other one was exposed to the direct sunlight in the greenhouse (Table XVI). As the experiment was conducted during the winter months the light was at no time exceptionally bright. The cultural con- ditions, except for the variation in light intensity, were maintained the same for both series. The light readings were taken daily, sometimes two and three times a day, with the Clements photometer charged with a 72 Journal of Agricultural Research Vol. XVI, No. 2 printing-out photographic paper. The percentage of intensity was de- termined by means of a standard print made at noon of a bright sunny day in the fall of 191 5. Table XVI. — Results showing the effect of illumination on the physiology and morphol- ogy of urediniospores of Puccinia graminis tritici on wheat Ex- peri- ment No. Intensity. Daily limits. Total aver- age. Maxi- mum. Mini- mum. 35- 0 7-S 16.9 28.7 3-S 15.4 46.7 6.6 14.0 12-5 1-7 6.6 10. 0 2.0 3-1 2.0 0.9 1-3 Degree of infection. Result. Spore dimensions Size limits. Modes. Heavy. . , ....do... ....do... Weak. . . . ...do... Moderate 25. 2S-40. 32X16. 96-23 25. 60-40. ooX 16. 00-23 22. 40-40. 32X 16. 32-32 22. 72-35. 84X 16. 96-22 21.76-36.48X17. 2S-22 23. 04-40. 64X 14. 72-22 32.64X19.84 33.28X19-84 32.00X19.84 29. 76X19-53 29. 12X19- 84 29.76X18.88 The rust consistently developed better in fairly high intensities than in the lower ones. The size of the urediniospores, as given in Table XVI, responded in similar manner. The color of the uredinia in the shade varied from antique-brown to Sudan-brown, while of those in the light ranged from Sudan-brown to argus-brown — that is, somewhat lighter in shade than in the open. It appears that in as much as the photosyn- thetic activities of the host plant are affected by the light intensity in so much does the function and structure of the rust fungus depend on the same factor. Effect of Excessive Nitrogenous Fertilization The preliminary results obtained by the writers seem to indicate that an excessive amount of sodium nitrate, inhibiting the growth of the host,, also inhibits the development of the rust and dimimishes very percepti- bly the size of the urediniospores, as is shown in Table XVII. This is in accord with Sheldon's {10) carnation rust experiments which showed that the kind of soil that favored the growth of the host also favored the attack of the rust, and that, as a rule, the period of incubation of the rust was inversely proportional to the vigor of the host. The plants were considerably shriveled by the chemical and badly dried two weeks after application. The rust, however, had made a fair start on one blade out of the eight inoculated and developed uredinia of moderate size and ex- tent. The uredinia were darker in color than those developed under normal conditions. Jan. 13, 1919 Morphology of Urediniospores of P. graminis 73 Table XVII. — Effect of excessive nitrogenous fertilization on the physiology and mor- phology of urediniospores of Puccinia graminis tritici on wheat Fertilizer. Condition of host. Degree of infection. Result. Spore dimensions. Size limits. Modes. Sodium nitrate .... Control Shriveled and dried up Healthy and thrifty. . . Moderate Heavy. . . 1 8 20 20 20. 8CJ-36. 80X 16. 00-23. 04 25.28-40.32X16.96-23.68 28.48X19.52 EXPERIMENTS ON CULTURAL METHODS It was thought well worth while conducting a few experiments to ascertain the effect of the age of the host plant at the time of inoculation on the rust growth and on the size of the urediniospores, also to find out the length of time during which urediniospores retain their vitality and what is the relation of the age of the fungus to the morphology of the uredinio- spores. The results obtained (Table XVIII) show that the suscepti- bility of the host is little dependent on its age, and that urediniospores retain their vitality for a considerable length of time with no perceptible variation in size. Table XVIII. — Results showing the effect of age of host plant on the morphology of urediniospores of Puccinia graminis avenae Experi- ment No. Host plant. Avena sativa . do do do do do Age of plants inocu- lated. Days. 7 7 14 35 Spore dimensions. Size limits. • 00-35 • 04-35 • 96-34 • 96-35 • 76-37 ■ 72-35 . 52X16. 00-22. 08 . 52X16. 96-22. 08 . 88X14. 72-22. 72 . 84X16. 00-22. 40 . 12X16. 64-21. 76 . 84X16. 00-22. 40 Modes. 2g. 44X19. 20 29.44X19- 52 29. 44X19. 20 29.44X19. 20 29. 44X19. 20 29. 12X19. 20 EFFECT OF THE AGE OF HOST PLANTS ON THE DEVELOPMENT OF THE RUST FUNGUS AND SIZE OF THE UREDINIOSPORES Oats were here used as host plants, because they were found to thrive better under greenhouse conditions than wheat, barley, or rye. Inocu- lations were made when the plants were 7, 14, 21, and 35 days old, count- ing the age from date of sowing. The 7 and 21 days series were later duplicated. In all cases the plants were inoculated with a uniform amount of fresh urediniospore material of P. graminis avenae and cultured under similar and normal conditions. The plants i week old were slightly more vigorously affected (Table XVIII) at first, but at the end of 10 days the infection was heavier on the older plants, and especially so on those that were three weeks old at the time of inoculation. , From Table XVIII it will be seen that the 74 Journal of Agricultural Research Vol. XVI, No. 2 size of the urediniospores was remarkably uniform regardless of the age of the host; nor was there any difference in the shape and color of the spores. The junior author has also obtained very successful infection on mature plants of more than a hundred different varieties of wheat, grown in the greenhouse and artificially inoculated with P. graminis tritici. This latter work was conducted at the Kansas Agricultural Experiment Station in cooperation with the United States Department of Agriculture. ElfFKCT OF THE AGE OF THE) RUST FUNGUS ON TftB VITALITY AND MORPHOLOGY OF THE) UREDINIOSPORES Fromme (4, p. 504) states that De Bary found the length of time during which urediniospores of P. graminis retain their vitality to vary between one and two months, and that Bolley obtained a 5 per cent germination with urediniospores of P. graminis after exposure to air and sunlight during the month of August. The object of this experiment was to determine the vitality of the urediniospores after different periods of association with their respective hosts and effect of the length of associa- tion on the rust morphology. For the determination of the first phase of the experiment inoculations were made (Table XIX) with rust material at different stages of the development of the uredinia. Transfers were made when the uredinia were merely beginning to break through the epidermis and two and four weeks afterwards. There was no apparent difference in the degree of infection produced by these methods of inoculation. Table XIX.- -Results showing the effect of age of the fungus on the morphology of urediniospores of Puccinia grmninis Ex- peri- Biologic form. Age of spores meas- ured. Spore dimensions. ment No. Size limits. Modes. I 2 P. graminis tritici . . do Days. 8 14 22 63 7 20 40 8 14 25. 60-39. 68X16. 96-22. 72 25. 60-40. 32X16. 32-23. 36 23- 36-39- 04X 15- 68-22. 72 24. 32-40. 96X16. 64-23. 04 24. 96-35. 52X16. 96-23. 36 23. 04-35. 20X 16. 96-22. 08 23- 04-35- 52X 16. 96-22. 08 22. 40-34. 24X13- 76-19- 84 20. 48-33- 92X14- 08-19. 52 M 32. 64X 19. 84 32. 96X19. 84 30. 72X19- 20 32.64X19- 52 29.44X20. 16 29. 12X19- 52 29.44X19- 52 28.48X16.96 27. 52X16. 64 3 4 5 6 do do P. graminis avenae . . . do 7 8 9 do P. graminis secalis do In determining the effect of the age of the fungus on the morphology of the spores, measurements were made at different stages of the develop- ment of uredinia, beginning 7 days after inoculation and ending with 63 days. Three biologic forms of P. graminis were used in this experi- Jan. 13, 1919 Morphology of Urediniospores of P. graminis 75 ment and, as shown in Table XIX, there seemed to be no appreciable effect on the size of the spores, although the size of the uredjnia gradually and persistently became larger, which was due to the additional shed- ding of spores and coalescence of adjacent uredinia. The color of the uredinia became darker with age and the spores lost their coherent floccose consistency and by the least disturbance were separated from the uredinia. GENERAL DISCUSSION The data presented in this paper provide ample e\ddence to show that the morphology of biologic forms is but slightly and only tem- porarily changed in response to biotic and physical factors. Resistant host plants and unfavorable cultural conditions affecting the normal development and vigor of the rust fungus may also affect the size of its urediniospores. But as soon as the unfavorable factors are removed the fungus resumes its normal functions and regains its original structure. No host which is congenial to a given biologic form can, under favor- able cultural conditions, exert any perceptible influence on the mor- phology of the rust spores. P. graminis avenae appears to deviate from this rule in so far as shape and size of urediniospores are concerned. The shape varies considerably on any host to which the rust may be confined, while the spore sizes appear to be peculiar of the particular host the rust parasitizes. The attempts to split up the various biologic forms of P. graminis studied into a number of different morphological strains by culturing them for long periods of time on several different but definitely con- genial hosts have utterly failed. The attempts to unify the spore sizes of different biologic forms by culturing them continuously for a con- siderable length of time on the same host were also unsuccessful. Adverse environmental conditions, such as resistant host varieties, affect the virulence and spore size of the rust fungus. Excessive heat is more injurious to the rust growth and affects the size of urediniospores more effectively than does very low temperature. High humidity during the incubation period appears to be an indispensable condition; the difference in humidity later is probably of lesser importance. Defi- ciency of soil moisture and sunlight, and other ecological factors affect- ing the host plant unfavorably, appear to be equally unfavorable to the rust parasite. ' The results show that P. graminis is quite stable and can not be ex- pected to change rapidly. This is true both of its parasitic capabilities and of its morphologic characters. The facts presented in this paper give additional support to the rapidly accumulating body of data which show that the biologic forms studied are fairly constant. Whether this will apply equally well to the large number of forms recently found on varieties of wheat is a question which can be answered only by future investigation. y6 Journal of Agricultural Research voi. xvi. No. a SUMMARY (i) The amount of spore material used for inoculation has no percepti- ble effect on the result of infection or size of spores, except in so far as a more extensive area of and a greater certainty for successful infection may be secured. (2) The optimum length of incubation period in the moist chamber is 48 hours, thereby securing all certainty of infection without causing a tendency to supersensibility. (3) The superficial layer of each uredinium contains larger spores, and when this layer is removed the remaining spores are considerably smaller. But if the uredinium is allowed to produce a new crop of spores those on the surface again attain the same dimensions as the original ones. (4) In spore measurements 100 spores, obtained from a number of uredinia, are representative of their group. Modes are a practical basis for comparison. (5) Biologic forms are constant not only parasitically but also morpho- logically. As a general rule the morphologic differences between the various biologic forms are fully as great and distinct as between many established species of fungi. The morphologic stability of a biologic form is exhibited in the constancy of size, shape, and color of the uredinio- spores of the particular form. The stemrust of oats (caused by P. graminis avenae) is an exception to this rule in so far as the shape and size of urediniospores are concerned, these being very plastic. (6) Common hosts which are congenial to different biologic forms lack the ability to unify them, as they are unable to exert any influence on the spore morphology. Uncongenial hosts, on the other hand, almost invariably tend to decrease the size of uredinia and spores. (7) In computing data and comparing results it is necessary to take into consideration the ecological conditions under which the rust had been cultured — that is, cultural conditions should be kept as far as pos- sible uniform; or proper allowances should be made for any variation before final conclusions are drawn. (8) Adverse environmental conditions unfavorable for the host are also unfavorable for the parasite, affecting the virulence and spore size of the latter. (9) The optimum atmospheric temperature for the development of the rusts studied appears to range between 66.5° and 70° F. Sufficiency of water and plentiful light are indispensable for the best growth of the rust. (10) The age of the host seedlings, provided they are healthy at the time of inoculation, has no determining affect on the virulence of infec- tion or size of the urediniospores. (11) The length of association of a rust with its host, after the first uredinia have burst the epidermis until teliospores are formed, does not impair the viability of the urediniospores, nor does it exhibit any marked and consistent effect on their size. Jan. 13. 1919 Morphology of Urediniospores of P. graminis 77 LITERATURE CITED (i) Butler, E. J., and Hayman, J. M. 1906. INDIAN WHEAT RUSTS. Mem. Dept. Agr. India, Bot. Ser., v. i, no. 2, 58 p., 5 pi. (4 col.). (2) Christman, a. H. 1905. observations on the wintering op grain rusts. In Trans. Wis. Acad. Sci., v. 15, pt. i, p. 98-107. (3) Freeman, E. M., and Johnson, Edward C. 1911. THE rusts of grains IN THE UNITED STATES. U. S. Dept. Agr. Bur. Plant Indus. Bui. 216, 87 p. 2 fig., i pi. Bibliography, p. 79-82. (4) FrommE, F. D. 1913. THE CULTURE of CEREAL RUSTS IN THE GREENHOUSE. In Bul. Torrey Bot. Club, V. 40, no. 9, p. 501-521. Literature, p. 5x9-521. (5) Jaczewski, a. von. 1910. STUDIEN USER DAS VERHALTEN DES SCHWARZROSTES DES GETREIDES IN RUSSLAND. In Ztschr. Pflanzenkrank, Bd. 20, Heft 6, p. 321-359, 8 fig. (6) Johnson, Edward C. 19 11. timothy RUST IN THE UNITED STATES. U. S. Dept. Agr. BuT. Plant Indus. Bul. 224, 20 p. (7) 1912. CARDINAL TEMPERATURES FOR THE GERMINATION OP UREDOSPORES OP CEREAL RUSTS. (Abstract.) In Phytopathology, v. 2, no. i, p. 47. (8) Long, W. H. i914. influence of the host on the morphological characters of puc- ciNiA ELLisiANA AND pucciNiA ANDROPOGONIS. In JouT. Agr. Re- search, V. 2, no. 4, p. 303-319. (9) Salmon, E. S. 1903. on specialization op parasitism in the erysiphaceae. i. in bcih. Bot. Centbl. Bd. 14, p. 261-315, pi. 18. (10) Sheldon, John L. 1905. THE EFFECT OF DIFFERENT SOILS ON THE DEVELOPMENT OF THE CARNA- TION RUST. In Bot. Gaz., v. 40, no. 3, p. 225-229. (11) Stakman, E. C. 1914. a STUDY IN CEREAL RUSTS: PHYSIOLOGICAL RACES. Minn. Agr. Exp. Sta. Bul. 138, 56 p., 9 pi. Bibliography, p. 50-54. (12) and Jensen, Louise. 191 5. INFECTION experiments WITH TIMOTHY RUST. In Jour. Agr. Research, v. 5, no. 5, p. 211-216. Literature cited, p. 216. (13) and PiEMEiSEL, F. J. 1916. INFECTION OP TIMOTHY BY PUCCINIA GRAMINIS. In JouT. Agr. Research, V. 6, no. 21, p. 813-816. Literature cited, p. 816. (14) I917. BIOLOGIC FORMS OP PUCCINIA GRAMINIS ON CEREALS AND GRASSES. In Jour. Agr. Research, v. 10, no. 9, p. 429-496, pi. 53-59. Literature cited, p. 493-495- (15) Ward, H. M. 1902. on the relations between host and parasite in the bromes and THEIR BROWN RUST, Puccinia dispersa (Erikss.). In Ann. Bot., v. 16, P- 233-315- ADDITIONAL COPIES OF THIS PUBUCATION MAY BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 25 CENTS PER COPY Subscription Price, 83,00 Per Year V Vol. XVI JANUARY 20, 1919 No. 3 JOURNAL OP AGRICULTURAL RESEARCH CONXE^NXS Paga Variations and Mode of Secretion of Milk Solids - - 79 JOHN W. GOWEN (Contribution fron Maine Agricuttaral Experiment Station} New Biologic Forms of Puccinia graminis - - - 103 E. C. STAKMAN, M. N. LEVHTE, and J. G. LEACH (Contribution from Minneeota Agricultural Experiment Station) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCL^TION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS ^A^ASHINOTON, D. C. WAaHINQTON l QOVEnNMeNT PniNTlNQ OFFICE :«»!• EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chairman Physiologist and Associate Chief, Bureau of Plant industry EDWIN, W. ALIvEN Chief, OMce of Experiment Stations CHARLES L. MARLATT Entomologist and Assistant Chief, Bureau of Entomology FOR THE ASSOCIATION H. P. ARMSBY Director, Institute of Animal Nutrition, The Pennsylvania Stale College E. M. FREEMAN Botanist, Plant Pathologist, and Assistant Dean, Agricultural Experiment Station of the University of Minnesota J. G. LIPMAN Director, ^^gv Jersey A griculturml Experi- ment StSton, Rutgers 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa. J0I1N£ OF AGRICDITIAL RESEARCH Vol. XVI Washington, D. C, January 20, 191 9 No. 3 VARIATIONS AND MODE OF SECRETION OF MILK SOLIDS ^ By John W. Gowen ,^- 7 *^' Assistant Biologist, Biological Laboratory, Maine Agricultural Experiment Station ''"•^■r^ INTRODUCTION ^; The chief contributors to the subject of the physiology of milk forma- tion in the mammary gland have chosen as the foundation for their theories the grounds of analogy with the mode of formation of the secre- tion in the two types of glands, sebaceous and salivary, and as positive evidence have cited histological studies of the mammary tissue. On such grounds the conclusions are likely to be weak. As yet there has been little attempt to use the store of accurate mathematical data on the composition and variation of the milk constituents for the analysis of this problem. The investigation reported in this paper is an attempt to analyze the variations and associations of the constituents of Holstein-Friesian milk to furnish definite mathematical evidence bearing on the problem of the kind of mechanism liberating these constituents to form the fluid known as milk. The specific problems and the viewpoints taken in this paper may be best understood by considering the natural divisions into which milk secretion falls. The mammary functions may be divided into two main divisions. The first of these has to do with the formation of the materials of milk before the constituents are finally brought together as milk. The second has to do with the release of the complete product, milk. The first of these needs only concern us. This problem may be again narrowed to exclude the genetic differences which most certainly exist between individual cows. Given the constituents of cow's milk, our problem is thus limited to the maimer in which these constituents are released into the milk ducts. Is it through the secretory action of the gland cells or is it through the destruction of the whole or a part of these cells? Toward the analysis of this problem and related problems the data on the variations and associations of the constituents of cow's milk have been collected. The special problems bearing on the question will be discussed in coimection with the data of the later sections of the paper. ' Papers from the Biological Laboratory of the Maine Agricultural Station No. 121. This paper is the fourth of a series of studies on milk now being conducted in the Biological Laboratory of the Maine Station. Journal of Agricultural Research, Vol. XVI, No. 3 Washington, D. C. Jan. 20, 1919 ay Key No. Me.-i4 (79) 8o Journal of Agricultural Research voi. xvi.no.s MATERIAL AND METHODS The Holstein-Friesian Association (75)^ has, as part of its semiofficial advanced registry work, collected a considerable amount of data on the yearly production of Holstein-Friesian cows. The majority of these records contain the following data: The name, advanced registry num- ber, and herd-book number are given, together with the volume where the last entry record was made, as the requirements of the semiofficial test include the making of the 7-day official test. The rest of the ani- mal's record includes age at calving, length of record, weight of milk, percentage of butter fat and total weight of butter. Some of these records, fewer than we could desire but still far more ample than those of any other known breed, give the total solids for the milk produced. These records will furnish the material for the analyses of the problems previously indicated. The objection may be raised that these data are not accurate, since they are taken from this type of record. It is realized that there may be some justice in the criticism; yet the inaccuracy should not be magni- fied. Any advanced registry system must be subject to all the criticism that may be brought against these particular records. Criticism can not be made on the ground of conscious inaccuracy due to poor management on the part of the association officials, as every record is carefully checked for inaccuracies. Each record is under oath as to its accuracy by both parties, the tester, and the owner. The only justifiable criticisms which can be brought are those of the personal equation type, errors from the variation in the values as read by two different men. These errors can not at any time be very great. They constitute that group of errors which are as likely to go one way as another — that is, they should counter- balance. The methods used are, in general, those of any adequate statistical treatment of a quantitative subject. The constants for the distribu- tions, means, standard deviations, and coefficients of variations are cal- culated by the usual formula for grouped distributions and without the use of Sheppard (27) correction. The correlations are calculated from the correlation surfaces by the usual Bravais formula. The necessity ot correcting for the effect of age and quantity of milk in the comparison with the amount of butter fat and solids-not-fat have made necessary the use of partial correlation coefficient to measure such association for a constant value of the disturbing variables. These constants have been calculated from the ordinary correlations, by the method devised by Pearson {21). VARIATION OF MILK, BUTTER FAT, AND SOLIDS-NOT-FAT Some study of the variation of the milk and butter fat have been made, notably those by Gavin (jj), Vigor {34), and Pearl {20), where definite variation constants have been determined. These studies in- ' Reference is made by number (italic) to "Literature cited", p. 99-102. Jan. 20, 1919 Var iations and Mode of Secretion of Milk Solids 81 elude chiefly the determination of the relation of this milk and butter fat as it varies within itself and as it varies with age. They do not con- sider the relation of the variation of the other solids; in fact, the avail- able material has been wanting. Such variation constants are highly desirable; in reality, indispensable for the succeeding studies that are to follow. These Holstein-Friesian data are exceptional in that they are accurate data taken from one breed under the best of conditions. The material as tabled is taken from volumes 18 to 28 of the Advanced Registry of the Holstein-Friesian Association (75) for their semiofficial year records. All of the records for milk, total solids, butter fat, and solids-not-fat are for 365 days. The constants are calculated from the grouped frequencies of the formed correlation tables (IV to IX). It is evident from the correla- tion table for age and percentage of solids-not-fat that one error probably exists in the records either from a typographical cause or from an error in the determination. I shall therefore give the constants both for this animal included and for the distribution where it is omitted. Table I shows the means, standard deviations, and coefficients of variation of the variates. The two values of some of the constants in this table are, first, those for the whole population of the Holstein-Friesian milch cows, and second, those for the population which also records the values for the total solids. Table I. — Fundamental constants /or Holstein-Friesian mean production of milk Character. Amount of milk pounds. . Do do... Age years. . Do do Butter fat pounds . . Do do Solids-not-fat do Do do Total solids do Do do... Butter fat per cent. . Solids-not-fat do Do do... Total solids do ... . Do do... Num- ber of individ- uals. 1,387 334 1,387 334 335 1,387 335 334 335 334 1,387 335 334 335 334 Mean. 15,417-44 ± 67 15,149-70 ±111 4- OS ± 4-49 515-37 528.01 1,302.86 1,301. 20 1,814.78 1,812.92 ± 12 3-44I± 8.6i2± 8. 6o4± 12.02 ± 12. 0141b 7667 3272 0369 0838 9956 4334 5828 5489 9929 9723 0058 0134 0124 0206 0202 standard deviation. Coefficient of variation. 3,741-59 ±47 3,016. iS ±78 2. 04 ± 2. 27 ± 108.43 ± 2 134-36 ± I 260. OS ± 6 258. 71 ± 6 352-59 ± 9 351.46 ± 9 -3i8± .364± -338± .56o± -547± 9111 7223 0261 0592 8256 7204 7769 7523 1884 1731 0040 0094 0088 0146 0143 24. 268±o. I9-909± - 50.37I± - 50. S56±i. 2I.039± . 25. 446± . 19-959± - 19.S82± . I9-429± - 19. 386 ± . 9. 228± . 4.22I± . 3-923± - 4-657± - 4. 549± . 3285 5398 7919 6218 5720 3334 5404 539° 5250 5246 I191 IIOI 1025 1215 1188 The following facts are easily deducted from Table I : The Holstein- Friesian cows making the semioflticial record have a mean milk production of slightly over 15,000 pounds of milk, containing about 520 pounds of butter fat and 1,300 pounds of solids-not-fat. The ratio of the solids- not-fat to the butter fat is approximately 2.5 to i. Taken in the form of percentages, the Holstein-Friesian milk contains slightly over 1 2 per cent of total solids, composed of 3.43 per cent of fat and 8.60 per cent of solids- not-fat. The mean age of the group is slightly more than 4 years. The animals who have total-solids records are, on the average, about >^' year 82 Journal of Agricultural Research Vol. XVI, No. 3 older than those without such records. This is no doubt due to the progressive tendency to test young animals and to select animals for high production. The animals whose total-solid records were determined are found in the earlier herd books. It is interesting to compare these values with the milk of other breeds. To facilitate this, Table II has been drawn up. The data for this table have been gathered from many sources, chief among which are the papers of the Agricultural Experiment Stations and the analyses of public chemists. Each tabulated value is, in general, the mean of a considerable number of observations and may be considered close to the true value. Unfortunately it is not possible to obtain the original data so that the other variation constants could be obtained. Table II. — Mean milk constituents of the different breeds c- Breed. Molltaler Blond vich Angler Jeverland Holland East Friesian. .. . Lova Rhine Breitenburg .... Red Holstein . . . Wesermarsch. . . . Schwyz Simmental Westerwold Glan Alderney Jersey Guernsey Holstein- Friesian Ayrshire Shorthorn Polled Jersey . . . French Canadian Dutch Belted. .. . Brown Swiss. . . . Bed Polled South Devon . . . Kerries Dexter Holstein-Friesian Total solids. Per cent. 13. 22 12. 75 12. 51 11.86 II- 54 11.80 12. 12 12.34 12. 07 11.85 12. 76 13-27 12-99 13-57 13. 60 14-39 13. 61 11. 78 12. 46 12. 61 13-93 13-32 12.31 12. 61 12. 66 12.93 13. 10 12.58 12. 02 Fat. Per cent. Solids-not- fat. Per cent. 9-39 9 09 9 00 8 77 8 04 8 71 8 81 8 98 8 80 8 61 9 16 9 22 9 20 9 41 9 79 9 27 9 08 8 46 8 84 8 91 9 26 9 32 8 91 8 99 8 99 9 21 9 08 9 II 8 60 Ratio of solids-uot- fatto butter fat. 2-43 2.48 2- 56 2.83 2- 63 2.81 2.66 2. 67 2. 69 6.65 2- 52 2. 28 2. 42 2. 26 2-57 1. 81 2. 00 2-55 2.44 2. 41 1.98 2-33 2. 62 2-45 2. 47 2. 26 2- 63 2. 50 the ^_ "The references to the data combined in this table will be found in the following numbered papers of "Literature Cited": 1, 7, 10, 12, 14, 19, 24, 30, 35, 36, 39, 40. These data are not entirely satisfactory, representing, as they do, data collected under a great variety of conditions. This heterogeneity is un- fortunate. Two errors are easily discernible : The fat percentage of the Holstein-Friesian is about o. i per cent too low, and the fat percentage of the Guernsey from unpublished data on 4,900 animals for a year's test is 4.9 instead of the 4.53 of the above list. However, this is the only Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids 83 material available where any number of animals are tested for their total solids and percentage of fat. The belief is held that even with these discrepancies the table will give a fair comparative \'iew of the average composition of the milk of the various breeds included. Taken at their face value, the data show that the butter fat percentage in the different breeds varies between 3.05 and 5.12 per cent. Similarly, the total solids are shown to vary between 1 1.54 and 14.39 and the solids-not-fat between 8.04 and 9.79. This would make the average composition of the Hol- stein-Friesian breed rather lower than most of the other breeds, both in the percentage of butter fat and in the total solids. If we consider now the ratio of the solids-not-fat to the butter fat when the milk is constant, the values of the ratios run between 1.8 and 2.81. This means that the breed considered in the data has a high proportion of solids-not-fat. There appears to be an association between the percentage of fat charac- teristic of the breed and the content of the solids-not-fat carried in the milk — that is, the Jerseys, with their high fat percentage, also have an increased amount of the solids-not-fat over the other breeds, and the Holland, one of the lowest breeds, also has the lowest amount of solids- not-fat. This increase does not go up in direct proportion to the amount of fat present in the milk, as a glance at the proportion of the two will show. It will remain for a later section to show how these constituents vary within the Holstein-Friesian race. Data have been tabulated to show the differences in the milk of different species of animals (Table III). Table III. — Mean milk constituents of different species of animals '^ Species. Sow Goat Ewe Indian buffalo . . Bitch Ass Mare Man Cow Colostrum of cow Colostrum of man Total Fat. Solids-not- solids. fat. Per cent. Per cent. Per cent. 18.51 6.60 II. 91 11.80 3-54 8.26 18.52 7.17 "•35 16. 24 6.77 9-47 15.89 5-65 10. 24 9. 72 .90 8.82 10. 92 •99 8-93 II. 78 3.28 8.50 12. 01 3-44 8.61 22.88 2.30 20. 58 12. 91 2. 60 10.31 Ratio of solids-not- fat to butter fat. 1. 8 : I 2. 3 : I I. 6 : I I. 4 : I 1. 8 : I 9. 8 : I 9. o ; I 2. 6 : I 2. 5 : I 8. 9 : I 4. o : I » The references to the data named in this table will be found in the following numbers of the "I,itera- ture cited." s. 6, 8, 9, 18, 22, 25, 26, 29, 32, 33, 38. The data given above are open to the same criticism as that in Table II, and are to be taken with the same limitations. The milk of the different species varies considerably both in its butter- fat content and in its solids-not-fat. The lowest percentage of fat pro- duced is 0.90 per cent, found in the milk of the ass. The milk of the mare corresponds closely to this, 0.99 per cent. The highest percentage of 84 Journal of Agricultural Research voi.xvi. No. 3 butter fat is 7. 17 per cent, contained in the milk of the ewe, closely followed by that of the Indian buffalo and the sow. The milk of the dairy cow is about intermediate between these two extremes. The colostrum is lower in its fat content than is either of the normal milks of the same species. The solids-not-fat content varies from 8.61 per cent for the cow to 1 1. 91 per cent for the normal milk of the species included in the table. It reaches its highest value in the colostrum, where the cow's milk in- cludes as much as 20.58 per cent. The same general association is also seen in the milk of the different species that are present in the milk of the different breeds — that is, the milk of species containing a low per- centage of fat contains proportionately more solids-not-fat than does the species which contains the higher percentage of fat. The species con- taining a low percentage of fat contains less actual solids-not-fat per hundred pounds of milk than does the species containing a higher per- centage of fat. A survey of Table I shows that the most variable character in the Holstein-Friesians, is the age included in the tests. This has a very high coefficient of variation, the highest shown by any of the measured char- acters, emphasizing the need of a proper age correction when the amount of milk is to be studied for its hereditary behavior. The next highest variation coefBcient is that for milk (24.3). This is closely followed by the variation coefficients for butter fat, 21.0; solids- not-fat, 19.9; and total solids, 19.4 These last four coefficients may be considered as dependent variables and owe part of their variation to variations in other characters. Thus, the large constant of variation for the amount of milk is due in some part to the age differences in the animals included, for, as has already been shown, milk production rises in a logarithmic curve with increasing age. Again, as will be shown later, the relation of the milk constituents to the amount of milk is so close that a large part of their variation may be explained by variations in the amount of milk. As a matter of fact, these data are not needed here, for when the coefficients of variation for the percentages of butter fat, solids-not-fat, and total solids are considered, it is seen that the coefiticients are reduced to 9.2, 3.9, and 4.5, respectively, or coefficients which compare rather favorably with physical variables. The high coef^cients of the milk solids are thus shown to be due to the variations in amount of milk and not to variations in percentage contents of these constituents. FACTORS AFFECTING THE COMPOSITION OF MILK Already some work has been done on factors affecting the composition of milk by Wilson {37) and by Pearson {21-23). In his studies Wilson attempts to show that the percentage of butter fat is not dependent on the amount of milk. The methods used to draw this conclusion are, according to Pearson, open to criticism on the following grounds : The tail frequencies are clubbed together so that the real correlation can not be Jan. 20, I9I9 Variations and Mode of Secretion of Milk Solids 85 determined, and the means of the separate distributions lead us to a correlation ratio, which, while small, is highly significant in showing the dependence of butter -fat concentration on amount of milk. These studies limit themselves to the relation of butter fat to the milk. In dealing with this question the association of the percentage solids- not-fat with the quantity of milk produced will also be studied. Tables IV and V show the correlation surfaces for weight of milk and percentage of butter fat and the age of the cow and the percentage of butter fat. Table IV. — Correlation surface for amount of milk and percentage of butter fat as deduced from, individual year records of Holstein-Friesian cows Weight of milk (pounds). Percentage of butter fat. 0 00 q CO I fO NO 6 25 90 117 92 66 50 24 4 3 2 CO 1 to 4 I 4 II 9 8 4 3 2 4 NO 4 00 4 "3 7, 000— 9, 000 3 17 42 5 32 66 5 25 58 67 49 48 13 8 6 3 2 3 15 17 30 31 14 8 7 I 3 I I 24 119 307 400 371 295 154 96 32 25 15 5 9, 000-11, 000 I 17 12 9 20 7 7 3 4 2 II, 000—13, 000 2 4 3 4 3 3 2 I 3 5 5 I 2 2 I I 17,. 000— I s, 000 15, 000-17, 000 1 2 66 '107 17, 000-19, 000 19, 000-21, 000 55 16 16 6 5 5 5 79 49 25 8 4 I I 3 2 2 .... 21, 000—2 ^, 000 23, 000-2^, 000 25, 000-27. 000 2 I 27, 000-29, 000 I 29, 000-31, COO Total . . 3 22 82 2Qi; Alo: 479 284 130 45 19 II I 1,843 ^9b Table V. — Correlation surface for age at the beginning of test and percentage of butter fat contained in the milk for the individual records of Holstein-Friesian cows Percentage of butter fat. Age at test. 00 q I •0 k 00 i 0 i 4 4 1- r 4 NO 4 i 4 "3 0 Yr. mo. I'r. V20. I 6-2 0 I II 7 4 8 5 6 5 2 7 66 23 15 20 15 7 10 14 10 6 II 4 3 3 2 2 2 12 84 38 29 32 17 20 16 22 19 17 5 5 8 5 5 3 2 16 91 59 25 28 12 23 20 23 13 18 17 8 5 6 6 I 10 66 38 19 20 18 8 8 13 6 4 3 4 2 4 2 3 21 21 13 9 7 8 8 2 3 2 3 6 8 I 2 4 I 2 2 I I 4 2 2 2 53 354 2 0-2 6 2 6-3 0 2 4 2 I 2 I 2 2 T, 0-2 6 I I 3 6-4 0 123 81 4 0—4 6 2 I I 4 6-5 0 c 0- :; 6 I 77 72 80 5 6-6 0 2 6 0-6 6 51 49 40 23 18 6 6-7 0 I I 3 I 7 0-7 6 I 7 6— S 0 8 0-8 6 8 6-9 0 I I 2 I 2 I I 17 15 17 6 9 0- Q 6 I 9 6-11 0 I I II 0-15 0 Total 3 16 55 220 339 371 225 lOI 35 14 7 I 1,387 86 Journal of Agricultural Research voi. xvi, No. 3 A glance at these tables shows that there is little correlation between butter fat percentage and the weight of milk or age at test for the Hol- stein-Friesian cows. It seems well before tabulating these coefficients that we consider the effect of increased production in Holstein-Friesian cattle on the percentage of solids-not-fat contained in the milk. Two tables similar to those above are necessary for this comparison. These data are given in Tables VI and VII. Table VI. — Correlation surface for the amount of milk produced in one year and the per- centage of solids not fat contained in the milk of Holstein-Friesian cows Percentage of solids-not-fat. Weight of milk (pounds). 0 0 CO h 06 •3- t 06 X 06 00 06 0 t 00 t t 0 t 6\ 00 t 0\ 0 2 d I 0 d I 6 ■0 d I 6 6 6 0 0 s 3 0 I 6 13 24 21 13 6 4 17 IS 10 9 3 I I 9 6 4 4 3 I I I 4 I 3 7 7 7 5 4 I 2 s 10 10 4 2 I 5 10 16 19 18 S 2 I 2 78 I I 2 63 8S 74 59 23 6 13,000-15,000 I I (l) 2 I t - I I 59 6 - I I Total 10 31 34 84 28 335 Table VII. — Correlation surface for the variables age at test and percentage of solids not fat for the semiofficial year records of Holstein-Freisian cows Percentage of solids-not-fat. Age at test. 1^ 00 0 t 06 t CO -0 06 00 00 0 06 0 t d> 00 I 0 d I d- d t d d I 6 6 t 6 CO d vt d 0 d YT.mo.yr.mo. I I 3 2 6 4 4 5 5 2 3 I 7 I 9 6 3 4 I I IS 14 IS II 10 s 4 3 I 22 18 14 8 9 4 I 5 2 18 IS 7 10 4 2 2 I 12 3 6 I I 3 4 I I 87 I 6t S6 43 34 I 2 I S 6-6 6 6 6-7 6 7 6-8 6 8 6-9 6 I I I I I (i) Tft 6 II 6-12 6 2 I I - z I 10 Total 31 34 78 84 59 28 6 I I 335 It will be noticed that in both Tables VI and VII there is one value (in parentheses) far removed from the distribution of the other entries. It seems desirable to determine the correlations with this value included and excluded. Consequently, the correlations will be given both with and without it, since it is highly probable that some error has crept into the determination of the solids for this test. Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids 87 Table VIII gives the values of the correlation coefficients and the correlation ratios, together with the constants to show the approach to linearity of the regression lines. Table VlU.~A7ialytical constants for inter-individual variation of the constituents of cow's milk Character correlated. Weight of milk and percentage of butter fat Age and percentage of butter fat Weight of milk and percentage of solids- not-fat Age and percentage of solids not fat with- out doubtful observation Weight of milk and percentage of solids- not-fat Age and percentage of solids-not-fat 0977±o.oi56 0546± .0181 05S3± -0367 i6l2± .0359 0659 ± .0367 2I9I± .0351 o. i25i±o.oi55 • I32S± .0178 . n6i± .0364 . 234i± .0348 •I373± -0362 . 24S9± .0347 o. 1213 .1917 •l8S7 • 3084 • 2032 .1883 o. 006 1 ±0. 0024 .oi46± .0044 . oio4± -0075 . 0288it .0124 .oi4S± .0089 • 0I25± .0082 This table shows that the weight of milk produced in a year is nega- tively correlated with the percentage of butter fat and of solids not fat contained in this milk. In each case the correlation is low, in neither case being as great as -o.i. For the butter fat percentage the corre- lation-0.0977 ±0.01 56, although low, is highly significant, since the correlation value is 6.2 times its probable error; or, in other words, could be expected from random sampling only once in slightly more than 100,000 times. The correlation for percentage of solids not fat and milk, -0.0553 ±0.0367, is only about half that for the percentage of butter fat and milk. Further, this correlation can not be considered significant, as it is only 1.5 times its probable error, or about once out of three trials a correlation as great or greater than this due to random sampling would be expected. It will be noted that even where the abnormal observation is eliminated the correlation does not increase to a value where it becomes significant. The correlation between the percentage of butter fat and age is sUght (-o.0546io.0181) and in the same direction as that of butter fat and milk production— that is, minus. It may possibly be significant, since it is about 3.1 tiines its probable error. Even if it were significant, however, it would be scarcely detectable except in a large mass of data where statistical methods were applied. On the other hand, the correlation between age at test and percentage of solids-not-fat is significant, for, with the doubtful observation, the correlation is 4.4 times its probable error, and without this doubtful ob- servation the correlation is 6.2 times the probable error. Furthermore, the difference between the correlation of percentage of butter fat and percentage of solids-not-fat in cow's milk and the age at which the test is made ^ is probably a significant difference. The difference of the corre- lation of the percentage solids-not-fat and age and the correlation be- ' The probable error of the difference is calculated by the usual formula ±0.67449 Vo^H^ 92803°— 19 2 8g Journal of Agricultural Research voi. xvi, no. 3 tween the percentage of butter fat and age is slightly over 2.7 times its probable error when the doubtful observation is included in the data. The difference and its probable error for this is 0.1066 ±0.0400, or 2.7 times its probable error when the doubtful observation is included in the data. The difference and its probable error for this is 0.1066 ±0.400, or the difference is 2.7 times its probable error. When this doubtful value is not included in the calculations, the difference and its probable error becomes 0.1645 ±0.0395, ^^ 4-2 times the probable error, a value which certainly represents a greater effect of age on the solids-not-fat content of cow's milk than of age on the butter-fat content of the same milk. Much the same statement holds for the relation of the percentage of solids-not-fat and weight of milk produced and percentage of the solids- not-fat and age at test. The difference is of the same magnitude as is the difference between percentage of solids-not-fat and percentage of butter fat and age — that is, the difference is only slightly significant if we consider the correlation found in the presence of the doubtful value, and is markedly significant when this value is thrown out of the table. LINEARITY OF REGRESSION The analytical constants necessary to test the linearity of regression are given in Table VIII. In every case the correlation ratio is a somewhat larger numerical quantity than the correlation coefficient for the same table. These differences are sho\\Ti to be of little significance in view of the fact that S^ and rf — r^ are substantially zero. The difference be- tween the correlation ratios and the correlation coefficients are probably not significant. In only one case is the difference tf' — r'^ greater than three times the probable error (j) , and in this case the difference is only 3.3 times the probable error. For this one case the difference is in all probability not significant. For the other correlations the difference is certainly not significant. It may be concluded, therefore, that the re- gressions are linear and that the correlation coefficient represents the true correlation. The following conclusion may be drawn from the above analysis con- cerning the relations between the constituents of cow's milk and the varia- bles, age at beginning of the year test and amount of milk produced dur- ing this year test. 1. As the amount of milk given by the cows in this test increases, the percentage composition of the butter fat in this milk decreases. The amount of this decrease is statistically significant. Considered practically, this fall in butter-fat content could not be easily detected in the small samples usually handled. 2. There is a slightly significant fall of the percentage of butter fat con- tained in the milk as age advances. This slight fall may, however, be accounted for by the rise in milk production which occurs coincident with Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids 89 this increase in age. For the partial correlation between percentage of butter fat and age, holding the milk production constant, is 0.0105 ± 0.0181, or there is no significant correlation. 3. The quantity of milk produced for the year is entirely independent of the percentage content of the solids-not-fat, or put in another way, the factor or factors causing high or low milk production are separate and distinct from those causing a high percentage of these constituents in the milk. 4. Age is a prominent factor in bringing about the reduction of the percentage of solids-not-fd,t. This reduction is not due to differences in the amount of milk, as the milk held constant by the partial-correlation method gives practically the same correlation as when the milk produc- tion is not considered. 5. Decrease in the amount of milk raises the percentage of butter fat in a greater degree than it affects the percentage of the solids-not-fat. 6. Increased age has a marked effect in reducing the percentage of solids-not-fat. It does not so reduce the percentage of butter fat. CORRELATION BETWEEN THE BUTTER FAT AND THE SOLIDS-NOT- FAT IN COW'S MILK This question of the correlation between the butter fat and the solids-not-fat has considerable importance for the problems of the mech- anism of milk secretion in the mammary gland. Does this gland secrete a high content of butter fat when it secretes a high content of the other solids — ^lactose, protein, and ash? Should such an association exist, it becomes evident that the factors leading to a high fat content also lead to a high solids-not-fat content. Taken in connection with either theory to account for the presence of the organic constituents of milk, such a correlation indicates a regulatory mechanism which balances these constituents together in similar proportions in any given individual cow. Apart from the bearing on the problems of the milk secretion, such a correlation has wider significance. It predicates that the factors in in- heritance for this high content of one constituent also transmits the pro- duction of high content of the other solids. The problem thus becomes important for the student of inheritance of quantity and quality in cow's milk. To answer these questions for Holstein-Friesian cows, it is necessary to arrange the data for the yearly records of butter fat in a table of double entry or correlation table. In the arrangement of this table loo-pound inten^^als were chosen as the basis of division of both the solids-not-fat and butter-fat production of the year tests. In the first two observa- tions of the butter fat it is necessary to group these between 280 and 300 pounds and calculate accordingly. The results are given in Table IX. Observation easily shows that these two variates, solids-not-fat and butter fat of cow's milk, are highly correlated. The correlation coeffi- 90 Journal of Agricultural Research voi. xvi, no. 3 cient in this case is r = 0.8991 ±0.0071 and the correlation ratio r? = 0.9023 ±0.0069. As in the previous tables, the cell which contained the doubtful observation is indicated by parentheses. Where this doubtful observation is left out of consideration, the correlation rises slightly to r = 0.9095 ±0.0063, and the correlation ratio is 7^ = 0.9064 ±0.0066. Table IX. — Correlation surf ace for pounds of solids-not-fat and pounds of butter fat as deduced from individual year records Weight of butter fat (pounds). Weight of solids-not-fat (pounds). 8 1 1 8 h 0 1 i 0 0 i d 8 i 1 {2 700-800 I I 3 6 20 13 2 A 2 5 28 40 23 S 2 9 25 41 47 47 50 39 30 20 I,OOC— 1,100 1,100-1,200 5 24 43 13 2 1,200—1,300 \ 1,300—1,400 2 4 16 6 1,400-1,500 1,500-1,600 1,600-1,700 2 4 4 2 I I 1,700-1,800 I II 1,800-1 ,900 (2) 6 1,900—2,000 I 3 I 2,000—2,100 2,100—2,200 I 2,200—2,300 I I Total 2 a.A 1 T 0 C 122 45 14 2 I 335 These constituents of cow's milk are shown by these correlations to be highly correlated variates. This correlation can not be accounted for by the regression of solids-not-fat on butter fat, not being a linear regression, as even a glance at the values of the correlation coefficient and the correlation ratio will convince anyone that they are so nearly the same in value as to make it certain that the regressions are linear. Consequently it does not seem necessary to calculate the customary constants for this linearity, as both Zm and 17^ — r^ would be negligible quantities. This establishes the conclusion finally that butter fat and solids-not-fat contained in cow's milk are correlated variates. This correlation being positive, a rise in the amount of either constituent also means a rise in the other. Part of the correlation between the butter fat and solids-not-fat may be due to the rise in the amount of milk of the different individual cows. For the problem of the mechanism of the secretion of these constituents this is of especial importance. Tables X and XI give the correlation surfaces for the variables. Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids 91 Table X. — Correlation sutface for amount of milk and amount of butter fat as deduced from individual year records of Holstein-Friesian cows Weight of butter (at (pounds). 280-300. . 300-400. . 400-500. . 500-600. . 600-700. . 700-800. . 800-900. . 900-1,000. Total. Weight of milk (pounds). 63 85 (59) 6 74 59 23 2 44 105 122 45 14 2 335 Table XI. — Correlation surface for amount of m,ilk and solids-not-fat as deduced from, individual year records of Holstein-Friesian cows • Weight of milk (pounds). Weight of solids-not-fat (pounds). j s 'O I o_ d C^ d 8 I 6 8 13 700—800 . . . 2 2 9 9 4 800—900. 9 25 41 900—1,000. 16 33 14 I 000— I , I 00 8 32 37 8 I 100— I 200 I 10 38 22 2 47 47 50 39 30 20 1,200—1 300 I 300—1 400 4 17 25 13 I 400—1 ^00 ! 1,500—1 600 3 7 10 . . . . 1,600—1 700 1,700-1,800 I II I 800—1 900 (I) ■! 2 6 1,900-2 000 3 3 I 2,000—2 100 I I I I 2,200—2 300 ' I Total 2 20 63 85 74 59 23 6 I 2 335 These tables show that the variables butter fat and solids-not-fat are highly correlated with the amount of milk produced. The cor- relation coefficient for butter fat and milk is r = 0.8644 ±0.0093 and the correlation ratio 77 = 0.8638 ±0.0093. The correlation coefficient for solids-not-fat and milk is r = 0.9497 ±0.0036 and the correlation ratio is 77 = 0.9484 ±0.0037. As in the preceding case, these variates are 92 Journal of Agricultural Research voi.xvi.no.s highly correlated. The regressions are so obviously linear that it does not seem necessary to calculate any of the customary constants for determining this, other than the correlation ratio given above. In order to obtain the coefficients to measure the relation between the butter fat and solids-not-fat for a constant production of milk, it is necessary to resort to correlation of the first order given by the formula of Pearsons {21-23). ''l2~''l3''2i r 12.3 = Vi-^s Vi- ■''23 When the correlation between the butter fat and solids-not-fat for a constant quantity of milk is thus measured, it is found that the partial correlation coefficient is r 12-3 = 04964 ±0.0278 for the case where the doubtful observation (shown in parenthesis) is included in the calcu- lations and is r 12.3 = 0.5635 ±0.0252 where this doubtful observation is not included. These correlations show that the production by the mammary gland of butter fat and of solids-not-fat are correlated functions. This correlation being plus it means that an increase in the 'liberation of either constituent of cow's milk means a coincident increase in the other. This conclusion is important, as it shows that the factors responsible for the increase in the content of butter fat for a given volume of milk are in a high degree responsible for the increase in the solids-not-fat content in this same milk. It means that the physiology of the mammary gland in elaborating the milk solids is such that the release of a certain amount of butter fat to the milk also releases a proportionate amount of solids-not-fat. In order to account for this correlation, it is neces- sary to explain how the mammary gland sorts out the different elements into the milk to give the proportion of the butter fat and solids-not-fat. This question refers itself back to the fundamental one of how milk is secreted. DIURNAL VARIATION OP THE) CONSTITUENTS IN COW'S MILK Before discussing the direct bearing of these data on the problem of the milk secretion a few more important data must be presented. It is a well-known fact that the evening milk of a cow is, in general, higher in butter fat than the morning milk. The relation of the evening and morning milk for solids-not-fat is not so well known. Table XII gives this relation for two groups of cows : Those in the first half of their lactation period and those in the last half. This table shows the variation between morning and evening milk in the percentage composition of the same individual cow. The sample of two consecutive mornings' milk were made, and aliquot parts of each composited for analysis. The same holds true for the evening milk. Each percentage may therefore be said to represent the mean Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids 93 between two days of lactation for morning and for evening milk. The milkings begin in the morning at 4.45 and in the evening at 3.45. The interval therefore is shorter between the morning-to-evening milking. Table XII. — Daily variation of the constituents of cow's milk « Cow No. First half of lactation. Morning. Fat. Average 4. 078 P.ct 4 4 Solids- not-fat. '.ct. 35 49 48 55 76 79 34 05 00 8.646 Evening. Fat. ' Solids- not-fat. P.ct. 8. 36 8.60 8.77 8.59 8.64 8.85 8.41 9.29 9. 01 Last half of lactation. Morning. Fat. P. a. 4. 20 4.40 4.40 3. 20 6.80 4.90 3-50 3.60 4- 50 4-756 8.724 4-389 Solids- not-fat. 8.58 8.777 Evening. Fat. P.ct. 4.90 4.80 6. 20 4. 10 8.00 5. 20 3.80 4. 20 4.90 5. 122 Solids- not-fat. P.ct. 8.76 9. 18 9-55 7-95 9.96 9. 12 8.47 8. II 9. 00 8.90 o The author is indebted to the Chemistry Laboratory of this Station for the careful analysis given in Table XII. The average composition of morning milk in the first half of the lactation is seen by Table XII to be 4.078 per cent butter fat and 8.646 per cent solids-not-fat. The average composition of evening milk is 4.756 per cent fat and 8.724 per cent sohds-not-fat. Thus, the butter- fat constituent increases markedly in the evening milk over that of the morning. The increase of the sohds-not-fat is not as marked, although a slight increase does occur. The significance of this increase is supported further by the same kind of relationship exhibited by the morning and evening milk of the cows in late lactation. The numbers are not large, but the consistency of the increase composition of butter fat of the evening milk over that of the morning leads to the conclusion that this relation is certainly significant. For the increase of solids-not-fat the case is not so clear. It is possible that this increase is slightly significant, but this seems doubtful. In no case is this rise of the solids-not-fat as great as that of the butter fat. This, taken in consideration with the fact that the solids-not-fat are more than twice as great in amount as the butter fat, establishes the conclusion that the butter-fat composition of milk is affected to a much greater extent by these different times of milking than are the other solids. This conclusion is further emphasized by the work of Ingle {16) on the same question. In the mixed milk of a herd of 23 animals milk for a period of 18 weeks the average composition of the morning milk gA Journal of Agricultural Research voi. xvi. no. 3 was 2.97 per cent butter fat and 8.87 per cent solids-not-fat, and the evening milk was 4.31 per cent butter fat and 8.86 per cent solids-not- fat. The significance of these facts as above established on the problem of the mode of liberation of the constituents into cow's milk has been overlooked. Before 1850 the prevailing opinion held that the milk solids were filtered out by the mammary gland from the blood serum. This view was shown to be incorrect by the fact that lactose is not present in the blood and the fat percentage of the serum is not sufficient to account for the fat in a single milking. To replace this old theory, three major hypotheses have been put forth to account for the secretion of the mammary gland : (i) Cells of the gland break loose bodily and disintegrate in the alveoli to form the milk solids. (2) The portion of the cells toward the alveoli becomes loaded with solids, breaks loose from the basal portion, and disintegrates to form the milk solids. (3) The cells of the mammary gland secrete the materials of the milk solids without themselves breaking down. In opposition to the first theory, it may be said that no such extensive cell multiplication is witnessed in the mammary gland as would be necessary to replace the cell destruction called for on the theory. This disintegration, as pointed out by Heidenhain (ij) for the milk produced by some cows in one day would require the replacing of all cells in the udder at least five times a day, a replacement of cells unprecedented in our knowledge of cell division. The second theory, suggested by Langer and ably supported by Heidenhain (ij), Steinhaus (28), and Brouha (4), lays its foundation on histological evidence. According to this evidence, the gland cells lengthen out into the lumen of the alveoli. The projecting ends of these cells become loaded with nutrients similar to milk solids. These projecting ends disintegrate to allow the escape of these solids. The basal portions, including a nucleus, are left to rebuild the cell and to enable it to repeat the process. Steinhaus says that, in order to support this rebuilding, mitotic divisions are frequent, and that the daughter nuclei which lie on the outer portion of the cell often degenerate. The third theory lays its stress on analogy with the other secretory glands without other supporting evidence than the negative evidence of Bertkau (2), who says the disintegration appearing in the secretory cells is due to imperfect fixation and that no necrobiosis of any kind appeared. The above summary of the evidence for the three theories to account for the introduction of the solids into the milk shows how contradictory is the evidence so far presented. This contradiction, however, is not to be wondered at The examination of the cells of the actively lactating Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids 95 mammary gland of a Holstein-Friesian cow showed that they were quite small. Considered in the light of this small size, it is likely that obser- vations on the distal end of a cell might be called by one observer the destruction of this portion and by another the cell in its natural shape. This explanation of the confusion in interpretation of obser- vations in these cells is made further probable by the change of shape which cells undergo at different stages of lactation. Thus, in the mam- mary gland of a bitch when just emptied, Heidenhain says that the cells were high and columnar and in another bitch where milk had not been drawn for 48 hours the cells were flat. The weakness of the his- tological evidence is obvious. To the final solution of the problem it appears that other evidence beside the histological observations must be presented. Some evidence from the physiological side has been presented in the foregoing pages of the interaction of the four variables, age, quantity of milk secreted, diurnal variations, and the effect of the content of one solid on the relation of the other variable, butter fat, or solids-not-fat. These variables give criteria to the efficacy of the three contending hypotheses to explain the release of the milk solids. Consideration of the manner of metabolism and energy requirement for most bodily functions seems to furnish the explanation of the slight negative correlation of butter-fat concentration with the amount of milk produced, where no such correlation exists with amount of milk pro- duction and the other solids. Energy has been shown to be required for most bodily functions. There does not seem to be any reason to suppose that the mammary gland is any exception to this rule when milk is pro- duced. Such energy requirement would of necessity be dependent on the amount of production — that is, the high producer will require more energy and to produce "this energy will consequently take a slightly greater amount of fat that might have gone into the milk. The other solids would not be required to furnish any of this energy and conse- quently would show no effect of the amount of milk produced on their concentration. The correlations obtained show the associations that would be expected with this explanation. The conclusion seems justi- fiable that the energy required in the production of milk causes a slight reduction in the amount of fat present in the milk of high-producing cows. The maintenance of the fat concentration of the milk throughout life and the decline of the solids-not-fat apparently represent the normal conditions going on throughout the whole body. It is common knowl- edge that increase in age generally brings with it a relative decrease in the protein upbuild of the body and an increase in the fat. This in- creased metabolism of fat appears to extend itself to the mammary gland, as well as to other parts of the body, being just great enough to maintain the butter-fat concentration throughout life, whereas the g6 Journal of Agricultural Research voi. xvi. No. 3 relative decreased metabolism of the other solids causes a decrease in the concentration of these solids in the milk. By far the best evidence yet presented for the secretion theory for the liberation of the milk solids (the third hypothesis) is given by the diurnal variation of the constituents of cow's milk shown above. It is very difficult to see why the cell constitution should change in balance be- tween the solids-not-fat and butter fat between morning and evening milking on any other theory. Why should the cells discharged in the evening contain the ratio a to 6 of butter fat to solids-not-fat, whereas in the morning the ratio is changed to a markedly lower value of c to b? On the first and second theories the cell must contain a fixed quantity of solids-not-fat, while the butter fat varies in such a way that, in a longer time between the emptying of the gland, the cell accumulates less fat than in the shorter time; or, taken in another way, the cell accumulates relatively more protein as the interval between milkings is lengthened. Our knowledge of fat formation by other cells of the body makes it probable that either of these two possible alternatives for the formation of this milk fat on the cell-destruction hypothesis are inconsistent with the facts. In the formation of fat, the cell is first composed chiefly of protein material. In this protein material the fat is accumulated in ever-increasing amounts at the expense and crowding out of the protein constituents. This change of the ratio of the fat to solids-not-fat is just opposite to what must take place in the mammary gland on the cell-destruction hypothesis. The ratio of the fat to the solids-not-fat in the fat cells increases as the cells increase in age ; the ratio of the fat to the solids-not-fat in milk, derived from the cell breakdown on the two destruction hypothesis, decreases as the age of the cell increases. It is hard to believe that there is such a difference in fat formation going on in the body. It is much more likely that the mechanism of fat forma- tion in the two cases is the same and that the diurnal variation of the ratio of butter fat to solids-not-fat is only another phase of the changes which take place in known secretions. The large variations current in the amount of milk produced, the variations of the constituents with age, the great and characteristic differences in the composition of the milk of two cows of the same breed all add weight to the view that milk is secreted. By analogy, the mammary-gland mechanism for milk secretion would agree with practically all of the glands which secrete. There is no need to assume that rapid cell division is taking place in order to maintain the necessary number of cells for breakdown, as called for on the cell-destruction hypotheses, where no such amount of mitosis as would be necessary is witnessed microscopically. Further, the secretory theory has supporting evidence of whole granules being secreted into the saliva by the salivary glands in much the same way as is done in the secretion of butter fat. The data supporting the secretory hypothesis Jan. 20, 19 19 Variations arid Mode of Secretion of Milk Solids 97 are certainly strong, whereas the proof for the cell-destruction hypo- theses seems weak when analyzed in the light of the above facts, and, in truth, in some particulars is contrary to known facts. The conclusion that milk is a true secretion seems justified by what we know of the mechanism behind such glands. The data above presented give us a criterion to judge the value of any hypothesis for the origin of the milk solids from a common mother sub- stance, such as has been suggested by Thierfelder (jj) and later by Landwehr {17), to account for the derivation of casein and lactose from nucleoproteids or glycoproteids of the gland cells by splitting. The correlation of the solids-not-fat and fat might lead one to suppose such a common origin for some component of such solids and the fat. This can not be the case, however, as the correlation of fal: and of solids-not- fat with amount of milk and age precludes that possibility, for if such a common origin*occurred, the fat and solids-not-fat would necessarily be correlated to these other variables by comparable amounts. The milk components are not correlated equally with either milk quantity or with age; consequently, the hypothesis of a common origin is not tenable. The correlations furnish the necessary evidence for the determination of an important genetic relation between the hereditary factors for the concentration of the butter fat and the solids-not-fat. Since the data are taken from the presumably homogenous population of the advanced registry Holstein-Friesian cows, linkage of factors for milk solids can not be used to account for this correlation; for, granting the homo- geneity of the population, the factors are as likely to be in opposite chromosomes as in the same chromosomes — that is, calling one "a" and the other "b," the parental chromosome content would be: ab a b ab ab ab ab ab a b a ab a a ab a b b b b a b ab b or, reducing down, four chromosomes would be present in equal numbers ab ^ a ^ b ^ These bred together would give the random dis- tribution, considering that crossing over is as likely to occur one way as another. This, however, is not what actually happens in our case. The quantity of fat is correlated to the quantity of solids-not-fat per volume of milk. This must mean that some of the factors responsible for fat concentration are responsible for the concentration of sugar, protein, or ash in cow's milk. 98 Journal of Agricultural Research voi. xvi, no. 3 From the practical side of increasing the soHds content of cow's milk, the importance of the correlation between the butter fat and the solids- not-fat should be pointed out. This high correlation between these varia- bles allows us to use the determination of the percentage content of one as a means of predicting what the content of the other will be. Thus, butter-fat content is easily determined by almost any one familiar with the Babcock test; but solids-not-fat are not so easily determined nor so fre- quently recorded. We may, in trying to improve the solid content of milk select as breeders those cows which test well with the Babcock appa- ratus, and at the same time improve the solids-not-fat content of the milk. SUMMARY This paper is the fourth of a series of studies on milk now being con- ducted in the Biological Laboratory of the Maine Agricultural Experi- ment Station. The data for this study are taken from the semioflficial year record of the pure-bred Holstein-Friesian cows, compiled and super- vised by the Holstein-Friesian Association. (i) The means, standard deviations, and coefficients of variation are given for these year records. The mean annual production of these ani- mals was 15,417 pounds of milk, 528 pounds of butter fat, 1,303 pounds of solids-not-fat at a mean age of four years. The standard deviations are 3,742 pounds of milk, 134 pounds of butter fat, 260 pounds of solids-not- fat, and two years. The coefficients of variations are, respectively, 24, 25, 20, and 50 per cent. (2) Comparison is made of Holstein-Freisian milk with the milk of other breeds and other species. (3) Correlations are presented between the variables butter-fat percen- tage and amount of milk produced, butter-fat percentage and age at test, solids-not-fat percentage and amount of milk, and solids-not-fat and age at test. These correlations lead to the following conclusions: (a) As the amount of milk given by the cows in this test increases, the per- centage composition of butter fat decreases. The amount of this de- crease is highly significant, measured statistically. Considered practi- cally, this fall in butter-fat content would not be easily detected in small samples, (b) The correlation between the age at test and butter fat is not significant, (c) The correlation between the amount of milk pro- duced and the percentage of solids-not-fat is not significant; or, put in another way, the quantity of milk produced for one year is independent of the concentration of the solids-not-fat. This, from a genetic view- point, means that the hereditary factors for high or low milk production are separate and distinct from those causing a high percentage of solids- not-fat. (d) The correlation of age at test and solids-not-fat is — 0.2 191 ± 0.0351 — that is, as the age of a cow increases, the solids-not-fat percen- tage of the milk decreases, (e) The constants for the linearity of re- gression are given. They show the regressions to all be linear, (f) Jan. 20. 