tie abe ses paces : Plebeam a eee A: ae CER ne sim oak, Digitized by the Internet Archive in 2007 with funding from Microsoft Corporation http://www.archive.org/details/introductiontoba00smituoft if 7 Wo SAN n iL TRY Cyan ie Dose oy ‘ Th! TMA) 5. ate, ieG ae wu, mis — AN INTRODUCTION TO BACTERIALE DISEASES OF PLAN ES BY ERWIN:- 2. SMITH In Charge of Laboratory of Plant Pathology, Bureau of Plant Industry, United States Department of Agriculture, Washington, D. C. Member of the National Academy of Sciences and of the American- Academy of Arts and Sciences; Fellow of the American Philosophical Society; Ex-President: Society for Plant Morphology and Physiology (1901), Society of American Bacteriologists (1906), Botanical Society of America (1910), American Phyto- pathological Society (1917), etc. Ecrire l'histoire d'une science nouvelle, c'est se vouer a d’éternels recommencements—Madame Duclaux ny’ a ne < “Vv \ Nor PER Os aN PHILADELPHIA AND LONDON W. B. SAUNDERS COMPANY 1920 Copyright, 1920, by W. B. Saunders Company PRINTED IN AMERICA To the memory of Charles F. Wheeler and Volney M. Spalding: modest American men of science, strong idealists and splendid teachers. Wheeler was born in New York; studied in Mexico Academy and the University of Michigan; served in the Civil War; was 20 years in a Michigan country drug- store, which he made a center of fine intelligence; was instructor in botany in the Michigan Agricultural College for 12 years; and, finally, for 8 years research worker in the United States Department of Agriculture. Spalding was born in New York; pursued high school and academic studies in Ann Arbor, Michigan; was teacher of botany in the University of Michigan for 28 years, broken only by studies in Germany under Detmer, Pfeffer and Brefeld; was investigator for 6 years in the Carnegie Desert Laboratory at Tu¢gson, Arizona; and, finally, endured a long period of forced inaction in Southern California, retaining, however, his clear mind and his scientific interests to the end. Wheeler studied critically the flora of a State, Spalding changed the type of botanical teaching in our secondary schools. Each was my friend for nearly 40 years. The first showed me how to study flowering plants, opened my eyes to the wonders of wood and field and was my companion in a thousand delightful rambles. From him I had also my first lessons in French. The second taught me how to study the parasitic fungus and where to find its literature, often read- ing it with me when it was in foreign tongues. Each was devoted to the micro- scope and to the laboratory method. Each served his generation faithfully and now sleeps with his fathers. They reached a multitude of students whose gratitude remains, and so, in the lovely words of Simonides, they lie aynparvTw X PWpmEvor evhoyin PREFACE THE manuscript of this book was completed for publi- cation in 1915 and, in general, that year may be taken as the date of the outlook, but here and there, where it seemed most necessary, 1t has been revised down to the end of 1919. Those who seek for completeness in these pages will not find it. The book is in no senes a monograph, but only, as its title indicates, an introduction to the subject. While the book has been made primarily for laboratory use under the guidance of a competent teacher, who will add to it or subtract from it as he desires, it is believed that many persons not students may find in it various things of interest, and partly with this wider public in view it has been illustrated very fully. This book is the result of 35 years of reading and 25 years of diligent laboratory and field investigation. More than most books, it is the product of experiment. There is scarcely a line or a statement in it that has not required more than one experiment. It is also largely the product of a single labora- tory, that is to say, 8 of the 14 organisms here selected for special study were named by the writer (one with a colleague), two were worked out by others in his laboratory (Bacillus carotovorus and Bacterium maculicolum), and of the remaining four, all of Appel’s statements have been verified with addi- tions under Bacillus phytophthorus, many additions have been made to Pammel’s statements under Bacteriwm campestre, the entire body of description has been worked up for Bacterium mori, and some additions have been made to Bacillus amylovorus. Moreover, all but 35 of the 650 illustrations are from this laboratory. A majority of the photographs in the book were made by James F. Brewer and the remainder by the writer. Fre- quently we worked together. In case of particularly good vi PREFACE photographs involving special technic I have sometimes men- tioned the maker and the method used, since every student should learn to make his own photographs and photomicro- graphs if he wishes to excel. Nearly all the photomicrographs were made on the little upright stand (Fig. 55) because I wished to demonstrate to the student that excellent results can be ob- tained with very simple apparatus if one is only willing to take pains. The book was written at the earnest request of teachers and by their judgment it will stand or fall. It is the first treatise of its kind in the world and, therefore, I trust, that evidences of crudity will be excused. Not being a teacher, I have been in doubt many times how best to present the difficult subject. I shall be glad to receive criticisms and suggestions looking toward a second edition and also notes, photographs, cultures, fresh and dry specimens and separates of papers from all parts of the world, that I may be able to continue my monographic studies. ERWIN F. SMITH. WASHINGTON, D. C., August, 1920. CONBENTS PA A CONSPECTUS OF BACTERIAL DISEASES OF PLANTS DIsTRIBUTION AMONG FLOWERING PLANTS PERIOD OF GREATEST SUSCEPTIBILITY. WHat GOVERNS INFECTION... How Inrection Occurs Time BETWEEN INFECTION AND pba ARANCE OF THE Pianasu RECOVERY FROM DISEASE. AGENTS OF TRANSMISSION. : 3 EXTRA-VEGETAL HABITAT OF THE Patactens: MorRPHOLOGY AND CULTURAL CHARACTERS OF THE Padnerras ACTION OF THE PARASITE ON THE PLANT . . . 3 REACTION OF THE PLANT—COLOR-CHANGES, BLIGHTS, Ane AYS OVERGROWTHS. . . : : PREVALENCE AND Cnaceneeie AL Dra eainonion 5 METHODS OF CONTROL . PART Il METHODS OF RESEARCH REFERENCE Books. APPARATUS . ‘ A For Preparation of ealtuce Meier : For Isolation and Care of Cultures. For Preparation and Study of Sections . For the Hot House and Inoculation Berenice For the Photographic Room. Uses of Cutturre Mepta. PREPARATION of CutruRE-MeEpDIA TECHNIC OF ISOLATION. Care or CULTURES StTuDY oF CULTURES . Meruops or INOCULATION 3 TIME AND PLACE OF INOCULATION . - CarE OF INOCULATED AND ConTROL PLANTS. PREPARATION OF SECTIONS . Vil , DISTCRTIONS, 48 vill CONTENTS PaGE STATNING VUE THODS ea cc kh ck kk ie eae eer co ee OTC CARE OF SPECIMENS . . . eed vate dp ghee eae ne Mere he La) RREEPARATIONLOR MG USTRATIONS 9 50 4 2) mite ee Sie nin aes as eee etnnen mel. 2()) IhoOtOgraps sso. 2 eects 2. + 52 2) Re ee mec RhOtomMIcnOgTaAphS: ; oye ac El bs) “ke: Oe ee ce eer 25 letumiaeretPlates’ 20h aie. Bsc ae ee en te ort Plamarss oe 8 ste Te tee ee ere den er 9) een (9 Drawings. 230.9) soi gta po Se ee oe a2, cee Paintings . . A ene tec a ene SOE «Ul Ramee hg ay Ra e's EDO, Carp CATALOGUES AND eens OF aaa. Pradesh ef ARRON ey ent ee Rr (ea PART III SYNOPSIS OF SELECTED DISEASES bre (CucurBit Warr oo. 4 oy ene Ae 1S, Bypek:).. ME Re Me tn >i, Sigh aeneee By Cree Ra ciius cherie EFS. Md een | 3 here keslars Mech mic’, 0g. 1s ny > se ere he eet ee ee nei ae eT (-3) Determine .. . eee ee ee Aria ree 2s) 5 cag Soe te. ALSiey For the Oreanien oie Eg TR EAE Ss oes 0a ere A a NT 37 Morphology. ogs.5 See oe a a ee ee eee G4 Cultural characters. . . . A pean nny ortuee) ere Ar Eats} Response to Non- noeeeronel Bavaro CUNS hay ies ak eee lS For thes Diseaser cc. 78 eet elie ele Or sear con nar lence eta LES OP SOT iS sw ee Ae RO RE Roca ee Sleek es od ORR AOE ae com Be Ce OP FRISCOLO RY geen AE EAL ee rede aye a cc Scere FSO) Variability: 2226 S025 tate See se ena enn eens cee ee ‘Transmission: . 2) > Site see ee en eee ee ens emt ATT Literature... . LE AS EEE SON Dr ata lire WL caper me IIA II. Buack Ror or Ciste TFERS:. (Sy oehi Gos i else teen eens abies See te eh aA) Ay en 0s |. et Ae Gankt Ui ae mee mre eos re |S LAL ance Baca Pace (Panic) EF S. slat la oc ae ATE Pechmie.* 05066. 2) 2S ar aa ee ET Se a eres ee ee | Determine .. . Ae ee et emb egies celine lo. 4 Slay For the Oreanien Bade at got 25 TOS AT nme NN RL SA a COS Morphology . 3 <4. 3G! 5 oa a eS COR a remem UN. S15 Cultural Characters . . . RR nent satiee | Ny Response to Non- nuitvitionall Rhyironntent Aico aah k emkh a feet Ute eae W5)5) Fot the’Disease . «2 2. 2 40% 2° & 0 eee ee ia Signs; 9.0. 5 2 Ae iy Be eee eee ive Histology: a) 20. iy S266 0.4) ee en tats Variability .0.2). 40< 4 4>- 2, ee eel ae Transmission: . ., -< 53) 2. |) Ree eee eee 10 Means, of Prevention.: © 9°. 2+!..5)2') (A eee elie Drtevatures i. ek no eo Ml: Stewart's Dispasr or Maizm..... . > eee eee ce rE O DY POS ee a aye we te a ke ee eT) TV. CONTENTS Cause—A planobacter stewarti (EFS.) MeCutloch . Technic Determine For the pecatean Morphology . Cultural Characters Response to Nonenuetuenell Becowaent For the Disease . Signs. Histology Variability Transmission . Means of Prevention. Literature. : THE Brown Ror or Sanaior EAE Type. Gane Bacioanin m ealanacenr um EFS. Technic. Determine : ; For the Oreste i Morphology . Cultural Characters . Response to Nemetuntrancll Eeavinosenent For the Disease . Signs. Histology. Variability Transmission . Literature V. BaAcTERIAL Ganxun OF BOREAO: VL. Type. Cause eobacier: ni aaense EFS Technic. Determine. : For the Opeanian ; Morphology. Cultural Chaiacteis . Response to Non- auteitionsl Env eonment For the Disease . Signs. Histology. Variability Transmission . Host Plants . Literature. 3 Jones’ Sort Rot or 4 (Olan om mee Type. Guise Bacillus su otnwaris TL. R. Tories i Technic. . Xx CONTENTS Determine . For the organism Morphology . : Cultural Characters . Response to Non- ee ritionall Eigvinc nent For the Disease . Signs. Histology Vaniability Transmission . Literature. He Me A ote 2 Vl1l. BacrertaL Buack Rot oF THE PoTaTo . Type. : Cnnce Racine Pinopiihon US Out ore : Technic. Determine ; For the Cheatin : Morphology. : Cultural Characters . : Response to Non-nutrituonal Env ivonment a For the Disease . Signs. Histology . Variability . Transmission . Literature. VIIt. Tor Bean Banca, Type. Crib iaseale EFS : Technic Determine : : For the Organism . Morphology. Cultural Characters. . Response to Non- nutritional avaonmient For the Disease . Signs. Histology . Variability Transmission . Literature. 1X. McCuttocu’s Cnn LIFLOWER ~ Son. Type. ; nee Paciert ium geulenlinn Tracie Mecullocht Technic. Determine For the Organism . Morphology. : Cultural Characters . PaGcE . 243 . 243 . 243 . 246 . 249 . 251 . 251 251 . 251 . 252 252 . 253 . 253 = Zon . 263 . 268 . 268 . 268 . 269 . 272 . 274 . 274 . 274 acid 2a -) 208 . 280 280 . 285 5 AS ZO 5 Zag 2 ZAI ol 3 7B . 294 . 294 294 . 295 . 295 299 . 300 . 300 . 304 . 310 5 oil il 5 olil 2 alll . 312 CONTENTS Response to Non-nutritional Environment For the Disease . Signs. Histology. Variability . Transmission . Literature. s X. THe ANGULAR LEAF Shem OF iConmonm Type. Cause—B Geer dum walnaceatt um EFS . Technic Determine For the Onuntietn Morphology. : Cultural Characters . ; Response to Non-nutritional Havironm Git For the Disease . Signs. Histology. Variability . Transmission . Literature. . XI. THe MULBERRY TBs J Type. Orne Bacon mort iBos: er ‘and anabert Cnend. EFS : Technic. Determine ; For the Ores Morphology. ; Cultural Characters Response to Non nutritionall Bavigonmient For the Disease . Signs. Histology . Variability . Transmission . Literature. XII. Frre-BLIGHT OF Moen) Paean, Ounce Hire, Type. Ome Bacillh us amt loners (Tr. J. Buell aTWeieAn Technic Determine , For the Greame ’ Morphology. ; Cultural Characters . Response to N Grenuentionnll Enpiannent For the Disease . Signs. Histology. xil CONTENTS PAGE Watiability .¢ atyobyc). | 0 a eee eee a UPAMISTMISSIOD. oc. <. sh ay | ca. Sc. eu eet ea een en Ue ge mS CSE Eradication of the Disease... . = 2 &.) 2S eens ee ar So eiteratures ee): DP se SR SS ety XIII. THe Onive imaneeen PR ee ol PY er eee Pe Tate) 4 NA OGiee ote ok Se eae tere ee ee Gee erin Renae: EFS iy i Nh Me eras. Sr nel MINGGHINIG] Air eee hea a ee wc ores ah a yin a LH Determine . . : PEE St eos a iy (pungent eg” oft a AOE For the Coenen OPE Ti ayo a Ee | wel OG Morphology. 60%. 0... caey URES poe econ een Cultural Characters . . . Deets eae i 2 LOY) Response to Noncantntional Env Shove ant MR are. ye eae AOS Hortherwisease og et Le ead (9) Glens ec, Le ed ein a AOD) Histology. .:2¢.a::0<.2) 068s ee Se ee, ee AOD Variability: 033.3. <2 8 A ee TTANSMAISSION © «. -2) sy pecs ee ee ee or 51,0) Literature... . Se Gl Ree EE ee ee ee XIV. THe Crown Cie: pre Ea. RR yy a hg ae Gul: Ay peeve es; a SAT caret Abt Be AM ad al 3) Cause Bacienin nereciens Smith aud Townsend Zip eeh Alia rel tanec eee Maclean ee es Sk sch Tenn par ag ee eee he ree eae RE Determine... CoN fin Titel ats ae aa Cole SST SNe CCL SU la Oh REEL For the Oiraniem OEM re eC EG ee oa eel ING (Coy 2) 9) 000) Koya GEOR coe rowk AG begtiy oor eee’ <10 4..2...... 2.5.8 150 90. Buried and surface colonies of Bacteriwm campestre by direct transmitted HATA RES ciemetee re ice toc, cr SIO Te nae or oh nina A) Sec gee ar ee 151 91. Agar colonies of Bacterium campestre by oblique transmitted light. ..... 152 92. Cultures of Bacterium campestre and Bacterium phaseoli in Dunham’s SOUS Shc aes 5 oes eM lac igh oes tool ce ee ome a aa 153 XVill LIST OF ILLUSTRATIONS PAGE 93. Tyrosin crystals in milk fermented by Bacterium campestre...........- 153 94. Cross-section of a small cavity in a cauliflower stem, due to Bacterium CUTUPESITNE. as akan ce se s/o ak 20 e+ i eee ase ee ce ae 154 95. Cauliflower bundle in longitudinal section showing disorganization due to Bacterium Cam pestre. on 2.02.2 s vlan ee ope re eee ee 154 96. Turnip root in cross-section showing a bundle attacked by Bacterium CATH DESENE seen tse ett a gies Son's w S/d Steen ea oe 155 97. Beginning of a bacterial cavity in a turnip root...................... 156 98. Single cell of turnip root showing Bacterium campestre, occupying the imbercellular spaces... i2/6...: Siete eeste Reese hte ent eae 158 99. Longitudinal section showing a vessel and cells of a turnip root occupied Dy WBaclenumnCANUPestTes 2. ooo. 2 ile eae CRC tee eee ten 158 100. Large sweet-corn plant destroyed in the field by Aplanobacter stewarti (EPS) MeO 02 abs sie AS ee ee ee ee ee eee eae 160 101. Flint field-corn, showing white top due to Aplanobacter stewarti........ 162 102. Aplanobacter stewarti oozing from cut bundles (cross-section) of a sweet- (Lolwales\( 211) eee eens iettaieny tect sin org cic crop cd'o.0"s 2 0 vo ofo an am 163 103. Aplanobacter stewarti oozing into water from vascular bundle of a sweet-corn stem—loneitudinalisection. .2-. 25 2... 15 eee ie 163 104. Corn plant from Chula Vista, California, showing dwarfing, white top and suckering due to A planobacter stewartt ..................----- 164 105. Yellow spots on inner husk of sweet corn due to A planobacter stewartt.. 164 106. Cross-section of corn-husk from an inoculated plant showing bacterial MASSES AN “LHe ISSUES ee als eS Oe a ae vente als eRe rer ene ett 165 107. Stewart’s disease of sweet corn, a detail of figure 106................. 166 108. Infected small bundle at extreme base of a corn kernel. Cross-section.. 167 109. Cross-section of a larger bundle at the same level as figure 108......... 168 110. Longitudinal section showing infected vessels, etc., in periphery of a corm kernel at the leyel.or the tadiclest em. mene ee 169 111. Infected vessels in periphery of sweet-corn kernel at the level of figure 110. It shows Aplanobacter stewarti forming a small cavity......... 169 112. Inoculated young sweet-corn plant attacked by Aplanobacter stewarti... 170 113. Sweet-corn plant inoculated when young, and diseased when old....... 171 114. A, B. Two types of surface colony in Aplanobacter stewarti.........-.- 172 115. Agar plate showing buried colonies of Aplanobacter stewarti and one Conon ayer iio) lave Sumaikwoe, ING Caio cscccascrassesssoosesscacanss 173 116. Longitudinal section of two infected corn stems showing brown node and: yellowistripin oye ove ce Slee Aue aa hecr Cee eee ee EE fo 174 117. Appearance under the microscope of cross-section of empty and infected (Stained) bundles ammialzers te nays sensei eet ern ee 174 118. Single disorganized bundle of an infected maize stem, enlarged. Bac- fierial mass stainedidlee prec eer = sia sina eerste siete ie is 119. Potato attacked by Bacteriwm solanacearum EFS. From a field near Washington,, Ds (Gon. i ee ee nee ree tc ILe/7/ 120. Early Rose potato inoculated with Bacterium solanacearum. Late StAGe ose a Ss ee RE ee ee ee eee ar 178 121. Inoculated young tomato. Early stage. Leaves of inoculated shoot reflexed tah o's 1: ee ae eee 179, 122. A later stage of figure 121, showing incipient roots on inoculated stem.. 179 lao NO Ww bw bo 2 OV me Oo See bo bh bh Ooo I 130. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 3. Tobacco leaf attacked by Bacterium solanacearum . Inoculated young tobacco plant attacked by Bacterium solanacearum. .. 5. Dwarf nasturtium attacked by Bacterium solanacearwm............... . Dwarfing of Ricinus due to Bacteriwm sotanacearum . A, B. Sunflowers destroyed by Bacterium solanacearum............... 5. Stabs of Bacterium solanacearum in nutrient + 10 gelatin . Cross-section of a young potato tuber showing removal of starch from LIST OF ILLUSTRATIONS A. Common garden balsam attacked by Bacterium solanacearum. B. Shemgotesammerenlair es ed ..-/:5-cy ee venee aeeeee we eS ae fo alte se vag Seah Cross-section of a mature potato tuber showing vascular invasion by ES CLETUUNTUNS OLGNLO CORUM Ves ayctecieed Pee ee eee Ta ee pee Nee ee tpn meee aye Petiole of Tropaeolum majus (nasturtium) attacked by Bacterium solanacearum. Inoculation on stem at X . Dwarfing of Helianthus annuus due to Bacterium solanacearum. Checks CW THU OTL $2100 UN C2) pe ES Ger ce a ca etic Bre. aces eh eres eu comet ee 2. Appearance of Bacterium solanacearum on agar poured plates..... ; 3. Fluid character of colonies of Bacteriwm solanacearum on agar plates... . . Flagellate rods of Bacterium solanacearum: a, East Indian origin; b, ATITENT CAMMY OTIC 83.2), us.t Bite tate Renee es a ge en were cea te SU the areas infected by Bacteriwm solanacearum. . Infected potato stem in longitudinal section. eee Sue Bacon solanacearum confined to a single vessel . Beginning of a bacterial cavity (around a vessel) in stem of a potato plant inoculated on a leaflet with Bacteriwm solanacearum........... Empty and bacterially occluded vessel in a potato plant. Result of a SteMeIMOCUlatl Omer. sae pM es Tor ie Sie oie ass are ea eee ene Bacteria from same series as figure 139, highly magnified. . Tomato stem in cross-section, showing origin and struchine of tae incipient roots—result of an inoculation. . sheen Tyloses in vessels of a potato stem attacked iy Basescu: solaenecnrure. BACKER Aga te AGae Rake Rime rept re ee 7 eee We poe Ln ae OM begs Tomato plant attacked by Aplanobacter michiganense EFS, as result of MeCdle—pMeCke ml OCUlat OTe pera sere ce eer emule i tyre eet yar Tomato plants inoculated one month with a pure culture of A planobacter michiganense. Massachusetts organism. 1915, colony A. Check Olan GS sual ac komo unc eprgaee cecwckar ee MCN eM on a aM Di iid AIR oe Stems of tomato plants showing only a slight tendency to form roots when inoculated (over 3 months) with A planobacter michiganense .. . Tomato leaf showing irregular withering of leaflets due to A planobacter micmganensea sinoculatedemmimerstennys reir er-raee alesse = = Tomato stem in cross-section showing an incipient root destroyed from within (the black part) by A planobacter michiganense. . A, B. Tomato stem in cross-section showing large canritiiea dl in vile eulcen as a result of inoculating A planobacter* michiganense...............- Longitudinal section of a tomato stem attacked by A planobacter michi- GOMER? 10 WHE HEYEAHUOS MENON. 55 onc eo deo sno one ooo eno oes omen te Cross-section of a small group of sieve-tubes in a tomato stem showing sieve-plates, and disintegration of the phloem by Aplanobacter michi- GOMLCTUS CHR yee MRA ee ea OREO E, (PLE Oa rT As cae Td jooss Afro: «2% ) Suayc ese 209 XX LIST OF ILLUSTRATIONS PAGE 151. 1, 2. Green tomato fruits attacked by A planobacter michiganense. From a hothouse in Massachusetts in 19152722. 5.52 - se ee 210 152. Same series as figure 150, showing loss of green color and ae of stems before rupture. (See figure 153 for later stage. yee : ite 153. Crack on a tomato stem due to A planobacter eH anOnee eich was inoculatedotarther GO wa: css 5.5.45 espe aimee eee eee eee eee er tree 211 154. Green tomato fruit oozing A planobacter michiganense as result of a stem THO CULATION. x0. oo evoke hak xe a ow ene RD OE Sette pew encn acer ee 212 155. Tomato plant infected with Aplanobacter michiganense through broken PLOY OL HSiced Ge an a ee LR tits och OiSlo ty Hidtie ala pud'd Glu oy ond a hore 212 156. Stomatal leaf-infection due to Aplanobacter michiganense.............- 213 157. Longitudinal section of a tomato leaf showing bundle disorganization due to eA planobacter michiganense sr. eset ee ee ee 214 158. Detail of a leaf-bundle infection in a tomato sprayed with A planobacter LA [ELS eee AE PR SC ieee on Sid cha late & Has Hea SE OE 215 159. Rods of Aplanobacter michiganense highly magnified...........--..--- 216 160. Aplanobacter michiganense induced to grow in acid agar by presence of aliother organism: : ./t:./: 2). ye eae oe ee ee A if 161. The new Massachusetts potato disease (net-necrosis) by reflected Hight 50°52 shhe. a Sng ts Oe ee cane Se 218 162. Cross-section of stem-end and eye-end of a diseased potato tuber (net- TAOIRO SHES) MRO IBIS, 5 >oocc doo osdoaooaaneseussvascgdsace 220 163. Thin section of a third tuber photographed by transmitted light....... 221 164. Raw carrot destroyed by Bacillus carotovorus L. R. Jones. Check-half SOUNG oe Aitctotcs. oh s Maem ewee Deee gars eee ee eemettn Se earner ears ee 224 165. Like right half of figure 164, but after it was accidentally dropped. ... . 226 166. Separation of cells of carrot due to Bacillus carotovorus..........----- 225 167. A. Bacillus carotovorus streaked on flabby vs. turgid carrot—3d day; B. Ditto, 6th day. In each case one check is omitted.............- 227 168. Raw potato tuber attacked by Bacillus carotovorus...............----. 228 169: Potatoishootiattacked) byasacilis calotovonise ene aie ee ee 228 170. Green cucumber rotted by Bacillus carotovorus. Check-half sound..... 229 le Callanhlysrotiduer()stosSacillisicanatouonsme eens ite eer 230 172; Hnlareed cross-section on eure l/l ati bases jana oes sae ae 231 173. A detail of figure 172 highly magnified, showing bacterial disorganiza- tron ofthe cellawally yuan ean See oe nae ieee hee Cae eter 232 174. Cork layer formed by a potato tuber under a rot spot due to Bacillus COT OUOVOTUS Sho oo inc. ot eeee ene Ps alee nt RE ee ER kee ee Zao 175. Cross-section of a potato stem attacked by Bacillus carotovorus........ 233 762 hlacellate rodsiok SaculUsscanoLovonis seen a ee ee 234 177. Agar-poured plate colonies of Bacillus carotovorus (?). Stock culture 3a (ongeimmtyslalb oratory) eee ie ease eee eee ee 235 178. Do. Jones’ original stock of Bacillus carotovorus. 3a (received from Madison; Wisconsin: inl92 05) sea einen ene ae renner 236 179. ‘Gas from Bacillus carotovorus 1m potato jUIces: -- 2 eee ae 237 180. Bacillus carotovorus on gelatin—magnified surface colony 24 hours old, VV MUR io NoPE UMP BIN So baogsnodoavacodyooccc oc Genoese pEOK 239 181. Bacillus carotovorus on gelatin—fimbriate margin of a surface colony Ssidays old=liquefied ‘at lett sa.25 ean ena an eee ence nes LIST OF ILLUSTRATIONS Xxl1 PAGE 182. Bacillus carotovorus in gelatin—buried colonies 24 hours old, showing RO Od USE VOU LTO NW ENS ey. ss co5s coe Re oe teen es Sener = MS. ors 240 183. A, Bacillus carotovorus and B, Bacillus apiovorus in gelatin stabs....... 241 184. Behavior of soft rot bacteria in peptone beef bouillon with ten per Centmeiay alcohols. >i. <)> i. ae hee eee has, Si ae ee ne SEU Ie Se 242 185. Rot of celery due to Bacillus apiovorus Wormald.................+-+-- 244 186. Resistance of potato shoots to Bacillus aptovorus...............-.+055: 245 187. A, B, C. Three photomicrographs of flagella of Bacillus aroidew Towns- CESOVO TE he Sart Beare nr re ae Pe Te ies cl ch ck 7G re eee earn A CPR ESS 247 188) Bacilus-aroideae atter 8 days on Taw CaLrote: 20. ss. 15. -s---- ee o= 248 189. A, B. Gas in milk produced by Bacillus aroideae—all COz............ 250 190. Curling of potato leaflets when stem is attacked by Bacillus phytoph- UH OUP UES ofA) 6) Oe) Leet Oe he eI MRR et 2S rn of icahte we "bueiie oe Sod ae EEO LS a res Cee 253 191. Potato stems attacked by basal stem rot due to Bacillus phytophthorus— EA SM TT) ULI eee oh EN ee eR a eve a opm pe get. (hh iar 254 192. Same as figure 191 but after another.48 hours....................-.- 254 193. Inoculated potato plant destroyed by Bacillus phytophthorus—7 days... 255 194. Potato shoot attacked by Bacillus phytophthorus—43 hours........ 256 195. Shoots of White McCormick potato inoculated with Bac wipe: thorus in 1915. Control plants in the background................. 258 196: Sameasnoure 1955 but: twordayslaterwaees ies ees ai een 259 197. Woody base of figure 193 two days later. Sound mother tuber at TEL alGtLice 9 4 Shy anne Sere RP ROP team GRR ce EG I eae es cl eee 260 198. Bacillus phytophthorus: same as figure 195, but the inoculations are on GUC, MAGE HSMM HOO. oo aco oes ee oeceoosqeduareacuerenues 261 199. Same as in figure 195, but 19 days later—new shoots growing up from GEMM CLES LEONE Ctl ASC hsyuete ene Men eects ce aaa Bat el che, ee etc ectane ie 262 200. Enlarged cross-section of figure 193, well above the inoculated part. iy ACCOM MIRA ViCSSe ANN Reta Se Raho Lelnthpenie tes cut at da oc aeebeteky See wth 263 201. Bacillus phytophthorus attacking the cut surface of a raw potato....... 264 202. Lenticel infection in a potato tuber due to Bacillus melanogenes P.and M. 265 203. Very early stage of decay in potato tuber under a sound skin—lenticel IMeG MONE Meramec O2iprOss iar ase TR. eshte ree Sells ae PO aS siete eis 266 204. BPlagellate rods of Baculus phytophihorus 2.3. fos ee 267 205. a. Gelatin colonies of Bacillus carotovorus with (b) those of Bacillus phytophthorus for comparison. Natural size...:................--- 269 206. Gelatin plate cultures of Bacillus phytophthorus from a South Carolina BORE) RO LA a US Rea OS. c ie ry VRC Rg ae cee eR 270 207. Bacillus phytophthorus on gelatin—a surface colony magnified to show the SGA OP EMAS DENG AlAs Roe lolc o cc oo wo 6-d'o Gus Guach aemcuemcne oar eye Choos ic Once cei 271 208. Bacillus phytophthorus in gelatin—2 buried colonies, magnified......... 271 209. Bacillus phyt all buried colony in gelatin 5 days at 16° C., - showing lenticulate coronal colonies in the gelatin...............--. 202 210. Effect of ethyl alcohol on Bacillus phytophthorus and Bacillus carotovorus imme ptome WOUULOmey eos ee etpeeehS aer teh ARs) eRe ttle Es 273 211. Inoculations showing virulence of Bacillus phytophthorus (Appel 1) BG ersll> wears MP CMItmbeMCMIA se Mit els Fhe ace ee eles ae 275 212. Gelatin plate culture of Bacillus carotovorus. B. Cross-section of Tropaeolum stem attacked by Bacterium solanacearum.............. 276 Xxll LIST OF ILLUSTRATIONS Pace 213. Portion of an immature bean leaflet showing an early stage of infection with Gactenzum phascolt WES... ass ee eigen ine ee een 280 214. Pure-culture spray inoculation of Bacterium phaseoli on an immature OVEN TH (eH ASS ye et ee ak aS weet A f. S0o1s bio om & eto 0% 281 215. Bean leaflet attacked by Bacterium phaseoli. From a garden in Wash- IMe tO, OOS !s 3h). Ame eis oes ES ane epee ee eee 282 216. Spots on a bean leaflet due to a pure culture stomatal infection of 1914. Chiorophylltpersisting around the spotsieyauci err per eye eee 283 217. Distortions of bean leaves due to infection of the young veins by Bac- LETLUIN DROSCOM i ssc)< x 5 soo 5 gl ouhs ite UE el A eRe or 284 218. Portion of a bean pod enlarged to show earliest visible stage of stomatal INfECHLOMAS Sth Gay css sic Ssn a. £1 Rie Ve Reet ee era cee er 285 219. Pure culture pod inoculation of bean blight (Bacteriwm phaseoli). A singlespotienlarged «+ 4: :.. 5. Ordinary appearance of Beyonia phyllomaniaca.................-+4-- é - Non-proliferous and proliferous shoots of Begonia phyllomaniaca....... ¢ - Enlarged central part of a dwarfed proliferous leaf showing that the proliferations are not restricted to the main veins................-. . A, B. Structure of leaf buds of Begonia phyllomaniaca; C. Plant grown from an adventive shoot on a leaf blade; D. Enlarged view of stem- enbicelssandolarids) seer errant ej 5 pits a ee ah, Phe ed . A, B. Proliferation on shoot and leaf stalk of Plant No. 1, first series, front and back view; C. Proliferation and beginning cork formation on aunTaneheotaphe sania yay trae oats |e Sl heli ee earn oa! tte eas 5 , 431. Dwarfed proliferous leaf of No. 1, first series, and next four leaves SUOIE Mitra oth Pee et Senet Me ited SE rea aes g 584, ¢ 32. Plant No. 6, first series, showing proliferous leaves and internodes with non-proliferous ones above and below... . Plant No. 6, first series, showing pee ifoneers upper mince ee lent bide Z andenean ly smoothtaceowA. nextibelow tteses. 5-6 L5 ise.) -52-85-- . (1) Lower (free) and (2) middle (leafy) internodes of No. 6, first series; 580 XXX 438. 439. 440. 449, LIST OF ILLUSTRATIONS PAGE (3) Branch of No. 1, first series showing cork formation in stimulated 102 Hite iets neon eNO Sr Do ab od dig doo 0,7 Ob bce Gears 5. Plant No. 8, first series, showing old and new proliferations on the main URIS Cn oe she ee oa hohe coe. sages: due, cutis Seoul ee eee 5. A. Plant No. 8, first series, third branch, showing proliferous and non-proliferous internodes; B. Plant No. 9, first series, whole of main axis with two stimulated proliferous internodes; C. Shoot arising from rH LG] OVO} 0\ aed et a ERRMSG SBS og God oc ¢ o's be . Plant No. 3, second series, main axis, showing dwarfing of proliferous leaf (M), adventive shoots on middle internodes, cork. formation (CEG C sac Sieeccnb-0ts jie ok ee ee re eee ees Plant No. 9, second series, petiole and upper face of leaf M, blade proliferations mostly from the midmbsi): 27.2 25-5 ->.2.-...-- i ie A. Plant No. 9, first series, part of an internode enlarged with cork at C; B, Plant No. 9, second series, petiole enlarged. Both very pro- WiferouUsie.. cee whs Soke SES ae SE ee DE a ee ee eee A. Plant No. 10, second series, main axis, contrasting, especially, big proliferous and non-proliferous leaves; B. Cross-section of a petiole- trichome showlnes a bud sarisin gs Gon pee a a ane are . Plant No. 10, second series: middle back part (center) of proliferous leaf blade wi ot digs 440 enlarced mera eee 2. Plant No. 10, second series, middle leaf, from a branch showing effect of vicinity of midribs on number and size of the adventive shoots...... 3. Plant No. 15, second series, main axis, upper face of leaf-blade L and its) petioles hulltotradventiviershOOtsrsee setter teen . 1, 2. Plant No. 18, second series, main axis, upper and lower part of the PLOlikeroushinternod es enlare. cose ees een eens ee . Plant No. 1, third series of dried cuttings. Ck, cork; Tp, topmost small leaf when cutting was made; X, fallen leaf; Y, proliferous leaf :S¢, ‘stipules:s Proliferous leaf. Y isidwariedie-;21- pe eee . Plant No. 1, sixth series of cuttings, third, fourth and fifth leaves from the top. Stimulated proliferous leaf not dwarfed.................. . Parts of leaves enlarged showing leafy shoots originating from the edge of knife wounds;and needle’ pricks../; sees ae -o oe oe are = . A. Shoots arising from knife-slit in lower surface of a midrib; B, effect of dwarfing on limitation of production of shoots from edges of wounds; C, two abnormal fused leaves from an adventitious shoot........... 1, 2. Petioles developing adventive shoots from trichomes. © 3. Stimu- lated internodes which have developed adventitious shoots and cork formation. (Pl. No. 6, first series.) 4. Same as 1 and 2. 5. Midrib- trichomes developing shoots): 4) 25] heen eee ee eee . A, B, C. Further evidence of the development of shoots from petiole . A. Cross-section of a leaf and, B, cross-section of an internode, showing Superiicial ongin! of the adwventivershootsaee es: a eee eee een eee 2. Abnormal leaves from adventive shoots: also stem-glands (2, y) giving rise tosshoots.i.:). sed) ee ee ee A. eee eee 3. Like 451A but an earlier stage of shoot-development from the epidermal region (a, y)... oe le ee Ae Ca ee ee ee 589 591 592 598 614 Pee bE RIAL DISEASES OF PLANES PAK at A CONSPECTUS OF BACTERIAL DISEASES OF PEANTS All our knowledge of these diseases has come within a gen- eration. It began forty years ago with the announcement of the bacterial origin of pear blight by Professor T. J. Bur- rill of the University of Illinois (See Frontispiece), who has but recently passed away.' During the first half of this period progress was very slow and doubt universal, especially in Europe. In the early study of these diseases a few men were far in advance of their generation, as always happens when a new science un- folds. Photographs of the leading workers of that period, all of whom are still living, are shown in Fig. 1. All were made at that time with exception of Savastano’s which reached me too late and is shown separately as Fig. 308. It is now twenty-four years since I ventured the statement,’ that ‘‘there are in all probability as many bacterial diseases of plants as of animals.’ This statement was received with much skepticism, not to mention active opposition, but time has more than borne out my statement, and there is now no one left to dispute it. To-day I will venture another and broader generalization, to wit: It appears likely that event- ually bacterial diseases will be found in every family of plants, from lowest to highest. This prediction is based on the fact that although the field is still a very new one, with no workers in most parts of the world, such diseases have been reported 1 Born in Massachusetts, April 25, 1839; deceased in Ilinois, April 14, 1916. 2 Am. Nat., vol. 30, p. 627. 1896. 1 BACTERIAL DISEASES OF PLANTS Savastano Iie, Il Cavara Arthur Wakker Waite CONSPECTUS: INTRODUCTION ' Fre. 2.—Fruit, fruit-stalk, and leaves of mango attacked by the South African bacterial disease. (After Ethel M. Doidge.) 4 BACTERIAL DISEASES OF PLANTS from every continent, and are already known to occur in plants of one hundred and fifty genera distributed through more than fifty families. DISTRIBUTION Following Engler’s arrangement, I will list these families that you may see how wide is the distribution of bacterial diseases in plants and how utterly wrong were those who said that there were no such diseases, and also those who conceded a little but said that they were very rare and restricted to the soft underground parts of a few bulbous and tuberous plants, and generally preceded by fungi (German writers and their follow- ers). In this list, I have included only the flowering plants, but some of the eryptogams are also subject to bacterial attack. The number following the family name indicates the number of bacterial diseases known within the limits of the family. The total of the figures, however, will not give the number of bacterial parasites, because some of the diseases overlap. TABLE I SHOWING THE FAMILIES OF FLOWERING PLANTS ARRANGED SERIALLY FROM LOWEST TO HIGHEST. THOSE CONTAINING GENERA SUBJECT TO BACTERIAL DISEASES ARE UNDERSCORED, AND WHEN SEVERAL DISEASES HAVE BEEN RECOGNIZED THEIR NUMBER IS ALSO GIVEN 1. Cycadaceae 17. Gramineae 14 34. Juncaceae 2. Ginkgoaceae 18. Cyperaceae 35. Stemonaceae 3. Taxaceae 19. Phoenicaceae 36. Melanthiaceae 4. Pinaceae 2 19. Palmae 37. Liliaceae 3 5. Gnetaceae 20uiGuclanthacenes 38 .Convallariaceae 6. Typhaceae 21. Araceae 39. Smilacaceae 7. Pandanaceae 22. Lemnaceae 36. | ~ ¥ nore 19ee9eE « n) & 7 8. Sparganiaceac 23. Flagellariaceae 37 SE ye C : Ae. : : 9. Potamogetonaceae 24 se aloskionacese ne | 10. Naiadaccae 94. Restionaceae bundles, some of w ‘In ic stalk from Tr fruit- oozing ana f a ban 10n O 5.—Cross-sect the slime of Bacillus muse Rorer together with the drops are st Photo. by James F. fie. hich AT cule as om the v fr 3. « x Nn. 1 brow alnec er. Brew 12 BACTERIAL DISEASES OF PLANTS _ find in the maize stems that extensive breaking down of the pith and bark into vast cavities which is so common, for example, in tobacco and tomato stems. WHAT GOVERNS INFECTION ¢ Within the plant we may suppose, from certain indications, that abundant juiciness is one of the factors governing the in- fection of immature tissues. To this may be added an abun- dant supply of well-adapted food and, in some cases, probably the absence of inhibiting substances, which may appear later. As parts approach maturity, the intercellular air-spaces become much larger and the water content becomes relatively less. Along with this, acids, sugars, proteids, amino-acids, ete., are consumed and converted into substances less well adapted to the needs of the meristem-parasites, if not wholly inimical. In young shoots of potato and tomato, or of pear and apple, as contrasted with old ones, or in the roots of carrots as com- pared with the leaves, or in juicy carrots as compared with flabby ones, or in rapidly growing cabbages as compared with slow-growing ones, we know that there is an excess of water, and this alone appears to be sufficient to explain the difference in behavior of their respective parasites in old versus young parts. When, however, we come to ripening fruits, such as the pear and the plum, it would seem that they are still juicy enough to favor the growth of almost any bacterium; we are forced, there- fore, to the hypothesis of chemical changes within the fruits to account for the failure of inoculations, and this throws some doubt on the preceding hypothesis. Asa rule (there are striking exceptions), parasitic micro-organisms are rather sensitive to changes in their environment, e.g., to drying, exhaustion of food- supplies, multiplication of their own by-products, conversion of an easily assimilable substance into one less assimilable or actually harmful, appearance of esters, new acids, ete. But why speculate! Much additional experimenting must be under- taken before we shall have precise and full data.. We are still largely in the observational stage and experiments are needed. ! ‘In the above connection the following list of fruit acids may be of some use: CONSPECTUS: WHAT GOVERNS INFECTION 13 The parasites of ripened tissues do not require so much water, are able to convert starch into sugar, or have a special liking for some other element of the plant tissue. Externally, a number of factors favor infection. One of these is excessive shade, either of clouds or of fobage, and an- other is high temperature. When these two factors are accom- panied by excessive rainfall, high winds, wet earth, and heavy dews, the conditions are ideal for the rapid dissemination and the destructive prevalence of a variety of bacterial diseases of cultivated plants. The bean blight due to Bacterium phaseoli, the angular leaf spot of cueumber due to Bacterium lachrymans (Figs. 6 to 9), the black spot and canker of the plum due to Bac- terium pruni, and the lark-spur disease due to Bacterium del- phinu, are all favored by heavy dews and by shade. In hot, wet weather in midsummer, pear blight due to Bacillus amy- lovorus often bursts out like a conflagration and sweeps over whole orchards. In warm, moist autumns bacterial diseases of the potato may destroy almost or quite the entire crop over extensive districts. Fruit Acids found A Dleieme ners ae Malic only. Banana ‘sae ss. Probably malice only. Cantaloupe............ Malic none—probably all citric. Cherry: sees -:.-. . Mahe only. Cranberry: were rns 6 Citric probably predominates—malic also present. (Gunna. tee eae tee Citrie probably predominates—malic sometimes pres- ent. Gooseberry: 2. 4.2.2. Malice and citric. Reaches =e eerobablyannalrcromlye LELERT Rs did ager tne came eee ects Malic only in some varieties; citric probably predomi- nates in others with small amounts of malic. Rersimnmonmeer a4. se Probably malice only. Jeliviot."; oe See eee Malie only. Pomepranates 2.2.2... - Probably all citric—no malic or tartaric. Quin Genesee ace Malic only—no citric, Raspberry (red)........ Probably citric only—maliec, if present, in traces only. Watermelon........... Malic, no citric. Jour. Amer. Med. Assoc., vol. Ixix, No. 17, Oct. 27, 1917, p. 1433. Bigelow, W. D., and Dunbar, P. B.: The acid content of fruits, Jour. Industrial and Engineering Chem., 1917 (9, 762). 14 BACTERIAL DISEASES OF PLANTS Fig. 6.—Angular leaf-spot on cucumber due to stomatal infections produced by spraying-on Bacterium lachrymans Smith and Bryan. It shows breaking of tissue around old spots. Time, 12 days. Fre. 7.