Bacteria in Relation to Plant Diseases BY ERWIN F. SMITH In charge of Laboratory of Plant Pathology, Bureau of Plant Industry, U. S. Department of Agriculture VOLUME THREE VASCULAR DISEASES (CONTINUED) WASHINGTON, D. C. Published by the Carnegie Institution of Washington 1914 CARNEGIE INSTITUTION OF WASHINGTON Publication No. 27, Vol. three Copies of wer» first iMued AUG 4 1914 }1YZ PRESS OF GIBSON BROTHERS. WASHINGTON, D. C. CONTENTS. Page. Vascular Diseases — Continued. Cobb's Disease of Sugar-cane 3 Definition 3 Host-plants 3 Geographical Distribution 3 History 3 Signs of the Disease 15 Etiology 2i Inoculations 21 Series I, 1903 21 Series II, 1903 27 Series III, 1903 27 Series IV, 1903 3° Series V, 1904 31 Series VI, 1905 33 Series VII, 1906 46 General Remarks on the Inoculations 46 Acid Canes Less Susceptible 47 Conditions Favoring Spread of the Disease . 48 The Red Stain 49 Relation to Sereh 52 Morbid Anatomy 52 The Parasite — Bacterium Vdscularum RGS. ... 54 Resume of Salient Characters 66 Treatment 68 Table of Susceptible and Resistant Varieties. 69 Pecuniary Losses 70 Literature 71 The Sereh Disease of Sugar-cane 72 Literature 79 Top-rot of Sugar-cane in Java 81 Literature 84 The Humid Gangrene or Polvillo of Sugar- cane in Argentina 85 Literature 88 Stewart's Disease of Sweet-corn (maize) ... 89 Definition 89 Host-plants 89 Geographical Distribution 89 Signs of the Disease 89 Etiology 91 Field Observations in 1902 94 Inoculation Experiments 96 Series I and II, 1902 96 Series III to VII, 1902 no Series VIII and IX, 1903 1 1 1 Series X, 1905 112 Infected Seed as a Means of Distribution 114 Field Observations of 1903 114 Page. .Stewart's Disease of Sweet Corn (maize) — Continued. Etiology — Continued. Susceptible and Resistant Varieties 117 Series XI to XV, 1903 121 Summary 123 Experiments of 1908 124 Series XVI to XIX, 1908 125 General Remarks on Series XVI to XIX 127 Series XX to XXIV, 1908 128 Series XXV, 1912 129 Morbid Anatomy 130 The Parasite — Bacterium stewarli KFS 132 Resume of Salient Characters 144 Treatment 145 Pecuniary Losses 146 History 147 Literature 147 Smith's Disease of Amaranths Bacterium ama- ranthi EFS 148 Literature 150 Van Breda de Haan's Wilt of Peanut 151 Literature 153 English Disease of Mangolds and Sugar- beets 154 Literature 154 Blight of Young Rose Shoots 154 Bacteria in Vessels of Flax Stems 154 Rathay's Disease of Orchard-Grass — Aplano- bacler rathayt EFS 155 Description of Disease 155 Comment 157 Personal Observations 157 Literature 160 The Grand Rapids Tomato Disease — Aplano- bacter michiganense EFS 161 Preliminary Studies 161 Notes on Cultural Characters 162 Additional Observations, Inoculation Experi- ments, and Inferences 163 Resume of Salient Characters 164 Literature 165 Spieckermann's German Potato Disease 166 Literature 167 Vascular Diseases of Banana 168 1. Earle's Jamaican Disease 168 2. Smith's Cuban Disease 168 3. Rorer's Trinidad Disease — Bacillus musae Rr. 170 4. The Panama Disease 172 5. South American Diseases 173 6. Old World Diseases 173 Literature 173 111 IV CONTENTS. Pagb. Vascular Diseases — Continued. Brown Rot of Solanaceae 174 Definition 1 74 Host-plants 1 74 Geographical Distribution 1 75 Signs of the Disease 1 75 Etiology 1 78 Synopsis of Inoculations 182 Morbid Anatomy 190 The Parasite — Bacterium solanacearum EFS. . . 193 Resume of Salient Characters 199 Treatment 201 Pecuniary Losses 202 History 203 Appendix 207 The New Zealand Disease 207 The Australian Disease 207 The Dutch East Indian Disease 209 The Malay States Disease 211 The Mysore Ring- Disease 212 The Ceylon Disease 214 The African Disease 214 The Russian Disease 214 The French Disease 214 The Italian Disease 215 The German Ring- Disease 215 The English Disease 216 Malkoff's Disease of Sesamum 216 Literature 218 Wii.t-Diseases of Tobacco 220 The Dutch East Indian Disease 220 History and Etiology 220 Pace. Vascular Diseases — Continued. Wilt-Diseases of Tobacco — Continued The North American Disease 227 History 227 Morphology and Cultural Characters of the parasite 231 Etiology 233 Experiments of 1907 233 Experiments of 1908 233 Experiments of 1909 236 Losses; Treatment 237 The Japanese Disease 238 Honing's Sumatran Studies 244 American Studies of the Sumatran Organism . 263 The Russian Disease 265 The Italian Disease 266 The French Disease 266 The Indian Disease 267 The South African Disease 268 Literature 270 Schuster's German Potato Disease — Bacterium xanthochlorum Schuster 272 Observations by Schuster 272 Experiments in Washington 274 Literature 276 The Dutch Disease of Wallflower 277 Literature 277 The Italian Disease of Stock — Bacterium mal- thiolae Briosi and Pavarino 277 Literature 279 The Brazilian Disease of Manihot — Bacillus maniholus Arthaud Berthet 280 Literature 281 Addendum 282 LIST OF ILLUSTRATIONS. PLATES. Plate. Faces Page. i. Cross-sections of Diseased Cane-stems, showing Ooze of Bacterium vascularum and Red Stain 16 2, 3. Cane Plants Destroyed by Bad. vascularum as a Result of Needle-priek Inoculations on Leaves 2 to 3 feet from the Nodes 24, 26 4. Longitudinal Section, showing Top-rot of Cane Due to the Introduction of Bad. vascularum by Needle- pricks 4 months earlier on Leaf-blades lower down 28 5. Bacterial Ooze from Vessels of Cane-plant No. 40, Inoculated with Bad. vascularum (second time through plant) 30 6. Two Plants of Sweet Maize Dried Up and Destroyed by Bad. stewarti (natural infection) 90 7. First Cases of Stewart's Disease Obtained from Pure-culture Inoculations — early signs 98 8. Sweet-corn Plants Drying Up from Stewart's Disease, 66 days after Inoculation — late signs 98 9. Longitudinal Radial Sections through Stems of Inoculated Sweet-corn, showing Nodal Stain, Pockets in Parenchyma, and Yellow Bundles (dark lines in the photograph) 100 io. Base of Inoculated Plants XLII and XLVI or XLVII Affected by Stewart's Disease, showing Nodal Stain, Absence of Stain in Cortex, Externally Sound Roots, etc 102 1 1. Cross-sections of Inoculated Cane-Stems, showing Red Stain and Yellow Ooze. Also Cultures of Bad. vascularum, Bad. stewarti, and Aplanobader michiganense (colored) 138 1 \a Cross section of two spikelets of Dadylis glomerata attacked by Rathay's disease 156 1 ib. Poured plate agar cultures of Aplanobader rdthayi 160 12 to 15. Grand Rapids Tomato Disease. Hot-house Plants Inoculated from Pure Cultures 162 16. Inoculated Chinese Banana Plants Destroyed by Bacillus musae 170 17. One clump from Plate 16; Inoculated Parent Plant Killed, Sucker Diseased 170 18. Inoculated Moko Plantain Destroyed by Bacillus musae 1 70 19. Red Banana Inoculated with Bacillus musae, showing Characteristic Break of Leaves at Top of Plant 170 20. (1) Cross-sections of Diseased Fruit-stalks of Banana. (2) Longitudinal Section of a Sucker, show- ing Rhizome and Base of Pseudo-Stem with Infected (Black) Bundles 170 21. (1) Section of Root-stock of Red Banana, showing Blackened Vascular Bundles. (2) .Same Rhizome as fig. 1, but here there are Bacterial Cavities as well as Blackened Bundles 1 70 22. (1) Pseudo-Stem of Chinese Banana in Cross-section, showing Bacterial Destruction of Inner Leaves. (2) Same as fig. 1, but a Cross-section at the Base of the Pseudo-Stem, showing many Infected Bundles (black specks) 17° 23. Potatoes Attacked by Bad. solanacearum; also Cultures of this Organism, of Bacillus solanisaprus, of Bacillus phytophthorus, and of Aplanobader rathayi (colored) 174 24. Solatium tuberosum, Solatium commersoni, and Lycopersicum esculentum, attacked by Bad. solanacearum . 176 25. Potato-plants from a Field in Virginia, showing Effect of Bad. solanacearum 176 26. 27. Two Plates illustrating the Progressive Effect of Bad. solanacearum (South Carolina strain) upon the Tomato (left-hand shoot infected by needle-pricks) 178 28. Tomato-plant which did not Wilt on being Inoculated with Bad. solanacearum, although other Signs developed 178 29. Another Tomato-plant Somewhat Resistant to Bad. solanacearum 178 30. District of Columbia Potato from which was Isolated a very virulent Strain of Bad. solanacearum 180 31. Potato Shoots Inoculated with a Pure-culture of Bad. solanacearum from Plant shown in Plate 30 188 32. Effect of Liquid Air on Bad. solanacearum. Checks at left 198 33. Inoculated Tobacco-plant No. 8 (1905), showing the Granville Tobacco Wilt 228 34. Leaf from an Inoculated Tobacco-plant, showing irregular Action of the Wilt 228 35. Diseased Leaf from Inoculated Tobacco-plant No. 34 (1905) 228 36. Group of 9 Inoculated Tobacco-plants Destroyed by the North Carolina Tobacco Wilt 228 37. Petri-dish Agar Poured-plates of the North Carolina Tobacco-wilt Organism 228 38. Collapsed Tobacco-plant from the Second Set of Inoculations No. 31 (1905) 230 39. Surface and Sectional Views of Stems of Inoculated Tobacco-plants 35 and 36 (1905) 230 40. Results Similar to Plate 39 Obtained by Inoculating Tobacco with Bad. solanacearum from a Potato . 230 41. Tobacco Wilt; also enlarged spikelets of orchard grass showing Rathay's disease (colored) 232 42. Brown Stripes on Tobacco-stems Attacked by North Carolina Tobacco Wilt (1908) 234 43. The Jimpson Weed, Datura stramonium Attacked by the Granville Tobacco Wilt (rgo8) 236 44. Tobacco wilt (action of Medan organism on cream) and for comparison Aplanobader michiganense, Bacterium campeslre, and Bacterium mori 264 45. Loss of virulence in Bacterium solanacearum 282 (Total Plates 47.) v VI LIST OF ILLUSTRATIONS. TEXT FIGURES. Fig. Page, i . Oozing of Bad. vascularum from Cut Surface of Australian Sugar-cane 5 2. Cobb's Figures of Bad. vascularum 6 3. Cane-plants Dwarfed by Bad. vascularum 9 4. Bacterial Cavity under Terminal Bud in Sugar-cane Attacked by Bad. vascularum 15 5. Radial Longitudinal Section through Leaf-sheath of Sugar-cane No. 4, Attacked by Bad. vascularum . 15 6. Detail from fig. 5, showing how the Bacteria reach the Surface through Stomata 16 7. Cross-section of fig. 5, including two vascular bundles 17 8. Inoculated Cane No. 9, showing Sidewise Pushing of the Terminal Bud 17 9. Cane Stripped of Leaves to show Dwarfing and Twisting of Terminal Bud (zigzag growth) due to Bad. vascularum 18 10. Pushing of Buds in Inoculated Purple Cane No. 38 18 1 1 . Longitudinal Section Showing Pushing of Buds and Red Nodal Stain in Stem of Sugar-cane Attacked by Bad. vascularum 19 12, 13. Microscopic Appearance of Reddened Vascular Bundles of Sugar-cane Attacked by Bad. vascularum. 19, 20 14. Pure Culture of Bad. vascularum Plated in 1902 from Australian Cane 20 15. Nanism of Inoculated Sugar-cane 22 16. Leaf of Sugar-cane showing White Stripes due to Bad. vascularum 23 17. A Pure Poured Plate Culture of Bacterium vascularum from inoculated Cane No. 9 25 18. Contents of Red Slime from Interior of Sugar-cane 9 months after Inoculating with Bad. vascularum 45 19, 20. Cross-sections of Vascular Bundles of Sugar-cane, showing Parts Occupied by Cobb's Organism. 49,50 2 1 . Early Stage in Occupation of a Bundle, Cane-plant No. 6 51 22, 23. Longitudinal Sections through Inoculated Cane No. 6, showing Destruction of the Vascular Bundles. 52, 53 24. Longitudinal Section through Inoculated and slightly Diseased Purple Cane No. 30, showing Red Bundles as Dark Lines 54 25. Cross-section of Vascular Bundle of Sugar-cane Occupied by Bad. vascularum (the dark portions surrounding the yellow slime were a deep red color) 55 26, 27. Bacteria (highly magnified) from Different Parts of Inoculated Cane-plant No. 6 55 28, 29. Flagellate Rods of Bad. vascularum 55, 56 30. Rods of Bad. vascularum from a Young Culture on Nutrient Agar 56 31. A Pure Culture, Agar Poured Plate, of Bad. vascularum from Plant No. 11 57 32. Streak Cultures of Bad. vascularum on Potato Agar 58 33. Magnified Colony of Bad. vascularum on Glucose Gelatin 59 34. Growth of Bad. vascularum on Cane-juice-gelatin with and without Peptone 59 35. Tear-drop Formation of Bad. vascularum on Slant Gelatin with Cane-juice 60 36. Streak Cultures of Bad. vascularum on Potato 60 37. Bacterium vascularum in a Petri-dish Agar Poured-plate (one-half exposed to sunlight) 66 38. Crystals from an Agar Streak Culture of Bad. vascularum 67 39, 40, 41. Cross-sections of Maize Stems, showing Bad. stewarti Oozing from Vascular Bundles. Figs. 39, 40, from plants shown in pi. 6 (natural infections), fig. 41, from artificially infected plants... 91,92,95 42. Radial Longitudinal Section through Basal Part of Stem of Diseased Sweet Corn from Long Island . . 98 43, 44. Yellow Bacteria from Smear Preparations Made from Stems of Long Island Sweet Corn 98 45, 46. Periphery of Portion of Corn-cob in Cross-section, showing Bacteria under and in Kernels .... 1 14. t 15 47. Section through a very immature Corn Kernel and its Supporting Tissues (the area occupied by Bad. stewarii in the base of the Kernel is drawn in solid black 1 116 48. A Detail from fig. 47, showing Bacterial Cavities and the Occlusion of Vessels 119 49. Cross-section of the Infected Corn-husk shown in Vol. II, fig. 14, Inoculations of 1902 119 50. A Detail from fig. 49, in the Vicinity of *, i. e., from another section in the series, showing stomatal Ooze of Bacteria 119 51. Surface View of an Infected Corn-husk showing Bad. stewarti oozing from a Stoma. (For appear- ance in section of a similar stoma, see fig. 50) 120 52. Longitudinal Tangential Section through a Diseased Corn-husk, showing Substomatic Chambers filled by Bad. stewarti 120 53. Bacterially Spotted Husks, same as Vol. II, fig. 14, but from a Naturally Infected Plant, i. e., one grown in the hot-house in 1908 from diseased seed 121 54. Occlusion of Single Spiral Vessel by Bad. stewarti on the Periphery of a Cob (longitudinal view). . 129 55. Cross-section of Stem of Sweet Corn from Long Island, showing one Bundle with Occlusion of a Single Vessel by Bad. stewarti 129 56. Section similar to fig. 55, but from Sweet Corn Inoculated by the Writer in Washington, in 1902, and showing entire Xylem Portion of the Bundle Occupied by the Bacteria (phloem free) 130 57. Section through lower part of a Diseased Sweet Corn Kernel, showing Relation of Parts, etc 130 LIST OF ILLUSTRATIONS. VII Fig. Page. 58. Details from base of sweet corn kernels showing bacterial occupation 131 59. (a) Bad. stewarti from Margin of a Hanging Drop after 52 Hours in Bouillon at 3o°C. (b) Bait. slewarli from Yellow Slime on Potato after 3 Days at 300 C 131 60. Flagella of Bad. stewarti 1 33 61. (a) Gelatin-stab-culture of Bad. stewarti (non-liquefying), (b) Gelatin-stab-cultures of Liquefying Non-pathogenic Yellow Organism from Surface of Corn Kernels 133 62. Agar-streak-cultures of Bad. stewarti 134 63. Top of an Old Agar-stab-culture of Baet. stewarti enlarged to show Crystals in Surface Growth 134 64. Bad. stewarti on Potato (at left), with Bad. campestre for Comparison 135 64a. Cirri of Bad. Stewarti oozing from stomata (pedicel of ear) 147 65. Bacterium amaranthi occupying Intercellular Spaces in the Pith of an Amaranth 148 66. Interior of a Pith-cell Occupied by Bad. amaranthi 149 67. Xylem Vessels of Amaranth Occupied by Bad. amaranthi 149 670. Photomicrograph of Bad. amaranthi in the tissues 150 68. Cross-section of Root of Javan Peanut, showing Bacteria Confined to the Bundles 151 69. Cross-section of Stem of Peanut from Java, showing Bacteria Restricted to the Xylem Part 151 70. A Detail from fig, 69 152 71. Cross-section of Petiole of Peanut, showing Bacteria in Vessels and Formation of Cavities 152 71a. Danish Orchard grass attacked by Rathay's disease 158 716. Austrian Orchard grass showing Rathay's disease: stem pushing out sidewise from leaf-sheaths . . 158 71c. Rathay's disease: cross-sections of culms, showing bacterial layer between leaf and stem, etc. . . 158 ■jid. Cross-section of upper part of Dactylis showing dense bacterial growth between the leaves 159 71c Small portion of margin of leaf, showing multiplication of bacteria in intercellular spaces 159 71/. Detail of Dactylis leaf more highly magnified, from a section similar to that shown in fig. 71c . 159 7ig. Capsules of Aplanobader rathayi 159 71/;. Rods of A planobacicr rathayi adhering to the surface of a leaf of Dactylis 159 72. Cross-section of a Tomato Stem from Epidermis to Pith, showing effect of the Grand Rapids Dis- ease— Cavities in Phloem 163 73. A Detail from Margin of Bacterial Cavity in the Outer Phloem shown in fig. 72 163 74. Longitudinal Section through Phloem of Tomato Stem (fig. 72, inner cavity) showing Bacterial Masses wedging apart the Sieve-tubes 1 64 75. Grand Rapids Tomato Disease, showing Avidity of the Bacteria for Meristematic Tissues 164 75a. Streaks of ( 1 ) Apt. rathayi and (2) Apl. michiganense on steamed potato 165 76. Cross-section of Banana Fruit-stalk, showing Bacillus musae oozing from the Cut Bundles 169 77. Rorer's Bacterial Disease of Banana — Leaf Inoculated in Washington 169 78. Behavior of Bacillus musae in Gelatin-stab-cultures (compare with fig. 1 1 1) 1 70 79. Longitudinal Section of Leaf-stalk of Banana, showing some Parenchyma Cells Occupied by the Bacteria and Others Free 171 80. (a) Longitudinal Section through Bundle in Leaf-stalk of Banana, showing Parasitism of Fusarium cubense. (b) Longitudinal Section through Banana Bundle Attacked by Bacillus musae 172 81. Bad. solanacearum occupying Vessels in the Root of an Egg-plant from Porto Rico 175 82. Section through External Part of Infected Tuber of Inoculated Potato Plant No. 14 (1896), showing Bacterial Infection Restricted to the Vascular System (V) 1 76 83. Cross-section of Small Potato-tuber Infected naturally by Bad. solanacearum, showing Partial Dis- appearance of Starch and Restriction of Bacteria to Vicinity of the Vascular System 1 76 84. Cross-section of Underground Tuber-bearing Stem of Potato Infected in the Vascular Ring by Bad. solanacearum, the Movement of the Bacterial Growth being toward the Tuber 177 85. A Detail from fig. S4, showing the Character of the Bacteria occupying the Vessels 177 86. Cross-section of .Stem of Inoculated Potato Plant No. 14 (1895), showing Vascular Bundle Occupied by Bad. solanacearum, and Cavities in the outer Phloem 1 80 S7. Transmission of Bacterial Brown Rot to Potato by Insects 181 88. Cross-section of Bundle of Datura stramonium from Plant 23 (1895), showing Bacterial Cavity. . 182 89. Cross-section of Stem of Solatium nigrum from Inoculated Plant No. 34 (1896) showing Bacterial Occlusion of the Bundles 183 90. A Detail from One of the Bundles in fig. 89, showing the Bacteria more Clearly 184 91. Radial Longitudinal Section of Stem of Nightshade (fig. 89), showing Destruction of Bundle 184 92. A Detail from fig. 91, showing bacterial Occlusion of a Pitted Vessel 185 93. Cross-section of Stem of Physalis from Plant No. 57 (1896), showing Bacteria in Bundles. . 185 94. A Radial Longitudinal Section from the same Physalis Stem as fig. 93 186 95. Cross-section of Petiole of Potato from Inoculated Plant No. 23 (1901) 1S7 96. Tomato Plant No. 10 (1903), 7 Days after Inoculation with Bad. solanacearum 188 VIII UST OF ILLUSTRATIONS. Fig. Page. 97. Tomato Plant Xo. 64 (1904), 3 Days after Introducing Bad. solanacearum into the Stem by means of a few Needle-pricks (control on left) 1 89 98. Cross-section of Stem of Tomato No. 26 (1895), showing Bacterial Cavities and Incipient Roots 190 99. A Detail from one of the Cavities in fig. 98 1 90 100. Longitudinal Section of Stem of Inoculated Potato No. 5 (1896), showing Bad. solanacearum Restricted to a Single Vessel 191 101. A Detail from the Same Stem as fig. 86 192 102. Cross-section of Stem from Potato No. 5 (1896), 10 Days after Inoculation 193 103. A Detail from fig. 102 194 104. A Detail from fig. 82 of Inoculated Plant No. 14 (1896), showing Starch-grains Undestroyed by the Bacteria in which they lie Embedded 1 95 105. The same as fig. 104, but by Polarized Light 196 106. A Detail from fig. 83 at x, showing Starch Undestroyed in Cells Occupied by Bail, solanacearum. . 196 107, 108. Details from fig. 83 at A and B, showing Nature of the Tissues, varying Extent of Infection, and Absence of Starch in the Vicinity of the Bacteria 197 109. (a), (/>), (c) Flagellate Rods of Bad. solanacearum Stained by Different Methods ; (d) Non-flagellate Rods of Bad. solanacearum Stained with Silver Nitrate 198 1 10. Figure Designed to Show Transluceney of an Old Culture of Bad. solanacearum in Milk 199 111. Gelatin Stabs of Bad. solanacearum 199 1 12. Agar-poured-plate of Bad. solanacearum (South Carolina) 200 1 13. Agar-poured-plate of Bad. solanacearum (Porto Rico) 201 1 14. Australian Sore Eye or Spewey Eye of Potato 208 1 1411. Margin of a hanging drop showing Bacillus solanincola 215 1146, c. Sesamum plants showing Malkoff's disease (after Malkoff) 217 1 ij,d. Bacteria from Malkoff's disease of Sesamum (after Malkoff) 217 1 14c Histology of Sesamum petiole attacked by bacteria 217 1 1 4/. Bacteria from Austrian disease of sesamum 217 115. Tomatoes following Tobacco in a North Carolina Field and Subject equally to the Granville Wilt. 221 116. Cross-section of Petiole of a Tobacco-plant Attacked by the Granville Wilt (North Carolina) 228 117. A Detail from fig. 116 228 118. Bacterially Infected Tobacco-stem Attacked by Fusarium in Hot-house, 5 months after Inoculation 229 119. Granville Tobacco Wilt: Leaf showing Dead Apex and Brown Veins in the part not dead 230 120. Cross-sections of Base of Petiole of Inoculated Tobacco Plant No. 34 (1905) 231 121. Midrib of Inoculated Tobacco Sliced Longitudinally to show Vascular Stain Due to Bacteria. ... 231 122. Agar-plate from Interior of Plant 31, showing the Presence of a Secondary Soft-rot Organism 231 123. Aerial Roots on Tobacco Induced by Inoculating with North Carolina Tobacco-wilt Bacterium. . . 232 1 24. Crystal from an Old Culture of North Carolina Tobacco Bacterium in Litmus Milk 233 125. Cross-section of Base of Stem of Datura stramonium (plate 43), showing Extent of Gray-white Bac- terial Ooze from the Vascular System about one-half Hour after Cutting 233 126. Field of Tobacco in Japan Attacked by Bacterial Wilt 239 127. Agar Cultures of Bacillus nicotianae (stab and streak) 240 128. Agar Plate of B. nicotianae, showing Ringed Colonies and Brown Stain around them 240 129. Rods of Bacillus nicotianae Uyeda 242 130. Agar-poured-plate of Bacillus nicotianae 242 131. Cross-section of Stem of Japanese Tobacco Received from Uyeda, showing Bacteria in Vessels. . . . 243 132. Bacteria from a Stem of Japanese Tobacco Attacked by Wilt, for comparison with Bad. solanacearum. 243 133. The same, Stained for Flagella, and more highly magnified 243 134. Honing's Figure showing Effect of a Bacterial Saprophyte on the Sumatran Tobacco Bacterium. 259 134a Flagella of Sumatran tobacco organism 264 1346. Tobacco plant wilted by motile bacteria, check on Fig. 134 a. i. e , inoculated from same culture. . 264 133. Potatoes Inoculated with Bacterium xanlhochlorum 275 136. Stem of Vicia faba 10 weeks after inoculation with Bacterium xanlhochlorum 276 137 Brazilian manihot wilting from attack of bacterial disease 280 [38 Manihot: (a) stem showing bacterial blister and exudate; (6) cross-section of stem showing upper pari diseased > icteria from same 281 (Total figures 155.) Bacteria in Relation to Plant Diseases By ERWIN F. SMITH VASCULAR DISEASES-Continued. COBB'S DISEASE OF SUGAR-CANE. Synonyms: Gumming of Cane, Sereh in part (?), Top- rot in part (?), Polvillo (?). DEFINITION. This is a specific communicable disease of sugar-cane, due to a yellow, one-flagellate schizomycete. The most conspicuous signs are dwarfing, etiolation, stripes on the leaves, reduction of sugar-content, decay of the terminal bud, and the appearance in the fibro- vascular bundles of a yellow slime and a red stain. In early stages of the disease this slime is inconspicuous, but in later stages it is one of the most striking features of the disease (see plate n, fig. 2), and frequently gives trouble in the sugar-factory, gumming the ma- chinery, interfering with the clarification, and delaying the boiling in the vacuum-pans. HOST PLANTS. So far as known, the disease is confined to the sugar-cane (Saccharum offlcinarum). GEOGRAPHICAL DISTRIBUTION. This disease occurs in New South Wales (Cobb, Greig Smith), Queensland (Tryon, Cobb), Fiji Islands (Clark), Mauritius (Boname), Java (Went), Borneo (Kriiger), New Guinea (Cobb), and Brazil (Dranert). From various statements in Spegazzini's paper on Polvillo or humid gangrene of the cane I believe this disease is also present in Argentina, Tucuman province (see Polvillo, p. 85). It is probably not in North America. No com- plaints have been received from the cane-fields of Louisiana. Dr. Went did not find it, or hear of it, in his tour of inspection in the Dutch West Indies in 1902. It has not been reported from the British West Indies nor from Porto Rico. The writer did not see it, or hear of it, in Cuba in 1904, nor was it seen in 1907 by John R. Johnston, assistant in the Laboratory of Plant Pathology, who spent six months in the West Indies, visiting Cuba, Jamaica, Trinidad, Guiana, Venezuela, Barbados, and Porto Rico, and who was instructed to look for it especially. Recently a top-rot of sugar-cane has been reported from Cuba by Cook and Home, but their account of it leaves very much to be desired. It does not occur in the Sandwich Islands (Cobb). Sereh and cane-gummosis are said by Dr. E. J. Butler to be "unknown or rare" in India (letter to our Secretary of Agriculture, April 21, 1903). It is a disease most prevalent in the Southern Hemisphere, but one likely to occur wherever cane is grown. The Javan top-rot as described and figured by Wakker is not this disease. Possibly, however, it may have been confused with the Javan heart-rot or with the pokka-bong. HISTORY. Dranert was probably the first person to describe this disease in a scientific publication, and certainly the first one to find bacteria in connection with it. His observations were made more than 40 years ago in the great sugar region of the province of Bahia, in Brazil (S. lat. 130). This disease had been present at the time of this first paper (1869) to a very alarming extent for about 6 years, and recognized as a distinct disease for a much longer period. During the "past three years," in the comarca of Nazareth, near the city of Bahia, 3 4 BACTERIA IN RELATION TO PLANT DISEASES. he says "the crops have been almost entirely destroyed by this disease." During the same time the disease made very serious inroads into the cane-fields of the northern parts of the province. He also says that the disease occurs farther south, in the province of Santa Catharina (S. lat. 260 to 280), although a part of the injury to cane in that region is ascribed to unfavorable climate, the cold being occasionally too severe in winter (40 C.) and the mean annual temperature too low, i. c, 21.50 C. at Blumenau, whereas cane requires a mean of 240 C. He also seems to think the disease occurs in Cuba, but his only evidence appears to be "much complaint of a sugar disease some years ago." At that time cane was generally reproduced in Brazil by planting the tops of the culms, including the terminal bud. The disease is said to have been most prevalent in a rainy year, and to have caused least complaint in a very dry year. "Repeated investigations at the instigation of the Government have thus far led to no results." Inasmuch as the disease had been ascribed to insects by one of the Commissions, Dranert first examined for borers, Coccus sp., etc., but could not in any way connect them causally with the disease. The disease occurred when they were absent and vice versa. On the other hand, as a result of microscopic examinations, he found various minute vege- table organisms in the languishing cane, and to them he ascribed the disease. Accepting Hallier's peculiar view of polymorphism, he believed all these to be various stages of one organism, and experimenting in the same crude way as Hallier, believed he had demon- strated that one grew out of the other. He speaks mostly of an alga as the cause of the disease, but this is only because he believed that to be the terminal stage in the develop- ment of the microscopic organisms (bacteria) seen by him in the yellow gum. These latter were considered to be the spores of the algae, or the reduced forms of the spores. He figures a micrococcus, a cryptococcus (yeast), and one or more fungous or algal (?) bodies as stages of this parasite. Owing to the imperfect technic of the time he was unable to obtain from his cultures results of any value and some of his reasoning is of the post hoc sort. Nevertheless, I believe we may safely accept such of his statements as are based on field observations, and also some of the simpler conclusions he drew from his microscopic examinations. He figures nothing resembling Bacterium vascularum, but his micrococcus is about the right size and it is very easy to understand how he should have mistaken short rods for a coccus, especially with the crude microscope he is likely to have used. There is no doubt whatever in my own mind that the Brazilian cane-disease studied by him was really that subsequently described from Australia by Cobb. He saw the top-rot, the bacterial slime, and the red stain in the bundles. I translate from his short papers in Hallier's Zeitschrift all that is pertinent to the signs of the disease: On the contrary, as an infallible sign of the disease there appears in the first place a red stain in the wood and the surrounding cambium tissue of the nodes. As the disease progresses this stain spreads in the same bundles through the whole shaft of the cane, while the parenchyma for a time retains its natural clearness. In the fully developed disease a yellow, thick, fluid substance flows out of the bundles. This substance hardens in the air, but dissolves in water, and under the micro- scope, with very high magnifications, is seen to have only a granular structure. When dissolved in water one can make out very minute cells lying in amorphous heaps, or attached to each other in necklace fashion. He goes on to tell how after six to eight days, in the expressed cane-juice, these little cells, which he identifies and figures as a micrococcus, grow out into an alga which produces swarmspores and ferments the cane-juice, with production of hydrogen and carbon dioxide and organic acids. This "alga "he afterwards found in the diseased cane and identified it as the cause of the disease. The yellow masses filling the bundles are regarded as its spores. Farther on he mentions the fact that under the influence of this disease the leaves turn yellow and the terminal bud rots. The "spore dust" might enter the plant, he thinks, through insect injuries. COBB S DISEASE OF SUGAR-CANE. In a second brief communication (an excerpt from a letter to Hallier), after speaking of the discovery of a cryptococcus (yeast) in the juice freshly squeezed from diseased cane, Dranert goes on to say: "In the same juice there are, however, in much greater numbers, that little cell (fig. 58, a), probably the same that you name Micrococcus." A little farther on we have the following confirmatory sentence: "From such cane the yellow material was collected which, dissolved in water, appears as a micrococcus." He then goes on to state as facts what in the light of our present more exact knowledge we know to be only infer- ences drawn from inexact observation, to-wit, how these micrococci in 24 hours grow out into those algal threads he has already described and figured. Renewed attention was called to this disease in 1893 by Dr. N. A. Cobb, who found it present to an alarming extent in eastern Australia. Dr. Cobb observed this disease in the Richmond River district and the Clarence River district in New South Wales (S. lat. 290 to 300). He has also seen gummed cane from one district in Queensland and says: " It is altogether improbable that it is confined to a small district in Australia." The chief complaint had been from the Clarence River region: "There is scarcely a farm on the lower Clarence where gumming is not abundant, and on many of them its ravages are only too apparent." As to the earliest appearance of the disease we have the following: A farmer on the Lower Clarence told me that he saw gummed cane sixteen years ago [1876] on his farm; I have no doubt of it. The disease is probably nothing new; in fact, is very likely as old as the sugar-cane plant itself. I think it very likely that the disease occurs wherever cane is grown. Tryon, in Queensland, also found a number of persons who remem- bered having seen the disease earlier, one man as early as 1884, to a slight extent in Rappoe cane. The signs of this disease observed by Cobb in Australia are best stated in the author's own words : When a cane crop is gummed it presents a variety of symptoms that vary according to the severity of the disease. When only slightly gummed the crop appears to be in a fair condition. Here and there, however, will be seen stools containing one or more stalks with dead tops. The base of the arrow [terminal shoot or flower-stalk] in such cases will be found to be rotten, and usually one or more cavities of considerable size are to be seen near the top of the stalk, filled, or partially filled, with offensive matter. At first one is inclined to attribute these cavities to the inroads of grubs or borers. This F'S- I.* idea is soon seen to be mistaken, for there is no entrance or outlet to the cavity, nor are there any traces of excrement, both which facts prove conclusively that the cause lies in some other direction. The tissue about these cavities is generally brown, black, or dark red in color, and reeking with a slimy offensive substance which varies from nearly colorless, through yellow, to brown. Plants whose tops have died from gumming often shoot from buds half way down, but this symptom is not peculiar to gumming. It occurs also in plants nipped by frost or by borers. If a stock which has died at the top in the manner described be cut into pieces with a very sharp knife, in such a manner as to leave the cut surface quite smooth, a honey-colored gummy matter will, in a few minutes, be seen to ooze slowly out and form in droplets on the end of the cut fibers [see fig. 1]. This gum is sometimes nearly transparent, sometimes rather opaque, and varies also in color from nearly colorless into various tints of yellow according to the stage reached by the disease. This gummy matter is usually more abundant near the top of the stalk than near the bottom, or at least oozes out more freely. In the course of an hour or thereabouts these droplets of gum become so large as to run together and form large drops, and if two or three dozen cuttings from badly diseased stalks be laid in a closely covered box over night one may in the morning collect from their ends a teaspoonful of yellow mucilaginous gum. *Fig. I. — Oozing of Bacterium vascularum in drops from cut surface of diseased Australian sugar-cane. After Cobb. BACTERIA IN RELATION TO PLANT DISEASES. The gum which thus oozes out in such quantities, if allowed to do so, at last becomes dry, owing to the evaporation of its water. In this state it appears as bright yellow stains on the end of the cutting. Sometimes the gum oozes out in a state so nearly dry that it hardens as it issues from the fibers into a yellow, coiled-up, hair-like body, and inasmuch as each fiber gives rise to one such body, all the fibers together originate a yellow, mossy appearance on the end of the cutting. If another stalk, which is apparently sound, be taken from a stool in which one or more stalks have been already blighted by gumming, as above described, such stock will be found to exhibit the same svmptoms, except that the cavities and rottenness at the base of the arrow are wanting. More- over, if other stalks be removed from shoots of cane standing nearby in the same field, but which, as yet, show no outward symptoms of this disease, the chances are that some of them will also be found to be more or less gummed. In some cases the amount of gum is so small as to ooze out in but a trifling quantity, to be detected only with a magnifying glass. Finally, the quantity of gum may be so small as not to ooze out at all; in such cases a good microscope is necessary to demonstrate that the gum is present. "Gumming" is considered by Dr. Cobb to be a good descriptive term for this disease, although he states that the substance is not a true gum. I continue to quote from Dr. Cobb's paper: After the cane is cut and ready for the mill, gummed stalks can be recognized not only by the yellow dried-up gum that exists on the cut ends, but also by their color. Gummed stalks generally have an over-ripe appearance. Green and yellow canes, when badly gummed become yellowish or orange, or even somewhat purple in color. The ribbon canes show similar alterations in their yellow stripes while their purple or black stripes tend to take on a reddish cast. The waxy bloom, usually to be seen on perfectly sound cane, has disappeared on badly gummed cane. All symptoms of gumming seem to be more pronounced when they occur in plant cane than when they occur in ratoons.* An explanation of this fact will be offered later on. In the sugar mill the juice of gummed cane may be recognized by the greater amount of lime required for its clarification, and there seems to be reason to suppose that the crystallization of the jellies is slower and less perfect when they are derived from gummed cane. The sieves used to separate the fiber from the juice are apt to clog up when gummed cane is being crushed. They are easily cleaned with soda. The foregoing symptoms are those connected with cane so attacked as to produce a marketable crop. In some cases, however, the gumming prevents the growth of the sets. The plants may reach a height of a foot or two feet, but they then die back and shoot again from the base, or from buds half-way down the stalk. In such a case the loss is total or nearly so. The gum presents the same features in this case as in milder ones. Fig. 2.f So much for the gross appearance of this disease. A microscopic examination gave the following additional facts, which seem to have been made out very satisfactorily : A microscopic examination of a thin slice across a gummed cane shows at once that the disease is not general, but local. The gum, except in certain cases, is confined to the fibers; in fact, to the sap-vessels, these latter being plugged up with gum. A cross-section of a healthy fiber shows the sap-vessels as empty spaces, the sap having flowed out [?] in the process of cutting the section; a cross-section of a gummed cane on the other hand shows the sap-vessels to be filled with yellow granular matter, in other words, gum. This confinement of the gum to the sap-vessels is one of the most striking microscopic features of gummed cane. In advanced cases, and in the more tender tissues at the top of the cane, the gum is not so local in its distribution; it may, under such circum- stances, be found outside the fiber. *Plant-cane is the first year's crop from buried cane or cane cuttings. Ratoon-cane is the second crop from a stool or any subsequent crop. In the rich cane-lands of Australia sometimes as many as ioor 15 successive crops are obtained without renewal of the root-system. In Cuba it is customary to plant some cane annually, but ratoon crops are also common. fFic;. 2. — a, Bacterium vascularum from sugar-cane stained with methyl violet without heat, and mounted in water; b, Bad. vascularum from a culture on cane-sugar agar; c, Bact. vast alarum from sugar-cane, stained with fuchsin and mounted in balsam. X2,7oo. After Cobb. COBB S DISEASE OP SUGAR-CANE. Lenses of high power show the gum to be swarming with microbes of the form known as bacilli [fig. 2]. When the gum is fresh and yellow in appearance, the microbes are all of one kind whose features are well shown in the illustrations. * * * This microbe appears to be one not hitherto described, and I propose to call it Bacillus vascularum, in consequence of its occurrence in the vessels of the sugar-cane. Each microbe has about it a small amount of gummy matter, which is a product of its growth. The gum described above as issuing from the sap-vessels of the cane has, therefore, two component parts, namely, microbes and a viscous gummy matter. This gummy matter appears to be a new substance, and to it I have applied the name vasculin. This substance, which is probably made up of the bacteria themselves or of soluble substances in great part derived from the outer wall of the bacteria, is described as follows: Vasculin, the substance formed by the growth of the Bacillus vascularum as it occurs in sugar- cane, is a yellowish, non-cry stallizable, viscid substance, having an almost imperceptible acid reaction. The taste is that of a slightly soured solution of gum arabic. A short time after oozing out from the ends of cut cane, by solution it converts ten times its weight of water into a fluid of the consistency of mucilage as used for adhesion purposes. Though soluble in water it is insoluble in alcohol. The addition of absolute alcohol to the raw substance converts it into a hard mass, but this is only owing to the abstraction of water ; on placing the hardened mass in water it soon resumes its former con- sistency and appearance. Vasculin is not coagulated by alcohol. Watery solutions of the yellow exudate from the vessels of the cane were tested in various ways. Lime-water precipitated it and the precipitate was redissolved on addition of hydrochloric acid. The exudate was also precipitated by the hydrates of potassium, sodium, barium, and strontium, but not by ammonium hydrate. All of the above precipi- tates were redissolved by hydrochloric acid. Watery solutions were also precipitated by lead acetate and ferric chloride, but not by ferrous sulphate, barium chloride, or silver nitrate. The dissolved substance has little or no action on polarized light. The raw gum contains only a slight amount of nitrogen. Badly gummed cane contains less sugar than very slightly gummed cane according to the tests shown in table 1. TablB i. — Analyses of Very Slightly and Badly Gummed Cane for Comparison. Per cent of cane sugar Per cent of fruit sugar Per cent of other organic matter and soluble ash. . Per cent of total solids Quotient of purity Very slightly Badly gummed. gummed. 12. 50 10. 50 .82 .88 1.9' 2.36 15.2^ M . 74 82. 10 76.40 Some of the same chips were pressed by hand, and the juice thus expressed was tested for sugar, with the following results : Very slightly gummed. Badly gummed. Per cent of cane sugar I4.90 I 1 .50 The raw juice squeezed out by hand having stood 48 hours, was tested withdecinormal solution of sodium hydrate. Very badly gummed. Slightly gummed. N 100 c.c. required of — NaOH 3.8 c.c. 3.2 C.C. 10 The greatest care was taken that the two lots of canes subjected to analysis should be strictly comparable in all respects other than the amount of gumming. 8 BACTERIA IN RELATION TO PLANT DISEASES. To test the pathogenicity of the organism Dr. Cobb inoculated a few sugar-canes. He writes as follows: In order to show whether the disease is caused by the microbes healthy plants were inoculated with gummy matter from diseased cane. I found it to be unnecessary to first make pure cultures in the way usually adopted, for the reason that a pure growth of microbes could be obtained without this precaution. Plenty of cane could be found containing no other organism in its interior tissues and advantage was taken of this fact in making inoculations. This is good as far as it goes, but to complete the proof and satisfy the skeptic, pure- culture inoculations are necessary- Concerning the pathogenic nature of his Bacillus vascularum, Dr. Cobb also has the following: What we wish mainly to know is whether the microbes are the cause of the disease. As a result of hundreds of careful examinations, I am able to say that the disease of gumming, as described, never occurs "without the yellow gummy matter in the sap-vessels. The result of even a much greater number of examinations made with equal care showed that the yellow gummy matter never occurred without the microbes, indeed it is very evident that the gum is a product of the growth of the microbes. This leads to the conclusion that the disease never occurs without the microbes being present, and it may be added that many cases were examined in which, in spite of long and careful search, none but the merest traces of other foreign organisms could be found. This is very strong evidence that the microbes are the cause of the disease, but it does not amount to proof. Dr. Cobb's methods of inoculation are quite carefully described. Pure cultures were not used, but all inoculations were direct (see this monograph, vol. I, p. 9). They were made in healthy canes by means of a needle which was thrust into vertical slits made with a needle-chisel. The surface of the cane was first covered with hot wax, the chisel was thrust through this a half inch or more into the cane, then the needle-thrust followed. The bacteria on the tip of the needle were obtained directly from the fresh ooze out of the ends of cut bundles. The following precautions were used: Needle and chisel were steril- ized by heat and protected till ready for use. Cane was selected which was occupied apparently only by a pure growth of this yellow organism; the rind was removed, and the segment was placed in a large glass-stoppered jar the inside of which had been smeared with glycerin over night ; all the operations were performed in a room as free from drafts and dust as possible. When all was ready the protected segment of diseased cane was shortened with a sterile knife and the yellow ooze from the freshly exposed surface of the bundles was then touched with the inoculating needle and some of the slime was transferred to the interior of the healthy canes. The lips of the wounds were then covered with more hot wax. This paper contains no hint as to the result of these inoculations. All that relates to the morphology of this organism is given in the three cuts here reproduced from Dr. Cobb's paper. The magnification is said to be X 2,700, which would make the organism about the same size as Bacterium campestre. Judging from these figures it is also variable in form like the latter. We are told by Dr. Cobb next to nothing about the cultural characters of his Bacillus vascularum. All I can find is the following: Cultures on agar-agar containing about 5 per cent cane-sugar gave at first roundish colonies on the surface, having to the unassisted eye no structure either radiated or concentric. In the course of a few weeks the yellowish-white and somewhat opalescent growth had extended several inches along the edge of the tube on the surface of the agar-agar and between the tube and the agar-agar, without causing any liquefaction. On gelatin the growth was much slower and remained circular and almost imperceptibly concentric. Its color, &c, were as on agar-agar. If the "&e."is entirely inclusive, then we must assume that this organism did not liquefy gelatin. From its slower growth on this medium, it is not unlikely that litmus neutral gelatin exerts on it a retarding influence the same as it does on Bact. campestre. Figure 3, which shows the dwarfing effect on cane, is borrowed from Cobb's paper. COBB S DISEASE OF SUGAR-CANE. In 1894, a correspondent of The Sugar Cane described a serious disease of cane at Pernambuco, Brazil (lat. 8° S.). His account makes it still more certain that the Brazilian disease is none other than the one here under consideration. The Pernambuco disease is described as follows: It may interest some of your readers to remind them of some features of the disease which for many years threatened the existence of the sugar industry in the Province of Pernambuco, and in other provinces of Brazil. Unfortunately, in one sense, before the observations which are the subject of this note were made, the evil had so far diminished that it was difficult to obtain sufficient specimens to permit of a thorough investigation, and other cir- cumstances cut short the proposed in- vestigation almost at the commencement. The unfortunate "Otaheite" cane, or "Cayanna" cane, as it is called in Brazil, was here as elsewhere the prin- cipal victim, and nine-tenths of the cane grown was of this kind. A green striped cane called here Imperial, and a small yellow cane, called Creoula, also suffered. The most striking symptoms were: Gum. — The formation of a bright yellow, gummy substance, exuding appar- ently from the ends of the fibres on cutting the cane across. Sometimes this yellow gum, which turned orange colour on drying, appeared only after half an hour or more, at other times it appeared immediately on cutting the cane, and in such quantities as to drip from the cut ends, and in very bad times is said to have frequently choked the strainers of the raw-juice tank. Premature death. — Indicated by the early drying up of the leaf tips; by the successive joints becoming shorter and less in diameter as the cane grew, giving it the appearance of the joints having been telescoped one into another; by some internal parts turning red, denoting fermentation, which may have set in be- fore or after the cane had succumbed, and by the death of the eyes. Any or all of these symptoms may have occurred together, and some canes, which to all outward appearance were perfectly healthy and well developed, Fig. 3. contained a quantity of the yellow gum, together with a normal percentage of sugar. The canes which did not die before maturity gave no ratoons, and the juice was most difficult to work, refusing to crystallize in the ordinary way, sometimes in any way. The greatest difficulty was encountered in the "old process" factories, probably on account of the great heat accompanying open evaporation. The Usines, with their lower temperatures, could work better, but they also at times got the "devil in the house," especially if they did not take care to use but little lime in the defecation. The lime was frequently reduced to 10 grammes per hectolitre, and in this way the juice worked best. The appearance of this disease has not been assigned to any distinct causes. All the varieties attacked have been cultivated for many years in the most primitive manner. The usual method still generally followed in Brazil is to dig shallow narrow holes very close to one *Fig. 3. — Field of sugar-cane in New South Wales. The plants in the foreground grew from sets which were slightly diseased by Bacterium vascularum when planted; those in background grew from healthy sets. After Cobb. IO BACTERIA IM RELATION TO PLANT DISEASES. another in fields barely cleaned of the brushwood, still less of weeds, unploughed, undrained, needless to say never manured, seldom weeded, the canes being never trashed, the ratoons uncared for. After taking off one or two ratoon crops, the land is abandoned for a period of not less than 3 years. The seed cane is frequently taken from any old abandoned piece; no selection is ever made. All the themes as to causes suggested by the planters and others were flatly contradicted in actual practical experience; some said, for instance, that the salt air was to blame, and the disease did first make its appearance on the coast, but when it afterwards crept farther up country it should have died out, which it did not. Some suggested exhausted soil, but the disease appeared in canes grown from uninfected sources in virgin and rich soil. Others put it on degenerate seed cane, and certainly to judge from the seed cane often used any imaginable disease might result, but the offspring of fine healthy cane brought from far inland districts also suffered. The planting of pieces of cane and tops also made no difference ; the former is the almost invari- able custom here. In a wet season more cane was usually attacked than in a dry season. It is probable that, as has been suggested in other cases, the responsibility of its appearance must be laid on a combination of causes inducing general debility and aptitude for catching disease, rather than to any one particular cause. These elements of disease, if they may be so called, are like malaria in a swamp, always present and only awaiting suitable subjects. In the absence of definite evidence as to the cause of the disease, improved methods of cultiva- tion, manuring, advisedly not indiscriminately done, regeneration of stock by selection, the rearing of seed-cane in nurseries, were advised. The method adopted, however, was more heroic; as the planters could not find a cure, they threw out the Cayanna and other canes affected, and introduced varieties of foreign origin. The dark claret coloured cane with a still darker stripe, called here " Louzier," was the principal kind introduced, also the "Lalanjore," and some 70 others. (As the names of these when first received were much mixed up, it is possible that " Louzier" is not the correct name for this cane.) None of these new varieties had been attacked, though now growing 15 years under precisely similar circumstances as regards bad methods of cultivation, and the Cayanna is again being success- fully cultivated on lands which ten years ago could not grow it. As it is a far better cane as regards yield and early maturity than the new ones, it is to be hoped this improved state of things will continue. In 1894, M. Boname reported Cobb's bacterial disease of sugar-cane as common in the Mauritius, especially on a variety called La canne Bambou. In the beginning there are no external signs. As the disease progresses growth ceases, the head shortens, the 5 or 6 green leaves remaining at the top grow paler, bleach, become yellow, and dry out. Sometimes shoots (ailerons) in greater or less abundance are pushed out of the upper nodes, but more often the whole cane dries out and dies. When the leaves begin to turn yellow, if the stem is cut transversely, little masses of viscid grayish yellow matter appear on its cut surface and dry yellow. This gum is generally more abundant toward the top of the stem. In early stages of the disease the gum is extruded only from isolated points. In bad cases the gum is very abundant and the gummy foci unite to form large, sticky masses covering a considerable part of the section. The gum is not disseminated through all the tissues of the stem. It is seen only in the interior of the two large vessels which form the center of the nbro-vascular bundle, and is not found in the sugar-cells. Among other results, chemical analysis proved the amount of sugar to be less in diseased canes than in healthy canes grown under otherwise identical conditions, as shown in table 2. Table 2. — Reduction of Sugar in Diseased Canes. Kind of cane. Sugar per 100 c. c. of juice. Sugar per ioo canes. Healthy 18.71 24 . 30 22. 51 25.27 14.52 .8.43 17.30 19.23 Slightly gummed Healthy cobb's disease of SUGAR-CANE. I I There is no record of any cultures or inoculation experiments. In 1895 in an article on gumming of cane in the Annual Report of the Queensland Department of Agriculture, Tryon states that the disease occurs in Queensland, and says of Cobb's Bacillus vasculanim: "Personally I also have failed to produce the disease in healthy plants by the method of inoculation with the pure culture of the bacillus." But Tryon also says that Cobb says someone else in New South Wales, whose name is not mentioned, has succeeded in inducing the disease by means of pure-culture inoculations. The writer of this abstract asked Dr. Cobb for details, but was unable to learn the name of this experimenter, if such there was. Tryon studied this disease in the Burnett district and Wide-bay district (S. lat. 250 to 260), i. c, on the eastern coast of Australia, a few hundred miles north of the localities examined by Cobb. His description of the signs of the disease on the foliage is in some respects confirmatory of Cobb's. They are in many respects so strikingly like those obtained by the writer from pure-culture inoculations that I quote in full, as follows: When the presence of the disease has been determined by the use of sets derived from a badly "gummed" crop, some of them will fail after having emitted attenuated shoots, that fail to reach the surface. Others will give rise to slender and weakly plants, which apparently struggle to survive, whilst a few will develop foliage with the individual leaves narrower and more irregular in size than if they were healthy, with the central ones yellow-green and more or less crinkled and contorted, with, at times, the central leaves interlaced in a tangled mass. A few of these leaves, again, espe- cially the inner ones, may exhibit bright rust-red streaks in their tissue, and this may constitute a conspicuous feature. If any such plant is cut longitudinally through the point of growth, it will be generally observed that the central shoot beyond where it joins the cane proper, instead of arising in a straight, erect manner, is more or less bent and contorted, its upward tendency having been apparently hindered. Moreover, the young cane itself, immediately below the shoot, will present one or more cavities, each containing a semi-fluid tenacious pale brown substance. Should the "sets" be the progeny of a crop of cane affected but slightly by the disease, nothing very abnormal may be noticed until the cane proper has commenced to form. Then, although the bulk of the plants will appear well-grown and perfectly healthy, others, though at first fully devel- oped, will evince the presence of the affection in varying degree. The first symptom in a plant of its occurrence will then probably be afforded by a thin, pale longitudinal stripe arising in one of the outer leaves, in which the green colouration being discharged may gradually be giving place to brown. If this plant is divided by a longitudinal cut through its growing apex, nothing abnormal may yet be recognized. A plant in which the disease has made further progress will exhibit brown stripes of dead tissue one side of the central nerve, or along the margins of the outermost leaves, whilst on two or three of the inner ones will have appeared elongated rust-red streaks. If this plant be cut as before, very marked changes will be noticed in the tissue immediately below the growing apex. These are afforded by the presence of several cavities having ill-defined walls partly filled with an odorous yellowish substance of the consistence of pus, as well as of spots where the external tissue is becoming soft and brown to mark the site where subsequent ones may arise. These cavities occur in the intervals between nodes, whilst the denser tissue of which these latter are composed may exhibit — in small number — specks, or thread-like lines of a red colour. When still further advanced we may have a strong, healthy-looking plant, with a stem measuring some 2 feet 6 inches in height from the ground to where the green foliage commences. In this several of the outer leaves, and the central ones as well, have longitudinal brown stripes or bands of dead tissue extending for the greater part of their length; and, as will be seen by a longitudinal cut, the joints or internodes immediately below the apex, to a distance of from 1 to 2 inches, are quite hollowed out, and there is considerable discolouration and softening of the tissue generally and incipient decay, both in them and in the nodes also. In such a plant the central shoot, now almost dead, may readily be pulled out. * * * The disease may appear at any period in the growth of the cane, and even when it is fourteen or more months old; but these late manifestations probably generally arise when the malady is of spontaneous origin. * * * In the first instance the central leaves are quite normal in appearance, and perfectly green and turgid, but the lateral leaves are marked by broad brown longitudinal bands of dead tissue, the lowermost having evidently prematurely died. The eyes on that part of the stem from which the leaves have naturally fallen have shot out, and the slender shoots thus formed are already dead; the uppermost eyes, however, are still alive, and have not as yet sprouted. * * * 12 BACTERIA IN RELATION TO PLANT DISEASES. As an illustration of a still further advance in the progress of the malady, a plant may exhibit the following symptoms: The central shoot and leaves are already quite dead, though still flaccid, and readily yield to a slight pull. There occurs also in those, on either side of it, that are still partly green, a brown band of dead tissue, proceeding along each margin; this band widens in the case of the outer leaves, whilst the outermost of all are all involved in it, being quite dead. The cane itself, instead of exhibiting that yellowish colour indicative of the fact that it is already ripe, is of a dull bright-green colour above; nearer the ground it is clouded, with the same hue, but at the extreme base evinces little unusual in this respect. All the buds except those situate lowest on the cane have sprouted, some having given rise to peculiar elongated slender shoots; beneath the leaf-sheaths the buds are already dead or apparently dying. * * * On cutting any of these canes across, small bright droplets of a canary-yellow thick adhesive substance will arise from the pores distributed over the surface of the section, and in many instances will run together and coalesce. When the disease is advanced to the stage above described, some of the buds within an inch or so of the decaying summit of the shoot may shoot out and develop a tuft of narrow leaves; but no further growth takes place in the cane itself, though the latter may be some time before it actually dies. * * * All the canes that arise from a single stool may not be affected simultaneously and to an equal extent. As an instance of this, the following occurrences were remarked in the case of an affected plant : All the canes had been checked by cold when they had experienced from six to seven months' growth, otherwise the plant was remarkably robust. Three of them had the central shoot, and several of the lateral ones already dead. A fourth had the leaves as well as the central shoot quite green, and all of these canes exuded droplets of gummy substance when their stems were cut across. A fifth cane — the stoutest of all — was entirely wanting in the gummy exudate, as was seen when after lopping off its top and allowing it to remain still connected with the ground, none of this gum was observable even after the lapse of some hours. In addition to these five canes, there were several suckers, some of which had formed cane, and were almost as high as the stalks that surrounded them. These, however, even when arising alongside gummy canes, were themselves apparently quite free from disease, as no "gum" emanated from their cut ends. In October 1895, in the Agricultural Gazette of New South Wales, Dr. Cobb reported on his inoculation experiments. The eane-disease was produced, apparently at least, by means of his Bacillus vascularum, but the inoculations were too few to be very conclusive, because they were made in a region much subject to this disease and also because not enough plants were held as checks. Under the circumstances, i, c, much diseased cane in the vicinity, not less than 100 canes should have been inoculated and as many more held as checks. Cultures derived from single colonies should also have been used for purposes of infection rather than ooze from diseased canes, although the author quite agrees with Cobb that the organism often occurs in practically pure culture in the interior of the diseased canes. The reader will be able to judge of the quality of the evidence by reading the following, from Dr. Cobb's report, which contains all that is pertinent: Six inoculations were made, one a check — that is, pricked with a sterilized needle only. The stools were in charge of Mr. C. N. Stevens, Manager of the Colonial Sugar Refining Company's mill at Harwood. At the time of inoculation all the stools appeared to be healthy. The canes [which were inoculated in August 1893] were forwarded to me at Sydney in September, 1894, and examined with the following result: 1. Mr. Stevens reported the check lost. 2. Of the inoculated canes, four were still alive, the other was dead. All the four living canes were gummed — one slightly, but very manifestly; two very markedly; the fourth dying, and evidently from gum, though fungi had made their appearance. The gumming was most marked in the neighborhood of the point of inoculation and above it, though it appeared throughout the culm. Concerning the dead and dying canes, Mr. Stevens writes: "The dead canes have probably some time or other been broken off or shaken at the root, which would account for their demise." The fact that one of the still living stalks, as reported above, was dying, and to all appearances from gumming, would suggest that death may have been caused by gumming. It is impossible to say, lmwever, from the dead and dying canes in their present dry condition, what was the cause of death. The canes were in live different stools. Mr. Stevens writes: "All the canes in those stools are now COBB S DISEASE OF SUGAR-CANE- 13 more or less gummed." The question arises: Would these five canes have become diseased if they had not been inoculated? As stated above they appeared at the time of inoculation to be perfectly healthy, and this, of course, was the reason why they were chosen for the experiment. This fact — their apparent healthiness — is in favor of their remaining healthy. The gumming in the canes for- warded to me was most marked near the point of inoculation and above it. This fact is in favor of the disease originating from the inoculation. As reported below, canes were also inoculated with a fungus. In the same stool two stalks were inoculated with microbes. In the case of this stool the two stalks inoculated with the fungus showed no trace of gumming, though they were examined minutely. * * * This fact is again strongly in favor of the gumming which appeared in this stool having originated from the [bacterial] inoculation. Against these facts and deductions must be set the fact that a large part of the cane on the Lower Clarence, where these experiments were made, suffers from gumming. I think it may fairly be said that the canes taken for experiment were not very liable to become gummed, as they were in good garden soil not before under cane, and had good care and were derived from what were supposed to be sound crops. To the writer the strongest evidence adduced by Cobb is that the gumming was most pronounced in the vicinity of the inoculations and upward therefrom. I might also add that after seeing gummed cane and examining the yellow ooze microscopically, I became at once less skeptical, and am now [this paragraph was written in 1902] strongly inclined to believe that Dr. Cobb is correct in his conclusion as to the cause of this disease, although his inoculation experiments are not entirely conclusive. At the close of this paper, which is largely a repetition of statements made in the previous one, Dr. Cobb says that as a result of his former publication it has been shown that cane in Java, New Guinea, Brazil, Mauritius, etc., either suffers now or has in the past suffered from what is beyond reasonable doubt the same disease — gumming. In 1902, R. Greig Smith published, in Australia, a paper on "Gummosis" of the sugar- cane. He isolated the organism and described its growth on various culture-media, but said nothing of any inoculations. His studies were mostly on the nature and origin of the gum and were carried on along the lines of qualitative chemistry. Through the reactions of the gum, which were for the most part the same as those of the bacterial slime obtained from pure cultures on agar, he came to the conclusion that the two were identical and consequently that the gum in the diseased canes was of bacterial origin and not a patho- genic secretion of the plant upon which the bacteria lived saprophytically. The following paragraph is quoted from his paper : When a pure culture of the bacterium is smeared over the surface of neutral cane-gelatin or agar in a suitable vessel and incubated, a luxuriant growth is obtained after the lapse of about a week. The culture is precisely similar in appearance and consistency to cane-gum. Both have the same soft buttery appearance and gummy consistency and both slowly mix with water to form a pale yellow opalescent solution. The opalescence is due to the suspended bacteria, from which [solution] it is rather difficult to separate [them] without at the same time removing the gum. The separation can not be effected by filtering through paper, and the usual coagulating agents coagulate both gum and bacteria. Aluminum hydrate, for example, forms an insoluble compound with the gum. An attempt was made to clarify the suspension by heating it up to three atmospheres in the autoclave, a proceeding which was successful with another gum-forming bacterium, but the suspension was unaltered. Eventually the bacteria were separated by filtration through porous porcelain. A clear solution of the gum was thus obtained, but at the same time it was noted that a considerable amount of the gum remained on the porcelain, adhering to the bacteria, even after the suspension had been boiled for some time to diffuse the gum. The filtered and clear solution was tested simultaneously with an opalescent suspension, and it was found that the two behaved similarly to the addition of the various reagents, from which we must conclude that in the suspension the bacteria are inert, and for purposes of identification of the gum it is unnecessary to separate them. He gives the following table (table 3), showing the chemical reactions of the gum taken from cane and of the bacterial slime formed in laboratory cultures: 14 BACTERIA IN RELATION TO PLANT DISEASES. Table 3. — Chemical Behavior of Cane Gum and Bacterial Slime. Substance. Lead acetate Basic lead acetate Ammoniacal lead acetate. Barium hydrate Calcium hydrate Copper sulphate Ferric chloride Hydrochloric acid Alcohol Aluminum hydrate. . . Dilute acetic acid. . . . Glacial acetic acid. . . Acid mercuric nitrate Xantoproteic reaction Sulphuric and phosphotungstic acids Acetic and tannic acids Acetic acid and potassium ferrocyanide Hydrochloric acid and potassium mercuric iodide Sodium hydrate and copper sulphate Cane-gum. Precipitate ....Do ....Do ....Do ...Do ....Do ....Do Opalescence Slimy voluminous precipitate in presence of salts Coagulation No precipitate Precipitate Precipitate soluble in excess; no color reaction Color reaction Precipitate Opalescence Opalescence Precipitate on standing No biuret reaction, but pre- cipitate Bacterial slime. Precipitate. Do. Do. Do. Do. Do. Do. Opalescence. Slimy voluminous precipitate in presence of salts. Coagulation. Opalescence Precipitate. Precipitate soluble in excess; no color reaction. Color reaction. Precipitate. Opalescence and precipitate. Opalescence. Precipitate. No biuret reaction, but pre- cipitate. The weakness of the solutions, owing to the limited amount of eane-gum at the dis- posal of the experimenter, accounts, he says, for the absence of perceptible precipitates on the addition of dilute acetic acid, and a mixture of acetic acid and tannic acid. No reac- tions were obtained with KI3, AgN03, KOH, BaCl2, NaOH, cone. HC1, picric acid, and Adamkiewicz reagent. On precipitating the aqueous suspension [of the gum] three or four times with alcohol an opales- cent alcoholic solution is obtained, from which the gum can be precipitated by small quantities of neutral salts, such as NaCl. This fact, together with the reactions obtained with some of the albuminoid reagents, suggested the similarity of the gum to the Mucins. That the Zoogloea-slime of bacteria consists of mucin, or a substance nearly allied to it, has already been suggested, but there are many points of difference between the bacterial slime and the mucins. The crude gum obtained by precipitating the cultures with alcohol contains 6.1 per cent of ash and 3.08 per cent nitrogen in the ash-free, dry substance. Repeated precipitation with alcohol, and also filtration of the gum from the accompanying bacteria, would undoubtedly lower this percentage. It is, therefore, apparent that the nitrogen content alone is sufficient to distinguish the slime from mucin or the allied muciuoids. In connection with this work Greig Smith made a variety of observations on the cultural characters of the organism, and citations will be found under the heading "The Parasite." When Dr. Went visited the West Indies for the Dutch government in the winter of 1902, the writer asked him to look particularly for this disease, but he reported that he did not find it, although he said he had seen it in Java, where he had not regarded the organism as a parasite. In 1904, following some very successful inoculation experiments, the writer published a short paper on this disease, in the second part of the Centralblatt fur Bakteriologie. This was subsequently translated into English and published in full by Cobb in his Third [Hawaiian] Report on Gumming of the Sugar-cane (pp. 12-22). Cobb's Hawaiian report closes the history of this subject so far as known to the writer. It contains a summary of Cobb's earlier papers, the translation referred to, a review of Greig .Smith's work, and some additional observations which will be made use of elsewhere. See also in this connection "Sereh" (p. 72), "Top-rot" (p. 8i), and "Polvillo" (p. 85) cobb's disease op sugar-cane 15 SIGNS OF THE DISEASE. The most conspicuous signs of this disease are dwarfing, striping of the leaves, dying of the tops, decay of the heart (terminal bud), and the appearance of a yellow slime or gum in the bundles of the stem and leaves. Many of the bundles are also stained red. Microscopic examination shows that this gum contains millions of bacteria. Cobb and Boname agree that there is also a reduction of the sugar-content. Fig. 4.* Fig. 5.f The disease is primarily one of the vascular system, but in advanced stages the paren- chyma is attacked, especially the soft tissues just below the terminal bud, and cavities are formed which are filled with the yellow bacterial slime. Sometimes these cavities contain as much as a teaspoonful of the slime (fig. 4). In the later stages of the disease, the *Fig. 4. — Rot of sugar-cane clue to Cobb's disease (Bacterium vascularum). Longitudinal section through the upper part of stem of plant No. 6 just under terminal bud, showing a closed bacterial cavity which was filled with yellow slime (bacteria). Enlarged 3 times. tFic 5. — Radial longitudinal section through a leaf-sheath of sugar-cane from inoculated plant No. 4, showing general occupation of the intercellular spaces by bacteria, which are oozing to the surface through stomata, 2 months from date of inoculation, and several feet from places inoculated (see text). Outer face of sheath at left. Section taken between vascular bundles which are filled with the bacteria. For condition of bundles in sheath as shown on cross-section, see fig. 7. Drawn with Abbe camera, Zeiss 3 mm. 1. 40 n. a. apochromatic objective, and No. 2 compen- sating ocular. Slide 466 H 9. For a detail at x, see fig. 6. 16 BACTERIA IN RELATION TO PLANT DISEASES. interior of the leaf-sheaths is rusty brown and covered with the sticky bacterial slime, which is also sometimes seen oozing from other portions of the leaf. This slime oozes from the stomata (figs. 5, 6, 7). In very bad cases the leaf-sheaths above the terminal bud are completely stuck together, so that the growing shoot can not elongate naturally, but is forced to bend on itself repeatedly and push out sidewise through the sheaths (figs. 8, 9). The gumming together and pressure of the outer leaves around the terminal bud result in the doubling, twisting, and bulging of the main axis and eventually the stopping of the terminal growth. In the most pronounced cases the terminal shoot enveloped in its wrap- pings has a club-shaped appearance. In such cases there is sometimes a development of lateral shoots (figs. 10, 11) and of aerial roots. A very interesting sign, also mentioned by Tryon in his paper, is that of the red bundles. We have heard much about red bundles in connection with the "Sereh" disease of sugar- cane in Java, and, indeed, this reddening has been known as a subordinate (oxidation) phenomenon in many diseases of the sugar-cane, but never has the writer seen it more beautifully developed than in canes inoculated with pure cultures of Bacterium vascularum. In all the "common green" canes, diseased as a result of these inocu- lations, many red bundles were present (see plates 1, fig. 4, and 11, fig. 1). It was a very striking sign of the infection and could be ascribed to no other cause. The writer re- gards it as a reaction on the part of the plant. According to Prinsen Geerlings, there exists in the cellu- lose of the normal sugar-cane a neu- tral, colorless substance, not easily soluble, which becomes yellow in an alkali but changes to red and finally to brown when brought into contact with air. The nature of this substance is unknown. In the majority of these red bundles bac- teria were no longer to be seen: In place of them was a red formless mass (figs. 12, 13). Red and yellow bundles were inter- mingled in the stems, however, and often the same bundle would be both red and yellow, i. e., variegated, the yellow parts being filled with bacteria, c. g., lower part of fig. 12. The same phenomenon has been observed in maize inoculated with Bacterium stewarti, except that in the latter case the variegated bundles were yellow and brown. The red pigment was most pronounced in the nodes and immediately under them (fig. 11). This was observed in many canes. Without exception there was more pigment in the upper part of the internode than in the central or basal portion, but by far the greater part was in the nodes, where often nearly all of the bundles were as red as blood. Probably this localized pigmentation is due to greater aeration through the leaf-traces centering in and immediately under the nodes. f In the sugar-cane, as in the sweet corn, the pigment does Fig. 6.* *Fig. 6. — A detail from fig. .s at X, showing the bacteria multiplying in the intercellular spaces and pushing toward the inner surface of the leaf sheath, i. c, in the direction of the arrow, st, stoma. fWcnt has shown that the leaf-traces take a downward direction soon after entering the node. PLANT BACTERIA, VOL. 3. PLATE I. Cross-sections of sugar-cane inoculated with Bacterium vascularuin. Figs, i, 2, 3 show bacterial ooze from vascular bundles of freshly cut stems. Figs, i and 2 are from plant 6, fig. 3 from plant 8. X3. Fig. 4, section in vicinity of a lower node, showing dark red stain in vascular strands. From plant 6. X 3- Figs. 5, 6, 7. 8, sections of stem, photographed after being under alcohol for some weeks. The white spots are hardened masses of bacteria. The dark spots are reddened bundles; on one side is a bacterial cavity opening to the surface, X 1-75 circa. Plants inoculated by needle-pricks, Feb. 6, 1903, on the blades of the leaves, 2 to 3 feet away. Time about 2.5 months. cobb's disease of sugar-cane. 17 not appear in the first stages of the disease, and the writer is inclined to think that the reddening of the bundles is a later stage than the yellowing. Valeton makes the same observation respecting Sereh. Plates poured from bundles showing red ooze yielded only Fig. 7.* Fig. 8.| the yellow colonies of Bacterium vascularum, indicating that this red ooze was due not to red bacteria, but to a red reaction on the part of the host-plant. Perhaps the formation of this pigment would not be as noticeable in all varieties of sugar-cane as in "common *Fig. 7. — Cross-section of a leaf-sheath of inoculated sugar-cane No. 4, showing bacterial occupation of bundles and of intercellular spaces of the parenchyma. This is not an inoculated leaf, but one higher up and infected from the vascular bundles of the stem. There was abundant bacterial ooze on the inner face of this sheath (right side). Slide 466K12, upper row, second section from the left. Drawn with Zeiss 8 mm. apochromatic objective, No. 12 compensating ocular, and Abbe camera. tFic. 8. — Inoculated cane-plant No. 9 at the end of 10 weeks, showing the doubled-up terminal bud pushing through sidewise, owing to the fact that the leaves above it were gummed together by the bacteria. Buds at base pushing abnormally. i8 BACTERIA IN RELATION TO PLANT DISEASES. Fig. 9.' green" cane. It is not a sign peculiar to this disease, but appears in diseases of an entirely different nature. The writer saw well-marked cases of it at the Har- vard Gardens, on the Soledad Estate near Cienfuegos in Southern Cuba, in the progeny of canes im- ported from the East Indies, but there was no accompanying yel- low bacterial ooze. Possibly this disease was Sereh. In order to be sure one is deal- ing with the gum- disease, one must find ft i the yellow bacterial slime in the bundles. This is usually an easy task, except in the case of very resis- tant canes. When one is in doubt samples of the suspected canes should be steamed and then examined (Clark). A very conspic- uous sign of the dis- ease in the "common green" cane used for the writer's first set of inoculations was striping of the leaves. In the inoculated ijtwN? leaves white or yel- lowish-white stripes extended upward and downward from the pricks. In places, in the white stripes, there were very often rust-brown spots and streaks. Later these became reddish- brown and finally dull-brown and dead, but the etiolation always preceded the Fig. lo.f drying out. Similar c< t *F ig. 9. — Cobb's disease of sugar-cane : Plants 6, 8, and 1 2 stripped of leaves to show abnormally short internodes and zigzagging of shoot-axis on itself in efforts to grow through the gummy inclosing leaf-sheaths. These plants are shown in pis. j and 3. Nos. <> and u have sent out basal shoots. The bacterial cavity in fig. 4 was from No. 6. fFiG. 10. — Common purple cane stripped of leaves to show abnormal pushing of buds. Plant No. 38, inoculated May 5, 1903, photographed Aug. 8, 1903. cobb's disease op sugar-cane. 19 white or pale stripes appeared as secondary signs in the uninoeulated leaves of the same shoot and in young shoots from stools bearing inoculated diseased canes. Etiolation has been less marked in the other varieties tested, but they have in every case been more resistant to the disease. All of these conclusions are based upon pure-culture inoculations made in Washington hot-houses, the writer never having seen the disease in the field. Since premature development of the male in- florescence is one of the striking signs in sweet maize attacked by Bacterium stewarti, and since an incli- nation to bloom is also a conspicuous feature of sugar-cane attacked by Sereh, the writer has been at some pains to learn whether it occurs in Cobb's disease. Cobb does not mention it, neither does Tryon himself, but in Tryon's paper "arrowing" is mentioned by several planters as one of the gen- eral signs of deterioration in the widely planted Rappoe or Rose Bamboo cane, and these men were planters in just those districts of Queens- land where the "gum dis- ease" prevails extensively. I quote from them as follows respecting this variety, which, at that time, was the common cane of Queensland : Moreover, the plant was tending to become shorter lived, a feature that appeared to be evinced by its frequent arrow- ing. Arrowing seemed to be the last event in the life of the plant — an indication that it had arrived at its term of life. For- merly an "arrow" was quite a curiosity, especially in the case of cane grown upon rich scrub soil. In fact it might be said that Rappoe generally did not arrow. Now it arrowed com- monly. (F. Young, of Fairy- mead.) Until three seasons ago he scarcely remembered to have ever seen an arrow ; now, if the plants were mature, nearly the whole plantation arrowed. (H. O. Nott, of Windemere.) f'g- 'I.* Fig. 12.| por some years past the cane has arrowed to a larger extent, especially in the sandy flats, and much more so than in for- mer years. (Mr. McGuigan, Island Plantation, Mary River.) *Fig. 11. — Longitudinal section through basal part of inoculated sugar-cane 8 or 12, showing red nodes (dark cross-stripes) and buds pushing at X,X, X, X. Photographed about May 6, 1903, i. e., 3 months from date of inoculation. fFic. 12. — Longitudinal section through a mottled (red and yellow) bundle from the stem of a sugar-cane inocu- lated with Bad. vascularum, showing how the bacteria fade out above into an amorphous red mass. Slide 310 (17. For a detail see fig. 26. 20 BACTERIA IN RELATION TO PLANT DISEASES. Others, as might be expected, state that they have observed no evidence of deterioration in the Rappoe cane. Arrowing is considered by one of these men as a sign of early ripening. Another had observed 15 years before that the cane would arrow where the land was dry and sandy and nowhere else on the plantation. To the following question : Have you noticed any ten- dency toward premature blossoming in sugar-cane attacked by your bacterial gum-disease? Cobb replied as follows: No; I can not say that I have noticed any earlier blossoming, but it might have occurred without my having noticed it. The chances of blossoming are reduced by the method of growing cane, the object being to prevent blossoming. Nevertheless the tendency might be there and, as the actual blossoming is prevented by the harvest, the tendency might escape notice. There is a tendency to what might be termed premature ripening of the cane, that is, the stalk takes on the ripe colors at an earlier stage. This of course is a feature of many plant diseases. ^00 mm Fig. 13.* Fig 1 4.f *FlG. 13. — Longitudinal section of stem of sugar-cane inoculated February 6, 1903, showing red stain inan affected bundle. There are no bacteria in the part here shown except the vague outlines of some at level of X. Slide 310 (20, lower left-hand corner. fKiG. 14. — Petri-dish poured-plate of Bacterium vascularum in nutrient agar, stock 716. This plate was made directly from yellow slime taken from the interior of "gummed" sugar-cane received from New South Wales in October 1902. Plate poured Oct. 16, photographed Oct. 30 by transmitted light. The organism grew slowly (temperature about 230 C). COBB'S DISEASE OF SUGAR-CANE. 21 ETIOLOGY. This disease is caused by Bacterium vascularum (Cobb) Greig Smith, a honey-yellow, one-flagellate organism, which forms the yellow slime always present in the vessels of diseased plants. It is a short rod occurring singly, in pairs, fours, or eights (end to end), and it often exists in practically pure culture in the fibro-vascular bundles of the diseased sugar-cane. The constant presence of this particular yellow organism in the diseased cane goes a long way toward establishing a causal relation, or would if it could be shown that nothing else is constantly or usually present. The writer knows, however, from personal observation, that other bacteria are sometimes present in the vessels, and Dr. Cobb's paper leads one to suppose that "traces" of other organisms are usually present in some part of each diseased cane. There are many parts of the cane, however, where Bad. vascularum exists in pure culture (Cobb, EFS.), and the fact that no other organism is necessary to the production of the disease has been proved conclusively by the writer's successful pure- culture inoculations. In October 1902, the writer received from the Richmond River district, Australia, through the courtesy of the Commissioner of Agriculture of New South Wales, three diseased canes, from two of which he had no difficulty in obtaining pure cultures of the organism by means of Petri-dish poured plates. No attempt was made to get cultures from the most badly decayed of these canes, because in places the bacterial ooze was nearly white, indi- cating an extensive mixture of organisms in the stem.* Nine Petri-dish poured plates were made from the interior of each of the other canes. One set of plates gave in addition to Cobb's organism a mixture of other things, mostly white bacteria of several sorts, but also colored colonies and molds; the other set of plates yielded practically pure cultures of Cobb's organism (fig. 14). Cuttings from healthy canes were obtained in November 1902 from southern Georgia and were planted in a hot-house in Washington. Before planting, the canes were shortened at each end and inspected critically by the writer. All were free from Cobb's disease and otherwise sound. Shoots from these canes were inoculated early in February 1903, with slime from potato subcultures of the yellow organism derived from the Australian cane. These inoculations succeeded beyond expectation, the canes developing local signs in a few days. Stripes extended up and down from the pricked part of the leaves and in one variety constitutional signs of a pronounced and typical sort appeared a few weeks later, the same being preceded by the enormous multiplication of the honey-yellow bacterium, first in the bundles of the inoculated leaves and later in those of the main axis of the cane and those of the uninoculated leaves. From the interior of the stem of several of these inoculated plants the organism was again obtained in pure culture. Inoculations made with sub- cultures of the organism thus isolated from the artificially infected cane were likewise successful, and the bacterium was again isolated from the diseased cane in pure cultures. INOCULATIONS. SERIES 1. 1903. The first set of inoculations (12 in all) was made on February 6, into common green cane. The inoculations were made by means of needle-pricks on the leaf blades about 1 to 2 feet from the stem. Two leaves were inoculated on each plant, those selected being about the tenth or eleventh from the base of the plant. About 20 to 30 fine pricks were *Respecting these canes Dr. Cobb makes the following statement in his Third Report: "There can not be the slightest doubt as to the identity of the organism cultivated by Dr. Smith and inoculated by him into American canes. * * * The second shipment was attended to personally by myself after it had been forwarded to me by my friend Mr. A. C. Barry, manager of the Broadwater sugar-mill, Richmond River, who at my request selected the canes and forwarded the same to Sydney. They were there unpacked, and sealed up with sealing wax and repacked at my laboratory. I examined the gum. The organism was the same species as that described in 1803 and reported on subsequently as the cause of the newly discovered disease for which I proposed the name "Gumming of the sugar-cane." 22 BACTERIA IN RELATION TO PLANT DISEASES. made on each leaf, some in the midrib, but the majority in the tissue on either side of it. The pricked area covered about 4 sq. cm. Three subcultures from as many separate yellow colonies on the plates poured from the Australian cane were used. Many leaves were pricked as checks, but these never developed any disease. Until nightfall the inoculated leaves were protected from the light by paper, but the air was not shut off. A semitropical temperature was maintained in the greenhouse and the plants grew rapidly, an important factor. Three weeks after the bacteria were introduced by the needle-pricks the inoculated leaves showed white stripes, in which reddish or brown dead spots and stripes soon appeared. These stripes had their beginning in the inoculated part of the leaf and slowly spread upward and downward. For some weeks there were no further signs of infection; then white stripes appeared on other leaves (uninoculated ones on the same shoots) and, somewhat later, the red or brown stripes, with shriveling of the leaf -parenchyma. At this time the plants were considerably dwarfed, and this nanism finally became very marked (fig. 15). The condition of these plants at the end of two months (April 3) was as follows: (1) The leaf 1 b is dead from the punctures down to the stem on each side of the midrib. The distance from the pricked area to the top of the sheath is 19 inches. The length of the sheath itself is about 1 foot. The leaf is dried out on the margins and brown above the pricked area for a distance of 2.5 feet. The terminal one foot of the leaf is bright green, and the midrib is green throughout its whole length. The signs on leaf / a, which is about the same length as the other, are the same, namely, dry brown margins about 0.25 inch or more in width extending down to the sheath and upward to within a foot of the tip. The only marked difference in the upper end is that the extreme tip has lost its chlorophyl and a portion of the leaf inside of the dry brown margins is also yellow. The midrib is green throughout. The yellow bacterial gum is oozing out of the green midrib about 9 inches above the top of the sheath, i. e., from the pricked portion. Two or three little, bright yellow gummy masses are present on the inner face of the leaf at this point. This leaf was severed from the stem 3 inches above the sheath. Photograph made. The stem of the plant 1.5 feet from the ground is about an inch in diameter. It has 17 leaves and the height of the plant is about 7 feet. The secondary signs of the disease are conspicuous on most of the upper leaves. They have ap- peared as white or yellowish- white longitudinal stripes, which in some cases have already be- come reddish-brown, not unlike the signs I obtained some years ago by inoculating Bad. hyacinlhi into amaryllis leaves. They extend in many cases from the base of the leaf -blade, in some instances half-way up, and in others entirely to the apex. This striping shows on live of the upper leaves. One of the most conspicuously striped leaves was cut off and photographed (fig. 16). (2) This plant is about 6 feet high and bears 13 leaves. The cheek-leaf is normal; that is, only the tissues in the immediate vicinity of the punctures have turned white. The tissue in the pricked area as a whole is bright green and normal in appearance. This is the plant from which a pricked (inoculated) leaf was cut off some weeks ago and found to contain the organism. That leaf (the foot-long stub) now has dry-shriveled nearly to the sheath. This is 2 a. The other inoculated leaf (2 b) is dried out and has become brown on the margins from the pricked area down to the sheath and up to within 1.5 feet of the tip on one side, and entirely to the M'n; 15. Row of inoculated badly dwarfed sugar-cane (at X) with a row of taller, sound, uninoculated cane of same age to either side. Hot house, Washington, D. C, 190.?. Drawn from a broken negative. cobb's disease op sugar-cane. 23 tip on the other side. The midrib is bright green the whole length. The needle-punctures were made about a foot from the leaf-sheath. Secondary signs are visible on three of the upper leaves of this plant as long yellowish stripes, interrupted in one of the leaves. In places on one leaf the stripe is dead and reddish-brown for a distance of 8 inches and a breadth of one-sixteenth to one-eighth inch. On another there is a marginal dead stripe 2 feet long. (3) Leaf j a is dead entirely from the tip down for a distance of 3 feet, and the rest of the way it is dead on the margins, but the midrib is green. It was pricked about 18 inches from the top of the leaf-sheath. The leaf-sheath itself is about a foot long. Leaf 3 b is dead at the tip and down for a distance of 3 feet and the midrib is dead a foot farther down, but the margins are green. In the lowest foot both midrib and margins seem to be normal. There are no secondary signs on this plant, which is 6 feet tall and bears 13 leaves. (4) Leaf 4 a is dead from the apex down a distance of about 3 feet, while for an additional 2 feet it is dead on the margins but green in the midrib. Punctures made about 9 inches from leaf-sheath. Leaf-sheath is about 9 inches long. Leaf 4 b is dead from the apex down a distance of about 4 feet. A foot farther down it joins the stem. The lowest foot of the leaf is dead on both margins but green in the midrib. The plant itself is about 5 feet high and shows conspicuous secondary signs; 18 inches from the ground the diameter of the stem, including the leaf-sheaths, is about half an inch. It bears 12 leaves. Two of the upper leaves are dead almost all the way down and two more show brown striping on one side. The upper two are flabby. Plant brought in and photographed. It is the larger of the two of this date. Bad. vase ul arum was plated from the stem of this plant. A white gas-forming organism was also obtained from two places in this stem, 8 inches apart. (5) This plant is about 4 feet high and is rather small. Diameter of the stem a foot from the ground is a little more than 0.25 inch. Both inoculated leaves are dried up throughout. The plant bears 10 leaves, not counting some small basal ones which are dried out. It shows distinct secondary signs, which consist of whitening and striping of the leaves. (6) Plant 6 is about 6 feet high. At a foot from the ground the diameter of the stem is nearly an inch. It bears 14 leaves. Inoculated leaf-blade 6 a, which is about 5 feet long, has dried out throughout, except about a foot at the extreme base, and even here there are brown spots on the margins running down to the leaf-sheath. The midrib, how- ever, is bright green. Leaf-blade 6 b is dead throughout, except about 9 inches of the mid- rib at the base. The sheath itself is also drying out. This plant shows also conspicuous white, yellow, and brown striping on 3 of the upper leaves (secondary signs). (7) Plant 7 is about 6 feet high. A foot from the ground the stem is 0.75 inch in diameter. It bears 13 leaves. Leaf 7 a, which is about 5 feet long (or 6 feet if the leaf-sheath be included), is dead throughout, including everything except the base of the leaf -sheath. Leaf-blade y b, which is like y a in size, is also dead throughout, including most of the leaf-sheath. There is distinct etiolation of the margins of two of the uninoculated upper leaves, and there are also red lines and specks in these etiolated parts for a distance of a foot in one leaf near the base and a less distance on the other. (8) The diameter of the stem of this plant a foot from the ground is about 0.625 inch. It bears 13 leaves. The check-leaf pricked on March 2 with more than 100 needle punctures at 6 inches from the stem, is bright green in the pricked area, the only dead parts being the tissue immediately around the punctures, which is white. Leaf o' a, which is about 4 feet long (5 feet with sheath), is dead throughout, except extreme base of sheath. Leaf-blade S b is dead for a distance of 4 feet down. The basal 1 foot is dead on the margin but green in the midrib. There are distinct signs of secondary infection on the upper leaves which are Fig. 16.* *Fig. 16. — Middle part of one of the uninoculated higher leaves of the common green cane, showing etiolation due to secondary infection with Bacterium vascularum: Margins green and normal in appearance, midrib green. To either side of the midrib are white stripes with red-brown dead spots and stripes in them. The white striping continued to the apex of the leaf. In this stage of striping bacteria are usually present in some of the bundles of the affected part of the leaf. Drawn from a broken negative. 