1919 Variations and Mode of Secretion of Milk Solids 99 These conclusions give us two variables which influence the concentration of butter fat and solids-not-fat differently. This difference in action of these variates proves that the butter fat and the solids-not-fat can not have a common mother chemical from which they are derived from split- ting. (4) Correlations are presented between the variables, pounds of milk, butter fat, and solids-not-fat. Each variable is highly correlated, the correlation ranging from r = 0.8644 ±0.0093 to /- = 0.9497 ±0.0036. In each case the regressions are linear. The partial correlation between butter fat and solids-not-fat for a constant value of the milk is found to be 0.5635 ±0.0252. This correlation, together with those above, fur- nishes the data necessary to establish the conclusion that some of the factors responsible for high concentration of butter fat are also respon- sible for high concentration of some of the solids-not-fat in cow's milk. Another important practical conclusion may be drawn from this correla- tion ; that if it is desired to improve either the butter fat or solids-not-fat concentration in a given herd the determination of the concentration of either solid will also result in an increased concentration of the other solid. (5) Data on the diurnal variation of cow's milk are presented. These data show that the morning milk is between 0.678 and 0.723 per cent lower in butter fat than in the evening milk throughout the whole lac- tation. No appreciable difference occurs in the solids-not-fat. These data offer criteria between the theories to account for the secretion of the milk solids. In the cell-disintegration theories the cell must contain a fixed quantity of solids-not-fat, while the butter fat varies so that in the longer interval between milkings the cell accumulates less fat than in the short time; or, taken the other way, the cell contains relatively more protein and sugars than fat as the interval between milkings lengthens. This is contrary to our knowledge of fat formation, for it is commonly accepted that first comes the cells composed largely of protoplasm and that as time goes on this cell is more and more loaded with fat at the ex- pense of the protoplasm. Unless these mammary cells behave very differ- ently in the formation of this fat than other body cells, this variation is enough to discredit seriously the hypothesis of cell disintegration to ac- count for these milk solids; and in fact, to make it an absurdity. Fur- thermore, so far as our knowledge of the variations of secretory glands goes, the variations of the milk fall in well with the secretory hypothesis to account for these solids. LITERATURE CITED (i) AsHCROFT, William. 1905. THE MILKING TRIALS OF 1904. In Jout. Bfit. Dairy Farmers' Assoc. V. 19, p. 88-117. lOO Journal of Agricultural Research voi. xvi. no. 3 2) Bertkau, F. 1907. ein beitrag zur anatomie und physiologie der milchdruse. in Anat. Anz., Bd. 30, No. 7/8, p. 161-180, 7 fig. Literatur, p. 179-180. 3) BlakEman, John. 1905. ON TESTS FOR LINEARITY OF REGRESSION IN FREQUENCY DISTRIBUTIONS. In Biometrika, v. 4, pt. 3, p. 332-350. 4) Brouha. 1905. RECHERCHES SUR LES DIVERSES PHASES DU DEIVELOPPEMENT ET DE l'activit^ DE tA MAMELLE. /m Arch. Biol., t. 21, p. 459-603, pi. 18-20. Travaux renseignes, p. 591-596. 5) BuTTENBERG, P., and TetznER, F. 1904. EIN BEITRAG ZUR kennTnis DER ziEGENMiLCH. In Ztschr. Untersuch. Nahr. u. Genussmtl., Bd. 7, p. 270-272. 6) Elsdon, G. D. 1916. NOTE ON HUMAN MILK. In Analyst, v. 41, no. 480, p. 74. 7) Farrington, E. H. 1905. dairy cow demonstration of THE LOUISIANA PURCHASE EXPOSITION. 64 p. [Fort Atkinson, Wis.] 8) Fischer, K. 1908. tJBER ziegenmilch und ziegEnbutter. In Ztschr. Untersuch. Nahr. u. Genussmtl., Bd. 15, Heft i, p. 1-13. 9) Fuller, J. G., and Kleinheinz, Frank. 1904. ON the daily yield and composition of milk FROM EWES OF VARIOUS BREEDS. In Wis. Agr. Exp. Sta. 21st Ann. Rpt. [i903]/o4, p. 48-50. 10) Fuller, Valancey E. 1901. PAN-AMERICAN MODEL DAIRY. FINAL REPORT. Jersey Advocate and Dairyman, v. i, no. 37, Sup. 2 p. 11) Gavin, William. I913. STUDIES IN MILK RECORDS: ON THE ACCURACY OP ESTIMATING A COW'S MILKING CAPABILITY BY HER FIRST LACTATION YIELD. In Jour. Agr. Sci., V. 5, pt. 4, p. 377-390- 12) Haecker, a. L. 1907. DAIRY HERD RECORD FOR TEN YEARS. In Nebr. Agr. Exp. Sta. Bui. loi, p. 1-27, fig. 1-5. 13) Heidenhain, R. 1883. DIE MiLCHABSONDERUNG. In Hermann, Handbuch der Physiologie. Bd. 5, F. I, p. 380. 14) HoFMANN, K., and Hansen, J. 191 1. LEISTUNGSPRUFUNGEN MIT verschiedenen rinderschlagen. In Landw. Jahrb., Bd. 40, Erganzungsbd. i, p. 210-305, 345-430, 14 pi. Abstract in Exp. Sta. Rec, v. 26, no. 9, p. 879-880. 1912. 15) Holstein-Friesian Association op America. 1907-17. Advanced Registry Year Book. v. 18-28. 16) Ingle, Herbert. 1903. VABQATiONS IN THE COMPOSITION OF cow's MILK. In Trans. Highland and Agr. Soc. Scotland, s. 5, v. 15, p. 135-182. 17) Landwehr, Herm. Ad. 1887. ueber die bedeutung des thierischen gummis. In Arch. Physiol. [Pfluger], Bd. 40, p. 21-37. 18) Leather, J. Walter 1901. THE composition OF INDIAN COWS AND BUFFALOES MILK. ^n Analyst, V. 26, no. 299, p. 40-42. 19) Lloyd, Fred J. 1904. THE MILKING TRIALS OF 1903. In Jour. Brit. Dairy Farmers' Assoc, V. 18, p. 95-121. Jan. 20, 1919 Variations and Mode of Secretion of Milk Solids loi (20) Pearl, Raymond. I914. ON THE LAW RELATING MILK FLOW TO AGE IN DAIRY CATTLE. In ProC. Soc. Exp. Biol, and Med., v. 12, no. i, p. 18-19. (21) Pearson, Karl. 1896. MATHEMATICAL CONTRIBUTIONS TO THE THEORY OF EVOLUTION. III. Regression, heredity, and panmixia. In Phil. Trans. Roy. Soc. London, s. A, v. 187, p. 253-318. (22) 1905. MATHEMATICAL CONTRIBUTIONS TO THE THEORY OP EVOLUTION. XIV. On THE GENERAL. THEORY OF SKEW CORRELATION AND NONLINEAR REGRESSION. Drapers' Co. Research Mem. Biom. Ser. 2, 54 p., 3 pi. (23) I910. NOTE ON THE SEPARATE INHERITANCE OF QUANTITY AND QUALITY IN cow's MILK. In Biometrika, v. 7, pt. 4, p. 548-550. (24) Richmond, H. Droop. 1910. THE composition of milk. In Analyst, v. 35, no. 411, p. 231-237. (25) Sanna, Andrea. 1905. COMPOZIONE QUANTITATIVA MEDIA DEL LATTE PECORINO DELLE CAM- pagnE mERIdionali della sardegna. In Staz. Sper. Agr. Ital. v. 38, fasc. 4, p. 289-292. (26) Schlossmann, Arthur. 1897. UEBER ESELSMilch. In Ztschr. Physiol. Chem., Bd. 23, Heft 3, p. 258-264. • (27) Sheppard, W. F. 1907. the calculation of moments of a frequency — distribution. In Biometrika, v. 5, pt. 4, p. 450-459. (28) Steinhaus, Julius. 1892. DIE MORPHOLOGiE dER milchabsonderung. In Arch. Anat. u. Phy- siol., 1892, Sup. Bd. Physiol. Abt., p. 54-68, pi. 5-7. (29) SuTHERST, Walter F. 1902. THE composition OF COLOSTRUM. In Chem. News, v. 86, no. 2223, p. 1-2. (30) Svoboda, H. 1904. VERGLEICHENDE UNTERSUCHUNGEN UBER DIE BESCHAFPENHEIT UND MENGE DER AHLCH DER BEIDEN KARNTNER HAUPTLANDESRASSEN. In Oesterr. Molk. Ztg., Jahrg. 11, No. 14, p. 191-193; No. 15, p. 205-207. (31) ThiERFELDER, Hans. 1883. zur physiologiE DER MiLCHBiLDUNG. In Arch. Physiol. [Pfliiger], Bd. 32, p. 619-625. (32) Trillat and Forestier. 1902. SUR LA COMPOSITION DU LAiT DE brebis. hi JouT. AgT. Prat., ann. 66, sem. 2 (n. s. t. 4), No. 28, p. 38-39. (33) VlETH, P. 1885. ON THE COMPOSITION OF MARES' MILK AND KOUMISS. In Analyst, V. 10, Dec, p. 218-221. (34) Vigor, H. D. 1913. the correlation between the percentage of milk fat and the quantity op milk produced by ayrshire cows. /» sup. jout. bd. Agr. [London], ii, 28 p. (35) Whitley, S. R. 1906. THE MILKING TRLU-S. In JouT. Brit. Dairy Farmers' Assoc, v. 20, p. 135-169. (36) 1909. THE MILKING TRIALS OF 190S. In Jour. Brit. Dairy Farmers' Assoc, v. 23, p. 101-144. I02 Journal of Agrictdtural Research voi. xvi, no. 3 (37) Wn^ON, James. 191O. THE SEPARATE INHERITANCE OP QUALITY AND QUANTITY IN COWS' MILK. In Sci. Proc. Roy. Dublin Soc, n. s. v. 12, no. 35, p. 470-479. (38) WiNDiSCH, Richard. 1904. BEiTRAGE zuR KENNTNis der bufpelmilch. Ill Ztschr. Untcrsuch. Nahr. u. Genussmtl., Bd. 8, Heft 5, p. 273-278. (39) WOLL, F. W. 1899. THE COMPOSITION OF sow's MILK. In Wis. Agr. Exp. Sta. i6th Ann. Rpt. [1898]/ 99, p. 267-270. (40) I9OI. ON THE AVERAGE COMPOSITION OP MILK OF PURE-BRED COWS OF DIF- FERENT BREEDS. In Wis. Agr, Exp. Sta. i8th Ann, Rpt. [1900]/ 01, p. 85-97. NEW BIOLOGIC FORMS OF PUCCINIA GRAMINIS* [PRELIMINARY PAPER] By E. C. Stakman, Head of Section of Plant Pathology, Department of Plant Pa- thology, Botany, and Department of Agriculture, University of Minnesota; M. N. LEVINE, Field Assistant, Bureau of Plant Industry, United States Department of Agriculture; and J. G. LEAch, Shevlin Fellow, University of Minnesota COOPERATIVE INVESTIGATIONS BETWEEN THE AGRICULTURAL EXPERIMENT STATION OF THE UNIVERSITY OF MINNESOTA AND THE BUREAU OF PLANT INDUSTRY OF THE UNITED STATES DEPARTMENT OF AGRICULTURE Several biologic forms of Puccinia graminis on wheat (Triticum spp.) have been described. Originally P. graminis tritici was supposed to be the only form capable of attacking wheat varieties. None of the com- mon wheats (T. aestivum) was resistant to this form, although several varieties of durum (T, durum), emmer {T. dicoccum), and einkorn (T. monococcum) were either resistant or almost immune. The first demonstration that there was more than one form of stemrust on wheat was made in 191 6 when P. graminis tritici-compacti was de- scribed.^ This form proved to be especially interesting and significant because it could not infect the hard spring wheats normally, but developed well on soft wheats. It was also found that many of the hard winter wheats were resistant to the new form. The range of parasitism of the second form was therefore narrower than that of the ordinary P. graminis tritici. But in 191 8 Melchers and Parker^ found that Kanred, Kansas P. 762 (CI 5146),^ Kansas P. 1066 (CI 2879), and Kansas P. 1068 (CI 5880), three selections from the Crimean group made at the Kansas Experiment Station, were almost immune to P. graminis tritici. These selections were also found to be moderately resistant to P. graminis tritici-compacti, and they therefore seemed to be resistant to the stemrust of wheat. Later Melchers and Parker ^ found a form which infected Kanred and the two other selections nor- mally. Levine and Stakman ^ and Leach ^ had begun an intensive study of the parasitic capabilities of forms of P. graminis on varieties ' Published, with the approval of the Director, as Paper 144 of the Journal Series of the Minnesota Agricultural Experiment Station. 'Stakman, E. C, and Piemeisbl. F. J. a new strain op puccinia graminis. (Abstract.) In Phytopathology, v. 7, no. i, p. 73. 191 7. • Melchers, Leo E., and Parker, John H. three varieties of hard red winter wheat resist- ant TO STEM RUST. In Phytopathology, v. 8, no. 2, p. 79. 1918. • CI = Cereal Investigations. ' Melchers, Leo E., and Parker, John H. Another strain op puccinia graminis. Kans. Agr. Exp. Sta. Circ. 68, 4 p. 1918. • Lbvinb, M. N., and Stakman, E. C. a third biologic porm op puccinia graminis on wheat. In Jour. Agr. Research, v. 13, no. 12, p. 651-654. 1918. ' Leach, J. G. a comparathtb stttdy op the parasitism op puccinia graminis tritici and puccinia graminis tritici-compacti. To be published as master's thesis. University of Minnesota. Journal of Agricultural Research, Vol. XVI, No. 3 Washington, D. C. Jan. »o, 1919 ra (103) Key No. Minn. 36 I04 Journal of Agricultural Research voi.xvi. No. 3 of wheat and other species of Triticum, and also found a form which developed normally on Kanred, P. 1066, and P. 1068. It was clear from these results that the new forms could therefore do something which neither of the two other forms could do. But none of the known forms could infect White Spring emmer (Minne- sota 1 165), durum (Mindum, CI 5296), and several other varieties, mostly durums. It was perfectly evident from the work with P. graminis tritici-compacti ^ and the two other new forms that the biologic specializa- tion within the genus Triticum could only be determined by testing many species and varieties and that there was a strong probability that forms of rust would be found which were capable of attacking varieties resistant to all known forms of stemrust. This is exactly what has been found. A form was found which infected White Spring emmer and Mindum normally. The work has continued until about a dozen forms have been found up to the present time (Oct. i, 1918). About 25 varieties and strains of Triticutn aestivum, T. durum, T. compactwm, T. dicoccum, and T. rnonococcum are being used as diflfer- ential hosts, and no variety so far tried is resistant to all of the rust forms except Khapli (CI 4103), an emmer originally imported from India. Some of the forms are very virulent on many varieties, while others are weak and can attack only a few varieties successfully. Some forms differ from each other only in their action on one or two varieties; but these differences are definite and consistent. No attempt has yet been made to name the recently discovered forms. The factors governing the distribution of the forms are not at all clear. Material has been collected from 27 States and most sections of the country are represented. Two distinct forms have often been isolated from the same lot of material, and at least four have been found in Minnesota. The fact that there are so many biologic forms of stemrust on wheat seems to be of profound significance in at least two ways. It is an additional reason for eradicating the rust-susceptible varieties of bar- berry (Berberis spp.), and it is of the greatest importance in the work of breeding wheats for rust resistance. Many of the virulent forms seem to occur in the Northern States, where everyone will now concede that the barberry is of tremendous importance in the persistence of stemrust from year to year. Eradicate the common barberry and there is reason to believe that these forms may gradually die out entirely, or at least be reduced to a condition bordering on impotence. The fact also that in the South and on the Pacific coast, where barberry does not rust commonly, the forms of rust seem to be more uniform than in other regions certainly lends some color to the view that the barberry may have some effect on their devel- opment. A hypothesis that forms may have originated by hybridiza- 1 Leach, J. B. op. cit. Jan. 20, 1919 New Biologic Forms of Puccinia Graminis 105 tion on the barberry may be worth investigating. Of course mutation and adaptation must be considered also in any attempt to explain how so many forms originated. The fact that the same variety of wheat may be immune in one locality and susceptible in another is clearly explained. Formerly recourse was taken to the theory that the environmental conditions changed the physiologic processes and materials of wheat varieties so fundamentally that the resistance of the plants broke down. The real explanation of this phenomenon however is the fact that there are many biologic forms of the rust fungus. This has actually been demon- strated in field experiments. There is also preliminary evidence to show that the same thing may be true of Pvccinia triticina on wheat. Methods for breeding for rust resistance must be changed funda- mentally— if indeed it is worth while to do such work at all until more is known about the specialization of the rust fungus. The breeder must know and work with those forms of rust which occur in the region for which his new variety is intended; and even then breeding must be very largely a regional or even a local problem. For instance, in the breeding plots at the Minnesota Agricultural Experiment Station cer- tain varieties were practically immune to stemrust, but rust forms have been found within 50 miles of the plots which can attack these varieties so heavily as to make them worthless for rust resistance. The discovery of so man)'' forms of stemrust on wheat complicates the rust problem seriously. Extensive experiments are under way to determine the number, characteristics, and distribution of biologic forms as well as their constancy and probable origin. ADDITIONAL COPIES OF THIS PUBUCATION MAT BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE "WASHINGTON, D. C. AT 6 CENTS PER COPY Stosckiption Price, Per Year, $3.00 Vol. XVI JANUARY 27, 1Q19 No. 4 JOURNAL OF AGRICULTURAL RESEARCH CONTENTS Page Influence of Salts on the Nitric-Nitrogen Accumulation in the Soil - - - - - - _ - - 107 J. E. GREAVES, E. G. CARTER, and H. C. GOLDTHORPE (Cootrlbatkm trom UUh Agricultural Experiment Station) PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COUEGES AND EXPERIMENT STATIONS ^V:ASHINGXON, D. c. WASHINQTOM : QOVERNMENT rRINTINO OFFICE : l«l« EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT FOR THE ASSOCIATION KARL F.KELLERMAN, Chairman H. P. ARMSBY PhysiMogist and Associatt Chkf, Bureau of Plant Industry EDWIN W. ALLEN Chief, Qfici of Experiment Statitms CHARLES L. MARLATT Entomoloffisi and A tsistant Chief, Bureau of Entomology Director, Institute of Animal Nutrtiitm, Th* Pennsylvania State College E. M. FREEMAN Botanist, Plant Pathologist, and Assistant Dean, Afffi'^turaJ Experiment Station of the Univertily of Minnesota J. G. LIPRL\N Director, New Jersey Agricultural Experi- ment Station, Rutgers 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa. JOIMAL OF AGRICETIML ISEARCH Vol. XVI Washington, D. C, January 27, 191 9 No. 4 INFLUENCE OF SALTS ON THE NITRIC-NITROGEN ACCUMULATION IN THE SOIL By J. E. Greaves, Chemist and Bacteriologist, E. G. Carter, Assistant Bacteriologist, and H. C. Goldthorpe, Assistant Bacteriologist, Department of Bacteriology, Utah Agricultural Experiment Station INTRODUCTION Salts which may occur in soils and those applied to them in various operations influence the number, species, and actixaty of the microflora of a soil. These factors are in turn reflected by the yields obtained from the soil. Some substances applied to a soil serve as food for the growing plant; others increase plant growth but not through the direct furnishing of food. This latter effect may be due to a change brought about by the salt on the physical, chemical, or bacterial properties of the soil. The substance may alter the physical properties of the soil to such an extent that the bacterial flora is modified ; this in turn may increase or decrease the crop produced upon the soil. Other substances may react chemi- cally with constituents within the soil and in so doing liberate substance which can be directly utilized by the growing plant. Again, they may directly modify the microflora and fauna of the soil both as to numbers and physiological efficiency. Or, in some cases, all three changes may be wrought by one and the same salt. The question therefore arises as to what effect this or that fertilizer or soil amendment is going to have upon the bacterial activities of the soil. Furthermore, there are millions of acres of land in arid America which contain varying amounts of soluble salts. Some of these soils contain such large quantities of these so-called "alkaUs" that no vegetation is found upon them. Other soils contain onl}^ a medium amount of soluble salts, and the vegetation is composed chiefly of alkali-resisting plants. Still other soils contain much smaller quantities of soluble salts, and they become injurious only when the soil is improperly handled. The reclaiming of the heavily charged soils and the maintaining of the others in a productive condition can be carried on successfully only when we understand the influence of salts upon the growing plants and their action upon the biological, chemical, and physical properties of the soil. The purpose of this investigation is therefore to determine the relative toxicity of various substances found in or applied to a soil, as measured in terms of bacterial activities of the soil; also to determine the stimu- Jouraal of Agricultural Research, Vol. XVI, No. 4 Washington, D. C. Jan. 27, 1919 qz jQy Key No. Utah-io io8 Journal of Agricultural Research Vol. XVI, No. 4 lating influence of various substance upon bacterial activity and the manner in which the stimulation is exerted. The results thus obtained can be directly compared with those obtained for higher plants. Then, if a correlation between the two, the lower and the higher plants, be obtained, it should give a quick method of testing not only alkali soils but also other soils containing various soluble constituents. Further- more, it makes possible the studying of balanced solutions within the soil by means of bacteriological tests, thus getting in a short time com- parative results which with higher plants would be obtained only by an enormous amount of work and time. Knowledge thus obtained can be used in the reclaiming of the alkali lands of the arid West. A careful review of the literature has been made elsewhere and, hence, is not in- cluded here {3)} EXPERIMENTAL WORK The soil used in this work, taken from the College farm, is of a sedi- mentary nature. It was deposited by streams flowing into the valley, laden with debris derived from the near-by mountains, which are com- posed largely of quartzite and limestone. A physical and chemical analysis of the soil is given in Table I. Table I. — Physical and chemical composition 0/ soil 1 Physical composition. i Chemical composition. Soil. Per cent. Constituent. Per cent. Coarse sand (above i mm.) Fine sand (i to 0.03 mm.) Coarse silt (0.03 to o.oi mm.) Medium silt (o.oi to 0.003 mm.). . Fine silt (0.003 to o.ooi mm.) .... 17.69 37-39 15- 19 10.36 10.32 Insoluble matter 66.69 •55 • 49 7.41 4.15 2-93 3-49 .25 .07 7. 62 2. 18 Potash (KjO) Soda(Na2 0) Lime (CaO) Magnesia (MgO) Clay (below o.ooi mm.) Ferric oxid (Fe^Os) Moisture and loss 9-05 Alumina (AI3 O2) Phosphorus pentoxid (P2OS). . . Sulphur trioxid (SC3) Carbon dioxid (CO2) Humus Total nitrogen •IS The soil used, therefore, was a sandy loam very high in acid-soluble constituents, but the water-soluble constituents were not excessive. The calcium and magnesium contents were very high and mainly in the form of the carbonate. The soil was well supphed with phosphorus and potassium, and there was a fairly large quantity of iron present. In fact, all of the elements of plant food were present in abundance, with the exception of nitrogen, which was low. The soil was very productive, and previous work had shown the ammonifying and nitrifying powers of the soil to be about the average for the soils of the arid regions. The ' Reference is made by number (italic) to " Literature cited, " pp. 134-135. Jan. 27. 1919 Influence of Salts on Nitric Nitrogen in Soil 109 nitrogen -fixing powers of the soil were above the average, and previous work had shown it to have an intensely interesting bacterial flora. Several hundred pounds of the soil were thoroughly mixed, stored in a large box, and kept as near field conditions as possible so that all the work could be done on the same soil. As the soil was needed in the work, portions were brought to the laboratory, air-dried in the dark, then weighed in loo-gm. portions into sterile covered tumblers. To each of these were added 2 gm. of dried blood. The whole was then carefully mixed, and the salt in most cases added from a carefully stand- ardized stock solution. This, together with sufficient sterile distilled water to make the moisture content up to 20 per cent, was thoroughly mixed in the soil. Each series, together with sterile blanks, was incu- bated at 28° to 30° C. for 21 days, and then the nitric nitrogen deter- mined as follows {4, p. 200) : The contents of the beaker, together with 500 cc. of distilled water and 2 gm. of alum, were placed in quart Mason jars and agitated for five minutes in a shaker. An aliquot part (100 cc.) of the supernatant liquid was pipetted off, and, together with 2 cc. of a saturated solution of sodium hydroxid, was evaporated to about one-fourth of its original volume to free it from ammonia. To this were added 50 cc. of ammonia-free water, 5 gm. of "iron-by-hydrogen" and 30 cc. of sulphuric acid (sp. gr. 1.35). If less than 40 mgm. of nitric nitrogen is to be determined, it is well to take a correspondingly smaller quantity of iron and sulphuric acid. The neck of the reduction flask was fitted with a 2 -hole stopper, through which passed a 50-cc. separatory funnel and a bent tube which dipped into a vessel containing water in order to prevent mechanical loss. The acid was slowly added and allowed to stand until the rapid evolution of hydrogen was over. It was then heated to boiling for 10 minutes. The contents of the side vessel were returned to the reduction flask before the reaction was complete, thus insuring the complete reduction of any nitrates which may have been carried over with the first violent evolution of the hydrogen. The contents of the reduction flask were transferred to Kjeldahl flasks, neutralized with sodium hydroxid, and distilled into standard acid. The excess of acid was titrated back with standard alkali, lacomoid being used as an indicator; controls were made on all the reagents, including the alum used as a flocculant. In every case at least four determinations were made with each con- centration of the salt, and, in the absence of agreement between deter- minations, the series was repeated so that the results as herein reported are in every case the average of four or more closely agreeing determina- tions. Hence, experimental error has been reduced to as near a mini- mum as possible in this kind of work. The solutions of the salts were prepared by weighing gram-molecular quantities of Merck's best grade of the respective salts into 1,000 cc. of no Journal of Agricultural Research Voi. xvi,no.4 sterile distilled water and then quantitatively determining the amount present. In those cases in which the analysis showed the concentration wrong, it was corrected, so that we have a definite knowledge of the quantity of salt added to the soil, as the varying results reported by different investigators can in many cases be interpreted by the unknown variation in salts added. The solution thus prepared was then added to the soil in such quantities as to make the am.ount of the anion and of cation the sam^e and directly comparable the one v/ith the other. The comparatively insoluble salts, calcium carbonate, calcium sulphate, etc., were carefully weighed and intimately mixed with the soil. The ar- ranging of the v/ork in this order gives us as nearly absolute results as can be obtained by the present bacteriological methods, and at the same time gives us directly comparable results, which after all is what we have to look for in this work. The salts tested were the chlorids, nitrates, sulphates, and carbonates of sodium, potassium, calcium, magnesium, manganese, and iron. INFLUUNCI3 OF SODIUM SAINTS The compounds used in this series were sodium chlorid, sodium sul- phate, sodium nitrate, and sodium carbonate. They were in concen- trations such that equivalent quantities of sodium in the various forms could be directly compared. The strengths varied from o to 1,380 p. p. m. of soil, and represented the actual proportion of sodium in the various forms applied to the soil. The results are reported as percentage, considering the nitric nitrogen produced in the untreated soil in each case as 100 per cent. This method of reporting the results makes them more directly comparable than if stated as milligrams of nitric nitrogen formed in 100 gm. of soil. The average nitrifying power of the untreated soil was 53 mgm. of nitric nitrogen per 100 gm. of soil. The results are given in Table II, and in every case are the average of at least four and sometimes of several times this number of closely agreeing deter- minations; hence, they should represent very closely the comparative influence of the various sodium salts upon nitrification. Sodium chlorid is the only one of the sodium salts tested which in- creases the accumulation of nitric nitrogen in the soil. In this regard nitrification differs widely from ammonification, for in the latter both sodium nitrate and sodium carbonate stimulate. However, sodium chlorid is a much more active stimulant of the nitrifiers than it is of the ammonifiers, and it stimulates in much higher concentration, being the most. active when the soil contains 230 p. p. m., and is not toxic until the quantity in the soil exceeds 460 p. p. m. The toxicity rapidly increases above this concentration, and at a concentration of 1,380 p. p. m., the nitric nitrogen present had been reduced to 16.4 per cent of what it was in the original soil. Jan. 27, I9I9 Influence of Salts on Nitric Nitrogen in Soil III TablS II. — Percentage of nitric nitrogen formed in lOO gm. of soil containing 2 gm. of dried blood and varying amounts and forms of sodium salts [The untreated soil is taken as loo per cent] Amount of sodium. P.p.m None 3-6 7-2 14-4 28.8 57-5 II5-0 230.0 460.0 920.0 1.380.0 Percentage of nitric nitrogen formed in presence of — Sodium chlorid. 100. O 102. 4 102. 5 100. 6 103. I 114. 7 139.6 142. o 136. 2 57-5 16. 4 Sodium sulphate. 100. O 87.8 60.3 57-1 86.2 74.0 55-1 65-4 63.2 63. o 50.8 Sodium nitrate. 92. 88. 94. lOI. 75- 71- 69. 48. 17- — 14. o Sodium carbonate. 100 100. 79 76 88 94 79 73 76 63 58 Although sodium carbonate is toxic in the lowest concentration tested, yet its toxicity does not increase as rapidly as does that of the chlorid, for at the highest concentration it still produced 58.8 per cent of nitric nitro- gen. The action of sulphate and carbonate nearly parallel each other throughout the entire series. Sodium nitrate probably stimulates slightly at 28.8 p. p. m., but above this concentration the nitric nitrogen rapidly decreases and when the concentration of sodium in the form of sodium nitrate reaches 1,380 p. p. m., there is an actual loss of nitric nitrogen from the soil. It is quite evident from these results that the order of toxicity of these salts are as follows: Sodium sulphate, sodium carbonate, sodium nitrate, and sodium chlorid; but if we consider them at the highest concentra- tions the order becomes sodium nitrate, sodium chlorid, sodium sulphate, and sodium carbonate. The results for sodium chlorid confirm the findings of C. B. Lipman (. quired to produce the maximum effect vary greatly with the salt. These facts are summarized in figures i and 2. Only 6 of the compounds tested, sodium sulphate, sodium carbon- ate, potassium sulphate, potas- sium carbonate, calcium carbon- ate, and ferric nitrate , failed to increase the nitric-nitrogen con- tent of the soil. The i8 others all increased the nitric-nitrogen content of the soil. There is no correlation between the stimula- tion of the ammonifying and nitrifying processes of the soil. This is remarkable when we re- member that the speed of the latter is undoubtedly controlled and dependent upon the other. And the results herein reported probably indicate that there are other side reactions taking place which are influenced by these salts but which are not measured by these methods. Averaging the molecular weights for the 12 compounds acting as the strongest stimu- lants, we find them to be con- siderably lower than the average molecular weight of those which exert little stimulating influence. We really find some of the com.- pounds with the lowest molec- ular weight — for instance, sodium chlorid — the greatest stimulants. Hence, it would seem thatGrutzer's generalization \ pjlfcj^^ for animal stimulants does not ^ hold for either t«he ammonifying Some of -Graphs showing molecular concentrations at which the highest stimulation is noted or nitrifying organisms, the strongest stimulants for plants, sodium chlorid and calcium sul- phate, increase to the greatest extent the nitric-nitrogen content of the soil. Therefore it is certain that the increased plant growth is due to a Jan. 37. i9»9 Influence of Salts on Nitric Nitrogen in Soil 1 29 great extent to the increased available plant food yielded by the accel- erated bacterial activity of the soil. The quantity of the salt necessary to produce maximum stimulation varies greatly with the salt. It is usually the case that those compounds which are the greatest soil stimulants must be added in larger quan- tities to produce maximum stimulation than those which are not as active stimulants and which produce their greatest effect at lower con- centrations. Fig. 2. — Graphs showing the percentage of stimulation at the above noted molecular concentrations (see fig. i), the untreated soil bclnj counted as producing loo per cent of nitric nitrogen. RELATIVE TOXICITY OF THE VARIOUS SALTS The salts used in this work may be compared as to toxicity from three viewpoints: First, the lowest concentration of the salt at which a toxic effect is noted toward the nitrifying organisms; second, the molecular concentration at which nitric-nitrogen accumulation is reduced to three- fourths normal; and third, the percentage of nitric-nitrogen produced in the presence of the largest quantity of the various salts, which is 2 X io~^ mole of the salt in 100 gm. of soil. These results are reported in figures 3, I30 Journal of Agricultural Research vo1.xvi.no.4 4, and 5. Not one of the compounds tested was toxic at the lowest con- centration tested, 78 X lo'^ mole. All of the others became toxic at some fe: is b S?- J<) ^ ft J^ Hi ?5 55 1^ tl^l^l^ tVll 3 ^ ^ ^^^^^^^^ Fig. 3.— Graphs showing the molecular concentrations at which the various salts are toxic to nitrification of the concentrations tested. In 1 1 out of the 20 cases tested the point of toxicity for the ammonifiers and nitrifiers were the same, whereas Jan. 27, 1919- Influence of Salts on Nitric-Nitrogen in Soil 131 in the remaining cases the quantity required to become toxic to the ammonifiers was much greater than it was for the nitrifiers. In only I ^ ^ ^ ^ <;■;'? "^ «; > _^ '^ ^ /TNOj Ca/VOj CaCOj /eC/j //4C/ :hyx/o-^ ^i:^aY/o-^ ^i^sjr/o-f ■2^ ^x/o-.f 2X/0'^ tX/O'* Fig. 4.-Graphs showing the molecular concentrations which reduce the nitrification to three-fourths normal three instances were the salts more toxic to ammonifiers than to nitrifiers. 132 Journal of Agricultural Research Vol. XVI, No. 4 It is evident from these results that while the increased osmotic pres- sure exerted by the salts added to a soil plays an important part in the retarding of the bacterial activity, it is not the only factor nor probably the main one. The principal factor is probably a physiological one caused by the action of the substance upon the living protoplasm of the cell, changing its chemical and physical properties so that it can not function normally. However, we do not find a relationship between the toxicity of the compound and its power to precipitate colloids. It appears, therefore, that while the precipitation of the colloidal cellular F CaCQ, CaSOf. >. \ 'h h. > 'h 'h >,}>, y^ ^ I I I I Fig. s. — Graphs showing the percentages of nitric nitrogen produced in loo gm. of soil to which had been added aXio"' mole of the various salts, the untreated soil being counted as producing loo per cent. material often causes death of the organisms, it is not necessarily the determining factor in the toxic action of these salts. As can be seen from figure 4, it is not necessarily those compounds which become toxic at the lowest concentration which have the greatest far-reaching effect upon the bacterial activities of the soil. This con- dition holds for both the ammonifying and nitrifying organisms. It re- quires in almost every case more of the specific salt to reduce ammonifi- Jan. 27, 1919 Influence of Salts on Nitric Nitrogen in Soil i ^^ cation to three-fourths normal than is required to produce the same effect upon the nitrifiers. It is evident from these results that the common soil alkalis, calcium chlorid, sodium carbonate, sodium sul- phate, and sodium nitrate, are very toxic to nitrifying organisms, and if present to any great extent, will greatly reduce the nitric-nitrogen con- tent of the soil. The toxicity of the compound to ammonification was found to be controlled largely by the cation, but no such a relationship is found to exist in the case of the nitrifiers, as can be seen from figure 5. The toxicity of the compounds to ammonification was found to be controlled largely by the cation, but no such relationship is found to exist in the case of the nitrifiers (fig. 5). SUMMARY The toxicity of the chlorids, nitrates, sulphates, and carbonates of sodium, potassium, calcium, magnesium, manganese, and iron as deter- mined by nitrification is detennined by the specific salt and not by the electro-negative ion, as was the case with the ammonifiers. With the exceptions of the manganous chlorid and sulphate and the chlorids of iron and sodium, the salts tested all became toxic at a lower concentration to the nitrifiers than to the ammonifiers. The quantity of a salt which can be applied to a soil without decreasing the nitric-nitrogen accumulation in the soil varies with the salt, and for the soil under investigation it is in the order of decreasing toxicity of the salts as follows : Sodium sulphate, sodium carbonate, calcium carbonate, potassium sulphate, potassium carbonate, ferric nitrate, sodium nitrate, magnesium sulphate, ferric sulphate, calcium nitrate, potassium nitrate, potassium chlorid, magnesium nitrate, manganous carbonate, manganous chlorid, manganous sulphate, ferric carbonate, magnesium chlorid, man- ganous nitrate, ferric chlorid, magnesium carbonate, sodium chlorid, calcium chlorid, and calcium sulphate. It is not necessarily those compounds which become toxic in the lowest concentrations which are most toxic in higher concentrations, as the toxicity of some salts increase more rapidly than the toxicity of others. It is quite evident from the results reported that the increased osmotic pressure exerted by the salt added to the soil plays a minor part in the retarding of the bacterial activity. The main factor is probably a physiological one due to the action of the substance upon the living protoplasm of the cell, changing its chemical and physical properties so that it can not function normally. The common soil "alkalis," calcium chlorid, sodium sulphate, sodium carbonate, and the less common one, calcium nitrate, are very toxic to the nitrifying organisms, and if present in soil to any great extent will greatly reduce the nitric-nitrogen accumulation in such a soil. 134 Journal of Agricultural Research voi.xvi.no. 4 Sodium sulphate, sodium carbonate, calcium carbonate, potassium sul- phate, potassium carbonate, and iron nitrate failed to increase the nitric- nitrogen accumulation in a soil. All of the others, however, in some of the concentrations tested acted as stimulants. The extent of the stimu- lation and quantity of salt necessary for maximum stimulation varied with the specific compound. Naming them in the order of increasing efficiency, they are: Sodium nitrate, magnesium sulphate, ferric sulphate, calcium nitrate, potassium nitrate, potassium chlorid, magnesium nitrate, manganous carbonate, manganous chlorid, manganous sulphate, ferric carbonate, magnesium chlorid, manganous nitrate, ferric chlorid, magne- sium carbonate, sodium chlorid, calcium chlorid, and calcium sulphate. The last two increased the nitric-nitrogen accumulation of the soil 67 and 97 per cent, respectively. Those compounds which are the strongest plant stimulants are also the most active in increasing the nitric-nitrogen accumulation of the soil and it is very likely that the effect upon the plant is due mainly to the action of the compound upon the bacteria which in turn render available more plant food. Many of the nitrates caused large losses of nitric nitrogen from the soil; this is due to the stimulation of other species which transform the nitric nitrogen into protein nitrogen and not to denitrification. Magnesium nitrate, ferric nitrate, calcium nitrate, and manganous nitrate are very active stimulants of the nitrogen-fixing organisms. In some cases these compounds increased nitrogen fixation many times over that in the normal soil. The ammonifying powers of a soil containing alkalis are a better index of its crop-producing powers than are the nitrifying powers. LITERATURE CITED (i) Deh^rain, p. p. 1887. SUR LA PRODUCTION DES NITRATES DANS LA TERRE ARABLE. In Ann. Agron., t. 13, p. 241-261. (2) Greaves, J. E. 1910. EFFECTS OF SOLUBLE SALTS ON INSOLUBLE PHOSPHATES. In JoUT. Biol. Chem., T. 7, no. 4, p. 287-319. Bibliography, p. 318-319. (3) 1916. THE INFLUENCE OF SALTS ON THE BACTERIAL ACTIVITIES OP THE SOIL. In Soil Sci., v. 2, no. 5, p. 443-480. Literature cited, p. 476-480. (4) and Hirst, C. T. 1917. SOME FACTORS INFLUENCING THE QUANTITATIVE DETERMINATION OF NITRIC NITROGEN IN THE SOIL.. In Soil Sci. V. 4, no. 3, p. 179-203, I fig., pi. I. References, p. 200-203. (5) Griffiths, A. B. 1889. A TREATISE ON MANURES. 393 p. London. (6) Harris, F. S. i915. effect of alkali salts in soils on the germination and growth op CROPS. In Jour. Agr. Research, v. 5, no. i, p. 1-53, 48 fig. Literature cited, p. 52-53. Jan. 27, 1919 Influence of Salts on Nitric Nitrogen in Soil 135 (7) Hopkins, C. G., and Whiting, Albert L. 1916. SOIL BACTERIA AND PHOSPHATES. III. Agr. Exp. Sta. Bul. 190, p. 391-406. (8) LiPMAN, Chas. B. 1912. TOXIC EFFECTS OF " ALKALI SALTS " IN SOILS ON SOIL BACTERIA. II. Nitri- fication. In Centbl. Bakt. [etc.], Abt. 2, Bd. t,^, No. 11/14, p. 305-313, 2 fig. (9) Storer, F. H. 1887. agriculture in some of its relations with chemistry. ed. 7, 3 v. New York. (10) Storp, Ferd., Konig, J., Bohmer, C, Cosack, C, and Weigmann, H. 1884. UEBER DEN EINFLl'SS VON KOCHSALZ- UND ZINKSULFATHALTIGEN WASSER AUF BODEN UND PFLANZEN. In Ccntbl. Agr. Chem., Jahrg. 13, p. 76-87. (11) Voelcker, Augustus. 1867. field experiments of crude GERMAN POTASH-SALTS AND COMMON SALT ON MANGOLDS. In Jour. Roy. Agr. Soc. England, s. 2, v. 3, p. 86-91. (12) V/hEELER, H. J. 1905. PLANT PECULIARITIES AS SHOWN BY THE INFLUENCE OF SODIUM SALTS. R. I. Agr. Exp. Sta. Bui. 104, p. 40-92, illus. (13) Hartwell, B. L., etal. 1907. CONCERNING THE FUNCTION OF SODIUM SALTS. In R. I. Agr. Exp. Sta. 19th Ann. Rpt. 1905/06, p. 189-316. ADDITIONAL COPIES OF THIS PUBLICATION MAY BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 6 CENTS PER COPY Subscription Price, $3.00 Per Year. A Vol. XVI FEBRUARY 3, 1919 No. 5 JOURNAL OP AGRICULTURAL RESEARCH CONXKNXS Pace Pbysoderma Disease of Com ------ 137 W. H. TISDALE (CooMtmUaa from Bbreau ol Plant Isdnatisf) PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOOATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASHINOTON, D. C. wASKUMcrroN c oovenMMeMT f>gy FOR THB ASSOCIATIOW J. G. LIPMAN Director, New Jersey Agriculiural Experiment Station, Rtitffers College W. A. RILEY Entomologist and Ckiif, Dtvtsion of Ettto- mology and Economic Zoology, Agricul- tural Experiment Station of the University of Minnesota H. P. ARMSBY Director, Institute nf AnirruU SutrHion, The Pennsylvania State CoUege 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. JOINAL OFAGiaaiTDRAlRESEARCH Vol. XVI Washington, D. C, February 3, 1919 No. 5 PHYSODERMA DISEASE OF CORN By W. H. TiSDALE Assistant Pathologist, Cereal Investigations, Bureau of Plant Industry, United States Department of Agriculture INTRODUCTION In recent years the Physoderma disease of corn (Zea mays) has been reported as doing considerable damage in the southern part of the United States. The uncertainty as to the distribution of the disease and its economic importance, together with the lack of a knowledge of the life cycle and parasitism of the causal organism and the possibility of its becoming a serious pest in the Com Belt, led the Office of Cereal Investi- gations to undertake an exhaustive investigational study of the problem. This work was undertaken by the writer in December, 191 6. Since that time certain phases of the problem have been more or less completely developed, while others are in need of further study. HISTORY OF THE DISEASE Shaw (Sy in 1 91 2 reported the occurrence of the disease in India as early as 1910, and gave a short description of the causal organism. At the annual meeting of the American Phytopathological Society at Cleve- land, Ohio, 1 91 2, Barrett^ reported the occurrence of the disease in Illi- nois in 191 1. In a personal interview Barrett stated that he received specimens of diseased com from Ohio and North Carolina. Barre (j, p. 23) states that the disease was known to be present in South Carolina as early as 191 1,. and since that time has been doing considerable damage. Reports of the occurrence of the disease in Georgia in 1910 have come to the writer indirectly, but he has never been able to confirm them. There is no reason, however, to doubt its cocurrence in Georgia at that time, since it is now known to be so widespread throughout the country. Prof. J. M. Beal, of the Mississippi Agricultural College, noted the disease in Mississippi as early as 1914. Mr. A. P. Spencer, of the Florida Agricul- tural College, claimed that considerable damage was caused by it in Lake County, Florida, in 191 5. In the summer of 191 5 Melchers (d) collected ' Reference is made by number (italic) to "Literature cited," p. 154. 2 Barrett, J. T. physoderma zeae-maydis shaw in Illinois. Not published. Reference to title only in Phytopatholoay, v. 3, no. i, p. 74. 1913. Journal of Agricultural Research, Vol. XVI, No. 5 Washington, D. C. Feb. 3, 1919 rg Key No. G-163 (137) 138 Journal of Agricultural Research Vol. XVI, No. s specimens of the disease at Manhattan, Kansas, but, not being sure of its identity, kept the specimens until 191 7 before making his report. A number of farmers throughout the South have told the writer that the disease has been present on their farms for many years. It was no doubt present in this country a long time before being reported by pathologists. DISTRIBUTION AND PREVALENCE Tn the third issue of the Plant Disease Survey Bulletin (9, p. 52), Sep- tember 15, 1 91 7, the writer published a map showing the known distribu- tion of Physodermazeae-madyis and the localities in which there was notice- able damage. The disease at that time seemed to be rather thoroughly Fig. -Map showing the distribution of Physodertna zeae-^maydis in the United States. Broken lines, P. zcae-maydis present: solid line, P. zeae-maydis causing damage. distributed throughout the Southern States as far north as North Carolina and Tennessee and west to the Mississippi River. Only single instances of its occurrence were known in Illinois and Ohio. Melchers ((5) since that time has reported its occurrence in Kansas as early as 191 5. In Sep- tember and October, 191 7, in cooperation with the Office of Plant Disease Surv^ey, a detailed study was made of the prevalence and importance of the disease in representative localities of the infested area of the South- east, not including Florida. The survey was also extended to deter- mine the distribution of the disease throughout the United States. The disease was found to be prevalent practically throughout all localities where the detailed survey was made, very few fields being entirely free from it. The writer found diseased corn plants on the Blue Ridge Mountains of North Carolina at an elevation of about 3,000 feet. How- ever, it was much less abundant than on the lowlands of the State. In the more extensive surv^ey the disease was found to occur in Arkansas, Feb. 3, 1919 * Physoderma Disease of Corn 1 39 Delaware, Indiana, Kentucky, Maryland, Minnesota, iMissouri, Nebraska, New Jersey, Oklahoma, South Dakota, Texas, Virginia, and West Vir- ginia. It was also found in other sections of Kansas, southern Illinois, and Ohio. The disease, however, has not yet been found beyond the eastern part of Texas and Oklahoma, northward to southeastern South Dakota and Minnesota. Likewise the northern border of the infested zone extends from southern South Dakota and Minnesota through southern Illinois, Indiana, Ohio, West Virginia, and Virginia, and along the coast regions of Maryland, Delaware, and New Jersey (fig. 1). The disease is apparently much less prevalent in the area west of IMississippi, and north of North Carolina and Tennessee. The nature of the survey, being less intensive, may be responsible to a certain extent for this con- clusion. It is possible that the disease has spread almost, if not quite, to its northern and western limits as permitted by certain weather factors. FXONOMIC IMPORTANCE The nature of the fungus causing the disease is such that its ability to produce serious injury to corn is limited by weather conditions, and in any ordinary season only local damage need be expected. How- ever, in certain humid sections of the South, where the time for com planting may extend over a period of three or four months in each year, weather conditions will more than likely be such as to favot a serious development of the disease on some of the plantings. This holds true especially for the South Atlantic and Gulf coasts and lower I\Iississippi Valley. Barre (2, p. 23) says: The com disease caused by Physoderma sp. as mentioned in the report last year has caused serious loss again this year. This disease was collected during the past season at a number of widely separated points in the state and seems to be more wide spread than ever before. Tliis disease certainly deserves some attention and it is hoped that an investigation of its life history and habits can be undertaken in the near future. The disease w^as known to be causing loss to the corn crop in Florida and Mississippi in 191 5. During the survey of 191 7 the most pronounced losses were found along the Atlantic and Gulf coasts and in the Mississippi Valley. In the lowlands of North and South Carolina, and in the Gulf and Delta sections of Mississippi, frequent reports of as much as 5 per cent loss were given by the sur\^ey men. In some cases the damage was estimated at 6 to 10 per cent of the crop. The v/riter visited a few fields in the eastern part of South Carolina where the damage was perhaps as much as 10 per cent. Fields of this kind, however, were seldom found. These estimates were based entirely on grain loss, whereas the foliage, v.hich is not considered of great importance except where the plants are used for silage, etc., was often badh' injured. Smaller areas sustaining con- 140 Journal of Agricultural Research ' voi. x\^, No. 5 siderable damage were reported throughout the Southeast and as far north as the Mississippi Valley sections of southern Illinois and Missouri. These areas were usually very limited and were confined for the most part to low, wet lands. Considering the infested area as a whole, how- ever, the percentage of damage, at least in the year 1917, was not very great. In 1918, up to July 15, the disease had not developed to any great extent in the South. This fact was due, no doubt, to the very dry season in that section, which will be discussed later. However, mid-July is not too late for considerable injury to develop should conditions so change as to favor the disease. FACTORS INFLUENCING THE DEVELOPMENT OF THE DISEASE Seemingly the more important factors in the development of the disease are moisture and temperature. The fungus requires considerable moisture, with a fairly high temperature, for a high percentage of germi- nation and infection. If these conditions are realized before the corn plants are more than half -grown, the disease probably will become severe if there is abundant spore material present. The following information regarding these factors has been noted : (i) Serious injury has been confined largely to the South, where the summer temperature is continuously high, and to localities in which there has been considerable rainfall during the early growth of the com crop. Plants may become infected in the later stages of growth, but the damage is not likely to be great in cases of this kind. The warm summer showers which may occur daily for a week or more furnish ideal conditions for the development of severe attacks by P. zeae-maydis. For instance, in the coast district of the Carolinas, where the disease was most severe in 191 7, there was considerable rainfall in early summer. This would also hold true for the Delta in Mississippi and for southwestern Tennessee, where there was considerable rainfall in early June. (2) Where the seasons were dry the disease was more pronounced on corn growing near water, or on low, wet land where the atmosphere was moist. Plants growing under these conditions are more likely to retain the sheath and bud water until the spores can germinate and produce infection. Conditions of this kind were noticeable at the South Carolina Station in 191 8, where corn on the low bottom lands had considerably more infection than highland corn. In the lowlands the foliage of plants is less subject to drying by winds. (3) Where the early corn season was dfy and the late season wet the disease was more severe on late com, and the reverse. Striking exam- ples of the former were noted at the Mississippi Station in 191 7, where there was less than 10 per cent infection on early corn and as high as 40 per cent infection on late corn, and at the Kansas Station, where early corn was almost free from the disease, while late corn showed considerable Feb. 3. 1919 Physoderma Disease of Corn 141 infection. The latter, however, might have been influenced by tempera- tures. Early corn at the Pee Dee Station at Florence, S. C, was dwarfed by dry weather in 191 8 and was not attacked to any great extent by the fungus. At Clemson College, S. C, very early and very late com sus- tained considerable injury in 191 7, while com of intermediate ages suffered much less damage. The midsummer was very dry at this station, while the early and late seasons were rather wet. (4) Apparently the more vigorous plants in certain cases sustain the severest attacks. These plants, however, will not continue to look vig- orous after the disease has had time to develop. The fact that these vigorous plants are capable of shielding the free water which is held behind the sheath and around the growing point, or bud, from the drying effects of the wind and sun offers more favorable conditions for spore germination and no doubt accounts for the greater percentage of infec- tion on plants of this type. In low, wet fields small plants are injured the same as large ones. This greater injury to large, vigorous plants was more noticeable in dry territory. (5) Where seasons were wet — for instance, in the sections where greater damage was done in 191 7 — there was httle noticeable difference in the amount of infection on corn growing on high and on low lands. At the South Carolina Station the most severe injury was caused to very early com grown on comparatively high land. As previously men- tioned, the early season was fairly wet at this point. (6) The disease was found on the Blue Ridge Mountains of North Carolina at an elevation of about 3,000 feet, where it is claimed that the summer nights are always cool. In 191 7, corn foliage was killed by frost on September 1 1 at this point. Very little of the disease was found at this elevation, however, even on wet lands. The disease is probably held in check to a certain extent by low temperatures which prevail at that elevation. A similar explanation was offered by the writer, in a summary of the survey work which was given by Lyman (4, 5), for the absence of serious injury by the disease in the Northern States. It was also thought probable at that time that the disease had reached its northern limits. This supposition was drawn from the results of temperature studies of the germination of sporangia in the laboratory. Since that time, however, further investigation has shown that the sporangia of the fungus will germinate at a considerably lower tempera- ture than was then supposed. However, the minimum temperature at which they are known to germinate is rather high (23° C.) as will be explained later, and it is probable that this temperature does not occur commonly during and immediately after the cold rains of early summer in the north. So far as is known at present, it would require a tempera- ture not lower than 23° C, continuously for three days, with sufficient surface water for germination, in order for severe attacks to develop provided the sporangia are present on the plants. There is a question 142 Journal of Agricultural Research voI.xvi.no. s as to whether these conditions are realized to any great extent in the Northern States, and it is hoped that the disease will not become a serious one in the Corn Belt. (7) The rare occurrence or absence of the disease farther west is no doubt due to the semiarid conditions which exist there. The moisture requirements suitable for the development of an epidemic of the disease perhaps are seldom, if ever, realized in this section. However, further investigations are needed to determine in detail what the weather condi- tions are for the given sections and to study the possibilities for further development and spread of the disease in the Com Belt. HOSTS So far as is known, all varieties of corn, including pop corn and sweet corn, are susceptible to the disease. Of the numerous varieties obsei-ved in the South there seems to be little, if any, difference in the degree of susceptibility shown by them. P. zeae-maydis also occurs on teosinte (Euchlaena mexicana), a near relative of the corn plant. It is possible that the disease was introduced from Mexico or Central America with this plant. The fact that it has been found in considerable quantities on corn in comparatively isolated fields where corn was never grown before and where no corn products were applied to the land suggests the possi- bility that there are other hosts for the fungus among wild plants. SIGNS OF THE DISEASE The disease occurs on the blade (PI. A), sheath, and culm (PI. B),and in rare cases it has been seen on the outer husks of the ears. Infection is usually more abundant on the lower half of the plant. Its first appear- ance on the thin parts of the blades resembles the early stages of the corn rust caused by Puccinia sorghi. It is first evidenced by slightly bleached or yellowish spots, which become darker within a few days when sporangia are formed. This darkening continues until the spots are brown to reddish brown, with a somewhat lighter margin. These spots are very small, seldom becoming more than i mm. in diameter, except where two or more of them coalesce. The spots may m some instances be so numerous as to give the entire blade a rusty appearance. For this reason the disease is often considered a true rust by persons who are not familiar with its nature. This rusty appearance is not uncommonly seen in bands across the blades, owing to the nature of infection, which takes place through zoospores in the bud water. On the midrib of the blade and on the sheath the spots become considerably larger. Often a single spot will measure 0.5 cm. across. They are irregular in shape and sometimes may be almost square in outline. This is due to the fact that they are definitely limited by the cell walls. In the very early stages these spots are evidenced by a color which is a somewhat darker green than the normal tissue surrounding them. This seems to indicate a Feb. 3. 