—Cucumber stem showing white bacterial film and cracks due to Bac- terium lachrymans. Inoculated by spraying May 6, 1915. Photographed May 20. x 14. CONSPECTUS: HOW INFECTION OCCURS 13 HOW INFECTION OCCURS As I have already described elsewhere how infection oc- curs,! I will dwell on it here only for a moment, offering a few examples. The commonest way of infection is probably through wounds. In Italy, the olive tubercle due to Bacteriwm savastanoi has been observed to begin very often in wounds made by hail- FGsrS: iti, (8). Fie. 8.—A beef peptone gelatin (+10) stab culture of Bacterium lachrymans after 12 days at 20°C. In the unliquefied part the colonies along the needle track are very small showing that it 1s aérobic. Fig. 9.—Two different illuminations of a small gelatin colony of Bacterium lachrymans to show the characteristic margin. X 14. stones. In South Africa, crown gall is said to be disseminated in the same way. In this country and also in Sumatra, Bac- terium solanacearum enters the plant more often than other- wise through broken roots. A tomato or tobacco plant with 1Smith, Erwin F.: “Bacteria in relation to plant diseases,” Carnegie Inst. Washington, Publ. 27, Vol. 2, pp. 51-64, 1911. 16 BACTERIAL DISEASES OF PLANTS unbroken roots will thrive in a soil deadly to one that has been root-pruned. I have myself observed this. We may suppose that substances attractive to the particular bacteria diffuse into the soil from the broken roots, following which they enter the plant. Resistant plants may be supposed to diffuse indifferent or repellant substances. All infections must be chemotactic. More interesting perhaps are those diseases which begin in natural openings, 7.e., in places where the protective covering of the plant gives place to special organs such as nectaries, water- pores, and stomata. All the pome fruits subject to fire-blight are lable to blos- som infection. The bacteria multiply first in the nectaries of the flower and pass down into the stem by way of the ovary and pedicel. Blossom blight of the pear is a very conspicuous and common form of the disease, as everybody knows. Thou- sands of blighted blossom-clusters may be seen in any large orchard subject to this disease. Blossom-blight arises from “hold-over” blight (see Figs. 282 and 283), the visiting insects acting as carriers. In the black rot of the cabbage due to Bacterium campestre, the majority of the infections begin in the water-pores. These are grouped on the margins of the leaf at the tips of the ser- ratures. From this point the bacteria burrow into the vas- cular system of the leaf and so pass downward into the stem and upward into other leaves. In the black spot of the plum, due to Bacterium pruni almost or quite all of the leaf and fruit infections are stomatal. < 10! other Western States is a seed-borne infection, and so is the very similar barley disease described by Jones, Johnson and Reddy. The yellow disease of hyacinths is carried in the bulb. Potato tubers from diseased fields may infect healthy fields. Apple grafts have transmitted crown-gall. Slightly infected trunks and limbs of trees (hold-over pear blight, walnut blight, canker of the plum) may infect shoots, leaves, blossoms, or fruits CONSPECTUS: AGENTS OF TRANSMISSION 21 the following season. The soil around the infected plant may serve, it is believed, for years as a source of infection to other species (crown gall), or to other individuals of the same kind (various leaf-spots). Occasionally, however, a parasite seems to die out of certain soils (Bacterium solanacearum, Bacillus tracheiphilus). The pear blight organism probably dies as quickly in soils as it does in a majority of the blighted branches. Pear blight or cucurbit wilt by soil-infection is not known. Among extraneous agents, wind and water have been sus- pected. Ihave never seen any clear indications of purely wind- borne infection, not even when contiguity seemed to invite it, but water and, of course, in driving rains, the wind, also, often carries parasites and furnishes conditions favorable to infection (citrus canker, angular leaf-spot of cotton, and bacterial canker of the tomato due to Aplanobacter michiganense). Horne has shown that the olive tubercle in California may be transmitted from the surface of diseased branches to sound branches by rain or dew (see Fig. 300). Honing, in the tobacco fields of Sumatra, has traced infection several times to the watering of plants from infected wells, and has cultivated the parasite from the water. I have discovered experimentally that to obtain in abundance several sorts of bacterial leaf-spots, e.g., those occur- ring on bean, cotton, peach, plum, carnation, larkspur, sorghum, geranium, etc., the leaves must be kept moist to the same extent they would be in case of prolonged dews or frequent light show- ers. In nature such conditions are necessary to enable the bacteria to penetrate the stomata and begin to grow. In case of water-pores, however, the plant itself furnishes the water neces- sary for infection, if the nights are cool enough, 7.e., if the air remains near enough to saturation to prevent for some hours the evaporation of the excreted water from the leaf-serratures. Every plant with functioning water-pores awaits its appropriate bacterial parasite. The genus Impatiens is a good example. I have looked for one on it in vain but I am sure it must occur. Man and the domestic animals, especially through the agency of the dung-heap, infallible repository of all sorts of discarded refuse, undoubtedly help to spread certain bacterial diseases of plants (potato rots, black rot of cabbage, etc.). De, BACTERIAL DISEASES OF PLANTS ee os Fie. 14.—Bacterial black chaff of wheat. Stalk bent double to show diseased (black-striped) upper part and sound (pale green) middle part. Wheat No. 268, collected June 28, 1917, on the Rhodes farm at Manhattan, Kansas. Photo- graphed July 3, 1917, by James F. Brewer, using a W. and W. panchromatic plate and a yellow color screen CONSPECTUS: AGENTS OF TRANSMISSION 23 Fie. 15.—Montana spring wheat. Crop of 1917. Coll. Nv. 318. Diseased glumes showing bacterial exudate of the black chaff organism, Bacterium trans- lucens var. undulosum 8., J.and R. X 15. 24 BACTERIAL DISEASES OF PLANTS Fra. 16.—Black chaff of wheat. A glume infection done by Lucia McCulloch in the summer of 1917 with No. 20, from McKinney, Texas. Inoculated June 21 from a pure culture. Photographed July 9. The tiny beads are bacterial masses oozing from stomata. X 13. CONSPECTUS: AGENTS OF TRANSMISSION 25 Birds probably transmit some of these diseases on their feet or in other ways. In connection with the bud-rot of the coconut palm in the West Indies, I suspect the turkey-buzzard, but the evidence is not complete. Long since, Merton B. Waite obtained (once in Florida, once in Maryland) the strongest kind of circumstantial evidence going to show that pear blight may be spread by birds. Respecting insects, molluscs, and worms, the evidence is complete. They often serve to carry these diseases. I have summarized our knowledge in another place’ and will here content myself with a brief statement calling renewed atten- tion to the subject. We had very good evidence of the transmission of one bac- terial disease of plants (pear blight) by insects long before the animal pathologists awoke to the importance of the subject,” but it cannot be said that they have ever paid much attention to it, although it antedates by two years the work by Theobald Smith and Kilborne showing that Texas fever is transmitted by the cattle tick (Jxodes bovis Ry.). That discovery also belongs to the credit of the United States Department of Agriculture, and the two together may be said to have laid broad and deep the foun- dations of this most important branch of modern pathology. Waite isolated the pear blight organism, grew it in pure cultures and proved its infectious nature by inoculations. With such proved cultures he sprayed clusters of pear flowers in places where the disease did not occur and obtained blossom-blight, and later saw this give rise to the blight of the supporting branch, found the organism multiplying in the nectar, and re-isolated it from the blighting blossoms. On some trees he restricted the disease to the sprayed flowers by covering them with mosquito netting to keep away bees and other nectar-sipping insects. On other trees where the flowers were not covered he saw bees visit them, sip from the inoculated blossoms and afterwards visit blossoms on unsprayed parts of the tree, which then blighted. 1Smith, Erwin F.: “Bacteria in relation to plant diseases,’’ Carnegie. Inst. Washington, Publ. 27, Vol. 2, p. 40, 1911. 2 Waite, Merton B.: Results from recent investigations in pear blight, Bot. Gaz. 16, 259; Am. Assoc. Adv. Sci., Proc., 40, 315, 1891. 26 BACTERIAL DISEASES OF PLANTS Fie. 17.—Black chaff of wheat. Agar plate poured (June 2, 1917) from a leaf stripe of No. 20, McKinney, Texas. Photographed June ll. X 4. Colonies yel- low and smooth on surface (as photographed), and also by direct transmitted light; but by oblique transmitted light they are like Figs. 18, 19. CONSPECTUS: AGENTS OF TRANSMISSION Fig. 18.—Bacterium translucens var. undulosum Smith, Jones and Reddy, rom black chaff of wheat. No. 662, Monticello, Illinois. Surface and buried ellow colonies on + 15 beef-peptone agar-poured plate. A pure-culture glume solation of June 21,1918. Photographed July 1 by oblique light to show internal narkings—surface smooth. X 10. BACTERIAL DISEASES OF PLANTS Fra. 19.—Bacterium translucens var, undulosum S., J. R. from black chaff o wheat. No. 678, El Reno, Oklahoma. Surface and buried yellow colonies on +15 beef-peptone agar plate. Plated June 24, 1918 (from a glume). Photo graphed July 2, by oblique light; surface smooth. At a2 an intruding colony. x 10. CONSPECTUS: AGENTS OF TRANSMISSION 29 Fic. 20.—Bacterium translucens var. undulosum 8., J. R. from black chaff of wheat. No. 252, Republic, Missouri. Beef-peptone gelatin-poured plate (+9) from colony 52. Photographed by oblique light (from direction of the arrow) to show the small dry liquefaction pits. For the two at z, enlarged, see Fig. 21. 30 BACTERIAL DISEASES OF PLANTS Finally he captured bees that had visited such infected blossoms, excised their mouth parts, and from these, on agar-poured plates, obtained Bacillus amylovorus, with colonies of which he again produced the disease. These experiments were done in several widely separated localities with identical results. I saw them and they made a great impression on me. The writer has since proved several diseases to be transmitted by insects, notably the wilt of cucurbits, and here the trans- mission is not purely accidental, but there appears to be an adap- tation, the striped beetle (Diabrotica vittata) chiefly responsible for the spread of the disease being fonder of the diseased parts of the plant than of the healthy parts. This acquired taste, for it must be that, works great harm to melons, squashes, and cu- cumbers. Whether the organism winters over in the beetles, as I suspect, remains to be determined. Certainly the disease appears in bitten places on the leaves very soon after the spring advent of the beetles, 7.e., before they have had opportunity to become infected from newly wilted cucurbits: In the summer of 1915, Mr. Frederick V. Rand, assistant pathologist in my laboratory, undertook, at my suggestion, two series of experiments on Long Island, N. Y., to determine the truth or error of this hypothesis. His results, which have afforded a striking confirmation of my views, may be summarized in brief as follows: In two cucumber fields where 75 per cent of the plants contracted the bacterial wilt disease in 1914 and where, up to September 1, 800 plants or about one in four contracted it in 1915 (later cases no doubt occurred but no further record was attempted owing to the appearance of the downy mildew), 180 plants kept inside of 50 large insect cages distributed at uniform distances through the two fields remained entirely free from the disease, except in two cages. In one of these two cages Diabrotica vittata was purposely introduced when the plants were only 2 to 3 inches high, and before there was any of the disease on the check plants. In this cage all of the plants contracted the disease which first appeared in bitten places on the leaves. In the other cage, a single beetle of that species penetrated accidentally later in the season (when the disease was quite prevalent outside on the checks) and gnawed and infected a single plant before it was discovered and removed, the other, unbitten, plants in the cage remaining healthy. The beetles were collected in one of the two experimental fields, remote from other plantations, at a time when the check plants were small and all still free from the disease. It is believed, therefore, that they hibernated in the vicinity and that their last source of infection was diseased plants of the preceding year, ‘.e., that they carried the wilt organism over winter in their bodies. That not all hibernated beetles transmit the disease is shown by the fact that some were liberated at the same time in three other cages but the plants remained healthy, and by the additional CONSPECTUS: AGENTS OF TRANSMISSION 31 Fig. 21.—Black chaff of wheat. Two small surface colonies on +9 beef pep- tone gelatin. Enlarged X85 to show thin pale fringe in the dry pit of liquefaction. Plate poured January 30, photographed February 9, 1918. on BACTERIAL DISEASES OF PLANTS Fic. 22.—Colonies of Bacterium iranslucens var. undulosumS., J. R., the cause of black chaff of wheat. Same as Fig. 21 but three days later. At the upper right side of the lower colony, lifted above the surface of the gelatin, is a bacterial tendril which has made 6 turns. X 85 CONSPECTUS: AGENTS OF TRANSMISSION 33 fact that on the checks the disease first appeared on a few only of the many bitten plants, and from these few was subsequently spread to many others by the beetles, the disease appearing everywhere first, in bitten leaves, a few days after they were gnawed by the beetles. From these experiments we may conclude: 1. Striking confirmation of my statements respecting summer distribution of this disease by Diabrotica viltata. 2. Freedom of plants from disease when protected from this beetle by wire screens, although presumably growing in infected soil. 3. Inability of aphides and flea beetles to cause the disease, since they entered the cages to some extent but did not act as carriers. 4. Evidence that the disease is not air-borne or water-borne. 5. Proof that the disease is not transmitted by way of the soil—at least not in the absence of insects. 6. Strong circumstantial evidence that Bacillus tracheiphilus winters over in “bacillus carriers,” 7.e., in certain beetles which function as the spring distributors of the disease.! In 1897 I observed and proved experimentally that molluses sometimes transmit brown rot of the cabbage, and in 1913 I saw indications in Southern France which lead me to think that snails are responsible for the spread of the oleander tubercle, i.e., | saw them eating both sound and tubercular leaves, and found young tubercles developing in the eroded margins. Parasitic nematodes break the root-tissues and open the way for the entrance of Bacterium solanacearum into tobacco and tomato, as was first observed by Hunger in Java and later by myself in the United States. One of the serious problems of plant pathology is how to control the nematode, Heterodera radi- cicola, not only because of its wide distribution on a great variety of cultivated plants and the direct injury it works but also on account of the often very much greater injury it causes through the introduction into the roots of the plant of bacterial and fun- gous parasites. The man who shall discover an effective field remedy will deserve a monument more enduring than bronze. Parts of our Southern States in particular are overrun by this parasite. In the hothouse, of course, it may be controlled by steaming the soil, and in other ways. Much remains to be done before we shall know to what ex- tent fungous parasites function as carriers of parasitic bac- teria. H. Marshall Ward sought to explain the presence of 1In most beetles, as shown by Rand’s further studies (which will appear in Phytopathology) the ingested bacilli are promptly destroyed: in a few, they per- sist for a long time and are voided in the feces, which are then infectious. 3 34 BACTERIAL DISEASES OF PLANTS bacteria in diseased plants by supposing that they must enter the plant through the lumen of fungous hyphae. In this he was wrong, certainly, if it be stated as a general proposition, since many bacteria are able to attack and do attack, unassisted, but it appears to be clear that in some cases the two types of parasites work together, the fungus invading first, and the bac- terium following hard after and often doing the major part of the damage, as in potatoes attacked by Phytophthora infestans. The reverse of this also occurs, the bacterium entering first and the fungus following, as in crown gall followed by Fusarium. Parasitic bacteria are soon followed by saprophytic bacteria, which complete the destruction of the tissues, and, if the dis- ease is somewhat advanced, cultures from the tissues may yield only the latter (potato rots, lettuce rots, etc.). Also, as in animals, one bacterial disease may follow another and the second be more destructive than the first, e.g., fire-blight on the apple following crown gall. EXTRA-VEGETAL HABITAT OF THE PARASITES Here is perhaps the place to say a few words about the non- parasitic life of the attacking bacteria. All are able to grow saprophytically, 7.e., on culture media of one sort or another, and probably all live or may live for a time in the soil. Very few, however, have been cultivated from it. The vast mixture of organisms present in a good earth rather discourages search. In some of the unsuccessful attempts failure may have been due to having undertaken isolations at not exactly the right time, or in not Just the right place, or on not just the proper medium, but more often prob- ably to the swamping tendency of rapidly growing saprophytes. How long a parasite is able to maintain its virulent life in a soil must depend largely on the kind of competitors it finds. I have used the term virulent, because it is conceivable that an organism might remain alive in a soil long after losing all power to infect plants, just as we know it can in culture media. Bacterium solanacearum causing brown rot of Solanaceae and of many other plants, Bacillus phytophthorus causing basal stem- CONSPECTUS: EXTRA VEGETAL HABITAT 35 rot and tuber-rot of the potato and Bactertum tumefaciens, causing crown gall, sometimes certainly live in the soil, and the soundest plants when set in such soils, especially if wounded, are liable to contract the disease, if they belong to susceptible species. The root-nodule organism of Leguminosae, which I have not considered here, also lives in many soils, as every one knows. MORPHOLOGY AND CULTURAL CHARACTERS OF THE PARASITES Most of the plant bacteria are small or medium sized rod- shaped organisms. They have rounded ends and are of variable length but are seldom more than ly in diameter and sometimes less than 0.54. Very few parasitic coccus forms are known; in fact, none are very well established, but animal diseases due to coeci occur and presumably there are such plant diseases. Some of these bacteria are Gram positive, others are not; few, if any, are acid fast. All takes stains, especially the basic anilin dyes, but not all stain with the same dye equally well. Most of the species are motile by means of flagella—polar or peritrichiate; a few are non-motile, genus A planobacter.!_ Some develop conspicuous capsules, others do not. Few, if any, pro- duce endospores. Under special conditions long filaments and chains are frequent. Under abnormal conditions many _ be- come club-shaped, y-shaped, or otherwise branched. Lohnis believes (Jour. Agr. Research, Vol. 6, July 31, 1916, p. 675) that all bacteria have an amorphous stage, but such is not my belief. Grown pure on culture media in mass, they are either yellow, pure white, or brownish or greenish from the liberation of soluble pigments. Red or purple plant parasites are not known. We formerly supposed that there were no green fluo- rescent species capable of parasitism, but now several are known: e.g., the organism causing the lilac blight of Holland (Bacterium syringae[C. J. J. van Hall] EFS), with pure cultures of which the writer was the first to obtain typical infections, at Amsterdam in 1906 (garden of the Willy Commelin Scholten Laboratory, courtesy of Johanna Westerdijk) and afterwards in 1Smith, Erwin F.: “‘ Bacteria in relation to plant diseases.” Carnegie Inst. Washington, Publ. 27, Vol. 1, p. 171, 1905; Ibid. 27, Vol. 3, pp. 155, 161, 1914. 36 BACTERIAL DISEASES OF PLANTS the United States; Bacteriwm lachrymans, the organism causing the angular leaf-spot of cucumber (Figs. 6 to 9 and 23 to 26); Bacterium aptatum Brown and Jamieson, causing leaf-spots on Tropaeolum and on beet; and some of the lettuce spot organisms (Bacterium viridilividum. Brown, Bacterium marginale Brown). . Some species produce gas (chiefly CO, and H), liquefy gelatin, consume asparagin, destroy starch, and reduce ni- trates; others do not. Their fondness for sugars and alcohols is quite variable. Some are extremely sensitive to sunlight and dry air (Bacillus carotovorus, Bacillus tracheiphilus, Bacterium solanacearum, Bacterium malvacearum); others are remarkably resistant, remaining alive and infectious on dry seeds for a year (Bacterium campestre, Aplanobacter stewart, Aplanobacter rathayi, Bacterium translucens). Some are strictly aérobic, others can grow in the absence of air, if proper foods are available. Some are very sensitive to acids, alkalies and sodium chlorid, others are not. Some have wide ranges of growth from O°C. upwards. Some will not grow at or near 0°C., others will grow at or above 38°C. Very few, however, will grow at blood temperature, certain ones even in plants or on culture media are killed by hot summer temperatures, and none are known definitely to be animal parasites, unless we except Bacterium tumefaciens. My own animal experiments with this organism have been limited largely to efforts to produce tumors in fish and salamanders. Many of the trout died early of what appeared to be septicaemia and from the dorsal aorta of one of these fish the crown-gall organism was re-isolated in pure culture on agar poured plates and with subcultures from one of the colonies crown galls were induced on sugar beets. Other trout have yielded, both in the abdominal wall and in the eye-socket, what I regard as small tumors but no metastases have been observed. According to Friedmann, Bendix, Hassel and Magnus, Bacterium tumefaciens causes a purulent menin- gitis in man and also an ulceration of the intestinal mucosa (Zeits. f. Hygiene u. Infektionskr., April 23, 1915), but Jensen of Copenhagen has contradicted this, having shown that Friedmann’s supposed pure culture was contaminated, and Friedmann himself now admits that he was in error. CONSPECTUS: MORPHOLOGY AND CULTURAL CHARACTERS ot Fig. 23.—Angular leaf-spot of cucumber. Under-surface of an inoculated leaf showing the ‘‘tear-drop”’ exudate of Bacteriwm lachrymans Sm. and Bry. Planar enlargement by James F. Brewer. Inoculated May 6, 1915. Photographed May 12. X 4 circa. 38 BACTERIAL DISEASES OF PLANTS Fic. 24.—Buried and surface colonies of Bacterium lachrymans Sm. and Bry. on a rather thickly sown +10 peptone-beef-gelatin plate: the cause of angular leaf-spot of cucumber. Poured May 12, 1915. Photographed May 17. X 10. CONSPECTUS: MORPHOLOGY AND CULTURAL CHARACTERS 3$ Fic. 25.—Surface and buried colonies of Bacterium lachrymans Sm. and Bry., on thin-sown plates: A. Peptone-beef gelatin (+10). Plate poured May 12, 1915. Photographed May 19 with oblique lighting to show the peculiar margin atz. X14. B. Peptone-beef agar (+15). Photographed by direct transmitted light to show the internal structure of the smooth white surface colonies. Plate poured August 6, 1916. Photographed August 9. x 14. 40 BACTERIAL DISEASES OF PLANTS Fic. 26.—A, Film of Bacterium lachrymans floating on Cohn’s solution and full of crystals. Inoculated November 23. Photographed November 28, 1914. x 13. B. Bottom of an agar slant culture of Bacteriwm solanacearum from Tropaeolum, showing crystals formed in the agar. Slant at S. Photographed September 23, 1914. x< ie CONSPECTUS: ACTION ON THE PLANT 4] ACTION OF THE PARASITE ON THE PLANT In some eases it is hard to draw the line between parasitism and symbiosis or mutualism. Probably we shall find more and more of these transition states; undoubtedly there are many. I have included Ardisia in my list of genera and have excluded the genera of legumes subject only to root-nodules. But a nodule on the root of a legume, so far as the local condition is 4 or pay a *, A! aus 4 HIG 27. Fie. 28. Fic. 27.—Ardisia leaf showing swollen, white, bacterially invaded leaf-teeth. 16 nat. size. Fig. 28.—Bacterial cavity in leaf-tooth of Ardisia crispa. concerned, is a disease as much as a leaf-spot, and, if Nobbe and Hiltner’s statements are to be credited the general effect of the root-nodule organism on the plant may be excessive and injurious and not to be distinguished from a disease. ! In the tropical East Indian Ardisia, which is one of the strangest cases of mutualism known to me, and on which Miehe 1Smith, Erwin F.: “Bacteria in relation to plant diseases,’’ Carnegie Inst. Washington, Publ. 27, Vol. 2, 1911, p. 131, last paragraph. 42 BACTERIAL DISEASES OF PLANTS has done a beautiful piece of work, we have perhaps something akin to what occurs in the root-nodules of legumes. This is a common hothouse plant, grown for its ornamental red berries and thick evergreen foliage (Fig. 27). Here the bacterial injury is local and internal. The bacteria are most abundant in the leaf-teeth where they form pockets or cavities (Fig. 28) and multi- Fig. 29.—Section of tooth of Ardisia, showing awater- of Pavetta angustifolia from Java. One show- pore (S) connecting with the bac- ing bacterial leaf knots; the other free. Re- terial cavity. duced. (Courtesy of Johanna Westerdijk.) ply enough to make the leaf-serratures appear blanched or yel- lowish and slightly swollen, but never enough to kill them, or cause the leaves to become yellow and fall. There are no superficial indications of disease, except that the leaf serratures gradually enlarge slightly, lose chlorophyll and become white. In smaller numbers the bacteria oecur in other parts of the plant, including the inner parts of the seed from which they are trans- CONSPECTUS: ACTION ON THE PLANT 43 mitted to the seedling, whose leaf-serratures, infected probably through their water-pores (Fig. 29), in turn become the chief focus?of the bacterial multiplication. Apparently the bacteria are always present, and we do not know what would happen to wy oN igerearons Ml WY ant Ay) ily Ress Ani a iM iq i a) ‘ ‘a r\ Jone, Bil, Ere, 32: Fie. 31.—Cross-section of leaf of Pavetta angustifolia showing a veinlet and a small bacterial nodule. The veinlet has a closed cylinder of xylem. pockets in the young nodule are drawn in solid black. chyma. The bacterial At c.c. are masses of collen- It looks as if the organisms entered from the upper surface very early through the palisade tissue. Drawn November 21, 1914, from an unstained sec- tion lying in water. Fira. 32.—Structure of one of the leafknots shown in Fig. 304. Stained with anilin blue. Ardisia plants grown without them, nor do we know how to obtain such plants (1915). It would be an interesting experi- ment to see if they could be produced without the bacteria and to watch their behavior. 44 BACTERIAL DISEASES OF PLANTS Ardisia plants, so far as I have been able to observe, grow very slowly. Query: Are they dwarfed by the presence of the bacteria, or on the contrary if deprived of them would they be unable to grow at all? I set one of my assistants at work on the problem, asking her to heat Ardisia seeds in water at tem- peratures between the killing point of the seeds and that of the bacteria (which is somewhat lower). The seeds heated for the Fie. 33.—Part of another leaf-nodule of Pavetta angustifolia (Fig. 30A) more highly magnified, showing many small cavities. right time at the proper temperature were not killed but germi- nated and grew, although with extreme slowness as compared with those growing from untreated seed, so that even after a year they had scarcely a leaf to show but only a swollen bud and some roots. Sections from the leaf-teeth of such plants showed them to be free from bacteria, and this makes it seem probable that Ardisia plants actually require the bacteria. A CONSPECTUS: ACTION ON THE PLANT 45 repetition of this experiment gave the same results—good growth of checks and astonishingly slow growth of plants from the heated seed. We are now (1918) growing Ardisia plants from surface- sterile seeds in flasks of glowed sand in nitrogen-free media. ! The bacterial nodules on the leaves of the East Indian Paveitas (first described by Zimmermann) are apparently of a similar nature, but here the bacterial foci are scattered over the surface of the leaf, often, however, with astonishing regu- larity (Figs. 30 to 34). - Fic. 34.—Bacteria from a nodule on leaf of Pavetta angustifolia. > 1000. Stained with anilin blue. The action of such organisms as I have just mentioned differs probably from the behavior of active parasites in that they liberate much weaker toxins and enzymes, can attack only very actively growing parts, and also give off compensating nitro- genous substances. Not yet proved for Ardisia (1915) but proved apparently for the Pavettas by Dr. F. C. von Faber whose paper is in Jahrb. f. wiss. Bot., 1914, Bd. 54, p. 243.2 'Dec., 1919. These plants have remained alive for 18 months, but the foliage is paler and the plants have made less growth than checks in ordinary soil. The experiment is not conclusive because at the end Dr. Jodidi found a trace of nitrogen in the sand (0.5 mg. per kilo). > Since this was written Miche claims to have proved it for Ardisia, but owing to,the war I have not been able to obtain his paper, which is in Ber. d.d. Bot. Ges., xxxlv Bd., 1916, pp. 576-580. 46 BACTERIAL DISEASES OF PLANTS The active parasites produce toxins freely, poisoning the tissues, and enzymes converting starches into sugars, com- plex sugars into simpler ones, and so on, for their nutrition. They also neutralize and consume plant acids, and feed upon amino bodies and other nitrogenous elements of the host. As a result of their growth, many of them liberate both acids and alkalis, to the detriment of the plant. The solvent action of their products on the pectin compounds of the middle lamellae separates cells and leads to the production of cavities in the cortex, pith, phloem and xylem. There is also, or may be, a mechanical splitting, tearing or crushing due to the enormous multiplication of the bacteria within confined spaces. The whole intercellular mechanism of soft plants may be honey- combed and flooded in this way, and if the cavities are near the surface the tissues may be lifted up or the bacteria may be forced to the surface through lenticels or stomata in the form of tiny beads or threads (pear, plum, bean, maize, sugar-cane, cotton, mulberry, ete.), or by a splitting process. The splitting in plum fruits and peach fruits, due to the black spot, results, however, from local death of the attacked tissue with continued growth of the surrounding uninjured parts. I now doubt if any of these plant parasites consume true cellulose. A majority of the forms known to cause plant diseases are extra-cellular parasites occupying chiefly the vessels and inter- cellular spaces, causing vascular diseases, soft rots, spot dis- eases, etc.; but intra-cellular parasites also occur, e.g., Bac- tervum leguminosarum' causing root-nodules on legumes, and Bacterium tumefaciens causing crown gall. The former multi- plies within the cell myriadfold, prevents its division, destroys its contents including the nucleus, and enormously stretches the cell wall so that the cell becomes much larger than its normal fellow cells and is packed full of the bacteria. The latter does not multiply abundantly in the cell, does not enlarge it greatly, does not injure its viability, and would be a harmless messmate were it not for the fact that it exerts a stimulating effect on the nucleus, compelling the cell to divide again and again. !'This is a polar flagellate organism—usually it is 1-3 flagellate. CONSPECTUS: ACTION ON THE PLANT Al Fic. 35.—A. O’Gara’s Aplanobacter agropyri on Agropyron smithii (the west- ern wheat, grass). Utah, photographed from dried material collected by P. J. AS BACTERIAL DISEASES OF PLANTS In 1911 from Maryland carnations! and again in 1913 from Danish orchard grass? the writer called attention to a new type of bacterial disease in which the principal growth of the parasite is on the surface of the plant, that is between closely appressed organs. Since then O’Gara has described a similar disease from wheat-grass in Utah® and Hutchinson from wheat in India.* See Fig. 35. THE REACTION OF THE PLANT We now come to the reaction of the plant. What response does it make to this rude invasion? ‘Twenty years ago we might have said, ‘‘ With rare exceptions, the plant is passive or nearly so,” but that would have been a superficial observation. In every disease we must suppose that the plant makes some effort to throw off the intruder, although often its forces are paralyzed and overcome very early in the progress of the disease. One of the most conspicuous results is lessened growth In some of my plants recovering from brown rot due to Bac- terium solanacearum,? a month after external signs of the disease had disappeared the check plants were twice the size of the inoculated ones, and there was still a very decided dif- 1 Smith, Erwin F.: “ Bacteria in relation to plant diseases,’ Carnegie Inst. of Washington, Publ. 27, Vol. 2, Fig. 4, Oct. 30, 1911. 2 Smith, Erwin F.: A new type of bacterial disease, Science, n.s., Vol. 38, p. 926, Dec. 26, 1913. For a fuller account with figures see ‘* Bacteria in relation to plant diseases,’ Carnegie Inst. of Washington, No. 27, Vol. 3, pp. 155-160. 3’ O’Gara, P. J.: A bacterial disease of western wheat-grass, Agropyron Smithit, Phytopathology, Vol. 6, No. 4, August, 1916, p. 341. 4 Hutchinson, C. M.: A bacteria! disease of wheat in the Punjab. Memoirs of the Department of Agriculture in India, Agr. Research Inst., Pusa, October, 1917, Bacteriological Series, Vol. 1, No. 7. *>Smith, Erwin F.: “Bacteria in relation to plant diseases,’’ Carnegie Inst. Wash- ington, Publ. 27, Vol. 3, 1914, Plate 45—D. O’Gara in 1916. The slime dries brownish yellow and masses of it may be seen adhering to various parts of the spike; B. The same showing a knee-shaped culm bending; C, D. Hutchinson’s wheat disease of the Punjab (India) said to be due to a polar flagellate schizomycete (Pseudomonas tritici Hutch.). All the spikelets are stuck together or overgrown with a mass of lemon yellow slime. In D there is bending of the culm. (After Hutchinson.) CONSPECTUS: REACTION OF THE PLANT 49 ference after more than two months. See also Fig. 169 where the development of the potato shoot inoculated with Bacillus carotovorus has lagged behind its twin shoot. Even more striking retardation results were obtained by the writer and Mr. Godfrey (summer of 1918) on Ricinus communis and on Helianthus annuus, using the same organism (Figs. 127, 131). On potato. plants attacked early by Bacteritwm solanacearum the tubers remain small. On maize attacked by A planobacter stewarti the ears are imperfect. Olive shoots inoculated and infected by Bacterium savastanoi are always dwarfed (Figs. 299 and 300), and frequently the crown-gall dwarfings are very conspicuous. The dwarfings of melon and squash plants at- tacked by Bacillus trachetphilus are also conspicuous. Unin- oculated sugar-cane stems soon surpass in height and vigor those successfully inoculated with Bacterium vascularum. I do not know how to explain this checked growth unless it be due to the paralyzing effect of absorbed toxins. Changes in color are also conspicuous. The attacked parts may become greener than normal, or fade to yellow, red, brown or black. In tomato fruits there is often a retarded ripening on the attacked side, with persistence of the chlorophyll. In certain leaf-spots also the leaf green persists in the vicinity of the spot while the rest of the leaf becomes yellow (bean- blight). Crown galls on daisy are greenish. On the contrary thé teratoid parts of crown galls on tobacco and on cauliflower are often blanched. The male inflorescence of maize attacked by Aplanobacter stewarti ripens prematurely and becomes white Gig TOI). Distortions of various kinds appear (leaves of bean, lilac, larkspur, hyacinth, mulberry, Persian walnut, etc.). The leaves of tomato plants attacked by Bacteriwm solanacearum are bent downward; so are the fronds of the coconut palm when at- tacked by the bacterial bud-rot (Fig. 4). The leaves of potatoes attacked by Bacillus phytophthorus are sometimes bent upward and almost always the leaflets are rolled upward, from the edges. Knee-shaped curvatures of the culms appear on Dactylis attacked by Aplanobacter rathayi, in the buds of the sugar-cane attacked by Cobb’s disease, on Agropyron attacked 4 50 BACTERIAL DISEASES OF PLANTS by O’Gara’s disease (Fig. 35B), and on wheat attacked by Hutchinson’s disease (Fig. 35D). Organs may be developed in excessive number or out of place, as roots or leafy shoots in crown gall, witch-brooms on Pinus, and incipient roots on the stems of tomato, tobacco, chrysanthemum, nasturtium, etc. Hunger found a bud on a tomato leaflet which he attributed to the stimulus of Bac- terium solanacearum but this may have been natural (see Fig. 36) and an old paper by Duchartre! who first discovered adventi- tious buds on the leaves of the tomato. Fig. 36.—Cross-section of middle part of a tomato leaf in the region of the midrib showing leafy shoots originating from the suleus. Variety Livingston’s Dwarf Aristocrat. I have rooted and. grown these foliar shoots into mature fruit-bearing plants. Photographed June 23, 1916. X 4. In various diseases the plant removes starch from the vicin- ity of the bacterial focus which it endeavors to wall off by the formation of a cork-barrier, and in this effort it is sometimes successful, if the parasite is growing slowly (Figs. 136, 174). In other cases (hyperplasias) starch is stored in the diseased parts. The most conspicuous response of the plant, however, is in the form of pathological overgrowths—cankers, tubercles, and tumors. Some of these are very striking, e.g., those on the ash, olive, citrus, beet (Bact. beticolum?), pine, oleander, and on a multitude of plants attacked by crown gall. In some of these 1 Duchartre, P.: Sur des feuilles ramiféres de Tomates, Ann. d. Sci. Nat. 3 Sér. Bot., Tome 19, Paris, 1853, pp. 241-251, Plate 14. 2See “Tuberculosis of Beets”? in “Crown Gall of Plants: Its Cause and Remedy.” Bull. 213, U. S. Dept. Agr., Bu. Pl. Ind., Washington, D. C., 1911, pp. 194-195, and plate XXXIV. CONSPECTUS: REACTION OF THE PLANT 5a growths, which are hyperplasias, there is a great multiplication and simplification of the parenchyma and a great reduction of the vascular system, but in crown galls produced in the torus of the sunflower (which is a very vascular tissue) there is an excessive number of vessels. There are also various other phenomena, chemical and physical, nearly related to what takes place in certain insect galls, that is, increase of sugars, starches, enzymes, acids; and structural simplifications and reversions to more primitive forms. What I mean by reversions may be seen by consulting my figures illustrating insect galls. In crown gall, cell-division under compulsion proceeds at such an abnormally rapid rate that the cells are forced to divide while still immature, and in this way masses of small-celled, unripe (anaplastic) tissue arise (Figs. 353, 354) and these develop tumor-strands (Figs. 319 and 823 to 325) in which secondary tumors form—phenomena suggestive of what occurs in malig- nant animal tumors (Consult text of No. XIV and various plates and figures and, especially, read Jensen’s recent (1918) Danish paper referred to under Literature of No. XIV). PREVALENCE AND GEOGRAPHICAL DISTRIBUTION Economically considered, bacterial diseases of plants may be classed as major or minor. Most of the leaf-spots would fall into the latter class. Various soft rots, blights and vascular diseases, being wide-spread and destructive to plants of great: economic importance, may be classed as major diseases. Cankers and tumors would fall midway in such a grouping. Occasionally a minor disease, e.g., lettuce rot, celery rot,’ under conditions favorable to the parasite may assume great Importance. This is especially true of leaf-diseases which attack the fruit, e.g., the black spot of plum and peach due to Bacterium pruni, the bean-blight due to Bacterium phaseoli, the angular leaf-spot of cotton due to Bacterium malvacearum, the African mango dis- ease due to Bacillus mangiferae, the black chaff disease of wheat 1“ The loss from this disease in the field where 1 gathered the specimens was 150 crates out of every 700 crates packed.’’ (Dr. J. Rosenbaum, Hastings, Fla. Letter of April 4, 1916.) 52 BACTERIAL DISEASES OF PLANTS due to Bacterium translucens var. undulosum (see Figs. 12, 14, 38) and citrus canker due to Bacterium citri. It will be of interest to mention a few of these diseases with particular reference to their distribution and prevalence. Dutch East Indies —The tobacco disease of Sumatra and Java is probably the most destructive, if the Sereh of sugar- cane is not bacterial. Each of these diseases has caused enor- mous losses. Each threatens or has threatened anindustry. The tobacco disease occurs also in the West Indies, in the United States, and probably also in South Africa. If Janse’s root dis- ease of Erythrina, the coffee shade tree of Java, is also bacterial, as he supposed, then there is another great bacterial plague in that region, for hundreds of thousands of trees have died, and another species has been substituted as a shade tree. The brown bast disease of rubber trees (Hevea brasiliensis), which is a tumor disease of the bast of suspected bacterial origin, is widespread and has attracted much attention in recent years. There is also a bacterial disease of peanuts. West Indies.—Here the most destructive disease is the bac- terial bud-rot of the coconut palm, which occurs all around the Caribbean, and threatens the entire destruction of a profitable industry in Cuba. There is also the bacterial disease of bananas and plantains, but the most wide-spread and destructive Musa disease of the Western Hemisphere is the Panama disease, due to Fusarium cubense EFS.' Australia—Cobb’s disease of sugar-cane has probably at- tracted more attention in Australia than any other bacterial trouble, although bacterial rots of the potato are also very destructive. The cane disease in both Queensland and New South Wales has in many cases destroyed the output of whole plantations and greatly discouraged planters. This disease occurs also in Fiji, and probably in South America. According to G. F. Hill, the citrus canker occurs in the Northern Territory of Australia (Bull. N. T., Austr., 18, 1918). 1 On this subject see papers by Elmer W. Brandes as follows: (1) Ann. Rep., Porto Rico Agr. Exp. Sta. for 1916, pp. 29-31; (2) Distribution of Fusariwm cubense EFS, the cause of Banana wilt, 20th Report Mich. Acad. of Sciences, 1918, pp. 271-275; and (3) Banana wilt, Phytopathology, Sept., 1919, pp. 339- 389, 14 plates and 5 text figures. CONSPECTUS: PREVALENCE AND DISTRIBUTION 53 Japan.—The tobacco wilt, which has destroyed many fields, is probably the worst Japanese disease. This is believed by Hon- ing and by the writer to be identical with the tobacco wilt of Sumatra and of the United States. The citrus canker occurs and several other interesting bacterial blights have been re- ported from Japan, including one on the basket willow (Iig. 37). Frag. 37.—Agar poured plate colony of the schizomycete causing the Japanese basket-willow disease. Photographed by oblique transmitted light to show inter- nal structure. x 10. China.—In the interior of China there is a destructive wilt disease of tobacco (Frank N. Meyer), but its nature is unknown. A Fusarium cultivated from it in my laboratory did not cause the wilt when I inoculated it copiously into the soil near broken roots of young tobaccos nor yet when I introduced it into deep wounds made in the stems of young vigorous plants at the sur- face of the earth. o4 BACTERIAL DISEASES OF PLANTS The Philippines.—In Luzon, citrus canker, a bud-rot of coco- nut, brown rot of potatoes and egg plants, a leaf spot of tobacco, bean blight, a bacterial rot of bananas and Musa textilis, and some other diseases occur. Most of the islands are pathologically unexplored. According to Reinking (The Philippine Journal of Science, Vol. XIV, Jan., 1919, pp. 131-151) the coconut bud- rot of the Philippines is due to Phytophthora faberi Maubl., Bacil- lus coli and a schizomycete resembling B. coli isolated from the rotting palm bud may aggravate the rot but cannot initiate it except under very favorable conditions of moisture and pre- vious injury. India.—The brown rot of Solanacee is common and destruc- tive. Citrus canker is common especially in the Punjab (Hutch- inson, ina letter to the writer). There is also a bacterial disease of the opium poppy. Most of Asia is a terra incognita. South Africa.—The mango disease in recent years has greatly reduced the exports. Potato and tomato wilts are common. There is a serious tobacco disease, probably bacterial. Crown gall is common and injurious on shade and orchard trees. An- gular leaf-spot of cotton is prevalent. Other bacterial diseases occur, including several on citrus. Nothing is known about the greater part of Africa. South America.—There is a serious disease of sugar-cane in Brazil and another in Argentina, both of which I believe are of bacterial origin and identical with Cobb’s disease, but this remains to be proved. Bondar has reported a destructive mani- hot disease. The bud-rot of the coconut occurs in the north. The banana disease of Guiana, however, is due to Fusarium cubense. Most of South America, like Asia, is unexplored. United States and Canada.—Potato rots of which we have several distinct forms, probably cause the greatest losses, one year with another. Following these I should think pear and apple blight. Perhaps the latter should be placed first, for the destruction of an acre of potatoes would scarcely equal the value of a single fine pear tree, and thousands are destroyed every year. In California, which was formerly free from pear blight, the losses in the last twenty years have been enormous, amounting to about one-third of all the full-grown orchards and to a money- —t CONSPECTUS: PREVALENCE AND DISTRIBUTION oe loss estimated at $10,000,000 for the five years preceding the efforts for its restriction begun in 1905 by the United States Department of Agriculture. This is a very conservative esti- mate considering the number of trees destroyed. In the San Joaquin Valley in California, “in the short space of three years, from 1900 to 1904,” according to O’Gara, ‘‘almost half a million pear trees were lost by blight. Practically no attempt was made to check the disease and one of the greatest industries of the San Joaquin Valley vanished like a dream.’’ Very serious losses from this disease are experienced every year in the East, or were until growers became generally familiar with methods of control. In certain seasons bacterial diseases of barley and oats injure these crops to a considerable extent. In our southern states the wilt disease of tobacco and the tomato, due to Bacterium solanacearum, has made it impossible to grow these crops on many fields. In the northern United States the cucurbit wilt is wide-spread and destructive, but cucurbits are, of course, a minor crop. Blight of beans due to Bacterium phaseoli is another common and troublesome dis- ease. In certain seasons and on some varieties the angular leaf-spot injures cotton very seriously (see Part III, No. X). The wide prevalence and destructive nature of the bacterial black chaff of wheat in the United States west of the Mississippi River in 1915, and since, adds another to our serious bacterial diseases. This blights the leaves, shortens the head and shriv- els the kernels (Fig. 38). In the study of this disease in my labo- ratory during the last three years we have discovered a second bacterial disease of wheat previously confused with the “black chaff,’ the basal glume rot (Figs. 39, 40), due to Bacterium atrofaciens McCulloch, a green fluorescent organism which causes a black rot at the base of the kernel. The walnut blight has done much damage in California and recently it has been reported from New Jersey (Cook) and from other parts of the Eastern United States (MeMurran, Bull. 611, U. S. Dept., Agric). This disease occurs also in Chili, South Africa (Miss Doidge: Letter to the Author), New Zealand and Tasmania. The bacterial disease of alfalfa has been serious in parts 56 BACTERIAL DISEASES OF PLANTS Fic. 38.—Kernels of Russian winter wheat attacked and shriveled by the black chaff organism, Bacterium uwranslucens var. undulosum, 8., J. R., Coll. No. 2714, Kansas, 1917. All kernels from one head and all but 8 shriveled. Bacterial films can be seen on the shriveled kernels, especially those of the middle row. | - ~ CONSPECTUS: PREVALENCE AND DISTRIBUTION ee Beco ere PSicamin ne a Cee NC AB he gy, an te RG a i A roe 9 Tig Paks eRe oe cig Sn SH Pen miag ane sie Fig. 39.—Head of wheat from Kansas crop of 1917, Coll. No. 478, showing basal glume rot, a new disease, due to Bacterium atrofaciens McCulloch, a white organism causing a green fluorescence in media. ~I 5S BACTERIAL DISEASES OF PLANTS Fie. 40.—Glumes and kernels of wheat blackened by Bacteriwm atrofaciens McCulloch, the cause of the basal glume rot, crop of 1917, Coll. No. 285 (New York) and No. 399 (Canada). CONSPECTUS: PREVALENCE AND DISTRIBUTION 59 Fic. 41.—Bacterial canker on leaves of grape-fruit from Florida: (1) A natura! infection, 1914; (2) an inoculation of 1914. Both leaves were deposited in the Pathological Collections, B. P. I1., U. S. Department of Agriculture, in June, 1914, by H. E. Stevens of Florida. No. 2, designated as a “ pure-culture inocula- tion,” was supposed by Stevens to have been caused by his fungus (Phoma or Phyllocticta) which is present, but is not the parasite. 60 BACTERIAL DISEASES OF PLANTS Fig. 42.—Citrus canker on leaf of seedling grape-fruit. Inoculated by the writer with Bacterium citri (Hasse) Jehle, isolated from a grape-fruit leaf received from Mississippi in 1915. Time, 16 days. Cankers not yet ruptured. X 5. CONSPECTUS: PREVALENCE AND DISTRIBUTION 61 of the West, but I have not heard of its occurrence in the East. It is most injurious early in the season, 7.e., on the first cutting. Alfalfa is now, according to Piper! the third most important forage crop in the United States, only timothy and red clover exceeding it. There is a physiological ‘‘ white spot” on alfalfa (O’Gara) not to be confused with Sackett’s disease. Fic. 43.—Citrus canker due to Bacterium citri (Hasse) Jehle: A, on Citrus decumana (grape fruit); B, on Citrus trifoliata. Disease introduced into America recently from Eastern Asia. Recently in Florida the citrus canker (see Figs. 41 to 44) has caused orange growers a great scare and strenuous efforts are on foot to suppress it. During the last four years, under pressure from the citrous States, the General Government of the United States has made five appropriations for this purpose 1 Piper, Chas. V.: Forage plants and their culture, N. Y., The Macmillan Co., 1914. 62 BACTERIAL DISEASES OF PLANTS as follows: Special, January, 1915, $35,000; special, February, 1916, $300,000; general, in Department of Agriculture appro- priation bills, 1916-17, $250,000; 1917-18, $430,000; 1918— 1919, $250,000.! This disease occurs also in Japan, China and the Philippines (Water T. Swingle) and was certainly intro- duced into the United States on imported citrus plants. It now occurs (or did oceur in 1916) in every Gulf State. It should not be confused with the somewhat similar looking Costa Rican pseudo-canker (Figs. 45, 46) which is of non-bacterial origin, nor with the verrucosities -due to Cladosporium citri (Fig. 47). The true bacterial canker is usually sur- rounded by a narrow water-soaked area best seen by holding the leaf up to the hght (Fig. 48) and is swarm- ing with bacteria, whereas the pseudo- canker shows no such border and con- tains at most only some fungous threads well corked out. Holland and Denmark.—In_ Hol- land the yellow disease of hyacinths Fic. 44.—Cross-section of Will eventually put an end to hyacinth- grape-fruit leaf showing a growing for export if means cannot be young canker inoculated by had for its control, since the land fc Gee tune, 1° suited 10 hyacinths is limited in amount. Black rot of cabbage oc- curs in Holland and Denmark, and is common now also in many parts of the United States. It was probably imported into the United States from Denmark on cabbage seed. Some years in nurseries about Amsterdam the lilac blight has been troublesome. In Denmark Rathay’s disease is said to be rather troublesome on orchard grass grown for seed. ‘On April 30, 1918, in the Florida Plant Commissioner’s Office, Department of Citrus Canker Eradication, 183 persons were employed, including a divisional inspector, district inspectors, assistant district inspectors, foremen and inspectors. During the year 1918 over 2,000,000 grove trees were inspected and six times as many nursery trees. CONSPECTUS: PREVALENCE AND DISTRIBUTION 63 6 Pic. 45.—Pseudo-canker on Citrus aurantium, probably scab due to clado- sporium citri. Some mycelium is present but no spores. The cankers have healed, being cut off from the rest of the leaf by a cork layer. Costa Rica, 1913. 64 BACTERIAL DISEASES OF PLANTS Sandwich Islands.—There is a serious banana disease but its cause is not known (1915). It attacks the Chinese banana. There is a serious potato disease and a bad shade tree disease, both of unknown origin. The mosaic of sugar-cane occurs. Great Britain and Germany.— Until recently in these countries not much critical study was given to bacteria as a cause of plant Fig. 46.—Costa Rican peudo-canker of citrus. A detail from Fig. 45, further enlarged to show absence of any translucent border such as that shown in Fig. 48. Photographed by transmitted light. xX 6. diseases, but now good students are at work. Potato rots are probably the most destructive bacterial diseases. Appel has described one and Spieckermann another. The _ bacterial potato rot Pethybridge and Murphy described from Ireland? is like the German ‘‘black leg.’”’ Wormald has described a rot of celery which is common also in the United States (Fig. 49). 1 Proc. R. Irish Acad., vol. xxix, Sect. B, No. 1, 1911. CONSPECTUS: PREVALENCE AND DISTRIBUTION 69 Fig. 47.—Verrucosities on an orange leaf from Florida, due to the fungus Cladosporium citri. Photo by Brewer, but on a Seed’s plate which shows no dis- tinction between the pale green of the leaf and the dull yellow of the scabs. See page 121. oO 66 BACTERIAL DISEASES OF PLANTS Potter has written on a rot of swedes and Paine on a rot of mushrooms and a leaf-spot of Protea. France and Italy.—Potato diseases are common and at times very destructive. Olive tubercle, common also in California, and all around the Mediterranean, is prevalent in spots. Vine diseases, especially Maladie d’Oleran and crown gall, do con- siderable damage. Pear blight seems to be absent in France, but has been reported from several places in Italy. Mul- berry blight occurs. The destructive Italian rice disease, Fic. 48.—Bacterial citrus canker enlarged and photographed by transmitted light to show translucent border. X 6. The tiny white specks are oil glands. brusone, is not due to bacteria as reported, but to a fungus (Piricularia). Not much exact work has been done on bacterial diseases of plants either in France or Italy. Spain and Portugal——These countries are a terra incognita. Russia.— A few years previous to the late war there was a great awakening in Russia. - Jj] DRYING RACK = (WNP = © ELECTRIC BULBS “r FAUCETS PHOTOGRAPHIC ROOM == WINDOWS (DARK ROOM,19°x2'6") Z ELECTRIC SWITCHES CAMERAS WINDOW WINDOW Pope ss BY eos Som Fie. 61.—Diagram of photographic room, printing room and dark-room used by the writer. two parts: (1) an ante-room on the west side, used for making prints; (2) the dark-room proper, used only for the development of negatives. The ante-room is long and narrow with a sink at the northwest corner (behind the corridor door), the re- mainder of the west wall space being occupied by a printing and developing table. The printing is done by means of a battery of large size, fixed tungsten lights, in the southwest corner. The EEE EE METHODS OF RESEARCH: APPARATUS 97 developing is done under a red (bulb) drop light near the sink. There is a door at either end (both opening inward): one gives entrance from the bright photographic room, the other from the rather dark central corridor of the building. Both should be hinged on the side nearest to the developing table. The size of this ante-room is 5 feet 8 inches by 9 feet. The dark-room proper takes the remainder of the space (9 feet 4 inches by 9 feet), but one corner of it is occupied by the labyrinth so that the actual working space is considerably reduced. The labyrinth (2 feet wide) begins in the northeast corner of the ante-room next the door opening into the dark corridor. As an additional precaution against the accidental entrance of light, this corridor door is provided with a push lock to be used whenever necessary. On the right-hand side, as one enters the dark-room proper, behind the south wall of the labyrinth, is a narrow space occupied its whole length on one side (the north side) by the loading shelf. On the east wall, facing the observer as he enters the dark-room, is the developing shelf. At his left against the north (corridor) wall is the fixing shelf and in the angle between the two shelves is the deep sink (36 inches long by 20 inches wide). The dark-room consists, therefore, of two rectangles, the larger 5 by 9 feet occupied on the outer (east) side by the long developing shelf and the sink just referred to; and at one end (the north) by the shorter fixing shelf, and at the other (south) end opening into the smaller rectangle (3 feet 10 inches long by 3 feet wide) which is occupied, as already stated, by the loading shelf. This abuts against the labyrinth wall, and above and below it are other shelves for the storage of boxes of dry plates and plate holders. There are also shelves under the developing shelf for extra trays, and under and above the fixing shelf. Two persons can develop in this dark-room without inter- ference and the loading shelf is also long enough to allow two persons to use it at the same time. There is, however, no waste space and everything is within convenient reach. A step or two brings you to any part of the room. The subdued light by means of which exposures must be developed may be sunlight or electric light, passed through a 7 98 BACTERIAL DISEASES OF PLANTS series of screens, 7.¢., ground glass, orange glass or orange paper and red glass. An opaque screen also should be provided and all the screens should be counter-weighted to slide up and down easily inside a window frame. There should be at least two of these windows. Our room has three. Two are over the developing shelf and a third is in the south wall between the developing shelf and the loading shelf. The developing shelf should face the source of light at a convenient height rather than receive its light from one side. The washing sink (1 foot deep) should be close to the de- veloping shelf to avoid waste of time and on the other side of it should be a roomy fixing shelf. By ‘‘roomy”’ I mean large enough for several trays. The loading and fixing shelves and the developing table are each 36 inches high. The loading and fixing shelves are 18 and 16 inches wide. The developing table is 20 inches wide and the surface of this table consists of two removable frames of slat-work. Under it is a shallow lead-lined sink sloping to the left and emptying into the deep sink. The window-ledges are 814 inches above the top of this table. There should be plenty of storage shelves in the dark-room (under the fixing shelf and the developing shelf, and over them also) for trays, bottles, beakers, graduates, etc. The loading shelf should be in the darkest part of the room, 7.e., as far away from the red lights as possible and behind the door or labyrinth, so that there shall be a minimum of danger from fogging when boxes of dry plates are opened. I open boxes of dry plates, always, with the opaque screen drawn low over the red light, especially if they are orthochromatic plates. The washing and printing are best done in adjoming rooms (see Fig. 61). Dark-rooms, like kitchens, are most convenient if everything is handy; they should not, therefore, be very large, and consequently some sort of ventilation becomes necessary, especially if they are much used. In the top of our own room I have inserted a hood in the bottom of which is placed an electric fan which can be turned on at will and which rapidly pumps the foul air out of the room, the fresh outside air flowing in through METHODS OF RESEARCH: APPARATUS 99 the labyrinth. This cost only a small sum and has proved very satisfactory. The electric bulbs for the red lights are outside but may be turned on or off without leaving the dark-room, by means of a double push button at Z (Fig. 61). USES OF CULTURE-MEDIA Culture-media are needed every day in the routine work of the laboratory and are required for several distinct purposes: (1) for the isolation of organisms from mixtures or directly from diseased tissues; (2) for the long-continued growth of organisms without loss of virulence; (3) for differential descriptive pur- poses; (4) for cultures adapted to chemical analysis. For the first purpose we must study the nature and needs of the various parasites, and when they differ from the common sorts must cater to them, devising media exactly suited to their requirements, or at least better adapted to the needs of the para- site than to those of the accompanying saprophytes. This frequently requires considerable study, as to range of toleration of acids, alkalies, salts, N.-compounds, sugars, alcohols,: ete., but standard media should be tried first. For first isolations I always try +15 peptone-beef-agar poured plates. If this fails, then +7 beef-peptone agar may be tried, and other media, such as steamed potato and special agars, e.g., dextrose potato agar or whey agar. The second kind of media varies a great deal with the or- ganism and can be discovered only after prolonged study of the parasite on a variety of substrata. Some observations on such media will be found under the various diseases described in Parc hhh. The third sort need not be, and in fact cannot be, media of universal value. They are good only for the particular purpose in mind, and the future will see a large increase in their number. What we seek here are media that will bring out not necessarily good growth, or any growth at all, for that matter, but differ- ences in behavior when a variety of bacteria are tested in it, that is, changes in gross appearance, morphology, pigmentation, pre- cipitates, pellicles, crystals, weak vs. dense clouding, medium 100 BACTERIAL DISEASES OF PLANTS reactions (acid, neutral, alkaline), using neutral litmus and phe- nolphthalein, ete.—and here a medium generally neglected by bacteriologists may be just the one needed. The student should not regard the chart sanctioned by the Society of American Bacteriologists as in any sense a finality—there are no last words in science, at least not in bacteriology—and he must be always on the lookout for simple and effective means of differentiation. Often, when the colonies of two organisms look exactly alike on +15 peptone-beef agar, we try potato agar, prune agar, string- bean agar, starch agar, whey agar, or some kind of gelatin medium, and find a difference. In this connection see Fig. 184. For the fourth purpose, to avoid complications, one would naturally select, first of all, well-aérated, simple synthetic media. Carefully considered (tested) formulae for the preparation of various culture-media will be found in “ Bacteria in Relation to Plant Diseases,’ Vol. I. For Meyer’s mineral solution con- sult: WbideaViol: nies pe 250: PREPARATION OF CULTURE-MEDIA It is impossible to pursue the study of bacterial diseases of plants without the use of at least some forms of culture media, and the student should know how to prepare with his own hands all necessary substrata. It is a useful training of the hands and of the judgment even for those who are not to use it later in research. Culture-media may be divided into two classes: (1) complex organic substances and (2) simple synthetic preparations. The first, though they are harder to prepare, are still used more generally than the second, and are in the main better adapted to the growth of parasitic micro-organisms than are the more exact media compounded out of simple chemical substances. The latter, however, may be expected eventually to take the place largely of the more or less variable animal and plant compounds now in general use. At the same time the writer would like to register an objection against the discontinuing of any of the cultural substances now in use until such time as we have well- recognized suitable substitutes. At present we need all of them, METHODS OF RESEARCH: PREPARATION OF CULTURE MEDIA 101] especially for the study of parasites. Before proceeding to a discussion of these cultural substances, the student should read quite carefullythat part of the preceding chapter on Apparatus, and also what he can find on the making of culture media in other text books. In the preparation of culture-media one must be governed by the substances and the apparatus at hand. The simplest sub- stances to prepare are cylinders and slices of potato, carrot, and other vegetables, and these are still of great use in the study of diseases of plants, and not to be discarded for purely synthetic media. These vegetable substances may be used either cooked or raw. If cooked, they are best kept in cotton- plugged test tubes in the form of slant cylinders, standing with the bottom immersed in a small quantity of water or free on wet cotton. If raw, they may be cut into the form of cubes or slabs and placed in deep Petri dishes. We will first discuss the proper preparation of such media. 1000 circa. Fie. 99.—Longitudinal section of root of an inoculated turnip showing Bac- terium campestre occupying a vessel and cells at the left. X cirea 800. The in- oculation was made on the leaves by needle pricks. CN ee THE BLACK ROT OF CRUCIFERS: TRANSMISSION 159 Transmission.— Greenhouse slugs may be used, feeding them first on infected leaves and then on sound plants. Also larvae of the cabbage butterfly. Can the disease be spread by aphides? If opportunity exists, collect seeds from diseased plants and try to isolate the organism from them (rather difficult) and to get infected seedlings from them. Why should a seedsman collect and disseminate seeds from stock he knows to be diseased ? The writer has seen a serious outbreak of the disease on parts of a cabbage field that received as a manure the diseased refuse from a storehouse in which brown-rotted cabbage had been wintered over (see No. VII). He has seen an entire crop ruined and the organism introduced into the soil of a field previously free from it by setting it out with plants from an infected seed bed. (See U.S. Dept. of Agr., Farmers’ Bull. No. 68.) MEANS OF PREVENTION Use of seed derived from healthy plants. Seed beds on land free from the disease. Care in transplanting that roots shall be wounded as little as possible. LITERATURE For literature, ete., consult: Black rot of Crueciferous Plants in ‘“‘ Bacteria in Relation to Plant Diseases,”’ Vol II, pp. 300-334, Carnegie Institution of Washington, 1911. See also Jbid., Wolly Biase AaoG. i, LS lO Ge 1141S, 00,1 Lon 116, 117. The first important paper on the subject was published in 1893 by Prof. L. H. Pammel (Bot. Gaz., Jan., 1893). The organism was first named Bacillus campestris by Pammel in 1895 in Bull. No. 27, Iowa Agr. Col. Exp. Station, pp. 130-134. The last paper is by Walker and Tisdale: Observations on Seed Transmission of the Cabbage Black Rot Organism. Phyto- pathology, Vol. 10, No. 3, March, 1920, pp. 175-177. These authors have proved introduction of the disease into Wisconsin on seed imported from north Europe. They have also established that the disease can be reduced to negligible proportions by soaking the seed for 30 minutes in 1-1000 mercuric chlorid water. II. STEWART’S DISEASE OF MAIZE Type.—This is a vascular disease confined principally to sweet corns, especially those rich in sugar and ripening early, but it has been seen by the writer upon several varieties of field corn. The foliage shrivels gradually, the lower leaves usually first (Fig. 100); the male inflorescence develops pre- ‘e! Fre. 100.—Large sweet-corn plant destroyed by Aplanobacter stewarti. . A natural infection. Bundles of the stem occupied by the yellow slime. District of Columbia, 1903. maturely and is white (Fig. 101); and on cross-section or lon- gitudinal-section of the stem a yellow slime oozes from the vascular bundles (Figs. 102 and 103); stooling also sometimes occurs (Fig. 104). Infection takes place principally in the seed- ling stage through stomata and is greatly favored by actively functioning water-pores situated on the young leaf-tips. The 160 STEWART’S DISEASE OF MAIZE: TYPE 161 organism is extremely abundant in the vessels and is much inclined to come to the surface of the husks through stomata (Figs. 105, 106, and 107); thus flooding the kernels, but it may occur also inside the kernels, particularly at their junction with the cob (Figs. 108 to 111). Some of the infected plants are de- stroyed in the seedling stage (Fig. 112), but many of them reach a height of several feet before showing secondary signs (Fig. 113). Itisatypical example of a seed-borne infection. Noth- ing is known as to the occurrence of this disease outside of the United States. Miss Doidge has not seen it in South Africa. The exact distribution of the disease in the United States is un- known but it occurs from New York and Maryland to California. Cause.—It is due to Aplanobacter stewarti (HFS) McC. This is a non-motile, non-flagellate, non-sporiferous, inadhesive, or moderately viscid, yellow, slow-growing, non-liquefying, non-milk-curdling, non-nitrate-reducing, non-gas forming, non- starch-consuming, chloroform-tolerant, sodium chlorid-tolerant, aérobic, rod-shaped schizomycete, growing on the surface of agar-poured plates in the form of small, flat, circular or nearly circular pale colonies which become yellow with age.' It reddens lavender-colored litmus milk slightly and does not grow in Cohn’s solution. Its growth on steamed potato is thin and soon at an end (contrast with Nos. II, VIII, or X). Why is this? In Dunham’s solution containing methylene blue the bacterial precipitate should be blue. Its minimum temperature in +15 peptone beef bouillon is above 9°C. At this temperature there was no clouding in 14 days. The checks at room temperature clouded heavily the second day and formed a pellicle the third day. It is not sensitive to dry air, and like No. II retains its vitality and its virulence for a long time. It is rather tolerant of weak organic acids. On the kernels the majority of the bacteria are destroyed by exposure for 15 minutes to 1:1000 mercuric chlorid water: 1 Sometimes the surface colonies on agar have depressed centers (Fig. 114B). No mention was made of these in Volume III of my monograph because I was not then certain that they belonged in the life-cycle of this organism, but recently Lucia McCulloch, of my laboratory, has proved them to be infectious. She has also proved my former statements respecting the motility of this organism to be incorrect (see Phytopathology, August, 1918, p. 440). BACTERIAL DISEASES OF PLANTS ae -. er —— Ng a ig uf at ‘ Se 8 "os cs * a ad ; we a age ay LE ae Oe i) Fiq. 101.—Blue Squaw flint corn from a field on Arlington Farm, July 16, 1915 (an early sort): No. 1, slightly diseased; No. 2, badly diseased, showing white top (prematurely dead male inflorescence) and dry pale leaves, due to A plano- Photo by James F. Brewer. bacter stewartt. STEWART’S DISEASE OF MAIZE: CAUSE 163 first plunge the seeds momentarily into alcohol, rinse them very lightly and dry quickly or plant at once. Why this last direction? Why also first into aleohol? It fills the infected vascular bundles with a yellow slime which oozes on cross- section (unless the plants have been frosted). Why not then? Fig. 102.—Cross-section of a diseased sweet-corn stalk showing A planobacter stewarti oozing from the bundles. Planar enlargement. Technic.—Isolations may be made from externally sound upper internodes of the maize stem, by the first method de- scribed under No. I. Often pure cultures may be obtained directly from the cut stem by streaks on steamed potato or Fic. 103.—Water mount of a sweet-corn stem in longitudinal section showing A planobacter stewarti oozing from a vascular bundle like smoke from a chimney. (After F. C. Stewart.) nutrient agar if the surface sterilization has been thorough, but if so made, subsequently they should be plated out. It is more difficult to isolate from the interior of kernels. Such kernels should be soaked in 1:1000 mercuric chlorid water for 30 to 60 seconds, to inhibit, rather than to kill, surface 164 BACTERIAL DISEASES OF PLANTS organisms. The bases may now be removed, crushed in a sterile mortar and allowed to soak in bouillon for some hours before plates are poured. Some of the latter should be sown heavily. Keep the tubes and pour a second set of plates next day; pour AAP. fg Z ' Fie. 104. Hines elOS: Fie. 104.—Corn plant showing very pronounced dwarfing, premature develop- ment of male inflorescence and stooling due to Aplanobacter stewarti. Vessels full of the yellow slime. From Chula Vista, California, in 1915. Fra. 105.—Spots on inner husk of a sweet-corn ear as a result of bacterial cavi- ties due to Aplanobacter stewarti. Plant from infected seed. Spots bright yellow. also from dilutions of the same, if clouding has developed. There are various non-parasitic, motile, yellow schizomycetes on the surface of corn kernels, and only those non-motile forms which behave properly in the agar, gelatin, nitrate bouillon, litmus STEWART’S DISEASE OF MAIZE: TECHNIC 165 milk, Uschinsky’s solution and Cohn’s solution need be tried further. For inoculation purposes select first of all seedling plants and inoculate from young potato or agar streaks on the leaf- tips when the plants are 2 to 3 inches high and show only 2 or 3 unfolding leaves. The inoculations may be made by spraying or by touching the leaf-tip with an infected platinum needle. After inoculation the young plants may be placed y Fie. 106—Corn husk in cross-section showing vessels and intercellular spaces of the parenchyma (dark areas) filled with A planobacter stewarti. Stoma oozing bacteria at X. See Fig. 107. either in cages or under the greenhouse bench. The essential is damp earth and a moist shaded place where the water-pores at the leaf-tips will function freely. Examine from time to time to make sure that drops of water remain on the leaf-tips. If nec- essary, wet down the greenhouse thoroughly so as to saturate the air. After 30 hours set on the bench and withhold water for a day, if the soil looks wet. Change the plants frequently to larger pots and transplant into the garden at the end of June (May in the South) when the plants are about 15 inches tall, and make 166 BACTERIAL DISEASES OF PLANTS Frc. 107.—A detail from Fig. 106 X showing A planobacter stewarti separating cells of the corn husk and filling the substomatie chamber. Photomicrograph by the writer. STEWART’S DISEASE OF MAIZE: TECHNIC 167 the final examinations in September before frost supervenes. One or more of the following sensitive varieties may be used: Black Mexican, Golden Bantam, Crosby’s Early, Cosmopolitan, Pocahontas. Along with these, white and yellow field corns should be inoculated for comparison, taking pains to secure the names of the latter. The seedlings should be ready for inoculation about 8 days after planting, which, in the North, should be toward the end of May, if the seedlings are to be transplanted into the open field. Uninoculated check-plants should be held. These should be grown at some distance from the inoculated plants (preferably Fic. 108.—Cross-section of a small bundle at the extreme base of a kernel of sweet corn showing A planobacter stewarti in the single vessel. in an adjoining house) and should be transplanted to the other side of the garden. Even then, some cases may be expected unless the seed corn is beyond suspicion, and the house free from insects. Determine For THE orGANIsM. Morphology.—Size in microns, form (Ziehl’s earbol fuchsin or amyl Gram may be used for staining), aggregation of elements, motility (hanging drop), question as to occurrence of flagella (hanging drop and van Ermengem’s silver-nitrate stain; in case of the hanging-drop method, boil the 168 BACTERIAL DISEASES OF PLANTS culture and reéxamine), absence of endospores (heat, stains), presence or absence of capsule, occurrence of involution forms, reaction to Gram’s stain (diaphragm wide open). Cultural Characters.—On agar (Figs. 114, 115), on gelatin, on steamed potato, in bouillon, nitrate bouillon, Cohn’s solution, Uschinsky’s solution, lavender-blue litmus milk. Fermenta- tion tubes in peptone water with various puresugars and alcohols. =e be ‘ Fig. 109.—A larger bundle at the same level as Fig. 108, showing A planobacter stewarti occupying many of the vessels. Growth in acid plant juices, e.g., green tomato juice full strength and diluted with an equal volume of water (titrate with phenol- phthalein and N/20 sodium hydrate to determine the acidity). Compare with No. II or No. VIII. Non-nutritional Environment.—What is the optimum tem- perature for growth? the maximum?. the minimum? Can you get any clouding of +15 peptone beef bouillon at 9°C.? STEWART’S DISEASE: NON-NUTRITIONAL ENVIRONMENT 169 Contrast with No. IX. Effect of sunlight? of dry air? of freezing? of weakacids? of weak sodium hydrate? of chloro- Fig. 110—Longitudinal section of outer layers at base of a sweet-corn kernel (level of the radicle) showing presence of Aplanobacter stewart between cell-walls and in the vessels. Fig. 111.—Aplanobacter stewarti forming a cavity in the periphery of a sweet- corn kernel. Same section and same level as Fig. 110. form in bouillon? Maximum toleration of sodium chlorid in bouillon? (Begin with 5 per cent. Contrast with No. I.) FoR THE DISEASE. Signs.—How soon does the disease ap- pear in the inoculated leaves? How many days between the 170 BACTERIAL DISEASES OF PLANTS local appearance of the disease on the leaf-tips and signs of gen- eral infection in the plant? On well-grown plants the earliest signs are flagging and shriveling of the lower leaves, and “‘ white top,” 7.e., the premature development and drying-out of the male inflorescence. This is conspicuous at a distance (Fig. 101). Watch for these signs and try to correlate them with the pres- ence of bacteria in the vascular system of stem and leaf. Can you find any macroscopic evidence of the presence of the disease on the inside (surface) of the leaf-sheaths? or in the ear, especially on the husks? Contrast with Nos. I and II. Look for dwarfing effects. Is the plant as tall as its fellows? Are the ears well filled? Are the roots generally sound? Do the leaves become yellow before they dry out? Does the plant rot? or break over? Describe the disease. Histology.—Select, section and stain (in Ziehl’s carbol fuchsin) a number of leaf-tips, some days after inoculation (4 to 7 days). Can you find distinct evidences of Ba ae infection? Have the bacteria en- ote eee Fen tered through ordinary stomata, leaves in the seedling stage and OF through the water-pores? promptly destroyed by 1000 cirea. did you obtain from your trials on young vs. old plants? Can you increase susceptibility by overwatering or decrease it by liming the soil? or by the liberal use of potash and phosphates? What do you conclude with reference to the effects of tempera- ture? Does the optimum temperature for the plant coincide with that of the micro-organism? Have you found any non-sus- ceptible varieties? A good non-susceptible tobacco would just now be worth its weight in gold! Why is the disease common in our Southern States and unknown or hard to find in our Northern States? Can you determine its presence in states north of THE BROWN ROT OF SOLANACEAE: VARIABILITY 199 Virginia and Texas? Specifically: is it in California, Maine, Wisconsin, Michigan, Ohio, Kentucky or New York? Does it occur in Canada? If not in these places, why not? Transmission.—Have you seen any indication leading you to think that insects spread this disease? In 1896 I obtained very successful infections on potato, using the Colorado potato beetle (Leptinotarsa decemlineata). Many narrow, dark, bacteri- ally infested streaks started in the bitten places and passed Fre. 141.