24 BACTERIA IN RELATION TO PLANT DISEASES. whitening in stripes along the margin. Four leaves show these signs and a fifth shows median white striping with dead brown patches toward the base, in the stripes. (9) Plant 9 is about 4 feet high and about half an inch in diameter at 18 inches above the ground. It bears 14 leaves. Leaf g a, which is about 5 feet long, is dead throughout, except possibly the extreme base of the sheath. Leaf g b, which is about 5 feet long, is dead throughout, except a small portion of the basal part of the midrib and a part of the sheath. The plant shows distinct secondary signs. One entire upper leaf, which is about 6 feet long, is dead throughout. Another large leaf is drying out on one margin for a distance of 3 or 4 feet and the apex is flabby. A third leaf is dry from the base upward to the tip on one side. A fourth small central leaf is flabby. (10) No. 10 is a small plant. Height 3.5 feet; diameter a foot from the ground 0.25 inch; number of leaves 11. Inoculated leaf 10 a is dead throughout, including all the visible part of the leaf-sheath. Leaf 10 b is dead throughout, including all the visible part of the leaf-sheath. The top of the plant is also dead ; that is, three leaves. Three additional upper leaves are dying in stripes. Photograph made. It is the smaller of the two plants photographed to-day (April 3). (11) No. 11 is nearly an inch in diameter at 1 foot from the ground. It is about 5 feet high. It bears 14 leaves. Leaf 1/ a is dead throughout, unless it be a portion of the leaf-sheath. Inocu- lated leaf 11b, which is about 6 feet long, is dead and dry throughout, except the basal 1 foot of the midrib and a portion of the middle part of the sheath. This plant also shows conspicuous secondary signs. Of the upper leaves, five are distinctly striped and the two uppermost are flabby. One long leaf is dead throughout, except the basal 1 foot, and even this part is dead on the margin, the midrib being the only live part. There is conspicuous etiolation of the margins of some of these leaves. This sign precedes the drying out. There are also distinct, narrow, brown or red-brown interrupted stripings in the basal portion of the etiolated part. One of these upper leaves was pricked with about 150 needle-pricks (sterile) at 3 feet from the leaf -sheath on March 2, as a check: No striping has resulted. The needle-punctures themselves have a narrow white margin around them but the tissue between the pricks, which are set in close together, is still bright green. I mean by this that the pricked area is not over 1.25 inches broad (width of the leaf) by 1.5 inches long, and yet with all this number of punctures in it there has been no general death of the tissue, and no up or down striping such as took place within a few days in case of every one of the inoculated leaves. (12) Plant 12 is about 3.5 feet high; the diameter at 1 foot from the ground is about 0.5 inch; it has 12 leaves. Inoculated leaf 12 a is dead from the tip down a distance of about 3 feet. From there to the sheath, a distance of another foot, the midrib is green, but the margins are dead. Inoculated leaf 12 b, which is 4 feet long, is dead in the upper 1 foot and dead on both margins all the rest of the way down to the sheath, but the midrib in this part is green. This plant also shows distinct secondary signs on five leaves. One uninoculated leaf is dead from the apex down a distance of 3 feet. Another is dead on both margins for a long distance down and also has reddish-brown, narrow, interrupted stripes in the green part farther down, and a third has conspicuous etiolation near the midrib on one side, and there are a great number of narrow interrupted reddish-brown spots in this etiolated part. The two rows of cane to either side of this row are at least 3 feet higher and are altogether thriftier, showing no signs whatever comparable to those just described (fig. 15). Neither of these vigorous rows to either side are outer rows, but they are at the same time not of the same variety, one of them being Purple Striped cane and the other Striped Green cane. At the end of 2.5 to 3 months all of the inoculated leaves and some other large leaves were shriveled and the tip of the terminal bud had gone over into a bacterial decay. All of the inoculated plants succumbed. The leaves at this time were not all dead and the well- developed lower part of the stem was green and sound externally. The internodes were shortened and the basal buds were either much swollen or beginning to push. The plants stood in a dry house and had not received an excessive amount of water. I )uring the pro- gress of the disease some shoots and roots pushed out from the nodes at the base of the cane. When the first signs appeared on the inoculated leaves one was removed and thin cross- sections were examined under the microscope for the presence of the bacteria. These were found in great numbers in the bundles about 18 cm. above and for some distance (10 cm., perhaps) below the pricked area. Later the bacteria became very abundant in the stems. They were also found in bundles in the etiolated parts of the uninoculated leaves. Plates of nutrient agar poured from several plants and from different heights in the stem yielded PLANT BACTERIA, VOL. 3. PLATE 2. Cobb's disease of sugar-cane. Plant No. 6, inoculated on the blades of two leaves by needle-pricks. Feb. 6, 1903, with a pure culture of Bacterium vascularum derived from Australian cane. The vessels of the stem from its base nearly to the terminal bud were filled with the yellow bacteria. The leaf-sheaths on the inside were also very gummy from the presence of bacterial ooze. Photographed May 1903 Variety, Common Green cane. COBB S DISEASE OF SUGAR-CANE. 25 in pure culture great numbers of the same yellow organism which had been isolated from the Australian cane (fig. 1 7). The bacteria were present in very great numbers and usually in pure culture in the yellow bundles of these stems. The extent of this stem infection (resulting, be it remembered, from a few needle-pricks on leaf-blades several feet removed from the nodes) will be better appreciated if transcripts are inserted from notes made at the time the canes were cut. For this purpose some observations made at the end of the third month will suffice. Brought in to-day (May 6, 1903) 4 sugar-cane plants, inoculated Feb. 6. They are all badly diseased. Photographs were made of No. 6 (plate 2) and of Nos. 8 and 12 together (plate 3). No. 6. — The height of this plant is about 70 inches to the curve of the top leaves. The height to the youngest leaf is 40 inches, i. e., the plant is small for its age (6 months, nearly). Both of the inoculated leaves are dead and dried, including the leaf-sheaths. Of the leaves lower down one is dead and dry and has fallen off, and three others are partly dead — that is, dead at the tips and along one margin but green in the midrib and on the other margin. Farther up, three large leaves are dead throughout, except the sheath. Four others are dead at the tips and in the case of one for 4 feet downward. The small terminal leaf is also dead at the tip. The diameter of the stem at the surface of the earth is 0.68 inch. The diameter of the stem a foot from the ground is 1 inch, including the leaf- sheaths. At the base of this plant there are three young shoots which are healthy in appearance, and live buds in various stages of pushing, one of them being an inch long. Signs of disease in the shape of bacterial slime are present under the leaf-sheaths a few inches from the base of the stem, the inner surface being gummy and wet. The inter- nodes are short; the longest ones are only about 2 inches in length, but the surface of the lower in- ternodes looks healthy. Gum- ming of the inner surface of the sheaths continues up a distance of several feet, and on the inner surface of one sheath the bacte- ria are abundant enough to look yellowish; the organisms have also broken through from the interior of the stem in three places and come to the surface as yellow slime on as many internodes. The sheaths are more or less rusty-brown spotted, especially in the gummy parts on the inner surface. There is no indication of rottenness, but a considerable quantity of gum covers the inner face of many of the leaf-sheaths, while the main axis is still green. Aphides are not present. Most of this bacterial slime has come from the interior of the leaf-sheath, but some from the interior of the stem. There is a tendency in this, just as in two other plants brought in to-day, for the terminal bud to push out sidewise. In the interior of the plant above the terminal bud there is much red-brown spotting of the tissues and much bacterial gumming. This rusty -red spotting of the inner, rolled-up leaves — those that are protected from the light and from surface contaminations — is very conspicuous. Terminal bud seems alive, but is crowded down on itself (zigzag) by the drying up and gumming together of the sheaths above it. The base of the uppermost young leaf is alive and wraps around another smaller leaf, the top of which is dead and gummy. Fig. 17. *Fig. 17. — A pure agar culture of Bacterium vascularum poured from the interior of plant No. 9. at 200 to 280 C. Age 8.5 days 26 BACTERIA IN RELATION TO PLANT DISEASES. On cutting across the basal secondary shoots an inch from the stem their tissues seem to be sound (hand-lens). The main axis was then cut open and examined. The stem was first cut crosswise in the middle of an internode about 6 inches from the base and then in other places. Some of the vessels are gummy (yellow) and others are distinctly red stained. This red staining, as in the stems previously dissected, is much more conspicuous immediately below a node than it is above a node. A few minutes after cutting, the yellow bacterial ooze was very distinct from many of the bundles. There are cavities in the upper part of the stem. In other places, as well as those first recorded, the red stain is greater just below the node than it is above. The middle 3 inches of the stem was slit longitudinally and found to contain a closed bacterial cavity which is 0.75 inch long (estimated) and 0.1875 mcn wide in the widest part, full of pale yel- low slime. Slit the basal 3 inches longitudinally and found the disease in the base in the shape of reddened vascular bundles and yellowed ones from which ooze the yellow bacteria. The longitudinal section shows marked red-browning in the vicinity of the nodes and some red vessels in the internodes and, guile interestingly, there is in many cases a red and yellow stain in the same vascular bundle — red in one place and yellow in another, showing clearly that the red stain is closely associated with the yellow slime. This material was saved in alcohol and may be identified by the fact that it is cut in the form of thin slabs. No. 8. — The height of this plant is about 58 inches to the curve of the top leaves. It is badly diseased, both of the inoculated leaves being dead to the stem and also all of the lower leaves, while the upper leaves are dead at the tips and dried up along one or both sides, but with the midrib living. The terminal leaflet is flabby. The stem is very short. This plant is especially interesting from the fact that 6 inches above the ground the main axis is doubled and twisted and wrinkled crosswise in a swollen mass, the terminal growth being stopped by a drying and gumming of the tissues above the terminal shoot. Lower down the terminal shoot zigzagged and tried to push out sidewise in the same manner as shown in fig. S. Around this swollen, bulged portion the leaf-sheaths are rusty- brown or pale green and very gummy, the bacterial slime occurring in sufficient quantity to show itself distinctly yellow. The gum does not string up much, but it is sticky to the fingers and so copious that both of my hands are covered with it. The interrupted rusty-brown striping is con- spicuous in the leaf-sheaths that are buried deep in the interior of the shoot, and some very interesting and conspicuous examples of this have been saved in alcohol. The terminal bud is not killed — only gummy and glued up and squeezed together (endwise) by the effect of the disease on the sheaths outside of it. There is an abundant yellow bacterial ooze from cross-sections of the stem of No. 8. Some of the vascular bundles are also reddened. There is distinctly more reddening of the vascular bundles immediately under the nodes than there is immediately above the nodes. These red vessels are mixed in with those giving rise to the yellow ooze. Slit the stem longitudinally. Some of the vessels are bright yellow from the presence of the bacteria and others are red. These are mixed in. Specimens put into alcohol. I am inclined to think that the reddening of the bundles is a later stage in the disease than the yellowing. Three cross-sections were cut from No. 6 and one from No. 8 (smaller) and a photograph (X3) made of them together, designed to show the yellow bacterial ooze and also the red bundles (plate 1). A photograph was also made of a longitudinal section of No. 6, showing the bacterial cavity (fig. 4). On the same negative is a longitudinal section of No. 12 (fig. 11), showing red stain in the nodes, and red and yellow bundles in the internodes. No. 12. — This plant is a little smaller than No. 8. The two inoculated leaves are dead and dry and all the upper leaves are dead, including the terminal one. Lower down there are two partially green leaves. Both, however, are dead at the tip and have dead stripes on the margin. This plant has been allowed to stand too long and some of the outer, dead leaf-sheaths are moldy, which was not the case with either of the others. The inner sheaths are rusty-red spotted, as in case of the others, and there is much bacterial gumming between them. In some cases the spotting is quite brilliant — almost a vermilion red. The gumming here also is abundant enough to stick up my fingers very markedly. About 6 inches from the ground there is the same effort of the shoot (terminal bud) to wrinkle, swell, and push out sidewise. Above this swelling (6 inches to a foot) the sheaths surrounding the young leaves are very gummy, and there can be no doubt that this sidewise bulging is due to the gumming and pressure of the outer leaves around the terminal bud. Unwrapping the youngest leaf visible, which was dead at the tip, I find inside of it a younger leaf, also dead at the tip, and the terminal bud is in worse condition than in either of the other plants. The basal buds to the number of seven have begun to push. These three stems (6, 8, and 12), stripped of their leaves, were photographed together (fig. 9), and 8 and 12 were photographed together before removal of the leaves (plate 3). PLANT BACTERIA, VOL. 3. PLATE 3. Cobb's disease of sugar-cane. Plants 8 and 12, inoculated Feb. 6. 1903, with a pure culture of Bacterium vascularum plated out of sugar-cane received from New South Wales. Inoculated by needle-pricks only, each plant in two leaf-blades at some distance from the stem. Most of the leaves dead and dry when photographed, stem bundles filled with the honey-yellow slime. The terminal bud pushing out sidewise may be seen near the base of each cane. Photographed May 1903. About one-twelfth natural size. Com- mon Green cane. COBB'S DISEASE OF SUGAR-CANE. 27 On cutting open the stem of No. 12, red bundles were found to the extreme base. These are mixed in with yellow ones, from which there is a distinct bacterial ooze. Slit the basal 4 inches longitudinally. Here again the stem shows a series of red cross-stripes corresponding to the nodes. In the internodes some of the bundles are bright yellow, others are red, and still others are mixed (mottled) red and yellow, and there are bacterial cavities. The red stain is evidently a later stage, just as the black stain is a later stage in sweet corn attacked by Bacterium stewarti. No. 5. — This plant is a little smaller than No. 12. All the basal leaves are dead. Both of the inoculated leaves are dead, including the sheaths. The terminal leaf is flabby and all the other leaves are more or less diseased, being dead at the tips and with dead margins for a long distance down. The base of the plant for a foot up inside the sheaths is very wet and gummy, just like the others. These sheaths are rusty spotted. There is a peculiar smell to the disease not unlike that of some bacterial cultures, but I am not able to define it any closer. The inner sheaths are green and some of them are badly rusty spotted. The color of the spots is a brick red. Cut open the stem at the base; the vascular system is stained a reddish-brown. This reddish- brown staining is very conspicuous. The terminal bud appears to be normal. The tissues in its vicinity appear to be sound in places, diseased in others. Other plants in this series gave similar results. These were examined in April or else later on, in May and June (plate 4). January 5, 1904, only one good ratoon cane has come up from the stools. In addition there are two small canes and a shoot a foot high. The large cane is free from signs of disease at the base, or nearly free. The little shoot is free. Had these stools been healthy they would have given many tall shoots. SERIES II, 1903. Fourteen shoots of Louisiana cane, Variety No. 74, were inoculated February 9, 1903, from two potato cultures 1 1 days old by making needle-pricks on two leaves of each plant, as in case of the first series of inoculations. At the end of 5 weeks all showed local signs of the disease and six of the shoots also developed constitutional signs, but on the most of them these were not pronounced. The greater number of these plants proved very resistant, i. c, subsequently they grew out of the disease, although they were somewhat dwarfed by it. At the date of inoculation these canes were the most vigorous ones in the patch, but when they were cut out for exam- ination, on July 20, the largest and best canes were the sixteen uninoculated ones. These big canes were small and inferior at the time the others were inoculated. From the inoculated canes dwarfed cuttings, containing red vascular bundles, were saved for planting, but no yellow slime was present in the bundles of these stems ; at least none that could be detected with the hand-lens, and only one reddish-yellow bundle was seen. SERIES III. 1903. Eight shoots of common purple cane planted in the greenhouse on November 15, 1902, were inoculated with Bad. vascularum on May 4, 1903. The organism used for these inoculations was from cultures 7 days old on potato cylinders kept at room-temperatures. These were subcultures made from colonies on Petri-dish plate No. 9, poured April 16, 1903, from plant No. 11 of the common green cane inoculated February 6, 1903. The plate was absolutely a pure culture of Bad. vascularum . The inoculations were made by spreading three or four 3 mm. loops of the organism over about 6 or 8 sq. cm. of leaf-surface, generally about a foot above the top of the leaf- sheath, and then making about 100 to 150 needle-pricks through this area. After the pricks were made, two or three more loops of the organism were rubbed over the pricked area. Two leaves on each plant were inoculated. Leaves near the top of the plant were chosen when possible. The canes were quite tall at this time — averaging perhaps 6 feet, and many of the leaves reached to the top of the greenhouse (10 ft.?). The inoculated areas were protected from the sun for a day by cuffs of manila paper. Five checks were made. The 28 BACTERIA IN RELATION TO PLANT DISEASES. inoculated plants were given numbers from 27 to 34 inclusive, and the inoculated leaves were labeled 27a, 27ft; 28a, 286, etc. For these inoculations tubes Nos. 1 and 3 of April 27, 1903, were used. On May 5, 1903, finished inoculating the row of common purple cane. Four plants, Nos. 35, 36, 37, and 38, were inoculated in just the same way as those of preceding day, with one exception; in this case the plant was inoculated in the stem and on one leaf instead of two leaves. This was plant No. 38. Tube No. 4 of April 27, 1903, from poured plate No. 6, of April 16, 1903, was used for these four plants. On May 14, 1903, all these plants showed the effects of the inoculation. A distinct discoloration about the punctures was noticed three days earlier and to-day the punctured area is dead, entirely or in great part, on at least eight of the inoculated leaves. All the other inoculated leaves show a marked reddish-brown streaking or spotting in the punctured areas. The check leaves show no such discoloration. The punctures on these leaves show simply as small white points. On June 13, 1903, common purple cane No. 33, inoculated May 4, 1903, from tube No. 3, April 27, was broken over by accident and brought in for examination. It is a shoot about 9 or 10 feet high, with a thick stem and large green leaves, but the basal ones are mostly dead (normal). The inoculations were on the blades of two leaves about midway down the stem, and the pricks were about 8 inches from the sheath on both leaves. The midrib of each inoculated leaf is alive throughout. To either side of the midrib on one leaf are dead areas for a distance of 2 to 3 feet. The other leaf is much the same, except that on one side the dead portion is not so long, perhaps 18 inches. Above this, however, in the green part are yellowish and rusty- yellow dead stripes for a distance of another 18 inches. The outer edges of the sheaths are also dead nearly or quite to the nodes. The ends of both leaves are wholly alive. Made cross-section of the sheath at the extreme base and examined it under hand-lens for bac- teria. In one they seem to be present in small quantities, but nothing very definite in either one under the hand-lens. These must be looked at under the microscope to determine the presence of bacteria in the vessels of the inoculated leaves. Cut the stem 6 inches above the base. No evidence under the hand-lens of bacteria in the bundles. Cut 6 inches higher up and examined again. No bacteria visible in the stem when examined under the hand-lens. Cut another 6 inches up. Here, also, the stem appears to be perfectly sound under hand-lens. Cut another 6 inches up. The whole stem appears here also to be sound. We are now getting into the vicinity where the inoculated leaves join on to the stem. The whole stem appears to be sound. Slit the 6-inch segment below the last longitudinally and examined. No evidence of disease. Cut another 6 inches up (cross-section). One red bundle in the internode, which is indicative of the disease. Slit this segment longitudinally and examined: Distinct reddening at both the included nodes, but very slight in comparison with the effect on the common green cane. At each node there are a dozen or more red bundles. No ooze. This is a very resistant variety. Cut another 6 inches up. No evidence of disease. Slit the stem longitudinally. No evidence. The whole upper part of the stem for a distance of several feet was now slit longitudinally and examined for the presence of bacteria. No stain in the bundles; no evidence of disease. The disease is confined to the two inoculated leaves, to the two nodes which bear these leaves, and to the immediate vicinity of these two nodes. On subsequent examination there seems to be a very light red stain on one side of two nodes next farther up. These also were put into alcohol for further study. A microscopic examination showed the bacteria to be present in the bundles of the midrib of the inoculated leaf-blades and in the bases of the leaf-sheaths. On August 8, 1903, two of the plants of common purple cane which were inoculated with Bat /. vascularum, May 5, 1903, broke off with their own weight. The two plants are Nos. 37 and 38. No. 37 was inoculated in two leaves, while No. 38 was inoculated in one leaf (386); and through various leaf-sheaths into the stem (38a). On examination the conditions were as follows: PLANT BACTERIA, VOL. 3. PLATE 4. I m Rot of sugar-cane due to Bacterium vascularum. Leaves removed and top of cane split longitudinally to show advanced bacte- rial decay. This decay at the top includes all but the surface of the cane: Nodes red, cavities in the internodes full of yellow bacteria. A little far- ther down the cavities disappear. Base of cane sound. Inoculated Feb. 6, 1903. Photographed June 15, 1903. cobb's disease of sugar-cane. 29 Externally, plant 38 shows much more disease than 37. Plant 38 is about 7 feet high and is almost entirely dead as regards leaves. The basal part is devoid of leaves for a distance of 2 feet from the ground and the stem is about 1.5 inches in diameter. The first leaf is partially green, but is beginning to die along the margin. The next leaf is entirely dead. The sheath of this leaf was pricked through when the stem was inoculated. The next two leaves are about half dead, while the next (which is the inoculated one, 38 b) is about three-fourths dead. Two or three of the leaves higher up show traces of green, but the entire terminal bud, com- prising about 1 5 leaves, is entirely dead and dry. All these leaves are rotted at the base, so that the whole mass of them came loose from the stem. On removing the leaves from the stem, 10 or 1 1 of the uppermost buds are seen to be well started, some of the upper shoots being 4 inches long. Half of the internode into which pricks were made has a water-soaked appearance and there is a large black wound at one end of it. The outside of this internode is sticky to the touch. None of the internodes above this point are healthy. They show water-soaked areas all along one side. In the lower foot of this stem there are no red bundles, but above that point one or two red bundles appear. The number of diseased bundles increases from this point up, until at a distance of 4 feet from base of stem there are at least 30 diseased bundles. Some are black, others red, while still others are yellow, and after a few minutes standing show a yellow ooze from the cut surface. The upper 1.5 feet of this stem was photographed in toto to show development of shoots (fig. 10). The diseased bundles in the stem are all on one side, which corresponds to the needle pricks in the stem higher up. In the internode next below the pricked one there is a large group of diseased bundles, black, red, and yellow. Yellow ooze shows quite distinctly from about 20 of these. There are at least 100 bundles affected in the inoculated internode. About half of these are black and a few red, but there are a large number which show the yellow bacterial ooze. All of the internodes above this point, 12 in number, are very badly diseased. About one-quarter of the bundles are entirely black in these, and nearly all the others show yellow ooze. There is a small cavity along one side of the stem in three of these internodes, which contains yellow slime. The plates poured from the first internode below the inoculated one miscarried. I did not split the stem longitudinally, as I wished to save it for planting. On sectioning: Two cuttings apparently not diseased; four cuttings slightly diseased; five cut- tings badly diseased. Plant 37 is about 8 feet high and externally looks to be a very healthy cane. Both inoculated leaves are entirely dead, but with one or two exceptions all the other leaves seem all right except one or two of the upper, which were torn in the ventilator. The basal part of the stem is 1.5 inches in diameter. On cutting the cane, the first red bundle appeared about 2 feet above the base of the stem. In the cut a foot higher there seemed to be no diseased bundles. None showed farther up at all. Cuttings saved for planting: 3 very slightly diseased (1 or 2 bundles red) ; 6 not at all diseased. On January 5, 1904, the remainder of these canes were cut out, with the following results: 27 a and b. — Very large cane, no trace of red bundles, nor other signs of disease in either of the nodes into which inoculated leaves ran. Another cane, apparently inoculated, is sound. J4 a and b. — Excellent cane about 10 feet long. A few red bundles run in at. two nodes, which probably bore the inoculated leaves. 3 •. . .*" B . • . W'"> A Hkfe^>_ 1 Fig. 40.* among which the disease previously existed, even to a slight extent. Only one of the field experi- ments is worth reporting in detail. It is as follows: In 1896 thirteen hills of Manhattan sweet-corn were planted in one row. In each of the first seven hills there was placed, at time of planting, a hand- ful of dirt taken from soil in which the disease was prevalent the preceding season. The remaining six hills were left untreated for comparison. When the plants were a few inches high they were thinned to l< iur in a hill. A few of the plants in the inoculated hills began to wither before they were a foot high, and from this time on they withered one by one, until on July 20, when the kernels were "in the milk, " all of the inoculated plants except two were either dead or dying. At this date, not a single plant in any of the uninoculated hills showed any symptoms of the disease; but later in the season several of the planl ; became affected. How they came to be affected is not known. While this exper- iment was not wholly satisfactory the results tend to show that the disease is communicable. M'ii.. 40. Same as fig. 39, but from another plant and enlarged 6 times, to bring out bacterial ooze more distinctly. STEWART'S DISEASE OF SWEET CORN (MAIZE). 93 Several attempts were made to inoculate sweet-eorn by puncturing the stem near the ground and inserting a small quantity of the diseased tissue of an affected plant. In some of the large varieties the inoculated plants remained healthy to the end of the season. In the smaller varieties the disease usually appeared in from two to four weeks after inoculation, but the uninoculated plants used as a check, likewise, invariably became affected to a considerable extent, so that no trustworthy informa- tion could be obtained from such experiments. Finally it became evident that the plants must be grown in pots of sterilized soil if the inoculation experiments were to furnish results of any value. A quantity of soil was thoroughly sterilized in steam sterilizers and placed in large pots. On July 3 Early Cory sweet-corn (grown in Iowa) was planted in the pots and inoculation experiments with pure cultures of the yellow germ were started. The pots were allowed to remain uncovered [out of doors] ; otherwise, all precautions were taken to prevent contamination. Nevertheless, an undoubted case of the disease was found in one of the check pots on August 3, and later several others were found. This meant that diseased seed had been used and the experiment was worthless except to prove that infection may be brought about by the germs which cling to the seed. This appears to have been the only inoculation experiment attempted by Mr. Stewart with pure cultures. At least it is the only one mentioned, and no statement is made as to how many, if any, of the inoculated plants contracted the disease or how near these were to the check plants. When the rainfall was not sufficient they were watered with sterile water. Three unsuccessful attempts were made to produce the disease in yellow dent field-corn by inocu- lation. On August 20, 1895, ten plants of yellow dent corn (variety unknown) were inoculated by puncturing the stem at the surface of the soil with a sterilized scalpel and then inserting into the punc- ture a small quantity of the yellow substance taken directly from the interior of the stem of a diseased sweet-corn plant. These plants were under observation until frost (about October 7), but none of them showed any symptoms of the disease. On July 12, 1897, twenty plants of yellow dent corn, variety Golden Dent, were inoculated in the same manner as in the experiment of 1895. None of these plants developed outward symptoms of the disease, but a month after inoculation it was found that in several of the plants the yellow bacillus had ascended a few of the tibro-vascular bundles, where it was visible to the naked eye as far as the third node above the point of inoculation. It was notice- able, however, that it occurred only in bundles which had been ruptured by the needle used in inocu- lation. In 1897 a 50-foot row of the same Golden Dent corn was inoculated by placing in the drill, at time of planting, a liberal quantity of soil, in which diseased plants had grown the preceding season. Not a single plant developed the disease. Pop-corn, also, has resisted all attempts at inoculation. In 1897 a 50-foot row of pop-corn, var- iety Maple Dale, was inoculated by putting diseased soil in the drill at time of planting. None of the plants became diseased. On July 8, 1897, twenty plants of the same variety of pop-corn were inocu- lated by puncturing the stem and inserting diseased tissue into the wound, as in the experiments with sweet-corn and field-corn. None of the plants became diseased, but as in the case of field-corn the germ could be seen in some of the bundles up to the third node. Oats, inoculated by means of diseased soils, and teosinte (Euchlaena luxurians) , inoculated both by puncture and diseased soil, gave negative results. Stewart believed the plants became infected through their root-system. He says in so many words: Since the cause of the disease is entirely within the tissues and probably gains entrance through the subterranean parts of the plant, the application of fungicides to the parts above ground must be absolutely without avail. Stewart observed the disease in Early Cory, Manhattan, and many different varieties of sweet corn on Long Island. In 1897 he found it very prevalent as early as June 12, among plants which were 8 to 10 inches high. In some plants he was able by means of the microscope to demonstrate the presence of the bacteria in the bundles before the yellow slime could be detected with the naked eye, and before there were any outward manifestations of the disease other than dwarfing. In plants dead of the disease he found an abundance of bacteria in all parts of the stem "up to the tassel and in the ear." It is easily demonstrable, he says, that insects are not 94 BACTERIA IN RELATION TO PLANT DISEASES. responsible for the disease. Stewart also makes the following interesting observations on the relation of weather to the progress of the disease : If plants are examined in periods of wet weather it will be found that the amount of the yellow substance which they may contain in their vessels without showing outward symptoms of the disease is much greater than it is in dry weather. * * * It is interesting to observe the effect of alternat- ing periods of wet and dry weather. For about one month preceding July 12, 1897, it was very dry on Long Island- — so dry that in the latter part of the period some crops suffered severely. During this time the corn disease was very destructive. Then came about three weeks of rainy weather fol- lowed by a short period of dry weather. Many plants which were partially dead revived during the rainy season and promised to outgrow the disease, but as soon as the rains ceased they suddenly collapsed. In 1898, as a result of observations in Michigan, I pointed out the extreme probablity of the bulk of the infections taking place not through the roots but in the seedling stage of the plant through the water-pores. Thereafter I contented myself with a study of the cul- tural characteristics of the organism which had been sent to me by Mr. Stewart for that pur- pose, hoping the latter would go on and complete the gaps in his infection experiments. This he did not do. In 1902, therefore, having an opportunity to study the disease on Long Island, the writer renewed his cultures and afterwards inoculated many plants in the city of Washington, where the disease was not then known to occur naturally. The results of these inoculations were very convincing, but before proceeding to an examination of them the reader will be interested in some statements regarding the origin of the cultures used for making these inoculations. FIELD OBSERVATION IN 1902. My observations on Long Island are recorded in the following transcript from a note book: Notes of July 16, 1902, on Stewart's Sweet Corn Disease. Found disease at William M. Croucher's place, on the Merrick Road, 1.25 miles from Jamaica, Long Island, New York. The disease occurred on a good quality of truck-land, loamy and not wet. This land rents for $10 per acre per year. Nothing in the surroundings to explain the appear- ance of the disease. Mr. Croucher, who is perhaps 30 years old and has been a trucker all his life, says he has seen the disease ever since he can remember. He says some varieties of sweet corn are much more subject to it than others. The Early Cory is one of the sorts most subject. His field of about 3 acres was in good condition as to cultivation. The plants were tasseling in part. The field was not badly diseased. He had already pulled out some of the diseased stalks, and I found 15 or 20 more. So far as I can judge, the disease is to be told by the shriveling of the leaves and by the pre- mature blossoming of the male flowers. At some distance the male flowers on the diseased plants looked white and dry. Those on the healthy plants were green or purplish. Every plant of this sort, namely, with white and dry spikelets and shriveling leaves, proved on cutting open to be full of bac- teria, although the stems were green and sound externally and in most cases all the bases of the leaves. The plants were also well provided with roots. As a rule such plants were somewhat dwarfed. I was surprised at the complete occupation of the vascular system — the whole length of the stem except in the extreme top. A cut anywhere else across the stem was followed by a yellow ooze from the vessels even as far up as a foot from the top, and undoubtedly the organism could have been detected still farther up with the aid of the compound microscope. The ooze from the bundles was yellow, especially in the lower part of the stem, e.g., one foot from the ground. In the upper part of the stems it was usually very pale. This I interpret to mean not two organisms, but two stages of one, the lower down, yellower ooze consisting of older organisms and growing perhaps in the presence of a greater amount of air. (See p. 58, and also Yellow Disease of Hyacinths, vol. II, p. 345.) Infection by boring or gnawing insects was out of the question in case of these plants. The surface was smooth and unbroken. There were no insect injuries and no ooze of the bacteria to the surface. Except in the extreme base of the stem (of which more later) the bacteria appeared to be confined quite strictly to the bundles. The tissue between was sound, that is, normal in color and free from any appearance of disease. The bacteria were not only very abundant in the bundles of the stem, but wire also in the green bases of many of the shriveled leaves. Whether these leaves were infected from below (from the stem) or from above (through their water pores or stoma ta) could not be determined by a field examination. Tips of many of the shriveled STEWART'S DISEASE OF SWEET CORN (MAIZE). 95 leaves have been saved in alcohol for microscopic study. The bases of many of these same leaves were still green and certainly contained bacteria in their bundles. If bacteria are present in the tips of these leaves it would not prove infection by way of the water-pores, but if they are not present, it would mean that the leaves were infected from the stem or at least not first at their apex; in other words, the movement of the bacteria in the foliar bundles might be assumed to be upward and not downward. This last inference I regard as probable for several reasons, particularly because the leaves are well up on the stem and because the stem-bundles are so full of bacteria that we must sup- pose the infection of the plants to have taken place several weeks ago and considerably lower down. I am inclined to think that the drying up of these leaves is due to secondary infection and to the very general plugging of the vascular system of the stem.* If my observations and inferences are correct, the primary infection took place when the corn was small and through the water-pores of the first leaves, which have long since fallen. The condition of the extreme base of the stem in these plants Fig. 41. t points strongly to this conclusion. It is sound externally but within it is more badly diseased than any other part. Here the connective tissues are brownish and gummy, and there are small cavities, i. e., the bacteria have been present for a considerably longer time than farther up the stem, have escaped from the bundles, and have stained the tissues. Sections of two such stem-bases have been preserved in alcohol for further study (fig. 42). None of the roots were diseased externally and all seem to be sound within, but not enough were examined to venture any positive general statement. Higher up the stem only an occasional bundle is stained brown. On longitudinal sections the affected bundles are distinctly yellow against the background of white pith. One characteristically diseased stem was saved dry and another was sectioned and put into alcohol. Under the compound microscope the slime from these maize plants appeared to be all one thing, whether pale yellow or bright yellow, and subsequent experiments confirmed this inference. For *These inferences were confirmed by a study of the leaf-tips already mentioned. On microscopic examination it turned out that in more than one-half of the dead leaf-tips (13 out of 25) bacteria were not present. fFiG. 41. — Cross-section of stems of inoculated sweet corn, showing Bacterium steward oozing as a yellow slime from the vascular bundles. Plants inoculated in the seedling stage in the hot-house on the tips of the leaves and grown in the field (Potomac Flats) nearly to maturity, before the disease developed. Photographed Oct. 7, 1902. X 6. 96 BACTERIA IN RELATION TO PLANT DISEASES. example, on July 16, at Mr. Croucher's, stem-segments 5 or 6 inches long and 0.5 too. 75 inch in diameter were cut from the middle part of 8 typically diseased sweet-corn plants and carried to New York, where, through the courtesy of the pathologist, Dr. James Ewing, I had opportunity to examine them the next day under proper bacteriological conditions in a laboratory of the Cornell University Medical School. The male inflorescence was drying out in these 8 plants, but the stems were green and normal in external appearance and each plant still bore some green leaves or at least green leaf-sheaths. The yellow slime oozed abundantly from the vascular system when the stems were cut. By direct transfer, without the intervention of poured plates, a pure culture of Bacterium stewarti was obtained from each one of these 8 stems. All that was done was to work rapidly, in a clean room, in still air, and to ex- clude surface bacteria by fire. The very simple steps in the process were as follows: (1) The stem was rolled over and over several times in a Bunsen flame until its surface was judged to be sterile or nearly so; (2) it was then shortened an inch at one end (in the flamed part) with a butcher-knife heated nearly red hot and used hot; (3) after some minutes the yellow bacteria oozed from the cut surface of some of the bundles and often I hastened this oozing by squeezing the stem; (4) one or more, usually several, of these droplets were touched with a sterile platinum needle which was then thrust several times into the agar. The organism grew promptly and each one of the eight tubes turned out to be a pure culture of Bad. stewarti. Plate-cultures were made from some of them and all remained under observation more than a year during which time none of them became contaminated. Descendants of these agar-stab cultures furnished the material used for making the inoculations, after I had satis- fied myself by culture on various media that this was actually the same organism as that formerly received by me from Mr. Stewart and subsequently lost through inattention. It should also be noted here that stabs made from the bright yellow slime and the pale yellow slime yielded cultures which could not be distinguished. Cover-glass smear-preparations, stained with Loeffler's alkaline methylene blue and with Ziehl's carbolfuchsin.were also made from each of these 8 stems (figs. 43, 44), after which they were sectioned and put into 95 per cent alcohol for subsequent study. It is a remarkably interesting fact that 8 different maize plants should have yielded the same organism in pure culture by direct transfer, but it does not stand alone (see Cobb's Disease of Sugar- cane, pp. 12, 25, and Wilt of Cucurbits, vol. II of this monograph, p. 287). INOCULATION EXPERIMENTS. SERIES I AND II. 1902. Two series of inoculations were made in August 1902, at nearly the same time. The experiments did not get under way until late in the season owing to the interference of other work, and they were consequently cut short by frost, but they continued long enough to yield conclusive results. First Series. This embraced two kinds of sweet corn, viz, Perry's Hybrid and Early White. The principal dates were as follows: August 2. — Planted Perry's Hybrid. August 4. — Planted Early White. .1 u«ust 9, 5 p. m. — Inoculated each sort by placing on the tips of the leaves by means of the platinum wire a small quan- tity of the bacteria from young (48-hour) cultures on slant agar. .1 ugust 19. — Shifted to 6-inch pots. August 28. — First cases (two). August 28. — Transplanted from the hot-house to the field. .September 8. — Growing well and as large again as when transplanted. Three additional cases. I ktober 7. — More cases. October 10 to 27. — Numerous cases. ier 29. — -First hard frost. November 1. — Closed experiment. Second Series. This embraced three kinds of sweet corn, viz, Perry's Hybrid, Early Red Cory, Early White Cory. The principal dates were as follows: August 7. Planted. ust ;,;, /.;, 75, 16. — The plants were inoculated at sundown on each of four days by spraying upon them in the rery fine mist a portion of 50c. c. of sterile water, containing each time the slime from two young slant agar cultures. / 28. — Transplanted from the hot-house to the field. September 8. — Growing well; twice as large as when transplanted. 1 ' ober 7. — First cases. October 10 to 27.— Many cases. October 29. — First hard frost. November 1. — Experiment closed. STEWART'S DISEASE OP SWEET CORN (MAIZE). 97 The conditions following inoculation in both series were made as favorable for infection as I could render them without injury to the plants, i. c, they were such as the plants would be subject to in the field during wet, warm, and cloudy weather. The plants of Perry's Hybrid (first series) were 3 to 5 inches high and had three well- developed small leaves when inoculated. Those of the Early White (first series) were 1 to 3 inches high when inoculated, in most cases with two leaves, but occasionally the tip of a third leaf was visible, while in a number of instances only one leaf was to be seen. As soon as inoculated the plants of this first series were placed under the bench in obscure light for nearly two days (2 to 5 p. m. August 9 to 10 a. m. August n), and the floor of the greenhouse was very thoroughly wet down with water, so that the air of the house should be unusually moist. At 10 a. m., August 10, the plants were examined. The tip of nearly every leaf bore a little drop of exuded water and the conditions appeared to be very favorable for infection unless it were that the excess of fluid poured out should wash away all the infectious material, which may have been the case in some instances. The morning was cloudy and the temper- ature 2Q°C.,the same as the day preceding, but probably somewhat higher in the hot-house. At 10 a. m., August 1 1, the pots were taken from under the bench and placed on the bench. Many of the leaves still bore drops of water at their tips, especially the younger ones. The plants had grown about 2 inches and the new growth was rather pale green, owing to the obscurity. The earth in the pots was very wet and occasionally there was a trace of mold on the surface. Roots in quite a number of the pots had come to the surface, owing to the darkness. The sky was overcast and the temperature was 29° C. The gardener was directed not to water the plants that day. At 2h3om p. m. the temperature of the lab- oratory was 310 C, the sun was shining, but there was not much wind, and the air was full of moisture. In the second series, the first inoculation was made with cultures 24 hours old, the second with cultures 48 hours old, the third with cultures 3 days old, the fourth with cul- tures 4 days old. A good atomizer was used which broke the fluid into an extremely fine mist or fog. The plants were small when they received the first spraying. They were 3 to 6 inches high when they received the fourth spraying; each spraying was made at sunset. On the morning after each spraying it was observed that little beads of fluid stood on the tips of many leaves (see this monograph, vol. I, fig. 73), so that here also it was judged that the conditions favored infection. Local signs were first visible at the tips of the inoculated leaves and within a few days in some cases, but there were no constitutional signs until the nineteenth day after inoculation (first series) and then only in two plants. In case of the sprayed plants, 52 days intervened between the last spraying and the first cases, i. e., fewer bacteria entered the plant, and a longer time was requisite for infection. The plants of both series were removed from the hot-house to the open field on the same day. They were then 18 inches to 2 feet high, healthy in appearance, and with leaves from one to two inches broad. The pots were well watered before removal, and after careful transplanting each hill of plants was watered and the wet earth was then covered with loose earth to prevent rapid evaporation. The day was sunny and warm, but there had been heavy rains the previous afternoon and evening, and the plants did not suffer from the re- moval. Not a single one of them died as a result of the transplanting. They were set in a freshly plowed and harrowed, fertile, level field from which a crop of potatoes had just been harvested. No other corn plants were near them, but at some dis- tance there were several acres of healthy field corn of several varieties. The hills were set at a good distance apart each way and the field was freely cultivated so that no weeds grew. Three additional cases appeared in the first series on September 8 (twenty-ninth day after inoculation), and thereafter no more for a month. Meanwhile the plants grew vigor- 9§ BACTERIA IN RELATION TO PLANT DISEASES. ously. It was observed, however, on September 20, that many of the plants had begun to show the staminate inflorescence. This was judged to be premature, since these plants were only 2.5 feet high, i. e., they were plants dwarfed and prematured by the disease, but show- ing as yet no other unmistakable signs. On October 7, cases began to appear again in the first series and then also for the first time in the second series. For the sake of conclusiveness, even at the risk of being tedious, the results of an examination of some of these cases will be described in detail, after which the whole experiment will be summarized in a table. Record of the Diseased Plants. I. Photographed (plate 7, fig. 1) and dissected. From vessels toward the base of the stem the characteristic yellow slime oozed out. With proper caution two pure cultures were obtained on slant agar by direct transfer; a third was contaminated by a green-fluorescent organism. II. No record. III. IV. Sep- tember 8, photo- graphed together (plate 7, fig. 2). Externally the plants give no in- dication as to the cause of the disease. The larger plant is 2 feet high without stretching up the leaves. The outer- most four leaves are shriveled and brownish ; the upper ones are wilted. The smaller plant is about 18 inches high. All of its leaves are shriveled except the upper- most one, which is wilted. The stem of the larger plant was cut off 6 inches from the roots. All the inner (undeveloped) leaves are free from the organism, but the outer two or three are infected. The same is true of the smaller plant. The bacteria ooze out in the form of chrome yellow masses. Cut higher up, the bacteria are less and less abundant, the infection of the large leaves being clearly from the base upward. Cut within 1 inch of the roots, the larger plant shows a pretty general infec- tion of the vessels through all parts of the stem and a browning of some of the bundles. The same is true of the smaller plant except that at this level the browning is less conspicuous. The basal 1 inch of the larger stem was then slit through lengthwise and inspected. The most badly diseased part was the extreme base. Here there was much browning and some softening of the Fig. 43. f Fig. 42.* Fig. 444 *Fig. 42. — Radial longitudinal section through a maize stem (sweet-corn) attacked by Bacterium stewarti, show- ing a badly diseased (browned) area in base of stem, i, e., in that part of the stem first occupied by the bacteria; also browned vascular bundles in the internode, and sound exterior. Long Island, July 16, 1902. Compare with results of pure-culture inoculations obtained in Washington and shown on plate 10. tFic. 43. — Bacterium stewarti stained 10 minutes in alkaline methylene blue and washed in water. A cover-glass (smear) preparation made directly from the yellow ooze out of the stem-vessels of a sweet-corn plant collected on Long Island, July 16, 1902. Figure made with Zeiss 2 mm. apochromatic 1.30 n. a. objective, No. 12 compensating ocular, and Abbe camera, and drawing then reduced one-half. X 1,000. Compare with drawing made from slide stained with carbol fuchsin. JFiG. 44.— Cover glass (smear) preparation of Bacterium stewarti, direct from stem of Early Cory sweet corn. Long Island, July 16, 1902. Stained 5 minutes in carbol-fuchsin. Staining irregular and wall often not well defined (compare with vol. II, fig. 122, where a similar effect is visible). No spores. X 1,000. PLANT BACTERIA, VOL. 3. PLATE 7. Bacterium stewarti in sweet;'corn. (i) First'case of disease in hot-house, inoculations of 1902. Photographed Aug. 29. i. c, 19 days after'placing the bacteria on the plant. (2) Third and fourth cases from first series of inoculations. The largest plant 2 feet high without.lifting the leaves. Vessels full of the yellow bacteria. Variety Early White, planted Aug. 4, inoculated Aug. 9, photographed Sept. 8, 1902. PLANT BACTERIA, VOL. 3. PLATE 8. Sweet corn attacked by Bacterium stewarti. (1) Plant No. xviii, inoculations of 1902 (see notes). Stem dwarfed, leaf-blades dead, male inflorescence prema- turely developed. Bactetia abundant in the stem. Photographed Oct. 14 1902. (2) Plant xx. or xxi. stem dwarfed, with basal branches and foliage dead, male inflorescence dry and white. Photographed Oct. 14. 1902. STEWART'S DISEASE OP SWEET CORN (MAIZE). 99 tissues and slight indications of small cavities. Some of the bundles in this longitudinal section were distinctly yellow from the presence of the bacteria. The smaller stem slit in the same way showed the same thing, except that there was more browning and injury at the base of the stem. Some of the bundles were distinctly yellowed by the bacteria and others were browned. In the bases of the badly diseased leaves, in some places, the bacteria had escaped from the vessels and had made yellow pockets in the neighboring parenchyma, just as Bacterium hyacintki does in the bulb-scales of the hyacinth (vol. II, plate 20). Material was saved in alcohol. The disease in these plants is the typical " bacteriosis " of sweet corn as it exists on Long Island. Eight Petri-dish poured plates were made from each plant into acid (mulberry) agar, but the organism did not grow. V. No record. VI, VII, VIII, IX, October 7 : The external appearance of each of these four plants was the same, viz, a sound green stem and sound roots, but shriveled leaves, the reason for which was not apparent. On cutting, the vascular system of each stem was found badly diseased, the vessels being filled with a yellow slime. The bacteria were traced up the stems for many centimeters, but the bundles in the first decimeter of the stem were most fully occupied. The tissues were most badly diseased in the extreme base of the plant, i. c, in the first two or three nodes and short internodes, the same being that part of the stem nearest the point of union with the particular leaves which had been inoculated. In the internodes of this part of the stem a few of the bundles were brown, but most of them were bright yellow from the presence of the bacteria. In the extreme base of the stem there was a very decided browning of all the tissues; in two cases this browning also included the next two nodes. On cross- section of the stems the bacteria oozed from the bundles as beads of yellow fluid (fig. 41). X, XI, XII, XIII, October 10: Four additional suspects were brought in for examination. In each the staminate inflorescence was dead. The tallest of these plants was 3.5 feet. The average height of the plants in the field on this date was 3 to 3.5 feet. A few were much shorter and a few were over 4 feet high. Numerous bundles in the stem of each one of these four plants contained the yellow slime of the bacteria in abundance. This was visible to the unaided eye in bundles at a distance of more than a foot from the root. On longitudinal section the first two to three nodes of the stem were decidedly brown, the internodes being white. Plants XIV to XXIV were brought in and examined on October 14. They averaged 3.5 to 4 feet in height. Roots were abundant and sound externally in all. The leaves were more or less shriveled. Several of the plants had small weak shoots coming up from the base. XIV. Basal nodes brown, but infection less widely spread and less conspicuous than in the fol- lowing cases. No indication of yellow vessels in the internodes. XV. Stem cut 3 inches from base : Numerous bundles from which yellow slime oozes ; also some brown bundles. Stem split longitudinally: Brown nodes, bundles in internodes either brown or yellow; infection extends far up into the upper leaves; lower nodes of cobs brown within. XVI. Stem cut 3 inches from base: To the naked eye one-half dozen infected bundles. Split longitudinally: Basal nodes brown, but only a few of the internodal bundles are infected; two upper internodes not yellow; all lower internodes are brown. Ear affected with larva?; doubtful if bacteria present. Comparatively little infection of upper part of stem. XVII. Basal parts of stem show yellow bundles. XVIII. This plant was photographed separately (pi. 8, fig. 1). Cut 3 inches from base: Vessels filled with yellow slime. This plant has several little ears coming out near the ground; these are browned in the nodes and yellow (gummed) in the internodes. Specimens of this and other cobs were saved in alcohol. Split lengthwise : Nodes at base brown with yellow bundles in the internodes; the upper nodes grade from brown into yellow. Infection traced to just below the staminate inflores- cence. On longitudinal section the bundles in the upper internodes show as distinct yellow stripes. XIX. Cut plant about 3 inches from base: It has numerous bundles showing the yellow ooze. Cut 1 foot from the base: Still shows yellow bacteria. Cut 2 feet from base: Still shows yellow bac- teria. The yellow slime appears to be, to a small extent, in the roots close to the stem. On longi- tudinal section, brown in all nodes (7) up to tassel, not in ear. XX. Either XX or XXI was photographed separately (pi. 8, fig 2). Cut about 3 inches from root: Numerous bundles show yellow ooze. Brown in nodes at base of stem, where there are also small bacterial cavities. Bundles between the basal nodes are either brown or yellow. Infected at all nodes, the two upper nodes yellow instead of brown. The five basal nodes of the largest ear are yellowish from presence of the organism, and there is yellow slime in the internodal bundles ; the infec- IOO BACTERIA IN RELATION TO PLANT DISEASES. tion extends as far as the base of the cob and runs out into some of the husks around the cob. In the main axis visible infection ends just below the male inflorescence. XXI. Cut 3 inches from root: A great many bundles are infected. Base cut longitudinally: Lower nodes brown ; bundles of the internodes either brown or yellow ; roots slightly infected near their juncture with the stem. All nodes infected; lower, brown, grading into yellow in the upper part of the stem. Infected visibly to just below tassel. In the cob, the base and a few bundles further up are visibly infected. XXII. Basal parts of stem show yellow bundles. XXIII. Cut 3 inches from roots : Bundles packed with yellow slime. Split longitudinally (basal part). Browned in nodes and some yellow bundles in the internodes. Upper nodes as in XXI. XXIV. Basal nodes brown in longitudinal section (3 inches above root). No indication in these lower internodes of yellow bundles, but numerous plainly yellow bundles in an internode 6 inches above the roots. Plants XXV to XXXV were brought in and examined on October 17. All of the plants were well provided with roots which looked sound on the surface. The steins were green in all cases and normal in external appearance, but the leaves were shriveled and the tops were dried out as well in those standing as in those accidentally broken off. The cause of the disease was not apparent. XXV. Cut close to root: No plain evidence of infection. This plant was broken off accident- ally, and therefore brought up at this time. One of the basal nodes is brown. No distinct evidence of the disease in the internodes. This brown node was examined microscopically with doubtful results. XXVI. Brought in because broken off at root. Section cut about 3 inches above the root : In the internode no distinct signs of disease, but three nodes above it are brown. In the internodes between there is no distinct yellowing of the bundles. Infection, if any, is slight. Several of these plants seem to confirm Stewart's statement that the brown stain is not a sign of the disease. Later, found a yellow slime-infested bundle in one of the basal internodes. XXVII. A few brown vessels in an internode about 2 inches from base of the stem. The lower nodes are brown. Infection slight. One node 8 inches up, one node a foot up, and a third node 16 inches up are brown. A hasty examination shows no appearance of infection in the internodes between these nodes. XXVIII. One of the lower nodes slightly browned. No evidence of disease in internodes. The upper nodes look all right. Hasty microscopic examination gave doubtful results. Basal part slit through, shows brown nodes; no evidence of disease in the internodes. XXIX. Section 3 inches from base of stem contains a great many bright yellow bundles. Leaf- sheath shows yellow spots corresponding to masses of bacteria which have oozed out of the bundles into the parenchyma. Slit longitudinally, the base of the stem shows brown nodes and yellow bundles in the internodes. The disease extends very plainly two-thirds of the way up the stem and runs out into the cob, and occurs to a slight extent in the remainder of the stem. XXX. No plain evidence of infection except the brown stain. Base slit through shows brown nodes. No evidence of yellow ooze in the internodes. XXXI. Cut 3 inches from the root, the bundles show a yellow ooze. Longitudinal section through this base shows brown nodes with some yellow bundles in the internodes ; not in the ear. The browning is less conspicuous in the basal nodes than in some of the other plants. The nodes higher up are not brown. Visible infection is confined to the basal parts. XXXII. Section 3 inches from base shows in the internode numerous bundles from which there is a yellow ooze. In a leaf-sheath about 9 inches up, the bacteria have escaped from the bundles and formed bright yellow patches in the parenchyma. Corresponding to this escape from the bundles is a water-soaked patch 2.5 inches long by 0.75 inch broad (saved in alcohol, flat between glass slides) . The yellow slime is also in the base of the ear. The bacterial ooze comes out of bundles close under the tassel. Basal part of the stem slit shows brown nodes, and yellow bundles in some of the internodes. XXXIII. This plant, which is about 4 feet high, has one good-sized ear and several small ones. Cut about 3 inches from the base, many of the bundles show yellow bacterial ooze on cross-section. Nothing in the two abortive ears. Longitudinal section through the basal 3 inches of the stem shows 3 brown nodes with yellow bundles in the internodes. The infection extends the whole length of the stem, the basal half dozen nodes being brown and the upper ones yellow. The internodes show yel- low stripes, corresponding to diseased bundles, for a long distance up the stem. The leaf-sheaths of leaves 1 foot to 2 feet above the root also have the bacteria in their bundles. PLANT BACTERIA, VOL. 3. PLATE 9. Longitudinal sections through stems of 10 sweet-corn plants, showing invasion by Bacterium steivarti. Plants inoculated when young by placing bacteria from a pure culture in the drops of fluid extruded from the leaves and resting on their tips. Signs of disease in about 2.5 months, when the plants were 3 to 4 feet high. Bacteria confined principally to the vascular bundles. Nodes brown, internodes white, except infested bundles, which were usually bright yellow, but occasionally brown. In X, X, bacteria have escaped into the parenchyma, forming bright yellow pockets. In c, the brown stain involves the whole of the lower internodes. About natural size. STEWART'S DISEASE OF SWEET CORN (MAIZE). IOI XXXIV. Cut about 3 inches above the root, numerous bundles are brown and others show a yellow ooze. Split longitudinally, the base of the stem shows brown nodes with internodes having bright yellow bundles and brown bundles. The first 4 nodes above the basal 3 inches are brown. The disease extends visibly up to the tassel. It also extends into the base of the ear, but only to a slight extent. The bacteria are abundant in the middle internodes, some of the bundles being brown and others bright yellow. Further examination shows the bacteria as far up as the middle part of the male inflorescence. The bacteria are also in a leaf-sheath midway up the stem. XXXV. Cut at 3 inches from root, bundles full of yellow slime. Cut a foot above the root, bundles full of yellow slime. Cut 2 feet above the root, no evidence of bacterial masses under a hand- lens. Split longitudinally through the base of the stem, the lower 3 nodes are brown in the center, but the outer portion of the stem is white, as has been the case in all of these plants. The lower inter- nodes have some brown bundles and some yellow ones. The bacteria extend up into the base of a good-sized ear. The nodes above the first 3 vary from brown to yellow, the lower ones being brown and the upper ones yellow from the presence of the bacteria. It is quite clear that the infection was from below upward, i. c, the basal nodes are the worst affected. A second ear on the same plant shows the disease in the basal part of the cob. The bacteria are present in the bundles of a leaf- sheath 15 inches from the ground, and the node from which this leaf-sheath originates is yellow from the presence of the bacterial slime. Also, in a leaf-sheath 2 feet from the ground, the bacteria are present. Longitudinal sections through the central internodes show long yellow stripes, and in at least one place the bacteria have escaped from the bundles and have made a yellow spot (cavity) in the parenchyma (specimen saved in alcohol). Plants XXXVI to LI were brought in and examined on October 21. This bundle of diseased plants gave no external indication as to the cause of the trouble. The drying out of the leaves much resembled the effect of frost, but there had been no frost. When the male inflorescence is not mentioned it was broken over. Photographs were made showing the appearance of the stems in cross-section and longitudinal section, and also the appear- ance of the diseased husks. XXXVI. Plant 3.5 feet high, stem green and sound externally, leaves dried up, tassel white (dry), roots abundant and sound on the surface. This plant has two small ears, one of which is infected at the base. Cut 3 inches from the base of the stem, several of the bundles show a distinctly yellow ooze. Slit longitudinally, the six lower nodes are brown, those higher up are yellow (gummy). The internodes toward the base of the stem show some black bundles and others which are bright yellow. Internodes half way up the stem also show distinctly yellow bundles. The bacterial slime apparently runs out toward the top of the stem, i. e., it is not visible under the hand-lens. XXXVII. Plant 3 feet high; male inflorescence dead. This plant branches at the base. Cut the main stalk 3 inches from the ground; nothing definite in the internode. Slit the base longi- tudinally, find 3 nodes decidedly black. No traces in the other branch. The basal internodes of the main stem have large black streaks and have been injured by a borer. No evidence of yellow striping in the bundles. This is the first plant observed to be affected by root insects. No evidence of bac- terial disease farther up the stem. XXXVIII. Plant 3.5 feet high, broken over. The vessels of the bundles in internode 6 inches up the stem are full of yellow slime. A large ear about 1 foot up shows no signs of the disease, but there are numerous bright yellow bundles in the main stem under this ear. A smaller ear cut near the base and slit longitudinally shows the infection in the lower part of it, in the bundles, in the form of bright yellow stripes. The lower nodes are brown and the upper ones yellow for more than two- thirds of the way up the stem. The bacteria can be traced in the internodes of the stem readily under the hand-lens nearly to the top, the infection becoming less and less as one goes higher up the stem. XXXIX. Plant 4 feet high. Stem cut 3 inches from base, yellow bacteria very abundant in the bundles. Slit longitudinally: The lowest 3 nodes are brown; the internodes have bright yellow bundles; 3 of the nodes next higher up are brown, and then 4 more are distinctly yellowed. The bac- teria are present in abundance in the base of a good-sized ear, but not in the kernel part as far as can be seen with the naked eye. The disease disappears in the upper part of the stem. It is visible over half way up the stem. The bacteria are abundant in the internodes in the lower and middle parts of the stem as yellow stripes. They are in the bundles. In places bacteria have also oozed out of the bundles into the parenchyma, making small bright yellow spots (dark in photograph). (See plate 9.) XL. Plant 4 feet high. Disease absent from base of one small ear. The bacteria are abundant in the base of the larger ear, which is farther up than the smaller one. On cutting the pedicel they ooze out immediately as bright yellow masses (samples saved in alcohol). The bacteria are also present in the husks of the ear and have in some cases escaped from the bundles into the parenchyma 102 BACTERIA IN RELATION TO PLANT DISEASES. of the green husk, making bright yellow spots (small cavities). The bacteria are traceable in the cob of this ear as far as the kernels, but not distinctly into them. Two inches from the base of the stem the internode shows diseased vessels. The lower 2 nodes are brown and the internode between shows yellow bundles. The 5 nodes next above this basal portion are also brown. The infection is clearly visible in the internodes to a distance of 2 feet above the ground as yellow bundles and is traceable still farther up the stem, but becomes less abundant; the disease (yellow slime in the bundles) dis- appears in the second node below the tassel. XLI. Plant 4 feet high. Cut 3 inches from the base, internode shows many yellow bundles. Basal part slit longitudinally, its 3 nodes are brown; the internodes contain black and yellow bundles. One large ear and two small ears, each with bright yellow bacterial ooze visible in the bundles. It extends visibly to the base of the cob in the larger ear. The 3 nodes next above the basal cut are brown. Three nodes next farther up are distinctly yellow from the presence of the bacteria. The disease runs out into the sheath of a large leaf half-way up, but I can not detect the bacteria with any certainty in the nearly dried up blade of this leaf (hand-lens). Disease plainly visible in inter- nodes half-way up the stem in the form of bright yellow bundles from which yellow slime oozes when they are cut. As in all the cases hitherto observed, the infected bundles become less numerous or less distinctly diseased as one proceeds from the root to the male inflorescence at the top of the plant, showing quite clearly that the infection must have proceeded from the base of the stem upwards. In this plant also there are places where the bacteria have oozed out of the bundles and affected the surrounding par- enchyma of the internodes. These spots or pockets in the parenchyma are bright yellow. XLII. Plant 3 feet high, male inflorescence white, leaves dried, stem green and sound externally. Cut 3 inches from base, the internode contains many bundles from which there is a distinctly yellow bacterial ooze. Basal part slit longitudinally, the two nodes are brown. The browning, as in all cases hitherto reported, is central and does not extend entirely out to the periphery of the stem. The hard outside portion immediately around this brown area is sound (plate 10). The basal internodes show numerous yellow bundles. The nodes farther up are brown or yellow — lower ones brown, upper ones yellow i. c, from presence of Bad. stcwarli. The internodes in the middle of the stem show some brown bundles and many yellow ones. One large ear and two smaller ones, each infected at the base. The bacteria are not distinctly visible in the middle leaves, and in the stem they disappear, so far as can be determined with a hand-lens, about a foot below the male inflorescence. XLHI. Plant 4 feet high, leaves dried up, roots plentiful and sound externally. Basal part of the largest ear shows distinct infection, but not very abundant. Stem cut 3 inches from the base: Numerous yellow bundles in the internode. The plant has a great many healthy roots. (All the plants are well provided with roots.) Base slit longitudinally shows the 2 nodes brown and the internodes with brown and yellow bundles. There are 3 small ears and a large one. One of the small ears is diseased at the base. The other three appear to be sound. Three of the nodes in the main axis next above the basal cut are brown. The fourth one, which gave rise to the large ear, shows bright yellow bacterial ooze in one place. One of the nodes next farther up is distinctly yellow with bacterial ooze, and those still farther up show no distinct evidence of the disease. The internodes half- way up the stem are diseased, but the yellow ooze is much less abundant and in fewer bundles than farther down the stem. Some of the leaf -sheaths half-way up the stem have diseased bundles. XLIV. Plant 4 feet high, male inflorescence white and dry, leaves dried up, roots abundant and sound externally. Cut 3 inches from base, there are many yellow bundles in the internode. Slit longitudinally through this basal part, there are 2 brown nodes connected by white internodes striped with yellow bundles. The 4 nodes next farther up are brown. The next 3 are in an earlier stage of infection, i. e., yellow from the presence of the bacteria, but not yet brown. Four of the 5 small ears are infected at the base. The middle internodes of the stem have numerous yellow bundles. XLV. Plant only 14 inches high, leaves and male inflorescence dry, roots sound in appearance. Cut 3 inches from the base: Internodes contain brown bundles. Slit longitudinally, the nodes are brown and the internodes contain both brown and yellow bundles; farther up the nodes are yellow and the internodes greenish-white with yellow bundles. Visible infection disappears toward the top. XLVI. Plant 3 feet high, roots sound externally, male inflorescence white and dried out, all leaves dry. Good-sized cob, basal part infected. As in a number of other instances, the bacterial infection is most abundant on the periphery, on the lowest, outermost side of the ear. Stem cut 3 inches from the base, the internode shows numerous yellow bundles. Sliced longitudinally, the base of the stem shows 3 brown nodes and yellow stripes in the white internodes. The inner husks of the ear are badly infected (in the bundles), and in many places (100 to 200) the bacteria have made their way into the parenchyma between the bundles in quantity so as to show as yellow masses. The bacteria have also oozed out on the inner side of the husk, in several instances, in yellow masses, wet- PLANT BACTERIA, VOL. 3. PLATE 10. Stewart's disease of sweet-corn. Section through the base of two sweet-corn stems (plants xi.ii and xi.vi or xtvn). attacked by Bacterium stewarti. as the result of a pure-culture inoculation in the seedling stage, showing brown stain in the nodes, sound cortex, healthy roots, and a bud pushing- STEWART'S DISEASE OF SWEET CORN (MAIZE). 103 ting its surface. These masses when touched with the finger string up decidedly (1 cm. or more). Six of the husks are infected in this way. There are about 180 small bacterial pockets in the par- enchyma of a single husk; in another husk there are more than 100. Specimens of these husks were saved in alcohol and one of the husks was photographed (vol. II, fig. 14) but the contrast is less than in nature. Samples were saved in alcohol from the base of the cob. It is doubtful whether the bac- teria can be traced out into the kernels. The first 2 nodes above the basal cut are brown, several far- ther up are brown with a yellow ooze. The next 4 are yellow. The infection is plainly visible with a hand-lens in bundles 3 inches below the base of the male inflorescence. The middle internodes con- tain numerous bright yellow bundles. The sheaths of the middle and upper leaves also contain yel- low bundles, from which there is a bacterial ooze. The disease is not distinctly visible in the leaf- blades, which are dried up. There is less of the bacterial infection toward the top of the stem. XLVII. Plant 4 feet high, roots sound externally, all leaves dry except part of an upper one. Stem on the outside green and sound. Cut 3 inches from the base, there are a great many yellow bundles from which the bacteria ooze out. Basal part slit longitudinally, there are 3 brown nodes, and the white internodes contain yellowed bundles. The plant bears 3 ears, the bases of which are diseased, i. e., have vascular bundles occupied by the bacteria. Above the basal cut 2 nodes are brown, 3 others farther up are yellow from the presence of the bacteria. The middle internodes show distinctly yellow bundles. Organism present in the bases of the middle leaf-sheaths but not very abundant. There are quite a good many places in the internodes where the bacteria have escaped from the bundles and made yellow spots in the white parenchyma. Bacteria are plainly visible in the stem as far up as a foot under the male inflorescence, but not nearly so many vessels are occupied as lower down. XLVIII. Plant 4 feet high, roots abundant and sound externally, stem green and normal on outside, male inflorescence dead and white, all leaves shriveled except the uppermost one. Cut 3 inches from the base : A great many yellow bundles in the internode. Slit the base of the stem longi- tudinally; the nodes are brown; the white internodes contain bright yellow stripes corresponding to the bacterially infested bundles. Farther up 3 nodes are brown and 3 others next higher are yellow, showing when cut a distinct bacterial ooze. The infection extends into the basal part of the male inflorescence. The plant has 3 ears — 1 large and 2 small. The base of each one shows a distinct yellow ooze from the bundles on section. The husks around the ears are also infected, the bundles are yellow and there are yellow spots and water-soaked spots in the parenchyma corresponding to places where the bacteria have oozed from the bundles. The middle internodes of the main axis have yellow bundles, and here also there are yellow spots in the parenchyma where the bacteria have oozed out. The bacteria are not abundant in the middle leaves. XLIX. Plant 4 feet high, all the leaves dried out, stem green and sound externally. Bacteria not abundant in the middle leaves. I can not see any yellow slime with the hand-lens on cross-section of the sheaths. Plant cut across an internode 3 inches from the base, there are a great many yellow bundles; in fact, nearly every bundle of the stem shows a yellow ooze. This part of the stem slit longitudinally shows 2 brown nodes connected by white internodes with yellow bundles. The numer- ous roots are sound externally, but some show the bacterial ooze on cross-section near the stem. The roots do not appear to be much infected. The most badly infected node, as in many other cases, if not all, is the basal one. The disease disappears (hand-lens) some distance under the male inflores- cence, perhaps a foot. Above the basal cut 3 nodes are brown, 2 nodes farther up are yellowish, and the middle internodes show yellow bundles in abundance. The plant has 2 ears each slightly infected at the base in the bundles. L. Plant 3 feet 9 inches high, good roots, all the leaves dry, stem sound and green externally. The sheaths of the middle leaves are plainly infected in a few bundles. The same is true of the leaf- sheaths up to the top of the plant. They are all more or less occupied by the yellow bacteria. The disease is visible in the stem close under the male inflorescence, that is, 1 inch under the first branch of it. The yellow bacteria are distinct in the base of all three cobs. Stem cut across an internode 3 inches from the base : Nearly every bundle shows a yellow ooze. Slit longitudinally, its lower 3 nodes are brown and the internodes are greenish white with yellow bundles. Above this, 3 nodes are brown, the next 2 are yellow and show the bacterial ooze distinctly. The bundles in the middle internodes are packed full of the yellow bacteria. LI. Plant 3 feet 9 inches high, roots sound externally, stem green and sound outside, all the leaves dry. The yellow slime is present in the middle and upper leaf-sheaths, exclusive of the two uppermost. The upper part of the stem for a foot under the male inflorescence appears to be free (hand-lens). Three ears, all badly infected at the base. This infection extends up into the part which bears the kernels, and the bases of a few of the kernels seem also to be attacked (these have been saved in alcohol). Cut stem 3 inches from the base; every bundle appears to be occupied by the yellow slime. Slit longitudinally, the nodes are brown, the internodes white with yellow bundles. 104 BACTERIA IN RELATION TO PLANT DISEASES. Above this cut 3 nodes are brown and the others farther up are yellow and show the bacterial ooze distinctly on section. The middle internodes are white, with numerous yellow bundles. The plants LI I to LXX were brought in and examined on October 27. All the plants had good roots. The ears were in the edible "green-corn" stage. The tops had been broken off in many cases. When not otherwise recorded, the parts, except leaves and male inflorescence, were normal in appearance. Most of these plants were 3 to 4 feet high. There had been a slight frost and a few of the leaves here and there may have been killed as the result of the frost, but I think it unlikely, because in the same hill one plant would have dry leaves and another not, and also because all of the remaining plants had more or less green leaves. Many of these leaves were 3 to 3.5 inches wide. The leaves of these 19 plants were dried out and dead. The male inflorescence where it was present was white and dry. The stems in all cases were green and normal in appear- ance and many of the leaf-sheaths were alive, the blades being the dry part. LI I. Cut 3 inches from base, nearly every bundle is occupied by the yellow bacteria. All the middle internodes contain many yellow bundles. The organism is abundant in the bundles at the base of each one of the five ears. It can be traced into the upper internodes of the main axis, but is less abundant there. The basal 3 inches slit through shows much less browning in the nodes than in any plant hitherto examined, but the basal node is brown. LIU- This is a very small plant. It has had a bad injury underground, and the height of the plant is less than a foot. The injur}' referred to cut the stem at least half in two. No distinct indica- tion of the disease in the upper part of the stem. The 2 basal nodes are brown, but the internode is whitish. I can see no yellow or brown bundles in the internode. Probably the dwarfing was due to the extensive stem injury. Not examined under the compound microscope. LIV. Stem cut 3 inches from the base, nearly every bundle shows an abundant yellow ooze. There are 4 ears; all show presence of bacteria at the base in the form of yellow ooze. Slit the base of the stem longitudinally. There are 2 brown nodes with yellow stripes in the internodes. I also find the organism moderately abundant 6 inches under the male inflorescence. Cut still higher up, that is, immediately under the male inflorescence, the bacteria are still present. (Turned over the rest of the plant to my colleague, Mr. Merton B. Waite, who stated that he found bacterial infection in the bundles of every node and internode.) LV. Cut 3 inches from the base, the internode contains yellow and black bundles, but they are much less numerous than in the preceding. Sliced longitudinally, the 3 lower nodes are brown; the internodes are white, with yellow and brown bundles. Cut 1 foot higher, there is almost complete freedom from visible infection in the internode. This plant is much less infected high up than the pre- ceding. The disease runs out entirely, so far as one can see with a hand-lens, at 1.5 feet under the male inflorescence. One small ear and one large one; neither show any infection. LVI. This is a small plant, not more than 3 feet high. A cut 3 inches from base shows numer- ous points of infection. There must be at least 50 bundles from which there is a yellow bacterial ooze. The ear shows disease in a number of the inner husks in the shape of infection of the bundles, water-soaked spots in the parenchyma and a yellow bacterial ooze on the inner surface of the husks. The inner face of certain husks is the only place where the bacteria have been observed coming to the surface. The base cut longitudinally shows 3 brown nodes and many yellow bundles. There are 3 brown nodes above this cut and 3 yellow ones higher up. The internodes higher up show numerous yellow bundles. The yellow bundles run out into the base of the cob. LVII. Two ears, partially destroyed by insects and smut, show yellow bundles in the unde- stroyed base. Cut 3 inches from the base, the main stem shows many bundles with the yellow bac- teria oozing out in great quantities. The base slit longitudinally shows 3 brown nodes, the cut bundles exuding yellow ooze. The internodes show very many yellow bundles. The disease appar- ently runs out in the first node below the tassel. A small ear near the base of the stem also contains bacteria in the bundles of the husks and in the base of the cob. LVIII. Cut 3 inches above the base, the stem shows bundles with a yellow ooze, rather few in number. Plant cut 1 foot higher, in the internode, shows a yellow ooze from about 40 bundles. One ear which was badly gnawed by larvae shows many yellow and brown bundles in the uninjured base, and some of the inner husks show the yellow bacteria oozing out. The middle internodes of the main axis, slit longitudinally, show yellow and brown bundles. When cut 8 inches under the male inflores- cence, 2 or 3 bundles in the internode show a yellow ooze. Cut 0.5 inch under the male inflorescence, the bacterial ooze is still plainly visible in one bundle. Above the basal cut there is 1 brown node and STEWART'S DISEASE OF SWEET CORN (MAIZE). 105 2 yellow ones, and the internodes show many yellow bundles and some places where the bacteria have oozed out into the parenchyma, forming bright yellow spots. The basal part cut longitudinally shows 2 brown nodes ; some of the bundles of the internodes are blackened and a few yellow ones show the bacterial ooze. L,IX. Cut 2 inches from the base, the stem shows a few yellow bundles with the characteristic ooze. The longitudinal section shows one node slightly brown. One bundle in the internode is very markedly yellow in color; several other bundles are slightly colored with the yellow bacteria. There are 2 ears, both badly gnawed by larvae (southern corn worm), but both show numerous yellow bundles at the uninjured base. A cross-section in the internode 1 foot above the first cut shows about 20 bundles giving out the yellow ooze. There are brownish yellow nodes and yellow bundles in the inter- nodes for a distance of a foot up from the root. Farther up the disease is less distinct, and after one reaches the height of about 3 feet the bacterial slime ceases to be visible to the naked eye or with the hand lens. L,X. Cut 3 inches from the root, there is a yellow ooze from a great many bundles. Cut a foot higher, the number of visibly infected bundles is still a good many. Cut another foot up, I see a yellow ooze from only about one dozen bundles. Cut immediately under the male inflorescence, another foot up, the visible infection is reduced to 3 bundles. There are 3 ears, all gnawed by larva, but all showing the yellow bacterial infection at the uninjured base. The base of the stem cut longi- tudinally shows 2 very brown nodes and internodes with many yellow bundles and some very brown ones. The middle internodes sliced longitudinally show numerous yellow bundles. Above the lower (brown) nodes are yellow nodes, from which the bacteria can be seen to ooze on section. LXI. Main stem sliced across a node 3 inches from the base, there is yellow bacterial ooze from many bundles. Cut a foot higher, distinct yellow ooze can be seen from about 15 bundles. This portion of the stem slit longitudinally shows yellowish gummy nodes and bright yellow bundles in the internodes. The base cut longitudinally shows the 2 lower nodes markedly brown, the one above brownish yellow, the internode showing yellow bundles. Visible infection runs out about 3 inches under the male inflorescence. There are 2 medium-sized ears and 3 very small ears. Yellow bac- terial ooze shows only in the base of the largest ear (cob). The upper part of this plant contains a much less quantity of the yellow bacteria than many of the others. LXII. A small plant, that is, about 3 feet high. Cut 3 inches from the base, many bundles of the internode show the yellow ooze. The basal portion slit longitudinally shows 2 brown nodes, with some yellow bundles in the internodes. One of the inner husks of the ear is infected, and the dis- tinctly yellow bacteria have oozed out of the spots and lie free on the inner surface of the husk. There are numerous yellow bundles in the base of the cob. The middle internodes sliced longitudinally show distinctly yellow stripes corresponding to the infected bundles. Farther up the stem there are numerous areas where the bacteria have oozed out into the parenchyma. In the node just below the male inflorescence there are numerous yellow bundles. LXIII. Cut 4 inches from base, there is a distinctly yellow bacterial ooze from a great many bundles, at least 100. Stem sliced longitudinally for a distance of 1.5 feet above the basal cut, the lower nodes are brown and the upper ones are yellowish brown. The internodes are greenish white, with many yellow bundles. The infection can be traced as far up as the slicing. Stem above this broken off. One medium-sized cob, badly infected. At least 4 bundles show yellow ooze in the very tip of the cob. This cob bears no well-developed kernels, probably because it is so badly infected. There are numerous yellow bundles in the husks, and numerous areas where the bacteria have oozed through to the surface (inner face) of husk. One small ear, gnawed by insects, shows yellow bundles at the uninjured base. The base of the stem cut longitudinally shows 3 brown nodes; the whitish internodes show yellow stripes (bundles) with bacteria oozing out in many places. LXIV. Cut 3 inches from base, yellow slime oozes from nearly every vascular bundle. Basal portion slit longitudinally, the 3 basal nodes are brown and the greenish-white internodes have bright yellow bundles. The disease runs into the base of one of the ears. The bundles of the leaf-sheaths are occupied, and the bacteria have also made spots in the parenchyma and oozed out as yellow slime on the inside of the leaf-sheaths. A section of stem about a foot above the basal cut shows plenty of bacteria in the vessels ; bacteria are also present as a yellow ooze from the bundles a foot above the last cut. One small ear shows yellow bundles very near the tip of the cob. The husks also show numerous water-soaked spots where the bacteria have oozed from the vascular bundles. Another small cob higher up the stem is slightly infected at the base. Slit the middle section of the stem longi- tudinally and examined the internodes. There are many yellow bundles. To the unaided eye the disease runs out in the upper 18 inches of the stem. LXV. Top broken off. An abundance of good roots, as in case of nearly every one of these plants. Cut 3 inches from the base, there is a yellow ooze from nearly every bundle. Base slit longi- tudinally shows the lowest 3 nodes brown, the internodes white, with numerous bright yellow bundles. 106 BACTERIA IN RELATION TO PLANT DISEASES. Stem above the basal cut slit longitudinally for a distance of a foot, the nodes are yellowish brown, and the internodes have bright yellow bundles. There are 2 small ears ; both show infection at the base of the cob, and in the husks of one are numerous water-soaked spots, from which the bright yellow bacteria have oozed to the inner surface. LXVI. Cut 3 inches from the base, there is a yellow ooze from nearly every bundle. Slit the basal part longitudinally; the three nodes are brown; the greenish-white internodes show distinctly yellow bundles. Plant cut 1 foot higher, there is a bright yellow ooze from many of the bundles at the upper cut. Slit this foot-long piece longitudinally ; it contains 2 nodes which are yellowish brown ; the yellow stripes (yellow bundles) in the greenish white internodes are conspicuous. Cut 8 inches below the male inflorescence, there is a yellow bacterial ooze from about 30 bundles as far up as this (29 inches from the base of the stem). Slit this upper, 14-inch, portion longitudinally; the nodes are distinctly yellow and show a yellow ooze; the greenish white internodes bear conspicuous yellow bundles. In some places the bacteria have oozed out of the bundles and formed yellow spots in the parenchyma of the internodes. On examining microscopically the bacteria are seen to be in nearly or quite every bundle 4 inches below the male inflorescence (3^, inches from base of stem). The male inflorescence was broken over about 1 inch above the point from which the section was cut. There were 2 ears, both of which showed infection at the base. The bacteria are single or joined end to end in twos or fours. They are enormously numerous, and I fail to see any distinct motility. They seem to be rather more than twice as long as broad. LXVII. Three abortive ears and one ear with kernels. The three small ears show slight infec- tion at the base, while the large ear shows distinct yellow bundles at the base of the cob, and all the husks when cut near the base show a yellow ooze from the bundles. When cut 3 inches from the base, the stem shows a yellow ooze from nearly every bundle. The basal part split longitudinally shows 3 brown nodes, with many yellow bundles in the internodes. The first 3 nodes above the basal cut are also brown, while the remaining nodes of the stem show a yellow bacterial ooze. There are numerous yellow bundles in the lower internodes. The disease apparently runs out (hand-lens) at the node next below the tassel. LXVIII. Cut 3 inches from the base: There is an immediate yellow ooze from nearly or quite every bundle. Cut the stem a foot higher : There is a yellow ooze from nearly every bundle. Slit the basal portion longitudinally; the 2 nodes are brown; the greenish white internodes contain yellow bundles. One large and one small ear. Both show numerous bundles, from which yellow bacteria ooze ; these are near the base of the cob. The bundles of the leaf-sheaths also show yellow ooze. Slit the middle portion of the stem longitudinally; the white internodes contain numerous yellow bundles and in places the bacteria have escaped from the bundles into the parenchyma, forming small pockets from which they ooze out on section as yellow slime. Cut 20 inches farther up, the infection is incon- spicuous at this height ; there are a few bundles plainly diseased in the leaf-sheath surrounding the stem, but I can not see any in the stem. Six inches lower, just under the node, there are at least 40 bundles infected. This is at a distance of about 2.5 feet from the ground. The disease is present in a number of the leaf-sheaths half-way up the stem. About a foot below the tassel at least 20 bundles in the stem show the yellow bacterial ooze, and a number of the bundles in the surrounding leaf-sheath also show the yellow ooze. The bacteria have oozed out into the parenchyma to a greater extent in the internodes of this plant than in any other plant examined. They occur as small bright yellow spots in the white pith. LXIX. A large plant with 2 large and 2 small ears. The bacteria are present in a leaf-sheath half-way up the stem. They are also present in the base of the blade of the same leaf. The 2 smaller ears show yellow bacterial ooze from the bundles at the base of the cob. With a hand-lens I can not detect any bacterial slime in the leaf-sheath next higher up, nor in the one above that, but I presume a microscopic examination would show its presence there. (Later: The microscopic examination was made and showed that the bacteria were present in 6 bundles of this leaf-sheath.) Nothing certain as to any infection in the 2 larger cobs. Not examined with compound microscope. Cut main stem 3 inches from the base; there is an immediate yellow ooze from nearly or quite every bundle — 150 or more. (Examined later under the compound microscope and counted 184 bundles, of which at least 1 50 were infected.) Slit the stem longitudinally the whole length, a distance of only 3 feet; the basal 3 nodes are brown, the next 3 are yellowish; the internodes are greenish white, with bright yellow bundles and yellower spots in the parenchyma between the bundles where the bacteria have formed small cavities. The infection continues distinctly up to the last internode but one, where it becomes less conspicuous and is not visible under the hand-lens. As in all the plants thus far examined, the bacteria become less abundant toward the top of the stem. This plant bears numerous good roots, as in case of nearly all the rest. The basal part slit longitudinally shows 3 brown nodes and whitish internodes with yellow bundles. I examined in cross-section a dozen or more roots quite carefully. If there is any infection whatever, it is not visible under the hand-lens except in the case of one root from which there is a slight yellow ooze. None of the roots show any external signs of disease. STEWART'S DISEASE OP SWEET CORN (MAIZE). 