1919 Physoderma Disease of Corn 143 stimulating effect caused by the presence of the invading fungus. A few days later these spots are dark brown in the center, owing to the forma- tion of the dark brown sporangia of the fungus. This change in color spreads until the entire spot is a dark or chocolate brown. These infec- tions are often so abundant as to coalesce, and sometimes the entire sheath may become brown (PI. 10). Where the infections are as numer- ous as this, the entire leaf often is killed before the plant is mature. However, the disease is usually more abundant on the parts of the sheath which are beneath the overlapping parts where the moisture is held. The disease often is accompanied by a reddening of the sheath and mid- rib, and especially the latter, which may almost entirely mask the brown spots. After the plants begin to mature, the epidermis becomes loose over these areas and they appear as brown blisters. This dry epidermis breaks easily and the spores are liberated as a brown spore dust. The entire parenchyma tissues of the invaded parts are destroyed by the disease, leaving the vascular system as so many free threads after the spores have been liberated (PI. 11, C, E). On the culms the spots are very much like those on the sheath and midrib. They are usually more abundant at the nodes and just below the nodes, where spores are more hkely to lodge and where free water is held by the sheath. The culms often are completely girdled at the nodes and are very easily broken by the winds after the tissues have been invaded. The disease is responsible for considerable lodging of corn in the South in the early stages of matur- ity. Only the lower nodes as a usual thing become so thoroughly in- vaded by the fungus as to be easily broken (PI. ii,A,B), Considerable damage may result from severe attacks of this kind. After the plants have fallen, the pith at the infected nodes will be found to be filled with a brown mass of spore material (PI. 1 1 , D) . The signs of the disease on teosinte (Eicchlaena mexicana) are very similar to those on corn, and therefore a separate description will not be necessary. The pronounced signs of the disease have led farmers to apply various significant terms to it in the way of common names. The writer has heard the following names applied to it: " Rust," "corn measles," "corn pox," "dropsy," "frenching," and "spot disease." None of these terms, however, is in general use, and some of them — for instance, "rust" and "frenching" — would be incorrect, as corn is known to be affected by other distinct diseases called by these names. The name "falserust" has been suggested as a desirable common name for the disease. There would be a strong tendency, however, on the part of the layman to drop the word "false," thus causing a confusion with the true rust. Further- more, the disease on the sheath and the culm bears very little resemblance to a rust. Since no satisfactory term suggests itself at present and since the scientific term " Physoderma," seems to be gaining favor as a common name, the author suggests that this tenn be retained. 144 Journal of Agricultural Research voi. xvi.no. s CAUSAL ORGANISM There still remains some doubt as to the correctness of the classification of the causal organism. According to the description of Cladochytrium and Physoderma as given by the leading mycologists, the organism evidently belongs in one of these genera. The essential difference between the two genera lies in their method of reproduction. The genus Clado- chytrium may have both thick-walled sporangia, or so-called resting spores, and thin-walled sporangia, or presporangia, while the genus Physoderma is characterized by having only thick-walled sporangia (resting spores). As the species on corn is not know^n to produce the thin-walled sporangia, its thick -walled sporangia definitely place it in the genus Physoderma. Shaw's (iiitjp>>t''.'.>ili^'**^*>^^ d % Journal of Agricultural Research /. Vol. XVI, No. 5 Physoderma Disease of Corn a. Plate 15 / Y h A d ''%^ ^-/->.*?'ii*v.".53iWWrtv%;g ^M^' ""'^^ ^ '-•/ Journal of Agricultural Research .9 Vol. XVI, No. 5 PLATE IS Physoderma zeae-maydis: Mycelial stages within the host cells. a-d. — Drawings from ordinary high-power m^nifications showing the fibers and enlarged cells of the mycelium. e-g. — Drawings magnified with oil-immersion lens. b, d, g. — Notice the yoting sporongia at the ends of the short hyphae. PLATE 1 6 Physoderma zeae-^maydis: a-€, Mycelial fibers penetrating the cell walls of the host tissue. /, g, Different types of reproductive bodies. Notice the double nucleate condition in figure g. Physoderma Disease of Corn Plate 16 CO b % « ^ y V <^- .-^ .^O! ■:^'*vi ...^^^ <\ ^^ ^ s / ■'/' Journal of Agricultural Research Vol. XVI. No. 5 Physoderma Disease of Corn PLATE 17 Journal of Agricultural Research Vol. XVI, No. 5 PLATE 17 Physoderma zeae-maydis: Photomicrographs showing the different stages of the devel- opment in the host tissue (teosinte). A. — Notice the reproductive bodies connected by the very fme threadlike hyhae in the central cells of the figure. B. — Host cells filled with matiu"e sporangia. ADDITIONAL COPIES OF THIS POBUCATION MAT BE FROCURED FBOU THE SUPEHINTENBENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 20 CENTS PER COPY Subscription Price, Per Year $3.00. A Vol. XVI KKBRUARY lO, 1919 No. <5 JOURNAL OF AGRICULTURAL RESEARCH CONTENTS Pag* Injtiry to Casuarina Trees in Southern Florida by the Mangrove Borer - - - - - - - 155 THOBIAS E. SNYDER (Contribntlon (rom Boraso ol Bntomolocy) Life-History Observations on Four Recently Described Parasites of Bruchophagus funebris - - - - 165 THEODORE D. URBAHNS (C«attlbaaoa ttom Boreau ol Entoxoology) PUBLISHED BY ACTHOMTY OF THE SECRETARY OF AGRICULTURE. WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS VSTASHINGTON, O. C. WASHIHOTOH : OOVERNyENT PRINnfiQ OFFICE t ISIS m M EDITORIAL COMMITTEE OF THE tTNTTED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THB DEPAXTMSIVT KARL F. KELLERMAN, Chairman Physiologist and Associate Chief, Bureau of Plant Industry EDWIN W. ALLEN Chief, Office of Experiment Stations CHARLES L. MARLATT EtUomolosist and A ssistont Chief, Bureau of Entomology FOR THB ASSOCIATIOn J. G. LIPMAN Director, New Jersey A ffriculturcU ExperimetU Station, Rutgers College W.A.RILEY Entomologist and Chief, Division of Ento- mology and Econontic Zoology, Agricut-. tural Experiment Station of the Unhersi^ of Minuetota H. P. ARMSBY Director, Institute of Anitrud Nutriticm, The Pennsylvania State College All cwrrcspondence 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 Statbns should be addressed to J. G. Lipman, New Jersey Agricultural Experiment Station, New Brunswick, N. J. • JOim OF AGRICCITIIAL RESEARCH Vol. XVI Washington, D, C, February io, 1919 No. 6 INJURY TO CASUARINA TREES IN SOUTHERN FLORIDA BY THE MANGROVE BORER By Thomas E. Snyder Specialist in Forest Entomology, Forest Insect Investigations, Bureau of Entomology, United States Department 0/ Agriculture INTRODUCTION In southern Florida many thousand casuarina, or "Australian pine," trees (Casuarina equiseiijolia Forster) have been and are being planted for shade and ornament along roads and avenues, on reclaimed swamp land, on golf courses, along the seashore, and as windbreaks for fruit trees (PI. 18, A). The tree makes a rapid growth, is not affected by salt spray from the ocean, and is utilized for the same purposes as eucalyptus trees in California. It is indigenous to tropical Asia and Australasia and, in addition to southern peninsular Florida and the Florida Keys, it has been introduced throughout the West Indies and other tropical regions of North and South America. Reports of serious injury to casuarina trees in Florida by a bark- and wood-boring insect {Chrysohothris tranquebarica Gmelin) * led to special investigations by the writer which resulted in the discovery that this buprestid beetle was a common and destructive enemy of the red man- grove {Rhizophora mangle Linnaeus), and that, therefore, the mangrove was the source of the trouble affecting the casuarina trees. The fact that this beetle has so changed its normal habits as to attack and breed in a plant so different botanically from its common host, to- gether with the economic importance of this changed habit to property owners who have made extensive plantings of the casuarina, has ren- dered the subject of special scientific interest and practical importance. The first reports of insect injury to the casuarina came from Hobe Sound and Miami Beach in April, 191 6. These and other localities in southern Florida were visited by the writer in May, 1916, March and April, 1917, and April and May, 1918, in order that a thorough investiga- tion of the insect, the conditions relating to its attack, and the methods of combating it might be made. • Determination by Mr. W. S. Fisher, Bureau of Entomology. Journal of Agricultural Research, Vol. XVI, No. 6 Washington, D. C. Feb. lo, 1919 rf Key No. K -7s (155) 156 Journal of Agricultural Research voi. xvi, No. e CHARACTER AND EXTENT OF THE INJURY It was found that the mangrove borer attacks only living red mangrove and casuarina. The casuarina trees attacked range from 2 to 6 inches in diameter; those over 5 years old usually are not attacked, except high in the tops or branches. Small casuarina trees are attacked near the base as a rule. In case of small trees the trunk may be girdled before the larvae attain their growth, and in most cases the damage is done before the presence of the insect is noticed. Many casuarina trees were killed at Miami Beach in 1915 (PI. 18, B) and more in 1916. The infestation in 191 7 at Miami Beach was apparently less than in 191 6, it having been estimated that among trees planted during the winter of 1916-17, within half a mile of the mangrove swamp, not more than i tree out of 20 was lost. In the mangrove swamp along Biscayne Bay many red mangrove trees were found in 191 6 to have been killed by the borer. In 191 7 a great accumulation of dead and stag- headed mangrove trees which had been gradually killed by the borer was noted, and many newly infested trees. In 191 8 many additional mangrove trees were found infested and it was noted that the infestation extended for many miles north of Miami. The dead trees and the stag-headed, partially killed trees, many of which are of large size, are strikingly evident against the sky line. At Hobe Sound, Jupiter Island, Fla., which is farther north than Miami Beach, quite a few casuarina trees were killed in 191 5; the trees are nearly 5 years old and, hence, not so liable to attack. At this locality the red mangrove is low and scrubby, being apparently too far north for favorable growth. In the swamps near by the borer was found in the red mangrove, but the infestation was not heavy. On the ocean keys or reefs south of Miami the red mangrove apparently is not infested by C. tranquebarica. At Adam Key, about 27 miles south of Miami, neither the red mangroves nor the casuarinas which have been planted there are infested, and no damage to mangrove by the borer has been noticed. On Key Biscayne, just south of Miami, there was for- merly a heavy infestation in the casuarinas, but the trees have now reached an age at which they are out of danger of further attack. In- fested red mangroves apparently do not occur in swamps continuously from Miami Beach to Hobe Sound ; therefore there are broken centers of infestation. No infested trees have been found south of Key Biscayne. STAGES, HABITS, AND SEASONAL HISTORY OF THE BEETLE Although C. tranquebarica was collected by Mr. H. K. Morrison at Key West in 1886 and by Mr. E. A. Schwarz on cordwood of red mangrove at the same locality in 1887 and although the beetle has been known to science since 1787, it appears that nothing has been recorded regard- ing its various stages, seasonal history, habits, etc. Feb. 10. I9I9 Injury to Casuarina Trees 157 Because of its thorough establishment in the red mangrove it is evi- dent that this beetle was not introduced into Florida with the casuarina; in fact, specimens had been collected at Key West before the casuarina was planted in Florida. The beetle's habitat is the West Indies, where the red mangrove tree is also native. In India the casuarina is a common tree, but the red mangrove does not occur. C. tranquebarica, despite its specific name,^ does not occur in India. Tranquebar is on the east coast of Madras. THE ADULT The adult of the mangrove borer (PI. 20, D; 21) is metallic greenish bronze and has two lighter-colored and one smaller basal impressions on each elytron. There are also impressions on the thorax. Adults can be told from those of any other speciesofChrysobothris found in the United States by the fact that the eyes are nearly contiguous on top of the head. The female is larger than the male, and the front of the head is green. The length ranges from 13.5 to 17 mm. The smaller, more active male ranges in length from 12.5 to 14 mm.; the front of the head is bright red. There are other sex differences in the last ventral segment of the abdomen (PI. 19, A) and, of course, in the genitalia. Adults of both sexes are fond of bright sunlight and are commonly found flying from 10 a. m. to 3 p. m. (central time) in open places in the swamps and on the casuarina trees. Oviposition takes place in either morning or afternoon. Both male and female beetles feed on the tender, succulent bark of the trees which they infest. They may be found resting on the trunks of trees in the bright sunlight chewing through the outer bark to the cambium. The beetles, owing to their rapidity of movement, strong powers of flight, and shyness, are probably able to survive enemies and live for two or three weeks, or possibly a month or so. They are difficult of detection when resting on the bark of red mangrove, but when flying in the sunlight they are conspicuous on account of the bright-green color of the body. The beetles are never active unless the day is warm, sunny, and not windy. As the beetles are strong fliers and are fond of flitting from one sunny tree trunk to another, and as they lay many eggs each, it is probable that one female may be responsible for the death of many trees. On April 13, 1918, in a mangrove swamp along Biscayne Bay, oppo- site Miami, Fla., females were found ovipositing at i.io p. m. (central time), and the operation observed. After a short exploration of the bark, made with extended ovipositor (PI. 19, B), a proper crevice was ' Fisher. W. S. Chrysobothris tranquebarica Gmel. vbrsus imprbssa Fabr. In Proc. Ent. Soc. Wash.. V. 20, no. 8, pp. 173-177 November, 1918 (1919). 158 Journal of Agricultural Research vo1.xvi.no.6 found under loose bark and the beetle remained with its ovipositor in the crevice for one and one-half minutes. During this time there was a perceptible pumping motion near the basal end of the ovipositor, and 4 eggs were laid in an irregular row. The tree is attacked anywhere from the large aerial roots to high up on the trunk, but usually in the middle trunk. THE EGG The egg (PI, 19, C) may be compared in shape to a scallop ohell, and one end, which is broader than the other and flattened, is irregularly ribbed. It is white and ranges from 1 to 1.5 mm. in length; the average width is approximately 0.75 mm. The red mangrove has the bark separated into plates; in the process of growth loose bark occurs at the dividing lines (PI. 19, C). The eggs are inserted under this thin outer layer of loose bark in an irregular longi- tudinal row. Four eggs are the largest number that have been found together. Eggs occur singly and in twos and threes. One female may lay eggs in several trees. Twenty-three full-sized eggs were dissected from one female, many eggs being in the distended oviduct, and many immature ovules were present. The period of incubation was not determined but probably one week is required. Young larvae 5.5 mm. in length were found on April 23, 1 91 8, in a red mangrove tree near Miami Beach. THE LARVA The larva^ is white and a typical " flatheaded" borer (PI. 20, B; fig. i). It is of the common Chrysobothris type, moderately compressed, and sparsely covered with coarse, light-colored bristles. The first thoracic segment is large and oval; the second wider and shorter than the third; the third wider than the first abdominal, which is narrower than the second abdominal ; the third to eighth abdominal are of about equal width, the ninth and tenth successively narrower; the lateral folds of the second to ninth abdominal segments are well developed ; the dorsal plate of the first thoracic segment is marked with a well developed, inverted V of grooves and pointlike rugosities; the ventral plate has a well devel- oped groove extending back three-fourths of the distance from the ante- rior margin, and rugosities which tend to form ridges. The length is 30 mm. and the width of the first thoracic segment 7 to 8 mm. The young larvae upon hatching from the eggs bore through the cambium to the surface of the wood and as they feed on the cambium and grow they extend the burrows horizontally, spirally, or longitu- dinally (Pi. 20, A). The entire length of the burrow is packed with boring dust. The length of the larval period is nearly one year. When ' Description by Mr. H. E. Burke, Bureau of Entomology. Feb. lo, 1919 Injury to Casuarina Trees 159 full grown or mature, the larva ranges from 29 to 35 mm. in length. At this stage it bores into the wood to a considerable depth and exca- '•-4. ■■t ^^■^ -^ ^^ Ji %^^^ -% ,-*^ Fig. J.—Chrysobothris tran^Hebarica: Larva, dorsal, lattraj, and ventral views. X 5. vates its pupal cell. A hole for the exit of the beetle is also excavated by the larva from the pupal cell to or near to the surface and is there i6o Journal of Agricultural Research Vol. XVI, No. 6 finally packed with coarse boring chips. In some large, heavily infested red mangrove trees as many as three pupal cells per linear 2 inches were found. THE PUPA The pupa is white and of the shape characteristic of buprestid pupae (PI. 20, C; fig. 2). It is of the common Chrysobothris type, with the head resting on the breast and the legs and wings folded on the ventral surface. The developing insect gradually acquires characters of the adult beetle. The size varies with the individual and there is also a sex difference; the length ranges from 15 to 20 mm. KiG. 2. — Chrysobothris tranquebarica: a, Female pupa, ventral view; b, same, dorsal view. The average duration of the pupal period is about two weeks. When the adult becomes mature it chews its way out through the plug of wood fiber, cuts an oval hole through the bark, and escapes. This hole is often mistaken by property owners for the point of entrance of the borer. SEASONAL HISTORY One year is required for the development of the mangrove borer from egg to adult. Adult beetles first begin to emerge about the ist of April. The period of maximum activity of th^ beetles on the wing is from the Feb. lo, I9I9 Injury to Casuarina Trees i6i middle of April to the ist of June, but a few stragglers are found as late as August. Most of the eggs are probably laid from the middle of April to June. The larvae seem to be full grown by August, and the majority form the pupal cells before winter. The species probably passes through a dormant period, or one of comparative inactivity, during the months of December, January, and February, as mature larvae under the bark or in the pupal cells. On March 19, 191 7, such mature larvae, together with pupae and immature adults, were found in infested trees at Miami Beach. On April 4, 191 7, many pupae were changing color, indicating that they would soon transform to the adult stage. In 191 8, on April 8, mature larvae, pupae, and adults were in pupal cells in infested trees at Miami Beach. The first eggs were found on April 13, 1918, and the first young larvae on April 23, at Miami Beach, in infested red mangrove trees. PREDATORY ENEMIES AND PARASITES The flicker {Colaptes auratus) and the red-headed woodpecker (Melan- erpes erythrocephalus) pick out larvae and pupae from infested trees, and often obtain a high percentage of the insects infesting a few trees. Pre- dacious beetle larvae account for other borers. On April 3, 191 7, larvae of a predacious trogositid beetle (Tenebroides sp.y were found under the bark of a red mangrove tree infested by C. tranqueharica, in a swamp near Miami Beach. On April 9, 191 8, in the same general locality, larvae of an elaterid beetle (Adelocera sp.)^ were found under the bark of a red mangrove tree infested with the beetle. Presumably they were predacious enemies of the mangrove borer. Two species of hymenopterous parasites have been found. One species, Atanycohis rugosiventris Ashmead,^ was found to be fairly com- mon at Miami Beach in 191 7 and 191 8. Its cocoons occur in a mass at the end of the larval burrow of the beetle. Adults were found emerging from the cocoons on March 19 and April 10, 1917, and on April 9, 1918. The other species, A. lahena n. sp.,^ constructs a single cocoon in the pupal cell of C. iranqtiebarica, in infested casuarina trees. Notwithstanding the numerous natural enemies of Chrysobothris tranque- harica it is evident that reliance can not be placed upon them to control this borer without help from man. CONTROL OF THE BORER In view of the large number of casuarina trees which have been and are being planted in southern Florida and the varied uses to which they are adapted, it will be seen that the problem of controlling this injurious borer is important. Since 191 6 owners of these large plantations have ' Detennination by Dr. Adam G. Bbving, Bureau sf Entomology. ' Determination by Mr. J. A. Hyslop, Bureau of Entomology. « Determination by Mr. S. A. Rohwer, Bureau of Entomology. 1 62 Journal of Agricultural Research voi.xvi, no. e been acting upon the advice of the Bureau of Entomology in efforts to prevent injury, but the problem is greatly complicated at Miami Beach by large areas of heavily infested red mangrove trees in near-by swamps. In 1916 and 1917, at Miami Beach, badly infested young casuarina trees were removed or topped, and borers were killed in the pupal cells by cutting them out. Some trees were sprayed with poisoned kerosene emulsion. Supporting stakes of red mdngrove were removed. In 191 7 the infestation appeared to have been reduced, but in 1918 it was again severe. In the red mangrove swamps there appeared to be a steady yearly increase of infestation. The infestation at Hobe Sound, the farthest north that C. tranqueharica has yet been found, has not been so severe. The casuarina trees are new (May, 191 8) about 5 years old and of large size. In May, 191 6, when these trees were younger and the injury more severe, the trunks were thoroughly and repeatedly sprayed with the poisoned kerosene emul- sion. About 900 casuarina trees growing in avenues were sprayed at a cost of approximately 10 cents per tree. As the old formula, used at this time, contains a larger proportion of sodium arsenate than is neces- sary, the cost per tree can be lowered. The outfit consisted of three men and a team of mules to haul the standard orange-tree spray pump. Almost any good spraying outfit, however, would answer the purpose of spraying the trunks of small trees. In addition to spraying, the rough bark at the bases of trees at Hobe Sound was scraped and the borers killed by cutting them out of the pupal cells. The infestation of 191 6 was less and there was a still further decrease in that of 191 7, after the use of the same control methods, A few borers were still found in the tops of the casuarina trees in 191 8 but these have been cut out. The infestation in the low scrubby red mangrove tree here is not and has not been heavy. METHODS RECOMMENDED FOR COMBATING THE INSECT Investigations have shown that many trees can be saved by carrying out the following methods of control : All badly damaged casuarina trees should be cut and burned between September and March to kill the insects before they emerge. The trees may be entirely removed, cut off near the ground, or merely topped so that they will sprout from the stump and make new growth. Since the borer usually attacks the young trees near the base, where there are rougher bark and more suit- able places for egg laying, care should be exercised that no infested stumps remain. Trees only slightly damaged and showing evidence, in the rapidly healing wounds, of recovery should not be cut. The wounds will soon heal, and as the trees grow will disappear. Casuarina trees between iK and 6 inches in diameter, growing in prox- imity to mangrove swamps or near other infested casuarina trees, should be examined carefully in September and March and the young larvae Feb. lo. 1919 Injury to Casuarina Trees 1 63 killed by spraying the affected part of the trunks with poisoned kerosene emulsion ^ made in accordance with the following formula, recently revised by Mr. F. C. Craighead : Standard miscible oil pint i Water gallons 5 Sodium arsenate poimd .... X Dissolve the arsenate in water, stir, then add i pint of miscible oil. From April to June, when large numbers of the adult beetles are flying and feeding on the bark, they should be killed by spraying the tree trunks with the poisoned kerosene emulsion. No pruning of casuarina trees should be attempted between April and August, since the consequent flow of sap will attract the flying beetles to the trees. Mangrove stakes should not be used to support young, recently set- out trees, as they will attract the borers. According to the host-selection principle ^ as advocated by Dr. A. D. Hopkins, the beetles that breed for one or two generations or more in the casuarina will be much more likely to reinfest this host than they are to go back to the original host; and, since the beetle became established in the mangrove before the casuarina was introduced, it is to be expected that only occasional individuals, among the thousands of beetles that breed in the mangrove, will deposit eggs on the casuarinas. It is of primary importance, therefore, to keep as many of the beetles as possible from reaching maturity in the casuarinas. 1 Craighead, F. C. a new mixture for coNTRoi,LrNG wood-boring insects — sodium arsenate EMULSION. /« Jour. Econ. Ent., V. 8, no. 6, p. 513. 1915. * U. S. Department OF Agriculture, program of work [igibj/igij, p. 353. Washinfiton, 1916. PLATE i8 A.— Casuarina trees planted along the water front, Belle Isle, Miami Beach, Fla., June, 1918. Photographed by W. E. Brown. B.— Casuarina trees disfigured and killed by the mangrove borer {Chrysobothris tranquebarica) at Miami Beach, Fla. (164) Injury to Casuarina Tree^; Plate 18 Journal of Agricultural Research Vol. XVI. No. 6 Injury to Casuarina Trees Plate 19 Journal of Agricultural Research Vol. XVI, No. 6 PLATE 19 Chrysobothris tranquebarica: A. — Sex differences in the last abdominal segment. X 9. Drawn by E. Armstrong. B. — Lateral and dorsal view of ovipositor. X 9. Drawn by E. Armstrong. C. — Bark of red mangrove (Rkizophora mangle) showing how it is divided into plates. Natural size. The eggs are superficially inserted under the thin outer layer, where the bark is loose, at a crack. Eggs X 4- PLATE 20 Chrysobothris tranquebarica: A. — Larval burrow in cambium of Atistralian pine (Casuarina equiseiifoUa), Miami Beach, Fla. Note how the biurow is packed with frass, the exit hole and the cam- bium growing over the wound. Natural size. B. — Larvae, ventral and dorsal views. X 3. C. — Pupa, dorsal and ventral views. X 2}4- D. — Female and male adult beetles. X 2^- Photographed by Mr. William Middleton. Injury to Casuarina Trees PLATE 20 Journal of Agricultural Research Vol. XVI, No. 6 Injury to Casuarina Trees Plate 21 Journal of Agricultural Research Vol. XVI, No.G PLATE 21 Chrysobothris tranqiiebarica: Adult male, dorsal view. X 7. LIFE-HISTORY OBSERVATIONS ON FOUR RECENTLY DE- SCRIBED PARASITES OF BRUCHOPHAGUS FUNEBRIS By Theodore D. Urbahns Entomological Assistant, Cereal and Forage Insect Investigations, Bureau of Entomology f United States Department of Agriculture INTRODUCTION The parasitic Hymenoptera referred to in this paper were observed, together with others, while the writer was making a detailed study of the chalcis-fly Bruchophagus funebris infesting the seeds of alfalfa (Medicago saliva) and red clovter {Trijolium pratense). Observations and notes were made concerning the life habits of these new parasites, as the opportunity presented itself, to determine any economic value which one or more of these species may have in the control of Brtichopliagus funebris. LIFE-HISTORY SUMMARY OF THE HOST The host insect, Brtichophagus funebris Howard, completes its develop- ment from the egg to the adult stage within the seed of alfalfa, red clover, or wild species of Medicago. Upon reaching maturity the adult gnaws an opening through the seed shell and makes its escape. B. funebris hibernates in its larva stage within the infested seeds remaining upon the field. It passes through several generations in a single season. METHOD OF STUDYING THE PARASITES In order that the development of these parasites could be observed, it became necessary to dissect several thousand alfalfa seeds under the microscope, locate parasite larvae, and remove them together with their host for study. Each host, with its parasite, was then transferred to a single little cage consisting of an 8-mm. cork with a small cavity in one end and covered by a medical capsule. Here each parasite could be observed from day to day until it had completely destroyed its host, developed, passed through the pupa stage, and transformed to the adult stage. LIODONTOMERUS PERPLEXUS GAHAN The two species of Liodontomerus discussed in this paper belong to the hymenopterous superfamily Chalcidoidea, family Callimomidae, and sub- family Monodontomerinae. The genus Liodontomerus was erected by Mr. A. B. Gahan,^ of the Bureau of Entomology, for specimens of Liodon- ' Gahan, a. B. descriptions of new genera and species with notes of parasitic hymenoptera. /« Proc. U. S. Nat. Mus., v. 48, p. 155-168, Dec. 16, 1914. p. 159: Liodontomerus. new genus. Journal of Agricultural Research, Vol. XVI, No. 6 Washington, D. C. Feb. 10, 1919 rd Key No. K-7S (165) 1 66 Journal of Agricultural Research voI.xvi,No.6 tomerus perplexus Gahan reared by the writer from Bruchophagus funebris infesting alfalfa seeds at Yuma, Arizona. Liodontomerus perplexus was first reared by the writer from alfalfa seeds collected at Yuma, Ariz., during August, 1912. It was again reared Sep- tember 20, 1912, from El Centro, California, and from Chino, Cal., on November 4, 191 2. Infested alfalfa seeds dissected and subjected to a microscopic examination soon showed this species as being parasitic upon Bruchophagus funebris. In the year 191 3 it was first reared on July 19 from Corcoran, Cal.; on July 25, from Glendale, Cal.; and in 1914 the first rearing dates from new localities were July 24, Brawley, Cal., and September 8, Red Bluff, Cal. On August 4 it was reared from B. funebris infesting bur clover {Medicago hispida nigra) at Tulare, Cal. Examinations of various chalcids reared by different members of the Bureau of Entomology from alfalfa seeds infested by Bruchophagus fune- bris showed that Liodontomerus perplexus was reared by C. N. Ainslie at Newell, South Dakota, November 15, 1913, and at Mitchell, S. Dak., in 1914. A single specimen was labeled "Red Oak, Iowa." Liodontomerus perplexus was described by Mr. Gahan as a new species * from the type specimens reared by the writer from Bruchophagus funebris infesting alfalfa seeds at Yuma, Arizona, in August, 191 2. STAGES OF HOST SHOWING PARASITISM Liodontomerus perplexus is primarily parasitic upon the larva stages of Bruchophagus funebris. It feeds externally upon its host and frequently destroys the entire host larva with the exception of the head. In excep- tional cases this parasite has been found to be parasitic upon the pupa stage of B. funebris. Of 97 larvae of L. perplexus under observation 86 were parasitic upon the larva stage and 9 upon the pupa stage of their host. A single specimen of this species was found to be a secondary para- site and feeding upon the larva of a different species after the latter had destroyed the host larva. HIBERNATION The larvae, which become fully developed late in the summer, or in the fall, mostly hibernate until the following spring. This takes place within the alfalfa seeds in which the host was attacked. While hibernation is normal in the larva stage, occasional individuals have been observed to hibernate in the pupa stage under the mild climatic conditions of the Southwest. APPEARANCE IN THE FIELD This species does not seem to appear in the field in large numbers as early in the season as might be expected. In southern California and western Arizona it becomes active in April and slowly increases in numbers throughout May. In August the abundance of adults is probably great- est, while a few individuals may be found as late as November (Table I). 'Gahan, A. B., op. cit., p. 159. Feb. lo, 1919 Life History of Parasites of Bruchophagus funebris 1 67 Table L.— Dates of emergence of adults of Liodontomerus perplexus which developed from larvae spending the uinier in hibernation March 1-15 March 16-31 April 1-15 April 16-30 May 1-1$ May 16-31 June 1-15 June 16-30 July i-is July 16-31 August I-] August 16-31 September 1-15 Percentage OVIPOSITION The adult female locates the green and tender seed pods of alfalfa in which seeds are infested by lar\-se of Bruchophagus funebris, inserts the ovipositor through the seed pod, and deposits an egg upon or near the host larva within the green seed. I..\RV.\ Development.— The different larval instars were not studied by the writer through lack of time for this particular subject. Field and lab- oratory observations, however, showed that the larvae develop very rapidly upon their host and under favorable conditions require from 8 to 12 days to make their growth. They do not always transform to the pupa stage as soon as they become full grown. Dormant period.— After the larva of Liodontomerus perplexus has become fully developed upon its host within the alfalfa seed it may at once enter the pupa stage, but if the seed is exposed to dry climatic con- ditions a dormant period in the larva stage frequently follows. This dormant period may begin at almost any time throughout the summer and continue right on into hibernation. Transformation to the pupa stage is then delayed until the following spring. In the laboratory a few larvae that became dormant in the summer continued so throughout the following winter and through the next summer, and hibernated again the second winter before transforming to the pupa. A hibematmg larva was taken from the field on December 18, 1913- It remained m the larva stage until March, 191 5, then transformed to a pupa, and emerged as an adult on April 19. 1915- This particular habit is undoubt- edly of great value to the species and enables it to be carried over the long continued dry seasons of the desert sections of the Southwest. 98354°— 19 2 i68 Journal of Agricultural Research Vol. XVI, No. 6 Description. — ^The larva of Liodonlomerus per plexus (fig. i) varies in color from white to smoky gray. The length averages 1.5 mm. and the thickness averages 0.7 mm. The general appearance is grublike, while a side view shows the general shape, suggesting an interrogation mark. The head of the larva shows the eye lobes and on each a small tubercle. The front of the head contains about eight fine setae. Mandibles, slightly chitinous, are usually inconspic- uous, but sometimes distinctly visible. Segmentation of the 13 body segments is very marked. The body is covered with bristle-like setae which are from 0.04 mm. to 0.1 mm. in length. Two rows, and a broken third row, are present in the first segment. The second and third segments each bear one row with a broken second row. The other segments each bear one row encircling the segment. Setae on the dorsal portion of the body are much coarser than those of the ventral side. The last segment is dorso-ventrally bilobed and bears setae on each of the lobes. Fio. I. — Liodonlomerus perplexus: Larva. PUPA Pupation. — After the pupa has completed its development within the larval skin the latter breaks open along the antero-dorsal margin and is slowly worked back to beyond the tip of the abdomen by a slight movement of the newly formed pupa. Descpjption. — The pupa (fig. 2) is white when newly formed. It is about 1.5 mm. long and 0.5 mm. thick. The eyes are at first white, but after a few days turn to pale brown. The head and thorax bend slightly forward. The antennae, legs, and wing pads are folded close to the body and the ovipositor sheath is bent back across the end of the abdomen. In the last few days of the pupa stage the pupa turns almost black, with dark-brown eyes and pale-brown antennae, legs, and ovipositor. Length of pupal period. — The length of the pupal period varies greatly according to the season during which -pupation occurs. Hiberna- ting larvae under observation began entering the pupa stage as early as March; others did not pupate until July and August, and a few remained in the larva stage until the following year before pupating. Twenty-six pupae, which proved to be males, averaged 23.7 days in the pupa stage; and 31 pupae, which proved to be females. Fig. 2. — Liodonlomerus perplex- us: Pupa. Feb. 10, 1919 Life History of Parasites of Brtichophagus funebris 1 69 averaged 27.9 days in that stage. The longest pupal period observed was 45 days and the shortest was 8 days. These observations were made in the laboratory under natural temperatures. It is very probable that under the most favorable field conditions the pupal period may require even less time than the minimum period recorded. ADULT Emergence. — ^The adult (PI. 22, A), upon emerging from the pupal skin, finds itself surrounded by the thin seed wall and within the alfalfa seed pod. It proceeds at once to gnaw a small irregular opening through the seed in which the host has been destroyed, then through the seed pod, and thereupon escapes. ReIvATive proportion of sexes. — Both sexes of this species seem to be well represented in all of the localities from which specimens were reared. A count made of 859 adults showed 121 to be males and 738 females, or a ratio of i to 6.92. AduIvT variation. — Some adults of this species vary from the true type in that they show a stigmal cloud in the forewing. In a few individuals this clouded area was very conspicuous. SEASONAL HISTORY Observations show that about 30 days, under very favorable conditions, are required for the complete development of a single generation and that in alfalfa seed fields of Arizona and southern California there may be as many as three generations in a single season. Other individuals sub- jected to different local conditions may require an entire season for their development. PARASITIC IMPORTANCE This species appears to be a parasite of considerable economic impor- tance in helping to reduce the ravages of Bruchophagiis funebris in alfalfa seed throughout the western Arizona seed-gromng districts. It is appar- ently not present in sufficient numbers throughout the California, Idaho, and Utah seed-growing sections to be of value in reducing the destructive work of the seed chalcis-fly. LIODONTOMERUS SECUNDUS GAHAN Ltodontomerus secundus was first collected by the writer on September 5 1 91 4, at Albany, Oregon, where it was found ovipositing in the green ovaries of florets on red-clover heads. On September 1 6 the writer reared specimens from red-clover seeds infested by Brtcchophagus funebris at Caldwell, Idaho; and on September 23 it was reared from infested red-clover seeds taken at Albany, Oreg. Microscopic examination of the seeds showed that this species was parasitic upon the larvae of B. funebris. It was also pres- ent among chalcids reared from red clover in 1915 at Elk Point, South Dakota, by C. N. Ainslie. lyo Journal of Agricultural Research Vol. XVI, No. 6 Liodontomerus secundus was described as new by Mr. A. B, Gahan ^ from specimens reared by the writer from infested red-clover seeds taken at Caldwell, Idaho. HIBERNATION Examination of red-clover seeds infested by Bruchophagus funebris revealed larvae of Liodontomerus secundus hibernating in the lar\^a stage within the seeds which had been destroyed by their respective host larvae. The hibernating larvae pupated in the months of April, May, and June, spending from 24 to 40 days in the pupa stage under laboratory conditions before emerging as adults. LARVA The larva (fig. 3) is smoky white in color and averages 1.72 mm. long and 0.8 mm. in thickness. The general shape is grublike. The head, of medium size, shows the eye lobes with a tubercle apparently more distinct in this species than in L. per- plexus. Pointed mandibles show a slight tinge of brown. The body is covered with pubescence about o. 055 long, the pubescence longest on dorsal portion of first tv,o body segments. Pubescence finer than on larvae of L. perplexus. PUPA The pupa (fig. 4) is white when newly formed, but later it turns smoky white and finally brownish black. The eyes turn brown. It averages 1.6 mm. long. The sheath of the ovipositor is folded around the end of the abdomen and back half wa}' along the dorsal side. ADULT The adults (PI. 23, B) emerge from the infested red-clover seeds in spring. Some continue to emerge from the old seeds as late as July. They apparently have two or more generations in a single season and are active in the fields until late in fall. Observations show that both sexes are well represented in localities where the species was taken. Fig. 3. — Liodontomerus secun dus: Larva. Liodontomerus secun- dus: Pupa. ' Gahan, A. B. descriptions op some new parasitic hymenopter.\. In Proc. U. S. Nat. Mus., v. 33, P.t95-2i7. May 26, 1917. p. io8: Liodontomerus secundus, new species. Feb. lo, 1919 Life History of Parasites of Brttchophagus funehris lyi EUTELUS BRUCHOPHAGI GAHAN Eutelus bruchophagi belongs to the superfamily Chalcidoidea, family Pteromalidae, and subfamily Pteromalinae. This insect was first reared by the writer from alfalfa seeds infested by Bruchophagus funehris, collected at Blackfoot, Aberdeen, and Caldwell, Idaho, and from Nephi, Gunnison, Manti, and Salt Lake City, Utah, in September, 1914, and from Susanville, California, on September 12, 1917. Mr. T. R. Chamberlin, of the Bureau of Entomology, reared males of this species from alfalfa seeds collected at Salt Lake City in June, 1 914. Upon careful examination of the infested seeds this species was soon found to be parasitic upon B. funehris. Specimens reared by the writer at Nephi, Utah, were described by Mr. A. B. Gahan, of the Bureau of Entomology, as a new species.^ HIBERNATION Fig. s. — Eutelus brucho- phagi: Larva. Fig. 6. — Eutelus bruchophagi: Pupa. Eutelus hruchophagi hiber- nates in the larva stage within the infested alfalfa seeds and seed pods remaining on the fields. The warm spring days hasten pupation, and a few weeks later the newly formed adult gnaws an opening through the seed wall and makes its escape. LARVA The lar\^a (fig. 5) is grublike in appearance and averages 1.5 mm. in length. Its body is white with a glossy surface free from pubescence and having a clouded appearance under the epidermis. The head is of medium size and the eye lobes rather shallow. The setae on the eyes are distinctly visible. The anal segment is bilobed and shows three very fine setae. PUPA The pupa (fig. 6) is white when newly formed and after a few days shows the eyes turning to salmon brown. It averages 1.5 mm. in length, and turns black before changing to the adult stage. ADULT The adults (PI. 22, B) live for one or two months under favorable condi- tions and locate, for oviposition, on the newly-forming seed pods of alfalfa 'Gaham, a. B. op. cit., 1917, p. 2ia. 172 Journal of Agricultural Research Vol. XVI, No. 6 plants which have become infested by Bmchophagus funehris. Appar- ently there are at least two generations in a single season. The specimens reared showed a much larger percentage of males than of females. TIMEROMICRUS MACULATUS GAHAN Trimeromicrus mactdatus (PI. 23, A) belongs in the hymenopterous superfamily Chalcidoidea, family Pteromalidae, and subfamily Ptero- malinae. Specimens reared by the writer were determined by Mr. A. B. Gahan of the Bureau of Entomology as belonging to a new genus. The genus Trimeromicrus was therefore erected by Mr. Gahan ^ for this species. This species was first reared by the writer from alfalfa seeds infested by Brtichophagus funehris and taken at Yuma, Arizona, in September, 1 91 2. Larvae of this species were later dissected from alfalfa seeds where they were found to be parasitic upon the larvae of B. funehris. It was later reared from the following localities : El Centre, Cal., September, 1912. Glen dale, Cal., September, 1912. Chino, Cal., November, 1912. Corcoran, Cal., July, 1913. Tulare, Cal., Jime, 1914. Red Bluff, Cal., September, 1914. San Diego, Cal., August, 1915. Susanville, Cal., September, 1917. Examination of the undetermined collections and the field notes made by different members of the Bureau of Entomology showed that this species was also reared from infested alfalfa seeds as follows : Tempe, Ariz., July, 1911, E. G. Sm3rth. Buckeye, Ariz., July, 1912, R. N. Wilson. Newell, S. Dak., August, 1913, C. N. Ainslie. Salt Lake City, Utah, September, 1915, T. R. Chamberlin. Mesilla Park, N. Mex., June, 1909, C. N. Ainslie. Sacaton, Ariz., June, 1909, C. N. Ainslie. Casa Grande, Ariz., June, 1910, V. L. Wildermuth. Wellington, Kans., August, 1910, E. G. Kelly. Brawley, Cal., March, 1911, V. E. Wilder- muth. Trimeromicrus maculatus was described as a new species by Mr. A. B. Gahan ^ from the specimens reared by the writer from Bruchophagus funehris infesting alfalfa seeds at Yuma, Arizona. HIBERNATION This species, like the others mentioned, hibernates in the larva stage within the infested alfalfa seeds. It frequently hibernates as early as September. In early spring the larvae change to pupae, remain so for about 30 days, and the adults emerge from the seeds by the time the new seed pods are forming. 1 Gahan, A. B. descriptions of new genera and species with notes of parasitic hymenoptera. In Proc. U. S. Nat. Mus., v. 48, p. iss-168. Dec. 16, 1914. p. 161: Trimeroinicrus, new genus. ' Op. cit., 1914, p. 162. Feb. lo. 1919 Life History of Parasites of Briichophagus funebris 1 73 LARVA The larva (fig. 7) varies from white to smoky gray, averages 1.6 mm. in length, and is somewhat grublike in general shape. The head is of medium size and faintly shows the eye lobes. Each eye lobe shows a very fine seta. The thirteen body segments are subequal, the first one back of the head being the largest and the others decreasing in size to the anal seg- ment, which is bilobed, the upper lobe containing three very fine setae. The dorsal portion of the first twelve body segments also shows indica- tions of very short and fine setae in some specimens. Parasitic habit. — The larva of this species was found to attach itself externally upon the larva of its host. In the course of a few days the host larva apparently dies and the parasite makes a rapid growth, feeding upon the body contents of the dead host. PUPA The pupa (fig. 8) is white when newly formed. It is 1.6 mm. long and about 0.6 mm. wide. The head is placed slightly for- ward and the appendages are folded close to the body. The entire pupa is enclosed in a thin pupal skin. During the last few days of the pupal period the pupa turns almost black. FlZZ-Trimeromicrusma- Fio. S.-Trimeromicrus Pupation.— The duration of «♦/«/«..- Larva. macuiatus. ■ fup^. the longest pupal period observed was 15 days and the shortest was 6 days. The average number of days in the pupa stage as observed in the laboratory was 9. RELATIVE proportion OF SEXES The localities from which this species was reared showed both sexes well represented. A count, made of 322 adults reared from various localities, showed 85 males and 237 females, or a ratio of i male to 2.67 females. ECONOMIC IMPORTANCE Trimeromicrus maculatus is apparently well established in the Yuma Valley of Arizona, where it was found to destroy about 7 per cent of the larvae of Bruchopkagtis funebris infesting alfalfa seeds. Apparently it is also well established in the Honey Lake Valley of northeastern California. PLATE 22 A. — Liodontomerus perplexus: Adult female. B. — Eutelus bruchophagi: Adult female. (174) Parasites of Bruchophagus funebris Plate 22 Journal of Agricultural Research Vol. XVI, No. 6 Parasites of Bruchophagus f unebris Plate 23 Journal of Agricultural Research Vol. XVI, No. 6 PLATE 23 A. — Trimeromicriis ttiaculatus: Adult female. B. — Liodonto-ynerus secundus: Adult female. ADDITIONAL COPIES OV THIS PUBUCATION MAT BE PROCTJllED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. 0. AT 10 CENTS PER COPY Vol. XVI KKBRUARY 17, 1919 No. 7 ^ JOURNAL OF AGRICULTURAIy RESEARCH CONTENTS Page Cyanogenesis in Andropogon sorghum - - - - 175 C. T. DOWELL (Contribution from Oklaboma Agricultural Experiment Station) Effect of Certain Compounds of Barium and Strontium on the Growth of Plants - - - - - - -183 J. S. McHARGUE (Contribution from Kentucky Agricultural Experiment Station) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASHINGTON, D. C. W«*HlMaTON I QOVERNMENT PRINTINO OFFtCe : I9I» ''Ai;' W^'^'^j. EDITOJRIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chah^man Physiologist and Associate Chief, Bfireau of Plant Industry EDWIN W. ALLEN Chief, Office of Experiment Stations I • CHARLES L. MARLATT Entomologist and Assistant Chief, Bureau of EntotHoiogy FOR THE ASSOCIATIOSr J. G. LIPMAN Director, New Jersey A oricuUural Etperiment Station, Rutgers College W. A. RILEY Entomologist and Chief, Division of Ento- Ttwlogy and Economic Zoology, Agricul- turcl Experiment Station of the University of Minnesota H. P. ARMSBY Director, 'Institute of Anianal Nutrition, The Pennsylvania State College All correspondence regarding articles from the Department of Agriculture should be addressed to Karl F. Kellerman, Joiamal 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. JOINAI OF AGRICCITIIRAL RESEARCH Vol. XVI Washington, D. C, February 17, 191 9 No. 7 CYANOGENESIS IN ANDROPOGON SORGHUM By C. T. DowELL Chemist, Oklahoma Agricultural Experiment Station INTRODUCTION It is a prevalent belief among farmers and also among certain writers on the subject of sorghums (Andropogon sorghum) that when the sorghum is cut and cured it is no longer poisonous to stock. While this is a strong belief among farmers and is stated as a fact by certain writers and inves- tigators, yet there are other writers and investigators who have claimed that curing has no effect on the power of sorghum to poison stock. In fact, the literature on this subject is quite conflicting in its statements. For instance, Churchill (jY states that sorghum is rendered safe for feed- ing by curing. Turrill (8) states that in curing the sorghum is rendered harmless. On the other hand, Schroder and Dammann (7) and also Brunnich (2) claim that the sorghum is not rendered harmless in the curing process. Furthermore, the well-known fact is recalled in this connection that linseed cake and certain varieties of beans are known to contain hydrocyanic (prussic) acid in the form of glucosid. Peters, Slade, and Avery (6) are not sure whether sorghum is rendered suitable for feeding by curing, and stated that the subject should be further inves- tigated. During this past summer reports came to this Station through the newspapers of several cases of poisoning caused by sorghum which had been cut for some time. This information, the fact that several inquiries were made by farmers as to whether or not it would be safe to feed sor- ghum which had been cut during dry weather, and the lack of definite information in the literature caused me to take up the present investi- gation. There are several questions that should be investigated. The first and probably the most important is to determine whether or not the glucosid ■ is decomposed and the prussic acid liberated when the sorghum is cured; second, to determine whether or not the enzym become-s inactive in the process of curing as claimed by Peters, Slade, and Avery (6); tkird, to determine the effect of the presenoe of substances such as glucose and ' Reference is made by number (italic) to " Literature cited, p. i8i." Journal of Agricultural Research, Vol. XVI, No. 7 Washington, D. C. Feb. 17, 1919 rk Key No. Okla.-i (175) 176 Journal of Agricultural Research voI.xvi.no. 7 maltose on the liberation of the hydrocyanic acid from the glucosid; and, fourth, to determine whether or not the hydrocyanic acid may be present in more than one form as has been claimed by Willaman {10). While these are the main points studied, there are several others, possibly of minor importance, that were studied. EXPERIMENTAL WORK Four different samples of sorghum were used. One was a sample ob- tained from Mr. Ed. Singleton, of Chickasha, Okla., and was a part of a lot of sorghum which had been cut when it was about 2% feet high and at a time when there was an extreme drouth in the southwestern part of the State. This was a part of some sorghum which had been fed to 12 head of cattle, 10 of which had died within one hour. This will be called sample i. Sample 2 was cut at the same stage of growth by Mr. P. A. Gould, of Stillwater, but had not been subjected to as extreme drouth as sample i, as it was cut at the beginning of the dry weather. Sample 3 was a second-growth sorghum which had grown after heavy rains had fallen and there had been plenty of moisture in the ground all during its growth. This sample was cut fresh each time as it was needed and was about knee-high at the time of cutting. Sample 4 was a volunteer sorghum cut from the Experiment Station farm. This sample was in the dough stage when cut, but it had been subjected to the dry weather of the summer and had grown quite vigorously after the rains had fallen. The method of determining the hydrocyanic acid was a modification of that used by Viehoever and Johns (9) and by Knight (5). In the case of the dry samples Nos. i and 2, the sorghum was cut into fine pieces and then run through a feed mill. Samples 3 and 4 were cut a little at a time, this part being thoroughly wet and bruised in a large iron mortar. The bruised portions were placed in water in the digestion flask. At first each of these samples were kept in the digestion flask in a water bath at 40° C. for two hours, the apparatus being so arranged that any hydro- cyanic acid which passed off would be collected in sodium hydroxide. After this period of digestion the water bath was removed, and 100 cc. were distilled as rapidly as possible, the hydrocyanic acid being collected in sodium hydroxid. It was found by two or three trials that all of the hydrocyanic acid was driven over by distilling 100 cc. At first the distillate was evaporated in vacuum as directed by Viehoever and Johns (9), but since this required such a long time it was decided to carry on the evaporation by placing the distillate in a flat-form evapora- tion dish on a water bath which was heated by an electric hot plate. A current of air from an electric fan at a low speed was directed across the evaporating dish. It was found that under these conditions the solu- tion was usually at aboiit 60° C. and in no case did the temperature go above 70° C. With such an arrangement the evaporation could be made easily within two hours. After the distillate was evaporated almost to Feb. 17, I9I9 Cy anagenesis in Andropogon sorghum ijy dryness, freshly prepared ferrous sulphate was added and acidified with 30 per cent nitric acid as directed by Viehoever and Johns (9). Instead of filtering the Prussian-blue precipitate into a Gooch crucible, as was done by Knight (5), it was filtered in the ordinary way and washed thoroughly with dilute nitric acid and then with water. The precipitate and filter paper were then placed in a flat-form platinum dish and heated slowly to dryness in an electric muffle furnace, and then heated strongly to burn the precipitate and oxidize the iron. The dish containing the resi- due, consisting of the ash of the filter paper and the ferric oxid, was weighed. From the weight of the ferric oxid the amount of hydrocyanic acid was calculated, and from this the percentage of hydrocyanic acid in the dry sorghum. The percentage of moisture in the different samples of sorghum was found by drying at 105° C. No effort was made to determine whether or not this method would give accurate results, but it was thought that the results would be as accurate as those obtained in using Knight's method (5); the colori- metric method of Viehoever and Johns (9) and of Francis and Con- nell {4) could not be used, since a colorimeter was not available. More- over, it was thought that this method would give results sufficiently accurate for comparative purposes. In order to determine whether or not a part of the hydrocyanic acid was lost in the drying, sample 3 was cut and digested as described above, and then some of it was allowed to dry in the laboratory for 2X days and was then placed on top of a Freas oven overnight. The temperature on top of this oven was 33° C. A part of this sample was used for the determination of hydrocyanic acid and another for the determination of the water still present. In order to determine the effect of the rate of drying on the loss of hydrocyanic acid, if any, another part of sample 3 was dried at 50° C. within 24 hours. The results obtained here are given in Table I under experiments i, 2, and 3. In order to determine the effect of the presence of glucose and maltose on the liberation of the hydrocyanic acid, portions of sample 2 were digested in a solution containing i per cent of dextrose and i per cent of maltose. The results of two trials here are given in Table I under experiment 5. To determine whether or not a part of the hydrocyanic acid existed in the form of nonglucosidic acid, as has been claimed by Willaman (10), portions of samples 2 and 3 were digested, and 200 cc. distilled off. Then 50 cc. of 10 per cent sulphuric acid were added to the digestion mixture, which had a volume of about 800 cc, and another 100 cc. was distilled. This last distillate was evaporated, and tests were made for hydrocyanic acid, with negative results, as indicated in Table I under experiment 6. It has been pointed out by Auld (z) that with most feedstuffs digestive conditions would be unfavorable for the action of the enzym on the 178 Journal of Agricultural Research Vol. XVI, No. 7 glucosid, but he points out very correctly that a shght acidity is the best condition for the action of the enzym and that this acid condition might be found in the paunch of ruminants when certain feedstuff s are used. This being true, it was important to knovv the acidity of the juice of the sorghum. The juice was pressed from portions of samples 3 and 4, and portions of this juice were diluted very much and titrated with sodium hydroxide, phenolphthalein being used as the indicator. These results are given in Table I under experiment 7. The acids present in samples i and 2 were not determined, but it is quite probable that all the acids present were nonvolatile and remained in the dry sorghum. Several other determinations of lesser importance were made, these results being also given in Table I. Table I. — Results of experiments showing the cyanogenesis in dry and fresh sorghum under various conditions Experi- ment No. Description of experiment. Percent- age of hydro- cyanic acid found. la lb 2a 2b 3a 3b 4a 4b 4C 4d 5a 5b 6a 6b 7a 7b 8 9a 9b 9C loa lob iia lib 12a 12b 13a 13b 13c Sample 3. Digested in water at 40° C. for i hour Sample 3 . Same asia Sample 3. Dried for 2)/^ days in the laboratory, then dried for 16 hours at 33 ° C Sample No. 3 . Same as2a Sample 3. Dried at 50° C. for 24 hotirs. Sample thoroughly dried . Sample 3. Same as 3a Sample 2, ph:s emulsion digested at 40° for 2 hours Sample 2. Same as 4a, except no emulsion present Sample i. Same as 4a Sample i. Same as 4b Sample 2 in a solution of i per cent of dextrose and i per cent of maltose Sample 2. vSame as 5a Sample 3. Digested for i hour at 40° C, distilled off 200 cc, then added 10 cc. of 10 per cent sulphuric acid and distilled another 100 cc. Test for hydrocyanic acid in last distillate Sample 2. Same treatment as 6a Titrated juice from sample 3. Normality found to be o.oij N Titrated juice from sample 4. Normality equals o.ojoy N Sample 4. Digested for 2 hours at 40° C Sample 2. Digested for 2 hours in 5 per cent tartaric acid Sample i. Treatment same as 9a Sample 3. Treatment same as 9a, except sample was ground under 5 per cent tartaric acid Sample 3. Kept at 40° C. for 15 minutes Sample 2. Same treatment as loa Sample 2. Treated witli water at 80° C. and kept at tliis tempera- ture for I hour Sample 2 . Treated with water at 90° C. and kept at this tempera- ture for I hour Sample 2. Kept in air bath at 70° C. for i hour Sample 2. Kept in air bath at 115° C. for i hour and 30 minutes. . Sample i. Kept for i hour in Njioo sodium hydroxid made acid and distilled Sample 3. Kept in solution of sodium hydroxid {N 1 100) for i hour. . Sample 3. Treatment same as 13b, except the sodium hydroxid was NI50, and the solution was made slightly acid with tartaric acid and kept for i hour at 40° C o. 0221 . 0228 .0050 . 0069 . 0109 . 0070 . 0222 • 0130 .0514 .0450 .0038 .0059 None. None. . 0087 . 0119 . 0018 None. . 0281 .0177 .049 . 0040 . 0124 . 0087 . 