—Photomicrograph showing origin and structure of two incipient roots in an inoculated diseased tomato stem. Bacterium solanacearum occurs in some of the vessels at the lower left side. What stimulus sets the roots growing? rapidly down the stems, both stems and tubers being de- stroyed. If you have opportunity watch infected fields closely and if you obtain clues make some experiments. Hunger in Java incriminated several insects and also thought Phytoph- thora nicotiana paved the way for this parasite. Are plants on wet soils more lable to it? Do the roots of the infected plants usually bear nematode galls? Are plants on limestone soils free from it? Of course, one susceptible crop should not closely follow 200 BACTERIAL DISEASES OF PLANTS another. There should be a long rotation on infected lands, using non-susceptible species—clovers (?), grasses, etc. In this connection it is very important to know whether any of our common forage crops are susceptible and also whether many of our American weeds are subject to this disease, and might act as hold-over hosts. Honing found susceptible weeds in Sumatra; Stanford and Wolf have found them in North Carolina. Long ago I found it readily inoculable into Datura stramonium (the jimson-weed). Has anyone found it naturally Fig. 142.—Tyloses in vessels of a potato stem attacked by Bacteriwm solanace- arum. At X is a vessel occupied by the bacteria. on this plant? In this connection read Stanford and Wolf’s papers. ‘The disease has been reported to me several times from Florida as occurring on ‘‘new land.” LITERATURE For literature, ete., consult Van Breda de Haan’s Wilt of Peanut; Brown Rot of Solanaceae; and Wilt Diseases of Tobacco in “Bacteria in Relation to Plant Diseases,” Vol. III, pp. 151- 153, 174-219, and 220-271, Carnegie Institution of Washington, 1914. Jbid., Vol. I, plates 4, 24, 25, 26,27, and Fig. 10; and Vol. Pie. 1k THE BROWN ROT OF SOLANACEAE: LITERATURE 201 Stanford, E. E. Studies on Resistance of Tomatoes to Bacterial Wilt. N. C. Ag. Exp. Sta. 40th Ann. Rept., 1916-— 1917, pp. 92-93. See also Stanford, E. E. and Wolf, F. A. ‘Studies on Bac- terium solanacearum.” Phytopathology, Vol. VII, No. 3, June, Ike pp. 155—-165.° 1 Fig: The first note on this disease was by Prof. T. J. Burrill in 1890. The first paper relating the disease to a definite micro- organism was by the writer in 1896: ‘‘A Bacterial Disease of the Tomato, Eggplant, and Irish Potato.’ U.S. Dept. Agric. Bull. No. 12, Div. Veg. Phys. and Path., Washington Govt. Printing Office. Here the name Bacillus solanacearum first appears. The first paper proving the disease to occur in tobacco was also by the writer: ‘‘The Granville Tobacco Wilt.” U. 5. Dept. Agric., Bu. Pl. Ind., Bull. 141, part II, Washington Govt. Printing Office, 1908. The last notes are by Smith and Godfrey, Brown Rot of Solanaceae on Ricinus, Science, N. S., Vol. XLVIII, July 12, 1918, pp. 42-43; and by Smith and McCulloch, Bacterium solanacearum in Beans, Science, N. 8., Vol. L, Sept. 5, 1919, p. 238. V. BACTERIAL CANKER OF TOMATO Type.—This disease (Figs. 143 and 144), which for want of a better name I first called The Grand Rapids disease, after the locality in Michigan from which it was first sent to me and where it occurred seriously over large fields, is an infectious par- enchymo-vascular wilt of the tomato (and probably also of the potato), somewhat resembling the brown rot of Solanaceae due to Bacterium solanacearum and often confused with it, but differ- ing in a number of particulars, e.g., it is highly infectious through the above-ground parts; there is less brown stain in the bundles and not so strong a tendency to develop incipient aérial roots (Fig. 145); there is a slow shriveling of the leaflets one after another (Fig. 146) rather than a sudden general wilt of the leaf; the petioles are not reflexed; the meristem is attacked and cor- roded into cavities, e.g., the heart of the incipient roots (Fig. 147); the phloem is specially susceptible to disorganization (Figs. 148 to 150); and there is a strong tendency of the bacteria to come to the surface through fissures on the shriveling leaves, fruits and shoots (Figs. 151 to 154), thus affording an abundant surface slime for the above-ground infection of neighboring plants (through stomata); infection through broken roots has also been observed (Fig. 155). The disease spreads easily from one plant to another often by stomatal infection (Figs. 156 to 158) and is very destructive. It is, I believe, primarily a phloem disease. I think also that it is a seed-borne infection. I have seen its yellow slime close under the seeds in the middle of green tomato fruits, both in the vascular bundles of the peri- carp and in those of the placenta, and also once in the base of an immature seed, but I have not yet actually traced it into or plated it from the ripened seeds. Whether or not it actually occurs in the interior of seeds capable of germination, the fre- quent extensive invasion of the outer part of the tomato fruit is certain to bring about a surface contamination of the seeds. It occurs in the Northern United States both under glass and 202 BACTERIAL CANKER OF TOMATO: TYPE 203 Fig. 143.—Tomato plant inoculated 24 months with a pure culture of the non-motile, yellow Aplanobacter michiganense plated from a New York tomato, showing slow, irregular wilting of the leaflets. Photographed Nov. 25, 1912. This and Fig. 144, made in 1915, may be compared with pure culture inocula- tions of 1909 shown on plates 12 to 15 “Bacteria in Relation to Plant Diseases,’ Vol. IIT. OF PLANTS BACTERIAL DISEASES 204 ‘SJOYRIT oY} JO SUI[PATIYS IBpNSo1 -Il aAIOSGQ, “punorsyovq oy} url syuvyd YooyD “queyd o7BUI0Z syjyosnyousseY B WO] CTGT Ul poxeld “(py AUO]OD) asuaunbryoiue Lajapqgounjd y Jo oI [no oind & IM syorid-o[poou Aq YQuout suo peyepnooulr syueyd OJVWO TL — FFL Oly BACTERIAL CANKER OF TOMATO: TYPE 205 in the field. I have had it from Michigan, Western New York and Eastern Massachusetts. So far it has not been reported from the Southern States, but I believe I had it once from Texas without recognizing it as distinct from the brown rot. It undoubtedly occurs in Europe. It should be looked for in England, France, Belgium, Germany and Italy. os ¥ + é ¥ iS € (a ct Fig. 145.—Stems of tomato plants inoculated with Aplanobacter michiganense October 5, 1909, and photographed January 17, 1910, when most of the foliage had shriveled. These show slight tendency to formation of adventitious roots, as compared with those attacked by Bacterium solanacearum. Compare with Fig. 122 inoculated only 12 days. Cause.—This disease is due to Aplanobacter michiganense EFS. This is a rather short, viscid, yellow, non-motile, non- sporiferous, non-gas-forming, aérobic, very slowly liquefying, non-nitrate-reducing, rod-shaped schizomycete (Fig. 159) form- ing slowly on beef-peptone agar-poured plates small circular 206 BACTERIAL DISEASES OF PLANTS colonies which become darker with age but are always yellow (not orange colored). Katherine Bryan has also isolated a form which spreads on agar. It stains by Gram, but is not acid- fast. It grows copiously in milk with slow coagulation,: form- ing a thick pellicle and a broad yellow rim. A very little acid seems to be produced, as litmus in milk becomes gray or purplish Fria. 146.—Tomato leaf showing irregular wilting of leaflets due to 1000. — disease in the field (the writer has not had) you should ex- amine particularly for carriers of infection (insects, ete.), for evidence of under-ground infection, 7.e., through the root-system, and for transmission on seeds taken from diseased plants. The disease was so prevalent and destructive at Grand Rapids, Michigan, that seemingly it must have begun early in the life of the plants. It escaped from my control in one of the Department of ~J BACTERIAL CANKER OF TOMATO: TRANSMISSION Zit Fic. 160.—A planobacter michiganense: uniformly sown on +15 peptone-beef agar-poured plate, showing growth of colonies inhibited everywhere except in the vicinity of the central white intruding colony which is an alkali-producing schizomycete. At x there is an inhibiting mold colony, probably an acid producer. There was a brown stain in the agar between and around the yellow colonies. The colonies on the inner part of the periphery were fluorescent. Those on the outer one-fourth were not fluorescent. Photographed December 13, 1915. Shghtly enlarged. 218 BACTERIAL DISEASES OF PLANTS Fig. 161.—The Berkshire, Mass., potato disease (net-necrosis). Slice of tuber photographed by reflected light. February 28, 1919. x 4. BACTERIAL CANKER OF TOMATO: TRANSMISSION 219 Agriculture hothouses and infected various check tomato plants and also a bed of West Indian plants (Solanum mammosum) said to be resistant to Bacteriwm solanacearum. This unwelcome infection was attributed to spatterings from the gardener’s hose. I first called attention to this method of dissemination in 1914. The experiences of growers in New York and Massa- chusetts show that it is capable of doing much damage to hot- house tomatoes. Host Plants.—It is very important to determine whether this parasite has other hosts than the tomato. I believe it occurs also on the potato but the evidence is not yet conclusive. In the winter of 1918-19 I received potatoes from Berk- shire Co., Mass., said to be fair samples of a great many occurring in that locality. These tubers were sound externally but the outer one-half inch or more of their flesh was mottled with numerous brown spots, forked lines and streaks (Fig. 161). On studying sections under the microscope, no distinct lesions were observed but foci of bacteria were found in the center of some of the spots. In all the tubers I examined, the stem end of the tuber was always badly diseased, but often the eye-end was free (Fig. 162). When the flesh of the tuber was examined in thin section (1-3 mm.) by transmitted light, the brown spots and streaks were seen to be surrounded by a narrow clear zone indicating disappearance of the starch in the surrounding tissues (Fig. 163) which was confirmed by tests with iodine. Several organisms were cultivated out on potato. Some made a pale whitish slime at first, becoming distinctly yellow, but in other cases pure white cultures were obtained, and in many instances nothing whatever. Inoculation tests on tomato and potato gave nothing definite. This disease was not discovered by the planters in the field but during the winter in the stored tubers (variety, Green Mountain). This new disease has received the name of “ Net- necrosis.’ By some it has been ascribed to frost injuries, but I cannot think the phenomena as it occurred in Massachusetts in the winter of 1918-1919, and as shown on the accompany- ing plates, was due to freezing. When the plates were made I believed the disease due, probably, to bacteria, but now I 220 BACTERIAL DISEASES OF PLANTS * es i i | Fic. 162.—Cross-sections of a Green Mountain potato tuber from Erwin E. Maynard, Savoy Center, Berkshire Co., Mass., showing net-necros Stem- end diseased; eye-end free. Photographed April 3, 1919. BACTERIAL CANKER OF TOMATO: HOST-PLANTS ae Fic. 163.—Like Fig. 161, but from a thin section photographed by trans- mitted light to show narrow clear spaces (starch destruction) around the browned vascular bundles. X 4. Dee BACTERIAL DISEASES OF PLANTS have no definite opinion as to its cause. Possibly it is of fun- gus origin. The disease appeared again the following year in Berkshire County, according to Mr. Maynard, but less seri- ously. Such tubers give spindling plants. My illustrations should be compared with those of Jones, Miller and Bailey in “Frost Necrosis of Potato Tubers”’ (Agr. Exp. Sta. of Univ. of Wis. Research Bul. 46, Oct., 1919) which appeared since the above was in type. LITERATURE The first paper definitely relating this tomato disease to a particular organism was by the writer in 1910, Science, N.S., May 20. For literature, etc., consult The Grand Rapids Tomato Disease in “‘ Bacteria in Relation to Plant Diseases,” vol. II, pp. 161-165. In this connection, read also what is said con- cerning Spieckermann’s potato disease, [bid., pp. 166-167. Spieckermann and Kotthoff’s full paper on the ring rot of the potato is in Landw. Jahrbticher. Bd. 46, Heft 5, 1914. See also Paine, Sydney G. and Bewley, W. F. “Comparison of the Stripe Disease with the Grand Rapids Tomato Disease” in Studies in Bacteriosis. IV.—‘Stripe’ Disease of Tomato. The Annals of Applied Biology, Vol. 6, 1919, Nos. 2 and 3, pp. 200-202. VI. JONES’ SOFT ROT OF CARROT, ETC. Type.—This is a rapid bacterial wet-rot of storage paren- chyma (roots, rhizomes, fruits and fleshy stems). It seldom attacks well-developed green parts, nor does it develop vigor- ously in storage tissues unless they are turgid. It was described in 1901 by Prof. L. R. Jones from carrot roots grown in Vermont, but he obtained it on the fleshy parts of many other plants by pure-culture inoculation, and it is nowknown to be widespread in nature on a variety of hosts. Probably it occurs all over the world but its geographical distribution remains to be worked out. We owe most of our knowledge of this disease to Prof. Jones, but others have also studied it critically in recent years, notably, Harding and Morse, and to some extent also the author of this book. Since the appearance of Jones’ first paper the same organism has been isolated from soft rots on other plants by several plant pathologists, who have studied and described it under other names. Furthermore, several other very closely related if not exactly identical soft-rot organisms have been discovered, de- scribed and named. M. C. Potter’s white rot of turnips is due to this organism, and his paper appeared in 1899 (two years earlier than Jones’ paper), but he described as its cause a polar flagellate organism and his name, therefore, cannot be substituted. The parasitic action of all of these morphologically and cul- turally similar soft-rot schizomycetes is essentially the same and Harding and Morse believe all of them to be one species but I am not entirely committed to this belief. They enter the plant through wounds and rapidly disintegrate the susceptible parts into a soft, wet pulp (Figs. 164 to 166), having first poisoned the tissues by means of their by-products. They advance into the weakened tissues by way of the intercellular spaces and separate 223 224 BACTERIAL DISEASES OF PLANTS the cells one from another by dissolving the middle part of the cell-wall, which is of a different composition from the outer part. The protoplasm of such separated cells is collapsed and dead, but the bacteria are not found inside the cells except in late stages of the disease. The first indications of disease in a carrot root are the ap- pearance of water-soaked (translucent) places around the in- fected wounds. These spots are visible in from 18 to 36 hours Fig. 164.—Bacillus carotovorus L. R. Jones, streaked for 3 days on raw carrot. Kept on the table at 23°C. in a large covered dry culture dish. The inoculation was from a rotting raw potato which was inoculated from a gelatin colony. The carrot was first washed, then soaked in 1:1000 mercurie chlorid water, and cut with a cold sterile knife. The left (check) part remained sound. after inoculation when the roots are held at 20° to 24°C. With- in two or three days this tissue breaks down, shrivels and exudes drops of a gray fluid swarming with the bacillus. Sometimes a thin, gray bacterial film also covers the surface. When a 2- mm. loop of a bouillon culture is placed on a slice of carrot in a covered Petri dish the water-soaked appearance may sometimes be seen in 6 hours at 20° to 23°C. In nature the rot usually JONES’ SOFT ROT OF CARROT, ETC.: TYPE 99n begins at the crown or at the root-tip. The disease continues in the stored carrots which often decay very rapidly and in large numbers. The core of the carrot rots more rapidly than the outer part of the root, and flabby roots are much less susceptible than turgid ones (Fig. 167). The attacked roots of half-long orange carrots are stained a dark brown, this color commencing within 24 hours; those of the long-orange carrot are not stained or only slowly and slightly stained. Inoculated parsnip roots are ” PS - 5 I 5 = . = Fig. 165.—Same as inoculated half of Fig. 164, but after it had been dropped. Tissue entirely soft rotted except a thin external layer. 45 nat. size. changed to a clay color deepening to cinnamon brown. The spots on green tomato fruits are turned dark. Jones observed no stain in other inoculated rotting plants. Decay of the cruci- ferous roots was accompanied by an offensive odor. Decaying onions also emitted a bad odor. The disease has been seen in the United States occurring naturally or has been obtained artificially by pure-culture in- oculations on the following plants: carrot, parsnip, celery, 15 226 BACTERIAL DISEASES OF PLANTS lettuce, cabbage, cauliflower, turnips, radish, cucumber, musk- melon, potato, tomato and pepper (ripe and green fruits— faster in the latter), eggplant (ripe fruits), hyacinth (leaf and scape), and onion (bulb and leaf). Jones’ inoculations failed on ripe oranges, bananas, pears and apples; on cauliflower, sweet potato, beet, asparagus; and Fig. 166.—Photomicrograph showing separation of cells of carrot due to the action of Bacillus carotovorus. Inoculated from a beef-bouillon culture. Time, 2 days. 1915. Organism 3a, in the laboratory several years. repeatedly on Irish potato tubers (once successful, however). They also failed on yourg carrot, parsnip and lettuce plants, and on the petioles and stems of the tomato. Most of his early inoculations were made in my laboratory in Washington early in the year (February to April) on roots and fruits from the mar- 5 nella ETC: SOFT ROT OF CARROT, ’ JONES sera ay a OO Kay, ee be Bao Pha E ; note . SS PON righ ame time, € c arrots inoculated at thes ‘ c aph of two e ‘ © A. Shows photogr Fie. 167. BACTERIAL DISEASES OF PLANTS me eee all ine eee Fie. 168. Fic. 169. Fic. 168.—Surface and side view of a raw potato tuber (Green Mountain) streaked 4 days, at 23°C., with Bacillus carotovorus from a gelatin colony (38a, long in my laboratory). has penetrated. Photographed Jan. 16, 1916. Fig. 169.—Potato plant, variety Green Mountain, one shoot of which has The right-hand figure shows the depth to which the rot been inoculated 7 days with Bacillus carotovorus. and wilting with internal brown streaks. phytophthorus. Photographed Feb, 8, 1915. Inoculated shoot dwarfed Organism less active than Bacillus from same (4-day) bouillon culture of Bacillus carotovorus. No. 1 was flabby, No. 2 was turgid. Left check omitted. Time, 3 days. B. Same as A, but at the end of 6 days at room temperature (25°C.) in a large culture-dish. The inoculated half of the flabby carrot now shows a slight rot at the top where an unusually large amount of the cloudy bacterial fluid was deposited, 7.e., much more than on the badly rotted piece. Experiment of May, 1915. Four days later there was little change. Right check omitted. This was still sound. ta JONES’ SOFT ROT OF CARROT, BTC.: TYPE 229 ket. Time of year, absence of turgor, varieties used, or gradual loss of virulence on the part of the organism might make a differ- ence with potato tubers. Or it may be that what is here de- scribed as Bacillus carotovorus is a composite of two or more species. His conclusion at that time was that it did not attack the potato. According to my observations the organism does not lose virulence readily, and the culture I have (which came originally Fra. 170.—Green cucumber inoculated by longitudinal stabs introducing Bacillus carotovorus from gelatin colonies. Sliced and photographed January 20, 1915, i.e., after 6 days at 23°C. Interior soft rotted. Sliced (check) cucumber from the same lot on the right side, entirely sound. 1} nat. size. from Jones, but bas been transferred many times in my labora- tory) rots raw potato tubers readily (Fig. 168) and also attacks the soft green stems of this plant (Fig. 169). Its disintegrating action on many tissues other than those of the carrot, ¢.g., green cucumber fruits (Fig. 170), is very rapid. I have also obtained with it a calla lily rot resembling Townsend’s rot (Figs. 171 to 173) and a rot of young leaves of carrot. 230 BACTERIAL DISEASES OF PLANTS Fic. 171.—Calla lily rot due to needle-pricks introducing Bacillus caro- tovorus. Leaf-stalk inoculated 48 hours at Y. The rot extended intern- ally beyond Y. Another leaf-stalk of the same series rotted entirely across and fell over the third day. In potato tubers a protec- tive layer of cork is often devel- oped under the rotting area (Fig. 174). Inthe shoots of potato it is not partial to the vascular system but nevertheless may sometimes be found in vessels at a considerable distance above the place of inoculation (Fig. oye Cause.— The cause of this dis- | ease is Bacillus carotovorus L. R. Jones. This is a gray-white,’ non-capsulate, non-sporiferous, actively motile (2 to 5 flagel- late), peritrichiate (Fig. 176), slowly liquefying (gelatin, but not coagulated egg albumen or Loffler’s solidified blood serum), nitrate reducing, milk curdling (by an acid), aérobie and facul- tative anaérobic, gas-forming (with muscle sugar, dextrose, saccharose, lactose and mannit but pot with glycerin nor with 1 Wormald says yellow on Soyka’s milk rice (.l¢.). The quality of white- ness is variable as is that of any other color. Lagree with Wormald that it is yellowish in contrast with the pure white of the rice medium, about as yellow as steamed potato cylinders, but I would eall these white rather than yellow or more exactly following R» nearly pale cream color, becoming cream color in old cultures. It is a matter of opinion. Very few white organisms are as white as white rice but nothing would be gained by calling all of them chromogens. For preparation of this useful medium see Eyre’s Bacteriological Technique. JONES’ SOFT ROT OF CARROT, ETC.: CAUSE 23 1 potato juice in fermentation tubes,! the gas beirg 20 per cent CO, and 80 per cent explosive), heat-sensitive (thermal death-point 51°C.—even 10 minutes in bouillon at 47°C. re- tards growth), dry-air-sensitive (sometimes even to 2 minutes’ exposure—Jones), sunlight-sensitive (10 minutes of direct sun- light and 2 hours of diffused sunlight, fatal—Jones), not ex- ceedingly frost-sensitive (frozen in +15 peptone beef bouillon, in 1919, 13 per cent survived) short, rod-shaped, catenulate, or filamertous (up to 200u or more—Jones) schizomycete Fria. 172—Cross-section of Fig. 171 at Y, moderately enlarged to show char- acter of the bacterial rot. The tissue of the attacked part X did not hold the stain. (greatest observed variation in diameter 0.6 to 0.94, usual diameter 0.7 to 0.8), growing on the surface of agar-poured plates in the form of round, raised, smooth, gray-white, wet- shining colonies (2 days) having entire well-defined margins and a transient flaky areolation (X10) with a slight fluo- rescence, the buried colonies being globose, oblong or spindle- shaped with irregular margins (125), but, if thin sown, 1 Repeated in 1919 in potato juice in fermentation tubes with contradictory results, using Jones 3a (branched) and also 3a received from Wisconsin in 1920. The latter sometimes gives a little gas and at othertimes not. Dae BACTERIAL DISEASES OF PLANTS the surface colonies (Jones 3a, stock long in my laboratory) are sometimes more or less irregular in outline and may send out finger-like branched projections (Fig. 177). Fearing that in some transfer the labels of Bacillus aroideae and Bacillus caro- tovorus might have been interchanged I sent to Wisconsin in 1920 for another culture of 3a and this in agar-poured plates a «he Fig. 173.—Detail from Fig. 172 at X. Much enlarged to show the bacteria dis- integrating the swollen cell-wall and confined to the intercellular spaces. gives round colonies (Fig. 178). The two stocks also differ in amount of gas formed from potato juice (Fig. 179) and in other ways. On the +10 gelatin-poured plates the margins of the young surface colonies (125) are thickly set with parallel filamentous outgrowths (Figs. 180, 181), this fimbriate margin being about 50u wide on the second or third day. The buried colonies JONES’ SOFT ROT OF CARROT, ETC.: CAUSE 233 Fic. 174.—Cross-section of a McCormick potato tuber taken 12 days after inoculation, i.e., when the rot had subsided, showing (at X-X) the formation of an inhibiting layer of cork under the rotted area. This tuber was streaked with Bacillus carotovorus at the same time as Fig. 168, and rotted as well at the begin- ning. The rotted part (at top) is full of starch grains. From the sound part (below) the starch has been removed to form the cork-layer. Compare with Fig. 136. Fic. 175.—Cross-section of a potato stem inoculated with Bacillus carotovorus for comparison with Bacillus phytophthorus. Section far above the point inocu- lated. Vessel full of bacteria. 204 BACTERIAL DISEASES OF PLANTS in gelatin plates (X125) are irregularly spherical, often more or less clumpy, uniformly granular and with sharp margins which tend to become hazy. Sometimes the buried colonies send out colorless root-like growths (Fig. 182). Stab cultures liquefy first at the surface but eventually throughout (Fig. 1834 ). There is a fragile imperfect white pellicle and a copious white precipitate, the fluid becoming strongly alkaiine. Peptonized beef bouillon clouds very rapidly, especially when neutral to phenolphthalein (in 6 hours at 30°C., when in- oculated with a l-mm. loop). If undisturbed there is formed a very thin imperfect pellicle which shakes down readily leaving a scanty interrupted rim, easily washed away. The white : ‘ . a « bit: > We x f. SS a <8 f 3 = ‘ . f- \ Bat: * ~ > j fi © 2 ‘ - 4 . n ‘ : a : { ’ . ot ig 5 « s a ‘ Fig. 176.—Flagellate rods of Bacillus carotovorus. From a 2-day agar streak. Van Ermengem’s silver nitrate stain. In the upper right there are two bacterial rods lying together. x 1000. precipitate is not copious. It (8a) gives in +15 peptone bouillon a heavier clouding than B. phytophthorus. Growth in Dunham’s solution is feeble. Growth in Uschinsky’s solu- tion is abundant and long-continued and the fluid remains more or less acid throughout: there is a copious precipitate (15 pr 20 times that in bouillon), but only a delicate easily fragmented pellicle. In milk a curd separates about the fourth day. It has the odor of cheese curds and there is little or no peptonization of this curd. Litmus milk is reddened and the litmus is, or may be, subsequently reduced. Other pigments are reduced, such as methylene blue (in Dunham’s solution with grape sugar, not without). JONES’ SOFT ROT OF CARROD) at Ce @2200is i eye) > Fig. 177.—Agar poured plates of Bacillus carotovorus? (Jones 3a?) on +15 peptone beef agar at 25°C. for 10 days. Photographed May 3, 1919. Culture long in my laboratory and frequently transferred along with other soft rot isolations. Possibly confused in some transfer with Bacillus aroideae. This is the culture that was infectious to calla lily, and various other statements in the text respecting B. carotovorus are based on this organism. BACTERIAL DISEASES OF PLANTS Fig. 178.—Original stock of B. carotovorus (Jones’ 3a, received from him in 1920). Photographed after 3 days at 25° on + 14 beef-pepton agar, showing surface and buried colonies. Y is a buried colony beginning to come to the sur- face, X is a young thin colony. Photographed, X 10 by oblique transmitted light for comparison with Fig. 177. The colonies are smooth on the surface and show internal wavy markings by oblique transmitted light. Still infectious to carrot. ~ JONES’ SOFT ROT OF CARROT, ETC.? CAUSE Ze The growth on steamed potato cylinders forms a slightly raised, wet-shining, smooth, cream-white covering, with a slight evolution of gas and the conversion of starch into amylodextrin. Fra. 179.—Fermentation tubes with potato juice: A. Jones 3a (original Bacillus carotovorus) from Wisconsin in 1919. Gas in 24 hours. The gas in 48 hours and in 15 days is also indicated. B. Jones 3a (long in my laboratory). No gas in 4 days. Amount of gas in 15 days is indicated. Good growth in both tubes. 8B rots calla lily, A does not. B also rots young carrot tops. Gas is also formed from steamed carrot cylinders and the gray- white bacterial layer is seldom thick enough to hide the orange 238 BACTERIAL DISEASES OF PLANTS or yellow color of the substratum. Both these substrata become alkaline as early as the third day and increasingly so later on; both are softened, especially the carrot which often may be shaken apart in water after a week (Jones) or even in much less time. The maximum temperature for growth is between 38° and 39°C. The optimum temperature for growth is between 25° and 30°C. The minimum temperature for growth on steamed vegetables is above 4°C. Raw vegetables have not been re- ported upon.' No appreciable growth was obtained on any medium at 0.6° to 1°C. (20 days) but there was a slight growth on nutrient gelatin at 2° and at 3°C. Growth after 5 days at 12°C. on steamed vegetables (potato, carrot, turnip, rutabaga) was about one-third that on the same substrata at 20° to 24°C. Except as already noted the cultures were free from strong odors. Not much indol is produced. Neutral bouillon gives the best growth, but the organism tolerates sodium hydroxid in bouillon down to below —40 on Fuller’s scale and malice acid up to a little beyond +30. The organism is sensitive to it own acid products. In peptone water containing grape sugar, swollen, vacuolate and knobby involution-forms occur. Tolerates sodium chlorid up to 6+ per cent but not 7 per cent in +15 peptone beef bouillon. Grows well in +15 bouillon with 5 per cent NaCl. Jones 3a (culture received from him in 1919—descend- apt of his original isolation of Bacillus carotovorus) tolerates ethyl aleohol up to 7 per cent in +15 peptone bouillon; grows promptly and well in the presence of 5 per cent. In further ex- periments it grew readily and formed a heavy pellicle in the presence of 10 per cent ethyl aleohol and made some growth in the presence of 11 per cent, but. would not grow in the presence of 12 per cent. See Fig. 184 where the behavior 1In October, 1915, growth and rot were obtained by the writer on raw potato and carrot at 5°C. inoculating (3a) from a potato culture, but neither at 5°C. nor at 8°C., inoculating from bouillon. The first rot from the bouillon inoculations was at 9° to 11°C. and that feeble (5 days). JONES’ SOFT ROT OF CARROT, ETC.: CAUSE 239 of six soft-rot organisms is shown in +15 peptone beef bouillon containing ten per cent of Squibb’s absolute ethyl alcohol viz. 3a recently from Jones (year 1919), 3a long in my laboratory (the branched 3a of Fig. 177), Potter’s organism (No. 79 of Jones’ laboratory), Spieckermann’s organism (No. 78 of Jones’ laboratory), Bacillus phytophthorus (Appel I) and Bacillus apiovorus Wormald. Fie. 180. Geel sile Fra. 180.—Surface colony of Bacillus carotovorus on + 10 beef-peptone gelatin after 24 hours at 18°C., showing marginal fringe. X 110, about. Actual dia- meter of colony, 0.4 mm. Fra. 181.—Margin of a surface colony of Bacillus carotovorus after 3 days on gelatin, showing bacterial fringe pushing out into the gelatin. The darker band behind the fringe was liquefied and full of bacteria which exhibited, as a whole, a Weaving or swaying motion. Medium magnification. Nore.— Not having myself-worked-over all of these soft-rot organisms critically, 7.e., through a series of years, the following conclusions on the synonomy are expressed tentatively. Forms apparently identical with Bacillus carotovorus are Bacillus oleraceae Harrison on cauliflower and Bacillus omnivorus van Hall on iris. Under the name of Bacillus apiovorus 240 BACTERIAL DISEASES OF PLANTS Wormald in England has described a schizomycete which at- tacks celery producing a soft-rot (Figs. 49 and 185) but is not active on potato shoots (Fig. 186). This, he is now inclined to think, is also identical with Bacillus carotovorus, but I am in doubt and shall keep it separate for the present. Very closely related forms are Bacillus aroideae Townsend (Fig. 187) on calla lily and Bacillus melonis Giddings on musk- melon. ‘Townsend’s organism differs in the form of its colonies on agar (they are, however, like those shown in Fig. 177) and in some of its fermenting powers, t.e., acid without gas from dextrose, lactose, saccharose and mannit. Its effect on raw Fra. 182.—Buried colonies of Bacillus carotovorus in +10 beef-peptone gelatin plates after 24 hours at 18°C., showing colorless root-like extensions. The several small dark spots ringed with light are due to irregularities in the gelatin or to dirt on the eyepiece. 135 cirea. carrot at the end of 8 days is shown on Fig. 188. Gidding’s organism produces abundant gas from milk (99 per cent. CO.) in the closed end of fermentation tubes; lquefies blood serum; does not produce gas with dextrose, saccharose, lactose, maltose or mannit, and has a maximum temperature of about 45°C. I think Bacillus aroideae and Bacillus melonis are identical. At least an organism isolated by us from rotting calla lily and identified as Bacillus aroideae produces gas in milk (Fig. 189). With his original isolation (now lost) Dr. Townsend made no tests in fermentation tubes containing milk. I have been inclined to think that Bacillus carotovorus and Bacillus phytophthorus are not strictly identical, and have kept JONES’ SOFT ROT OF CARROT, ETC.: CAUSE 2 | them separate in this volume, but further comparisons are necessary. Technic.—It is not difficult to isolate this organism, since very often it occurs almost unmixed in the decaying tissues. If the advancing margin of the rot is selected and the surface organisms are destroyed by pressing a hot spatula on the part selected (which may be the sound surface near the rot), one may then dig through the burned surface and into the rotted area with little danger of external contamination and the certainty of ob- Fic. 183.—A, Bacillus carotovorus L. R. J., and B, Bacillus apiovorus Wormald, in gelatin stabs at the end of 5 days at 20°C. taining on the poured plates almost or quite a pure culture of the parasite. The organism is easily identified by its rapid disintegrating action on raw carrots or turnips and by its cultural peculiarities. As here described it can not be distin- guished with certainty from Bacillus phytophthorus (No. VII) by its behavior on raw potato, por by its growth in thin-sown gelatin plates, as I formerly supposed (compare Figs. 206 and 212A with a, 6 of Fig. 205). For inoculation experiments, roots of various kinds may be selected and also fleshy above-ground parts. The work may 16 I joddy #4) oy}) ALopBALOgey AUL UL SUC] ‘VE “Gg S(GIGBT UL SouOLr “YY TT WOdy ‘DE) sNLOdOJOLDO SND “Y :SUSIUBSIO JOL JOS SNOLIBA YIM UOT] G d ‘ ly r a . . . . \ O bn 4 . . . ON UISUODSI AA) UISTURSIO S.10}}0q “fp tsnsoaoidy snppoog “q ‘(snsoyjyydojhiyd snjyjwovg) ‘(UOJ AUOJ[OD pPoyouRag “VOUT LoJFV SyooM J ‘OYoOo]R pAYyJo oynyposqw s qqmbyg Jo yuoo dod QT YBa ‘UoT]MOG Joog ouoydod GT-+ Jo soqny—FST “ply OF PLANT DISEASES _ 1000. one-half. Time, 8 days.. Inoculated in 1915. 248 BACTERIAL DISEASES OF PLANTS ments can be tried, 7.e., effect on the gas of shaking, in the presence of a strong solution of sodium hydroxid or potassium hydroxid. Is part of it absorbed? Is the absorbed part, roughly, one-fifth? Do not conclude too hastily. Allow time. Will the remainder Fig. 188.—A. Effect of Bacillus aroideae Townsend, on raw carrot, B. check The left part was stirred up a little with a glass rod. explode when brought into contact with a flame? Is it hydro- gen? From what media are acids produced? Can the organism develop acid without gas (try glycerin)? Is more than one JONES’ SOFT ROT OF CARROT, ETC.: CULTURAL CHARACTERS 249 acid produced? Distill a flask-culture which has become acid and determine whether the steam is acid to neutral litmus paper. Use a large flask with a shallow layer of liquid. Collect the steam in water and make tests for nature of the acid. Is it acetic acid or only CO,? Boil the residue and determine whether it becomes more acid on concentration. Can you identify the residual acid? Is it lactic acid? Contrast with B. phytophthorus in +15 peptone bouillon with from 5 to 10 per cent of ethyl alcohol added (by means of a sterile pipette) after sterilization. Study nitrogen nutrition, reduction of nitrates, formation of hydrogen sulphide, ammonia, indol. Production of enzymes— starch-converting, proteolytic, cytolytic, etc. Whatispectinase? Try the following experiment: Inoculate the center (surface only) of several agar plates and when the growth has become 34 inch in diameter cut out the agar with a sterile knife in such a way as to remove all of the bacterial growth without touching it and transfer the agar bottom down to slices of raw carrots, tur- nips, etc. If you have done the work properly there will be no growth of the bacteria on the raw surface and yet it will rot. Why? Demonstrate absence of bacteria in the decaying tis- sues and describe their appearance under the microscope. Is the milk curd a normal cheese curd? Is gas ever produced from milk? Try it in the closed end of fermentation tubes. Hold checks. On what raw media and steamed substrata is the brown pig- ment produced? What is the nature of this compound? Is it a host reaction or a bacterial excretion? It is important to isolate the organism from carrots (natu- rally rotting), from Gidding’s melon rot and from Townsend’s calla lily rot for comparison. Do so by all means if you have the opportunity, or send the material to some one who will. Much additional work remains to be done on the soft-rot bacteria. Non-nutritional Environment.—Action of heat, cold, dry air (very sensitive), sunlight (very sensitive), acids, alkalies, germi- cides. Behavior in vacuo, and in neutral gases such as hydrogen, nitrogen, carbon dioxide. 250 BACTERIAL DISEASES OF PLANTS Fig. 189.—A. Fermentation-tube milk culture of Bacillus aroideae. The closed arm is full of gas (all CO.) and so is the U. From x upward, whey; and downward, curd. Curd also in base of the closed arm. Inoculated January 15, 1915. Photographed February 10, 1915. B. Same as A but 48 hours later when all the gas has been absorbed by ad- dition of a strong solution of sodium hydroxide. SOFT ROT OF CARROT: NON-NUTRITIONAL ENVIRONMENT Bol Why does 5 per cent grape-sugar retard or inhibit growth? Why does it ear y kill off the cultures grown in media containing it, For THE DISEASE: Signs.—Write a description of the signs of this disease drawn from your own inoculations on carrots and other vegetables. How many hours from inoculation to the first appearance of the soft rot? Study the progress of the rot as related to: (1) copious vs. sparing inocu ation; (2) moist vs. dry air; (3) cool vs. warm air; and (4) flabby vs. turgid tissues. At what cool temperature does the rot cease? Above what tem- perature does it cease? Compare this organism with No. IV, especially on raw potato, and with No. VII. Submit photographs or good drawings or both. Remember you cannot have too many striking and conclusive illustrations of the various diseases. Histology.—F ix, embed, section and stain early stages and late stages of the rot on various plants to determine location and action of the organism. Are the cells invaded or is the action of the organism entirely extra-cellular? Do not conclude too hastily. How are the middle lamellae destroyed, 7.e., by tear- ing or by solvent action? What is the composition of this mid- dle part of the cell-wall? Is cellulose destroyed? How are the bacterial cavities formed? Are the cells killed in advance of the bacterial invasion? Does the water-soaked area surround- ing a bacterial nidus stain the same in sections as the sound area just beyond (see Fig. 172 at x)? Under the microscope unstained, does it look the same? What differences in the cell- wall? in the cell contents? In the attacked parts just previ- ous to disintegration are the cell-walls swollen? Make good permanent preparations showing various stages of the tissue- disintegration. Variability There appears to be considerable more resis- tance in some varieties of the attacked species than in others. Is this accidental or inherent? Why are green tomato fruits more subject than ripe ones? Why is the core of the carrot rotted sooner than the outer part? Why are young (seedling) carrots exempt? Are they, really? Why are the green parts of susceptible plants not more generally attacked? Read what 2a BACTERIAL DISEASES OF PLANTS you can find on this subject and make some experiments. The man who is continually trying out his ideas by means of careful experiments is the one who makes discoveries. Reading alone will not serve; it makes a full man, but not a fruitful one. Transmission.—Nothing is known respecting special carriers of this disease. One should certainly avoid throwing diseased refuse into manure piles and into streams, and rotation of crops should be practised. Carrots should be dried and sunned as thoroughly as possible before storage, which should be at alow temperature. LITERATURE Read Jones: ‘‘ Bacillus carotovorus n. sp., die Ursache einer weichen Faulniss der Mohre.” Centralb. f. Bakt., 2 Abt., VII Bd., 1901, pp. 12 and 61; also Jones: ‘‘A soft rot of carrot and other vegetables, etc.” 13th Report, Vermont Experiment Station, 1901; and Jones, and Harding and Morse: ‘‘ The bac- terial soft rots of certain vegetables’? (23d Annual Report, Vermont Agricultural Experiment Station, 1910, where other literature is referred to.) Consult “Bacteria in Pelation to Plant Diseases,’ Vol. I, Figs. 2, 3, and 88. and some statements in the text; also Jbzd., Vol. II, text statements (see index). Read Spieckermann’s ‘‘ Beitrag zur Kenntnis der bakteriellen Wundfaulnis der Kulturpflanzen,’” Landw. Jahrb., 31 Bd., Berlin, 1902, p. 155, and Harrison’s ‘‘A bacterial rot of the potato caused by Bacillus solanisaprus’’ Centralb. f. Bakt., 2te. Abt., XVI, Bd., 1907, pp. 34, 120, 166, 384. See also Townsend’s paper: A Soft Rot of the Calla Lily,”’ U. 8. Dept. Agr., Bureau of Plant Industry, Bulletin No. 60, 47 pp., 9 pls., 7 text figures, 1904. And Gidding’s paper: “A Bacterial Soft Rot of Muskmelon, Caused by Bacillus melonis n. sp.,’? Vermont Agricultural Ex- periment Station, Bul. No. 148, pp. 366-416, with 14 text figures, 1910. The first papers on this subject and the last one also are by Prof. L. R. Jones. VII. THE BACTERIAL BLACK ROT OF THE POTATO (Syn. Black leg, Basal Stem-rot and Tuber-rot ) Type.