107 LXX. There are 4 ears, 3 abortive. The abortive ear nearest the base of the stem is conspicu- ously diseased only at the extreme base. Cut an inch higher up, I can see no evidence of infection with a hand-lens, except in some of the bundles of one of the outer husks. The best ear, which is only a nubbin, shows distinct infection at its base. Cut 1 inch higher up, the bacteria are still present, both in the base of the cob and in the husks. One very small ear shows numerous yellow bundles at the base and also in the husks. Main stem cut 3 inches from the base, there is a yellow ooze from nearly or quite every bundle. Cut 1 foot higher and examined at the upper part, numerous bundles show the yellow ooze. Cut 1 foot higher and examined at the upper cut, there is no distinct infection. Cut 4 inches higher, that is, 1 inch under the male inflorescence, I can not see any distinct signs of infec- tion. The basal portion of the main axis shows 4 brown nodes and the greenish white internodes have conspicuous yellow bundles. The foregoing include all of the plants examined up to the date of the first hard frost, the night of October 29. The plants were covered with paper bags at 10 o'clock p. m., but too late to protect them, i. e., when the temperature was between o° and i° C. The leaves of all the plants were frosted. On October 31 most of the plants had recently frosted green foliage, but 16 of them had dried-out white foliage, and these 16 were pulled up separately and taken into the laboratory for examination. My notes on these plants are as follows : All the plants, with one exception, had abundant and sound roots. The male inflores- cence, where present, was dried up, and all the leaves were dried and shriveled. The plants were from 4 to 4.5 feet high. The stems of all the plants were green. LXXI. The stem cut 3 inches above the root shows no bacterial ooze from the bundles. The 6 lowest nodes are distinctly brown, while the 4 upper nodes are yellow, and there are very many bundles in the upper part of the stem which show the yellow bacterial ooze. A hasty microscopic examination of the portion of the stem between the second and third nodes shows no bacteria in the bundles. There are two ears, one small, which does not show, clearly at least, any bacterial ooze; the larger ear, cut crosswise at the base, that is, across the cob, shows two or three bundles very distinctly marked with the yellow ooze — possibly several more, but not clearly seen. In a transverse section of the stem 6 inches below the male inflorescence there are numerous bundles which distinctly show the yellow bacterial ooze. It is especially noticeable around the periphery. LXXII. This plant shows very slight signs of the disease. In a cross-section 3 inches from root no ooze could be found. Likewise in a longitudinal section of the base there is no ooze. The lowest node is discolored. The second node is discolored a little. No other nodes show any discoloration whatever. The only bacterial ooze found is in one node about half-way up the stem and in a cross- section in the base of the cob, the yellow slime running out also into a few of the inner husks. LXXIII. A transverse section of stem 3 inches above the base shows numerous bundles from which there is a yellow bacterial ooze. In the longitudinal section the lower nodes are brown, with yellow bundles in the internodes. There is one small undeveloped ear, which shows no bacterial growth. A somewhat larger ear badly eaten by insects shows in the uninjured part numerous bundles with yellow bacterial ooze. A few yellow bundles are present in the second node below the tassel. LXXIV. This is a very severe case. A cross-section 3 inches from base shows a large number of bundles with a bright yellow ooze. In a longitudinal section of the base these bundles may be traced the entire length by their marked color. The 3 nodes of this base are very badly discolored, being nearly black. In the section above the basal cut, 2 of the nodes show brown discoloration, while the upper 3 show yellowish discoloration. At least 2 yellow bundles can be seen in a transverse section 6 inches below the tassel, in the internode between the tassel and the first node below. There are 2 ears, a small undeveloped one which shows bacteria present at its base, and a larger one which shows numerous yellow bundles at the base, the majority of which run out into the husks. LXXV. In a cross-section 3 inches from base of the stem numerous yellow bundles are seen. A longitudinal section of the base shows 3 badly discolored nodes, the internodes having many yellow bundles. The first 3 nodes above the basal cut are distinctly brown in color, while the upper nodes are yellowish. The disease can be traced to a node about 1.5 feet below the tassel. There is one ear on the plant, the base of which shows numerous yellow bundles, nearly all of which run out into the husks and not into the cob. LXXVI. A small plant. There is one ear on this plant, the base of which shows numerous bundles from which the yellow bacteria ooze. One of the inner husks has numerous water-soaked spots, from one or two of which the bacteria have broken out to the surface on the inner side of the husk. The upper nodes of the stem show on section a distinct yellow bacterial ooze from the bundles. 108 BACTERIA IN RELATION TO PLANT DISEASES. The plant was broken off about 1.5 feet below the tassel. A eross-seetion 3 inches from the base shows very few bundles with the yellow ooze. A longitudinal section of this base shows but one bundle clearly marked with the yellow ooze. Several more longitudinal sections were made, and other bundles were found which showed the yellow discoloration. LXXVII. There are two medium-sized ears, neither of which shows any bundles infected with bacteria. A transverse section of the stem 3 inches above the base shows a brown discolored area on the periphery, but there are no bundles which show the bacterial ooze from the cut surface. In longi- tudinal section the nodes at the base of the stem are quite brown, but the disease, if present, runs out about half-way up the stem, as the 3 uppermost nodes appear to be perfectly normal. A hasty micro- scopic examination of the basal part of the stem shows 1 or 2 bundles infected with bacteria. LXXVIII. A transverse section of the stem 3 inches above the root shows numerous bundles from which there is a yellow bacterial ooze. The basal nodes are slightly brown, and there are many yellow and brown bundles in the internodes. The nodes higher up are yellow, and there are 2 or 3 yellow bundles in the uppermost node of the stem. In all the internodes above the basal cut yellow bundles can be seen. In the third internode above the basal cut the bacteria have oozed from a bundle into the parenchyma. This is also the case in some of the upper internodes. This plant is very badly infected all the way up to the uppermost node. There is one developed ear, which shows the bacterial ooze at the base of the cob in several places. The bacteria disappear one-third the way up the cob. None of the husks shows any signs of the bacteria. LXXIX. On the surface, at the very base of the stem, there are 3 places which are blackish- brown, and the epidermis is dead. The discoloration does not extend inward to any great distance. A transverse section of the stem 3 inches above the base shows a few bundles from which there is a yellow bacterial ooze. In longitudinal section the lower nodes are distinctly brown, while the upper nodes are yellow. In the node just below the tassel, yellow bacterial ooze can be seen issuing from at least one bundle. In the middle internode numerous yellow bundles can be seen. The one ear shows numerous places where the bacteria have come out in an ooze. A few of the husks show a very marked wet spot, i. e., bacterial ooze extending through several of the husks in the same place. LXXX. A cross-section 3 inches from the base shows no bundles colored by the bacteria. A longitudinal section of this base contains 3 nodes of a slightly brown color, but no bundles can be seen that contain the bacteria. The first two nodes above the basal cut are brown. Those higher up show no distinct coloration. With a hand-lens it is impossible to tell whether there are any bacteria present ; but a microscopic examination of material taken from one of the upper nodes shows the pres- ence of the bacteria. A cross-section at the base of the one ear fails to show any bacteria with a hand-lens. LXXXI. This is a smaller plant than most of the preceding. A cross-section 3 inches from the base shows very many bundles with the yellow ooze. These bundles may be traced in the longi- tudinal section from the cut to the extreme basal end of the stem ; the 2 nodes are brownish in color. A cross-section of the stem a foot above the first cut shows numerous bundles containing the yellow bacteria. Another foot higher, which is 6 inches below the male inflorescence, the cross-section shows possibly half a dozen bundles containing the bacteria. All of the nodes are somewhat browned. The upper 3 or 4 contain much yellow slime. The plant has two ears, both of which at the base show numerous bundles from which there is a yellow bacterial ooze. In the larger ear most of the yellow bundles run out into the husks, but a few of the bundles can be traced at least one-third of the way up in the center of the cob. In the smaller cob all of the infected bundles seem to run out into the husks. LXXXII. On surface around the basal nodes of this plant there are numerous brown cracks. A transverse section of the stem 3 inches above the base shows nearly every bundle filled with the yellow bacteria. Split the stem longitudinally — the 6 basal nodes show a brownish color, while all of those higher up are yellowish from the presence of the bacteria. There are numerous yellow bundles in all the internodes. At one place in the uppermost internode the bacteria have oozed into the par- enchyma. There is one developed ear, the pedicel of which shows numerous bundles containing the bacteria. These bundles appear mostly to run out into the husks, where a surface ooze may be seen in numerous places. There is also a place in one of the lower internodes where the bacteria have oozed out of a bundle into the parenchyma. I.XXXIII. A cross-section 3 inches from base shows numerous bundles with the yellow bac- terial ooze. There are 3 nodes below this cut, all of a brown color. The internodes show several bundles very distinctly marked with the yellow bacteria. They are especially noticeable on the edges (periphery). A longitudinal section of the stem above this cut shows 2 nodes of a brown color and all the upper nodes yellowish in color (the normal color is white or greenish white). In places in the internodes the bacterial masses are plainly seen in the bundles. In other places they can not be so clearly seen with a hand-lens. There is one ear which shows many infected bundles at the base. The greater number of these bundles run out into the husks. STEWART'S DISEASE OF SWEET CORN (MAIZE). IO9 LXXXIV. A cross-section 3 inches from the base shows 20 or more bundles with the bacterial ooze. In longitudinal section the base shows 2 slightly browned nodes. The bundles in the inter- nodes are considerably colored by the yellow bacteria. In making this longitudinal section several roots were cut longitudinally. The presence of any yellow material in these roots could not be seen. A longitudinal section of the stem above the basal cut shows 5 brown nodes. The internodes show about half a dozen bundles that are plainly marked (yellow) with the bacterial disease. Two or three yellow bundles can be traced up to the node immediately below the tassel. This plant has one ear, which shows yellow bundles at the base running out into the husks. A few of the infected bundles are visible as far out as the middle of the cob. LXXXV. A cross-section 3 inches from the base shows a few bundles occupied by the bacteria. Longitudinal section at base : here 2 discolored nodes and several bundles in the internodes with yel- lowish slime. Above the basal cut there are also 2 brown nodes, the remainder being yellowish. Yellow bundles can be seen in all the internodes. There are two ears, eaten by insects, but on section they show the yellow bacterial ooze issuing from the bundles at the base. LXXXVI. This plant possesses very few roots. At surface of the nodes from which the roots spring there are numerous dark brown spots. There are several of these spots in the second and third internodes. In some cases the brown spot has a hole in the center as if, perhaps, it had been eaten by some insect. These holes, together with the brown discoloration, extend inward, in one case as far as the center of the stem. A cross-section 3 inches from the base shows a goodly number of bundles with the bacterial ooze. A longitudinal section of the base shows 3 badly browned nodes, the stain evidently not caused by the outer injury before described. One node, however, shows a dis- coloration of an entirely different nature, which apparently comes from one of the aforesaid black spots on the outer surface. The internodes show bundles with the bacteria. Longitudinal section of the parts above the basal cut shows 3 nodes of a brown color, the remaining upper ones being yellowish or green. Some bundles in all of the internodes show bacteria. There is one ear, which shows at its base a few bundles infected with the yellow bacteria. On November 1, all the remaining plants were examined in the field one by one, but it is not worth while to describe any of them minutely, since it would be only a repetition of what has preceded. The results obtained are summarized in table 7. Table 7. — Number of Cases by Varieties and the Time when the Signs were First Observed. Date. Inoculated August o, in seedling stage, by placing bacterial slime on tips of young leaves. Cases. 1 Date. Inoculated August 13, 14, 15. and 16. in seedling stage, by spraying (atomizing) upon the plants water containing the bacteria. Cases. Aug. 28 Sept. 8 Sept. 8 Oct. 7 Oct. !0 Oct. 10 Oct. 14 Oct. 14 Oct. 17 Oct. 17 Oct. 21 Oct. 21 Early White (in greenhouse) Early White (in the field) 2 2 I 2 1 3 2 2 3 2 8 1 Oct. 7 Oct. 7 Oct. 14 Oct. 14 Oct. 14 Oct. 17 Oct. 17 Oct. 21 Oct. 21 1 1 2 I 5 3 1 5 Early White Cory. . . Early White Early White Cory Early White Earlv White Cory Early White Cory Early White After light frost— very little indication of frost injury. Oct. 27 Oct. 27 7 1 Oct. 27 Oct. 27 3 7 Early White Cory Plants pulled, examined, and found diseased after a heavy frost which confused external signs. Oct. 31 Oct. 31 Oct. 31 Nov. 1 Nov. 1 7 1 2 36 Oct. 31 Oct. 31 Nov. 1 Nov. 1 Nov. 1 1 5 32 40 43 Early White Cory Pirlv White Early White Cory Total Total "5 ■5' IIO BACTERIA IN RELATION TO PLANT DISEASES. Not all of the plants showed bacterial slime. On sectioning and examining with the hand-lens, about 160 were apparently free from the yellow bacteria, and all of these showed absence of brown stain in the nodes, except in some cases a very slight stain at the extreme base of the stem, which may have been due to incipient or arrested infection. It is not known why a portion of these plants escaped. The following explanations are offered: (i) Possibly most of the remainder were slightly infected and some undoubtedly would have devel- oped the disease in course of another month if they had not been killed by frost. (2) The infectious material was washed off these plants by copious flow of fluid from the water-pores during the seedling stage. (3) The inoculated leaves died and dried out before the bacteria had opportunity to reach the stem by way of the foliar bundles. (4) The bases (nodes) of these particular plants were exceptionally hard and offered unusual physical resistance to the movement of the bacteria. This is a very important point, as we shall see later. (See also ante, Cobb's disease of sugar-cane, p. 37). (5) The juice of these plants was more acid, or in some other chemical way was more injurious to the bacteria than that of the plants which became diseased. Total number of plants found diseased to such an extent that a microscopic examina- tion was not necessary to detect the bacterial slime, although it was made frequently, 266. Add to the above 30 that were plainly diseased when examined hastily under a compound microscope, and 18 doubtful cases which should probably be classed with the diseased, i. e., in which the bacteria could probably have been demonstrated by a more careful examina- tion (only a few minutes study was given to each one) , and we have the following result : Diseased plants, 296; doubtful plants, 18 (probably diseased); sound plants, i. e., not any visibly infected, unless the occasional slight brown stain at the extreme base be so inter- preted, 159; total, 473; per cent of diseased, excluding doubtful, 62. In this connection it must not be forgotten that the experiments were begun late and were cut short by a frost. Probably if they had been gotten under way 3 weeks sooner or if the frost had held off that much longer there would have been a considerably greater number of cases. At the end of the experiment all of the refuse was carefully gathered together and burned. Undoubtedly some of the organisms were left in the soil, but no diseased plants were left to be blown about or eaten by animals. Only the smaller roots were left in the soil. SERIES III TO VII, 1902. Employing the same strain of organism and susceptible varieties of sweet corn and making the inoculations during the seedling stage with young cultures in the same way as in Series I, but growing the plants to maturity in small pots in the hothouse under very different physical conditions, a very different result was obtained, most of the plants failing to take the disease. Of course one might attribute the failure to loss of virulence on the part of the organism, but, in the present state of our knowledge, this would be purely hypo- thetical, especially as no great amount of time intervened between the two experiments. I am, therefore, inclined to ascribe it rather to the very slow growth of the plants, this view being more in accord with other facts observed by the writer, e. g. , see Series XI to XV and XX to XXIV. Certainly the hard, dry tissues of these plants would afford a much greater physical obstacle to the movement of the bacteria than would soft juicy tissues. We do not know what chemical substances are stored in the plant during such phases of growth, but if acids are stored in excess, as might well be the case, then there would be also chemical difficulties in the way of general infection of the plant. All such problems remain to be worked out. The facts are as follows: STEWART'S DISEASE OF SWEET CORN (MAIZE). Ill August 20. — The following varieties of sugar-corn were planted: Red Cory, Early Red Cory, Early White Cory, Perry's Hybrid. There were many pots of each. All stood on a bench in one of the hot-houses. August 30. — Tubes of slant agar, Nos. 9 to 16, were streaked directly from tubes 1 to 8 of July 17 (the agar-stab-cultures brought from Long Island). September 3. — Masses of the yellow slime from these eight agar-streak-cultures were placed on the tips of the young leaves by means of a platinum loop. On some plants one leaf only was inoculated, on others two. The young plants were 1 to 2 inches high. The work was done between 1 and 3 o'clock on a cloudy afternoon. As soon as inoculated the plants were placed under the bench in the dark. September 4, night. — The plants remained under the bench about 28 hours. They were then put on the bench. September 6. — The plants are now about 3 inches high; there are some indications of infection at the leaf-tips, but noth- ing absolutely sure. October 18. — The first case was brought in and examined to-day. It is the variety known as Red Cory. The vessels are full of the yellow slime. October. — The second case, a very typical one, was found in the Early Red Cory. A plant of Early White Cory, acci- dentally broken by the gardener, was examined at the same time, but did not appear to be affected. December 16. — Those plants nearest to maturity were removed and dissected to-day. The culms were so hard that they could be cracked like pipe-stems. The plants were from 18 inches to 4 feet high and quite spindling. Most of the stems were green throughout (internally) and so brittle that they broke apart with a noise when bent at the nodes; even many of the internodes snapped like a dry stick, although the stems were not dead. The diameter of the stems at the base varied from 0.25 inch to 0.5 inch; most were under 0.5 inch. They had produced very few good ears. The plants were specked somewhat with Puccinia maydis, and many of the leaves had dried out, but mostly as a result of unfavorable conditions rather than by reason of the disease. January 5. — The remainder of the plants were examined to-day. These plants also were very brittle. Many of them were green throughout, though the pith is ordinarily white. Combining the early counts with those of Decem- ber 16 and those obtained to-day, we have the results given in table 8, which should be premised by the statement that yellow bacterial slime was found in the vascular bundles of the stem of all plants marked "diseased." Table 8. — Results of the Inoculations. Series and variety. III. Perry's Hybrid. .. IV. Early Red Cory. . V. Early Red Cory. . VI. Early White Cory VII. Red Cory Summary Diseased. '9 >4 Free. 45 44 30 105 98 Total. 5° 49 39 114 103 322 355 * Several with black bundles. As a result of examinations with the compound microscope, 6 of the 14 doubtful cases were found to contain bacteria, making a final total of 25 cases. Subtracting 60 check plants from the whole number, we have approximately 8 per cent of diseased plants, against 62 per cent obtained in the previous experiment. Three of the check plants became diseased. These plants were first crowded together for 2 months, or more, on the west side of the house, became too large for the pots, and grew very spindling. This crowding was due to the fact that we had no other place to set them. They were then moved into the middle section of the hot-house, which was much too warm for them. Some of them were repotted and others not. There they remained for about 6 weeks. They were then shifted back again to the cooler, north end of the house, middle bench, where they had room to grow and plenty of light, but they never overcame the original crowding and stunting. This interrupted growth had much to do, I suspect, with the slow progress of the disease. These plants stood at first on the bench, where the very successful infections were made earlier in the month of August, and the checks were close to the inoculated plants, so that they were not entirely safe. Only a few of the affected plants were badly diseased. SERIES VIII AND IX, 1903. In 1903, at the writer's instigation, an experiment was made to determine whether the disease could be communicated to field-corn and to pop-corn. This was entirely in the hands of Mr. James Birch Rorer, one of my laboratory assistants, whose notes are as follows : 112 BACTERIA IN RELATION TO PEANT DISEASES. July 8, 1903. — Set out to-day on the Flats 10 rows of corn, each hill containing 4 plants. The rows run from east to west and are as follows, beginning at the north end of the plot: 1. Uninoculated: 2. Inoculated (June 20 and 23) by touching water from Nelson's Yellow field (yellow flint) 12 hills. water- pores with loop of pure culture of Bad. stewarti: Mottled Pearl pop-corn 12 hills. Nelson's Yellow field 16 hills. Hominy (white flint) 12 hills. Mottled Pearl pop-corn 18 hills. Boone County White (white dent) 12 hills. Improved Clairage 18 hills. Improved Clairage (yellow dent) 12 hills. Boone County White 18 hills. Hominy 18 hills. September 30, 1903. — Cut out all of the Nelson's Yellow field corn, both inoculated and uninoculated. All the plants are more or less dead. The tassels are dried up and the leaves brown. There are some good ears. Many stems in both lots show an abundance of yellow ooze from the bundles. A section from each stem has been put into alcohol for future examination. This variety seems to be a much faster grower than any of the other varieties, which as yet are quite green and the ears are not yet ripe. Could it be the disease that hastened the ripening or is it a varietal characteristic? Only one of the five varieties proved susceptible to this disease, viz, Nelson's Yellow field corn. Why were the control plants of this variety also attacked ? On making inquiries I learned certain facts which appear to answer this question sufficiently. When the inocu- lations were made the check-plants stood some 30 feet away, but after a few days it became necessary to move the plants into another hot-house, where there was not much spare room, and where the inoculated and uninoculated plants stood much crowded and close together for a number of days. The infection of the check-plants probably dates from this time, as the gardener's hose would spatter all of the plants indiscriminately and that would dis- tribute over the control plants any bacteria remaining on the surface of the inoculated ones, or on the soil of the pots. The cultures used for inoculating were derived (June 19) directly from those direct transfers made by the writer on July 17, 1902, the organism being alive in each one of these 8 tubes. The Mottled Pearl pop-corn, Boone County White, and Improved Clairage were planted June 15 and were inoculated June 20. Inoculations were made from slant agar- cultures. A loop of the organism was touched to each drop of water exuding from the water-pores situated on the tips of the leaves. The drop became clouded. At this time the first leaf was just beginning to uncurl, so that the tip of the second leaf was exposed. The inoculated plants were put under the east bench in the pineapple house, where they remained from Saturday afternoon until Monday morning. They were covered with a framework of netting to keep out mice, and the top of this was covered with manila paper. The temperature of the house went up to 410 C. on Sunday and was down to 200 C. during the night. When the plants were taken from under the bench and put in the light the margins of the leaves were covered with drops of exuded water and all the leaves were some- what bleached. The White Hominy and Nelson's Yellow field corn were planted June 1 7 and inoculated June 23, when the plants were from 2 to 4 inches high. In most of them one leaf was uncurled, so that the tips of the second and third leaves were often exposed, and three leaves were inoculated on some plants. The work was done in the afternoon, in the same manner as in the preceding series, except that the White Hominy was inoculated from a 24-hour-old bouillon-culture derived from the slant agar; the plants were placed under the bench until the morning of June 25, when they were uncovered and placed on the bench. The leaves were more or less etiolated. The result of this experiment shows that the organism had not lost its virulence for susceptible varieties (Nelson's field corn) at the end of 1 1 months' growth on agar. The thoroughness with which the work was done also indicates that the other varieties experi- mented with are not susceptible to this disease. SERIES X, 1905. In the spring of 1905 the inoculation experiments were repeated on several varieties of sweet corn and field corn grown for the purpose in a bed in one of our hot-houses. The STEWART'S DISEASE OF SWEET CORN (MAIZE). 1 13 writer controlled the cultures used and made notes on the results. The inoculations were made by Miss Alice C. Haskins, one of the laboratory assistants. In this case all of the plants were inoculated by needle-punctures, mostly in the blades of upper leaves. No hypodermic injections were made. Owing to unavoidable delay on her part the plants were not inoculated until they were about half-grown, i. c, 3 weeks later than I had planned, and when it might be presumed that they would be able to complete their growth before any general infection of the plant could take place. The needle-pricks were made March 10 to 14. The cultures used were grown on slant agar and on potato and were subcultures from an isolation of the previous year. Altogether 57 plants were inoculated — 32 sweet-corn and 25 field-corn. With one or two exceptions, two leaves were inoculated on each plant. The bacterial slime was mixed with water; a portion of this was spread on the leaf -blade with a platinum loop and punctures were made through the smeared portion ; a little more of the fluid was then spread over the punctured area. The following notes were made by the writer : March 20, 1905. — A few of the plants show slight indications of infection streaking down from some of the pricks. One plant also shows two narrow water-soaked stripes near the pricked area, but not centering in any of the pricks, as though it were stomatal infection due to the material placed on the leaf. This was visible the day before yesterday and this morning. Not visible this afternoon. The sweet corn is in full blossom. Some of the field-corn plants are coming into blossom, others not. April 8, 1005. — The first case was brought in to-day from hot-house No. 4. It is plant No. 18. Two inoculations were made on the base of two uppermost leaf-blades, which are now dead, together with the whitish, dwarfed male inflorescence. The plant, which is a small one, bears 2 small ears and 3 green leaves. The progression of the dry leaves is the reverse of that observed in the field, but otherwise the disease looks typical. On cutting open the base of the stem it is free from infection, but the bundles in the green leaf-sheaths of the inoculated leaves contain a pale yellow ooze, which under the microscope is bacterial and of the typical appearance. The yellow slime occurs also in the vessels of the upper part of the stem, mostly in the peripheral bundles. This plant is one of the sweet corns. It was inocu- lated March 13, from a potato culture (tube 1, March 10). The plant has shown signs of the disease for 4 or 5 days »'. e, dry leaves and a white top. The inoculation took place just before flowering time. April 29, 1905. — A majority of the inoculated plants show dead stripes in the pricked leaves. In some cases the entire pricked leaf is dead. Quite a number of the plants are dead at the top. May 1, 1905. — Plant 19. Bacteria in vessels of the stem 18 inches from the top. Top dead. All the basal leaves are green. Plant 20: Basal leaves green, only the pricked ones are dead. Top dead and white. Bacteria present in stem 18 inches from the top. Plant 23 : The upper three leaves of this plant which were inoculated now have dead stripes. All the other leaves are sound. Top dead. No bacterial ooze visible with hand-lens in cross-section of upper part of stem. Cut it in three places. . Plant 33: Leaves above and below pricked ones are green. One pricked leaf is dead. The other is dying. Bac- teria not visible in stem under hand-lens. Plant 37: One pricked leaf is dead and two-thirds of the blade of the other. All the other leaves are healthy. Bacteria are not visible in the upper part of the stem, with the hand-lens. The above-mentioned 5 plants are sweet corns. May 8, 1905. — Four plants were cut out this morning and examined. No. 2 : The middle, inoculated leaves are now dead. Their sheaths are infected by the bacteria. The top leaf is green. The male inflorescence is dead. The basal leaves are green. The middle portion of the stem contains the yellow bacteria in some abundance. No. 5 : The upper leaves and top of this plant are dead. The lower leaves are green. Cross-sections of the stem 2 feet from the top of the plant show a vellow ooze in a few of the bundles. No. 27: The male inflorescence is dead, but it bears some kernels of ripe corn, so that it may have died naturally. The top of the plant and the basal leaves are green. The inoculated leaves are dying. There is a visible stem infection only near the junction of the inoculated leaves and this infection is confined to a few bundles. No. 35 : The male inflorescence and the upper 4 leaves are dead. The lower leaves are green. The yellow bac- terial ooze is distinct in a few bundles in the upper part of the stem. Nos. 2,5,27, and 35 are sweet corns. As in the plants already cut and described, the stem-infection is slight in comparison with the stem-infection in the plants inoculated 3 years ago in the seedling stage by way of the water-pores. The inoculations were made too late in the growth of the plants. Mav 13, 1905. — Five plants were cut out and examined to-day. Plant 17- Top dead; base green; pricked leaves dead. The bacterial infection is present but slight. Plant 22 : Top dead; pricked leaves dead; the lower leaves green; stem green. On cross-section of the upper part of the stem S or 10 bundles show a distinct yellow ooze. Plant 24: Top dead, including inoculated leaves. Slight infection of the stem, that is, yellow bacterial ooze from a few bundles, mostly peripheral ones. . Plant 26: Top dead; pricked leaves dead; lower leaves green; stem green. A few bundles in the upper part of the stem show a yellow ooze on section. . Plant 5 1 (field corn) : Cut out and dissected. No indications of stem-infection when examined under hand-lens. H4 BACTERIA IN RELATION TO PLANT DISEASES. Nothing is visible in the field corn inoculated at the same time as the sweet corn, i. c, there are no signs of disease. May 22, 1905. — Cut and examined field-corn plants labeled 8, 10, 11, 12, 39, 40, 41, 42, 44, and 46, which were drying up (the corn is ripening). None of them showed any signs of disease in the stem except numbers 42 and 44. In the stems of these two plants a few of the vessels were found occupied by the yellow slime, but only a few, the infection being slight and confined to the vicinity of the inoculated leaves. This also shows that it is possible to inoculate the disease into field corn, but in general it appears to be much less susceptible than the sweet corn. The variety was not Nelson's Yellow. As we have seen, it is a frequent movement of the bacterium to grow out into the husks surrounding the ear and then come to the inner surface, thus affording a very favorable opportunity for the contamination of the surface of the kernels. In this connection the following field observations are of interest : INFECTED SEED AS A MEANS OF DISTRIBUTION. In the summer of 1903 the writer found this disease in two places in Washington under circum- stances which pointed to seed-corn as the carrier of the pathogenic organism, and the Congressional Seed Distribution as the agent of dissemination. Although these in- ferences were not clinched by the actual demonstration of Bacterium stewarti on or in the suspected seed- corn, the circumstantial evidence is nevertheless so strong as to be worth recording at some length. This evidence is the more credible be- cause the organism is often found in the vascular bundles of the cob, and may penetrate from these into the base of the kernel (see figs. 45, 46, 47, 48), and because, as already recorded, it occurs very frequently in the inner husks and shows a strong tendency to come to the sur- face of these organs as a wet slime, which might easily contaminate the surface of the kernels and has been seen so contaminating them by Stewart on Long Island and by the writer in Washington (figs. 49- 50, 51, 52> 53)- FIELD OBSERVATIONS OF 1903. On July 18, 1903, the writer, greatly to his surprise, discovered one typical case of the Long Island sweet-corn disease (Bad. stewarti) in the garden of Mr. Brown at Takoma Park, Fig. 45/ *Fig. 45. — Bacterium stewarti in sweet corn. Flats, 1902. Vertical section through two kernels and a portion of the cob, showing the relation of parts: A, Floral bract; B, C, Scales of the perianth. Those bundles of the cob in which the bacteria are abundant are drawn in solid black. Bacteria also occur sparingly in the base of each kernel at X, X. This section pas les nearly at right angles to that shown in fig. 57 and, therefore, does not include the embryo. Slide 478 (6, stained with pyronine and methyl violet. Drawn from a photomicrograph, but with various details strengthened. Section torn in places, as at A. For the appearance of D under a high power see fig. 46. STEWART'S DISEASE OF SWEET CORN (MAIZE). 115 Maryland, 8 miles from the experimental grounds of the Department of Agriculture. The variety was Cosmopolitan and was grown from seed obtained from the Department of Agriculture. Mrs. Brown said the variety came up badly and some stalks died while small. This plant was about 3.5 feet high, with three suckers. The main stalk had a dead- white inflorescence (all of the flowers were dead and also the upper part of the rachis). The stem was green, but every leaf on the plant was dead or dying. The death of the leaves began with the lower ones, and the cause was not apparent on the surface. When the stem was cut 4 inches from the base, there was a copious yellow ooze from the vessels. I cut it 5 inches under the male inflorescence and found yellow bacteria there also, but less abundant. The base of the stem, when slit longitudinally, showed brown nodes and white internodes, with yellow and brown bundles. Two of the suckers ap- peared to be healthy. The bundles of the third sucker at a foot from the ground contained the yellow bacteria in abundance. It seemed likely that the dis- ease was introduced into this garden on the seed, and an inquiry was made as to its origin. It formed part of the Congressional Seed Dis- tribution of the spring of 1903.! It was bought from a reputable seed firm in Philadelphia, for whom it was grown under contract by a seedsman in Ohio. A large amount had been contracted for, but only a small quantity was furnished. The reason for this failure to deliver the seed is given in the following letter of November 5, 1902, addressed to Mr. A. J. Pieters, of the Department of Agriculture: Dear Sir: We regret extremely to have to advise you that yesterday's mail brought a letter from the grower of the Cosmopolitan sweet corn stating that this variety is rotting and he will only be able to deliver 20 per cent of the quantity ordered. Our last previous report on this variety was 80 per cent and it is very disappointing to now be advised of such a terrible "fall down." Yours very truly, On inquiring as to where I could find more sweet corn grown from this same suspicious seed, Mr. Pieters told me that he had himself directed some of it to be planted along with many other varieties for a trial test on the Potomac Flats (one of the Department farms) south of the Washington Monument. An inspection of these trial plots by the writer *Fig. 46. — A detail from fig. 45, showing in cross-section a vascular bundle from a corn-cob, the xylem part of which is occupied by Bacterium stewarti. fAlso in 1913, Mrs. Enlows, one of my assistants, found this disease at Parsons, West Virginia, in sweet corn grown from seed distributed by the Department of Agriculture (Congressional Seed Distribution of 1913). n6 BACTERIA IN RELATION TO PLANT DISEASES. T 5Ee>> revealed an astonishing condition. On July 21,15 Per cent of the Cosmopolitan corn was diseased by Bad. stewarti and a month later there were 21 additional cases, making a total for this variety of 26 diseased plants to 7 sound ones (80 per cent), 7 other plants having disappeared prior to July 21, probably as a result of disease during the seedling stage or soon after. Many other varieties in the plot were more or less diseased. The subject was of so much interest that the writer made a trip to Wakeman, Ohio, to learn something more concerning the nature of the "rot" which on good land could destroy four-fifths of a crop of seed-corn. The field on which the Cosmopolitan corn had been grown was then planted to other crops and nothing could be learned from an inspection of it. It was a level field of good char- acter. The owner of the field, the man who grew the corn, evidently knew much more than he was willing to ad- mit ; in fact he was so suspicious and secretive when questioned con- cerning the nature of this rot that nothing of any value was obtained from him, other than the unavoid- able inference to be drawn from the way in which he resented all in- quiries. That the rot in his field was due to Bad. stewarti the writer has not the slightest doubt, but it would have been much more satisfactory and helpful to agriculture if he had been willing to cooperate in settling the question. To return now to the trial plots on the Flats. These were about a quarter mile from my experiments of 1902. Maize had been grown in many varieties, in large tracts, on this farm for several years and this was the first spontaneous appearance of the disease. The chance of its having come from my experiments of 1902 seemed extremely slight. The most reasonable explanation of the appearance of the disease in such a severe form was Tig. 47. — Bacterium stewarti in sweet corn. Cross-section of outer portion of a cob, showing how the bacteria netrated into the base of the immature kernel. They are only in the vascular bundles and their immediate vicinity (solid black parts). Plant inoculated in August 1902, on tips of the leaves in the seedling stage. Material collected and fixed (in strong alcohol) Oct. 27. Figure drawn with the help of the Abbe camera, but numerous nuclei omitted and also certain indistinct tissues in the angles and around the margins of the lunulate area in which the scale of magnification is placed. Slide 235 B 14. For a detail see Fig. 48. STEWART'S DISEASE OF SWEET CORN (MAIZE). 117 that several varieties of seed-corn were infected when planted, yielding some diseased plants early in the season, and that other varieties were infected therefrom. This is the more likely because in some varieties there were frequent skips, whole hills being missing. Inas- much as this trial plot furnishes the best evidence possessed by the writer on the interesting question of varietal resistance, all the data are given in table 9 for what they are worth, i. e., as a suggestion toward future experimentation. SUSCEPTIBLE AND RESISTANT VARIETIES. Table 9. — Cases by Varieties on July 21 in Department of Agriculture Trial Rows, Southwest Corner of the Farm on the Potomac Flats, beyond the Stables. Short rows, about 20 plants in each row, some more, some less. Trial No. 853 854 855 856 857 858 859 860 86l 862 863 Source. Seed and Plant Introduction 9573 Seed and Plant Introduction 9447 Burpee Ferry Free Seed Distribution 278 (Clarke, Conn.), double row. Burpee Emerson Free Seed Distribution 346, purchased from Burpee and grown by Clark, Ohio, double row. Free Seed Distribution 228(Clark, Conn.) double row. Free Seed Distribution 370, western seed (Waterloo, Nebr.), double row. Free Seed Distribution 375, western seed, double row. Variety. Malakoff Malakoff Golden Bantam Oakview Early Market Extra Early Red Cory Fordhook Emerson's First Crop. . Cosmopolitan Metropolitan Hys' Metropolitan. . . . Crosby's Early No. of eases. Per cent, diseased. 4 20 2 10 5 25 10 5° 7 '7 2 10 3 15 5 15 2 5 5 12 3 9 Long rows, originally 40 plants per row, reduced to an average of 32 per row, according to my count. 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 882 Free Seed Distribution 230 (Clark, Conn. ) Free Seed Distribution 245, Robinson (Waterloo, Nebr.). Free Seed Distribution 271 (Clark, Conn.) Free Seed Distribution 305, Robinson. . . A. N. Clark (Conn.) Holmes (Harrisburg) Ferry. Many skips, only 6 plants remain. Free Seed Distribution 369, western seed A good row by the side of two very poor rows, 869 and 870. Tall corn. Free Seed Distribution 37 1 , western seed Free Seed Distribution 352, Phillips (To- ledo, Ohio). Free Seed Distribution 373, western seed. A. N. Clark, Conn Do Free Seed Distribution 227, Clark, Conn. . Free Seed Distribution 270, Clark, Conn. . Free Seed Distribution 276, Clark (seed- house floor). Free Seed Distribution 229, Clark, Conn Free Seed Distribution 275, Clark, Conn A. N. Clark, Conn Crosby's Early. ....Do ....Do .... Do .... Do Holmes' Premo Early Sweet or Sugar. Champion Early Black Mexican (tall corn) Potter's Excelsior. (Fine growth; full row.) Egyptian (tall corn; the one case a dwarf). Potter's Excelsior Country Gentleman (4 skips) Country Gentleman ...Do ...Do Hickory Improved (diseased plants, very small). Hickory Improved (both large plants, slightly affected). Hickory Sugar 10 T5 4 2 1 o o o 1 (slight) 2 2 4 33 25 25 30 33 20 66 6 '5 o ♦And some skips. tSkips and inferior plants. n8 BACTERIA IN RELATION TO PLANT DISEASES. Table 9 — Continued. Originally 40 plants per row, now an average of 35. Trial No. Source. Variety. S83 Free Seed Distribution 372, western seed 884 ' Free Seed Distribution 231, Clark, Conn. 885 Free Seed Distribution 272, Clark, Conn . 886 Ferry 887 Free Seed Distribution 374, western seed . 888 Beadle 889 Free Seed Distribution 269, Clark, Conn. 890 Free Seed Distribution 210, Cushman. . . 891 A. N. Clark, Conn | 892 Free Seed Distribution 277, Clark 893 Ferry Worst row in field; only 4 plants left. 894 Eastman j A half row, of which no plants now remain, i. e., after pulling out these 4. 895 Burpee 1 A half row, i. e., about 16 plants. Old Colony ....Do Old Colony (one skip) Old Colony (four skips) Zig Zag Evergreen Beadle's Mammoth Evergreen. . . Stowell's Evergreen ....Do ....Do Late Mammoth (several skips) . . Mammoth (many skips; all dwarf) Golden Hybrid Sheffield. No. of cases Per cent, diseased. O 0 0 0 0 0 O O 0 0 3 8 0 0 0 1 0 3 2 I 6 20 Field corns. 896 897 898 899 900 901 Free Seed Distribution 253, Woods Truckers' Favorite (very tall) . Free Seed Distribution 40, Woods. Free Seed Distribution 42, Woods. Free Seed Distribution 44, Woods. No firm name (half row) No firm name (half row) Total cases. White Dent (very tall). White Dent. ...Do No name. . . ....Do o o o o 3 7 140 o o o o '4 33 Those varieties most subject were: Oakview Early Market, Crosby's Early, Cosmo- politan, Extra Early Red Cory, Holmes' Premo, Early Sweet or Sugar, Black Mexican, Golden Hybrid, Golden Bantam, Hys' Metropolitan, 900, 901, and Malakoff. All early sorts except Black Mexican, which is medium and very sweet.* Those sweet corns nearly free were: Champion Early, Potter's Excelsior, Egyptian, Country Gentleman, Old Colony, Zig Zag Evergreen, Stowell's Evergreen, and Sheffield. All of these are late varieties except Champion Early and Sheffield. On August 22, in my absence, Mr. Rorer began for me a second count of cases (new cases) in the trial rows on the Flats, but was unable to finish it. His notes, as far as they go, are given below: 853 [Malakoff]. — No live stalks at all. Tassels all dried up and leaves on every stalk entirely dry and brown. No good ears at all. Most of them are smutted. Cases, 12. Nearly every stalk was diseased by the bacteria. 854 [Malakoff]. — Presents same general appearance as 853. Cases, 11; sound, 2. 855 [Golden Bantam]. — Looks same as 853 and 854. Cases, 1 1 ; sound, o. Two stems were entirely rotted away, so that condition of bundles could not be made out. In all these cases almost every bundle showed yellow ooze. 856 [Oakview Early Market]. — All dead. Only 6 stalks. Cases, 5. One stem too badly rotted to make anything out as to bundles. 857 [Extra Early Red Cory]. — A little better-looking row. The stems are taller and some of the leaves are more or less green. None look very healthy, however, so have cut all out. Cases, 23; sound, 13. 858 [I'ordhook]. — Very bad-looking lot. All stems entirely dead and leaves dry and shriveled. Cases, 13; doubtful 2; sound, 1. Two stems too much decayed to make out bundles. *In his bulletin Stewart states that on Long Island the disease is most destructive to early varieties. According to observations made by the writer in 1909 at Kensington, Maryland, the variety Pocahontas is also much subject to the disease. This is a small early sort. STEWART'S DISEASE OF SWEET CORN (MAIZE). 119 Fig. 49. f Fig 504 *Fig. 48. — Section of periphery of a sweet-eorn cob, showing Bacterium stewarti filling the vascular bundles and forming closed cavities in base of kernel. For orientation see fig. 47. Drawn under Abbe camera. Slide 235 B 14. fFiG. 49. — Cross-section of husk of sweet corn occupied by Bacterium stewarti. Bacteria oozing to the surface on many parts of the husk. From a pure-culture inoculation made in August on the leaves in the seedling stage (Potomac Flats experiment). Material collected and fixed in alcohol Oct. 21, 1902. Stained with carbol-fuchsin. Drawn with Zeiss 8 mm. apochromatic objective, No. 12 compensating ocular and Abbe camera. Slide 477 A 2, lower row. The stoma shown in fig. 50 lies on the next section at X. JFig. 50. — Husk of sweet corn, showing Bacterium stewarti making its way to the surface through a stoma. These bacteria undoubtedly came from the crowded vascular bundle shown in fig. 49. Slide 477 A 2, lower row. 120 BACTERIA IN RELATION TO PLANT DISEASES. 859 [Emerson's First Crop]. — Very bad looking. Cases, 8; sound, o. 860 [Cosmopolitan]. — All stems dead, with dried tassels and brown leaves. Cases, 21; sound, 7. 861 [Metropolitan]. — A slightly better stand, but most of the plants show the external signs of the disease. Cases, 18; sound, 12. Some of these cases showed only one or two bundles affected. 862 [Hys' Metropolitan]. — Looks about like 861. Many plants show external signs of disease, but there is a better stand than in the first four or five rows. Cases, 14; sound, 14. Of the diseased stems in this lot 5 or 6 had only one or two bundles diseased. 863 [Crosby's Early], — Looks diseased, but is a rather better stand than others. Cases, 15; sound, 15. Some of the cases only slightly diseased. A little of this seed corn remained undistributed in the hands of the Free Seed Distri- bution officials of the Department of Agriculture. As soon, therefore, as possible after the discovery of the above-mentioned facts respecting infection of the trial rows, i. " ? 3? < < > T ? « Fig. 51. Fig. 52. t The place selected was an old field on the Arlington estate, one which certainly had not been planted in maize since before the Civil War, i. e., for more than 40 years, and perhaps never. This field is about a mile and a half from the trial plots just described, on the other side of the Potomac River, and there was very little communication between the two farms, i. e., the management used different teams and tools and another set of farm laborers. Here, if anywhere, one might expect to get the uncomplicated effect of the seed, since we *Fig. 51. — Surface of a corn-husk highly magnified, showing a single stoma with Bacterium stewarti oozing to the surface. The conditions immediately under such a stoma are shown in figs, so and 52. Slide 477 B 1. From a plant inoculated in the seedling stage. Collected Oct. 21, 1902, Potomac Flats. fFiG. 52. — Surface view of husk of sweet-corn attacked by Bacterium stewarti. Surface slightly curved, so that the microtome knife has removed the greater part of the epidermis (crinkly-walled cells) laying bare the deeper tissues; R, remnant of epidermis; Se, sub-epidermal tissue; Sc, sclerenchyma strand; St, remnants of stomata, about all that is left of the three upper ones being a portion of the mother cell on the left side (see figs. 50 and 51 for orientation); Sst, substomatic chambers filled with bacteria. Plant inoculated in August in the seedling stage by placing bacteria on the tips of the leaves. Collected October 21, 1902, from Potomac Flats. Slide 477 A 8, stained with carbol fuchsin. STEWART'S DISEASE OP SWEET CORN (MAIZE). 121 have no evidence of any general distribution of this organism in agricultural soils, rather the contrary, outside of districts where sweet-corn is grown, and since in many bacterial diseases, both plant and animal, we have an enormous amount of evidence tending to show that the pathogenic bacteria are not carried far by the wind ; or, if so carried, do not induce disease. I reasoned as follows: If the case or cases of this disease at Takoma Park and those which appeared in the trial-rows on the Flats were due to local causes, i. c, to organisms already present in the soil, and not to infected seed, then the plants grown from the same lot of seed-corn on Arlington Farm should be healthy. If, on the contrary, Bacterium stevuarti was present in a living condition on any great number of the kernels, then the result would be the same as at Takoma Park and in the trial-rows, i. e., many cases of the disease, and the disease would appear first in those varieties carrying the organism into the field, however much it might become infec- tious to neighboring varieties later in the season. The second of these two supposi- tions is exactly what happened. My notes on this experiment are as follows : SERIES XI TO XV, 1903. About 3 acres of sweet corn were planted at the Arlington Farm on July 29 and 30. The seed came from Mr. Pieters, and was the same as that used for his trial rows, i. e., of same origin. The ground was not in very good condition, not having been harrowed enough. The land slopes gently from east to west. There is a ditch on the west side and the west end is perhaps 25 feet lower than the east end. The rows ran east and west. The plots were not check-rowed, but seed was dropped by hand, the hills being made about 3 feet apart. Five standard varieties of corn were planted — Old Colony, Potter's Excelsior, Cosmopoli- tan, Country Gentleman, and Crosby's Fig. 53.* Early. Beginning at the south end, the plots were as shown in table 10 (between the plots a row was skipped, making a distance of 6 feet only between varieties). The italicized varieties are those suspected of harboring Bad. stevuarti on their kernels. The other varieties are some of those which were nearly or quite free from disease in Mr. Pieters's trial-rows on July 2 1 and were not suspected. On leaving the city for the summer the writer left word to have them re-examined in August, but unfortunately this wasnotdone. *Fig. 53. — Inner husks of Golden Bantam sweet-corn, showing yellow spots (black areas) due to Bacterium stewarti. Vascular system badly diseased. Slime oozing on the kernels. A natural infection obtained by planting diseased seeds which formed part of a Congressional seed distribution. Hot-house experiment of 1908 (see text). 122 BACTERIA IN RELATION TO PLANT DISEASES. September 28, 1903. — Looked at the sweet corn on Arlington Farm. There are large patches in the middle of the field where the seed did not come up at all (stiff soil) . There is not a good stand anywhere except the first two rows on the south side. Some of the plants look as though they were going to be diseased. Cut out 6 typical cases from the Crosby's Early plot and 2 cases from the Cosmopolitan. There is unquestionably much more disease, but the plants will be allowed to remain a little longer, so that the signs may become more evident. Table 10. — Varieties Planted and Behavior on the Other Farm. Variety. Amount planted. Per cent found diseased in Mr. Pieters's trial rows on the other farm. July 21. August 22. Old Colony 12 rows 24 rows 12 rows 0 3 *I5 No record. No record. Sn Country Gentleman 3 , No record. 24 te>i *30 per cent, if the missing plants are included as diseased. tOne only of the six strains of this variety, and the one showing least disease in July, were not examined in August. The others October 19, 1903. — Tenny and Rorer cut out and examined every plant in the 14 rows of Old Colony sweet corn planted on July 29 at the Arlington Farm. The corn at the lower (west) end and in the first two rows (south side next the fence row) was about 3 or 4 feet high and well tasseled out and looked fairly well. In the upper (east) and central part of the plot, however, the corn had made a very poor growth, many of the plants being only about a foot high and less than half an inch in diameter (due to poor soil). The examination resulted as follows: Old Colony. Stalks found to be healthy, 6,235; stalks found to be diseased, 65. Total, 6,300; diseased, 1 per cent. Many of the diseased stalks showed only two or three yellow bundles, while only a few were very badly diseased. Some of the most badly affected were put into alcohol. October 2 4 and 27, 1903. — The other varieties of corn were cut and examined by the writer, assisted by Messrs. Tenny, Rorer, and Deane B. Swingle, with the following results: Cosmopolitan. Stalks found to be healthy, 1,905; stalks found to be diseased, 366. Total, 2,271; diseased, 16 per cent. It appeared on the start that there were many more cases in the lower moister land, where the corn had made the best growth, than on the side hill or the level land on the top. When classified in this way the result shown in table 1 1 was obtained : Table i i . — Effect of Slow Growth on General Infection. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent, diseased. ( 1 ) Low, moist ; at foot of hill ; corn well grown 754 605 546 233 58 75 987 663 621 24 9 12 (3) Flat* at top of hill Owing to the labor involved and the bad weather the entire plot was not counted, but only aver- age portions of it. The same method was followed with the remaining varieties; the only variety counted in full being Old Colony. Country Gentleman. Healthy stalks, 881; diseased stalks, 32. Total, 913; diseased, 3.5 per cent. Stewart's disease of sweet corn (maize). 123 The variations in the plot were not great, as may be seen from selected portions (table 12). Table 12. — Showing that Kind of Soil Makes no Difference when Seed is not Infected. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent, diseased. 445 436 17 15 462 451 3.68 3.33 (2) North side; middle of field, better growth than to Crosby's Early. Healthy stalks, 2,171; diseased stalks, 437. Total, 2,608; diseased, 17 per cent. Arranged in three groups according to amount of soil-moisture and vigor of the plants, the same results follow as in the case of the Cosmopolitan : Table 13. — Showing that Moist Soil is Favorable to Development of the Disease when the Organism is Present. Kind of ground. (1) Middle of field (east and west) on hill-slope (2) West side, moist end (3) North side, near west end, i. e., in lowest, moistest part of the field Healthy stalks. ','45 456 570 Diseased stalks. 165 76 196 Total. 1,310 532 766 Per cent, diseased. 12.6 '4-3 25.6 This corn was larger than any other variety except the south two rows of Old Colony. Potter's Excelsior. Healthy stalks, 2,227; diseased stalks, 44. Total, 2,271; diseased, 2 per cent. Arranged in groups according to soil-moisture and size of plants, the above count gave prac- tically the same results as the Country Gentleman : Table 14. — Showing that Moist Soil has no Injurious Influence in Absence of the Parasite. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent, diseased. (1) Lower, moister part of field (west), near badly dis- 764 920 543 21 12 1 1 785 932 554 2-7 '•3 2.0 (2) About 8 rods from west end. (These plants about same size as neighboring portion of Crosby's Early, (3) Upper (east) end of field; higher ground. (Plants averaged only 1.5 feet in height; stand not very good) SUMMARY. The above facts may be summarized as follows : Table 15. — Per cent of Cases by Varieties and Soil. Variety. Irrespective of nature of soil. On moist parts of field. Old Colony... 2 16 3-5 '7 No record. 2-7 23.6 3-7 25.6 124 BACTERIA IN RELATION TO PEANT DISEASES. The results at Arlington agree substantially with those obtained from the trial-rows on the other farm. In both Cosmopolitan and Crosby's Early they point unmistakably to infection of the plants from the seed-corn. In the other three varieties the cases are so few that they may have arisen from proximity to the two diseased varieties. It is not diffi- cult to understand how this could have taken place in blocks of healthy plants sandwiched in between diseased ones, provided some of the latter developed the disease early in the growing season. If the disease was actually derived from the seed-corn there probably would have been some cases during the seedling stage, and fragments of these soft plants full of the bacteria would have been blown upon neighboring plots, or dragged by cultivators, or carried on the feet of men and horses, or bitten into by insects, or washed about by rains and dews. There are ways enough to account for the dissemination of the bacteria and the infection of a few plants where the distance is only a matter of a few feet. When plants are well past the seedling stage there is much less danger of infection, even though the infectious material is then much more abundant. That a larger number of cases did not occur in the Crosby's Early and the Cosmopolitan is attributable to the poor soil and slow growth as compared with that on the Flats and probably also to the gradual dying out of the bacteria on the seed-corn between spring, when the first plantings were made, and midsummer, when the last plantings were made. The experiment is equally interesting, however, if we assume all of the seed-corn to have been free from Bact. stewarti. In such event the organism causing the disease must have been present already in the soil of the field. If for the sake of argument such a supposition be granted (and it can be granted for no other reason), it certainly stretches the limits of probability to assume also that the organism was prevalent only in those narrow strips where Crosby's Early and Cosmopolitan corn happened to be planted. If the organism was present in the soil it is likely to have been there a long time and to have been pretty uniformly distributed, just as it is in the infected soils on Long Island. Granted these premises, then we may assume that 3 of the 5 varieties tested are very resistant to the disease, and are, therefore, desirable varieties to plant on infected land, and important to use as one parent in originating resistant varieties by cross-breeding. The other two varieties, on the contrary, should never be planted on infected land. Subsequent experiments showed that an abundance of moisture stimulating rapid growth is very favorable both to primary infection during the seedling stage, and also to the general distribution of the bacteria through the stem of the plant later on. On the contrary, dry soil and dry weather following planting interferes with infection, and any decided check in growth later in the season interferes with the movement of the organism through the plant by hardening the basal nodes and thus rendering it difficult for the organism to get past them. In infected plants, however, the disease shows sooner, accord- ing to Stewart, in periods of drouth than in rainy weather. The writer has observed the same thing in diseased Cucurbitaceae (see this monograph, Vol. II, pp. 216, 284). EXPERIMENTS OF 1908. In 1908, Golden Bantam sweet corn attacked by Bacterium stewarti was sent to me from a garden in Falls Church, Virginia, with the statement that the seeds were obtained from a Philadelphia seedsman, who was believed by the planter to have sold him bad seeds, because nearly all the plants of this variety were badly diseased, while other varieties obtained elsewhere showed no disease or only very little. I asked the dealer where he procured the seed of this variety and he said it came from Mr. - — , Wakeman, Ohio, the man to whose farm I traced the disease in 1903. This interesting fact led me to procure a half-bushel of this particular lot of seed (all the dealer had left) for experiment. The first endeavor was to isolate Bacterium stewarti from the surface of the kernels. In this effort we failed. The organism may have been STEWART'S DISEASE OF SWEET CORN (MAIZE). 125 present, but not alive after so many months. Several kinds of yellow bacteria were obtained in abundance from the numerous poured-plates, but none corresponded exactly to Bacterium stewarti. Many resembled it in the agar-poured-plates, but all of the hundred or more colonies selected as hopeful differed in some particular, i. c, either they grew differently on potato, or coagulated milk, or liquefied gelatin, or clouded Cohn's solution, or refused to grow in Uschinsky's solution, or formed gas, or rapidly reddened or blued litmus milk, etc. They were checked up against three strains of Bacterium stewarti obtained from as many different localities. One form in particular, designated as " Nv" which appeared as though it might be Bacterium stewarti (before additional cultural tests were made which threw it out) was pricked into the leaves of young plants of this variety of sweet-corn copiously, but no disease resulted.* This failure to obtain Bacterium stewarti believed to be present on the kernels is prob- ably to be attributed to the presence in abundance of non-parasitic yellow bacteria on the surface of the corn, while the right organism occurred in a viable form in very small numbers or not at all, so that on the poured-plates it either did not appear or occurred in such small numbers as to be overlooked, the problem of finding it on agar-poured-plates under such conditions being a difficult one. Probably if we had cultured from crushed kernels we would have obtained the organism. This conclusion is borne out by the following experiments. SERIES XVI TO XIX, 1908. On July 23, 1908, a center bed extending the whole length of one of our large hot-houses was planted to this corn. Hitherto corn had never been planted in this house. Orange trees of large size stood in this earth and had done so for many years. The trees were grubbed out, the soil was spaded, a little bone meal was added, and then the suspected corn was planted thickly in rows crosswise of the bed, at a distance apart of about 18 inches. The bed was divided crosswise into four portions as follows: (1,2) two plots planted with selected sterilized seed ; (3) one plot planted with untreated seed just as it came from the original sack; (4) one plot planted with the most inferior looking kernels in the sack (also untreated). The crop was harvested on October 6 to 9. Cross-sections of every stem were examined under the hand-lens by the writer. Some were also examined under the compound micro- scope and many were put into alcohol for future use. The results were as follows: (1) Selected (hand-picked) good-looking seed, soaked 15 minutes in 1:1000 mercuric chloride water, rinsed in tap-water, and immediately planted ( 1 1 rows) : Number of healthy plants, 1,090; number of diseased plants, 13. Total, 1,103; Per cent diseased, 1.2. Remarks. — None of the affected plants were badly diseased. The record is as follows: Eleven slightly diseased, 2 of which had open wounds on the stem above. Only one bundle visibly infected in most of these. Bacteria visible only in the basal part of the stem. The twelfth had 3 diseased bundles, and the thirteenth, which contained several bundles occupied by yellow bacteria, had one whole internode above soft-decayed. Possibly those with bruised or wounded stems should have been omitted from the record. None had yet reached the stage of shriveling leaves. (2) Selected (hand-picked) good-looking seed, soaked 10 minutes in 1 : 1000 mercuric chloride water, rinsed in tap-water, and immediately planted (12 rows): Number of healthy plants, 1,277; number of diseased plants, 23. Total, 1,300; per cent diseased, 1.8. Remarks. — Two plants with 3 bundles infected; 19 slightly diseased (a few bundles in the lower part of the stem) ; 2 badly diseased, both having many bundles occupied by the organism and the leaves dried out. In one, the stem was infected the whole length, and toward the base nearly every bundle was occupied by the yellow slime. *In 1904, Miss Florence Hedges, of my laboratory, obtained similar negative results on a much larger scale, using a yellow organism supposed at first to be Bad. stewarti, but afterwards found to behave differently on certain media. Three hundred sweet-corn plants were inoculated very thoroughly on the leaf tips when the seedlings were extruding water, but no disease resulted. 126 BACTERIA IN RELATION TO PLANT DISEASES. Assuming the kernels to have been the carriers of the organism, the failure of the relatively long soaking in mercuric chloride water would seem to indicate either that the bacteria escaped destruction because they were inside the kernels, or else because they were protected from the action of the germi- cide by being massed together in dried-down hard crusts, the former being the more probable explana- tion. Certainly all those freely exposed on the surface of the kernels must have been destroyed. (3) Unselected, unsterilized seed (20 rows) : Number of healthy plants, 1,548; number of diseased plants, 156. Total, 1,704; per cent diseased, 9.0. Remarks. — Of these plants 24 were slightly diseased; 25 moderately diseased; and 52 badly dis- eased. No remarks on the remainder. Comments on some of the badly diseased will be of interest as showing the extent of infection : One badly diseased: Bacteria as far up as the cob, and also far above the cob in the top of the stem. One badly diseased: Bacteria have oozed through and formed water-soaked yellowish stripes or spots on the pedicel of the cob, and have also oozed through the husks, forming many water-soaked and yellow patches on at least half a dozen inner husks. The bacterial slime was on the inner surface of these husks and also on the kernels. Had photograph made of the husks (fig. 53). A similar oozing through the husks was found yesterday in a plant removed from this same plot to send to Prof. H. H. Whetzel, of Cornell University. Miss Lucia McCulloch examined the slime on the kernels with the compound microscope and found it to be made up of bacteria of the typical sort. These are just as good examples of disease of the cob as any I obtained on the Potomac Flats in 1902. Two badly diseased: Every bundle apparently occupied. Infection extends up to male inflorescence, a distance of 3.5 feet from the base, and out into the husks, which are sticky to the touch from the extruded bacterial slime. Two badly diseased : Husks badly spotted by the bacteria, and bacterial slime abundant in the stem far above the husks. One badly diseased: I find the bacterial ooze bright yellow and viscid on the inside of some of the leaf-sheaths in masses sufficient to scrape up with the knife. It has also oozed out in certain places on the internodes. These places are long, water-soaked, yellowish stripes. Until yesterday I had never seen it forming water-soaked stripes on the sur- face of the internodes. The ears of this plant are also diseased, there being many water-soaked places and many yellow spots on the inner husks. One badly diseased: Here the bacteria have formed pockets in the leaf-sheaths, and have oozed out on the surface of the stem as a conspicuous bright yellow slime, which is viscid enough to string out in cobwebby threads. I have just stretched them out 6 inches. One badly diseased: Here again the bacteria have oozed from the inner surface of the leaf-sheaths, especially the lower ones, and have formed a great number of yellow pockets in the inner husks. The bacteria have oozed onto the inner surface of the husks in large yellow masses which are sticky. The infection is very striking. Seeing such ears one readily appreciates how impossible it is for the surface of the kernels to remain free from infection. One badly diseased: Nearly every bundle at the base of the stem is occupied. The cob is not visibly infected, but the stem near the cob is infected. Two badly diseased : The smaller one has no ears, but the inner face of the leaf-sheaths contains water-soaked and yellow spots, and there is a copious yellow bacterial ooze on the inner face of at least three such leaf-sheaths. The larger plant shows no ooze from the leaf-sheaths, but the ear is infected in the same way as those previously described, *. e ., there are numerous yellow spots with bacterial ooze on the inner face of the husks. One diseased badly: There are water-soaked stripes on the surface of the internode, but no bacteria have actually come through. Four diseased badly : Every bundle is occupied in each stem. One diseased badly: Every bundle at the base of the plant is occupied by the yellow slime and bacteria are visible in the bundles at least 3 feet from the ground, probably all the way up to the male inflorescence. Yellow ooze is visible also on the inside of some of the leaf-sheaths and also on cross-section from the base of the cob. One badly diseased: Every bundle of the stem is occupied. The bacteria have oozed to the inner surface of some of the leaf-sheaths. One diseased badly: In the husks as well as in the stem. One badly diseased: Bacterial pockets in lower leaf-sheaths. The husks of the ear are diseased. One badly diseased: Here for the first time in my study of this disease I find evidence of bacterial ooze from the pedicel of the cob (surface). It is water-soaked and yellow and there is ooze from a half-dozen or more stomata in the form of short cirri. One of these cirrus threads is many times as long as broad. This material is so good that I will put it into alcohol by itself (fig. 64 a). One badly diseased : Yellow bacterial ooze on inside of leaf-sheaths. Three badly diseased: In the largest one of the three there are water-soaked stripes on the internodes, and also a badly diseased ear, the husks containing numerous yellow bacterial pockets from which slime has oozed to the inner face of the husk. The lower leaf-sheaths are also crusted with yellow bacterial slime on the inner faces. One badly diseased : Every bundle of the stem occupied. Four badly diseased: Nearly every bundle occupied at the base of the stems. Also spots on the inner face of the leaf-sheaths where the bacterial growth is bright yellow in patches, also many bacterial patches on the inner husks. One badly diseased: No ears. One badly diseased: Husks of the ear spotted with water-soaked and yellow places. One very badly diseased: Like most of these it has no living leaves. A badly infected ear. Many yellow spots and much bacterial ooze on the inner husks. Nearly every bundle is occupied at tie base of the plant and I can count at leasl a dozen bundles full of bright yellow slime at a distance of 6 feet from the ground and about 4 inches under the male inflorescence. badlj diseased: Every bundle of the stem occupied. No ear. One badly diseased: Every bundle apparently is occupied at the base of the stem. Bacterial ooze from inside of some of the leaf-sheaths and on the husks. One moderately diseased: Has perhaps 20 or 25 bundles occupied. STEWART'S DISEASE OF SWEET CORN (MAIZE). 1 27 One very badly diseased: Husks and cob included. One slightly diseased: 3 or 4 bundles show yellow ooze and four are browned. Two badly diseased: Cobs infected. Hundreds of little yellow spots on the husks. One diseased: About 20 or 30 bundles infected at base. One badly diseased: No ears. One badly diseased: The husks as well as the stem. The leaf-sheaths show bright yellow bacterial ooze on their inner surface. (4) Selected, bad seed, untreated (six rows) : Number of healthy plants, 284; number of diseased plants, 29. Total, 313; per cent diseased, 9.3. Remarks. — No notes on the first 25, some of which were badly diseased. The twenty-sixth, twenty-seventh, and twenty-eighth were slightly diseased. In the twenty-ninth four bundles were observed to be infected. GENERAL REMARKS ON SERIES XVI TO XIX. July 28. — The germinated plants are 1 to 2 inches high. The best germinations are in the plot treated with mercuric chloride 1 : 1000 for 10 minutes. The next best are those treated in the same way 15 minutes. The untreated ones (unselected seed) are decidedly poorer. The poorest plot is that planted with the selected bad seed. October g. — The experiment gave about 8 times as many cases on the untreated as on the treated. The contrast in the amount of infection in individual plants on the untreated plots and on those treated was very striking. Many stems of the former had nearly every bundle occupied to the top of the plant, and also many ears involved, and all of the leaves were dried out, whereas in the latter there were only a very few plants which showed any infected bundles except near the base of the plant, and most of the leaves were yet green. The total bacterial multiplication in the plants grown from unsterilized seed was several thousand times as much as in those grown from the sterilized seed. The infection of the ears was as abundant and striking in this experiment (untreated plots) as in any I have ever made. Several of these ears were put into alcohol. They had a very large number of yellow spots on the husks (fig. 53) and the bacterial slime had oozed from some of them and was sticky to the touch. In one it was so viscid that the slime was stretched out a distance of 31 inches before the cobwebby thread ruptured. In the untreated plots I did not find a single row that had no diseased plants in it. This experiment indicates to me quite clearly that the seed corn was the carrier of this disease. Probably if I had extended the soaking in mercuric chloride to 20 minutes, the check plots would have been entirely free from the disease instead of nearly free. I feared to do this lest I might destroy the germinating power of the corn. The soil was the same, the corn was the same, the water was the same, the experiment was controlled in the hothouse, and the only known variable factor was the germicide. Moreover, the corn was already under suspicion because of its source and of its behavior at Falls Church, Virginia. The experiment serves to establish more firmly the conclusion drawn from an earlier experiment respecting the existence of infected fields at Wakeman, Ohio. It further empha- sizes the wrong done by seedsmen to the general public in selling corn grown on fields infected by this organism, since it serves to distribute the disease broadcast over the country. Wherever such corn is planted it not only gives a poor crop, but also serves to cause disease in a succession of crops on the same land as a result of soil infection. The time between planting and the first appearance of cases was about 60 days. The plants were crowded in the rows much like fodder corn and grew spindling, many of the stems being brittle. Had the plants grown uncrowded in a moist field the number of cases would undoubtedly have been tripled or quadrupled, as a rapid watery growth greatly favors the multiplication of the bacteria inside the stems, whereas stunted, hard basal nodes often prevent the organism from getting out of the base of the plant. Probably a spring planting would also have given more cases than the midsummer planting, since the dried-out bacteria gradually perish with lapse of time. 128 BACTERIA IN RELATION TO PLANT DISEASES. The effect of stunting in preventing general infection is shown quite clearly in Series III to VII (1902) and in the field experiments at Arlington in 1903. It is also shown in the following experiment. SERIES XX TO XXIV. 1908. This experiment was begun July 14-15, 1908, on another part of the grounds of the U. S. Department of Agriculture, out of doors, in a vacant cold frame between two hot- houses. The same Golden Bantam sweet corn was planted as in Series XVI to XIX, but the soil was different, and the other conditions were such that it was known from the start that the plants would be badly dwarfed. In fact, the experiment was made for this very purpose, so that the effect of slow growth might be compared with more rapid growth such as was anticipated and obtained to some extent in the preceding experiment. The corn was planted rather thickly in new 6-inch pots. Good hot-house soil was used. This was prepared from well-rotted sods thoroughly mixed with old cow manure which had been on the Department grounds in compost heaps for the previous 9 months. The cold frame was divided into five compartments and planted as follows : (1) Two hundred and seventy-five pots, planted with selected good seed, about 6 seeds per pot, more rather than less, soaked 8 minutes in 1 : 1000 mercuric chloride water, and then washed in sev- eral waters. On July 21, 621 plants were visible. About one-half the seeds germinated. (2) Two hundred and seventy-five pots, planted with selected good seed, about 6 seeds per pot, more rather than less, soaked for 30 minutes in 1 : 200 formalin water. The formalin delayed the germination. On July 21, 481 plants were visible. About one-third of the seeds germinated. The above two plots were planted on July 14 and the following next day. (3) Two hundred and seventy-five pots, planted with selected good seed, about 6 seeds per pot, more rather than less, soaked for 1 hour in 1 : 2 hydrogen peroxide water. On July 21, 603 plants were visible. About one-half germinated. (4) Six hundred pots, planted with unselected, untreated seed, about 8 seeds per pot, more rather than less. On July 21, 875 plants were visible. Less than one-fourth germinated. (5) Three hundred and eighty-five pots, planted with selected bad seed, about 1 2 seeds per pot. On July 21, 502 plants were visible. An average of about 1 in 6 finally germinated. The pots stood close together on clean sand. The average height of the plants when harvested was between 2 and 3 feet, i. e., they were much stunted by lack of room for the roots. Many of the seedlings showed, soon after germinating, what were regarded at first as suspicious signs, and plants in 74 of the pots were marked, but nothing developed later. The suspicious signs were water-soaked streaks on the young leaves. These turned out afterwards to be due to excessive pumping out of water through the water pores and were not infections. Some of the leaves recovered promptly; others retained yellowish stripes fora long time, but none contracted Stewart's disease. On September 1 (forty-seventh day) a careful examination of all the plants showed no cases. The corn had begun to tassel. It was at that time also badly dwarfed and yellowish from lack of room for the roots. The tallest and greenest plants were next to the hot-house, where they were somewhat shaded from the morning sun, and did not, therefore, dry out as quickly. The first cases were observed at the end of about 60 days. The plants were harvested September 29. The per cent of cases by plots is shown in table 16. Table 16. — Effect of Slow Growth on Development of the Disease. No. of plot and kind of seed. Treatment. Healthy plants. Diseased plants. Total. Per cent, diseased. ( 1 ) Selected best Soaked 8 minutes in i: 1000 mercuric chloride water. 1 : 200 formalin, one-half hour. 1 : 2 hydrogen peroxide, one hour. Untreated 736 620 721 ".533 811 3 4 3 8 9 739 624 724 i,54i 820 0.4 0.7 0.4 0.5 1 .2 (2) Do (3) Do (4) Average kernels out of bag. Do STEWART'S DISEASE OP SWEET CORN (MAIZE). 129 Here the overmastering influence was the lack of room to make a vigorous juicy growth Many of the stalks were brittle as pipe-stems when cut. SERIES XXV, 1912. In the spring of 1912, Lucia McCulloch, of my laboratory, inoculated sweet corn and field corn in the hot-house by spraying on a water suspension of Bad. steward when the plants were about a foot high. Soon after, they were set out on the grounds of the U. S. Department of Agriculture, where they grew well and showed but slight signs of disease. When cut in September the field corn was free from disease. Of the sweet corn about one-half of the stems were infected, but most of them only slightly. Of the whole number (about 50) only 5 or 6 were badly diseased. The season was Fig. 54.* Fig. 55. t a growing one and the organism was infectious. The feeble results, therefore, are to be attributed, I believe, either to resistance on the part of the variety used or to the lateness of the inoculation, i. c, after the plants had passed out of the seedling stage. *FlG. 54. — Cross-section of the base of a sweet-corn kernel, showing a single spiral vessel filled with Bacterium stewarti, the result of an inoculation made about 2 months earlier by placing a trace of a pure culture on the tips of the leaves of the seedling plant. Material fixed in strong alcohol, embedded in paraffin, sectioned on the microtome, stained with carbol-fuchsin, differentiated in 50 per cent alcohol, and mounted in Canada balsam. Figure drawn under the microscope with the aid of an Abbe camera. Slide 235 A 8. For orientation see fig. 47. fFlG. 55. — Cross-section of internodal bundle of a sweet-corn stem showing Bacterium stewarti occupying a single vessel. Material obtained on Long Island. New York, July 16, 1902, fixed in 95 per cent alcohol, embedded in paraffin, sectioned on the microtome, and stained with carbol-fuchsin. Slide 249 C 2. 130 BACTERIA IN RELATION TO PEANT DISEASES. MORBID ANATOMY. The lesions in this disease are strikingly like those induced by other organisms of this group, e. g., Bacterium hyacinthi and Bad. vascularum. The vascular system is occluded by the bacteria to an astonishing degree. So far as I know, scarcely anything comparable to it in extent and severity occurs in the animal body, the nearest approach, perhaps, being certain septicaemias (figs. 54, 55,56). There is this astonishing difference, however, due to the very different character of the circulation in plants and animals, that while in many cases the general septicaemias of the animal body are rapidly fatal, a few days sometimes sufficing, this disease of maize progresses slowly in spite of the presence of enormous numbers of the bacteria, and destroys the plant apparently only when the water-conducting tissues (vascular Fig. 56.* Fig 57. t bundles) have become blocked up by the invading organism to such an extent that transpiration greatly exceeds water-supply. An early death as the result of the action *Fig. 56. — Cross-section of an internodal bundle in sweet-corn, showing restriction of Bacterium stewarli to the xylem p;irt of the bundle. The result of a pure-culture inoculation made in the seedling stage by placing the bacteria on the tips of young leaves. Material collected and fixed in October 1902. The entire stem at this level contains 287 vascular bundles, of which all but 10 are occupied by the bacteria. In every instance (?) the bacteria are still confined to the xylem part of the bundle. Slide 248 A 6. fFlG. ,s,7. -Diagrammatic longitudinal section through lower half of a sweet-corn kernel attacked by Bacterium stewarti, the areas occupied by the bacteria being drawn in solid black (lower left-hand side). 5, envelope of the grain; St, starchy part; E, scutellum; P, plumule; R, radicle; B, base of kernel. The starchy part was very soft when embedded and is squeezed together and upper part torn away. A detail from X is shown in fig. 5S, A . Slide 485 (7. X 14. F'rom Golden Bantam corn grown from diseased seed in the summer of 1908. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 131 of toxic substances does not occur. Stewart's disease is preeminently a vascular disease, but is not confined to the vessels exclusively, as he supposed. The disease, like many other bacterial diseases, begins in the parenchyma and ends in the parenchyma. In the earliest stages of the disease, in order to enter the vascular system the organism must first penetrate Fig. 58.* the epithem or multiply in the cellular tissues under the substomatic chamber (vol. I, fig- 75) ! only in the rare event of infectious wounds made directly into the vessels can it be otherwise. In the late stages of the disease, also, as already noted under "Etiology," one very frequently finds conspicuous bacterial pockets in the soft parenchyma adjacent to the bundles. The histological character of these pockets is shown in figs. 48, 49. Their small size and their relative abundance can be seen in fig. 53 and plate 9. The writer has not observed in sweet corn any cavities at all compar- able to the large ones frequently seen in sugar-cane attacked by Cobb's disease. Many organs of the plant are subject to invasion by this organism, i. c, roots, nodes, internodes, leaf-sheaths, leaf-blades, male inflorescence, female inflores- but there are no hyperplasias. Occasionally small cence ; \?s \ t> *w — 1 F.g. 59. t shoots push from the base of the plant (plates 6, 8, 10), but these are not very numerous, nor frequent. In the ears the bacterial slime occurs in the cob, in the kernels (sparingly), and in the husks, often very abundantly in the latter, as may be seen by consulting my notes under "Etiology." From the husks the surface of the kernels is readily infected. *Fig. 58. — A. A detail from fig. 57, at X, showing cavities in the vascular region of the outer layers of a corn kernel occupied by Bacterium stewarti. Stained with methyl green and acid fuchsin. B. Similar infection, but from another kernel and in cross-section. The slides prepared contain numerous serial sections and were numbered from the base of the kernel upwards, 1, 2, 3, etc. The sections on slides 1 to 12 contain bacteria, i. e., to a point above the level of the base of the radicle and close to the scutellum. Besides the central bacterial cavity, spiral vessels in the upper part of the drawing are also sparingly occupied. The heavily shaded cells are part of the enveloping sclerenchyma. Slide 485 A 1 (6, second row from top, second section from left, stained with pyronine and methyl violet. The bacteria are deep red, the sclerenchyma is blue. fFiG. 59. — A. Bacterium stewarti drawn unstained from the margin of a hanging drop. Organism grown in +15 bouillon for 52 hours. B. Bacterium stewarti from yellow slime on a potato culture 3 days old at 300 C. Rods motile, many dividing. Drawn unstained from a hanging drop of water. 132 BACTERIA IN RELATION TO PLANT DISEASES. The slime finds its way to the inner surface of the husk more readily than to the outer surface, but sometimes it reaches the latter also. It generally comes to the surface through stomata (figs. 49, 50, 51, 52). The only conspicuous parts in which the slime has not been seen are the floral organs and the silicified outer parts of the stem. Even the latter state- ment must be modified somewhat as a result of the observations recorded in 1908, since rarely the bacteria may be seen issuing from small cracks in the siliceous covering of the internodes, especially where protected by the leaf-sheaths. Probably the stigmas (silks) are never infected, they are so fugitive, but the male flowers might well be infected, since the writer has traced the infection well up into the main axis of the male inflorescence a number of times. As yet we do not know how the wall of the vessel is pierced. It is probable that cellu- lose is not destroyed. THE PARASITE. Bacterium stewarti EFS.* as it occurs in the vessels of the maize plant and in young cultures on ordinary culture media is a short rod under i/i in diameter and generally less than 4/i in length, with rounded ends (fig. 59). It occurs singly, in pairs, or fours, joined end to end. According to Stewart the organism usually occurs in pairs with a plain con- striction. "A pair varies in length from 2.5/x to 3.3/4 and in width or diameter from 0.65,11 to 0.85/1." My own measurements do not differ materially. The following are average measurements taken from organisms grown in various media: Measurements made July 21,1 909, from potato-cultures 4 days old gave the following results : Amyl Gram stain gave 1.3 to 2.imX 0.4 to 0.611; carbol-fuchsin gave 0.9 to 2.0^X0.3 to 0.7^. On some of the slides the carbol-fuchsin seemed to have contracted the protoplasm into small lumps. Agar streak-cultures 2 days old gave the following results : Amyl Gram, 0.9 to 2.0/j X 0.4 to o.7m; carbol-fuchsin, 0.9 to 1.7^1 X 0.4 to 0.6/i, Taken direct from the vessels of the maize plant, the following measurements were obtained: Bacterium stewarti, stained in the tissues (material from Jamaica, Long Island), measured 1.1 to 1.5^X0.5^1. A smear preparation from the Long Island sweet-corn stained by carbol-fuchsin measured 0.9 to 1.4/1X 0.4 to 0.55^. Stained in section from a corn husk taken from the Flats experiment, slide 477 c i.the organism measured 1 to 1.4MX 0.4 to 0.5M- The general appearance of the organism is shown in figs. 59 and 60. The organism on media is slightly viscid at times and undoubtedly possesses a capsule, although no efforts have been made to demonstrate it by means of special stains. The only very viscid masses seen are those mentioned under examination of diseased plants in 1908. The organism is motile, especially in young cultures, and the writer has demonstrated a polar flagellum (fig. 60). Usually, at least, only one flagellum is present on the end of each rod. Long chains or filaments have not been observed. So far as known the organism does not produce endospores. Small clumps and bunches of the bacteria (pseudozoogloeae) occur in various media as follows: Uschinsky's solution, nitrate bouillon. The organism stains readily with various anilin dyes. The writer has tried the follow- ing with fairly good results: Carbol-fuchsin, Loeffler's alkaline methylene blue, amyl Gram. Gram's stain gave negative results or at least not a deep stain. The rods were visible with wide-open diaphragm, but were only a pale blue. When cultivated in salted peptone water containing methylene blue the bacterial pre- cipitate was stained a deep blue, the pigment in the fluid remaining unreduced and bright blue or greenish-blue in color. Repeated in 1908 with same result, i. e., bacteria distinctly blue both to the naked eye and under the microscope. *Syn. Pseudomonas stewarti EFS. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 133 Fig. 60.' This organism grows slowly on gelatin without liquefaction. In a stab-eulture in nutrient gelatin No. 478 at the end of 41 days, at 170 to 220 C, there was a thin line of growth along the needle-track, best toward the top, and a dense, rather dry, slightly rough- ened, bright, buff -yellow surface growth 7 mm. in diameter. Fig. 61 a shows its appearance in gelatin stabs. In +10 nutrient gelatin, in rather thin sowings in Petri-dish poured-plates, at the end of 7 days, at io° to 200 C, the surface colonies of Bacterium stewarti under the Zeiss 16 mm. objective and 12 comp. ocular were small, not perfectly circular, not uniformly granular, more or less finely fissured, the margins not entire and sometimes even more or less decid- edly notched; the buried colonies were globose-lobulated and less than 1 mm. in diameter. This may not be a con- stant phenomenon. In streaks on Loefner's solidified blood-serum Bad. stewarti made a good bright buff-yellow growth, but without liquefaction. The cultures were under observation for 32 days. On agar plates it grows slowly, forming small round yellow colonies, the buried ones being very small. In agar streak-cultures it gives a smooth trans- lucent yellow slime which is usually paler than that of Bad. hyaciiithi (fig. 62), and frequently lobed on the margins. Old streak-cultures are usually thin and somewhat dry, and the older bacterial layer generally bsars numerous small raised, wet-shining, yellow colo- nies on its surface. Jagged X-shaped crystals and short prismatic crystals are not uncommon (fig. 63). In agar stab-cultures at the end of 16 days there was a moderate growth the whole length of the needle- track and a distinct surface growth some millimeters ' in diameter. Even where there were several stabs in one tube the growths did not cover the entire surface of the agar or coalesce ; the color was between saffron- yellow and deep chrome (Ridgway). At the end of 5 weeks the surface growth was a deep yellow and not at all viscid ; there was a good growth the whole length of the stab; the agar was unstained, and there were prismatic crystals in the upper part of it. At the end of an additional 9 weeks, during which the cultures were in the refrigerator, the surface growth was chrome yellow. In stab-cultures in nitrate agar (stock 718) the organism made a good growth, the surface layer being at first whitish. At the end of 24 days the agar was not stained, and no gas had been formed in the agar. A few crystals were present in the uppermost layers of the agar. The stab had made a good growth the whole length, but it thinned out slightly toward the bottom. There was a very distinct Fig. 61. f *FiG. 60. — Flagella of Bacterium stewarti. Stained by van Ermengem's method from dilute Uschinsky's solution. Slide*, August 7, 1904. fFiG. 61. — a. Gelatin stab-cultures of Bacterium stewarti after 10 days at about 200 C. Gelatin not liquefied. b. Gelatin stab-cultures of a non-pathogenic yellow liquefying organism from the surface of sweet-corn kernels. Sum- mer of 1908. Two-thirds to three-fourths liquefied. 134 BACTERIA IN RELATION TO PLANT DISEASES. surface growth, which was plainly yellow and rough granular (possibly ? from the presence of the crystals). It covered only a part of the surface and was between Ridgway's buff- vellow and deep chrome in color. A month later there was a very good growth, the surface was wet-shining and not then granular, but covered with little denser masses of the bacteria, resembling colonies growing on the older growth. In stab-cultures in 6 per cent glycerin agar the organism grew nearly or quite the whole length of the stabs, but best toward the top. The surface growth increased slowly, so that finally most of the surface was occupied. The growth was pale yellow, particularly at first. Surface smooth or slightly irregular. Streak-cultures also grew. On silicate jelly (see vol. I) Bad. stewarti made a very feeble growth. The streak- cultures were under observation 2.5 months. In 2 days there was a very scanty, rough, yellowish growth along the line of the streak which did not increase much. A number of organisms were tested. The only other one that failed eventually to make a good growth on this medium was Bad. vase itl arum , al- though quite a good many had difficulty in start- ing off, and had made little or no visible growth at the end of 6 days. Bad. steward behaves on potato much like Bad. hyacinth i , i. e., out of the water it makes a moderate amount of yellow growth which lg' "' soon passes its maximum (see this monograph, vol. II, plate 17, fig. 2). The fluid around the cylinder never becomes thickened by the continued development of the organism, and the surface growth ceases for the most part after a week or two, owing to the exhaustion of the small amount of sugar and other soluble foods and the inability of the organism to take its food readily from starch. No gas bubbles appear. The litmus reaction is alkaline. The iodin- starch reaction is always strong in potato-cylinders on which this organism has grown, even when tested immediately under the bacterial layer, although it must have a slight action on starch, since the reaction is generally a deep purple-blue rather than a pure blue, such as the check-tubes yield. The cell-walls of the potato are not softened. The following are notes on a series of S cultures (from as many plants) on potato-cylinders in test-tubes: Fig. 62.* *FlG 62.— a. Streak-culture of Bacterium stewarti on a +15 agar slant 3 days at 27° C, September 1902. the -aim on +15 agar slant 8 days at 260 C, October 1908. fl'iG. 63. — Crystals formed in old nutrient agar-culture of Bacterium stewarti. Photographed down upon the (nail-head) of the culture. The dark body in the center is the denser growth along the needle-track. Crystals confined to the surface. X4. The culture had been at a temperature of about 14° C. for many weeks. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 135 A ugusl 24, 1902. — Inoculated. August 2g. — All the cultures resemble each other closely. The potato out of the water is slightly to distinctly grayed, and bears a thin, decidedly yellow slime. In one tube it was recorded as bright yellow. September. — The potato-cylinders are grayed to a greater extent than they were and the organism is yellower in some tubes than in others (variability in potato-cylinders). When compared with the check-tubes there is an incon- spicuous pink tint in the potato (?). Repetitions in 1909 and 1913 (held 5 months) failed to give this tint. Fig. 64 shows appearance on potato contrasted with that of Bacterium campestre. On coconut cylinders Bad. stewarti made only a moderate amount of growth. Its color was buff -yellow and crystals were formed. Repeated in 1913 the following results were obtained : Moderate to good growth ; pale buff -yellow slime and precipitate ; no crys- tals. In the milk of the coconut there was a moderate growth with yellow rim and precipi- tate, and subsequently the fluid became yellow (checks remained colorless). When old there was a copious yellow growth with a wide yellow rim; the fluid was pale yellow. Though acid when inoculated it was now feebly alkaline. There were no crystals. On cylinders of yellow turnip there was a thin buff-yellow slightly iridescent growth. At the end of a week this was decidedly less than in corresponding tubes of Bad. campestre. Growth ceased, practically, after the second week. There was a buff-yellow precipitate in the fluid surrounding the cylinders, but no thickening of the water with bacterial slime. There was no browning or softening of the substratum. The culture was alkaline to litmus at the end of the fifth and ninth weeks. Repeated in 1913 with same results, except recorded as not iridescent. When old the growth was orange-ochraceous, feebly alkaline, wet-shining, not viscid, moderate in amount, and free from crystals. The appearance of this organism when grown on rutabaga was much the same as on yellow turnip. On the seventh day the growth was about one-tenth as much as that of Bad. hyacinthi and one-twentieth to one-thirtieth as much as that of Bad. campestre. On the eighteenth day streak-cultures yielded a thin buff-yellow growth covering the whole surface exposed to the air ; this was not smooth and not dense enough to hide the slight irregularities of the substratum. Its surface was slightly iridescent, and when examined under the hand-lens fine striae were visible. The water contained a moderate amount of buff-yellow precipitate, but was not filled with a solid slime. In old cultures there was no increased growth, no brown stain, and no softening of the tissues. The iridescence persisted. The cultures were alkaline, at least after a time (thirty-fourth and sixty-fourth days), and had a feeble, peculiar smell. On the thirty- fourth day the thick slime would not wet litmus paper until water was added. When tried in 1913 on white turnip cylinders standing in water (stock 5778), there was a moderate to good, pale yellow, flaky, iridescent surface growth, and a pale yellow precipi- tate. When old the slime was ocher yellow, slightly alkaline, not viscid, wet-shining, not copious, and free from crystals. On radish cylinders (tests of 19 13), there was a thin, whitish, iridescent surface growth with a scanty yellowish precipitate and either no browning or slight. Steamed cylinders of sugar-beet standing in distilled water proved a very good medium for Bad. stewarti. It grew on this substratum abundantly and without retardation. The color of the growth on the cylinders was a deep buff-yellow, and there was a copious buff- yellow precipitate in the fluid at the bottom of the tube. Fig. 64. *Fig. 64. — Bacterium stewarti (a), and Bacterium campestre (b), after 30 days on cooked potato at room-temper- ature. The former grew only during the first few days, ;'. e., was not able to use the starch as food ; the latter continued to grow for weeks, owing to its diastasic action on starch. Bad. stewarti shows a little slime on the upper part of the potato (dark part) and a small amount of precipitate in the water (at right). Bad. campestre has covered the potato and filled the water solid. Photographed November 2, 1908. 136 BACTERIA IN RELATION TO PLANT DISEASES. In tubes of peptonized beef bouillon at the end of about 18 days (room-temperatures of spring) there was a feeble, whitish rim, a well-clouded fluid, about 10 mm. breadth of pale yellow precipitate, and no distinct pellicle (stock 791). Repeated in 1908 in stock 3398. At end of 10 days, when notes were taken, the tubes were well clouded with a moderate amount of pale yellow flocculent precipitate. There was also a well-defined but rather scanty whitish rim. In a fluid prepared by grinding green cabbage-leaves, taken from old, slow-growing hot- house plants, and extracting the juice under pressure without addition of water, there was a very prolonged and copious growth without retardation. A portion of this fluid was steril- ized by forcing it through a Chamberland filter and the rest by discontinuous steaming. The reaction of each was +40 on Fuller's scale. Bact. phaseoli and Bad. campestre refused to grow in this fluid, but when it was solidified by adding agar-flour, Bad. campestre grew upon it very copiously and for a long time, although it was started with much difficulty. Bad. stewarti was retarded at first, but afterward made a prolonged and copious growth in three out of four tomato juices undiluted with water. In the fourth it refused to grow. In stocks 333 and 334, obtained respectively by squeezing green full-grown or nearly full-grown fruits, and small green fruits (one-twentieth to one-quarter grown) there was a good growth on the fifth day. The acidity of these two stocks was respectively +55 and +59 of Fuller's scale. In stock 33 1, which was the steamed juice of ripe fruits, there was a greater retardation, the tubes becoming clouded between the eighth and fifteenth day. The acidity of this med- ium was +64. Stock 332, in which the organism would not grow, was +68, i. e., a little more acid. It consisted of the steamed juice of fruits which were yellowish green, i. e., full- grown and beginning to ripen. It would seem, therefore, if we may trust the titration, that +64 is near the limit of toleration for the acid of the tomato.* Bad. campestre and various other organisms refused to grow in these fluids. In a 2 per cent agar containing the juice of 10 grams of acid mulberry fruits in 490 c.c. of water (autoclaved) Bad. stewarti made no growth, i. e., 8 Petri-dish poured plates made with yellow slime from each of two recently infected plants. On the 16 plates not a colony appeared. That the organism was living in each case was shown by subsequent cultures from the dilutions. The work was defective in that the acidity of the agar was not deter- mined by titration. Two mulberry agars were in the laboratory at the time, one made from green fruits of Moras alba, the other made from ripe fruits of Morns rubra, but there is no record as to which was used. In a potato broth of half strength (1:4) which titrated +30 on Fuller's scale, Bad. stewarti grew readily, while Bad. phaseoli and Bact. campestre refused to grow, and Bad. hyacinthi was much retarded. Bad. stewarti also grew in this broth without retardation when sufficient malic acid had been added to it to make it +45. Bact. hyacinthi, Bact. phaseoli, and Bact. campestre, refused to grow in this fluid. Bacillus amylovorus and Bact. dianthi grew in it readily. This experiment was repeated in 1908, using Bad. stewarti from three sources and dilute potato broth rendered +41 by malic acid. Each grew well. The tubes each received two 3-mm. loops from peptonized beef bouillon 3 days old. Each was well clouded at the end of 48 hours. On the seventh day each tube had a copious pale precipitate and a pale rim. On the eleventh day the tubes were still well clouded with a pale flocculent precipitate and a pale buff-yellow rim, 1 to 2 mm. wide. In beef broth concentrated by boiling until it was quite yellow and strongly acid ( + 80), Bat !. stewarti grew for a long time and very luxuriantly, but clouding did not appear until the eighth day, although the inoculation was from a solid culture. In this medium the fol- lowing bacteria refused to grow: Bact. hyacinthi, Bact. phaseoli, Bact. campestre, Bacillus amylovorus. "This acid is said to be tartaric acid. Experiment repeated in 1913: No growth in +45 Or +60. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 137 This experiment was repeated in 1908, using a beef broth free from peptone and concen- trating it until it titrated +73. Bad. stewarti from three sources was tested. Each tube was inoculated with three 3-mm. loops from bouillon. There was no clouding for the first three days or more, but afterwards a heavy growth. On the thirteenth day the tubes were heavily clouded with a buff -yellow rim (2 to 3 mm. wide), a thin pellicle, scarcely visible, and considerable buff-yellow flocculent precipitate. Tubes 10 days old but inoculated from a solid culture (potato) were heavily clouded with heavy yellow rims (2 to 3 mm.) and a wide buff-yellow precipitate. Bad. stewarti grows in milk for a long time without precipitation of the casein, or suffi- cient production of alkali to render the milk translucent. There is no visible change in the milk, even after several months, although the organism forms on the wall of the tube, at the surface, a distinct yellow bacterial rim and an abundant bright yellow precipitate. There is a change, however, in the milk, as shown by the litmus reaction. In litmus milk there is a gradual slight development of acid. The color of the litmus, on the start a deep lavender blue, becomes a lilac color or dull purple (pi. 11, figs. 3, 4, 5). Usually this color does not appear until after about two weeks, and sometimes a longer period intervenes. In the end the litmus is more or less reduced, sometimes entirely. This reduction may occur early, but often not until after several to many weeks. Frequently the acid reaction is obscured by reductions. The organism produces no lab ferment, and not enough acid to cause coagula- tion. No crystals were observed in milk-cultures. In 8 tubes of litmus milk (stock 697), inoculated in 1902, the litmus was entirely reduced at the end of 16 days ; there was then a slight yellow precipitate ; a pale yellow rim was begin- ning to form at the surface ; the milk was entirely fluid. At the end of a month there was no separation of the casein, the litmus was still entirely reduced in two tubes and nearly reduced in the others, there being, however, a slight pinkish hue at the top. The check remained unchanged. At the end of 10 weeks there was a yellow precipitate about 10 mm. broad, a yellow rim 6 to 7 mm. wide, and in some of the tubes a yellow pellicle or partial pellicle. The amount of litmus reduction varied a little ; it was complete in several tubes and nearly so in the others. The milk in the tube showing most color was pinkish vinaceous (Ridgway) in the upper half. At the end of 14 weeks the reduction was still quite marked, but not complete. The returning color of the litmus was various shades of lilac, i. c, quite distinct from the unchanged deep lavender of the check tubes. At this date the milk was still fluid ; there had been no separation of the casein; the bacterial rim was about 10 mm. wide and dirty yellow. Two of the cultures were slightly viscid, one decidedly viscid, and 5 not viscid. Streaks on slant agar made at this time from these tubes yielded 8 pure cultures of Bad. stewarti. These milk cultures were also alive at the end of 7 months, as determined by cultures in bouillon and on potato. The same results with litmus milk were obtained in 1908. There was good growth, slow change from blue to lilac color, and no separation of the casein. In the following medium Bad. stewarti did not grow. Distilled water 1 ,000, dipotassium phosphate 2, magnesium sulphate 0.1, ammonium phosphate 0.1, sodium acetate 5. Bad. campestrc grew in it feebly. Repeated several years later (1908) with same result. In Uschinsky's solution, Bad. stewarti grew for a long time and very copiously. It is a good medium for differential purposes. The general appearance of cultures in Uschinsky's solution was as follows : The growth was slightly retarded and sometimes rather feeble at first, then copious and long continued; rim whitish at first, then distinctly yellow and of variable width; a yellow pellicle, sometimes bearing yellow colonies; copious chrome-yellow to buff-yellow precipitate, fluid gradually becomes slightly fluorescent, but less distinctly so than in case of Bad. pruni; pseudozoo- gloeae are present. In one set of tube-cultures made in 1902, in stock 702, there was at the 138 BACTERIA IN RELATION TO PEANT DISEASES. end of two months a thin yellow rim 5 mm. wide; a thin yellow pellicle over the whole sur- face, fluid moderately clouded, translucent, a bright yellow precipitate 1.5 cm. broad, and no crystals visible; fluid feebly fluorescent. At the end of another month the fluid in each one of the seven tubes was turbid and the precipitate slowly increasing, it being then 17 mm. wide and about 4 mm. deep; the liquid was slightly fluorescent, and plainly to strongly alka- line to neutral litmus. It was not viscid. At the end of four months these cultures were still living; they were distinctly fluorescent and not at all viscid ; all had a deep chrome yellow rim, and all but one had more or less pellicle ; pseudozoogloese were present. Repeated in 1908 with similar results: On twenty-third day heavily clouded with specks of yellow on the surface (floating islands), and a granular pale buff -yellow precipitate. On shaking, a great number of small flocculent particles filled the fluid. On nutrient starch-jelly the addition of the following substances gave no increased growth: Lactose, maltose, dextrin, mannit, glycerin. Cane-sugar and galactose, on the con- trary, greatly stimulated growth. On the slant surface of the jelly containing the cane-sugar there was a copious, smooth, wet-shining, buff-yellow, sirupy growth. On that containing the galactose there was 100 times as much growth as on the jelly containing glycerin, and nearly as much growth as on that containing the cane-sugar. Bad. stewarti did not grow in —80 peptonized beef-bouillon (sodium hydrate), but clouded —40 bouillon the first day or the beginning of the second. Probably there was some error in the former titration, as a repetition in 1908, using —35 bouillon, gave no growth, nor would it grow in —30 peptone beef -bouillon when tested in 191 2. The maximum toler- ated dose of sodium hydrate in bouillon, therefore, is not known with certainty, but is, I believe, under —30 of Fuller's scale. On the acid side +30 bouillon clouded the first day and +60 is believed to have been clouded feebly after a time; growth also occurred in +80 bouillon (acid of beef-muscle), in +64 tomato juice, +40 cabbage juice, and in potato broth reinforced with malic acid from +30 to +45, as has been stated already. The range of toleration, therefore, for alkalies and acids is greater on the part of this organism than on the part of many related forms, e. g., Bad. hyacinthi. Salted bouillon. — The organism will tolerate a considerable amount of sodium chloride, i. e., nearly or quite as much as Bacillus coli. In 1909, in experiments with two strains (McCulloch and Galloway), Bacterium stewarti grew promptly in +15 peptone bouillon containing 5 per cent c. p. NaCl, and after a time in bouillon containing 8 per cent. The experiment was repeated in 191 2 with similar results. There appeared, after a time, to be a trace of bottom growth, even in 10 per cent salt bouillon, but this should be repeated. Cokn's solution. — The organism did not grow in Cohn's solution when inoculated into it from young fluid cultures. Repetitions in 1908 gave the same results. Fermi's solution. — The organism grows feebly in Fermi's solution. Reduction of nitrates. — Bad. stewarti has no action on potassium nitrate. It is not reduced in bouillon, either with or without the presence of grape-sugar. The growth of Bad. stewarti in nitrate bouillon was feeble in comparison with that made by Bad. hyacinthi or Bad. campestre. Aerobism. — So far as tested Bad. stewarti is strictly aerobic. It produces no gas and does not grow in the closed end of fermentation-tubes when free from air. It is able, however, to get along on a relatively small amount of air, as shown by experiments in vacuo, etc. In an atmosphere of nitrogen Bad. stewarti behaved on coconut cylinders the same as Bad. hyacinthi. At the end of 15 days, when the sealwas broken, there was no visible growth, although the check-tubes showed a distinct yellow color in 48 hours and continued to develop typically. Fxpo- sure to the nitrogen also caused distinct retardation of growth after the seal was broken, the yellow slime being first visible on the fifth to the eighth day thereafter. In another test, using white-turnip cylinders, there was no growth during the 15 days exposure, and none subsequently. The checks grew promptly. In a third experiment the absorption of the oxygen by the pyrogallol was very slow, occupying 5 clays. During this time there was some growth, but at the end of 15 days it was paler yellow and only about one-thirtieth as abundant as in the check-tubes. In an atmosphere of hydrogen for 16 days there was either no growth or only a trace of growth, according to the medium used: PLANT BACTERIA-VOL. 3. Wis*;*:: -*%;»; F 5 BACTERIUM STEWARTI. BACTERIUM VASCULARUM. APLANOBACTER MICHIGANENSE. CD Cross-section of inoculated sugar cane (nodal region) showing red and yellow ooze; (2) Do. internodal region of stem. Bacterium stewarti : (3) Litmus-milk culture after 11 days at 25°C. 'milk fluid); (4) Do., another stock of milk, after 11 days at 27°C. (5) Check on tubes, 3, 4, 6, and 7. Aplanobacter michiganense: (6) Litmus-milk, 8 days at 27°C; (7) Do. after 11 days at 27°C, litmus reduced, milk fluid; (8) Potato streak culture after 48 hours at 25°C; (9) End of 3rd day at 25°C. (10) Check tube of litmus- milk for comparison with 11 and 12. Bacterium vascularum : (11) Litmus-milk inoculated March 25, 1905, from 16, October 6 (organism originally plated from inoculated cane No. 40) and painted May 2, showing gradual increase in alkalinity with a slight yellow precipitate; (12) Do. an older litmus-milk culture, tube X inoculated March 25, 1905, painted June 10 (organism out of cane No. 40); (13) Do. potato cylinder standing in distilled water, inoculated October 19, 1905, from June 7, painted October 24 (descended from cane plant No. 40). STEWART'S DISEASE OF SWEET-CORN (MAIZE). 1 39 (i) In the first experiment there was no growth on potato and a decided retardation after breaking the seal; in beef-broth there was either no growth, or only a trace of growth, but no retardation on exposure to the air; on slant agar there was a distinct, very feeble growth, consisting of several hundred tiny whitish colonies, best seen under the hand- lens. On exposure to the air these colonies enlarged and became burl-yellow. (2) In the second experiment, which also lasted 16 days, there was no growth in +15 beef-broth, and no retarda- tion of growth on subsequent exposure to the air. In peptone-water with grape-sugar and methylene blue there was a trace of growth, and no marked retardation on exposure to the air. In salted peptone-water (Dunham's solution) with rosolic acid there was no growth, and not much afterward. In Uschinsky's solution there was no growth and none afterward in the air. Each of these tubes received a 2-mm. loop from a cloudy culture 5 days old in peptonized beef-bouillon neutral to phenolphthalein, and it was known by previous tests that the organism grew well in these media. During 10 days' exposure to an atmosphere of carbon dioxide there was no growth. On subse- quent exposure to the air there was no retardation of growth on the coconut, some retardation on potato, and no growth in beef-broth. In a second test in carbon dioxide (14 days) there was no growth whatever on potato or in bouillon. The checks grew. I find no record of the subsequent behavior of the exposed tubes. Growth in vacuo depends entirely on the degree of exhaustion. In a vacuum with the mercury at 2.25 inches, the remnant of the oxygen having been absorbed by pyrogallol in caustic-potash water, there was no growth during the 9 days' exposure, and a distinct retardation of growth after removal to the air. The four corresponding check-tubes (2 coconut, 2 potato) showed a distinct buff -yellow growth in 48 hours. In a second test on coconut, potato and carrot, and in alkaline beef-bouillon, the jar being sealed with the mercury at 3 inches and the remnant of the oxygen not removed, there was a slight, retarded growth, paler than on the checks and less in amount. The check-tubes showed a good growth at the end of the third day, and a plainly visible one in 48 hours. On the twelfth day, when the seal was broken, conditions were as follows: Carrot. — Merest trace of growth. Potato. — About 0.33 to 0.50 as much growth as on check; potato not grayed. Coconut. — Thin cream-colored growth. The check is buff-yellow and contains several times as much growth. Beef -broth. — Very feebly clouded. The check is twice as cloudy and contains twice as much bacterial precipitate and this is yellower. All of these cultures made additional growth on exposure to the air. No acid reaction was obtained when Bad. stewarti was grown in bouillon containing glycerin or ethyl alcohol. In culture-media in the presence of air, this organism breaks up the following substances with- out gas, but with the formation of a small quantity of non-volatile acid : grape-sugar, cane-sugar, galactose, mannit. In a repetition of these tests in 1908, using Bad. stewarti from three sources and streaking on litmus agars containing only agar, water, Witte's peptone, and the specified sugar or alcohol, and inoculating copiously (two 3 mm. loops) from bouillon cultures 3 days old, the following results were obtained: End of 48 hours — distinct growth, but no reddening either with plain agar or that containing dextrose, lactose, galactose, maltose, mannit, or glycerine. Most growth on that con- taining the cane-sugar, and on this a trace of red at the extreme top of the slant. On the eleventh day the conditions were as follows : (1) Plain agar. — Bluer than checks. Rather feeble growth. (2) Dextrose agar. — Two strains bluer than checks, except for dab of red in one at extreme top. Third (a weaker strain in other media) dull red throughout. (3) Lactose agar. — Two are bluer than checks, except dabs of red at extreme top of slant. Third (weaker strain) purple- red in upper two-thirds (in upper one-third 4 days earlier). (4) Galactose agar. — Each dull purple-red. Redder than checks but not bright red. The checks were a blue purple. (5) Maltose agar. — Each tube is bluer than the checks. This was also true 4 days earlier. (6) Mannit agar. — Distinctly bluer than the checks, but a dab of red at extreme top of one tube (not the weaker). This was not present 4 days earlier. (7) Glycerin agar. — -The litmus is now a dull purple-red. It was neutral 4 days earlier. Slight acid formation, per- haps C02. (8 ) Sacchirose agar. — -Each uniform in color and bluer than the checks, except for a dab of red at the extreme top of each slant where the agar is drying out. Here would seem to be an acid masked by an alkali. In salted peptone-water (Dunham's solution) containing rosolic acid and a slight amount of hydrochloric acid (color of fluid a pale orange-yellow) there was no decided change during the first two weeks, but on the twenty-eighth day the fluid was pale red, and subsequently geranium red. This medium would serve to differentiate Bad. stewarti from Bad. hyacinthi and Bad. campestre, cultures of the former being colorless and cultures of the latter geranium red at the end of the second week. 140 BACTERIA IN RELATION TO PLANT DISEASES. In cultures in peptone-water containing acid fuchsin and a little hydrochloric acid (to counteract the alkalinity of the Witte's peptone) the red color persisted a long time, fading very gradually; the moderate amount of bacterial precipitate was yellow and unstained. When Bad. stewarti was inoculated into salted peptone-water containing indigo carmine, there was no immediate reduction, but the blue color disappeared after about 2 weeks. In the case of Bad. hyacinthi and Bad. pericarditidis (B. pyocyaneus pericarditidis) the blue color persisted for a long time, gradually changing to green, and then fading. Table 17. — Growth of Bacterium stewarti in Various Media. Inoculations of August j, icjoS. McCulloch and Galloway Strains. Medium. Strain. Growth. August 5. August 7. August 14. (1) Stock 3410: Distilled water, cane-sugar, ammonium tartrate. Do McCulloch Galloway Both (4 tubes). Both No clouding. . . ....Do ....Do Thinly clouded; rolling clouds on shaking. No clouding. . . ....Do ....Do ....Do .... Do ....Do Well clouded. . . Clear Feebly clouded. Clear. Do. Faintly cloudy. Clear. Do. Clear. Clear. Well clouded. ....Do ....Do Same as on Aug. 5. Clear... (3) Stock 3407: Distilled water and cane- sugar. Do (5) Stock 3409: Distilled water, cane-sugar, and ammonium citrate. (6) Stock 3408: Distilled water, cane-sugar, and asparagin. (7) Stock 341 1 : Distilled water, cane-sugar, and ammonium lactate. ....Do (McCulloch (McCulloch (Galloway Both. . . . ....Do ....Do ....Do ■••■Do ....Do Well clouded.. •For composition, see p. 141, footnote 3 (t). Table 18. — Growth of Bacterium stewarti in Various Media. Inoculations of August 5, iqoS (from Bouillon Transfers made from an Agar Slab, No. 1, November 16, igof), Miss Hedges' Strain.] Medium. Growth. August 7. August 14. August 17. (1) Uschinsky's solution (stock 3414). (2) Beef broth Thinly clouded ...Do Well clouded; feeble rim; moderate yellow precipi- tate. Cloudy. Moderately cloudy; no rim or pellicle. Clear. Milk lilac color; buff-yellow rim and pellicle; no sepa- ration of casein. Clear Well clouded; faint, pale rim; no pellicle; no visible fiocculence ; 4 mm . breadth of buff-yellow, fine preci- pitate. Milk lilac; reduced some- what; remained fluid; formed a bright buff-yel- low rim and considerable yellow precipitate. Clear. (3) Nitrate bouillon (stock 3399)- (4) Cane-sugar, distilled water and ammonium lactate (stock 541 1). (5) Litmus milk, blue Well clouded Clear No reddening Clear . (7) Cane-sugar and ammo- nium citrate (3409). (8) Cane-sugar and ammo- nium tartrate (3410). (9) Caiu'-sugar water (3407) . (10) Potato ..Do Do. Moderately cloudy. Feebly clouded. Moderate buff-yellow growth. Clear. Do. Do. Thinly clouded Clear Thin, buff-yellow growth. (11) Tomato juice + 60 (12) Cane-sugar and aspara- gin-water. (13) Synthetic (stock 3406). . . ffhis third strain w;is plated in 1907 by Miss Hedges and transferred by Miss McCulloch Aug. 3, 190S, from agar-stab (No. 1, Nov. 16. 1907) which had remained alive in cool box 8.5 months— agar dried out one-half. Tube labeled: "Fr. I, July 31. 1907. through 1313 of Nov. 12, '07." STEWART'S DISEASE OF SWEET-CORN (MAIZE). Table 19. — Growth of Bacterium stewarti in Various Media. 141 Each tube was inoculated with 2 big 3 mm. loops of the cloudy fluid from a 6-day-old peptonized beef-broth culture. (From notes of August 17, 1908.) Medium. Stock 3410: cane-sugar, ammonium Galloway (2 tubes) tartrate, water. Stock 3410 1 McCulloch Do. * ! Hedges (2 tubes) . . . Stock 341 1 : cane-sugar, ammonium Galloway (2 tubes) . lactate, water. Stock 341 1 McCulloch (2 tubes) Do. Hedges (2 tubes) . Stock 3408: cane-sugar, asparagin, Galloway (2 tubes) . water. Stock 3408 McCulloch (2 tubes) Do.. . Hedges (2 tubes). . . No. 1, Aug. 3. No. 2, Aug. 14. Stock 3413: tomato juice, titrating Galloway (3 tubes) . No. i.Aug. 14. Date inoculated. Date reinoculated. No. I, Aug. 3. . Aug. No. 2, Aug. 14. Same as above. No. 1, Aug. 5. No. 2, Aug. 14. No. i, Aug. 3. No. 2, Aug. 14. No. 1, Aug. 3 . No. 2, Aug. 14. No. 1, Aug. 5. No. 2, Aug. 14. No. 1, Aug. 3. No. 2, Aug. 14. No. 1, Aug. 3. No. 2, Aug. 14. +60. Stock 3413. Do.... Stock 3407: cane-sugar in water. No. 2, Aug. 3. . No. 3, Aug. 3.. McCulloch (3 tubes) Same as above. No. 1, Aug. 5. . No. 2, Aug. 14. . Hedges (2 tubes). Galloway (2 tubes) Stock 3407. Dot. ■ ■ Stock 3409: cane-sugar, ammonium citrate and water. Stock 3409 Do Stock 3406: sy-ntheticj Stock 3406 Do Stock 3415: beef-bouillon +73 Stock 3415 Do§ Stock 3424 : beef-bouillon — 3 5 Stock 3424§ Do Stock 3427: Dunham's solution with methylene blue.§ McCulloch (2 tubes) Hedges (2 tubes) . . . Galloway (2 tubes) . McCulloch (2 tubes) Hedges (2 tubes). . . Galloway (2 tubes) . McCulloch (2 tubes) Hedges (2 tubes)... McCulloch (3 tubes) Hedges (3 tubes). . . Galloway (3 tubes) McCulloch (3 tubes) Hedges (3 tubes).. . Galloway (3 tubes) . 3 tubes, one of each strain. No. 1, Aug. 3. No. 2, Aug. 14. No. 1, Aug. 3. No. 2, Aug. 14. Same as above. No. 1, Aug. 3. No. 2, Aug. 14. Same as above. .... Do ....Do ....Do ...Do Aug. 15 (a. m.). .... Do ....Do ... .Do ....Do .... Do ....Do M Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Aug. 14. Same as above. ....Do ...Do ....Do ....Do Behavior. Both thinly clouded. Do. Thinly clouded. Clear. Both clear. Do. Both clear. Check tubes of Aug. 14 in bouillon have clouded. Both clear. Both clear. One cracked on side and fungus has en- tered tiny crack. Both clear. All clear. Do. Both clear. Both thinly cloud- ed. Rolling on shaking. Thinly clouded. Clear. Both clouded, the reinoculated one more so. Both clear. Do. Do. Do. Do. Do. Clear. Do. Do. Do. Do. Do. All clear. •Conclusion: Bacterium stewarti will grow in this medium but has some difficulty in getting started. Must be inoculated copiously tMediuro evidently not entirely free from nitrogen. JComposed of: distilled water 1. 000, dipotassium phosphate 2.0: magnesium sulphate 0.1; ammonium phosphate 0.1; sodium acetate 5- n- §September 22: Stock 3415 heavily clouded; stock 3424. doubtful, if any, growth; stock 3427. color much duller than in the check, due to clouding. Nitrogen foods. — The behavior in Usehinsky's solution indicates ability to use sodium aspara- ginate and ammonium lactate, one or both, under the conditions present. The inability to reduce potassium nitrate shows that nitrogen is not obtained from this source. Peptone and the nitrogen in beef-juice are both foods. Tests made in 190S with Bact. stewarti from three sources, inoculating copiously, gave the fol- lowing results: (1) No growth or doubtful: Asparagin water (2 per cent asparagin); ammonium lactate and cane-sugar in water; ammonium citrate and cane-sugar in water; ammonium tartrate and cane-sugar in water. (2) Feeble clouding: Asparagin and cane-sugar in water. 142 BACTERIA IN RELATION TO PEANT DISEASES. In table 17 are given the notes of August 5, 7, and 14, 1908, on the growth of two strains of Bacterium stewarti in various media, inoculated August 3 from 4-day-old bouillon cultures proven to be alive by inoculation therefrom to common bouillon which had clouded. The two strains used were (1) McCulloch, isolated from Fairfax, Virginia, corn in 1907; (2) Galloway, isolated from Golden Bantam corn from Dr. B. T. Galloway's Maryland farm, 1908. One tube of each medium was used, except acid tomato juice, of which two were used, each tube receiving all the fluid that could be taken up on a 3 mm. loop — a drop. Temperature 300 C. Table 18 gives notes on a third strain, and table 19 summarizes the results of reinoculations with each one of the three strains. Bad. stewarti produces only a moderate amount of alkali, and in some media, e. g., milk, this is wholly obscured by the moderate production of acid. The following media are usu- ally rendered alkaline to litmus : Beef -bouillon, plain peptone agar, potato cylinders, maltose agar. Potato cylinders are usually grayed by Bad. stewarti, but strips of lead acetate paper were not browned by exposure over them for 9 days. Cultures on rutabaga and on yellow globe turnip did not brown the paper nor stain the substratum (64 days) ; white radish was not browned in 64 days. Indol. — In one set of experiments a slight indol reaction was obtained ; in two others made sub- sequently there was no pink color on adding sodium nitrite and sulphuric acid, but a slight pink color appeared on heating the tubes at 80° C. for a few minutes. The latter were cultures 14 days old in peptone-water, and in peptonized Uschinsky. The question of indol formation must therefore be left an open one. Pigments. — The yellow pigment is probably similar to that in other yellow species of bacteria, i. e., lipochrome (see vol. II, Bad. hyacinthi). No special studies have been made. Its color varies from buff -yellow to chrome, but is much paler when the supply of free oxygen is scanty. The brown pigment formed by this organism does not appear as readily in culture-media as that of Bad. campestre. For instance, cultures of the latter stain cruciferous substrata dark brown in the course of 6 or 8 weeks, while in the same media (radish, rutabaga, yellow globe turnip) Bad. stewarti produces no stain. The organism causes a brown stain in the host-plant, but this is perhaps a host reaction. This develops slowly and is usually most conspicuous in the parts longest occupied, i. c, in the lower nodes. Crystals. — Crystals occur in old agar cultures. They are prismatic (fig. 63) or jagged X-shaped. They were once seen in cultures on coconut. Contamination (?). Enzymes. — Our knowledge of the enzymes produced by Bad. stewarti is very imperfect. Cavi- ties are formed in the host-plants, and there must therefore be some substance capable of dissolving the middle lamella although not necessarily an enzyme. The question of the existence of a cytase is an open one. In test-tube cultures, the tissues of potato, coconut, rutabaga, yellow globe turnip, radish, and carrot were not softened. Cane-sugar is inverted, but invertase was not formed in the absence of sugar (beef-broth without peptone). Bacterium stewarti has only a slight action on potato-starch and therefore very little diastase is produced. In this particular it resembles Bad. hyacinthi and differs widely from Bad. campestre. No trypsin and no lab-ferment are produced. Old cultures cause a copious evolution of oxygen from hydrogen peroxide. One gram of grape-sugar in 10 c.c. of +15 nutrient agar did not retard the growth of Bad. stewarti in streak-cultures; on the contrary, growth was stimulated from the start, being in 48 hours four times as great, and at the end of a week at least five times as great as in the check-tubes. Vitality. — Bacterium stewarti lives for a considerable time on culture-media, especially on agar, in milk, and in Uschinsky's solution. It was dead on agar in the ice-box at the end of 17.5 months. It was alive under similar conditions on the same medium at the end of 14 months. It lived in litmus milk for 7 months and in Uschinsky's solution for 4 months. It remained alive in a variety of culture- media for more than 2 months. Mixed cultures and infections. — In the host-plant the organism often occurs in pure cultures. Nothing is known respecting mixed infections, or the effect of other organisms on this one in mixed cultures. In pure culture the organism does not readily lose its power to infect maize. Germicides. — Very little is known respecting the behavior of this organism toward antiseptics and germicides. STEWART'S DISEASE OF SWEET-CORN ( MAIZE). 143 In test-tubes in beef-broth standing over chloroform the organism grew vigorously with only a slight retardation and remained alive for more than 2 months. Under the same conditions Bad. campestre refused to grow. In streaks on slant nutrient agar containing phenolphthalein and 0.5 per cent potassium formate, considerable growth appeared at the end of 48 hours and a faint pink tint on the third day, none on the second. At the end of the tenth day this color was still very faint and after 3 weeks transfers from the tubes failed to grow. The organism also grew (after a second inoculation) in neutral beef-bouillon containing 1 per cent potassium formate. Infected kernels were nearly sterilized by exposure for 15 minutes to 1 : 1000 mercuric chloride water (see p. 125). Heat. — The thermal death-point of Bad. stewarti in +15 beef-bouillon (10 minutes' exposure) is approximately 530 C. In Uschinsky's solution it is a little higher. The maximum temperature for growth is about 39° C. It grows in the thermostat on most media at 360 to 37° C, but not so well as at room-temperatures of 240 to 250 C. It refused to grow in the thermostat at 40° C, in +0 beef-bouillon and in Uschinsky's solution. The minimum temperature for growth was determined as follows: In the earlier experiments tubes of Uschinsky's solution and of alkaline beef-broth (382) were inoculated with a 3 mm. loop from a fluid culture 3 days old and then exposed in the ice-box for 18 days at 2. 8° to 4.50 C. The checks clouded the second and third days, but during all this time there was no clouding of the tubes in the ice-box, and probably no growth because, contrary to Bad. hyacinthi, on removal to room-tempera- tures the fluids did not cloud any sooner than the check-tubes. Subsequent experiments (summer of 1909) showed the minimum temperature to be about 90 C. The exposure was for 34 days in the ice-thermostat with the results shown in table 20, four tubes (two strains) being placed in each compartment in +15 peptonized beef -bouillon inoculated from young cloudy bouillon-cultures. The checks at room-temperature clouded promptly. The tubes in compartments I, II, III clouded on removal to room-temperature and showed a yellow precipitate. They were first exam- ined on the ninth day. The optimum temperature for growth is above 300 C. Table 20. — Result of Minimum Temperature Experiments in the lee Thermostat. Compart- ment. Temperature. Tenth day. Eighteenth day. Thirty-fourth day. I. II 1°± C All clear All clear All clear. Do. 3.5° to 5-9° C; mostly under 50 . .Do Do . . . and above 4° C. III Range6.5°to 10.5°; ave. 74 observa- ....Do ....Do Do. tions, 9° C. IV RangeS.2°toii°C.; ave. 74 observa- . .Do. . All clear, but on shak- No true clouding; clear until shaken, then many pseudo- ing some tiny zooglo- tions, 9. 40 C. ese in one tube of the 4,* indicating a very feeble and abnormal growth on the bottom. zoogloea? in fluids inocula- ted with McCulloch strain, and a few (100 or more) in fluids inoculated with Gal- loway strain, ;' e., at mini- mum temperature motility is cut out and growth consists of compact small pseudozoogloeas, lying on bottom of tubes (side when tilted). v Range 8.3° to 12. 30 C; mostly 10° to Zoogloeae in bottom of tubes; these cloud true clouding in top layers. ii°C. the fluid feebly on shaking; not a normal growth. VI. Range 1 1.6° to 15.2° C. ; mostly 14° C. Bouillon thinly clouded; also small flocks visi- ble on shaking; this clouding was visible on 6th (?) day. *Some days later the other tubes showed the same phenomenon. ^4 BACTERIA IN RELATION TO PLANT DISEASES. Effect of Acids.— Bacterium stewarti is rather resistant to acids. It tolerates much more of lactic, malic, or tartaric acid than Bad. hyacinthi. (See observations under growth in acid plant juices, etc.) Dry Air. — The relative resistance to dry air is not known. Its resistance is probably con- siderable. Sunlight. — Stewart exposed a portion of an agar-poured plate to bright sunlight for 3 hours, nearly all of the organisms being destroyed. Under the covered portion colonies came up thickly in 4 days at 23° C. The writer has made no experiments. Effect on Animals. — The organism is not known to be pathogenic to animals. The writer has made no experiments. It is frequently fed in great quantity to cows (in corn stalks) . Additional cultural tests were made in 19 13 with the following results: Beef bouillon over chloroform: Growth retarded for some days, but after two weeks about equal to growth in ordinary bouillon. Alkaline peptone beef-bouillon ( — 40 stock .5700, —35 stock 5792, and -30 stock 5791: No growth (Jan. 18, Feb. 24, April 3, May 28). The tubes were inoculated heavily both from agar and bouillon. Cabbage juice ( + 17): A good growth. After several months a copious, dirty yellow, moder- ately alkaline growth, free from crystals (May 28). Tomato juice (+60): No growth. Cohn's solution: No growth in 6 weeks. Uschinsky's solution: A good growth. Asparagin (2 per cent in distilled water): No growth in 3 months. Asparagin (2 per cent in river water) : Slight growth after 1 1 days in tubes heavily inoculated from agar cultures; none in 2 months in tubes inoculated from peptone beef-broth cultures. Peptone water (stock 5705, 2 per cent Witte's) : Growth. No indol reaction after 10 days; slight indol reaction after 22 days. Tested with sodium nitrite and sulphuric acid. Cane-sugar with asparagin (stock 5704): Growth in 2 tubes out of 10 after 2 months. Temperature: Thermostat 24 hours at 270 to 280 C. Good clouding. Ice box, compartment 6 (temperature 140 to 14.50 C). Clouded in 48 hours and cleared in 14 days. In compartment 5 (temperature 8.4° to 120 C., average about io° C.) zoogloea? visible in 8 to 10 days. Not then clouded, but a faint clouding after 22 days. In compartment 4 (temperature 8° to ii° C.) slight clouding in 29 days (McCulloch strain); in 39 days (Galloway strain). In com- partments 3 to 1 (temperature 6.50 to io° C, 3.70 to 5.40 C, and 0.90 to i.5°C. respectively), no cloud- ing or zoogloese in 39 days, but when removed to room temperature the tubes clouded in less than 7 days. RESUME OF SALIENT CHARACTERS. POSITIVE. Yellow organism causing a vascular disease of maize, especially of sweet corn; short rod with rounded ends, generally less than 4X1/J, often 1 to 2^X0.5 to 0.7^; occurs singly, in pairs or fours, joined end to end, or as small pseudozoogloeae (in various media) ; motile by means of a polar flagellum (sometimes more than one is present) ; stains readily with various anilin dyes, often not uniformly with carbol-fuchsin ; very slow growth in gelatin, surface- growth dense, rather dry, slightly roughened, bright buff-yellow; good bright buff-yellow growth on Loeffler's blood-serum; slow growth on agar plates, forming small, round, yellow, surface colonies, buried colonies very small; smooth, translucent growth on agar streaks, frequently lobed on the margins; jagged X-shaped or prismatic crystals often present in old agar-cultures ; good growth whole length of the stab in stab-cultures in agar; good growth in nitrate agar, whitish at first; very feeble growth on silicate jelly (Fermi's solu- tion) ; moderate amount of yellow growth on potato cylinders accompanied by graying of the potato; very little action on potato starch; moderate buff -yellow growth on coconut cylinders ; thin buff -yellow slightly iridescent growth on cylinders of yellow turnip ; growth on rutabaga resembling that on turnip; excellent deep buff-yellow growth on sugar-beet cylinders; feeble whitish rim and pale yellow precipitate in peptonized beef-bouillon which becomes well clouded; prolonged and copious growth in cabbage-leaf juice titrating +40; growth in tomato-fruit juices (titrating +55, +59, and +64, Fuller's scale) was retarded at first but later was prolonged and copious; good growth in potato broth titrating +30, STEWART'S DISEASE OP SWEET-CORN (MAIZE). 145 on Fuller's scale and +45 (by addition of malic acid) ; a retarded but prolonged and luxuriant growth in concentrated (4-73, +80) beef-broth; a good growth in 4-30 beef -bouillon ; slow production of acid (lilac color) in litmus milk, followed frequently by the reduction of the litmus; milk cultures alive at end of 7 months; long and copious growth in Uschinsky's solution, yellow rim and pellicle, latter sometimes bearing circular yellow colonies — a very good medium for differential purposes ; growth on nutrient starch-jelly greatly stim- ulated by the addition of cane-sugar or galactose; growth stimulated by addition of 10 per cent grape-sugar to +15 nutrient agar; feeble growth in Fermi's solution; strictly aerobic (so far as known) ; cultures on coconut cylinders live some days in an atmosphere of nitro- gen, but make no growth; cultures on white turnip cylinders killed by 15 days' exposure to nitrogen; in atmosphere of carbon dioxide no growth, but organism is not killed on some media, e. g., coconut cylinders. Growth in vacuo depends on amount of air remaining; grape-sugar, cane-sugar, galactose, and mannit are broken up with formation of a small quantity of non-volatile acid; old cultures in Dunham's solution containing rosolic acid and a slight amount of hydrochloric acid become pale red and subsequently geranium-red; Dunham's solution containing indigo-carmine lost its blue color after about 2 weeks; slight amount of indol formed; organism causes a brown stain in the plant (host reaction?) and produces small cavities in parenchyma. Cane-sugar is inverted. Alive on agar in ice-box (i2-i5°C.) at end of 14 months and 17 months; lived in litmus milk 7 months and in Uschinsky's solution 4 months. Vigorous and only slightly retarded growth in bouillon over chloroform. Tolerates much sodium chloride. Thermal death-point approximately 53° C. ; maximum temperature about 390 C; optimum temperature above 300 C; mini- mum temperature 8° to 90 C. Retains virulence on media for a year, or more, and as long on, or in, infected kernels. Absorbs methylene blue. Group No. 212.2222523. NEGATIVE. No long chains, filaments, or endospores have been observed ; not stained by Gram's method; no reduction of methylene blue in salted peptone water; no liquefaction of gelatin or Loefner's blood-serum; agar not stained; no gas formed in nitrate agar or any other medium , no filling up of the water in cultures on potato cylinders ; no growth in tomato- fruit juice titrating +68 Fuller's scale (once none in that titrating 4- 60) ; no growth in mul- berry agar (acid) ; no precipitation of the casein or coagulation in milk cultures ; no growth in —80 peptonized beef-bouillon (sodium hydrate); no growth in —30, —35, —40 beef- bouillon; no growth in Cohn's solution; no reduction of nitrates; no clouding of closed end or production of gas in fermentation-tubes ; little or no growth in an atmosphere of hydrogen ; no production of hydrogen sulphide observed in cultures on potato, rutabaga, or yellow globe turnip ; invertase not formed in the absence of sugar ; very little diastase produced ; no trypsin or lab ferments formed; no growth at 400 C, in -f-o beef-bouillon, or in Uschinsky's solution. No growth in peptonized beef-bouillon after long exposure to carbon dioxide. Asparagin not a carbon food. No acid reaction in bouillon containing glycerin or ethyl alcohol; no indol production in 10 days. It was dead at room temperatures after 6 months on potato; 7 months in litmus milk; 8 months in sodium chloride bouillons; 15 months in agar, potato, and beef bouillon, 16 months on peptone beef gelatin. It was dead in the refrigerator on agar after 17.5 months. In carbon dioxide no growth. Any organism producing gas, liquefying gelatin, blueing litmus milk or throwing down the casein, reducing nitrates, growing well in Cohn's solution, or refusing to grow in Uschin- sky's solution may be set down at once as something else. TREATMENT. The proper treatment for this disease remains to be worked out. Four lines of inves- tigation will occur at once to the reader who has followed the text to this point. First and foremost, compulsion applied to the growers of sweet corn for the trade. They must be 146 BACTERIA IN RELATION TO PLANT DISEASES. compelled to practice field hygiene. In bringing this about seedsmen generally (middle- men) should cooperate with State authorities. Second, the origination of resistant varieties by cross-breeding and selection. By this method alone, wisely pursued, it ought to be possible to overcome the disease, but not immediately. Third, treatment of the seed-corn with germicides, such as mercuric chloride, formalin, etc. Fourth, bactericidal powders or sprays applied during the seedling stage when the water-pores are functioning and the plants are specially liable to contract the disease. Probably the plants will prove very sensitive to chemicals during this stage of growth, and one should therefore proceed with caution lest the damage wrought be greater than that likely to result from the disease. The first and third methods are the more feasible. These directions are intended primarily as suggestions to plant pathologists rather than as advice to growers, although some of the latter might be able to work out their own methods of treatment. To prevent the introduction of the disease into new fields, growers of sweet corn for seed, and seed-firms generally, should cooperate with the farmer. The former ought not to put on the market seed-corn likely to transmit the disease, that is, corn grown on land subject to this disease, and the farmer as a matter of ordinary precaution ought to treat his seed-corn with some germicide before planting it, unless he knows beyond doubt that it was derived from healthy plants. The thermal death-point of the organism is so high (530 C.) that probably hot-water treatment of the seed would not be effective. Soaking the seed 20 minutes in 1 to 1,000 water-solution of corrosive sublimate in glass vessels or in wooden pails or tubs is the method most likely to destroy the organism on the surface of the kernels without serious injury to the latter. It will slightly retard the germination of a portion of the kernels, but should not destroy any of them, if the treated seed is immediately rinsed in clean water on taking it out of the germicide, or dried at once and planted without rinsing. The writer, as the result of experiments made in 1909 (see vol. II, p. 196), has no hesitation in advising exposure of suspected seed to mercuric chloride water (1 : 1,000) for 20 minutes just prior to planting. This will destroy most of the bacteria on the seeds and will not prevent germination or seriously delay it. The dry seed-corn should first be wet with alcohol, then covered with the germicide, removed at end of 20 minutes, rinsed slightly, and planted at once, or else dried quickly. The corn must not be soaked in advance of treatment; nor must the rinsing be prolonged. Such treatments, of course, will not reach the organism when it is lodged in the interior of the kernel. As an added precaution, therefore, all shrunken, suspicious kernels should be removed from the seed-corn before it is treated. This may be done by running the kernels through a fanning mill and by a little subsequent hand- picking. The latter should not be neglected. To recapitulate: The greatest pains should be taken to secure only sound seed-corn, but in the present indifferent state of the seed-trade even the best should be treated with mercuric chloride before planting. On fields subject to the disease only resistant varieties should be planted. Manure containing corn stalks from diseased fields, or gathered from animals pastured in such fields, should never be used on land designed for com. PECUNIARY LOSSES. Nothing very definite can be expressed under this head. There are no general statis- tics available, only individual instances. The disease is a bad one on Long Island, and, if the writer's inferences under "Etiology" are correct, also in at least one locality in Ohio. That the disease may take from 10 to 70 per cent or more of the plants in experimental plots, not artificially inoculated, is also shown by the record of the trial plots mentioned on pages 117 to 1 20. The general experience on Long Island is summarized by Stewart as follows: Occasionally, an entire crop has been ruined and losses of from 20 to 40 per cent have been fre- quent; but in the majority of cases the loss has been so slight as to pass unnoticed by the farmer, although one familiar with the disease could readily detect it in almost any field of early sweet corn on Long Islandjluring'the past season. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 147 The disease is on the increase in the United States and has been, I believe, disseminated widely in recent years by ignorant and unscrupulous seedsmen. It is likely to be found now in any State. HISTORY. The history of this disease begins with Stewart's bulletin (1897), unless we are to assume that BurrhTs bulletin "On a bacterial disease of corn" (1889) relates in part to the same subject. I have not included the latter in the bibliography of this disease, although perhaps I should have done so. Some parts of his description correspond very well to Stewart's disease, c. g., irregular distribution of the disease in the field; dwarfing; brown nodes; the base of the stalk most badly affected ; death of the leaves as a whole, the lowest showing worse; and gumming of the ears; but if he really had this disease under observation, why is there no mention of the most conspicuous sign, i. c, yellow slime in the internodal bundles? The internodes appeared to him to be healthy in plants having the browned nodes. Along with these signs, as part of the same disease, are mentioned other less characteristic signs, i. c, decaying roots, the lower ones most affected ; corroded spots on the roots ; brown spots on the leaf-sheaths, most conspicuous within, and occasionally bordered by red. These brown spots are "half rotten" discolorations, the disease in this stage being evenly distributed through the field. There is a gelatinous bacterial substance on the roots and inside the leaf-sheaths. The description of the organism which he isolated and with which he obtained infections is what finally decided me to exclude it. This was a white peritrichiate schizomycete subse- quently identified by Dr. Theobald Smith as Bacillus cloacae. Fig. 64 a.* LITERATURE. 1898. Stewart, F. C. A bacterial disease of sweet corn. New York Agrie. Exp. Sta. Bull. No. 130, Dec. 1897, pp. 423-439, plates i-iv, Geneva, N. Y. [Bull, distributed in Jan. or Feb. 1898] Reprinted in 16th Ann. Rept. Bd. of Control N. Y. Agric. Exp Station for 1897, pp. 401-416. 1898. Smith, Erwin F. Notes on Stewart's sweet- corn germ, Pseudomonas stewarti, n. sp. Proc. Am. Assoc. Adv. Science, vol. 47, meeting at Boston, Aug. 1898. Published at Salem, Dec. 1898, pp. 422—426. Also a separate. 1899. Halsted, Byron D. Sweet corn smut and bac- terial disease, in Mycological Notes, Torrey Bull., New York, Feb. 1899, p. 77. Reports occurrence in 189S of bacterial disease due to Pseu~ domonas steu'arii in one variety of sweet corn at New Brunswick. N.J. 1901. Smith, Erwin F. The cultural characters of Pseudomonas hyacinthi, Ps. campestris, Ps. phaseoli, and Ps. stewarti — Four one-flagellate yellow bacteria parasitic on plants. Bull. 28., Div. of Veg. Physiology and Pathology, U. S. Dept. Agric, Govt. Printing Office, Washing- ton, D. C, 1901. 1903. Smith, Erwin F. Completed proof that Ps. stewarti is the cause of the sweet-corn disease of Long Island. Abstract of an address delivered Dec. 30. 1902. before the Society for Plant Morphology and Physiology. Science, n. s., vol. xvu. No. 429. March 20, 1903. Also a separate. 1909. Smith, Erwin F. Seed corn as a means of dis- seminating Bacterium stewarti. Science, n. s., vol. xxx, No. 763, Aug. 13, 1909, pp. 223-224. [See also various figures and observations in vols. I and II of this monograph.] *Fig. 64a. — Pedicel of ear of sweet corn, showing bacterial ooze from stomata at X X. Experiment of 1908. Photographed from alcohol in 1914, X6. Originally the cirri were much more conspicuous and then also water-soaked places were visible on the surface. See text, p. 126. SMITH'S DISEASE OF AMARANTHS. This disease of amaranths was discovered by the writer in October 1897, in a neglected garden at Mumford, New York. The plants were weeds standing on what had been a cab- bage seed-bed. They were small and seemed to have dried up when half grown. There was no surface indication as to the cause of the disease. The stems were browned internally and there were cavities in the parenchyma in the region of the vascular ring. These cavities were full of bacteria. They were small, actively motile rods with rounded ends. Fungi were not observed. There was an abundance of the diseased plants, but unfortunately only a small amount of material was collected and the disease has not come again under my observation. No plate cultures were made, but the organisms taken from the interior of two stems were streaked on four tubes of Loeffler's blood-serum — all that happened to be avail- able. It was my intention to keep the organism alive and make some inoculations, but during a winter crowded too full of other duties the cul- tures were allowed to die. The organism may be known as Bacterium ama- ranthi (syn. Pseudomonas amaranthi), but my knowl- edge of it is very incomplete. The bacteria made at first a dirty white or pale yellowish-white, wet growth on the slant tubes of Loef- fler's blood-serum. This subsequently became a yel- low growth, and there was a rather copious yellow pre- cipitate in the fluid at the bottom of the V. At first the growth was slower and looked unlike that of Bacterium campestre, but afterward on this medium the color was the same or very nearly the same. No liquefaction was observed. ( )n potato cylinders which were inoculated from a single pale yellow colony on the side of the streak in one of the tubes of blood-serum, the manner of growth and the amount of growth closely resembled that of Bacterium hyacinthi, but the color was quite different. The growth on the potato was homogeneous, wet-looking (shining), and did not obscure the sur- face of the potato. It produced a thin, translucent growth not at all resembling the all- pervading growth of Bad. campestre on this medium. It grew best out of the water. No •Fig, 65 Cross section of an amaranth stem, showing a small portion of the pith in which arc intercellular spaces occupied by Bacterium amaranthi. One cell is filled with crystals of calcium oxalate. Not much disorganiza- tion of tissues in tins stem. Collected at Mumford, New York, October 1X97. Slide 450 B 3, lower row, left-hand section. 148 Fig. 65.* SMITH S DISEASE OF AMARANTHS. 149 gas was formed. Its color on potato was at first pale yellow and then distinctly ochraceous a color lying between ochraceous and ochre-yellow (Ridgway's plate v, 7 and 9), i. e., there was much more buff in it than in cultures of Bad. campestre. It was cultivated at room temperatures ranging from 180 to 240 C. The substratum was soon stained a de- cided gray (not brown), drab -gray (Ridgway's plate 11, 13). I have a note saying that several tubes of potato inoculated with Bacterium campestre the same day were not grayed (fourth day). At the end of 25 days a little of the potato from immediately under the thin layer of bacteria blued litmus paper decid- edly. On mashing old potato cul- tures in iodine potassium-iodide water there was a copious brown-pur- ple reaction, showing that the starch had been acted upon only a little. The organism grew on sugar- beet cylinders, producing consider- able yellow slime, but the culture was dead at the end of 5 months. In its morphology, so far as examined, it closely resembled the ordinary forms of Bact. hyacinth-i, being a short rod with rounded ends. No long chains, filaments or endospores were observed. Its general morphology and relation to the tissues is shown in figures 65, 66, and 67. So far as I have been able to deter- mine from an inspection of the microtome sections the vessels are occupied (a small number only in the material cut), the inter- cellular spaces in the pith (frequently), and also occasionally large pith-cells. The latter have pits in their walls, and appar- ently the bacteria have forced their way through these thin places into the interior of these cells. Surrounded by free cells these bacterially occluded cells present a striking appearance when stained deep red with fuchsin. The bacteria were found fre- quently in crystal-cells. I have observed no bacteria in the cortex, which is made up largely of eollenchyma. Fig. 67. t Fig. 66.* *Fig. 66. — Pith-cell of amaranth occupied by Bacterium amaranthi. Other similarly occupied pith-cells occur and it is believed that the bacteria gained an entrance from the intercellular spaces by way of thin places in the wall (pits), some of which are shown in the middle of the figure. Slide 450 B 5. |Fig. 67. — Xylem vessels of amaranth stem occupied by Bacterium amaranthi. Also margin of a cavity. Slide 450 B 2, lower row, left-hand section. Drawn with Zeiss 2 mm. 1.30 n.a. apochromatie objective, No. 12 comp. ocular, and Abbe camera. 150 BACTERIA IN RELATION TO PLANT DISEASES. Dr. G. P. Burns has stated to me that he saw this disease in two species of cultivated amaranths at Ann Arbor, Michigan, in 1901, and plated out a yellow organism, but, having read my note, dropped work on it. Had he continued we might now know something more definite concerning the biology of the organism and its ability to produce the disease from pure-culture inoculations; also to what extent the biological characters here assigned to it are correct. The only reference in literature is, I believe, the following: Fig. 67.* LITERATURE. 1901. Smith. Erwin F. The cultural characters of Pseudomonas hyacinthi, etc. Bull. No. 28, Div. Vcg. Phys. and Path., U. S. Dept. Agr., i). 153. FlG '■; 0. Photomicrograph showing appearance of Bacterium amaranthi under a high magnification (X 2000). Slide 450 B 5. Vascular bundle. VAN BREDA DE HAAN'S WILT OF PEANUT. In December 1905, J. van Breda de Haan prepared a paper (Teysmannia, 1906) con- cerning a bacterial disease of the peanut Arachis hypogwa, known as "hamawedang,"and observed by him in October of that year in the departments of Koeningan and Cheribon, Cheribon Residency, Java. The following is an abstract of this paper: The soil in this portion of Java is chiefly red or red brown, sometimes black-sandy, very porous, and occasionally clayey. Often the stiff clay cracks open in times of drouth. Irrigation is largely practiced and rotation of crops, c. g., sugar-cane is planted for cuttings every third year, then katjang tolok (Phascolus radiatus), after which the peanut (katjang soeoek, katjang holle) is planted, followed by rice. Much care is usually given to the culture of katjang soeoek, low beds of about a yard wide by 4 yards in length with shallow channels be- tween them being used for this purpose. The channels render irriga- tion easy and also allow of thorough drainage and easy access to the plots for weeding, etc. The fields are irrigated every week or less often. There is no trouble any- where from ground- water. The variety of Arachis hypogaa used chiefly is the katjang holle or Bandoeng, which ripens in about 3 months from date of planting. It is planted chiefly in the East Monsoon in fields that can be irrigated. The product is sold to Chi- nese merchants, who in recent years have paid Fig. 68.' F.g. 69. f good prices for it, making it a very profitable crop, which accounts for its wide cultiva- tion ; and in view of this fact it can readily be seen how a disease which formerly scarcely drew attention should now be considered a serious problem, especially because the yield of katjang holle has noticeably decreased, being sometimes but half a normal crop. Insect injuries were not numerous in the fields examined by van Breda de Haan, and the only fungus disease mentioned is a black spotting due to Scptoglwum arachidis Raciborsky, which is said to have no connection whatever with this disease. *Fig. 68. — Bacterial disease of peanut (Arachis) from Java: Cross-section of root, showing bacteria confined to the vascular bundles (the heavily inked portions). fFic. 69. — Bacterial disease of peanut (Arachis) from Java: Cross-section of stem from epidermis to pith, show- ing the bacteria restricted to the xylem (heavily inked parts). Drawn by Katherine Bryan. 151 152 BACTERIA IN RELATION TO PLANT DISEASES. Plants attacked by this bacterial disease look as though hot water had been poured over them. The foliage of the plants withers sometimes very suddenly — all the leaves simul- taneously. The leaf loses its fresh green color, becomes flaccid and droops, then dries up quickly. Usually the progress of the disease is so rapid that the leaf does not dry yellow, but retains a dull black color. After the wither- ing of the leaves, the leaf-stalk and the stems also dry up, and soon nothing remains but a dry mass of stems and leaves. If pulled up, the roots of such a plant are seen to be colored brown, the side roots being black and more or less rotten. If the plants are not attacked in an early stage of growth the fruits show no signs of the disease exter- nally, except in size, being some- what smaller. When, however, young plants become diseased, their fruits often show brown spots on the surface of the pods, the fruits themselves being more wrinkled than when normal and often spongy, or with the seed decayed. Neither the type of soil nor the altitude seems to have any connec- tion with the disease. The disease occurs both on porous black soil and on stiff clay soil and it is in the mountains as well as on the plains. It occurs in patches, rarely an entire bed being affected. Sometimes it is on the borders of the beds, sometimes in the center. The first signs of it do not appear until the crop is 2 to 2.5 months old. It never ap- pears until after the first weeding — end of the first month — but it has also ap- peared in undiminished virulence in fields purposely left unweeded. It is a root-disease, but does not seem to be due to soil-infection, i. e., fields planted after a diseased crop are not worse attacked than other fields. If it ever spreads in a concentric manner it is only after it has become very well established. Microscopic examination of dis- eased roots from 5 fields showed bac- teria in large numbers throughout the entire root-system, including the wood parenchyma, and in advanced stages Fig. 71. f 'Fig. 70. — Bacterial disease of peanut (Arachis) from Java: A detail from the xylcm part of tig. 69 to show the morphology of the bacteria. Figure drawn by (Catherine Bryan. \FlG. 71 — Bacterial disease of peanut (Arachis) from Java: Cross-section of an immature petioleat the extreme top of tin- plain showing bacterial occupation of the vessels and the formation of small cavities in their vicinity, the rest of the tissues free. Drawn by (Catherine Bryan. VAN BREDA DE HAAN'S WILT OF PEANUT. 153 of the disease the bacteria had spread also into the vessels and into the parts of the plant above the ground. The bacteria were not found in the leaf-stems or the fruit-stems, but in the wood of the main stem and of the lower branches. In the attacked parenchyma- cells the starch had disappeared. No other harmful organisms were found on the inside of the diseased plants. The death of the plant, therefore, is ascribed to the presence of the bacteria in the wood parenchyma and in the wood vessels. "The hama wedang must thus be a root-disease caused by bacteria. * * * Some plants show a predisposition to the disease. This greater susceptibility to the hama wedang, I think, must be ascribed largely to the use of inferior seed. " The growers do not reserve their own seed, but sell the entire crop and buy their seed from the Chinese merchants, who supply seed which represents the inferior product, less desirable for the oil-mills. The organism was not cultivated and no measurements are given. Through the courtesy of Dr. van Breda de Haan the writer received alcoholic material of peanuts affected by this disease, and a study of these has enabled him to show the gen- eral morphology of the schizomycete and its relation to the tissues of the host-plant (figs. 68, 69, 70, and 71). There is no reasonable doubt that it is a genuine bacterial disease, nor that it should be classed among those acting through the medium of the vascular bundles. Since this text was prepared for publication, Honing, in Sumatra, has reported the occurrence there of a bacterial disease of the peanut (probably this one), which he attributes to Bacterium solanacearum (for literature see "Wilt of tobacco"), and in the winter of 1912-13 Fulton, of North Carolina, stated that this same organism causes a disease of the peanut in that State (Annual meeting Am. Phytopathological Society, at Cleveland, Ohio). LITERATURE. 1906. Breda de Haan, J. van. Rapport over Ziekte in den aanplant van Arachis hypogwa (Kat- jang holle) in de Afdeelingen Koeningan en Cheribon der Residentie Cheribon. October 1905. Teijsmannia, Batavia, vol. 17, I9°6. pp. 52-63- 191 1. Honing. 1913 (See under Tobacco Wilt.) (See under Fui.ton, H. R, and JR. Winston Tobacco Wilt.) ENGLISH DISEASE OF MANGOLDS AND SUGAR-BEETS. A disease of mangolds and sugar-beets ascribed to bacteria has been reported from Saxlingham, Norfolk, England, by R. H. Biffen, of the University of Cambridge. The first signs are on the leaves, which turn brown and die, while the inner leaves become a sickly yel- lowish-green and are much wrinkled. The signs of the disease usually appear first on the edge of the leaf-stalk. The roots of diseased plants are dry and tough, and all the vascular bundles in both roots and leaf-stalks are purplish-black and plugged with a mucilaginous mass containing a large number of bacteria, "which in all probability have been responsible for the disease." The tissue surrounding the bundles becomes brown and dried. The bundles of healthy plants are cream-colored. The disease does not actually kill the roots, however, as specimens removed from the field and grown in the greenhouse lived, but were stunted and poorly developed. Mr. Biffen thinks that this is the disease described by Kramer on fodder-beets in Russia [Austria] and by Soraueron sugar-beets in Germany. Its external signs, he says, are also similar to those of a sugar-beet disease in the United States in which the sugar-content of affected roots is smaller than that of normal roots. Probably what we call "curly-top" is here referred to. There is no mention of cultures or inoculations, but it is stated that the disease is still under investigation. From a conversation with Mr. Biffen in 1906, the writer inferred that not much additional attention had been given by him to the etiology of this disease. LITERATURE. 1901. Biffin, R. H. A disease of mangolds and sugar-beet. Cambridge University Department of Agriculture. Third Ann. Rep. on experiments with crops and stocks, etc., pp. 87 and 89 (in all, 1 page). BLIGHT OF YOUNG ROSE SHOOTS. In June 1907 Mr. Charles F. Wheeler brought to me from Lanham, Maryland, a few rose shoots, showing a blackening and death of the unopened buds and of the pedicels. No fungus was found, but bacteria were present in abundance. On studying paraffin- embedded, stained sections of the pedicels, most of the bacteria appeared to be confined to the vascular bundles. The organism was not isolated, or at least not studied in pure culture. In the summer of 1909 Dr. William T. Councilman, pathologist of Harvard Medical School, observed the same or a similar disease on some of his roses at York Village, in Maine. The affected stems were cut off and the disease did not reappear that season. He described the bacteria to me as filling the vascular system. BACTERIA IN VESSELS OF FLAX STEMS. In Dutch literature Dr. Ritzema Bos has mentioned finding bacterial plugs in vessels of dying flax. In this he was mistaken, according to his statement in reply to my letter asking for further information about it. J54 RATHAY'S DISEASE OF ORCHARD GRASS. A disease of orchard grass (Dactylis glomerata) associated with bacteria and called bac- teriosis has been described by Rathay. He found it on grass growing in the shade of a deciduous forest on the Vienna sandstone formation 430 meters above the sea. The locality is not given, but is presumably somewhere in the vicinity of Klosterneuberg. It was not found in fields or on any other grass. According to Wiesner, these plants received light only one-eleventh to one-thirtieth the intensity of ordinary daylight, which resulted in a shade- form with short stems and a spindling inflorescence. In 1897, 1898, and 1899, from the end of May to the beginning of July, many plants of Dactylis glomerata were found having one to several affected shoots. The following account is condensed from Rathay's paper. DESCRIPTION OF DISEASE. The diseased culms were distinguished by the following peculiarities: (1) Dwarfing through incomplete elongation of the upper internodes. (2) Presence of a very viscid lemon-yellow slime consisting of bacteria which, in a layer some- times as much as 0.09 mm. thick, envelops wholly or in part the uppermost leaves, the upper part of the stem, and different parts of the inflorescence. All or any one of these organs may be affected. Repeatedly the yellow slime was observed to occur on the outer surface of the higher internodes, as well as on the outer and inner side of the leaf-sheaths enveloping them, and there were cases in which an internode which bore 2 to 3 unopened, enveloping leaf-sheaths was covered with a bacterial layer, while each of the enveloping leaf-sheaths was also covered with the slime on the inner as well as the outer side. Thus it often happened that one could make out distinct bacterial layers separated by the leaf-sheaths. (3) On the portions of the epidermis covered with the bacterial slime, failure of the cuticle to give the characteristic brown reaction when treated first with alcoholic iodine and then with a watery solution of iodine followed by concentrated sulphuric acid. (4) Presence soon of little yellow granules, which are found in the place of the chlorophyll bodies in the cells under the diseased areas in the epidermis. (5) Appearance later of citron-yellow bacterial slime in the intercellular spaces of the paren- chyma and in one or more vascular bundles of the parts of the stem which are covered by the bac- teria. Thus far the bacteria in the leaves, both blade and sheath, have been found only in the vessels of the wood, not in the intercellular spaces. (6) Penetration of the bacteria into the intercellular spaces of the parenchyma (Grundgewebe) of the stem through the solution, in places, of the middle lamella, and the isolation of the cells thereby. (7) Knee-shaped bendings by means of which often one or more branches of the axis of inflores- cence push out laterally from the highest leaf-sheaths which inclose the latter. This phenomenon is caused, on the one hand, by the viscidity of the lemon-yellow bacterial slime on the inner side of the leaf-sheath, by which slime the distorted spikes are stuck fast and, on the other hand, through the elongation of the under part of the stem [see "Cobb's disease of sugar-cane," fig. 8]. (8) Premature drying of all the organs covered by the lemon-yellow bacterial layer. This dry- ing is usually accompanied by a secondary infection with Cladosporium herbarum or a species of Sporidesmium. It is of importance to note that no bacteria have ever been found in the subterranean portions of plants of Dactylis glomerata or in any parts of normal plants. Likewise all search for a similar phenomenon on other grasses (Poa nemoralis, Brachy podium silvaticum, Triti- cum caninum) growing in the vicinity of the diseased Dactylis glomerata, and developing at the same time, has been in vain. The bacterial slime reddens blue litmus paper and has no characteristic odor. The existence of the slime in pure culture is suggested by the microscopic examination, which shows that the bacteria composing it are morphologically alike. Cultures on various agars and gelatins demonstrate, however, the presence of other organisms. In fact, on these sub- 155 156 BACTERIA IN RELATION TO PLANT DISEASES. strata the lemon-yellow organism did not come up at all; only contaminating organisms appeared. The citron-yellow colonies were obtained, however, by stirring a small amount of the slime from diseased plants into a drop of sterile water on a sterile cover-glass and trans- ferring this to slices of sterile potato lying in glass dishes. By making several parallel streaks, one finally obtains pure cultures. The first streaks contain bacteria in great num- bers ; the last, on the contrary, only isolated colonies. Some of the latter were lemon-yellow colonies of an organism agreeing in all respects with that found on the diseased plants. The isolated bacterium, and also that examined from the plant, is short-ellipsoidal, 0.66 to o.qqm long and slightly less wide. It has a distinct capsule both on media and in the plant and is non-motile in hanging drops made with organisms from both sources. It stains with Loffler's methylene-blue, with carbol-fuchsin, and with Gram's stain (both sources). It is not acid-fast (both sources). When treated with iodine solution there is no granulose reaction, either with slime taken from the plant or with pure cultures. No spores have been observed. In fluid media, like bouillon, decoction of Dactylis glomcrata, with and with- out peptone, there forms in the course of 7 days, on the surface of the liquid, an interrupted pellicle of pale citron-yellow color. There is also a precipitate of similar particles, but the fluid itself is clear. The organism grows rapidly on sterilized potato, but makes a very slow growth on different nutrient agars and gelatins, such as bouillon-gelatin, Dactylis decoction-gelatin with or without peptone, and Dactylis decoction-agar with peptone. On potato it grew equally well, whether the slices were weakly alkaline or neutral or made slightly acid by the addition of diluted lemon juice. Potato cultures made at a warm sum- mer temperature (probably 200 to 250 C.) often showed a lemon-yellow streak on the second day and in the next days a considerable portion was covered. The cultures were then wrinkled, easily separable from the substratum, and of a deep lemon-yellow color; the margins were coarsely crenate in places. In this condition it reminds one of Bacillus vul- gatits (Fliigge) Migula, and B. mcsentericus (Fliigge) Lehm. and Neum., but is distinguish- able from them by its vivid lemon-yellow color. Finally, the bacterial growth covers the whole surface of the potato. Although the colonies are yellow the individual bacteria are colorless. The coloring matter exists in the colonies neither in the form of granules nor crystals. It is not soluble in water or alcohol, and with concentrated sulphuric acid does not give the blue reaction characteristic of lipochrome. The organism does not liquefy gelatin. Whether there is any fermentation of sugars or any production of indol has not been determined. It has not been tested for the reduc- tion of nitrates to nitrites or the production of hydrogen sulphide. It is not sensitive to direct sunlight and thrives in sunlight under a Senebier bell-jar filled with a solution of potassium dichromate, as well as under a similar jar filled with ammonium cuproxide. It is not told how these tests were made, i. c, whether plates were poured or fluid cultures used. If only the latter, then the statement is not to be depended upon. All the inoculations, both in mature plants and seedlings, failed (methods not given) ; still the number of cases investigated satisfied Rathay beyond a doubt that the bacteria are the primary cause, and it is equally certain he thinks that special unknown conditions are necessary for infection. It is only in the woods, where Dactylis glomcrata flowers late, only from the end of May to the beginning of July, and only on the upper leaves, internodes, and inflorescence, that the disease has been observed. Dactylis glomcrata is the only grass among several growing in the same place on which this disease appears. The disease always makes its first appearance on the surface and only in later stages is the organism found in the intercellular spaces and the fibro-vascular bundles. It has never been found in the cell-contents. It is in the highest degree probable that the bacterium can gain a foothold only so long as floral organs of the plant are enveloped by the leaf -sheath, since no new infections are observed after the inflorescence has emerged. Infection is made possible only by the unexplained disappearance of the cuticle and the drying up of the underlying tissues. PLANT BACTERIA, VOL. 3. PLATE 1U. Cross-section of two spikelets of Dactylis glomrrata, showing the floral organs widely separated by the yellow slime of Aplaiwbacter rathayi EFS. All of the amorphous parts (B, B) are masses of the bacteria. Photographed with Zeiss planar lens. F 20. and Zetnow's fluid filter screen. Slide 934 C 3, top row, second section from left. Stained with Ribbert's capsule stain. rathay's disease oe orchard grass. 157 COMMENT. This interesting paper leaves many particulars undetermined. The disease as described has some points of similarity to Cobb's disease of sugar-cane and to Stewart's disease of sweet corn. If it is like the latter, then we may suppose that infection occurs earlier in the season at the base of the plant and that Rathay was in error in supposing the occlusion of the vascular system of the stem to be a secondary or late infection. If, on the contrary, as he states, the organism attacks the parts first observed by him to be diseased and makes its way across tissues from the surface to the interior, it would seem to be like Burrill's disease of broom corn and then we might suspect aphides or similar insects of being carriers of the disease. The failure of cultures on agar and gelatin is probably attributable to defective tech- nique, very viscid growths, such as Rathay describes, not lending themselves readily to plate cultures without some preliminary rubbing up in water or bouillon (see B. trachei- philus, vol. II, pp. 287, 294). It is probable, as he believes, that the great mass of the yellow slime on the plants was an unmixed or nearly unmixed growth. It is not likely that the wrong organism was isolated, and it is probable that if young cultures had been inserted into young, growing stems and leaves by needle-pricks successful infections might have been obtained. The last paragraph of the paper promises a full account of the disease and of the organ- ism later on, the same to be properly illustrated. Death, however, cut short the distin- guished author's labors, and, so far as known to the writer, nothing has since been published on this interesting disease. Through the courtesy of Prof. Dr. Julius Wiesner, of Vienna, I learn that this disease occurs on the Kahlenberg between Stephaniewarte and the track of the Kahlenberg- (Zahnrad-) Bahn, i. c, near Vienna. Rathay's material is preserved in the Lehr Anstalt fur Wein- und Obstbau at Klosterneuberg. PERSONAL OBSERVATIONS. After the above comments were in type the writer received from Dr. F. K^lpin Ravn, of Copenhagen, heads of Dactylis glomerate (collected in Denmark), attacked by a bacterial disease which appears to be identical with that described by Rathay. Dr. Ravn's note is as follows : As samples without value I send you some specimens of Dactylis glomerata, infected by bacteria. The disease is reported in Kirchner: Krankheiten und Beschadigungen, 2 Aufl., p. 163, but bas- so far as I know — not been studied more closely. Last summer my assistant, Mr. F. Lind, dis- covered the disease in this country and found that it is widely spread and is of some significance for the growing of seed of Dactylis. Mr. Lind continues his field studies, but we would be much obliged to you if you might have time to report to us your opinion as to the identity of the bacterium after cultural characters and comparison with others. The heads of these plants are dwarfed, distorted, and gummy-yellow from the presence of enormous masses of surface bacteria, and often imprisoned within the leaf-sheaths which are stuck together (fig. 71a). The glumes, which are badly attacked, are lemon yellow or water-soaked, and the sheaths and stems are also involved more or less. On some spike- lets only a portion of the glumes are attacked and these not in all parts. (For an early and late stage of the disease on the spikelets see plate 41 , figs. 9 and 10.) The yellow slime is also abundant between the upper unfolded leaves and the stem (B, B of fig. 71c?). This yellow slime reddens blue litmus paper. Microtome sections show extensive occupation of the floral organs (pi. 1 1 a) . The bac- teria are present mostly between and surrounding these organs, as in the gum-bud disease of carnations (see this monograph, vol. II, fig. 4). In many cases they have been seen occupying intercellular spaces (figs. 710, 71/), and more rarely vessels. The organism most abundant on these plants is a yellow, non-motile, non-sporiferous schizomycete, which so far has refused to grow readily on any sort of agar, but which grows 158 BACTERIA IN RELATION TO PLANT DISEASES. promptly and very well on cooked potato where it is viscid after some days. We have tried to grow it with negative or unsatisfactory results on standard +15 beef peptone agar, Fig. 71*. Fib 71a.* S Fig. 7U.J corn meal agar, potato agar, banana agar, carrot agar, Loefncr's malt extract agar, and standard beef peptone agar with oxalic acid (prepared for fungi). Neither have we been I 1 '.. 7i«. — Stems of Dactylis glomerata from Denmark, showing dwarfing and distortion due to Rathay's disease. [G. 716. — Stem of Dactylis glomerata from Austria, photographed in 1913 from alcoholic material collected by Rathay. Leaves stuck together above and culm below pushing side-wise out of the sheath. Original in Museum of Lehr Anstalt at Kin 1. rneuberg. tFlG. 71c. .1. Cross section of stem of Dactylis glomerata, showing bacterial layer (white mass! entirely sur- rounding the culm, and separating n from the enveloping leaf-sheath. B. Cross-section from another plant, showing partial separation of an inner and outer leaf-sheath by the bacteria (white line at the top). Both were cut and photo- graphed in Washington from Rathay's male rial. rathay's disease of orchard grass. 159 able to grow it in +15 beef peptone bouillon. At the end of 16 days small circular yellow colonies appeared on some of the agar plates, and transfers grew fairly well on the surface of agar stab-cultures. It also grew slowly on agar-stabs when intro- duced along with the white organ- ism mentioned below. It did not grow readily in nitrate bouillon. In the plant it is quite inclined to be associated with a white lique- fying organism which grows as a very short rod usually in pairs or fours with a plain constriction. This white organism grows in agar, where it is motile in early stages of growth on the margin of the colo- nies. It was not clearly motile when taken from a 5-day potato culture, and here T's and Y's were seen. It stains by Gram. The yellow organism is alcohol fast, but not acid fast. It is Gram positive, and during the viscid stage Fig. 7 le.f Fig. 7IA.** Fig 71/4 on potato possesses a capsule. The yellow slime from a 7-day culture on potato was feebly alkaline to neutral litmus paper. The color on potato resembles that of Aplano- bactcr michiganense (pi. 11, figs. 8, 9) and the substratum is grayed. *Fig. 7if the drawing in cork (potato skin); the oval dark bodies are starch-grains; there are very few in the vicinity of the bundle, bul this is not due to their destruction by the bacteria (see figs. 104. 105). The bacteria lie within and between the cells, and have already eroded a considerable cavity. Drawn under the Abbe camera from material which whs cm mi the microtome after infiltrating with paraffin. Cells shriveled by the strong alcohol. Slides 156(2 and 1 56 ' fFiC 83. Cross-section of a small developing potato tuber diseased by Bacterium solanacearum, collected at noutb, Virginia, October 10 >$. Tuber infiltrated with paraffin and cut on the microtome; section stained with carbol fuchsin. The skin and subjacent layers are unbroken, the infection having taken place by way of the under- ground stem. The unshaded parts denote absence of starch and the dark blotches the presence of the bacteria. (For details at .1, H, see ti>;s. 107, 108.) The most crystal-sand cells are on the left side. There are a few starch-bearing cells at X. For a detail of this part see fig. 106. Slide 349(15. PLANT BACTERIA, VOL. 3. PLATE 24. x: _ u r; ~ -j o c = •* — ■-on. c « ■ rt ntJ 5 = x x.2 a O c ° y'si ™ « u „ Si- u o o ■- = 1 "■■0*3 §^ ■5 3° u-1 « £ c a .BflO ,« ^* rt ,£~o *3 ■- W -5E -a «o "' , 0 tc PLANT BACTERIA, VOL. 3. PLATE 25. Two potato-plants badly diseased by Bad. solanacearum (a natural infection). Portsmouth, Virginia, Oct. 1905. All of the tubers were internally brown rotted, i. e . in the vicinity of the vascular ring. The stems were free from superficial ulcers and also the tubers, but in some of the latter the internal brown stain shows through, as in the front tuber and lower tuber of fig. 2. BROWN ROT OF SOLANACEAE. 177 treme tips of the growing shoots, which are flabby or shriveled (plate 30). In less woody plants the branches will be shriveled, or flaccid, as shown in plate 24. In the potato plant the organisms pass down through the vascular system of the stems into the underground parts and cause an internal brown rot of the tubers. This rot of the tuber begins in the vascu- lar system at the stem end and gradually extends to the oppo- site end by way of the vessels, rot- ting the tuber from within and causing the appearance of dusky patches on the smooth surface of the tubers in advance of any rupture of the superficial cork- layer (pi. 23, figs. 9, 11). On cross-section these patches are seen to be due to the internal brown stain centered in the vas- cular ring and showing through the more superficial layers of white tissue. In later stages of the disease the cork-layer is ruptured and the rot continues as a mixed infection due to the entrance of other organisms di- rectly from the soil. In tubers less far advanced in the rot, cross-sections often show the de- cay pretty closely restricted to the vascular ring (figs. 82, 83), from which there is always a gray-white or dirty white bac- terial ooze (vol. I, plate 24, top). In yet earlier stages of the dis- ease the brown stain and bac- terial decay are found only in the stem end of the tuber (vas- cular ring) or only in the vascular bundles of the underground stem leading to the tuber, the surface of this rhizome and the whole of the attached tuber being entirely sound (figs. 84, 85). On plants attacked with great virulence and very early no tubers are formed. The tubers on plants infected in the middle of the growing season are small and few and are found at harvest time in all stages of decay (pis. 24, 25). Often on such plants, as first noted by Dr. Halsted, tubers not larger than a pea, together with their rhizome, will be found sound superficially but brown-rotted in the vascular ring; in other cases, as already noted, the Fig. 84. : Fig. 85. f *Fio. 84. — Cross-section of underground stem of a potato-plant leading to a tuber sound on the surface but bac- terially rotted in the vascular system, i. e., a tuber such as that shown in fig. 83. Rhizome fixed in Carnoy's fluid, embedded in paraffin, and sectioned on the microtome. Slide 348 a 1, stained with carbol-fuchsin. The bark and pith are entirely free from bacterial occupation. The bacteria are confined principally to the vessels of the woody part of the bundle, but there is a cavity in the outer phloem (at the top) and there are various small bacterial foci in the inner phloem. The V-shaped marks in parenchyma-cells (use lens) denote crystal-sand. Material collected at Portsmouth, Virginia, October 1905. Drawn with a Zeiss 16-mm. apochromatic objective, No. 4 comp. ocular, and the Abbe camera. (For general appearance of the bacteria in these vessels see fig. 85.) fFio. 85. — Bacterium solanacearum from one of the vessels in fig. 84 (cross-section rhizome, Portsmouth, Virginia, potato). Slide 348 Ai. 178 BACTERIA IN RELATION TO PLANT DISEASES. entire tuber will be sound and only the rhizome affected — this through the whole length of its vascular system or only at that end farthest from the tuber. On the other hand, when plants are attacked late in the growing season, the tubers are more numerous and larger, and the majority of them, especially if borne on long rhizomes, may escape infection or be diseased only slightly at the stem end when dug. In tubers of this sort the rot is apt to continue after digging, but exceptionally it might, perhaps, remain dormant, or nearly so, until the season for planting. In plants attacked by this disease the juice of the stems will be found to have an alkaline reaction to litmus paper and, if examined microscopically, will be seen to be swarming with bacteria, which are very easily cultivated. On tomato shoots this organism causes the early development of great numbers of incipient roots in the form of small nodules. Hunger was the first to point out this sign and his statements have been confirmed repeatedly by the writer (see pi. 27). Sometimes swelling of the inoculated parts of the tomato stem and the appearance of these incipient roots, with dwarfing, and the changed position of the leaves are the only external signs of disease in inoculated plants (pi. 28). These nodules are visible in the first figure of an inocu- lated tomato published by the writer (in 1896) and were observed repeatedly in the field in 1895, but inasmuch as they develop naturally on sound stems under very moist conditions, e. g., when a stem lies for some time on the earth, the influence of this disease in stimulating their premature development was overlooked. Anyone may satisfy himself easily, how- ever, that Hunger's statements are correct. For instance, if he will select a young, rapidly growing tomato plant, the stem of which is entirely free from such developments, and will inoculate one of two nearly equal branches, that one which was inoculated will promptly develop the roots, while the other will remain free (pis. 26, 27) or will develop them only much later as the result of a general infection of the stem. This is true even when the younger of the two shoots has been the one purposely selected for inoculation. Moreover, these roots always appear first in parts of the inoculated stem nearest the punctures. Wounds made by a sterile needle do not cause them to appear. ETIOLOGY. The cause of this disease is a dirty white or brownish-white schizomycete which the writer named Bacillus solanacearum in 1896. Further studies have shown that it is usually motile by means of one polar flagellum. It should be classified therefore as Bacterium sola- nacearum or Pseudomonas solanacearum, if one follows Migula's system. This organism was first isolated and described by the writer, unless Burrill's statements relate to this species (see History and Literature), and the statements here given rest chiefly upon his own observations. The disease is sometimes very readily induced in susceptible species by simple needle-punctures, without hypodermic injection. At other times the writer has experienced many failures, using in some instances, at least, what appeared to be equally genuine material.* In recent years the irregular behavior of plants inoculated with this organism has been a source of much perplexity. Some have contracted the disease with great rapidity ; others not at all; others have shown some signs of the disease and then recovered, or have been seriously injured only in the inoculated shoots, or only after a great length of time (pi. 29). The reason for these marked differences has not been made out clearly. It seems to depend partly, at least, on the amount of water in the stem. Frequently the punctured areas have been separated from the rest of the plant by the development of a protective cork-layer. In general, infections have been more successful in young, rapidly growing, soft, watery plants than in slow-growing, woody ones or in mature ones; infections have been more suc- *One labors under difficulties in the isolation of this organism. On agar plates its colonies are best distinguished from other while colonies by waiting a week or two for the appearance of a brown stain in the colony, and yet this treatment seems to weaken the virulence of the organism. Plant bacteria, vol. 3. PLATE 26. Tomato-plant No. 17 (1903), showing two equally developed young, smooth shoots, one of which was inoculated by needle-pricks with Bacterium solanacearum (South Carolina strain) immediately after photographing (see plate 27, for condition 9 days later.). Photographed and inoculated July 16, 1905. PLANT BACTERIA. VOL. 3. PLATE 27. Tomato plant No. 17, nine days after inoculation; leaves of the inoculated branch reflexed, and incipient roots well developed. Check branch at right side normal. PLANT BACTERIA, VOL. 3. PLATE 28. Tomato-plant inoculated with Bacterium solanacearum (District of Columbia strain) on September 6, 1904, and photographed September 30, at two-thirds natural size. No wilt resulted. Observe swollen stem, shoots pushing, reflexed leaves and incipient roots which are absent above and below. The needle-pricks were made in the middle (swollen) portion of the stem. PLANT BACTERIA, VOL. 3. PLATE 29. Tomato plant two-thirds natural size. Inoculated on Sept. 6, 1904, with Bacterium solanacearum (District of Columbia strain) which had been grown in the thermostat at 37.5° C. Photographed Sept. 30; pricked part swollen and nodular, leaves reBexed but not wilted, has wilted, shrivelled, and fallen otf. The extreme top BROWN ROT OF SOLANACEAE. 