0220 .0051 0136 Feb. 17, I9I9 Cyanogenesis in Andropogon sorghum 179 DISCUSSION OF RESULTS Any discussion of the experimental results will necessarily be of the nature of a summary. A comparison of the percentage of hydrocyanic acid found in experiments la and ib w-ith those in 2a and 2b shows that approximately three-fourths of the acid is set free in the process of drying. This goes to confirm the common belief that sorghum is safe for feeding after it has been dried. At the same time the results show that not all of the hydrocyanic acid disappears. A comparison of experiments 2a and 2b with 3a and 3b shows that the rapidity with which the sorghum is d*^ed determines the percentage of the hydrocynaic acid that is re- tained by it. This point is of considerable importance in Oklahoma on account of the fact that farmers quite frequently cut their sorghum during drouths after it has been partially dried while yet standing; and after it is cut, being already partly dry, it dries very quickly. Under such conditions a large percentage of the hydrocyanic acid would be retained in the fodder. Sample i was cut under such conditions. A glance at experiments 4, a, b, c, and d, will show that the enzym which is present in the sorghum is still active and that the addition of emulsin does not cause the hydrocyanic acid to be liberated in greater quantity. A comparison of the amount of hydrocyanic acid found in experiments 5a and 5b with experiment 4 shows that the addition of such a small quantity as i per cent of dextrose and i per cent of maltose seems to hold back or prevent the liberation of about three-fourths of the acid. This is an extremely important result from the practical standpoint. Dextrose and maltose were selected because of the fact that they are formed by the action of the pytalin on the starches in the paunch. This retention of the hydrocyanic acid in the presence of these sugars may be assumed to be due either to a reaction between the sugars (aldehydes) and hydrocyanic acid or to a lessening of the activity of the enzym by the sugars. This would lead to the suggestion that in case there is any doubt about the poisonous nature of the sorghum one should feed some concentrate before feeding the sorghum. In this way a considerable quantity of dextrose and maltose would be produced by the salivary digestion and would tend te prevent liberation of the hydrocyanic acid of the sorghum which is fed afterwards. At the time of this experiment I had not read Peters, Slade, and Avery's work (6), in vrhich they showed that it was possible to give very large doses of hydrocyanic acid without any harmful effects provided at the same time a somewhat proportionate amount of dextrose was given. It has been claimed by Willaman, as has already been stated, that the hydrocyanic acid exists in the sorghum in two forms — glucosidic and nonglucosidic. It seems natural to suppose that the nonglucosidic acid would not be liberated under the conditions that existed in my i8o Journal of Agricultural Research voi. xvi.no. ? work, that is the digestion was carried on in a very faintly acid solution, the acidity being due to the acids present in the sorghum. If this assumption is made, the results in experiments 6a and 6b seem to show that no nonglucosidic hydrocyanic acid exists in the sorghum. Of course it is possible that the nonglucosidic acid was distilled over in the first ICO cc, but this would not be in harmony \vith Willaman's suppo- sition that it is the hydrocyanic acid that is obtained in 5 per cent of tartaric distillation that causes the poisonous effect and which is a non- glucosidic acid. Furthermore, the fact that no acid was found in the distillate from sample 3 when it was ground under 5 per cent of tartaric acid and distilled from the acid solution shows that nonglucosidic acid is not present. The results in experiments 9a and 9b show that when a dry sorghum is digested with 5 per cent of tartaric acid a considerable percentage of the hydrocyanic acid is not liberated, and when this is taken in con- nection with experiment 9c, one may conclude that the water was ab- sorbed by the dry substance more rapidly than was the acid and that some hydrocyanic acid was set free before the acid came in contact with the glucosid. It is seen from the acid concentrations as found in experiments 7a and 7b that the contents of the paunch would be faintly acid in reaction when the green or the dry sorghum is eaten. It might be argued that the acidity would be neutralized by the alkalinity of the saliva ; but when the acidity as found here is compared with the alkalinity of the saliva it is seen that, when the alkalinity of the saliva is taken into account, and assuming a normal saliva flow, the contents of the paunch w^ould still be slightly acid, a condition most favorable for enzym action. This acid condition would exist until rumination talkes place, when the acid would be neutralized. A comparison of the results of experiment loa and lob with that of experiment i shows that all the hydrocyanic acid is liberated within the first 15 minutes of the digestion. Willaman and West (ri) and other investigators have shown that hydrocyanic acid gradually disappears from sorghum during its growth, so that but Httle is present in the mature plant. It was thought that this might not be true if large am^ounts of the acid had been formed as a consequence of dry weather, in the sorghum at some stage of growth. Sample 4 had been stunted by dry weather, but it is seen from experiment 8 that nearly all of the hydrocyanic acid had disappeared. The per- centage of hydrocyanic acid found in this sample should be compared with that of sample i , which was doubtless greater still before the sample was dried. No discussion is needed of the experiments 11 to 13b, inclusive. The reason for making experiment 13c, v^^as that it was thought that possibly as shown in my work, the enzym is rendered practically inactive by dilute alkaline solution, and it might be that the hydrocyanic acid would not Feb. 17. I9I9 Cyanogenesis in Andropogon sorghum i8i be liberated on this account in the paunch ; but when it later entered the true stomach, where the solution would become slightly acid, the hydro- cyanic acid would be set free. The result under experiment 13c seems to show that this is true. Digestion first with N/ioo sodium hydroxid, as shown in experiment 13b, prevents the liberation of the hydrocyanic acid. Certainly, then, digestion with A^/50 sodium hydroxid would prevent the liberation of this acid, and yet it is seen by acidifying this solution and allowing further digestion that more than one-half of the hydrocyanic acid was given off. LITERATURE CITED (1) AULD, S. J. M. 1 9 13. CYANOGENESIS UNDER DIGESTIVE CONDITIONS. In JoUf. Agr. Sci., V. 5, pt. 4, p. 409-417. Bibliography, p. 417. (2) Brunnici^J. C. 1903. HYDROCYANIC ACID IN FODDER-PLANTS. In Jouf. Chem. Soc. [London], V. 83, pt. 2, p. 788-796. (3) CHURCmLiv, O. O. 1914. FORAGE AND SILAGE CROPS FOR OKLAHOMA. Okla. Agr. Exp. Sta. Circ. 34, 15 P- (4) Francis, C. K., and Connell, W. B. 1913. THE COLORIMETRIC METHOD FOR DETERMINING HYDROCYANIC ACID IN PLANTS WITH SPECIAL REFERENCE TO KAFIR CORN. In Jour. Amer. Chem. Soc, v. 35, no. 10, p. 1624-1628. (5) Knight, G. W. 1914. determination of PRUSSIAN BLUE IN TEA. In Jour. Indus. and Engin. Chem., V. 6, no. 11, p. 909-910. (6) Peters, A. T., Slade, H. B., and Avery, Samuel. 1903. POISONING OF CATTLE BY COMMON SORGHUM AND KAFIR CORN. Nebf. Agr. Exp. Sta. Bui. 77, 16 p. (7) Schroder, Johannes, and Dammann, Hans. 1911. zur kenntnis DER aus verschiedenen hirsearten ENTWICKELTEN blausaurEmEngen. In Chem. Ztg., Bd. 35, no. 155, p. 1436-1437. (8) TuRRiLL, William Bertram. 1914. POISONING by sorghum halepEnsE. In Roy. Bot. Gard. Kew, Bui. Misc. Inform., 1914, no. 6, p. 229-230. Reprinted without author's signature in Jour. Dept. Agr. Victoria, v. 14, pt. 11, p. 653. 1916. (9) ViEhoever, Amo, and Johns, Carl O. 191 5. ON THE determination OF SMALL QUANTITIES OF HYDROCYANIC ACID. In Jour. Amer. Chem. Soc, v. 37, no. 3, p. 601-607. (10) WiLLAMAN, J. J. 1917. THE ESTIMATION OF HYDROCYANIC ACID AND THE PROBABLE FORM IN WHICH IT OCCURS IN SORGHUM VULGARE. In Jour, Biol. Chcm., V. 29, no. I, p. 25-36. (11) and West, R. M. 1916. EFFECT OF CLIMATIC FACTORS ON THE HYDROCYANIC ACID CONTENT OF SORGHUM. In Jour. Agr. Research, v. 6, no. 7, p. 261-272. Literature cited, p. 272. EFFECT OF CERTAIN COMPOUNDS OF BARIUM AND STRONTIUM ON THE GROWTH OF PLANTS By J. S. McHargue Chemist, Kentucky Agricultural Experiment Station INTRODUCTION Although it has been known for more than a century that plants are able to extract appreciable amounts of the relatively insoluble compounds of barium contained in soils, very little scientific investiga- tion has been made to determine whether or not the compounds of this element have any specific function in the vegetable economy. Because compounds of barium are poisonous when taken into the animal body, there appears to be a general impression that these compounds would exert a similar influence upon plants. In a former investigation ^ the writer has shown that small amounts of barium can be readily detected and determined quantitatively in the ash of tobacco, com, potatoes, and a number of other plants grown under normal conditions in the field. Since soils contain only very small amounts of barium, necessarily in the form of relatively insoluble com- pounds, it is a question of considerable scientific interest how and why it is that notable amounts of this element are absorbed and apparently assimilated by plants, under normal conditions of growth. The object of the present investigation was to determine the effect of some of the well-known compounds of barium and of the closely related metal, strontium, upon the growth of plants. EXPERIMENTAL WORK Preliminary experiments consisted in growing plants in nutrient solu- tions to which were added certain compounds of barium, soluble as well as insoluble. It soon developed that plants could be grown in a nutrient solution containing moderate amounts of barium nitrate or carbonate, whereas an equal amount of the chlorid or sulphate produced a decided toxic effect. After having determined that the plants selected for the water-culture experiments were tolerant of barium carbonate and nitrate, it was decided that a method more nearly approximating the normal conditions under which plants are grown would be a better procedure. Accordingly the plan was adopted of growing the plants in barium-free sand contained in earthenware pots to which the necessary basal plant- food ration could be added, together with the desired compounds of barium. 1 McHargue, J. S. Thb occurrence op barium in tobacco and other plants. In Jour. Amer. Chem. Soc, v, 35, no. 6, p. 826-834- 1913- Journal of Agricultural Research. Vol. XVI, No. 7 Washington, D. C. Feb. 17, 1919 rh K;ey No. Ky.-? (183) 98355°— 19 2 1 84 Journal of Agricultural Research Vol. XVI, No. 7 COWPEAS In the first series of experiments twelve i -gallon earthenware jars were filled with a clean quartz sand that contained very little plant food. To each of the pots of sand was added the following basal plant- food ration: lo gm. of calcium carbonate, lo gm. of tricalcium phos- phate, 5 gm. of magnesium carbonate, 4 gm. of potassium nitrate, 2 gm. of potassium chlorid, and 2 gm. of sodium thiosulphate. In addition to this plant food, varying amounts of barium carbonate were added to all the pots except the first, which served as a check against any other one pot in this series of experiments. Cowpeas ( Vigna sinensis) were planted in the sand in the pots, and during the time the plants were making their growth the sand was kept moist with clear hydrant water. Previous to starting the experiment, 100 liters of hydrant water were evaporated to dryness and the residue thus obtained examined for barium, but none was found. In another experment 25 liters of water flowing from the drain tiles on the Experiment Station fann were collected and evaporated. The residue thus obtained was exam- ined for barium compounds, but none were found. The cowpea plants were allowed to grow until they were about 10 to 12 inches tall. They were then taken up in such manner as to pre- serve the roots intact, and the adhering sand was washed off as well as possible. The photograph reproduced in Plate 24, A, was taken two weeks after planting; that shown as figure B, after the plants were removed from the sand in which they grew. Table I shows the amount of barium carbonate added to each pot and also the weight of the air-dry plants that grew in each of the pots. Tabids I. — Effect of barium carbonate upon the growth of cowpeas — First series Pot. No. I (control) 2 3 4 5 6 Quantity of barium Weight carbon- of 10 ate air-dried added to plants. soil. Gm. Gm. None. 9- 15 o-S 12. 00 I II. 20 2 10. 15 3 9- 50 4 IO-55 Pot No. 7- 8. 9- 10 Quantity of barium carbon- ate, added to soil. Gm. Weight of 10 air-dried plants. Gm. II. 40 10. 90 11. 15 10. 80 11.65 " This pat received no calcium carbonate, and all the plants died. From the results obtained in this experiment it is to be observed that there were appreciable increases in the yields of all the plants grown in the presence of barium carbonate and calcium carbonate over that of the control pot. In the absence of calcium carbonate, Feb. 17. I9I9 Effect of Barium and Strontium on Plant Growth 185 however, the action of the barium carbonate was strongly toxic, as shown by the failure of the plants in pot 1 1 . The efifect of the barium compound upon the growth of the cowpeas is more strikingly shown in Plate 24. In figure A the pot on the right is the control, which received no barium compound. The pot in the middle received the same plant food as the control and 10 gm. of barium carbonate in addition. The pot on the left received 5 gm. of barium carbonate, but no calcium carbonate. It received the same amount of tricalcium phosphate as the other pots. One object in mind in this experiment was to ascertain whether there would be a tendency on the part of the plants in this pot to substitute barium for calcium in their growth. The peas germinated and came through the sand, made a stunted growth for a few weeks, and then died. The difference in the growth of the plants in the pot in the center and the one on the left shows very strikingly the toxic effect of barium carbonate in the absence of calcium carbonate. This experiment affords a very striking example in the plants in the center pot of the protective action of calcium carbonate on plants when grown in the presence of a toxic substance. Figure B of Plate 24 shows the effect of barium carbonate on the growth of the roots of the cowpea plants grown in pots 1,2, and 8; the plants on the right were the control and received no barium carbonate, the plants in the center received 0.5 gm. of barium carbonate, and plants on the left received 6 gm. of barium carbonate. It will be observed that the plants which grew in the presence of barium carbonate have made a markedly increased root growth over the control. It is also to be borne in mind that the plants in the center received only 0.5 gm. of the barium compound, whereas the ones on the left received 6 gm. or 1 2 times as much as the former, thus indicating that a very small amount of barium carbonate produces as great effect on the root growth as much larger amounts. The compounds of strontium have many chemical and physical prop- erties similar to those of barium and calcium. It was thought that a few comparative experiments showing what effect like compounds of barium and strontium might have upon the growth of plants would be of some interest in this connection. Therefore in the series of experiments that follow plants have been grown in the presence of both barium and stron- tium compounds and compared with similar plants grown in the presence of calcium compounds. OATS In a second series of experiments oats {Avena saliva) were grown in sand under conditions similar to those in which the cowpeas v/ere grown in the previous experiment, with the same basal plant food ration as before. 1 86 Journal of Agricultural Research Vol. XVI, No. 7 After the young oat plants had reached a height of about lo inches they were thinned to the same number of plants in each pot and as near equal in size as possible. The oats were brought to maturity and har- vested and, after thoroughly air-drying, the grain was threshed and the weights of the air-dry grain and straw produced in each pot were deter- mined. There were two control pots in this experiment, and the average weight of the grain and the straw from these two pots was taken as a check against other pots receiving compounds of barium or strontium in this series. Table II. — Effect of certain barium and strontium compounds upon the growth of oats — Second series Pot No. and treatment. Control pot i Control pot 2 Average Pot 3-I-2 gm. of barium carbonate Pot 4-1-5 gm. of barium carbonate Pot 5-I-2 gm. of strontium carbonate. .. Pot 6-i-S gm. of strontium carbonate. .. Pot 7-I-2 gm. of barium carbonate and 2 gm . of strontium carbonate Pot 8-f-5 gm, of barium sulphate Weight of grain. Gm. 19-25 17.40 18. ?,Z 18.50 20. 65 18. 40 21. 10 16. 50 II. 00 Gain or loss in weight of grain over control. Gin. Weight of straw. -l-o. 17 + 2.32 -f .07 + 2.77 -1.83 -7-33 Gm. 39-25 34-50 36.88 40. 00 44-75 37-15 46.25 37-25 24-75 Gain or loss in weight of straw over control. + 3- " + 7-87 + .27 + 9-37 + -37 -12. 13 Table III. — Comparison of weight and percentage of nitrogen, phosphorus, and potas- sium per pot — Second series Pot No. and treatment. Control Pot3-|-2gm. of barium carbo- nate Pot 4-I-5 gm, of barium carbo- nate Pot 5-f 2 gm of strontium car- bonate Pot6-|-5gm. of strontium car- bonate Pot 7-H2 gm. of barium carbo- nate-|-2 gra. of strontium car- bonate Pot 8-f-5 gm. of barium sul- phate Nitrogen. Gm. 0.3217 .3608 . 4120 •3367 •4579 -2739 - 1727 Per cent. I- 755 1-95 2. 00 1.83 2. 17 1.66 1-57 phosphorus. Gm.. o. 0752 0777 0847 0773 0886 0644 0418 Per cent. O. 41 -42 .41 .42 -42 -39 Gm. o. 0522 .0518 •0599 •0534 .0570 . 0429 .0286 Per cent. .285 .28 .29 .29 .27 .26 .26 In Table II is given the amount of barium or strontium compounds added to each pot, and the air-dry weights of the grain and the straw produced in each of the experiments. Table III gives a partial analysis Feb. 17. i9«9 Effect of Barium and Strontium on Plant Growth 187 of the grain showing the important constituents contained in the grain produced in each experiment. Both barium carbonate and strontium carbonate have increased the percentage of nitrogen as well as the total weight of nitrogen when applied separately. Applied together, there is diminution. Barium sulphate has diminished both percentage and total weight of nitrogen. The weights of grain and straw produced in this series of experiments show increased yields in all pots receiving either barium carbonate or strontium carbonate separately. The pot in which there was a mixture of the two carbonates shows a decrease in the yield of grain, while the yield of the straw is practically the same as that ^f the control. In the pot receiving barium sulphate there is a very marked decrease in both the grain and the straw, which shows the toxic effect of this compound when compared with the carbonate. The analysis of the grain produced in each of the pots for nitrogen, phosphorus, and potassium shows a sHghtly greater content of each of these elements where there was an increase in the yields of the plants over that contained in the control. The last two pots in the series. No. 7 and 8, show a marked falling off in their nitrogen, phosphorus, and potas- sium content when compared with the controls and the other pots in this series. The maximum increase in protein — that is, NX6.25 — ^amounts to 2.60 per cent over that of the control, and the grain containing it was grown in the presence of 5 gm. of strontium carbonate. The next highest result was obtained where 5 gm. of barium carbonate were present. The phosphorus and potassium content appears to be less affected by barium and strontium compounds than does nitrogen. SPRING WHEAT In the third set of experiments spring wheat (Triticum aestivum) was sown in pots containing sand to which was added the same basal plant- food ration as that added to the pots in the experiments with cowpeas and oats. The quantities of barium and strontium compounds added are given in Table IV. In addition to the barium and strontium carbonates certain pots received small amounts of what was claimed to be a very active commercial radio-active fertilizer. The amount of this material added to each pot is given in Table IV and is in accordance with the recommendations of the company marketing this material. After the young plants had reached a height of 6 or 8 inches, they were thinned to the same number of plants in each pot and were brought to a state approaching maturity. Unfortunately, when the wheat grains were in the dough stage, a careless attendant left the ventilators of the greenhouse open over Sunday and the sparrows came in and consumed a part of the grain growing in each pot ; hence, the results for the grain in this series of experiments were discarded. The straw was allowed to ripen i88 Journal of Agricultural Research Vol. XVI. No. and was harvesbed. When thoroughly air-dried it was weighed. The results appear in Table IV. Table IV. — Effect of barium carbonate and stronttu-m carbonate on the growth of wheat — Third series Pot No. and treatment. Pot I (control), no barium added Pot 2 (control), no barium added Pot 3+2 gm. of barium ca^onate Pot 4+2 gm. of barium carbonate Pot 5+2 gm. of strontium carbonate Pot 6+2 gm. of strontium carbonate Pot 7+2 gm. of barium carbonate+2 gm. of stron- tium carbonate Pot 8+2 gm. of barium carbonate+2 gm. of stron- tium carbonate Pot 9+2 gm. of barium carbonate+0.7 gm. of radio- active material Pot 10+2 gm. of barium carbonate+0.7 of gm. radio active material Pot 1 1 +0.7 gm. of radio-active material alone Pot 12+0.7 &™- ^^ radio-active material alone. . . . Weight of dry straw. Observed. Average. Gm. 41- 15 40.75 34-25 34.25 38-50 37-75 32-25 35-75 37-75 36.75 37-35 36.00 Gain or loss over control. Gm. } 40. 95 } 34- 25 } 38. 12 34.00 37-25 } 36. 67 Gm. — 6. 70 -2.83 -6.95 -3-70 -4.28 The results in this series of experiments shoAv a loss in the weight of the straw over the average weight of the straw in the control pots; however, the greater loss occurs in the barium pots. The strontium pots show a loss of one-half of that of the barium pots. The radio-active fertilizer, when used alone or in combination v>ith barium carbonate, did not affect the yield of the straw greatly, the yield in each case being less than that of the control. WINTER WHEAT In a fourth series of experiments winter wneat was sown in pots of sand containing the same basal plant-food ration as in previous experi- ments. Strontium nitrate was the compound subjected to experimenta- tion in this series. The amounts added are given in Table V, which also gives the yields and average weight of the grains of wheat produced in in each experiment. The results obtained in this series of experiments show abnormal yields in both grain and straw which probably are due to the large amounts of nitrate radical present rather than to the strontium ion, since strontium carbonate has in no instance given such marked increase in yields. Feb. I-. 1919 Effect of Barium and Strontium on Plant Growth 189 Table V. — Effect of sirontinm nitrate on the growth of winter wlieat — Fourth series Pot No. and treatment. Number of giains per pot. Weight of grain per pot. Average weight per grain. Weight of straw. Pot I (control), no strontium nitrate 372 292 Gm. 8. 8872 7- 7650 Gm. 0. 0239 . 0266 Gm. 34.50 27.50 Pot 2 (control \ no strontium nitrate Average 332 8.3261 . 0252 31.00 Pot 3+5 gni. of strontium nitrate 369 555 II. 9065 19. 6108 -0323 -0353 44- 50 52.00 Pot 4-I-5 gm. of strontium nitrate Average 462 15- 7586 •0338 48.25 Pot 5 + 10 gm. of strontium nitrate ; . . . 561 17-6505 . 03146 62. 00 The results obtained^ in the analysis of the grain for nitrogen, protein, phosphorus, and potassium are interesting (Table VI). It will be seen that with the addition of strontium nitrate there is a decided increase in the nitrogen content of the grain and a decrease in the phosphorus, while the potassium content remains practically constant. Table VI. — Percentage of nitrogen, protein, phosphorus, and potassium contained in the grain produced in each of the foregoing experiments Pot No. Nitrogen. Protein (NX6.2S). Phosphorus. Potassium. Pot I (control) 1.68 I. 71 10. 50 10.68 0.36 •35 0. 18 Pot 2 (control) • 24 Average 1.695 IO-59 •355 . 21 Pot ? 2.77 2-73 3.00 17-31 17.06 18.75 . 22 •23 •23 . 22 Pot 4 .18 Pot t; . 21 Having obtained unusual results in the yields and in the nitrogen con- tent of the grain in the previous series of experiments, another, the fifth, series of pot experiments, similar to the ones that have been described, was carried out. This series was planned as a further check on the effect of strontium carbonate on the growth and the nitrogen content of wheat. The amount of strontium carbonate added to each pot and the yields of grain and straw produced are given in Table VII. The seeds in pots 9 and 10 came up, and the stunted plants struggled for existence for the greater part of the time the other plants in this series v/ere making a complete growth. The plants never reached a height of more than 10 inches, thus showing that strontium can not replace calcium in the growth of plants. 190 Journal of Agricultural Research voi.xvi. no. Table VII. — Effect of strontiiivi carbonate on the growth of wheat — Fifth series Pot No. and treatment. Weight of grain. Weight of straw. Gain or loss in — Grain. Straw. Pot I (control) Gm- 10. 00 9. 00 Gm. 36.50 34.50 Gm. Gm. Pot 2 (control) Average 9-5° 35.50 Pot 3+5 gm. of strontium carbonate 9. 00 10. 50 42. 00 40. 00 Pot 4+5 gm.. of strontium carbonate * Average 9-75 41. 00 + 0. 25 + 5-50 Pot 5-I-10 gm. of strontium carbonate II. 50 13.00 39-50 40. 00 Pot 6+ 10 gm. of strontium carbonate Average 12.25 39-75 + 2.75 +4-25 Pot 7 -(-20 g^ of strontium carbonate 9-5° 10. 00 36.00 35-50 Pot 8-|-2o gm. of strontium carbonate Average 9-75 35-75 + -25 + -25 Pot 9+10 gm. of strontium carbonate, no cal- cium carbonate >None. (a) Pot lo-f 10 gm. of strontium carbonate, no cal- cium carbonate "Not weighed. The results in the fifth series of experiments agree very closely with those of the other experiments in which strontium carbonate was used, both with respect to yields and the results obtained in the analyses of the grain (Table VIII). They also show conclusively that the increased yields obtained in the fourth series of experiments in which strontium nitrate was used were due to the greater amounts of nitrate being present which was assimilated and thus produced grains of wheat that contained 8 per cent more protein than was found in the control experiments, which showed a protein content equivalent to that of wheat grown under normal conditions. The last two experiments in the fifth scries show conclusively that strontium wll not replace calcium in the growth of plants. They also show, however, that strontium carbonate in the absence of calcium carbonate fs apparently less toxic towards plants than barium carbon- ate in the absence of calcium carbonate. It will be recalled that in the first series of experiments, in which an attempt was made to grow cow- peas in the presence of barium carbonate without calcium carbonate, all the plants died soon after coming through the sand, whereas in the case of the wheat plants in the presence of strontium carbonate and the absence of calcium carbonate the plants did not die soon after they Feb. 17. I9I9 Effect of Barium and Strontium on Plant Growth 191 were up, but maintained a struggling existence during the greater part of the time other plants in the series were making a normal growth, thus indicating that strontium carbonate is less toxic in the absence of cal- cium carbonate than barium carbonate. Table VIII. — Percentage of nitrogen, protein, phosphorus, and potassium in the grain grown in the pots in the fifth series of experiments Pot No. and treatment. Nitrogen. Protein (NX6.23). Phosphorus. Potassiu m. Pot I (control) I. 69 1.66 10. 56 10.38 0.31 0 .27 Pot 2 (control) t8 Average 1.68 10.47 .29 19 Pot ^ 1.68 1.79 10. 50 11. 10 •35 19 19 Pot 4 Average 1.74 ID. 80 •34 19 Pot 1; I. 61 I. 69 10. 06 10.56 .27 •31 19 17 Pot 6 Average 1.65 10.31 .29 t8 Pot 7 I. 64 1.68 10. 25 10.50 .27 •31 19 20 Pot 8 Average 1.66 10.38 .29 19 CORN In a sixth series of experiments com plants {Zea mays) were grown in pots of sand containing the usual basal plant-food ration. To these pots were added var^dng amounts of barium and strontium compounds as shown in Table IX. Three com plants were allowed to grow in each pot until the plants had tasseled and bloomed. As was to be expected, the corn plants were dwarfed on account of greenhouse conditions, the plants reaching a height of about 3 feet. After making their maximum growth the stalks were cut from the roots at the top of the sand. The fodder was stripped from the stalks. The roots were taken up and washed as free from adhering sand as possible. The dififerent parts into which the plants were divided were kept separate, and after thoroughly air-drying, the weight of each of the parts determined and from thence the air-dry weights of the entire plants were computed. These results are given in Table IX. The results in this series of experiments agree in a general way with those obtained in previous experiments with wheat and oats, where the same compounds of barium and strontium have been applied in equal quantities and under similar conditions. 192 Journal of Agricultural Research Vol. XVI, No. 7 Table IX. — Air-dry weights of the corn plants in each of the experiments Pot No. and treatment. Air-dry weights. Gain or loss in weight v^hen com- pared with the controls. Roots. Stalks. Fodder. Entire plants. Roots. Stalks. Fodder. Entire plant. Pot I (control) Gm. II. 50 8.00 Gm. 13.25 lo- 30 Gm. 17. 60 IS-3S Gm. 42.35 33-65 Gm. Gm. Gm. Gm. . ... 9-7S 11.77 nfi. 00 Pot 3+2 gm. of barium carbonate. . . . 10. 65 12. 10 14-25 8.25 18.75 19.30 43- 6s 39-65 Pot 4+2 gm. of barium carbonate 11.38 11.25 A-i.f,i -0.52 + 2. 55 +3- 6s Pot 5+2 gm. of strontium carbonate. . 13.00 11-75 IS- 80 14.50 23- SO 20. 80 Pot 6+2 gm. of strontium carbonate . . 47- OS Average 12.38 IS- IS 22. 15 49.68 + 2.63 +3-38 +5-68 + 11.68 Pot 7+2 gm. of barium carbonate and 2 gm. of strontium carbonate Pot 8+2 gm. of barium nitrate Pot 9+5 gm. of barium sulphate Pot IO+2 gm. of barium chlorid Pot II + 5 gm. of barium carbonate. . . Pot 12 + 5 C™- of strontium carbonate. 14- 7S II. 50 10.75 11.50 13- S 13-8 13-00 13- 2S 8.00 7-50 12. 00 14- 5 20.25 18.7s 14- 25 19.25 20. 00 20. 7 48. 00 43- SO 33-00 38.25 45-50 48. 40 + 5- 00 + 1-75 + 1.00 + I.7S +3- 75 +3- 45 + 1.23 + 1.48 -3-77 -4-27 + -23 + 2-73 +3- 78 + 2.28 — 2. 22 + 2.78 +3-53 +4-23 + 10.00 + S-SO — S-00 + 0- 25 + 7- so + 10.40 It will be noted again that the maximum increase in yield occurred in the presence of strontium carbonate, while equal amounts of barium carbonate produced only a very slight increase in the yield of the entire plants. It is interesting to note that all of the roots in the corn experiment show some increase in yield over that of the controls, while in the weight for the stalks there are an equal number of minus and plus differences. In the weights of the fodders there is only one experiment in which the fodder produced is less than the control. In the weights for the entire plants barium sulphate gave a very decided negative difference. Both the sulphate and the chlorid reduced the yields in the stalks very de- cidedly. Table X. — Analyses of the corn fodder from the preceding expervinents [The results are expressed as percentage of the moisture-free substance) Pot No. and treatment. Crude ash. Insoluble residue (silica, etc.). Ferric oxid (FezOs). Lime (CaO). Magnesia (MgO). Potash (K2O). Composite sample from pots r and 2 (con- trol) 9. 71 9-66 9- IS 10. 20 9- IS 10.54 11. 07 12. 10 9-53 I- IS .84 .91 1. OI I. 14 1.38 I. 21 1-23 .78 ao.44 •39 .60 •44 .26 •.sS •73 •83 •14 1.03 .89 .92 1. 00 1. 14 1-34 1.30 .90 .90 0. 92 •49 •56 .67 •67 .82 •58 .60 •SO 3.2J 3.88 3- so Pots 3 and 4+2 gm. of barium carbonate . Pots 5 and 6+2 gm. of strontium carbon- ate Pot 7+2 gm. of barium carbonate and 2 gm. of strontium carbonate 3.61 Pot 9+5 gm. of barium sulphate 3.96 Pot To+2 gm. of barium chlorid Pot 11+5 gm. of barium carbonate Pot 12+5 gm. of strontium carbonate. . . . 3-91 4-34 3-86 « The irregularities occuring in the iron determinations are probably due to iron-oxid scales which may have come from the paint on the mill hopper, as some such scales were obscived in some of the samples. Feb. 17, 1919 Effect of Barium and Strontium on Plant Growth 193 TablB X. — Analyses of the corn fodder from the preceding experiments — Continued. Pot No. and treatment. Soda (Na20). Barium sulphate (BaS04). Stron- tium sulphate (SrS04). Phos- phorus pentoxid (P2O6). Nitrogen (N). Protein (NX6.2S). Composite sample from jxjts i and 2 (con- trol) 0.86 •72 •43 .18 .29 .24 .24 •23 •37 0. 22 .22s .24 .27 .27 •32 .28 .28 •27 1. 16 .96 .81 •93 1.47 1.44 .98 1.63 •93 7.22 Pots 3 and 44-2 gm.of barium carbonate. •059 Pots s and 6-I-2 gm. of strontium carbon- ate •IS .16 5- "5 S-8s 9-17 9.00 6.06 Pot 7+2 gm. of barium carbonate and 2 gm. of strontium carbonate •03s •044 Pot 8+2 gm. of barium nitrate Pot io-(-2 gm. of barium chlorid Tr. . 02 Pot ii+s gm. of barium carbonate Pot i2-t-s gm. of strontium carbonate • 20 S-84 The results of the analyses of the fodders that were produced in each of the experiments show no very striking differences in the mineral com- position of the fodders in any of the experiments (Table X). It will be obser\^ed that only very small amounts of barium and strontium have been taken up by the plants growing in the presence of compounds of each of these elements. SOYBEAN In Plate 24, C, are shown four jars of soybean {Soja max) plants that were grown in cultural solutions. The plants in the jars on each end have been grown in a cultural solution containing no barium compound, whereas the two pots in the center have been grown in a similar solution containing barium nitrate. The plants in the two jars in the center received their sulphur from a solution containing taurin, while the plants in the end jars received their sulphur from a solution of mag- nesium and potassium sulphates. The differences to be observed in the growth of the two sets of plants is attributed to the presence of the barium nitrate v/hich appears to have retarded the growth of the roots, stems, and foliage of the two sets of plants in the center. When the very small amounts of the barium compounds that occur in the soil and the relatively insoluble state in which they occur are taken into consideration, one is led to wonder how it is that plants are able to extract even as much barium as can be determined in the ash of normal plants. Since no barium was found by careful examination of the residue from the evaporation of 25 liters of water flowing from a tile drain on the Station farm, although the presence of barium in the soil of the area drained had been proved by extracting 0.0508 gm. of barium sulphate from the hydrochloric-acid solution from 500 gm. of an average sample representing the first foot of soil from this field, it would appear that the roots of plants do not obtain their barium from the percolating soil water, but rather by some kind of selective 194 Journal of Agricultural Research vo1.xvi,No. ? action upon the soil particles. A determination of total barium in the soil of another field near by gave 0.08 per cent of barium sulphate, obtained by decomposing the soil with hydrofluoric and sulphuric acids. CONCLUSIONS From the results obtained in the different series of experiments in this investigation the following conclusions are drawn. (i) Barium compounds in the absence of calcium carbonate are poisonous to plants; but barium carbonate in the presence of an excess of calcium carbonate apparently exerts a distinct stimulating influence on the growth of the plants studied. (2) There is no tendency for barium to replace calcium in the growth of plants when calcium carbonate is omitted from a plant-food ration under the conditions of these experiments. " (3) Strontium compounds have in most instances given larger increased yields than barium compounds. (4) Strontium carbonate can not be substituted for calcium carbonate in the growth of plants under the conditions studied, though strontium carbonate is less toxic to plants in the absence of calcium carbonate than barium carbonate. (5) Neither barium nor strontium compounds can be looked upon as important plant foods, although the presence of small amounts of the carbonate of each of these elements has given increased yields that are noteworthy in most instances. (6) Barium and strontium carbonates accelerated the growth of the roots of such plants as were examined. (7) Increasing the amount of strontium nitrate gave a corresponding increase in the nitrogen content of wheat. (8) No barium compounds were found in the residue obtained upon evaporating 25 liters of drainage water collected from the drain tiles on the Station farm, which would indicate that the barium found in plants is taken up in place by the plant roots. PLATE 24 A. — Effect of barium on the gro\\th of cowpeas with and without calcium carbonate. B. — Stimulating effect of barium on root growth of cowpeas. C. — Effect of barium on the gtowlh of soybeans. Effect of Barium and Strontium on Plant Growth Plate 24 Journal of Agricultural Research Vol. XVI, No. 7 Vol XVI FEBRUARY 24. 1919 No. 8 JOURNAL OP AGRICULTURAL RESEARCH CONXKNTS Pago Apple-Sc&ld -----_.._ 195 CHARLES BROOKS, J. S. COOLEY, and D. F. FISHER ( Contrlbotfam tn»n Boreaa of Plant taduatry ) Angular-Leafspot of Tobacco, An Undescribed Bacterial Disease -- - - - - _-. _ 219 F. D. FROMME and T. J. MURRAY (Contxllnitlon from Virginia Agricultural Kq>eriment Station) PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOQATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASHINGTON, D. C. WA9H!NaT0N : OOVEHHMEMT PHINTIHQ OFHCE 1 1016 W:v^'XliM^^S&^:M!iSMM EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF^GRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chairman Physiologist and Associate Chitf, Bureau of Plant Industry EDWIN W. ALLEN Chief, OMce of Experiment Stations CHARLES L. MARLATT Entomologist and Assistant Chief, Bureau oj Entomology FOR THE ASSOClATIOIf J. G. LIPMAN Director, New Jersey A grictdtural EiperitMut Station, Rutgers College W. A. RILEY Enlotrtologist and Chief, Division of Ento- mology and Economic Zoology, Agricul- tural Experiment Station of the UniversUf of Minnesota H. P. ARMSBY Director, Institute of Animal Nutrition, The Pennsylvania State College All correspondence regarding articles from the Department of Agriculture should be addressed to Karl F. Kellerman, Journal of Agricultiu-al 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. JOIMAL OF AGffiETlML RESEARCH Vol. XVI Washington, D. C, February 24, 1919 No. 8 APPLE-SCALD By Charles Brooks, Pathologist, and J. S. Cooley and D. F. Fisher, Assistant Pathologists, Fruit-Disease Investigations, Bureau of Plant Industry, United States Department of Agriculture INTRODUCTION The present paper gives a report of studies on the nature and control of apple-scald, including experiments upon the relation of orchard and storage conditions to the development of the disease. The literature upon the subject of apple-scald and the apparatus ^ and methods ^ used in these experiments have been rather fully reported in earlier publica- tions, RELATION OF CHARACTER OF FRUIT TO SCALD DEVELOPMENT MATURITY It is generally recognized that immature apples (Malus sylvestris) scald worse than mature ones.^ A striking example of the fact was obtained in storage experiments at Wenatchee, Wash., in the winter of 1 91 7-1 8. The apples of the different pickings were from the same trees and were approximately alike in every respect except in maturity. The first picking of the various varieties was made when the ground color of the fruit was very green and when the red varieties had de- veloped bHt a slight blush, the second picking when the ground color was beginning to show yellow and most of the apples of the red varieties had become deeply colored. The apples were stored in commercial box packages. One or more boxes of fruit were used under each storage condition of every experiment. The final notes for the Rome Beauty and Stayman Winesap were taken on March 19 and for the other varieties on Match 12. The Rome Beauty and Stayman Winesap were allowed to stand in cellar storage five days before the notes were taken, and the other varieties were held in a laboratory at 20° C. for four days before note taking. The results are given in Table I. In all cases there was less scald on the well-colored than on the poorly colored fruit, and in most cases fruit picked at the proper maturity was almost entirely free from scald. ' Brooks, Charles, and Coolev, J. S. temperature relations of apple-rot fungi. In Jour. Agr. Research, v. 8, no. 4, p. 139-164, 25 fig., 3 pi. 1917. * EFFECT OF TEMPERATURE, AERATION, AND HUMmiTY ON JONATHAN-SPOT AND SCALD OF APPLES IN STORAGE. In JouT. Agr. Research, v. 11, no. 7, p. 287-318, 23 fig., pi. 32-33, 1917. Literature 0> cited, p. 316-317. ' Ramsay, H. J., McKay, A. W., Markell, E. L., and Bird, H. S. the handling and storage oP APPLES in the pacific northwest. U. S. Dept. Agr. Bui. 587, 32 p., 7 col. pi. 1917. CD Journal of Agricultural Research, Vol. XVI, No. 8 ■■ * Washington, D. C. Feb. 24, 1919. rv '*^ Key No. G-173 Q_ (195) 196 Journal of Agricultural Research voi. xvi. No. s Table I. — Effect of maturity of fruit upon susceptibility to apple-scald Bx- peri- ment No. Variety. Storage condition. Date of picking. Maturity at time of picking. Per- cent- age show- ing scald March, 1918. I 2 3 4 5 6 Rome Beauty .... do . .. .do fCold storage con- 1 tinously. [Cold storage 2 months, then in cel- [lar storage. [Cold storage till Jan- l uary 25, then in [ cellar storage. JCold storage con- \ tinuously. [Cold storage 2 1 months, then in [ cellar storage. [Cold storage till Jan- i uary 25, then in [ cellar storage. [ Coldstoragetill Feb- 1 ruary 11, then in [ cellar storage. . ...do \Oct. 8 /Oct. 24 lOct. 8 (Oct. 24 loct. 8 [Oct. 24 \Oct. 9 /Oct. 25 ]0ct. 9 [Oct. 25 lOct. 9 [Oct. 25 ISept. 27 Oct. 30 [Sept. 22 {Oct. 2 [Oct. I [Sept. 22 \ Oct. 2 [Oct. 12 [Sept. 22 {Oct. 2 [Oct. 12 Rather immattu-e Well colored Rather immature . Well colored Rather immature . Well colored Rather immature . Highly colored Rather immature . Highly colored . . . Rather immature . Highly colored.... Rather immature. Well colored Immature Well colored Rather overripe . . Color green Color yellowing. . . Rather overripe . . Color green Color yellowing . . . Rather overripe . . 20 0 40 0 95 Stayman Winesap . . .do 5 65 0 30 do 0 90 7 8 Baldwin 5 50 Bellflower 0 90 40 9 rGrimeSjheavily ir- \ rigated. fGrimes, lightly ir- \ rigated. \ do 30 95 95 30 50 25 10 i I . do J Scald prevention on eastern-grown fruit is apparently not as readily accomplished. In an earlier report ^ the writers found little contrast in susceptibility to scald on eastern Grimes apples, a part of which were picked on August 11, when the fruit was quite green, a part August 28, when the apples were in condition for commercial picking, and a part on September 21, when the fruit was quite yellow. This experiment was repeated in 1917 on Grimes apples from Vienna, Virginia. The first picking was made on August 2 1 , when the ground color of the fruit was green, and a second picking on September 14, when the apples were becoming yellow and were at their best for commercial picking. The fruit was stored in moist chambers at various temperatures ^ in special storage boxes at Washington, D. C, and notes taken at various times on the development of scald. The results are given in figure i. ' Brooks, Charles, and Coolby, J. S. effect of temperature, aeration, and humidity on jona- THAN-SPOT AND SCALD OF APPLES IN STORAGE. In JouT. Agr. Research, v. ii, no. 7, p. 287-318, 23 fig., pi. 32-33. 1917. Literature cited, p. 316-317. » Temperature equivalents: o' C.-3a* F ; 5* C.-4i* F-: is* C.-S9* F : 'o* C.-6S' F.; 25* C.=77* F.; 30*C.-86*F. Feb. 24, 1919 Apple-Scald 197 The results at the higher temperature are in agreement with those of the preceding year, indicating little difference in susceptibility to scald between the well -colored and poorly colored Grimes, but at 0° the latter finally developed about twice as much scald as the former, giving further evidence of the greater susceptibility of green fruit when held at tempera- tures low enough to prevent ripening. 30 I I / / EASTERN AND WESTERN FRUIT Experiments were made to determine the relative susceptibility to scald of eastern and western Grimes of practically the same degree of ma- turity. The western Grimes were shipped from Wenatchee, Wash., to Washing- ton, D. C, in well- iced pony refrigera- tors. The eastern ap- ples were placed in storage the day after picking. Part of the western apples were from trees that had been heavily irri- gated. These apples were large, most of them 3 to 3X inches in diameter. The re- mainder of the west- em apples were from trees that had re- ceived very little ir- rigation and were small, ranging from Fig. I. — Graphs showing the effect of maturity upon susceptibility of Grimes apples to scald. The graphs show the percentage of scald on the two lots of apples at the ends of 8 and i6 weeks, respectively. The ones marked " G " give the results on the iruit picked on August 2 1 and those marked " R" the results on the fruit picked September 14. The dotted lines show the percentage of scald after the apples had been removed from storage and had stood in the laboratory at a temperature of 20° C. for three days. 2X to 2% inches in diameter. Most of the eastern apples were from 2K to 2K inches in diameter. All of the apples were held in moist chambers in the storage boxes already mentioned. Ten apples were used in each test. The results are shown in figure 2. The heavily irrigated western apples were somewhat less susceptible and the lightly irrigated ones much less susceptible to the disease than the eastern apples. While the eastern and western fruit did not receive exactly the same treatment, the results as a whole indicate that western Grimes apples from a region of intense sunlight are less susceptible to scald than eastern apples of practically the same maturity. 198 Journal of Agricultural Research Vol. XVI, No. 8 EFFECT OF IRRIGATION UPON SUSCEPTIBILITY TO SCALD A Study of figure 2 gives some evidence that apples from heavily irrigated trees are more susceptible to scald than those from lightly irrigated ones. In another experiment heavily and lightly irrigated Grimes apples of the same maturity were held in commercial cold storage at Wenatchee, Wash., till February 1 1 , and then in cellar storage till March 1 2 . The apples were stored in commercial box packages, two or more boxes of fruit being used under each storage condition of each experiment. The results are given in Table II. Under all of the different conditions of picking the heavily irrigated apples showed a greater sus- ceptibility to scald than the lightly irri- gated ones, the former averaging about twice as much scald as the latter. There were more large apples in the heavily irrigated lots than in the lightly irrigated ones, but this fact seemed to have but little influence upon the results, as heavily irrigated apples of a particular size were scalded worse than lightly irrigated ones of the same size. Table II. — Influence of irrigation upon susceptibility of apples to scald 7S/V/^/?^7-(//?£' C^A/r/G'/?/9£>S I^IG. J. — Graphs showing the relative susceptibiUty to scald of eastern and western Grimes apples. The graphs show the percentage of scald at the end of the given week. Those marked " E " give the results on the eastern apples, those marked "WI" the results on the heavily irrigated western apples, and those marked "WD" the results on the western apples receiving practically no irrigation. The dotted lines show the percentage of scald after the apples had been removed from storage and had stood in the laboratory at a temperature of 20° C. for three days. Expe- riment No. Variety and condition. Rather poorly colored Grimes apples picked on September 22 . Fairly well colored Grimes apples picked on October 2 Rather overripe Grimes apples picked on October 12 Percentage scald. Heavily irri- gated. 95 95 30 Lightly irri- gated. SO 25 10 Feb. 24, 1919 Apple-Scald 199 9i*e^AS RELATION OF TEMPERATURE TO APPLE-SCALD A rather full discussion of the^ relation of temperature to apple-scald has already been published by the writers.^ The results given in figures 3 to 10, inclusive, of this papei' confirm and extend the statements of the earlier report. As in the earlier experiments, the apples were stored in moist chambers and ten or more apples were used in each test. The experiment was started on August 21. A study of the figures shows that the optimum for scald production is approached at 15° C. and the maximum apparently reached at 25°. With all of the different varieties tested scald failed to develop at either 25° or 30°. This fact gives evidence that scald is not purely an old-age characteristic and that it can not be mainly due to the ac- cumulation of carbon dioxid, for both the aging and respiring of the fruit are accelera- ted by these high tem- peratures. A comparison of the results at 15° and 20° shows that in several cases (fig. 3, 4, 5) there was a shift in the opti- mum as the experiment advanced. Scald ap- peared first at 20° and for several weeks was worse at this temperature than at 15°, but later became decidedly worse at the lower temperature. A particular degree of scald usually developed 8 to 12 weeks later at 5° than at 15° and several weeks later at 0° than at 5°. Scald was worse at 5° than at 0° in all cases except with the very green Grimes (fig. 3) and fairly green Rome Beauty (fig. 8). In all of the above temperature experiments the apples were placed in moist chambers. The relative humidity was practically 100 per cent, the carbon dioxid from i to 3 per cent, and there was practically no air movement. In all of the various experiments and at all of the different temperatures similar apples were held in open containers in an atmos- phere having less than 0.5 per cent of carbon dioxid, a relative humidity ' Brooks, Charles, and Cooley, J. S. EFFECT OF TEMPERATtTRE, aeration, and humidity ON JONATHAN-SPOT AND SCALD OF APPLES IN STORAGE. In Jour. AgT. Research, v. ii, no. 7, p. 287-318, 23 fig.i pi. 3a-33. 1917- Literature cited, p. 316-317. Fig. 3. — Graphs showing the effects of temperature on apple-scald at the end of 4, 6, 7, 9, 13, and 16 weeks. The dotted graph shows the amount of scald that was evident after removal from storage at the end of the given week and holding the apples at 20° C. for 3 days. The apples were Grimes from Vienna, \'a., picked on August 20. 200 Journal of Agricultural Research Vol. XVI, No. 8 of 85 to 95 per cent and an air movement of >^ to X "lile per hour. With two exceptions, both in the case of very green apples at 0°, the fruit held in the open remained free from scald to the end of the various experi- ments, indicating that other factors are even more important than tem- perature, and that a solution of the problem of scald prevention should be found^either in the composition or rate of movement of the storage air. INFLUENCE OF AIR COMPOSITION UPON APPLE-SCALD HUMIDITY It did not seem probable that a reduction in the relative humidity from 100 per cent to an average of 90 per cent as mentioned above could be responsible for the complete elimination of scald, but it seemed desirable to have further tests on the point. Table III gives the results of va- rious experiments in which the humidity was varied, with little or no change in temperature or other environmental factors. In cases where it was necessary to in- troduce outside air this was brought to the tem- perature of the fruit before being allowed to come in contact with it. A study of the results from the various experi- ments reported in Table III shows that, in gen- eral, only about half as much scald developed on apples exposed to dry air as on those exposed to saturated air. It does not seem, however, that high humidity can be the primary cause of the disease, for in no case was scald entirely prevented by dryness, and in every case where the air was stirred, the disease was practically eliminated, even in the presence of the highest humidities. The withering of the apples in the dry air makes this method of partial prevention an imprac- tical one, and the fact that the disease can be prevented without drying naturally raises the question whether the beneficial efifects noted from the use of moisture-absorbing agents may not be at least partly due to their power to absorb some substance other than water, or to the fact that the evaporation of the water assists in the elimination of some dis- tinctly harmful substance. Fig. 4. — Graphs showing the effects of temperature on apple-scald at the end of 2, 3, 4, 5, 7, 12. and 16 weeks. The dotted graphs show the amount of scald that was evident after removal from storage at the end of the given week and holding the apples at 20° C. for 3 days. The apples were from the same trees as those of figure 3, but were picked 24 days later, on September 13, and the experiment was started on September 14. Feb. 24, 1919 Apple-Scald 201 Table III. — Influence of humidity upon apple-scald Ex- peri- ment. No. Al A2 A3 Bi B2 B3 Ci C2 C3 C4 cs Treatment. Air saturated, passed slowly over wet filter paper and through wash bottles of water Same as No. i, but air-dry, bubbled slowly through sulphuric acid and glycerin Same as No. i, but air in motion at rate of about yi mile per hour Air saturated, wet filter paper in bottom of container and the entering air bubbled through water Air-dry, calcium chlorid in bottom of con- tainer and the entering air passed over calcium chlorid and bubbled through glycerin Apples in open, exposed to air having a relative humidity of 85 to 95 per cent and a constant movement of >i to^niile per hour Saturated air, renewed slowly Same as No i, but air-dry Same as No. i, but with air circulated by air pump Same as No. 2, but air renewed 10 to 15 times more rapidly Apples in open package Percentage of scalds. Grimes I ^°'''' Imperial, j Arkansas. atis°C.! 50 23 At2rc 20 8 Ato°C.'At2j°C. 60 32 at 0° C. 55 25 Experiment A. — Grimes apples of the lot described in the legend for figure 5 were stored at 15° C. for 7 weeks. In all three cases cited the carbon dioxid of the storage air was held at 3 to 4 per cent by the constant introduction of air containing 3 per cent of this gas. The rate of renewal was such that a volume of air equal to that in the container was carried in once in every 24 hours. In No. 3, however, in addition to this slight air movement, the air was kept in constant motion at a rate somewhat less than yi mile per hour by means of a closed-circuit connection with an air pump. Experiment B. — Grimes apples of the same lot as mentioned in Experiment A were used, but they were held in commercial cold storage for eight weeks before the experiment was started. The contrasted results were obtained after three weeks' storage at 15° C. With Nos. i and 2 the apples were held in unsealed jars and fresh air drawn in rapidly for about 10 minutes every second day, the volume of air carried through being several times that of the container. Experiment C. — The apples used in this experiment were York Imperial and Arkansas of tie same lots as described in the legends for figures 9 and 10, respectively. The contrasted results were obtained after 20 weeks of storage at the temperatures given. With No. i the apples were held in a closed container and fresh air introduced continuously at a rate such that a volume of air equal to that in the container was carried in once in 24 hours. The air was kept saturated with moisture by means of wet filter paper in the bottom of the jar and by bubbling the entering air through water. No. 2 was handled exactly as No. i with the exception that calcium chlorid was placed in the bottom of the jar and the entering air was passed over calcium chlorid and bubbled through glycerin. No. 3 was treated the same as No. i with the exception that the air of the container was kept in motion at a rate somewhat less than yi mile per hour by means of a closed-circuit connection with a rotary air pump. No. 4 had practically the same degree of dryness as No. 2 (evidenced by the withering of the apples), but this was secured by drawing in fresh air at a rate 10 to 15 times faster than in the case of No. 2 without using any drying agent either in the container or with the entering air. With No. 5 the apples were held in the open, exposed to air moving at the rate oi yito yi mile per hour, and having a relative humidity at 2^° C. of 70 to 80 per cent and at 0° C. of 85 to 90 per cent. 202 Journal of Agricultural Research Vol. XVI, No. 8 CARBON DIOXID Perhaps the most natural assumption in regard to apple-scald is to consider carbon dioxid as the responsible agent. The writers have made numerous experiments looking to the establishment of this hypothesis, but these have resulted in proof that carbon dioxid is not a causal agency in the produc- tion of the disease. The nature and results of the experiments are shown in figure 1 1 . The results give con- clusive evidence that an accumulation of carbon dioxid is not responsible for the production of scald. In 2 of the lo differ- ent tests the amount of scald was slightly decreased with a de- crease in amount of carbon dioxid, but with the other 8 it was either unchanged or decidedly increased. The results as a whole indicate that, while an accumulation of the gas may sometimes be an accompaniment of apple-scald, carbon dioxid itself really tends to prevent rather than aggravate the develop- ment of the disease. Fig. s. — Graphs showing the effects of temperature on apple-scald at the end of 2, 3, 4, 5, 9, and ii weeks. The dotted graph shows the amount of scald that was evident after removal from storage at the end of the given week and holding the apples at 20° C. for 3 days. The apples were of the same lot as those of figure 4 and were picked on the same day, but were held in commercial cold storage from September 14 to October 15, and were transferred to the storage boxes for the above experiment on the latter date. The weeks of storage as given on the graphs are counted from October 15, the time of starting the special experiment. Tabi,E IV. — Effect of storing apples in carbon dioxid for short periods on development of scald Ex- peri- ment No. Al. A2. Bi. B2. Treatment. Apples in 100 per cent of carbon dioxid at 30° C. for 3 days, then at 15° in moist chamber for 8 weeks. Apples in moist chamber at 15° continuously for the above-mentioned periods without carbon-dioxid treatment. Apples in 100 per cent of carbon dioxid at 15° C. for 6 days, then in moist chamber at 15° for 11 weeks. Same as Bi, but continuously in moist chamber without carbon-dioxid treatment. Percent- age of scald. O o 40 Feb. 34, X919 Apple-Scald 203 Apples stored in higher percentages of carbon dioxid than those given in figure 11 soon developed a disagreeable alcoholic taste, but if they were removed after a few days' exposure to the gas they were found to have but little, if any, of this objectionable taste and to have developed a decided resistance to scald. The results of two experiments of this sort are given in Table IV. The apples were Grimes of the lot described in the legend of figure 7. In the first experiment the taste of the apples was slightly affected by the exposure to carbon dioxid, but in the second experiment the apples exposed to carbon dioxid had as good a taste as those held continuously in moist chambers. In both cases the treated apples developed color in storage very much more slowly than the untreated. It would seem from the results that the carbon dioxid had produced a very decided inhibition of the activities of the apple, and thus led to scald prevention. OXYGEN In the experiments r Fig. 6. — Graphs showing the eHect of temperature on appel-scald at the end of s, 7, 9, II, 13, 16, and 19 weeks. The dotted graphs showthe amount of scald that was evident after removal from storage at the end of the given week and holding the apples at 20° C. for 3 days. The apples were from heavily irrigated Grimes trees at Wenatchee, Wash. They werepirkedonSeptcmbcr27, shipped to Washington, D. C. , in iced pony refrigerators, and the experiment started on October 3 . with carbon dioxid re- ported in figure ii the oxygen of the air was usually slightly below normal, but with the ex- ception of (c) and (d) under B there was never a deficiency of more than I or 2 percent. With(c) the average carbon di- oxid content of the air after the first two weeks of the experiment was 6 per cent and the average oxygen content 8 per cent, while with (d) the average carbon-dioxid content for the period v/as 14.5 per cent and the average oxygen content 6.9 per cent. In both cases any pressure or suction was prevented by a small U -tube opening closed with oil. The results given in figure 1 1 give no evidence that these deficiencies in oxygen had any tendency either to increase or decrease the amount of scald. The apples seemed normal at the end of the experi- ment, with the exception of a very faint trace of an aromatic musty flavor. In an earlier paper ' experiments were reported indicating that slight increases (increasing the percentage from 21 to 24) in the oxygen content 1 Brooks, Charles, and Coolev, J. S. effect op temperature, aeration, and humidity on jona- THAN-spoT AND sc.\LD OF ^\PPLES IN STORAGE. In Jour. Agr. Research, v. ii, no. 7, p. 287-318, 23 fig., pi. 33-33. 1917. Literature cited, p. 316-317. 98356°— 19 2 204 Journal of Agricultural Research Vol. XVI, No. 8 8 of the air also had no appreciable effect upon the development of scald. During the past season this test was repeated, using higher percentages of oxygen. The air was slowly renewed in the manner de- scribed in Table III and was not stirred. The temperature was 15° C, except E, which was 0° C. Five apples were used in each test. The re- sults are given in Table V. The results have not been consistent. An increase in the percentage of oxygen in the air gave a de- cided decrease in the amount of scald on Newtown, Pippin, and Rome Beauty apples that had been held several months in cold storage before the experiment was started (B and C), but failed to do so on Grimes apples that were exposed in similar atmospheres from the beginning of their storage life (A, D, and E). As a whole, the results are in decided contrast with the uniformly beneficial effects reported later as resulting from air circulation. Table V. — Influence of increase in oxygen upon the development of apple- scald 1 /«5;^«%%y 1 t /am5£?r-S' 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 /^i*irrA-sw'/34KS'^^r^^/f£;^ot'MJi I i / / / / \ \ /t? /»«fis*-^ J Vw \ 1 ' A L— — — ^'"O**^ 1 ' jT -'" /o^srn^ >S^ 1 ' jT ^ >>^ ^N^ ''^^""^ '/^^ - -— ^ V 1 [ ' fc.i' 1 S' /^' SO' Fig. 7. — Graphs showing the effect of temperature on apple-scald at the end of 7, 10, 12, and i8 weeks. The dotted graphs show the amount of scald that was evident after removal from storage at the end of the given weekand holding the apples at 20° C. for 3 days. The apples were from very lightly irrigated Grimes trees at Wenatchee, Wash. They were picked on October 3, shipped to Washington, D. C, in iced pony refrigerators, and the experiment started on October 9. Ex- peri- ment No. Variety and treatmtut. Composition of air supplied. Percent- age of scald. Grimes apples of lot described in legend for figure 3. Results after 8 weeks. NewtowTi Pippin from Hood River, Oreg. In cold storage till Jan. 26. Experiment started on this date and ended 12 weeks later. Rome Beauty from Vienna, Va. In cold storage till Jan. 26. Experiment started on this date and ended 12 weeks later. Grimes apples from Vienna, Va., picked Aug. 26, 1918. The experiment was started Aug. 27 and the results obtained after 8 weeks. ("Same as D, but at 0° C. and the \ results obtained after 16 weeks. 4 per cent of carbon dioxid, 28 per cent of oxygen. 4 per cent of carbon dioxid, oxygen normal. Normal air (21 per cent of oxygen). 32 per cent of oxygen Normal air (2 1 per cent of oxygen) . , .do > do }■ do 45 40 65 10 80 5 65 45 50 35 38 Feb. 24, 1919 Apple-Scald 205 S' /S° SO' as' Fig. 8. — Graphs showing the effect of temperature on apple-scald at the end of 12, 14, 20, and 26 weeks. The dotted graph shows the amount of scald that was evident after removal from storage at the end of the given week and holding the fruit at 20° C. for 3 days. The appels were Rome Beauty from Vienna, Va. They were picked on October 3, and the experiment was started October 4. AIR MOVEMENT AS A PREVENTIVE OF SCALD AIR CIRCULATION The value of aeration in the prevention of apple-scald was pointed out by the writers in an earlier report^ and the previous data of the present paper have given confirma- tory evidence on this point. Other experi- ments were made in which the effect of air circulation apart from air renewal was test- ed. The air move- ment was obtained by connecting the con- tainers to rotary air pumps. A continu- ous circulation in a closed circuit was thus secured. The rate of movement was less than ]/i and probably more than yz "li^G per hour. (See Table III for methods used in keeping the composition of the air constant.) The results are given in Table VI. The results are strik- ing. With all of the different air composi- tions and all of the dif- ferent lots of apples a gentle air movement practically eliminated scald, while similar ap- ples held in stagnant air of like composition be- came badly scalded. The writers attribute the beneficial effects of the air movement to the breaking up of layers of dead air adjacent to the skin of the apple, thus disseminating harmful gases that might otherwise hang in the tissues of the apple. ' Brooks, Charles, and Cooley, J. S. effect of temperature, aeration, and humidity on jona- Than-spot and scald of apples in storage. In Jour. Agr. Research, v. u, no. 7, p. »87-3i8, 23 fig., pi. 32-33- 1917- Literature cited, p. 31^-317. / ** / ^'soivrs/fs ! f 1 1 ^ 1 ^en<£-£^ ** 1 \re tv£-£-A-s- J ^^ kl Fig. 9. — Graphs showing the effect of temperature on apple-scald at the end of 8, 12, 16, and 20 weeks. The dotted graph shows the amount of scald that was evident after removal from storage at the end of the given week and holding the fruit at 20° C. for 3 days. The apples were York Imperial from Vienna, Va. They were picked and packed in barrels on October 2 and placed in commercial cold storage the following day. They were removed from storage on December 4 and the above experiment started the same day. 2o6 Journal of Agricultural Research voi.xvi. no. s Table VI. — Effect of air movement upon apple-scald Ex- Variety and treatment. Treatment. Percentage of scald. peri- ment No. Grimes. Arkan- sas. York Impe- rial. Grimes apples of lot de- scribed in figure 3 af- , ter 8 weeks' storage at i 15° c. York Imperial and Ar- kansas apples of lots described in figures 9 and 10. Results ob- tained after 20 weeks' , storage at 2.5° C. [Grimes apples of lot de- l scribed in figure 5 af- [ ter b weeks at 15° C. With 4 per cent of carbon di- oxid; air stirred. With 4 per cent of carbon di- oxid; air not stirred. With 2 per cent of carbon di- oxid; air stirred. With 2 per cent of carbon di- oxid; air not stirred. Air; air (0.2 per per cent of car- bon dioxid and 0.5 per cent of oxygen) stirred. Air' air not stirred 0 40 2 60 3 65 2 80 A Apples in open; air movement J^ to M iiiile per hour. Apples in moist chamber; air not stirred. 