—This is a wide-spread and very destructive soft rot of the potato and some other plants. Vessels are sometimes occupied, but it is a disease of the parenchyma rather than of Fre. 190.—Curling of potato leaflets due to Bacillus phytophthorus (Appel 1). Spring of 1915. Second set of needle-prick stem inoculations. the vascular system, being found, according to Dr. Appel, only occasionally and exceptionally in the vessels of the potato plant. The first above-ground signs of the disease are either sudden 253 254 BACTERIAL DISEASES OF PLANTS wilting or a slow yellowing of the lower leaves and a stricter habit of growth in the upper ones, the leaflets of which (Fig. 190) are or may be more or less ineurled (upward). If one examines the base of such shoots they will be found to be black-spotted res LOW Hire lG2s Fic. 191.—Stems of Green Mountain potato inoculated 48 hours at XY with Bacillus phytophthorus Appel, from a 2-day agar-streak culture. Stems black and rotting in the pricked area. Hothouse experiment of January 23, 1915. Tubers half grown. Organism from Germany. In the laboratory since August, 1906. Fic. 192.—Same lot of plants as Fig. 191, but 4 days after inoculation (at X). Stems black and nearly rotted off in the pricked area; bundles infected upward for long distances and with a brown stain coming to the surface. end more or less softened (Figs. 191, 192) at the surface of the earth or just below it, and hence the German name Schwarzbein- igkeit (black leg). Generally at first this blackening and ulcera- tion are restricted to the base of the stem but upper parts soon BLACK ROT OF THE POTATO: TYPE 200 wilt, blacken, shrivel, and fall over (Fig. 193), and often the tubers decay. The disease is readily inoculable into the soft upper part of shoots (Fig. 194) and may run out on the petioles in black lines exactly as in case of Bacter- ium solanacearum (No. IV) or of Bacil- lus amylovorus (No. XII). It is also readily inoculable into the base of potato shoots when not too old (Figs. 195, 196) and then progresses in the same way as the natural infections. Old shoots are less susceptible than young ones (Figs. 197, base, and 198) and very often when the basal parts of stems are inoculated they rot across and break over without much down- ward movement of the bacteria (Fig. 198), the underground parts sending up new dwarfed shoots to take the place of those destroyed (Fig. 199). Prior to shriveling, especially as the season advances, all the green parts of the potato (stems, leaves, flower stalks) may show black spots due to the organism and from these pure cultures of it may often be ob- tained (Appel). The bacteria neither show any special tendency to multiply in the vascular bundles, although some- times found there (Fig. 200), nor, in F1G. 193.—Potato plant ; = inoculated by needle pricks on the base of the shoot (at X) and wilted by Bacillus phytophthorus (Appel 1). Plant inoculated January 30, 1915 from a 16-day culture on a steamed potato. Photographed on the 7th day, 45 natural size. Of 9 shoots on 6 plants all of which were inoculated only one failed to become diseased, and this was one of 4 from the same tuber (perhaps it was older than the other 3 shoots). The disease progressed much faster upward than downward as shown by the tiny shoot which is still unaffected though coming out of the base of the blackened stem at the earth’s surface. Earlier stages of the disease resembled Figs. 191 and 192. 256 BACTERIAL DISEASES OF PLANTS Fic. 194.—Potato shoot from same lot as Fig. 1938 (Bacillus phytophthorus) but from another plant and inoculated at the top. Hothouse temperature ranging from 65°F., to 95°F. Needle-pricks at X, from a 2-day agar-streak culture. Time, 43 hours. German organism (Appel I). BLACK ROT OF THE POTATO: TYPE DASY my top-inoculated plants have I seen any strong tendency of the disease to extend into the tubers such as we see in case of potato shoots inoculated with Bacterium solanacearum. The disease occurs in its worst form during warm moist summers and autumns, but may continue on the tubers through the winter, if the temperature in the store-houses or pits is sufficiently high. In the tubers, the disease does not begin in the vascular ring (contrast with No. IV). In the laboratory raw potato tubers may be rotted very quickly (contrast again with No. IV) by streaking the organism on their cut surface (Fig. 201). Lenticel infections occur on the tubers (Fig. 202). Starch is not de- stroyed and the infection is chiefly intercellular (Fig. 203). Dr. Appel’s principal studies were made upon the potato but he also isolated his organism from diseased comfrey (Sym- phytum officinale). He successfully inoculated it into yellow lupins, horse beans (Vicia faba), green tomato fruits, slices of raw carrot, etc. This disease occurs all over Germany, in Ireland, and in various parts of the United States (Maine, Virginia, South Carolina, Wisconsin). Its distribution is probably co-extensive with the culture of the potato, and I now regard it as one of the most serious diseases of the potato. Cause.—The basal stem-rot is due to Bacillus phytophthorus Appel (not sufficiently distinguished from Bacillus carotovorus Jones, which name is earlier). This is a white, rapid-growing, non-sporiferous, Gram negative, motile, peritrichiate-flagellate (Fig. 204), promptly liquefying, nitrate-reducing, aérobic and facultative anaérobic, acid-forming, gas-forming (but not in the potato), milk-curdling (by formation of an acid), alkali- tolerant, sodium chlorid-tolerant, chloroform-tolerant, dry- air-sensitive, rod-shaped or filamentous schizomycete, forming quickly on agar-poured plates circular, grayish white or, by transmitted light, slightly bluish white, well-developed colonies; and on very thin-sown gelatin plates characteristic, rapid-grow- ing, big, circular (Figs. 205 and 206), opaque-white, fringed (fimbriate-margined) colonies (Fig. 207), floating in a pit of liquefaction (thick-sown plates liquefy too rapidly for these examinations). The buried colonies in gelatin plates appear as 17 OF PLANTS BACTERIAL DISEASES 258 ‘punorsyoRq oy} Ur syuryid jouyUOD “sARp G aoyye poydeasojoyg ‘(siwoA G ALOJBIOGeE AUL UT UIBIS UBULIOD) GIG] ‘T oung snwoyjydopiyd snjpong YIM syotad-o[poou Aq poxenoout oOyeJOd YoOruULIODoJY o}IY AA JO SJOOYQ—'GH] “YLT BLACK ROT OF THE POTATO: CAUSE 259 Checks at either side ., alter 7 days. 5 but two days later, 7 ¢ 19 and in the background. Wie ig. —Bacillus phytophthorus: Same as F 196. HG 260 BACTERIAL DISEASES OF PLANTS shown in Figs. 208 and 209. The organism is white on most media but on Soyka’s milk rice it is pale pinkish cinnamon verging toward vinaceous pink in old cultures (R.). Streaked on slices of raw potato it grows rapidly and characteristically, forming a white slime surrounded by a dark (black or brown) border in the disintegrating flesh of the potato. The disin- tegrating potato shoots also are often very black. Bouillon is Fic. 197.—Base of Fig. 193, 2 days later. The mother tuber, the young tubers and the woody base of the stem are still sound. clouded very quickly and gelatin stabs develop a prompt funnel of liquefaction. Potato juice clouds quickly even in the absence of air (closed end of fermentation tubes) but no gas is formed. Ethyl alcohol in peptone bouillon retards or hinders growth (Fig. 210A). The organism does not form in- dol, and does not grow in Cohn’s solution. It produces a non- volatile acid from dextrose, saccharose, lactose, galactose, and 261 CAUSE ELORATOE ny vy) THE BLACK ROT OF SuOT}R]NOOUL OY, jnq suOTyRy_NOOUL ‘OSRoSIp OY} OF DIURYSTISOA PosvotOUL MOYS Ao], youd -o]poou Joye sAep 9 poydeasoyoyd pur Col “(9 os ot *SUI0}S OY} JO Z Z ouny) dopyo sav] A se squid jo yo] J UO TOYJoOSOYR poprey ) GG Uot AM poye_Nooul IWBS— SOL “Ol OF PLANTS BACTERIAL DISEASES eS | ee ee sO AS aE [Nal AZ the NM fame e dan | Kes (| [\ZAr Ache’ oy} 10 vuAyouoivd ayy Jo AvM Aq Joypo ‘Seseq-Wle}s prBvy oy} YSno1yy uMOp ssed 0} a_qvun AT[OUM SUISTUBSIQ, ‘“oJooAUIOZIYPS ay} Aq padOrsap osoy} JO vo, UT syooys AYyBeYy gnq Suypurds dn ques pry 19072 S + a9, NG ota ¥- ay LS ed 2 > eA “a ears a aval . saseq SUOIS poABO@pUN ay} UM ‘FZ 9UNL UO spe yng ‘OB] PUB CHT ‘SSI sv oWBg—'GET “OIA BLACK ROT OF THE POTATO: CAUSE 263 maltose, and small quantities of gas from innosit (muscle sugar), lactose and mannit. A volatile acid also distills off from pep- tone dextrose cultures (flasks 15 days old). It is a common be- lief (of German origin) that the organism loses virulence readily, but in nine years, in the strain originally received by me from Dr. Rudolph Aderhold in Berlin, and designated m my labora- tory as ‘“‘Appel I,” I have not observed any loss of virulence. The last inoculations made with it by me in June, 1915, on rapidly growing potato shoots by needle pricks, yielded striking Fic. 200.—Cross-section of potato stem several inches above the inoculated base. Enlarged to show the bacteria (Bacillus phytophthorus) occupying a vessel. infections, the tops of the 18 inoculated shoots being entirely destroyed in 5 to7 days. A contaminating non-parasitic coecus form is common (Appel). This is, probably, what Dr. A. B. Frank figured, and supposed to be the parasite. Technic.— Because saprophytes quickly follow parasites in the decaying potato, it is often difficult to isolate the latter, Bacillus phytophthorus being no exception. This sufficiently explains why the causes of such an insistent and annually re- current phenomenon as the rot of potato tubers remained so long undetermined. Not knowing that the same organism could not both begin and complete the destruction of the potato tuber, 11 tested it again on potato tops in April, 1919 (13th year in my laboratory) with the same striking results. See Fig. 211. It was also infectious in 1920. 264 BACTERIAL DISEASES OF PLANTS Reinke and Berthold, Kramer, Frank, Wehmer, and, prior to Appel, all the Germans who studied potato rots (mostly by means of the microscope) were led astray by the non-infectious saprophytes (starch-destroyers, gas-producers, endospore-bear- ing rods, coccus forms, etc.) which soon swarm in the tissues and complete their destruction, but cannot be induced to begin the rot except under very abnormal asphyxiating conditions. Then, of course, almost any refuse-loving organism will grow in the dead tissues, since the potato is a good culture medium for many things. In this connection, read Wehmer’s paper in Sri Fig. 201.—Streak culture of Bacillus phytophthorus Appel on raw potato 36 hours at about 23°C., showing a soft rot bordered by a dark stain. Inoculated from a 48-hour culture on steamed potato. Tuber soaked 30 minutes in 1:1000 mercuric chlorid water before cutting with a sterile knife. The check 14 remained sound. Laboratory experiment of 1914. 14 nat. size. Centralblatt f. Bakteriologie, 2te Abt., IV Bd., 1898, pp. 540’ 570, 627, 694, 734, 764 and point out his fallacies. Appel by his masterly paper (1903) let a flood of light into an obscure situation, because while the writer had proved conclusively 7 years earlier (1896) that a part of the potato rot of the United States was due to a schizomycete, this organism (Bacterium solanacearum) had not been subsequently isolated BLACK ROT OF THE POTATO: TECHNIC 265 with certainty from European potatoes, and its existence did not account for all of our own potato rots, and particularly for those common in the more northern parts of the United States in which the distribution of Bacterium solancearum remained (and still remains) unknown, although it was isolated by us nearly every year from various species of plants received from our Southern States. Fra. 202.—Potato tuber showing lenticel infections due to Bacillus melan- ogenes. At the bottom on the left side and in the center, various infections have fused. From a plunge inoculation by the writer in 1912. Following the appearance of Appel’s paper, his organism was found in the United States by the writer and others (Morse, Jones, ete.), and two or three similar organisms were soon described, e.g., Bacillus solanisaprus Harrison. Morse has substituted van Hall’s name, Bacillus atrosepticus, for Appel’s name but a careful re-reading of van Hall’s Dutch paper (May 266 BACTERIAL DISEASES OF PLANTS 21, 1902) leaves me in doubt. No one now has transfers from van Hall’s original culture, I believe, so as to enable one to clear up the doubtful points and under the circumstances it is best, I think, to retain Appel’s name, especially as van Hall made very few inoculations under natural conditions and as he says of his organism: “On artificial media the parasite loses its virulence very quickly.”’ Moreover, if May 21, 1902, or some Fic. 203.—Stained section of a potato tuber in the vicinity of an infected lenticel (stage of Fig. 202) showing bacteria dissolving the middle lamella and wedging apart the starch-bearing cells. later date is the actual date of publication of van Hall’s thesis, then Appel’s name is at least 2 months earlier than van Hall’s name and the latter, even if synonymous, does not have priority. To isolate this organism, the surface through which it is proposed to enter for cultures should be burned with a hot knife or spatula, or soaked for twenty minutes in 1:1000 mercuric BLACK ROT OF THE POTATO: TECHNIC 267 chlorid water. By preference the entrance should be through sound tissues close to the advancing margin of the rot, from which scrapings may then be made for the poured plates. One should not be discouraged if the first plates yield only saprophytes, but should try in several other places on the same plant or on other plants. The organism grows readily and is easily identified by its behavior in Cohn’s solution, litmus milk, thin-sown gelatin plates, surface of raw potato, ete. Spore-bearing organisms and coccus forms may be eliminated from consideration on the start, also those schizomycetes that produce gas from potato eae 3 ; _* * } -d * _ anced ‘ a » . ee $e * Fie. 204.—Flagellate rods of Bacillus phytophthorus Appel. Stained from a young (2 day) agar culture of Appel I by van Ermengem’s silver nitrate method. Photographed March 29, 1915. x 1000. (Compare with Fig. 176.) juice in the closed arm of fermentation tubes, and all vile-smelling forms. For inoculation it is best to select the base of young shoots of the potato in rapid growth, or the soft tops of older plants, using needle-pricks from young cultures on agar, potato or gelatin. Tubers to be inoculated should be freshly dug (or at least not flabby), sound and flawless, and may be kept either in the open air of the laboratory or in damp air under bell-jars. If many checks are held it is sufficient to wash the surface free from dirt. If none are held, then the part through which the needle 268 BACTERIAL DISEASES OF PLANTS enters must be soaked in 1:1000 mercuric chlorid water for 40 minutes. In wet autumns, in moist soil, there is often a wholesale rotting of potato tubers due to the entrance of the parasite through the lenticels and it is a very instructive experiment to demonstrate lenticellate infection in the laboratory. Sorauer was, perhaps, the first person to see bacteria enter the potato tuber in this manner. The writer saw it many years ago (1886) and has obtained on potato tubers in recent years very typical and beautiful infections by way of the lenticels (Fig. 202), using Bacillus melanogenes (which as received by me was a mix- ture of Bacillus phytophthorus and Bacillus solanisaprus). For this purpose one should select smooth, sound, recently harvested tubers, wash clean and plunge for 30 hours under distilled or autoclaved or even non-sterile tap water, to which a young agar-streak culture of this organism has been added. If the variety is susceptible, numerous infections centering in lenticels should appear within a few days, and soon the interior of the tubers should be wholly disintegrated. The sterility of the water used is of no great consequence so long as it does not contain parasites, and so long as the surface of the tuber itself is not sterile, the aim being to set up conditions like those obtaining in ordinary infections in wet earth. Of course, checks to which the parasite has not been added should be held and these will remain sound unless their surface happens to be contaminated with this or some similar parasite, but the tubers must not be asphyxiated. What proportion of the wholesale rot of potato tubers in the soil in wet autumns is to be ascribed to Bacillus phytophthorus, Bacillus solanisaprus, and similar bacterial parasites, and what to mere saprophytes following asphyxiation cannot be determined until a great many more studies have deen made of the flora of rotting potatoes. Determine FoR THE ORGANISM: 1. Morphology—Size in microns, form, aggregation of elements (Do chains or filaments occur? See No. VI), motility on margin of a hanging drop (How long BLACK ROT OF THE POTATO: MORPHOLOGY 269 does motility persist in cultures?), presence and distribution of flagellee (use Hugh Williams’ stain), absence of endospores (try heating for 10 minutes at 80°C., and spore stains). Any capsule? Reaction to Gram’s stain (examine with the dia- phragm wide open). Is the organism acid-fast? Do involution forms occur? Test in dextrose peptone water. 2. Cultural Characters—Growth on very thin-sown +19 beef-peptone gelatin plates at 20°C. is very characteristic (If the plate contains only 4 to 6 bacteria, they should grow quickly, forming big circular opaque white colonies (Fig. 206) in 3 or 4 days. Compare with No. VI (Fig. 212A.) The rate of Fig. 205.—a. 48-hour gelatin colonies of Bacillus carotovorus L. R. Jones, grown at same temperature and on same batch of gelatin as b. Natural size. b. Gelatin colonies of Bacillus phytophthorus Appel. Photographed natural size after 48 hours at 18°C. liquefaction in gelatin stabs resembles that of Bacillus carotovorus. Appearance in agar plates, streaks and stabs. Growth on steamed potato. Behavior on slices of raw potato (select smooth, sound tubers, wash, soak 40 minutes in 1:1000 mercuric chlorid water, slice when dry with a sterile knife, and put into shallow-covered culture dishes or deep Petri dishes). Why does the black stain not appear also in the cultures on the cooked potato? What is tyrosinase? Does it play any part here? Why does the dark stain disappear in later stages of the rot in raw tubers (Fig. 201)? Growth in bouillon, nitrate bouillon, Cohn’s solution, Uschinsky’s solution, Fermi’s solution (Does this medium become yellowish or greenish?). Growth in milk (Is the casein thrown out of solution by an acid or by a lab ferment? What is the odor of the fermented milk? Does this 270 BACTERIAL DISEASES OF PLANTS Fic. 206.—Thin-sown +10 beef-peptone gelatin plates of Bacillus phy- tophthorus isolated from a South Carolina potato in 1917. Colonies exactly like “Appel 1” from Germany. 45 natural size. BLACK ROT OF THE POTATO: CULTURAL CHARACTERS 271 organism produce a bad odor on any medium?); What is the early and late behavior of this organism in lavender-cclored litmus milk (watch closely from the start)? On a proper use of Fria. 207.—Bacillus phytophthorus (Appel I). On +10 beef-peptone gelatin held for 3 days at 16°C. All but the corona has liquefied. Actual diameter of colony, 1 mm. Margin fringed like Bacillus carotovorus (see Figs. 180, 181). Photographed February 4, 1915. Buried colony below at left. The specks scattered about are due to dust on the eyepiece. litmus in milk, consult ‘‘ Bacteria in Relation to Plant Diseases,”’ Vol. I, pp. 48, 196. Test shake-cultures in beef-peptone agar (for gas-bubbles which will appear only if muscle sugar is Fic. 208.—Bacillus phyiophthorus (Appel I). Small buried colonies from same gelatin plate as Fig. 207 but enlarged about one-third more. One colony out of focus. present); try peptone water in fermentation tubes with all the common sugars and alcohols. From which is acid only pro- duced? From which both acid and gas? 272 BACTERIAL DISEASES OF PLANTS If fermentation tubes are not available, shake-cultures may be made in litmus-peptone agar containing 5 per cent of the carbon compound to be tested (examine early and frequently for gas-bubbles and change of color). Study behavior in potato juice in fermentation tubes. The closed end should cloud. Is any gas formed? I have never seen any. Test suitable cul- tures for indol, for hydrogen sulphide. What is the nature of the acid, or acids, formed by this organism? Can they be driven off by boiling (test the vapor with neutral litmus paper and ob- Fig. 209.—Small buried colony of Bacillus phytophthorus (Appel I) after 5 days in gelatin at 16°C. The fringe looked like individual bacteria but con- sisted of lenticulate colonies as determined by staining in situ. Colony about 0.4 mm. in diameter. Not all the buried colonies showed this fringe. serve the litmus reaction of the concentrated fluid)? Is lactic acid produced? Determine toleration of acids. The organism is sensitive to acids (Appel). 3. Non-nutritional Environment.—Effect of heat? of dry air? of sunlight? of freezing (salt and pounded ice)? of salted bouil- lons (try 5 per cent first)? of chloroform in bouillon? of weak acids? of sodium hydrate (try —40 first)? of germicides? Can you get any growth on culture-media at 5°C. or at 40°C.? Try several sorts, e.g., potato broth, peptone-beef BLACK ROT OF POTATO: NON-NUTRITIONAL ENVIRONMENT 2/3 Fic. 210.—A. Bacillus phytophthorus in +15 peptone-beef bouillon with varying per cents of ethyl alcohol (4 to 7 per cent). Clouded check tube at the right, time 6 days. The others clear. B. Bacillus carotovorus (old 3a) ditto. All showing growth. The white at the bottom of the tubes is a reflection, not a precipitate. C. Same as B, but photographed against a white background. Introduced to show how the camera may deceive. 18 274 BACTERIAL DISEASES OF PLANTS bouillon, milk, steamed potato, nutrient agar, ete. Inoculate by needle punctures susceptible tubers (of more than one vari- ety) and place one or more of each sort at the following tempera- tures: 5°C., 8°C., 20°C., 30°C. Repeat if you are not satisfied. What do you conclude? Is there any practical application? If you have time, try also dry storage vs. damp storage at differ- ent temperatures, inoculating as'before. Select tubers of some variety whose flesh reddens or browns quickly on exposure to the air, pare, grate quickly, squeeze the juice at once through a cheese cloth into a narrow, tall jar, divide into two equal portions, and steam one immediately (to destroy the action of the oxidizing enzyme); allow the other portion to oxidize freely in a shallow dish for 6 hours or more, then steam. The two lots may now be tubed, re-sterilized and comparative inoculations made, using, for each: a, one carefully measured l-mm. loop from a very young fluid culture; b, the least quantity that can be withdrawn by dipping the end (149 centi- meter) of a platinum needle into the culture fluid. Watch the early stages of growth critically. What do you conclude? FOR THE DISEASE: (1) Signs.—What is the period of incuba- tion—on stems of different ages? on tubers? Time between local rot on the base of the inoculated shoots and a general ap- pearance of disease? What changes occur in the foliage? How soon after the above-ground signs do the tubers begin to rot? This is best studied in the open field in summer and autumn. Can the tops be destroyed without causing a rot of the tubers? How do youaccountforthis? Isthebrown or black stain ever ab- sent from the stems when this organism is present? There isa bacterial rot in which the brown stain is absent (Appel). To what is this colorless rot due? Describe the disease. What are the signs on the tomato? Try inoculating green tomato fruits. Is it a common disease of the latter? Can other plants be infected? Try all the plants that are rotted and not rotted by Bacillus carotovorus. Study the flora of many naturally rotting potatoes, especially the advancing margin of the rot, for the presence of this organism. Histology.—Section diseased stems and tubers and make per- manent mounts. Do the bacteria follow the vessels, or only BLACK ROT OF BOAO HISTOLOGY > ) Test on White McCormick potatoes showing continued virulence of Bacillus phytophthoris (Appel I) after 1: ihil years on culture the ») VIG. 16 shoots at the right were inoculated by needle pricks in Checks at left. The L got the same laboratory: tops, April 10, 1919, and photographed April 18. media in my Rose and EK. ( Jobbler results on 21 shoots of Blush, My experiments lend no countenance to the statement that b. phytophthorus loses virulence readily. in 1920 (14th yr. ). 276 BACTERIAL DISEASES OF PLANTS a Fig, 212.—A. Thin-sown (+10) peptone-gelatin poured plate of Bacillus carotovorus. Kept for 4 days at 18°C. Compare with Figs. 205, 206 of Bacillus phytophthorus. BLACK ROT OF POTATO: HISTOLOGY rarer occupy them incidentally to the general destruction of the paren- chyma? Compare with sections of potato plants attacked by Bacterium solanacearum. Study the disintegration of the tuber. Are cavities formed? What is the earliest stage of the rot? What becomes of the starch? Of the cell-wall? Is any gas formed out of the tissues of the potato? Compare with Bacillus carotovorus (No. VI). Inoculated, rotting tubers placed at 5°C. for 10 days offer a good opportunity to study the reduction of bacterial growth and the development of a protective cork barrier (See also No. VI). Cut sections until you have made out the newly formed cork layer clearly. Use some good cork stain if necessary. Make sections through lenticels on the tuber in very early stages of infection to show the bacterial penetra- tion. Cut onthe microtome from paraffin-infiltrated material. These sections should be made not much later than the third day, v.e., as soon as a trace of infection is visible on the surface (using a hand lens). 1000. young spots. Is it always stom- atal? Is it generally so? In well- developed spots on the pods, determine the manner of extrusion of the bacteria. Is it always stomatal or may it be through rifts in the tissues? (Sometimes the damp chamber may be of assis- tance in determining this.) Still using the pods, make cross- sections (free-hand and on the microtome) of spots in various stages of development to determine what tissues are invaded by the bacteria and how this invasion takes place. Make perma- nent preparations. Young pods will cut much better than old ones, but tissues of all parts, and of all plants, for that matter, will THE BEAN BLIGHT: HISTOLOGY 295 cut better if the air is pumped out of them when they are first put into the acid-alcohol or other fixative. Do the same things with the leaf spots. Is there any increase in the number of chloroplasts in cells surrounding the leaf spots? To what is the russet or rusty-red phenomenon due? What becomes of the cell-walls? How is the middle lamella dis- posed of? Ziehl’s carbol fuchsin is a good stain. Is the tissue killed in advance of bacterial occupation? What are your reasons for thinking so? Fic., 228.—Agar-poured plates of Bacterium phaseoli (Idaho isolation). Inner one-half of each exposed to bright sunlight on ice: a, 2 minutes; b, 5 minutes, and then incubated for 6 days. Much less sensitive than Bacterium malvacearum. Compare with Fig. 249. Photographed June 16, 1915. 14 nat. size. Does the organism frequently penetrate the seed coats? On stem-inoculated plants how far can you trace the bacteria in the vascular bundles? Is the phloem attacked? Variability.—Under field conditions different varieties of beans show marked differences in susceptibility. If the student has opportunity he should study variability in the field and garden, being always on the lookout for resistant varieties and hardy individuals in susceptible varieties. Make careful (and legible) pen notes of what you have seen. Transmission.—We know nothing concerning living carriers of the organism but, owing to the free oozing of the bacteria to the surface, any bird or insect might act as a carrier from diseased to healthy surfaces, as in case of fire-blight of apple 296 BACTERIAL DISEASES OF PLANTS and pear (No. XII), the only subsequent agent necessary being rain or dew. That a considerably greater number of hours of continuous moisture (than under experimental, fresh- culture, hothouse conditions) is necessary to insure infection under natural field conditions, is shown, I think, by Halsted’s observation that in a New Jersey bean field nine-tenths of the infections were on the west side of the pods, 7.e., on that side Fig. 229.—Bacteriuwm phaseoli: Buried and surface colonies on an agar poured plate. Idaho isolation. Plate poured August 24, 1914. Photographed August 28. 10. Oblique lighting. likely to remain damp longest because shaded from the morning sun. (In this connection read comment on black spot of the plum in “Bacteria in Relation to Plant Diseases,” Vol. II, p. 62). Can you verify Dr. Halsted’s observations? Do you think that what he saw could have been due to driving rains from the west? In that case the infection should sometimes occur most abundantly on the east side of the pods. Watch for this. Barlow proved by transfers to culture media that Bactervum THE BEAN BLIGHT: TRANSMISSION 297 phaseoli can live over winter on naturally infected seeds (kept both in the pods and in sterile test tubes) and also determined that such seeds were not injured beyond the power to germinate and grow, but does not state that he traced the disease on into the seedlings derived from such seeds. It has not yet been proved experimentally that this disease is commonly carried on the seed, unless we may assume that Edgerton has done so, but such I believe to be the case (read what is said under Nos. II and III, and respecting Rathay’s Disease of Orchard Grass in “Bacteria in Relation to Plant Diseases,’ Vol. III, p. 160, and make all the observations and experiments you can). My reasons for this belief in addition to Barlow’s statement, and Edgerton’s, are the facts drawn from my own observations that the bean bacterium is not very sensitive to dry air, and that it may pass entirely through the walls of the pericarp and infect the seeds without destroying them, 7.e., as they are approaching maturity. Such seeds, which are usually more or less distorted, should be saved in large numbers in sterile tubes and tested from time to time through the autumn, winter, and spring, to determine: (1) whether Bacterium phaseoli can be cultivated from many of them; and (2) especially whether seedlings grown from such seeds (in autoclaved soil and watered with boiled water) do commonly become infected. Here is a definite, interesting, practical problem, easy of solution—given a bean field containing an abundance of the disease and a student with some aptitude for research. Five hundred or a thousand diseased bean pods would not be too many to save for such an experiment. A little preliminary observation will determine what pods should be selected, 7.e., how badly diseased they must be externally to warrant belief that the pericarp has been perforated. Bean cotyledons are often distorted and bear rusty spots when they emerge from the soil. Is any part of this phenomenon the disease in question, or is it all due to fungous infection? If Barlow’s conclusions can be generalized, as seems prob- able, we shall have a very simple and practical way of dealing with this disease. 298 BACTERIAL DISEASES OF PLANTS Fic. 230.—Buried and surface colonies of Bacterium phaseoli (Idaho isolation) in a thin-sown +15 peptone-beef-agar poured plate. The smooth, wet-shining, yellow, surface colonies show internal markings by direct transmitted light. Plate poured April 11, 1919. Photographed April 18. Xx 10. Temperature 25°C. THE BEAN BLIGHT: TRANSMISSION 299 If the organism is generally carried on the seed, then seed treatments are in order, and these should be made anyway, until it is known that they are of no avail. In this connection see Part I, page 69. Another important subject for field study is the discovery or production of good resistant varieties. The subject is very hopeful if the right persons take hold of it. Fic. 230*.—Agar plate of Bact. phaseoli showing a common form of colony referred to in the text. Organism isolated by Florence Hedges in 1917 from a Maryland bean pod and used many times for successful inoculations. Plate poured May 22, 1920, and photographed June 4. X 5. LITERATURE Bracu: Bull. 48, N.S., N. Y. Agr. Exp. Sta. (Geneva) 1892. SmiruH: Proc. Am. Assoc. Adv. Sci., Vol. XLVI, 1897, pp. 288- 290. Species here named Bacillus phaseolv. SmiTH: Bull. 28, Div. Veg. Phys. and Path. U. 8. Dept. of Agric., 1901. HAnsrep: Bull) 151, N. J. Agr. Exp. Sta., 1901. Bartow: Bull. 136, Ontario Agr. Exp. Sta., 1904. SACKETT: Bull. 1388, Colorado Agr. Exp. Sta., 1909. EpGerton and Moretanpb: The bean blight and preservation and treatment of bean seed. La. Agr. Expt. Sta. Bull. 139, Baton Rouge, La. 1913. Rapp: Aged bean seed, 2 control for bacterial blight of beans. Science, n. s., Vol. L, Dec. 19, 1919, p. 568. See also “‘ Bacteria in Relation to Plant Diseases,’’ Vol. I, hie '62- Volo il, Rie. 15 and pl. f7 (Figs 3). IX. McCULLOCH’S CAULIFLOWER SPOT Type.—This is a disease of cauliflower and cabbage, char- acterized by a copious stomatal blotching, spotting or specking of the green leaves, both on the veins and in the parenchyma. When the midrib and the veins are attacked early and seriously, the leaves become variously puckered and distorted (see also Nos. VIII and XI). Seen by reflected light, the spots are at first water-soaked, then brownish to purplish gray, but by trans- mitted light they appear thin and almost colorless in the center, with a dark border. The spots on cabbage are darker than those on the cauliflower. The individual spots, which occur in great numbers, are usually small (mere points to areas 1 to 3 milli- meters in diameter, seldom larger), and are circular when quite small but soon become more or less irregularly angled, owing to limiting veins. By coalescence, elongated, irregular, and much larger spots occur, making the leaves look ragged. The badly spotted leaves also turn yellow and fall off (8 to 5 weeks after infection). The disease was not observed naturally infecting the bleached flowering parts of the cauliflower in the Virginia material, nor was it obtained by spraying such parts, except sparingly on some of the larger flowerstalks, but later was obtained on cauli- flower heads from Florida and produced on the cauliflower heads in one of our hothouses by pure culture inoculations. Probably the stomata do not function as readily on the bleached parts as on the green parts and consequently are less likely to be entered by the bacteria. All of the infections, so far as observed, are stomatal, each small round spot having a single stoma in its center, below which is a bacterial pocket. By spraying water-suspensions of young agar-streak cultures upon cauliflower plants and cabbage plants, numerous infections were obtained (Fig. 231) but mostly on the under surface of the leaves. When the infectious spray was confined to the upper surface of the leaves very few spots 300 MCCULLOCH’S CAULIFLOWER SPOT: TYPE 301 Fre. 231.—Cauliflower leaves attacked and spotted by, Bacterium maculi- colum, McCulloch’s cauliflower parasite. A pure-culture inoculation. Time, 13 days (May 19 to June 2, 1909). The infections are stomatal. 302 BACTERIAL DISEASES OF PLANTS were obtained. Time of day might make a difference. The old and the very young leaves appear to be partially or wholly immune (see observations under Nos. VIII and X). Fie. 232.—Spotted cauliflower head from Sanford, Florida, April 3,1916. Gray to black discoloration of the epidermis and deeper tissues of the inflorescence. Plates were poured by Miss McCulloch on April 4 and Bacteriwm maculicolum was obtained and used for successful inoculations. The period of incubation is short, numerous spots being visible within 3 to 6 days from the time of spraying, and always beginning, as in the spots on the naturally infected field cauli- flowers, in the substomatie chamber. MCCULLOCH’S CAULIFLOWER SPOT: TYPE Fic. 233.—Peptone-beef-agar poured plate (+15) of Bacterium maculicolum plated April 28, 1916, from spot on a cauliflower leaf which was inoculated April 24 with the Sanford, Florida, organism. Photographed May 2, with oblique trans- mitted light for internal colony markings, the surface being smooth. X 10. 304 BACTERIAL DISEASES OF PLANTS Little is known regarding the geographical distribution of this disease. It was first received at the Laboratory of Plant Pathology in Washington on cauliflower leaves from Southeast- ern Virginia in April and May, 1909. Subsequently (1912) something on cauliflower resembling it was received from Pal- metto, Florida, and Miss McCulloch succeeded in isolating an organism that culturally seemed right but no inoculations were made. Later (1916) from cauliflower heads grown at Sanford, Florida (Fig. 232), the organism was plated out (Fig. 233) and with it pure culture inoculations were obtained (Fig. 234). In 1918 it was received from Prof. H. H. Whetzel on cauliflower leaves collected in Ithaca, New York (Fig. 235). Atfirst Prof. Whetzel thought that the disease was only a peculiar form of the black rot (see No. II) but since the leaves were without marginal infection and the petioles were not diseased, and the spots did not yield any yellow organism, he sent the leaves on to me to know what it was. Miss McCulloch obtained from it her Bacteriwm maculicolum, with pure cultures of which she secured typical infections on cauliflower in one of our houses, and from such infections again obtained in pure culture typical colonies of the organism (Fig. 236). In April, 1919 we received it from the New Orleans market (courtesy of Lex R. Hessler) on cauliflowers said to have been grown in California. This disease probably occurs also in Australia, that is, in 1900 I received specimens of cauliflower leaves and cabbage leaves from Prof. D. McAlpine in Melbourne, bacterially spotted with what I now believe to be this disease. His letter states that it is the cause of serious damage. The conclusion respecting occurrence of the disease in cab- bages depends upon artificial inoculations made in Washington in 1909 and 1910. We owe our knowledge of this disease to the researches of Lucia McCulloch, carried on in my laboratory during the years 1909, 1910, 1911 and later. No one else appears to have written upon it. Cause.—The cauliflower leaf-spot is due to Bacterium maculi- colum McCulloch. ‘This is a moderately growing, white, motile (even after 4 months in beef bouillon at 0.5°C. to 1.5°C.), 1-5 Fig. 234.—Bacterium maculicolum: Cross-section of cauliflower inflorescence (buds) showing depth of black spotting. From a pure-culture inoculation made November 20, 1916, using the Florida organism. Collected and fixed December 7. Slide 1310B5, second section, upper row. Carbol fuchsin stain. 16 mm., 4 oc. 50 bellows. 20 306 BACTERIAL DISEASES OF PLANTS polar flagellate (Fig. 237), non-sporiferous, Gram negative, non- acid-fast, liquefying (gelatin, not L6éffler’s blood serum), milk- clearing (by a lab ferment with formation of tyrosin), non-gas- forming and aérobic (in fermentation tubes in peptone water with dextrose, saccharose, lactose, maltose, glycerin or mannit), non-nitrate-reducing, green fluorescent (a pale yellowish-green stain in milk, peptone beef bouillon, peptone beef agar, peptone beef gelatin, Fermi’s solution and Uschinsky’s solution), alkali- tolerant (NaOH down to —25 in beef bouillon), acid tolerant (in bouillon up to +34 for oxalic acid, and +36 for malic acid and citric acid), sodium chlorid-sensitive (beyond 2 per cent—at 4 per cent in bouillon motility ceases, and at 5 per cent there is scarcely any growth); chloroform-tolerant; grows below 0°C. but is injured by freezing in bouillon (70 to 90 per cent killed) }, dry-air sensitive (very), sunlight-sensitive (very), heat-sensitive (very), short rod-shaped, catenulate (on agar and in 4 per cent. NaCl bouillon) or filamentous schizomycete (0.8 to 0.9u in diameter), growing on +15 peptone beef agar plates in form of round, smooth, flat, shining, translucent, opalescent, white, entire-margined colonies, becoming | to 3 mm. in diameter in 3 or 4 days at 23°C., at which time or earlier the inner structure is coarsely granular and reticulate under the hand-lens. Later the inner structure becomes finely granular and the reticulations disappear. In thin-sown plates at the end of 7 days the surface colonies are 6 to 8 mm. in diameter, and after 15 days 12 to 15 mm. in diameter. With age the colonies become dull or dirty white, and slightly irregular in shape, with undulate or faintly crinkled margins into which run indistinct radiating lines. The buried colonies are small and lens-shaped. On +10 nutrient beef-peptone gelatin plates, after 3 days at 17° to 18°C., the well-isolated colonies vary from mere points to round growths 2 mm. in diameter. The gelatin is liquefied 1 The statement on p. 13 of Bull. No. 225, Bureau of Plant Industry, is erroneous. Fie. 235.