1 79 cessful in hot wet weather than in cool dry weather, and with cultures recently plated from diseased plants sent in from various parts of the United States than with organisms which have been longer removed from the plant, i. c, cultivated in the laboratory on various nutri- ent media for 6 months or a year. One of two inferences seems to be warranted: (i) the hosts vary enormously from plant to plant and time to time, or condition to condition, in their ability to resist this disease; or (2) the organism readily loses its virulence, for reasons yet unknown, when subjected to ordinary cultural conditions. Both inferences may be true. The subject requires for its elucidation more time than the writer has been able to devote to it. That the failures noted were due to accidental substitution of similar-looking non-infectious organisms was sought as a first explanation and might answer for a single failure, but hardly, one would think, for repeated failures, since the same care was exercised as in the case of other organisms where no such phenomena appeared. Descendants of the most virulent strain the writer ever had in the laboratory (vol. I, pi. 26) lost all infectious power in the course of a year, although apparently preserving the same cultural peculiarities. This strain was isolated from the plant photographed on plate 30. When virulent cultures are used on susceptible plants the time elapsing between the puncture and the first appearance of the disease is usually not more than 8 to 10 days and is sometimes as short as 2 or 3 days on watery shoots in very hot weather. All of the success- ful inoculations of 1895 were made with the organism taken from tomato plants grown in Mississippi. The very successful inoculations of 1896 were made with cultures derived from an egg-plant grown at Charleston, South Carolina. In both years infections were obtained on tomatoes and potatoes, the resultant disease being identical with that observed in the field. The organism was demonstrated in the vessels of the plants in enormous numbers at long distances from the points of inoculation (fig. 86, about 15 inches, time 18 days; fig. 82, about 3 feet, time 42 days). Pure cultures were isolated from the interior of such plants and the disease was again produced by means of these cultures. Needle-punctures on leaf- lets of potato produced the disease as readily as those into the stems, the only difference being that the organism had a longer distance to travel and consequently the tubers were not reached and destroyed so quickly. Sometimes the organism was observed to pass out into the under portion of the midrib of the potato leaf, blackening it for a distance of several inches in advance of the staining of the upper surface or the wilting of the foliage ; at other times it ran out in long, narrow black lines on the upper surface of the leaf. The most rapid downward movement of the bac- teria in inoculated potato stems was observed during hot weather in July 1896: In 16 days from the date of an inoculation by needle-pricks on the upper part of a stem the bacteria had passed downward a distance of rather more than 2 feet and caused signs of wilt in another stem from the same root. In another tall shoot pricked lower down, ;'. e., in the more woody tissues 6 inches from the earth, the signs on other shoots from the same root were slower to appear, i. c, they did not develop until after the twentieth day. Signs of the disease were observed to pass up and down stems on the pricked side much faster than sidewise. In 1895 a tomato plant which was of large size when inoculated showed local signs after a few weeks, but general signs developed only after a long time. On then making sections, the bacteria were found in the vascular system in enormous numbers and at long distances from the point of inoculation. A similar case occurred in 1903, another in 1905, and still another in 1909. The latter was 12 feet high and full of fruit when it finally wilted. It was inoculated on a terminal shoot when about 4 feet high. It grew well for 3 months after inoculation, showing at first only a feeble wilt of a few leaves, from which it soon recovered. When dissected the upper 6 feet of the stem was free from bacteria. They were very abun- dant, however, in the vascular bundles of the lower 6 feet of the stem, including some of the roots. Cavities had formed in the tissues only near the point of inoculation. The stem was sound externally. The wood was stained brown in the part occupied by the bacteria. i8o BACTERIA IN RELATION TO PLANT DISEASES. In 1901 the writer again obtained successful infections. The plants inoculated were potato, tomato, etc. The source of infection was a tomato plant received that summer from South Carolina. The inoculations were by means of needle-pricks on leaves and the upper parts of growing shoots. The infectious material was fluid from the bottom of slant agar- cultures (the first subcultures from colonies on poured plates). The period of incubation varied from 3 to 7 days. Successful inoculations were made in the summer of 1903 with pure cultures obtained from a diseased potato plant gathered at Norfolk, Virginia. Inoculations were carried out successfully in 1904 on tomatoes, potatoes, etc., with pure cultures obtained from a potato- plant growing in the District of Columbia, near Washington. Partially successful inocu- lations were carried out in 1905 on potatoes with pure cultures obtained from a tomato stem received from Florida (vol. I, pis. 24 and 25), and on tomatoes with subcultures of colonies plated from Florida pota- toes. For details concerning these results and for the various failures see synopsis of the in- oculations. Plants in the field are attacked in all stages of growth. There seem to be, how- ever, varietal dif- ferences in suscep- tibility as well as great individual differences. The writer succeeded in pro- ducing the disease by allowing the Colorado potato- beetle (Doryphora TO-lineata) to feed on diseased plants and then transferring them for a few hours to healthy plants. The beetles were obtained from healthy potato fields where the disease did not afterwards appear, and the only possible source of infection was the diseased potato leaves and stems on which they were fed, and which were the result of pure-culture inoculations. The interior of these leaves and stems swarmed with bacteria and it was impossible that the mouth-parts of the beetles should not have become contaminated. The infected plants did not contract the disease immediately, but signs appeared in from 7 to 9 days as narrow brown streaks extending downward rapidly inside of the stems and leaf-stalks. There were numerous infections on each plant and all of them appeared to originate in slight wounds made by the jaws of the Fig. 86.* *FlG. so. —Cross-section of a small portion of a potato-stein, showing bacteria in vessels, and also three bacterial cavities in the outer phloem. From plant No. 14, May 27, 1895, inoculated by needle-pricks, using a pure culture of Bacterium solanacearum. The activity of the organism is shown by the following facts: The distance (downward) from lln mi *' w Potato-shoots inoculated by needle-pricks August 8, 1904, with a pure culture of Bad. solanacearum plated from the stem of the Woodridge, D. C, potato shown in plate 30. Photographed Aug. 16. Notice particularly the dark stripes extending down inside the stems. The left-hand shoot was pricked in a petiole (the black one at the left midway). The right-hand shoot received one needle-puncture in an upper internode. The middle shoot received 10 needle-pricks in an upper internode. BROWN ROT OF SOLAN ACEAE. 189 June 27, 1905. — Potato plants (2 varieties) were inoculated from tubes Nos. 4, 5, 6, June 22, from as many colonies (stem of tomato, Hastings, Florida). Made 85 inoculations on shoots and leaves on 26 plants. Result. — Positive in part; slow. On July 13 the Blush showed fewer cases than the Early Rose. Plants on this date 2 to 2.5 feet high. The difference in resistance appeared to be lodged in particular plants. The Blush was more resistant than Early Rose. Each culture was virulent. (See vol. I, plate 24. The source of the culture is there stated to be potato, but the potato organism from Hastings appears to have been tried only on tobacco except as below, unless certain of the notes have been lost.) July S. — Eight tobacco plants were inoculated in stem and leaf with potato-cultures A and B.July 6 (from two separate colonies, descended from potato tuber, Hastings, Florida). Result. — Failure. The experiment was followed until September 6, when 6 plants were examined microscopically. Bacterial multiplication and brown stain slight and confined to pricked parts. July S. — Thirty-four young tomato plants 12 to 16 inches high were used in this experiment (variety test). Potato cultures A and B, July 6 (from 2 separate colonies descended from potato tuber, Hastings, Florida), were used. Fig. 97.* Result.- — Most of the plants grew to be 6 to 9 feet long, fruited well, and were alive 4 months after the inoculations. They doubled in size within 6 days after the inoculations, and the inoculated shoots had elongated about 12 inches. The plants continued to grow rapidly. On July 25 they were 3 times as large as on July 8. A few cases developed very slowly, most failed, except that the inoculated parts were swollen and browned and adventive roots pushed out. Of many varieties, Livingston's Dwarf Aristocrat was most susceptible In October a plant with large pear-shaped fruits contracted the disease and the bacteria were found filling vessels of stem and branches 4 feet from the needle-pricks. September 23. — Tobacco (24 large plants) was used for this experiment. The plants were inoculated by needle-pricks in the stem, introducing potato-cultures A to D, September 21 (potato tuber, Hastings, Florida.). Result. — Slow progress of disease; pith blackened. No distinct wilt. September 23. — Tomato plants were inoculated with potato cultures A to D, September 21 (potato tuber, Hastings, Florida). Result. — Adventive roots developed and some shoots were swollen and stunted; wilt developed not at all or very slowly. September 30. — Tomatoes (20 plants, 10 to 12 inches high) were inoculated by needle-pricks with beef-bouillon cultures A to D, September 20 (potato tuber from Hastings, Florida). Result. — Failure. *Fig. 97. — Tomato plants pricked with a delicate needle at X, X on July 5, 1904. When used on the left-hand plant the needle was sterile, when used on the other (No. 64) it was flamed and then dipped into a 3-day-old culture of Bacterium solanacearum (District of Columbia strain). Photographed July S. 190 BACTERIA IN RELATION TO PLANT DISEASES. September 30. — Tobacco (24 plants) was inoculated by needle-pricks with beef-bouillon cultures A to D, September 20 (potato tuber from Hastings, Florida). Result. — Failure. October 26. — Tomatoes (3 plants about 18 inches high) were inoculated with potato cultures Nos. 1 and 2, October 20 (growth black on the potato) ; alive October 23. The organism was not plated out, but descended from potato cultures Nos. 1 and 2, October 19, which were streaked directly from diseased tuber from Portsmouth, Vir- ginia. It was probably the right organism — it showed the typical jet-black growth on potato. Result. — Failure. October 26. — Tobacco plants were inoculated with same cultures used for above. Result. — Failure. October 2S. — Tobacco plants were inoculated with potato cultures Nos. 3 and 4, October 23 (from Portsmouth, Vir- ginia, tuber); alive October 27. Result. — Failure. (For successful inoculations on tobacco see Wilt Diseases of Tobacco, p. 227.) October 30. — Tomatoes (2 vines, 18 inches high), were inoculated with potato culture No. 2, October 23 — jet-black (from Ports- mouth, Virginia, tuber). Result. — Failure. November 24. — Tomatoes (5 vines) were inocu- lated with potato cultures Nos. 1 to 5, November 10, each from a separate colony on plates of October 19 (potato tuber, Portsmouth, Virginia). Result. — Failure. The cultures were alive; the transfers made from them on date of inoculation grew well. A single needle-prick introduc- ing into a susceptible plant a small quantity of a virulent culture is suffi- cient to produce this disease, as shown on plate 31, right-hand figure. MORBID ANATOMY. The premature and excessive de- velopment of roots on tomato shoots as a result of this disease has been mentioned under "Etiology." For a section through such incipient roots, showing their origin and relation to infected tissues see fig. 98. An en- larged view of bac- teria from this section is shown in fig. 99. In rapidly fatal cases there are no hyperplasias in con- nection with this dis- ease other than the adventive roots on tomato stems, but in stems of tomato and potato inoculated with feebly virulent cultures there may be considerable enlargement in the vicinity of the punctures. Such stems *Fig. 98.— Cross-section of a small portion of stem of tomato plant (No. 26) inoculated with Bacterium solana- cearum < >i 1 June 8, 189.5, and fixed in strong alcohol on July 3. The figure shows vessels occupied, the formation of small bacterial cavities, and a well-developed incipient root (R), the growth of whieh has been stimulated by the pres- ence cil the bacteria at a distance. Several much more rudimentary roots are depicted in the upper portion of the fig- ure (J?'). At the left, above the lower cavity, the bacteria fill the intercellular spaces around a few cells (X). The bast fibers are not distinct in the section itself, which was stained with special reference to the bacteria. There are no bac- teria immediately under or in the budding root (R). Slide 1 1 1 (2. For further illustrations of these budding roots see \ ai ions plates. fl'ic. 99. — Bacterium solanacearum from inoculated tomato plant No. 26, 1895. An optical plane from one of the cavities shown in fig. 98. F.g. 99. f BROWN ROT OF SOLANACEAE. 191 frequently attain a diameter twice that of the stem a few inches above or below the pricked area (pis. 28, 29). In such enlarged areas there is usually developed around the punctures a considerable area of protective cork-tissue, and an excessive amount of bundle-tissue is formed. This malady is chiefly a disease of the vascular bundles (figs. 81, 84, 90, 100, 101, 102, 103), but the organism is not so strictly confined to the bundles as in case of the wilt of cucurbits and the brown rot of the cabbage. There is, however, the same enormous multi- plication of the parasite inside of the vessels. The walls of the vessels are dissolved in their thin places or else are ruptured, and cavities are produced in the surrounding tissues, the bacteria being found in the in- tercellular spaces but also often inside of cells without one's being able to make out clearly how they got there (see vol. II, p. 76). The parenchyma, especially in soft parts, such as the pith of young potato shoots and tomato shoots, succumbs quite readily, a large portion of the interior of such stems break- ing down into a soft, wet rot which sometimes oozes through to the surface. The bacteria first invade the intercellular spaces, which are generally occupied quite fully in advance of the complete destruction of the cells. The middle lamella is then dissolved. The sepa- rated cells are squeezed into a variety of shapes by the multi- plication of the bacteria and finally are crowded aside alto- gether, leaving behind cavities partly or fully occupied by the parasite (figs. 86, 88, 91, 93, 98). In woody parts such as stems of old potato plants or tomato plants, and of well- grown egg-plants, there is usually a marked browning of the lignified tissues and an enormous multiplication of the bacteria in the vessels and the adjacent parts, but there is no such general collapse as in case of soft stems. Lignified tissues are not dissolved. In the potato tuber, brown-walled cavities beginning in the rudimentary vascular tissue are numerous. These cavities, which are full of bacteria and remnants of the broken-down tissue, gradually enlarge and coalesce until all of this part of the tuber is involved. Subsequently they reach the surface (pi. 23, portions of figs. 8 and 10) and open up the tuber to all sorts of external destructive agents. The starch in the Fig. 100.* *Fig. 100. — Longitudinal section of a potato stem, No. 5, 1896, inoculated June 1, collected June 11, showing Bacterium solanacearum restricted to a single spiral vessel. Drawn from a photomicrograph. The fibers.at the left polarize in the same way as the spiral thickenings. Slide 138, Vernier readings on Zeiss photomicrographic stand 13.4X2.0. Stained w-ith carbol-fuchsin and washed in 50 per cent alcohol. X210. 192 BACTERIA IN RELATION TO PLANT DISEASES. tubers resists well and appears to be but slowly acted upon by this organism (figs. 104, 105, 106). In young tubers, however, such as that one shown in figs. 83, 107, 108, there may be considerable areas of the starch-bearing tissue in which no starch occurs. These are most extensive on that side of the tuber longest occupied by the bacteria, and the only explanations which seem to fit the case are that (1) the potato plant has withdrawn the starch preparatory to the formation of a cork-barrier as Appel has described for B. phytoph- thorus in potato, or that (2) excreted products of the bacteria (enzymes or other sub- stances) have killed these cells or have so paralyzed their activities that the plant has not been able to make use of them for the storage of starch. A very little is sometimes found (figs. 105, 106). That the starch was once stored and then dissolved by diastasic action of the bacteria is contrary to all that we know of its behavior, both in vitro and in older tubers. In the inoculated plants of the Jamestown weed (fig. 88), of the black nightshade (fig. 91), and Physalis philadelphica (fig. 94), there was the same enormous multiplication of the bacteria in the vascular system, even at long distances from the point of inoculation, and the tissues were destroyed in the same way, with the formation of extensive bacterial cavities. Several observers have reported finding an excess of crystal-sand in plants at- tacked by this organism. This substance occupies particular cells and consists of numerous discrete tetrahedral crystals of calcium oxalate (fig. 88). It occurs naturally in a large part of the Solanaceae, the amount varying with particu- lar organs and with the age of the parts; it is, therefore, not easy to determine whether there is an excess of it in dis- eased tissues, but there seems to be. In most of the draw- ings crystal-cells are depicted diagrammatically by minute arrow-marks ( < < ). In the Solanaceae we have a bicollateral bundle, the two phloems being flanked or subtended by thick-walled bast-fibers, as shown very clearly in the cross-section of a potato-rhizome (fig. 84). In the plants which I have examined there is also usually a large pith and considerable subepi- dermal collenchymatic tissue, which is usually somewhat shrunken and torn on fixing in alcohol and does not always show distinctly as collenchyma in the accompanying draw- ings, but may be seen very clearly in the cross-section of a potato petiole shown in fig. 95. Both phloems are frequently invaded by the bacteria and numerous cavities are produced in this tissue, as shown in the figures already referred to. In later stages of the disease, cavities are also common in the bark-parenchyma and in the pith. Indeed, the disorganization has proceeded very far in such stems as that shown in cross-section in fig. 1 of vol. II. ' Pig. ioi. — Cross-section of small part of same potato stem as fig. 86, showing vessels occupied by Bacterium solana- cearum, and surrounding tissues mostly free, except as they have flooded out of the vessels during the preparation of the section. These hitter bacteria occur only on under side of section and are omitted from the drawing. Drawn from a photomicrograph by the writer. Section stained with carbol-fuchsin and differentiated in 50 per cent alcohol. The lignified connective tissue is represented by solid black walls and that of the vessels by fine dots. XSoo. Slide 171. Fig. 101. BROWN ROT OF SOLANACEAE. 193 THE PARASITE. Bacterium solanacearum EFS.* is a medium-sized rod with rounded ends. It multi- plies by fission. It often occurs in pairs with a plain constriction (fig. 109). It is usually 1.5 to 3 times as long as broad. When crowded in the plant it may be so short as to some- what resemble a micrococcus. It stains readily. When taken from young cultures in pep- tonized beef-bouillon and stained with methyl violet many of the rods are 1.5/u by 0.5/x. Longer and shorter rods occur and thicker and thinner ones, the measurements depending to a considerable extent upon the age of the culture and the kind of stain employed (fig. 109). It is motile at times both in the plant and in cultures, and is often very actively so in young cultures. Owing to imperfect preparations it was at first supposed that the flagella were per- ipheral, but further studiesof these slides and of others subse- quently prepared indicate that the or- ganism is usually motile by means of a single polar flagel- lum, which is much longer than the rod (fig. 109). Pseudo- zoogloeae are com- mon in fluid cultures and inclined to ac- cumulate in the upper layers. Spores have not been ob- served. Longchains and filaments have not been observed. On August 8, 1904, two 3-mm. loops of the District of Columbia organism taken from a slant potato-agar culture 3 days old (tube 2, Aug. 5) were put into 15 c.c. of sterile water, and then the faintly clouded fluid was studied in a hanging drop under high powers of the microscope. The organism was actively motile. It was a short rod, single or in pairs, rarely in fours, the doublets with a plain constriction in the middle. The length of each element of the doublet was not more than 1.5 to 1.8 times its breadth. In old potato cultures examined in 1904, the organism was a short rod with rounded ends, single or in pairs, with a plain constriction. Involution forms were not *Syn. Bacillus solanacearum EFS.; Pseudomonas solanacearum EFS. fFiG. 102. — Cross-section of stem of potato (No. 5, 1S96) inoculated with Bad. solanacearum June 1 ; fixed in alco- hol June 11, after being badly wilted for 3 days. The culture used was a tube of peptone-water inoculated directly from the interior of an egg-plant stem. The heavily shaded parts are the ones occupied by the bacteria. There are 16 crystal-cells marked by arrow heads (use lens). The parts of the stem not heavily shaded are free from the bacteria. viz, all the bark, most of the pith, and considerable portions of the woody ring. Slide 166(2. Figure somew-hat diagrammatic, especially in the collenchyma and phloem. Compare with fig. 1, vol. II, which shows cross-section of a potato stem in a later stage of the disease. The vessel at X is shown highly magnified in fig. 103. 1 in m Fig. 102. f 194 BACTERIA IN RELATION TO PLANT DISEASES. observed. This has been the general form of the organism seen nearly every summer since 1895 in tomato stems and potato stems collected in various southern States, and in cultures made therefrom. In glycocoll solutions Honing has seen involution forms (p. 253). This organism is often not so pure a white as Bacillus amylovorus or Bacillus trachei- philus. In the host plant and on certain media it may be described as pure white at first, but soon grayish-white, dirty white, or brownish-white, becoming brown, since it produces a soluble brown pigment which modifies its appearance. On steamed potatoes this stain is usually developed to such an extent that the cultures soon become brown or even nearly black (3 to 10 days); the water also is browned around potato cylinders. This pigment, which is produced in the dark as well as in the light and which on potato is not infrequently as black as tar (pi. 23, figs. 8, 10, and pi. 41, fig. 4), is soluble in water and glycerin and slightly in methyl alcohol on long stand- ing; it is insoluble in ethyl alcohol (exp. 1896, 1905*), ether, chloroform, xylol, and carbon bisulphide. The browning did not occur in the absence of sugar and alkali. The pigment is not destroyed by weak acids or alkalies. It behaves in some re- spects like a humous compound, i. c, it does not dialize readily and is precipitated by compounds of iron and calcium. This organism grows well in pepton- ized beef-bouillon, with or without the addition of sodium carbonate, and yields much more precipitate than similar cul- tures of B. tracheiphilus; a brown stain appears, and this was especially noticeable in case alkali was added; tendency to form pellicle rather slight. Old peptone- water cultures do not brown. In +15 peptonized beef-bouillon the District of Columbia organism at the end of 20 days showed good clouding with an enormous number of small pseudozoogloea?, but with no rim or pellicle ; the dirty white or pale brownish precipitate covered a breadth of 13 mm. on the bottom of the test-tube. In Dunham's solution cultures of the same age were well clouded, but less so than the bouillons, with only about half as much of the dirty white precipitate. Cultivated in milk there is no precipitation of the casein, but after about 3 to 7 weeks the fluid becomes transparent (fig. no, a). Milk was intensely alkaline to litmus paper as early as the twentieth day. The organism is able to live in milk for at least 48 days. The casein of the transparent milk is thrown down by hydrochloric acid (1896, repeated in 1905), and by strong solutions of sodium chloride or copper sulphate. In litmus milk no acid is developed, but after 2 or 3 days the fluid becomes slightly bluer, and this alkalinity increases gradually from day to day until the milk is an intense deep blue. On the twenty-eighth ci solanacearum (District of Columbia strain) in gelatin-stab. On adding water to the alcohol the- pigment was slowlj soluble. 1 1 ' 1 < • 103. Cross-section of a very small portion of a potato-stem (a detail from fig. 102), showing a single vessel occupied by Bacterium solanacearum, the surrounding tissue being free from the bacteria. The lignified wall of the vessel is indicated by line dots. Slide 166(2. Zeiss photomicrographic stand, vernier readings 14.7X2.8. Plant inocu- lated with pure peptone-water culture made direct from the interior of a diseased egg-plant. Fig 103-t BROWN ROT OF SOLANACEAE. 195 day such milk was approximately Ridgway's indigo blue ; after 3 months it was dark hya- cinth blue (pi. 23, fig. 7) ; but the fluid was neither viscid nor gelatinous. Litmus in milk is sometimes a little reduced (white) in the bottom of the tubes. The bacterial precipitate is white. In another set of tubes of litmus milk examined at the end of the seventh week the color was recorded as a clear intensely dark blue. All these milks were cream-free. Ljtmus-laetose-agar streaked with this organism becomes slowly a deeper blue. There is never any reddening, but sometimes the litmus is partially reduced and the agar finally browns. The behavior in cream-free litmus milk is like that of the culture figured on pi. 41. The streak of the District of Columbia organism on slant litmus-lactose-agar after 10 days at about 270 C. was white, wet-shining, and there was a copious whitish precipitate in the V; the upper half of the agar was blued very decidedly, in the lower half the litmus was reduced. The surface of the slant beyond the streak was iridescent. The agar was not then browned, but later (seventh week) it was browned decidedly and the surface growth was a dirty brownish-white ; the extreme upper part of the agar continued bluer than the check-tube, but in the lower part the formation of the alkali was masked by the brown stain and the reduction processes. The organism does not liquefy gelatin (experiments of 1895-96, repeated in 1904 and in 1905), at least not when made as described in vol. I of this monograph. The surface colonies in gelatin are small, circular, thin, thin-edged, smooth, white, wet-shining; the buried col- onies are globose, yellowish or brownish, and smooth, with well- defined margins. In stab-cultures (fig. 111) the upper part is best de- veloped, but growth was not rapid at 240 C. and ceased at io° C. The Virginia organism grown in gelatin- stabs for 7 days at 220 to 250 C, and then 2 days at 150 C, developed a thin, white, wet- shining surface growth which was roundish and about 3 to 4 mm. in diameter. Growth was visible the whole length of the stab, but was best at the top. There was no stain, no gas, no liquefaction, no development of crystals. The District of Columbia organism grown for 10 days in gelatin-stabs at 250 C. was similar in all respects to the above. The + 10 gelatin in old cultures becomes dusky or browned, especially in the upper part of the stab. In agar the buried colonies remained small and were irregularly round or oblong, with a roughened margin. The surface growth was white or gray-white at first, then brownish. The general form of the surface colonies after some days on +15 nutrient agar at 250 C. is shown in figs. 112, 113. As a rule the agar was stained brown by growing this organism on it (pi. 23, fig. 2, and pi. 41, fig. 8, from tobacco, will answer for potato and tomato). In thin sowings on peptonized beef agar the organism from the South Carolina tomato, used for successful inoculations in 1901, formed colonies about 2 to 3 mm. in diameter at the Fig. 104/ *Fig. 104. — Cross-section of a few cells of a potato tuber rotted by Bacterium solanacearum. Plant No. 14, 1S96, inoculated June 15. A detail from fig. 82 made under x, showing cells occupied by the bacteria. The starch-grains which lie buried in the bacterial mass appear to be uncorroded; they polarize, but I was unable to detect any rings of light and dark substance in them. Section stained with carbol-fuchsin. Drawn with the Abbe camera. Slide 156(2. 196 BACTERIA IN RELATION TO PLANT DISEASES. end of a week (temperature 2 8° C). days later they were brownish and the after the first 10 days. On steamed potato this organism was white at first, then a dirty yellowish-white, and finally brown or even nearly black. The poorest agar-streaks, with perhaps one exception, were on al- kaline agar containing 6 per cent glycerin. Inagar-stabs the growth was best on the surface and in the upper part of the needle-track. No gas-bubbles or acid reac- tions were observed as the result of cultivation in any of the ordinary culture-media, but in each case there was a gradually increasing alkalinity. Grown in fermenta- tion-tubes in 2 per cent alkaline peptone-water with addition of grape-sugar, fruit-sugar, cane- sugar, milk-sugar, galactose, mal- tose, or dextrine, there was no gas production, no detected acid de- velopment, and no clouding of the These were circular, white, and wet-shining, and 9 agar was also browned. They did not enlarge greatly Fig. 106-t Fig. 105.* closed end of the tubes. The same result was obtained with the juice of potato tubers diluted with water and sterilized in fermentation-tubes (1909 organism from tobacco). Culture-fluids containing grape- sugar, fruit-sugar, and cane-sugar browned decidedly after some weeks. In 1904, using the District of Columbia organism, clouding was heaviest with cane-sugar. All of the writer's numerous experiments go to show that Bacterium solanacearum is a strict aerobe (the fermentation-tube experiments were repeated with the same results in 1904). If it is ever capable of growing in the absence of air we do not know on what media or under what cir- cumstances. All of the common media have been tried and in all of them it requires free oxygen for respiration. *Fig. 105. — Border of a small cavity in the vascular region of a potato tuber attacked by Bacterium solanacearum show inn 7 uncorroded starch-grains lying in one cell embedded in a mass of bacteria. Slide 156(3. Vernier readings on Zeiss photomicrographic stand, 12.5 X8.1. Plant No. 14, 1896, inoculated on the stem June 15. Drawn by polar- ized light. The dark patches are masses of bacteria. In. 106. — Detail from fig. 83 at x, showing uncorroded starch-grains embedded in a cell occupied by Bacterium solanacearum. The dotted parts of the starch-grains are the only ones stained by the carbol-fuchsin. Light and dark 1 -nigs can not be made out in these grains, but they polarize the same as grains in the middle of the tuber, remote from the bacteria. Drawn with 2 mm. 1.30 n. a. Zeiss apochromatic objective, 12 compensating ocular and Abbe camera. Slide 349(15- BROWN ROT OF SOLAN ACE AE. 197 The organism is not sticky, or only slightly so occasionally on agar, and diffuses out of the vessels of the host-plants readily in 75 per cent alcohol, but not, according to Hunger, when 1 per cent nitric acid is added to the alcohol. It possesses a decided odor when grow- ing on potato, i. e., a smell like that of some rotting potatoes, and likened by one person to the odor of sour bran. The starch of steamed potatoes is not destroyed to any great extent, but a little of it is converted into amylodextrine, yields a red reaction with iodine. There 1. e. was no growth, or very slight, in Cohn's solution; very little growth appeared on silicate jelly (with Fermi's solution) at the end of 6 days. The *nh Fig. 107.* •■?..- ^t^^i \^^fm&^immi%^ Fig. I08.f *Fig. 107. — Portsmouth, Virginia, potato tuber. A detail from fig. 83 at A, showing more extensive bacterial occupation than in fig. 83 at B, and complete absence of starch on the periphery. A few starch-grains occur inside of cells at Si. (center). Only four cells containing crystal-sand are present. Cavities have begun to form and additional ones are present beyond C (.right margin), where also are a few cells containing starch-grains (see fig. 106). In various places, as at X (use lens), some change has taken place in the walls of the cells and they are made out with difficulty. In other places, as on the left side, cells which appear to have unbroken walls are occupied by the bacteria. The walls of the cells destitute of starch seem to take the stain somewhat heavier than those of cells in which it is present. Camera drawn, but slightly diagrammatic. Slide 349(15. fFio. 108. — A detail from fig. 83 at B, showing character of the tissues and absence of the starch in the vicinity of the bacterial foci (at the center). Tissues fixed in Carnoy's solution. Camera drawn, but slightly diagrammatic i. e., it is probable if the slide were taken and compared cell by cell with the drawing, some omissions and some errone- ous lines would be detected. 198 BACTERIA IN RELATION TO PLANT DISEASES. organism reduces potassium nitrate to nitrite in bouillon; does not produce indol in peptone-water; and does not stain by Gram's method, i. c, is only slightly blue after washing (diaphragm wide open). Cultures were easily obtained from a bouillon-culture after exposing it for 20 minutes to minus77°C.,but quantitative experiments with liquid air show that a large proportion of the rods are killed by a single freezing (pi. 32). The minimum temperature for growth is about io°C; optimum temperature 350 to 370 C. (?); maximum temperature not determined (about 410 C.*); the thermal death-point (South Carolina organism, 1896) is above 510 C. and below 530 C, being probably about 520 C. In 1904 all 10-minute exposures of the Dis- trict of Columbia organism at 520 C. remained sterile but not those at 510 C. The organism grew readily and for a long time in the thermostat at 370 C, i. e., in peptonized beef-bouillon held at 370 C. the South Carolina organism was alive in one test after 3 weeks' exposure, and in another after 7 weeks' exposure. At 380 to 400 C. the District of Columbia organism Fig. I09.f clouded + 15 peptonized beef-bouillon in 48 hours and grew well, but not so freely as at room temperature. After 10 days' exposure in the thermostat at this temperature, streaks to slant agar gave only a discrete growth (separate colonies), indicating that the cloudy bouillon was then only thinly occupied by living bacteria. It also lives for a considerable time in peptone-water and in bouillon at 200 to 250 C. This bacterium does not live for many weeks ♦Exposed in Feb. 1904 in the thermostat at 39.4" C. the Virginia organism clouded peptonized beef-bouillon of the following reactions: + 25, +15, o, and refused to cloud —20 bouillon, Dunham's solution, or Uschinsky's solution. The experiment was twice repeated with the same results. fFiG. 109. — (a) Bacterium solanacearum (District of Columbia strain). From a slide (2a) of August 9, 1904. Flagella stained by Lowit's method. (B)Bacl. solanacearum (D. C. strain) showing flagella stained by van Ermengem's silver nitrate method. Slide of August 12, 1904. (c) Freehand sketches of flagella from three slides of Bad. solana- cearum, stained in 1896 by van Ermengem's silver nitrate method. Slides X, XX, and XXX (South Carolina organ- ism, No. XXX taken from inoculated Physalis No. 57, the others from cultures). The numbers correspond to the fol- lowing vernier readings on Zeiss photomicrographic stand 32532 used by the writer: (1) 12.8X5.35; (2) 12.2X6.8+; 14.4X7-3; (4) 20.1 X7-3! (5) 20+X7-2; (6) 18.5X7-15; (7) 13-6X5-6; (8) 20X5; (9) 19-3X6.75; (10) 18X44; (") 17.3X0.9; (12) 15.4X6.6; (13) 16.1X6.9; (14) 15.6X575; (15) 13-8X5-6 artefact (?); (16) 16.35X11.35; (17 and 18) 14.17 X6.35. Numerous flagellate rods are visible on slide XXX at 14.17 X6.35 and all seem to be one-flagellate, but it requires a high power and a bright light to make them out as all are feebly stained: One might also add. keen eyesight 1 I1" paration of Bad. solanacearum (D. C. strain) stained August 12, 191)4, by van Ermengem's method from a young agar culture. Flagella wanting or not visible. This organism was extremely pathogenic at first (see pis. 30, 3 1 . and vol. 1, pi, 26), but afterwards lost much of its virulence. The bacteria are thicker than when stained without mordanting. PLANT BACTERIA, VOL. 3. PLATE 32. Four Petri-dish poured-plates of Bacterium solanacearum (Virginia strain), showing the effect of freezing. .1 and B, plates poured before freezing; C and D, corresponding plates inoculated with the same carefully measured amount of the bouillon culture, but after freezing, i e., as soon as the culture was thawed. The plates were incubated for some days at 300 C. The large dark colonies are on the surface, the large pale colonies are between the bottom of the dish and the agar, the dot-like growths are colonies buried in the body of the agar. Photographed Feb. 20, 1904. BROWN ROT OF SOLANACKAE. 199 Fig. 110/ in steamed potato cultures, especially if the brown stain is well developed, and the well- browned colonies on agar are usually dead. Two weeks is about the limit of vitality on cooked potato ; sometimes cultures were dead at the end of one week. An actively motile bouillon-culture frozen in liquid air for 20 hours was still motile upon thawing out. Exam- ined in a hanging drop within 20 minutes hundreds of the rods were active. This was not a Brownian movement, but a rapid darting motion which often carried the rods out of the field. However, a distinctly less number seemed motile than before the exposure and poured plates (Vir- ginia organism) demonstrated 50 per cent to be dead. The organism grew well in acid bouillon (-{-33, acid of beef juice), but less rapidly at first than in nitrate bouillon (+15). After 3 months the bouillon was stained brownish. There was then an interrupted, dirty, gray-white pellicle, a dirty, brownish-white precipitate, and numerous small crystals. The Virginia organism did not grow well on No. 602, a rather acid agar made from the juice of sugar-beets diluted with water. The Virginia organism after growing in peptonized Uschinsky's solution for from 9 to 14 days had developed no pellicle, rim, or pseudozoogloea?, but only a thin clouding, with a small amount of brownish-white precipitate. Around the surface growth, in the agar in contact with the air, a white amorphous sub- stance develops. This substance is finely granular under the microscope and dissolves in acetic acid. There appear to be many degrees of virulence, and possibly there are several strains of the organism. RESUME OF SALIENT CHARACTERS. POSITIVE. Cause of a vascular disease in solanaeeous plants — potato, tomato, egg-plant, etc. — and from recent studies by Honing and others it would seem also of a disease in plants of several other families. vShort rod, often termo-like, motile, flagella polar; organism in the plant often easily mistaken for a coccus; dissolves middle lamella, cellulose (?); plugs vessels, attacks phloem, forms numerous closed cavities in parenchyma of hosts ; causes pre- mature development of adventive roots in tomato stems. The feebly virulent strains induce slight local enlargements ; surface colonies on agar rather slow-growing, roundish, white at first, then brownish; clouds bouillon with formation of numerous flocculent particles which accumulate in top layers; culture fluids containing grape-sugar, fruit-sugar, or cane-sugar brown decidedly after some weeks; growth in acid bouillon ( +33, acid of beef-juice) is feeble at first in comparison with that in +15 bouillon, lll.t *Fio. no. — Drawing designed to show the clearing action of Bacterium solanacearum on milk (a sub-culture from colony B, Florida potato): A, tube of milk inoculated July 6, 1905, and drawn August 4; B, uninoculated check-tube. A has become translucent slowly without a previous precipitation of the casein; B is opaque. fFic n 1. — Gelatin stabs of Bacterium solanacearum (Virginia organism) after about 16 days at room temperature. No liquefaction. Inoculated Feb. 2, 1904. Photographed Feb. iS. 200 BACTERIA IN RELATION TO PLANT DISEASES. but afterwards excellent; produces a brown stain in host, on agar, gelatin, etc.; pigment soluble in water and glycerin; streaks on silicate jelly (with Fermi's solution) feeble at end of sixth day (250 C.) ; grows readily on steamed potato cylinders, producing a pale brown to dark brown stain and an alkaline reaction ; odor on potato feebly disagreeable ; has slight action on potato starch ; slowly converts milk into an alkaline translucent fluid ; gradually changes the lilac color of litmus-milk to a deep indigo or hyacinth blue ; litmus- lactose-agar slowly becomes a deeper blue; slight reducing power on litmus; aerobic; reduces nitrates to nitrites ; portion of rods killed by freezing, others retain vitality and motility ; injured by acids (Hunger, EFS.) ; thermal death-point 520 C, approximately; minimum temperature io° C, approximately; optimum 370 Cor below; maximum 410 C, approxi- mately; alive in milk after 48 days; browned growths are usually dead; dies early on steamed potato, usually the first or second week. Chains occur (Honing, p. 247). Group No. 21(^.3333823. NEGATIVE. Spore-formation ; capsules ; long chains and filaments (so far as observed) ; liquefaction of gelatin (at least during first weeks) , blood-serum ; starch-de- struction (or feeble) ; precipita- tion of casein from milk; acid from milk; gas-formation (all media) ; anaerobism (so far as known) ; acids from sugars and alcohols (so far as known) ; dendritic growth; green fluo- rescence; vile odors; wrinkled growth on potato; disintegra- tion of cooked potato-cylinders ; action on pigment of dilute acids (10 per cent) and alkalies (5 per cent) ; pigment insoluble in ethyl alcohol (absolute), sul- phuric ether, chloroform, turpentine, benzine, xylol, benzole, carbon bisulphide; cultures not viscid, or only exceptionally and slightly so ; growth in Cohn's solution absent or feeble ; Gram's stain; indol. Non-motile (Hutchinson). Any organism which produces spores, stains well by Gram, gives a decided pink reaction in old peptone-water cultures with sulphuric acid and sodium nitrite, evolves gas, liquefies gelatin readily, acidifies cream-free milk, develops a lab ferment, grows abundantly in Cohn's solution, grows well anaerobically in bouillon with grape-sugar or cane-sugar, has a thermal death-point above 530 C, or fails to blue (cream-free) litmus milk and to brown steamed potato on long standing, may be set down at once as something else. This organism may be distinguished horn Bacillus phytophthorus and its allies by the fact that it does not liquefy gelatin, at least for several weeks, does not redden cream-free litmus Fig. 112.* *FlG. 112. — Colonics of Bacterium solanacearum on +15 standard nutrient agar after 8 days at 23° to 270 C. Plate poured June 18, k>i 13, from the interior of a diseased tomato stem, received from South Carolina. Natural si/e. BROWN ROT OF SOLAN ACEAE. 20I milk (compare pi. 23, figs. 1, 1a, 3, 5 with fig. 7); or grow in closed end of fermentation tubes in peptone-water with common sugars. There are other differences, e. g., behavior on cooked potato (compare pi. 23, figs. 6, 6a with pi. 41, fig. 4) ; appearance of colonies in thin-sown gelatin plates; and, finally, behavior in the plant. TREATMENT. Fields on which this disease has appeared should be planted for some years to crops which are not subject to it. Inasmuch as the organism appears to be a wound-parasite often disseminated by insects, etc., prompt attention should be given to the destruction of these pests. Tand subject to nematodes should not be planted to Solanaceae if this bacterial organism also occurs in the soil. When transplanting to such soil is necessary, it should be done early and with great care not to injure the roots. Plants with broken roots should not be set on such land. In the transplanting of large plants manyroots are certain to be broken. The prompt removal of diseased plants is also recommended in early stages of the disease. Such a course reduces to a minimum the amount of infectious material sub- ject to insect depredations and liable to contaminate the soil further, a precau- tion which must always be an important matter in all diseases disseminated in this way. It is probable that the dis- ease is sometimes spread from field to field in "seed" potatoes. For this rea- son potatoes designed for planting should be sorted over several times at intervals of some weeks with the greatest care, all suspicious tubers being rejected. There should also be a final inspection at planting time. It would be still better to secure tubers for plant- ing from fields known to be free from this disease. The tubers from infected localities should be cut across the stem- end at planting time and a further re- jection made of all suspicious ones. The sound ones should now be exposed before planting to dry air for a day or two, i. e., long enough for the develop- ment of a protective cork-layer on the cut surface. By following this method of careful selection and by a rotation of crops, being very careful never to plant potatoes immediately after potatoes, a certain Portsmouth, Vir- ginia, planter, who is a very successful potato-grower and who was formerly much troubled by this disease, believes he has overcome it. At least it is not now troublesome in his fields. In 1905, the writer searched for it in vain on this farm in a large field planted from such carefully selected tubers, but found it readily on the same farm in another (rented) field planted with a variety of unselected tubers on land which was in potatoes the preceding year. In case of fields of potatoes attacked by this disease, the crop should be harvested as soon as fit to dig, should be stored in a cold, dry place, and should be marketed and con- sumed as early as possible. In no case should the crop be allowed to remain in the ground *Fig. 1 13. — Colonies of Bacterium solanacearum on + 15 standard nutrient agar, grown for 7 days at 300 C. Plate poured June 10, 1902, from the interior of a diseased tomato stem received from Porto Rieo. Natural size. These colonies became brown and stained the agar. ^^Bj • *■* *• • r' ' , " ' #••" ' - .* • V \ • • •f *,-••♦ '■ 1 '- # • • • •• ^ ;,:; • * . ■#■* • ■ "#:H <» ■ > • •*~x ; ■ •#" -.' 'Mli ■ 1. • * V * • • ."•• ®4M ■ • .*,•. ff'&gk aJv teL... Fig. 113.= 202 BACTERIA IN RELATION TO PLANT DISEASES. until rainy weather has set in. The cardinal rules should be: Dig early, dry thoroughly, store in a cool, dry place after removing all suspicious-looking tubers, and use as soon as pos- sible. The crop can not rot from this disease if stored at 8° C. (46 + °F) or under. Moist land should be underdrained or planted to other crops. Tomatoes and other plants subject to the disease should either be germinated where they are to stand or else transplanted quite young and with the greatest care to avoid crushing or breaking the roots. Seedlings having the galls of eel-worms on their roots should be rejected; also over- grown seedlings, the latter because their roots are likely to be broken in transplanting. To recapitulate : Inspect tubers at the stem-end by cutting some days before planting and reject all that show any trace of this disease. If possible procure seed tubers from localities where this disease does not occur. Plant on land not infected by this organism. If infected land must be used, select that not subject to root-nematodes, and in transplanting seedlings to such land do it early in their growth, and break the roots as little as possible. Practice rotation, but not of one susceptible species after another. Drain the moister portions of the field. Burn over or steam the soil selected for a seed bed. Potatoes grown on infected land shoidd be sold in the summer or autumn, and always stored in a cool dry place. Destroy insect enemies. Be on the lookout for resistant varieties. Read what is said under "Wilt-Diseases of Tobacco" (p. 238). PECUNIARY LOSSES. This disease has destroyed a great many fields of tomatoes and potatoes in the South, and has put an end to commercial tomato-growing in certain sections, e. g., southern Missis- sippi, southern Alabama, and parts of Florida. The following letter, dated May 25, 1905, from a tomato grower in North Florida, is like many others received by the U. S. Department of Agriculture from various parts of the South in recent years: Under separate cover I send you a diseased tomato plant. Out of a crop of 30,000 plants I have lost fully one-third, and many more seem to be becoming affected. The first thing noticeable is the tender tips of the plant wilting, but nothing can be seen outwardly or even by cutting into the plant at the top, but by cutting into the roots, or stem, for a short distance above the ground, the wood immediately under the bark and the interior of the roots are found to be discolored and bad. The disease has spread with great rapidity during the past few weeks and is still increasing, and it seems about to wipe out the entire crop. It is not confined to any one field and is equally distributed on soils of widely different character, some of it high, light, and some of it low, black soil. Also, the disease is equally distributed on fields where tomatoes were never grown till this year. Another man writes as follows under date of May 16, 1903, respecting this disease: Our truck farmers around this section, Tyler, Texas, who have hundreds of acres of tomatoes, are complaining that the blight is destroying some of their fields, thus entailing a loss of thousands of dollars. ( )ne planter at Norfolk, Virginia, lost 3,000 barrels of potatoes by this rot in 1908. Additional statements respecting losses may be found in the chapter dealing with the history of this disease. If Bad. solanacearum also plays any considerable part in the frequent and widespread outbreaks of potato-rot in the northern United States, as now seems likely, but has not been proved, then the annual losses can be reckoned only by hundreds of thousands of dollars. The northern distribution of this organism is unknown. The subject of potato-rots is still surrounded with a good deal of uncertainty. The fungus Phytophthora infestans undoubtedly causes great losses in the cool, moist regions of Europe and occasionally in the more northern parts of this country, where the writer has seen BROWN ROT OF SOLANACEAE. 203 the foliage of whole fields destroyed in a week, but there is no basis for the assumption that this is the only or the principal cause of the rot of potatoes either in this country or abroad. Species of the fungus form-genus Fusarium also cause, in the United States as well as in Europe, serious diseases of potatoes, one of which begins in the field as a vascular disease of the stems and continues in cellars as a dry rot of the tubers.* The great bulk, how- ever, of the soft rot of potatoes, in this country and also in Europe, is due to bacterial organisms, either acting independently, which is often the case, or following Phytophthora and Fusarium. Several are known to induce decay and very likely a dozen or more dis- tinct kinds of schizomycetes will be found capable of destroying the tubers in wet seasons when the lenticels open in the soil. When this happens all sorts of soil bacteria gain an easy entrance to the tubers under conditions likely to be very favorable to their destruction. This attack of the tubers in a badly aerated, wet soil, with the lenticels wide open, is a very different affair from the active development in the parts above ground, and under normal conditions, which is manifested by Bacterium solanacearum, or by Bacillus phytophthorus, which causes the only other bacterial decay of potatoes which has thus far been described with any great degree of accuracy. Since this was written, Harrison, in Ontario, has described Bacillus solanisaprus, an organism strongly suggestive of Appel's organism but not absolutely identical (see Basal Stem-rot in vol. IV), and Pethybridge and Murphy in Ireland have also described their Bacillus melanogenes, so that in general it may be said that our knowledge of the bacterial diseases of the potato is much further advanced than it was ten years ago, when the rough draft of this chapter was written. HISTORY. According to Hunger this disease was first recognized by Comes in Italy in 1882. There is much uncertainty, however, concerning this. Professor Comes undoubtedly observed a serious bacterial disease of tomatoes in the vicinity of Naples in 1884 or earlier and recog- nized it as such; but his descriptions of it, and of the fluid cultures made therefrom, do not enable one to speak with any degree of certainty either concerning the signs of the disease or its exact cause. It may have been due to B. phytophthorus or to some other organism. His name, if retained, should apply to a grape bacterium. The disease formed brown or black cankers on the stems near the earth, the whole plant being finally destroyed. Bark and pith were disorganized and granular ; the vessels of the wood with their surrounding cells, but not the medullary rays, were occupied by a yellowish or brownish granular mass. "In the disorganized tissues, "says Comes, "and in the gummy granulations was observed the presence of myriads of microbes, similar to those of Bacterium gummis, found by me always in tissues of woody plants affected by gummosis. " In 1890, before the Society for the Promotion of Agricultural Science, Prof. T. J. Burrill, of the University of Illinois, described very briefly a disease of potatoes which had attracted his attention as something new, and which was probably this disease. The examinations were made on tubers shipped from the south in June, many of which were rotting. Only bacteria were found. Several species of these were isolated, and suc- cessful infections were obtained with the one presumed to be the potential factor in the rot, but these experiments are not described. The organism causing the rot was a termo-like bacterium actively motile, oval to short cylindrical in outline, occurring single, in couples, or rarely in chains of 3 or 4 links, its rods measuring about 0.7 X 1 to 1.5/*; it was non-sporifer- ous, non-liquefying, and formed non-characteristic zoogloeae in the potato. How the organisms gain access to the subcortical tissues without some previous puncture of the skin of the tuber has not been ascertained, but some observations seemed to show that the same bac- terium infests the leaves and culms, causing injury and death to these parts and possibly sometimes reaching the tubers through them. *See Smith and Swingle, Bull. 55, Bureau of Plant Industry, U. S. Dept. Agric, Feb. 16, 1904. 204 BACTERIA IN RELATION TO PLANT DISEASES. In 1 891, Dr. Byron D. Halsted reported a widespread and destructive decay of pota- toes in south New Jersey, part of which appeared to be due to bacteria. The potato tubers, it is said, Developed well-formed ulcers, and slices of these quickly turned to almost a coal black, especially near the surface beneath the skin, and over all rapidly grew [oozed?] an almost velvety layer, consist- ing entirely of masses of bacteria. This entry probably belongs here, or if not here, then most likely under Basal Stem-rot. Early in 1892, Dr. Halsted published a bulletin from the Mississippi Experiment Sta- tion giving an account of the Southern tomato-blight, as observed by him in that State in 1 89 1. This disease he ascribed without hesitation to bacteria, basing his conclusions on numerous microscopic examinations. Dr. Halsted noted the wilting of young leaves and growing tips of the stem, the water-soaked appearance of certain tissues, the watery greenish disorganized pith, the brown stain in the vascular system, and under the microscope in freshly gathered material the presence of large numbers of bacteria, while all traces of fungi were absent. Inoculations into growing plants failed. A blight of potatoes was also observed and studied by Dr. Halsted at Ocean Springs, Mississippi. The above-ground signs were similar to those on the tomato. Below ground the stem is more or less darkened, in patches brown, and occasionally almost black. The old "seed" potato is a soft rotten mass, and the few new potatoes usually small and decayed, invariably at the stem end and almost always at the eye, appearing at first "watery" and afterward brown. It is an interesting fact to be stated in passing that the earth adheres very closely to the surface of the decaying portions. It seems evident from the extended study of these diseased potato plants, hundreds being examined, that the rot passes from the main stem to the tubers, and probably comes originally from the "bud." Often a lateral underground branch including its minute potatoes, as large as peas, is entirely softened. Upon making halving sections of the tubers it is seen that the most diseased portion is in a circle which includes the base of each eye. This portion is a soft, grow- ing layer, rich in nitrogenous substances, and furnishes the avenue through which the disease spreads from one eye to another. From this decayed tract a milky juice quickly accumulates upon the sec- tion that is made up very largely of bacteria and contains no other form of fungus [consult this Monograph, vol. I, pi. 24]. Dr. Halsted believes the disease is transmitted to the growing plant from the mother- tuber. Cross-inoculations from potato to tomato in the field failed. The tomato-plants were then full-grown. Direct infections under bell-jars in moist air succeeded. These were made many times over and the checks usually remained sound. For this purpose Dr. Halsted used slices of tomato-fruits and potato-tubers and also short pieces of tomato- stems and potato-stems. The decay was rapid. The milky ooze from the potato rotted tomato, and that from the tomato rotted potato, but under the same conditions melon- tissues were also rotted when inoculated with this material, and Bacillus phytophthorus, Bacillus uuiouis, or some similar organism must have been present. The disease as observed in the field must, however, I think, have been due to Bacterium solanacearum. Several attempts were made to infect seedling tomatoes by planting them on soil con- taining diseased material, by soaking seeds in it before planting, and by spraying infected fluid on the plants, but as the results were not conclusive it is not necessary to make further mention of them. According to a note by Director S. M. Tracy prefaced to this paper the losses from this disease in Mississippi had been serious for several years, amounting in some cases to from a quarter to half the plants. In 1890 the losses were widespread and severe. In 1 89 1 the disease appeared at Ocean Springs, Mississippi, in the tomato fields on May 1. (In 1914, Rolfs found it for me on potato shoots at Gainesville, Fla. April ij.) In 1892, in Garden and Forest, Dr. Halsted reported occurrence of this southern tomato disease in tin- north. The material was received from F. L- Stevens, Syracuse, New York, where ,} fields were affected. In the field most seriously injured about 3 per cent of the plants were dead, 8 per cent badly affected, and many more showing traces when the BROWN ROT OF SOLAN ACEAE. 205 inspection was made. This may have been, however, the Grand Rapids disease, due to Aplanobacter michiganense (see p. 161). The same year, in two of the Cornell University Experiment Station bulletins, Bailey and Corbett made a brief mention of a disease which was serious upon some of their tomatoes that year. Specimens were submitted to Dr. Halsted, who thought the disease was "prob- ably identical with the Southern one" (Bull. 45), and "distinct" (Bull. 43). Professor Bailey adds : Apparently the same disease has been reported to me from three localities in this State during the last two seasons and in two cases it had practically ruined the crop. Probably this was the Grand Rapids disease due to Aplanobacter michiganense. In 1893, Professor Burrill published another note on the new potato disease, the most important part of which is here cited: The present note is simply to substantiate as correct what was simply surmised before. The micro-organisms taken from the different portions named of the plant not only appear alike and grow alike in artificial cultures, but the results are identical when cross-inoculated into the living tissues. * * * When the aerial stems are infected, pure artificial cultures are as easy to make as from the tuber itself, and we have had abundant opportunity to do it. [These cultures and inoculations are not de- scribed.] It now appears that in most instances at least the tubers in the ground are infected by way of the rhizomes and aerial stems. * * * The leaves are primarily infested and very commonly first at the tips or margins, then are progressively destroyed, becoming first watery, or semi-trans- lucent, then brown, and afterward dry and curled. The supporting petioles and stems are in turn invaded, but initial spots may also be found on these. It has not been ascertained whether or not previous mechanical rupture of the epidermis is necessary, but all observed facts indicate the contrary. In 1895, Dr. Halsted wrote as follows about conditions in New Jersey: Bacteriosis of the tomato prevails in many parts of the State, and is not, as yet, well understood. In 1895-96, the writer isolated the organism causing this disease and studied it many months. He obtained typical infections on tomato and potato plants, inoculating from pure cultures by means of needle-pricks on stems and leaves; he determined by cross- inoculations the identity of the disease as it occurs on potato, tomato, and egg-plant, obtained successful inoculations on various weeds {Datura, etc.), failed to obtain the disease in tobacco or peppers, perhaps because these plants were not growing rapidly, determined experiment- ally that the organism was non-infectious to the cucumber and some other plants, and demonstrated that the disease could be conveyed by insects. As a result of these investi- gations he described the disease and figured it, named the organism causing it, and for the first time described the latter according to well-recognized bacteriological methods. According to Selby, the disease is serious in parts of Ohio. One tomato-grower in Clermont County stated on July 23, 1896, that "in my three acres there is scarcely a sound plant." In 1898, P. H. Rolfs, at Lake City, Florida, published short notes on this disease in two of the Florida Experiment Station bulletins. The disease was known to be present in Florida in several fields. It was inoculated successfully into tomatoes, which were all destroyed. Egg-plants appeared to be more resistant than tomatoes, but suffered quite severely. Peppers seemed to be free from this disease. Some potatoes on the Experiment Station were carefully treated with fungicides, but no good result could be seen from the use of this material. In Bulletin 47, of the Florida Station, it is said: That the disease is not indigenous to Florida I think is evident from the fact that it rarely occurs in new portions of the State until after vegetables have been grown for two or three years. The loss 206 BACTERIA IN RELATION TO PLANT DISEASES. from this disease varies from a few plants to at times every plant in small areas — ofttimes amounting to as much as 80 per cent to 90 per cent of the whole field. There seems to be no special preference for either high or low land. The same year, in Proceedings Florida Horticultural Society, Rolfs made some observa- tions on the distribution of the disease in Florida and again reported successful inoculations. Last summer at the Experiment Station [Lake City] several plots were set aside for studying the effects of this disease. A plot of egg-plants, another plot of tomatoes and a plot of peppers, each one- hundredth of an acre, were planted contiguously and the disease [was] started in the corner of the egg- plant field, this corner being the most remote from the tomatoes and the peppers. The disease mani- ested itself in less than a week on the plants inoculated. For a time it was confined to this corner but before long sporadic cases occurred throughout the plots of egg-plants and tomatoes, until within the course of about three weeks the entire plots of egg-plants and tomatoes were destroyed. In no case was a pepper plant hurt. * * * Soon after the egg-plants and tomatoes had all been destroyed the field was cleared of all these plants and of all the debris left from the plots. The plots were then kept free from vegetation for two weeks, when they were again planted to egg-plants and tomatoes just as before. The disease now appeared in various spots over the entire field. We then took out the plants as soon as they showed signs of the blight and the place occupied by the plant was filled by another one, so that it gave us a full field, all of it being in plants that showed no blight, but neverthe- less the blight continued to appear until cold weather cut short the growth of both egg-plants and tomatoes. * * * During last winter these plots were planted first to radishes, and next to lettuce. None of these plants showed any indications of the disease. As soon as the weather permitted, egg-plants and tomatoes were planted in the same plots as last year. These have now begun to show signs of blight- ing which makes it seem quite probable that the disease can remain over winter in the field. In 1900, F. S. Earle, then of the Experiment Station at Auburn, Alabama, also wrote upon the disease as follows: This serious disease of the tomato has so far only been observed in the southern part of the State. It is very destructive in Mobile and Washington counties. * * * Each succeeding crop suffers worse than the last. * * * As the result of rather wide experience with it in Mississippi I am of the opinion that direct underground infections do take place as suggested by Dr. Smith. * * * Contagion carried by winged insects may well be the means by which the disease first becomes introduced to new fields, but this method of infection can hardly account for the spread of the disease from year to year in somewhat regular concentric circles from such new centers, especially as it usually takes almost every plant in its path. Insect infection would not either account for the facts reported by me in the 6th Annual Report of the Mississippi Station, pp. 53-61, where, in a large tomato field that was under observation, the disease was very largely confined to a narrow strip of wet, seepy land, running diagonally through it, while the drier land on either side was nearly exempt. As the disease is thus so markedly a soil disease, the possibility of soil treatment as a remedy at once suggests itself. Very few experiments are recorded in this direction. In the Mississippi experi- ments mentioned above in one case heavy applications of kainit seemed beneficial and in another case there was apparent benefit from the use of lime. Marked benefit also seemed to follow the use of lime in an experiment at Deer Park, Ala. (see Ala. Bull., 92 : 109). These experiments, however, need confirmation. Sulphuring the soil does not seem to have been tried. Spraying the plants and the surface of the ground with Bordeaux mixture gives no result. In his report for 1907-8 Stevens, of the North Carolina Experiment Station, stated that the wilt disease of tomatoes due to Bad. solanacearum was widely prevalent in North Caro- lina and constantly spreading into new territory. The newer Dutch East Indian studies are reported under Wilt-Diseases of Tobacco (pp. 224, 244). APPENDIX. In conformity to the writer's custom of keeping things separate until it has been defi- nitely established that they are identical, the following diseases are put by themselves, although it is not unlikely that some of them are due to Bacterium solanacearum. There is the more reason for suspending judgment in some of these cases, because in recent years through the labors of Dr. Appel, in Berlin, we have come to recognize a widespread disease of potatoes similar in some respects to the one described by the writer, as due to an entirely different organism (see Basal stem-rot of potato, vol. IV), and also because a full description of Bad. solanacearum has not been available. [Recently, I have ceased to have doubt concerning identity of the American and Dutch East Indian Disease. See Wilt-Diseases of Tobacco.] THE NEW ZEALAND DISEASE. Kirk is authority for the statement that Bad. solanacearum causes a disease of potatoes in New Zealand. It is not widespread. THE AUSTRALIAN DISEASE. In 1894, the entomologist, Mr. Tryon, reported the appearance in Queensland of a new and destructive disease of potatoes and tomatoes, ascribed to a bacterial parasite which fills the vascular system, wilts the plant, and destroys the tubers. The leaflet, which is only a brief excerpt from an unpublished MS. report of Mr. Tryon to the Department of Agriculture at Brisbane, mentions no inoculation experiments and contains no description of the organism. The reader is given no information concerning it, other than what may be inferred from the following statements: "A small, living microbe, having an average length of less than one ten-thousandth of an inch, resembling in appear- ance the bacillus of chicken cholera and other organisms." (The italicizing is mine.) In the tubers the disease first appears as "an indistinct, translucent line running parallel to the outer margin" of the tuber when cut across. "They soon, however, commence to rot, decay starting in the more superficial portions of the tissue, and the potato substance is eventually converted into an offensive, odorous, tenacious, whitish slime." (Italics mine.) This leaflet deals chiefly with suggestions for treatment and does not materially help the pathologist or bacteriologist. In June 1895, Mr. Tryon stated that he had had an opportunity of reinvestigating the new potato disease and that the microbes are "scarcely distinguishable from those which are met with in diseased sugar-cane. " "They are found clogging up the vessels of stems, roots and rhizomes, and, in the initial stages of the disease, nowhere else." The disease here referred to is Cobb's gumming of sugar-cane, due to a honey-yellow bacterial organism. Without further description the organism is therefore designated Bacillus vascularum solani. In 1895, Helms reported on several potato diseases occurring in the Clarence River dis- trict in New South Wales, in south latitude 290 to 300. The paper is agricultural rather than bacteriological. Judging from the plates, one of these diseases (No. 1) is like Prillieux's potato disease ascribed to Bacillus caulivorus, and another (No. 3, p. 328) is possibly like that due to Bad. solanacearum. No cultures or inoculations are mentioned, and positive or even presumptive identification from the figure is impossible. If it were infected with Bad. solanacearum, the tuber in longitudinal section might be expected to show decay at the stem end, but it is not so represented. In 1899, Tryon identified his Australian disease as that described by me and intimates that his name, Bacillus vascularum solani, should have been used in my bulletin on "The brown rot of tomatoes, egg-plants, and potatoes," published in 1896. I would have used 207 2o8 BACTERIA IN RELATION TO PLANT DISEASES. Mr. Tryon's name, or some portion of it, had I at that time had any means of knowing what he meant by it. This I did not know, and do not yet know. His organism certainly can not be like three very different organisms. It would appear that Mr. Tryon had written a considerable manuscript on this disease and had submitted it to the Department of Agri- culture of Queensland, but that unfortunately they allowed it to remain unpublished. He now makes citations from this unpublished paper, but these are not sufficient to clear away the uncertainties. For all we know from anything he has published, this Australian disease may be due to Bacillus phytophthorus, or to some undescribed species. Mr. Tryon has never published a proper (exact) description either of the disease or of the organism causing it. Certain statements of his do, indeed, make the reader think of the brown rot, but there are others which certainly can not be applied to Bad. sola- nacearum, e. g., the "sticky, tenacious" slime which is said to choke up the vessels; the "froth-like" viscid exudate from the eyes of the tuber; the "foetid odor," the opening of the lenticels; the gradual decay of the stems from the base upward ; the frequent destruction of the organism by the temperature of the Queensland summers, etc. Mr. Tryon is either describing mixed infections, or else a different disease. Why not "Schwarzbeinigkeit" due to Bacillus phytophthorus? We shall never know the specific cause of this Australian potato disease until some bacteriologist takes hold of the problem, isolates and describes the organism in ways recognized as proper, and demonstrates his ability to reproduce the disease with one particular organism by means of pure- culture inoculations. A few words as to Mr. Tryon's name. In my judgment the name Bacillus vascularum solani is unusable because it can not be attached, by means of anything he has yet published, to any particular organism. It is a trinomial, it was published with- out description, without isolation of the organism in pure culture, without proof that the bacteria actually under observation and named by him had anything to do with the disease, with- out proof that the disease itself was actually due to bacteria, and finally without careful description of the disease, e. g., such descriptions as would have enabled anyone to identify it with the North American disease. His name, therefore, for reasons sufficiently set forth in volume I of this monograph, can be regarded only as a nomen nudum. Professor McAlpine, of Melbourne, has sent me a photograph (fig. 114) of a potato- tuber decaying at the eyes, this disease being known in Australia as "sore eye" and "spewey eye." This disease is associated with bacteria and probably due thereto. Some years it causes great losses, it is said. Perhaps it is the disease observed by Tryon. Living material sent to me was too badly decayed when received to make anything out of it. It appeared to be a brown bacterial rot (only a little Fusarium was present), and the vicinity of the vascular ring was stained darker than any other part. Fig. 114.* *Fig. 114. — Potato tuber, showing the bacterial "sore eye" or "spewey eye" of Australia. From a photograph sent by Prof. D. McAlpine, of Melbourne. This is perhaps Tryon's disease. There is a slimy ooze from the eyes and the earth usually sticks in them. BROWN ROT OF SOEANACEAE. 209 THE DUTCH EAST INDIAN DISEASE. A disease of tomatoes prevalent in Western Java and rapidly fatal was identified by Dr. F. W. T. Hunger of Buitenzorg in 1901 as due to B. solanacearum Smith. The follow- ing observations are from Hunger's paper (see Bibliography) : This disease has not been so prevalent as in the United States, but nevertheless whole plantations have been destroyed by it in Java. The course of the disease is rapid. Hunger experimented with diseased and healthy plants, exposing them to the sun with- out water and then watering them. The diseased plants recovered slowly from the wilted condition, and after some days not at all. The undiseased recovered promptly on watering. In two instances he observed buds out of place, namely, on the leaves, and he is inclined to think that these signs were due to the disease. The adventive root-formation occurs so regularly that there can be no doubt as to this being a sign of the disease. The roots develop regularly on the stem and they may appear also on the leaf-stalk. The vascular ring of the stem was stained brown and bacteria were abundant in the vessels. Whenever a tomato plant showed signs of the disease above ground he always found the root-system diseased. These roots were more or less browned, and when well along in the disease were blackish and soft, and filled with bacteria. In early stages the browning was confined to the xylem wedges of the root. Tissues put into alcohol of various strengths gave the result described by Smith, viz, the bacteria diffused out of the vessels regularly in 75 per cent alcohol, and also to a less extent in 90 per cent, but were hindered in their diffusion by absolute alcohol. Hunger found that by adding 1 per cent nitric acid to the 75 per cent alcohol he obtained a solution in which diseased tissues behaved as in absolute alcohol. He found the pith of diseased plants greatly changed, i. c, from white to spotted gray, with formation of bacterial cavities. The whole pith of diseased stems may disappear, the remnants being found on the inner edges of the vascular ring. Groups of tetrahedral crystals, the so-called crystal-sand, are stated to be much more common in diseased than in healthy plants. Hairs are absent from the vicinity of those parts of stems which are sending out adventive roots [the writer has not observed this in the North American disease]. These roots spring from the pericycle. Hunger believes that the formation of tyloses which he observed abundantly in the vessels of diseased plants is induced by the presence of the bacteria. These tyloses are always filled with bacteria. They soften the walls of the vessels in places and then the tyloses are formed; the evidence adduced in proof of this statement is not conclusive, but nevertheless Hunger is probably right (see similar observations by the writer on mulberry shoots attacked by Bad. mori, vol. II, fig. 30, of this monograph). Hunger also notes that in Tryon's paper there is not the least description of his Bacillus vascularum solan i. He says: The bacteria found by me as cause of this tomato disease agree in every particular with Bad. solanacearum Smith, so that I hold the two for identical. Hunger believes that he has determined by inoculation with the tomato organism the occurrence of the same disease on Nicotiana tabaewn and Capsicum annuum. He says that he has repeatedly obtained the same signs of disease in these plants as in sick tomatoes. He inoculated it into tobacco, using cultures from tomatoes, and obtained the slime-disease (see Wilt Diseases of Tobacco, p. 222). His inoculation experiments were as follows: Tomato seeds were germinated on filter-paper and the young plants then brought into water- cultures, which were infected with pure cultures of Bad. solanacearum. The young plant grew as well as the check-plant, and a subsequent examination showed that there were no bacteria in its interior. In a similar experiment, varied only by the fact that the root-system was wounded in various ways, viz, by needle-pricks and by teazing out the end of some of the young roots, the plants became infected after a week and the bacteria were demonstrated in the vascular bundles. In one of the plants the bacteria were found in the vessels of the leaf-stalks of two of nearly the youngest leaves. Tyloses had not yet formed in these vessels. 2IO BACTERIA IN RELATION TO PLANT DISEASES. In another series of experiments the young plant was placed in the earth and when growing rapidly was watered daily with water containing a pure culture of Bad. solanacearum. No infection took place. In a similar experiment, varied only in that the roots were purposely injured, the plants became infected after 1 1 days with typical signs and a microscopic examination showed the presence of the bacteria in the tissues, vessels included. In a third experiment a number of leaves of two tomato-plants were plunged into infected water for some days to see if the bacteria would enter through the water-pores. After 3 days the leaves were investigated microscopically. They were not infected and the writer draws the conclusion that they can not be infected in this way, but this seems not strictly warranted, because of an additional state- ment: "Certainly I found rather commonly Bacillus solanacearum in the large, intercellular spaces under the water-pores, but not at all in the corresponding water-vessels." A similar result would not be uncommon at the end of 3 days in case of the cabbage infected through the water-pores by Bacterium campestre, but a like conclusion would be erroneous, and it seems not unreasonable to suppose in this case that after a longer period had elapsed, say 9 days or 12 days, Dr. Hunger might have obtained different results and come to an entirely different conclusion, since in the cabbage after that time the bacteria would have entered the vascular system and begun to move downward in the veins of the leaf (see this monograph, vol. II, p. 308). A plunge-experiment similar to the preceding but varied by the fact that the leaves were bruised and wounded more or less before being put into the infected water gave positive results. After 5 days a distinct infection was observed, and a microscopic examination showed the presence of the bacteria even in the stem of the plant, these having passed down through the leaves from the wounded places. In one specimen there was already the beginning of adventive root-formation on the stem. Hunger also smeared portions of leaves and stems with pure cultures of Bad. solanacearum, and placed these plants under large bell-glasses with water, so that the atmosphere was always moist. No infections were obtained; the time, however, was rather short (8 days). The conclusions drawn are that sound tomatoes are not attacked by Bad. solanacearum, and that infections do not take place through natural openings, such as water-pores. According to Hunger, the greatest care should be exercised in planting that the roots be not broken or crushed, and to this end the plants should be removed from the bed and put into the field early, that is, before they have made long roots, which must necessarily be broken in transplanting. He believes that crushing the roots in planting is a common cause of infection. He cites a case in point at Batavia. The disease occurred seriously in the field, and on examination and inquiry he found that the field had been planted not in the ordinary careful manner by an experienced gardener, but that the planting was delayed beyond the proper time and then performed hurriedly. The plants were too large when transplanted ; therefore many roots must have been broken, and they were set out by the gardener with help of two house servants, the coachman, and water-carrier. Hunger often found the root-system attacked by a species of Phytophthora, which he says plainly made a passage-way for the bacteria. He is inclined to think that an imper- fectly drained soil is very favorable to the development of this disease. Of this I, too, think there can be no doubt. He made some experiments to determine the effect of animal and plant parasites on the outbreak of the disease. In his first experiment, which consisted of pot-cultures in disinfected earth, the surface of the earth was infected with Phytophthora nicolianae from tobacco. The plant was then watered with water which had been infected with a pure culture of Bad. solanacearum. After 7 days the Phytoph- thora had overrun the entire surface of the earth and had begun to attack the lower end of the stem. After 26 days the plant developed the bacterial disease, and the infection plainly originated from the lower end of the stem. In a second set of experiments, made in the same way in disinfected earth, a mole-cricket was buried in the soil of each pot, and the plants were regularly watered with infected water. Ten of the 1 1 plants were soon destroyed outright by the insects, the young stem being bitten off underground. < >ne plant remained normal up to the sixteenth day, when it showed the first signs of the bacterial dis- ease; the following day it died. Upon investigation the mole-cricket was found dead in the soil, but the tap-root had been bitten by it, and it is probable that the infection took place through this wound. In a third series of experiments, in sterilized earth, the plants were infected with eel-worms (Heterodera radicicola), by mixing with the earth small pieces of roots which had been attacked by BROWN ROT OF SOLANACEAE. 211 them but which were free from Bad. solanacearum. The plants were watered with ordinary water; they grew well, blossomed, and bore for the most part small fruits. The disease did not develop. In another experiment, identical with the preceding, except that the plant was watered with water containing a pure culture of Bact. solanacearum, the disease developed after 27 days and the adventive root-formation was already under way. The plant died just one month after the beginning of the experiment, and both a macroscopic and a microscopic examination demonstrated the typical phenomena of the bacterial disease. As already stated, Dr. Hunger believes the disease is induced mostly through the inter- vention of Heterodera. He thinks the bacteria are carried up the vessels by the water- movement, but he does not bring sufficient proof in support of this latter statement. He states that infection is not limited to the vessels, but may occur throughout the entire vascular bundle, and also in the phloem and the parenchyma. The sieve-tubes may be plugged by the bacteria. He thinks the adventive roots are due to the action of the bacteria on the phloem. By placing wet moss around a stem showing these adventive roots, he induced them to develop further and on removing this part of the plant and setting it out in the earth he obtained a new plant, which grew well for some time, but afterwards developed the disease. He obtained his pure cultures used for infection from Petri-dish poured plates on agar. This agar had the following composition: water 1 liter; agar-agar 20 grams; glucose 10 grams; peptone 10 grams; magnesium sulphate 0.5 gram; potassium phosphate, 0.5 gram. Dr. Hunger states in a number of places that the color of the surface colonies on this medium was "leverkleurige," that is, liver-colored. They were small and round, and spread slowly in a concentric manner. In the writer's experiments colonies of Bact. solanacearum were not brown (liver color) until after a week or two. He states that the least amount of acid in the agar exerts an injurious influence on the bacteria, but that the addition of alkali favors their growth, for example, the addition of sodium phosphate. He found the organism sensitive to sunlight, development being abso- lutely hindered by it, and a long exposure destroying it. He states that Bact. solanacearum is early contaminated in the plant by the presence of other organisms. These impurities consist of saprophytic bacteria, and especially a yeast. In size and shape this yeast, which he seems to have found quite common, corresponds exactly to van Breda de Haan's statement respecting his coccus from tobacco plants, viz, round and 8/x in diameter. We may assume either that this was what van Breda de Haan saw in his diseased tobacco, or that his measurement is erroneous. If for the 0.008 mm. of the text we should read 0.0008 mm., then it is easy to understand how the short rod might have been taken for a coccus, especially with objectives of only medium magnification. On plate-cultures Hunger found he could distinguish colonies of this yeast readily from Bact. solanacearum by the fact that the latter appeared bluish-green by transmitted light. There is some discussion of methods of prevention, but no certain ones are pointed out other than avoidance of planting on diseased land. It is suggested that possibly some resistant species of this family may be found upon which the tomato could be grafted. THE MALAY STATES DISEASE. In 1 910, Keith Bancroft, Assistant Mycologist, reported on a bacterial disease of potato and tomato occurring in the federated Malay States as follows : Specimens of potato plants have been recently received from Taiping (Perak) exhibiting the well- known " bacteriosis " caused by Bacillus solanacearum E. Smith. The same disease has been known for two or three years to occur in Kuala Lumpur on tomato plants cultivated in vegetable gardens. These two crops are but little cultivated in the Malay States, so that the disease has little more than a passing interest. It is, however, interesting to record its occurrence in this country, and a brief note will be given of the symptoms of the disease and of some methods of treatment which are likely to prove effective. 212 BACTERIA IN RELATION TO PLANT DISEASES. The disease is well-known in the United States of America where it has been carefully studied by Dr. E. Smith on potato, tomato, and egg-plant {Solarium melongena). It has also been recorded on potato in Scotland, in the north of England, and in Mysore, and recently in Ceylon. The symptoms of the disease appear to be constant in the different countries in which it is known to occur, and they may be briefly reviewed thus: Attacked plants show a wilting and shriveling of the leaves; soon afterwards brown streaks appear on the stem and spread downwards to the parts underground. Finally, the whole stem rots. A browning of the affected parts is very characteristic of the presence of the bacterium ; the browning will be found to be first evident in the vascular ring on examining the cut surface of an affected stem. The vascular bundles are first affected, the supply of water to the leaves is checked and the conse- quent wilting and shriveling makes its appearance. The disease in the potato tubers is very well-marked by the presence of a circular, usually incom- plete, brown ring which is situated at some little distance from the surface and which corresponds in position with the vascular ring of the tuber. This ring darkens and expands until the whole of the starch-producing area of the tuber is affected. The tuber decays and the bacteria are liberated in the soil where they can infect other healthy tubers. Dr. E. Smith considers that the rapid spread of an epidemic is due to insects of different kinds feeding alternately on healthy and infected plants. Under these circumstances it is advisable to spray the plants with an insecticide. A vegetal wash, e. g., tobacco wash, should yield good results. This may be made by infusing half a pound of tobacco leaf in water for about six hours, straining off and pressing the tobacco and again infusing; the extract is added to a solution of half a pound of soft soap in water, the whole being made up to ten gallons. The wash is applied by means of a spray. Any coarse tobacco leaf may be used. Infected plants should be taken up along with their roots and burnt. They must on no account be thrown on to a manure heap. In the case of the potato plant it is advantageous to lift the crop early when the disease has made its appearance; in this way many of the tubers may be saved. Plants belonging to the potato family should not be planted on soil which has yielded infected plants for at least two years, since the bacterium probably attacks a large number of members of that family (Solanaceae). THE MYSORE RING DISEASE. In 1909, Leslie C. Coleman published a short paper on a "Ring disease" of potatoes {Solatium tuberosum), of which the following is an abstract: The Bangadi or ring disease of potatoes has been reported from various localities in India. It was first noticed in Bombay Presidency in the vicinity of Poona, and later in other parts of the Presi- dency. In Mysore it is found practically in every locality where potatoes are grown. The extent of its ravages is very much greater than is generally supposed. The disease is readily to be recognized by the sudden wilting of the potato plants in an affected field. Usually one or two leaves first show the wilting, but within a few days the whole plant wilts down and dries up. If the tubers of an affected plant are cut through, some of them will almost cer- tainly show a brown ring a short distance in from the surface. This ring will be found, usually at least, to begin at the point where the tuber is attached to the underground stalk which bears it and to spread from that point around to the other end of the potato. In its first stages, therefore, the ring is not complete, but is rather simply indicated by a more or less distinct brownish streak in the neighbor- hood of the point of attachment of the tuber. If a diseased potato be cut and squeezed slightly, a series of small creamy-white slimy drops are to be seen exuding along the course of the brown ring, and if these drops are examined they are found to contain myriads of bacteria shaped like small and very short rods. If a very thin slice of such a potato is examined microscopically these tiny rods are found chiefly in the vessels of the tuber which they plug quite full. If the stem of a diseased plant is exam- ined it is found also to be plugged with masses of the bacteria. This plugging up of the vessels from the roots up to the leaves causes the wilting of the plant. The questions investigated in regard to this disease are: " (1) Are these minute bacteria the real cause of the disease? (2) If so, how do they get into the potato plants, and how do they increase to such numbers as to block up the vessels?" The reasons given for answering the first question affirmatively are as follows: " (1) They are to be found in large numbers in the vessels of every potato plant suffering from the disease. (2) In the earlier stages of the disease they are the only organisms to be found in the vessels. Later, when the whole plant or tuber begins to rot, other bacteria, as well as certain kinds of higher fungi, are to be found associated with them. (3) It has been proved by direct experiments that these bacteria are BROWN ROT OF SOLANACHAE. 213 capable of causing the disease. These experiments were carried out as follows : The bacteria were removed from diseased potatoes and grown in the laboratory in such a way that they could be sepa- rated from all other forms of bacteria. When by careful testing it was found that we had masses of these bacteria growing cjuite free from any other form, such masses were used to give the disease to healthy potato plants. Small pricks were made in the leaves and leaf -stalks of healthy plants with a fine needle which had been held in a flame to kill off all bacteria that might be on it. Then masses of these bacteria were stroked over these pricks so that the bacteria could enter into the substance of the plant. After a few days it was noticed that the leaves and plants infected began to wilt and in the course of 2 to 4 weeks the plants were quite dead. Moreover, when the stems of these plants were cut across and examined under the microscope these same bacteria were found inside the vessels. Some of the tubers attached to these plants also showed the characteristic brown ring. We have thus shown clearly that these bacteria are the real cause of the ring disease of potatoes." Dr. Coleman states that there are at least two common ways of infection as determined by his experiments: (1) Through the planting of seed-tubers which already have the dis- ease, and (2) the presence of these bacteria in the soil of the plot where the potatoes are planted. An experiment was also conducted which showed that potatoes harvested from a plot where the disease was present, even though they did not show the brown ring, would produce the disease if planted in soil unquestionably free from the bacteria — the young plants wilting throughout the plot soon after coming up. A similar, if not identical, disease is found attacking the potato in America. Dr. Erwin F. Smith of the Agricultural Department, Washington, has studied it thoroughly and has come to the conclu- sion that insects have a great deal to do with the spread of the disease. In all my experiments I have kept a very careful watch for any instance of the spread of the dis- ease by insects. * * * On the whole, however, insect attack has been insignificant, and although I have examined very many wilted plants indeed, I have never yet seen one in which the infection could, with any likelihood, be traced to the gnawing of insects. Dr. Coleman states that because in case of the above-mentioned potato-disease in Amer- ica Dr. Erwin F. Smith has found that it also attacks egg-plants (brinjals) and tomatoes, he instituted experiments to ascertain whether tomatoes and brinjals can be given the disease. The results already obtained show that both these plants can be infected with it by means of inoculation with pure growths of bacteria, or with bacteria taken direct from diseased potato-tubers. In one successful experiment with tomato-plants, 8 comparatively young plants were inoculated with pure growths of the bacteria; all the 8 contracted the dis- ease and within 3 weeks were practically dead. Three larger plants (bearing fruit) inocu- lated at the same time with the same growths of bacteria remained practically unaffected and could not after 3 weeks be distinguished from an uninoculated plant of the same age (see last paragraph on p. 179). Similar results have been obtained with brinjal plants — the younger plants contracting the disease, whereas larger plants resisted it. From the results obtained the following directions should be strictly adhered to, if possible : (1) Only such potatoes should be used for seed as have been grown on a plot which has been quite free from the disease; (2) where the seed is cut before planting, all pieces that have the least trace of brown spots or a brown ring should be discarded; (3) if the disease has been prevalent on a certain piece of ground, potatoes should not be again planted there for at least one year. Probably two years would be better, but a definite statement in this con- nection can not be made until the experiments in progress have been completed. Also, brinjals and tomatoes should not be planted on such infected ground. Spraying the plants with a fungicidal or insecticidal spray is a waste of both time and money, as the disease is not transmitted, at least to any appreciable extent, by insects; nor can it be carried through the air from plant to plant. Another and a very effective means of checking the disease would be by the introduction of a disease-resistant variety of potato. Experiments in this direction are in progress. The description of the organism is withheld by Dr. Coleman for a separate paper. 