'Apples in open; air movement J's to }4 mile per hour. Apples in moist chamber; air not stirred. With 3 per cent of carbon di- oxid; air stirred. With 3 per cent of carbon di- oxid; air not stirred. B 3 80 ^5 80 7 60 0 20 0 5 0 Normal air* air not stirred IS Apples in open; air movement fs to }4: ™ile per hour. Apples in moist chamber; air not stirred. With 6 per cent of carbon di- oxid; air stirred. With 6 per cent of carbon di- oxid; air not stirred. With 3 per cent of carbon di- oxid; air stirred. With 3 per cent of carbon di- oxid; air not stirred. Air (air with 14.5 per cent of carbon dioxid, 6 per cent of oxygen); stirred. Air (air with 0.6 per cent of carbon dioxid, 0.8 per cent of oxygen); stirred. Air not stirred; i per cent of carbon dioxid. 0 75 5 18 I 50 I 3 60 C INTERMITTENT AERATION In the experiments reported in Table VI the air was kept in constant circulation, but this continuitly of the movement is apparenty not essential to the prevention of apple-scald. In an earlier paper ^ experi- ' Brooks, Charles, and Cooley, J. S. effect of temperature, aeration, and humidity on JONATHAN-SPOT AND scAtD OF APPLES IN STORAGE. In Jour. Agr. Research, v. 11, no. 7, p. 287-318, 23 fig. pi. 33-i2, 1917. Literature cited, p. 316-317. Feb. s4, 1919 Apple-Scald 207 ments were reported in which scald was entirely prevented on Grimes apples at 15° C. by drawing the air rapidly through the container for a lo-minute period three times a week. During the past season this experiment was repeated but at 5^ C. and with York Imperial and Arkansas apples. The amount of apple- scald developed after 20 weeks is given in Table VII. Table VII. — Effect of intermiftent aeration on apple-scald Ex- peri- Treatment. Percentage of scald. ment No. Arkan- sas. York Imperial. I Air renewed continuously, a volume of fresh air equal to that in tlie container being passed in every 24 hours 85 50 2 Air renewal every second day, a volume of fresh air equal to twice that in the container being passed in in 10 minutes 30 The control of apple-scald was not as complete as in the earlier experi- ments, but a limited amount of air had a greater beneficial effect when passed into the container within a period of 10 minutes than when dis- tributed over a period of 48 hours. With a slow rate of air movement the amount of scald was found to vary with the length of time the movement was continued, as shown in the results given in Table VIII. Table VIII. — Relation of period of aeration to the development of apple-scald Ex- peri- ment No. \'ariety and previous treatment. Treatment. Percent- age of scald. A I A2 A3 Bi B2 B3 Grimes apples of same lot as de- scribed in legend for figure 6 after 9 weeks' storage at 15° C. ....do ....do Grimes apples of same lot as de- scribed in legend for figure 7 after 18 weeks' storage at 0° C. ....do ....do In moist chamber continuously.. In open continuously; air move- ment yi to X miles per hour. Alternately 2 weeks with same treatment as No. i, then 2 weeks as No. 2. In moist chamber continuously . . In open continuously; air move- ment >i to X miles per hour. Same treatment as No. i for 8 weeks, then same as No. 2. 65 iT o 20 The rate of air movement was probably but little above the minimum for scald prevention, and the results show a direct relation between the duration of the movement and the amount of apple-scald. 208 Journal of Agricultural Research Vol. XVI, No. 8 TEMPERATURE CHANGES AS A MEANS OE AERATION Apples held at a constant temperature have usually scalded worse than those exposed to temperature changes, the beneficial effects of the fluctuating temperature apparently being due to the aeration of the apple tissue thus obtained. Experimental results on this point are given in Table IX. All of the apples were held in moist chambers and were therefore poorly aerated. Table IX. — Influence of temperature changes upon apple scald Ex; Percent- peri- ment No. Variety and previous treatment. Temperature. age of scald. I Rather immature Grimes apples At 5° C. continuously for 16 weeks. . 38 of lot described in figure 3 . At 0° C. continuously for 16 weeks. . OS At 5° C.for 4 weeks; then at o°C. 6 for 12 weeks. At 0° C. for 4 weeks; then at 5° C. 20 for 12 weeks. At 5° C. for 8 weeks; then at 0° C. 10 for 8 weeks. At 0° C. for 8 weeks; then at 5° C. 60 for 8 weeks. 2 Grimes apples of lot described in At 5° C. continuously for 12 weeks. . 40 figure 7. At 0° C. continuously for 12 weeks. . 0 At 5° C. for 8 weeks; then at 0° C. 5 4 weeks. At 0° C. for 8 weeks; then at 5° C. 35 4 weeks. 3 Grimes apples of lot described in figure 6 after 9 weeks of storage . At 15° C. continuously 65 25 Alternately 2 dAys each at 5° C. and 25° C. (Average temperature, 15° c.) The results in experiments i and 2 indicate that the amount of scald was decreased by moving the apples from one temperature to another during the first weeks of storage. The apples were given no aeration at the time of change, and a probable explanation of the beneficial effects resulting from shifting the apples from one temperature to another seems to be some sort of renovation of intercellular air condi- tions accompanying the temperature changes in the tissues. The apples stored first at 5° and then at 0° had less scald and were of better quaUty than those stored first at 0° and then at 5° or than those stored continuously at 0°. In experiment 3 the apples were held part of the time at a temperature (25° C.) that has been proved to be too high for the production of scald. Other experiments have been made in which aeration has been combined with high temperature with decidedly beneficial results in scald pre- vention. In an earlier paper ^ an instance was reported in which scald 1 Brooks, Charles, and Cooley, J. S. effect of temperature, aeration, and humidity on jona- THAN-SPOT and scald OF APPLES IN STORAGE. In Jour. Agr. Research, v. 11, no. 7, p. 287-318, 23 fig., pi. 3^-33. 1917. Literature cited, p. 316-317. Feb. 24, 1919 Apple-Scald 209 was prevented by one thorough aeration for 24 hours at 20° C. and then by storing at 5° C. In the winter of 1 917-18 some striking results on this point were again obtained. Of two lots of Grimes apples from Wenatchee, Wash., picked from the same trees and placed in commercial cold storage at the same time, one lot consisting of 10 boxes was brought out twice for aeration and note- taking, remaining at a temperature of 20° C, the first time for 4 hours (after 5 weeks' storage), and the second time for 48 hours (after 10 weeks' storage) . The second lot consisting of 1 2 boxes was left in cold storage continuously. At the end of 17 weeks' storage the amount of scald on the fruit in the former lot ranged from 5 to 30 per cent, averaging 15.5 per cent, while that in the latter lot ranged from 50 to 80 per cent, averaging 65 per cent. The two aerations at laboratory temperature were apparently suffi- cient to reduce the scald to one -fourth that on apples held . so continuously in cold storage. ^ i AIR-COOLED CELLAR i}^'^ STORAGE k It has already been ^bo pointed out that in the fi experimental storage boxes apple scald was o prevented at all tem- peratures from 0° to 30° C. by a gentle air movement. Other ex- periments were made under more nearly commercial conditions, in which air-cooled cellar storage was compared with commercial cold storage. The experiment was made at Wenatchee, Wash. In the fall the door and window of the cellar were kept open at night and closed in the day, and throughout the winter frequent ventilation was given. Hygrothermograph records showed that in Octo- ber the average temperature of the cellar was 12° C. (53.6° F.) and the average relative humidity 60 per cent; in November the average tem- perature was 8° C. (46.4° F.) and the average relative humidity 78 per cent. From the first of December to the middle of March the tem- perature stood fairly constantly at 5° C. (41° F.) and the relative humidity at 86 per cent. In the cold-storage plant the average tem- perature for November was 2.5° C. (36.5 °F.) and the average relative humidity 84 per cent; for December the average temperature was 0.28° C. wetfer/rs \ /^ \ \A sUsf£A:s\. k Fig. 10. — Graphs showing the effect of temperature on apple-scald at the end of 8, 12, and 16 weeks. The apples were Arkansas from Mid- dletown, Va. They were picked and packed on October 17 and placed in commercial cold storage the following day. They were removed from^ storage on December 4 and the above experiment started the same day. 2IO Journal of Agricultural Research Vol. XVI, No. 8 M: z I O.OJ "" > \3.o • \6. O /O ^O 30 '^^O SO eo TO 3 O 6. a 3.S SO O.e /<2 s = s ™ M: 0.03 /.4i 3. 9 S. 7 IIMIIIIIIIillll)iBMBB)BBBBIIi^MBM^HBMiill4lllilllllllgBpM HnB SB i^S =i "■"""'" ""■" ^. 7 0.2 SI I m /^ {2 O 03 O. S 2.2 wmmamwmmmmwmmammmmtmwi^makm {a a. o3 3. o o.2 inch of animal charcoal in bot- | i torn of jar. Same as No. 3, but with apples packed in excelsior. ... Same as No. 3, but with apples packed in sawdust In sealed moist chamber; air renewed slowly I 81 In the open i o In unsealed moist chamber; apples not wrapped 70 In imsealed moist chamber; apples wrapped in usual commercial manner. Same as No. 4, but with wrappers impregnated with para- ffin. Same as No. 4, but with wrappers impregnated with vase- line. Same as No. 4, but with wrappers impregnated with cocoa butter. Same as No. 4, but with wrappers impregnated with para- ffin (50 per cent), vaseline (50 per cent). Same as No. 4, but with wrappers impregnated with bees- wax (30 per cent), vaseline (70 per cent). Same as No. 4, but with wrappers impregnated with cocoa butter (75 per cent), vaseline (25 per cent). Same as No. 4, but with wrappers impregnated with cocoa butter (80 per cent), olive oil (20 per cent). Same as No. 4, but with wrappers impregnated with bees- wax (30 per cent), olive oil (70 per cent). None Paraffin Vaseline Cocoa butter Paraffin (50 per cent), vaseline (50 per cent) Beeswax (30 per cent), vaseline (70 per cent) Cocoa butter (75 per cent), vaseline ^25 per cent) Cocoa butter (80 per cent), olive oil (20 per cent) Beeswax (30 per cent), olive oil (70 per cent) Fruit coated with wax. Fruit with bands of wax. 70 20 70 50 2 3 5 3 A. — The apples were Rome Beauty of the same lot as described in the legend for figure 8, but they were held in commercial cold storage for 12 weeks. They were en- tirely free from scald at the time of starting the experiment. They were stored at 15° C. in nine liter jars. The results were obtained after 12 weeks' storage. B. ^Newtown Pippins of tlie same lot as described in Table V, B were used in this experiment. The results were obtained after 12 weeks' storage in moist chambers at 15° C. The special wrappers were prepared by dipping the usual apple wrappers in hot waxes and oils of the given composition and then allowing them to drain and cool. C. — All conditions were the same as in B except that part of the apples were prac- tically covered with a thin coating of wax, others had narrow bands of wax, and still others had no wax in any form. All were wrapped with ordinary apple wrappers as in commercial packing and stored in moist chambers. 2i6 Journal of Agricultural Research voi. xvi, no.s As a whole, the results in Table XIII give most remarkably clear-cut and complete evidence that apple-scald can be prevented by the absorp- tion of the gases (other than carbon dioxid) thrown off by the apples themselves in storage. The beneficial effects of the substances used in the experiments described under (A) may have been partly due to their water-absorbing power, but this could hardly be true of those used under (B) and (C) . One of the particularly striking features brought out is the fact that the various substances have had a beneficial effect in direct proportion to their absorbing power. Excelsior greatly reduced the amount of scald, but sawdust entirely prevented the disease. Paraffin is distinctly the most inactive of all the waxes and oils used, and it was the only one that did not furnish practically complete control for the disease. Apple-scald can evidently be prevented by substances having a compara- tively limited capacity for taking up gases if the absorbing surfaces are placed in rather close contact with the skin of the apple. NATURE OF APPLE-SCALD The foregoing experiments have approached the apple-scald problem from several different angles, and the results give considerable evidence as to the real nature of the disease. Apple-scald is not necessarily an old-age phenomenon, but is due to the long-continued action of more or less abnormal storage conditions, conditions that cause the produc- tion or prevent the elimination of certain waste products. Most varie- ties of apples may be exposed to such unfavorable conditions for several weeks without developing scald and without showing any ten- dency to the disease if later stored under more nearly normal conditions; but they finally reach a certain critical period at which time they are not scalded, yet have developed a tendency to scald that can not be eradicated by removing the agencies that were originally responsible for the trouble. In the experiment reported in Table VIII, B, apples that were held under conditions favorable to scald for eight weeks showed no sign of the disease when removed to a warm temperature for a few days, yet these apples developed scald later, under storage conditions that did not produce scald on fruit that had never been exposed to unfavorable conditions. With apples that have been shifted from one storage place to another it is evident that the conditions existing at the time of the development of scald may not be the ones that are responsible for the occurrence of the disease. Apple-scald seldom, if ever, becomes evident while apples are held continuously at o° C. (32° F.), but cold-storage apples may be found to be badly scalded after a few days at a higher temperature. As was pointed out in an earlier paper,^ the real cause of this sudden appearance of the scald is not the sudden change of temperature. The disease already existed, but the cells were unable to carry out their death processes while a temperature of 0° was maintained. 'Brooks, Charles, and Cooley, J. S. effect of temperature, aerationj and humidity on jona- Than-spoT and scald of apples in storage. In Jour. Agr. Research, v. ii, no. 7, p. 287-318, 23 fig. pl. 33-33. 1917. Literature cited, p. 316-317. Feb. 24. 1919 A pple-ScaCd 217 SUMMARY The foregoing experiments furnish conclusive proof that apple-scald is a preventable disease. The following are some of the more salient facts that have been experimentally established. (i) Well-matured apples are much less susceptible to scald than immature ones. (2) Apples from heavily irrigated trees scald worse than those from trees receiving more moderate irrigation. (3) The rapidity of development of apple-scald increases with a rise in temperature up to 15° or 20° C, the optimum often shifting from 20° to 15° C. during the storage period. (4) Apple scald has not occurred at temperatures of 25° or 30° C. (5) It has been found possible to store apples in air saturated with water vapor without the development of scald. In several different experiments scald was considerably reduced by decreasing the humidity, but the beneficial effects were apparently not entirely due to the decreased moisture in the air. (6) Accumulations of carbon dioxid (i to 6 per cent) have not favored the development of apple-scald, but tended to prevent it. (7) Apples susceptible to scald have been made immune by storing for a few days in an atmosphere of pure carbon dioxid. (8) Increasing the percentage of oxygen in the air has not given consistent beneficial effects upon apple-scald. (9) A constant air movement of from yi to % mile per hour has always either entirely prevented apple-scald or reduced it to a negligible quantity. The intensity of the air movement was apparently more important than the continuity and the circulation of the air more impor- tant than its renewal. (10) Scald has been greatly reduced by shifting apples from one temperature to another. The beneficial effects are attributed to the aeration of the apple tissue thus obtained. (11) Thorough aerations during the first eight weeks of storage have been more helpful than later ones. (12) Apples have scalded less in air-cooled cellar storage than in unventilated commercial cold storage. (13) Apples packed in boxes or ventilated barrels have scalded much less than those in tight barrels,especially when the storage room received an occasional ventilation. (14) Scald was greatly reduced on rather immature apples by a delay in storing, if the fruit was well aerated during the delay, but was increased by the delay if held under conditions that allowed Httle or no ventilation. (15) Ordinary apple wrappers have had no effect on apple-scald, and par- affin wrappers but little; but wrappers soaked in various mixtures of olive oil, cocoa butter, vaseline, or beeswax have entirely prevented apple-scald. (16) Apple-scald is due to volatile or gaseous substances other than carbon dioxid that are produced in the metabolism of the apple. They can be carried away by air currents or taken up by various absorbents. ANGULAR-LEAFSPOT OF TOBACCO, AN UNDKSCRIBED BACTERIAL DISEASE^ By F. D. Fromme, Plant Pathologist and Bacteriologist, and T. J. M.vrs.ay,^ formerly Associate Bacteriologist, Virginia Agricultural Experiment Station INTRODUCTION About the first of August, 191 7, the Virginia Experiment Station received a petition from 52 tobacco growers in HaHfax County, asking assistance in combating a tobacco disease which threatened serious losses to the crop. Diseased tobacco plants (Nicotiana tabacum) were later received from a correspondent at South Boston, the leaves of which were covered with spots which were different from any previously seen by us, the most distinctive feature being the irregularly angular shape. Numerous motile bacteria were found in crushed tissue mounts and freehand sections of spots, and the organism was readily obtained in pure culture from poured plates of beef-peptone agar. The same organ- ism was obtained later from material which the writers collected in the field from five different places in Halifax County. Several inspection trips were made during the remainder of the season of 1 91 7. On August 10 the disease was found on the tobacco plants in most of the fields along the road between South Boston and Republican Grove, a distance of 30 miles. It was found later in the northern part of Halifax County, at Clarkton and other points, in the southern part of Campbell County, at Brookneal and Naruna, and at Charlotte Court House, in Charlotte County. Inquiries through county agents extended the distribution to include Mecklenburg, Pittsylvania, Henry, and Patrick Counties, involving the greater part of the flue-cured-tobacco belt in Virginia. FIELD APPEARANCE OF THE DISEASE The epiphytotic was well advanced by August 10. Many fields were found in which practically every plant was affected, and in some fully 50 per cent of the crop was estimated by the growers to be unfit for harvest. Late plantings were found to be less severely spotted than the early plantings, and the most forward and vigorous plants were invariably more seriously affected than the less vigorous ones. The distribution of the spots on the plants was a distinctive feature and one that readily separated them from "frogeye" (caused by Cerco- ' Paper 53 from the Laboratory of Plant Pathology anfl Bacteriology, Virginia Agricultural Experiment Station. * Now Bacteriologist, Washington Agricultural Experiment Station. Journal of Agricultural Research, Vol. XVI, No. 8 Washington, D. C. Feb. 24. 1919 rj Key No. Va. (Blacksburg)-3 (219) 220 Journal of Agricultural Research voi. xvi, no. s spora nicotianae E. and E.)- The heaviest spotting was found on the top and middle leaves, while the bottom or sand leaves were but slightly affected. Frogeye, on the contrary, is found chiefly on the sand leaves. The field evidence indicated that the vertical distribution of the spots on the plants was determined by their stage of growth at the time of infection, and that leaves which had attained a certain stage of growth were not susceptible to infection. In some fields the infection was heaviest on the top leaves and in others on the middle leaves. Fre- quently the spots were found to be most numerous on one side of the plants, indicating the probable dissemination of the inoculum by wind- blown rain. The consensus of the statements of the farmers placed the first appear- ance of the spotting between the third and fourth weeks in July, follow- ing a protracted period of rainfall. The tobacco at this time was at about the stage for topping. No one had seen any of the spotting earlier in the season, and there had been no evidence of it in the seed beds. One farmer who had planted two fields from the same seed bed stated that the disease was much worse in the field on new ground than on old ground. There was no evidence from any source that continuous cropping with tobacco, as practiced on many farms, had been conducive to heavier infection. Some of the farmers had been troubled with the disease in the previous year, 1916, and a few stated that they had seen it occasionally over a period of 10 or 12 years, but never so generally destructive as in 191 7. Prof. T. B. Hutcheson, of the Virginia Experiment Station, informs us that he has known this spotting in Charlotte County for 12 or 15 years. Opinions among the farmers as to the cause of the disease were cen- tered on the wet weather, the fertilizer, especially the supposed deficiency in potash, and the seed. The disease was found on both the flue-cured and sun-cured types of tobacco, and no differences in the susceptibility of varieties were apparent. Evidence that the disease is not occasioned by lack of potash and that it is not a fertilizer phenomenon, except in a secondary way, was obtained from a study of the distribution of the spotting on the fertilizer plots at the experimental substation at Charlotte Court House. Early in Sep- tember the disease was prevalent on all plots which had received appli- cations of acid phosphate alone or in various combinations with sulphate of potash and nitrate of soda. It was the opinion of several observers who went over these plots that they were uniformly spotted. None of the spotting was present, however, at this time, on any plots which had had no applications of phosphorus and which had received only nitrate of soda or sulphate of potash, or both. There was no spotting on the control plots. The tobacco plants on those plots which had received phosphorus were larger, more vigorous, and matured three weeks earlier than those which had had no phosphorus. The superintendent of Feb. 24, 1919 Angular-Leaf Spot of Tobacco 221 the substation informed us that the tobacco on these backward plots became spotted later with the advent of additional rainfall and cool nights, and that the second growth tobacco from the early harvested plots also became diseased. CHARACTER OF LOSSES A demonstration acre of tobacco at the substation at Charlotte Court House was severely spotted in 191 7, much more so than in any previous years. The yields from this acre were obtained to determine the varia- tion in yield and grade between 191 7 and the average of preceding years. The data obtained are included in Table I. The yield of the average year was determined from demonstration acres for the years 191 3, 191 4, and 1 91 5. Complete data for 191 6 were not available, but the total yield for this year, 1,005 pounds, shows a close agreement with the three preceding years. The effect of the disease is shown both in a reduction in yield and in grade. The yield from the acre in 191 7 was 212 pounds less than that of the average year, or approximately 80 per cent of the average. The yields in the different grades for 191 7 show a loss in weight in all of the three highest grades and a strong increase in the lowest grade. There were approximately 40 per cent more sand lugs in 1 91 7, in proportion to the higher grades, than in the average year, and the percentage of longs was slightly greater. The gain in the sand lugs was made up by losses in the two middle grades, good lugs and shorts. The total loss in weight was 20 per cent, and the loss in grade approximately 40 per cent. Table I. — Variation in yield and grade between severely spotted tobacco in igij and the average of three preceding years of light spotting Grade. Average year. Average year. Difiference between 191 7 and average year. Sand lugs. Good lugs . Shorts Longs . . . . Pounds. 495 155 190 Pounds. 205 290 346 Per cetit. 58.9 18.5 22. 6 Per cent. 19. 4 27. 6 32- 9 20. I Pounds. + 290 — 290 -191 — 21 Total . 840 1,052 Per cent. +39- 5 — 27. 6 -14.4 + 2.5 DESCRIPTION OF THE SPOTS The spots are found only on the leaves, none on stems or floral parts. They are scattered over the entire leaf surface, between the larger veins, or are crowded in irregular groups. They are usually bordered by viens whicji act as barriers against further enlargement. More than 500 spots may be counted on the average leaf of a heavily infected plant. They are about equally prominent on the upper and lower leaf surfaces. The 222 Journal of A gricultural Research voi. xvi. No. 8 size of the spots varies from a pinhead up to 8 mm. in diameter at the widest part, and the average diameter of the mature spot is 4 mm. The most striking feature is the irregularly angular shape and the uneven jagged outline (PI. 25-27). The center of the spot is tan or reddish brown, with a darker thin border and often a suggestion of zonation which is never conspicuous. A small, dark center is usually seen. In earlier stages the spots are darker in color, almost black ^ at first, and are circular or only slightly angular (PI. 25, A). The center of the spot becomes dry and thin with age, paler in color until almost white, and may drop out, leaving irregular holes which are sometimes so numerous with the conflu- ence of spots that a skeleton leaf composed only of the viens remains. Most of the tobacco is harv^ested before this stage is reached. No sharply defined halo is present, but a narrow clear zone on the border is seen by transmitted light. The tissue bordering the spots is yellowed, but this diffuses gradually into the normal green. Usually the spots are more numerous on one side of the midrib. In stained sections of fixed material the organism is found in great num- bers throughout the shrunken tissue included in the spot. It is found both within and betv/een the cells, and vacuolated cells are completely filled with it. INOCULATION EXPERIMENTS Proof of the pathogenecity of the organism isolated from leafspot material was obtained through inoculations on seedling tobacco plants (Warne variety). This work was carried on in the greenhouse during the winter months. In all, some 150 plants were inoculated, and fully 96 per cent developed spots. All five isolations of the organism pro- duced infection. Infection was readily obtained by atomizing the plants with aqueous suspensions of the organism, by swabbing the leaves with the cotton plug of bouillon cultures and by puncture with contaminated needles. It was found necessary to place the plants in moist chambers for 24 hours subsequent to inoculation. Some plants which were inocu- lated and left in the open greenhouse failed to develop any spots, and none of the many control plants became infected. No leaf injury is nec- essary for infection. Apparently the organism gains entrance through the stomata on either the upper or lower surface of the leaf. The inoculum has been recovered in pure culture a number of times, and these re- coveries have been used for reinfections. Secondary infections have never developed, but this is a common experience with plant pathogens under the dry atmospheric conditions of the greenhouse. As many as 200 spots were obtained on a single leaf through inoculation with the atomizer, but the average ran much lower than this. Still heavier infections were obtained by swabbing with contaminated cotton plugs. The spots obtained were typical of the spots seen in the field, but were smaller. They averaged about 1.5 mm. in diameter, and ' One farmer stated that his first impression of the spot was that someone had spattered ink on the leaves. Feb. 24, 1919 Angular-Leaf Spot of Tobacco 223 many were no larger than 0.5 mm., mere pin pricks. The spots were frequently grouped on a limited area of the leaf and were often found on one side of the midrib only. The discrepancy in size between the field spots and those developed in the greenhouse is apparently due to the difference in the size and vigor of the plants. The plants did not grow vigorously in the greenhouse and at maturity were not more than half as large as plants in the field. The largest spots in the greenhouse were always found on the most vigorous plants and on the most rapidly growing leaves. The incuba- tion period was also shortest with the same plants and leaves. This varies between 4 and 10 days, with the average about 7 days. When plants which have developed a number of leaves are inoculated, the first spots are seen on one or two leaves near the top and intermediate in age. Later, within a few days, spots may appear on two or three older leaves immediately below these, but no spots develop on old, full- grown leaves, nor on very young ones. Young leaves become infected however, when the inoculum is rubbed in with the fingers or a cotton plug. The young leaves are closely set with trichomes, and these seem- ingly serve as a mechanical protection against inoculation with the atomizer; the spray is caught and retained on them. The spots attain their maximum size within a few days after their appearance. They are largest on the younger leaves and may be mere pin pricks or flecks on the older leaves. Table II. — Results from the inoculation of tobacco plants with the angiilar-leafspot organism Plant No. Number of spots per leaf on leaf No.o I 2 3 4 5 I. . 2 14 I 68 2 13 19 5 17 39 51 63 5 I 2 33 1 21 14 23 T. I 5 4 c 6 . 14 157 29 8 9 7 . 30 8 6 Q 44 I 18 I I 3 6 5 3 4 4 41 16 I 58 30 I 37 23 18 14 Total spots 277 193 280 76 74 Total flecks 45 •- o Leaves are numbered from the top downward. The plants bore from lo to 14 leaves. Bold-face figures indicate flecks or spots that are very small and are visible only in transmitted light. 224 Journal of Agricultural Research voi. xvi, no. s The records of one inoculation series are given (Table II) chiefly to show the vertical distribution of the spots with reference to the position of the leaves and their relative ages. These plants were inoculated on January i6, 191 8, with an atomizer containing an aqueous suspension of a strain of the angular-leafspot organism isolated from material from Republican Grove, Va., on August 10. The plants were covered with moist chambers for 12 hours subsequent to inoculation. They were at the blooming stage and were topped just before inoculation. The spots were first visible on January 22, and the coimts were made on February i. Table II shows that the spots were about equally distributed over the first, second, and third leaves, with a few on the fourth leaves and none on those older and lower on the stem. Flecks developed on the fourth and fifth leaves, but not on younger or older leaves. COMPARISON OF ANGULAR-LBAFSPOT WITH OTHER LEAFSPOTS OF TOBACCO The angular-leafspot can not be assigned with certainty to any of the previously described tobacco leaf spots of bacterial causation. It has some features in common with the "whitespot" of Delacroix {2, 3),^ caused by Bacillus maculicola Del., but the descriptions of this disease and of the organism are too meager to afiford an adequate basis for com- parison. "Blackrust," a disease of Deli tobacco described by Honing (4), differs from angular-leafspot in several important features, and the causative organism. Bacterium pseudozoogloeae Honing, is readily distin- guished from the angular-leafspot organism. The spot which Wolf and Foster (7) have recently described under the name "wildfire" from North Carolina appears to differ strikingly from the disease under discussion. The most noteworthy points of difference being found in the broad, distinct halo which borders the wildfire spots, in their circular form and zonated interior, and in size. Wildfire spots are 2 to 3 cm. in diameter, while those of the angular-leafspot are only 4 mm., on the average. Some contrasting features between Bacterium tabacum Wolf and Foster, the wildfire organism, and the angular-leafspot organism are given in Table III. Features in common between the two diseases are found in their sudden appearance and rapidity of spread, and in the relation between rainfall and epiphytotics, although this seems a common feature of bacterial leafspots of tobacco. It seems quite probable that the disease to which Wolf and Foster refer as "speck" is identical with our angular-leafspot. They state that speck is caused by a lack of potash. ' Reference is made by number (italic) to "Literature cited," p. 227-128. Feb. 24, 1919 Angular-Leaf Spot of Tobacco 225 Table III. — Comparison of Bacterium, angulatum and Bact. tabacum Bad. angulatum. Bact. tabacum. 1. Size, 0.5X2-2.5 At 2. 3 to 6 polar flagella 3. lyiquifies gelatin rapidly 4. Forms acid with saccharose and dex trose. 5. No growth in closed arm of fermenta tion tubes. Size, 1.2X3.3 At. I polar flagellum. Liquifies gelatin slowly. Forms acid with saccharose, dextrose, lac- tose, and glycerin. Growth in closed arm of fermentation tubes containing dextrose and saccharose. An unsigned note in the Yearbook of the Virginia Department of Agriculture and Immigration (5) contains a report of a field investigation of a spotting of tobacco leaves (evidently angular-leafspot) which was prevalent in Pittsylvania and Mecklenburg counties in 191 7. The spotting is assigned to microorganisms, and it is stated that the disease becomes serious only under certain conditions which affect the resistance of the tobacco plant. The most important of these are considered to be rainfall and excess of nitrogen in the fertilizer or soil. A liberal supply of potash is said to decrease the severity of the disease, but considerable damage was noted even with heavy potash applications. The same disease is said to occur in North Carolina and Maryland. Two other leafspots, the causes of which have not been assigned to bacteria, and which appear somewhat similar to angular-leaf spot, are "rust" of tobacco in Connecticut (7, p. 366-^67, pi. 31, b, c) and Pock- enkrankheit (6, p. 56) of tobacco in Europe. Clinton's figures of rust, especially that shown in his Plate 31, c, look much like angular-leafspot. He states that rust is usually found on leaves affected with calico and believes it may be caused by scorching of the sun. Pockenkrankheit is ascribed to excessive transpiration accompanying decreased water supply. OCCURRENCE OF THE DISEASE It seems quite probable that angular-leafspot is a disease of rather general distribution and one of long standing which has not been suf- ficiently destructive to attract extensive notice, except in seasons un- usually favorable for its development. Our experience indicates that rainfall accompanied by subnormal temperatures favors infection by the leafspot organism and that any combination of conditions which pro- motes a rapid, succulent growth of the host favors the development of the organism within the leaf tissue. It seems quite probable that in- fection may be common in some seasons, but that in the absence of the predisposing growth factors little development ensues, and no damage results. 226 Journal of Agricultural Research voi. xvi, No. s DESCRIPTION OF THE ORGANISM The caustive organism of angular-leafspot appears to be an unde- scribed species, and a description is therefore appended. Bacterium angulatum, n. sp. As it occurs in the plant and also on media, the organism is a short rod with rounded ends, single or in pairs, 0.5 m wide by 2 to 2.5 m long. No spores are produced, and no capsules have been demonstrated. It is motile by means of a small tuft of flagella at one pole, demonstrated by the Van Ermengen silver-nitrate method. The number of flagella varies from about three to six, and they are slightly longer than the body of the bacterium. It stains readily with the ordinary dyes, and is Gram-negative and not acid-fast. TEMPERATURE RELATIONS The best growth is obtained at temperatures between 17° and 20° C. There was no growth at 37.5° C. CULTURAL CHARACTERS Agar plates. — Colonies are visible in 48 hours at 20 to 22° C. They increase slowly in size, being less than i mm. in diameter at three days. After seven days the largest are 4 mm. in diameter and the average about 3 mm. The maximum size attained is 8 mm. They are roimd, smooth, convex, shining, opalescent at first, later becoming dull white with a slight creamy cast and develop an opaque center with a clear margin. They are finely granular under the compound microscope, with a slightly undulate margin. Buried colonies are lenticular. Agar slants. — Growth is slight in three days, the line of the stroke being about I mm. broad, and is not more than 5 mm. broad after one month. The growth is filiform, slightly raised, shining, smooth, and slimy. Considerable white sediment is formed at the base. The medium attains a slight pale-green fluorescence. Gelatin plates. — Colonies are visible in 48 hours as small points similar to those on agar. Liquefaction is rapid, beginning in cuplike hollows in 48 hours. The cupules are 5 to 10 mm. broad in 3 days. Thickly sown plates are completely liquefied in 48 hours. Gelati.n stabs. — Liquefaction is infundibuliform and begins in 24 hours. As liquefaction progresses, the upper part becomes stratiform, and the lower maintains the blunt funnel form. Liquefaction is complete within 15 days at 18° to 20° C. Beep bouillion. — Uniform heavy clouding occvirs within 48 hours. No surface scum or pellicle is produced, and there are no zoogleae. A grayish precipitate forms in old cultures. Beep bouillion with sodium chlorid. — Two per cent sodium chlorid produced only a slight inhibition of growth in 48 hours. Heavy clouding was present at seven days with 2 per cent of sodium chlorid, but gro^vth was practically inhibited with 4 per cent of the salt. Potato c\'Linders. — The form of growth is similar to the agar slant but with a slight dull yellow pigment. Milk. — Inoculated milk clears slowly and without coagulation. The protein is digested. Clearing begins within seven days in definite layers from the top downward, and is complete within three weeks. The liquid is only faintly translucent at this time and is near Ridge way 's pale fluorite green. Litmus milk. — Lavender-colored litmus milk becomes blue from the top down- ward in definite layers. The color change begins on the second or third day and is complete within 14 days. During two months the medium remained dark blue and liquid. Feb. 24. I9I9 Angular-Leafspot of Tobacco 227 Fermentation tubes.— The tests were made in basal solutions of i per cent pep- tone, to which was added i per cent of the following carbon compounds: Saccharose, dextrose, lactose, maltose, glycerin, and mannit. Clouding occurred in the open ends of all tubes in 48 hours, but the closed ends remained clear with a distinct line across the inner part of the U. Tests with neutral litmus paper gave an acid reac- tion with saccharose and dextrose, while the others were neutral or faintly alkaline. No gas was formed with any of the compounds. The tests for acid production with dextrose and saccharose were repeated, with loo-cc. portions of 2 per cent of each in 2 per cent peptone water. After 10 days the reaction was determined with phenolphthalein as the indicator. Both solutions showed acid production in excess of the controls as follows: Saccharose control, +6.6; saccharose inoculated, +11. o; dextrose control, +8.8; dextrose inoculated, +12.6. Reduction op nitrates. — Nitrates are not reduced. Indol. — A moderate indol production was obtained in Dunham's solution. Uschinsky's solution. — Clouding w£i.s evident after 48 hoiurs and was only moderate at seven days. The medium did not change color and no sciun or pellicle was formed. Aerobism. — The organism appears to be strictly aerobic. Following the chart of the Society of American Bacteriologists, the group number is 211.23220^ 33. SUMMARY A leafspot disease of tobacco which was prevalent in the flue-cured belt in Virginia in 191 7 is described under the name "angular-leaf spot." The disease has apparently been present to some extent for several years and may have a wide distribution. The disease is caused by a specific organism, which is described as "Bacterium angulatum." Rainfall is an important aid to infection, and the development of the organism within the tobacco leaf is apparently dependent to a marked degree on those predisposing factors which promote a rapid, vigorous growth of the host. The disease produced losses in both yield and grade. These were calculated in one field as a 20 per cent reduction in yield and a 40 per cent reduction in grade. LITERATURE CITED (i) Clinton, G. P. 1915. CHLOROSIS OK PLANTS WITH SPECIAL REFERENCE TO CALICO OP TOBACCO. In Conn. Agr. Exp. Sta. Ann. Rpt. 1914, p. 357-424, pi. 25-32. (2) DELACROIX, Georges. 1905. LA ROUILLE BLANCHE DU TAB AC ET LA NIELLE OU MALADIE DE LA MO- SAiQUE. In Compt. Rend. Acad. Sci. [Paris], t. 140, no. 10, p. 678-680. (3) 1906. RECHERCHES SUR QUELQUES MALADIES DU TABAC EN FRANCE. In Ann. Inst. Nat. Agron., s. 2, t. 5, p. 141-232, 17 fig. {4) Honing, J. A. I914. DE"zWARTEROESTE" DER DELI-TABAK. (black RUST OF DELI-TOBACCO.) Bui. Deli- Proef Stat, i, 16 p., 2 pi. Literatuur, p. 16. 1 A very slight fluorescence is imparted to the medium with agar stroke. 228 Journal of Agricultural Research voi. xvi. No. s (5) Leafspot Disease of Tobacco. 1918. In Va. Dept. Agr. and Immigr. Yearbook, 1917/18, (Bui. 126), p. 55-56. (6) Peters, Leo, and Schwartz, Martin. I912. KRANKHEITEN UND BESCHADIGUNGEN DBS TABAKS. Mitt. K. Biol. Anst. Land. u. Forstw. 13, 128 p., 92 fig. (7) Wolf, Frederick A., and Foster, A. C. 1918. tobacco wildfire. In Jour. Agr. Research, v. 12, no. 7, p. 449-458, 2 fig., pi. 15-16. Literature cited, p. 458. PLATE 25 A..— A tobacco leaf showing an early stage of the angular-leafspot. B. — Angular leaf spots on a tobacco leaf. About natural size. Angular- Leafspot of Tobacco Plate 25 Journal of Agricultural Research Vol. XVI, No. 8 Angular-Leafspot of Tobacco Plate 26 Journal of Agricultural Research Vol. XVI, No. PLATE 26 A. — ^Upper surface of a tobacco leaf affected with the angtxlar-leafspot. B. — Lower siirface of a tobacco leaf affected with the angular-leafspot. PLATE 27 A. — Angular leaf spots on a tobacco leaf as seen in transmitted light. B. — ^Atypical angular leaf spots on a narrow leaf of tobacco. The spots are blanched and rounded. Angular-Leafspot of Tobacco Plate 27 ^^HHBr^_ c - . £l A 1 k.- 3^. B Journal of Agricultural Research Vol. XVI, No. 8 Vol XVI MARCH 3, 1919 No. 9 JOURNAL QP AGRICULTURAL RESEARCH CONXE^NXS Pa«e Two Species of Pegbmyia Mining the Leaves of Dock - 229 S. W. FROST ( Contribution from Cornell Agricultural Ezperimeat Station ) Influence of Foreign Pollen on the Development of Vanilla Fruits - - - - - - > - - 245 T, B. McClelland (Contribution from States Relations Service) A Blood>Destro3ring Substance in Ascaris Lumbricoides - 253 BENJAMIN SCHWARTZ ( Contribution from Bureau of Animal Industry ) PUBLISHED BY ADTHORITY OF THE SECRETARY OF AGRICTJITDRE, WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WA.SMINGTON, r>. C. WASHINaTON : OOVERHMENT POIKTINa OFFICE ; ItIS ^^i ','>?J EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chairman Physiologist and Associate Chief, Bureau of Plant Industry EDWIN W. ALLEN Chief, Office of Experiment Stoiiont CHARLES L. MARLATT Entomologist and Assistant Chief. Bureau of Enlomologf ]fOR THE ASSOCIATION H. P. ARMSBY Director, Institute of Anitwd Nutrition, The Pennsylvania State College J. G. LIPMAN Director , New Jersey A gricuUural Experitaet^ Station, Rutgers College W. A. RILEY Entomologist and Chief, Drrision of Ento- mology and Economic Zoology, Agricul- turol Experiment Station of ti* Unioertity of Minnesota All correspondence regarding ai1:icles 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa. JOIMAL OF AGRIQllTDRAL RESEARCH Vol. XVI Washington, D. C, March 3, 191 9 No. 9 TWO SPECIES OF PEGOMYIA MINING THE LEAVES OF DOCK ^- By S. W. Frost - Instructor in Entomology, The Pennsylvania State College INTRODUCTION The life history and habits of two leaf-mining Anthomyids {Pegomyia spp.) are presented in this paper for the first time. The docks Rumex crispus h. and R. obtusifolius L. are extensively mined by several species of Pegomyia. Among these are two ^, P. calyptrata Zett. and P. affinis Stein., which occur commonly throughout the United States. P. calyp- trata Zett., is by far thie more common of the two species. The adult is readily distinguished by a bluish-gray thorax and a reddish-yellow abdomen. P. affinis Stein., on the other hand, is less common. It occurs about Ithaca, N. Y., abundantly in early summer, but later in the season no eggs, larvae, or adults have been found. This species is distinguished by its inconspicuous gray color. THE MORE COMMON SPECIES, PEOQMYI A CALYPTRATA HISTORICAL REVIEW P. calyptrata Zett. was first described by Zetterstedt (1846).^ since that time there have been but few references to it. Stein (1897),^ (1907),^ and Pandelle (1901)'' refer to this species, but mention nothing regarding its habits. This species has undoubtedly been noticed by many, but its identity has been unknown. In looking over some unidentified material in the collection at Cornell University the writer found specimens reared by Prof. Comstock as early as 1882. Dr. A. O. Johannsen, also of Cor- nell, noticed the same species in 191 3 mining dock, but did not publish his observations. ' Contribution from the Entomological Department of the Cornell University Agricultural Experiment Station. 2 The writer wishes to acknowledge his indebtedness to Dr. Robert Matheson, of the Department of Entomology, Cornell University, for many helpful suggestions and the criticism of this paper. ' There are at least two other species of Pegomyia occurring in this country, Pegomyia bicolor Wied. and Pegomyia winthem.i Meig., which mine Rumex spp. These occur in the northern United States and Canada, but do not appear to be common at Ithaca, N. Y. * Zetterstedt, J. W. diptera scandinavl/E. v. 5, p. 159 Limdae. 1846. 5 Stein, P. nordauerikanische anthomyiden ... In Berlin. Ent. Ztschr., Bd. 42 (1897), Heft 3/4 p. 239-241, 286. 1898. 5 Becker, Th., BEza, M., KERTi:sz, K., Stein, P. katalog der palaarktischen dipteren. v. 3, p. 701. Budapest. 1907. ' PANDB1.1.E, L. ETTJDEs SUR LES MtJSCiDES DE FRANCE. In Rev. Ent. France, t, 20, no. i, p. 294, 1901. Journal of Agricultural Research, Vol. XVI, No. 9 Washington, D. C. Mar. 3, 1919 rl Key No. N. Y. (Cornell^ (229) 230 Journal of Agricultural Research voi. xvi, N0.9 DISTRIBUTION p. calyptrata Zett., occurs in Europe as well as America, although it appears to be exceedingly rare in the former. Stein (1906)* neg- lects to list this species in his paper. Zetterstedt (1846)' mentions its occurrence in Sweden, stating that one specimen was taken near Lund by Mr. D. Dahlbom and a second at Vadstena by himself. Two specimens were also taken at Altenburg, Germany. In America it is a common species, especially in New York State, where the author has found it widely distributed. Stein (1897)' mentions its occurence in Washington, Minnesota, Illinois, Pennsylvania, and Massachusetts. To these the writer adds New Jersey and New York, having taken the the species at Ithaca, Binghamton, and Tarrytown, N. Y., and Orange, N.J. HOST PLANTS P. calyptrata mines exclusively in the leaves of several species of Rumex. Adults have been reared from R. ohtusifolius and R. crispus. Both species of Rumex are equally susceptible to the attack of the miner. R. acetosa is evidently a third host plant. Eggs of P. calyptrata were found on this plant. They hatched and the young larvae entered the leaf, but the writer did not succeed in rearing adults. A large num- ber of adults of other species were reared from larvae mining the leaves of garden beets (Beta vulgaris), spinach (Spinacia oleracea) and Swiss chard {Beta vulgaris var. cicla), as well as many weeds, such as Chenopo- dium album, Amaranthus retroflexus, and Atriplex pattila; but P. calyp- trata was not obtained from any of these. A number of experiments were performed to induce the larvae of P. calyptrata to mine in the leaves of other plants. Three eggs were care- fully removed from a dock leaf and placed on a beet leaf. Two days later the eggs hatched, but the larvae died without entering the leaf. In a second experiment four second-stage larvae were dissected from R. ohtusifolius and placed on Chenopodium album. The following day the four larvae were found dead. In a third experiment two third-stage larvae from R. crispus were placed on C. album. The following day one larva had entered the leaf and formed a small blotch mine, but the next day the larva died. These experiments seem to substantiate the fact that P. calyptrata mines solely in Rumex spp. LIKE HISTORY AND HABITS Eggs. — The eggs are glossy white, and in the field are laid usually in groups of three to five, occasionally in groups of six or seven, but 'Stein, P. die mir bekannten europaischen pegomyia-arten. In Wiener Ent. Ztschr., Jahrg. 35, Heft 2/3/4, P- 47-107- i9o6- 2 Zetterstedt, J. W. diptera scandinavl.*. v. s, p. 1775, 1846; v. 12, p. 4751, iSss- Lundae. * Stein, P. nordambrikaniscbb anthomyiden ... In Berlin. Ent. Ztschr., Bd. 42 (1897), Heft 3/4 p. 239-241, 286. 1898. Mar. Species of Pegomyia Mining Dock 231 seldom singly. They are laid in neat transverse rows on the undersur- face of the leaf. In captivity, however, the eggs are scattered over both surfaces of the leaf and are frequently laid singly instead of in groups. The number of eggs occurring on a single leaf is surprising. As a rule, one finds only 5 or 6 groups, but it is not uncommon to find more. In one instance the writer found 20 groups of eggs on a single leaf, 65 eggs in all. On another leaf, 16 inches long, he found 18 groups of eggs, 47 eggs in all. It is interesting to note that all the larvae from these eggs did not mature within the leaf on which they were laid, but migrated and started new mines on other leaves. The length of the e^gg stage is given in Table I. Table I. — Incubation period of the eggs of Pegomyia calyptrata Kxperinient No. A112 Am A113 A117 A122 A139 A132 A116 As... Ai34 A141 his.. Eggs hatched. May 13 May 14 ...do.... May 20 May 22 May 25 May 30 May 19 May 17 May 30 June s June 26 Length of egg stage. Days. LARViE. — The eggs hatch in from two to six days, and the young larvae immediately enter the leaf, making small holes through the lower epidermis. All the eggs of a single group hatch at the same time, and the larvae feed in a common mine, which is at first linear. The larvae mine side by side, progressing only in a forward direction. They keep close together, and all change their direction of mining at the same time, leaving behind them a short linear path (Pi. 28, D). In about a day, although no definite time can be set, the larvae begin to enlarge their mine laterally, forming a blotch. They still remain in a common mine, but separate in different directions! It is not an unusual sight to see several such blotches on a leaf. Each represents a number of larvae that have hatched from a single group of eggs. These increase in size until they interfere with each other, and a large blotch is produced, covering the entire area of the leaf. Many of the larvae are naturally forced to abandon their mines and form new ones in other leaves. The presence of nearly mature larvae in small blotch mines is an indication that they have entered fresh leaves (Pi. 28, H). 12,2 Journal of Agricultural Research Vol. XVI. No. 9 The writer has removed a larva from its mine and watched it form a new one. At first the larva cuts a short slit in the epidermis of the leaf. Then by inserting the mouth hooks in this slit and working them back and forth the lower and upper epidermises are separated. The larva then pushes the anterior end of its body into the small opening which it has made. After the first two segments have been forced into the leaf, it is only a matter of a few minutes before the larva works its way completely within it. This operation is accomplished with many vig- orous twists of the body as it is drawn into the leaf. Larvae of the third instar bury themselves completely in the leaf in less than 20 minutes. The feeding habits of the larvae are most interesting and can be very conveniently watched under a microscope by means of transmitted light. The pharyngeal skeleton (fig. i, A-D) bearing the mouth hooks, is loosely joined within the first and second segments and is capable of great freedom of movement. These move very rapidly when the larv^a is feeding and are very effective tools for tearing away the parenchyma of the leaf. There are two distinct types of movement which the mouth . hooks possess. First, a lateral movement ; the mouth hooks are turned perpendicular to their normal position and work in the plane of the mine. They strike to the right for a short time, then to the left, separating the upper and lower epidermises but not tearing away any of the tissue. The second movement of the hooks is a vertical one. As the hooks are held in their normal position, they are thrust downward, and pieces of the parenchyma are torn loose from the bottom of the mine. In the first case the two epidermal layers are only separated, while in the second the parenchyma of the leaf is actually removed. Table II. — Length of the larval period of Pcgomyia calyptrata Experiment No. A 126 A 129 A 13s A 35 A 225 Eggs hatched. May- May May July Oct. Puparium formed. June 7 June 10 June 8 July 12 Oct. 21 Length of larval period. Days. 16 10 9 13 From Table II it will be seen that the larval period varies somewhat. This variation is undoubtedly due to weather conditions. During warm weather the larvae mature rapidly, but during cooler weather the miners become inactive, and the larval period may be prolonged several days. Unfortunately the table is not complete enough to show this. How- ever, it shows a tendency for a longer larval period in early May and October than in the latter part of May and July. Mar. 3, 1919 Species of Pegomyia Alining Dock 233 Fig. 1.— .4, pharyngeal skeleton of first instar, Pegomyia calypCrata Zett, ; B, pharyngeal skeleton of second instar.P. calyptrala; C,ma.ndihulnr sclerite of second instar, P. calypirata: D, pharyngeal skeleton of thjrd instar, P calyptrata; E, pharyngeal skeleton of first instar. P. affinis Stem.; F, mandibular sclerite of first instar, P. affinis; G, pharyngeal skeleton of second instar, P. affinis; H, pharyngeal skeleton of third instar, P. affinis. 234 Journal of Agricultural Research voi. xvi, no. 9 Formation of the; puparia. — The mature larvae escape through the epidermis of the leaf, but seldom make their exit through a definite hole. Usually the upper epidermis become dry and parchment -like, rupturing itself and allowing the larvae to escape. Sometimes the larvae cut circular holes through the epidermis. They fall to the ground and ordinarily penetrate the soil to a depth of 2 or 3 inches. If the ground is hard, they do not enter, but form their puparia beneath leaves or other rubbish. The depth to which the larvae penetrate varies considerably. To determine this, a few experiments were performed with full-grown larvae. For this purpose a root cage was used, having a space of X inch between the glass and the back. This space was filled with loose sandy soil. The surface of the earth was covered to prevent the larvae from eacaping. This dark- ened the top of the soil and slightly altered normal conditions. Twenty-seven mature larvae were placed on the surface of the soil. The following day eight puparia were found on top of the earth. The remainder of the larvae either failed to transform or escaped. In a second experiment 32 mature larvae were placed on the surface of the soil. Four larvae transformed on the surface, six were found at a depth of 2 inches, one at a depth of 5 inches, and two at a depth of 6 inches. These experi- ments, though few in number, give some idea of the depth to which the larvae penetrate loosely compacted soil. In captivity many of the larvae formed their puparia within the leaves. Mined leaves were collected in large numbers and put in receptacles to obtain puparia. In most cases the puparia were found in the leaves or between the leaves from which they had issued. A few of the larvae wandered about in an attempt to find a more suitable place in which to transform. Evidently many of the lar\^ae under natural conditions never enter the soil. Adults. — While working with the adults, the writer had an oppor- tunity to experiment with several types of rearing cages. A cage similar to the Riley cage, covered with cheesecloth on three sides and with glass on the fourth, gave good results when large numbers of individuals were used, but proved useless for individual pairs of flies. Glass cylinder cages (PI. 29, B) }delded better results for this purpose, but were unsat- isfactory because the adults escaped when the cylinders were lifted. The most satisfactory type of cage proved to betheFiskecage (Pi. 29, A,C), used a great deal in the rearing of parasites by the United States De- partment of Agriculture at Melrose Highlands, Massachusetts. The small opening at the top readily permits one to introduce food, while the ring at the bottom prevents the flies from escaping. In spite of all the precautions in handling and feeding the adults, they did not live long in captivity. One male was kept alive for 18 days. Table III gives the length of life of the males and females as obtained from pairs that were kept in captivity. Mar. 3, 1919 Species of Pegomyia Mining Dock 235 Table III — Length of the life of the adults of Pegomyia calyptrata Females. Males. Issued. Died. Lived. Issued. Died. Lived. Days. Days. July 2 July 5 3 July 2 July 5 3 July 3 July 19 16 July 3 July 21 18 July 19 July 20 July 20 July 27 Aug. 16 Aug. 23 Aug. 16 Aug. 23 7 Do. Aug. 24 Do. Aug. 22 6 Aug. 22 Sept. 2 II Aug. 22 Sept. 2 II Aug. 26 Aug. 30 Aug. 26 Aug. 30 4 Aug. 31 Sept. 10 10 Aug. 31 Sept. 7 7 Sept. I Sept. 3 2 Sept. I Sept. 3 2 Sept. 23 Sept. 28 5 Oct. 10 Oct. 12 2 Oct. 10 Oct. 12 2 May 6 May II 5 Aug. 16 Aug. 22 6 Copulation frequently takes place a few hours after the female issues. Flies that had issued during the night were observed in several cases to copulate during the following morning. In order to induce copulation, good results were obtained by placing pairs in 6-dram vials. They could be conveniently watched, and mating took place quite readily. Pairs that were in larger cages usually died before copulation took place. Copulation usually lasted about 40 minutes; in one case it lasted for i hour and 15 minutes. It is still unknown whether a second or later copulations take place. The number of eggs laid was obtained only by dissection. About 62 ripe eggs were found in a fertilized female. All these are evidently laid during one oviposition period. Number of generations. — The writer has been unable to determine accurately the number of generations a year. This is due to the repeated difficulty he experienced in attempts to keep the adults alive for any considerable period of time, as well as the failure to obtain females to oviposit. Only four of many females experimented with laid eggs, and of these none laid more than five eggs. Under such conditions it was impossible to rear the generations through from eggs to adults. How- ever, the number of generations was followed quite accurately by observ- ing conditions in the field and comparing them with conditions under observation at the insectary. The various generations overlap one another, and it is impossible to tell, from outside conditions alone, when one ends and the other begins. The terminations of the first generation can be plainly seen. For example, in the spring of 1916 the first flies to issue from puparia kept out of doors over the winter appeared on May 4. The adults continued to issue until May 16. The females began laying soon after emerging; 236 Journal of Agricultural Research Vol. XVI. No. 9 those issuing from puparia in captivity laid in four or five days. This checks with condition in the field, for the first eggs were observed out- side on May 7. The larvae matured in from 9 to 16 days. Toward the end of the larval period they mined rapidly, producing conspicuous blotches on the leaves. The maturing of the first-generation larvae could thus be easily distinguished. After this it was impossible to fol- low the number of generations by observation in the field alone. One can go out in the field any time during the summer and find eggs, larvae, puparia, and adults all at the same time. From a number of eggs laid in the spring by the females, overwinter- ing as puparia, the writer succieeded in rearing adults of the first genera- tion. These adults laid a few eggs, but none of them hatched. By bringing in eggs from the field at this time the writer reared adults of the second generation. This was continued through the summer, and a fair idea of the number of generations was obtained. During 1915 and 1916 the writer obtained three and a partial fourth generation. The majority of the fourth generation were overtaken by cold weather and perished. From Table IV it will be seen that some of the puparia fonned in June and July, as well as those formed in September, did not give forth adults the same year that they were formed, but overwintered as puparia, and the adults issued the following spring. This seems to be a provi- sion of nature to insure the continuation of the race the following year in case all the individuals of any generation should perish. Table IV. — Length of the pupal stage of Pegomyia calyptrata Experiment No. A150. A168. A165. A9... A14. . A167. A169. A182. A17.. A27. . A26.. A28. A31. Number Puparia Adults Number of of puparia. formed. issued. adults. 6 June 5 July I I 9 /Jiuie 9 l.do [uly 5 2 9 4 luly 6 I 9 /..do \..do lulv I 6 s 29 .Ttily 3 I 9 4 June II luly 6 I S /..do \..do luly 3 4 c? Tuly 4 3 9 29 June 13 July 3 I cf lO ...do luly .'; I 9 17 June 16 luly 3 I ^ 39 /June 25 \..do luly 16 I , p. 239-241, 286. 