—One of a number of spotted and blotched New York cauliflower leaves received from Prof. H. H. Whetzel in the fall of 1918. The leaves were yellowish and the spots pale green to black. These gave an organism culturally the same as Bacterium maculicolum McC., and this produced the characteristic lesions when sprayed upon cauliflowers in one of our houses, and from one of these Fig. 236 was obtained. Good infections were also obtained with it in Feb., 1920. MCCULLOCH’S CAULIFLOWER SPOT: CAUSE 307 Ene, PR 308 BACTERIAL DISEASES OF PLANTS in cup-like hollows. The margin of the smaller colonies is entire, that of the larger ones is fimbriate. Thickly sown plates are entirely liquefied in 2 days at 15° to 16°C. Peptonized +15 beef bouillon, if inoculated from young vigorous bouillon cultures, clouds thinly in 6 hours and moder- ately to heavily in 24 hours at 24° to 25°C. The growth is best at the surface, where a fragile white pellicle forms. There is no rim, and no pseudozoégloeae are formed. In two days there are heavy clouds and a flocculent white precipitate which is slimy and finally viscid. The medium becomes slightly greenish and small erystals appear in the sediment. In acid bouillons, pseudozoégloeae may occur and the rods become very short (spheroidal). In —17 beef bouillon both filaments and involution forms were seen at the end of 2 weeks. A fragile white pellicle forms also on Fermi’s solution and Uschinsky’s solution, and pseudozoégloeae occur in the latter. In Cohn’s solution it grows without green fluorescence, rim, or pellicle, but with the formation of large feathery crystals. The organism blues litmus milk in well-defined strata from the top downward; no acid is formed, the cultures being dark blue by reflected light even after 6 months. The color-changes in milk likewise proceed from the top downward in definite layers. In 15 to 20 days the whole tube is yellow with a greenish tinge; it is translucent but without de- struction of the fat or separation of whey and curd. In 4 months when evaporated from 10 ce. to 5 ec. the milk is reddish brown and rather thick. The organism is a cool-weather parasite. Its optimum temperature is 24° to 25°C., its maximum (in bouillon and on agar) is 29°C., and its minimum below 0°C. Of 20 bouillon cultures exposed for 10 minutes at 47°C. none grew; of 12 bouil- lon cultures exposed for 10 minutes at 46°C., one clouded after 11 days; exposure of bouillon for 10 minutes at 45°C. killed the bacteria in some of the tubes (less than one-half) and retarded development in the others 3 to 5 days, 7.e., many were killed; finally, exposure in beef bouillon for 315 days at 33° to 36°C. destroyed the organism, 7.e., prevented subsequent clouding at MCCULLOCH’S CAULIFLOWER SPOT: CAUSE Fig. 236.—Surface and buried colonies of Bacteriwm maculicolum plated from the New York cauliflower Three buried colonies coming to the surface. Photographed by oblique transmitted light to show internal colony markings. Plate made from a spot on an inoculated plant. »X 10. 310 BACTERIAL DISEASES OF PLANTS optimum temperatures. No growth could be obtained in the plant or on agar or in bouillon at or above 29°C. (84°F.). The organism is rather long-lived on media but loses viru- lence readily. It stains deeply with carbol fuchsin and by amyl Gram. There is a feeble production of ammonia, indol and hydrogen sulphide. A few cultures in litmus milk showed a slight reduction of the litmus at the bottom. It is extremely sensitive to dry air and to sunlight. Four minutes exposure to direct sunshine killed all organisms in the insolated half of the thin-sown agar plates, exposed bottom up on ice, although the colonies developed as usual on the covered half of each plate. Taken from young, well-clouded bouillon cultures, and dried in the dark at 22° to 25°C. on cover glasses which were then dropped into suitable bouillon, the bacteria were dead on 75 per cent of the covers in 24 hours, on 90 per cent in 48 hours, and on all at the end of 5 days. Technic.—The organism causing this disease grows readily in +15 agar-poured plates when the temperature is under 29°C. (not at all at or above this temperature)! and there are no difficulties in the way of isolation, other than surface con- taminations, which are held in check, more or less, by short ex- posures (20 to 30 seconds) in 1:1000 mercuric chlorid water, after which the spots are crushed in bouillon for the poured plates. 1 T felt so sceptical about 29°C. as the maximum temperature that in January, 1919, I asked Miss McCulloch to do over for me this part of her work, which she did with the following results: Tests of Bacterium maculicolum for maximum temperature. Isolations used: ( Sanford, Florida, Col. 2 of April, 1916. | Floral Stalk, Col. 1, December, 1916. a Leaf, Col. 2, December, 1916. | Mid-rib, Col. 6, March, 1917. B ii Ithaca, New York, Col. 3, November, 1918. ‘ | Greenhouse, Cols. 10, 11, 13 of January, 1919. () 18—25°C: All of the above isolations clouded +15 peptone beef bouillon moder- ately in 16-18 hours. (2) 28.5-30°C. (Incubator heated by electricity and as workmen were chang- ing wires the current was sometimes off.) ‘““A” strains, which have been in artificial media from two to three years, clouded slightly in 18 hours. MCCULLOCH’S CAULIFLOWER SPOT: TECHNIC oe For the inoculations, growing cabbage and _ cauliflower plants 6 to 12 inches high may be used. These, preferably, should be made in infection cages, by spraying the under surface of the leaves with water containing suspensions of young agar- streak cultures. If proper conditions are obtained, good in- fections in large numbers should be available for study by the end of the first week. By ‘‘proper conditions” are meant: (1) moist conditions for 48 hours; (2) growing plants; (3) temperatures under 29°C. (84°F.); and (4) use of bacterial cultures which have not lost their virulence or become attenu- ated by harmful laboratory conditions. Miss McCulloch several times obtained numerous infections on both cauliflower and cabbage plants by spraying, but none in hot weather and none or few with the descendants of old cultures. It is possible, however, that some of her isolations may represent feeble strains of this parasite. Under our rather dry hothouse conditions secondary in- fections were not observed. That is, the infections were limited to the leaves actually sprayed. Determine For THE ORGANISM. Morphology.—Size in microns. Ab- sence of endospores (try heat and spore stains). Motility on the margin of a hanging drop. Persistence of motility in old cultures. Number and distribution of flagella (use van Ermengem’s or Hugh Williams’s method). Conditions “B” strains were not clouded in 18 hours. After three days there was faint clouding in “B”’ strains. In six days the growth in “B” strains is less than in room temperature cultures in 18 hours. (8) 29.5-30.5°C. ‘“A”’ strains were slightly clouded in 24 hours. “B” strains not clouded. “B” strains not clouded in four days. (4) 30-31°C. ‘“A” strains clouded faintly in 24 hours. “B” strains not clouded in four days. 312 BACTERIAL DISEASES OF PLANTS under which chains and filaments are formed. Involution forms. Under what conditions do they occur? Cultural Characters.—Behavior on thin-sown agar-poured plates. Ditto in agar-streak cultures and stab cultures. Char- acter of growths on thin-sown gelatin-poured plates. Ditto in stab cultures and streak cultures on gelatin. Growth in streaks on L6ffler’s solidified blood serum. Behavior on potato. Growth in milk and litmus milk (examine every day). Growth in synthetic media—Cohn’s solution, Fermi’s solution, Uschin- sky’s solution, etc. Action in peptone water on various sugars and aleohols in fermentation tubes. Is any gas produced? Is there ever any clouding of the closed end? Is any acid formed in the open end? Test in appropriate media for pro- duction of indol, ammonia, hydrogen sulphid, amylase, lab ferment, proteolytic ferment. Non-nutritional Environment.—Thermal death-point (begin with 45°C.).. Maximum temperature for growth (try first in +15 peptone beef bouillon at 30°C.). Optimum temperature for growth (use measured loops into bouillon and examine every three hours). Lowest temperature at which growth occurs (try first at 1°C. and continue experiment for six weeks). Effect of dry air (using young bouillon cultures first). Effect of insolation (expose on ice to a bright sun for 5 minutes and hold half of each plate covered as a check). Effect of freezing (salt and pounded ice). Behavior in salted bouillons (try 5 per cent first). Behavior in bouillon over chloroform. Tolera- tion of sodium hydroxide in bouillon (begin with —22). Tolera- tion of organic acids in bouillon—tartaric, citric, malic, ete. (begin with +30). Action of fungicides. FoR THE DISEASE. Signs.—Describe early, middle and late stages of the disease and determine how long it takes to produce these conditions on sprayed plants at given tempera- tures. Make inoculation experiments at the same time in two houses (or places) having temperatures 10 degrees apart: one 20° “to: 25°@., the other at: 305 toes, C5 Ane. Migs: Vie- Culloch’s statements as to impossibility of obtaining growth or infections at or above 29°C. entirely correct? In that case, MCCULLOCH’S CAULIFLOWER SPOT: SIGNS ole where does the organism summer over? Can you obtain the disease on the bleached flowering parts? Histology.—F ix in Carnoy’s solution pieces of cauliflower leaves and heads showing small spots, carry them through the paraffin-infiltration process, section on the microtome, stain and study. What are your conclusions regarding manner of infec- > : a / u € fs ; ; : ® \ q ¢ te ‘ * i ; oe fe ‘ Ry . int Cre ll Lee 1 ca ee o. > a 4 8 a nae 3 4 . 4 -- he Le Fig. 248.—Flagellate rods of Bacteriwm malvacearum cultivated from an angular leaf spot. Arizona cotton. Stained by van Ermengem’s silver method. x 1000. good growth. At the end of 60 days (same temperature) less than one-half the gelatin was liquefied. On Léffler’s solidified blood serum, there is a copious growth (paler yellow than that of Bacterium phaseoli) with slow lique- faction. At end of 15 days in tubes containing Bacterium phaseoli all the substratum had changed color (darkened) and most of it had liquefied; whereas in tubes containing Bacteriwm malvacearum there was very little change in color (the bulk white) and not one-twentieth part had liquefied. At the end of 30 days the difference in color and amount of liquefaction was 326 BACTERIAL DISEASES OF PLANTS still marked. Experiment repeated in 1919 with the same dis- tinct differences (Fig. 258). On steamed potato cylinders standing in water there is at first a thin, pale yellow, wet-shining growth, which soon becomes copious and entirely fills the fluid, making it solid. The color Fie, 249.—Agar-poured plates of Bacteriwm malvacearum showing appearance 5 days after exposure (on ice) of the right half to bright sunlight for 2 minutes: A. Bacteria isolated from Turkestan cotton in 1909; B. Bacteria isolated from Arizona cotton in 1914. is then Naples yellow to wax-yellow (R,), becoming brownish with age. The potato grays more or less, and the starch is con- sumed (see No. II and No. VIII). Experiment repeated in 1915 using the Arizona organism with the same result. The THE ANGULAR LEAF-SPOT OF COTTON: CAUSE out growth was copious, smooth, glistening and the color after a month was mostly between Ridgway’s light cadmium and his empire yellow (R»), the dark stain ranging from his orange citrine in the slime to his mouse gray in the potato, and all but an insignificant residue of the starch was consumed. In peptone-beef bouillon there is a moderate and persistent clouding, the best growth at first in unshaken tubes being at the top. There is a pale yellow rim and a moderate maize yellow precipitate. There are some pseudozodgloeae. It grows moderately in Uschinsky’s solution with a pale rim and considerable flocculence. It grows feebly or not at all in Cohn’s solution. The thermal death-point lies between 50° and 51°C. but is never 50°C. (tests of 1919 in +15 peptone beef bouillon). The maximum temperature lies between 36° and 38°C.—repeated in 1919 it grew at 35° and not at 37° and after 14 days at 37° the tubes did not cloud at room temperature (6 days). It grows slowly at 10°C. There was no growth in +15 peptone-beef bouillon in 6 weeks at 8°C. Repeated in 1919 faint clouding after 10 days at 8.5° to 10.5°C.; the tubes at 6° to 8°C. remained clear for 21 days (as long as tested). Tubes of plain milk inoculated in 1919 gave after 483 days the result shown in Fig. 259. At this time there were no tyrosin crystals. The whey, sterilized at 54°C., or kept in the thermostat at 38-44°C., caused prompt precipitation of the curd when added to sterile milk and subsequent transfers from these tubes to suitable media showed them to be free from bacteria. In litmus milk the litmus is blued and the casein is thrown down slowly. The litmus may be reduced, but is not reddened, so that the existence of a lab ferment is inferred. The precipi- tated curd is not promptly digested but subsequently most of it disappears (2 months), and tyrosin crystals not visible at first (80 to 40 days) are then abundant (Fig. 260). Compare with No. II (Fig. 93). This organism is extremely sensitive to light, even 2-minute exposures (on ice) being enough to clear the sunlit one-half of thickly sown agar-poured plates, and even 1-minute exposures 328 BACTERIAL DISEASES OF PLANTS Fra. 250.—Agar poured plates showing effect of freezing on Bacterium malva- cearum: Arizona organism. £8. Check plate. A. Same quantity of the culture, sowed after freezing for 1 hour in +15 peptone-beef bouillon. THE ANGULAR LEAF-SPOT OF COTTON: CAUSE 329 destroyed the greater number (Turkestan organism; Arizona organism). Contrast with No. VIII. In various ways this organism resembles Bacterium campestre, Bactervum phaseoli and Bacterium citri but I did not succeed in cross-Inoculating it to cabbages, to beans or to oranges. Fur- Fig. 251.—Part of an agar-poured plate of Bacterium malvacearum enlarged to show fugitive motiling of the surface colonies. From one of the leaf spots shown in Fig. 240. Time, third day. Temperature 23°C. Plating of March 20, 1915. x 14. ther comparisons should be made not only with Bacteriwm cam- pestre, Bacterium citri, and Bacterium phaseoli, but also with Bacterium pruni, Bacterium juglandis, and Bacterium translucens, all of which are closely related. Indeed, some of these names are perhaps synonyms, but this can be settled only by many cross- inoculations and much further study. 330 BACTERIAL DISEASES OF PLANTS Technic.—Sometimes there are difficulties in the way of isolating this organism, owing to the occurrence on the cotton plant of yellow saprophytes which somewhat resemble it. These are often found in the spots or on the surface and are perplexing Fig. 252.—Bacterium malvacearum: part of an agar-poured plate, enlarged to show fugitive mottling of the surface colonies. From one of the leaf spots shown in Fig. 240. Time, third day. Temperature 23°C. Plating of March Pape, MS)NSy, 1000. Determine production of indol using Bacillus coli for com- parison and testing at end of 10 days and 20 days (heat the tubes in a water bath at 80°C. for five minutes, if necessary). Non-nutritional Environment.—Minimum temperature for growth (try first at 2°C.)? Maximum temperature (try first at 34°C.)? Can you obtain any growth at 1°C. or at 37°C.? Optimum temperature (try first in peptone bouillon at 27°, 30°, and 33°C., inoculating copiously from bouillon and examining every 3 hours)? Is the organism sensitive to dry air (to have all the bacteria freely exposed, use young bouillon cultures) ? Effect of sunlight (expose on ice for 10, 20, 30, and 40 minutes in thin-sown agar-poured plates, preferably after a storm, 7.e., 356 BACTERIAL DISEASES OF PLANTS when the sky is free from clouds, dust or haze, keeping one-half of the plate covered). Effect of sodium chlorid in +15 peptonized beef bouillon (begin with 5 per cent and increase to 10 per cent, comparing on the one hand with Bacillus carotovorus and Bacillus coli and on the other with Nos. I and V). OL Fig. 274.—Surface and buried colonies of Bacterium mori on agar poured plates. a. Photographed by direct transmitted light. 6b. The same by oblique transmitted light, showing the internal markings. These colonies are smooth on the surface and glistening white by reflected light but pale brownish by direct transmitted light. X is a thin colony on the bottom of the plate. Time, 5 days. Photographed February 10, 1919. X 10. Growth in test tubes of unshaken bouillon over chloroform (Compare with No. II). Nothing is known respecting the action of germicides. Can’t you make some tests? FoR THE DISEASE. Signs.—Period of incubation in young stems and leaves. Time from appearance of water-soaked THE MULBERRY BLIGHT: SIGNS BOF spots to the blackening of leaves and shoots. How far down the stems in advance of external signs can you trace an internal stain? In what tissues? Effect of the bacteria on the tree as a whole. Describe the disease. Make photographs or drawings. Histology.—Cut, freehand, various cross-sections and longi- tudinal sections of affected stems, and examine at once in water. Fix, embed, section and stain suitable pieces of stem and leaf in early stages of the disease to show the bacterial invasion. Make permanent preparations. Study ooze of the bacteria through lenticels; section young stems some inches below external signs of the blight for presence of tyloses in the affected Fig. 275.—Agar surface colony of Bacteriwm mori showing internal markings when viewed by transmitted oblique light. Colony smooth on the surface. x 23. vessels. Can you find any in normal shoots of this age? Com- pare with tomato and potato stems attacked by No. IV or VI, which also show tyloses. What substances cause their produc- tion (see Figs. 357, 358 and 359 for tyloses induced by purely chemical means)? How many inches can you trace the bacteria downward in the vascular system below the lowermost external indications of the disease? Can the tyloses be traced farther than the bacteria? What stem-tissues are specially involved? For answer to this question examine both the soft terminal part 358 BACTERIAL DISEASES OF PLANTS of blighting shoots and the harder basal part. Is the pith in- volved? Are the medullary rays occupied? Where are the bacteria located outside of vessels, 7.e., within cells or between them? In the leaves do the bacteria make special use of the vessels? Is the parenchymatic tissue killed in advance of its occupation? Parts long diseased are hard to embed on account of entrance of air; you will, therefore, collect unshriveled soft stems and leaves, and fix without delay, using the air pump. Variability.— Little is known. Transmission.—Nothing is known respecting carriers of the bacteria or the natural methods of infection. The organism enters readily through wounds and probably also through stom- ata and lenticels. Settle the latter by experiments, if you have the opportunity. As in fire-blight of the pear, the copious ooze of bacteria to the surface of the blighting leaves and shoots affords abundant material for infecting other parts of the same tree and for transmission to neighboring trees, and it is advisable, therefore, to remove all bighting branches promptly. LITERATURE For earlier notes by the writer read: Science, N. S., Vol. XXXI, May, 1910, p. 792; and Phytopathology, Vol. 4, 1914, p. 34. For Macchiatti’s paper consult Malpighia, Anno. V. Fase. VII-XII, Genoa; 1892, pp. 299-303. . For Boyer and Lambert’s paper consult Comptes Rendus hebd. des Sé de. |’ Acad. des Scr., Paris. Tome CXVII, Aug. 21, 1893; pp. 342-343. Read Doidge’s paper ‘‘ The South African Mulberry Blight”’ (The Annals of Applied Biology, July, 1915, p. 113). See also ‘Etudes sur les maladies du Murier en 1913” by G. Arnaud and Ch. Seerétain, Annales du Service des Epiphyties. Tome II, Memoires et Rapports, Ministére de L’ Agriculture, Paris. Librairie Lhomme, 1915, pp. 233-249, 11 text figs. Consult also “ Bacteria in Relation to Plant Diseases,’ Vol. LiLo ahigs: 3 anc 30. XII. FIRE-BLIGHT OF APPLE, PEAR, QUINCE, ETC. \ (Called also pear blight, apple blight, quince blight, ete.) Type.—Fire-blight, so called since the time of Wm. Coxe (1817), is a time-limited, rapid, parenchymatic decay, chiefly of the pear, the apple, quince, and other pome fruits (Figs. 276, 277, 278). From stone fruits it was first described in 1902 by L. R. Jones who found it on the cultivated plum (Prunus sps.). It was also found independently on the plum by Merton B. Waite. It has been seen on the loquat (Eriobotrya), and on the cherry. It occurs also on certain wild genera, e.g., Crataegus, Amelanchier, Heteromeles (Waite). O’Gara has reported it from the apricot in the Northwest. Heald also has reported finding it on the apricot in Texas. In 1915 the writer cross-inoculated it to apricot readily. He also saw it escape naturally from inoculated pears to a neighboring apricot (Fig. 279) and with the organism plated from a dying apricot twig produced typical blight on pear shoots. Munn has recently reported it as inoculable into the blossoms of the strawberry (Phytopathology, vol. 8, p. 33). Can you find it occurring naturally on the strawberry? Search in the vicinity of blighting pear and apple orchards. It begins by destroying the blossoms, green fruits, and young shoots, including young leaves which are specially favorable places for its development (Fig. 280), but it passes quickly downward, chiefly by way of the bark parenchyma, into the inner bark of the larger branches and of the trunk, which often are girdled and killed. It gets its common name from the con- spicuous black or brown appearance of the blighted branches, the dead persistent leaves of which look as if scorched (Fig. 278). The blackening of the leaves is a host reaction and occurs in the absence of the bacteria, but the bacteria often attack leaves as well as stems, passing from the stem through the petiole into the leaf blade (Figs. 280, 281). As the fruits ripen and as the inner (living) bark of the shoots becomes firm, in late summer or 359 360 BACTERIAL DISEASES OF PLANTS early autumn, the blight ceases to spread, and the organism, in a majority of cases, dies out of the blighted (killed) trunk and limbs, but in a variable per cent of cases the bacteria persist in certain patches, forming what Mr. Waite, who discovered it, has termed ‘‘hold-over blight,” and Prof. Whetzel ‘‘cankers.”’ From these spots (Figs. 282 and 283), which ooze living and Fig. 276.—Fire-blight on a pear tree. Healthy and blighted branches, seen close. Maryland, July 1, 1914. virulent bacteria, especially during the increased sap-flow of the spring, and which are visited by bees and other pollen-gathering and nectar-sipping insects, the exudate being sweetish, accord- ing to O’Gara, the bacteria are carried to the blossoms of neigh- boring trees and a new outbreak is started, the organism, brought by these insects, growing first in the nectar of the flowers or in FIRE-BLIGHT OF APPLE, PEAR, ETC.: TYPE 361 Fig. 277.—Branch of an apple tree showing blighted flowers, fruits and shoots due to Bacillus amylovorus. Time of year may be judged from the size of the green apple below. Season of 1914. 362 BACTERIAL DISEASES OF PLANTS Fic. 278.—A pear tree showing limbs recently blighted by Bacillus amylovorus (from hold-over blight) and at X the old blight, 7.e., that of the previous season. Washington, D. C., 1914. FIRE-BLIGHT OF APPLE, PEAR, ETC.: TYPE 305 bitten or punctured shoots. Sackett believes that we have greatly underestimated the number of cases of hold-over blight, especially on the smaller limbs and twigs, since in a total of 83 blighted pear twigs, examined by him in the winter and spring of 1910 and 1911 in Colorado, 25 per cent contained living blight bacteria. These cultures were taken at the border line joining sound and blighted tissues. On the contrary, working Fic. 279.—Fire-blight on apricot. A natural infection from an inoculated pear tree in one of our hothouses. Photographed June 7, 1915. in Pennsylvania on pruned branches allowed to lie on the earth, Fulton found that the bacteria were dead in nine-tenths of them at the end of a week (35 branches tested). More tests should be made in various localities. Here is a good opportunity for useful experiments — Secondary infections are very common during the growing season, owing to the abundant and fluid nature of the bacterial OF PLANTS BACTERIAL DISEASES FIRE-BLIGHT OF APPLE, PEAR, ETC.: TYPE 365 slime which oozes through natural openings (stomata and lenticels) to the surface of stems, fruits (Fig. 284) and leaves (Fig. 285) in great abundance, where it is visited by insects, and from which is also drips readily to other parts of the tree In the shoots it is primarily a bark disease (Figs. 286, 287). It disintegrates the green fruits by multiplying in the inter- cellular spaces and dissolving the middle lamellae (Fig. 288) and is enormously abundant in such fruits. In cultures I have not observed rapid loss of virulence. A strain carried along on media in my laboratory for 7 years (1908-1915) blighted pear shoots readily, even of a so-called ‘“‘blight-proof”’ sort, when inoculated by needle pricks in June, 1915. Another strain on media 5 years was infectious in 1920. Fire-blight occurs destructively every year in some part of the United States (Fig. 289), and in certain years (as in 1914 and 1915) sweeps over the whole country. It has been known for a hundred and forty years in the eastern United States where it was probably first present on wild shrubs, but its distribution in other parts of the world remains uncertain. It has, how- ever, been reported from Italy. I have been told also that it now occurs in northern Japan on pear and apple, especially on the Red Astrachan (Gentaro Yamada). In Cornwall, England, I saw what looked at a little distance like fire-blight on apple twigs, but on. going into the orchard I found that the twigs had been smothered by lichens. The disease can be controlled by prompt, intelligent and severe pruning (Fig. 290). This is the only remedy known. To avoid spreading the disease by means of the pruning tools they must be disinfected after the removal of each infected limb. The cut surface should also be disinfected (see Part I, page 71). O’Gara recommends 1:1000 mercuric chloride in water applied Fig. 280.—Blight on pear leaves due to Bacillus amylovorus. Introduced to show how petioles and midribs often blight in advance of the leaf-blade. In these leaves both petioles were blackened on their surface (except at X) and were exuding bacterial droplets from numerous stomata, but enough fluid was still passing through their vascular bundles to keep the leaf-blades green and turgid except an extremely small portion of the base of B, and a much larger but still relatively small area along the midrib of A. The invasion came from the bark of the shoot, which was blighted. 366 BACTERIAL DISEASES OF PLANTS Fig. 281.—Growing shoot of Clapp’s Favorite pear inoculated 5 days with a pure culture of Bacillus amylovorus plated from an apple twig. Bark blighted and exuding drops of bacterial slime. Blight running out on petioles (a, a, a) and on leaf-blade (b). Terminal leaves still green and turgid but in another 24 to 48 hours they would have become brown and shriveled. Lowest visible blight at X. Internally the bacteria were traced under the microscope 3 inches farther down the stem. May, 1915. The blue-black stripe of blighted bark and the pale green of the normal part of the shoot photographed exactly alike, both with Ham- mer’s double-coated orthochromatic plates and with W. and W. panchromatic plates. The eye-contrast was finally obtained on the latter plate by using a green color screen. FIRE-BLIGHT OF APPLE, PEAR, ETC.: TYPE 367 by means of a sponge. It is most easily applied on trees that have been trained to a vase shape (Fig. 290). Fhose trees trained to pyramidal form with a single central stem (Fig. 278) are often killed by the first attack of fire-blight. Fig. 282.—Fire-blight canker on apple (Waite’s hold-over blight). Spring condition—bacteria exuding and likely to be visited by bees and other pollen- gathering and nectar-sipping insects. Bark discolored on the right side. After W hetzel. Cause.— Pear blight or fire-blight is due to Bacillus amylo- vorus (Burrill) Trevisan. The original name, Micrococcus amylovorus, was given to it by Prof. Burrill, under the assump- tion that it destroys starch but such is not the case. It isa white, motile, peritrichiate-flagellate (Fig. 291), non-sporiferous, non-odorous, non-acid-fast, Gram negative, non-nitrate-reduc- 368 BACTERIAL DISEASES OF PLANTS Fic. 283.—Bark of an apple tree in spring showing Bacillus amylovorus oozing to the surface from a patch of ‘‘hold-over”’ blight. Enlarged 6 times from a photographic print furnished by Prof. H. H. Whetzel. Planar by James F. Brewer. FIRE-BLIGHT OF APPLE, PEAR, ETC.: CAUSE 369 ing, rather slowly liquefying (gelatin, not L6ffler’s solidified blood serum), more or less viscid (often quite slimy from pear and apple fruits), butyrous on agar and potato (D. H. Jones) aérobic and facultative anaérobic (with grape-sugar, fruit-sugar, cane-sugar, or malt-sugar), non-gas-forming, sodium Fic. 284.—Green pear fruit (Duchess) rotted by Bacillus amylovorus. Result of a pure-culture inoculation. Ooze of bacteria through stomata can be seen in many places above the central area. Below is a larger amount of bacterial ooze from cracks in the vicinity of the needle-wounds. chlorid-tolerant, sunlight-sensitive, dry-air-sensitive, rod-shaped schizomycete, growing on the surface of agar-poured plates in the form of circular, small, entire-margined, more or less opalescent white colonies, and in the depths as smaller, some- times ringed, fringed or hazy-margined colonies (our pathogenic 24 370) Fig. 285.—Blight- ing pear petiole. A detail from Fig. 280 at Y, showing more distinctly the stomatal bacterial exudate (Ba- cillus amylovorus ) from the blackened petiole. On the right side at X the tissue is still green and turgid. BACTERIAL DISEASES OF PLANTS cultures of 1905—not those of 1915; although both were from apple). On thin-sown +15 peptone-beef agar plates, held at 20°C. the homogeneous wet-shining surface colonies may reach a diameter of 5 or 6 mm. by the end of the sixth day (Fig. 292). Their ap- pearance when viewed by reflected and ob- lique transmitted light is shown, on Fig. 293. In tubes of nutrient agar there is a good growth the whole length of the stab. On gelatin plates the colonies are circular and inclined to pile up rather than to spread widely. In some gelatins a rather prompt pit of liquefaction appears around the colony (Fig. 294). The color of inoculated milk remains unchanged but after some days a soft curd settles, and later this is more or less completely digested (Fig. 2954). IRfa lab ferment is produced it must be very sensitive to heat (15 minutes at 57°C.). It does not redden litmus milk, but precipitates the casein usually within a few days (4 or 5). There is often a partial reduction of the litmus, the medium being bluer than the check at the top, paler blue at the bottom (in the curd) and wholly reduced in the middle, ¢.e., changed to a pale brownish white. It grows rapidly in bouillon especi- ally if cane-sugar is added. In beef bouillon or potato broth there is not only a prompt apd heavy clouding with presence of pseu- dozoégloeae (turbidity) but if undisturbed a slight granular pellicle forms. Clouds potato juice in closed end of fermentation tubes without gas. Indol reaction scanty or negative. Tolerant of small quantities of malic acid and citric acid but not of lactic acid. I now doubt if malice acid actually FIRE-BLIGHT OF APPLE, PEAR, ETC.: CAUSE ofl stimulates growth, as Waite supposed. Test. Unneutralized acids of gelatin inhibit growth. Tests may be made. Growth is best when the gelatin is made neutral or nearly neutral to phenolphthalein by use of sodium hydrate. Acids are formed from various sugars. In Uschinsky’s solution no growth, or slow growth, unless peptone is added: growth copi- ous, not viscid (D. H. Jones). In Cohn’s solution no growth or slight (Repeated in 1915 with the same result). The opti- mum temperature is 30°C. Growth at 3°C. is very slow (D. H. Jones), and there is no growth at 0.5°C. Exposure in Fic. 286.—Cross-section of a young pear shoot showing cavities in the bark due to Bacillus amylovorus. The bacteria diffuse out of such cavities very readily on staining. Inoculated by the writer in May, 1915. beef broth at 483°C. for 10 minutes retards growth and at 43.7°C. kills (L. R. Jones). The thermal death point in bouillon lies between 45° and 50°C. (D. H. Jones). In liquids all are killed by ten minutes’ exposure to 55°C. (O’Gara). In a recent inoculation (Oct., 1919) using our 1915 isolation from apple, the organism clouded at 43°C. (but with retardation) and finally at 44°C. One also of the four tubes exposed at 45°C. clouded (after 6 days) but none at 48°C. (15 days), when exposed for 10 minutes in the water bath in +15 peptone beef bouillon. The checks grew promptly and the six uninoculated tubes re- Ye BACTERIAL DISEASES OF PLANTS mained sterile. In a repetition of the experiment there was no clouding either at 45° or 46°C. As arule it lives long on culture media. Drying on cover glasses up to 5 days has no appreciable effect (L. R. Jones, D. H. Jones). Tolerates HCl in bouillon (+4) up to +16 and NaOH down to —6 (D. H. Jones). Opti- mum reaction for growth + 0 (D.H. Jones). It resists freezing fairly well. In tests made in 1919, 15 per cent survived. It is identified readily by its behavior under bell jars, when streaked, Fig. on raw green pears, which should rot; and on ripe ones, which should not rot (Waite’s method). This test may be made also in late winter or early spring (in advance of the growing season) on vigorous shoots of the pear by bringing them into a warm room and standing the lower parts in a jar of water until they have begun to sprout (Waite’s method, verified by Katherine Golden). Then with a sharp knife remove the tops making a slant stroke, on which should be rubbed the organism to be tested. If it is a virulent strain of Bacillus amylovorus, the FIRE-BLIGHT OF APPLE, PEAR, ETC.: CAUSE ale shoots will soon begin to show the blight. To keep the cut sur- face from drying out, the jar of water should be covered with a tall bell jar; it should also be protected from direct sunshine. The shoots should be of sensitive varieties, e.g., Clapp’s Favorite or Flemish Beauty, and the organism must be virulent. The following unsupported or wholly erroneous statements respecting the pear blight organism have gained more or less Fig. 288.—Section from a disintegrating pear fruit (like Fig. 284), showing Bacillus amylovorus between and in the cells. Photomicrograph by the writer. x 1000. currency, vz., that it is a Micrococcus, that it is non-flagellate (the flagella are hard to stain), that‘it is a rapid destroyer of starch, that it produces gas and more specifically carbon dioxide or hydrogen, that it produces butyric acid, that it will not grow on agar, that it does not liquefy gelatin, that it is yellowish, that it is red or reddish, that it cannot be found swarming in the leaves, that it causes a disease of peach trees and of Lom- bardy poplar trees, that it commonly passes the winter in dead limbs or in the earth, that it cannot be cross-inoculated from apple to pear and quince or vice versa, that it never enters the 374 BACTERIAL DISEASES OF PLANTS plant in the absence of wounds. Many other misconceptions exist among horticulturists and the uninformed multitude, e.g., that the disease is due to ‘‘thunder and lightning”’ or to ‘‘frozen sap,” but the above include about all that the writer has observed in the writings of scientific men. Technic.— Knowing the biological peculiarities of the pear- blight organism, isolation is not difficult if one attempts it only from freshly blighted fruits or shoots and makes his transfers only from the advancing margin of the diseased parts, using the methods described under No. I. Then usually the poured plates are pure cultures. Isolation from older blight is more difficult, and often it is impossible owing to the prompt death of the organism in the blighted dry tissues. The commonest invaders on the plates are non-parasitic yellow colonies and sometimes only these appear. For inoculation purposes, immature pear and apple fruits are very convenient since they are everywhere available in May and June. They may be inoculated by needle stabs or other wounds at any stage of growth preceding that internal change which takes place when they have reached full size and begin to ap- proach maturity—fruits one-fourth or one-half grown are very suitable. They may be inoculated either on the tree, which is the more natural way, or under bell jars, especially if they are sliced. In the former case they must be protected from insect visitation by covering with a double fold of mosquito netting or with surgeon’s gauze. For inoculations on shoots, it is convenient to have half a hundred small pear trees in pots, some of which must be growing freely. Others which are growing feebly should, however, be inoculated for contrast. Slow-growing and rapid-growing shoots on various kinds of pear trees in the open may also be inoculated (the best time is May—June) but they should be covered with netting to keep off insects and thus avoid the spread of the disease. In the same way if the blossoms of pear, apple or quince are inoculated, which may be either by means of an atomizer, a platinum needle, or a pipette drawn to a fine point, the clusters must be covered, unless it is desired to study the transfer of the OF HT Y > f, FIRE-BLIC “OUTR, aNd ‘ZO6T Jo Jouruns pur ees " “Te Fy surids \\ q Uo Aq yYdeasojoyd wv wor “suds SUIMOT[OJ oy} opvuUl oy} UL 4YSITqG-o1y AG podoajsop ‘oyUO_A aT AotIwBA “pavyoIO Wed SEXO T—'G8Z “OT 376 BACTERIAL DISEASES: OF PLANTS Fia. 290.—Photographs from a Maryland pear orchard showing result of proper eradication of pear blight. Views made in March, 1912. Blighted limbs removed 6 years earlier. From photographs by Merton B. Waite. FIRE-BLIGHT OF APPLE, PEAR, ETC.: TECHNIC old disease by insects. Avoid wounding the flowers in making the inoculations. For varietal contrast, Seckel, Duchess, Douglas, Winter Nelis or Kieffer (which blight slowly) may be compared with Bartlett, Howell, Flemish Beauty or Clapp’s Favorite, (which blight rapidly). There is also a so-called ‘‘blight-proof”’ pear derived from the Chinese Sand pear, which is not blight-proof Fic. 291.—Flagellate rods of Bacillus amylovorus. Stained by van Ermen- gem’s silver nitrate method. Isolation of 1908 from apple. > 1000. An isolation of 1915 from apple stained by Miss Bryan also*showed peri- trichiate flagella of the same type. (Fig. 296), and which may be tested. No entirely resistant sorts are known to the writer but recently Reimer has discovered several. These are stocks of Pyrus ussuriensis and other Asian sorts (collected in China by Frank N. Meyer and by F. C. Reimer). These are now being tested on a large scale in the open field in several localities in the West by Reimer, and in the Kast by the United States Department of Agriculture. O’Gara BS BACTERIAL DISEASES OF PLANTS states that stocks of Kieffer and Winter Nelis on the Pacific Coast are quite resistant, the blight on sensitive varieties often stopping at the point of union when these stocks have been used. Out of season the only means of obtaining material suitable for inoculation is by the use of a forcing house, by use of young Fig. 292.—Surface and buried colonies of Bacillus amylovorus from agar poured plates. (a) The buried colonies have vague, fuzzy margins. X 7. Age, 4 days. Plated from an apple, in 1905. It was with descendants of this isolation that Dr. Rudolph Aderhold, of Berlin, obtained his infections. From the unlike appearance of the buried and surface colonies he thought at first, so he told me, that I had sent him a contaminated culture. (b) Plated from an apple limb in 1915. Buried colonies not fuzzy but also infectious (see Figs. 281, 296). seedlings, or on cut shoots by the simple means described under Type, which, however, sometimes fails. As material for inoculation, streaks on agar or potato may be used, or bouillon or potato-broth cultures. FIRE-BLIGHT OF APPLE, PEAR, ETC.: TECHNIC SYA) en Fig. 293.