214 BACTERIA IN RELATION TO PLANT DISEASES. THE CEYLON DISEASE. In 1909, Petch reported very briefly on a wilt disease of tomato plants occurring in Cey- lon, during a period of wet weather, and first visible when the plants were in bearing. The wilted leaves decayed and fell off, and finally the stems decayed also. There is very little evidence of disease in the stem when the leaves begin to droop,- but if it is cut across near the base the woody part immediately round the pith is found to be brownish, and minute white or yellowish drops of liquid ooze out from this region. These drops consist of enormous numbers of bacteria, and if sec- tions of the stem are examined under a microscope, they are found to issue from the vessels of the wood which are completely filled by them. * * * The bacterium is, as far as can be ascertained without elaborate bacteriological investigation, Bacillus solanacearum. THE AFRICAN DISEASE. The writer's knowledge of the African potato-disease here referred to was obtained from a conversation with Mr. George Milton Odium, a student of scientific agriculture, who for some time had charge of plantations for English capitalists at Umtali, in Rhodesia, where he saw the disease. He described it to me as a bacterial rot, beginning as a black stain in the vascular system; the foliage wilts, the stems shrivel and blacken, or have black stripes in them, and the tubers decay. He has known whole fields to be destroyed quite suddenly. The microscope always shows great numbers of bacteria in the diseased plants. In his opinion the disease is the same as that described by the writer. THE RUSSIAN DISEASE. In 1899, Iwanoff reported the occurrence in 1898, in all of the fields for a distance of 26 miles around St. Petersburg, of a very destructive bacterial disease of potatoes closely resem- bling that due to Smith's Bacillus solanacearum, ii not the same. The organism present in the stems in the earliest stage of the disease is described as a "small, short, oval-cylindric, actively motile rod of medium size (in general rather variable) 1.5 Xo.5^. " Two organisms isolated from the diseased stems in pure culture were not infectious, but direct infections were successful. The tissues were browned, the bacteria passed up and down the stem from the point of inoculation by way of the vessels, the pith was badly disorganized, the starch was not attacked, young plants were killed much sooner than old woody ones, the juice of diseased plants was alkaline, and in the pith and the bark parenchyma there was an increased produc- tion of calcium oxalate. The tubers were small and few, but were not observed to be dis- eased. The disease spread with great rapidity. A field which contained only scattering cases on August 8, showed all plants diseased on August 15, the only green things in the field, and in many similar fields, being weeds. Possibly this should be referred to B. phytophthorus. THE FRENCH DISEASE. In 1901, in two publications, Delacroix stated that Smith's disease of the potato was prevalent in France. I translate as follows: The bacteria are found far up in the stem and in parts apparently still living. They are espe- cially numerous in the vessels. This bacterium seems to me not different from the Bacillus sola- irum of Krwin F. Smith. Its cultural characters are the same; the signs of the disease observed in the United States on the potato, tomato, and egg-plant are exactly those I have myself seen. Afterward Delacroix attributed the disease to a green fluorescent bacillus, B. solanincola I klacT.. which is different from Bad. solanacearum, but non-pathogenic in my hands. Here again, it is uncertain whether we have to do vfithBacillus phytophthorus, Bacto ium 5 ,' macearum, or some third organism. The writer obtained a culture of B. solanincola from Prof. L- R. Jones, to whom it was given in Paris by Delacroix, but either it never possessed BROWN ROT OF SOLANACEAE. 215 any pathogenic properties, which is quite probable, or else had lost them by cultivation. Nothing Delacroix afterward published on this disease served to clear up the question.* In 1902 Gueraud de Laharpe published a note on a French potato-disease said to be due to Bacillus solanincola. He makes the following observations on the subject: For several years the culture of the potato in France has decreased more and more, so that now in some departments it is insignificant or has been entirely abandoned, owing to the extremely difficult conditions. The year 1901 was particularly disastrous. The Central and Western departments suffered most. Within a few days toward the close of July whole fields were destroyed, leaving of the crop only faded leaves and blasted stalks, while the tubers, when formed at all, were no larger than walnuts. The disease appears in general about July 15 upon Jaune de Holland, an early variety, and a little later on late varieties, such as Richter's Imperator. The early varieties suffer most. It is encountered especially in soils which have borne potatoes repeatedly, but it is also found on newly cleared forest or vineyard soil, even on soils which have not borne potatoes within the memory of man. Though it seems to prefer calcareous soil, it causes much injury also in clayey and sandy regions. 'HI Xoomm Tyloses are said to be formed in the stems. Delacroix advises soaking sound seed-tubers for an hour and a half in 1 part of commercial formalin diluted with 20 parts of water. Delacroix's most susceptible varieties are 40-day Halle and Long Yellow Holland. THE ITALIAN DISEASE. In south Italy, according to Comes, a disease of tomatoes due to his Bacterium gummis is common. The stems rot usually from the base and the tissues are filled with bacteria. Bad. gummis is now regarded as insufficiently described and it is not known to what the name was applied, as the organism is said to occur in a variety of plants. This is probably a disease of late spring, summer, and autumn. The writer searched in vain for it in the market-gardens about Naples in the early spring of 1906. A great many fields of potatoes were seen, but there was no trace of this disease. Voglino has also reported a disease from Turin, which he ascribes to Bad. solanacearum. THE GERMAN RING DISEASE. Appel and Kreitz describe a ring-disease, which possibly is caused by Bacterium solana- cearum. the most striking characteristic being a browning or blackening of the vascular bundles. In severe attacks the potatoes do not come up. On the seed-tuber small tubers appear and a great development of shoots, but these are too weak to break the ground and gradually die. When less severe the shoots come up, but are stunted. On subterranean parts are brown cracks which soon become glossy, black spots appear on the leaves, and the plant dies. In the lightest form the affected plants can not be distinguished from sound ones until harvest. Then the leaves wilt and usually show black spots. Such plants *Fortunately, Delacroix left an abundance of material both dry and in alcohol to illustrate his potato disease. This, through the courtesy of Etienne Foex, the present director of the Station of Vegetable Pathology, in Paris, the writer had opportunity to examine critically in the autumn of 1913, i. e., since the above text was written. Numerous microscopic examinations were made, but no bacteria were seen either plugging vessels or corroding tissues and cer- tainly nowhere any in numbers sufficient to account for the disease. All of the plants were examined, and each in several places. In the stems and roots of those in alcohol (a large jar of material), nothing was found; but on the shriveled leaves there was a great abundance of Phytophthora infestans. On the dried material evidently collected at another time and place, there was no Phytophthora, but Alternaria solani was found on the brown shriveled leaves, or at least an Alternaria resembling it; and in the xylem vessels in the basal part of some of the stems, a fungus suggesting Fusarium, i. e, a delicate colorless mycelium abstricting colorless elliptical conidia in small numbers. Whatever Delacroix may have first identified as " Brunissure de la pomme de terre," due to his Bacillus solanincola , the museum material which he selected to illustrate it represents only fungous diseases of the potato. tFiG. 114a. — Margin of a hanging drop showing Bacillus solanincola Delacroix. From an agar culture given to Jones by Delacroix in August 1904. 2l6 BACTERIA IN RELATION TO PLANT DISEASES. bear almost a normal number of tubers, but are a menace to the crop, as the disease spreads from the small spots on the harvested tubers. There can be no injury unless the bacteria come into contact with the vascular bundles. Infection often occurs by abnormal growth of the young stem, resulting in cracks reaching even to the pith. Also through wounds made by animals or otherwise the bacteria easily gain access, c. g., the disease is more prevalent when cut seed-tubers are used. If such are employed they should be covered with moist sacks for two days that the formation of a cork-layer may occur. We made agar-poured plates in the laboratory in Washington from the browned vascular bundles of a potato sent from Miinchen, Germany, by Mr. W. A. Orton in 191 1, as affected by the bacterial ring-disease, but obtained only a white endospore-bearing motile schizomycete, non-infectious to growing potato stems. In Eriksson's book (Die Pilzkrankheiten der Landwirtschaftlichen Kulturpflanzen. Aus der Swedischen uebersetzt von Dr. A. Y. Grevillius, Kempen a. Rh., Reichenbachsche Verlagsbuchhandlung, Leipzig, 1913, pp. 8-9) the ring disease is attributed both in the text and in the figure-description to Bad. solanacearum. For further knowledge of this disease we must await Appel's full report. THE ENGLISH DISEASE. There are a number of statements by English agricultural writers to the effect that Bacterium solanacearum Smith causes a disease of potatoes in Great Britain. These statements, however, are too indefinite to furnish any reliable basis for judgment. MALKOFF'S DISEASE OF SESAMUM. In 1903 a bacterial disease of sesamum was described by Malkoff, from Sadovo in Bulgaria. Early in August 1902, in a plot in which the plants had been watered and had made more rapid growth than in a check plot, many leaves showed brown spots and dried up quickly. Sections revealed the presence of many bacteria. After 2 to 3 days the disease was widespread and the stems were also affected. These were dark brown to black, shrunken and covered with a slimy exudate which dried on the stem. This ooze was at first gray- white, but soon became dark brown. In a few days the stems became entirely black, bent over, and finally dried out. Sometimes the whole plant is attacked, sometimes a single shoot (figs. 114 b, c). The disease progresses rapidly and in 3 to 4 days all the leaves are attacked and the stem decayed. It appeared on the early Bulgarian sesamum first, but later occurred on the two Asiatic varieties. The disease was confined to plants which had been in blossom for some time. In the unwatered plot no plants were affected until after a rainfall in September. Inoculations of sound leaves and petioles with the juice of the affected plants, both with and without wounds, produced the characteristic signs in 4 to 7 days. In inoculations on plants growing in an unwatered plot, the disease also appeared (in 7 to 24 days) except in case of inoculations on the stem and under side of leaves without wounds. Two kinds of bacteria were isolated — a short rod and a long one. The first formed yellow col- onies, the second white ones, in 24 to 48 hours at ordinary temperature. Both were motile and non-sporiferous. Growth in bouillon, gelatin, and agar was very luxuriant and the gelatin was soon liquefied. Ten per cent sesamum leaves with peptone and gelatin was an excellent medium. Milk was not curdled by either form, but was peptonized after 8 days by the gray form. Inoculations were made with pure cultures and here and there the disease appeared, but it did not progress rapidly as the tissues were then hard. Those on leaves produced the disease in 7 to 8 days. This disease occurred throughout the sesamum-growing district in 1902, but was less widespread that year because of the dry season. In previous years, according to the growers, the entire harvest had often been lost through its ravages. It occurs principally in southern Bulgaria on the Turkish border. The author believes that the disease is dis- tributed on the seed. BROWN ROT OF SOLANACEAE. 21 Fig. 1 14 t. Fig. 114/.** *Fig. 1146. — Bacterial disease of sesamum: 1, normal; 2, partly, and 3, wholly diseased. (After Malkoff.) tFic. 114c. — 1, normal sesamum; 4, bacterially diseased sesamum. (After Malkoff.) JFig. ii4 Cross-section of petiole of a tobacco leaf, showing bacterial invasion of vascular system. From a natural infection in North Carolina. Material for sections received in alcohol from Prof. F. L. Stevens. Slide 291 a 6. fFlG. 117. — Cross-section of a tobacco stem diseased by Bacterium solanacearutn. A detail from slide 291 o 15, section X, »'. c, from the same source as tig. 1 16. A somewhat diagrammatic wash drawing. PLANT BACTERIA, VOL. 3. PLATE 33. North Carolina tobacco wilt. Tobacco plant inoculated on the stem by needle-pricks from a pure culture of Bacterium solanacearutn on July 13. 1905, and photographed August 1. About one-half natural size. PLANT BACTERIA, VOL. 3. PLATE 34. Tobacco-leaf from the hothouse, showing the typical red-brown shriveled spots of the Granville tobacco wilt. The remainder of the leaf was green. Bacterium solanacearum abundant in vascular system of the midrib and in many side veins. Plant inoculated in stem, by needle- pricks, on Sept. 23, 1905 using a pure culture of the North Carolina tobacco organism. Photographed Feb. 20, 1906. About one-third natural size. PLANT BACTERIA, VOL. 3. PLATE 35. Leaf from inoculated tobacco plant No. 34 (Granville tobacco wilt). Notice dead apex and four elliptical brown patches on the right side near the midrib between the larger veins. Cross-sections from the base of this petiole arc shown in fig. 120. The terminal portion of the midrib is shriveled. Bacteria were abundant in ils vascular system at X.and also in the side veins between the dried-out patches at X. X- Surface of leaf dry. Inoculated Sept. 23, 1905, on the stem. Photographed Dec. 11. One third natural size. PLANT BACTERIA. VOL. 3. PLATE 36. . a. c to o ti-o to >S j-j s .sS t/1 C5 -ail g o r ^ pj ti "g 3 o - OJ u ^ u. S -r w a» -*-! C3 v £ *S o -e C W c; X w .o 5 s Bti n a a o fc"0 U-w PLANT BACTERIA, VOL. 3. PLATE 37. WILT-DISEASES OP TOBACCO. 229 only recently diseased. Nothing resembling the Bacillus nicotianae Uyeda or the large micrococcus of van Breda de Haan was observed. Only a few of the many plants received from North Carolina were used for making poured plates. The foliage was removed from the remainder and the plants (most of which were in early stages of the disease) were then set out in a hothouse belonging to the U. S. Department of Agriculture, and carefully watched. For some weeks they showed no further indications of disease. They rooted readily and made new leaves. Afterwards all of these plants developed further signs of wilting (Vol. I, pi. 27, left fig.) and finally perished of this disease, the pith being rotted out as in the Sumatran disease. The vascular ring in these plants was stained dark brown or black, and the parenchyma was also blackened in places, both in the interior and on the surface of the green stems. In some instances cracks or cavities ap- peared on the surface, opening into the depths of the stem, and the tissues in and around these fissures were blackened. The bacteria were present in the diseased tissues in enormous numbers. When the plants had died they were removed and healthy tobacco plants grown from seed sown in the Department houses were set in their place. Theseplants were some months old and naturally the root-system was wounded considerably in transplanting them. To the writer's surprise all of these plants (a dozen or more) contracted the disease within a few weeks and finally died of it. The checks left in the other house remained free from the disease. Starting with single colonies of the North Carolina organism obtained from the Petri-dish poured plates, subcultures were made, and from these subcultures healthy tobacco plants in another house were inocu- lated in considerable numbers by needle-punctures in the stem or leaf. These plants readily contracted the disease, going through the same progressive stages as the plants obtained from the field. All the character- istic signs were present in these inoculated plants, one of which was figured in Volume I (pi. 27, right fig.). For the appearance of the remainder see plates $$ and 36. From the interior of several of these plants the organism was again obtained in pure culture by means of Petri-dish poured plates, and with colonies from these poured plates, or rather with subcultures therefrom, the dis- ease was obtained in tobacco plants a second time by needle-puncture inoculations. These were stem-inoculations on plants 13 to 36, most of which contracted the disease, but only slowly, i. c, not until they had become large plants. They were inoculated September 23, 1905, and up to the middle of January the signs were confined to yellowing and loss of the lower leaves, with the exception of one plant, which collapsed entirely (pi. 38). The affected leaves developed pale green spots and then dried out very irregularly. Often the "ears" died first, as described by van Breda de Haan; sometimes the apex died first (fig. 119), or one margin (pi. 34), or the leaf dried out irregularly (pi. 35). In all such cases the *Fig. 118. — Fusarium fruiting on the surface of a diseased tobacco stem in pinkish, pustular lines. Possibly McKenney's tobacco Fusarium. A secondary infection and the only case that occurred in the house. Plant inocu- lated on Sept. 23, 1905, with the North Carolina tobacco bacterium. Photographed Feb. 20, 1906. Stem dead. X2. Fig. 118/ 230 BACTERIA IN RELATION TO PLANT DISEASES. vascular bundles at the base of the green midrib were browned and occupied by the bacteria. Cavities were formed in the midribs of such leaves. This browning and bacterial occupa- tion was not confined, however, to the petiole or base of the midrib, but could be traced through the latter for a distance of i to 2 feet. In some cases also the browning and bacterial occupation were traced out into branches of the midrib. The appearance of some of the blackened midribs in cross-section and of similar petioles in longitudinal section is shown in figs. 1 20, 121. Poured plates made from the brown slush in the hollow stem (pi. 38) showed the pres- ence of an intruding soft white-rot organism, as was anticipated (fig. 122), and the inoculated bacterium was not made out clearly. The blackening and partial de- struction of the pith in two of these inoculated plants is shown on plate 39. Similar results obtained by inoc- ulating Bact. solana.cearumpla.ted from a potato are shown on plate 40. There was in some cases an abnor- mal pushing of roots from inoculated stems (fig. 123), the same as in tomato plants inoculated with Bad. solana- cearum (pis. 27, 28). Studies carried on at the same time, and afterward, indicated that the cultural characteristics of this or- ganism, so far as tested, did not differ materially from those obtained with Bacterium solanacearum plated from diseased potato and tomato stems obtained from various parts of the United States. On the other hand, cross-inoculations from tobacco to tomato and from potato to tobacco were not particularly successful. The tomatoes inoculated with the tobacco organism developed adventive roots on the stem, but no wilt appeared. The tobaccos inoculated with the potato organism did not wilt or show other external signs of disease, but the strain used was not a very virulent one. The following are the morphological and cultural characters of the North Carolina tobacco organism, so far as determined in my laboratory: Fig. 119. "Fig. in). Dark veins and large Brown stripes on tobacco stems attacked by the North Carolina tobacco wilt. These plants were inoculated from tomato stems (direct transfer of the bacterial slime) on Aug 5. 1908, by needle-pricks on leaves and stems higher up, the injuries here shown being accomplished from the vascular bundles downward and out- ward, the epidermis remaining unbroken. The tomatoes from which the bacterial slime was taken were wilting plants which contracted the disease through broken roots from soil in which diseased tobacco plants had been buried. Photo- graphed Aug. 27. Compare with plate 41, fig. 2. WILT-DISEASES OF TOBACCO. 235 August 2Q. — Yesterday I saw slight wilt on another Datura stramonium standing in the bed where the North Carolina tobacco was buried. It is a large plant and has been healthy hitherto. This morning I brought it in and had a photograph made (pi. 43). It is badly wilted. Stem sound externally; vessels filled with Bacterium solanacearum. Fusarium not present. Infections took place through broken roots. The bacteria are most abundant in the lower part of the stem and ooze out copiously on cross-section (fig. 125). August 31. — To-day, removed an additional wilted tomato plant from bed in the old rose house. The surface is sound except some small roots. The vessels in the lower half of the plant are filled with gray slime which under the microscope has the characteristic morphology of Bacterium solanacearum. Short rods ; many in pairs (dividing) . The plant is 3 feet long. The vessels are browned only in the lower part. Not many bacteria present in the upper part and no stain there. As one goes toward the root-system bacterial occupation increases. Infection took place unquestionably through the root system. This is the twentieth tomato plant to wilt. October 28. — Old rose house. Removed the remainder of the Daturas this morning. Some were in fruit. They have lost many of their leaves and others are brown spotted, but the cause of the dis- ease is uncertain. None of them except the 4 already mentioned appear to be attacked by the tobacco disease. Also pulled out the remainder of the tomatoes, none of which were wilted. Out of this bed I have removed 21 wilted tomato plants attacked by Bacterium solanacearum, and now these 9 which are not wilted. These 9 plants have sprawled over the ground, covering it with foliage, and have made a growth of 8 or 10 feet altogether, but have borne very little fruit. Only one or two green tomatoes were present, and the gardener says he has not seen fruit on them. It is not certain from this that the plants are resistant to the disease. It may be that they got such a good root start that the disease could not attack them successfully, or it may be they are resistant individuals. With a view of testing this I made cuttings from soft wood at the tops of 8 of these plants and removed all the leaves. I then planted them in the same bed after spading it up. There are 27 of these cuttings. If the plants are not resistant some of the cuttings ought to contract the disease, provided the organ- ism is still living in the soil. I also pulled out all the remaining Daturas and set in place egg-plants, two varieties of peppers, and some Connecticut tobacco. There are three rows of the egg-plants, 1 2 plants, var. Black Beauty. They were growing vigorously with leaves as large as my hand or larger, and perhaps a dozen such leaves. I pulled off all the leaves and root-pruned each one severely before setting it out. The peppers are Cayenne and Ruby King, 1 2 plants of each sort. Six of each sort were set out very carefully without disturbing the roots in the least, and 6 of each sort were thoroughly root-pruned and had all the leaves pulled off. Of the tobaccos, I set 6 in one part of the bed and 6 in another part of the bed. Three of each lot were transplanted without injury to the roots; the other 3 had all the leaves pulled off and most of the roots. In addition, I made 3 cuttings from a vigorous growing Datura, and set these out in the bed. Some tomatoes which were set out in a portion of this soil by Mr. Bisset, the gardener, about 2 months ago have made a very uneven growth. As a control I had a similar bed on the other side of the partition planted at the same time with a number of tomato plants, but in soil which had not received the diseased tobacco. These check-plants are very leafy, 3 feet or more in height and healthy-looking. Of the 24 in the diseased soil only 10 plants are good-looking ones; the others are dwarfed and have the terminal leaflets dead or dying. Nine of the 1 4 dwarfed ones are very badly dwarfed. These are only about a foot high, and have only about one-fourth as much foliage as the check-plants. I have seen no wilt on them, and I am not yet certain as to the cause of the dying of the terminal leaves and general dwarfing of the plants. November 16. — The rooted tomatoes, now about 3 months old,showno distinct indications of wilt, but they are dwarfed and sickly. They have made not nearly so good a growth as the check plants in similar soil but which had not received diseased tobacco. I am at aloss to know whether this disease is due to the slow action of the tobacco organism on the roots of the tomato or whether it is a distinct disease. The checks are free, although the two beds are separated only by a glass partition and 16 inches of space. The three cuttings from young Datura stramonium, have died, i. e., they did not root. The 12 transplanted tobacco plants show no indications of disease. The 12 pepper plants with un- pruned roots are continuing to grow normally. Of those with heavily pruned tops and roots, 10 appear to be recovering, one is dead, and another is dying. I have just pulled up the latter and find the base of the stem rotten. These plants are not recovering easily from the heavy top and root pruning. The 1 2 egg-plants, which were transplanted after being very heavily root and top pruned, are beginning to recover and are making a little growth. Of the 29 tomato shoots which we attempted to root in this bed, only 5 appear to be living ; the others have gradually rotted away. More mature cuttings should have been used. The stems hitherto examined have not shown any definite indications of bacterial infection in the vascular bundles. 236 BACTERIA IN RELATION TO PLANT DISEASES. EXPERIMENTS OF 1909 (1908 CONTINUED). March 22, igog. — None of the cuttings rooted. None of the peppers or egg-plants have con- tracted the wilt. Probably the stems were too woody when transplanted. The heavily pruned ones recovered very slowly. The older rooted tomatoes gradually got worse and worse, but with no definite wilt, or brown stain in the stems, or bacterial occupation of the vessels. The checks finally contracted the same disease. One of the tobaccos (a large plant now) has contracted the bacterial wilt. Its vessels are full of the gray slime, especially on one side. This was most abundant in the lower part of the stem, but was traced out into the leaves and into the top of the plant just under the seed pods, where a few vessels were browned and occupied by the bacteria, as shown by a microscopic examination. In the middle of the stem the bacterial slime was very abundant, but agar poured- plates sowed copiously showed at the end of the seventh day only intruders (a few colonies with finger- like radiations). The right organism was not recovered with certainty even on a second set of plates made the fifth day from the second dilution tube, which had been kept and was then clouded. The only possible inference is that the parasitic bacteria had been present in this part of the stem for some time and that they were now dead — supplanted by saprophytes. The outside of the stem, on one side over the most badly affected part of the vascular cylinder, bore long dark stripes and sunken places. This tobacco was one of those not root-pruned. From the interior of a basal branch of the same plant which was then wilting, another set of plates was poured on March 30 and these plates yielded the right organism. On May 6, 1909, a second tobacco plant wilted with characteristic signs, and poured plates made from the browned interior yielded the right organism. July 6, igog. — The experiments with tobacco, egg-plants, and peppers were closed out to-day with the following results: (1) Tobacco: 8 plants healthy, 4 diseased. From 2 of the diseased, as already stated, Bad. solanacearum was plated out; the other 2 had similar signs but no cultures were made. (2) Egg-plant: There was no definite wilt on these egg-plants during the whole of the time they stood in the bed; 9 of them are sound, 2 being in fruit and the rest in blossom; 1 is wholly rotted, and 2 are gone, ;'. e., perished earlier. (3) Pepper: There has been no indication of wilt in the peppers during the whole time they have stood in the bed. Like the egg-plants, the root-pruned ones never fully recovered. There are now 9 free-growing peppers bear- ing green and ripe fruits in abundance and 9 stunted peppers (the root-pruned ones) ; six plants perished ear- lier, but with no definite signs of this disease. There is a trace of brown stain in one or two places in the wood at the base of the stem of 3 of the root-pruned ones, and a microscopic examination shows the presence of tyloses in the browned vessels, but no distinct evidence of bacteria. The bed was now spaded up and prepared for large tomato plants. On April 24, 8 good-sized tomato plants were inoculated with the organism plated from the interior of the diseased tobacco on March 30. These inoculations were made from slant agar-cultures of April 2 1 by means of needle-pricks on the stem, 2 plants being inoculated from each culture, a large amount of material being introduced and 2 internodes of each shoot being pricked. On May 6, six additional plants of the same series were inoculated from a beef-broth culture of April 22 in the same thorough manner. The tomato plants were rapid-growing, rather tall individuals, propagated from cuttings. July 6, igog. — The tomato plants continued to grow rapidly and up to this date none of them have ever shown any trace of wilt, with the exception of one plant, which after a time showed one or two slightly wilted leaves that subsequently recovered their turgor. The tomato was a free-bearing, red, small-fruited, hothouse variety. Final Mote. — The organism appears to have died out of the soil which yielded so many diseased plants (tobacco and tomato) in 1908. It was moved to another hothouse and tomato, tobacco, and the Porto Rican spiny weed (p. 182) grown in it for a year, but with wholly negative results. In 191 2, Coleman, of Mysore, told me he had observed the same thing at Bangalore in connection with his work on the Indian potato-disease. After about 2.5 years an arti- ficially infected soil was no longer infectious. This is a highly important matter deserving of thorough study. Very likely the growth of certain soil saprophytes may serve to bring about destruction of Bad. solanacearum in certain soils, and if they could be obtained in pure culture, they might be sown to advantage on infected lands. Since this was written Honing has obtained some evidence bearing on this point (see fig. 134). 1 \ t haps, also, a proper course of rotation and cultivation would do much to free the soil. PLANT BACTERIA, VOL. 3. PLATE 43. Shoot of Datura stramonium attacked by the Granville tobacco wilt. The infection took place through broken roots as the result of transplanting to a bed in which diseased tobacco plants had been buried. The vessels of this plant, especially in the lower part of the stem, were swarming with the bacteria (see fig. 125J Photographed Aug. 1908. WILT-DISEASES OF TOBACCO. 237 LOSSES; TREATMENT. The Florida tobacco-disease, according to Mr. W. W. Cobey, has been present on a plantation near Quincy, Florida, for several years. The loss in 1905 was estimated at about $4,000, a total of about 4 acres of plants being destroyed. The disease was patchy, but occurred on many parts of the plantation, being much more widely distributed than in 1904. This was probably due to the fact that the refuse tobacco material of 1904 was composted and spread on the land in 1905, the parasite being undoubtedly present in some of the composted tobacco leaves. The losses around Quincy, Florida, in 1908 far exceeded those of 1905. Mr. Shamel, a tobacco expert of the U. S. Department of Agriculture, wrote to the Department as follows respecting the Florida tobacco -disease in June 1908: The Granville wilt has appeared to an alarming extent, especially in the fields of the Owl Com- mercial Company [Quincy, Florida]. I have sent plants to Stevens for identification and to Dr. Briggs in connection with some soil samples. The wilt does not attack the plants noticeably until from 3 to 4 weeks after transplanting. Then one leaf or a portion of a leaf begins to wilt, then follows the destruction of the entire plants. In some of the shaded fields on the Owl plantation from 25 to 70 per cent of the plants have been killed. On an affected field of last season, planted in potatoes this year, the potato plants have all been killed. It is the most discouraging factor I have ever seen in the way of a parasitic disease of tobacco. The writer examined some of the diseased plants from this locality in 1905 and again in 1908, but could find no Fusarium, only bacteria. The losses in North Carolina have been much greater than those reported from Florida. In the Thirtieth Annual Report of the North Carolina Experiment Station for the year ending June 30, 1907, Dr. F. L. Stevens reports that the Granville tobacco wilt — continues to spread and is reported as worse this year, and the loss is said to be 25 to 100 per cent of the crop in the infected region. The loss in one county is estimated this date at $20,000. It is estimated to be 40 per cent more destructive this year than last year. Reports to the writer in 1908 from North Carolina indicated more extensive losses than any previous year. Many planters harvested their tobacco in July, unripe and half- grown, in order to save some portion of it. In June 1908, Mr. Cozart, statistical agent for the U. S. Department of Agriculture, wrote as follows from Creedmoor, Granville County, North Carolina, respecting the pros- pect for a crop of tobacco : In making this report I am at a loss to estimate the tobacco crop. We have a fine growth, good size, and it promises to be a good crop, but we have a serious problem to contend with — the will tobacco. There is hardly a farm in this township free from it. It is very serious and spreading at an alarming rate; 20 per cent of our tobacco already lost by wilt, and if ratio continues, 30 to 40 per cent will be lost by the time it goes to knife. W. E. Graham, of Church Road, Virginia, reports (October 7, 1908) that the Granville tobacco wilt is getting worse on his place all the time, and further, that it is "confined mostly to gray lands, as I have seen very little on red soils." In his report for 1907-8, Stevens, of the North Carolina Experiment Station, reports the Granville tobacco wilt as occurring in Granville, Durham, Vance, and Ashe counties in North Carolina. According to the experience of Mr. Tunstall, land affected with the wilt left without tobacco for 10 years is still affected so that if tobacco is again planted it will become diseased. It is also said that there came under his personal observation one field in which the tobacco was very badly wilted, and in which, it is stated, tobacco had not been planted for at least twenty-five years. No statement is made, however, respecting 238 BACTERIA IN RELATION TO PLANT DISEASES. the planting on such fields during the interim of other crops which might act as carriers of the disease, or on the condition of the seed-bed from which this field was set. (For infection of a cabbage field with Bad. campestre from a seed-bed see this monograph, Vol. II, p. 329.) From these statements it is evident that under favoring conditions this disease may prove very detrimental to tobacco-culture. The reader will gather from what precedes, to recapitulate: (1) that only sound plants should be set out, and to this end the seed-bed should be on soil free from this organism; (2) that seedlings should be transplanted early and injured as little as possible in the removal; (3) that they should not be watered from infected wells, ponds, or streams; (4) that diseased plants shoidd be removed from the fields as promptly as possible, so that they may not become centers of subsequent infection; (5) that the fingers or knives should not be used on sound plants after use on diseased plants without a preliminary sterilization; and, finally, (6) that as far as practicable, badly infested fields should be devoted to other and non-solanaceous crops. Toma- toes, potatoes, peppers, or egg-plants must not follow the tobacco, nor should peanuts. THE JAPANESE DISEASE. In 1904, Uyeda described a Japanese tobacco disease which seems to belong here, at least so far as signs are concerned. He says : This disease causes much damage in Japan, especially in those cases in which the transplantation from the seed-bed is carried out rather late. Prolonged moist weather with following great heat will cause the development of the disease. The first symptom is the wilting and yellowing of the lower leaves, then follows the blackening of the stem and leaves, and finally of the root. The plants are generally killed in about two weeks after the first symptoms are observed. The natural infection seems to take place generally through the roots, but very frequently also by the wounds caused by topping and suckering the plants toward the end of July. A microscopical examination reveals an immense number of bacteria in almost a state of pure culture in the vascular bundles. The central part of the stem shows a rotten condition and contains a dark brown liquid. Finally the stem becomes hollow and the root is completely destroyed. The organism, it is said, was cultivated pure, apparently in bouillon, and infections are said to have been obtained in about 2 weeks by irrigating healthy tobacco plants in pots and in the field. Infections through the stomata are also said to have been obtained by spraying a pure culture of the organism upon healthy leaves of young tobacco plants about 25 cm. high, but no evidence of this is offered other than that after 8 days a blackening was observed along the veins of the leaves, which extended more and more, and a microscopical examination showed enormous numbers of bacteria in the pitted vessels. Egg-plants which were irrigated in the same way as the tobacco did not contract the disease. This, however, might have been due to old and woody stems (see p. 236). This organism is described as Bacillus nicotianae and is said to have the following characteristics : Agar-stabs and streaks blacken, especially in the parts exposed to air (fig. 127). Growth on gelatin in streak-culture is very slow, forming a uniform pellicle, white at first, gradually blackening, with feeble liquefaction of the gelatin after about 5 weeks. Gas is produced in glucose agar and glucose bouillon, and there is a rancid smell, but only a little acid is formed. Milk is cleared. On potatoes it produces a yellow pigment, gradually turning brown-gray. In colonies on agar the growth is dirty white: gradually the brown pigment is produced, diffusing beyond the colony, which itself remains generally gray and shows concentric rings as it enlarges (fig. 128). Growth is best at 320 C. The organism is facultative anaerobic. It produces no odor in peptonized bouillon. It is 0.6 to 0.9M by 1 to 1.2/x long. It is said to bear about 8 peritrichiate flagella. WILT-DISEASES OF TOBACCO. 239 The following statements are taken from the more recent paper by Uyeda (1905) : The disease is widespread in Japan and has been known for at least 25 years, having been de- scribed in 1 88 1 in Ensoroku, a book on tobacco culture. Since that time it has been written upon with special reference to methods of prevention by T. Kugahara in the experiment station of Huku- shima-Ken (1891) and at about the same time by Dr. S. Hori, in the Imperial Central Agricultural Experiment Station at Tokyo. Prof. Y. Kozai, director of the experiment station at Nishigahara, has also isolated a schizomycete, which he holds to be the cause of the disease. This is the same as that described by Uyeda, who did his work at that station. The disease is variously known to the Japanese as "stem-rot, " "black-leg, ' ' and "wilt-disease. " Uyeda 's studies were begun in 1899 and continued nearly 5 years. The disease is widespread in the province of Hitachi, and occurs also in the province of Sagami and Shinano. Fig. 126.* The signs of the disease are much like those of the Granville wilt. Compare fig. 126 with pi. 33, and pi. 41, figs. 1, 2, 7, with pi. 42, for brown stripes on the green stems. The disease occurs from June to September on plants of all ages. The first sign is the sudden wilt of the foliage, after which the leaves become yellow. The stem then becomes black and the roots decay. When the juice from diseased plants or a pure culture of the bacillus is placed on healthy tobacco leaves, they begin to blacken and to show brown spots within one or two weeks. The principal veins of the leaf are then hollowed out and destroyed. The infection experiments were made on young and old plants at Nishigahara. These are not described. Old leaves sometimes show wavy black spots bordering the veins, where the bacteria are said to have entered by the stomata. The organism often enters also by the roots or through wounds above ground. All the affected parts contain great quantities of bacteria and the reaction of the fluid containing them is alkaline The bark of the stem shrinks considerably as the disease progresses and black lines appear upon the surface. The tissues *Fig. 126. — Experimental field of tobacco at Ota, Japan Uyeda.) destroyed by bacterial wilt. Season of 1904. (After 240 BACTERIA IN RELATION TO PLANT DISEASES. of the root become separated and disorganized. At first in both stem and leaves the bacilli are found only in the vascular bundles. In the leaves the veins are first blackened and then the parenchyma is destroyed. In very recently diseased plants the vascular bundles are only stained locally, while the rest of the tissues are entirely sound. In the first stage of the disease only one side of the stem is black. Later the entire woody tissue becomes black. Sometimes the black stain does not appear until after the stems are cut. The bacteria gradually pass inward from the woody tissue into the pith and outward into the bark. The bark-parenchyma is badly disorganized; cell-sap, starch, chlo- rophyll, nucleus, and the rest of the cell-contents disappear; only the sclerenchymatic cells are very resistant. There is the same disorganization of the parenchymatic parts of the vascular bundles. Sometimes a cork-layer is developed between sound and diseased parts. Small cavities filled with an immense number of bacteria often occur in the stem. The veins of the leaf are often hollowed out by the bacteria. These are often also found swarming, it is said, in the leaf-hairs. When the roots are attacked the first sign is the separation of the bast from the wood. When for any reason the attacked plants are not at once destroyed the leaves are dis- torted. The disease causes much damage by its very rapid spread during the rainy season. The spread of the disease is also greatly favored by the high summer temperature. F,g. I28.f The organism said to cause this disease has been named Bacillus nicotianae by Uyeda. He states that it resembles Bacterium solanacearum, but to the writer it appears to differ very materially in its morphology and cultural characters (if we may assume all these statements to relate to one organism), although the signs of the disease are strikingly like those of the Granville (North Carolina) tobacco wilt. Uyeda's description of this organism differs from Bacterium solanacearum, as I have cultivated it, in the following particulars: \ ;ai lab and streak cultures of Bacillus nicotianae Uyeda, showing a brown stain in the upper part, the lower portion of each being unstained. (After Uyeda.) fPlC illus nicotianae I ryeda, from an agar- plate culture 2 weeks old, showing development of the brown stain in and around the colonies. (After Uyeda.) WILT-DISEASES OF TOBACCO. 24 1 Peritrichialc flagella: There are 4 to 8, which are 3 to 4 times the length of the rod. Endos pores: These are formed after a month in both fluid and solid media. They are found very often in the top of agar-streak cultures grown for 3 months at room temperatures, but not in the lower part of the streak. They are not described or figured. Early pellicle: This appears the third day on neutral peptonized beef-bouillon with subsequent brown stain of the fluid; the pellicle breaks upon shaking into parti-colored portions, blue and white. The pellicle becomes gray and rather granular, and after a month there is a black ring. Liquefaction of gelatin: In stab-cultures liquefaction begins to appear the second day. After 4 days there is a distinct funnel-form liquefaction. After about 3 weeks all of the gelatin is liquefied, and gradually stained gray-black. In streak cultures all of the gelatin is liquefied in 2 weeks. This contradicts an earlier statement by Uyeda. Color and rapidity of growth on agar- plates: The colonies appear within 24 hours at August tem- peratures. They assume a reddish color after about a week and then gradually change to a dirty gray and black. Frequently giant colonies appear on the plate cultures and these become ringed concen- trically. Rhomboidal crystals of ammonium magnesium phosphate are frequent in the agar. Disorganization of potato slices: This takes place within one or two weeks. The growth is yellow. Coagulation of milk by the formation of an acid: Milk inoculated with this organism becomes strongly acid and is coagulated. This contradicts an earlier statement. Later it is partially pep- tonized and becomes chocolate-brown and feebly alkaline. Thermal death-point: This is 540 C. At 53° the result was not uniform. In one place 550 is stated to be the "maximum" temperature. A microbism: In sugar-agar it grew well in the depths of the stab. In bouillon in fermentation- tubes it clouded the closed end. It also grew very well in an atmosphere of hydrogen. Acids: Acids were developed in various saccharine media. Gas: Scanty development of gas in glucose-agar and glucose-bouillon. Kind of gas not stated. Indol: A weak indol-reactiou was detected in young cultures. Stains by Gram. Reduces litmus in milk. Non-infectious to egg-plant and tomato. Other characteristics of the organism are as follows : It is a short rod with rounded ends usually 1 to 1.2 by 0.5 to 0.7/x (fig. 129). In the plant it occurs often isolated, but some- times in pairs. In cultures it is single, in pairs, or fours. Even in cultures 2 to 3 weeks old it rarely forms chains. It varies somewhat in size according to conditions of growth, e. g., in bouillon the rods are somewhat larger than on agar. Capsules occur in old agar cultures. On gelatin-plates, in 1 to 2 days, small colonies appear both on the surface and in the depths. Magnified 50 times the surface colonies are roundish with an irregular edge, some- what granular and darkest in the center. After 4 or 5 days the gelatin around such colonies is liquefied centrifugally and there is a bacterial sediment in the fluid. After 6 days the colony is somewhat beaker-form. After a week the colonies retain their original form, floating in the liquefied gelatin. The buried colonies are ellipsoidal and within a few days they fuse into a very small mass. In agar-plates (fig. 130) the surface colonies are not granular, but very smooth, moist and bright in their center. A membrane is often formed over the giant colonies. Rarely the concentric giant colonies have a serrate margin. At first the surface colonies are white. The buried colonies are elliptical or egg-shaped. They have a high sheen by transmitted light; by reflected light they are bluish-white and somewhat fluorescent. Growth on steamed potatoat July temperatures is rapid, forming after a time a greenish- yellow layer. In course of 1 to 2 weeks the substratum becomes gray-black. This pigment is soluble in water and is due to tyrosinase, an oxydizing enzyme. The black pigment from agar-streak-cultures is readily soluble in water, very little in alcohol or glycerin, and not at all in benzine, ether, or chloroform. This pigment is produced within a few days in agar, bouillon, gelatin, potato, and milk. In August, at 300 C. (room-temperature), the pro- duction of pigment was very rapid. Bouillon and milk are wholly blackened at a high temperature (350 C.) and a black ring is often formed on the wall of the tube at the surface of the fluid. 242 BACTERIA IN RELATION TO PLANT DISEASES. - » »» »■.. Fig. 129.* Growth on cooked carrots looks yellowish at first. After some days there is a dis- agreeable odor. On cooked radish there is a sharp odor in i to 2 weeks and the tissue is then disorganized. The organism makes only a slight growth in Uschinsky's solution. The optimum temperature is about 320 C. Methylene blue (1 per cent) in bouillon is reduced. Litmus is reduced. Nitrate is reduced to nitrite (tested with meta- phenyldiamin, also sulphanilic acid + a-naphthylamin). In young cultures in peptone-water or bouillon, indol was detected in small quantity. In 10 days in 5 per cent peptone-water the cultures became so black that indol could not be detected. Hydrogen sulphide is produced. The organism was found in the earth of a tobacco field to the depth of 3.5 decimeters. Uyeda has the following on enzymes. Invertase is formed (the filtrate from a bouillon-culture passed through a Chamberland bougie inverted saccharose in one day). A very slight quantity of diastase appears to be excreted. Cytase is believed to be produced. Tyrosinase was detected in fresh agar-streak-cultures and in bouillon-cultures by the addition of paraphenyl- endendiamin and /3-naphthol (Spitzer's reagent). With this there developed a pale red stain soon becoming black. If one adds a 1 to 5 per cent solution of tyrosin to a cul- ture of this bacillus the red-black color increases more rapidly than without it. Trypsin is also produced. This was demonstrated in gelatin-cultures several weeks old by adding some drops of chlorine-water (Neumeister's tryptophan reaction), the color changing to a red. In the same way bromine gives a violet color. The organism grew as well (luxuri- antly) in an asparagin-dextrose solution as in a peptone-dextrose solution. Bacil- lus nicotianae did not grow in a mineral solution (KH2P04 0.1, MgS04 0.03, NaCl 0.5 per cent) with dextrose or glycerin, and the following nitrogen compounds in 1 per cent doses : Ammonium tartrate, po- tassium nitrate, ammonium chloride. In the same mineral solution with 1 or 2 per cent asparagin and 1 per cent dextrose there was a good growth and the forma- tion of a pellicle when the medium was alkaline and no growth when it was acid. In the mineral solution with potassium nitrate and glycerin Bad. solanacearum devel- oped normally, it is said. In the writer's experiments it developed feebly as compared with the growth in bouillon. J The pigment formation is independent of magnesium salts. Fig. 130-t *Fig. i-'',. Rods of Bacillus nicotianae Uyeda, from a photomicrograph. X1200. (After Uyeda.) fFlG. 130. — Bat illus nicotianae Uyeda. from an agar-poured plate S days old. (After Uyeda.) {Repeated in 1914 using Medan III with the same results. When inoculated thinly no growth; when inoculated heavily a moderate growth with reduction of the nitrate. WILT-DISEASES OF TOBACCO. 243 Nicotiana rustica is not attacked, and the following varieties of N. tabacum are not much subject: Ohasama, Taketadate, Mitsuke, Kentucky White, Green River Prior. Inoculations succeeded on Physalis minimum, Capsicum longum, Amaranthus gangeticus, and Polygonum tinctorum; they failed on Solatium mclongena, Lycopersicum esculcntum, and Physalis alkekengi. No details are given as to methods of inoculation, phenomena produced, or number of experiments. Nitrogenous fertilizers predispose to disease; pot- ash salts do not. Early planting is the best protec- tion. Through the courtesy of Dr. Uyeda, who sent alcoholic material, I was enabled to study his dis- ease in serial sections. The bacteria closely resemble Bad. solanacea- rum in general appearance. They are enormously abun- dant in vessels of the wood, in inner and outer phloem, in the cortical parenchyma, andinthepith. I also found them to have made cavities Fig. 131.* resembling those due to Bad. solanacearum. They stained deep red with carbol fuchsin and were quite uniform in appearance. Spores were not observed. (Compare figs. 131 and 132 with those made from stems of the North Carolina to- bacco, figs. 116 and 117.) Also, with bacteria taken from this Japanese tobacco stem I made several attempts to stain theflagella. At first I got no indications of peritrichiate rods and many faint indications of Fig 132. f Fig. 1334 polar fiagella; nevertheless no well-stained ones, such as could be relied on for demonstration. *Fig. 131. — Cross-section of a tobacco-stem received from Uyeda, showing character of the bacterial occupation in the Japanese tobacco wilt (compare with fig. 1 17). The section is taken from the wood midway between pith and bark, the cambium being in the direction of the arrow. The vessels next beyond 1', 1", 1*. I", Fare also occupied by the bacteria, as well as many others. Middle section of slide 482, stained with carbol fuchsin. The contrast is greater than here indicated. At X are a few bodies which look like fungus spores. (For an enlarged drawing of the bacteria see fig. 132.) fFic 132. — Schizomycetes from the Japanese tobacco-stem shown in fig. 131. JFig. 133. — Klagella on bacteria taken directly from the interior of a tobacco-stem attacked by the bacterial wilt of Japan (Uyeda's disease). Out of alcoholic material received from Uyeda. Pitfield's stain. 244 BACTERIA IN RELATION TO PLANT DISEASES. Subsequently I tried again with somewhat better results, using Pitfield's stain (fig. 133). All the flagella appeared to be polar. I am therefore inclined to think that the description of Bacillus nicotianae Uyeda may not have been drawn wholly from one organism. HONING'S SUMATRAN STUDIES. Since completion of the preceding account of the tobacco-diseases, which includes notice of one paper by Honing (p. 224), 11 additional Dutch papers (1911 to 1913) dealing with the Sumatran disease have been received from Dr. Honing. These papers add much to our knowledge of that disease. They render it probable that it is identical with the Japanese disease and that both are due to Bad. solanacearum, first described by the writer from the United States, although some discrepancies remain to be explained. These Dutch papers are summarized as follows : (I) THE CAUSE OF THE SLIME-DISEASE AND ATTEMPTS TO COMBAT IT— II. If two bacteria agree culturally, in reaction to stains, to substrata, etc. (making due allowance for variable culture influence), but do not attack the same plants, then we may assume that they are two nearly related but not identical species. Uyeda was warranted in separating his tobacco bac- teria from Bad. solanacearum only on the ground that the latter does not attack tobacco, a supposition which must now be abandoned, as Smith himself has shown and as has been confirmed in Sumatra. Our experiments with different strains of the bacteria have shown a great difference in virulence. The following experiments (table 25) show the infectious nature of the bacteria from different sources and the effect of a previous infection of the soil. Each of the six rows contained 100 plants, of which the first 50 in each were inoculated by soaking the seedlings for a few minutes in water to which a bouillon-culture had been added. Table 25. — Honing s Field Results with Bacteria from Various Sources. Source of bacteria. No. of inoculations which died. Dead in control. IO 28 38 IO 20 '9 5 3 18 13 23 '5 4. Mucuna (Leguminosae) 6. Check (throughout) Rows 4, 5, and 6 were on land infected about 4 months before, and row 3, which also showed many cases in the control, was adjacent to the previously infected land; 2 of these 5 cultures were more virulent than the others. This experiment was repeated in pots with the same variable result, as shown in table 26. Table 26. — Honing s Pot Results with Bacteria from Various Sources. Origin of bacteria. No. inoculated. No. which became diseased. 10 IO 20 10 IO 29 0 IO 20 10 O O Control . . "Why the bacteria from tobacco a were not able to attack the seedlings I do not know. For the bacteria out of Pluchea the ease is different, because proof of their identity must yet beobtained." With the bacteria from tobaccos b, c, and Ageratum, the disease was visible in 5 or 6 days. The other plants were entirely sound after 24 days. WILT-DISEASES OF TOBACCO. 245 In another series of inoculations, 99 out of 103 plants contracted the disease, 13 checks remaining sound. These were as shown in table 27. Table 27 . — Result of Further Inoculations by Honing. Plants infected. Source of bacteria. Number inoculated. Number slime-sick. Plants infected. Source of bacteria. Number inoculated. Number slime-sick. Ag.* a Ag. b Ag. a, Ti .. T.b, T,.... T. c, Tl... T. c, T3.... 10 10 10 10 10 10 10 10 10 9 9 8 Tobacco r.d T. c, T3. . . . T. c, T,.... r.c, Ti.... 10 '4 12 7 10 '4 12 7 Do Solatium melongena: Violet fruited. . . White fruited. . . Pouzolzia Do .. Do... Do Do.., *Ag. = Ageratum ; T = Tobacco. The small letters denote plant selected. In 7 cases the strain was reinoculated into tobacco and reisolated before using, the sub-figures denoting the particular plant from which reisolated. Time alone can tell whether cultivation, rotation, disinfection, or selection of less susceptible races will prove most serviceable in combating this disease. Some planters have said that the slime-bacteria would not attack plants under ideal food condi- tions, but this can not be admitted. Inoculated potato plants (16 in number) from two sources contracted the disease within a week, although they were obtaining their food directly from the mother tuber. It is likely that any plant of susceptible sort, variety, or race will be attacked, no matter what the food conditions. It is impractical to prove absence of the slime-bacteria from the soil by the ordinary plate method, but cuttings of tobacco plants may be used. In an experiment in which soil-water from pots, which had been infected about 6 weeks before, was diluted with tap water in 5 dishes and 2 cuttings put into each, 9 of the 10 plants contracted the disease. In two tests made in the same way with suspected soil from two fields, each with 9 cuttings, 7 were positive for soil of one field, 2 only for that of the other. If such tests, therefore, are made, a considerable number of cuttings should be used. Because the soft tobacco stems rot easily, Honing substituted the harder stems of Ageratum and Pouzolzia successfully, but then had to wait longer. Although unquestionably the slime-bacteria are killed off in the soil by other organisms (other- wise there would soon be nothing else in the soil), nevertheless the fact remains that they may persist in the soil for years. The continuation of the experiments with chloride of lime and potassium permanganate yielded nothing very hopeful, as may be seen from the following experiment (table 28) on 1,392 plants in 5 similar plots: Table 28. — Effect of Soil Treatment on Sumatran Tobacco-Disease. Substance used. No. dead. No. sound. 297 3,8 299 268 215 9 2 1 3 10 3. Potassium permanganate, 3 kg. . . . This was the worst field. None, however, were very hopeful. The best plot gave n per cent more sound plants than the control. The remainder of this paper is taken up with discussion of morphological and cultural char- acters of the slime-bacteria, which are here omitted because treated more fully in one of the following papers. (2) THE CAUSE OF THE SLIME-DISEASE AND ATTEMPTS TO COMBAT IT— III. In the year 191 1 , up to April, Honing received of tobacco seedlings diseased by the slime-bacteria, 44 samples from 24 plantations. Many of these fields were visited and frequently the disease was found showing in the seedlings so slightly that they would have been selected for planting, especially those infected in the leaves. On a plantation where the leaves of the seedlings were supposed to have been injured by guano or Schweinfurth green, the actual cause of the injury proved to be this bacterium, and all stages were 246 BACTERIA IN RELATION TO PLANT DISEASES. observed between slight marginal injury and penetration of the bacteria through the side veins, into the midrib, and even into the vessels of the stem. The general conclusion from this examination may be stated as follows: "So long as the seedlings stand in the row they are more likely to be infected in the leaf than in the root. " Whether the attacked seedling is planted out depends a great deal on the age of infection. If it is somewhat advanced an intelligent coolie would reject it. On some plantations the rule is: " No seedlings shall be planted except those on which the lowermost leaves are still good. " But this rule involves the destruction of many good seedlings. Also, it is not always the lowermost leaves that are first infected. The best proof that such leaf-infected seedlings can yield infected plants is the finding of plants in the field with diseased stems and sound roots. Such plants have been seen as early as a month after planting. Diseased seedlings, however, are not the only cause of diseased tobacco in the fields. This was well shown in a field where a great many of the plants died. This field was planted in part, it is true, from an infected seed-bed, but also in part from a sound bed, which on higher land yielded sound plants. Here the soil of the field, which had stood under water, was infected. A similar case was observed on another plantation. The water used for irrigation may be also a source of infection. Up to this time the water of 7 wells used for this purpose has been examined and the slime-bacteria found to a certainty in 4 of them on 3 different plantations. Because of the organism's need of free oxygen these wells are believed to have become superficially infected by the falling into them of infected earth rather than from the ground-water. This is shown also by the fact that where a new well was made the seedlings watered from it remained free, while those watered from the old well became diseased. This and other cases cited tend to show that sound plants set on healthy land may become diseased by the use of infected water. By use of 1 to 1,000 potassium permanganate such water is disinfected and is not too concen- trated for watering transplants, but would burn seedlings in the bed. It has been communicated to Honing that 0.05 per 1,000 (50 grams per cubic meter) does not burn the seedlings. In using this substance it must be remembered that in water rich in organic matter potassium permanganate soon loses its disinfecting power and greater quantities must be used, concerning which no general rule can be laid down. There are some plantations on which it is not possible to grow healthy seedlings, all of the soil being infected. If one must make seedlings on such land, then not only the water used but also the soil of the seed-bed itself must be disinfected. Sound seedlings are of the utmost importance, because a large percentage of the dead plants have come from infected seedlings. Capsicum is less susceptible than Solatium species. In one case a tobacco strain which failed on Capsicum (18 plants) became infectious to Capsicum after passing it through Mucuna. (3) REPORT ON THE SLIME-DISEASE TESTS OF 1911. General infection of the soil is probably less common than has been supposed. The tobacco has not died on all the flooded lands. The disinfections with chlorid of lime and with potassium permanganate yielded no striking results. Water from 2 1 wells was examined, of which 7 on 5 plantations yielded the slime-bacteria. Bacillus solanacearum Smith has now been isolated and cultivated out of seedlings or field tobacco from 17 plantations. While the cause of the slime-disease is the same on all the plantations, the circumstances under which infection takes place are very different and are known only in part. (4) DESCRIPTION OF THE DELI STRAINS OF BACILLUS SOLANACEARUM SMITH, THE CAUSE OF THE SLIME-DISEASE. The most important questions resulting from a study of the literature, the inoculation experi- ments on tobacco and rotation of crops, and from a study of the cultures are the following: (a) Is there to be found a series of foodstuffs, by means of which this organism can be distin- guished from others by growth or absence of growth; also how to separate virulent from non-virulent forms? (b) Is the slime-bacterium in Japan, which has been described under another name (Bacillus nicotianae Llyeda), possibly the same as that in America and in Deli, so that means of combating it elsewhere recommended can be tried here? (c) Is perhaps Bacillus sesami Malkoff in Bulgaria [see p. 216] also the same bacterial sort as that in our tobacco and our Sesamum? MORPHOLOGY. Form and size. — The Deli strain of Bacillus solanacearum is somewhat more variable than Smith and Uyeda have announced (see pp. 193, 238). WILT-DISEASES OF TOBACCO. 247 The greatest length observed after staining with earbol fuchsin and measuring without a cover glass was 1.8/1. In old cultures they are shorter, i. e., 0.5/j, and then look more like cocci than rods. In nearly all the preparations the bacteria are single or united in pairs. Chain-form colonies, reported by Uyeda in 3-months-old cultures, were seen only in glycocoll-glucose solutions, where arose twisted threads of 20 or more cells, among which were swollen single cells which no longer stained like the others. Motility. — In 1 or 2 day old bouillon-cultures the bacteria often show self-motility which gradu- ally ceases. Agar-cultures appear less favorable for its demonstration. Also, when freshly isolated out of tobacco, the examination is mostly negative. Flagella-staining by Van Ermengem. — More than 3 fiagella I have not seen on a free-lying bac- terium, but whenever a pair lie close together there are more than 3. Capsules. — Contrary to Uyeda, I have not been able to find any capsules, not even in agar-cultures 2 to 3 months old stained with earbol fuchsin or by Friedlander's method. Spores. — According to Uyeda, B. nicotianae forms spores in cultures poor in nutrient substances, but B. solanaecarum does not. Since Uyeda neglects to say how he demonstrated the spores, speak- ing neither of germination nor of resistance to dry air or high temperature, and not even of staining, he must still show proof (see under Polar staining). For the Deli strain, spore-staining with earbol fuchsin and methylene blue yielded no positive result, not even after mordanting with chromate of potash (Chroomzure Kali). The slight resistance of these bacteria to heat makes the existence of spores very improbable. Tests were made of 5 strains grown for 16 days in glycocoll-glucose medium by exposing them for 2 minutes at 8o° C. and then during the next 3 minutes cooling the water down to 400 C. From each of the 5 cultures so treated 3 transfers were made to peptone bouillon, but all remained sterile. The experiment was repeated using 4 strains grown in bouillon 66 days. These cultures were plunged into water at 920 C, which in 2 minutes cooled to 83° C. and showed after 3.5 minutes a temperature of 400 C. Three transfers were then made to fresh media from each of the 4 strains, but the 1 2 remained sterile. The check tubes clouded in 24 hours, showing that each one of these strains was still living. Finally, 15 sterile strips of filter paper (3 from each culture) were wet with fluid from 5 cultures 26 days old, representing 5 different strains, placed in sterile Petri-dishes, and incubated in a thermostat at 360 C. for 45 hours, after which they were transferred with sterile forceps to sterile bouillon. In none of the 15 transfers was there any growth. Moreover, Uyeda's contention that spores develop best in exhausted media is in contradiction to the statements of Schreiber, and also those of Lehmann and Osborne. These authors find bacterial sporulation proceeds better in nutrient- rich material. Polar Staining. — Polar staining is very plain, even on material taken directly from the diseased tobacco. In order to avoid confusing this with occurrence of spores, slides were fixed in the flame and also with alcohol and colored with Loeffler's methylene blue, with Ziehl-Nielsen's earbol fuchsin undi- luted for some seconds, and diluted (1 : 10) during a minute, and with 0.01 per cent watery methylene blue solution during 5 to 30 minutes. The question now arises whether Uyeda mistook polar staining for endospores. Gram's Slain. — The Deli-tobacco bacillus is Gram-negative. There were tested at the same time Bacillus coli communis (negative) and Diplococcus enteritis (positive). PHYSIOLOGY. Generally 10 strains of the tobacco bacteria from different localities were investigated at the same time. After a couple of weeks they were again inoculated into tobacco to keep the grade of virulence and always afford comparative material. Bouillon (1 per cent Liebig's meat extract, 1 per cent Witte's peptone, 0.5 per cent salt, acid or neutral). — This is generally clouded throughout in 12 to 24 hours. Often the growth is plainly strongest above, which appearance disappears after two days. Some strains then develop a pellicle like Uyeda's B. nicotianae, but others behave like .Smith's B. solanaecarum, forming a pellicle first after 1 or 2 weeks. The Deli strains also stain the fluid brown, but not all of them to the same degree. A series of 36 cultures (3 from each strain) showed typical differences. The 3 cultures from one strain were colored brown after about 3 weeks, and those from 3 other strains became dark-brown during the same time, while cultures of the 8 other strains were only a little stained. On far more than 200 bouillon-cul- tures a black ring around the pellicle was never observed, but in all a gray- white precipitate. Gelatin. — The behavior of the Deli strains on gelatin is somewhat different from that Uyeda described for B. nicotianae and recalls rather that of B. solanaecarum; that is, the liquefaction proceeds more slowly. Stab-cultures are funnel-form above and thread-form below, and sometimes a pellicle spreads slowly over the surface. Later short projections develop along the track of the stab, and liquefaction may occur in the clefts of these outgrowths. 248 BACTERIA IN RELATION TO PLANT DISEASES. Liquefaction begins irregularly in the upper layer mostly after 3 to 5 weeks, and slowly continues. Consequently the variability is very great, just as is the browning. Entire liquefaction in 3 weeks occurs very rarely even with weak gelatin, 4 weeks is rapid, and 2 and 2.5 months the rule, and under paraffin oil more than 3.5 months are required. The melting-point of the gelatin exercises a great influence. Always the gelatin with the highest melting-point liquefied most slowly (Non plus ultra of Von Gehe & Co., Dresden), and this stained black, while other gelatins were not colored or only became brown. These tests were made on 58 stab cultures from 15 isolations out of tobacco, 3 stab- cultures from an Acalypha stem, and 2 cultures from an Agcratum stem. In Uyeda's gelatin-cultures liquefaction with B. solanacearum was less rapid than with B. nicotianae, i. e., 5 to 6 weeks in place of within less than 2 weeks. Because the rapidity of liquefaction is dependent on the melting-point and on the alkalinity of the gelatin, I can not say that I have found any great difference between the behavior of the bacteria investigated by Smith and by Uyeda. Moreover, not only are the different Deli strains unlike in their behavior on gelatin, but there are also distinct differences between sister cultures of the same strain. Because the time of liquefaction is three times as long for some of the Deli cultures as for others, the liquefaction difference between B. nicotianae and B. solanacearum pointed out by Uyeda is of importance only in case it is constant. Agar. — In the beginning I obtained no blackening of agar-cultures. Later some cultures were obtained which darkened agar. Potato. — As for Smith's organism and Uyeda's, the Deli bacteria blacken slices of potato after about a week. They become, for the most part, violet-black and not gray-black. .Sometimes the 4 per cent glycerin solution, above which the potato rested on a short glass rod, was colored brown. Milk. — In Migula it is stated of B. solanacearum: "Milk becomes soapy, strongly alkaline, the casein is not precipitated. " In his preliminary communication Uyeda says of B. nicotianae: "Sapo- nifies milk," but in his complete work: "Milk first coagulated, but the coagulum is gradually dis- solved and peptonized. " These two statements do not agree. The 20 isolations first investigated behaved for the most part alike, but different from the Ameri- can and Japanese bacteria. Of 95 cultures from 14 isolations out of tobacco, 3 out of Acalypha, 2 out of Agcratum, and 1 out of Physalis, all remained nearly unchanged, there was no precipitation of the casein, but also not much alkali was produced [clearly, or the milk would have become translucent as I have described, but perhaps the cultures were not held long enough. They should have been kept under observation for at least 8 or 10 weeks]. To litmus the cultures remained feebly alkaline, and were not distinctly acid to phenolphthalein. Only one isolation showed in 4 of the 6 cultures after 7 weeks, precipitation of the casein with weak acidity. Noticeable for the Deli isolations is the behavior in litmus milk. In 7 isolations [which are men- tinned specifically] each one of the 20 cultures rendered the milk alkaline after 3 days, which reaction progressed gradually. After 5 days each one was entirely alkaline. But two davs later 16 of the 20 cultures had begun to form acid, first a small edge which became gradually broader, until finally the whole column was colored red and the milk solid. [If this may be taken as the typical action of the Deli tobacco organism and not the effect of undetected intruding saprophytes, or other non-compara- ble circumstances, then we may conclude that it is not the same as Bad. solanacearum.] Neither in "Natura Milch, " from Bosch & Co., Waren, Mecklenburg, nor in boiled fresh Medan milk was any chocolate-brown color observed, at room-temperature or at 360 C. [see pi. 41, fig. 3]. Cultures were also made in other milks, with similar results. From his table (p. 229) it would appear that most were alkaline at first, and then acid with coagulation. A few remained alkaline, and one of these was an isolation which soon lost its virulence. The results in milk may be summarized as follows: ( 1 ) The Deli isolations coagulate and peptonize as a rule the milk of the zebu (except 4 cultures of 1 isolation of the 95 from 20 sources) just as Uyeda's B. nicotianae. (2) With sterilized European milk all the isolations show in the beginning an alkaline reaction, just as B. sola- rh iii-arum Smith (concerning the strength of the reaction without figures nothing can be judged). The term "soapy" 1 (1m not like. (3) Some (weakened?) isolations do not proceed any farther, the most begin afterward to ferment the sugar to form acid and to throw down the casein. This is just the opposite of what Uyeda states for B. sclanacearvm [and Smith Com lusion. — Because the Deli isolations have given three different results depending on the age of the cultures and on the kind of milk used, the possibility is not excluded that Uyeda actually had another bacterium, but it is just as likely that if he had worked with "Nutricia," "Landbouw," " Natura," or "Milkmaid" milk, he would have obtained the same results. Taken in connection with the numerous other points of agreement this is very probable [see pp. 263, 264]. Need oj ( h ygen. This is plainly shown by the much slower growth of cultures under paraffin oil, by the form of the gelatin-stab-cultures, by the slow clouding of the closed end of fermentation tubes in e pari ion with the open end, and by its slow growth in an atmosphere of hydrogen in a Botkin's apparatus. WILT-DISEASES OF TOBACCO. 249 Reduction of Methylene Blue. — In 92 bouillon-cultures from 12 isolations, containing 0.002 per cent methylene blue, 9 were bleached after 1 day, 31 after 2 days, 71 after 3 days, 85 after 5 days, and 87 after 14 days, except the uppermost 3 to 4 mm. The bleaching took place after 1 day in glucose bouillon (3 cultures from each of 3 isolations — out of tobacco, Mucuna, and Blumea). Reduction of Litmus in Milk. — Litmus is also wholly or partly reduced [see p. 264]. Reduction of Sodium Sclenite. — To obtain reductions the quantity of sodium selenite (Na2Se03) used must be small. Growth and reduction was obtained with all strains in 2 days, using 0.01 per cent in peptone bouillon agar, but no growth where 0.1 per cent was used. Reduction of Nitrate. — Nitrate is reduced when the Deli organism is grown in presence of glucose or saccharose, but not when starch is used as the carbon food, as starch is not assimilable by this organism. Diastase Production. — As mentioned above, starch is not acted upon. In this respect the organ- ism agrees with Smith's statements. Tyrosinase. — Each one of 8 isolations grew using tyrosin as carbon-nitrogen food, but less well than in peptone-water. None of the 34 cultures were actually blackened. "When one adds 1 to 5 per cent tyrosin solution to a culture of the bacillus, it takes on a red-black color quicker than with- out this addition" (Uyeda). On repeating this experiment the result was again negative, but the concentration of tyrosin was less. Without more ammonia or alkali Uyeda could not have made a 1 to 5 per cent solution, since according to Erlenmeyer only one part of tyrosin dissolves at 20° in 2,454 parts of water. Indol. — According to Uyeda, B. nicotianae produces a small amount of indol. The same is true of the Deli cultures. With sulphuric acid and potassium nitrite the reaction of 22 bouillon-cultures from 9 isolations was negative or nearly so. Repeating with 25 cultures from 10 isolations, 20 of the cultures, after heating, gave a permanent red color of extremely variable intensity. It also happened that a culture which gave no indol reaction like Uyeda 's B. solanaccarum, after inoculation into tobacco, gave a positive indol reaction like Uyeda's B. nicotianae. Yet is it doubtful whether indol is really produced. According to Crossonini the indol reaction of Ehrlich is much sharper with para- dimethylamidobenzaldehyd, and there are bacteria known as indol-formers on account of the old reaction, which when tested by the new method show no trace of indol. Following this suggestion, Honing tested 7 strains of the Deli tobacco-bacteria with the paradimethylamidobenzaldehyd in alcohol with hydrochloric acid, but all with negative results. Hydrogen Sulphide. — This substance is formed in very slight quantity, as Uyeda has also shown. vStrips of lead-acetate paper exposed for some days remained white or were only slightly blackened. Of 18 cultures [kind not stated] from 9 sources only one gave a positive reaction. After the careful boiling away of 32 cultures (10 isolations) the lead-acetate paper was black in only 10 tubes, and here only a little around the edge, while 22 remained negative. From one and the same source sometimes one culture was positive and another negative. Fat. — Neither with osmic acid nor with Sudan III in alcohol or glycerine could the formation of fat be shown. Glycogen. — This substance is not formed by the bacteria (reagent, iodine-potassium iodide), nor can it be used as a carbon food by the bacteria, as will be shown later. Disagreeable Odor. — This is noticeable from peptone-bouillon cultures, but varies greatly in cul- tures from different sources. Isolation Ts resembled Uyeda's B. nicotianae, while others produced scarcely any odor, like Uyeda's strain of B. solanaccarum. Gas Production. — Gas-formation does not occur in the closed end of fermentation tubes with glucose, saccharose, or mannit. For the most part even in the presence of oxygen there is no trace of gas-formation. Once very fine bubbles were detected in 24 glucose-bouillon cultures, 10 inoculated out of tobacco, 7 out of Physalis, 2 out of Ageratum, 4 out of Mucuna, and 1 out of Blumea. This is hardly a specific difference. Acid- and Alkali-formation. — With exception of milk (in the second stage; i. e., after the alkaline reaction), and with a nutrose-substratum, alkali is always formed. In normal growth the original always feebly acid solution became plainly alkaline. In feeble growth the alkalinity was feebler orthe fluid had not yet reached the litmus neutral point. Acid-formation has not been detected with any single sugar when the source of nitrogen was not casein. Temperature. — Uyeda's statement that heating to 550 C. is fatal and Smith's that exposure to 520 C. for 10 minutes kills, is true of the Deli strains. The tropical climate (Medan lies in +30 north latitude) appears not to have increased the maximum temperature. Bouillon-cultures exposed for 10 minutes at 550 C. appeared to be sterilized (5 isolations tested). We do not, with Uyeda, consider 3° C. difference in maximum temperature as specific, since 550 C. (for B. nicotianae) and 520 C. (for B. solanaccarum) are not the average of a great number of determinations with comparable material from different sources (see under "Spores"). Resistance to Drying. — This is slight, as already noted under "Spores. " 250 BACTERIA IN RELATION TO PLANT DISEASES. SOURCES OF NITROGEN AND CARBON. As a stock solution for these tests, Honing used Meyer's mineral solution.* All the substances tested were from Merck, and those marked "insoluble in water" were dissolved with as little as possible of Na2COa or HC1. In this stock solution, adding one substance as source of both nitrogen and carbon, the results given in table 29 were obtained with the Deli tobacco bacteria. The one substance that brought out distinct differences in the isolations was asparagin. All three cultures of Tg and Th showed growth and also one of Td. The rest remained entirely clear, even after 14 days. For reasons given, the auxanographic method could not be used at Medan; the inoculations, therefore, were all made into solutions after steam-heating to ioo° C. on 5 consecutive days. Potassium Nitrate. — With 1 per cent potassium nitrate (in the following solution: KHoPO.4,0.1 per cent; MgS04, 0.03 percent; and NaCl, 0.5, alkaline) Uyeda found B. nicotianae made no development, not even on addition of 1 per cent glucose or 1 per cent glycerin, while B. solanacearum gave a weak Table 29. — Ability of Deli Tobacco Organism in Meyer's Mineral Solution to obtain both Carbon and Nitrogen from One Substance. Substance tested. Albumin Asparagin Fibrin of blood: Alkaline Acid Gluten Glycocoll Guanine: Alkaline Acid Hemialbumose: Alkaline Acid Legumin: Alkaline Acid Leucine Nuclei'n VVitte's peptone Protein Protein (alkaline) Tyrosine Ammonium acetate. . succinate lactate. . . tartrate, citrate. . . No. of isolations out of — Tobacco. Ageratum. Physalis. 7 2 , 7 2 I 7 2 1 8 I 3 2 1 7 2 1 7 2 1 9 1 7 2 I 9 1 6 2 1 9 1 7 2 I 7 2 I 5 2 1 3 2 1 4 2 1 5 2 1 10 1 10 1 10 1 10 I 10 1 No. of cultures. 42 4" 42 27 27 3' 40 29 40 30 38 30 42 4' 33 3° 3' 35 3' 32 33 33 33 No. showing growth. O 7 o o o o o o o o o o o o 33 o o 34 o 32 33 30 30 growth in presence of the glucose and a normal one in the presence of the glycerin. This is wholly in agreement with the Deli isolations of B. solanacearum with potassium nitrate in glucose and does not show that Uyeda had two kinds of bacteria, since 4 of Honing's strains failed to grow like Uyeda's B. nicotianae, while several others grewlike B. solanacearum. In addition to these differences, Honing found a strain which grew well with potassium nitrate and inuline, while another would not grow. In the same way one strain grew with potassium nitrate and dextrine, while another would not grow; also one strain grew with potassium nitrate and sorbit, while another would not grow; and, finally, one strain grew with potassium nitrate and saccharose, while another would not grow. These facts, Honing thinks, should make one very careful in drawing conclusions from a small number of cultures made from a few strains. Potassium Nitrite. — This substance may also serve as food. Asparagin. — Uyeda's B. solanacearum did not grow with asparagin, while his B. nicotianae made a feeble growth. That the latter, which with asparagin alone made a weak growth, made none what- ever when 1 per cent glucose was added, should have indicated to Uyeda that bacteria are variable ♦Water, 1,000.00; KII.PO<, 1.00; CaCl2, 0.10; MgSO^ILO, 0.30; NaCl, 0.10; Fe2Cl6, 0.01. WILT-DISEASES OF TOBACCO. 251 like other things, because, in the first place, 1 per cent glucose is not a poison and second, in an alka- line solution the growth of B. solanacearum is favored. With an eye upon the variability of the Deli strains of B. solanacearum, I must ask once more: Is the difference constant that Uyeda found? Of 12 Deli strains, 8 showed no growth with asparagin and glucose; 4 grew badly and for the most part in a portion only of the tubes. Of these 8 strains, 5 were inoculated into tobacco, reiso- lated, and re-tested with asparagin and glucose, whereupon 4 of the 5 grew in part of the cultures. These same 8 glucose negative strains were also saccharose-negative, but after 5 strains had been "freshened" by inoculation into tobacco each one of the 5 grew in a part of the cultures set, and also with potassium nitrate the same thing was shown — more grew with saccharose than with glucose. In a repetition with saccharose the same result was obtained. Table 30. — Deli Tobacco Organism in Meyer's Solution with 0.25 per cent KNOz and Carbon Foods. Carbon source. Arabinose Glucose Levulose Mannose Galactose Erythrite Adonite Sorbit Mannit Dulcit Rhamnose Saccharose Maltose Lactose Raffinose Quercit Inosite Dextrin Starch Glycogen Inulin Lichenin Glycerin Sodium acetate. . butyrate succinate malate. . lactate. . citrate. . Per cent used. I I 1 I 1 I I 0.2 I I 0.2 1 1 1 0.2 0.2 1 0.2 I I I No. of isolations from — Tobacco. Ageratum. Physaiis 9 10 1 1 2 2 2 2 9 9 9 9 10 9 9 9 9 2 9 6 9 9 2 9 9 9 9 9 9 9 Acalypha. No. of cultures. 44 45 66 15 '5 '5 "5 47 44 48 42 62 47 47 52 44 '5 43 46 45 48 "5 5' 5' 52 39 41 40 42 No. of cultures in which growth occurred. '9 27 o 1 1 '3 12 5 38 39 46 23 5° 33 38 50 '7 '4 27 o o 7 o 44 o* o 20 '7 o 24 Ammonia Nitrogen. — With ammonia nitrogen and with a suitable carbon food the Deli strains of B. solanacearum grew well, as shown by the results with ammonium succinate, lactate, tartrate, and citrate; further by 13 of 21 cultures from 7 sources with ammonia and glucose; and also by the urea cultures (46 of the 52 with glucose and 44 of the 49 with saccharose). In acid solution of 1 per cent ammonium chlorid, B. nicolianae grew for Uyeda and B. solana- cearum did not, or on addition of 1 per cent glucose only feebly, B. nicotianae also then did not. With 1 per cent ammonium chlorid and 1 per cent glucose in Meyer's solution, not neutralized, no growth appeared in 17 of the 18 cultures of the Deli tobacco organism from 6 sources, and with 1 per cent glycerin in none of the 21 cultures from 7 sources, although otherwise the growth with glycerin was comparable with that of glucose, but easier. These results harmonize well with Uyeda's when he used acid solutions. Yet after neutralization with Na2C03 (litmus neutral) the growth was very strong, as well with glucose as with glycerin. Even when inoculated from old weakened selenit- agar cultures there was still growth with glycerin but no longer with glucose. It is thus plain that ammonia nitrogen can be used very well by B. solanacearum, something that is not evident from Uyeda's communications. * I'sing Medan III and Florida potato (1914), the writer obtained similar results with these sodium salts, i. e., no growth with the lactate, acetate or butyrate, and prompt moderate growth in all of the tubes (8 of each strain), using the malate, tartrate, or succinate. In Meyer's solution with ammonium citrate there was heavy growth, and with am. tartrate and am. succinate, moderate growth (4 days), but none with ammonium lactate (4 days). Both strains grew in Meyer + urea + grape or cane sugar. 252 BACTERIA IN RELATION TO PLANT DISEASES. Substituting asparagin for potassium nitrate, the results shown in table 31 were obtained. Table 31. — Deli Tobacco Organism in Meyer's Solution with 1 per cent Asparagin and Carbon Foods. Carbon source. Nothing Arabinose Glucose Levulose Mannose Galactose Erythrite Adonite Sorbit Sorbine Mannit Dulcit Rhamnose Saccharose Maltose Lactose RafFmose Quercit Inosite Dextrine Glycogen Inulin Lichenin Glycerin Sodium acetate. . butyrate. succinate malate. . citrate. . lactate. . No. of isolations from- Tobacco. 12 6 '4 8 2 2 6 6 6 6 6 14 6 6 6 6 2 6 6 6 2 6 6 6 6 6 6 6 Ageratum. Physalis. Acalypha. No. of cultures. 62 21 81 36 '5 15 '5 '5 21 21 21 21 21 79 21 21 21 21 '5 21 21 21 '5 21 21 21 21 21 21 21 No. of cultures in which growth occurred. 7 o 27 4 9 10 10 8 16 18 20 18 10 40 10 9 16 10 10 13 o 19 o 12 O O 21 21 12 O Glycocoll. — B. solanacearum with glycoeoll alone does not grow, but it grows on addition of a carbon food such as glucose, maltose, or mannit, although badly in a part of the cultures and not at all with levulose. When glycocoll was used as the nitrogen food the results given in table 32 were obtained. Table 32. — Growth of Deli Tobacco Organism in Meyer's Mineral Solution with 0.1 per cent Glycocoll and the Carbon Foods Named. Carbon source. No. of isolations from — No. of cultures. No. of cultures in which growth occurred. Tobacco. Ageratum. 9 9 9 9 8 38 49 13 O Levulose Mannit. . . Maltose 49 32 45 2^ Also here occurred some typical cases of change in ability to ferment. Always in sets of 3, grew TgTi in lactose o, in maltose o, in mannit 1 ; but TgTjTi in lactose 2, in maltose 3, in mannit 3. Of 3 cultures from AbTiTj none grew with glycocoll and lactose, while of 3 others inoculated some time later from the same tube all developed. In table 33 all the results are united. The figures relate only to the proportion of cultures that grew, not to the intensity of growth: Thus 1 denotes growth in 1 to 20 per cent of the cultures; 2, growth in 21 to 40 per cent; 3, growth in 41 to 60 per cent; 4, growth in 61 to 80 per cent; and 5, gr< iwth in 81 to too per cent. The fact that these figures differ so widely throws a strong light on the variability of B. sol, nun varum. WILT-DISEASES OF TOBACCO. '■53 TablB 33. — Growth of Deli Cultures of B. solanacearum in Meyer's Mineral Solution with Different Carbon and Nitrogen Compounds. Carbon source. Arabinose. Glucose. . . Levulose. . Mannose. . Galactose. Erythrite. Adonite. . . Sorbit. . . . Sorbine. . . Mannit. . . Dulcit Rhamnose . Saccharose Maltose. . . Lactose. . . Raffinose. . Nitrogen source. KNO3. Asparagin. Glycocoll. NHi 3 0 3 2 0 1 4 3 5 4 4 4 2 3 5 4 5 5 5 5 5 3 3 5 3 4 3 5 3 5 4 Carbon source. Quercit Inosite Dextrine Starch Glycogen Inulin Lichenin Glycerin (Sodium)* acetate. . butyrate. Ammonium tartrate (Sodium)* citrate. . lactate. . malate. . succinate Nitrogen source. KNO3. Asparagin. Glycocoll- NHj *Or ammonium compound in the fourth column with NH3. Honing summarizes his conclusions as follows : 1. Bacillus solanacearum Smith often loses its virulence quickly. 2. This loss makes its appearance not all at once, but gradually, first toward Capsicum annuum, later toward Nico- tiana tabacum, and finally for Solatium melongena and 5. lycopersicum. 3. These circumstances explain the contradiction in the papers of Smith and Uyeda. 4. In Deli up to this time the bacteria have been found by me in: Nicotiana tabacum, Physalis angulata, Indigofera arreela, Arachis hypogaea, Mucuna sp., Acalypha boehmerioides, Ageratum conyzoides, Blum'ea balsamifera, Syvedrelta nodiflora. 5. As the result of artificial inoculation the following have also become diseased: Scsamum orientate, Solatium tuberosum, S. lycopersicum, S. melongena, and Capsicum annuum. 6. Both in its morphological and in its physiological characters the variability of B. solanacearum is much greater than is at present supposed. 7. In cultures with glycocoll and glucose the bacteria form threads more than 40 cells long, which are mostly crooked, and here and there show strongly swollen, feebly-staining cells without at first showing loss of virulence. By inoculation from these cultures into bouillon the normal growth in the form of single and paired rods returns. 8. Thus far the Deli strains have formed neither capsules nor spores. For the existence of spores Uyeda has not offered the least proof. 9. The Deli strains show polar staining after fixation in alcohol or in the flame, and staining with carbol fuchsin, or water solution of methylene blue. When peptone is used the polar staining becomes indistinct or may disappear altogether. 10. Examined on a slide at the same time with B. coli communis and Diplococcus enteritis, the Deli strainsare Gram- negative. 1 1. The reduction of nitrate by B. solanacearum (and probably also by other bacteria) can not be studied using starch as a carbon food. 12. Sodium selenite interferes with the growth to a high degree, or even sometimes stops it altogether on addition of o. 1 per cent in peptone-bouillon-agar, but is less harmful with 0.01 per cent. The selenite is reduced. 13. The different results reached by Smith and by Uyeda when the organism is grown in milk may be partly explained: a, by difference in the bacterial strains; b, by difference in the age of the cultures; c, very probably by differ- ence in the composition of the milk. Because Uyeda in his complete paper announces a different conclusion concerning the milk cultures than in his preliminary communication, and because the behavior of the Deli strains is rather variable, one can ascribe no great worth to the discovered differences. 14. The trials with fresh milk show that coagulation takes place only rarely (4 out of 95 cultures) and that the reaction is feebly alkalin or neutral. With European milk in tins or flasks the reaction after 3 to 7 days is always alkaline. In most of the strains the reaction changed again 3 to 7 days later and the milk curdled. When inoculated from old weak cultures the reaction remained alkalin. 15- The tests with combinations of different carbon and nitrogen compounds sometimes gave constant results and sometimes variable ones, the latter even when inoculated out of one and the same culture. 16. Of the 18 substances containing both carbon and nitrogen sources, growth occurred only with tyrosine, pepton, ammonia, ammonium succinate, ammonium lactate, ammonium tartrate, and ammonium citrate, and in a few cases also with asparagin (7 of the 62 cultures, and the 7 belonging to 3 strains). 17. The following may serve as sources of nitrogen: asparagin, glycocoll, potassium nitrate, potassium nitrite, and ammonia. 18. As sources of carbon in the combinations tested the following are absolutely unusable: glycogen, starch, lichenin, sodium acetate, sodium butyrate. 254 BACTERIA IN RELATION TO PLANT DISEASES. 19. Growth never followed inoculation into cultures with KNO3 and glycocoll with levulose; with asparagin and levulose only in 4 cultures from 2 sources. This is not strange, because also with asparagin alone growth occurred only once. 20. Development in all the cultures containing asparagin, glycocoll, and KNO3 was extremely rare. Very often only one or two of the three cultures showed growth when these were inoculated from the same culture of bouillon, of KNOj or of glycocoll. 21. In general the number of cultures which succeeded with some of the alcohols was greater than with the cor- responding sugars. 22. In a great number of cultures visible growth was not synonymous with strong development. 23. Because the Deli strains of Bacterium solanacearum are parasitic as well for Nicotiana tabacum and Capsicum annuum (like Uyeda's B. nicotianae) as for egg-plant and tomato (like Uyeda's B. solanacearum Smith), and moreover because nearly all of Uyeda's designated cultural differences fall away I think that B. nicotianae Uyeda is identical with B. solanacearum Smith. (5) REPORT ON THE ATTEMPTS TO DISINFECT WITH CHEMICALS THE SEED-BEDS ON SLIME-SICK SOILS. On 20 plantations belonging to 1 1 companies tests were carried out, using potassium perman- ganate, formaldehyd, and carbon bisulphide. The seedlings on 6 of the plantations were destroyed by ants and crickets. Report is made of the results on each of the other 14 plantations which show some success with the disinfectants, but not brilliant nor uniform. The entire subject is summed up in the following conclusions: (1) That in spite of the disinfection of seed-bed and of the well-water used, when the seedlings were planted on infected ground infections took place. (2) That this infection, as far as could be made out, took place more through the leaves than through the roots, so that the infection must have been brought to them from without. (3) How this transmission took place (whether by animals or tools) must be followed up and experiments are already under way. (4) It must also be looked into closer whether the disinfection of the whole seed-bed ground gives an important advantage over the disinfection, simply, of the seed-beds, leaving the paths between untreated. (5) That far away, in most cases, the plants were larger and more vigorous on the treated than on the untreated seed-beds. (6) That in all cases where differences appeared after planting out, they were to the detriment of the untreated seed-beds, so that it certainly is desirable to repeat the tests. (6) A CASE OF SLIME-SICKNESS IN DJATTI SEEDLINGS. This was found in Djatta plants [Tcctona grandis] standing among diseased tobacco. The signs appeared to be those of the slime-disease. Plate cultures were set and three sorts of bacteria were isolated, one of which agreed culturally with B. solanacearum from slime-sick tobacco. Although the identification by cultural tests left no room for doubt, inoculations were made into 4 tobacco seedlings, all of which became diseased. Out of two of these plants the bacteria were again isolated and served for new inoculations along with a strain isolated from tobacco. With these three strains 15 young tobacco plants, on land not subject to the disease, were inoculated by a prick, 5 from each culture. At the same time 9 young Djatti plants were inoculated, 3 from each culture. All of the tobacco plants showed the disease at the end of a week ; the Djatti did not. Three days later the tobacco was removed and the earth disinfected, although the disease was still confined to the top of the plant, i. e., not yet in the roots. All the Djatti plants were now diseased, not only those inoculated from the Djatti cultures, but also the 3 inoculated from the tobacco strain. No difference in virulence was observed on Djatti plants, but on the tobacco DTi was much the more virulent. The checks were sound, both the Djatti and the tobacco. With other cultures from the same source the inoculation experiment was repeated 4 days later on a trial field on 15 Djatti seedlings with the surprising result that only one of them died, the others being not much affected. Six weeks later, however, two other cases were found in the plants inocu- lated with the Djatti bacteria. The disease was visible soon after a couple of showers and the bac- teria were demonstrated microscopically in the stems more than 2 dm. above the point of inoculation. Later with Dl\ and T four tobacco plants in pots were inoculated, all of which became diseased in 5 to 7 days. By inquiry Honing learned that on other plantations occasionally diseased Djatti had been observed. (7) ON THE CONNECTION BETWEEN SLIME-SICKNESS IN SEEDLINGS AND IN PLANTED-OUT TOBACCO. In "Cause of the slime-sickness and attempts to combat it — III," were cited some instances of direct connection between disease in the seed bed and in the field, even in cases where the disease in the seed-beds was only sporadic and sometimes was not known. In "Report on the slime-disease WILT-DISEASES OF TOBACCO. 