1897. Mar. 3, 1919 Species of Pegomyia Mining Dock 241 Table VI. — Incubation period of the eggs of Pegomyia affinis Experiment No. Eggs laid. Eggs hatched. Length of stage. A115 A119 A125 A130 A131 May II May 14 May 18 May 24 May 25 May 16 May 21 May 24 May 28 ..do Days. IvARVA. — The eggs hatch in from three to seven days. All the eggs of a single group hatch at the same time, and the young larvge feed to- gether in a common mine. At first this is linear, but soon the larvae separate in different directions and a blotch mine is formed which ob- scures the original linear track. The mines produced on the leaves can not be distinguished from those of P. calyptrata. Table VII. — Length of the larval period of Pegomyia affinis Experiment No. A2... A124. A124. Eggs hatched. May May ..do. A127 May 22 A143 May 31 Puparium formed. May 23 June 6 Jtine 8 June 7 June 13 Length of larval period. Days. 12 16 18 16 13 From Table VII it will be seen that the length of the larval period varies from 12 to 18 days. A larger number of records would, perhaps, show even greater variation. This variation is due, as in case of P. calyptrata, to weather conditions. The mature larvae escape from the leaves through the cracked surface of the dried and parchment-like mine. If the ground is not too hard, they penetrate to a depth of 2 or 3 inches; otherwise, they form their puparia beneath leaves or rubbish. Number of generations. — ^The writer is uncertain as to the number of generations. At first he confused the two species mining dock and thought there was but one. Later he noticed his mistake, but it was then too late to make definite observations. The few notes made seem to indicate that there are but two generations a year. A portion of the first-generation adults issued in from 12 to 18 days; the rest overwin- tered as puparia and issued the following spring. It is believed that all of the second generation overwinter as puparia and issue the follovdng spring. This seems to be the case, because the eggs and larvae of this species were not found after the end of June. 242 Journal of Agricultural Research Vol. XVI, No. 9 Table VIII shows the tendency of the first generation to produce some puparia from which adults issue the same year and others that over- winter and from which adults issue the following year. The species is Uke P. calyptrata in this respect. Table VIII. — Tendency of individuals of the first generation to overwinter as puparia Experiment No. A13 An Aio A12 A16 A19 Number of pupa- ria. Pupariujn fonned. June 9 . .do ..do June II June 24 July I Adults issued. July 3 May 6° July 3 May 16a May 60 ...do Number of adults. I A SH 8K 9 9A 9H Girth in sixteenths of an inch at i}^ inches from stem end. 26 27 24 26 26 26 26 28 28 29 30 27 30 26 30 30 28 30 32 554 27.7 Increase or decrease in circumfer- enceof ix)ds. > > > > > > > > > > > > > > > > > > > > > > Girth in sixteenths of an inch at I inch from blossom end. 18 20 22 18 20 20 20 20 21 20 22 21 20 24 24 22 28 26 27 30 443 22. 2 248 Journal of Agricultural Research Vol. XVI, No. 9 Typical specimens are shown in Plate 31, figures A shovsdng the whole fruits and figure B the same with sections made at the lines of measure- ments. From left to right the first of the paired fruits shows the develop- ment of V. planifolia when close-fertilized, while the second shows the results when vanillon pollen has been applied. The sections show that many more ovules have been fertilized near the apex of the ovary by the V. planifolia pollen, while the vanillon pollen has fertilized many more near the base than near the apex. This difference in location of the ovules fertilized accounts for the striking difference in form which results from the application of V. planifolia or vanillon pollen to the V. plani- folia stigma. Table III shows the typical close-pollinated vanillon fruit to be of nearly equal average girth at i inch from either end. Of the 54 fruits measured the two girths were equal in 12, the apical girth greater in 18, and the basal girth greater in 24 fruits. The girth at i inch from stem end averaged 0.5 per cent greater than at i inch from blossom end. Table III. — Comparison of the girth measurements of the fruit of -vanillon 9 Xvanillon^ V51.. V13.. V 13.. V13.. V34.. V13.. V13.. V51.. V52.. V52.. V52.. V13. V13. V34- V43- V43 Vsi- V13. Vi3- V13. VI3. VI3. Vi3- V13. V52. V13. V13. V52. V H2. V52. VS2. V34- V34- V43- V34. Girth in Girth in Length of sixteenths [n crease or sixteenths of an inch decrease in of an inch fruit in at I inch circumfer- at I inch from stem ence of pods. from blos- end. som end. 3 34 > 32 3K 39 > 36 3K 37 = 37 3K 40 = 40 3K 30 > 29 sH 41 > 39 3K 40 = 40 3H 38 > 36 3H 40 > 36 3H 40 > 37 4K 36 > 34 4 40 = 40 4 42 = 42 4 32 > 31 4 40 > 32 4 31 > 29 4 40 > 36 4K 42 < 43 4>< 44 ^ ■^ 45 4X 42 < 43 4^/4 41 > 40 4M 45 = 45 4K 42 < 43 4>^ 43 = 43 4^ 43 < 44 4^/2 42 > 38 4K 46 > 45 4H 41 < 45 4H 41 > 40 5 47 = 47 5 42 = 42 5 41 < 44 SK 35 < 36 sX 33 = 33 sX 37 . > 34 sK 35 < 36 Mar. 3, 1919 Development of Vanilla Fruits 249 Table III. -Comparison 0/ the girth measurements of the fruit of vanillon 9 Xvanil- lonS — Continued . V43- V43- V34- V43- V34- V34- V43- V34. V43- V34. V43- V43. V34. V43- V43- V43- V43- V43- Total... Average. I,enKth of fruit in inches. sK sK sH sH 6 6 6 6K 6K Gitth in sixteenths of an inch at I inch from stem end. 43 40 34 42 35 31 46 37 43 32 38 39 29 35 44 46 45 45 2, 126 39-4 Increase or decrease in circtunfer- ence of pods. > < > < < > < > < > < < < > < > > > Girth in sixteenths of an inch at I inch from blos- som end. 38 40 36 39 38 35 45 42 41 35 38 38 34 '42 48 44 47 43 "5 39-2 » This fruit which externally resembled a V 9 XPcf cross had ovules fertilized throughout its length which was true of no crosses of V43 9 X P . 27) the physical improvement of the soil following the use of manure not to the carbonaceous matter of the manure, but to the superior growth of plant roots induced by the nitrogen and mineral elements carried by the manure. His conclusions are based chiefly upon data from experiments at the Ohio Experiment Station covering a period of 24 years, in which the recovery of nitrogen, phosphorus, and potassium has been higher from sodium nitrate, acid phosphate, and potassium chlorid than from farm manure. As the Ohio experiments have been conducted on a soil depleted of its organic matter by a long period of tenant husbandry before the test began and the average yields of the tmtreated land diu"ing the period of the test have been only 7.85 bushels per acre of wheat, 14.70 bushels of com and 21.76 bushels of oats {12, p. 26), the results would suggest that we have been in the habit of overrating the benefit derived from any increased water-holding capacity of the soil caused by the application of manure, or even that the effect of the added organic matter upon this property may in reality be too slight to have any practical importance. Entirely satisfactory fields for studies designed to determine the effect of differences in the content of organic matter upon the water-holding capacity of soils are scarce; and with most field soils the bringing about of an appreciable increase through applications of manure as light as those 1 Published, with the approval of the Director, as Paper 150, of the Journal Series of the Minnesota Agri- cultural Experiment Station. ^ Reference is made by niunber (italic) to " Literature cited," p. 277-278. Journal of Agricultural Research, Vol. XVI, No. 10. Washington, D. C. Mar. 10, 1919. ro Key No. Minn.-37. (263) 264 Journal of Agricultural Research voi. xvi. no. 10 used in farm practice requires a rather long time. This is well illustrated at Rothamsted by the Broadbalk Field, which has been continuously in wheat since 1843. On plot 2b, which receives 14 tons of farm-yard manure every year, the first 9 inches of soil gained only 0.098 per cent of organic carbon in the 12-year period from 1881 to 1893 (9, p. 12^), and in 1893, after having received such an annual application for 50 years, or 700 tons per acre in all, the 9-inch layer of soil contained only 1.342 per cent more organic matter than the adjacent plot 3, which during the same period had been continuously cropped without any manure or fer- tilizers. By starting with a virgin prairie soil rich in organic matter and putting it under continuous clean cultivation, an appreciable lowering of the organic matter can be induced much more quickly, but even in this case a long period is necessary (d, p. 136). On many fields the great variation in texture from place to place, espe- cially in the portion of the soil mass below the reach of the plow, renders any comparison of the relative amounts of useful moisture a laborious task, the difiFerences shown in moisture retentiveness being more depend- ent upon differences in texture than upon any differences in the content of organic matter that may have been induced by dissimilar methods of manuring, cropping, or tillage. Detailed studies of the uniformity in texture of the plots or fields under comparison have usually been omitted, and, hence, it may easily be that many of the data published in support of the common belief are due simply to the coincidence that soils of a finer texture, while they retain more water, also usually have a higher content of organic matter. Under natural grassland conditions the heav- ier soils are the richer in organic matter and in general it appears that when conditions of surface drainage and climate are similar, the finer the texture of the soil the higher will be the organic-matter content when equilibrium between the processes of decay and those inducing an accu- mulation of orgamc matter have once been attained. Under the condi- tions of ordinary mixed farming, arable soils will approach more nearly to grassland than to forest conditions. An unusual opportunity for such a study is offered by some plots at this Experiment Station, laid out by Snyder (//) some 25 years ago. In the summer of 191 5, incidental to a study of the effect of different systems of cropping upon the composition, properties, and productivity of the silt loam soil of these plots, we obtained some data upon the water- holding capacity. It so happened that the season was characterized by weather conditions especially favorable for revealing any differences which might exist in the water-holding capacity of the soils of the various plots. DESCRIPTION AND HISTORY OF PLOTS The land, originally covered by a heavy growth of deciduous trees, had been cleared about 1856, and during the following quarter of a century formed part of a typical grain farm of that period, oats or wheat being Mar. lo, 1919 Organic Matter and Water-Holding Capacity of Soil 265 grown upon it every year without the use of clover or manure. In 1883 the university acquired the farm for experimental purposes and the next year the field was seeded to clover, from that time on being kept in a good rotation until in 1893 Snyder {11, p. 2) laid it out in six plots as a fertility experiment (fig. i, A). All available records indicate that the land included in the plots had been treated alike during the preceding 36-year period (i 856-1 892). Plots 2 and 3 were to be kept in 4- and 5-year rotations, including clover and receiving manure, and each of the others to be devoted continuously to the same grain crop and to receive .zr 2zz: jz jT jzz: 0! < h .w < >- u > t U ^ u > bJ TT ►, u uJ M 2 > oi K o o 3 ^ C -J 5 ul 1- U ■< u S o z => o E ^5 a. < UJ 2^ tiy OK 0^ UJ <; Ul 66 33 6 S3 (,' $5 O - f<"' ComposiUs A OAOAOA OAOAOA OaOaOA OAOAOA JT 2zr JOi' jr ^7 c 0 E N s 0 R G H u M B T U R N I p S A L F A L F A - 1 M A N G E L S 1 ^ n Fig. I. — Diagram showing arrangement of plots and crops on field J, University Farm, St. Paul, Minn, A is the original plan adhered to from 1893 to 1914, while B shows the cropping plan in 1915, C shows the arrangement of the samples taken for the two composites from a plot. neither manure nor fertilizer, No. i, 4, 5, and 6 being planted to wheat, com, oats, and barley, respectively. The construction of a bam in 191 2 upon part of plot i has rendered this useless for experimental purposes (PI. 36). The present discussion deals chiefly with plots 3 and 4, on the latter of which, beginning with 1893, there had been grown 22 successive crops of corn, without the application of any manure. On the other there had been only 6 crops of com, but 4 of barley, 7 of oats, and 5 of clover, while 25 tons per acre of manure had been applied, 5 tons with each crop of com except that of 1897. 266 Journal of Agricultural Research voi. xvi, no. io Plots 2 and 3 are 5 rods long and 4 rods wide, while the others are of the same length but of only half the width. Bach plot is separated from its neighbors by a strip 6 feet wide. The plots have been seeded to the center of this 6-foot strip, the outer edge around each plot being cut away at harvest time. The soil has been classified by the Bureau of Soils of the United States Department of Agriculture as Hempstead silt loam (jo, p. 26). The silt loam stratum extends to a depth of from 39 to 50 inches, below \\hich is a thick bed of clean gravel and coarse sand. The surface stratum is very uniform in texture as may be seen from the moisture equivalents reported in Table III. CULTURAL CONDITIONS The original plan of the experiment had been carried out until it was interrupted in the spring of 191 5, when in order to determine the relative productivity of the different plots we had all five plowed, prepared alike, and planted to the same crops — viz, corn {Zea mays), sorghum (Andro- pogon sorghum), turnip (Brassica rapa), alfalfa (Medicago saliva), and mangels {Beta vulgaris macrorhiza) — these being so arranged that each of the five appeared on every plot (fig. i, B). Weeds were very bad on all the plots except No. 4 but, by frequent use of hoe and horse culti- vator, these were kept down in all the crops except the alfalfa. On account of the unusually cool weather, the crops made very slow growth until early in July, and none of the plots at any time appeared to suffer from a lack of moisture. DISTRIBUTION OF ORGANIC MATTER IN .SURFACE FOOT The ratio of organic carbon to nitrogen in surface soils is so nearly constant that determinations of the latter serve to indicate variations in the content of organic matter. Table I shows the nitrogen content ' of the successive levels within the first foot of soil on the five plots. The percentages reported in the first part of the table (a) are for com- posite samples from six borings using a 4-inch plate auger and those in (b) for composites of 24 samples taken by means of a 1.5-inch soil tube of special construction. The latter are really the averages of the data from two sets of samples, A and B, in each of which composites of 12 individual samples were employed, these being distributed as indicated in figure i, C. Vae concordance of the data from these two sets, as illustrated by Table II, is such that in the present discussion no purpose would be served by reporting more than their averages. From these data it is evident that any marked differences in nitrogen content, and, hence, in organic matter found between samples from the same level on different plots are to be attributed to differences in crop history and not to the experimental errors of sampling. Mar. lo, 1919 Organic Matter and Water-Holding Capacity of Soil 267 Table I. — Nitrogen and organic matter in successive levels of the surface foot (a) nitrogen in CORCOSITES from 6 BORINGS WITH AUGER Depth of section. Plot 2. Plot 3. Plot 4. •Plot s. Plot 6. Inches. I to ^ Per cent. 0. 242 •239 . 206 •15s Per cent. 0. 242 ■234 •223 . 190 Per cent. 0. 176 .177 . 169 .130 Per cent. 0. 210 .214 • 194 .163 Per cent. 4 to 6 7 to 0 • 195 .187 10 to 12 Average . 2X1 . 222 .163 •195 . 171 (b) NITROGEN IN COMPOSITES FROM 24 BORINGS WITH TUBE I to 6 0.236 •235 . 212 . 190 •175 . 160 • 149 0-235 .241 .232 . 222 • 205 . 192 . 181 0. 180 .168 .163 •151 •145 . 126 .116 0. 208 . 207 •203 . 200 . 180 .158 • 149 0.193 .187 . 170 .152 . 128 .116 . 108 7 8 0 10 II 12 Average . 211 .223 . 162 • 195 .168 (C) ORGANIC MATTER IN SOME OF ABOVE SAMPLES « ( = =ORGANIC CARBON X 1. 724) I to 6 5-21 5. 02 4- 05 3-97 3-79 2.41 7 to 0 10 to 12 Averages 4.76 3-39 1 (d) RATIO OF ORGANIC CARBON TO NITROGEN I to 6. 7 to 9. 10 to 12 12. 9 13.0 12. 4 12.8 13-5 10.8 a The carbon was determined bi" combustion with copper oxid in a current of oxygen after previous treatment with phosphoric acid. Plots 3 and 4 show the extremes, the former having the highest and the latter the lowest nitrogen content and at every level plot 3 shows the higher value. The percentages on plot 2 are almost as high as those on 3 while the values for plots 5 and 6 fall between those for 3 and 4. In its low nitrogen content, plot 6 approached 4, the sections below the ninth inch containing even less than the corresponding ones on the latter. The nitrogen content of the first foot on plot 3 was 138 per cent and the organic matter 140 per cent of that on plot 4. These two plots, as they showed the extremes in nitrogen, and, hence, in organic-matter content, while being adjacent, were selected for the more detailed moisture studies. 106544°— 19 2 268 Journal of Agricultural Research Vol. XVI, No. lo Table II. — Nitrogen content of soil at different levels on plots 2 and j, illustrating the degree of concordance of the daiafrotn duplicate sets of composite samples Depth of section. Plot, Set A. Set B. Difference Plot 4, Set A. Set B. Difference Inches. I to 6 7 8 9 lO II 12 13 to 15 16 to 18 Per cent. O. 229 ■237 .230 . 227 . 212 .199 . 187 .165 Per cent, o. 240 .244 • 233 . 216 • 197 . 184 .174 • 143 . 114 Per cent. O. Oil . 007 .003 . on .015 .015 .013 . 022 . 007 Per cent. O. 182 . 166 . 167 . 160 ■ 147 . 124 . 120 . 096 .078 Per cent, o. 178 . 170 .158 . 141 . 142 . 127 . 112 .099 .087 Per cent, o. 004 . 004 . 009 . 019 .005 • 003 .008 .003 . 009 UNIFORMITY IN TEXTURE The moisture equivalents of the soil from the different levels are re- ported in Table III. Those for the first 12 inches were determined on composites made by combining equal weights of the duplicate samples reported in Table II, and, hence, they represent composites of 24 indi- vidual samples from each plot. The data for the second and third feet are from composites from 6 borings on each plot. Table III. — Moisture equivalents of soil at different levels Depth of section. I to 6 inches... Seventh inch. Eighth inch . . Ninth inch. . . Tenth inch . . . Eleventli inch Twelfth inch . Second foot . . Third foot Plot 2. Plot 3. Plot 4. Plot s. Per cent. Per cent. Per cent. Per cent. 23-9 24. 2 22.8 23-9 24. 0 24-5 23-7 23.6 24. 2 25-3 23.2 24.1 23-4 24-3 23-3 24. 0 23-5 24. 6 22. 7 23. I 23.1 24. I 23.0 23-4 22. I 23.8 22.3 22. 6 24. I 23.6 24. 0 23. 2 23-3 23.2 22. 5 23-7 Plot 6. Per cent. 24-3 24-3 24- 5 24. 2 24. o 23.2 23. o 24-3 22. As was to be expected from the content of organic matter, the lowest values within the surface foot are shown by plot 4 and the highest by plot 3. The differences in the moisture equivalent are very slight com- pared with those in nitrogen and organic matter; while the nitrogen in the surface foot of plot 3 exceeds that in plot 4 by 38 per cent and the organic matter shows a corresponding difference of 40 per cent, the mois- ture equivalent is only 7 per cent higher. In the case of the second and third feet, the crop history of the plots appears to have exerted no appre- ciable influence upon the moisture equivalent, the differences shown in these levels being within the limits of error in sampling. Mar. lo, 1919 Organic Matter and Water-Holding Capacity of Soil 269 The uniformity in texture of both the surface soil and subsoil from plot to plot makes the field unusually favorable for such a moisture study. The thickness of the silt loam layer overlying the gravel stratum men- tioned above varies from about 39 to 50 inches, in nearly all places it being less than 48 inches, and the variations from place to place on the same plots appear as great as those from one plot to another. While we regularly sampled the fourth-foot section along with the second and third, it showed such an extreme range in texture, ov/ing to the varying proportions of its two component layers, silt loam and gravel, that the data on this level are of no use in the present discussion. WEATHER CONDITIONS The weather of the crop season of 191 5 was favorable for the mainte- nance of a very moist soil, being abnormally rainy, cool, and cloudy (Table IV). In each of the first three months. May, June, and July, the precipi- -2111. MAY JUNE JULY 1 AUG. SEPT 1 <71H- * » t * * » * ► M *^ 4 It d I J h ii . >,. ^J.^^ ll u..^ ij 1 J JL J, N,.l „„ I 5 10 15 20 25 15 10 IS 20 25 15 10 IS 20 25 t 5 10 15 £0 25 Fig. 2.— Diagram showing the amount and distribution of the rainfall at University Farm, St. Paul, Minn., during part of the season of 1915. The dates of sampling are indicated by asterisks. tation was somewhat above the normal and in August it was just equal to the normal. For each of the months the mean temperatures varied from 6.1 to 4.2 degrees below normal and the percentage of possible sunshine in the first three months varied from i6 to 26 below normal, while the wind movement was slightly below and the relative humidity slightly above normal. The precipitation of the autumn of 191 4 and the following winter and first two months of spring had been nearly normal, that of April being 2.31 inches. The rainfall of the four months covered by the study came chiefly in the form of slow rains which caused but little run-off; excep- tions were provided by two heavy rains, one on July 6 and the other on July 14 (Table V and fig. 2). The dates of sampling happened to be such as to well illustrate the various conditions met with in a wet season. Thus on both July 7 and 15, the samples were taken only a day after a very heavy rain had fallen while on May 18 and August 4 they were taken one day after the last rain in a succession of days with moderate rains. 270 Journal of Agricultural- Research Vol. XVI, No. lo Table IV. — Weather of the crop seasons of igij and igi8 at St. Paul compared with the normal PRECIPITATION 0 (inches) Item. Ma)^ June. July. Aug. Normal 3-34 I. 01 .98 4-03 0.68 -1.18 3-49 2. 42 .41 3-36 . 00 Departure in 191 5 Departure in 1918 . 20 MEAN TEMPERATURE (° K.) Normal Departure in 191 5 . Departure in 1918. suNSinNE (per cent op possible) Normal Departure in 1915 . Departure in 19 18. 67 I -3 WIND VELOCITY (miLES PER HOUR) Normal Departvu-e in 1915 . Departure in 1918 . 9-5 • 4 .8 RELATIVE HUMIDITY (PER CENT) Normal Departure in 191 5 . Departure in 1918 . 70 I — 2 o At University Farm. From September i, 1914. to April 30, 1915, 11.86 inches, and for the same period in 1917-1918, 6.89 inches. Table V.- -Daily precipitation at University Farm, St. Paul, during the season of 191 5 Day. May. June. July. Aug. Sept. Day. May. June. July. Aug. I 0.39 •57 I. 04 •74 17 18 10 0. 16 .07 2 T 0.65 .04 . 12 •03 T I. 46 0. 29 T T •03 3 4 s 6 0. 26 .62 0. 04 .19 20 21 22 2-3 .62 ■s T . 10 T T •03 1.82 7 8 T 0. 10 .07 "t" 0. 18 •17 24 Q 25 26 .66 10 .40 T .40 .04 .18 .08 T .07 . 24 I. 40 T .96 .09 II T T . 10 .38 •15 .27 27 .29 12 28 29 .44 . 10 .06 13 14 15 16 . 20 . 22 2. 60 •57 .04 ■JI t Total... 4-35 4.71 5- 91 3^36 T= Trace. Mar. lo. 1919 Organic Matter and Water-Holding Capacity of Soil 271 MOISTURE CONTENT OF THE SOIL Between May i and September 2, plots 3 and 4 were sampled 15 times (Table VI) to a depth of i foot, the samples being taken in 3-inch sec- tions from three borings in a north and south line across each plot. In the case of each set one boring was in the corn, another in the sorghum, and the third in the mangels, each being close to a crop row. At the time of the first sampling in each month we sampled the second- and third-foot section also, both on these two plots and on the three others (Table VII). From Table VI it will be seen that on every occasion the surface foot of plot 3 contained more moisture than that of plot 4, and, except on the very last date, the same holds true for the four 3-inch sections. Throughout the first three months the difference ranged between 3 and 5 per cent, being greatest when the sampling occurred soon after the ces- sation of a rain. During the last half month, at a time when the crops were drawing most heavily upon the soil moisture and there was but little rain, the differences were much less, falling on the average to less than I per cent. There is no evidence that, in general, more water was retained in the second and third foot on plot 3 than on plot 4 (Table VII), although more was found on May i, which, however, was not long after the frost had disappeared from the subsoil and there had not been time for the down- ward percolation of the water from the melting snow and the Apri[ rains. With the three other plots, the surface foot was intermediate in moisture between plots 3 and 4, the relative moisture content varying roughly with the nitrogen content (Table VII). Only at the time of the first sampling did they, like plot 3, show a higher moisture content in the second and third foot than plot 4. The above remarks apply directly to the total water content, which includes both the nonavailable and the available. As the portion of the soil moisture available to plants for growth and for the maintenance of life appears to be approximately that in excess of the hygroscopic coefficient (i, p. 122; 2), and as the latter value is a little lower for the surface of plot 4, being only y.y compared with 8.1 for plot 3 (Table VIII), we regard the differences in useful water as slightly greater than those in the total water reported in Table VI. INFLUENCE OF ORGANIC MATTER UPON MOISTURE CONTENT From the above data it would appear that the greater amount of organic matter in the surface foot of plot 3 is responsible for the con- siderably higher content of both total and free water shown by it through- out most of the summer of 191 5. Any advantage possessed by one plot over another, due to topography, lies with No. 4. The surface of the field is almost level, but after very heavy rains and at the time of the melting of the snow in the spring the last water to disappear from the field is found upon that plot (Plate 36). 272 Journal of Agricultural Research Yo\. XVI, No. 10 Table VI. — Moisture content of the surface foot of plot 4 and the excess of moisture in that of plot ;^ A — MOISTURE CONTENT OF SOIL OF PLOT 4 Depth of section. May I. May II. May 18. June 10. July I. July 7- July IS- July July 23- 30- Aug. 4- Aug. 7. Aug. 14. Aug. 21. Aug. 28. Sept. 2. Inches P.ci. J24.2 » P.ct. /21.4 \26.1 f 2?.2 P.ct. 27.5 27.7 27-7 26. s P.d. 24.9 27-5 27.9 27.2 P.ct. 22.6 24-9 2S-7 25-2 p.ci. 28.7 29.1 29.1 28,5 P.ci. 28.3 27-9 28.5 28.0 P.ct. P.ci. 22.3 22.2 24.9 24.7 25-5 25-2 P.ci. 28.0 28.8 28.4 29.2 P.ci. 2S-I 26.4 26.8 27-3 P.ci. 20.8 235 243 24.2 P.ci. 19-3 22.2 23.0 23-S P.ct. 17. 1 20.2 21. 1 21.8 P.ci. 17.6 18.6 h-'-'Uss Average i to 12. . 24.9 24.6 27.4 26.9 24.6 28.9 28.2 24-S 239 28.6 26.4 23.2 22.0 20.1 19.0 B. — EXCESS OF MOISTURE ON PLOT 3 OVER THAT ON PLOT 4 I 4-4' 1 6.7 \ 3.6 1 5.4 I 4-3 4-9 5.8 6.7 4-3 4-9 3-3 5-1 2.4 S-o 3-7 3-3 4.4 4-2 3-8 4-3 5-2 S-i 3-1 30 4.8 4.8 3-9 2.3 4-3 3-9 2.1 3-2 S-o 4.6 2-5 • 7 3-9 3.8 3-3 1.4 2.5 3-6 2.S 2-3 1-3 1-5 2.1 I.O 0.6 3-0 2-9 •9 I-S 3-2 Average i to 12. . 4-7 S-o 5-6 4.4 3-6 4.1 4-1 3-9 3-4 3-2 3-1 2-7 i-S 1-9 I.I Table VII. — Moisture content of soil on plot 4 to a depth of j feet, at the first of each month and the excess of moisture at corresponding depths on the other plots, arranged to show any relatioti of these differences to differences in the nitrogen content of the surface foot A — MOISTURE CONTENT Date and depth of section. Plot 4. Excess on other plots. Plotj . Plot 2. Plots- Plot 6 MAY I. I to 6 inches 7 to 12 inches Second foot Third foot JULY I. I to 6 inches 7 to 12 inches Second foot Third foot AUGUST 4 I to 6 inches 7 to 12 inches Second foot Third foot SEPTEMBER I to 6 inches 7 to 12 inches Second foot Third foot P.ct. 24. 2 25-5 23. 2 20. 5 23-7 25-4 25-1 23-7 28.4 28.8 28.0 26. 5 19.8 20. o 21.4 P.ci. 5- I 4.4 3-8 4-9 3-7 3-5 1-3 •9 4-8 1.6 •1-3 I 2-3 . o I. o I. o P.ct. 2.6 2.8 3-3 4-9 .6 - -4 . 2 4. o 1. 1 . I -I. I 2.7 — I. o - -7 —2. I P.ct. 3-2 I. 2 I. 2 2.9 1-7 ■ . I .6 .8 — . I -1-3 -1.6 1-7 .8 I. o .8 P.ct. 3-3 . I 2.9 - .7 1.4 •7 1. 1 1. 1 1-7 -4 ■1-3 2-3 - .8 I. I •4 B — NITROGEN CONTENT I to 6 inches. . , 7 to 9 inches. . 10 to 12 inches o. 180 . 161 . 129 055 071 064 0.056 .051 .032 o. 028 . 042 • 033 0.013 . 009 - . 013 ' A single 6-lnch sample used instead of successive 3-inch samples. Mar. lo, 1919 Organic Matter and Water -Holding Capacity of Soil 273 It is of interest that the differences in moisture content are as great as would be computed on the assumption that the organic matter of this silt loam has the same water-holding capacity as some of the most absorbent peats, some of these, even when well drained, being able to retain 300 to 400 parts of water to every 100 parts of dry peat. The surface foot of plot 3 carries 1.37 per cent more organic matter than the corresponding level on plot 4 (Table I), from which might be computed a difference of about 5 per cent in water-holding capacity. Table VIII. — Hygroscopic coefficients of successive levels on plots j and 4 Depth of section. Hydroscopic coefficient. Plots. Plot 4. 8. I 7- 8. I 7- 8.2 7- 8.1 7- 8.1 7- 7-9 8. 7-7 7- I to 3 inches , . . 4 to 6 inches . . . 7 to 9 inches . . . 10 to 12 inches. First foot Second foot. .. . Third foot INFLUENCE OF ORGANIC MATTER UPON PROPORTION OF USEFUL MOISTURE The nitrogen content of the surface foot of plot 3 is 138 per cent and the organic matter 140 per cent of that on plot 4, while the hygroscopic coefficient is only 5 per cent the higher on the former. As a consequence the proportionate increase in free water is much greater than that in total moisture content, and that in growth water still greater. Thus, the average moisture content of the surface foot for the nine samplings in May, June, and July was 26 per cent on plot 4 and 30.3 per cent on plot 3, the free water 18.3 and 22.2, and the growth water 13.5 and 17. i, respectively, corresponding to increases of 15, 21, and 27 per cent. Thus, the difference in organic-matter content, owing to differences in the manuring and cropping of the two plots, caused a marked differ- ence in the amounts of useful moisture during the season of 191 5 as is well illustrated by a comparison of the ratios of the moisture content to the hygroscopic coefficient (Table IX). The advantages of expressing the moisture condition of soils by such ratios has been discussed in several recent papers (3, p. 55; 4, p. 453; 5» P- 2^^)- The expression "hygroscopic coefficient = lo.o; ratio = 1.7" indicates a moisture content of 17.0 per cent, a wilting coefficient of 15.0^ {8, p. 65), 7 per cent of free water, and 2 per cent of growth water. The ratios i.o, 1.5, and 2.0—2.5 appear to indicate, respectively, the minimum to which crop ' The exact figure is 14.7. 274 Journal of Agricultural Research Vol. XVI. No. lo plants can reduce the soil moisture {I) , the point at which root penetration practically ceases (7, p. 2yg), and the water-retaining capacity of well- drained arable mineral soils {3, p. 6g), and such an expression as the above makes all these relations apparent at a glance. The ratio may be used alone to indicate the relative moistness, while its combination with the hygroscopic coefficient expresses the moisture condition. Table IX. — Ratio of moisture content to hygroscopic coefficient at different levels on plots 2 and 4, in igi5 Ratio on plot 4. Excess of ratio on plot 3. Date. Firstfoot. Second foot. Third foot. First foot. Second foot. Third foot. May I 3-2 3-2 3-6 3-5 3-2 3-8 3-7 3-2 3- I 3-8 3-4 3-0 2.9 2.6 2-5 2.6 2.7 0.8 0 6 II 4 5 4 3 3 3 3 3 I 2 0 I 18 Tune 10 July I 3-1 3-2 . 2 . 0 ic 2T, •20 AuiJ. 4 3-5 3-5 . I 7 14 21 28 Sept. 2 2-S 2.9 . I The high ratios observed in the subsoil of these plots is probably due to the retarding influence which the substratum of gravel and coarse sand exerts upon the downward movement of water (j, p. 34-41). In a study somewhat similar to the one here reported, but made in eastern Nebraska in 191 2, one of us (Alway) found a similar influence of the organic matter upon the amount of useful moisture retained (4, p. 474) , but there the conditions were not so satisfactorily comparable, an exposed subsoil poor in organic matter being compared with an adjacent surface soil. MOISTURE RELATIONSHIPS AND PRODUCTIVITY IN LATER SEASONS Plot 3 showed itself far the more productive of the two plots in 191 5, as had been the case also in such of the preceding 22 years of the experi- ment, as the coincidence of the corn crop on plot 3 permitted a direct comparison of yields (Table X). In 191 6 spring wheat was sown upon all five plots and the yields were relatively unchanged. However, red clover (Tri folium pratense) was seeded with the wheat and while the stand of clover plants was even and moderately thick on all the plots, it was especially fine on No. 4, and in the following year the yield of hay was considerably the greater on this plot and the aftermath also was Mar. 10, 1919 Organic Matter and Water-Holding Capacity of Soil 275 heavier than on No. 3. The second growth, being too light to make a fair cutting of hay, was plowed under and the field seeded to winter rye {Secale cereale), which gave a good yield in 191 8, it being almost as heavy on plot 4 as on plot 3. The yield of hay, straw, and grain combined, for 1917 and 1918, amounted to 9,738 pounds per acre on plot 4, compared with 9,088 pounds on plot 3. Evidently the lessened water-holding ca- pacity on the former had no serious effect upon the crop yields. Table X. — Relative productivity of ploti j and 4 Season. 1896 1897 1901 1905 1909 1915 1916 1917 1918 Crop. Com: Grain, bush Stover, cw-t Grain, bush Stover, cwt Grain, bush Stover, cwt Grain, bush Stover, cwt Grain, bush Stover, cwt Grain, bush Stover, cwt Sorghum, as cut green, cwt Ttunips: Roots, tons Tops, tons Mangels: Roots, tons Tops, tons Wheat: Grain, bush Straw, cwt Clover hay, tons Winter rye: Grain, bush Straw, cwt Yield per acre. Plots. 61. 7 44.8 33-3 18.8 40. 6 20. 4 71. I 20.8 96.6 25. 6 79-7 56.0 16. 4 12. 9 3-4 20. 7 2.9 29. 6 35-2 2. o 40.3 26.7 Plot 4. 44.0 10. o 6.2 37-8 26. 4 26.6 12.8 48.9 31-4 55- o 44.0 12. 6 7-4 1-5 11. 4 20. 5 24-5 2.44 38-3 25.6 Produc- tivity of plot 4.<» 71 33 33 93 129 37 62 SI 123 69 78 77 58 44 55 62 69 70 122 95 a Yield on plot 3=100. The moisture content of the soil on the two plots was determined on four occasions during the past season, and, in general, plot 3 was found the more moist in the surface 6 inches (Table XI). When, as in Table XII, we compare the ratios of moisture content to hygroscopic coeffi- cient with those for 191 5 (Table IX) it is evident that there was little difference between the two plots in the moisture content of the whole three-foot section in 191 8; the subsoil on both plots was much drier than in the earlier year, so dry in fact that until the rains of November fell (Table XIII), there was no opportunity for loss of water by percola- tion from this section. The dryness of the third foot indicates that it had been fully occupied by the rye roots and hence that all the water 276 Journal of Agricultural Research Vol. XVI, No. 10 that entered the surface had been retained within reach of the roots. With forage crops like clover, where a still larger amount of moisture is required for maximum yields, the same would hold true. Table XI. — Differences in moisture content of soil of plots j and 4 in season of igi8 Date and depth of section. June II (1.82 inches of rain on June 8 to 10): I to 6 inches 7 to 12 inches Second foot , Third foot June 26 (Only 0.15 inch of rain since June 10): I to 6 inches 7 to 12 inches , July 18 (1.89 inches of rain on July 14-16, 2.78 inches since June 26): I to 6 inches 7 to 12 inches Second foot , Third foot Nov. 12 (2.32 inches of rain Nov. i to 8; 8.63 inches since July 18): I to 6 inches 7 to 12 inches Second foot Third foot Plot 4- Plot 3. Excess on plot 3. Per cent. Per cent. Per cent. 24. 8 27. 8 3-0 25- I 26. 4 1-3 19. 9 17- 7 — 2. 2 13- 0 12. 4 -0.6 10. 8 II. 4 0.6 14. 5 13- 4 — I. I 21. 6 23- 3 1-7 19. b 19. I -o-S 12. 0 10. 7 -1-3 II. 5 12. 0 0-5 25- 2 27. I 1.9 25- I 27. 6 2-5 24. 4 25- I 1-7 16. 0 16. 5 o-S Table XII. — Moistness of soil on plots in igi8 compared with that in igi 5, showing the much drier condition of the subsoil in the former year Plot No. and depth of section. Ratios in igiS. June II. July 18. Nov. 12 Ratios in 1915. July 7. Aug. 4. Sept. 2 Plot 3: Firstfoot... Second foot Third foot. . Plot 4: Firstfoot... Second foot Third foot. . 2.6 1.4 I- 5 2.7 1-5 1-5 2-5 2.6 2.9 2-S 2-5 a. 9 In general, it appears that percolation causes but little loss of the sum- mer rainfall in the case of soils as fine in texture as the silt loams when these are in grasses or small grains, the portion of the subsoil occupied by the roots intercepting and giving up to the crop any of the moisture that penetrates through the surface foot. On fields with a sharply rolling surface a lowered water capacity, due to loss of organic matter, might be accompanied also by greater difficulty of penetration, and hence by a sufl&ciently greater loss by run-off to cause a markedly lower crop yield. Mar. lo, 1919 Organic Matter and Water-Holding Capacity of Soil 277 The comparatively slight influence that the water-holding capacity of the surface soil alone exerts upon the productivity finds an illustration in the common observation that sandy loams provided with fine-textured subsoils, when properly farmed, produce as heavy yields as clay loams. The moisture available to a crop, in so far as the character of the soil determines the amount, depends upon the water-retaining capacity of the whole soil section penetrated by the roots of the crop, and not chiefly upon that of the surface stratum, and while cultural methods which lessen the organic-matter content of this stratum lower its water-retaining capacity, it forms such a small part of the whole moisture-retaining sec- tion that any change in the moisture supply thus induced may be too slight to have any distinct influence upon the productivity. SUMMARY The paper reports a detailed study of the moisture conditions found on two adjacent Minnesota plots, both of which had a silt loam soil, very unifonn in texture, but differing widely in content of organic matter as the result of great differences in cultural treatment. During the cool, wet summer of 1915, when cultivated crops were grown, the surface foot, and this alone, showed a very marked difference in the moisture content, especially in the available portion, the soil the richer in organic matter retaining the more water; but in the warmer and somewhat drier summer of 191 8, when winter rye was used, much smaller differences were found. It is concluded that in the case of a finer-textured soil, with a fine-tex- tured subsoil and a comparatively level surface, the differences in the watery capacity that may be caused by differences in manuring or in cultural operations exert but little influence upon the productivity. LITERATURE CITED (i) Alway, Frederick J. 1913. STUDIES ON THB RELATION OF THE NON-AVAILABLE WATER OP THE SOIL TO THE HYGROSCOPIC COEFFICIENT. Nebr. AgT. Exp. Sta. Research Bui. 3, 122 p., 37 fig. (2) Klein, Millard A., and McDole, Guy R. I917. SOME NOTES ON THE DIRECT DETERMININATION OF THE HYGROSCOPIC COEFFICIENT. In Jour. Agr. Research, v. 11, no. 4, p. 147-166. Lit- erature cited, p. 165-166. (3) and McDoLE, G. R. 19 17. RELATION OP THE WATER-RETAINING CAPACITY OP A SOIL TO ITS HYGRO- SCOPIC COEFFICIENT. In Jour. Agr. Research, v. 9, no. 2, p. 27-71, 4 fig. Literature cited, p. 70-71. (4) 1918. VARIATIONS IN THE MOISTURE CONTENT OP THE SURFACE FOOT OF A LOESS SOIL AS RELATED TO THE HYGROSCOPIC COEFFICIENT. In JOUT. Agf. Research, v. 14, no. 11, p. 453-480, 5 fig. Literature cited p. 480. 278 Journal of Agricultural Research voi. xvi. no. 10 (5) Alway, Frederick J., McDolE, G. R., and Trumbull, R. S. 1918. INTERPRETATION OF FIELD OBSERVATIONS ON THE MOISTNESS OF THE SUBSOIL. In Jour. Amer. Soc. Agron., v. 10, no. 7/8, p. 265-278. Literatiire cited, p. 278. (6) and Trumbull, R. S. 1910. A contribution TO OUR KNOWLEDGE OF THE NITROGEN PROBLEM UNDER DRY FARMING. In Jour. Indus. and Engin, Chem., v. 2, no. 4, p. 135-138. (7) Briggs, Lyman J. 191 5. DRY-FARMING INVESTIGATIONS IN THE UNITED STATES. In Rpt. 84th Meeting Brit. Assoc. Adv. Sci., 1914, p. 263-282, 7 fig., pi. 5. (8) and Shantz, H. L. 1912. THE WILTING COEFFICIENT FOR DIFFERENT PLANTS AND ITS INDIRECT DETERMINATION. U. S. Dept. Agr. Bur. Plant Indus. Bui. 230, 83 p., 9 fig., 2 pi. (9) Dyer, Bernard. 1902. RESULTS OF INVESTIGATIONS ON THE ROTHAMSTED SOILS . . . U. S. Dept. Agr. Office Exp. Sta. Bui. 106, 180 p. (10) Smith, William G., and Kirk, N. M. 1916. SOIL SURVEY OF RAMSEY COUNTY, MINNESOTA. U. S. Dept. AgT. Bur. Soils, Adv. Sheets — Field Oper., 1914, 37 p., 2 fig., map. (11) Snyder, Harry. 1897. EFFECTS OF THE ROTATION OF CROPS UTON THE HUMUS CONTENT AND THE FERTILITY OF SOILS. In Minn. Agr. Exp. Sta. Bui. 53, p. 1-12. (12) Thorne, Chas. E. 19 18. THE FUNCTION OF ORGANIC MATTER IN THE MAINTENANCE OF SOIL FERTIL- ITY. In Amer. Fert., v. 48, no. 4, p. 26-27. Organic Matter and Water-Holding Capacity of Soil Plate 36 Journal of Agricultural Research Vol. XVI, No. 10 PLATE 36 View of Field J., Minn. Agr. Experiment Station Fann, showing topography and sur- roundings, looking from plot 6 to the bam which occupies part of plot i. The photo, graph, taken on the morning of February 27, 1918, as the snow was disappearing, shows plot 4 to be slightly the lowest, the two streaks of ice from north to south, marked by crosses, both being on the plot. The water, held back by the snow bank at the left, had frozen during the night. ADDITIONAL COPIES OF THIS PUBUCATION MAY BE PROCURED FROM THE Sin'ERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 10 CENTS PER COPY SXTBSCRIPTION PRICE, $3.00 PER YEAR A Vol. XVI MARCH 17, 1919 No. 11 JOURNAL OP AGRICULTURAL RESEARCH CONTENTS Paca Fusarium-Blight of Potatoes Under Irrigatioii - - - 279 H. 6. MacMILLAN (CttDlribatiaaa from Btucan ot Plant Indnttiy ) PUBUSHED BY ADTHORITY OF THE SECRETARY OF AGRICDLTURB, WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASHINGTON, D. C. WASHINQTON : OOVERNMENT PRINTINO OFncC : ltl« V**''? "'i ■ ^''S!iA EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMEUT KARLF. KEI.LERMAN, Ch.\irman 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 H. P. ARMSBY Director, Institute of Animal Nutrition, The Pennsylvania State College J. G. LIPMAN Director, New Jtrsey A griculturai Experiment 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 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa. JOMAlOFAGliiamiliALffiSMCH Vol. XVI Washington, D. C, March 17, 191 9 No. 11 FUSARIUM-BLIGHT OF POTATOES UNDER IRRIGATION By H. G. MacMillan Assistant Pathologist, Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant Industry, United States Department of Agriculture HISTORY OF FUSARIUM-BLIGHT The Irish potato {Solanum tuberosum) has been one of the most profitable crops grown in the Greeley district of northern Colorado. In this fertile, irrigated section, one of the oldest in the coimtry, the potato gave large yields for many years before any serious setbacks occurred. From 1908 to 1912 the inroads of disease threatened the industry severely, and in 191 1 and 1912 the crops were failures. In 1 91 5 a laboratory was established at Greeley for the study of potato troubles, but since that year the yearly losses have been light. Fusarium- blight has been present, however, as a conspicuous malady. Fusarium-blight, or potato-wilt, is well recognized in nearly all the potato-growing regions of the country, except in the extreme North- eastern States. It is caused most frequently by the fungus Fusarium oxysporwn Schlect., though other species of this genus have been found involved. In 1899^ Smith {15Y first proved that species of this genus would cause plant-wilts, and in 1904 Smith and Swingle {16) described a potato-wilt and tuber-rot due to F. oxysporum. It was believed by them that the F. solani of Pizzigoni {10) and of Wehmer {ly) was iden- tical with their species. The confusion which existed over the definition of species was largely removed by the taxonomic investigations of Appel and Wollenweber (j), Carpenter {2), Sherbakoflf {14), and Wollen- weber (20). Manns (6), Orton (9), Pratt {11), Wilcox {18), and others have continued to reveal the great losses which species of Fusarium cause in the potato industry and have suggested methods of control. ' Smith, Erwin F. The watermelon disease of the south. (Abstract.) In Proc. Amer. Assoc. Adv. Sci. 43d Meeting, 1894, p. 2S9-290. 1895. The following papers are likewise contributory: Smith, Erwin F. The spread of plant diseases. A consideration of some of the ways in which para- sitic organi:ms are disseminated. In Trans. Mass. Hort. Soc. 1896, pt. i, p. 117-133. 1896. . The fungous infestation of agricultural soils in the United States. In Sci. Amer. Sup., v. 48, no. 1246, p. 19931-19982. 1899. ' Reference is made by number (italic) to " Literature cited," pp. 301-303. Joiunal of Agricultural Research, Vol. XVI, No. 11, Washington, D. C. Mar. 17, 1919 rm Key No. G-174 (279) 28o . Journal of Agricultural Research voi. xvi. no. h PREVALENCE AND LOSS Fusarium-blight has not appeared to persist in any one locality. Its visitations are sporadic. The losses are, therefore, not to be esti- mated in concrete terms. In Colorado in 191 7, a favorable season for the crop, about 46,000 acres of potatoes were grown. It is estimated that, owing to disease, 10 per cent of the acreage planted gave either reduced yield or no yield. In a good field most of the diseased plants soon pass out of sight and make no impression on the casual observer. Yet these occasional diseased plants probably resulted in a loss of 500,000 bushels in Colorado alone. At other times the disease spreads more generally, and whole fields go down and are lost. Since the part of Fusarium spp. in the creation of disease depends largely on environmental factors, it is important to note that the conditions which prevail in Colorado do not exist in the same way in other places. The descriptions of diseases caused by species of Fusarium already published do not, therefore, closely apply to Colorado, because the climate, coupled with the soil conditions and irrigation practice existing there, creates a condi- tion unknown in the East, where most of the work on Fusarium spp. has been done. DESCRIPTION OF DISEASED PLANTS The common manifestations of the disease are a wilting and rolling of a few leaves, followed quickly, slowly, or intermittently by the wilting of the remainder and usually the premature death of the foliage. Occa- sionally a single leaf will wilt, turn yellow, and die, whereas the remainder of the plant may continue healthy throughout thes eason. Frequently one of the two or more stems of a hill wilts and dies, while the others re- main turgid and healthy. The time of appearance varies greatly. It may be noticeable when the first leaves appear or at any time throughout the season up to maturity. A faint lightening in the color of the plant may indicate a gradual attack, and when the attack is severe, a large plant may pass from health to complete collapse in two days. Late in the season, three or four weeks before frost, plants turgid and unwilted, are found upon which all the upper leaves are rolled. They are a little lighter in color than normal plants, and have often been designated by the uninitiated as being diseased by leaf roll. Plate 41,6, shows a plant of this type; the leaves are rolled, but show no wilting. These plants may continue to lighten in color, the leaves to roll, and gradually to die. These conditions are due to the presence of Fusarium mycelium in the vascular tissues of the stem. Plate 40,D, shows a plant on which the leaves have rolled and are gradually dying. What has actually happened in this plant is that the fungus has created a physiological drouth which has extended over a long period. Upon examining plants in the earlier stages of wilt, the stem and roots are usually found clean and apparently healthy, but the seed piece has rotted, and remains Mar. 17, 1919 Fusarium-Blight of Potatoes under Irrigation 281 as a wet, jelly-like mass. Later in the season the seed piece practically disappears, and the roots and stem may become blackened and decayed. At first the stems may be wet and slippery, but in time they become dry, brittle, and friable. The main and lateral roots of plants having rolled leaves late in the season are often normal in external appearance. The term " Fusarium-blight " is preferred for the disease, because it is more applicable to all its stages. The name "Potato-wilt" has been used elsewhere, and so has " Fusarium-wilt," but their use is likely to cause confusion with other diseases, and are not accurately descriptive, as they limit the picture to wilt. As will be seen later, the term "Fusarium-blight" covers the diseases much more accurately. INOCULATION EXPERIMENTS The cause of Fusarium-blight is F. oxysporum. Other species of Fusarium are capable of producing similar phenomena in the potato plant, but F. oxysporum is the species commonly found in isolation cultures from diseased material. At various times cut potato tubers have been inoculated with spore susspensions of F oxysporum and kept in moist chambers in the laboratory. These tubers were always destroyed by the action of the fungus, while controls made in the same manner with sterile water remained normal. On July 17, 1 91 7, six smooth Early Ohio tubers were given surface sterilization. Each tuber was cut into two equal parts, one half being reserved for control and the other half for fungus inoculation. Glass rings of the Van Tieghem cell type were smeared on the edges with petrolatum and put down on the center of the cut surface of the tuber after it had dried slightly from cutting. Two drops of a heavy spore suspension from an authentic pure culture of F. oxysporum were placed within the ring under aseptic conditions, and the cell closed with a cover glass. The controls were prepared the same way with sterile water. These seed tubers were taken to the field and planted in a row at a depth of 3 inches, where they were subjected to natural field conditions rather than to the artificial environment of a flat. On August 13, 191 7, twenty- seven days after inoculation, the ground was carefully scraped away and the plants taken up. The six seed pieces inoculated with the fungus were badly decomposed. Four of them had disintegrated to a completely rotten mass. The other two had sprouted, and a small firm area remained by the stem. F. oxysporum was recovered from these in pure culture. The controls were uniformly healthy throughout. Plate 37, A, illus- trates a control and a diseased seed piece of this experiment. On August 10, 1 91 7, fifteen tubers were cleaned and sterilized. They were cut as before, and one half of them were inoculated with a spore suspension of F. oxysporum dropped into glass rings, the other half being treated as controls. Five inoculated and five control seed pieces were planted together in sterilized soil in each of three flats. They were 282 Journal of Agricultural Research voi. xvi. No. ix watered with sterile water. The flats were set on the edge of a field, where ordinary weather factors would act as normally as possible. These flats were noted finally on September 8, 191 7. The control plants were healthly throughout, with normal foliage and stems 10 inches long. In the first flat only one tuber of the inoculated seed sent up a pair of sprouts. One of these was dead and the stem blackened for 2 inches above the ground. Although the seed piece was thoroughly decayed, the other sprout appeared healthy, as shown in Plate 37, B, It is characteristic in every respect of the field blight. A closer view of the seed piece, together with a control seed piece from the same flat, is shown in Plate 37, C. In the second flat two seed pieces failed to germinate, owing to decay. One was dead, and the stem was blackened. In the third flat one seed piece failed to germinate, while four sent up sprouts. These had all wilted thoroughly, though death had not yet occurred. None of these plants had been subject to frost. Isolation cultures were made from all the diseased plants from the three flats, and F. oxysporum was recovered in pure culture in each case. On September 9, 1917, three flats were prepared for inoculation. Sterile soil was used. Seed pieces of the Pearl variety were inoculated with a spore suspension of F. oxyspor^im in the usual way. The spore suspension was made from cultures taken from the diseased seed pieces of the inoculation experiment of July 17. No controls were made, because of lack of space. Owing to the lateness of the season, these flats were taken to Fort Collins, Colo., where, through the courtesy of the Horticultural Department of the Agricultural College, they were placed in the greenhouse. The plants were not seen until November 3; at which time all were dead with the exception of three plants, then about to die. On August 13, 1 91 7, nineteen whole tubers which had been planted in the field for three weeks and which had sent up sprouts were inoculated. Fourteen of them were inoculated with a spore suspension of F. oxysporum poured into glass tubes entering the epidermis of the tuber; five were treated with sterile water as controls. In each case the plant had a healthy, vigorous start. On September 9 the plants were taken up and examined. The seed pieces of the controls were sound and the plants healthy; seven inoculated plants were healthy, though the seed piece was decayed; the other seven were wilted, the stem was blackened, and the seed piece was thoroughly decayed. On August 10, 1 91 8, sixty-one plants planted in sterile soil in flats were inoculated with F. oxysporum. Sixteen plants in flats in sterile soil were treated as controls. The method of handling was different from that used before. The seed pieces had been planted with the cut surface turned up, about a month previously, and the flats left in a cool place. They were watered periodically with sterilized water. By August 10 the Mar. 17, 1919 Fusarium-Blight of Potatoes tinder Irrigation 283 plants had germinated and sent up strong, vigorous sprouts. The seed pieces were solid and unusually well calloused. For the purpose of inocu- lation the soil was scraped away, and a small core about i cm. long was taken out of the upper surface of the seed piece with a small coring tool. The pit made by the removal of the core was filled with a spore suspen- sion of F. oxysponim and closed with a cover glass smeared with petro- latum. The controls were treated in the same manner, except that the pit was filled with sterile water. For the next 20 days the flats were ex- posed to approximately field conditions. The experiment was discon- tinued on August 30. At that time the inoculated plants had wilted to the ground, while the control plants were normal. Upon examination of the underground parts of the inoculated plants the seed pieces were found to be wholly decayed, and the main root was infected. The roots were not destroyed nor was the main root decayed, but the vascular tissue was woody and filled with mycelium. Isolation cultures made from 40 of the inoculated plants gave F. oxysporum in pure culture. INOCUI^ATIONS ON MATURlS PLANTS On August 13, 1 917, inoculations were made on Early Ohio potato plants in the field for the purpose of approximating the disease in its mature stage. The plants were in good soil and had shown no signs of blight from natural infection, though the seed pieces had been attacked. The method employed was very simple. The soil was carefully scraped away from the stem to a depth of 3 or 4 inches, and a slit was made with a scalpel lengthwise through one stem of the plant. A wedge of raelilotus stem upon which F. oxysponim had been cultured, and which bore my- celium and spores plentifully, was inserted in the slit, and the whole being covered with soil. Forty-six plants were inoculated with the fungus, and 26 were treated as controls. On September 18 these plants were taken to the laboratory for examination. All of the plants had two or more stems, but in the case of the plants inoculated with F. oxysporum only the treated stem showed any injury. The plants treated as controls recovered from the mechanical injury, and the wound healed. Plate 38, B, shows a control plant and the method of inserting the wedge. Of the inoculated plants 3 were lost, 4 showed no infection, 2 showed weak or doubtful infection, and 37 showed positive infection. Stems showing infection were typical of the natural blight in every respect. The stems were dead, blackened, and shattered in most cases.. Plate 38, A, illus trates two stems of the same plant; the stem at the left was inoculated; the one at the right was not, and shows no injury. Of the 37 stems show- ing positive infection 20 were selected at random and isolation cultures made. These yielded pure cultures of F. oxysporum in 18 cases, the 2 others being badly contaminated. 284 Journal of Agricultural Research voi. xvi, no. ir MODE OF INFECTION Hitherto infection of potato plants by F. oxysporum through the root hairs and small rootlets has been accepted as the usual method. Smith and Swingle {16, p. 13) said this occurred, and Manns (6, p. 306) reasserted the fact. In case of the cowpeas and cotton, Orton (7, p. 10; 8, p. 8) found this manner of infection in both cases. Cromwell (5, p. 425) sup- posed root infection to be the means of entrance of Fusarium irache- philum Smith, causing the wilt disease of soybean. Jones and Oilman (4, p. 7) found the roots of cabbage to be attacked by Fusarium. These numerous instances would call for careful examination of the roots of in- fected plants. During the years 191 6 and 191 7, in only 6 plants out of many hundreds examined was this method of infection determined as probable in the case of fusarium-blight of the potato in the Greeley dis- trict. In 1 91 8 the soil temperature at a depth of 6 inches was 6° F. above the average for the month of June of the preceding two years. Plants of the Charles Downing variety, planted during the last of May or early June, were badly diseased in some fields by being attacked through the fine roots and root hairs by F. oxysportim. This one variety was more severely attacked than any other, even in fields containing several varie- ties. Most other varieties were not assailed in this manner at all, except a few scattering Early Ohio plants. Higher temperatures seem to be necessary for root infection. Infection from seed tubers containing the Fusarium organisms in the vascular bundles has been very seldom found. Wollenweber (79, 20) has shown that F. oxysporum overwinters in potato tubers, where it causes the familiar vascular discoloration. With the sprouting of the eyes when the seed piece is planted the organism infects the new plants and pre- sumably causes wilting and death. One of the most extensively advo- cated control measures has been aimed to avoid this kind of infection. No trouble has been experienced with this method of infection in the last three years. Field experiments tending to show the nonseverity of this method of infection will be given below. SEED-PIECE INFECTION In the Greeley district and in other parts of Colorado potato seed pieces become infected with the Fusarium organism from the soil. The cut seed is vastly more liable to attack than the whole seed, and the decay follow- ing infection will begin two or three days after planting. It is justifiable to assume that in the average field nearly all cut seed pieces are infected. Fields have been examined in which hundreds of seed pieces were dug a few days after planting, and less than 5 per cent were found to be free from infection. The infection occurs through the large open wound of the cut surface, lightly protected by callus. The interior loose parenchyma at the center of the tuber, farthest from the active tissue of the vascular Mar. 17. 