—A. Surface and buried colonies of Bacillus amylovorus (isolated from apple) on a +14 beef-peptone agar plate. Photographed February 5, 1919, by direct transmitted light. > 10. B. From the same plate but photographed by oblique transmitted light. x 10. 380 BACTERIAL DISEASES OF PLANTS Determine For THE ORGANISM. Morphology.—Size in microns (when growing rapidly in media the rods are often several microns long—3 to 6 or more), form, aggregation of elements, 7.e., chains, filaments, pseudozoégloeae, etc., motility (on margin of hanging drop), absence of endospores, presence and distribution of flagella (use V. A. Moore’s modification of Léffler’s flagella stain, or van Ermengem’s stain). Try Gram’s stain; acid-fast stain; Capsule stains. Do involution forms occur? Cultural Characters.—Appearance of colonies on thin-sown agar plates (Figs. 292, 293) and on gelatin plates; in stabs and streaks on agar, ditto on gelatin; behavior in peptone bouillon, in potato broth; try also malated and sugared broths. Growth in nitrate bouillon, Cohn’s solution, Uschinsky’s solution, milk, litmus milk. Is a lab ferment produced in milk? Behavior in peptone water in fermentation tubes with various sugars, alco- hols, and acids. Try it also in fermentation tubes with potato broth and other plant juices, e.g., apple or pear broth. Is there any clouding in the closed end? Test milk in fermentation tubes. Can you get the results shown in Fig. 295? Determine the nitrogen nutrition of this organism. Non-nutritional Environment—Maximum temperature for growth; minimum? Thermal death-point? Effect of sunlight; of dry air (killed quickly), of freezing (salt and pounded ice), of salted bouillons, of chloroform in bouillon, of acids, of alkah, of germicides. Read Reimer’s papers and experiment with vari- ous germicides. For THE DISEASE. Signs.—Period of incubation (examine morning and night each day); signs in flowers (especially in early stages of the disease—first 72 hours); in shoots (observe that the bark of the shoot may be entirely blackened on the sur- face from the bacterial action and yet for a time the terminal leaves may remain green and turgid. Why?); on leaves: a, di- rect effect, 7.e., dark lines running out along the petiole, midrib or side veins, due to bacterial infection from the shoot; 6, in- direct effeet—black specking, flagging, reddening or browning, due to stem injuries farther down. Note the persistence of the FIRE-BLIGHT OF APPLE, PEAR, ETC.: SIGNS 381 leaves. Learn the appearance of ‘‘hold-over blight.”’ Usually on smooth trunks such patches may be detected readily by the practised eye in the absence of the exudate since their color is somewhat different from that of the normal bark—redder, browner. Under rough bark detection is more difficult and a gouge should be used. Is the dark color of the blighted leaves and shoots a bacterial stain or a host reaction? Kill leafy pear shoots in various ways and see what results follow in the leaves. To what extent are the roots attacked? Write a description of the disease. Fic. 294.—Buried and surface colonies of Bacillus amylovorus after 3 days at 21°C. on +10 peptone beef gelatin. The bulk of the surface colony is floating in the middle of a pit of liquefaction. Isolated from apple in 1915. Photographed by the writer. X 7. Histology—How many centimeters in advance of visible blight can the bacteria be traced down the blighting shoot? What does this teach you relative to pruning for removal of the disease? Is the organism motile in the tissues? Is the wood attacked or only the bark? To determine this, examine in various places from the soft extremity of the blighted shoot downward. Any differences (compare with No. XI)? O Gara, who has had much experience, states that the bacteria may occur in the rich sap wood of the Bartlett, Howell, and other pears and in that of the Spitzenberg apple. According to Reimer this apple is no longer planted in South Oregon. because of its great susceptibility to blight. When the bark only is Bye BACTERIAL DISEASES OF PLANTS involved which suffers most, phloem or cortical parenchyma? Is the ecambium also attacked? What is the color of the nner bark in patches of ‘“‘hold-over”’ blight? What tissues are honeycombed with bacterial cavities? When the organism Fig. 295.—A. Fermentation tube of milk containing Bacillus amylovorus. Col. 6, isolation of 1915 from apple. Inoculated August 18, 1919. Photographed Sept. 27. Milk cleared and curd digested in closed end. 8B. Check tube. runs out into leaves what petiolar tissues does it invade? In petioles I have observed it wedging apart cells of the bark parenchyma and also forming cavities in the xylem part of the vascular tissues. FIRE-BLIGHT OF APPLE, PEAR, If possible, embed and section on the microtome early stages of blossom blight. Can you make out: a, multiplication of bacteria in the nectary; b, invasion of the ovary and pedicel? Is there any choice in the tissues invaded? Study also blight- ing shoots and fruits. The bacterial slime is often abundant enough to drip from the hands when such fruits are handled after cutting. Is the organism always inter- cellular or does it sometimes also enter the cells? There is a difference of opinion on this point. Do not decide too hastily. Is the cell-wall destroyed? What becomes of it? Does the organism commonly come to the surface on attacked stems? on fruits? Make permanent stained prepara- tions showing relation of the organism to the various tissues. Contrast with No. I. Variability—How long does an at- tacked shoot live? Using a_ susceptible variety, study effect on rapidity of blight of: a, rapid vs. slow growth, which may be correlated with time of year (May-— June vs. August-September); b, amount of rainfall or water given; c, moderate vs. high temperatures; d, light vs. heavy inocula- tions, e, kind of cultivation and manuring. Rich soils and alkali soils are said to favor the development of the disease (O’Gara). If you have opportunity to study blighting orchards look for varietal differ- ences. Some commonly cultivated pears are nearly immune, e.g., the Douglas, a seedling of the Kieffer (V. B. Stewart); others are very susceptible. The same is true of apples. Make inoculations on re- sistant and susceptible varieties and record the results. IDNs HISTOLOGY 383 Fie. 296.—Shoot of ‘ Bhght-proof’’ pear (hy- brid between the Chinese Sand pear and some com- mon pear). Inoculated 5 days with a pure cul- ture of Bacillus amylov- orus plated in 1915 from a blighted apple branch. Bark blighted (browned) and beads of slime oozing from the in- bacterial terior through stomata. May, 1915. This variety blighted rather freely as the result of needle prick inoculations. 384 BACTERIAL DISEASES OF PLANTS Whatever you do, make full and legible notes. Transmission.—On account of its large dependence on ani- mals (chiefly on insects) for distribution, this is one of our most interesting diseases. It is quite easy for any student who has the organism and a blossoming apple or pear tree to start blos- som-infection and demonstrate transmission of the disease by bees. This was discovered by Waite. According to O’Gara the disease may be transmitted by at least 50 kinds of insects visiting pear flowers. The disease is common, however, on nursery stock not in blossom, and here bees and flies are probably not the common agents of transmission. Sometimes birds carry the germs on their bills (sap-suckers) or on their claws, which often break and scratch twigs. O’Gara believes that the disease may be in- troduced sometimes through growth cracks (see No. XIII). D. H. Jones has shown that aphides by their punctures may transmit the bacteria, especially on apple trees. Some beetles also are carriers: Scolytus (D. H. Jones); and some bugs other than aphides: Lygus (V. B. Stewart). More recently (1915), A. C. Burrill also has proved the disease to be transmitted by aphides (A. avenae), and by an apple leaf-hopper (Hmpoasca malt). The disease may also be spread by means of pruning tools. Waite saw a nursery block of 10,000 Bartlett trees destroyed by pear blight which was transmitted in the Spring on pruning tools. There was some ‘‘hold-over”’ blight in the nursery and when. the tops of the trees were cut down to the dormant inocu- lated buds the tools were contaminated and the blight was distributed to nearly every tree. In the West, on the apple, it has been found to enter through wounds made by crown galls (O’Gara). It may also enter and blight trunks through soft water-sprouts which for this reason should not be allowed to grow around the base of the tree. According to O’Gara 80 per cent of the initial fire-blight infections in California and South Oregon were through water-sprouts and low fruit-spurs. Heald, in Washington State, has found the disease entering the plant (apple and pear) commonly through the leaves as if by water- pore or stomatal infection (1915). Do apple and pear leaves have water pores? Section a leaf-tooth and see. V. B. Stew- FIRE-BLIGHT OF APPLE, PEAR, ETC.: TRANSMISSION 385 art (1915) showed that it may enter through wounds made by hailstones (compare with No. XIII and XIV). The disease is probably carried sometimes on nursery stock, but not often, I think, owing to the fact that it dies out rather quickly in the dead shoots. Probably this fact also accounts for its not having been introduced into other parts of the world, if we may assume, as seems probable, that the eastern United States is the original home of the disease, and that it has not occurred until recently in any other pear or apple-growing region of the world. Was it introduced into Japan from the United States? Eradication of the Disease Complete freedom from fire-blight may never be hoped for, any more than from any other widespread and highly infectious disease, but there are certain palliatives which if properly appbed will reduce its destructiveness to insignificant proportions. These fall into two main categories: (1) tree surgery; (2) resis- tant varieties, or rather immune stocks for sensitive varieties, since the latter include all our finer sorts of pears and apples, the discarding of which is not to be thought of. It has been demonstrated conclusively by Waite and others that the spring blight is distributed principally by bees and other insects which obtain the infection from oozing patches of ‘‘ hold- over” blight. In the control of this disease it is, therefore, of prime importance to search the trunks, limbs and roots in late autumn or winter and remove all blighted spots. Such removal is equally important if the blight is wind-distributed, as main- tained by Stevens and his associates (Science N.S., Vol. XLVIII, pp. 449-450). If this is done thoroughly over a wide area there will be very little spring-blight. If the eradication of the canker, or hold-over blight, has been neglected the next best thing is to cut out the spring- andsummer- blight thoroughly as fast as it appears, including the neglected cankers, disinfecting the tools from limb to limb and tree to tree, since if you neglect this you will inevitably distribute the bacteria by means of your saw, gouge, knife and p uning shears. Both tools and tree-wounds should be disinfected. Mercuric 25 386 BACTERIAL DISEASES OF PLANTS chlorid, otherwise known as corrosive sublimate or bichlorid of mercury, is generally recommended for this purpose. It is made up in the proportion of one ounce of the poison to one thousand ounces of distilled water, clean rain water or boiled well water. This may be applied as O’Gara recommends by a sponge attached to the wrist or to the buttonhole by means of a string about 2 feet long. The effectiveness of this germicide is destroyed by contact with a metal container. It must be kept, therefore, in glass bottles or wooden pails, never in tin pails. Tools may also be dipped into 5 per cent carbolic acid water or into 1 part of formalin diluted with 9 parts of water. The hands must be kept out of both substances. For notes on Reimer’s newer germicides for fire-blight, consult Part I, page 71. Eventually in this country the pear blight problem, which is a very serious one, will be solved by discarding altogether the susceptible French seedling stocks (Pyrus communis) on which most of our valuable sorts are now worked, and substituting resistant stocks. The most hopeful substitutes are certain East Asian species, notably Pyrus ussuriensis, P. ovoidea, P. calleryana, and P. variolosa. All of these species are very resistant to Bacillus amylovorus, and the main question now appears to be which are the most resistant, and which will prove most satis- factory in. other respects, 7.e., grow equally with the graft, giv- ing to it a firm union and a long life. For details the reader is referred to F. C. Reimer’s very interesting paper ‘‘ Blight Resistance in Pear Trees and Pear Stocks”’ (1916 Ann. Rept. Pacific Coast Assoc. of Nurserymen. Also a separate pp. I-8). More recently (July 3, 1919) Mr. Reimer, who is superintendent of the Southern Oregon Branch Station of the Oregon Agricul- tural College Experiment Station (Post Office Talent, Oregon), has written me as follows: “In my pamphlet I made the following statement regarding Pyrus ussuriensis: ‘This species appears to be immune to pear blight, at least under the conditions in Southern Oregon.’ “This statement was based on inoculation work performed with only a very limited number of wild forms of this species. Since that statement was published we have collected at this Station a very large number of types of this species and have repeatedly inoculated them with the pear blight organism. The results based on four seasons’ work may be summed up as follows so far as Southern Oregon is concerned: FIRE-BLIGHT OF APPLE, PEAR, ETC.: ERADICATION 387 “1, Pyrus ussuriensis is more resistant to pear blight than any other known species of Pyrus. 2. Many forms of Pyrus ussuriensis have so far proved immune to pear blight, failing to blight, even in the tips of the young, tender, vigorous shoots, when inoculated. 38. Some forms of Pyrus ussuriensis blight only in the young tender shoots when inoculated, the blight usually king such shoots back only from one to ten inches, and very rarely as much as fifteen inches, in case of extremely vigorous trees. In such eases the disease is usually confined to the cur- rent season’s growth, although very rarely and in the most extreme cases It slightly enters the previous season’s wood. The disease in these forms has always been confined to that portion of the branch less than one-half inch in diameter, and very seldom has progressed into wood more than one-fourth inch in diameter. ‘These results were obtained in a region where pear blight is extremely viru- lent, on very fertile, irrigated and thoroughly cultivated soil, which produces a very vigorous growth, and where every effort has been made to induce the trees to bight. Up to the present time no natural infections have ever been found on the young trees of Pyrus ussuriensis.”’ LITERATURE For literature, consult various writings of Burrill, Arthur, Waite, Whetzel, L. R. Jones, D. H. Jones (Bull. 176, Ont. Ag. Col.), Stewart (V. B.), Sackett, Bachmann, Fulton, Heald, O’Gara, Merrill, Reimer, Stevens, ete. See also “Bacteria in Relation to Plant Diseases,” Vol. I (1905), plates 28-31, and Fig. 61. For various brief notes on the organism see also the index to Jbid., Vols. I, II, III. The student should learn early how to use literature and should be a wide and eager reader not only of all the newer things but also of old books and papers. Search out all the pear-blight papers from the above hints and make a respectable, chronological bibliography. Aderhold’s note is in Aderhold and Ruhland’s paper on “Die Bakterienbrand der Kirschbiume,” Arb. a. d. Kaiserl. Bio. Anstalt f. Land. u. Forstw., V Bd., 6 Heft, pp. 334-336. Reimer’s paper ‘“‘A new disinfectant for pear blight”’ is in Monthly Bulletin of the [California] State Commission of Horti- culture, Vol. VII, No. 10, Sacramento, Oct., 1918, pp. 562-565. P. J. O’Gara’s paper ‘‘ Pear Blight and Its Control upon the Pacific Coast’’ was published in the Medford Mail Tribune, Medford, Oregon, 1910, and a separate was issued, 8 vo., pp. 1-34. A pear grower’s observations on the disease as it occurred in New Jersey in the first years of the 19th century may be found in Wm. Coxe’s book. Who was Coxe? 388 BACTERIAL DISEASES OF PLANTS The name Micrococcus amylovorus was published by Prof. Burrill in 1882. M. amylivorus is a typographical error of 1883. Fra. 296*.—Illustrations of Pyrus ussuriensis Maxim. Resistant to fire-blight. 1. Rosetted terminal growth at close of season. 2. Leaf showing bristled serratures (34 nat. size). 3. Fruit, showing reflexed persistent calyx lobes (44 nat. size). 4. Ripe fruit cut open, showing easy separation of core. 5. A cul- tivated variety, called Man Yuan Hsiang, (about 24 nat. size). All from photographs by F. C. Reimer. XIII. THE OLIVE TUBERCLE (Syn. Olive knot) Type.—This disease is a conspicuous overgrowth. It occurs on wild and cultivated olives, forming large or small, irregular, spongy or cheesy knots or excrescences (Figs. 297, 298) which decay rather quickly. Attacked limbs are dwarfed or killed, and occasionally the whole plant is destroyed, particularly if small and on irrigated land, but more often the trees are only stunted and rendered unfruitful. New outgrowths often occur around the old dead knots, and also similar growths at a distance from them. Roots, trunk, branches, and leaves, are subject. Often when a terminal shoot is attacked it ceases to grow, even if it had been very vigorous (Figs. 299, 300), and the branch is continued by one or more of the lower side shoots, the terminal shoot dying. Once attacked a tree seldom or never recovers, that is, the disease persists from year to year, and invades new shoots and lower parts of the old (Fig. 301). No tumor-strand occurs and the secondary tumors have the structure of the tissue in which they are lodged, 7.e., the disease is a granuloma. The bacteria are abundant and easily visible, being lodged first between the cells and eventually in intercellu- lar, irregularly branched cavities, around which the tissue often has’a water-soaked or brownish appearance (Figs. 302, 303). During rainy weather the bacteria ooze readily to the surface of the tumor in great numbers (Horne) whence they are washed to other parts of the same tree and carried probably to other trees, entering through wounds to form other tumors. Deep tumors may also arise at a distance from the first tumor and these are due to bacteria which have migrated from the primary tumor by way of the spiral vessels of the inner wood which in such cases are browned, more or less disorganized, and occupied by the gray-white slime of the bacteria (Figs. 304 X, 305). The knot contains or may contain both wood and bark, the vessels. 389 390 BACTERIAL DISEASES OF PLANTS Fia. 297.—Olive branches from Genoa, Italy, bearing tumors due to Bacteriwm savastanot. Collected for the writer by P. J. O’Gara in 1905. THE OLIVE TUBERCLE: TYPE 39] being greatly distorted and reduced in number while the paren- chyma is in excess as in crown gall. Often a large part of the tumor, as in Fig. 306, is composed of bark, and then the tumor is of a soft “cheesy” character. The structure of a young tumor developing on the under surface of a leaf is shown in Fig. 307. Fic. 298. Fig. 299. Fic. 298.—Pure-culture inoculations of Bacterium savastanoi in olive shoots in 1903. Left-hand shoot Gentile, others Nevadillo Blanco. Time, 57 days. The organism used was plated from a California olive knot. 19 nat. size. Fic. 299.—Pure-culture inoculation of Bacteriwm savastanoi made in 1910, on an olive shoot at X, which became dwarfed and died. The lower tumors are later surface infections derived from bacteria that oozed from X. Photographed in 1912. 26 nat. size. This disease has been known since the time of Theophrastus (Savastano) and occurs in all olive-growing regions around the Mediterranean, and also in California, Argentina (Haumann- Merck) and other parts of the world. This same disease, or a very similar one, occurs on the European ash (Frazinus excelsior) in France, Germany, Austria 392 BACTERIAL DISEASES OF PLANTS Fic. 300.—Olive tree from one of the Department of Agriculture pathological hothouses showing result of an inoculation of Bacterium savastanoi (at X). Ter- minal shoot stunted and many secondary infections, due to slight (natural) wounds and the free use of the gardener’s hose. The artificial inoculation was made November 1, 1910, and most of the lower tumors developed the following year. Photographed January 31, 1912. THE OLIVE TUBERCLE: TYPE 393 and Italy. Noack in Germany described this ash disease in 1893 and attributed it to bacteria, solely on the basis of his micro- scopic examinations. Subsequently Vuillemin claimed it to be the same as the olive tubercle with- out, however, giving his reasons. The writer first saw the ash disease in the vicinity of Vienna in 1913. It persists from year to year on the trunk and limbs, attacking chiefly the bark, making rough cankerous thickened patches as large as one’s hand or larger, but also stimulating the growth of the wood so that the cankered part of the trunk or branch may be twice the diameter of the Fie. 301. Fic. 302. Fic. 301.—Olive tubercle. A detail from Fig. 300, showing secondary surface infections. The primary inoculation was in 1910 with a pure culture isolated from material collected by Florence Hedges at Portofino, Italy, in 1910. Photo- graphed December 5, 1912, department of Agriculture hot-house. Tubercles 6 to 12 months old. One-half natural size. Fic. 302.—Cross-section of a young, cheesy olive tubercle (pure-culture in- oculation), showing small brown bacterial areas with water-soaked borders. normal parts above and below it. The micro-organism present in these ash cankers has been studied critically in my laboratory by Nellie A. Brown and myself and is scarcely distinguishable from the olive-tubercle organism morphologically and culturally 394 BACTERIAL DISEASES OF PLANTS in a variety of media and yet is not absolutely identical. With it we have produced typical small cankers on American and Euro- pean species of ash, especially on the European Fraxinus ex- celstor, but no tumors on the olive although repeated inoculations were made on young olive shoots both in 1914 and 1915. The ash organism, therefore, should be regarded probably as a variety of Bacteriwm savastanoi rather than as identical with the olive organism, or as a distinct species, but further studies and comparisons should be made. The oleander in Europe and in some parts of the United States is also subject to a bacterial overgrowth on leaves and shoots, which by various observers has been thought to be due to the same organism as the olive tubercle, but my observations and inoculation experiments lead me to think it is not due to the olive-tubercle organism. ‘This is also Petri’s opinion. Cause.—In 1886 Archangeli described the olive tubercle, giving the name Bacterium oleae to something observed in it but without what we should now consider to be a proper de- scription, 7.e., it was named from the microscope, without cultures or proofs of its infectiousness by inoculation, and with the state- ment that it probably had nothing to do with the disease, which was ascribed by him to other causes. Savastano’s inoculation experiments (1887-1889), repeated and confirmed by Cavara, first proved the olive tubercle to be due to bacteria, but neither one of these men described the organism sufficiently. Savastano called his cultures Bacillus oleae-tuberculosis, but, following Tre- visan, systematic writers generally have spoken of Bacillus oleae (Arch.) Trevisan as the cause of the olive tubercle. Subse- quently Schiff-Georgini isolated and described very carefully a non-infectious, white, spore-bearing, peritrichiate, filamentous, potato bacillus from the olive-tubercle, called it Bacillus oleae (Arch.) Trev., and claimed to have obtained tubercles repeatedly by inoculating it into olive shoots (1905). On the contrary, Berlese (1905) considered a yellow organism isolated by him to be Bacillus oleae (Arch.) Trev., and the cause of one type of olive tubercle. Several saprophytes occur commonly in olive tubercles, one of which at least is yellow, 7.e., that seen by Ber- lese. Savastano must also have had a yellow organism in some THE OLIVE TUBERCLE: CAUSE 395 of his cultures, since he describes the olive-tubercle organism as yellow on media, which it is not. According to Petri this yel- low, capsulate, peritrichiate, liquefying bacillus (which he calls Ascobacterium luteum Babes) splits olive oil, forming freely a fatty acid, and is more tolerant of acids and of meat extract than the olive-tubercle organism. There being nowhere in literature any proper description of the olive-tubercle organism and in certain quarters much scepticism as to the bacterial origin of the growth (vide Robert Hartig’s Lehrbuch, 1900, p. 211, and Alfred Fischer’s Vorles- Fic. 303.—Same series as Fig. 302, but further enlarged to show irregular bacterial fissures with water-soaked borders. ungen, 2 Aufl., 1903, p. 277), the writer undertook (in 1903) to examine the whole question experimentally. For this purpose olive tubercles were obtained from Genoa, Italy, and from Cali- fornia, and cultures and inoculations in parallel series were in- stituted, he results being: (1) repeated demonstration of the pathogenicity of a particular organism; (2) the non-pathogenicity of all the others, including the common yellow species of bacillus and Schiff-Georgini’s white species which was obtained from Kral of Prague who had received it from Kornauth of Vienna to whom it was given by Schiff-Georgini; and (3) a description 396 BACTERIAL DISEASES OF PLANTS of the parasite, which, on account of the reigning confusion respecting the nature of Bacillus oleae (Arch.) Trevisan, was given a new name Bacterium savastanoi, in honor of Luigi Savastano (Fig. 308), who first proved the olive tubercle to be a bacterial disease. With these introductory remarks we may proceed to a description of the organism. Fig. 304.—Cross-section of an olive petiole showing the brown channel of bacterial infection at X. Cut between a primary stem-tubercle (due to needle- pricks introducing Bacteriwm savastanoi) and a deep (unruptured) secondary tuberele on the leaf. See next figure. The olive tubercle is due to Bacterium savastanot EFS. This is a slow-growing, white, non-sporiferous, motile, 1 to 4 polar-flagellate (Fig. 309), aérobic, non-liquefying (gelatin and Léffler’s solidified blood serum), non-gas-forming (see Petri’s statement), non-nitrate-reducing, sunlight-sensitive, heat- sensitive, acid-forming (with grape sugar and_ galactose), THE OLIVE TUBERCLE: CAUSE 397 non-milk-curdling, chloroform-tolerant, sodium chlorid-sensi- tive, acid-sensitive (but not to the acid of Cohn’s solution), alkali-sensitive, slightly viscid (7th day on agar, 3rd day on steamed potato), rod-shaped, or occasionally short filamentous or catenulate (up to 10u or 15y, rarely 40) schizomycete, forming on the surface of +15 peptone-beef agar poured-plates slow- Fig. 305.—A detail from Fig. 304 in the channel of infection, showing the bacteria. Tissues slightly out of focus. 1000. growing colonies, translucent at first then pure white, which on thin-sown plates at 22°-23°C. may be 1.5 to 3 mm. in diam- eter at the end of the third day (2 to 5 mm. at the end of the 7th day) and are circular, flat, smooth, glistening, and entire, or nearly so (Fig. 310), the internal structure under the micro- scope being amorphous or fine granular; the buried colonies are quite small and often biconvex. Young and _ perfectly smooth surface colonies may also show for a short time a 398 BACTERIAL DISEASES OF PLANTS reticulate or fish-scale inner structure, or an opaque white center with a translucent margin. On gelatin the marginal growth of surface colonies or streaks is unlike that of the body of the colony, being undulate-erose, frilled, lobed or incised (Figs. 311 and 312). With 1 per cent dextrose added to the gelatin the colonies are frequently ring-marked (Fig. 313). In +15 bouillon there is a thin clouding but no rim, pellicle or floeculence during the first 4 days; later there is or may be a thin pellicle. In neutral bouillon no rim or pellicle was observed. In undis- turbed tubes of 2 per cent Witte’s peptone water after 5 or 6 days there is a white pellicle which falls as a whole on gentle Fia. 306.—Cross-section of an olive twig at the level of a*Small tubercle. The open place is a bacterial cavity. Tumor composed chiefly of bark parenchyma, the pith and wood cylinder being undisturbed. Result of a pure-culture inocula- tion using Bacterium savastanot. Photographed in 1904. shaking. On steamed potato there is often a soluble brownish stain (tawny or tawny-white). This stain also occurs in some other media, 7.e., water containing peptone and dextrose. Potato starch is acted upon a little, the iodine reaction being purple while that in the checks is blue. According to Petri, potato starch is converted into amylodextrine and maltose. The growth, except as influenced by the above-mentioned brownish stain, is white on all media. Milk is gradually ren- dered translucent (Compare with Nos. IV and XI). Lavender or lilac-colored litmus milk becomes blue. No acid is ever THE OLIVE TUBERCLE: CAUSE 399 formed in litmus milk either with or without cream, nor is the casein of milk precipitated. The organism has only a very slight action on olive oil (Petri, Smith and Brown). Cane- sugar is inverted (Petri, Smith and Brown) ‘There is only a slight indol reaction. The organism stains readily with Ziehl’s ecarbol fuchsin, but not by Gram. It is not acid-fast. It grows readily and for a long time in Cohn’s solution (very often in the form of long rods, sometimes in chains) without fluores- cence (Petri says with it) and with the formation of numerous crystals of ammonium magnesium phosphate. These crystals Fria. 307.—Section through an olive leaf showing structure of a young tubercle developing from the lower surface. Result of a needle-prick inoculation. Palisade tissue undisturbed. become conspicuous in the thin pellicle if the tubes or flasks are left undisturbed for a few days. In Cohn’s solution with 1 per cent dextrose, rods in clumped masses occur. In Us- chinsky’s solution the bacteria are motile and elongated. Repeated in 1919: thinly clouded on 4th day; best growth in top !4 cm.; on 8th day a thin white pellicle, no fluorescence, very thinly clouded; motile, short filaments were present; after 6 weeks still clouded, not fluorescent. The organism grows from 1°C. or below, to 35°C., or a little above. It will not grow in bouillon at 38.5°C. and is always killed in + 15 pep- tone-beef bouillon by 10 minutes exposure in the water-bath at 400 BACTERIAL DISEASES OF PLANTS 50°C. (10-ce. portions in thin test tubes 17 mm. in diameter inoculated from young peptone bouillon cultures). Repeated twice in 1920 with same result, following exactly Petri’s meth- ods. The checks grew promptly. The 20 heated tubes re- mained clear (20 days). However, 50°C. is not the thermal death-point. That is still lower, 7.e., between 43°C. and 46°C. (Miss Brown); over 45°C. (Miss Elliot). It grows very slowly below 5°C. (Petri). Fia. 308.—Prof. Luigi Savastano. (Photograph made in Naples at the time he was studying olive tubercle.) A non-volatile acid is promptly formed from dextrose and galactose and this acid appears to be unfavorable to further growth. Saccharose is, on the contrary, very favorable to growth and less acid reaction is visible when it is used in litmus agar. Air is necessary for the production of acid from dextrose and galactose, 7.e., there is no growth or production of this acid in the closed end of such fermentation tubes as yield it in the ry open end. Lactose or maltose added to litmus agar does not THE OLIVE TUBERCLE: CAUSE 401 increase growth, and an alkaline reaction develops the same as on plain litmus agar (even in 30 days there is no acid reaction). Experiment repeated in 1915 with the same result. Litmus- mannit-agar first blues then becomes slowly purple, or red, like the litmus-dextrose or litmus-galactose agar. Litmus- glycerin agar remains neutral or nearly so for a week or more, Fie. 309.—Flagellate rods of Bacterium savastanoi EFS, stained by van Ermen. gem’s silver nitrate method. > 1000. and then becomes slowly purple, but never red: repeated in 1915 with a good growth of the organism and the same result (tubes under observation 67 days). Merck’s peptone from flesh retards growth (Petri); prevents all growth (Smith and Brown). Liebig’s meat extract retards growth (Petri). Beef bouillon is less favorable to growth than Witte’s peptone in water with saccharose (Petri). A little gas is produced in the closed end of fermentation tubes in Uschin- 26 4()2 BACTERIAL DISEASES OF PLANTS Fic. 310.—Surface and buried colonies of Bacterium savastanot on +15 beef- 92° peptone agar at end of six days at about 23°C. Photographed March 8, 1915. x 10. The shghtly irregular outlines are characteristic. , THE OLIVE TUBERCLE: CAUSE 403 sky’s solution containing 3 per cent xylose and the fluid be- comes acid (Petri). We could not get this result with our xylose. Growth in Winogradsky’s solution (nitrogen-free medium) with 3 per cent glucose, arabinose or xylose, is good, but in the same with 3 per cent saccharose, lactose or mannit, is scarcely appre- ciable (Petri). We could not verify these statements. 1000. heavy (6 to 8 three-millimeter loops); moreover, the plates should not be discarded until the end of the third week. Those colonies which come up on the poured plates from.the 4th to the Sth day or later are more likely to be the organism sought than those which appear during the first three days. The plates may be kept at 20° to 30°C. 28 434 BACTERIAL DISEASES OF PLANTS A very good procedure is to flame lightly and pare away the exterior of the tumor with sterile knives, then remove some of the sound-looking interior, plunge for 3 seconds into mercuric chlorid water (1:1000), then wash for about the same time in distilled water, and crush it thoroughly in a little sterile water or bouillon, using a cold sterile knife-blade on the bottom of a sterile Petri dish (or if great care is taken it may be done cs oe ‘ . Ps : : me : . ‘mig Pes , ee 4 Fig. 332.—Thin sections of crown gall of the daisy. Tissues stained by means of gold chlorid. Nuclei out of focus are visible in each photograph, and at X there is a Y-shaped rod. inside a thick-walled tube in bouillon), using considerable force. The cloudy fluid in the dish should now be pipetted and the mashings scraped and poured into a tube of peptone water, beef bouillon, or autoclaved water, and allowed to stand for has ruptured through to the surface. Stem inoculated by needle-pricks in two leaf axils using Bacterium tumefaciens plated from a tumor on hop. Inoculated September 29, 1916. Photographed November 24, 1916. Nat. size. (See next page.) THE CROWN GALL: TECHNIC Fig. 333.—A. Inoculated crown-gall teratoma on cauliflower. shoot above the naked tumor has a tumefied sarcomatous interior. » / The pale €55) (URE B. Same as A, but a side view. At X the sarcomatous tissue in the shoot 436 BACTERIAL DISEASES OF PLANTS Fig. 334.—Crown-gall teratoma on Ricinus communis (eastor oil plant). Inoculated with hop strain of Bacteriwm tumefaciens in two places by needle- pricks March 25, 1916. Photographed April 29, 1916. 64 nat. size. Leaves reflexed and dying. Secondary tumors on petiole at X. The tumor-bearing plant was of the same age and size as the check when it was inoculated. THE CROWN GALL: TECHNIC 437 oo ee eee ee Fig. 335.—Crown gall on common tobacco, bearing leaves and flower-buds. Leaves twisted, fasciated and tumefied at their base. Inoculated by the writer 438 BACTERIAL DISEASES OF PLANTS some hours to diffuse. Then make the plates, pouring some directly from the tube containing the mashings and others from dilutions of it (several drops of the cloudy fluid into the second tube of bouillon, and after it has stood for an hour, with some shaking, !¢ ec. from this tube into a third tube of bouillon). Rarely does one obtain colonies on plates poured from the dilutions when first made. Moreover, if your material is scanty you will of course save not only the remains of the tumor but also your original tube and the dilutions made therefrom, and pour another series of plates next day, but if there is a green stain or if gas is forming in the tube containing the mashings, then pour only from the dilutions. For these poured plates use +15 peptone-beef agar. On the poured plates all yellowish, orange, greenish, or pinkish colonies, all branching white colonies, and all circular white colonies, if opaque, are negligible. Only those colonies that come up slowly, that remain for a considerable time small, circular, raised and glistening-translucent (watery) need be considered (Figs. 351, 352). Even following this advice some of the colonies selected for the sub-cultures (which may be on agar or potato or in bouillon) may not prove to be infectious, therefore it is advised to experiment with quite a number of eolonies, and to examine them by transmitted light, rejecting all that show a narrow clear zone about the colonies even if they look right by reflected lhght. For inoculation purposes the student has choice of many kinds of plants since many are susceptible. For class work the young and rapidly growing roots of turnips or sugar-beets, the soft shoots of tomatoes, Pelargoniums, castor oil plants, or Paris daisies, and the crowns of young peach, almond, or poplar are recommended. Shoots and crowns of the hop or the European grape, if growing satisfactorily, may also be used; July 7, 1916, on the cut surface of the middle of an internode of the main axis near the top of the plant just before blossoming time, by needle pricks, using a pure culture of the hop strain of Bacteriwm tumefaciens, which had been passed through sunflower. Photographed August 4, 1916. Natural size. There were 11 flower-buds on this date but a day or two later they began to fall off without opening. The big side branches developed after the inoculation. THE CROWN GALL: TECHNIC 439 Fig. 336.—Crown galls on tobacco. A. Teratoma containing perhaps 100 leafy shoots. Inoculated July 29, 1916, by needle pricks on the cut end of the stem (middle of an internode) introducing the hop strain of Bacteriwm tumefaciens from a 48-hour agar culture. Photographed September 1, 1916. 34 natural size. B. Like A but inoculated March 1, 1918 (from hop through sunflower) on a cut internode of the main axis. The white part of the tumor was very smooth, free from chloroplasts and covered by an epidermis, 7.e., the malignant part lay deeper. Twelve of 13 inoculated internodes contracted the disease. Photo- graphed May 8, 1918, natural size, nearly. 440 BACTERIAL DISEASES OF PLANTS tine, BS7 THE CROWN GALL: TECHNIC 44] Fic. 337.—Crown-gall witch broom on the cultivated carnation due to Bacterium tumefaciens (a natural infection). There was no marked tumefaction at the base of the shoots but, as the sections showed twisted tissues, plates were poured and white colonies were obtained. Six of these were sub-cultured and tested by the writer on Ricinus and tobacco. Colonies 1, 3, 4, 5 and 6 failed to produce any growths, but colony 2 gave sarcomatous tumors on both plants and also on carnation. Photographed November 11, 1916, 64 nat. size. 442 BACTERIAL DISEASES OF PLANTS Fig. 338.—Crown-gall teratoma on Pelargonium. From a pure-culture inoculation of Bacterium tumefaciens made by the writer. Photographed January 11, 1916. 4. Time 12 weeks. THE CROWN GALL: TECHNIC 44 Fig. 339.—A. Section of crown gall on tobacco due to Bacterium tumefaciens. Teratoma developing on the cut end of an inoculated internode. Tumor below and a dwarfed shoot above developing out of it. B. Tissue of leaf from A (at X) showing the palisade tissue reversed, i.¢ facing away from the sky. 444 BACTERIAL DISEASES OF PLANTS Fig. 340. THE CROWN GALL: TECHNIC 445 Fig. 340.—A. Crown-gall teratoma produced by needle pricks on cut inter- node of Nicotiana tabacum using hop strain of Bacterium tumefaciens passed through sunflower in 1915. Photograph shows fused tumefied leaves, and sprouts growing out of all parts of them. At XY, X, X, shoots 3 and 4 inches long were cut away. Inoculated March 1, 1918, with 14-day agar-streak culture. Photographed May 8, 1918. Natural size. B. Same phenomena as in A, but on a petiole of Nicotiana sylvestris. Hop, colony 1, check on flask N. Inoculated January 16, 1917. Phctographed March 2, 1917. X 2. There is a continuous tumor on the margin of the petiole with leafy sprouts in five places (X — X). 446 BACTERIAL DISEASES OF PLANTS Fia. 341.