255 tests in 191 1," the number of examples were somewhat increased. Here are added some observa- tions of 1 91 2, made on tobacco seedlings received from 1 1 sources and set on the trial grounds. The conditions of the experiment and the results are given in table 34, omitting only the dates of planting (February 14 to March 30). Table 34. — Effect of Occurrence of Disease in Seed-bed on Planted-out Tobacco. Planta- tion. 1 2 3 a )b 4 5 6a 66 7 9 10 1 1 a n b 8 a Condition of seed bed. No disease. Quite a good many diseased. ....Do Less sick than in 3a A few diseased Many diseased Very few diseased .... Do Quite a good many diseased . ...Do Few diseased ....Do _.. Few diseased (specimens with bacterial stripe). Sound out of the same infected beds. Very few diseased (specimens with bacterial stripe). Sound out of the same sick beds Prediction as to outcome. Few dead A good many dead. Many dead Less dead Not many dead Many dead Few dead ....Do.... Quite a good many diseased. .... Do No remarks Few dead All dead Few dead All dead. Many dead . No. planted. No. Dead. 36 27 72 12 54 22 54 1 I 72 21 72 42 54 6 54 7 54 21 54 15 72 '4 72 '3 15 15 20 4 21 20 45 23 P. ct. dead . 75 17 41 20 20 58 1 1 13 39 28 19 18 100 20 95 51 Remarks: Predic- tion fulfilled (+) not fulfilled (-). — (Toa-toh and drought). + + + + + + + + Toa-toh bibit + + + + + The plats 11b and 86 are perhaps the most interesting. Naturally, sources of error were not wholly excluded. The factors which may have influenced the result are : (a) (b) The soil was not wholly free from the slime-disease. The persistent drought. (c) A toa-toh plague, especially for 1 to 9; the latter had toa-toh in the seedlings. Ul) The fact that the seedlings from all sorts of ground, loose red earth to low white clay, were planted out on the same piece of clay ground, (f) The small number of plants. All things taken together, it is plain that the dying in the field tobacco was more prevalent according as more sick seedlings occurred in the seed-beds. (8) ON THE ASSERTED NON-SUSCEPTIBILITY OF NICOTIANA RUSTICA FOR THE SLIME-DISEASE. Two ways stand open for him who would obtain a race of resistant Deli tobacco : The first looks to the discovery of fine sound specimens on slime-sick soil ; the second to the crossing with less sus- ceptible or wholly resistant varieties of tobacco. LIpon the first method more later. In order to cross the Deli tobacco with immune sorts we must first have these, and if obtained, it remains to be seen whether the desired peculiarity is transmitted in full measure to the descendants and is constant in the cross. If this should fall out according to wish, then comes the question whether the type obtained is of a sort that the planters will try, or whether it must be further im- proved, retaining at the same time the desired resistance. Tests were also made on a number of ornamental tobaccos, seed of which was received from Haarlem, viz., Nicoliana affinis, N. atropurpurca grandiflora, N. colossea, N. glauca, N. saudcrae (in 6 colored varieties), and N. silvestris. Both the Djatti and the tobacco strains were tried and all of the inoculated plants became diseased. Nicoliana latissima is also subject. The finer sorts of foreign tobacco of the species N. tabacum, so far as tested in Deli, are badly attacked by the slime-sickness, so that the chance of obtaining a reistant variety from these seems not better than from Deli tobacco. Of the coarser sorts it has been announced in literature (Uyeda) that one, namely, the Boer tobacco (N. rustica), is non-susceptible to the slime-disease. There were obtained, therefore, from the Agricultural Institute at Pusa in India, where this tobacco is commonly cultivated, seeds of different varieties to the number of 20, which varieties were tested by inoculation, only one of 200 plants remaining sound. The details are given in table 35. 256 BACTERIA IN RELATION TO PLANT DISEASES. Table 35. — Result of Inoculating B. solanacearum into 20 Types of Nicotiana rustica. Type. Pot trials, infected 3 days after planting. Field trials, infected 10 days after planting. Slime-siek after infection with bacteria from — Slime-sick in the controls (of each 4 examples). Slime-sick after infection with bacteria from — .Slime-sick in the controls (of each 8 examples) . Djatti stem (of each 3 examples). Tobacco stem (of each 3 examples). Djatti stem (of each 2 examples). Tobacco stem (of each 2 examples). I 11 III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0 0 0 0 0 0 O 0 0 O O 0 O O 0 1 O O 0 o 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 OOOOOOOOOOOOOOOOOOOO "The possibility remains that Uyeda possessed another truly immune variety, yet his communi- cations on other infection experiments which have been described earlier, make this highly improbable." (9) ON THE VARIABILITY OF BACILLUS SOLANACEARUM SMITH. During the search for combinations of carbon and nitrogen foods by means of which the slime- bacteria could be easily recognized, a variable behavior was often observed in media inoculated from the same culture. That this did not depend on faults of technic is shown by the fact that transfers of the bacteria into far more than 200 cultures of bouillon always succeeded; also all grew when inoculated into pep- tone-water (33 cultures), solution of ammonium succinate (32 cultures), ammonium lactate (33 cul- tures), asparagin with sodium malate (21 cultures), and sodium succinate (21 cultures). In the test-tubes in which visible growth occurred the originally feebly acid reaction of the solu- tion had become neutral, or alkaline (if growth was strong), while the tubes that remained clear always retained the feebly acid reaction to litmus. Experiments were then made by inoculating into solutions whose acidity or alkalinity had been increased slightly, with the results given in table 36. Table 36. — Growth of B. solanacearum in Meyer's Mineral Solution 'with 0.2 per cent Glycocoll and Glucose or Mannit with Variable Reactions (Cultures always in Sets 0/3). Carbon food. Per cent N/io sulphuric acid or sodium hydroxide added. Source of bacteria used. No. of tubes clouding. A,. A,T. A.T. T,. Glucose, I p. ct Do.. 2.5 H2SO4 3 3 1 2 2 0 0 0 0 0 3 5 3 2 (of 2) 3 2 (of 2) 3 0 3 3 3 1.25 HiSO,... Do .. 2 1 O 3 * Do... 1.25 NaOH 1 >S04 2 3 6 -4 7 0 0 0 0 O 2 3 3 0 0 3 1 3 3 With 1.25 p. ct. N/ioNaOH With 2.5 p. ct. N/10 NaOH Temperature and light are said to play no role in this variability. The test-tubes had been cleaned with acetic acid, but some soaked in chromic-acid cleaning mixture ga\re the same result.* With asparagin and potassium nitrate the results shown in table 39 were obtained. Table 39. — Variable Behavior of Different Strains of Sumatran Tobacco Organism to Combinations of Carbon and Nitrogen Foods (Tests always in Sets of 3 Tubes each). Feebly acid solution. Strain oi B. solanacearum used. Feebly acid solution. Strain of B. solanacearum used. A, AiT. A-T. T, T,. A.. A,T. A:T. T,. TV Asparagin: 0 0 0 1 0 0 0 3 2 2 1 1 0 2 2 3 3 3 0 0 3 2 3 3 3 0 0 [ 1 2 3 2 3 0 3 KN03: 1 2 1 3 0 0 3 1 3 3 2 0 0 3 1 3 3 3 0 0 3 2 3 3 3 0 0 2 0 1 3 3 0 0 0 Ervthrite Galactose Inosit Ervthrit Galactose Inosit Levulose Lichenin Mannose Levulose Lichenin Mannose Further studies showed that media which failed to show growth when inoculated with small doses of the bacteria would do so when inoculated with larger quantities, as shown in table 40. vSimilar results were obtained in neutral asparagin-glycocoll-KN03, or urea solution, with glucose or mannose, the heavier inoculation yielding the better results. When measured quantities of the bacteria were used the results shown in table 41 were obtained. *Medan III and Florida potato (1914) grew in Witte's peptone water + H2S04 (1 :ioooo), but not in the same acidulated 1:1000 (3 days test at 270 C). 258 BACTERIA IN RELATION TO PLANT DISEASES. Table 40. — Growth of Strain A2T in Glycocoll-glucose Solution after Inoculation with a Small and a Large Loop of Culture Fluid, the Latter Carrying about 5 times as many Bacteria as the Former {Tubes in Sets of 3). Glycocoll + glucose solution. Day, and small or large loop. 2 3 4 s 6 10 Small. Large. Small. Large. Small. Large. Small. Large. Small. Large. Small. Large. With 2.5 p. ct. N/10ILSO4 With 1.25 p. ct.N/10 H.SO4.... Neutral O 0 0 O 0 0 O 0 0 0 0 O 0 0 0 0 3 2 3 1 1 0 0 2 3 3 3 2 1 1 I 0 O 2 3 3 3 3 2 3 2 0 0 2 3 3 3 3 3 3 2 1 0 2 3 3 3 3 With 1.25 p. ct. N/10 NaOH. . . With 2.5 p. ct. N/10 NaOH. . .. Table 41. — Time of Clouding in Neutral Glycocoll-glucose Solution when Inoculated with Variable Numbers of Sumalran Tobacco Bacteria (10 Tubes used in each Case). Days required for appearance of growth. Number of bacteria used for inoculation of each tube. 300 3.000 30.000 2,400,000 6 10 '5 20 O 0 O 0 0 1 1 1 4 9 10 10 This disease has now been observed in Sumatra in 15 species of plants belonging to five families. The author summarizes his conclusions as follows : 1. With a great number of carbon and nitrogen foods B. solanacearum shows growth only in a part of the cultures inoculated. 2. Light and temperature have no influence, and small variations in alkalinity have slight influence on this variability. 3. More cultures succeeded after a larger quantity of inoculating material was used. Only a very slight number of individuals, one out of many dozens, is in condition to adapt itself to the new nutrient conditions. 4. The phenomena fall under the concept of modification; the acquired peculiarities are not constant, when the bacteria are once more inoculated into tobacco, reisolated, and again brought into the same solution. There is perhaps another explanation for the variable behavior of this organism in unfavorable solutions, viz, accident. On this supposition all the bacteria are equally capable of growth, but only those actually grow which happen to be lodged under the protecting cover of their fellows, *. e., those in the center of small pseudozoogloese masses. If no masses large enough to protect the first stages of growth are introduced then those particular tubes will remain sterile. This is indicated to me by the fact that Honing often observed the first indications of growth in glycocoll solutions "as little points on the wall of the tube which may grow slowly for some days before the fluid begins to be clouded." Glycocoll solution was used because Bad. solanacearum was easily identified in it on account of the development therein of the peculiar chains and involution forms already described. (10) ON THE DECAY BACTERIA FROM SLIME-SICK TOBACCO, DJATTI, AND SOME OTHER OF THE SLIME-SICK SUSPECTED PLANTS. In December 191 2 Honing published another paper, with the above title, in which he reported on the common bacteria found by him in Sumatra associated with the bacteria causing wilt of tobacco and other plants, but themselves not able to cause any disease when inoculated into tobacco. The following sorts were isolated and studied by him and the new species are described: WILT-DISEASES OF TOBACCO. 259 Micrococcus luteus Lehm. and Neum., Micrococcus pyogenes albus (Rosenbach) Lehm. and Neum., Micrococcus pyogenes {M. bicolor Zimmerman), Bacterium medanense n. sp., Bacterium stalactitigenes n. sp., Bacterium langkatense n. sp., Bacterium deliense n. sp., Bacterium shuffneri n. sp., Bacterium zinnoides n. sp., Bacterium sumatranum n. sp., Bacterium patelliforme n. sp., Bacterium aurantium roseum n. sp., Bacterium rangiferinum n. sp., Bacillus mycoides Fliigge, Bacillus mesentericus, and Corynebacterium piriforme n. sp. These generic names are used in the manner of Lehmann and Neumann, some of the forms being non-flagellate, others polar-flagellate, and others peritriehiate. The most interesting discovery recorded is that of the inhibiting action of some of these saprophytes, c. g., B. mycoides, and especially B. mesentericus, on the growth of Bad. solanacearum (fig. 134). The following is an abstract of Honing's last paper. (I I) HOW SHALL ONE OBTAIN A RACE OF TOBACCO THAT IS IMMUNE TO THE SLIME-DISEASE? Probably the application of some form of selection in tobacco-culture in Deli is as old as the culture itself. The founder of the industry, Nienhuijs, had seed imported from Java, Cuba, and elsewhere, and selected that which gave the best results on the new soil. Obviously he selected with a practised eye and lucky hand: A valuable new variety, the Deli tobacco, being the result. No one knows the age of Deli tobacco in its present form. Perhaps it is the result of one or more accidental crossings among the sorts imported into Deli; perhaps it originated through a splitting — caused or not by crossings in Deli — whereby arose combinations of char- acters which the first Deli plants really owe to crossings that took place years before in Java or Cuba. Perhaps, also, the new type arose suddenly or by degrees through ex- ternal conditions, which in Deli differ from those of the original habitats. Of this we know nothing, and prob- ably never shall be able to determine. A similar ignorance concerning the time and manner of origin of varieties is, alas, the rule. Of almost all culti- vated plants having a great number of known varieties, the origin lies in the dark, and only of forms which have been isolated in recent years does one know a part of their history. Only this is certain: In Deli a variety has arisen which did not previously occur in the older tobacco lands and is not found there at present, unless introduced from Deli. Wherever Deli tobacco grows outside of Sumatra: in Java, the United States, Cuba, Hawaii, everywhere one speaks of Sumatra tobacco, which would not be the case if, in the older tobacco districts, the Deli form had been recognized as a variety indigenous there. Had such been the case, so much trouble would not have been taken on all sides to make the variety nourish. Thanks to the favorable results in the years 191 1 and 191 2, one hears less said now than was the case three or four years ago, regarding the degeneration of the Deli tobacco. For *Fig. 134. — Drawing of a Petri-dish agar-poured-plate showing the inhibiting action of Bacillus mesentericus on the Sumatra tobacco bacterium. This experiment was made as follows: The letter A was painted on the bottom of the dish with a spore-bearing culture of Bacillus mesentericus. When this was dry it did not show. A tube of melted agar was then inoculated heavily with Bacterium solanacearum and poured into the dish. After some days, the letter A appeared and some surface colonies of Bacillus mesentericus which were derived from bacteria flooded from the letter A at the time the plate was poured. Bacterium solanacearum has also grown, but only at a distance from the former. The legends are: i, Bacterium solanacearum; 2, Bottom growth of Bacillus mesentericus; 3, Surface growth of Bacillus mesentericus. (After Honing.) Fig. 134.* 260 BACTERIA IN RELATION TO PLANT DISEASES. the benefit of those who still believe in the degeneration of the Deli tobacco, it is perhaps of service to state that the first Deli planter, fifty years ago, lost a whole field through "the death." At that time the tobacco was immune to the slime-disease even less than now. From this we may conclude that the slime-disease bacteria were here earlier than the culture of European tobacco. Always and everywhere goes hand in hand with extensive culture the multiplication of parasites, animal as well as vegetable, as the result of the one-sided activity, and always and everywhere it has cost much labor to keep a crop on the same level and still more to improve it. Therefore, especially in recent years, men have begun to select examples more resistant to disease, and have found these in forms which, in other respects, are unfit for culture, so that they have tried through crossing, followed by systematic selection, to produce the desired combination. In both ways, that is by selection and by crossing, the attempt must be made to produce from the Deli tobacco a less susceptible race. The selection can begin immediately; for the crossing one must wait the discovery of immune varieties, which must not be far re- moved from the Deli tobacco in quality. And because a variety, which elsewhere is little or not at all susceptible, may be extremely sensitive when subjected to the different soils and climatic conditions in Deli, one must not rely upon the literature, but all varieties must be investigated in Deli in regard to their susceptibility. This year tests have been made with 87 sorts or varieties, among which were good varieties from Java, the United States, Cuba, Mexico, Brazil, Paraguay, Santo Domingo, the Philippines, Japan, and Hawaii. In a later report these experiments will be described in detail. The following are the results in brief: (1) All of the 87 sorts or varieties investigated were susceptible to the slime-disease. (2) The 7 strains of Deli tobacco, which were inoculated at the same time as the foreign tobaccos, did not make a bad showing. They grew better after the inoculation in the stem than a great number of foreign sorts. (3) Two sorts of Philippine tobacco and one Japanese variety, although diseased, gave the impression of being less severely attacked, so that it is worth while to repeat the experiment on a larger scale with these three varieties another year. (4) The disease progressed more rapidly when the inoculation was made with a great number of bacteria than with a few. This last-mentioned fact affords perhaps the explanation of the remarkable phenomena which occurred in the disinfection-tests in 191 1 . In the different experiments the results of the disinfection were at first gratifying, but in the end, when the plants were examined, it appeared that not any or only a very slight advantage had been gained. But in the mean- time in one of the experiments on a trial plot which had been kept under longer and closer observation there were on each plant five to eight leaves more ("scrubs" and basal leaves). Thus disinfection was insufficient, but the important lessening of the number of bacteria had been advantageous. Now it appears that the rapidity with which the disease progresses in a plant is depen- dent on the number of invading bacteria [see pi. 31, middle and right figs.]. This affords perhaps the explanation of the above-described phenomena in the disinfection-tests of 191 1. The chance of finding among foreign tobaccos one which is immune in Deli appears to be not very great. However, the fact that among the 1 19 examined varieties or sorts not a single resistant one was present does not mean that none exists. The investigation, which fortunately does not consume an undue amount of time, must be made with still other sorts. Only, one must not raise too high his expectations of finding among the known varieties one which in Sumatra is immune to Bacillus solanacearum. And this is surely an additional reason for immediately subjecting our Deli tobacco itself to a severely conducted selection. There are various reasons which make it more than probable that the Deli tobacco is not a clearly circumscribed type, but that it consists of a mixture of several types — how WILT-DISEASES OF TOBACCO. 261 many can not be estimated. Apparently there are only a few, and in any event they are not of such a kind that one can go out into the field and select them. The Deli seed is much purer in Sumatra than that, for example, which was grown some years ago in America, when Shamel found 1 1 types in one field. The "Deli seed " with which this field in Connecticut was planted came from Florida and may thus have been mixed with other sorts. Proofs that in the Deli tobacco hereditary differences still appear are but two, namely, the giant plants and the .Strain B 1-5, which in 1906 was obtained in a mosaic-disease test, undertaken with Helvetia and a couple of other varieties. These plants, B 1-5, blossomed in 1907 about 10 days earlier than the others, formed thus a premature type, and this year the sixth generation again blossomed 10 to 12 days earlier than the others. Dr. Diem found in 1907 that the average difference in height between B 1-5 and the race B 1-1 was almost half a meter to the disadvantage of B 1-5, which also produced on an average four leaves less. Moreover, these differences, which indeed that one year were somewhat greater than in other years, have persisted, so that B 1-5 is to be considered a separate type. Besides these two examples there are perhaps more. One often finds in the plantations plants which vary. When it is a single example which is distinguished by broad or small leaves, there is no reason to consider it immediately a separate type. On the contrary, when one gets this impression from a whole plot (afdeeling) it proves that two forms, a broad-leaved and a small-leaved type, have been planted side by side. I remember such a case in an upland plantation in 191 1, and I am not the only one who was subject to this impression. Up to the present there have been but few experiments in which selection has been exercised in the sense of pedigree-culture, such as was advised as early as 1907. One gener- ally restricts himself to the maintenance of a fine stock from which the less vigorous plants are removed; thus a form of "mass-selection." Unquestionably this is one means of keep- ing the culture up to the mark, but the chance of making any advance in this way is ex- tremely slight. To be sure by this method the seed of the best plants is always collected and when there happens to be among them a couple of extra good plants which will produce better offspring, the seeds of these are also gathered, and are not lost. But this seed of the uncommonly good plants becomes diluted with the quantity of common seed which out- number them by some hundreds or thousands. On sowing, the descendants of the best plants are lost in the great mass. In case the circumstance should arise that the descend- ants of such a plant with large leaves should grow somewhat less rapidly in their youth, something more happens, namely, these will disappear during the thinning out, with the result that in the crop, the same as the previous year, but few examples of the better type will appear. If now, again, on a good piece of ground a portion of these be selected for seed plants, then is one just as far advanced as the year before — a single plant of the better type here and there in the great mass. Had one, however, kept separate the seed of a couple of extra fine plants, then it would soon be evident whether the wished-for characteristics were hereditary or not. A concrete example will make this still clearer. On two plantations there has been made this year, at my request, a beginning in selection-tests for immunity to the slime- disease. In one plot on one of these plantations, where there had been much damage from the slime-disease, there were in the worst field 40 plants which had remained intact (zich goed gehouden hadden), and which were guarded from cross-pollination by gauze sacks. The plants had the greatest chance of becoming infected, since the mortality on this spot had amounted to at least from 60 to 80 per cent, and the rest, especially toward the last, appeared to be very much diseased. At the present time it (the disease) is not evident on the plants, which appear to the eye to be sound. It is possible that by chance they have not come into contact with virulent bacteria and possibly in case of an infection they might be very susceptible, but there is also the chance that some of them would be somewhat more resistant, and a single one much more resistant than the average, to say nothing of 262 BACTERIA IN RELATION TO PLANT DISEASES. immunity. Since all this is not evident to the eye, too small a number of plants should not be isolated, and the seed from each must be watched, kept, and sown separately. Suppose the case that one plant of the 40 was by inheritance so much more resistant than the others that under exactly similar circumstances and on the same kind of soil mortality among its offspring, instead of being from 60 to 70 per cent, was only 5 per cent, while all the rest were barely alive as the result of infection. Now, what would happen if the seed of the 40 plants were mixed ? The mixed seed would give a crop which under the same conditions and on the same kind of soil and with the same amount of infection as the year before, would again show 60 to 70 per cent dead plants. The one superior plant has no noticeable influence on this mixture with the 39 others, and the result is that one must say: "This is just as bad as last year. This method amounts to nothing. Away with it!" If one had, on the contrary, kept this seed separate and planted its offspring separately, then it would be evident that of the 40 selected mother-plants there were 39 worthless ones and but one which would be of service for further testing. Thus in two respects the above-described method of work (that is, the method having, as its special purpose, the obtaining of an immune or more resistant race of tobacco) differs from the one customarily employed: (1) The seed-plants are not selected from the best soil, but preferably from the "sick soil" or places where the loss from the slime-disease has been very great. The chance of resistant individuals becoming conspicuous there is much greater than in places where not a single plant has been exposed to the disease, and thus no plant gets the opportunity of showing its ability to withstand adverse conditions. Twenty or thirty plants remaining sound in a field in which there is much disease give more chance of results than a thousand from a Held without any disease. (2) The seed from every one of the carefully selected plants is watched and sown separately. Pedigree-culture is introduced. That these methods involve extra labor it is useless to deny, but without work one accomplishes nothing. In great companies it will certainly be desirable to have a separate "seed assistant," but by "seed assistant" must be understood something other than a person who only sees to it that the company obtains the prescribed number of packages or bottles of clean seed from well-cared-for, active plants. In my opinion he must be a man who tries to select the types most suitable for certain kinds of soils. For this is one of the great differences between the modern culture and the old: one chooses the plant-type according to the kind of soil, well knowing that the soil may be improved but not changed in character. Since it is now known that in Europe for clay, sand, and peat soils, or for com- binations of all kinds of soils, definite varieties of grains, potatoes, cabbage, apples, etc., are used, why should not the same principle be applied to Deli tobacco? Only when it is certain that the Deli tobacco is a constant variety will efforts in this direction have no sense. But the complete uniformity of the Deli tobacco is anything but decided. Every planter knows instances of his ability to recognize in the curing-room the tobacco from a certain field by its color, texture (soepelheid), or by almost indescribable differences. vSometimes variations in rainfall are the cause of the difference, but sometimes there are variations occurring every year, caused by an entirely different composition of the soil. In most cases the crop thus differentiated is less fine. Would it not be more profitable to apply at once a system of strict selection with reference to the soil ? A principle that was very much applied in the past and still is followed is this: Use for an upland plantation seed from a lowland plantation, and vice versa. The purpose of this restoration (bloedverversching) has never been clear to me. Different companies have also resumed growing their own seed, which is continually used for the same plantation; a single company does this for every field. That is surely a step in the right direction. But one must, in my opinion, go still further and give attention to the kind of soil rather than to chance boundaries of the plots or experiments, so that one plantation must try WILT-DISEASES OF TOBACCO. 263 to cultivate two, and if need be three, types which are continually kept for the same kind of soil. This is directly opposed to the principle of "blood-refreshing." As it is agreed that in this "blood-refreshing" the habitat and the climate have influence on the charac- teristics of the offspring, something which is not proved but which we grant for the moment, why should one use seed from a hill plantation which always has sufficient, sometimes too much, rain, for cultivation on the coast where the tobacco stands every chance of being obliged to go through a long period of drought ; that does not seem to be logical. If from the Deli tobacco several types can be isolated, and this seems probable, never- theless one must not suppose that they will be precisely alike in their ability to endure drought or much moisture. Should not something be established in this direction on the coast plantations, where all the growers are again complaining? What has been said here about selection in general is also of value in regard to the selection for the purpose of obtaining a race more resistant to the slime-disease. Also in this connection one will do well to take into consideration the kinds of soil. What will happen I can not assert, but the possibility is not excluded that some one will obtain a more resistant race which is of no value for his neighbor who has different soil, and is perhaps still more susceptible than that which he has been accustomed to plant formerly. What makes this selection difficult is the fact that one can never see in a plant what he has there, although always its value as a seed-plant can be judged the following year in the descendants just as leaf-production is best judged in the curing-house. Two plants of similar height, with exactly the same number of leaves of about the same length and breadth, in short, plants which every one would consider equally valuable, may bring forth offspring which differ to an important extent. A plant with 30 leaves may belong to a family which brings forth on an average 30 leaves, but it may also be an exceptionally fine specimen of a family which produces on an average not more than 25 or 26, or, finally, an inferior example of a family which averages ^ leaves per plant. All this indicates the necessity of sowing separately purely pollinated seed from each plant. In such pedigree-cultures two or three years must elapse before we know whether the selected lines are constant. This affords the great advantage of comparing pure lines with the ordinary crop for two consecutive years, not only in the field but also in the curing-house. AMERICAN STUDIES OF THE SUMATRAN ORGANISM. Through the courtesy of Dr. Honing the writer received cultures of the Sumatran tobacco organism in the summer of 19 13. One was sent direct, another via Amsterdam, where the original tube was retained and a transfer sent on. Later a third came direct. The three cultures reached Washington in good condition and proved infectious to tobaccos, on which the external signs and the tissue disorganization could not be distin- guished from those due to the North American tobacco-wilt organism. Black stripes appeared on the stems, with yellowing and irregular drying-out of the leaves, the vessels in which were stained black and filled with the bacteria, especially those of the mid-rib. In the sections which I saw, the bacteria were confined to the inner wood, in which bacterial cavities had formed. Some of these sections were made 5 or 6 inches from the needle pricks. Up to the end of the sixth week (when the first experiments were broken off) the be- havior of this organism in our tubes of milk and litmus milk corresponded closely to that of the Granville tobacco-wilt organism, i. c, there was no development of acid or separation of the casein, but only a progressive bluing of the litmus.* Subsequently one of our stocks of litmus milk reddened. I thought at first that the reddening of the milk was due to an unsuspected acid-forming anaerobe, which was not killed by the steamings, and which was unable to make its presence visible except under the protecting influence of some active aerobe, such as Bad. solanacearum. With this in mind we made tests in deep tubes of agar in the following way : Stabs were made the whole length of the agar from litmus milk which had been inoculated with the Medan organism and which *Plain milk tube-cultures 7 weeks old were translucent and brownish. In another set a pencil could be seen behind those 8 weeks old, although the casein was not precipitated. 264 BACTERIA IN RELATION TO PLANT DISEASES. had reddened. On the surface of the agar a greedy aerobe was then sown (Bacillus subtilis) with the idea that if an anaerobe were present in the milk it would develop in the depths of the agar, whereas if only Bad. solanacearum were present the growth would not appear along the line of the stab or would occur only in the top of the stab. The second of these supposi- tions proved to be the true one, indicating that the litmus-milk cultures which reddened contained only an aerobe, viz, the Medan organism. Owing to the fact that the media which reddened had been made from uncentrifuged milk containing cream, whereas our ordi- nary litmus milk is always made with centrifuged cream-free milk, it then seemed likely that the acid must be due to the action of the bacteria upon the cream, not on the milk sugar, and this was borne out by subsequent tests. When we inoculated skimmed (cream-free) lit- mus milk we always got a gradual blueing of the litmus, just as in case of the American organ- ism, and never any subsequent reddening. On the contrary, whenever we added cream to the skimmed milk or prepared the litmus milk out of milk containing cream, we got a red- dening which began always in the layer of cream at the top and gradually progressed until the whole tube became reddened (pi. 44, figs. 2, 3, 4). There can be no doubt I think, therefore, that the discrepancy be- tween Honing's results and my own is not a real one. The Medan organism, like the American one, blues cream-free litmus milk. The Medan one reddens mixtures of cream and milk containing litmus, and probably the American tobacco organism would do the same thing, although we have not tested it.* In repeated tests we have never ob- served any reddening in cream-free litmus milk, using Honing's organism, but al- ways a reddening when cream was added. Coconut oil added to cream-free lit- mus milk is also split by Medun III with the production of an acid, but under the same conditions no acid was produced from olive oil, peanut oil, or cottonseed oil. Whenever a thin layer of sterile cream, paraffin, or any of the above- mentioned oils was placed on the sur- face of the inocu- lated litmus-milk, Fig. I34i.j Fig. I34a.f so as to interfere with the absorption of oxygen, there ensued a distinct reduction of the litmus, but this reduction was not observed when the surface of the litmus milk was freely exposed to the air— then the lavender blue milk became and remained a uniform deep blue. "The 'inly American strain I have tested is the Florida potato organism (1914): This dots not redden creamed litmus milk. In each of 4 trials the numerous inoculated tubes became anil remained bluer than the checks. At the end of j weeks this strain had grown well in Uschinsky's solution and very feebly in (John's solution. It is motile. (■FlG. t340. — Flagella of Sumatran tobacco organism. Culture received from Honing. JFlG. 1346. — Voting tobacco plant wilted by motile bacteria, a cheek on Fig. I.143- Honing, J. A. Hoe moet men trachten een tabaksras te verkrijgen, dat immuun is tegen slijmziekte? Mededeelingen van Het Deli Proefstation te Medan, Jaargang VIII, ie Af- levering, August I9'3i PP- 12-21. 1913. JENSEN, Hi. Slijmziekte. Mededeelingen van "Het Proefstation voor Vorstenlandsche Tabak." No. 5, Batavia, 1913, PP- 48-60. No infections with Uyeda's B. nicotianae — 60 plants inoculated. Fulton, H. R., and J. R. Winston. A disease of peanut plants caused by Bacterium solana- cearum. Phytopathology, vol. 3, No. 1, February 1913, pp. 72-73. Mere mention. Through courtesy of Dr. Fulton I have received the following note: In August 1912, peanuts {Arachis hypogaea) of the Spanish variety growing in Granville county. North Carolina, on soil known to be infected with Bacterium solanacearum (Erw. Sm.), were observed to be dying to the extent of 15 per cent of the planting. Examination of stem and root of the affected peanut plants showed discoloration and disorganization of tissues closely similar to those characteristic of Granville wilt of tobacco. The newly affected parts contained numerous bac- teria. Pure cultures were obtained from which both young and old peanut plants were successfully inoculated on several occasions, the disease induced being similar to that observed in the field. The organism was reisolated from such artificially infected plants, and this culture again used for infecting the peanut. Young and old tobacco plants were inoculated with the original cultures from peanut, with the production of dis- ease similar in all respects to Granville wilt of tobacco, and the typical organism was reisolated from these artificially infected tobacco plants. Cultures from naturally infected tobacco and pepper plants produced the disease in peanut. Cultures of three strains of the bacterium from peanut were compared with cultures from tobacco and from pepper on various media, such as potato agar, nutrient gelatin, potato strips, peptone broth, glucose broth, beef broth, potato broth, milk, and litmus milk. The cultural features on these several media were similar for alt the strains tested, and agreed with the original descriptions for Bacterium solanacearum. In size and shape, and in reaction to the ordinary stains, these several strains agreed with Bacterium solanacearum. The facts lead to the conclusion that peanut is susceptible to attack by Bacterium solanacearum. 1913. Hutchinson, C. M. Rangpur Tobacco Wilt. Memoirs of the Department of Agriculture in India. Bacteriological Series, vol. I, No. 2, July, 1913. Agric. Research Institute, Pusa. Calcutta and London, pp. 67 to 83, with 12 plates (5 colored). 1913. DE Jong, A. W. K. Eenige Opmerkingen over de Bacterienziekte (Pelepes) bij Katjang Tanah. Dept. van Landbouw, Nijverheid en Handel, Medeelingen van het Agricultuur Chemisch Laboratorium, No. Ill, Buitenzorg, 1913. PP- 56-59. No advantage was obtained from flooding infected fields for some weeks. Very hopeful results were obtained Irom the planting of selected sound seed. [See also Literature under Brown Rot of Solanaceae (p. 218) and Wilt of Peanut (p. 153).] SCHUSTER'S GERMAN POTATO ROT. OBSERVATIONS BY SCHUSTER. In 1912, Schuster described a polar-flagellate, green-fluorescent schizomycete from rotting potato tubers obtained from six different localities in Germany, as Bacterium xantho- chlorum n. sp., and asserted it to be the cause of the disease. The organism when inoculated also blackened, it is said, and destroyed leaves and stems of Vicia faba, and rotted other plants. It is by no means as active an organism on potato as Bacillus phytophthorus. It does not attack the stems of potato, and from Schuster's own account it seems to be a rather weak parasite except at high temperatures (most active, he said, at 36. 50 to 380 C.), or under other abnormal conditions, e. g., heavy doses of the bacteria, under bell-jars in very moist air. In dry air at room-temperatures the inoculations soon dried out and the tuber as a whole remained sound. At times it showed a decided tendency to occupy the vessels, c. g., in Vicia faba, and in early stages of the tuber-rot of the potato. All of Schuster's inoculation experiments appear to have been made under abnormal conditions, i. e., in laboratory rooms, under bell-jars, etc., in a saturated air. His Bact. xanthochlorum causes a slow, gray or yellow, wet-rot of the tuber, often proceed- ing from the vascular bundles in or out, and from the margin of this rot it is easy to isolate pure cultures. Often the tubers are not much injured on the surface. The rotted parts smell of ammonia. It is not able to enter the potato tuber through the opened lenticels [another indication of weak parasitism]. In general it is a wound parasite, but it may enter, it is said, the green parts of Vicia faba through stomata. It was inoculated successfully by Schuster into carrot roots, young tobacco stems (300 to 360 C.)j Lupinus nanus douglasi, Physalis alkekengi, and Campanula raphunculus. Fodder beets, sugar beets, yellow lupins, tomatoes, and pelargoniums proved insusceptible. This organism, according to Schuster, has the following morphological and biological characteristics : As a rule it is actively motile, but once a non-motile strain was isolated [this may have been some related organism]. The flagellum is polar. Sometimes two flagella are present, rarely three occur. The straight ones are 4.5 to 6ju long, i. c, two to three times the length of the rod. They stained poorly with carbol-fuehsin, but readily with Pepler's stain followed by carbol gentian violet. Hinterberger's modification of Van Ermengem's stain was also used successfully. At temperatures under 50 C. and above 400 C. no flagella formation could be detected. In bouillon motility was observed for 18 days, but on potato at 200 C.it ceased after one day. On agar-streak-cultures motil- ity ceased after one day at 370 C. but was visible after 20 days at 200 C. Under normal conditions of growth the organism is a slender rod with rounded ends, 1.5 to 3X0.75M, occurring for the most part singly or in pairs. It stains readily with basic anilin dyes but not by Gram. On agar at 360 C. and at higher temperatures, long threads are produced within 24 hours. These long rods may show constrictions, but do not afterwards separate into short elements. Short, plump rods were observed at 2° C. Spores do not occur. Involution forms (plasmoptyse) occur. Pseudozoogloeas occur. The yellowish-green fluorescent pigment is soluble in water. After cultivating this organism on potato agar, carrot agar, or wheat-decoction agar for 3 months the color disappeared, but always could be restored by inoculation into alkaline bouillon, even after 6 months of colorless growth on the potato agar. Glycogen was detected in bouillon-cultures at the end of the second day using potassium iodide. No acids are produced; the cultures are always alkaline [see below]. Ammonia, methylamin, and trimethylamin occur. Trypsin, amylase, tyrosinase, and a hemicellulase are produced. A soluble toxine active on the protoplasm is also produced. 272 SCHUSTER S GERMAN POTATO ROT. 273 Cultures in bouillon containing methylene blue (0.2 c.c. bouillon-culture: 1 : 1,000 m. b.), lost their virulence on exposure to diffuse daylight for 4 hours, while similar cultures kept in the dark showed an increasing virulence. The methylene blue in 1 per cent quantity in bouillon is reduced in 3 to 4 hours. Indol occurs (distinct in bouillon after 10 days). Hydrogen sulphide is produced slowly. Nitrates are reduced. The organism liquefies gelatin, but not rapidly. It does not liquefy blood- serum. It acts on starch slowly and incompletely. It grows best at 35. 50 C. [see below]. The range of temperature is not known. Schuster says slight growth at 20 C. and at 400 and 44° C. Good growth at 27.5° C. and pretty good at 360 C. Moderate at 370 C. and at 17.5° to 18. 5° C. Growth at all temperatures from 16° to 2° C. is recorded as slight. The organism is aerobic and facultative anaerobic. Schuster gives a table of its action on sugars, alcohols, etc., but I am not able to make much out of it, except that cane-sugar, grape-sugar, milk sugar, arabinose, mannit, peptone, and asparagin favor growth, whereas ammonium chloride, ammonium phosphate, ammonium tartrate, ammonium sulphate, glycerin, caffein, arbutin, potassium nitrate, potassium phosphate, citric acid, potato starch, and cellulose do not. With ammonium chloride and the substances named immediately thereafter, there was a weak growth, with those italicised there was no growth whatever or only the slightest. These experiments were made in flasks in 100 c.c. portions of Arthur Meyer's nitrogen- free mineral solution* to which were added usually 10 grams of the substance to be tested (caffein 0.3 gr., arbutin 3 gr.). Cane-sugar, glycerin, mannit, and asparagin gave pellicles and no precipitate; grape sugar and arabinose gave pellicles and precipitate ; milk sugar and peptone gave clouding and precipitate without pellicle. These flasks were each inoculated with one loop from a bacterial suspension of a 24-hour agar-culture and were kept at about 200 C. Gas is not produced from dextrose. At room temperatures milk is slowly coagulated (eighth day) and the curd is peptonized, but at 340 C. these changes did not occur. At both temperatures the upper layers (Fettschicht) became greenish-yellow. If a trace of sodium selenite is added to gelatin which is then inoculated with this bacterium the culture becomes blood-red in 12 hours. This organism causes in nutrient agar a greenish-yellow fluorescence. On agar-plates the surface colonies are circular, slightly raised, thinning at the edges, fine granular, with a denser center. The buried colonies are smaller and not characteristic. On alkaline agar-strokes the growth is visible in 24 hours; it forms along the needle track a dry, thin, yellow- white to greenish layer, exhibiting a large number of cross-folds forming a wrinkled mem- brane. In thick sowings the folds may be lifted up vertically a millimeter above the surface, and the layer is then moist and watery or slimy [see below]. In agar-stab-cultures there is only a slight growth along the needle line. This is best at the point of entrance where after 2 or 3 weeks there is a thick mulberry-shaped piling up of yellowish bacterial masses while the surface is covered with a white skinny layer. On Appel's slant potato- juice-agar there was a very abundant development of the yellowish-gray bacterial masses within 24 hours which later fused into a thick slimy layer. In the expressed juice of cooked carrots, with 2 per cent agar, there developed along the lower part of the streak a scanty growth in the form of a thin, white, weak, moist layer. On gelatin plates very small colonies in the form of round drops appear in 1 to 2 clays. Lique- faction begins in 48 hours as a pit in which the nearly smooth-edged colony floats in the form of small lumpy masses lying one over the other. The center of the colony is considerably thickened and dark brownish. The buried colonies are globose, granular, and liquefy more slowly. In gelatin-stab-cultures near the surface there is a funnel-form liquefaction extending gradually to the walls of the tube and not far downward along the needle track, where growth is slight. Judg- ing from Schuster's figures liquefaction the first day is in the form of a narrow (steep) funnel; that on the eighth day touches the walls and resembles a champagne glass, i. e., the liquefaction is restricted to the upper quarter of the tube. Sometimes, however, after very copious inoculations there appeared along the needle-track liquefaction centers where clumps of bacteria developed, but elsewhere the needle-track was very little changed. In Appel's potato-juice gelatin (Arb. Kais. Biol. Anst. in, p. 396) there was rapid growth in stab- cultures with intense liquefaction, the entire contents of the tubes being liquefied within a few days, the bacteria being heaped up in an irregular globose mass on the bottom of the tube. The non-motile organism already referred to liquefied gelatin more slowly. In neutral nutrient bouillon a strong general clouding takes place within 24 hours. A white pellicle forms on the surface and the fluid is filled with small particles (Krumeln). There is a copious white precipitate which on shaking holds together. Grape-sugar bouillon is very favorable for the growth of this organism. It did not grow well in beer. ♦Distilled water, 1,000; KH2PO„ 1.0; CaChO, 1.0; MgSO<+7 HA 0.3; NaCI, 0.1 ; FejClj, 0.01 . 274 BACTERIA IN RELATION TO PEANT DISEASES. From a liter of alkaline potato juice inoculated 8 days a clear, bacteria-free fluid was obtained by passing it through a Chamberland filter. When double its volume of absolute alcohol was added a copious precipitate was obtained, which was tested after further purification for its effect on cell-walls of potato tubers. The walls became swollen and the typical rot appeared within 36 hours at 370 C. Here again high temperatures were necessary, i. c, such as usually do not occur in the field or in storage. I translate as follows: Experiments with enzyme injection at different temperatures showed after 3 days at 20 to 28° C. no action; at 290 C. beginning of rot; at 300 to 35° C. feeble rot spots; at 36° to 380 C. strong wet rot; at 400 to 450 C. again feeble decay; at 6o° C. no decay. At 350 C. the swollen cell walls became twice their normal width in 18 hours and the cells sepa- rated wholly or partly along the line of the middle lamella the third day. After 8 days the starch- grains were also acted upon, resembling the skeletons obtained by exposing starch to saliva at 400 C. This remainder, which does not fall apart, is believed to be amylodcxtrin. Corrosion from the margin did not occur. The enzyme which dissolves the middle lamella is designated Xanthochlorum hemicellulase. When thin sections of a potato tuber are put into infected potato juice and examined under the microscope, the protoplasm can be seen to separate from the walls of the cell after 10 minutes, and after 40 minutes it lies as a dark-brown lump dead in the middle of the cell ; therefore the bacteria excrete a toxin as well as an enzyme. According to Schuster, the action of this organism on the stems of Vicia faba is very rapid even at room-temperatures; piteh-black spots appear at the place of inoculation and within 24 hours the stems rot and fall over, but it is not clear to the writer whether these inoculations were done on plants under bell-jars, or in the open, i. e., under normal condi- tions.* vSchuster's figures 8, 9, and 10 indicate a vascular infection. The rapidity of stomatal infection on leaves of Vicia faba is something hitherto unheard of, viz, penetration of the stomata with brown staining and bacterial occupation of the vessels of the leaflets, of the petiole, and even of the stem, within 48 hours, and would seem to indicate drenching of the leaves with hacteria and exposure to other abnormal conditions, i. c, to air saturated with vapor of water [see below]. The spiral vessels are the first to be attacked; the pitted vessels are then occupied, the bacteria entering them through the pits; subsequently larger or smaller cavities are formed in the vascular bundle. When cultivated from stems of Vicia faba on alkaline potato agar at 300 C, a weak reddish stain appears which soon turns black on addition of paraphenylendiamin and itf-naphthol (Spitzer's reaction). This red reaction is plainer in cultures to which 1 to 5 per cent tyrosin solution has been added. The rot on lupins was white, never black as on Vicia. The plants were injected and kept moist under bell-jars. The plants in the open were never destroyed by the inoculation, the growth of the bacteria soon ceased and the wounds dried out, but here pustule-like intumescences were observed [see below], and these are ascribed to the action of the organism, since the control plants remained free whether pricked or not (see this monograph, Vol. II, fig. 27). In Physalis also the rot was white. EXPERIMENTS IN WASHINGTON. The writer repeated certain of these experiments with a green-fluorescent organism brought in 191 1 from the Biologisches Anstalt in Berlin by Dr. Wollenweber and said to be Bad. xanthochlorum Schuster. This culture, it should be stated, did not come directly from Dr. Schuster, who had then left the laboratory, but was a transfer by another assistant from a stock culture of this organism. The organism which I have thus received and studied liquefies gelatin in the manner described and rots potato tubers as described, only much more slowly at 260 C, and not at all at 375°C., nor will it grow in culture media at that temperature. Also on Vicia faba *Dr. Wollenweber, who was at the Biologisches Anstalt during this period, tells me they were made under bell-jars in the laboratory. SCHUSTER S GERMAN POTATO ROT. 275 exposed to normal conditions in a hothouse it is not parasitic. I could not get any infec- tions either by rubbing it into stomata or by needle-puncture into leaves or stems. The organism grows in milk about as described, but on agar-streaks ( + 15) I did not obtain the cross-folds and am inclined to regard this as an accidental phenomenon due to dry agar or to some other peculiarity of medium. The organism was tried on various sugars and alcohols in the presence of river-water and Witte's peptone (1 per cent). Contrary to Schuster, it ferments grape-sugar and galactose with production of an acid, but a similar solution with cane-sugar gave an alkaline reaction (fermentation tubes, 11 days). The same results were obtained in two repe- titions (twenty-eighth day and later) . The other car- bon foods tested in fermen- tation tubes were fructose, lactose, maltose, mannit, and glycerin. All of these gave an alkaline reaction (eleventh day). In two repetitions the same result was obtained after longer periods. There was no gas formation and no clouding in the closed end. As stated, the organism did not rot potato tubers at 37°C, nor could we get any evi- dence of growth above 340 C. in + 15 peptone-bouillon or on steamed potato. It grew better at 300 C. than at 340 C. but less well than at26°C. It grows at i°C. Its thermal death-point is 510 C, approximately. It grows in bouillon over chlo- roform without retarda- tion. It grows in +15 peptone-bouillon with 4 per cent sodium chloride and slowly with 5 per cent, but not with 6 per cent. It does not grow in Cohn's solution. It grew in pep- tone bouillon from —30 to +25. It grew about as well in — 15 and —30 as in o or +25. It grew well in Uschinsky's solution, forming a copious pellicle and after a time a pale green fluorescence. f It is sensitive to dry air (dead on cover slips at end of 4 days). It liquefied gelatin in the manner described by Schuster but did not liquefy Loeffler's blood-serum, although it grew well upon it. It is quite sensitive to sunlight. Fig. 135.* *Fig. 135. — -Potato tubers inoculated with Sehuster's Bacterium xanthochlorum and exposed for six days to the dry air of the laboratory at 27° C. Photographed Sept. 11, 1912, after slicing to show the very slight rot. The specks in the rotted part are needle-punctures. In right lower figure there is rot in vascular ring at top near the pricks. fMiss Clara Jamieson isolated from Bad. xanthochlorum a non-fluorescent strain which retained this characteristic in subcultures on various media as long as studied (some months). On agar-plates the non-fluorescent colonies looked exactly like the fluorescent ones. 276 BACTERIA IN RELATION TO PLANT DISEASES. Several of the inoculated potato tubers exposed dry at room-temperature (260 C.) showed a slight vascular infection (rot) extending in one instance a distance of 2 cm. from the pricked area, but only visible close to the bundles. The check-pricked tubers remained sound. The experiments were made in August, i. e., on I tubers recently harvested (fig. 135), and part of a lot which rotted quickly when exposed to B. melanogenes. Miss Nellie Brown repeated this experiment for me on several potato tubers with the same result. Dr. Schuster's figures also call for some interpreta- tion or explanation. I am inclined to think they do not represent what actually took place in the tissues; by this I mean that some of them are suspiciously diagram- matic. Schuster's text figure No. 11, for instance, can hardly represent a stomatal infection in Yicia faba. Here the bacteria are represented as forming a branch- ing strand which has entered through the stoma and penetrated to the opposite side of the leaf in a very un- usual way, i. e., by boring its way through cell- walls. It has conspicuously avoided the intercellular spaces, and the surrounding cells are not only unoccupied by the bacteria but also uncollapsed and apparently unin- jured and the leaf has retained its turgor. I am told that Dr. Schuster did not make these drawings, but he ought, at least, to have supervised them, since profes- sional artists seldom have an eye for details of plant structure. In an unusual case like this, a photomicro- graph would have been more convincing. The subject is left in such shape that some careful bacteriologist should repeat all of Schuster's experi- ments, and make others; only in this way shall we finally come to know what weight to give his statements. On a few of the many plants of Viciafaba inocu- lated by me where the needle-inoculations were not far apart, and one above another, the rapidly growing stems cracked open with callus-formation not unlike that figured by Schuster for Lupinus, but there was no evi- dence of disease due to the bacteria. These stems were about 15 inches high when inoculated and were 5 feet tall with numerous vigorous leaves and blossoms when the photograph was made (fig. 136). Fig. 136/ LITERATURE. 1912. 1912. Schuster, Julius. Zur Kenntniss der Bak- terienfaule der KartofTel. Arbeiten aus der K. Biologischen Anstalt f. Land. u. Forst- wirtschaft, VIII Bd., 4 Heft, pp. 452-492. 1 plate, 13 figs. Berlin, 1912. Smith, Krwin F. Isolation of pathogenic potato bacteria: A question of priority. Phytopathology, vol.11, p. 213, Oct. 1912. 1913. Brown, Nellie A., and Jamieson, Clara O. A bacterium [Bad. aptatum] causing a disease of sugar-beet and nasturtium leaves. Jour. Agric. Research, U. S. Dept. of Agric., vol. 1, No. 3, Dec., 1913, with 3 pis. (one-colored), pp. 189-210. That part relating to Schuster's organism begins on page 209. *Fig. 136. — Stem of Vicia faba, 2.5 months after inoculating with Schuster's Bacterium xanthochlorum, showing tissue cracked open, with callus formation along the line of needle-pricks. X 1.75. THE DUTCH DISEASE OF WALLFLOWER. In the summer of 1900, in Holland, van Hall found a disease of the wallflower (Cheiranthus) which was characterized by the constriction of the upper part of the tap root, followed by yellowing of the leaves, which gradually fell off, first the lowest, then those farther up the stem. Cross-sections of the constricted portion of the tap root showed the wood to be brown or black, and the vessels of the wood to be occupied by bacteria, which were traced upward into the branches. Professor Beijerinck proved.it is said, that all of these were one sort. " Infection experiments have not yet been published, so that this disease must be placed with the incompletely known ones." Later Briosi and Pavarino published on a similar Italian disease of stock (Matthiola), as described in the following chapter. LITERATURE. 1902. Van Hall, C. J. J. Ziekte der Zomcrviolieren, Cheiranthus animus. In "Beijdragen tot de Kermis der Bakterieele plantenziekten." Academisch Proefschrift, Amsterdam, 1902, p. 72. THE ITALIAN DISEASE OF STOCK. In 1912 Briosi and Pavarino described a vascular and parenchymatic disease of the common stock (Matthiola annua L-), prevalent in parts of Italy and attributed it to a green- fluorescent bacterium. The following translation gives the substance of their paper: Two different species of erueifers are commonly designated by the name " Violaeeiocea:" The Cheiranthus chciri L-, which is the common violaeeiocea, and the Matthiola annua L., commonly called quarantina. Of this latter there are some varieties of notable commercial importance, cultivated on a large scale, especially in many sections of the Lignria, where entire fields are covered with them, and furnish abundant and splendid winter flowers for exportation. Some years the Matthiola quarantina shows signs of a grave disease, which spreads rapidly and causes such damage in some localities that it is necessary to suspend its cultivation. The signs of the disease begin with the appearance on the leaves of spots of a pale green color, of indefinite contour, which at first are observed with difficulty. Following this there appear small brown spots of irregular contour, and more or less punctiform, scattered over the petiole. Frequently the leaves, especially the young ones, become more or less deformed, not attaining the normal dimensions, and their margins curl toward the upper surface. The inflorescence is arrested in its development and becomes rachitic, and so the flowers lose their commercial value. The researches were made on plants gathered by us, in various localities such as Loano [on the Riviera, about halfway from Genoa to Ventimiglia], where some years the disease is active and widespread. Morbid anatomy. — The disease is not limited to the leaves, but extends to all the organs of the plant : flowers, branches, stalks, and roots. Sectioning young branches, it is found that the infection invades first the woody vessels, which show yellowing or browning and are more or less altered. The plant forms a zone of corky tissue which twists and circles about the infected vessels, to prevent the spread of the disease in a transverse direction, and limit the diffusion of it in the tissue. Later the disease is able to invade all the wood as far as the pith, which, in some cases, is seen to be strongly corroded. In the stalk the infection appears first in the primary wood, from which it extends interiorly toward the pith, and externally toward the secondary wood, as is clearly shown by the yellowing and browning of the walls of the vessels and of the fiber, and by the yellow, brown, or black coagu- lated substance which fills up the lumen of the cells of the diseased tissues. 277 278 BACTERIA IN RELATION TO PLANT DISEASES. In the roots the infection begins generally in the woody bundles of the central cylinder, and from there spreads to the secondary wood; and the attacked elements (vessels, fiber, etc.) color as usual either yellow or brown, becoming filled at the same time with the clotted, blackish substance. In tlic leaves the infection manifests itself by the pale spots above described, which are due to the disorganization of the chloroplasts through work of the microorganism, which multiplies rapidly in the cell. With the advance of the disease the protoplasm of the cell contracts and shrinks, caus- ing the collapse of the cell walls; from this time on the tissue decays and there appear in the mesophyl tin- brown and depressed spots which we have already noted on the petiole in advanced disease. Microscopic examination shows in the cells of the diseased tissues numerous motile microorgan- II), isolated and united in colonies, but does not reveal any trace of mycelium. We have cultivated this organism in many nutritive media, proceeding in the following manner: We took small pieces of the leaves, branches, stalks, and roots, carefully washed them with water and soap, and afterwards disinfected them with a solution of 1 : 1000 mercuric chloride; they were then put into sterile distilled water and passed finally through alcohol and ether. The pathological pieces thus treated were put into tubes containing nutritive media of our preparation, and from all the infected organs thus treated and sown in the different nutritive media, we obtained always the development of a particular microorganism, which presented the following morphological and cultural characters: Microscopic aspect and color ability. — This microorganism has the form of a small rodlet — length 2 to 4/u, width 0.4 to o.6ju, with the ends slightly rounded. It stains well with all the anilin stains, even cold, but especially with gentian violet; it resists Gram completely. In broth cultures we observed vivacious vibratory and rotary movements of the microorganism, which appeared in the form of rodlets or of spherules, according to the position in which observed. Relation to oxygen. — In anaerobic conditions the microorganism does not develop at all, or only with great slowness, which indicates that it is prevailingly aerobic. Behavior in regard to temperature and to nutritive media. — It develops well at room temperature (150 C. circa), but more rapidly in the thermostat in the various nutrient media, and especially in neutral conditions. Gelatin cultures. — In 24 hours at room temperature it forms, in stab-cultures, a cup of initial liquefaction which progresses rapidly in cylindrical form until all the gelatin is liquefied. On the surface of the culture there is then formed a pellicle sufficiently thick that it detaches itself on agita- tion, falling to the bottom as a whitish, mucilaginous precipitate, and the liquid becomes a beautiful light green color in the upper part of the tube. Agar. — On glycerinated streak-agar cultures there develops, after 48 hours of incubation, a whitish surface growth little extended and slightly elevated, not shining. In stab-cultures the development is still more scanty; after 4S hours' incubation, the stab-growth is scared v visible; growth soon ceases. In plain agar the organism develops rather better. In streaks, in 24 hours, it forms a wet- shining, surface growth, rather elevated, with lobed margin, of a whitish color, and with a tendency to occupy all the free surface of the tube. In stab-cultures the growth extends to the bottom of the tube, at the surface enlarging into the form of a whitish disk, in the center of which appears in time a roundish, yellow spot. Broth-cultures. — In alkaline broth it develops poorly, with slight turbidity and formation of a scanlv deposit; in the upper part of the tube the liquid assumes a pale green color. hi plain broth the development is more rapid and vigorous; in 48 hours of incubation the entire culture is uniformly clouded, with an abundant dirty white sediment, which in time becomes a pale yellow. At the surface and along the walls of the test-tube a pellicle forms which detaches itself easily; the liquid assumes a light green color. Potato-cultures. — At the temperature of the incubator there develops, in 48 hours, a gray-white growth, wet, rather elevated, with a tendency to spread. With age the culture assumes a granular aspect with a pale yellow color, tending toward brown. Chemical activity. — In the tubes an offensive gas develops, but not hydrogen sulphide, as we wire able to verify by exposing above the cultures a small strip of lead acetate paper previously exposed to the vapor of ammonia. . 1 rtijn ia! reproduction of the disease. — To prove that the disease was really due to the pathogenic action of the microorganism of our isolation, we infected some vigorous plants of Matthiola, spraying tin 111 with broth cultures, very much diluted. At the end of a few davs we obtained the reproduction of the disease on the aerial part of the plants sprayed, with the same external characters and the same anatomical alterations (in the leaves and in the young branches) which we have described in the plant naturally diseased. THE ITALIAN DISEASE OF STOCK. 279 The infection rapidly spreads on the leaves and branches, extending to the inflorescence, which dries up completely. The plants can throw out other floriferous branches, but of poor development and with few flowers. We tried also infection through the roots, bathing with a pure-culture solution the soil of some pots in which were healthy plants, but not a single sign of disease was observed at the end of two months. For the infection of the roots perhaps wounds are necessary. We are able to establish that the more common manner of infection is through the stomata, as plainly shown by the pale yellow coloring which spreads from the stomata to the surrounding cells, in which we observed the usual disorganization of the chloroplasts and of the protoplasm. From the organs infected artificially we repeatedly made cultures in various nutritive media for the identification of the morphological and cultural characters of the microorganism of our preceding isolation and description. This organism must be regarded as a new species, to which is given the name Bacterium mal- tliiolac n. sp. The same year Briosi and Pavarino published a second paper (Bacteriosi della Matihiola annua L-), which is exactly the same as the one just reviewed, except for the following additions : The stab growth in plain agar is described as ciliated (cigliato). . 1 gar plates. — (Natural size.) After 48 hours' incubation the surface colonies are round, margins smooth. They are slightly raised, but not transparent, and of a whitish color. The buried colonies may be roundish, but for the most part have the form of a whetstone. Enlarged 30 diameters. — The surface colonies appear roundish, radiate, with transparent contour, and of a yellowish color. The buried colonies may be roundish, but for the most part are like a whet- stone, always smaller and of a darker yellow color. Under plain broth the sediment is described as viscous. Milk-cultures. — At a temperature of 300 C. the coagulation is complete on the third day with decidedly acid reaction to litmus. Specific and practical methods for the cure of this disease are unknown. We made tests with the usual sprays, using copper sulphate as the base (Bordeaux mixture), but the results obtained were negative. Perhaps such treatments might be effective applied as preventives sometime before the flowering period, with additional sprayings afterward. A good practice to follow is that of extirpating the plants as soon as they present the symptoms of the disease. The extirpated plants should not be permitted to accumulate on the land, where when dried they can be easily transported to a distance by the wind or by animals, but should be carried out of the field and destroyed by fire. Likewise, in order to prevent the infection being carried back by means of stable refuse, these extirpated plants must not be thrown on the manure heap. The rotation should be modified so that the culture of the stocks is not repeated on soil where the disease appeared the preceding year. To prevent the disease being transmitted from one year to another, it is rather useful to employ accurate seed selection, using only seed that has come from healthy plants, and from fields remaining immune to the infection. And since it has been demonstrated that germs of bacterial diseases frequently are able to continue to live on the seed during the winter season, it would be useful also in the present case to disinfect the seeds, immersing them for 15 minutes in a solution of 1 :iooo mercuric chloride, or in a solution of formalin 1 150 for 20 minutes. This practice ought to be completed by the selection, according to the method of Nilsson, in the field of those individuals which show more resistance to the disease. Finally, it would, perhaps, render the plant more resistant to the disease to avoid abuse of organic fertilizers, and to have recourse to use of potassium fertilizers, and especially the superphosphate. The writer has had no opportunity to study this disease at first hand. Some samples of stock supposed to show this disease were received from Italy, but it proved to be some- thing else. LITERATURE. 1912. Briosi, G. and Pavarino, L. Una malattia 1912. Briosi, G. and Pavarino, L. Bacteriosi della batterica della Matihiola annua L. (Bac- Matthiola annua L. {Bacterium matthiolae n. terium matthiolae a. sp.). Atti della Reale sp.). Atti dell' Istituto Botanico della R. Accademia dei Lincei, Rendiconti, vol. xxi, Universita di Pavia, Serie II, vol. xv, Milano, Fasc. 3, Rome, August, 1912, pp. 216-220. 1912, pp. 135-141, 2 pis. (colored). THE BRAZILIAN DISEASE OF MANIHOT. In 191 2 Gregorio Bondar, of the Agronomical Institute at Campinas in Brazil, described a disease of Manihot that appears to belong here. I translate from his Portuguese leaflet, with some hesitation, as follows: The sweet mandioea (Manihot palmata), varieties white mandioca and aipim mandioca, has been with us subject to a disease which apparently has not been met with in other countries. This rot of the stalks and the shoots (brotos) is caused by bacteria, Bacillus manihotus Arthaud- Berthet. The disease was studied at the Agronomic Institute in Campinas by the [former] Director, Dr. Arthaud-Berthet with our collaboration. The microbiological study was verified in the bac- teriological laboratory by the specialist Dr. A. Perrier. In the Mandioca aipim the effects of the disease are as follows: The voung shoots (brotos) rot internally, wither, and die. By the transparency of the bark one can perceive dark subcortical lines which follow the bundles. Removing the bark one observes that these lines start from the sub- terranean stalk, which becomes rotten in a line corresponding to the shoot (broto) or presents the bundles (canaes) black, infected. In the young shoots (brotos), the bundles (canaes) presently decompose (fig. 138,6), injuring ^ the circulation of the sap and causing the withering. The disease may attack the shoot on a single side, but presently gen- eralizes itself in other bundles (canaes). In young planta- tions the disease occasions the death of the plants. In the white Mandioca the signs of the disease differ: The shoots present subcortical swel- lings which are produced in the bundles by the coagulation of the latex (fig. 138,0,6). These swellings are elongate. The subcortical accumulation £*£. -■^'A. iff- • il" . Fig. 137.* breaks the bark and the gummy substance exudes, forming small superficial crusts, of a light yellow color (amarello-clara) at the beginning, and afterwards reddish by oxidation. If the diseased shoots were (estiverem) grown on the stalks of the past year the black markings of the disease run through even to the subterranean part without, however, forming gummy swellings. The roots do not manifest any signs of disease. In a planting of ten months we observed that these roots were able to send out the shoots three or four times, which succumbed successively to the bacteria, until the roots were completely exhausted, dying directly afterwards. We made numerous artificial inoculations of the disease. The healthy shoots, inoculated arti- ficially with virus from the diseased plant, presented the same symptoms of disease after 15 or 20 days' incubation of the bacteria. The cuttings, infected with the bacteria before planting, died, rotting in the earth without budding. Of 20 infected cuttings not a single one grew. The experimental work on the disease, as also many observations, were made in collaboration with the Chefe de Culturas do Instituto, Sr. Joao Herrman. Attacking old stalks, with the roots formed, the bacteria bring about exhaustion of the accumu- lated starchy substance. The roots of the diseased stalks are unlit for food, for they are very hard, even when boiled two or three times as long as the ordinary mandioca. The chemical composition varies greatly also. The anaylsis made by Sr. R. Bolliger,in the Agro- nomic Institute in Campinas, revealed a noticeable diminution of the starchy substance: Instead of *Fig. 137. 280 -Brazilian manihot wilting from bacterial disease. (After Bondar.) THE BRAZILIAN DISEASE OF MANIHOT. 28l 73.13 per cent of starch in the dry material as in the normal mandioca, there was scarcely 63.90 per cent in the diseased mandioca. The observations show that the disease attacks very often the cuttings (mudas) of mandioca, when they are made very long before planting. In this case the tips of the stems begin to rot. As the circulation of the sap occurs even in the cut stems, the conta- gion propagates itself in the bundles (canaes) without leaving external signs. The slips cut from such stems die for the greater part, and on the other hand, are liable to con- taminate others. The disease is grave, contagious, easily propagated, inoculable, and can be transmitted by insects. If care is not taken it may cause con- siderable injury to the cultivation of this plant of ours, which is of general use. With some cultural precautions the malady may be avoided or les- sened in severity. Curative treatment can not be applied, since the disease is internal, and generalizes itself directly in the plant. The preventive measures which we advise, in order to avoid the pro- pagation of the plague, are as follows : 1. Plant resting shoots (mudas) absolutely not suspected, coming from healthy plantations, rejecting all doubtful stalks. 2. Cut the cuttings (estacas) with the greatest care, without tear- ing or wounding the tissue, in such a manner as to reduce the surface and the possibility of contamination. 3. Do not plant the mandioca in soil infected by preceding diseased plantings, but cultivate other things not subject to contamination by the same bacteria. 4. Plant the cuttings as soon as possible, in order to avoid the contamination which occurs easily during the period of slow growth. 5. Plant resistant varieties: Mandioca parda and Mandioca azul resist better than the Mandioca branca and aipim. The bacteria are found in pure colonies under the bark, in the young swellings. The isolation of the microbe, likewise the artificial inoculation of the plant, show, fortunately, that there are present no other bacterial diseases. A more extensive account of this disease was prepared, but its publication has been delayed. LITERATURE. c Fig. 138. 1912. Bondar, GrEgorio. Uma nova molestia bacteriana das hastes da mandioca. No. 4, April 1912, pp. 15-18, 3 figures, Brazil. Chacaras e Quintaes, vol. v. *Fig. 138. — Stem of manihot attacked by bacterial disease, a. Bacterial blisters and exudate, b. Cross-section of stem showing location of the diseased part (at the top), e . Bacillus manihotus A. Berthet, from a diseased manihot plant. X 1,000. (After Bondar.) [The photographs and drawings in this volume, as in Volumes I and II, were made, with a few exceptions, by James F. Brewer. The writer, however, selected the parts to be drawn, or photographed, and finally checked up under the microscope nearly or quite all of the drawings. With exception of figs. 1 and 2 of plate 11, the colored figures were also made by Mr. Brewer. The color chart, referred to in various places as "Ridgway, " is the first edition of Robert Ridgway's Nomenclature of Colors for Naturalists.] ADDENDUM. The following observations were made too late for insertion in the body of the text: (i) A planobacter michigancnse vs. Apl. ratkayi. — Both liquefy gelatin slowly, but the latter liquefies the more rapidly and the surface growth (streak) floats about in the nearly clear fluid as a twisted ribbon. Apl. michigancnse, on the contrary, clouds the fluid. (2) Peanut wilt. — In June, 1914, we obtained bacterial wilt of peanuts using both Medan III and Florida potato (isolation of 19 14). Inoculations were by needle pricks on stems; the time between inoculation and appearance of disease was about two weeks; great numbers of bacteria were seen in the tissues. Subsequently the wilt of peanut was also obtained by inoculating from a pure culture of the Creedmore, N. C, tobacco organism. (3) Tobacco wilt. — This was obtained in June, 19 14, using Florida potato (1914), Florida tobacco (1914) and Creedmore, N. C, tobacco (1914). The inoculations were by needle pricks from pure cultures (subcultures from poured-plate colonies) on stems and leaves. The time between inoculation and appearance of disease was about 4 to 10 days. (4) Bad. solanaccarum — Effect of drying. — On cover slips Medan III and Florida potato (1914) were dead at the end of 6 to 9 days, i. e., refused to grow when thrown into peptone-beef bouillon. These cover slips were wetted from young peptone water cultures and were kept in the dark in a covered Petri-dish at room temperatures (of June). Sub- sequently tests with the Creedmore tobacco organism (19 14) and the Florida tobacco organism (1914) showed all dead on the 6th day or earlier. The tests were begun on the 5th day, at which time the organism was alive on a few of the cover glasses, i. e., 1 out of 8 in Creedmore and 2 out of 8 in Florida. Tests on the 6th, 7th, and 9th days showed all dead. The covers (64 in all) were wet from cloudy 24-hour-old peptone water cultures and kept in the dark, in sterile Petri-dishes, in the well ventilated laboratory at room temperatures (of July). The covers were thrown into tubes of + 14 peptone bouillon. (5) Bacterium solanaccarum in litmus milk A- cream. — Medan III always reddens this medium. It may therefore be known as var. Asiaticum nov. var. None of the American isolations of 19 14 (Florida potato, Florida tobacco, and Creedmore tobacco) have done so. (Experiments continued 4 to 8 weeks, using subcultures from 15 colonies.) (6) Bacterium solanaccarum in Meyer's solution + Am. lactate. — With Medan III and Florida potato (1914) at the end of 4 weeks there had been no growth. (7) Bacterium solanaccarum in Meyer's solution + Asparagin. — At the end of 4 weeks, 4 of the 8 tubes of Medan III clouded. All of the Florida potato (1914) remained clear. (8) Bacterium solanaccarum in Meyer's solution + KN03 + Sodium acetate; Do. Do. Do. + Sodium lactate. Do. Do. Do. + Sodium butyrate. Neither Medan III nor Florida potato ( 1 9 1 4) would grow in these media. Test of 4 weeks. (9) Bad. solanacearum inoculated on Livingston' s Dwarf Aristocrat tomato. On page 189 (exp. of 1905) the statement is made that of many varieties tested the above named was most susceptible. Fearing this might be an accident, the experiments were repeated in July, 1914, using four strains of the bacteria: Medan III, Florida potato (1914), Florida tobacco (1914), and Creedmore tobacco (1914). In all 24 plants were inoculated, 6 from each strain. All except Medan III were wilting or wilted on the third day with enormous multiplication of bacteria in the tissues. The inoculations were made on young plants showing two good leaves (besides the cotyledons) and four when collected. The pricks were made with a fine needle ; most of the punctures were made on petioles. Older plants were not available. The check plants remained sound. These plants contained 93 per cent of water. This experiment was repeated a few days later with similar results. (10) Loss of Virulence. Medan III, lost much of its virulence during 12 months culti- vation in the laboratory without reinoculation into plants, i. e., then it was less active on tobacco and on tomato than the recent (1914) isolations from American sources (PI. 45). 282 PLANT BACTERIA. VOL. III. PLATE 45. Loss of Virulence in Bacterium solanacearum. Livingston's Dwarf Aristocrat tomato after infection with Bad. solanacearum. Each plant was inocu- lated on one petiole by means of delicate needle pricks from a 3-day agar culture. Plants in hot- house subject to ± 350 C. (July temperatures), and alike. A B, end of 5 days; CD, end of 7 days. (1) Florida potato (1914). Organism on culture media about 2 months, and still actively virulent. There were two more in this set, both badly wilted. (2) Medan III (1913). Organism on culture media about 12 months. It was isolated by Honing in the summer of 1913 and was extremely virulent when received, and for several months thereafter, but is now weak (see page 179). Only 2 of the 6 plants showed any trace of wilt — those in center of figure. (3) Creedmore tobacco (1914). Organism on culture media about 5 weeks. In this set were 6 plants, all badly wilted. (4) Same as 2. In each of the 6 plants the inoculated petiole was now wilting. NDEX. African disease of potato 214 Alternaria solani, in French disease of potato (Delacroix) 215 Amaranths, Smith's disease of 148 (See also Bacterium amaranthi.) description 148 geographical distribution . . 148, 150 literature 15° tissues attacked 149 Aplanobacter michiganense 165, 205 (See also tomato, Grand Rapids disease.) acid, production of 164, 165 acids, sensitiveness to. . . 164, 165 aerobism 165 agar, slow growth on 165 alkali, toleration of 164 colonies 161 comparison with Aplanobacter rathayi 165, 282 cultivation, difficulty of 165 cultural characters 162 dextrose, acid from 164 dissemination 163 drying, resistant to 165 fermentation tube cultures 164, 165 gelatin, slow growth on 165, 282 Gram positive 165 group number 165 growth in or on, acid beef-bouillons 165 akaline beef bouillon 164 beef-bouillon 162, 164 Cohn's solution 162, 165 cornmeal agar stabs 162 cream-free milk 162 litmus milk 162, 164 Loeffler's blood-serum 165 milk 162, 164, 165 nitrate bouillon 162 nutrient agar 161, 162, 164, 165 nutrient gelatin 162, 164, 165, 282 potato cylinders 162, 164 sugars 1 64, 1 65 Uschinsky's solution 165 infection, point of 161 through stomata 1 63 inoculations on potatoes 1 64 tomatoes 161, 163 isolation of 1 6 1 , 1 64 levulose, acid from 164 literature 165 litmus in milk, reduction of 162, 164 milk, copious surface growth on 165 morphology 161 motility, lack of 165 Porto Rican weed subject to 163 potassium nitrate, no reduction of 165 resume of salient characters 164 sodium chloride, sensitiveness to 164 starch, slow action on 164 substomatic chambers, occupied 163 thermal death-point low 165 tissues attacked 161 virulence, loss of 165 viscidity 165 Pagb. Aplanobacter rathayi 160 (See also Orchard grass, Rathay's disease.) acid, effect on 156 agar, slow growth on 156, 158 associated with white organism 159, 160 capsule 156, 160 Cohn's solution, no growth in 160 coloring matter 156 comparison with Aplanobacter michiganense . 165, 282 cultivation, difficulty of 156, 157, 158 cultural characteristics. . 156 cultures, separable from substratum 156 dextrose fermented 160 fermentation of sugars 156 gelatin, growth on 156, 165, 282 Gram's stain . 156, 159 growth in, or on agar and gelatin, slow 156, 158 beef bouillon 156, 159 cane-sugar water 160 Cohn's solution 160 colonies of associated white organism 160 Dactylis broth plus peptone 160 Dactylis decoction 156 grass broth 160 litmus milk ... 160 milk 160 nitrate bouillon 159 nutrient agars .... 156, 157, 158, 159 nutrient gelatins. . . 156, 157, 160, 282 peptone water plus potassium nitrate 160 potato 156, 158, 160 potato broth plus potassium nitrate 160 group number 160 hydrogen sulphide, production 156 indol production 156 inoculations 156 inoculations into tomato 160 iodine, reaction 156 isolation 156 lactose fermented slowly 160 lemon juice, effect 156 lemon yellow color 156 liquefaction of gelatin, slow 160, 282 Rathay's experiments 156 literature 160 litmus, reaction 160 longevity r . . . . 160 milk, behavior in 160 morphology 156, 157, 160 motility, lack of 156, 157 nitrates, no reduction of 160 pellicle present in 156 potato, used for isolation 156, 158 pseudozoogloeae 165 resistance to sunlight 156 saccharose fermented slowly 160 seeds, disease disseminated on 160 spores, not seen 156 stains, reaction to 156, 160 sugars, fermentation 156 sunlight, resistance to 156 viscidity . 155, 157, 158 vitality, retention of 160 white organism associated with 159, 160 yellow pigment, nature of 156 283 284 INDEX. Appel, Bacillus phytophthorus, formation of cork barrier. 192 "black-leg," cause of 175 ring disease of potato, Germany 166, 215 (See also Solanaceae, brown rot, ring disease of potato, Germany.) Arachis hypogaea, wilt 151, 153, 253, 271 (See Bacterium solanacearum, peanut-wilt organism; peanut wilt, Sumatra; peanut wilt, United States; peanut wilt, van Breda de Haan's.) Arrowing of sugar-cane io^ 20 Arthaud-Berthet, Brazilian disease of Manihot 280 Ashby, Panama disease of banana reported from Jamaica 1 73 Australian disease of potato and tomato 207 Bacillus aeruginosus (tobacco canker?) 266 (See also Tobacco, wilt diseases, French disease.) cultural characteristics 266 Gram's stain 266 inoculations 266 morphology 266 synonomy 267 Bacillus amylobacter, tobacco root-rot, Comes 270 Bacillus amylovorus, compared with Bact. solanacearum, color 194 growth in beef-broth concentrated and strongly acid 136 growth in potato broth with malic acid 136 Bacillus atrosepticus 175 Bacillus brassicaevorus, synonomy 267 Bacillus eaulivorus 17S, 207 synonomy 267 Bacillus cloacae from maize 147 Bacillus coli, sodium chloride, toleration for 138 Gram's stain, reaction to 247 Bacillus fluorescens liquefaciens, synonomy 267 Bacillus fluorescens putridus, synonomy 267 Bacillus glagae 75 Bacillus manihotus 280 (See also Manihot, Brazilian disease.) inoculations 280 literature 281 occurrence, pure, in subcortical swellings 281 Bacillus marcescens 87 Bacillus megaterium, isolated from diseased tobacco . 269 Bacillus melanogenes 175, 203, 276 Bacillus mesentericus, associated with Bact. solanacearum in Sumatra. . 259 resemblance to Aplanobacter rathayi 156 Bacillus musae 170 bacterial ooze 171 biological characters 171 cavities formed by 170, 172 growth black on potato cylinders 171 growth on gelatin 171 inoculations on 1 70, 1 7 1 manila hemp 1 7 ■ solauaceous plants 171 tomato 1 7 ' isolation 1 70 longevity, feeble 1 7 ' pathogenicity, quick loss of 171 similarity to Bacillus solanacearum 171 tissues attacked 170, 171, 172 (See also Banana, Rorer's Trinidad disease.) Bacillus mycoides, associated with Baet. solanacearum in Sumatra 259 Bacillus mycoides roseus, isolated from diseased tobacco 269 Bacillus nicotianae Uyeda, (See also Tobacco, wilt-diseases, Japanese disease. ) acid, effect of 242 acid-production 238, 241 alkali, effect of 242 Pagb. Bacillus nicotianae Uyeda — continued. anaerobism 238, 241 Bacterium solanacearum (potato and tomato-wilt organism), relation to 244, 254 (See also "comparison, etc.") Bacterium solanacearum (Sumatran tobacco-wilt organism), relation to 244, 254 (See also "comparison, etc.") blackening of, agar cultures 238 bouillon 241 milk 241 capsules 241, 247 colonies on agar 238, 241 colonies on gelatin 241 comparison (Honing's) of Sumatran tobacco-wilt organism with Uyeda's description of B. nico- tianae and Smith's and Uyeda's descriptions of Bact. solanacearum. . . . 247, 248, 249, 250, 251 (See also "similarity, etc.") comparison (Smith's) with Bact. solanacearum Smith 240, 242, 243, 244 (See also "similarity, etc.") crystals, ammonium magnesium phosphate 241 cultural characters 238, 241 comparison with Bact. solanacearum, Smith's observations 240, 242 cy tase, production of 242 description, Uyeda's 238 possibly not drawn from one organism 244 diastase, presence of 242 egg-plant, non-infection of 241 endospores 241, 243, 247 enzymes, presence of 241, 242 flagella 238, 241, 243 gas-production 238, 241 Gram's stain, positive 241 growth on or in, agar 238, 241 asparagin-dextrose solution 242 carrot 242 gelatin 238, 241 glucose agar, gas from 238 glucose bouillon, gas from 238 milk 238, 241 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, dextrose, and ammo- nium chloride 242 mono-potassium phosphate plus magnesium sulphate, sodium chloride, dextrose, and ammonium tartrate 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, dextrose and aspara- gin 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, dextrose, and potas- sium nitrate 242 mono- potassium phosphate plus magnesium sul- phate, sodium chloride, glycerin, and ammo- nium chloride 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, glycerin, and ammo- nium tartrate 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, glycerin, and potas- sium nitrate 242 peptone-dextrose solution 242 peptone-water, blackened 242 peptonized bouillon 238, 241 potato, yellow on 238, 241 radish, sharp odor on 242 Uschinsky's solution 242 hydrogen sulphide, production of 242 indol production 241, 242 inoculations, Jensen's, failure of 271 INDEX. 285 Page. Bacillus nicotianae Uyeda — continued, inoculations, Uyeda's, on Amaranthus gangeticus 243 Capsicum longum 243 egg-plant 238 Lycopersicum esculentum 243 Physalis alkekengi 243 Physalis minimum 243 Polygonum tinctorum 243 Solanum melongena 243 tobacco 238 invertase, presence of 242 liquefaction of gelatin 238, 241 literature 270, 271 litmus, reduction of 241, 242 maximum temperature 241 methylene blue, reduction of 242 morphology 238, 241 nitrate, reduction to nitrite 242 North American tobacco wilt, not found in 229 odor 238, 242 optimum temperature 238, 242 pellicle, early appearance 241 pigment, solubility 241 pigment-production 238, 241 effect of temperature 241 potato and tomato- wilt organism, relation to . . . 244,254 (See also "comparison, etc.") resistant varieties 243 similarity of tobacco-wilt organisms (Sumatra and Japan) and Bact. solanacearum Smith, de- scribed from potato and tomato 244, 254 (See also "comparison, etc.") soil, depth of penetration in 242 spores 241, 243, 247 staining, earbol fuchsin 243 staining, Gram's method 241 Sumatran tobacco-wilt organism, relation to . 244, 254 (See also "comparison, etc.") temperature relations 238, 241 thermal death point 241 tomato, non-infection of 241 tomato and potato-wilt organism, relation to . 244, 254 (See also " comparison, etc.") trypsin, production of 242 tyrosinase, presence of 241, 242 Bacillus phytoph thorns, Australian disease of potato and tomato 208 Bacterium solanacearum, characters distinguishing from 200 cause of Schwarzbeinigkeit 175 formation of cork-barrier 192 French disease of potato 214 growth on slices of raw potato 166 parasitism, active 203 pathogenicity compared with that of Bact. xantho- chlorum 272 Russian disease of potato 214 Bacillus prodigiosus, mixed culture inoculations on tobacco 267 Bacillus pseudarabinus 50 Bacillus putrifaciens putridus (tobacco pith-rotr). . . 266 (See also Tobacco, wilt-diseases, French disease.) cultural characteristics 267 inoculations 267 morphology 267 Bacillus sacchari Janse, aerobism 76 copper sulphate, effect 76 Debray 's work 76 growth in culture media 76 isolation 74 Janse's work 74.75 normal sugar-cane and other plants, presence in. . 75 resemblance to B. subtilis 76 Pagb. Bacillus sacchari Janse — continued. Sereh, causal relation to 75 spores 76 Bacillus sacchari Spegazzini 86 comparison with B. marcescens 87 comparison with Micrococcus prodigiosus Cohn . . 87 cultural characteristics 87 inoculation experiments 87 isolation from Polvillo 86 Janse's name earlier 86 morphology 87 red pigment produced 87 relation to Bact. vascularum 88 resemblance to B. sorghi 87 spores 87 Bacillus sesami 218 Bacillus solanacearum 178 (See Bacterium solanacearum.) Bacillus solanincola 175, 214, 215 Bacillus solanisaprus 203 Bacillus sorghi 87 Bacillus subtilis, growth on agar with Bact. solanacearum from Sumatran tobacco 264 isolation from top-rot of sugar-cane 83 resemblance to B. sacchari 76 Bacillus tabacivorus (tobacco collar-rot?) 266 (See also Tobacco, wilt-diseases, French disease.) cultural characteristics 266 inoculations 267 morphology 266 Bacillus tracheiphilus, compared with Bact. solanacearum, color 194 growth in beef-bouillon 194 Bacillus vascularum 8 (See Bacterium vascularum; Sugar-cane, Cobb's disease.) Bacillus vascularum solani 207, 208, 218 Bacillus vulgatus, resemblance to Aplanobacter rathayi 156 Bacterium amaranthi 148 (See also Amaranths, Smith's disease.) alkali, production 149 cells occupied by 149 color of 149 gas production, not seen 149 growth on, Loeffler's blood-serum 148 potato cylinders 148 sugar-beet cylinders 149 isolation 148 literature 150 morphology 149 starch, slight action on 149 tissues occupied 149 Bacterium aurantium roseum, associated with Bact. solanacearum in Sumatra 259 Bacterium campestre, browning of bundles 90 chloroform, marked effect of 143 comparison with Aplanobacter michiganense . 161, 164 growth in or on, beef-broth concentrated and strongly acid 136 cabbage juice 136 distilled water plus dipotassium phosphate, mag- nesium sulphate, ammonium phosphate and sodium acetate 137 Dunham's solution plus rosolic acid, differential . 1 39 Loeffler's blood serum 148 nitrate bouillon 138 potato broth 136 potato broth plus malic acid 136 potato cylinders, copious 67, 148 rutabaga 135 tomato juice 136 286 INDEX. Bacterium campestre — continued. growth on yellow turnip 135 infection through water-pores 210 pigment, brown 142 potato-starch, action on 142 retarding influence of litmus neutral gelatin 8 size compared with that of Cobb's Bacillus vas- cularum 8 starch, strong action on 60 wind, not borne by 68 Bacterium deliense, associated with Bact solanace- arum in Sumatra 259 Bacterium dianthi, growth in potato broth with malic acid 136 Bacterium gummis, tobacco-rot, Italy 266 tomato disease, Italy 203, 215 Bacterium hyacinthi, acids, toleration of 138, 144 alkalies, toleration of 138 growth on or in, agar 133 beef-broth concentrated and strongly acid 136 Dunham's solution plus rosolic acid 139 nitrate bouillon 138 nitrogen 138 potato broth 136 potato broth plus malic acid 136 potato cylinders 60, 134, 148 rutabaga 135 lesions produced by 130 minimum temperature 143 morphology 149 potato-starch, slight action on 142 Bacterium langkatense, associated with Bact. sola- nacearum in Sumatra 259 Bacterium matthiolae, (See also Stock, Italian disease.) aerobism 278 colonies 279 gas-production 278 Gram's stain 278 growth on or in, agar 278, 279 alkaline broth 278 broth 278, 279 gelatin 278 glycerin-agar 278 milk 279 potato 278 infection, manner of 279 inoculations 278 isolation 278 liquefaction of gelatin 278 literature 279 morphology 278 motility 278 reaction of media most fayorable 278 stains, reaction to 278 temperature relations 278 Bacterium medanense, associated with Bact. solana- cearum in Sumatra 259 Bacterium megatherium, renders tobacco bacterium non-infectious 267 Bacterium michiganense 165 (See Aplanobacter michiganense.) Bacterium mori 209 Bacterium patelliforme, associated with Bact. sola- uacearum in Sumatra 259 Bacterium phaseoli, growth in or on, beef-broth concentrated and strongly acid 136 cabbage mice 136 Hunger's agar, heaped up 57 potato broth [36 Page. Bacterium phaseoli — continued, growth in or on — continued. potato broth plus malic acid 136 potato cylinders 67 Bacterim pruni, growth in Uschinsky's solution. . . . 137 Bacterium rangiferinum, associated with Bact. sola- nacearum in Sumatra 259 Bacterium sacchari 50 Bacterium shuffneri, associated with Bact. solana- cearum in Sumatra 259 Bacterium solanacearum (Deli strain), [See Bact. solanacearum (Sumatran tobacco-wilt organism).] Bacterium solanacearum (Indian tobacco-wilt organism) 2 67 (See also Tobacco, wilt-diseases. Indian disease.) bipolar staining 267 culture medium, effect on virulence 267 growth on or in, agar 267 bouillon 267 gelatin 267 glucose bouillon 267 potato 267 inoculations 267 with mixed cultures 267 literature 271 longevity 268 morphological character, variations in 267 morphology 267 motility, denied 267 pigment 267, 268 effect on virulence 268 tissues, effect on 268 virulence, effect of pigment on 268 loss of 268 restoration of 268 variation in 267 Bacterium solanacearum (?) (Japanese tobacco-wilt organism), (See Bacillus nicotianae Uyeda.) Bacterium sclanacearum (Mcdan), (See Bact. solanacearum (Sumatran tobacco organism) ] Bacterium solanacearum (North American tobacco-wilt organism) 231, 234, 235 (See also Peanut wilt, United States; Tobacco, wilt- diseases, North American disease.) aerobism 233 browning on agar 232 browning on potato 232 comparison with Bact. solanacearum (potato and tomato) 230, 231, 234 comparison with peanut and pepper-wilt organ- isms 271 cultural characters 231 drying, effect on 282 Fulton's work on 271 gas-production, not observed 233 growth on or in, acid bouillon (plus 33 muscle acid) 232 beef-agar 231 Cohn's solution 233 gelatin 232 litmus milk 233 litmus milk with cream 282 milk 233 nitrate bouillon 233 potato 232 standard peptonized bouillon 232 incubation period 234 inoculations, (See also Tobacco, wilt-diseases, N. Am. disease.) direct, on tobacco (organism passed through tomato).. 234 INDEX. 287 Page. Bacterium solanacearum (N. Am. tobacco) — cont. inoculations, pure culture on peanut 271 tobacco 229 tomato 220, 230, 236 soil, on Datura stramonium 233 egg-plant 235 peppers 235 tobacco 181, 229, 235, 282 tomato 181, 233, 282 isolation 228 literature 270, 271 longevity 232 minimum temperature 233 morphology 231 peanut-wilt, cause of 271 potato and tomato-wilt organism like . . . 