1919 Fusarium-B light of Potatoes under Irrigation 285 region, is the weakest and least protected. There is a difference in the susceptibility of seed of different varieties, but what seems more im- portant is that seed of the same variety from different sources varies greatly in its power of resistance. The rot following infection may be swift, and the fungus will destroy the seed piece before germination begins. Plate 37, E, illustrates a seed piece upon which no eye has germinated, though the piece is nearly destroyed by rot. In this illustration two dark spots are to be noted in the vascular region, denoting vascular infection; 'yet no decay originated at that point. When the decay is slower, the seed germinates and sends up a vigorous shoot. Plates 37, D, and 38, D, illustrate cases of germination followed by seed-piece rot. The region adjacent to the active tissue is the last to decay because it is more resistant and because the decay begins in the loose parenchyma and advances toward the germinating point or place of attachment of the shoot. Where decay is delayed sufficiently to allow germination to take place, the decay works slowly through the active region, or it may stop temporarily. Plate 37, D, illustrates how the decay is delayed nearest the growing part and how it advances evenly toward this region. Plate 39, D, illustrates the base of a plant the seed piece of which had decayed thoroughly. The stem is cut away, showing the healthy tissue within and the absence of the parasite. The general good health of the roots should also be noted. Some plants appear to grow normally for a few weeks, after which symptoms of disease begin to appear. The color may or may not "change, and the leaves may show curling, rolling, or wilting. One lower leaf may turn yellow, wilt, and fall, while the remainder of the plant is a picture of health. In a single hill containing two or more sprouts the tip of one may wilt and the other remain healthy. Plate 40, C, represents a plant consisting of two stems, one of which is healthy, the other wilting. The stem at the left will die, while the stem at the right may live through the season and >neld normally. Upon taking up such a plant the decay of the seed piece will be shown to have advanced toward and into the wilted stem, while in all cases the root system is healthy in every branch. Plate 40, B, shows the top of a plant consisting of three stems. The top leaf and the one below it on the same stem are wilted. Neither the other leaves nor the color of the plant indicated anything abnormal. Plate 39, C, shows the seed piece and the three stems of the tops illustrated in Plate 40, B. The wilted leaf shown in Plate 40, B, is on the middle stem pictured in Plate 39, C, which is at the center of the decay. The stem at the left, healthy on Plate 40, B, is here shown with a slight sound area remaining in the seed piece. A stem which shows these symptoms in early summer when conditions are favorable may not at once succumb, but is usually doomed to an early death. Plate 39, B, shows a young plant in which the decay advanced continuously from the seed piece into 286 Journal of Agricultural Research voi. xvi. no. h the stem. Many plants are to be found which show the violent symp- toms, wilting, drooping, and death, within a few days. The great majority of plants in a field may advance to late maturity with no visible signs of Fusarium- blight. Entire fields have been ob- served which showed natural wilting caused by delayed irrigation; wet periods may occur, owing to excessive rain following an irrigation; and either of these conditions are conducive to the increased activity of the fungus, though recovery is possible and often occurs. In an entirely healthy field at any period of the season conditions may arise in which the blight gains the ascendancy, the plants wilting and dying in the course of a week. This may happen as late as September, yet infection did not occur immediately before the appearance of wilt, as the fungus had been present since the time of planting. Whole seed is protected by a sound epidermis underlain by an active vascular tissue, the best protection the seed may have. Whole seed germinates quickly and estabUshes a sound, vigorous plant weeks before the seed piece has been destroyed by fungi, and the plant becomes hable to attack. It is not unusual for whole seed to remain sound through the growing season, though the ultimate death of the nongerminated eyes, the wom-o^t vitality of the vascular region, and the dead epidermis make infection possible. Injuries in handling or planting, such as are received from picker planters, render infection comparatively easy. Clipping the stem end to inspect for vascular infection is a most reprehensible practice, as it breaks the epidermis and makes a wound in that part of the tube, tissue which is lowest in vitality and least in the power of seh-protection. OCCURRENCE OF THE CAUSAL FUNGUS In the case of seed pieces which obtained a favorable start and sent up Sprouts the decay is slow. In individuals where it takes weeks to decay, the decayed watery portion leaches away and a callus forms when the growing tissue is reached. Where field conditions are right, the fungus will continue its slow advance into the foot of the stem, causing no decay and shght or no discoloration. As conditions unfavorable to the plant arise, the fungus grows in the vascular bundles and causes discoloration. Plate 39, B, shows a 6-weeks'-old stem to the left portion of which the decomposed and dried piece clings. Discoloration of the pith is found only at the very foot of the stem, and the upper vascular bundles are free from any trace of the fungus. In the field this plant would be regarded as healthy. In plants of this type the lowest roots on the stem are cut off from supplying water,' and thereby cause some of the temporary queer symptoms to be noted in the foliage. The plant may recover, draw on the roots above more heavily, and continue growth. Nothing more may happen throughout the season; harvest may arrive, and the plant yield normally. In other cases where the soil is wet and compact the fungus is more active; it decays the foot of the stem, cuts off stolons, decays new potatoes, and finally kills the plant. Plate 39, A, pictures a Mar. 17, 1919 Fusarimn-Blight of Potatoes under Irrigation 287 plant taken from a field where irrigation water got beyond control and flooded a portion of the field. The fungus advanced rapidly, decayed the stem, and caused the death of the plant. The advance was so rapid that the roots were killed for only a short distance, remaining uninfected 2 inches from the stem. In severe cases action is rapid and universal; whole fields succumb, causing the well-known epidemics. When stems of rapidly killed plants are pulled up, they are black, soft, and wet, as is illustrated in Plate 38, D. This plant, naturally infected by F. oxyspo- rum, is strikingly like the plant shown by Link (5, fig. 7), as caused by artificial infection with this organism. Other organisms follow closely behind the species of Fusarium and complete the decay of any tissue not thoroughly invaded by that fungus. ISOLATION OF CAUSAL ORGANISMS During the growing seasons of 1916, 1917, and 1918 more than 1,500 cultures have been made in attempts to isolate the causal organisms. Plants in every condition, from newly planted diseased seed to new tuber infection at harvest, were used as sources of culture. The material was selected in the field, and taken at once to the laboratory. It was carefully washed under slowly running water and patted to comparative dryness between damp towels. The material was prepared for culturing by breaking it open and, with a sharp chisel-pointed platinum needle, transferring small pieces from the desirable areas to tubes containing sterile melilotus stems. In making these cultures too many precautions can not be taken to keep within very small areas with the needle. The line of demarcation between apparently firm tissue and diseased tissue is definite and narrow. Cultures made from the firm tissue immediately before the line, and on the hne, were usually pure, and sporulated readily. Tissue back of the Une gave many contaminations ; too far in advance gave no growth at all in culture. In decaying seed pieces it is well to keep within 2 mm. of the line of decay. In the green tissue of growing stems little trouble will be experienced if .the stems are not broken or torn before culturing; and, as infected stems soon become woody, a stiff sharp needle is necessary in culturing from them. Any blackened tissue will usually yield a cul- ture. These infected tissues invariably yield species of Fusarium, though, if decay has advanced to the point of disintegration, contami- nating organisms will be present. Stysamis stemonitis is frequently found in both attacked stems and seed pieces. Bacteria are rarely found in firm or semifirm tissue. FIELD EXPERIMENTS A series of experiments were performed with several lots of potatoes to determine as nearly as possible the origin of the disease developing during the growing season, basing the deductions upon the conditions 106545°— 19 2 288 Journal of Agricultural Research voi. xvi, no. h of the seed at planting time and the symptoms displayed during growth. These experiments were carried out in the field under conditions approxi- mating commercial field practice, and no methods of culture or treatment were used at any time after planting which would not have been used by a commercial grower. For the purpose of this experiment it was conceded that parts of the same seed potato, grown under like condi- tions, would follow within reasonable limits pretty nearly the same course of procedure in growth, disease symptoms, and general appearance. A difference in two plants from twin seed pieces must be accredited to different conditions encountered during the growing season after planting. Various lots of seed were assembled in 191 6 to test out this assumption. Among others, they consisted of one lot of certified Wisconsin Pearl, one lot of certified Wisconsin Rural, two lots of Early Ohio from the Red River Valley in Minnesota, one lot of Rural from the Carbondale District of Colorado. All tubers were cut from bud to stem end, dividing the tuber into two equal parts. All tubers above 6 ounces were cut into four pieces. These were cut in the field and planted immediately side by side in parallel adjacent rows. They were given as good care as possible during the growing season. The summer was excessively warm until July 30, 1 91 6, at which time 3.09 inches of rain fell. The remainder of the sum- mer was comparatively cool. Notes were taken four times during the summer: Once when the plants were about 6 inches high, then when they averaged 12 inches high, again when they were full grown, and finally when no normal change was to be expected. No reference was made to any previous note; other members of the force were asked to assist in the work, and every method employed by w^hich an impartial diagnosis could be made. KEY To TABLE I. Pbr the purpose of summarizing and presenting the performance of these lots of tubers, a new form of table, known as an aggregation table, has been constructed. This table must not be confused with a correlation table, which it resembles in general appearance, but not in context. In any single row of potatoes six different ultimate types of plant were recognized. These have been designated as "H," "HD," "DH," "D," "A," and "O." The meaning of these symbols are as follows: "H" denotes a plant which appeared healthy throughout the growing season; "HD" denotes a plant whicli was healthy during the first part of the season, but finished by being diseased; "DH" denotes a plant which gave manifestations of disease during the first part of the season, but finished by being healthy; "D" denotes a plant which was diseased throughout the season ; " A " denotes a plant so badly diseased as to be merely existing, with no hope of progeny ; "O" denotes no germination, or a case in which the seed piece suffered the maximum of disease and rotted in the ground. All plants in a row fell into one of these divisions. In comparing the plants from twin seed pieces in the two adjacent rows at the same time it is seen that in classification certain coincidences, or lack of tlicrn are significant. In the case of some plants classed as "H" in one row, the twins in the adjacent row Vv-ere "H" also; in more cases they differed for all the Mar. 17, 1919 Fusartum-Blight of Potatoes under Irrigation 289 other five groups. Originally in numbering rows in the field the even and odd fol- lowed naturally, so that in the tables the parallel adjacent rows are most easily desig- nated by the terms "even" and "odd." In the tables the "even" rows are read vertically — that is, the designating letters are placed across the top, and the aggregate totals for each class across the bottom; the "odd" rows are read horizontally, the let- ters at the left and the aggregate totals at the right. However, as noted before, many of the twins differ individually in their performance, so that in order to express this deviation the total of one class in one row is split up according to the numbers required to express the reciprocal in the other row. A concrete example is given in Table i A. In the even row there are 352 H, 4 HD, 5 DH, i D, 13A, and 12 O plants, a total of 387. In tlie odd row there are 320 H, 5 HD, 7 DH, i D, 23 A, and 31 O plants, a total of 387. In comparison with 352 H plants in the even row, twin for twin in the odd row, 296 plants are also H, while 4 are HD, 7 DH, i D, 20 A, and 24 O. Turning about, it is seen that, of the 320 H plants in the odd row, twin for twin in the even row, 296 are H, 3 HD, 3 DH, I D, II A, and 6 O. The same system follows for the other classifi- cations. It is revealed, then, how nearly alike twin seed pieces perform, for where they are alike the numbers appear in either the HH, HDHD, etc., squares down to 00. Differences are shown when they appear elsewhere. Table I. — Aggregation of seed piece performance of Irish potatoes A. — WISCONSIN PEARL [i equals 0.258398 per cent of 387] H HD DH D A 0 Total. No. Per cent. No. Per cent. No. Per cent. No. Per cent. No. Per cent. No. Per cent. No. Per cent. H 296 4 7 I 20 24 76. 49— 1.03 + i.Si — .26— 5-17- 6.20+ 3 I 0.78— .26— 3 0.78— I 0.26— II 2.84+ 6 I-SS+ 320 S 7 82.69— 1.29+ 1.81— HD DH D . 26— A 2 •52- I 5 .26- I. 29+ 23 31 5-94+ 8.01 + 0 2 •52- Total . . . 352 90. 96— 4 1.03 + s I. 29+ I .26— 13 3.36— 12 3- 10+ 387 100.00 B. — INFECTED WISCONSIN PEARL [i equals 3.70370 per cent of 27] H 21; 1 i 25 I 92.60— 3-70+ 3- 70+ HD I 1 ,.7o+ 1 i DH D A 1 0 1 ::..i. :.:...: ■ Total . . . 27 100.00 27 C. — CERTIFIED WISCONSIN RURAL NEW YORKER [i equals 0.223225 per cent of 444] H 89 20. 05— I 0.23— 24 5-41— I 0.23 — 19 4.28— 30 6.76- 164 36.94— HD DH 16 3-60+ 32 2 II 7.21 — •45 + 2.48— 2 •45 + 14 3-15 + 16 3.60+ 80 2 44 154 D •45+» 9.91 — 34- 68+ A 18 67 4- 05+ 15-09+ 2 4 •45 + .90+ 2 23 •45 + 5.18+ 11 40 2.4S— 9.01 — 0 I •23 — Total . . . 190 42- 79+ 2 •4S+ 88 19.82— 9 2.03 — 58 13. 06+ 97 21.8s- 444 ic;;. 00 290 Journal of Agricultural Research Vol. XVI, No. II Table I. — Aggregation of seed piece performance of Irish potatoes — Continued p. — INFECTED CERTIFIED WISCONSIN RURAL NEW YORKER [r equals 6.25 per cent of 16] H HD DH ! B A 0 Total. No. Per cent. No. Per cent. No. Per cent. No. Per cent. No. Per cent. No. Per cent. NO. ^1. H 6 37-5° 6.2c ..../... I 6. 25 I 6.25 9 56- 25 HD DH I 6.2s I j 6.2s I 6.25 3 18.7s D A I 2 10 6. 2% 12.50 I 3 6. 25 0 1 I 6.25 18.7s Total 2 12.50 I 6. 25 3 1 iS. 7? 16 100.00 E. — RED RIVER VALLEY EARLY OHIO. [i equals 0.259067 per cent of 386] H 141 2 29 2 7 62 36- S3- •S2— 7-5<-l- •52— 1.81+ 16. 06 4- 3 0.52— 25 6.^(5- I 14 363- 37 I 6 9-59— .26- I-SS+ 219 3 47 3 12 103 S6. 74— HD •78- DH II 1 2.85- I .26- 12. 18— D 1 .26- .78- A ■■■" : ::i:::::::; ::; I 3 .26- .78- 4 20 1.04- 5-18+ 3- II— 0 I .26- 16 j 4. 14+ 26. 42+ Total . . . 243 62.95 + 3 • 78- 52 13-47 + I .26- 19 4.92 + 68 J17.62- 386 100. 00 F. — INFECTED RED RIVER VALLEY E.\RLY OHIO. [i equals 2.857142 per cent of 35) H HD 16 45-71 + 4 11-43- 1 I 2.86- 3 5-71 + 2.5 65.71 + DH 2 5-71 + 2 5. 71 + D 1 A I 2 2.86- 5-7' + I 9 2.86- 0 4 11-43 — I 2.86— 2 S-7t + 1 Total . . . 22 62. So — I , 2.y6— 1 6 J17-14+ |....| 1 2.ii6- 5 14. 29— 35 100.00 G. — CARBONDALE RURAL NEW YORKER [i equals 0.3x25 per cent of 320] H 29 9.06+ 29 9.06+ I 0.31 + 5 1.56+ 27 8.44+ 91 28.44- HD DH 10 ■3-12 + 40 2 8 39 12. 50 .62 + 2.50 12. 19 I •31 + 3 .9.;- 16 5 • 00 70 2 24 133 21.88- 1) .62+ A 5 29 1-S6+ 9.06+ I I •31 + •31 + I 10 .31 + 3-12 + 9 2.81 + 53 16.56+ 7.50 0 I •31 + 41-56+ Total.... 73 22.8I + I •31 + 118 36.87 + 4 1. 25 19 S-94- los 32.81 + 320 100.00 H. — GREELEY LATE OHIO, [i equals i.iiiiii per cent of 90] H 45 I 6 50.00 1. 11 + fi.fi7 + T.II + 7 7.78- 3 3-33 + S ■;. 56- 61 I 14 67- 78- HD _ _- . 1.11+ DH i 6 6.67 + 2 2.22— 15-56— -T* 1 A I 1. 11 + 7 7- 78- 1 I 2 1. 11 + 2.22 + 2 12 2. 22+- Q 1 3 3-33 + 13- 33"+" ToUl.... 60 66.67- I I.II-i- 16 17-78- 3 3-33 + 10 IX. 11 + 90 100.00 Mar. 17. 1919 Fusarium-B light of Potatoes under Irrigation 291 Table I. — Aggregation of seed piece performance of Irish poiafocs — Continued I. — INFECTED GREELEY LATE OHIO [i equals 1.960784 per cent of 51] H HD DH 1 I) 1 i A 0 TotaL No. Per cent. No. c^[. No. c^l. |no. 1 Per cent. No. Per cent. No. Per cent. No. Per cent. H 26 50. 98+ I 1.96+ 2 ^ 1 2 3.92+ 33 64. 71 — HD DH 4 7-84+ 5 9. 80+ 1 9 D A 1 I I 1.96+ l.... I 8 1.96+ 0 6 11.76+ 1.96+ 1 1.96+ Total 36:-.o.co- 1 T 1.96+ 9 17.6s- ' . 2 3.92+ 3 5-88+ 51 ' •"' 17 6, |.... J. — GREELEY PEARL [i equals 0.7246377 per cent of 138] H 63 I 4 45-6S+ .72+ 2. go — 6 2 4-35- I-4S- 4 1 4 2.90— II I 2 I 3 13 7-97+ .72 + I-4S- .72 + 2. 17+ 9.42 + 88 4 6 I II 28 63. 77— HD . . .:.^°...|.::: DH :::::::: ::::i. ;;::::; D !■■■■ .72+ A S 10 3-62 + 7-2S- I 2 .72 + 1-45- I"" 2 3 1-45- 2.17 + 7.97+ 0 |.... Total 83 60. 14+ 11 7-97 + 4 9 6.52 + 31 22.46+ 138 K. — INFECTED GREELEY PEARL [i equals 1.8867924 per cent of 53] H 22 41-51 — 3 I S-66+ 1.89— 3 5-66+ 4 7-55- 6 II. 72 + 38 I 7. 170— HD 1.89— DH D 1 1 1 I I 1.89- 1.89- 5.66+ I 6 7 1.89— A 3 3 S-66+ S-66+ i ' ' 2 I 3-77+ 1.89- 11.32+ 0 I....1 t.... • 1 Total 28 52.83 + 4 7-55- 3 5-66+ ...i \ii 21 — II 20. 75+ 1 L. — INFECTED COLORADO PEARL 1 1 equals 0.6666666 per cent of 150] H "5 14 76.67- 9-33 + 14 5 9-33 + 3-33 + II II 2 1-33 + 131 19 87- 33+ HD \""t DH ! 1 D .. , 1 a::::::::::::: 1 0 1 ::::i:::::::. 1 Total 129 86- 00 ....1 !,...: 2 X.33+ 150 1 ! 1 M. — IDAHO-GROWN IDAHO RURAL [i equals 0.826446 per cent of i;i] H 105 S 86. 78- 4-13 + 9 7-44— 1 1 2 I.6S+ 116 5 95-87- HD DH D . . [ A 1 1 0 Total no 90.91- 9 7-44— 2 1-65+ 121 1 292 Journal of Agricultural Research voi. xvi, no. h WISCONSIN PEARiv (table; I, a-b) Aggregation Table I, A, illustrates the performance of the certified Wisconsin Pearls potatoes in 191 6. There was a total of 774 seed pieces planted, the result of dividing 387 tubers. The striking thing to be noted in this section of the table is the fact that with the majority of diseased plants in either row the twin was healthy. In only 1 1 cases did both twins fall outside of a healthy square, five of these being in the square 00. If vascular infection was to act here only the 5 twins in the 00 square could properly be said to come under control of it, because it is the only instance where the performance was the same for both twins. In the cases of the 80 pairs of twins, i of which was in some way diseased and the other healthy — that is, those in both rows where i twin was an H plant, exclusive of those in HH — it must be regarded that the disease was newly contracted. Before planting, all tubers were cut at the stem end to inspect for vascular discoloration, indicating the presence of a possible disease organism. Cultures were made from diseased tissue. Only 27 tubers, or 6.98 per cent, showed any discoloration. Tabulating then according to the place the tuber occupies in Table I, A, their places are shown in Table I, B. According to the old conception of the danger of planting diseased seed, the 27 tubers, planting the 54 plants here shown should have given some sign of disease. The 2 which fall without the HH square are healthy plants, for one-half of the tuber would indicate no tendency to disease be- cause of the vascular parasite, but because field conditions acted as in the case of 78 similarly situated plants, as shown in Table I, A. WISCONSIN RURAL (TABLE I, C-D) The certified Wisconsin Rural potatoes were treated in the same manner throughout, and were tabulated in the same way: 444 tubers were used, planting 888 hills, and their performance is shown in Table I, G. The plants in this table are well distributed except in the HD columns. The performance of the twins as representing the strictly inherent ten- dencies of the tuber seems not to be indicative of any considerable failure because of previous faults. There are 40 pairs in 00, and 30 and 67 pairs in HO and OH, respectively. The factors which placed the 40 pairs in 00 did not act on them as parts of 40 tubers, but as 80 individual plants, the same as took place with the 30 and 67 pairs, one-half of which were healthy. This variety is peculiar in showing such a contrast be- tween susceptibility to disease and vigor to survive and grow away from it. The tubers of this lot were planted within 10 feet of the lot represented in Table I, A. Table I, D, represents the place in which those seed tubers fell which showed discoloration in the vascular system. These 16 tubers (3.60 -f- Mar. 17, 1919 Fusarium-B light of Potatoes under Irrigation 293 per cent) are placed in the table according to the place they occupy in Table I, C, and it is regarded as of no significance that they fall where they do. EARLY OHIO (TABLE I, E-F) The Early Ohio seed obtained from the Red River Valley consisted of two lots. These lots were grown separately, but their performances were so nearly alike that they have been combined and presented in Table I, E, as one lot. This table shows plants falling in the DHDH, DD,AA, and 00 squares, a tendency not noted in the previous tables. In the case of the plants falling in DHDH, it would appear that some special weakness had devel- oped in the 22 plants grown from these 1 1 tubers which placed them there. Inherent weakness, then, can not be predetermined by mere examination of the tubers, because Table I, F, which represents the location of the tubers showing vascular discoloration (9.07 — per cent), placed according to their location in Tables I, E, has none represented in DHDH. These plants outgrew their earliest diseased condition, and finished the season in apparent healthy condition. Table I, E, does not indicate, however, any strong vigor on the part of this lot. Table I, F, represents the place the 35 tubers of Table I, E, which showed vascular discoloration fell, placing them according to their loca- tion in Table I, E. RURAL NEW YORKER (TABLE I, G.) One lot of seed of the Rural New Yorker variety was obtained from the Carbondale District of Colorado. It consisted of 320 tubers, free from vascular discoloration, and was regarded as stock of superior quality, selling at an advanced price. Table I, G, illustrates the almost complete failure of this seed through seed-piece infection and rot in the Greeley District. In this table, where so much disease is represented, the conspicuous absence of plants falling in the HD columns (i in the even row), and in the DD square, is significant. Root infection did not occur; no vascular discoloration was present in the seed. The great preponderance of plants in the 00 square and the O columns, shows clearly that a most serious inherent weakness is present in the seed to withstand infection from the soil. The number of plants that did not eventually become healthy, having previously grown and been diseased, are very few. There is a strong tendency to die or survive (H or DH), for the plants that are H or DH were vigorous at the end of the season. The others either failed, as in O, or gave evidence of a gradually decUning health, as in D and A. The lack of plants in the HD columns (i in the even row) is further evidence that a healthy plant maintains its position. 294 Journal of Agricultural Research voi. xvi, No. n LATE OHIO DISEASED SEED (TABLE I, H-l) In the fall of 191 5 several fields were visited for the purpose of staking diseased hills of potatoes. The hills selected all showed blackening of the stems and death of the tops, with many cases of rot in the tubers. At harvest the badly decayed tubers were discarded for the reason that they were in no condition to keep through the winter. The tubers showed a large percentage of vascular discoloration, and the remainder were believed to be infected, though not seriously. All came from hills affected by Fusarium-bUght. Cultures were made from the stem end of all showing discoloration, and all yielded species of Fusarium. The tubers were cut and planted as twins in adjacent rows and given the same culture as the lots mentioned above. Table I, H, shows the performance of such seed of the variety Late Ohio in the year 191 6. The complete absence of any plants falling in the D columns is the outstanding feature of this table. At best, only the 6 pairs represented in DHDH square could be said to show the results of vascular infection, especially from the occurrence of 5 of them in the DHDH square in Table I, I. Compared with Table I, E, which represents a healthy Early Ohio variety, the advantage in health is with the home-grown seed. A table representing the places in which the 50 tubers (56.66 + per cent) showing decided vascular discoloration fell, is shown by Tablel.I. PEARL DISEASED SEED (TABLE I, J-k) One lot of diseased seed consisting of 138 tubers. Pearl variety, acquired in the same manner as the Late Ohios, were cultured from the stem-end, and planted under the same conditions as other lots. Table I, J illus- trated the performance of this badly diseased seed stock. Two things are conspicuous here : The lack of plants in the D columns (one in DO) , and the comparatively large number in the O and the HD columns. These are not to be accounted for here altogether because of their vascular discoloration, because Table I, K, which represents where the 53 tubers (38.41 — per cent) fell which showed discoloration, does not account for the majority. Plainly these plants have been weakened by disease, their power of resistance lessened, and their vigor impaired. Examination showed that soil infection acted here to produce the disease, but a comparison of Table 1, J, with Table \, A, reveals the great weak- ness acquired by these plants, which made them so easily attacked. These tubers were extreme cases, being stock that would not ordinarily get into commercial seed. The circumstances surrounding this lot of of seed tends to explain the true reason why farmers of the Greeley Dis- trict prefer to plant newly introduced seed every two years. Table I, K, represents the places in which that seed fell which showed pronounced vascular discoloration, according to the place they occupy in Table L J- Mar. 17, 1919 Fusarium- Blight of Potatoes under Irrigation 295 PEARIv DISEASED SEED (TABLE I, L) In 1 91 7 one lot of seed, Pearl variety, was secured, each tuber of which showed positive vascular infection by species of Fusarium, as proven by isolation cultures. Conditions were generally more favorable for potato growth early in the 1917 season than they were iu the 1916 season, and less favorable late in 191 7 than in 191 6. Table I, I^, shows the perform- ance of this badly diseased stock. IDAHO RURAL (TABLE I, m) One lot of seed from Idaho, known as Idaho Rurals, and healthy throughout, were treated in the same manner. No tuber showed disease or infection in the vascular system. The performance of this lot of seed is illustrated in Table I, M. The similarity between Tables I, L, and I, M, is striking. The presence of plants in the HD columns is attributed to the unfavorable late season in 1 91 7. This is taken to account for the uniform health as represented for the diseased. Pearl variety in Table I, L. There is no reason to suppose that if mere chance had operated so as to have each seed fall where its twin fell, and vice versa, that the result would have been the same in any table. Each seed piece was sur- rounded by a different set of factors which operated to bring about disease or apparent health. For that reason it is unlikely that the results of a single year can be duplicated, though the average of similar years ought to strike a fair average. DISEASE RESISTANCE In the fall of 191 5 a field of potatoes was chosen upon which to conduct an experiment in disease resistance. It was one calculated to offer crop failure if one was reasonably possible. One end of the field was white with alkali, the soil was heavy, and drainage was poor. A portion of the field already had potatoes on it, supposedly of the Pearl variety. It presented a very ragged appearance owing to skips, diseased plants, and mixture. Two of the best-looking rows' were selected to work upon, and all the diseased and mixed plants were staked. After frost the staked plants were taken out by hand, and the remaining healthy plants were harvested with a machine. In 191 6 this seed was planted on an adjoin- ing plot. All the plants came up healthy and with increased vigor. Some plants succumbed to blight during the season, but at least 90 per cent reached harvest. Again in 191 6 several rows were inspected, the diseased and mixed plants staked, and the healthy ones harvested as before. These were planted in 191 7 in a plot adjoining the one used in 1 91 6. In comparison with other potatoes in the field, the vigor and health of the selected seed was notable. Very few diseased plants were to be found, and skips in the rows were rare. These plants promised 296 Journal of Agricultural Research voi. xvi. no. h well for another year, when a mistake was made in watering by the farmer, the ground became water-logged, and the entire field was lost through blight in the mature stage. SOIL CONDITIONS AND IRRIGATION Soil conditions materially assist the plant or the fungus. If the ground is well moistened and loose when the seed is planted, a strong vigorous start may be obtained by the plant which will carry it well beyond the immediate reach of the fungus. It has been the common practice to withhold irrigation until the new tubers begin to set. If the plant can endure withholding artificial watering until the new tubers set, it is well to delay, but to postpone it until the plant is suffering acutely, brings it to a condition from which it never wholly recovers. The fungus will make headway in a drouthy plant. After irrigation water has been supplied, it is expedient to cultivate deeply, because irrigation water packs the soil tightly. Too great an application of water on heavy soil may leave the soil puddled, in which condition it must remain for several days before cultivation is possible. If this is accompanied by a rising soil temperature, the ill effects are increased. Occasionally a heavy rain will puddle the soil late in the season preced- ing harvest. This may occur on ground irrigated too late. In such an event it is common for the plants to blight generally and die. The damage now is not in the death of the foliage or the death of the plant, but in the rot which will attack the new tubers. This is a black-rot which may enter by way of the stolons, a common method, or through wound or lenticel. When such tubers have begun to rot, they are a total loss. If the rot has not been detected in the field, it may occur later in the bin, causing a worse trouble. All the tubers of a plant may not be attacked, however, and in such a case control consists in getting them out of the ground without delay. Early varieties in which the new tubers have time to come to full ripeness are more susceptible than late varieties. At this time correct irrigation practice is unknown. No rule can be formulated, because each piece of ground requires different treatment. During some years it is expedient to "irrigate up," meaning to water the field immediately after planting. If the soil is too dry, irrigation is necessary for germination and will carry the plant for the maximum length of time before rewatering. Harm results if the water applied in addition to the soil moisture present creates an excess. Irrigation of a plowed field in which nothing has been planted is impractical, owing to the absence of row ditches, and the fact that a certain time must elapse before anything can be planted. Cultivation should be given after each irrigation, so long as it can be done without damaging the plants below ground. Sandy soils need less cultivation than heavy soils. The best Mar. 17, 1919 Fusarium-Blight of Potatoes under Irrigation ^97 judge of the soil on any farm is the farmer who has worked with it. Each parcel of land has its own pecularities, and advice on the handhng of land should be specific. The very best conditions obtainable for the potato should prevail throughout the season, and so long as the farmer can control the environmental conditions no trouble is likely to result. CONTROL Control of Fusarium-bUght has not been attained. Different methods have been employed, three of which offer reasonable hope of success. First selection of plants whose progeny will offer resistance to the invading organisms. For this purpose, experiments are being carried out vvrith standard accepted varieties known to be smted to the locality. It is possible to select for resistance and have it gradually evidenced m the performance of the plants, as in the field experiment noted above. That is satisfactory so long as some overwhelming circumstance does not intervene and wipe out the work of years. At best, resistance is but a relative thing. ■, ^ Second, control by seed treatment. The attempt has been made, not to kill something that may be on the seed as in the orthodox seed treat- ment but to coat the cut seed with a preservative or fungicide which would remain vital throughout the season, preventing infection. Could this be done, it would offer an easy solution to the problem. Expen- ments were carried out in 1917 to test out the effect of different solutions^ None of them gave satisfaction. Several lots of potatoes were treated and planted on May 18, 1918, with several different mixtures and com- pounds all of which for some reason or other were suspected of having some possible preservation value. The seed used was the Rural variety, and was cut in the usual way. The method of appUcation depended upon the nature of the fungicide, aijd this is noted under "Remarks" m Table II One lot was treated ^vith a spore suspension of F. oxysporum for comparison. On June 19, 191 8, a similar experiment was made with a few lots. The results of these experiments are given m Table 11, and are the data taken from counting 600 plants. 298 Journal of AgriczUtural Research Vol. XVI, No. II TablB II. — Effect of various seed treatment on germination of Irish potatoes PLANTED MAY l8; COUNTED JUNE 15 Treatment. Per- centage of ger- niina- tion. Remarks. Nicotine sulphate 75 68 90 0 35 40 0 60 70 85 97 Dipped. Dipped. Dusted. Dusted. several Sprayed. Dusted. Dusted. Dipped. Sprayed. Dipped i Dipped i Bordeau mixture Formula 5-5-50. Seed remained unusually firm. Seed killed by treatment and rotted by organisms. Strong spore suspension. Some seed killed Charcoal Hypochlorous acid a F . oxysporutn Iron sulphate Lithum carbonate Mercuric chlorid Thoroughly and quickly rotted. Solution, I to I 000 Mustard oil Controls n water Whole seed n 3 per cent solution of copper sulphate. PLANTED JUNE 19; COUNTED JULY 1 5 Charcoal F. oxysporum. Onion luice . . . Control, cut. .. Whole seed. .. 82 o 55 88 99 Dusted. Sprayed. Dipped. Expressed juice of onions. Dipped in water. Average field performance. No treatment. a SurrH, J. L. et al. antiseptic action op hypochlorous acid and its application to wound treat- ment. y» Brit. Med. Jonr., 1915, no. 2847, p. 129-136. July 24i 1915) In the first planting the charcoal treatment gave better germination than the controls, but fell behind in the second planting. None of the others were worth the trouble of treatment. The whole seed gave much better stands and of more healthy vigorous plants. Third, in applying the best-known cultitral practice to the propagation of the potato. For this no rules can be given. Each farmer should judge the condition of his land, its moisture content, tilth, and apparent needs. Rotation with grain and legumes is advisable, allowing the land to be cropped with alfalfa as many years as possible before potatoes are planted. Methods of irrigating and cultivation during the growing season should be investigated at the time for the field in question. Plate 41, A, shows a field planted with good seed, but owing to the dry- ness of the soil at planting time infection set in, and the fungus destroyed from 60 to 80 per cent of the seed, with the resulting poor stand. GENERAL DISCUSSION Infection of potatoes by F. oxysporum from the soil through the seed piece has never been recorded before, so far as is known. That it is of widespread general importance on alkali soils is believed, from conditions noted in several potato-growing regions of the West. In parts of the Mar. 17, 1919 Fusarium-Blight of Potatoes under Irrigation 299 San Luis Valley, where so many unfavorable conditions are at work, owing to subirrigation and a high water table, the large majority of the potatoes show signs of this infection. Other investigators have found vascular infection of the seed to be the cause of much trouble, and the seriousness of that manner of infection elsewhere can not be judged from these experiments. In the Greeley District, where the Fusarium-blight has been so serious for many years, a fortunate change has taken place. This is regarded as being due to the introduction of other crops, potatoes being brought into the crop rotation only once in four years or more. The use of seed beans, sugar beets, grain, and alfalfa in tlje definite rota- tion is extending the time between the same crops with corresponding advantage to each. The potato was desirable as a high-priced crop, and still is, and the percentage of loss is less with rotation. Alkaline soils are a favorable medium for Fusarium spp. Pratt (12) found them to be abundant in virgin desert soils. The prolific and lux- uriant growth of species of Fusarium on alkaline media in pure cultures is an indication of what may be expected in part in alkaline soils where humus is abundant. In disease investigations of this kind it was found desirable to conduct the experiments as much as possible in the field, for the reason that conditions there came about naturally, and the response was immediate and proportionate. Greater care must be taken to note and record every conceivable change of condition. In the gross the changes from day to day are observable and are recorded by suitable instruments; but the changes that occur in the plant are more delicate and rapid than gross observations indicate. Each square foot of soil has its own conditions, not distinguishable from the adjoining square foot perhaps, but of sufiflcient difference to be felt by the plant. The plant feels these things and responds. If resistant stock is to be selected, these changes and conditions should be known, and the finer symptoms indicated by the plant must be recognized for the purpose of anal3'sis. Temperatures of the soil are vital as regards infection. The critical temperature for infection has not been determined and it varies for the manner of infection. Seed-piece infection will occur at a considerably lower temperature than root infection. In the Carbondale District, at a higher altitude and in cooler soil than the Greeley District, only those plants show Fusarium-wilt symptom.s which have decayed seed pieces. Usually the seed piece remains sound throughout the season there, and the plants are free from blight. In the Greeley District the soil temper- atures are higher, and the seed pieces are generally attacked. Root infection occurs with temperatures higher than the average. As the plants get larger and shade the ground, and the roots penetrate deeper the danger from root infection is lessened. There has been a belief that less blight occurs when the potatoes follow alfalfa than otherwise, and that the older the alfalfa was the 300 Journal of Agricultural Research voi. xvi, no. h better would be the potatoes. The current reasons for this are many and varied, but the principal one given is that there is less blight in the soil. This may mean fewer fungus organisms in the soil, but that does not seem to be the case. In several instances potatoes grown from good stock on soil previously in alfalfa for nine years have been obsers'^ed as badly diseased as the same seed on soil only one year in alfalfa. The organism was present as abundantly as ever, and wherever the condition of poor cultivation or heated soil was present, the disease was manifest. The true value of alfalfa preceding potatoes lies in the fertilizer incre- ment and mechanical improvement added to the soil, and not to any dearth of Fusarium spp. The use of whole seed is suggested, not as a means of controlling the blight, but of avoiding it. By the use of whole seed is meant not culls and other small potatoes, but tubers in good condition, well selected, and preferably of iK-ounce weight or greater. Whole seed has been many times condemned as yielding quantities of unmarketable small potatoes, and from the horticultural point of view this is a serious fault. Under irrigation, however, the writer believes that whole seed can be made to yield nearly as many marketable tubers as cut seed. The increased stand resulting and the fact that no labor is required in cutting would promise a return commensurate with the initial increased cost of the seed. In one commercial field in 191 8 the yield from Avhole seed was 100 per cent greater than that from cut seed of the same variet3^ This field is shown in plate 41, B. The cut seed was planted on the left and the whole seed on the right. The photograph was taken at midseason. Sandsten {13) believes that whole seed is preferable to cut seed in dry-land farming because it prevents seed-piece rot. SUMMARY The disease of potatoes in the field caused by Fusarium spp., princi- pally F. oxysporum, whereby death of the plant or decay of any part of it is brought about, is to be regarded as different phases of the same disease. For that reason it is desirable to apply a generally applicable name covering all stages. The term " Fusarium-blight " expresses this adequately. Two methods of infection are recognized: Infection from the soil of roots and root hairs, and infection of the seed piece, whereby the plant becomes diseased. The latter method is regarded as the most serious and responsive to environmental conditions in the Greeley district of Colorado. Three methods of control are suggested, none of which have yet proved wholly effective. First, selection for disease resistance, a method shown to be effective only to a minor degree. Second, superior cultural conditions for the potato plant, whereby it may always maintain a degree of resistance to pathogenic organisms through activity and Mar. 17, 1919 Fusarium-Blight of Potatoes under Irrigation 301 health. Lengthened rotation periods employing other crops followed by alfalfa improve the nutritive and mechanical properties of the soil, while a judicious irrigation practice adapted to the particular field and season involved combined with suitable cultivation should constantly main- tain a steady and adequate, but never excessive, supply of moisture and insure suitable aeration. This is the method available to the farmer, so far as he knows what constitutes the best conditions for his land through- out a given season. Third, by the use of whole seed, free from wound or injury, thus preventing seed-piece infection, or at least maintaining the plant free from infection for the maximum length of time. The com- bination of the two last-named measures probably constitutes the most effective measures for control of Fusarium-blight. It is believed that more than one species of Fusarium is able to bring about each phase of the bhght. F. oxysporum in pure culture under suitably controlled and natural conditions has been found to do this. Three general stages of the Fusarium-blight are recognized. First, the stage in which decay and death of the seed piece and new plant occurs before the new shoot emerges from the ground. Germination may or may not have occurred. Second, the later stage, in which the young plant shows many and diverse symptoms of infection by Fusa- rium spp., often resulting in death. Some of these manifestations are not fatal, and recovery is possible. Third, the mature stage, resulting in death, usually at an advanced state of growth, often with infection and decay of the new tubers. Different varieties of potatoes show marked variation in their behavior under the same general conditions. There is an inherent weakness in different strains of the Rural variety toward Fusarium-blight, accen- tuated by the conditions under w^hich the seed was grown. The Pearl variety shows these weaknesses, but to a minor degree, unless brought to a poor condition by previous subjection to disease. Vascular infection of the seed is not the starting point of disease, but is one of the conditions assisting in bringing about decreased resistance to new infection from the soil. LITERATURE CITED (i) Appel, Otto, and Woi.i,Enweber, H. W. I910. GRUNDLAGEN EINER MONOGRAPHIE DER GATTUNG FUSARIUM (LiNK). ^ In Arb. K. Biol. Anst. Land u. Forstw., Bd. 8, Heft i, 207 p., 10 fig., 3 pL Verzeichnis der wichtigsten benutzen Schriften, p. 196-198. (2) Carpenter, C. W. 1915. some potato tuber-rots caused by species of fusarium. in jouf. Agr. Research, v. 5, no. 5, p. 183-210, pL A-B, 14-19- Literature, cited, p. 208-209. (3) Cromwell, R. O. i917. fusarium-blight, or wilt disease, op the soybean. in jout. agf. Research, v. 8, no. 11, p. 421-440, i fig., pL 95. Literature cited, p. 438-439- -202 Journal of Agricultural Research voi. xvi. No. n (4) Jones, L. R., and Gii^man, J. C. 1915. THE CONTROL OF CABBAGE YELLOWS THROUGH DISEASE RESISTANCE. Wis. Agr. Exp. Sta. Research Bui. 38, 70 p., 23 fig. Literature cited, p. 69-70. (5) Link, G. K. K. 1916. a physiological study of two strains op fusarium in their causal RELATIONS TO TUBER-ROT AND WILT OP POTATO. Ncbr. Agr. Exp. Sta. Research Bui. 9, 45 p., iUus. Reprinted from Bot. Gaz., v. 62, no. 3, p. 169-209. 1916. (6) Manns, T. F. 1911. THE FUSARIUM BLIGHT (wiLi) AND DRY ROT OP THE PATATo. Preliminary studies and field experiments. Ohio Agr. Exp. Sta. Bui. 229, p. 299- 337, pi. 1-15. (7) Orton, W. a. 1902. the wilt disease op the cowpea and its control. in u. s. dept. Agr. Btu. Plant Indus. Bui. 17, p. 9-22, i fig., 4 pl- (8) loio. COTTON WILT. U. S. Dept. Agr. Farmers' Bui. Z3>2» 24 P-. i^us. (9) I914. POTATO WILT, LEAF-ROLL, AND RELATED DISEASES. U. S. Dept. Agr. Bul. 64, 48 p., 16 pi. Bibliography, p. 44-48. (10) PiZZlGONI, A. 1896. CANCRENA SECCA ED UNiDA DELLE PATATE. In Nuovo Gior. Bot. Ital., n. s. V. 3, fasc. i, p. 50-53. 11) Pratt, O. A. 1916. A WESTERN FIELDROT OP THE IRISH POTATO TUBER CAUSED BY FUSARIUM RADicicoLA. In Jour. Agr. Research, v. 6, no. 9, p. 297-309, pi. 34-3?- (12) 1918. SOIL PUNGI IN RELATION TO DISEASES OP THE IRISH POTATO IN SOUTHERN IDAHO. In Joiu-. Agr. Research, v. 13, no. 2, p. 73-100, 4 fig-, pl- A-B. Literature cited, p. 98-99. (13) Sandsten, E. p. I918. POTATO CULTURE IN COLORADO. Colo. Agr. Exp. Sta. Bul. 243, 35 p. illus. (14) Sherbakoff, C. D. 1915. fusaria of potatoes. N. Y. Cornell Agr. Exp. Sta. Mem. 6, p. 89-270, 51 fig., 7 col. pl. Literature cited, p. 269-270. (15) Smith, Erwin F. 1899. WILT disease op cotton, WATERMELON, AND COWPIJA (nEOCOSMOSPORA NOV. GEN.). U. S. Dept. Agr. Div. Veg. Phys. and Path. Bul. 17, 72 p., 10 pl. (16) — and Swingle, D. B. 1904. THE DRY ROT OF POTATOES DUE TO FUSARIUM OXYSPORUM. U. S. Dept. Agr. Bur. Plant Indus. Bul. 55, 64 p., 2 fig., 8 pl. Literature cited, p. 61-62. (17) Wehmer, Carl. 1897. UNTERSUCHUNGEN UBER KARTOFFELKRANKHEITEN. 2. ANSTECKUNGS versuche MIT FUSARIUM soLANi. (die fusarium-faulE). In Centbl. Bakt. [etc.], Abt. 2, Bd. 3, No. 25/26, p. 727-742, pl. lo-ii. (18) Wilcox, E. M., Link, G. K. K., and Pool, Venus W. 1913. A DRY ROT OP THE IRISH POTATO TUBER. Nebr. Agr. Exp. Sta. Research Bul. I, 88 p., 28 pl. Bibliography. ^ Mar. 17, 1919 Fusarium- Blight of Potatoes under Irrigation 303 (19) Woi,l.ENWEBER, H. W. 1913. PILZPARASITARE WELKEKRANKHEITEN DER KULTUItPFLANZEN. In Bcf., Deut. Bot. Gesell., Bd. 31, Heft i, p. 17-34- (20) 1913. STUDIES ON THE FUSARIUM PROBi^EM. In Phytopathology, v. 3, no. i, p. 24-50, pi. 5. PLATE 37 Effect of Fusarium-blight on seed pieces of potato: A. — Early Ohio seed pieces: Control above; pieces inoculated with F. oxysporum below. B. — Early Ohio plant. See piece inoculated with F. oxysporum. C. — Early Ohio seed pieces: Control (left) and inoculated (right) seed pieces. The control shows the method used in inoculation. D. — Seed piece well decayed, resulting from soil infection. E. — Seed-piece rot in field. 304 Fusarium-Blight of Potatoes under Irrigation PLATE 37 Journal of Agricultural Research Vol. XVI, No. 11 Fusarium-Blight of Potatoes under Irrigation Plate 38 Journal of Agricultural Research Vol. XVI, No. 11 PLATE 38 A. — Inoculated and uninoculated stems of same potato plant. Stem at right shattered by F. oxysporum. B.— Potato plant (control), showing method of inoculating with wedge of melilotus stem. C. — Seed-piece rot in field. The yoimg potato plant has not yet been attacked. D. — Potato plant naturally infected by F. oxysporum in the field. PLATE 39 Potato stems showing seed-piece rot : A. — Stem split to show rotting due to organism entering through seed piece from soil. Note decay of roots from point of attachment outward. B. — Stem split to show slight discoloration at base where infection from soil-infected seed piece occurred. C. — Seed piece of potato plant shown in Plate 40, B. The center top leads to the center of decay. D. — Seed-piece rot in field. The seed piece is well decayed, but plant is unaffected and the roots are healthy. Fusarium-Blight of Potatoes under Irrigation Plate 39 Journal of Agricultural Research Vol. XVI, No. 11 Fusarium- Blight of Potatoes under I rrigation Plate 40 Journal of Agricultural Research Vol. XVI, No. 11 PLATE 40 Potato plants affected by Fusarium-blight: A -Potato plant late in season with rolled leaves. Fusariura blight. b!-Top of potato plant consisting of three stems. One leaf on one stem is wilted. ^C.-Potfto plant of two stems, one at left showing Fusarium blight, or wilt caused bv seed-piece infection; one at right healthy. D.-Plant with rolled leaves gradually dying from Fusarium blight. Severe case late in season. PLATE 41 A. — A field of potatoes showing the result of unfavorable cultural and soil condi- tions, by which seed-piece rot destroyed 60 per cent of the stand. B. A field of potatoes planted with whole seed (rows to right) and cut seed (rows to. left) at midseason. The hills planted with whole seed gave a 100 per cent greater yield than those planted with cut seed. Fusarium-Blight of Potatoes under Irrigation Plate 41 s i- "■ ,'■♦•«• ->i «k <.? -a««^^« Journal of Agricultural Research Vol. XVI, No. 11 Vol. XVI NI ARCH 24, 191 Q 3Sfo. 12 JOURNAL OF AGRICULTURAL RESEARCH CONTKNTS Paftt Effect of Certain Grain Rations on the Growth of the White Leghorn Chick ------- 305 G. DAVIS BUCKNER, E. H. NOLLAU, R. H. WILKINS, and JOSEPH H, KASTLE ( Contribution bom Kentucky AgricuKarai Experiment Station ) Ammonification of Manure in Soil - - - - - 313 H. J. CONN and J. W. BRIGHT ( Contribution from New 7ork State Asricultura] Experiment Station } PUBUSHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE, WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS VVTASHINOTON, D. C. ■ ■ ' — ~r — WAAHINOTON ! OOVERWMENT PRINTINO OWCE ! »»H mM ■!',?i -^■y^>. '!C^M EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCL/^TION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT KARL F. KELLERMAN, Chairman Physiologist and Associale Chief, Bureau of Plant Industry EDWIN W. ALLEN Chief, Ojffict of Experiment Stations CHARLES L. MARLATT Entomologisl and Assistant Chief. Bureau of Entomology FOR THE ASSOCIATION H. P. ARMSBY Director, Institute of Ajiimal A'ulrilicm, The Pennsylvania State College J. G. LIPMAN Director. New Jersey A friculturai Experiment Station, Rutgers College W. A. RILEY Entomologist and Chief, Division of Ento- mology and Economic Zoology, Agricul- tural Experiment Station of the University (>f Minnesota 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 H. P. Armsby, Institute of Animal Nutrition, State College, Pa, JOMALOFAGRIQimMLffiSEARCH Vol. XVI Washington, D. C, March 24, 1919 No. 12 EFFECT OF CERTAIN GRAIN RATIONS ON THE GROWTH OF THE WHITE LEGHORN CHICK f^t^'^'^'^f By G. Davis BucknER, E- H. Nollau, R. H. Wilkins, and Joseph H. KastlE ■^^'f]£ Department of Chemistry, Kentucky Agricultural Experiment Station ^^ In a former paper ^ from this laboratory evidence was presented to ' show that the lysin content of the proteins of certain grain mixtures fed to White Leghorn chicks was the limiting factor in their growth. The results of those experiments showed that on a ration consisting of wheat, wheat bran, sunflower seed, hempseed, cracked com, skim milk, cabbage, and sprouted oats, normal growth was obtained, while on a ration consisting of barley, rice, hominy, oats, gluten flour, butter fat, cabbage, and sprouted oats a condition of arrested growth resulted. The first-mentioned ration was supposed to contain a high percentage of the amino acid, lysin, as compared with the second ration, which was supposed to be low in lysin. These experiments were open to criticism because of the small number of chicks under consideration, the labora- tory conditions governing them and the possible inaccuracy in the numbers given for the amino-acid distribution of the grain mixtures fed. In view of this, an experiment was plaimed which would, as far as possible, eliminate these points of objection. In 1 91 5 an experiment was conducted by Buckner, Nollau, and Kastle, in which 14 one-day-old White Leghorn chicks were fed a ration consist- ing of 33 parts of ground soybeans and 67 parts of ground oats, supple- mented by 20 per cent of protein-free milk, sprouted oats, shredded cabbage leaves, grit, oyster shell, and a small quantity of sour skim milk. On this ration the chicks failed to thrive and grow and would eventually have died had not the grain ration been changed. It was changed to equal parts of wheat bran, sunflower seed, hempseed, barley, oats, and rice. On this diet a partial recovery was effected, yet the vigor and development of the normal White Leghorn chick of a similar age was not attained. One of us (Kastle) contended that this failure to grow 1 Buckner, G. D., Nollau, E. H., and Kastle. J. H. the feeding of young chicks on grain MIXTURES OF HIGH AND LOW LYSIN CONKNT. /n Amer. Jour. Physiol., V. 39, no. 2, p.162-171, i pi. 1915. Journal of Agricultural Research, Vol. XVI, No. la Washington, D. C. Mar. 34, 1919 rp Key No. Ky.-8 (305) 3o6 Journal of Agricultural Research Vol. XVI, No. 12 normally was caused by some toxic principle in the soybean, and in order to throw more light on this point the following experiment was planned. It was conducted in an airy, well-lighted basement room of the Kentucky Agricultural Experiment Station. Two lots of chicks, designated "A" and "B," consisting of 12 and 11 White Leghorn chicks, respectively, were selected entirely at random and fed rations composed of equal parts of soybeans, oats, wheat, ship stuff, sunflower seed, and cracked corn, supplemented by sour skim milk, sprouted oats, shredded cabbage, grit, and oyster shell. The food of lot A was given to them in the usual manner of feeding. In the case of lot B the grains were ground together, well mixed with a small quantity of distilled water, and baked in an electric oven. The temperature of the oven was kept at 420° F., and the mixture was frequently stirred. The baked feed was ground and fed to lot B in the same way as was the uncooked mixture to lot A. Table I gives the weights of the chicks, their increase in weight, and the number surviving each week. Tabi,E I. — Effect of uncooked and cooked rations on the growth of White Leghorn chicks LOT A July 6. 13- 20. 27. Aug. 3. 10. 18. 24. 31- Sept. 7. Date. 1915- Number of weeks. Number of chicks. Total weight of chicks. Gm. 528 I, 004 1, 266 1,832 2, 462 3.290 3.795 4,357 4,410 Average weight of chicks. Gm. 48.0 65- 9 91- 3 115. I 183.2 246. 2 329.0 379-5 435-7 490. o Average increase. Gm. 17.9 25- 4 23.8 68. I 63. o 82.8 50-5 56. 2 54.3 Average percent- age increase. LOT B July 6. 13- 20. 27. Aug. 3 . 10. 18. 24. 31- Sept.7. 1915- 12 12 I 2 11 3 8 4 8 5 7 6 7 7 7 8 4 9 4 554 788 I, 014 930 1.357 1. 655 2, 160 2,307 2, 062 2,275 46. 2 65-7 19- 5 42. 92. 2 26. 5 40. 116. 2 24. 0 26. 169.7 53-5 46. 236. 4 66. 7 39- 308.5 72. I 30. 329- 5 21.0 "6. 515- 5 186. 0 56. 566. 2 50-7 9- o Three chickens sick. Mar. 24. 1919 Effect of Grain Rations on Growth of Chicks 307 This experiment, as shown in Table I, covers a period of nine weeks in which both lots of chicks received identical treatment, except as to the preparation of the food. The growth of lot A was approximately normal as to weight and mortality, but their vigor and general condition was not good. In lot B, which received the cooked grain ration, a decided deleterious effect was shown by the weight and mortality record which can properly be ascribed to the ration. If we regard this ration in the light of the present-day conception of nutrition, it is balanced with reference to the dietary essentials, fat-soluble A being abundantly sup- plied in the sprouted oats, shredded cabbage leaves, and butter fat con- tained in the not-too-closely skimmed milk, while sufficient water-soluble B was obtained from the grain mixture before them as a dry mash at all times. The incomplete proteins of the grains were supplemented by the casein of the milk in the wet mash and the proteins in the cabbage and sprouted oats. These factors, with the mineral content amply supplied by grit, oyster shell, and milk, satisfy all conditions in making a com- plete diet. However, the food hormones were destroyed by heating the ration fed to lot B. The experim.ents of McCollum and his coworkers have demonstrated the nutritive limits of seeds. I^IcCollum states ^ in substance that when seeds are fed, supplemented by suitable inorganic salts and sufficient fat- soluble A, the limiting factor with respect to growth is the quality of protein. The data presented in this paper and the experimental work to be described corroborate this statement and throw further light on the following points : 1. That the soybean may enter into the dietary of the White Leghorn chick without having an accumulative deleterious effect. 2. The efifect of heat on the food value of certain grain mixtures. 3. That under approximately ideal conditions chicks which had been stunted by dietary measures and had survived by reason of greater vitality may remain in a fairly good state of health over a long period of time. The plan of the experiments was as follows: Four grain rations were selected so that two would contain grains the proteins of which were supposed to be high in lysin (one to be fed as a mash and one as a grain) ; the other two grain mixtures were supposed to contain proteins low in lysin and were to be fed in the same way as the first two. After this experiment had been started an effort was made to determine the amino- nitrogen distribution in these mixtures, but, owing to their large carbo- hydrate content, no satisfactory results have been obtained. The effort to analyze these complex grain mixtures is still in progress, and it is 1 McCoLLTJM, E. v., and Simmonds, N. a biological analysis of pellagra-producing diets, m. THE value of some seed PROTEINS FOR MAINTENANCE. In JoUr. Biol. Chem., V. 32, no. 3, p. 347-368, 12 charts. 191 7. 3o8 Journal of Agricultural Research voi. xvi, no. 12 hoped that their araino-acid make-up may be determined at some future time. In this experiment we selected at random from 600 i -day-old incubator chicks of the White Leghorn breed, three lots, each containing 60 chicks, which were kept under identical conditions except that the diets were different. The conditions governing these three lots of chicks were as follows : lyOt I was placed in a large brooder house which opened on a large grass run. The chicks were weighed individually on a torsion balance, sensitive to o.i gm. the day after they were hatched and were so weighed each succeeding week. The weekly individual weighings and mortality records were kept, the previous weight records of all chicks which had died being discarded, so that at the end of 28 weeks the weight records represented only those that were living at that time. No particular care was taken of these chicks except to see that they were properly watered, fed, and housed. They were fed a ration consisting of equal parts of finely ground soybeans, wheat, wheat bran, sunflower seed, and hemp- seed. This was fed as a dry mash and was kept before them at all times; and once a day, at noon, a wet mash of this mixture made with sour skim milk was fed. Once a day a coarsely ground grain mixture, consisting of equal parts of wheat, soybeans, hempseed, and cracked com, was thrown into the litter in order to make them exercise by scratching for it. Oyster shell, grit, and charcoal were before them at all times, while sprouted oats and shredded cabbage leaves were liberally fed once a day. It may be of interest to note that this ration is in every way identical, with the exception that soybeans were added in an equal quantity, with the ration fed to lot 3 of our previous paper,^ in which the growth and general physical condition were normal, even though the experiment was conducted under laboratory conditions. These chicks were closely watched, and any change in their physical condition and habits was noted. When the treading period began, the cockerels and pullets were separated to avoid any loss of weight occa- sioned by undue exercise. Lot 2 was kept under the same conditions, only this lot could not be separated into cockerels and pullets, because their external sexual char- acteristics were depressed to such an extent that they could not be dis- tinguished, and treading did not occur. A dry mash, which was kept before them at all times, consisted of equal parts of finely ground barley, rice, hominy, and oats, to which was added enough gluten flour and butter fat to raise the protein and fat content of this grain mixture up to that of the grain mixture fed as a mash to lot i. Once a day they received a wet mash made of this grain mixture moistened with protein- free milk. The grain ration fed in the litter consisted of equal parts of • BucKNER, G. D., NonAu, E. H., and Kastlb, J. H. 1915. op. cit. Mar. Effect of Grain Rations on Growth of Chicks 309 barley, rice, and hominy, to which was added enough pure butter fat and gluten flour to bring the fat and protein content up to the same level as that of the corresponding grain mixture fed to lot i.^ Lot 3 constituted the control for the two other lots of chicks. This lot was kept under conditions identical with those of lots i and 2, the cockerels and pullets being separated when treading began. These chicks received a ration known as the standard Cornell ration.^ Table II presents the weekly weight and mortahty records of these three lots of chicks, covering a period of 28 weeks, in which time there were no unusual weather conditions or epidemics among these chicks, so that these figures represent the degree of nourishment afforded by the rations fed to the separate lots. Table III. — Effect of various diets on the growth of White Leghorn chicks Week. Lot I. Average weight of- 25 cocks. 17 hens. Num- ber of chicks. Lot Average weight of cocks and hens. Num- ber of chicks. Lot 3 (control). Average weight of — 25 cocks. 17 hens. Mean. Num- ber of chicks. 3 4 S 6 7 8 9 10 II 12 13 14 IS 16 17 18 19 20 21 22 23 24 25 26 27 28 Cm. 41.0 70.4 100.9 146-5 184. 1 2s6. 6 296.5 363.4 420. 6 474.0 518.2 588.5 653-4 733-4 795-8 828.4 954-9 1,024.5 1,077.8 1,135-9 1,184.6 1,231. 6 1,214.5 1,204. 6 1,215-5 1,206. 7 1,268.6 1,250.4 1,294-3 Gm. 39- 66. 96. 138. 179. 236 276 318 382 423 460 523 582 63s 684 740 790 844 930 964 1,010 1,057 1,055 I, loi 1,094 1,079 1,084 1,135 1,182 Gm. 40. 2 68.4 98.8 142. 6 181. 6 246.5 286.5 340.9 401.5 448- 5 489.5 556.1 617. 9 684-6 740.0 784.2 872.5 934-3 1,004. 2 1,050- 2 1,097-4 1,144.4 1,135- 1 1,152-8 1,154-9 1,143-0 I. 176- 5 1,192-8 1,238.4 Gm 106 124 148 ISO 162 182 221 244 251 293 325 361 441 474 526. 543 565 615 663 734 763 781 Gm. 231- 292. 360. 442. 496. 537- 630. 674- 812. 902- 953- 980. 1,063. 1,154- 1,252. 1,295- 1,344- 1,396- 1.430- 1,442- 1,462- 1,480. 1.528- 1,560. 1,594- Gm. - 1 . 2 41 69 7 95 ■4 140 -6 191 . 2 248 9 288 8 354 9 395 6 409 9 488 4 506 9 622 3 682 2 695 4 717 2 780 9 841 7 889 3 922 8 956 7 992 6 1,038 6 1,047 I 1,066 0 1,082 5 1,075 6 1,090. 6 1, 120 Gm. 41.6 70.7 102. 6 151.1 211. 6 270.5 324.6 398.6 446- 2 473- 7 559-8 590-4 717-5 792-3 824- I 848.8 922. I 998.3 1,071.0 1, 109. I 1,150.5 I. 194.4 1.2^4-5 1,245-1 1,264. 1 1,281.3 1,301.9 1,325-4 1,357-5 48 48 48 48 48 48 48 47 47 47 47 47 47 45 43 43 42 In studying the growth record of lot i it will be seen that the average weights of the pullets closely followed the control throughout the 28 weeks, but that the cockerels were inferior in this respect after the third 1 At the expiration of these 28 weeks the ration was changed only by the addition of sour skim milk, which brought about profound anatomical and physiological changes. The combs and wattles developed, shortly to be followed by crowing and treading on the part of the cockerels. It is also of interest to note that their growth was greatly accelerated. The exact figures could not be counted on because of errors occasioned by change of student assistants helping in the weighings. ' NrxoN, Clara. FUEDIng young chickens. N. Y. State Col. Agr. Cornell Reading-Courses, v. 4, no. 88, p. 176. 1915. 3IO Journal of Agricultural Research voi. xvi, no. X2 week. Regarding their physical condition after the sixth week it was plainly to be seen that lot i did not have the same vigorous appearance as lot 3. This was most particularly noticed with regard to the general condition of the feathering which seemed slightly ruffled and imkempt as compared to lot 3. This can be seen only to a degree in the illustrations of these two lots (PI. 42 A, B). Their general vigor and development did not appear equal to that of lot 3, and these differences became more noticeable as they grew older, until, at the end of the twenty-eighth week, when the experiment was discontinued, it was plainly seen that lot I was in no way in the same physiological state as lot 3. It will also be noted from the data that after the seventeenth week lot i did not gain as much as lot 3 over the same period. During these 1 1 weeks the gain was 234.2 gms. as against 286.5 gnis. for lot 3 ; or, in other words, lot 3 made a gain 18.3 per cent greater than that made by lot i. It may be of interest to point out that the cockerels of lot i showed a greater variation in weight from the normal than the pullets. The results of the mortality record show the same number of deaths at the end of the experiment as in the control. In lot 2 we see that the rate of growth was retarded to such an extent that at the end of the 28 weeks the average weight of a chick was 809.4 gms. as against 1,238.4 gm. for those receiving the ration containing soybeans and 1,357.5 gms. for the control. The external sexual charac- teristics were rudimentary, and the feather tracts not properly developed and the fact that they retained the habits of the immature chick during this entire period is of added interest. This is shown in the illustration of this lot (PI. 42, C) which was taken the same day as those of lots i and 3, all having the same focal distance. The ration fed to lot 2 greatly in- creased the mortality so that only 19 remained at the end of the experi- ment, the weaklings having died earliest. Therefore we had only those with the greatest initial vitality which had lived for 28 weeks on a diet the biologic value of which was low. From this we would infer that the individual vitality of the animal plays a very important part in" deter- mining its ability to grow, and for this reason it is essential that a con- siderable number of animals should be used in experiments on nutrition. This would seem to follow when we realize that all of the chicks used in these experiments were pure-bred and came from the same parent stock. The chicks of lot 2 seemed to have a good appetite, and while they ate with apparent avidity, yet they always seemed to be in search of some- thing in their feed which they could not find. Regarding their ration, it will be seen that it is satisfactory with respect to every necessary dietary factor except the quality of protein. We are unable at this time to show wherein these proteins are limited, but hope to be able to prove this by experiments which are now in progress. Mar. 24, 1919 Effect of Grain Rations on Growth of Chicks 311 In conclusion we feel justified in making the following deductions from the results of these experiments : (i) The proteins of rice, oats, barley, hominy, and gluten flour are inefficient in promoting normal growth in the White Leghorn chick. (2) The results of these experiments seem to indicate that the proteins in the grains mentioned above have a retarding action on the develop- ment of the external sexual characteristics and their functions, which accompanies the arrested growth of the chicks. (3) We are unable to account for the apparent weakened vitality of the chicks of lot i, as shown by their weight record and general con- dition. However, we do not attribute it to any toxic action of the soy- bean. (4) Baking a grain mixture composed of equal parts of soybeans, wheat, wheat bran, sunflower seed, hempseed and cracked com, moistened with water, materially lowers its efficiency as a food, when all else enter- ing into the diet is sufficient. (5) The growth and development of any animal is largely dependent on its individual vitality, and for this reason it would seem desirable to use a large number in experiments on nutrition. PLATE 42 A. — Lot I at the age of three months. B. — Lot 3 at the age of three months. C. — Lot 2 at the age of three months. (312) Effect of Grain Rations on Growth of Chicks Plate 42 Journal of Agricultural Research Vol. XVI, No. 12 AMMONIFICATION OF MANURE IN SOIL By H. J. Conn, Associate Bacteriologist, and J. W. Bright, Assisian: Bacteriologist, New York State Agricultural Experiment Station FOREWORD A recent series of papers of the New York Agricultural Experiment Station (8-iiy contained the results of a study of the microscopic flora of the soil. The microorganisms of the soil were classified into a few large groups, some of which were further subdivided, and in a few cases the classification was carried as far as the recognition of species. This preliminary work was considered necessary before studying the different groups with the object of recognizing more of the individual species and learning their functions. A complete study of all soil microorganisms would be an endless task, and, indeed, rather unprofitable, provided the order of studying the different types were left entirely to chance. To begin a study of this kind, therefore, those organisms should be selected that are presumably impor- tant. It is difficult to judge, a priori, the importance of any particular microorganism in the soil, but a hint can be obtained by observing which types predominate in natural soil under conditions of considerable importance in practice. The organisms chosen for investigation in the present work were found to multiply in freshly manured soil. In such soil, ammonification and other forms of decomposition are vigorous and there is good reason to believe that the most rapidly multiplying organisms are of practical importance. Upon adding manure to soil several kinds of bacteria have been found to multiply strikingly, but many of them are difficult to recognize and especially difficult to describe so that others may recognize them. It has seemed unvvise to make a detailed study of any organism which could not be recognized by other workers; and the work has therefore been limited for the present to two types, both of which have been identified with previously described forms. The two bacteria investigated belong to the group of non-spore-formers (discussed in an earlier publication (lo) as one of the three large groups of soil microorganisms) and more specially to that division of this group described as rapid liquefiers {p. lo, 6-g). One of them is Pseudomonas fluorescens (Fliigge) Migula, described on page 6 of that bulletin. The second is described on page 8 of the same bulletin as the "orange lique- fying type," and has now been identified with Bacillus caudatus Wright. As a single polar flagellum is present, it is renamed "Pseudomonas caudatus (Wright)." 1 Reference is made by number (italic) to "Literature cited," p. 347-3S0. Journal of Agricultural Research, Vol. XVI, No. 12 Washington, D. C. Mar. 24, 1919 rr Key No. N. Y. (Geneva)-5 (313) 3 1 4 Journal of Agricultural Research voi. xvi. no. u The present paper is divided into two sections. The first shows the predominance of these two organisms in manured soil and gives the results of an investigation of their function in soil. The second gives a detailed description of the two organisms to aid in their identification by others. H. J. Conn. Mar. 24. 1919 Ammonification of Manure in Soil 315 I.— WHAT SOIL ORGANISMS TAKE PART IN THE AM- MONIFICATION OF MANURE ? By J. W. Bright INTRODUCTION The importance of the ammonification process in the soil has long been recognized, although there has been a tendency on the part of investigators to regard it as secondary in importance to nitrification in soil fertility. Gainey (19), however, has recently claimed that the fertility of a soil is limited by processes which precede nitrification — namely, ammonification — rather than by nitrification itself. The present work has been undertaken for the purpose of determining some of the organisms which actually cause the ammonification of manure in soil under natural conditions; to ascertain the extent to which they can carry on this ammonification; and to compare them with other organisms known to possess the power of ammonifying laboratory media. A survey of the literature suggests that the active ammonifying organ- isms in the soil are generally spore formers. This idea seems to be based principally upon the conclusion reached by Marchal (j6) that the spore- forming Bacillus mycoides is one of the most common of the soil organ- isms and the one that attacks protein most energetically. It should be noted, however, that he worked with a miscellaneous group of organ- isms and of his eight most important ammonifiers only one, the non- spore-former B. fluorescens liquefaciens , is a typical soil organism. J. G. Lipman (jj) assumed that the spore formers were important ammonifiers, as is evidenced by the fact that he referred to the B. suhtilis group and the streptothrices as being the most prominent ammonifying organisms numerically important in arable soils. Stephens and Withers {48) and C. B. Lipman {32) also assumed this when they decided to use B. suhtilis as the organism with which to do their experimental work on ammoni- fication. That this idea is still held by some soil bacteriologists is shown by the fact that in a recent investigation by Neller {39) (an associate of J. G. Lipman), the spore-forming organisms B. suhtilis, B. vulgatus, B. my- coides, and B. megatherium are used to represent "some of the more common species of soil organisms" causing ammonification in the soil. While it is undoubtedly true that a great many spore-forming organ- isms are capable of very active ammonification in manured soil, yet there is good reason to doubt their activity under natural conditions. Conn (6) has already pointed out that the spore formers probably exist in the soil almost entirely as spores rather than as vegetative cells and that their status as active ammonifiers in soil is doubtful. He further shows {10) that the non-spore formers not only exist in the soil in great numbers but that one group of them at least have proteolytic powers. 3i6 Journal of Agricultural Research voi. xvi, No. la One of this group, Pseudomonas fiuorescens , is known to be an ammonifier. This, together wdth the fact that the non-spore formers have been found to be especially abundant in freshly manured soil suggests that they may be among the important soil ammonifiers. The present work was planned to test whether this assumption is correct, and, if so, to obtain as rigid proof as possible of the ammonifying agency of the non- spore-forming organisms. TECHNIC The soil used throughout the series of experiments was Dunkirk silty clay loam ^ obtained from a plot on the station grounds. This soil was mixed with fresh horse manure or fresh cow manure, always in the proportion of 20 parts of soil to i part of manure. All samples were plated according to the usual methods, with at least two dilutions. The degree of dilution depended upon the char- acter of the samples to be plated. Four plates were made from each dilution used and the average count of the four plates was taken to represent the count for that dilution. Whenever possible, the count was based upon the dilution averaging between 30 and 150 colonies per plate. In some cases, "^however, it was necessary to take into account plates which varied from these limits. In a few cases where plates were lost on account of contamination or liquefaction, the count represents an average of three instead of four plates. The medium used in all the plating was "tap-water gelatin" made by dissolving 200 gm. of "gold-label" gelatin in i liter of tap water, adjust- ing the reaction to about Ph = 6.8, with bromthymol blue as the indi- cator, and clarifying with white of &gg. Nearly all of the plate counts were checked by direct microscopic examination of the soil according to the method described by Conn {12). An infusion of the soil to be examined was made by shaking up I gm. of the soil in 9.5 cc. of a fixative prepared by dissolving 0.15 gm. of gelatin in 1,000 cc. of hot water. Of this infusion o.oi cc. was meas- ured out with a capillary pipette and smeared evenly over an area of i square centimeter on a glass slide. This smear was then dried and stained with hot rose Bengal for i minute. For all pure culture studies the manured soil was placed in small Erlenmeyer flasks, 150 gm. per flask. These were then plugged with cotton and sterilized in the autoclave at 15 pounds' pressure for two hours. Subsequent platings proved that in this way all organisms and spores were killed. The infusion for inoculating the soil was prepared as follows: A freshly streaked culture of the organism was suspended in sterile water, and the number of organisms per cubic centimeter of this ' Described according to the system of the Bureau of Soils of the U. S. Dept. of Agriculture. (Marbut, Curtis F., Bennett, Hugh H., Lapham, J. E., and Lapham, M. H. Soils of the United States. U. S. Dept. Agr. Bur. Soils Bui. 96, 791 p., 1913. Carr, M. Earl, Lee, Ora, jr., Maynadier, Gustavus B., HaI/- I.OCK, D. J., and Frost, V. J. Soil Survey of Ontario County, New York. U. S. Dept. Agr. Bur. Soils, Adv. Sheets, Field Oper. 1910, 55 p., i fig., i map. 1912.) Mar. 24. I9I9 Ammonification of Manure in Soil 317 infusion determined by the above microscopic method. The infusion was diluted to the desired strength and i cc. of it introduced into each flask. The flasks were then incubated at room temperature and studied at specified intervals. All flasks were controlled by uninoculated flasks as controls. The method used for the determination of the ammonia produced was practically that of Potter and Snyder,^ which is an adaptation of the Folin^ aeration method. A number of alternating Kjeldahl flasks and absorption cylinders were set up in series so that a continuous cur- rent of air could be passed through the system. Twenty-five-gm. samples of the soils to be tested were placed in the Kjeldahl flasks and 200 cc. of N/30 hydrogen sulphate (HjSO^) were put in each absorption cylinder. The flasks and cylinders were so connected that the air from the end flask was driven over into its adjoining cylinder and absorbed in the standard acid. Arranged in this way each Kjeldahl flask and adjacent absorption cylinder with the cormecting tubes made one com- plete unit and any number of these units could be connected in the series. When the apparatus was set up and all was in readiness for the aera- tion, 2 gm. of sodium carbonate (NajCOg), and 50 cc. of ammonia-free water were introduced into each Kjeldahl flask. The flasks were then tightly stoppered, an^ the air was turned on at such a rate that about 6 liters of air per minute passed through the system. The aeration was continued for about two hours and the standard acid in the absorption cylinders titrated against N/30 sodium hydroxid (NaOH) to determine the amount of ammonia driven off from the soil. Care was taken to have the system absolutely air-tight and all rubber connections dry so that in passing from the flasks to the cylinders none of the ammonia would be absorbed by the water. Absorption in the standard acid was aided by using Folin ammonia tubes to break up the bubbles of air when they entered the absorption cylinders. The determination of the amount of free ammonia in soil has always been a difficult one. The accuracy of the results obtained is somewhat doubtful, as many of the protein substances present in soil are readily broken up by the reagents used in determining the ammonia piesent. Consequently the ammonia determinations in this series of experiments can not be regarded as absolutely true determinations of "ammonia production." Still other factors which might tend to destroy the accu- racy of the determinations are, first, that the organisms themselves might utilize the ammonia as rapidly as it is produced; and second, that it might escape into the air. The latter is improbable because the ammonia would be more likely to be absorbed by the water present in the soil. Controls of sterilized manured soil were always run at the same time as the inoculated soils, and in this way it was possible to determine whether or not the organisms in the inoculated soil affected the amount of ammonia production in any way. ' Potter, R. S., and Snyder, R. S. the determination of ammonia in soils. Iowa Agr. Exp. Sta. Research Bui. 17, 19 p., illus. 1914. ' Folin, Otto, eine methods zur bestimmung des ammonl\ks im harne und anderen thierischen PLUSSigkeitsn. In Ztschr. Physiol. Chem., Bd. 37, Heft 2, p. 161-176. 190a. 3i8 Journal of Agricultural Research Vol. XVI. No. i» RELATIVE NUMBERS OF NON-SPORE-FORMING AND SPORE-FORMING BACTERIA IN FRESHLY MANURED SOIL Work done by Conn {lo, table 3) on the flora of freshly manured soil, previous to the present series of experiments, offers striking evidence that the non-spore-forraing organisms predominate in such soil. During his work the manuied soil vv^as plated at inter\"als extending over a period of six months. On the third day it was found that almost 99 per cent of the entire flora was composed of non-spore-forming organ- isms. The present work on the flora of freshly manured soil includes experiments designed to verify these earlier results. The method of procedure in these later experiments was practically the same throughout, except for a few differences in the treatment of samples. Soil was mixed with fresh horse manure or fresh cow manure and, with the exception of the first experiment, the manured soil was then divided into two portions, one of which was placed in an open pot and one in a flask plugged with cotton. In the first experiment the manured soil was kept only in open pots. The moisture content of the pots was controlled somewhat by frequent additions of water to replace that lost by evaporation, but no allowance was_ made for this in the flasks. Platings were made at frequent intervals at the first of each experiment and at longer interv^als as the experiment proceeded. The experiment recorded in Table I was carried on under conditions exactly analogous to those under which Conn did his previous work, and its purpose was the verification of that work. The experiments recorded in Tables II and III were also carried on under similar conditions except that soil mixed with cow manure was used as well as that mixed with horse manure and samples were plated from plugged flasks as well as from open pots. Table I. — Plate counts of the microorganisms in manured soil in open pots. Experi- ment I [Counts indicate numbers of colonies per gram of soil] Time since adding manure to soil. Total count. Days. Average . 60, 000, 000 80, 000, 000 125,000,000 235,000,001, 45,000,000 43.000,000 35,000,000 50,000,000 55,000,000 85.000,000 45,000.000 95,000,000 18,000,000 20,000.000 Actinomycetes. Plate count. 4,000,000 6,000.000 5,000,000 6,000,000 5,000,000 4,000,000 12,000,000 13,000,000 12, 500,000 8, 500,000 13,000,000 8,500,000 5, 500, 000 5.000.000 Per- centage of total flora. II. I 9-3 34-3 36. o 32.6 TO. O 29.0 8.9 30-5 25- o 16.3 Non-spore formers. Plate count. 56,000,000 74.000,000 116,000,000 220,500,000 38,500,000 36,500,000 23,000,000 35,000,000 42, 500,000 76, 500,000 32,000,000 83,500,000 II, 500,000 15,000,000 Per centage of total flora. 92.5 92.5 92. 7 93- 5 85- S 85.0 65. 7 70. o 77-4 90. o 71. o 88.0 63- 8 75.0 81.6 Spore formers. Plate coimt None. None. 4.000,000 9, 500,000 1,500,000 2, 500,000 None. 2,000,000 None. None. None. 3,000,000 1,000,000 None. Per- centage of total flora. 3-3 3-9 3-4 S- 7 3- I 5- 7 Mar. 24, 1919 AmmonificaHon of Manure in Soil 319 Table II. — Plate counts of the microorganisms in manured soil in open pots and closed flasks. Experiment II cow MANURE [Counts indicate number of colonies per gram of soil] Total count. Actinomycetes. Non-spore formers. Spore formers. Treatment and time since adding ma- nure to soil. Plate count. Per- centage of total flora. Plate count. Per- centage of total flora. Plate count. Per- centage of total flora. Open pots: 164,000,000 93,000,000 98, 500. 000 127,000,000 55,000,000 490, 000, 000 251,000,000 52,000.000 412,000,000 67,000.000 395,000,000 245,000,000 9,500.000 10, 500,000 10, 000. 000 8, 500.000 7,500,000 35,000.000 40,000,000 7,000,000 190, 000. 000 16,000,000 160. 000, 000 50. 000, 000 5.6 11. 2 12.6 70 14-4 6.8 15.6 12.4 46. I 23- I 40. 6 22.6 155,000,000 82, 500,000 86,000,000 117,000,000 46. 000, 000 450.000,000 208.000,000 45,000,000 214.000,000 51,000,000 23 2 , 000, 000 186, 000, 000 94-4 88.6 87.2 92.2 83.8 92.0 83.2 86.7 52.1 76.2 58.7 76. 0 None. Trace. 2,000,000 1,000,000 1,000,000 5,000,000 3,000,000 500,000 7, 500, 000 500.000 3,500.000 3,500,000 0.0 2 days . 2 g 4 days 1.8 I. 2 I. 2 •9 1.8 ■7 •7 1.4 18.2 80.9 ■9 Closed flasks: 27,500,000 64, 000, 000 67,000,000 81,000,000 101,000,000 42,000,000 65,500,000 34,500,000 330,000,000 323,000.000 330,000,000 350,000,000 3,750,000 6,000.000 6,000,000 15, 000,000 15,000,000 12, 500.000 12,000. 000 9.000,000 290.000.000 300,000.000 225.000.000 190,000,000 18.3 10. 0 9.0 18.6 14. 6 28. 5 18.3 24-3 88.3 34-4 68.8 22, 500,000 57, 500.000 59,000.000 64, 000. 000 84,000,000 29,000,000 53,000,000 24, 000, 000 38,000,000 21,000,000 99, 000, 000 158,000,000 81.7 90.0 88.2 79.0 83.1 69. 2 81.0 69- S II. I 65.0 30.0 None. None. 1,800,000 2,000.000 2, 000, 000 1,000. 000 Trace. 1,500.000 2,000,000 2,000,000 6,000,000 0.0 2 8 2.4 2-3 2-3 ■7 6 2 8 days 9 days 6 6 15 days I. 3 6 32-3 66.1 I. 6 HORSE MANURE Open pots: 300, 000, 000 109,000,000 157,000,000 907, 500,000 775,000,000 625,000,000 67, 500,000 480, 000, 000 740, 000, 000 376,000,000 295,000,000 1, 705,000,000 9, 500, 000 18.000.000 12,000,000 22,500,000 56,000,000 65,000,000 6,000,000 85,000,000 115,000,000 100,000,000 95,000,000 700, 000, 000 3-4 18.4 7-4 2.8 7-S 10.4 8.9 17-7 15-6 26.6 32.2 41.0 390, 000, 000 87,000.000 144,000,000 880, 000, 000 713,000,000 556,000.000 61,000,000 392,000,000 622,000,000 273.000.000 198, 000, 000 1,000,000,000 96.6 79-8 91.7 97.0 91.6 89.0 90.4 81.7 84.0 72.6 67.2 S8.7 None. 2,000,000 1,500,000 2,000.000 7,500,000 4,000,000 500. 000 3,000,000 3,000,000 3,000,000 2, 000, 000 5,000,000 1 s 3 days 9 9 6 8 days 7 6 9 days 4 R 6 •^ 16.1 83.3 .6 Closed flasks: 63,000,000 88, 000, 000 82,000,000 78, 000, 000 336,000,000 810,000,000 75, 500,000 58, soo, 000 375,000,000 169.000,000 1,380,000,000 1,045,000,000 11,000,000 7,000,000 8,000,000 12,000,000 17-5 7.8 9.9 14.6 ^8 6, 52, 000,000 81.000,000 73,000.000 66. 000, 000 234,000,000 481,000,000 50,000,000 27,000,000 123,000,000 162,000,000 575, 000, 000 244,000,000 82. 5 92.0 89.2 84- S 70.8 S9-5 66.3 46. 2 32.8 96. 0 41.7 23-3 None. Trace. 750,000 750, 000 2,000,000 3,500,000 1,000,000 1 , 500, 000 2, 000, 000 2,000,000 5,000,000 I , 000, 000 I 3 I 3 days 9 9 6 4 3 S 6 8 days 24,000,000 30, 000, 000 250, 000. 000 65,000,000 800, 000, 000 800, 000, 000 32.4 SI- 3 66.6 2.8 57-9 76.6 4 Average 33-8 65-4 .8 320 Journal of Agricultural Research voi. xvi. No. Table III. — Plate counts of the microorganisms in manured soil in open pots and closed flasks. Experiment III COW MANURE [Counts indicate number of colonies per gram of soil] Total count. Actinomycetes. Non-spore formers. Spore formers. Treatment and time since adding ma- nure to soil. Plate count. Per- centage of total flora. Plate count. Per- centage of total flora. Plate count. Per- centage of total flora. Open pots: 58,000,000 73,250,000 497,500,000 93,000,000 102,500,000 36, 250,000 43 , 000, 000 17,500,000 40,000,000 96, 000, 000 187,000,000 75,000,000 64,000,000 8,000,000 5,000,000 15,000,000 12,000,000 12, 500,000 5,000,000 5,000,000 2. 500,000 12,000,000 15,000,000 20,000,000 20,000,000 10,000,000 13-8 6.7 3-0 12.9 12. 2 13-8 II. 7 14-3 30.0 IS- 7 10.7 26.7 14.4 48,000,000 68,000.000 482,500,000 81,000,000 go, 000, 000 31,250,000 38,000,000 15,000,000 28,000,000 81,000,000 167,000,000 55,000,000 52,500,000 82.7 93- I 97.0 87.1 87.8 86.2 88.3 8s-7 70. 0 84-3 893 73-3 83- 3 2,000,000 Trace. None. None. None. None. None. None. None. None. None. None. 1,500,000 3-S .0 . 0 .0 .0 .0 .0 .0 .0 .0 .0 a- 3 8 days 9 days i8 days Average 14-3 8s-3 •4 Closed flasks: 51,000,000 42,000.000 295,000,000 68, 000, 000 44,000,000 27,000,000 ^ 45,500,000 31,000,000 36,500,000 49, 500, 000 53,000,000 125,000,000 46,000,000 8,000,000 6,000,000 10,000,000 30,000,000 11,000,000 10,000,000 23,000,000 20,000,000 17,500,000 20,000,000 25,000.000 65,000,000 25,000,000 15-7 14-3 3-4 44.1 25- 5 37-6 50. 6 64.6 48.0 40.4 47-3 52- 0 54-4 41, 500,000 35.5°o,ooo 285,000,000 38,000,000 33,000,000 17,000,000 22, 500,000 11,000,000 19,000,000 39,500,000 28, 000, 000 60,000,000 21,000,000 81.4 84-5 96.6 55- 9 74-5 62.4 49.4 35-4 52.0 59-6 52.8 48.0 45-6 1,500,000 500,000 None. None. None. None. None. None. None. None. None. None. None. 3.9 I. 3 .0 .0 .0 .0 .0 ■ 0 3 days 7 days 8 days .0 .0 .0 .0 Average 38.3 61.4 •3 HORSE MANURE Open pots: 110,000,000 120,000,000 195,000,000 150,000.000 122,000,000 95,000,000 300,000,000 30,500,000 59,000,000 48,000,000 242,500,000 130,000,000 12,000,000 7,500,000 10,000,000 15,000,000 13,000,000 15,000,000 35,000,000 5,000,000 5,000,000 20,000,000 40, 500, 000 35,000,000 10. 9 6.3 5.0 10. 0 9-9 5-3 II. 7 16. 4 8.5 41-7 16. I 36. 9 97,000,000 111,000,000 185,000,000 135,000,000 1 10, 000, 000 80,000,000 365,000,000 25, 500,000 54,000,000 28,000,000 302,000,000 95,000,000 87.000,000 88 750,000 1,500,000 None. None. None. None. None. None. None. None. None. None. 500,000 I-O I. 3 .0 .0 • 0 .0 • 0 ■ 0 .0 .0 .0 .0 • s 92.5 95-0 90. 0 90. I 94- 7 88.3 83.6 91.5 58.3 83- 9 73-1 74.0 8 dyas Average 1 14.9 84.9 • 3 Closed flasks: 40,000,000 182,500,000 83,500,000 75,000,000 33,000,000 515,000,000 605,000.000 62, 500,000 780, 000, 000 67,000,000 43,500,000 100,000,000 40,000,000 4,500.000 8,000,000 10,000,000 22,500,000 13,000,000 375,000,000 150,000.000 32,000,000 600,000,000 50,000,000 27, 500,000 65,000,000 30,000,000 11. 2 4-3 12. I 30.0 39-4 72.9 24.8 51.2 76.9 74-6 63. 2 34,500,000 171,500,000 72,500,000 52, 500,000 20,000,000 140,000,000 455,000,000 30, 500,000 180,000,000 17,000.000 86 ■> 1,000,000 1,000,000 None. None. None. None. None. None. None. None. None. None. None. 2. S 1-7 .0 . 0 .0 2 days 94 87 70 60 27 75 48 23 25 36 35 0 9 0 6 I 2 8 I 4 8 0 7 days 8 days . 0 .0 .0 . 0 . 0 .0 .0 65.0 35,000,000 75.0 10,000,000 Average 46. 3 •3 Mar. 24, 1919 Ammonification of Manure in Soil 321 A survey of the results in Tables I, II, and III shows that the number of non-spore formers in the open pots of manured soil increased rapidly for the first few days (see Table I, column 5). In every instance the highest percentage of this group of organisms was reached within the first week after the addition of the manure and this maximum point was never less than 92.5 per cent, while in some cases it reached 97 per cent. The results in the flasks were much more erratic and, while the per- centage of non-spore formers often ran above 90 per cent of the flora, the lines of increase and decrease were not so well marked as they were in the pot experiments. This was undoubtedly due to the fact that con- ditions of aeration and moisture content were decidedly abnormal. By summarizing the three tables it was found that the non-spore-forming organisms averaged 74. i per cent of the entire flora in both the pots and flasks; the Actinomycetes 25.1 per cent, and the spore formers only 0.8 per cent. Table IV. — Results of the isolation of organisms from manured soil Source, Open pots. Closed flasks. * Sam- ple No. Kind of ma- nure. Time since add- ing ma- nure. Total coimt. Num- ber of organ- isms iso- lated. Num- ber which grew on agar.a Num- ber of non- spore form- ers. Num- ber of spore form- ers. Total count. Num- ber of organ- isms iso- lated. Num- ber which grew on agar.o Num- ber of non- spore form- ers. Num- ber of spore form- ers. I Horse. ...do... ...do... ...do... ...do... Cow... ...do... ...do... ...do... Horse. Cow... Horse. Cow... Horse. Cow... Days. 6 10 27 22 27 9 II 23 3 8 8 24 24 8 8 251,000,000 89,000,000 73,000,000 37,000,000 30, 000, 000 231,000,000 34 20 25 20 27 37 32 20 24 20 27 36 30 20 21 19 27 35 2 0 3 I 0 I 317,000,000 95,000,000 42,000,000 78,000,000 15,000,000 3 3 3 4 S 6 7 8 9 10 II 12 13 17 S 17 5 17 S 0 0 136,000,000 143,500,000 67,000,000 75.500,000 65,500,000 1,045,000,000 350,000,000 605,000,000 45.500,000 41 35 14 9 4 15 9 5 8 41 34 14 9 4 15 9 4 8 39 34 14 9 4 15 8 4 8 174,000,000 98, 500, 000 67, 500,000 251,000,000 I, 705,000,000 245,000,000 300,000,000 43 , 000, 000 35 15 7 8 13 II 3 lo 33 14 6 8 II 10 3 10 33 14 6 8 II 10 3 10 0 0 0 0 0 0 0 0 0 0 0 0 0 I 0 0 26s 254 247 7 162 160 157 3 o Plain nutrient agar was used as medium for isolated colonies. While the data accumulated in the preceding experiments indicated very strongly that the non-spore formers were the predominating organ- isms in the manured soil, yet the proof was not absolute, because it was based entirely upon the appearance of the colonies upon the plates. Those colonies which possessed the characteristic spreading or filamentous appearance of the typical spore formers were classified accordingly; but some non-spore formers may thus have been inadvertently recorded as spore formers, or some spore formers as non-spore formers. A number of isolations were made, therefore, from the plates poured during the series of experiments described above. All colonies which looked like 106546°--19 2 322 Journal of Agricultural Research voi. xvi, No. u spore formers were transferred to agar slants, as were a representative number of colonies of other types. About 97 per cent of these cultures grew and were subsequently examined under the microscope for spore formation. Table IV, which contains the recorded data from this experiment, shows that of the 254 organisms from the open pots which grew after isolation, only 2.8 per cent were spore formers, and of the 160 organisms from the flasks which grew after isolation only 1.8 per cent were spore formers. And this despite the fact that a special effort was made to include all those colonies whose appearance suggested that they might be spore-forming organisms. GROWTH OF PSEUDOMONAS FLUORESCENS AND PS. CAUDATUS COM- PARED WITH THE GROWTH OF BACILLUS CEREUS IN STERILIZED MANURED SOILS The organisms selected for the rest of the work were two non-spore formers, Pseudomonas fluorescens (Fliigge) Migula and Ps. caudatus (Wright) Conn, and a spore former, Bacillus cereus Frankland.^ The first two organisms are described in the second section of this article, and were chosen because of the frequency of their occurrence in manured soil. B. cereus, according to Conn (9) and Laubach and Rice (27), is a typical spore former occurring in soil and was selected for the purpose of comparison with these organisms. SOIL INOCULATED WITH THE THREE ORGANISMS SEPARATELY In the series of experiments designed to show the relative rates of growth of the three organisms under investigation, manured soil was sterilized in flasks and inoculated with pure cultures in suspensions of carefully determined strength. Samples from each series were plated at similar intervals, and an effort was made to make all results comparable. Microscopic counts were made of all the samples of soil inoculated with B. cereus in order to determine the number of vegetative cells actually present in the soil. As Ps. fluorescens and Ps. caudatus, on the other hand, grow well on plates, form no spores, and show no tendency to clump, a microscopic count of them was not so important as the plate count, and since they are so small as to be easily overlooked under the microscope a microscopic count proved even less accurate than the plate count. The results as set forth in Table VI, Experiment I, show that Ps. caudatus increased from a 13,300,000 plate count on the day of inoculation to a 1,720,000,000 count seven days later, or an increase of 132 times the original inoculation. The initial plate count of Ps. fluorescens was 4,390,000, and on the seventh day the count was 475,- 000,000, an increase of no times the original count. B. cereus, on the ' As identified by Conn. This organism agrees with the descriptions of Chester (2, p. 27S), and I.aiuence and Ford (25, p. 284-2S7). Mar. 24, 1919 Ainmonification of Manure in Soil 323 other hand, showed a much lower rate of increase and developed from an initial plate count of 1,800,000 (see Table V, Experiment I) to a count of 15,000,000 on the seventh day, an increase of 8.3 times the original inoculation. The microscopic coimt on the seventh day showed 36,000,000 vegetative cells, an increase of 20 times the initial count. In the four series Ps. caudatus showed its greatest increase in Experi- ment III (Table IV), on the ninth day, when it showed a count 913 times greater than its initial count ; Ps. fluorescens registered its greatest increase in Experiment IV on the fourteenth day, when it showed a count 530 times higher than the original count; and B. cereus made its greatest increase in Experiment IV (Table V) on the sixteenth day, showing a count 29 times higher than the original. Table V. — Multiplication of B. ceretis inoculated into sterile manured soil [Count indicates the numbers per gram of soil] Experiment No. and time since inoculation. Experiment 1: odays 3 days sdays 7 days Experiment II:'' odays sdays 8 days 17 days 23 days Experiment III: odays 2 days 4 days 6 days Sdays Experiment IV: o days 2 days 4 days 6 days Sdays 11 days 12 days 14 days i6days Plate count. ,000,000 , 750,000 ,000,000 600.000 000,000 000,000 000,000 000,000 000,000 500,000 000,000 000,000 000,000 500,000 000,000 000,000 000, 000 000. 000 000,000 Microscopic count. Groups. Vegetative cells. Spores. 5,000,000 6,000,000 36, 000, 000 2,000.000 2, 500,000 2, 500,000 2,500,000 2,000,000 1, 500,000 2, 500,000 1, 500.000 2,000.000 2. 500,000 2. 500,000 3,000,000 19,000,000 6,000,000 17,000,000 18,000,000 II, 500,000 16,000,000 14,500,000 5.000, 000 12,000, 000 11. 500.000 9,000.000 13,000,000 12,000,000 IS, soo. 000 21,000,000 Individuals. Vegetative cells. " 1,800,000 9,000,000 3,000,000 50, 000, 000 1 1,300,000 <• 116,000 13,000,000 10, 500,000 15,500.000 7, 500, 000 o 103,000 II, 500, 000 13, 500,000 9, 500.000 4. 500, 000 6,000, 000 7,000,000 6,500,000 6, soo, 000 Spores. None. 23.000,000 6,000,000 18, 000, 000 None. None. 33. 000. 000 16. 500,000 19,000,000 18,000,000 None. 30,000,000 24, 500, 000 27, 500,000 27,000,000 19,000,000 21,000, 000 26, 500, 000 32,000,000 <» Computed from the number of organisms in the infusion used for inoculation. & No microscopic count made. SOIL INOCULATED WITH A MIXTURE OF THE THREE ORGANISMS Table VII gives the results of placing the three organisms in compe- tition one with another by inoculating flasks of sterile manured soil with all of them together. Infusions were made from fresh cultures of each organism and the strength of these infusions determined by the micro- scopic method. After infusions of the proper strength had been obtained, equal amounts of each were thoroughly mixed and i cc. of the mixture 324 Journal of Agricultural Research Vol. XVI, No. 12 added to the flasks containing 150 gms. of sterile manured soil. These flasks were then incubated at room temperature and plates and smears made from them at regular intervals. In Experiment I, Table VII, the ratio between the numbers of organisms of Ps. fluorescens, Ps. caudatus ,a.nd B. cereus was i to i to i ; in Experiment II the ratio was i to 8 to 33; in Experiment III the ratio was i to 7 to 33. Although B. cereus was as abundant as the other organisms in Experiment' I and was much more numerous than they in the later experiments, it failed to appear upon any of the plates poured. The non-spore-forming organism.s multiplied very rapidly, and in Experiment II, Ps. fluorescens developed from an initial count of 30,000 to a maximum count of 560,000,000 on the third day, an increase of over 18,500 times its count at the time of inoculation. In the same experiment Ps. caudatus developed from an initial count of 180,000 to a maximum count of 1,190,000,000 on the seventh day, an increase of 6,600 times its count at the time of inoculation. The micro- scopic examination of the smears made during this series of experiments showed that the vegetative cells of B. cereus rapidly decreased in numbers and in a few days the organism could be identified only in the spore form, while the non-spore formers, especially Ps. caudatus, showed a steady increase in numbers for several days. Table Y I. —Multiplication of non-spore formers inoculated into sterile manured soil [Count indicates numbers per gram of soil] Pseudomonas fluorescens. Pseudomonas caudatus. Experiment No. and time since inoculation. Plate count. Microscopic count. Plate count. Microscopic count. Groups. Individuals. Groups. Individuals. Experiment I: 04,390,000 204.000,000 152, 000, 000 325,000,000 13,300,000 260, 000, 000 185,000,000 475,000,000 197,000,000 133,000,000 259,000,000 665,000,000 4.800,000,000 1, 720,000,000 492,000,000 1,340,000,000 1,105,000,000 1,614,000,000 1,254,000,000 Experiment 11:6 145,000,000 160,000,000 200,000,000 210,000,000 300,000,000 2, 700,000,000 I, 500,000,000 No count. 4.000,000,000 Experiment III: 1 1,600,000 88,000,000 188,000,000 247,000,000 328,000,000 a 1,000,000 140, 000, 000 260,000,000 315,000,000 350,000,000 79,000,0009 17^,000.000 236.000.000 310,000,000 1,440,000,000 1,340,000,000 1,460,000,000 1. 190,000,000 728,000,000 760, 000, 000 736,000,000 794, 000, 000 784,000,000 802,000,000 Experiment IV:<> 102,000,000 390,000,000 395,000,000 470,000,000 375,000,000 460,000,000 530,000,000 470,000,000 114,000,000 181,000,000 194,000,000 220,500,000 205,000,000 220,000,000 269,000,000 171,500,000 o Computed from the number of organisms in the infusion used for inoculation. b No microscopic count made. Mar. 24, 1919 Ammonification of Manure in Soil 325 Table VII. — Plate counts of the microorganisms in sterile manured soil inoculated -with a mixture of one spore former and two non-spore formers [Count indicates number of colonies per gram of soil] Ex periment I. i Experiment II. Experiment III Time j since inocu- lation. Pseudo- Pseiido- Pseudo- Pseudo- Pseudo- Pseudc^ p„,ill^. tnonas vionas ■mmtas tnonas monas monas cmidatus. ftuorescens. catidatus. fluorescens. caudatus. fluorescens. Days. 0 0- 250,000 0 100,000 a 180,000 0 30.000 1 50,000 « 1,380,000 0 240, 000 afiSo, 000 I None. 194,000,000 10,000,000 23,000.000 600,000 1,300,000 2 None. 188,000,000 89, 000, 000 106,000,000 No test. No test. 3 None. 260,000,000 297,000,000 560,000,000 24,000,000 6,900,000 4 80,000,000 240, 000, 000 490,000,000 150,000,000] 129.000.000 15,000,000 s 480,000,00c 150,000,000 575,000,000 150.000,000: No test. No test. 6 390,000.000 220,000,000 . 460,000,000 180,000,000 . No test. No test. 7 570,000,000 135,000,000 0 1, 190,000,000 250,000,000 0 No test. No test. OF AGRICULTURAI/ RESEARCH CONTENTS AND INDEX OF VOLUME XVI PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS ^'^ASHINOTON, D. C. WASHINQTON S OOVERNMENT PRINTINQ OPFIOB I IMt EDITORIAL COMMITTEE OF THE UNITED STATES DEPARTMENT OF AGRICULTURE AND THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS FOR THE DEPARTMENT FOR THE ASSOCIATION KARL F. KELLERMAN, Chairman H. P. ARMSBY Physiologist and Associate Chief, Bureau of Plant Industry EDWIN W. ALLEN Chief, Office of Experitnent Stations CHARLES L. MARLATT Eniomoloist and Assistant Chief, Bureau of Entomology Director, Institute of Animal Nutrition, The Pennsylvania State College J. G. LIPMAN Director, New Jersey Agricultural Expert" ment Station, Rutgers College W. A. RILEY Entomologist and Chief, Division of Ento- mology and Economic Zoology, Agricul- tural Experiment Station of (he University of Minnesota All correspondence regarding articles from the Department of Agriculture should be addressed to Karl F. Kellerman, Journal of AgriciUtural Research, Washington, D, C. All correspondence regarding articles from State Experiment Stations should be addressed to H. P. Armsby, Institute of Animal Nutrition, State College, Pa. INDEX Add — Page cx)mplete, effect on ash content of spinach . . 13-25 hydrocyanic, in Andropogon sorghum .... 175-181 hypochlorous, effect on germination of Irish potatoes 298 phosphate, effect on — ash content of spinach 15-25 nitrification of acid soils 27-42 prussic, in Andropogon sorghum 175-181 Acid soils, effect of lime, fertilizer, crops, and manure on nitrification 27-42 Acidity in wheat, determination with the hydrogen electrode 1-13 Actinomycetes in manured soil 318-322 Adelocera sp., natural enemy of Chrysobothris tranqueharica 161 Aerobic bacteria, effect of lime, fertilizer, and crops on number in acid soils 33-42 Age- effect on butter-fat content of cow's milk. , 85-102 of host, effect on morphology of uredinio- spores of Puccinia graminis 73-77 of rust fungus, effect on morphology of urediniospores of Puccinia graminis 74-77 A gropyron — smiihii, host of Puccinia graminis tritici. . . . 52-77 tenerum, host of Puccinia graminis tritici. . . 52-77 A grostis alba, host of Puccinia graminis agrostis 63-77 Alfalfa. See Medicago sativa. Alopecurns pratensis, host of — Puccinia graminis agrostis 63-77 Puccinia graminis avenae 58-77 Alway, Frederick J., and Neller, Joseph R. (paper): A Field Study of the Influence of Organic Matter upon the Water-Holding Capacity of a Silt-Loam Soil 263-278 Amaranthus retroflexus, host of Pegomyia calyptrata 230 Amino nitrogen in wheat, method for deter- mining 7-13 Ammonification of Manure in Soil (paper). . 313-350 Ammonium — nitrate, effect on nitrification of acid soils. . 27-42 phosphate, effect on nitrification of acid soils 27-42 sulphate, effect on nitrification of acid soils. 27-42 Anaerobic bacteria, effect of lime, fertilizer, and crops on number in acid soils 33-42 Andropogon sorghum, cyanogenesis 1 75-181 Anemia, relation to Ascaris infection 253-258 Angular-leafspot. See Bacterium angulatum. Angular-Leafspot of Tobacco, an Undescribed Bacterial Disease (paper) 219-22S Anthomyids, leaf-miners on dock 229-244 Apple-Scald (paper) 195-217 Apple-scald — cause 216 effect of — carbon dioxid 202-203 delayed storage 213-214 gas absorbents 214-216 Apple-scald — Continued, effect of — Continued. Page humidity 200-201 intermittent aeration 206-209 oxygen 203-204 temperature 199-200 ventilation 205-206, 212-213 A scaris — conocephala, action in intestines 256 lumhricoides. blood-destroying substance in 253-258 Ash Absorption by Spinach from Concen- trated Soil Solutions (paper) 15-25 Ash in corn grown with barium and strontium compounds 192 Atanycolus — labena, natural enemy of Chrysobothris tran- queharica 161 rugosiventris, natural enemy of Chrysobothris tranqueharica . . . 161 A triplex patula, host of Pegomyia calyptrata . . 230 Australian pine. See Casuarina equiseti folia. Avena sativa — host of Puccinia graminis 43-77 tolerance of barium and strontium com- compounds 185-187 Bacillus — caudatus. See Pseudomonas caudatus. cereus, growth in sterilized manured soil . . 322-325 fluorescens — liquefaciens. See Pseudomonas fluores- cens. liquefaciens minutissimus. See Pseudomo- nas fluorescens. nonliquefaciens. See Pseudomonas putida. putidus. See Pseudomonas putida. maculicola, cause of tobacco v/hitespot 224 megatherium, relation to ammonification of soil 315 mycoides, re\a.X.ionto ammonification of soils 315 pyocyaneum. See Pseudomonas fluorescens . subtilis, relation to ammonification of soils . 315 vulgatus, relation to ammonification of soils. 31s Bacteria — nitrogen-fixing, effect of salts 107-135 niunber of fluorescent in soil 337-338 spore-forming and non-spore-forming in freshly manured soil 3 18-322 Bacterial contents of acid soils as affected by lime, fertilizer, crops, and moisture 27-42 Bacterium — angulatum, n. sp. — cultural characters 226-227 description of — organism 226-227 spots 221-222 inoculation experiments 222-224 resemblance to — blackrust 224 wildfire 224 whitespot 224 temperature relations 226 (35x) 352 Journal of Agricultural Research Vol. XVI Bacterium — Continued. Page denitrofluorescens, nonliquefying denitri- fier 33S.338 pseudozoogloeae, cause of tobacco blackrust . 224 pyocyaneum, identical with Bacterium fluo- rescens 334 termo, denitrifier 335 tobacum, cause of tobacco wildfire 224-225 Barium — carbonate, effect on plant growth 183-194 chlorid, effect on plant growth 183-194 compounds, effect on plant growth 183-194 nitrate, effect on plant growth 1S3-194 sulphate — effect on plant growth 183-194 in corn grown with barium and strontium compounds 193 Barley. See Hordeum spp. Basic complete mixtiue, efi'ect on ash content of spinach 1 5-23 Basic slag, effect on ash content of spinach. . 15-25 Beet. See Beta vulgaris. Beta vulgaris — host of Pcgomyia calyptrata 230 var. cicla, host of Pegotnyia calyptrata 230 Biologic forms of Puccinia graminis 103-105 Black, Otis F., et al. (paper): Ash Absorp- tion by Spinach from Concentrated Soil Solutions 15-25 Blood-Destroying Substance in Ascaris lum- bricoides, A (paper) 253-258 Blood, dried, effect on nitric nitrogen in soil. 125-135 Bordeaux mixture, effect on germination of Irish potatoes 298 Bright, J. W., and Conn, H. J. (paper): Am.- monification of Manure in Soil 313-35° Bromus tectorum, host of Puccinia graminis avenae 58-77 Brooks, Charles, Cooley, J. S., and Fisher, D. F. (paper): Apple-Scald 195-217 Bruchophngus funebris, pest on Medicago sativa and Trifulium pratcnse 165-174 Buckner, G. Davis, Nollau, E. H., Wilkins, R. H., and Kastle, Joseph H. (paper): Effect of Certain Grain Rations on the Growth of the White Leghorn Chick 305-312 Butter fat, variation in cow's milk 80-102 Calcium — carbonate — effect on — ash content of spinach 15-25 nitric nitrogen in soil 114-135 nitrification of acid soils 27-42 in spinach 1 7-25 chlorid, effect on nitric nitrogen in soil. . 114-135 nitrate, effect on nitric nitrogen in soil. . . 114-135 sulphate, effect on nitric nitrogen in soil. 114-135 Calcium-magnesium ratio in ash of spinach. . 24-25 Carbon dioxid, effect on apple-scald 202-203 Carbon-dioxid-surviving bacteria in acid soils. 27-42 Carbonate — barium, effect on plant growth 183-194 calcium — effect on — ash content of spinach 15-25 nitric nitrogen in soil 114-135 nitrification of acid soils 27-42 in spinach 17-25 Carbonate — Continued. Page ferric, effect on nitric nitrogen in soil 118-135 lithum, effect on germination of Irish pota- toes 298 manganous, effect on nitric nitrogen in soil .116-135 of magnesium, effect on nitric nitrogen in soil 115-135 sodium, effect on nitric nitrogen in soil . . . 110-135 strontium, effect on growth of wheat .... 190-191 Carter, E. G., et al. (paper); Influence of Salts on the Nitric-Nitrogen Accumulation in the Soil 107-135 Casuarina eguisetifolia, host of Chrysobothris tranquebarica 155-164 Cercospora nicotianae, cause of frogeye in tobacco 219-220 Charcoal, effect on germination of Irish pota- toes 298 Chard, Swiss. See Beta vulgaris var cicla. Chenopodium album, host of Pegomyia calyp- trata 230 Chicks, White Leghorn, effect of grain rations on growth 305-3" Chlorid— barium, effect on plant growth 183-194 calcium, effect on nitric nitrogen in soil. . 114-135 ferric, effect on nitric nitrogen in soil 118-135 magnesium, effect on nitric nitrogen in soil 115-135 manganous, effect on nitric nitrogen in soil 116-135 mercuric, effect on germination of Irish potatoes 298 potassium — effect on nitric nitrogen in soil 112-135 in spinach 17-25 sodium — effect on — ash content of spinach 15-2S nitric nitrogen in soil 110-133 in spinach 17-25 Chlorin, effect on nitric nitrogen in soil 121 Chrysobothris tranquebarica — adult 157-158 control 161-163 egg 158 natural enemies 161 larva 158-160 parasite of Casuarina eguisetifolia 155-164 pupa 160 seasonal history 156-157, 160-161 Chrysolite, solubility of litne, magnesia, and potash in 259-261 Clover, effect on nitrification of acid soil 27-42 Colantes auratus, natural enemy of Chryso- bothris iranguebarica i6i Complete acid in spinach 18-25 Conn, H. J., and Bright, J. W. (paper): Am- monification of Manure in Soil 313-350 Conner, S. D., and Noyes, H. A. (paper): Nitrates, Nitrification, and Bacterial Con- tents of Five Typical Acid Soils as Affected by Lime, Fertilizer, Crops, and Moisture. . 27-42 Cooley, J. S., et al. (paper): Apple-Scald.. . 195-217 Com. See Zea mays. Cowpeas. See Vigna sinensis. Crops, effect on nitrification of acid soils 27-42 CyanogensisinAndropogonsorghum(paper). 175-181 Jan. 6-Mar. 31, 1919 Index 353 Page Dacnusa scaptomyzae, parasite of Pegomyia calyptraia 237-J38 Dactylis glomerata, host of Puccinia graminis avenae 58-77 Determination of Acidity and Titrable Nitro- gen in Wheat with the Hydrogen Electrode (paper) 1-13 Diboikriocepkalus latus, toxity 255 Dock. See Rumex crispus. Dowell, C. T. (paper): Cyanogensis in Andro- pogon sorghum 175-181 Drouth, effect on morphology of Puccinia graminis 44, 70-71 Durum wheat. See Triticum durum. Ecological factors, effect on morphology of urediniospores of Pucania graminis 43~77 Effect of Certain Compounds of Barium and Strontium on the Growth of Plants (pa- per) 183-194 Effect of Certain Ecological Factors on the Morphology of the Uredmiospores of Puc- cinia graminis (paper) 43-77 Effect of Certain Grain Rations on the Growth of the White Leghorn Chick (paper) 305-312 Einkom wheat. See Triticum mcmococcutn. Electrode, hydrogen, determination of acidity and titrable nitrogen in wheat 1-13 Elymus — canadensis, host of Puccinia graminis secalis . 56-77 condensatus host of Puccinia graminis trilici- compacii 54-77 glaucus, host of Puccinia graminis tritici- com,pacti 54-77 robuslus, host of Puccinia gramints secalis. . 56-77 virginicus, host of Puccinia graminis secalis. . 56-77 Emmer wheat. See Triticum dicoccum. Epidote, solubility of lime, magnesia, and potash in 259-261 Eucklaena mexicana, host of Physoderma zeae-maydis 142 Euielus bruchophagi — adult 171-173 hibernation 171 larva 171 parasite of Bruchophagus funehris 171-172 pupa 171 Ferric — carbonate, effect on nitric nitrogen in soil . 118-135 chlorid, effect on nitric nitrogen in soil. . . 118-135 nitrate, effect on nitric nitrogen in soil. . 118-135 oxid in com grown with barium and stron- tium compounds 192 sulphate, effect on nitric nitrogen in soil. 118-135 Fertilizer, effect on nitrification of add soils. . 27-42 Field Study of the Influence of Organic Mat- ter upon the Water-Holding Capacity of a Silt- Loam Soil, A (paper) 263-278 Fisher, D. F., et al. (paper): Apple-Scald. 195-217 Flicker. See Colaptes auratus. Flour, wheat, determination of acidity and titrable nitrogen 1-13 Fluorescent organisms, characteristics 338-342 " Frogeye," caused by Cercospora nicotianae 219-220 Fromme, F. D., and Murray, T. J. (paper): Angular-Leafspot of Tobacco, an Unde- scribed Bacterial Disease 219-228 Page Frost, S. W. (paper): Two Species of Pego- myia Mining the Leaves of Dock 229-244 Fusarium-blight of potatoes — causal fungus 286-287 control 297-298 description 280-281 inoculation experiments 281-283 mode of infection 284 resistance of host 295-296 seed-piece infection 284-286 soil conditions 296-297 Fusariimi-B light of Potatoes under Irrigation (paper) 279-303 Fusarium oiysporum — cause of Fusarivun-bhght 279-303 effect on germination of Irish potatoes 298 Gardiner, R. F. (paper): SolubiUty of Lime. Magnesia, and Potash in Such Minerals as Epidote, Chrysohte, and Muscovite, Espe- cially in Regard to Soil Relationships ... 259 «6i Glucoside in Andropogon sorghum 175-181 Goldthorpe, H. C.,et al. (paper): Influence of Salts on the Nitric-Nitrogen Accumulation in the Soil 107-135 Gowen, John W. (paper): Variations and Mode of Secretion of Milk Solids 79-103 Greaves, J. E., Carter. E. G., and Gold- thorpe, H. C. (paper): Influence of Salts on the Nitric-Nitrogen Accumulation in the Soil 107-135 Hemolytic nature of fluid of Ascaris 253-258 Holcus lanatus, host of Puccinia graminis agrostis 63-77 Hordeum — jubatum, host of Puccinia graminis 51-77 pusillum, host of Puccinia graminis avenae. 58-77 spp., host of Puccinia graminis 43-77 vulgare, host of Puccinia graminis trilici- compacli 54-77 Humidity, effect on — apple-scald 200-201 morphology of Puccinia graminis 70-77 Hydrocyanic acid. See Acid, hydrocyanic. Hydrogen electrode, determination of acidity and titrable nitrogen in wheat 1-13 Hydrogen-ion concentration of wheat, method for determining 1-13 Hypochlorous acid. See Acid, hypochlo- rous. Hystrii patula, host of Puccinia graminis seca- lis 56-77 Influence of Foreign Pollen on the Develop- ment of Vanilla Fruits (paper) 245-252 Influence of Salts on the Nitric-Nitrogen Ac- cumulation in the Soil (paper) 107-135 Injury to Casuarina Trees in Southern Flor- ida by the Mangrove Borer (paper) 155-164 Irish potato. See Solanum tuberosum. Iron salts, effect on nitric nitrogen in soil. . 118-135 Iron sulphate, effect on germination of Irish potatoes 298 Irrigation, effect on fusarium-blight of Sola- num tuberosum 279-303 Kastle, Joseph H., et al. (paper): Effect of Certain Grain Rations on the Growth of the White Leghorn Chick 305-312 354 Journal of Agricultural Research Vol. XVI Page Kelley, James W., et al. (paper): Ash Ab- sorption by Spinach from Concentrated Soil Solutions iS-aS Leach, J. G., et al. (paper): New Biologic Forms of Puccinia graminis 103-105 Levine, M. N., and Stakman, E. C. (paper): Effect of Certain Ecological Factors on the Morphology of the Urediniospores of Puc- diiia graminis 43-77 Levine, M. N., et al. (paper): New Biologic Forms of Puccinia graminis 103-105 Life-History Observations on Four Recently Described Parasites of Bruchophagus fune- bris (paper) 165-174 Light, effect on morphology of Puccinia graminis 44, 71-73 Lime — ■ effect on nitrification of add soils 27-43 in com grown with barium and strontium compounds 193 solubiUty in epidote, chrysolite, and mus- covite 259-361 Liodontomerus — perplexus — adult 169 hibernation 166 larva 167-168 oviposition 167 parasite of Brtichophagus funebris 165-169 pupa 168-169 secundus — adult 170 hibernation 170 larva 170 psiTzsitc ol Bruchoha^us fund>Tis 169-170 pupa 170 Lithum carbonate, effect on germination of Irish potatoes 298 LoliuTti temulenlum, host of Puccinia gram- inis avenae 58-77 MacMiUan, H.G. (paper) : Fusarium-Blight of Potatoes under Irrigation 279^303 Magnesia — in corn grown with barium and strontium compounds 193 solubility in epidote, chrysolite, and mus- covite 259-261 Magnesium — carbonate, effect on nitric nitrogen in soil 11S-135 chlorid, effect on nitric nitrogen of salts. . 115-135 nitrate, effect on nitric nitrogen in soil. . . 115-135 sulphate, effect on nitric nitrogen in soil. 115-135 Magnesium-calcium ratio in ash of spinach.. . 24-25 Malus sylvestris, scald 195-317 Manganous — carbonate, effect on nitric nitrogen in soil 116-135 chlorid, effect on nitric nitrogen in soil. . 116-135 nitrate, effect on nitric nitrogen in soil. . . 1 16-135 sulphate, effect on nitric nitrogen in soil. . 116-135 Mangrove, red. See Rhizophora mangle. McClelland, T. B. (paper): Influence of Foreign Pollen on the Development of Vanilla Fruits 245-353 Page McHargue, J. S. (paper): Effect of Certain Compounds of Barium and Strontium on the Growth of Plants 183-194 Manure, effect on ash content of spinach. . . . 15-35 Medicago sativa, attacked by Brucho- phagus funebris 165-174 Melanerpes eryihrocephalus, natural enemy of Chrysobothris tranquebarica 161 Mercuric chlorid, effect on germination of Irish potatoes 298 Milk, cow's — diurnal variation constituents 92-102 factors affecting composition 84-102 solids, variation and mode of secretion. . . . 79-102 variation of butter fat and solids-not-fat. . . 80-102 Moisture — effect on — morphology of Puccinia graminis 44, 70-71 nitrification of acid soils 27-42 soil — at different levels -268-269 effect of organic matter 271-277 effect on productivity 274-277 Monas termo. See Bacterium iermo. Morphology of urediniospores of Puccinia graminis 43-77 Murray, T. J,, and Fromme, F. D. (paper): Angular-Leafspot of Tobacco, an Undes- cribed Bacterial Disease 219-228 Muscovite, solubility of lime, magnesia, and potash in 259-261 Mustard oil, effect on germination of Irish fwtatoes 298 Nabtsferus, parasite Pegomyia calypiraia 238 Neller, Joseph R., and Alway, Frederick J. (paper): A Field Study of the Influence of Organic Matter upon the Water-Holding Capacity of a Silt- Loam Soil 263-278 New Biologic Forms of Puccinia graminis (paper) 103-105 Nicotiana tabacum, angular-leafspot 219-228 Nicotine sulphate, effect on germination of Irish potatoes 298 Nitrate- ammonium, effect on nitrification of acid soils 27-42 barium, effect on plant growth 183-194' calcium, effect of nitric nitrogen in soil. . . 114-135 ferric, effect on nitric nitrogen in soil 118-135 magnesium, effect on nitric nitrogen in soil 115-135 manganous, effect on nitric nitrogen in soil 116-135 potassium, effect on nitric nitrogen in soil . 112-135 sodium — effect on — ash content of spinach 15-25 nitric nitrogen in soil 110-135 in spinach 17-25 strontium, effect on growth of winter wheat 188-189 Nitrates, Nitrification, and Bacterial Con- tents of Five Typical Acid Soils as Affected by Lime, Fertilizer, Crops, and Moisture (paper) 27-42 Jan. 6-Mar. 31, 1919 Index 355 Page Nitric nitrogen in soil, effect on salts 107-135 Nitrification of acid soils 27-42 Nitrogen — amino, in wheat, method for determining. . 7-13 in plants grown with barium and strontiiun compounds 189,191,193 in wheat, determination with the hydro- gen electrode 1-13 ^ ratio to organic matter in surface soil 266-269 Nitrogen-fixing bacteria, effect of salts 107-135 Nollau, E. H.,etal. (paper): Effect of Certain Rations on the Growth of the White Leg- horn Chick 305-312 Nonglucoside in Andropogon sorghum 175-181 Noyes, H. A., and Conner, S. D. (paper): Ni- trates, Nitrification, and Bacterial Contents of Five Typical Acid Soils as Affected by Lime, Fertilizer, Crops, and Moisture 27-42 Oats. See Avena sativa. Oil, mustard, effect on germination of Irish potatoes 298 Onion juice, effect on germination of Irish potatoes 298 Optus guebecensis, parasite of Pegomyia calyptraia 237-238 Organic matter — effect on water-holding capacity of silt- loam soil 263-278 ratio to nitrogen in surface soil 266-269 Oxid, ferric, in corn grown with barium and strontium compounds 192 Oxygen, effect on apple-scald 203-204 Oxyhemoglobin in fluid of Ascaris 253-258 Pegomyia — affinis — distribution 240 life history 240-243 hosts 240 leaf-miner on dock 229-244 calyptrata — distribution 230 hosts 230 leaf-miner on dock 229-244 life history 230-237 natural enemies 237-238 hyoscyami, host of Nobis ferus 238 Spp., leaf-miners on dock 229-244 vicina. See Pegomyia affinis. Pentoxid, phosphorus, in corn grown with barium and strontium compounds 193 Pkleum pralense, host of Puccinia gramimis avenae 58-77 Phosphate — acid — effect on — ash content of spinach 15-25 nitrification of acid soils 27-42 in spinach 18-25 ammonium, effect on nitrification of acid soils 27-42 Phosphorus — effect on nitrification of acid soils 27-42 in plants grown with bariiun and strontium compounds 189, 191 in wheat extracts, method for determining. 10-13 pentoxid in corn grown with barium and Strontium compounds 193 Page Physoderma Disease of Corn (paper) 137-154 Physoderma zeae-maydis — control 152-153 description of causal organism 144 dissemination 150-152 germination of sporangia 145-146 hosts 142 overwintering of sporangia 149 symptoms 142-143 Pine, Australian. See Casuarina equisetifolia. Plant growth, effect of barium and strontiiun compounds 183-194 Pollen, foreign, influence on development of vanilla fruits 245-252 Potash— in corn grown with barium and strontium compounds iga solubility in epidote, chrysolite, and musco- vite 259-261 Potash-silica ratio in ash of spinach 18-25 Potash-soda ratio in ash of spinach 23-25 Potassium — carbonate, effect on nitric nitrogen in soil. 112-135 chlorid — effect on nitric nitrogen in soil 112-135 in spinach 17-25 effect on nitrification of acid soils 27-42 in plants grown with barium and strontium comjxjunds 189, 191 nitrate, effect on nitric nitrogen in soil. . . 112-135 sulphate, effect on — ash content of silage 15-25 nitric nitrogen in soil 112-135 Potato, Irish. See Solanum tuberosum. Potato-wilt of potatoes tmder irrigation 279-303 Protein — in plants grown with barium and strontium compounds 189-191, 193 Prussic acid. See Acid, prussic. Pseudomonas — aeruginosa — causing blue pus 334 denitrifier 335-337 caudalus — action on milk 346 action on sugars and glycerin 344-345 ammonifi cation of soil 329-332 chromogenesis 343-344 diastatic action on starch 346 growth in sterilized manured soil 322-325 liquefaction of gelatin 344 morphology 342-343 physiology 344 production of indol 346 reduction of nitrate 345 relation to oxygen 344 fiuorescens — action on milk 342 action on sugars and glycerin 339-340 ammonification of soil 329-332, 339 cultural characters 338-339 diastatic action on starch 341 growth in sterilized manured soil 322-325 liquefaction of gelatin 339 morphology 338 reduction of nitrate 340-341 relation to oxygen 339 356 Journal of Agricultural Research voi. xvi Pseudomonas — Continued. Page putida, nonliquefier 337 pyocyanea. See Pseudomonas aeruginosa. Puccinia — andropogonis, change of morphology with change of host 44-77 ellisiana, change of morphology with change of host 44-77 graminis — morphology 43-77 new biologic forms 103-105 agrostis, morphology of urediniospores. . . . 48-77 avenae, morphology of urediniospores 46-77 phleipraiensis, morphology of uredinio- spores 48-77 secalis, morphology of urediniospores. . . . 48-77 tritici, morphology of urediniospores 45-77 triiici-compacti, morphology of uredinio- spores 48-77 triticina, variation in biologic forms 105 Radio-active material, effect on tolerance of barium carbonate and strontium carbonate by wheat 187-188 Rations, cooked and uncooked, effect on White Leghorn chicks 306-309 Red clover. See Trifolium pratense. Rhizophora mangle, host of Chrysobothris tran- quebarica 15 5-164 Rumex — acetosa, host of Pegomyia calyptrata 230 crispus, host of Pegom,yia spp 329-244 oblusifolius, host of Pegomyia spp 229-244 Rye. See Secale ccreale. Salts- effect on nitric nitrogen in soil 107-135 toxity to nitrifying organisms 129-135 Schwartz, Benjamin (paper): A Blood-De- stroying Substance in Ascaris lumbricoides 253-258 Secale cerealc, host of Puccinia graminis 43-77 Silica in com grown with barium and stron- tium compounds 192 Silica-potash ratio in ash of spinach 22-25 Silt-loam soil, elTect of organic matter on water-holding capacity 263-278 Slag, basic, effect on ash content of spinach. . 15-25 Snyder, Thomas E. (paper): Injury to Casua- rina Trees in Southern Florida by the Man- grove Borer 155-164 Soda in com grown with barium and stron- tium compounds 193 Soda-potash ratio in ash of spinach 23-25 Sodium — carbonate, effect on nitric nitrogen in soil 110-135 chlorid — effect on — ash content of spinach 15-25 nitric nitrogen in soil 1 10-135 in spinach 17-25 nitrate — effect on — ash content of spinach 15-25 nitric nitrogen in soil 110-135 sodium content of spinach 15-25 in spinach 17-25 sulphate — effect on — ash content of spinach 15-25 nitric nitrogen in soil 110-135 in spinach 1 7-25 Soil — Page acid, effect of lime, fertilizer, crops, and moisture on nitrification 27-42 effect of salts on nitric-nitrogen accumula- tion 107-135 freshly manured, relative numbers of non- spore-forming and spore-forming bac- teria 318-322 silt-loam, effect on organic matter on water- holding capacity 263-278 Soil moisture, effect on morphology Puccinia graminis 71-77 Soil solutions, ash absorption of spinach from. 15-25 Soja max, tolerance of barium and strontium compounds 193-194 Solamimtuberosum,,iusariiaa-hhght 279-303 Solubility of Linie, Magnesia, and Potash in Such Minerals as Epidote, Chrysolite, and Muscovite, Especially in Regard to Soil Relationships (paper) 259-261 Soybean. See Soja max. Species, new 219-228 Spina cea oleracea — ash absorption from concentrated soil solu- tions 15-25 host of Pegomyia calyptrata 230 Spinach. See Spinacia oleracea. Spring wheat. See Triticum aestivum. Stakman, E. C, and Levine, M. N. (paper): Effect of Certain Ecological Factors on the Morphology of the Urediniospores of Puc- cinia graminis 43-77 Stakman, E. C, Levine, M. N., and Leach, J. G. (paper): New Biologic Forms of Puc- cinia graminis 103-105 Stemrust. See Puccinia graminis. Strontiiun — carbonate, effect on growth of wheat 190-191 compounds, effect on plant growth 183-194 nitrate, effect on growth of winter wheat. . 188-189 sulphate in corn grown with barium and strontium compounds 193 Sulphate — ammonium, effect on nitrification of acid soils 27-42 barium — effect on plant growth 183-194 in corn grown with barium and strontium compounds 193 calcium, effect on nitric nitrogen in soil. . . 114-135 ferric, effect on nitric nitrogen in soil 118-135 iron, effect on germination of Irish potatoes . 298 magnesium, effect on nitric nitrogen in soil 115-135 manganous, effect on nitric nitrogen in soil 1 16- 135 nicotine, effect on germination of Irish pota- toes 298 potassium, effect on — ash content of spinach 15-25 nitric nitrogen in soil 112-135 sodium — effect on — ash content of spinach 15-25 nitric nitrate in soil 110-135 in spinach 17-25 strontium, in com grown with barium and strontium compounds 193 Jan. 6-Mar. 31, 1919 Index 357 Page Swanson, C. O., and Tague, E. L. (paper): Determination of Acidity and Titrable Nitrogen in Wheat with the Hydrogen Electrode 1-13 Swiss chard. See Beta vulgaris var. cicla. Tague, E. L., and Swanson, C. O. (paper): Determination of Acidity and Titrable Nitrogen in Wheat with the Hydrogen , Electrode 1-13 Temperature, effect on — angular-leafspot of tobacco 226 apple-scald 199-200 morphology of Puccinia graminis 44-68-69 Tenebroides sp., natural enemy of Ckrysobolh- ris tranquebarica 161 Teosinte. See Euchlaena mexicana. Timeromicrus maculatus — economic importance 173 larva 173 hibernation 172 parasite of Bruchophagus futiebris 173-174 pupa 173 relative proportion of sexes 1 73 Tisdale, W. H. (paper): Physoderma Disease of Com 137-154 Tobacco. See Nicoliana tabacum. Toxity of salts to nitrifying organisms 129-135 Trichogramma minututn, parasite of Pegomyia calyptrala 238 TrifoUum praiense, attacked by Bruchophagus funebris 165-174 Triticum — aesthum — determination of acidity and titrable nitrogen 1-13 effect on nitrification of acid soil 27-42 host of Puccinia graminis 43-77 tolerance of barium carbonate and stron- tium carbonate 187-188, 190-191 Trj/icM»t— Continued. Page compaclum, host of Puccinia graminis tritici-compacii 54-77 dicoccum, — host of Puccinia gram.inis tritici-compacii. 54-77 resistant to Puccinia graminis 103-105 durum, resistant to Puccinia graminis. . . . 103-105 monococcum, resistant to Puccinia gram,inis 103-10S spp . , host of Puccinia graminis 103-105 True, Rodney H., Black, Otis F, and Kelley, James W. (paper): Ash Absorption by Spinachfrom Concentrated Soil Solutions. . . 15-25 Two Species of Pegomyia Mining the Leaves of Dock (paper) 229-244 Urbahns, Theodore D. (paper): Life History Observations on Four Recently Described Parasites of Bruchophagus funebris 165-174 Urediniospores of Puccinia graminis, mor- phology 43-77 Vanilla planifolia, influence of foreign pollen on development of fruits 245-252 Vanillon; effect of pollen on fruits of Vanilla planifolia 245-252 Variations and Mode of Secretion of Milk Solids (paper) 79-102 Ventilation, effect on apple-scald. . 205-206, 212-213 Vigna sinensis, tolerance of barium carbonate 184-185 Wheat. See Triticum aestivum. Wilkins, R. H. et al. (paper): Effect of Cer- tain Grain Rations on the Growth of the White Leghorn Chick 305-312 Winter wheat. See Triticum aestivum. Woodpecker, red-headed. See Melanerpes erythroce phalus . Zea mays — Physoderma disease 137-154 tolerance of barium and strontium com- pounds 191-193 o New York Botanical Garden Librai