230, 231, 234 reduction of nitrates 233 relation to Bact. solanacearum (potato and tomato) 230, 231, 234 similarity to peanut-wilt organism 271 pepper-wilt organism 271 Sumatran tobacco-wilt organism, behavior in cream-free litmus milk 264 signs of disease 263 soil, infection through (peanut) ... 271 soil, persistence of organism in 237 Sumatran tobacco-wilt organism, similarity in cream-free litmus milk 264 Sumatran tobacco-wilt organism, similarity in signs of disease 263 thermal death-point 233 tissues attacked 227, 230 tomato and potato- wilt organism like . . . 230, 231, 234 vessels occupied 227, 230 water-content of host, effect on inoculations ... 282 Bacterium solanacearum (Peanut-wilt organism) . 153, 253,271 (See also Peanut wilt, Sumatra; Peanut wilt, United States; Peanut wilt, van Breda de Haan's.) comparison with tobacco and pepper organisms. 271 cultural characteristics 271 Fulton's work on 271 Honing's report of 153, 253 inoculations, on peanut 271 tobacco 271 (See also Peanut wilt, United States.) isolation 271 literature 271 morphology 271 similarity to, pepper-wilt organism 271 tobacco-wilt organism 271 stains, reaction to 271 Bacterium solanacearum (potato and tomato) 178, 193 (See also Peanut wilt; Solanaceae, brown rot; Tobacco, wilt diseases.) acids, effect of 200, 2 1 1 acid production, not detected 196, 200 aerobism, strict 196 African disease of potato, Odium's observations on 214 agar, browned by 195 alkali, production of 194, 196 Australian disease of potato and tomato, Tryon's observations on 207 Bacillus musae, similarity to 171 Bacillus nicotianae, synonym of 254 not wholly like 244 (See also "comparison, etc.") Bacillus phy tophthorus, characters separating from 200 Bancroft, Malay States disease 211 bipolar staining of 267 Page. Bacterium solanacearum (potato, tomato) — cont. brown stain, accompanying loss of vitality 199, 200 in form of streaks in stems 176, 185 nature of 194 production of 200 casein, no action on 194 cell-wall, action on 191 Ceylon wilt-disease of tomato, Petch's 214 chains, seldom seen 193, 200 Cohn's solution, no growth in 197 Coleman, Mysore ring disease 212 colonies 195, 199 often dead when browned 199, 232 color 1 94 Comes, Italian disease of tomato 215 comparison with N. Am. tobacco organism. 230,231,234 comparison (Honing's) of Sumatran tobacco-wilt organism with Smith's and Uyeda's descrip- tions of Bact. solanacearum and Uyeda's de- scription of B. nicotianae. . 247,248,249,250,251 (See also "relation, etc.") comparison (Smith's) with B. nicotianae. . . . 240, 241, 242,243,244 (See also "relation, etc.") cucumbers not rotted by 174 cultural characteristics 194, 199, 211 culture-media, growth on, effect on virulence. ... 179 Delacroix, French disease of potato 214 dendritic growth, not characteristic 200 description of 193 diffusion from vessels 197 drying, sensitive to 282 Dutch East Indian disease of tomato, Hunger's observations 209 (See Solanaceae, brown rot, Dutch East Indian disease.) English disease of potato 216 enlargements, local, due to 199 entrance into host 181 fermentation-tube experiments 196 field inoculations on large scale with 187 filaments rare 193 flagella 1 93 Florida potato-wilt organism, bipolar staining 267 drying, effect of 282 growth in, Cohn's solution 264 litmus milk with cream 264, 282 Meyer's solution plus ammonium salts 251, 282 asparagin 282 potassium nitrate plus sodium salts 282 sodium salts 251, 282 Uschinsky's solution 264 inoculations on peanut 282 tobacco 282 tomato (Livingston's Dwarf Aristocrat) 282 water-content of host, effect on inoculations 282 freezing, effect on 198, 199, 200 French potato disease, Delacroix's observations 214, 215 de Laharpe's observations 215 gas, not produced by 196, 200 Gram's stain, reaction to 198 Grand Rapids tomato disease, not due to 161 green fluorescence, not characteristic of 200 group number 200 growth on or in, acid bouillon 199 beef-agar, peptonized 195, 199 beef-bouillon, peptonized 194, 199 beef-bouillon, peptonized, plus sodium carbonate 194 blood serum, solidified 200 288 INDEX. Bacterium solanacearum (potato, tomato) — cont. growth, on or in — continued. Cohn's solution 197, 264 Dunham's solution 194 gelatin '95. 200 glycerin-agar, alkaline 196 litmus-lactose agar '95, 200 litmus milk cream free '94> 200 plus cream (Florida potato organism) . . 264, 282 Meyer's solution plus ammonium salts 251, 282 asparagin 282 potassium nitrate plus sodium salts 282 sodium salts 251, 282 urea and cane sugar 251 urea and grape sugar 251 milk 194- 200 nitrate bouillon 198, 199 peptone water J94 plus various sugars • • !9° potato cylinders, steamed 194, 196, 199, 200 potato-juice '9° silicate-jelly with Fermi's solution 197. 200 sugars 196. 199 sugar-beet agar 199 Uschinsky's solution 264 plus peptone !99 Honing's observations, host-plants !99 involution forms !94 Sumatran disease of Sesamum 218, 246, 253 host-plants 174. 199 (See also Peanut wilt; Tobacco, wilt-diseases.) Hunger's observations 181, 197, 209 incubation period 1 79 indol-production 198 infection, difficulty of producing with strains long-isolated. 1 79 effect of, dependent on, age of plant '78, 180 moisture content '79, '82 rapidity of growth 178 temperature 179. 182 incubation period '79 through wounds 181, 182 (See also inoculations.) inoculations 174. 178, 179, 182, 205, 209, 210 feebly virulent strains 199 methods 178, 179, 190, 209, 210 needle-prick, single, result of 19° successful, strains used for 1 79 synopsis of 182 uncertainty of results 178, 179, 268 (See also infections.) inoculations on, Abutilon sp '86 Capsicum annuum 209 cucumber 183, 184, 185, 186 Cucurbita foetidissima 185 Datura cornucopiae 186 Datura fastuosa 186 Datura metalloides 186 Datura stramonium 183, 184, 186 Datura tatula (?) 184 egg-plant '83 Eltusine indica 185 heliotrope 185 peanut 282 pear-tree (Japan) 183 Pelargonium zonale 1 83 pepino 1 83 pepper 1 83 petunia 184 Physalis crassifolia 184 Physalis philadelphica 185 Pagb Bacterium solanacearum (potato, tomato) — -cont. inoculations, on — -continued. Portulaca oleraceae 1 85 potato. 179, 180, 182, 183, 184, 185, 186, 187, 188,189 Blush variety 189 Burbank variety 187 Early Rose variety 187, 189 Green Mountain variety 188 Ricinus communis 185 Solanum carolinense 183 Solanum dulcamara 1 86 Solanum nigrum 183, 184 tobacco. . . 183, 184, 186, 189, 190, 209, 230, 233, 282 tomato 179, 180, 182, 183, 185, 186, 187,188, 189, 190, 209, 210 green fruits 176 Honor Bright variety 188 Livingston's Dwarf Aristocrat variety. . . 189, 282 Red pear variety 188 Vigna catjang 185 (See also Tobacco, wilt-diseases.) involution forms 193,194 isolation, difficulties of 178 Italian disease of tomato, Comes' observations 215 Voglino's observations 215 Iwanoff, Russian disease of potato 214 Japanese tobacco-wilt organism, relation to. . 244, 254 (See also "comparison, etc.") killed by dry air 282 Kirk, New Zealand disease of potato 207 lab-ferment 200 liquefaction, gelatin 195. 200 Loeffler's blood-serum 200 literature 218 litmus milk, effect on 194 local enlargements due to 199 longevity, on or in, agar 199 dry air 282 milk 194. 200 steamed potato 198. 200 loss of virulence 188 Malay States disease, Bancroft's observations. ... 211 Malkoff, disease of sesamum 216 milk, clearing of 194 morphology 193. 199 motility 193. 264, 268 Hutchinson's observations on 200, 265, 267 movement of bacteria in inoculated plants 179 New South Wales potato diseases, Helms' 207 New Zealand potato disease. Kirk's observations . . 207 Odium, African disease of potato 214 odor 197. 200 oxygen, free, necessary for respiration of 196 peanut, cause of disease in 271, 282 Petch, Ceylon disease of tomato 214 pigment, action of dilute acids and alkalies on 194 produced by 194, 200 solubility of 194, 200 polar staining 267 pseudozoogloeae 193, 194 reduction of nitrates 198 relation to tobacco-wilt organism North America 230, 231, 234 Sumatra and Japan 244, 254 (See also "comparison, etc.") resistant Solanum 182 resume of salient characters 199 ring disease of potato, Germany 166, 215 (See also Solanaceae, Brown rot, ring disease of potato, Germany.) ring disease of potato, Mysore 212 (See also Solanaceae, brown rot, ring disease of potato, Mysore.) INDEX. 289 Bacterium solanacearum (potato, tomato) — cont. Russian potato disease, Iwanoff's observations. . . 214 sesamum, bacteriosis 218 (See also Solanaceae, brown rot, sesamum bacteriosis.) similarity to, Bacillus musae 171 tobacco-wilt organism (N. Am.) 230, 231, 234 tobacco-wilt organisms (Sumatra and Japan) 244,254 (See also "comparison, etc.") lack of, Sumatran tobacco-wilt organism in lit- mus milk with cream (Florida potato strain) . . 264 Spieckermann's potato- parasite, distinct from .... 167 spores, not seen 193, 200 spread of parasite in inoculated plant 179 stains, reaction to 193 starch, slight action on 192, 197 sugared fluids, browning of 196 Sumatran tobacco-wilt organism, relation to. 244, 254 (See also "comparison, etc.") Sumatran tobacco organism, variations from Florida potato organism in milk with cream. . 264 termo-like shape of 175, 199 thermal relations 198, 200 tissues attacked 175, 176, 191, 199 tobacco-wilt of Dutch East Indies, relation to. . . . 222 tobacco-wilt organism (N. Am.), like. . . . 230, 231, 234 tobacco-wilt organism (Sumatra), variation from Florida potato-wilt organism in litmus milk with cream 264 tobacco-wilt organisms (Sumatra and Japan) re- lation to 244, 254 (See also "comparison, etc.") virulence, degrees of 1 99 effect on, of growth on culture media 179 loss of 179, 188, 268 viscidity, not marked 197, 200 Voglino, Italian disease of tomato 215 water-content of host, effect on inoculations 282 white substance surrounding colonies on agar. . . 199 wrinkled growth absent 200 Bacterium solanacearum (Sumatran tobacco- wilt organism), American studies 263 acid produced from cream in litmus milk 264 browning of culture-media 263, 265 crystals 265 drying, effect of 282 flagella 265, 268 Florida potato-wilt organism, dissimilarity in litmus milk with cream 264, 282 growth on or in, agar with Bacillus subtilis 264 cream-free litmus milk, plus 264 coconut oil 264 cottonseed oil 264 olive oil 264 peanut oil 264 fermentation tubes 265 lactose not fermented by 264 litmus milk 263, 264, 265 litmus milk containing cream 264, 282 Meyer's solution plus ammonium salts 251, 282 asparagin 282 potassium nitrate plus sodium salts 282 sodium salts 251, 282 urea and cane-sugar 251 urea and grape sugar 251 milk 263 mineral solution with KNO3 and glycerin . 242 nitrate bouillon 265 nutrient gelatin 265 peptone water, plus sugars 265 glycerin 265 Pagb. Bacterium solanacearum (Sumatran tobacco) — -cont. American studies — continued, inoculations, on peanut 282 tobacco 263, 265 tomato (Livingston's Dwarf Aristocrat) 282 liquefaction of gelatin 265 litmus milk bluing of 263, 264 reddening of 263, 264 reduction of 264 motility, occurrence of 265, 268 nitrates, reduction of 242 pathogenicity 263, 265 similarity to North American tobacco-wilt or- ganism, cream-free litmus milk 264 similarity to North American tobacco-wilt or- ganism, signs of disease 263 similarity, lack of, to Florida potato-wilt or- ganism in litmus milk with cream 264 similarity, lack of, to North American tobacco- wilt organism in litmus milk with cream .... 282 stains, reaction to 265 virulence, loss of 282 water-content of host, effect on inoculations. . . 282 Honing's studies 224, 244 (See also Tobacco, wilt-diseases, Honing's Suma- tran studies.) acid, toleration of 256, 257 acid-production 249 in litmus milk 248 adonite as carbon food 251, 252, 253, 257 aerobism 248 albumin as carbon-nitrogen food 250 alcohols, growth in 249, 251, 252, 253, 254 alkali, production of 249 retarding effect of 257 toleration of 256, 257 alkali-production in litmus milk 248 ammonia-nitrogen used by 251 ammonium salts as carbon food 253 ammonium salts as carbon-nitrogen food. . 250, 253 amount of inoculating material used in making cultures, effect of 258 arabinose as carbon food 251, 252, 253 asparagin as carbon-nitrogen food 250, 253 asparagin as nitrogen food 252, 253, 254, 257 asparagin plus glucose as food 251 asparagin plus saccharose as food 251 associated organisms 258 Bacillus nicotianae, relation to 244, 254 (See also "comparison, etc.") blackening of agar 248 gelatin. 248 potato slices 248 browning of beef bouillon 247 capsules 247, 253 carbon, substances used as source of . 250, 251,252,253 carbon-nitrogen foods 250 253 chains in medium containing glycocoll and glucose 253 clouding, time of, relation to number of bacteria used in inoculation . 258 comparison of Deli strains with Uyeda's descrip- tions of B. nicotianae and Smith's and Uyeda's descriptions of Bacterium solana- cearum 247, 248, 249, 250, 251 (See also "relation, etc.") cultural characteristics 247 cultural differences in strains 250, 251, 252 dextrin as carbon food 251, 252, 253 diastase production 249 drying, resistance to 249 dulcit as carbon food 251, 252, 253 erythrite as carbon food 251, 252, 253, 257 290 INDEX. Tage. Bacterium solanacearum (Sumatran tobacco) — cont. Honing's studies — -continued. fat, formation of 249 fibrin of blood as carbon-nitrogen food 250 filaments in medium containing glycocoll and glucose . . 253 flagella 247 galactose as carbon food 251, 252, 253, 257 gas production 249 glucose, action on 249 glucose as carbon food 251, 252, 253 glucose plus asparagin as food 251 glucose plus potassium nitrate as food 251 gluten as carbon-nitrogen food 250 glycerin as carbon food . . . . 251, 252, 253 glycocoll as carbon-nitrogen food 250, 252 glycocoll as nitrogen food 252, 253, 254 glycogen as carbon source 249, 251, 252, 253 glycogen production 249 Gram's stain, reaction to 247, 253 growth in or on, acid glycocoll-glucose solution 257, 258 agar 248 alcohols 249, 251, 252, 253, 254 alkaline glycocoll-glucose solution 257,258 asparagin-glycocoll-potassium nitrate solution plus glucose 257 plus mannose 257 asparagin-solution plus carbon foods 257 beef bouillon 247 fermentation tubes 248, 249 gelatin 247 hydrogen 248 litmus milk . . 248 Meyer's solution, plus ammonium chlorid plus glucose 251 ammonium chlorid plus glycerine , . 251 ammonium salts 250 asparagin plus carbon foods 252, 253 asparagin plus sodium salts 252, 253 ammonia plus carbon foods 253 carbon-nitrogen compounds 250 glycocoll plus carbon foods 252, 253 glycocoll plus glucose with variable reactions. 256 glycocoll plus mannose with variable reac- tions 256 potassium nitrate plus carbon foods. . . 251, 253 potassium nitrate plus sodium salts. . . 251, 253 milk 248, 253 monopotassium phosphate plus magnesium sulphate plus sodium chlorid plus potas- sium nitrate plus dextrin 250 glucose 250 glycerin 250 inuline 250 saccharose. 250 sorbit. . 250 potassium nitrate solution plus carbon foods. 257 potato 248 sugars 249,251,252,253, 254 urea solution plus glucose 257 plus mannose 257 growth under paraffin oil 248 guanine as carbon-nitrogen food 250 heat, resistance to 247 hemialbumose as carbon-nitrogen food 250 host-plants . .. 226,253,254,255,282 (See also Inoculations. j hydrogen sulphide, production of 249 indol-production ... 249 inoculating culture media, number of bacteria used, effect 258 inoculating plants, number of bacteria used,effect 260 inoculations, Ageratum organism on tobacco. . . . 226, 244, 245 Page. Bacterium solanacearum (Sumatran tobacco) — cont. Honing's studies — continued, inoculations — continued. Djatti organism on Djatti plants 254 Nicotiana affinis 255 N. atropurpurea grandifiora 255 N. colossea 255 N. glauca 255 N. rustica 255, 256 N. sanderae 255 N. silvestris 255 ornamental and common tobacco ..... 254, 255 Mucuna organism on tobacco 244 Pluchea organism on tobacco 244 Pouzolzia organism on tobacco 226, 244 tobacco organism on Capsicum 246, 253 Nicotiana affinis 255 N. atropurpurea grandifiora 255 N. colossea 255 N. glauca 255 N. rustica 255, 256 N. sanderae 255 N. silvestris 255 ornamental tobaccos 255 tobacco organism (?), on potato 245, 253 tobacco organism, on Pouzolzia 245 tobacco organism (?), on Sesamum orientate. 253 on Solanun Iycopersicum 253 tobacco organism, on Solanum melongena 245, 253 on tobacco 225, 244 (?), 245, 255 inoculations, soil: tobacco organism on tobacco 225 inoculations, soil-water, on Ageratum 245 Pouzolzia 245 tobacco 245 inosite as carbon food 251, 252, 253, 257 inulin as carbon source 251, 252, 253 isolation from Ageratum 226 Mucuna 226 Pouzolzia 226 tobacco 225, 246 Japanese tobacco-wilt organism, relation to . 244, 254 (See also "comparison, etc.") lactose as carbon food 251, 252, 253 legumin as carbon-nitrogen food 250 leucine as carbon-nitrogen food 250 levulose as carbon food 251, 252, 253, 254, 257 lichenin as carbon source 251, 252, 253, 257 liquefaction of gelatin 247 literature 270, 271 litmus, reaction to (milk cultures) 248 litmus, reduction of 249 maltose, as carbon food 251, 252, 253 mannit, action on 249 as carbon source 251, 252, 253 mannose as carbon food 251, 252, 253, 257 maximum temperature 249 methylene blue, reduction of 249 morphology 246 variability in 253 motility 247 nitrate, reduction of 249, 253 nitrogen, substances used as source . . 250,251,252,253 nitrogen and carbon from one substance . . . 250, 253 nuclein as carbon-nitrogen food 250 number of bacteria. effect on growth in culture media . 258 effect on progress of disease 260 odor 249 peanut wilt, cause of 153, 253 pellicle 247 peptone as carbon-nitrogen food 250, 253 INDEX. 291 Page. Bacterium solanacearum (Sitmatrau tobacco) — cont. Honing's studies — continued. phenolphthalein, reaction to (milk cultures) . . 248 physiological characters, variations in 253 physiology 247 poiar staining 247, 253 potassium nitrate as nitrogen food. 250, 251, 254, 257 potassium nitrate plus glucose as food 251 plus saccharose as food ... 251 potassium nitrite as nitrogen food 250 potato and tomato-wilt organism, relation to 244 (See also "comparison, etc.") protein as carbon-nitrogen food 250 quercit as carbon food 251, 252, 253 raffinose ascarbon food 251, 252, 253 relation of tobacco-wilt organisms (Sumatra and Japan) and Bact. solanacearum Smith described from potato and tomato 244, 254 (See also "comparison, etc.") rhamnose as carbon food .... 251,252,253 saccharose, action on . ... 249 as carbon food. .... 251, 252, 253 plus asparagin as food 251 plus potassium nitrate as food. 251 saprophytes, associated with 258 inhibiting action ....... 259 similarity of tobacco-wilt organisms (Sumatra and Japan) and Bact. solanacearum Smith, de- scribed from potato and tomato 244. 254 (See also "comparison, etc.") sodium salts as carbon food 251, 252, 253 sodium selenite, reduction of 249,253 sorbit as carbon source 251, 252, 253 spores . . 247. 253 staining of culture media 247, 248, 249 starch, action on • 249 as carbon food 251, 253 strains, variability 244, 250, 251, 252, 257 sugars, growth in 249, 251, 252, 253,254 summary 253, 258 temperature relations 249 thermal death-point 249 tomato and potato-wilt organism, relation to . . 244 (See also "comparison, etc.") transmission of 254 tyrosin as carbon-nitrogen food 249, 250, 253 variability' effect of alkalinity on 258 effect of light 258 effect of temperature 258 in carbon and nitrogen-compound tests ... 253, 256,257,258 in cultural characteristics .250,251,252,253,256,257 in milk ■ 253 in toleration of acid 257 in toleration of alkali 257 in virulence 244 variations, transitory nature of 258 virulence, loss 253 Bacterium solanacearum var. Asiaticum nov. var 282 Hunger's observations 222 Bacterium stalactitigenes, associated with Bact. solanacearum in Sumatra 259 Bacterium stewarti 91, 132 (See also, Corn, Stewart's bacterial disease.) acids, toleration of certain 136, 144 acid fuchsin. effect of 140 acid production . I39.I42> J45 aerobism, pronounced 138 agar stabs, growth in 133 agar streaks 134 alkali produced 142 alkali, toleration of 138 P.tCR. Bacterium stewarti — continued. animals, no experiments with 144 asparagin, not a carbon food 145 cane-sugar favors growth 138 capsule 132 chains '32 chloroform, slight retarding effect of . . . . 143, 144, 145 colonies, appearance of 133 color on media 133. *34. 135, 137. 138 comparison with Apl. miehiganense on potato. ... 164 crystals 133, M2. '44 cultural characteristics 133 cytase, doubtful occurrence 142 description 132 dextrin, no increased growth with 138 dextrose, increased growth with 142 diastase 142. 145 discoloration of bundles 16 discovery of organism 91 dissemination through infected seed 114, 127 dry air, resistance to 144 endospores not seen 132 enzymes 142, 145 ethyl alcohol, no acid from 139 fermentation tubes, growth in 138, 145 filaments 132 flagella, polar. 91, 132 formalin, infected seeds treated with 128 galactose favors growth 138 gas production not seen. . . 138, 145 gases other than air, behavior in 138, 139 gelatin not liquefied 133 germicides 142 glycerin, no acid from 139 no increased growth with 138 some acid from 139 Gram's stain 132 group number 145 growth on or in. acid beef-bouillon. . 136, 138, 141, 145 agar. 133, M2, H4 agar in atmosphere of hydrogen 139 agar plus potassium formate and phenolphthalein 143 alcohols 139, 14S alcohol agars 1 39 alkaline beef-bouillon 138, 141, 144 alkaline beef- bouillon in vacuo. . . 139 ammonium salts i4°> l4I asparagin water . 141, I44> '45 beef-bouillon in atmosphere of carbon dioxide 139 beef-bouillon in atmosphere of hydrogen. . 138, 139 beef-bouillon over chloroform 143, 144. H5 beef- bouillon concentrated and strongly acid. . . 136 beef-bouillon plus ethyl alcohol 139 beef-bouillon plus glycerin 139 beef-bouillon plus peptone. 136, 144 beef-bouillon plus sodium chloride 138 cabbage-juice 136, 138, 144 cane-sugar 139, 142 cane-sugar plus asparagin 144 cane-sugar agar plus litmus . 139 cane-sugar starch jelly 138 cane-sugar water 140, 141 cane-sugar water plus ammonium citrate 140 cane-sugar water plus ammonium lactate. 140, 141 cane-sugar water plus ammonium tartrate . . 1 40, 1 4 1 cane-sugar water plus asparagin 140, 141 carbon dioxide 139. '45 carrot cylinders 142 carrot cylinders in vacuo 139 coconut cylinders 135. r42. '44 coconut cylinders in atmosphere of carbon dioxide 139 coconut cylinders in atmosphere of nitrogen 138, 145 coconut cylinders in vacuo 139 Cohn's solution 138, 140, 144 292 INDEX. Pagb. Bacterium steward — continued, growth on or in — continued. dextrin starch jelly dextrose agar plus litmus distilled water plus dipotassium phosphate, mag- nesium sulphate, ammonium phosphate and sodium acetate Dunham's solution plus methylene blue Dunham's solution plus rosolic acid in atmos- phere of hydrogen Dunham's solution plus rosolic acid and hydro- chloric acid 139, Fermi's solution galactose galactose agar plus litmus galactose starch jelly gelatin 133, 144, glycerin agar glycerin agar plus litmus glycerin starch jelly grape-sugar 139, hydrogen 138, lactose agar plus litmus lactose starch jelly litmus agars plus sugars litmus milk 137, 140, Loeffler's blood-serum 133, 144, maltose agar plus litmus maltose starch jelly mannit mannit agar plus litmus mannit starch jelly milk 137, 142, mulberry agar neutral beef-bouillon in atmosphere of hydrogen, neutral beef-bouillon plus potassium formate . . nitrate agar 133, nitrate bouillon 132, 138, nitrate bouillon plus grape-sugar nitrogen 138. nutrient starch-jelly plus sugars peptone water peptone-water plus acid fuchsin and hydrochlo- ric acid peptone water plus grape-sugar and methylene blue in an atmosphere of hydrogen peptone water plus salt and indigo carmine peptone water plus salt and rosolic acid peptone water plus salt and rosolic acid in an atmosphere of hydrogen peptonized beef bouillon 136, potato broth 136, potato broth plus malic acid 136, potato cylinders 134, 142, 144, potato cylinders in an atmosphere of carbon dioxide potato cylinders in an atmosphere of hydrogen . potato cylinders in vacuo radish 135, rutabaga 135, 142, saccharose 1 39, saccharose agar plus litmus silicate jelly 134, starch jelly plus sugars 138, sugars 138, 139, sugar agars sugar starch-jellies sugar-beet 135, tomato juice 136, 138, 140, 141, 144, turnip 13s, 142, turnip in atmosphere of nitrogen 138, Uschinsky's solution. . . . 132, 137, 140, 142, 143, Uschinsky's solution in atmosphere of hydrogen. vacuo 139, host-plants 89 hydrogen peroxide, seeds treated with 128 37 41 39 45 38 39 39 38 45 34 39 38 42 45 39 38 39 45 45 39 38 39 39 38 45 36 39 43 44 40 38 45 38 44 40 39 40 39 39 44 44 38 45 39 39 39 42 44 42 39 44 45 45 39 38 14 45 44 45 45 39 45 Pagb. Bacterium steward — continued. hydrogen sulphide, doubtful production of 142 ice box, growth in 143, 144 indigo carmine, effect of 140, 145 indol reaction 142, 144, 145 infected seed, treatment of 125, 127, 128, 146 inoculations 96 method, by needle punctures 113 by spraying 96, 97, 129 through water- pores 96, in, 112 on field corn 93, in, 112, 129 on oats 93 on pop-corn 93, 1 1 1 on sweet corn 92, 96, no, 112, 129 Series I and II 96 Series I and II, detailed results 98 Series I and II, tabulated results 109 Series III— VII no Series III— VII, tabulated results m Series VIII and IX in Series X 112 Series XI-XV 121 Series XVI-XIX 125 Series XX-XXIV 128 Series XXV 129 Stewart's 91, 92 inversion of cane-sugar 142 invertase 142, 145 isolation attempted from infected kernels 124 isolation by direct transfer from diseased stems. . . 96 kernels penetrated by bacteria 114, 116, 130, 131 lab ferment 142, 145 lactic acid, toleration of 144 lactose, no increased growth with 138 lesions produced 130 literature 147 litmus agars, behavior on 139 Loeffler's blood serum, not liquefied by 133 malic acid, toleration of 144 maltose, no increased growth with 138 mannit, no increased growth with 138 maximum temperature 143 mercuric chloride, seeds treated with. . . 125, 127, 128 methylene blue, vital staining with 132, 139, 145 milk, changes in 137, 140 minimum temperature experiments 143 mixed cultures 142 mixed infections 142 morphology 132, 144 motility 132 nitrates, not reduced by 145 nitrogen foods 141 nitrogen, no growth in 138 odor 135 organs invaded 131 oxygen, evolution of from hydrogen peroxide. . . . 142 parts of plant attacked 131 phenolphthalein, reaction of formate agar cultures . 143 pigments 142 polar flagella 91, 132 potassium formate, effect of 143 potato starch, slight action on 142 potato streak cultures, appearance 135 pseudozoogloeae 132, 137, 143 reduction of litmus 137 reduction of nitrates 138 resume of salient characters 144 rosolic acid, effect 139, 145 sodium chloride, marked toleration of 138, 145 stain in host-plant 16, 142 stains, reaction to 132, 144 starch, action on 134 sunlight, effect of 144 tartaric acid, toleration of 144 temperature relations 143, 144, 145 INDEX. 293 Bacterium stewarti — continued. thermal death-point 143 tissues attacked 89, 90, 91, 93, 131 trypsin 142, 145 Uschinsky's solution, good growth in 137 virulence, retention of 142, 145 viscidity 103, 126, 127, 132, 137 vitality 142, 145 wind, not borne by 121 yellow pigment, color dependent on air 139, 142 variable appearance of 142 Bacterium sumatranum, associated with Bact. sola- nacearum in Sumatra 259 Bacterium vascularum 54 (See also Sugar-cane, Cobb's disease of.) acid culture media, inhibiting effect on growth . 58 61, 63, 67 acid potassium phosphate, effect on growth 64 acidity of cane-juice, relation to bacterial growth. 47 aerobism 48, 64 air-hunger 58, 64 agar not stained by 56 alcohol, slime of, insoluble in 7. 14 alkali, effect on growth 58, 59, 61, 63, 67 alkaline reaction of cultures 56, 57, 60, 61 ammonium, effect on growth 64 ammonium salts as nitrogen foods 63 asparagin as nitrogen-food 63 bacterial slime oozing from leaves and stems ... 16, 22, 25.53.66 calcium, effect on growth 64 cane cuttings, disease transmitted by 48, 68 cane slices, steamed, growth on 60 cane-sugar, action of organism on 62 cane-sugar, as carbon food 57, 63 cane-sugar, effect on growth 64 capsules 67 carbon dioxide, effect on growth 64 carbon foods 63, 64, 67 chemical reactions of bacterial slime 7, 13, 14 citric acid, effect on growth 63 Cobb's studies 5,6, 7, 8, 12 (See also Sugar-cane, Cobb's disease of.) colonies, appearance of 8, 55, 57, 67 color of 54, 60 conditions favoring spread of 48 crystals, production of 56, 61, 67 cultural characters 8, 54, 67 culture media most favorable 65, 67 description of organism 7, 8, 54 dextrin, effect on growth 64 dextrose as carbon food 63 dextrose, effect on growth 64 effect on sugar-cane 3 fermentation-tube cultures 64 flagella, staining 54 gas-production, not observed 57. 64, 67 gelatin, browning of 59 color on 60 growth on 8 liquefaction of 57,59 tear drops on 59, 67 with cane-juice, growth on 65 with fruit-sugar, growth on 57 with glucose, growth on 57, 65 geographical distribution 3 glycerin, effect on growth . 63, 64 Gram's stain, reaction to 54 group number 67 growth, best medium for 65 growth best at 300 64, 65 growth inhibited at blood temperature 64 Pagb. Bacterium vascularum — continued, growth on or in, acid agars 63, 67 acid bouillons 61, 67 acid fluids, Greig-Smith's investigations 61 agar plus cane-sugar 56 agar plus fruit-sugar 56 agar plus peptone, levulose, potassium phosphate and tap- water 65 agar plus peptone, saccharose, potassium phos- phate and tap-water 65 alkaline agars 63 alkaline bouillons 61, 67 beef-agar 56, 66 beef-bouillon plus peptone 60, 67 beef-bouillon plus potassium formate 61 beet-cylinders 60 beet-juice agar plus peptone and saccharose ... 67 cane cylinders, steamed 47, 60, 67 cane-juice agar plus peptone 56 cane-sugar water plus peptone 62 carrot 60 cauliflower cylinders 60 coconut cylinders 60 Cohn's solution 67 distilled water plus dextrose, glycerin and so- dium asparaginate 64 Dunham's solution 67 Fischer's mineral solution 63 fruit sugar-agar 56 gelatin 8, 57, 59, 60, 65. 67 gelatin plus cane-juice 57. 58, 65 cane-sugar 57.58 fruit-sugar 57 glucose, Greig-Smith's description 57. 65 peptone, dipotassium phosphate and malic acid 57 peptone, dipotassium phosphate, malic acid and cane-sugar 58 peptone, dipotassium phosphate, malic acid, cane-sugar and sodium hydrate 58 glycerin agar 56 Hunger's agar 57 litmus lactose agar 57, 67 litmus milk 61 milk 66 nitrate bouillon 61 nutrient agar 54, 56, 66 poured-plates 54 slants 56 stabs 56 onion bulbs steamed 60 peptone water plus cane-sugar (fermentation tubes) 64 glycerin (fermentation tubes) 64 grape-sugar (fermentation tubes) 64 maltose (fermentation tubes) 64 mannit (fermentation tubes) 64 milk-sugar (fermentation tubes) 64 oxalic acid 63 potato, Greig-Smith's studies 65 potato agar 57, 66, 67 potato cylinders, steamed 60, 66, 67 saccharose agar plus peptone 56 silicate jelly 134 sugar-agars 56, 65 sugar-beet agar 56 turnip 60 Uschinsky's solution 62 gumming, constant association of organism with . 8, 2 1 gum, identity with bacterial slime 13 (See also Sugar-cane, Cobb's disease of.) gum-production, dependent on certain carbon foods 64 host-plants 3 indol reaction, faint 64 294 INDEX. Bacterium vascularum — continued. inoculation experiments, Cobb's . S, 12 inoculation experiments, Tryon's 1 1 inoculation, methods of 21, 27,31,33.46 inoculations, general remarks on ... 46 inoculations on Blanca cane 34 Caledonia Queen cane 33. 34. 46 Cinta cane ■ • ■ • 34 Common Green cane 21, 32, 34, 46 Common Purple cane 27 Crystalina cane 33. 34. 46 Jamaica cane 33- 34. 46 Louisiana cane. Variety No. 74. 27.34. \6 Striped Green cane 3 ' , 34. 4° inoculations, pure-culture .11,21 inoculations, variety tests. ... 31. 33. 46 ■ inversion of cane-sugar 62 isolation, methods of 54 isolation from artificially infected sugar-cane 2 1 , 24, 39, 46 Australian cane 21,05 red bundles ' 7. 5 1 red ooze 4° yellow ooze 46 lactose, effect on growth 64 lesions produced '3° levulose, effect on growth 64 liquefaction of gelatin 57. 59. 67 literature 7 • litmus, reaction 56, 57. 61, 67 longevity, effect of temperature on 66 in culture-media 65, 66 in the host 65 magnesium, effect on growth 64 malic acid, effect on growth 63 maltose, effect on growth 64 maximum temperature 64 measurements 54. 67 milk, acids not formed from 61 casein not precipitated . 61, 67 effect of organism on 61,62 surface growth not conspicuous 61, 62 tyrosin not formed in 62 with litmus becomes indigo blue 61 morphology 7. 21, 54, 67 motility, best seen in young cultures 54 motility, often absent in old growths .54 nitrates, not reduced by 61 nitrogen foods. 63, 67 one-flagellate 3.21,55, 5& optimum temperature 65 oxalic acid, effect on growth 63 pathogenicity s. 2 ' phenolphthalein, reaction of organism to 61 phosphates, effect on growth ... 57.64 potassium, effect on growth 64 potassium nitrate as nitrogen food 63 potato, growth on 60, 65 poured-plate cultures, description 54 I .seudozoogloeae 54. 61 pure-cultures from diseased cane 21 red bundles, isolation from 17. 51 reddening of bundles ... 16 (See also Sugar-cane, Cobb's disease of.) reduction of nitrates 61 relation to B. sacchari Spegazzini . .88 resume of salient characters 66 resuscitation from old diseased canes 46 saccharose, action on 62 saccharose, as carbon food 63 saccharose, effect on growth. 64 salts, effect on growth 64 Pagb. Bacterium vascularum — continued. slime, oozing from leaf-sheaths and stems 16, 22,25,53,66 Smith, R. Greig, studies 57,60,61,62,63,64 (See also Sugar-cane, Cobb's disease of.) sodium chloride, effect on growth 63, 64 spores j 54 stains, reaction to 54 starch, effect on growth 64 starch, slight action of organism on ' . . 60 substitution for B. pseudarabinus in Greig-Smith's experiments on red stain in gummed cane . . . 50 sugars as food 63, 64 sunlight, organism sensitive to 66 tear-drop formation on gelatin 59. 67 temperature, effect on longevity 66 temperature relations 64, 67 thermal death-point experiments 64, 65 tissues attacked 6, 7, 15, 24, 66 trimethylamin, production of 56 Tryon's studies 1 1 (See also Sugar-cane, Cobb's disease of) Uschinsky's solution, feeble growth in 62 vascular system attacked by 6, 15, 24, 66 vasculin 7 viscidity 56, 61, 66 vitality in stem and on media 65, 66 yellow slime, primrose to lemon color on potato 60 whitish in absence of air 58 Bacterium xanthochlorum, (See also Potato, Schuster's German Rot.) acid, toleration of 275 acid-production 272, 275 aerobism . . 273 alcohols, growth in. 273, 275 alkali, toleration of 275 alkali-production 272, 275 ammonia-production 272 amylase produced 272 anaerobism, facultative 273 biological characteristics 272 blood-serum, non-liquefaction of 273 chloroform, effect of 275 colonies on agar 273 colonies on gelatin ; 273 disintegration of potato-tuber sections in infected potato-juice 274 drying, sensitive to 275 entrance of organism into tuber 272 enzym, action on starch 274 effect on cell-walls 274 effect of temperature on 274 extraction of 274 injection into potato-tubers 274 fermentation-tube experiments 275 filaments, production of 272 flagella _ 272 effect of temperature on formation of 272 fluorescence, greenish-yellow 273 galactose, fermentation of 275 gas, non-production 273, 275 gelatin, liquefaction 273, 274 glycogen, production of 272 Gram's stain 272 grape-sugar, fermentation of 275 growth on, or in, acid bouillon . 275 agar (plus 15) 275 alkaline agar 273 alkaline bouillons . 275 alkaline potato-agar 274 alkaline potato-agar plus tyrosin 274 beer 273 bouillon over chloroform 275 bouillon plus grape-sugar 273 bouillon, peptonized, plus sodium chlorid 275 INDEX. 295 Page. Bacterium xanthochlorum — continued, growth on or in — continued. carrot-juice agar 273 Cohn's solution 27s fermentation tubes 275 gelatin 273, 274, 275 plus sodium selenite 273 Loeffler's blood-serum 273, 275 Meyer's solution, plus, alcohols 273 ammonium salts 273 arbutin 273 asparagin 273 caffein 273 cellulose 273 citric acid 273 glycerin 273 peptone 273 potassium salts 273 potato starch 273 sugars 273 milk 273, 275 neutral nutrient bouillon 273 nutrient agar 273 peptone-water plus cane-sugar 275 fructose 275 galactose 275 glycerin 275 grape-sugar 275 lactose 275 maltose 275 mannit 275 potato-juice-agar 273 potato-juice gelatin 273 Uschinsky's solution 275 growth, optimum temperature 273 hemicellulase produced 272 host-plants 272 hydrogen sulphide produced 273 incubation period 274 indol produced 273 inoculations on, Campanula raphunculus 272 carrot roots 272 fodder beets 272 lupines 272, 274 Lupinus nanus douglasi 272 pelargoniums 272 Physalis alkekengi 272, 274 potato tubers 272, 274, 275, 276 sugar beets 272 tobacco stems 272 tomatoes 272 Vicia faba 274, 275, 276 yellow lupins 272 involution forms 272 isolation 272 lenticels, inability of organism to enter 272 light, effect on virulence 273 light, sensitive to 275 liquefaction of gelatin 273, 274, 275 liquefaction of Loeffler's blood-serum, non-occur- rence of 273, 275 literature 276 methylene blue, reduction of 273 methylamin-production 272 milk, coagulation of 273 milk, peptonization of 273 morphology 272 effect of temperature on 272 motility 272 effect of culture media on 272 effect of temperature 272 nitrates reduced 273 non-fluorescent strain 275 Page. Bacterium xanthochlorum — continued. optimum temperature 273 parasitism, weak 272 pathogenicity 272 compared with that of Bacillus phytophthorus. 272 effect of temperature on 272 pigment, effect of culture media on 272 solubility of 272 potato starch, effect on 273 potato-tuber, disintegration of sections in infected potato juice 274 pseudozoogloeae 272 red stain on alkaline potato-agar, Spitzer's reaction 274 Schuster's observations 272 sodium chlorid, effect of 275 sodium selenite, action on 273 solubility of pigment 272 solution of middle lamella 274 spores, non-occurrence of 272 stains, reaction to 272 starch, action on 273 stomatal infection (Vicia faba) 272, 276 rapidity of 274 sugars, growth in 273, 275 temperature, effect of, on, enzym action 274 flagella-formation 272 milk cultures 273 morphology 272 pathogenicity 272, 274, 275 temperature, relations 273, 274, 275 thermal death-point 275 tissues attacked 272, 274, 276 toxin produced 272, 274 trimethylamin-produetion 272 trypsin produced 272 tyrosinase produced 272 vascular bundles occupied by 272 virulence, effect of light on 273 wounds, means of entrance 272 xanthochlorum hemicellulase 274 Bacterium zinnoides associated with Bact. solanacearum in Sumatra . . 259 Bailey, observations on New York tomato disease. . 205 Banana, Bengal disease 173 Cephalosporium 1 73 Fusarium 1 73 literature 173 Surinam disease, resemblance to 173 Banana, Central American disease. (See Banana, Panama disease.) Banana, Costa Rican disease. (See Banana, Panama disease.) Banana, Earle's Jamaican disease 168 geographical distribution 168 literature 173 organism isolated 168 Panama disease, relation to 173 signs of disease 1 68 Banana, leaf-blight 168 (See Banana, Earle's Jamaican disease.) Banana, Panama disease 172 bacteria in vessels 173 gas-forming 171 non- pathogenic 171 cause 173 Costa Rica, occurrence in 172 Earle's Jamaican disease, relation to 173 Fusarium in bundles 173 Fusarium isolated 171 geographical distribution 172, 173 inoculations . 172 leaf-curvature, characteristic of 172 literature 173 losses 172 296 INDEX. PagB. Banana, Panama disease — continued. parts of plant attacked 172 resistant varieties 173 Rorer's Trinidad disease, differences 171 signs of disease 172 splitting of leaf-sheaths 171 staining of bundles 173 Surinam, occurrence in 173 susceptible variety 1 7 ' Trinidad, occurrence in 173 vessels sometimes occupied by bacteria 171 wholesale prevalence of 1 72 Banana, Rorer's Trinidad disease 170 (See also Bacillus musae.) bacterial ooze 1 7 1 breaking over of leaves 17° cavities, bacterial 17°. 1T2 cells invaded by 1 7 1 diseased suckers, planting of 17° distinct from Panama disease 1 7 s inoculations 1 70, 1 7 1 inoculations on Manila hemp 17' solanaceous plants 17 1 tomato 1 7 1 isolation of parasite 1 7° literature 173 Panama disease, how different from. . . 171 resistant variety 1 7 r signs of disease 170 splitting of leaf-sheaths, not characteristic. . 171 staining of bundles 170 tissues attacked 170, 171, 172 varieties attacked 170 vessels occupied by bacteria I7°i '71 Banana, Smith's Cuban disease 168 bacteria sometimes present in 168 cause 1 69 comparison with Trinidad disease 171 inoculations 169 isolation of Fusarium 168 literature 1 73 signs of disease '7° splitting of leaf-sheaths 171 vessels, browning of 168 vessels, occupied by Fusarium 168 Banana, South American diseases 173 Banana, Surinam disease 173 (See also Banana, Panama disease.) Fusarium present 1 73 literature '73 resemblance to Bengal disease 173 Banana, Vascular diseases 168 (See Banana, Bengal disease ; Banana, Earle's Jamaican disease; Banana, Panama disease; Banana, Rorer's Trinidad disease : Banana, Smith's Cuban disease.) literature 1 73 Bancroft, rot of potato and tomato, Malay States . . 211 Basu, Bengal disease of banana 1 73 Benecke, Sereh observations 72. 73 Biffen, English disease of mangolds and sugar-beets. 154 "Black-leg" of tobacco 239 (See Bacillus nicotianae Uyeda : Tobacco, wilt-diseases, Japanese disease.) Blight of Young Rose Shoots. iS4 Bolliger, Brazilian disease of manihot 280 Bonamc, studies on "gummosis" of sugar-cane. 3,10, 1 5,63 (See Sugar-cane, Cobb's disease.) Bondar, Brazilian disease of manihot 280 Bos, Ritzema, flax stems, bacteria in vessels 154 Bowhill, tobacco-wilt. South Africa 268 Brazilian disease of manihot 280 (See Manihot, Brazilian disease.) Briosi, Italian disease of stock 277 Brown rot of Solanaceae 174 (See Solanaceae, brown rot; Bacterium solanacearum.) Page Burrill, bacterial disease of potato 203, 205 Bacterium solanacearum 178 brown rot of Solanaceae, observations on 181 Burrill's disease of maize 89 (See Corn, Stewart's bacterial disease.) Butler, E. J., Sereh and cane-gummosis in India. . . 3 Cabbage, brown rot 191 Canker of tobacco 266, 267, 270 (See Bacillus aeruginosus, Tobacco, wilt-diseases, French disease.) Capsicum annum, wilt. (See Pepper, blight.) Cephalosporium, Bengal banana disease 173 Ceylon disease of tomato (Bacterium solanacearum?) 214 Chlorosis of sugar-cane 81 (See also Sugar-cane, Top-rot, and Sugar-cane, Pokkah bong.) Cladosporium herbarium, orchard grass, Rathay's disease 155 Clark, Cobb's disease of sugar-cane, occurrence in Fiji 3 pecuniary losses in Fiji 70 Clinton, brown rot of Solanaceae reported from Connecticut 17s tobacco canker 267 Cobb. disease of sugar-cane 3 (See Sugar-cane, Cobb's disease of) studies on "gummosis" of sugar-cane. (See Sugar- cane, Cobb's disease of). Cobey, tobacco- wilt, losses 237 Coleman, Mysore ring disease of potato 212 (See also Solanaceae, brown rot, ring disease of potato, Mysore.) Collar-rot of tobacco 266, 267, 270 (See Bacillus tabacivorus; Tobacco, wilt-diseases, French disease.) Comes, Bacterium gummis 203, 2 15, 266 Italian disease of tomato 215 tobaeco-rot, Italy 266 Cook, top-rot of sugar-cane in Cuba 3 Corbett, observations on New York tomato disease. 205 Corn, Stewart's bacterial disease 89 (See also Bacterium stewarti.) bacterial occupation of the vascular system. . . 89, 90, 91,93,94, 130 bacterial ooze from cut stems, etc 89, 90, 91, 131 bacterial pockets 131 bacterial slime, enormous quantity of 126 homogeneity of 95 occurrence in stem 6 feet from point of infection. 126 paler in upper part of stems 94 bundles, stem, anatomy of 129, 130 cavities 91, 95, 131 cellulose not destroyed 130 cirri in 126 cob penetrated by bacteria 1 14 congressional seed distribution, disease spread by ._. 114, 115 constitutional signs, how soon visible 97 corn kernel, anatomy of 114, 1 16, 130, 131 crushed kernels, test cultures should be made from. 125 definition 89 dissemination through infected seed 1 14, 127 drouth, signs conspicuous during 94 Duggar's observations 89 dwarfing, due to 89, 90 influence on 1 1 1 early sweet varieties, most subject to. . . . 1 12, 1 13, 118 etiolation 90 etiology 91 field corn varieties subject to 1 12, 1 14 field observations on 94, 1 14 frost effects, resemblance 101 INDEX. 297 Corn, Stewart's bacterial disease — continued. fungicides 93, 146 geographical distribution 89, 93, 146 germicides 93, 146 germination of treated seed best 127 history 147 host-plants 89 hot-water treatment of seed 146 husks, anatomy of 1 19, 120 bacterial pockets in 103 spots and patches on 121 hyperplasias absent in 131 infected seed, dissemination through 114, 127 experimental plantings of 115, 121, 125, 128 treatment of 146 treatment with formalin 128 treatment with hydrogen peroxide 128 treatment with mercuric chloride 125, 127, 128 infection, accidental, manner of occurrence 112, 124 course of 90, 102 from base upward 96, 125 from top downward 113 insects, not due to 93. 94 means of, Stewart's theory 93 most serious in seedling stage. ... no, 113, 125, 129 slow to appear 127, 128 through seed 1 1 4, 1 2 7 through water-pores 94 time of 89 inoculations 96 (See Bact. stewarti.) inoculations, Stewart's 91, 92 result of late 113, 129 isolation of Bact. stewarti, attempted from infected kernels 124 by direct transfer from diseased stems 96 kernels, bacterial contamination of surface 114 penetrated by the bacteria 1 14, 131 leaf-tips, bacteria found in 95 leaves, effect on 89, 90 lesions produced 130 literature 147 local signs, how soon visible 97 losses, pecuniary 1 46 male inflorescence, premature development 89 male inflorescence, whitening of . 89,94 mercuric chloride, effect on infected seed. . . . 125, 127 method of obtaining pure cultures from 96 moist soil, effect of 122, 123, 124 morbid anatomy 130 nature of, markedly vascular 131 nodes, brown stain in 90, 100, 102 organism causing, description of . . 132 (See also Bact. stewarti.) organisms other than Bact. stewarti (on kernels) . . 12,1 parenchyma, bacteria in 91 parts of plant attacked 131 polar flagellate bacterium, cause of 91 pop-corn, experiments 93, 1 1 2 premature development of male inflorescence. . 19, 89 preventive measures 145 pure cultures from 96 resistant varieties 117 root-pressure ceases with frosting of leaves 89 roots, effect on not conspicuous. . . 90, 95, 105 seed, infected, dissemination through 114, 127 seed, treatment 146 shoots, production 131 signs of disease 89, 94 slow growth, hinders development of disease no, 1 1 1, 127, 128 Pags. Corn, Stewart's bacterial disease — continued. soil conditions, effect 123, 124 specific nature of organism causing 125 sprouting of basal buds 90 staining of tissues 89, 90, 95 stem, effect on 89 Stewart's studies, browning of tissues 90 discovery of Bact. stewarti 91 field observations 91 geographical distribution 89, 93 inoculations 93 means of infection 93 susceptible varieties 118 weather conditions, effect 94 stomata, bacterial ooze through 119, 120, 132 susceptible varieties 89, 94, 118 susceptibilitv, effect of slow growth no, 127, 128 time between infection and appearance of 90, 109, 127, 128 tissues attacked 89, 90, 91, 93 toxine, not conspicuous in 130 treatment 93, 145 trial plots, results on 117 imeveness of fields attacked by 91 varietal resistance, question of 117, 124 varieties resistant to 118 vascular infection, generalized 102, 106 vascular system, bacterial occupation of 89, 90, 9i.93.94. 130 viscidity of slime in 126, 127 water- pores, infection through 94 weather conditions, effect of 94, 124 whitening of male inflorescence S9, 94, 113, 115 yellow bundles 95 yellow ooze 90, 94 variability in color of 94, 95 Corynebacterium piriforme associated with Bact. solanacearum in Sumatra 259 Councilman, blight of young rose shoots 154 Cozart, tobacco- wilt, losses 237 Crossonini, indol reaction with paradimethylamido- benzaldehyd 249 Cucurbit wilt, vascular nature of 191 Dactylis glomerata, bacteriosis of 155 (See Orchard grass, Rathay's disease of.) Debray, Bacillus sacchari 76 De Jong, peanut-wilt, flooding infected fields for 271 planting, selected seed for 271 Delacroix, Bacillus aeruginosus 266 Bacillus caulivorus 175 B. putrifaciens putridus 266 B . solanincola 175,215 B. tabacivorus 266 French disease of potato 214 Tobacco, Fusarium disease 228 wilt-diseases 266, 270 de Laharpe, French potato disease 215 Deli tobacco organism [See Bact. solanacearum (Sumatran tobacco-wilt organism. 1] Dewar, tobacco- wilt. South Africa 268 Diem, Deli tobacco, variations 261 Diplococcus enteritis, Gram's stain, reaction to 247 Doryphora 10-lineata, distribution of brown-rot of Solanaceae by 1 80 Dranert, studies on hacterial disease of sugar-cane . 3, 70 Drost, South American banana diseases 173 Dutch disease of wallflower 277 Dutch East Indian disease of tobacco 220 [See Bacterium solanacearum (Sumatran tobacco-wilt organism); Tobacco, wilt-diseases, Dutch East Indian disease: Tobacco, wilt-diseases. Honing's Sumatran Studies.] 298 INDEX. Pagb. Dutch East Indian disease of tomato 209 (See Solanaceae, brown rot, Dutch East Indian disease.) Earle. banana leaf-blight 168 brown rot of Solanaceae 181, 206 Egg-plant blight 174 See Solanaceae, brown rot: Bacterium solanacearum.) Engelberts, Sereh, losses 72 English disease of mangolds and sugar-beets. . 154 English disease of potato 216 Eriksson, Ring disease of potato 216 Essed, banana disease 173 Fertilizers, relation to immunity 270 Flax, bacteria in vessels of stems 154 Florida potato organism 251, 264, 267 [See Bact. solanacearnm (potato and tomato).] Florida tobacco wilt 227 [See Bacterium solanacearum (North American tobacco-wilt organism); Tobacco, wilt-diseases, North American disease.] Fodder-beets, disease described by Kramer 1.54 French disease of potato , 214 French disease of tobacco 266 (See Bacillus aeruginosus, B. putrifacieus putridus, B. tabacivorus; Tobacco, wilt-diseases, French disease.) Fulton, peanut wilt . 153, 271 Fusarium, color produced on acid media by some species 51 Fusarium sp. seen in Australian disease of potato 208 Bengal banana disease 173 Panama disease of banana 173 potato diseases 203 Surinam disease of banana 173 tobacco-wilt 227, 228 Fusarium cubense 169, 173 isolated from Smith's Cuban banana disease 168 Panama disease of banana ascribed to 173 (See also Banana, Smith's Cuban disease.) German Ring disease of potato 166, 215 (See also Solanaceae, brown rot, ring disease of potato, Germany.) Graham, tobacco-wilt 237 Grand Rapids tomato disease 161 (See Aplanobacter michiganense ; Tomato, Grand Rapids disease.) Granville wilt of tobacco 174, 220, 227 [See Bacterium solanacearum (N. Am. tobacco-wilt organism) ; Tobacco, wilt-diseases, N. Am. disease] Griffon, Bacillus aeruginosus, synonymy 267 B. brassicaevorus, synonymy 267 B. caulivorus, synonymy 267 B. fluorescens liquefaciens, synonymy 267 B. fluorescens putridus, synonymy 267 immunity relation of fertilizers to 270 Gumming of sugar-cane 3 (See Sugar-cane, Cobb's disease.) "Gummosis" of sugar-cane 13 (See Sugar-cane, Cobb's disease.) Halsted. observations on bacterial disease of potato and tomato 204, 205 hamawedang 151 (See Peanut wilt, van Breda de Haan's.) Heart disease of sugar-cane 81 (See Sugar-cane, heart disease.) Helms, New South Wales potato diseases 207 Herrman, Brazilian disease of manihot . . 280 Heterodcra radicicola, effect on outbreak of Dutch Fast Indian disease of tomatoes 210, :i 1 Honing, Bacterium solanacearum, host-plants 199 Bacterium solanacearum, involution forms 194 cited 220 peanut wilt 153, 253 scsamum, sumatran disease 218 Page. Honing — continued. tobacco- wilt, Sumatran studies 224, 244 (Sep Tobacco, wilt-diseases: Honing's Sumatran Studies ) Hori, cited 239 Home, top-rot of sugar-cane 3 Humid gangrene of sugar cane 85 (See Sugar-cane, Polvillo.) Hunger, agar used for isolation of Bact. solanacearum .... 211 brown rot of Solanaceae 178, 181, 197, 209 (See Solanaceae, brown rot, Dutch East Indian disease.) cited 203, 220, 226 Dutch East Indian disease of tobacco 222 Dutch East Indian disease of tomato 209 (See Solanaceae, brown rot, Dutch East Indian disease.) Hutchinson, Bacterium solanacearum 267 motility denied 200, 265, 267 tobacco-wilt, India 267 Hypocrea sacchari 52 Immunity, relation of fertilizers to 270 Indian disease of tobacco 267 [See Bacterium solanacearum (Indian tobacco-wilt organism) ; Tobacco, wilt-diseases, Indian disease.] Irons, Solanum mammosum resistant to brown rot of Solanaceae 182 Italian disease of stock 277 (See Stock, Italian disease.) Italian disease of tomato 215 Italian rot of tobacco 266, 270 Iwanoff, Russian disease of potato 214 Iwanoski, Rot of tobacco, Russia 265 Janse, Dutch East Indian disease of tobacco 220 Sereh 52, 74. 75 Japanese disease of tobacco 238 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Japanese stem-rot of tobacco 220 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Javan top-rot of sugar-cane 3 Jensen, cited 220, 226 experiments with B. nicotianae 271 tobacco wilt, Dutch East Indies 224 Kirk, New Zealand potato disease 207 Kobus, Sereh, geographical distribution 76. 78 Kornauth, cited 218 Kotthoff, Spieckermann's German Potato disease. . 167 Kozai, tobacco-wilt 239 Kramer, disease of fodder- beets 154 Kreitz, ring disease of potato, Germany 215 (See also Solanaceae, brown rot, ring disease of potato, Germany.) Kriiger, Cobb's disease of sugar-cane, occurrence in Borneo . 3 Sereh 52, 73. 75 Kugahara, cited 239 Lehmann, spore-formation, rich medium favorable to, 247 Leptospora in diseased banana 173 Lounsbury, tobacco-wilt. South Africa 268 McAlpine, Australian disease of potato 208 McKennev, Panama banana disease 172 tobacco wilt, North Carolina 227 Maize, bacterial disease of 89 (See Corn, Stewart's bacterial disease of; and Bact. stewarti.) Burrill's disease of 89 (See Corn, Stewart's bacterial disease of; and Bacterium stewarti.) Stewart's disease of 89 (See Corn, Stewart's bacterial disease of; and Bac- terium stewarti.) Malay States disease of potato and tomato. ... 211 INDEX. 299 Page. Malkoff, Bulgarian disease of Sesamum 216 Mangolds, English disease of 154 Manihot, Brazilian disease of 280 (See also Bacillus manihotus.) black bundles 280 chemical composition of diseased plants 280 cuttings attacked 280 gummy exudate 280 inoculations 280 literature 281 parts of plant attacked 280 preventive measures 281 resistant varieties 281 signs of disease 280 starch-content, diminution in diseased plants. . . . 280 swellings, sub-cortical 280 tissues attacked 280 transmission by insects 281 treatment 281 Mean temperature required for growth of sugar-cane. 4 Medan III 251,268 [See Bacterium solanacearum (Sumatran tobacco-wilt organism), American studies.) Medan tobacco organism 242, 25 r, 263, 268 [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism), American studies.] Melanospora globosa, cultural characteristics 87 isolation from Polvillo 86 Mesentericus group, forms of, isolated from Appel's ring disease of potato 166 Micrococcus, luteus, associated with Bact. solanacearum in Sumatra 259 prodigiosus Cohn 87 pyogenes (M. bicolor Zimmerman), associated with Bact. solanacearum in Sumatra 259 pyogenes albus, associated with Bact. solanacearum in Sumatra 259 sp., isolation from top-rot of sugar-cane 83 Moko disease 171 (See Banana, Rorer's Trinidad disease.) Midler, sugar content of gummed cane 6.5 Murphy, Bacillus melanogenes 175 Musa chinensis, Rorer's Trinidad disease 170 (See also Bacillus musae; Banana, Rorer's Trinidad disease.) Musa paradisiaca, Rorer's Trinidad disease 170 (See also Bacillus musae; Banana, Rorer's Trinidad disease.) Mysore ring disease of potato 212 (See Solanaceae, brown rot, ring-disease of potato, Mysore.) New South Wales potato diseases 207 New Zealand disease of potato 207 North American disease of tobacco 227 [See Bacterium solanacearum (N. Am. tobacco-wilt or- ganism); Tobacco, wilt-diseases, N.Am, disease.] North Carolina tobacco wilt 227 [Sec Bacterium solanacearum (North American to- bacco-wilt organism); Tobacco, wilt-diseases, North American disease.] Odium, African disease of potato 214 Orchard grass, Rathay's disease 155 (See also Aplanobaeter rathayi.) bacterial slime 155, 157 between leaves and stem 158 cultures from 155 in floral organs 157 in leaf-tissues 159 litmus reddened by 155, 157 microscopical examination of 155 odor of 155 organisms composing 155 carriers of the disease 157 Page. Orchard grass, Rathay's disease — continued. chlorophyll bodies, effect on 155 Cladosporium, secondary infection 155 cuticle, effect on 155, 156 distortions of stem in 155, 157, 158 dwarfing 155, 157 geographical distribution 1.55, 157 glumes, lemon yellow or water-soaked 157 infection, point of, unknowii 156, 157 time of 155, 156 inoculations 156 knee-shaped bendings of stems 155, 158 leaf-sheaths stuck together 157 literature 160 occurrence, in meadows (Denmark) 157 in woods (Austria) 156 on surface first 156 seasonal 156 portions of plant attacked 155, 156, 157 Rathay's work 155 resemblance to Cobb's disease of sugar-cane, signs 157 Stewart's disease of sweet-eorn 157 resistance of other grasses 155, 156 secondary infection, with bacteria 155, 159 with fungi 155 signs of disease 155, 157 Sporidesmium (secondary infection) 155 tissues attacked 155, 156, 159 Osborne, spore-formation, relation of medium to . . . 247 Pavarino, Italian disease of stock 277 Peanut wilt, Sumatra. [See also Bacterium solanacearum (peanut-wilt organism); Peanut wilt, United States; Peanut wilt, van Breda de Haan's.] Honing's report of 153, 253 Peanut wilt, United States 153, 271 [See also Bacterium solanacearum (peanut-wilt organ- ism); Peanut wilt, Sumatra; Peanut wilt, van Breda de Haan's.] etiology 271 Fulton's work on 153, 271 inoculations, peanut organism on peanut 271 peanut organism on tobacco 271 pepper-wilt organism on peanut 271 potato-wilt organism (Florida) on peanut 282 tobacco-wilt organism (N. Am.) on peanut . 271, 282 tobacco-wilt organism (Sumatra, Medan III) on peanut 282 isolation of parasite 271 literature 271 signs of disease 271 soil, infection through 271 Peanut wilt, van Breda de Haan's 151 [See also Bacterium solanacearum (peanut- wilt organ- ism); Peanut-wilt, Sumatra; Peanut-wilt, United States.l altitude, effect of 152 etiology 153, 2.53, 271 literature 153, 271 parasite 153 parts of plant attacked 152 signs of disease 152 soil conditions, effect 152 tissues attacked 1,52 Pepper blight 1 74 (See also Solanaceae, brown rotj Pernambueo disease of sugar-cane 9 Perrier, Brazilian disease of manihot 280 Petch, Ceylon wilt-disease of tomato 214 Pethybridge, Bacillus melanogenes 175 300 INDEX. Page. Phytophthora infestans, French disease of potato (Delacroix) 215 losses caused by 202 Phytophthora nicotianae, effect on outbreak of Dutch East Indian bacterial disease of tomatoes. . . 210 Pith-rot of tobacco 266, 270 (See Bacillus putrifaciens putridus; Tobacco, wilt dis- eases, French disease.) Plantain, Rorer's Trinidad disease 170 (See also B. musae; Banana, Rorer's Trinidad disease.) inoculations 1 70, 1 7 1 isolation of parasite 170 literature 173 signs of disease 170 varieties attacked 170 Pokkah-bong of sugar-cane 3, 81 (See Sugar-cane, Pokkah-bong. J Polvillo of sugar-cane 3, 85 (See Sugar-cane, Polvillo.) Potato, African disease 214 Australian disease 207 "black-leg," resemblance to brown-rot 1 75 brown-rot, (See Solanaceae, brown-rot.) English disease 216 French disease 214 German ring disease 166, 215 (See also Solanaceae, brown-rot, ring disease of potato, Germany.) Malay States disease 211 New South Wales diseases 207 New Zealand disease 207 ring disease, Germany 166, 215 (See also Solanaceae, brown-rot, ring disease of potato, Germany.) ring disease, Mysore 212 (See also Solanaceae, brown-rot, ring disease of potato, Mysore.) Russian disease 214 Schuster's German rot 272 (See also Bacterium xanthochlorum.) blackening of tissues 272, 274 description of diseased, lupins 274 Physalis 274 potato-tubers 272 Vicia faba 272, 274 incubation period 274 inoculations. (See Bact. xanthocblorum.) isolation of parasite 272 literature 276 odor of rotted tubers 272 parts of plant attacked 272 Schuster's observations 272 stomatal infection (Vicia faba) 272 rapidity of 274 tissues attacked 272, 274, 276 tubers, diseased, appearance 272 Spieckermann's German disease 166 analyses oftubers, stems, etc 167 Aplanobacter michiganense, suspected cause. . . . 164 bacteria in diseased plants 166 Bacterium solanacearum, not cause of 167 brown rot of Solanaceae, relation to 167 cause of 1 67 diseased stems, microscopical appearance 166 diseased tubers, bacteria in vessels 166 diseased tubers, vascular ring, appearance of . . . 166 dissimilarity to Appel's ring disease 167 eyes of the tuber, blackening of 167 Fusarium, secondary infection 166 germination, effect on 167 inoculations 167 literature 167 Pagb. Potato — continued. Spieckermann's German disease — continued. parasite, cultural characteristics 167 description of 167 Gram's stain 167 optimum temperature 167 slow growth on media 167 progress of disease 166 signs of disease 1 66 similarity to Grand Rapids tomato disease. ... 164 slow softening of parenchyma in 166 softening of vascular ring 166 staining of vascular ring 166, 167 tissues attacked 166 tomatoes, occurrence on 167 varieties, many susceptible 167 vascular ring, effect on 166, 167 vessels occupied by bacteria 166 viscid organism cause of 167 wet-rot 166 yellow bacteria cause of 167 Potato-rot 1 74 (See Solanaceae, brown rot; Bacterium solanacearum.) Potato-rots, causes 203 recent great advance in knowledge of 203 Prillieux, Bacillus caulivorus 175 Prinsen-Geerligs, Sereh, geographical distribution 78 sugar-cane, cellulose, oxidizable substance in ... . 16 Pseudomonas, amaranthi . 148 (See Bact. amaranthi.) sesami 218 solanacearum 178 (See Bacterium solanacearum.) sp., isolated from ring-disease of potato 166 stewarti 132 (See Bact. stewarti.) vascularum. (See Bacterium vascularum.) Raciborski, tobacco-wilt, Dutch East Indies, cause . . 222 Rasa of tobacco 267 (See Tobacco, wilt-diseases, Indian disease.) Rathay's disease of orchard grass 155 (See Orchard grass, Rathay's disease, and Aplanobacter rathayi.) Ratoon-cane, nature of 6 Ravn, Rathay's disease of orchard grass 157 Ring disease of potato (See also Solanaceae, brown rot, ring disease of potato, Germany.) Germany 166, 215 Mysore 212 (See also Solanaceae, brown rot, ring disease of potato, Mysore.) Rolfs, P. H., brown rot of Solanaceae 205 Rorer, Panama disease of banana 173 Surinam disease of banana 173 Trinidad disease of bananas and plantains 170 Rose, blight of young shoots 154 Russian disease of potato 214 Russian disease of tobacco 265, 270 (See Tobacco, wilt-diseases, Russian disease.) Sackett, tobacco wilt, North Carolina 227 Schreiber, spore-formation, relation of medium to 247 Schuster's German potato rot 272 (See potato, Schuster's German rot.) Schwarzbeinigkeit, resemblance to brown-rot 175 Selby, brown rot of Solanaceae 205 Septogloeum arachidis 15 l Sereh disease of sugar cane 3, 52, 73 (See Sugar-cane. Sereh.) Sesamum, bacteriosis of 216 (See also Solanaceae, brown rot, Sesamum bacteriosis.) INDEX. 3d Shamel, Deli, tobacco impurity of seed 261 tobacco wilt, losses 237 Smith, R. Greig, on bacterial disease of sugar-cane. (See Sugar-cane, Cobb's disease, and Bact. vascularum.) Smith's Cuban disease of banana .168 (See Banana, Smith's Cuban disease.) Smith's disease of amaranths 148 (See Amaranths, Smith's disease and Bact. amaranthi.) Solanaceae, anatomy of 192 Solanaceae, brown-rot 174 (Tobacco-wilt diseases are indexed separately.) [See also Bacterium solanacearum (potato and tomato) ; Peanut wilt.] adventitious roots, tomato 178, 190, 199, 209 African disease of potato. Odium's observations. . 214 alkalinity of juice of attacked plants 178 animal parasites, as disseminators of. . . . 184, 210, 213 Appel, Ring disease of potato 166, 215 Australian disease of potato and tomato, Tryon's observations . 207 bacterial ooze 175, 177 Bacterium solanacearum, cultural characteris- tics 194, I9g description 193 (See also Bacterium solanacearum (potato and tomato.) Bailey's observations on disease of tomato 205 Bancroft's observations on disease of potato and tomato 211 basal stem-rot of potato ("black leg"), how distinguished from 176 liable to confusion with 175 beetles, disease spread by ... 181 "blackleg." (See basal stem rot.) blackening of tissues 179 broken roots, avoidance of, in transplanting 201 browning of tissues 174, 175, 177, 191, 209 Burrill's observations on disease of potato. . . 203, 205 calcium oxalate, excess of, in diseased plants 192 cavities, common in 176, 191, 199, 209 cell-walls, action on 19: cells occupied by bacteria in . 191 Ceylon disease of tomato, Petch's observations. .214 Coleman, Mysore Ring disease of potato 212 Comes' observations on disease of tomato. . . 203, 215 Corbett's observations on disease of tomato 205 cork-layer, protective, formation of 178, 191 crop cut short by 1 77 crystal-sand, excess of, in diseased plants 192, 209 definition 174 Delacroix's observations on disease of potato 214 de Laharpe's observations on disease of potato. . 215 diffusion of bacteria from cut vessels 197 dissemination by insects 180 drainage, effect on 210 Dutch East Indian disease of tomato 209 adventitious buds 209 adventitious roots 209, 211 Bact. solanacearum, cause of . 209 Bact. solanacearum, cultural characters 211 cavities in tissues 209 contaminating organisms. . . .211 crystals in diseased tissues . 209 culture-medium used for isolation of parasite . 211 diffusion of bacteria from cut vessels in alcohol . 209 drainage, effect on 210 effect of animal parasites on outbreak of disease . 210 effect of Phytophthora on outbreak of disease .210 Hunger's observations on ... 209 infection, manner of .211 infection through wounds .210 inoculation, methods of . . . 209, 210 inoculations on Capsicum annum 209 Nicotiana tabacum . 209 tomato 209, 2 10 Solanaceae, brown rot — continued. Dutch East Indian disease of tomato — cont. pith, effect on 209 prevalence 2oo prevention, methods 211 roots, diseased 209 infection through 2io signs of disease 209 tyloses 209 vascular ring browned 209 vessels occupied by bacteria .... 209 Dutch East Indian studies of Honing 224, 244 dwarfing „ ?8 Earle's observations on 206 early inoculations (1895, 1896) 182, 183, 184, 185 early stages, potato 177 English disease of potato \ 216 entrance of organism into host. . . ... 181 etiology .... 178 fluids, alkaline in j78 French disease of potato. Delacroix's observations 214 de Laharpe's observations 215 Smith's studies of Delacroix's material 215 geographical distribution i75 (See also Appendix, p. 207.) Grand Rapids disease, may be mistaken for. . 175, 186 Halsted's observations on disease of potato and tomato 204, 205 Helms' observations on disease of potato 207 history 20, Honing, bacteriosis of sesamum 2i8 Dutch East Indian studies 224, 244 host-plants ',„„ host-plants 174, 199 (See also Peanut wilt; Tobacco, wilt-diseases.) hot weather, increases virulence of 182 Hunger's observations 17S, 181, 197, 209 hyperplasias . . . ' 190 incubation period . 179, 180 infected soil, tomatoes grown in .' r8i infection, difficulty of producing with organisms grown long in culture media 179, 282 effect of age of plant . 178,180 effect of moisture 179, 182 effect of rapid growth . 178^282 effect of temperature I79> j82 incubation period [79 through wounds . 181,182,210 underground, in tobacco .181 underground, in tomato ^i (See also Bact. solanacearum, inoculations.) inoculations. (See Bacterium solanacearum.) insects, distribution by 180, 184, 210, 213 intercellular spaces occupied by bacteria in. .....' 191 isolation of parasite 205 difficulties experienced 178 Italian disease of tomato. Comes' observations 215 Voglino's observations 215 Iwanoff's observations on disease of potato 214 Kirk's observations on disease of potato 207 Kreitz, Ring disease of potato 215 literature ['_[ 218,270 McAlpine's observations on disease of potato. . . . 208 Malay States disease of potato and tomato, Bancroft's observations 2n signs of disease 2i2 treatment 2I2 Malkoff's observations on disease of Sesamum . . . 216 middle lamella, solution of .191 moisture, effect on 179, 182 morbid anatomy ' jqq 302 INDEX. Pace. Solanaceae, brown rot — continued. New South Wales, potato diseases. Helms' obser- vations 207 New Zealand disease of potato, Kirk's observations 207 Odium's observations on disease of potato 214 parts of plant attacked, egg-plant 176 potato 176, 177 tomato 176 pecuniary losses 202 pepper-blight 174 comparison of pepper, tobacco and peanut-wilt organisms 271 inoculations, Dutch East Indian tomato-wilt organism on pepper 209 Japanese tobacco- wilt organism on pepper . . 243 North American tobacco-wilt organism on pepper 235 pepper-wilt organism on peanut 271 potato and tomato- wilt organism on pepper. 183 Sumatran tobacco- wilt organism on pepper 246, 253 period of incubation 179, 180 exceptionally long 179 very short 282 Petch's observations on disease of tomato 214 pith, effect on 176, 192 plants subject to 174 (See also Peanut-wilt, and Tobacco, wilt-diseases.) Porto Rico, occurrence in 175 potato-tubers, effect on 177 internal brown rot of 177, 191 mixed infection in 177, 191 signs of disease in 177 tissues attacked 177, 191 production of, effect on 177 rains, favorable to 182 resistant Solanum 182 result of disease, potato 177 ring disease of potato in Germany 166, 215 Appel's observations 166, 215 bacteria isolated from 166, 216 cultural characteristics 166 dissimilarity to Spieckermann's German potato disease 167 infected seed tubers, use of 166 infected tubers from apparently sound plants. . 166 infection, manner of 166, 216 Kreitz's observations 215 Pseudomonas sp. isolated 166 signs of disease 166, 215 vascular ring, browning of 166, 215 ring disease of potato, Mysore 212 Coleman 's studies 212 geographical distribution 212 infection, manner of 213 non-relation of insects to 213 inoculations on brinjals (egg-plant) 213 potato 213 tomatoes 213 signs of disease 212 treatment 213 Rolfs' observations 205 root-infection, tobacco 1 8 1 tomatoes 181 roots, developed on steins in 17S wounding necessary for infection 181 rotation of CTOpS advised 201 Russian disease of potato, IwauolT's observations. 214 secondary signs 179 eed poti treatment of 201 Page. Solanaceae, brown rot — continued. Selby's observations 205 selection of "seed" potatoes 201 sesamum bacteriosis 216 Austria 218 Bulgaria, cultural characteristics of organisms 216 dissemination of 216 inoculations 2 t6, 218 isolation of bacteria 216 Malkoff's observations 216 signs of disease 216 varieties attacked 216 India 218 Sumatra, Honing's observations 218 shriveling of stems 175 signs of disease 174, 175,209 potato tubers 174, 177 sleepy disease of tomato, signs resemble 175 Smith, early studies of 205 Spieckermann's potato disease, not related to ... . 167 starch, removed from infected tissues (fig. 83). 176, 192 stem showing incipient roots (fig. 98) 190 stems, brown streaks in 180 Stevens' observations 206 stomata, infection through, doubtful 182 storage of potatoes at low temperatures advised . . 202 susceptibility, individual differences in 180 varietal differences in 180 temperature, effect on 179, 182 thickening of inoculated stems 178, 191 time of attack, effect, on potato 177, 178 tissues attacked 175, 176, 191, 192, 199 tobacco-wilt diseases, probable identity 220, 244 transplanting, care in 201, 202 treatment 201, 2 J 1 Tryon, on disease of potato and tomato 207 tubers, potato, infected by way of rhizome 177 vascular ring first attacked in 177 tyloses 209 underground infections in 181 vascular ring, browning of 174, 177, 209 vascular tissue, excessive 191 vessels occupied by bacteria 174, 17.5, 191,209 viscidity, not characteristic of 197 Yoglino's observations on disease of tomato 215 water-rich tissues more subject to. . 174, 178, 179,282 wilting 174, 175 yellowing of foliage 174,175 young plants specially sensitive to 174 Solanum mammosum, subject to Apl. michiganense . 163 Soltwedel, Sereh 73, 74, 75, 78 Sorauer, sugar-beet disease in Germany 154 South African disease of tobacco 268 (See Tobacco, wilt-diseases, South African disease.) vSpegazzini, Polvillo, geographical distribution 3 observations 85 Spieckermann's German potato disease 166 (See potato, Spieckermann's German disease.) Sporidesmium, Rathay's disease of orchard grass. . . 155 Stem-rot of tobacco 239 (See Bacillus nicotianae, Uyeda; Tobacco, wilt- diseases, Japanese disease.) Stevens, brown rot of Solanaceae 204, 206 cited 220 tobacco-wilt, losses 237 North Carolina 227 Stewart, observations on bacterial disease of sweet corn. (See Corn, Stewart's bacterial disease of.) Stewart's disease of sweet corn 89 (See Corn, Stewart's bacterial disease and Bact. Stewarti.) INDEX. 303 Pace. Stock, Italian disease 277 (See also Bacterium matthiolae.) browning of tissues 277 infection, manner of 279 inoculations 278 literature 279 losses 277 microscopic appearance of diseased tissues 278 morbid anatomy 277 parasite, description 278 (See Bact. matthiolae.) parts of plant attacked 277 progress of disease 277, 278 seed-disinfection 279 seed-selection 279 signs of disease 277 tissues attacked 277 treatment 279 Sugar-beets, disease described by Sorauer in Germany 154 Sugar-beets, English disease of 154 Sugar=cane, arrowing 19, 20 chlorosis 81 (See also Sugar-cane, Pokkah-bong; Sugar-cane, top- rot.) effect of copper sulphate on 76 gum formation 81 (See also Sugar-cane, Cobb's disease, and Sugar-cane, Sereh.) mean temperature required for growth of 4 Sugar=cane, Cobb's disease of 3 (See also Bacterium vascularum.) acidity of cane, effect on susceptibility 47 acidity of cane-juice, relation to bacterial growth . 4 7 adventitious roots 52 aeration and pigment stain 16 bacteria dormant or dead in cane . . . 30, 31, 46, 54, 65 bacteria first discovered in connection with disease . 3 bacteria, location in tissues 6, 15, 24, 52 bacterial ooze from leaves and stem . . 16, 22, 25, 53, 66 bacterial ooze, great abundance of 26 bacterial slime, identity with gum 13 (See also slime, gum and vasculin.) Bacterium vascularum, description .54 (See also Bact. vascularum.) Boname's studies, geographical distribution 3 signs of disease 10 reduction of sugar-content 15, 63 bundles, mottled red and yellow 16, 26, 27, 41 cavities formed in soft tissues 5, n, 15, 26, 52 check pricks, result of 28, 32 cirri from cut bundles 6, 30, 32 clarification of sugar hindered by 3 climate, effect on disease 4 Cobb's studies, (See also Bact. vascularum.) earliest appearance of disease 5 effect of moisture 49 geographical distribution 3, 5 inoculation experiments 8, 1 2 pecuniary losses in Australia and New South Wales 70 crystallization, effect on 9 decay of terminal bud 3, 5, 15 definition of the disease 3 destruction of bundles in 53 discoloration of tissues 3, 4, 5, 11, 12, 15, 16, 18 disease not air-borne 68 drainage, partial remedy for 49, 68, 70 Dranert's studies in Brazil 3, 70 dwarfing 3, 15, 22, 27 earliest appearance of disease 3,5 early signs of disease 34, 35, 36, 37 etiolation in 3, 10, 18 etiology 21 Page. Sugar-cane, Cobb's disease of — continued. geographical distribution of. .... 3, 4, 5, 9, 10, 1 1, 13, 14 gum, (See also vasculin.) chemical reactions 7, 13, 14 chemical reactions compared with those of bac- terial slime from cultures 13 comparison with the mucins 14 component parts 7 dissemination in tissues 6, 10, 15 identity with bacterial slime 13 nature and origin of 7, 8, 13 ooze from leaves and stem 16, 22, 25, 53, 66 separation from bacteria by filtration 13 signs of the disease 3, 15 Boname's observations 10 Cobb's observations 5, 6, 8 Dranert's observations 4 Tryon's observations 12 gummed cane, means of detecting 6, 69 microscopic appearance of 6, 24, 52 gumming of machinery 3,6 gumming of terminal shoot 16, 25, 26 gumming, signs of 3, 6, 15 (See also signs of disease.) history 3 host-plants 3 hyperplasias not present 52 immunity, cause of 47 infectious nature 48 intercelluar spaces, occupation by bacteria 53 inoculations 8, 1 2, 2 1 (See also Bacterium vascularum.) isolation of Bact. vascularum from artificially infected canes 21 naturally infected canes 21 juice of gummed cane, recognition of 6 lime, use of, in clarification 6, 9 literature 71 leaves, pale stripes on 23 local signs not followed by general infection. . . 27, 38, 4°. 42. 43. 46 losses due to 4, 6, 70 microscopic appearance of gummed cane ... 6, 24, 52 moisture, effect of 4, 49, 70 morbid anatomy 52 most disease on best land 69 nodes, red pigment most abundant in or under. 16, 26 odor of 27 organisms associated with Bact. vascularum in badly diseased cane 21, 23 parasite, description of 54 (See also Bact. vascularum.) parenchyma, cause of comparative immunity. ... 48 pecuniary losses 70 Pernambuco disease 9 crystallization, effect on 9 cultivation of cane, primitive method 9 gum in 9 preventive measures 10 resistant varieties 10 signs of 9 susceptible varieties 9 theories regarding cause 9 weather conditions, effect of 10 pigment, red 1 6, 53 (See also red stain.) plant-cane, signs of gumming in 6 planters responsible for spread of disease 68 polvillo, points of resemblance 85, 88 premature blossoming 20 development of axillary buds 11, 12, 16, 25, 52 drying up of attacked cane 9, 15 ripening 20 ratoons, signs of gumming in 6 3°4 INDEX. Page Sugar-cane, Cobb's disease of — continued. recovery from 27, 30, 46 red bundles 16, 26, 41, 53 (See also red stain J contents of 45, 52 fungus reported in 49, 51 isolation of Bact. vascularum from 17, 46 red ooze, cause of 46 red ooze, isolation of Bact. vascularum from 46 red stain 1 6 (See also red bundles.) ascribed to symbiotic action of B. pseudarabinus and a fungus 49 cause of 53 Greig-Smith's investigations of 49 reduction of sugar-content 3, 7, 10 remedies for 70 resistance of acid canes 47 resistance to disease, cause of 47 resistant varieties 47, 69 result of check punctures 28, 32 resuscitation of Bact. vascularum from old diseased canes 46 rusty spots and streaks on leaves n, 18, 22 23, 24, 25, 27 saprophytes following parasite in 21, 23, 26, 31 secondary infections, effect of slow growth 46 secondary signs 19, 22, 23, 24, 48 Sereh, relation of, to Cobb's disease 52 shoots, pushing of 24 shortening of internodes 24 shrivelling of leaves 24 signs of disease 3, 15 Boname's observations in Mauritius 10 Cobb's observations in Australia 5, 6 Dranert's observations in Brazil 3 observations in Washington 21 other observations in Brazil 9 Tryon's observations in Queensland n slime, bacterial 3, 7, 13, 14, 15, 24, 53, 66 (See also gum and vasculin.) chemical reactions of 7, 13, 14 comparison with the mucins 14 dissemination in tissues 6, 15, 24, 66 from cultures, comparison of chemical reactions with those of gum from cane 13 identity with gum 13 ooze from leaves and stem 16, 22, 25, 53, 66 sign of the disease 3, 15 slow growth of cane, effect on susceptibility. . 34, 37, 46 Smith, R. Greig, studies on the disease, (See also Bact. vascularum ) geographical distribution 3 gum, nature and origin of 13 gum-production, dependent on certain carbon- foods 64 preventive measures 69 soda, gummed sieves cleaned with 6 sodium chloride, poisonous effect of 63, 69 soil-conditions, relation to susceptibility of cane. . 69 solution of middle lamellae (fig. 6) 16 spread of disease, conditions favoring 48, 68 steaming cane for detection of 18, 69 stomata, bacterial ooze through 16, 53 striping of leaves 3, 1 1, 15, 18, 22, 23, 24 sugar, crystallization, effect of disease on 9 sugar, interference with clarification 3 sugar-content, reduction of, in gummed cane .7, 10, 15,62,63 sugar-content, relation to susceptibility of cane. . 69 susceptible varieties 9, 10, 47, 69 susceptibility, acidity of cane and 47 slow growth and 37, 46 soil conditions and 69 sugar content and 69 sweating of cane for detection of 69 Pagb. Sugar-cane, Cobb's disease of — continued. terminal shoot, distortions of 1 1 , 1 6, 25, 26, 5 2 tissues attacked 6, 7, 15, 24, 52 titration of juice of cane varieties 47 top-rot, points of difference 82 transmission of disease 48, 68 treatment 68 Tryon's studies, earliest reports of disease in Queensland 5 effect of drainage 70 effect of soil 70 geographical distribution 3, 1 1 inoculation experiments n pecuniarv losses in Australia 70 signs of disease n susceptible varieties 69 transmission 68 varieties resistant to 69, 70 Common Purple 47 Lalanjore 10 Louisiana No. 74 47 Louzier 10 varieties susceptible to 69, 70 Bambou 10 Cayanna 9 Common Green 47 Creoula 9 Imperial 9 Mauritius Ribbon 69 Oteheite 9 Rappoe 19 Rose Bamboo 19 vasculin 7 (See also gum.) characteristics of 7 production of 7, 8 reactions to chemicals 7 vessels, bacterial growth for 5 feet in 53 yellow bundles 16, 26, 41 yellow ooze, isolation of Bact. vascularum from . . 46 yellow slime, variability in appearance of 60 (See also slime, bacterial.) Sugar-cane, gumming 3 (See Sugar-cane, Cobb's disease of.) Sugar-cane, gummosis 13 (See Sugar-cane, Cobb's disease.) Sugar-cane, heart-disease 81 (See also Sugar-cane, Pokkah-bong; Sugar-cane, top- rot.) bacteria, presence of 83 molds, presence of 83 Sugar-cane, humid-gangrene 85 (See also Sugar-cane, Polvillo.) Sugar-cane, Pokkah=bong 3, 81 (See also Sugar-cane, top-rot.) aerial roots 82 bacteria, presence of 83 economic aspect of 82 leaves, abnormal development 82 moulds, presence of 83 preventive measures 84 red stripes : 82 Sereh, resemblance to 82 signs of disease 82 sprouting of buds 82 stem, thinning of 82 time of appearance 82 Sugar-cane, Polvillo 3, 85 acarids, possible carriers 86 animal parasites in deceased tissues 86 attack, point of 86 Bacillus sacchari Spegazzini, comparison with B. marcescens 87 comparisons with Micrococcus prodigiosus Cohn 87 cultural characters 87 inoculation experiments 87 INDEX. 305 Sugar-cane, Polvillo — continued. Bacillus sacchari Spegazzini — continued. isolation of 86 later name than B. sacchari Janse 86 morphology of 87 relation to Bact. vascularum 88 some resemblance to B. sorghi 87 spores of 87 bacteria in diseased tissues 86 bacteria in gum 86, 87 bacteria, isolation of 86 bacterial origin of disease 86 Cobb's disease of sugar-cane.'points of resemblance to 85,88 Diptera, possible carriers 86 earwigs, possible carriers 86 eel-worms, possible carriers 86 etiology 85, 86 fungi, isolation 86 fungus, in diseased tissues 86 fungus in gum 86 general effect of disease 86 gum. 85 gum, bacteria in 86, 87 gum. inoculations 87 Hemiptera, possible carriers 86 infections, artificial 87 inoculation experiments 87 leaves, effect on 85 Lepidoptera, possible carriers 86 literature 88 losses 85 Melanospora globosa, isolated from 86 microscopic appearance 86 mites, possible carriers 86 plant-cane, susceptibility 85, 88 propagation of disease 85 ratoon-cane, resistance of S5, 88 recovery from disease 86 red pigment 87 red stain 85,88 resistant varieties 86 sand-fleas, possible carriers 86 signs of disease 85, 88 soil conditions, effect 85 spread of disease 86, 87 sprouting 85 striping 85 susceptible varieties 86 terminal bud, effect on 85, 86, 88 transmission of disease 86, 87 weather conditions, effect 85 Sugar-cane, reddening of bundles 16, 17, 18 (See Sugar-cane, Cobb's disease.) Sugar=cane, Sereh disease of 3, 52, 73 aerial roots 73 atavism 75 Bacillus sacchari Janse 74, 75, 76 (See also B. sacchari.) bacteria on the surface of diseased cane So bacterial origin suggested 75 Benecke's observations 72, 73 blossoming, premature 19, 7.3 "bouquet" Sereh 74 causes suggested 7.5 cavities containing bacterial slime 79 cellulose, degeneration of 74 chlorophyll, effect on 73 Cobb's disease of sugar-cane, relation to 52 copper sulphate, effect of 79 cuttings, decay of 73 diseased, sound cane from 79 from mountain cane 76 time of appearance of disease in 73 transmission through 73 Pagb. Sugar-cane, Sereh disease of — continued. death of foliage 73 dwarfing 73. 75 Engelberts' observations 72 etiolation 73 etiology 72 fertilizers, unsuitable, effect 75 foliage, death of 73 geographical distribution 52, 72, 78 growth, effect on disease when rapid 74 gum, bacteria-like particles in 74 color of 74 consistency of 74 origin of 78 solution in potash 74 gummed bundles, isolations from 76 gumming, conditions unfavorable to 74 gumming of bundles 73, 74, 75 Heterodera sp 75, 77 Heterodera radicicola 75 history 72 Hypocrea sacchari 75 immunity 78 internodes, shortening of 73 isolations from gummed bundles 76 isolations from parenchyma 74 Janse's observations 52, 74, 75 Java, home of Sereh 72 Kobus' observations, geographical distribution . 76, 78 Kriiger's observations 52, 73, 75 leaves, conspicuous crowding of 73 dwarfing of 73 dying irregularly 73 etiolation 73 persistence 73 striping 73 literature 79 losses 72 moisture, effect on disease 74, 75 mountain cane, used for cuttings 76 nematodes 75 nucleated cells in phloem 74 odor accompanying decay of cuttings 73 oily or resinous drops in parenchyma 74 origin of term 72 parasites given as cause 75 parasitic nature of 77 parenchyma, granular substance in 74 gumming of 74 isolation of Bacillus sacchari from 74 oily or resinous drops in 74 shining, colorless, yellowish or red drops in ... . 74 staining of walls 74 yellow and brown spots 74 persistence of leaves 73 pigment, red 17, 73, 74 solubility of 74 planting, unsuitable methods, effect 75 planting of affected stock 75 Pokkah-bong, resemblance to 82 portion of plant most diseased 73 premature blooming 73 preventive measures 72, 76 primary Sereh 74 Prinsen-Geerligs, observations 78 progress of the disease 72 Pythium 75 red bundles 73, 74, 75 origin of 76 red stain 73, 74 resistant varieties 76 roots of diseased plants 77, 78 root-system, effect on 73 3°6 INDEX. Page. Sugar-cane, Sereh disease of — continued. secondary Sereh 74 shining, colorless, yellowish or red drops 74 shoots, abnormal production of 73. 75 shortening of internodes 73 sieve-tubes, plugging of 74 signs of disease 73. 76 slime, yellow, in vascular bundles 74 soil-conditions, effect 75 Soltwedel's observations 73. 74. 75. 78 spread of the disease 78 sprouting of buds below ground 73 starch in diseased canes 75 stooling, abnormally strong 73 striping 73 susceptible varieties 76 time of appearance of disease 73 transmissible Sereh 74 transmission through cuttings 73 Treub's observations 75 Tschirch's observations 73 Tylenchus sacchari 75. 77 Valeton's observations 17, 52, 72, 73, 74, 75 vascular bundles, gumming of 73. 74. 75 Wakker's observations 52, 73. 75. 77 water, lack of, effect 75. 77. 78 weather conditions, effect 75. 77 Went's observations 52, 72, 73, 75, 76, 77 yellow gum in vascular bundles 74 yellow slime in vascular bundles 74 Zeijlstra's observations 75. 78 Sugar=cane, top=rot 3. 81 (See also sugar-cane, Pokkah-bong.) Bacillus subtilis isolated from 83 Bacillus vascularum (?) isolated from 83, 84 bacteria, isolation 83 bacteria in diseased tissues 82, 83 cavities 81 Cobb's disease, points of difference 82 etiology, theory regarding 84 glassy appearance of stem 81, 82 growing-point, effect on 81, 82 gum 82 absence of bacteria in 82 infectious nature 84 inoculation experiments 83 isolation of bacteria 83 leaves, effect on 81 literature 84 Micrococcus, sp isolated 83 microscopic appearance 82 odor 81 preventive measures 84 red gum 82 red border-line 81, 82 signs of disease 81 sprouting of buds 81 stem, disorganization 81 susceptible varieties 8i lime of appearance 81 vessels, gum in 82 Wakker's observations 81 weather conditions, effect 84 yellow disease of hyacinths, comparison with ... 83 Sumatran slime-disease of tobacco 220, 222 [See Bacterium solanacearum (.Sumatran tobacco-wilt organism); Tobacco, wilt-diseases, Dutch East Indian disease; Tobacco, wilt-diseases, Honing's Sumatran studies.] Sumatran tobacco-wilt organism, American studies. [See Bac-t. solanacearum (Sumatran tobacco-wilt or- ganism).] Suniati;m tobacco-wilt organism, Honing's studies. [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism).] Page. Sweet corn, bacterial disease 89 (See Bacterium stewarti; Corn, Stewart's bacterial disease.) Stewart's disease 89 (See Bacterium stewarti; Corn, Stewart's bacterial disease.) Tobacco, "black-leg" 239 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) canker 266, 267, 270 (See Bacillus aeruginosus; Tobacco, wilt-diseases, French disease.) collar rot 266, 267, 270 (See Bacillus tabacivorus; Tobacco, wilt-diseases, French disease.) Dutch East Indian disease 220 [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism) ; Tobacco, wilt-diseases, Dutch East Indian disease; Tobacco, wilt-diseases, Honing's Sumatran studies.] Florida wilt 227 [See Bact. solanacearum (North American tobacco-wilt organism); Tobacco, wilt-diseases, North Ameri- can disease.] Granville wilt 174, 220, 227 [See Bact. solanacearum (North American tobacco- wilt organism) ; Tobacco, wilt-diseases. North American disease.] Japanese disease 238 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Japanese stem-rot 220 (See also Bacillus nicotianae, Uyeda; Tobacco, wilt- diseases, Japanese disease.) North American disease 227 [See Bacterium solanacearum (North American tobacco-wilt organism) ; Tobacco-wilt diseases, North American disease.] North Carolina wilt 227 [See Bacterium solanacearum (North American tobacco-wilt organism) ; Tobacco, wilt-diseases, North American disease.] pith-rot 266, 270 (See Bacillus putrifaciens putridus; Tobacco, wilt- diseases, French disease.) "pox-disease," Iwanowsky and Poloftzoff 270 root-rot, Comes 270 spot-disease, Iwanowski 270 stem-rot 239 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Sumatran slime disease 220, 222 [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism); Tobacco, wilt-diseases, Dutch East Indian disease; Tobacco, wilt-diseases, Honing's Sumatran studies.] Wilt=diseases 220 [See also Bacillus nicotianae Uyeda; Bact. solanace- arum (Indian tobacco-wilt organism); Bact. solanacearum (North American tobacco- wilt or- ganism); Bact. solanacearum (Sumatran tobacco- wilt organism).] brown-rot of Solanaceae, probable identity with 220, 244 Dutch East Indian disease 220 [See also Bact. solanacearum (Sumatran tobacco-wilt organism I ; Tobacco, wilt-diseases, Honing's Suma- tran studies.] bacteria, distribution in tissues. . . 220, 221, 222, 223, 225 staining of 220 brown stain 220, 22 1 cavities 220 cell-walls, action on 223 conditions, favoring 221, 222, 223, 224 \ INDEX. 307 Tobacco — continued. Wilt diseases — continued. Dutch East Indian disease — continued. dissemination 223 drainage, effect of 223,225 eel-worms, relation to 221, 222, 223 entrance of parasite into host 221 etiology 220, 222, 224, 244 fungi, presence of 220, 225 Glechia solanella, relation to 223 harvested tobacco, effect on 222, 224 Heterodera radicicola, relation to. . . 221, 222, 223 Honing's observations 224, 244 (See Honing's Sumatran studies.) host-plants 226, 253, 254, 255 Hunger's observations 222 infection, manner of 221, 222, 223, 224, 225 inoculations 222, 224, 225, 244, 245 (See also Honing's Sumatran studies.) insects, relation to 221 intercellular spaces occupied by bacteria. ... 221 isolation of parasite 225 Janse's observations 220 Japanese disease, probable identity with .... 244 Jensen's observations 224 literature 270, 27 1 Micrococcus sp., relation to 222 parasite, description of 221, 223 identity, probable, with Bact. solanacearum Smith 244 inoculations with 224 isolation 225 longevity in soil 225 spores, not seen 226 [See also Bact. solanacearum (Sumatran tobacco- wilt organism).] parts of plant attacked 221,222, 224 Phytophthora nicotianae, relation to 223 preventive measures 224, 226 reaction of juice of diseased plants 225 resistance to 224 roots, effect on 221, 222 saprophytes present 226 signs of disease 220, 221, 222, 224 soil, effect of 221, 223, 224, 225 persistence of parasite in 225, 245 starch, slight action on 221 "thick bellies," relation to 223 time of attack 222 tissues attacked 220, 221, 222, 223, 225 treatment 224, 226, 245 tyloses 223 van Breda de Haan's observations on 222 vessels occupied by bacteria. . . 220, 221, 222, 223 weather conditions, effect of 222 wounds, relation to 223, 224 yeast present 222, 225, 226 etiology 220, 244 French disease 266 (See also Bacillus aeruginosus; B. putrifaciens putridus; B. tabacivorus.) etiology 266 Fusarium disease, Delacroix 228 inoculations 266, 267 literature 270 susceptible varieties 266 Honing's Sumatran studies 224, 244 [See also Bacterium solanacearum (Sumatran tobaeco-wilt organism).] Albizzia, relation to disease 225 bacteria, parts of plant occupied by 224 persistence of, in soil 225 crossing tobaccos to obtain resistant race. . . . 260 Deli tobacco, degeneration of 259 origin of 259, 260 variations in 261, 262 Page. Tobacco — continued. Wilt diseases — continued. Honing's Sumatran studies — continued. drainage 225 etiology 244 field observations 245 food conditions, effect of 245 fungi 225 host-plants 226, 253, 254, 255 immune race of tobacco, how obtained 259 immune varieties of tobacco 255 immunity tests 255 infection, channels of, in plant 254 source of 246, 254 transmission of 254 inoculations, [See Bact. solanacearum (Sumatran tobacco-wilt organism).] irrigation-water, disinfection of 246 irrigation-water, infection of 246 isolation of parasite 225 literature 270, 271 parts of plant occupied by bacteria 224 planted-out tobacco, effect on, of occurrence in seed-bed 255 progress of disease, number of invading bacteria govern 260 reaction of juice of diseased plants 225 resistance, effect of soil 263 resistant tobaceo.method of obtaining . 255,260,263 resistant varieties 255 seed-bed, disinfection of 246, 254 seedlings, infected, use of 246, 254 selection, in tobacco-culture 259, 260 selection-experiments 261 selection-methods 262 soil, persistence of parasite in 225, 245 test of, for tobacco organism 245 treatment of 226, 245, 246, 254 soil-exhaustion 225 soil-infection, effect of 244, 246, 254 extent of 246 spread of disease, conditions favoring 225 susceptible varieties 255 transmission of disease 254 trap-plants for detecting Bact. solanacearum in soil 245 treatment 226, 245, 246, 254 variety tests 25s, 260 wells, Bact. solanacearum found in 246 Indian disease 267 [See also Bacterium solanacearum (Indian tobacco- wilt organism).] etiology 267 inoculations 267 literature 271 losses 267 middle lamella, solution of 268 organisms associated with 267 parasite, description of 267 tissues, disintegration of 268 toxins 267 wilting, cause of 267 Italian disease 266 literature 270 Japanese disease 238 (See also Bacillus nicotianae Uyeda.) blackening of bundles 240 of stem 238, 239 cavities 243 duration of disease 238 Dutch East Indian disease, probable identity. 244 entrance of bacteria into host, manner. . 238, 239 3o8 INDEX. PaGB. Tobacco — continued. Wilt diseases — continued. Japanese disease — continued. etiology 240, 244 fertilizers, effect of 243 geographical distribution 239 history 238 incubation period 239 infection, manner of 238 inoculations 238, 243, 271 (See Bacillus nicotianae Uyeda.) literature 270 losses 238 microscopic appearance 243 parasite, cultural characteristics 238, 241 (See Bacillus nicotianae.) description 238 morphology 238, 241 protective measures 243 resistant varieties 243 signs of disease 238, 239 tissues attacked 238, 240, 243 Uyeda's observations 238 vascular bundles, bacterial occupation of . . . . 238 weather conditions favoring 238, 240 wounds, infection through 238 literature 270 North American disease 227 [See also Bacterium solanacearum (North American tobacco-wilt organism).] adventitious roots 230 browning of tissues 229, 230 cavities 230 etiology 233 Fusarium 227, 237 incubation period, tomato 234 infected soil, effect 229, 233, 235, 237, 271 infection through wounded roots. . . 229, 234, 235 inoculations, peanut organism on tobacco 271 potato organism on tobacco 230, 282 tomato organism on tobacco 233 tobacco organism on tobacco 229, 282 [See also Bacterium solanacearum (North Ameri- can tobacco- wilt organism).] isolation of parasite 228, 229 literature 270, 27 1 losses due to 227, 237 McKenney's observations 227, 228 preventive measures 238 roots, infection through 229, 234, 235 Sackett's observations 227 signs of disease 229 similarity of peanut-wilt to 271 soil, infection through .... 229, 233, 23s, 237, 271 persistence of parasite in 237 Stevens' observations 227, 237 tissues attacked 227 treatment 237 vessels occupied by bacteria 227, 230 wounds, infection through 229, 234, 235 Russian disease 265 etiology 265 literature 270 signs of disease 265 South African disease 268 Bacillus sp. isolated 269 Bacillus megaterium isolated 269 Bacillus mycoides roseus isolated 269 etiology 269 gall worm, relation to disease 269 isolations from diseased plants 269 larva present in diseased plants 269 literature 270 Mucors isolated 269 potato moth, relation to disease 269 signs of disease 268 Pagb. Tomato, Australian disease 207 brown-rot (See Solanaceae, brown-rot). Ceylon disease 214 Dutch East Indian disease 209 (See Solanaceae, brown rot, Dutch East Indian disease.) Grand Rapids disease of 161 (See also Aplanobacter michiganense.) bacteria on surface of diseased stems 163 bacterial occupation of substomatic chamber. . 163 brown rot, how unlike 163 leaves not reflexed (pi. 14) 162 mistaken for 175 much less brown stain 161 stems less inclined to form roots (pi. 14) .... 162 cracking of stems 163 geographical distribution 164 infection, point of 161, 163 through stomata 163 infectiousness, high degree of 163 inoculations on, potatoes 1 64 tomatoes 161, 163 irregular drying of leaflets in 165 isolation of parasite 161 literature 165 meristematic tissue attacked 164 Porto Rican weed attacked by 163 sieve tissue attacked by 164 signs of disease 161 staining of tissues 161 stomatal infection, common in 163 tissues attacked 161 treatment 1 63 Italian disease 203, 215 Livingston's Dwarf Aristocrat, sensitive to brown rot 282 Malay States disease 211 sleepy disease 175 water-content 282 Tomato-wilt 1 74 (See Solanaceae, brown rot; Bacterium solanacearum.) Top=rot of sugar cane 3, 81 (See Sugar-cane, Top-rot.) Trap-plants 245 Treub, Sereh 75 Tryon, Australian disease of potato and tomato 207 Bacillus vascularum solani 207, 208 brown rot of Solanaceae in Australia 175, 207 studies on bacterial disease of sugar-cane, (See Sugar-cane, Cobb's disease.) Tschirch, Sereh, stumpy roots in 73 Tunstall, tobacco-wilt, persistence of parasite in soil . 237 Tylenchus sacchari 75, 77, 78 Ty rosin, solubility in water 249 Uyeda, cited 220, 226 tobacco wilt, Japan, observations on 238 Valeton, bacteriological investigations of healthy sugar-cane 75 Sereh, observations 52, 72, 73, 74, 75 Sereh, red pigment in, late stage of 17 van Breda de Haan cited 226 Dutch East Indian disease of tobacco 220, 222 peanut wilt 151 tobacco, diseased, coccus from 211 slime disease, inoculations 270 slime disease, underground infections 181 van Hall Bacillus atrosepticus 175 Dutch disease of wallflower 277 Vasculin 7 Voglino, Italian disease of tomato 215 INDEX. 309 Page. Wakker, Sereh 52. 73. 75. 77 top-rot of sugar-cane 81 Wallflower, Dutch disease 277 Went, Cobb's disease of sugar-cane, occurrence in Java 3, 14 heart disease of sugar-cane 81 sereh 52, 7=. 73, 75. 76. 77 top-rot of sugar-cane 83 Wiesner, Rathay's disease of orchard grass 155 Pagh. Williams, Goldsmith, Panama disease of banana reported from Jamaica . 1 73 Wilt-disease of tobacco 220 [See Bacterium solanacearum (North American to- bacco-wilt organism) ; Bacterium solanacearum (Sumatran tobacco- wilt organism); Tobacco, wilt- diseases.] Yellow disease of hyacinths 83 Zeijlstra, Sereh 75. 78 ERRATA. Page 16. Line 28, read Geerligs. Page 91. Last paragraph, first line, read caused by. Plate 26. Last line under the plate, for 1905 read 1903. Page 150. Under the cut for 67, read 67a. Page 228. Seventh line, after North Carolina add and Virginia. Page 256. Table 36. Second line of heading, for mannit read mannitol. Plate 44. Insert X, X, near bottom of tubes 1 and 3 respectively, to indicate pre- cipitate. Iteuocl Au(,uut 7, 'I9'H. •M.?.L. ^'H01 LIBRARY WH IflDY J S(f zx s -;