—A crown-gall teratoma on common tobacco showing an abnormal organ, shoot (?), leaf (?). Probably a stem, because its vascular system comes off the normal vascular cylinder and is itself an irregular cylinder. This growth is a blunt, cylindrical, curved, horn-like, white body, pale greenish at the swollen base and bearing 20 abortive green or greenish leafy organs (shoots), most of which are borne on longitudinal seams as if on rudiments of decurrent leaf wings. Pos- sibly it is a modified leaf as it does not arise in any leaf axil. The largest and green- est of the leafy outgrowths are 3 at X along a seam which extends to the top of the horn. Stem inoculated in the leaf axils November 26, 1916, with sub-culture of colony 2 (Bacterium tumefaciens) from carnation witch broom (see Fig. 337). Photographed January 24,1917. x 4. ( 44 rALL: TECHNIC ( CROW THE 341. Fig. 448 BACTERIAL DISEASES OF PLANTS Pre. 342. THE CROWN GALL: TECHNIC 449 likewise, soft shoots of young tobacco or the undeveloped disks of the sunflower. For study of the leafy tumors (teratomas), inoculations may be made in the leaf axils or on the cut inter- nodes of various plants. The writer has used chiefly tobaccos and the common hothouse Pelargonium. In the same way, for tumors containing roots, the tops of Impatiens balsamina (the common garden balsam) may be inoculated. For study of the tumor-strand and secondary tumors, the young rapidly erowing shoots of the Paris daisy (Chrysanthemum frutescens) are best, and the inoculations should be made toward the top of succulent stems which should continue to grow vigorously for at least 2 months. The needle should be thrust into the stems immediately under the leaves. Peklo in Bohemia (1915, |. ¢.) obtained very good tumor-strands in the stem of the sunflower by inoculating into the young flower disk, and the writer has verified his statements. Hop does not infect daisy. The result of the inoculations will be successful and inter- esting in proportion to the virulence of the organism and the activity of the plant. Well nourished rapid-growing plants yield much larger tumors than slow-growing ones. ‘To demon- strate killing effects of the gall use young sugar beets or young Nicotiana sylvestris, inoculating in the center of the crown. Cuttings of the Paris daisy, if made in the hothouse in September, should give plants suitable for inoculation in November and December. The slips should be end branches Fic. 342.—Crown-gall teratoma on orange due to Bacterium tumefaciens. Stem inoculated January 14, 1916. This is my No. 48 (hop strain through sun- flower, colony 1). It was made by needle pricks in the region of a dormant bud, but nothing in the way of a tumor developed either in 1916 or 1917. I looked at it many times. Other inoculated orange buds developed slight tumors bearing supernumerary buds and then died. The small firm dark green shoot marked X is an outgrowth of the stem. This appeared in 1917 but with no evidence of any tumor at its base. Three other shoots and about 12 buds developed from the tumor itself and all of them were soft and light green. This young, rapidly growing tumor appeared within 3 or 4 weeks of the time the photograph was made. The tumor is interesting as having remained dormant for two years and then begun to grow rapidly as an embryoma. The rough white part of the tumor is the naked sarcoma. Photographed March 2, 1918. X 3. Sections of leaf C and of shoot y between a and b were cut and examined for a tumor-strand but none was found. 29 450 BACTERIAL DISEASES OF PLANT Fie. 343. THE CROWN GALL: TECHNIC 451 not in flower-bud or in blossom. They should be set into shallow boxes in sand in close rows, and the fohage trimmed up considerably. Here they should remain for about 3 weeks, 7.e., until a callus has formed and roots begin to push. Bottom heat is not necessary, as they root readily. They should then be put into good soil in thumb pots and transferred from time to time (rather frequently) to larger pots so as to keep them growing rapidly. There should be no check whatever in their growth else they will bloom prematurely. Pelargonium slips should be treated in the same way. Peaches and almonds should be planted in similar boxes of sand after carefully cracking and removing the shell, without injury to the kernel. Hard- shell almonds as they come upon our markets are more likely to germinate than the bleached thin-shell almonds. Two or three months must be allowed for growth. Ricinus, tomatoes, tobacco, sunflowers, sugar-beets, and turnips should be grown from seed. Allow at least 2 months. Cuttings of willows and poplars may be rooted in the spring for use in houses the follow- ing winter or spring. Determine FoR THE ORGANISM. Morphology.—tIn various media, size in microns; form; aggregation of elements, 7.e., chains, filaments, pseudozoégleee; motility, presence and distribution of flagella (use Pitfield’s stain); absence of endospores; capsule stain; Gram’s stain; acid-fast stain; character of the involution forms. Cuitural Characters.—Size and appearance of colonies on thin-sown agar and gelatin plates; stabs and streaks on agar; ditto on gelatin; behavior in peptone bouillon (watch early Fic. 343.—Hard crown-gall embryoma, on mango (Mangifera indica). Ter- minal bud inoculated by the writer January 19, 1916, by needle pricks using the hop strain of Bacterium tumefaciens (sunflower Colony 1). This tumor contains 6 distinct centers of embryonic growth. For one of the larger ones (under the arrow) see Fig. 344A. Surface brown except the embryonic parts which were green. The leaves surrounded by the tumor, and appearing to grow out of it, are stem leaves. Time, 20 months (nearly). Photographed by James I’. Brewer, September 11, 1917. 34 nat. size. Actual size of the tumor 614 by 415 by 4 inches. OF PLANTS BACTERIAL DISEASES Fig. 344, THE CROWN GALL: CULTURAL CHARACTERS 453 Fie. 344.—A. Slow-growing crown-gall embryoma on mango. In the center (under X) a folded (twisted) green bud. Notice also a bud at the extreme left, one in the right upper corner, one below it, and one in the center under the main growth. These five I have counted as one of the six centers of embryonic growth. See Fig. 343. This embryonic portion was under observation many weeks but it developed no shoots. X 4.5. B. Cross-section of a very young orange fruit showing crown-gall tumors in the placental region. Bacteriwm tumefaciens (hop through sunflower) was in- oculated February 1, 1916, by needle pricks into the very young ovary. Two locules are infected, the others are normal. No shoots developed. Orange VI, fixed March 10, 1916. Slide 1198A6, stained with acid fuchsin and methyl green. The tumor tissue stains red. Actual diameter of the section (short way) 14 inch. Photographed with 75 mm. planar. 454 BACTERIAL DISEASES OF PLANTS Fic. 345.—Detail of Fig. 344B in tumor region. Free locule at left; infected locule at right; s, young seed the pedicel of which, here torn away, arises from the wall at x, a few sections above this one; ¢, t’, tumor tissue filling the locule. Between t, t/ and x on other sections the tumor tissue is continuous. It appears to have developed from the left side of the loculus out of an appendage like h. THE CROWN GALL: CULTURAL CHARACTERS 155 Fie. 346.—Same as Fig. 345 but from the right side of the locule and further enlarged. Normal septum at right. The rest is deep-staining tumor-tissue. Slide 1198A6; acid fuchsin stain. There are tracheids in the middle of this tumor to the left of the part here shown. Observe also the disorderly arrangement of the tumor cells. 456 BACTERIAL DISEASES OF PLANTS Se Fig. 347.—Crown-gall teratoma on sugar-beet. Plant inoculated by Nellie A. Brown, January 26, 1918, using a sub-culture of Bacterium twmefaciens plated from arose gall. The main axis of the beet is a long way off in the direction of the arrow. All here visible is tumor. Growing out of the rough tumor tissue at X is a small, bright-green, branched, fleshy (tumefied) shoot. Photographed J Nyovalll, AA, GOI. SK (5h THE CROWN GALL: CULTURAL CHARACTERS AD57 stages as well as later ones); growth in nitrate bouillon; Cohn’s solution; Uschinsky’s solution; milk, litmus milk; behavior in peptone water in fermentation tubes with various sugars and aleohols. Is there any clouding in the closed end? ‘Try also various plant juices in fermentation tubes. What acids are produced? Test for formic and acetic. Use flasks of river water with 1 per cent peptone, 1 per cent dextrose and a little ealcium carbonate. Examine at the end of 2 weeks, 6 weeks and 3 months. According to the chemists, aldehyd, aleohol and ace- tone are also produced. Determine its nitrogen nutrition. Non-nutritional Environment-—Maximum, minimum = and optimum temperatures for growth. Thermal death-point in peptone bouillon (10 minutes exposure). Effect of sunlight, of dry air, of freezing, of salted bouillon, of chloroform in bouillon, of acids, of alkali, of germicides. Determine conditions under which the involution forms are produced. Add various dilute organic acids (1 part acid to 9 parts water) in small quantity (5, 10, 15 and 20 drops to each 10 ec.) to 24-hour agar and bouil- lon cultures (holding check tubes), and compare for changes in structure of the organism, 7.e., appearance of involution forms (Y-bodies) after 24, 48, 72, etc., hours. Pour plates the 3d, 5th and 10th days, from both checks and treated tubes, using care- fully measured quantities of the fluids. Any reduction in num- ber of organisms in the acidified tubes? Any retardation in development of colonies on plates poured from such tubes? Oxidases (?) and peroxidases are said to be much more abun- dant in the galled tissue than in the normal tissue. Can you verify these statements? (See Bull. 213, p. 178.) Does this fact have any pathological significance? Is the formation by the micro-organism of acids and alkalies of pathological signifi- eance? (Consult Jour. of Agr. Research, Jan. 29, 1917.) How many distinct strains are there of the crown-gall or- ganism? I do not call every isolation of an organism a strain, but only such as possess distinct morphological, cultural, pathogenic or other characteristics. The two strains I am most familiar with are the Paris daisy strain and the Hop strain, but there are others. Jensen in Denmark isolated a strain from the Paris daisy which is unlike our strain in its serological reaction, 458 BACTERIAL DISEASES OF PLANTS Fig. 348. THE CROWN GALL: NON-NUTRITIONAL ENVIRONMENT 459 that is, the serum of animals inoculated with it will clump its cultures but will not clump the cultures of the American daisy strain, and vice versa. For tHE DisEease. Signs.—Period of incubation for primary tumor (I have observed well-developed small galls on the peach 18 days after needle puncture (Fig. 318), on the al- mond in 10 days; and beginnings on the daisy in 5 days. Under favorable conditions the beginning of galls on sugar- beets may also be seen as early as the 4th or 5th day. Fic. 349.—Flagellate rods of Bacteriwm tumefaciens (hop strain) stained by van Ermengem’s silver nitrate method. Photomicrographed by the writer. >< 1000. Time required for the development of secondary tumors in leaves of the daisy? The shortest time I have observed is 10 days from the time of stem-inoculation and commencement of the primary stem-tumor. Ordinarily, it is longer. For produc- tion of secondary tumors inoculate into leaf-traces immediately under the petiole in rapidly growing Paris daisy shoots. Is the tumor or tumor-strand (which is sometimes visible to the naked eye) green or greenish? How do you account for this? Is it ever brown or brownish? Is it under pressure? Fia. 348.—Top of Impatiens balsamina (the common garden balsam) showing “hairy-root”’ due to inoculation on July 26, 1916, with the hop strain of Bacterium tumefaciens. The stem was needle-pricked in the leaf axils. There was much red stain in the tumors and in the roots (red flowered variety) although the leaves and stems of this plant elsewhere were pale green. There was no red stain in tumors on the stems of white flowered balsams inoculated at the same time. Photographed August 22, 1916. Nat. size. 460 BACTERIAL DISEASES OF PLANTS Is it really a growth from the primary tumor, in the sense of a pushing in between tissues or only a change in more and more distant cells owing to the propagation of a chemical stimulus? Sometimes it would seem to be the one and sometimes the other. If the cells of the tumor do not change position, how do you account for the tissue distortions? Can you find the tumor strand in fruit trees? Can you cultivate the parasite from the tumor-strand? From the secondary tumors? What causes the browning of the cut surface of the tumors? Describe the appearance of the tumors. What effect, if any, do they have on the rest of the plant. Grow sugar beets or é a y ne <_* : : ae ” Fig. 350.—Y-shaped bodies of Bacterium tumefaciens from a young, pure culture treated with acetic acid. Colony 2, resistant daisy, 4 days on agar, then exposed 2 days to 10 drops of acetic acid water (1 ec. acid, 9 ec. water). Smeared and stained with Carbol fuchsin, March, 1915. tobacco (I used Nicotiana sylvestris) and inoculate the center of the big rosette of leaves rather early and observe the results. Is there ever stimulating action at a distance from the tumor? Observe in some of the photographs thickening of the wood on the tumor side of the stem. How do you account for it? Ex- amine the plant for stunting, curvatures, changes in color of leaves, death of parts, etc. Does the location of the gall make any difference? Histology—What is the structure of the earliest visible tumors (10 or 15 days from date of needle-pricks) as compared with structure of the tissue inoculated? How do you account THE CROWN GALL: HISTOLOGY Fig. 351.—Surface and buried colonies of Bacterium tumefaciens (Rose P) on +15 beef-peptone agar. Poured January 26. Colonies up January 31. Photographed February 1, 1917. X 9 circa. BACTERIAL DISEASES OF PLANTS THE CROWN GALL: HISTOLOGY 463 for the small size of the cells? (Young actively growing tobacco stems or daisy stems may be used for this purpose, making shallow pricks.) How many centimeters from the primary tumor to the remotest secondary tumor (using daisy)? On the sunflower shown on Fig. 319, sub. 4, it was 7 inches and on another it was 8 inches (time 5 to 6 weeks) but, of course, mean- while, there was stretching of the stem. Can you demonstrate the tumor-strand? Can you demonstrate the pseudo-stem structure in any of the secondary tumors occurring in leaves of the daisy, the primary tumor being on the stem? Is there any real difference between the structure of the secondary tumors in leaves and those produced on leaves by direct inoculation? Peklo states that he obtained root structure (secondary thicken- ings) in tumors on flower stalks of the sugar beet by direct in- oculation. Is the browned surface of the tumor composed of cork? What is the character of the vascularization of the tumor? Compare the number and direction of the vascular bundles with those of the normal stem and leaf in daisy—in the torus of the sunflower. How do you account for the distortions? For the difference in number of vessels in the two tissues? Does the tumor contain spiral vessels as well as tracheids? Are these abundant or rare? Normal to it or accidental? In cu- cumber leaves which contain spiral vessels and no tracheids I obtained crown galls containing tracheids and free from spirals. Are cambium and phloem normal constituents of the tumor? Can you demonstrate sieve tubes in it? Can you produce tumors without wounding the cambium, Are they vascularized? In such tumors (Fig. 329) how do you account for the tracheids? Are sieve tubes also present? Is there any tendency in these tumors toward the production of primitive and undifferentiated tissues—structures that occur in early stages of growth, or in Fic. 352.—Surface and buried colonies of Bacteriwm tumefaciens (hop strain) from Flask P) on +15 beef-peptone agar at end of 4 days at 25°C. Two buried colonies coming to the surface. The surface colonies are smooth and translucent glistening. Photographed January 13,1917. X14. In agar-poured plates made from old stock cultures the surface colonies of the hop strain are sometimes very unlike those shown on this plate, i.e, they may have a contoured surface and a sinuate margin with a radiate mottled internal structure, yet are infectious. 464 BACTERIAL DISEASES OF PLANTS Fig. 353.—Section of crown gall in Paris daisy showing spindle-shaped tumor- cells. Tracheids at theright. Slide cut and stained by Lucia McCulloch. Photo- micrographed by the writer. Medium magnification. THE CROWN GALL: HISTOLOGY 465 a ® ae e. # 1 Cee. eh ee * ry é . gee ee i ee EE Fig. 354.—Crown gall on Paris daisy: Photographed from another part of same tumor as Fig. 353. Non-malignant part of the tumor at the top. The middle and lower part is composed of large-nucleate round cells which are in very rapid division. The black dots are the deep-staining nuclei. 30 466 BACTERIAL DISEASES OF PLANTS related plants? See Figs. 353, 354, 415, 416, 417, 418, and Bulletin 255, Pl. LVI (for the wide medullary ray) for what I mean. Toward the production of giant cells? What is a giant cell? How does the structure of the teratoid crown gall differ from that of the non-teratoid gall? On tobacco internodes the writer obtained tumors bearing leafy shoots not only from the cambium but also from the protoxylem and from the bark. How do crown galls differ in structure from fungus galls? From insect galls? From nematode galls? How do you ac- count for these differences? For structure of the tumor and tumor-strand, cut cross-sections, and longitudinal sections of leaves and stems (between tumors and through them) from fixed material embedded in paraffin. Stain 6 to 24 hours in a 2 per cent aqueous solution of methyl green, and after rinsing in water gently so as not to wash off the sections, counter- stain 5 to 15 minutes in a 2 per cent aqueous solution of acid fuchsin (not basic fuchsin). Then pass very rapidly through graded alcohols into absolute alcohol, xylol and Canada bal- sam. The right amount of staining should be judged under the microscope as it is proceeding. Do not overwash the sections. Such sections may also be stained in various basic aniline dyes to demonstrate absence of the bacteria in the vessels and intercellular spaces, but the student will hardly be able to dem- onstrate the bacteria in the tissues, 7.e., inside the cells, by means of aniline dyes, unless he should have better success than the writer and his assistants have had. They may be demon- strated by allowing them to diffuse out of the cut tissues in bacteria-free water on slides free from bacteria, 7.e., clean flamed slides, which should then be dried and stained with Ziehl’s carbol fuchsin, which should also be free from bacteria. Both rods and Y’s can be demonstrated in this way. They should be studied under the 2-mm. oil immersion objective, using a No. 8 or No. 12 ocular. Certain bodies which at one time I identified as bacteria are best demonstrated in the tissues by cutting small slices (2 mm. thick) from young and tender galls and throwing them for 24 hours into 5 or 10 ce. volumes of a 5 per cent aqueous solution THE CROWN GALL: HISTOLOGY 467 Fig. 355.—Advancing margin of crown-gall tumor in the soft white pith of a sunflower disk Tumor hard grayish green and exceedingly vascular because derived from the very vascular torus (seed-receptacle). The white parts at the left are surrounded and compressed pith cells. Slide stained with acid fuchsin and counterstained with methyl green. Pith white, vessels (tracheids) blue, tumor tissue red. Consult also Jour. Cancer Research Vol. I, No. 2, Plate XIII, Bigs 52: 468 BACTERIAL DISEASES OF PLANTS of gold chlorid. They are then washed 3 minutes in water and placed for another 24 hours (in the dark) in a 0.25 per cent aqueous solution of formic acid. After this they are washed in water, passed through graded alcohols into xylol and embedded in paraffin in the usual way. Is there not at least a strong prob- ability that these rod-shaped bodies stained by the gold chlorid and supposed to be the bacteria are only normal constituents of the plant cell (mitochondria)? What are mitochondria? (Read a paper by the Lewises in Journal of Anatomy, Vol. 17, 1915, p. 339.) Have you observed any bacteria in the inter- cellular spaces of sound galls? Study the fauna and flora of old galls. Can you find Toumey’s organism ? Have you observed any excess of chloroplasts in the tumor or in the tumor-strand (daisy)? Any bleaching of tumors or shoots from tumors? Any floral pigment in tumors? Try Pel- argoniums, inoculating the tops of growing plants which are nearly ready to develop red blossom buds. ‘Try also red bal- sams, inoculating before the flower buds develop. Any starch? Any excess of sugar or of enzymes? Consult Figs. 353 to 356 for structure of the hyperplasial tumor tissue. Fig. 353 shows spindle-celled tumor tissue and Fig. 354 shows round-celled tumor tissue from the same gall. Fig. 355 shows both the crushing and invasive effect of a tumor which is excessively vascular, because arising from a very vascular organ—the torus of the sunflower. In Fig. 356 which is from Ricinus the glandu- lar epidermis is also involved. When a tumor is deep seated should the pushed-up and thickened cortex, the cells of which are normally oriented, be reckoned as a part of the tumor? If so, why any more than pushed up skin and muscle? Variability—We have found in various isolations from crown- gall of the Paris daisy marked differences in virulence (ability to produce galls), and from certain natural tumors on the sugar- beet (supposed to be crown gall) none of the many typical looking colonies on the agar-poured plates were infectious (we tried perhaps a hundred). From other similar looking natural beet tumors we obtained a very few infectious colonies, but these produced only slow-growing small tumors (Bull. 213, THE CROWN GALL: VARIABILITY 469 plate 36). Further studies must be made. In this connection read what Jensen says in his Danish paper (l.c.). Also two extremely virulent isolations, cultivated for several years in my laboratory, gradually decreased in virulence and finally lost all power to produce galls. Moreover, differences have been observed in the vigor of growth and harmfulness of galls occurring naturally on various fruit trees. The subject, therefore, is not only one of special interest to the pathologist but also one of much complexity and considerable discourage- ment to the nurseryman and tree inspector. Query: May a gall of little harm to one plant infect a soil injuriously for another plant? Should galled apple trees be planted on land that might later receive peaches, raspberries or grapes? Many such queries must be left to the future. The subject is one which invites careful and long-continued experimentation on the part of various experiment stations and boards of inspection. Query: Can you produce the disease on olives? On alligator pears? On onions or on garlics? On daisy with the hop organism? Transmission.—Everything we know about crown gall points to wounds as the usual, if not the only way of infection. Nothing is known respecting insect carriers. In some cases it would seem that the ‘‘heeling-in”’ of sound nursery stock in soil containing the organism has served to infect the youngtrees (O’Gara). How is the disease spread above ground on the limbs of trees? Everything points to nurserymen as the world-wide dis- tributors of this disease. Many of their soils are so badly in- fected that good stock cannot be grown in them. Mr. Waite has shown me young apple trees, in numbers, badly galled on the graft and almost or quite free in the stock, so that we could come to no other conclusion than that the disease was introduced into the nursery on the grafts. I have seen badly diseased pear stock that was shipped into the country from France, and badly diseased peach trees that were shipped into California from the eastern United States, and badly diseased gooseberries that were shipped from Iowa to the Atlantic Coast, and badly diseased roses that were shipped from Ohio to Florida. These are only 4 470) BACTERIAL DISEASES OF PLANTS Fie. 356.—Gland-inoculation of Bacterium tumefaciens on Ricinus at end of 27 days. The needle penetrated more than one layer of cells but the glandular epidermis appears to be involved and is dividing. Slide 1188. aS THE CROWN GALL: TRANSMISSION 471 a few out of many instances of such transfers that have come to my attention in recent years. LITERATURE Read Bulletins 213 and 255, Bureau of Plant Industry, U.S. Dept. of Agriculture (to be had from Superintendent of Docu- ments, Government Printing Office, Washington, D. C., price 40 cents and 50 cents respectively). See also “ Bacteria in Rela- tion to Plant Diseases,’ Vol. II, Figs. 24, 26, 28, 29, and Plates 5a, 5b, 7, 8, and 10; Phytopathology, Vol. I, pp. 7-11; and Brook- lyn Botanic Garden Memoirs I, 1918, p. 448. For suggested relations to cancer see also seven summaries by the writer: (1) ‘‘Le Cancer est-il une maladie du régne végétale?”’ in Proceedings of the ler Congrés International de Pathologie Comparée, held in Paris in October, 1912 (Tome II); (2) ‘‘Cancer in Plants” in Proceedings of the 17th International Congress of Medicine held in London, August, 1913 (volume de- voted to Section III, General Pathology and Pathological Anatomy); (3) ‘‘Studies on the Crown Gall of Plants: Its rela- tion to Human Cancer” (The Journal of Cancer Research, April, 1916); (4) “‘Further Evidence that Crown Gall of Plants is Cancer” (Science, N. S., June 23, 1916); (5) ‘‘Mechanism of Tumor Growth in Crown Gall” (Jour. Agr. Res., Jan. 29, 1917); (6) ‘‘Mechamsm of Overgrowth in Plants” (Proc. Am. Phil. Soc., vol. 56, 1917); (7) ‘‘Embryomas in Plants: Produced by Bacterial Inoculations” (The Johns Hopkins Hospital Bulletin, Sept., 1917). Read C. O. Jensen, ‘‘ Undersdgelser vedrdrende nogle svulst- lignende Dannelser hos Planter.’’ [Investigations concerning some tumor-resembling growths in plants.| Agl. Veterinaer-og Landboh¢jskoles Aarsskrift, Copenhagen, 1918. Serum laboratory No. LIV. This paper embodies ten years’ study of crown gall from the animal (cancer) pathologist’s standpoint. Read ‘‘ Crown Gall Injury in the Orchard”’ by Dean B. Swin- glesand Ho i! Morris, Bully 121, Agr. Eixp.. Sta.,. Bozeman, Mont., Jan., 1918, pp. 124 to 139, with 6 text figures. Their experimental work deals with the effect (injurious) of crown gall on apple trees and covers a period of 8 years. 472 BACTERIAL DISEASES OF PLANTS Read also Peklo, ‘‘ Ueber die Smith’schen Ritbentumoren.”’ Zeitschrift fur Zuckerindustrie in Bohmen. Jahrg. XXXIX, 5 Heft, pp. 204-219, Feb., 1915. Deals with both Bacterium tumefaciens and Bacterium beticolum, using cultures sent by the writer to Kral in Prague. The crown-gall organism was named Bacterium tumefaciens by Smith and Townsend in Science, n.s., Vol. xxv, April 26, 1907, pp.07 l-Ova: ek Deny MISCELLANEOUS I. NOTES ON SOME ADDITIONAL DISEASES The foregoing methods apply to the investigation of all bacterial diseases of plants and in case material is not at hand for the study of those diseases treated of in Part III, some of the following bacterial diseases may be available and in the hands of a good teacher will prove equally serviceable. In passing, I might say that I have abundant alcoholic material of several of the foregoing diseases which I shall be glad to give out in small quantity for the preparation of microtome sections for class use and that whenever I can do so I shall also be glad to furnish teachers of pathology with pure cultures of the various plant pathogenic schizomycetes considered in Part III of this book, but cannot promise to furnish photographs, stained slides or lantern slides, nor any of the organisms mentioned below. 1. Mango Leaf-, Stem- and Fruit-spot—Bacillus mangiferae Doidge. 2. Black Spot and Canker of Plum, Peach, etc.—Bact. prunt EFS. 3. Stripe Disease of Broom corn and Sorghum—Bact. andropogont EFS. 4. Jones, Johnson and Reddy’s Bacterial Blight of Barley— Bact. translucens, J., J. and R. Bacterial Disease of Banana—Bacillus musae J. B. Rorer. Lilac Blight—Bact. syringae (Van Hall) EFS. Wakker’s Disease of Hyacinths—Bact. hyacinthi Wakker. 8. Cobb’s Disease of Sugar Cane—Bact. vascularum Cobb. 9. Rathay’s Disease of Orchard Grass—A planobacter rathayv EFS. 10. O’Gara’s Disease of Western Wheat Grass—A pl. agropyrv O’Gara. 11. Woods’ Disease of Carnations—Bact. woodsii EFS. 12. Walnut Blight—Bact. juglandis (Pierce) EFS. 473 sleek 474 BACTERIAL DISEASES OF PLANTS 13. Coconut Bud Rot—Some form of Bacillus coli, according to John R. Johnston. 14. Larkspur Bhight—Bacillus delphinii EFS. 15. Alfalfa Stem and Leaf Blight—Bact. medicaginis (Sackett) EFS. 16. Stem Bhght of Field and Garden Peas—Bact. pisi (Sackett) EFS. 17. Citrus Canker—Bact. citri (Hasse) Jehle. 18. Lettuce Blight—Bact. aptatum Nellie A. Brown. 19. Metealf’s Soft Rot of Sugar Beet—A planobacter teutliwm (Metcalf) EFS. 20. Tubercle of Sugar Beet—Bact. beticolum Smith, Brown and Townsend. 21. Leaf Spot of Begonia. 22. Leaf Spot of Pelargonium—Bact. erodii Lewis. 23. Aderhold and Ruhland’s German Cherry Blght— Bacillus spongiosus Aderh. and Ruhl. 24. Barss’ Cherry Blight of Washington and Oregon (which is probably the same as No. 23). 25. Angular Leaf Spot of Cucumber—Bact. lachrymans Smith & Bryan. 26. Spieckermann’s ring rot of Potato. Aplanobacter sep- edonicum (Spk.) EFS. 27. Black Chaff of Wheat—Bacteriwm translucens var. un- dulosum Smith, Jones & Reddy. 28. Halo Blight of Oats—Bact. coronafaciens Charlotte Elliott. 29. Leaf Spot of Soy Bean—Bact. glycineum F. C. Coerper. 30. Velvet Bean Leaf Spot—Bact. stizolobii (Wolf) EFS. 31. Celery Blight—Bacillus apiovorus Wormald. 32. Basal glume rot of wheat—-Bacterium atrofaciens Lucia McCulloch. 33. Basket willow disease—Bacillus harai Hori & Miyake. 34. Tobacco Wildfire—Bacterium tobacum Wolf and Foster. II. SUGGESTION OF SUBJECTS FOR SPECIAL STUDY LARGER PROBLEMS 1. The natural immunity of plants. 2. Acquired immunity in plants. Effeet of hybridization. Search for resistant species and varieties. MISCELLANEOUS: SUBJECTS FOR SPECIAL STUDY 475 3. Carbon and nitrogen nutrition of the soft-rot organisms. Species relations. ‘Toleration of acids and alkalies. 4. Carbon and nitrogen nutrition of the yellow Bacterium (Pseudomonas) group. Species relations. 5. Field studies of soil relations of Bacterium solanacearum, especially to lime, phosphates and potash. 6. Climatic studies of Bacterium solanacearum. Determina- tion of its northern and western extension in the United States. 7. Geographical distribution of Bacterium solanacearum in Europe and in South America. Ditto Africa and Asia. 8. Exact determination of the causes of the bacterial potato rots of Australia and New Zealand—of France and Italy. 9. Comparative studies of the white organisms Bacillus tracheiphilus, Bacillus amylovorus, Bacterium andropogoni, Bac- tertum woodsii, and Bacteriwm mort. 10. Does Bacterium andropogoni attack maize as well as broom corn and sorghum? 11. Comparison of the above with Bacillus coli, Bacillus typhosus, and other related animal pathogenes. 12. Comparison of the soft-rot bacteria with B. lactis, B. coli, ete., in all their varieties. 13. Comparative study of the green fluorescent pathogenic species. 14. Studies of sub-species of various pathogenes. There are a good many. 15. Critical study of the chemistry of all the tumor-produc- ing-species, Bact. tumefaciens in its varieties, Bact. savastanoi, Bact. beticolum, ete., determinations to be made from young, middle-aged and old flask-cultures in various media. 16. Determination of all acids produced by plant pathogens. 17. Hydrogen-ion content of media as related to growth of plant pathogenic species. Conversion of Fuller’s scale and Clark’s seale to PH. 18. Adaptation of the newer culture media used by animal pathologists to plant bacteriology. 19. New differential media for separating closely related forms, such as the various soft-rot organisms and the members of the yellow Bacterium (Pseudomonas) group. 476 BACTERIAL DISEASES OF PLANTS 20. Comparative studies of Aplanobacter species. 21. Old world distribution of Stewart’s disease of maize. 22. Methods of distribution of parasitic species on seeds, rhi- zomes, tubers, corms and bulbs. Responsibility of the seedsman. 23. Distribution of bacterial parasites on nursery stock. Responsibility of the nurserymen and dealer. 24. Insect and other animal distributors of diseases and their control. “Bacillus carriers’ among insects. Man’sresponsibility. 25. The flora of rotting potato tubers—in the advancing margin of the rot and in remoter parts. 26. The saprophytic flora following each parasitic disease. 27. Occurrence of black chaff of wheat in Europe, Asia and South Africa. 28. Occurrence of bacterial barley blight in Europe and Asia. 29. Occurrence of Elliott’s oat blights in Europe and Asia. 30. Symbiotic diseases: Ardisias, Pavettas, etc. 31. Favoring organisms—bacterial, fungus, protozoan. 32. Antagonistic soil organisms. 33. Cause of tobacco mosaic and other mosaic diseases. 34. Nature of the sugar-cane disease in Brazil. 35. Nature of the sugar-cane disease in Argentina. 36. Nature of the East Indian cane-disease known as Sereh. 37. Nature of the serious Porto Rican stripe-disease of sugar cane. This occurs also in Java and Hawaii. One should plant sound cane and avoid rattoon crops. Insects spread it. 38. Nature of the destructive sugar cane disease of Fiji. (H. L. Lyons, Hawaiian Planters Record, Vol. III, July, 1910, p. 200 and F. Muir, /bid., p. 186). This causes tumors in stems and leaves. 39. Nature of the bark disease of rubber (Petch: Physiol. and Diseases of Hevea brasiliensis, London, 1911, Pl. LX). 40. Nature of Janse’s disease of Erythrina trees in Java. 41. Cultural characters of the bacterial organism causing tumors on the Aleppo pine and on other European pines. 42. Cause of peach yellows. Is it a leptome disease? 43. Cause of peach rosette and of pecan rosette. Are they mosaic diseases? Are they denitrification diseases? 44. Cause and control of bud-rot of the coconut. 45. Correlation of tobacco leaf spots. How many are there? N MISCELLANEOUS: SUBJECTS FOR SPECIAL STUDY 47 46. Bacterial diseases of cultivated orchids. How many? 47. Bacterial diseases of ferns; 48. of Algae; 49. of fleshy fungi. 50. Do plants harbor animal pathogens? 51. Is loss of virulence in cultures ever due to the death of an invisible symbiont? If not, why do some organisms lose virulence quickly and others retain it for many years? SMALLER PROBLEMS I have suggested many such problems in the preceding pages and others will occur at once to teachers and students. II. PRODUCTION OF TUMORS IN THE ABSENCE OF PARASITES On susceptible species of plants, in the absence of parasites, overgrowths of suitable tissues can be brought about in at least five ways: (1) by introduction of irritating foreign substances, that is by certain poisons, administered in stimulating rather than in killing amounts; (2) by slight freezing; (3) by mechanical irritation, that is by woundings; (4) by over-watering in a con- fined atmosphere; (5) by semi-asphyxiation with vaseline, etc. My attention was first drawn to this subject in 1909 as the result of observations on the formation of cell-ingrowths (tyloses) in the vessels of plants, especially of those attacked by bacteria, and, second, by experiments in 1916 with crown-gall products. In the vessels of mulberry shoots attacked by Bactervwm mori, in various plants attacked by Bact. solanacearum and in old parasitized stems of Cucurbita, Citrullus and Vitis, tyloses are very common but their cause has remained in doubt. They occur also in many other plants but I am speaking only of those in which I have studied them. To me everything goes to show that they are the host-reaction to by-products of invading organ- isms which may not be, necessarily, active parasites. I have produced them by pure-culture inoculations with Bact. mori in young shoots of the mulberry where they never occur naturally. (see ‘‘ Bacteria in Relation to Plant Diseases,’”’ Vol. II, Fig. 30) and also with Bact. solanacearum in very young shoots of the potato (Fig. 142). Here it is certainly not the bacteria per se that act as the stimulus but their soluble products, since the tyloses may occur at some distance from the advancing growth 478 BACTERIAL DISEASES OF PLANTS of the bacteria. This is also suggested by the fact that vessel- ingrowths in great numbers may be produced in the absence of parasites, by purely chemical means. The most striking ex- hibition of tyloses I have ever seen, a veritable pseudo-paren- chyma, was obtained in 1914-1915 by Caroline Rumbold in the vessels of chestnut wood, by injecting a 1599 g.m. solu- tion of lithium carbonate (Fig. 357 to 359). Here the effect was quite local both in time and place and there were other surprising phenomena, viz., the appearance in the bark of G 4, < rns ae She 4 +s ts }) i { Fig. 357.—Cross-section of chestnut wood, spring of 1914, showing large vessels filled with tyloses. Cutin 1915. Below is umaffected autumn wood of 1913. From Caroline Rumbold’s chestnut bark injections of spring of 1914 using 100 g.m. LisCO;. Photograph by the writer November, 1916. 16mm., 4 oc.. bellows at 35. Reduced 1s. numerous well-developed islands of wood, causing it to bulge out (Figs. 360, sub. 6 to 362) while in the normal situation in 1915 much less than the usual amount of wood was produced (Fig. 360 at 3). We may suppose the stimulus to have been either the alkali, an excess of carbon dioxide liberated from it, or both acting together. The same curious phenomenon—enorm- ous thickening of the bark (from 1 em. to 5 or 10 em.) with form- ation in it of numerous islands of wood—occurs in the brown bast disease of rubber trees (Hevea brasiliensis) in the Dutch'East MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 4/79 Fic. 358.—Chestnut wood of the year 1914 in cross-section, showing tyloses in the pitted vessels. Bark injected by Caroline Rumbold in the Spring of 1914, with 1499 g.m. Li.COs, cut and stained by her in 1915. Photomicrograph by the writer November, 1916. 8 mm. Zeiss apoc. obj., No. 4, comp. ocular and bellows at 35 on small upright stand. (See Fig. 55.) 480 BACTERIAL DISEASES OF PLANTS Fria. 359.—Same as Fig. 358, but from a longitudinal section which passes through the middle of a big vessel which is full of the proliferated cells. Wood at either side, also portion of a medullary ray. Photomicrograph by the writer. Same magnification as Fig. 358. Auerbach’s stain (C.R.1914, 6). MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 481 Indies, and here the phenomenon is probably due to the alka- line by-products of some undiscovered parasite. I also know from my crown-gall inoculations that a true cambium devel- oped in the bast of tobacco plants may give rise to tumor tissue and to shoots (‘‘Embryomas in Plants,’’ 1. c.) and from these results it would seem as though wood and bast respectively must be developed from cambium in a slightly alkaline medium and in Lo Spr ORR eh ewig Fie. 360.—Chestnut bark injection of 1914 by Caroline Rumbold showing in cross-section islands of xylem in the phloem: (1) 1913 wood (free,from tyloses) ; (2) 1914 wood (full of tyloses); (3) 1915 wood (free from tyloses); (4) cambium; (5) phioem; (6) islands of wood in the phloem; (7) more phloem; (8) cork; (9) injected (killed) area, 4509 g.m. LizCOs being the substance used. Photo- graphed by the writer from a section made and stained by Dr. Rumbold. a slightly acid medium and that to reverse the ordinary process it is only necessary to change the reaction. If this should prove true it would help perhaps to explain certain curious phenomena of wood and bast distribution observed in the stems of lianas and sometimes in other plants (See Jour. Agr. Res., Vol. VI, No. 4, Plates XX and XXI). By injecting a solution of sodium bicarbonate into cabbage stems I obtained hard woody cylinders 31 A482 BACTERIAL DISEASES OF PLANTS Fie. 361.—Detail in cross-section of the inner one-half of an island of wood which developed in chestnut bark following an injection of 1509 g.m. of lithium MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 483 in the pith (Fig. 363) but_no islands of wood in the bark. The experiment, however, should be repeated especially on plants with a thicker and firmer bark. In most if not all of my at- tempted bark inoculations the fluid ruptured to the surface and escaped. As in tyloses, so in crown gall we are forced to conclude that it is not the mere presence of the bacteria in the tissues that leads to the overgrowth, but rather the stimulus of certain prod- ucts of their metabolism. Theoretical considerations led me to ask the chemists of the Department of Agriculture to make analyses of flask cultures of Bact. tumefaciens and on the basis of their findings I experimented with various plants subject to crown gall, using dilutions (fluids, vapors) of irritating sub- stances said to be present in the cultures. With these I ob- tained many striking small overgrowths (hyperplasias) and little or no evidence of wounding. Such responses were obtained with ammonia (Figs. 364, 365), acetic acid (Figs. 366 to 372), aldehyd (Fig. 373), and formic acid (Figs. 374 to 377)—all said to be crown-gall products. All these tumors are chlorophyll free, even when arising in tissues full of leaf-green. Many years ago Hermann von Schrenk showed that in- tumescences could be obtained on cauliflower by the use of copper salts, and I have seen them on amaryllis sprayed with Bordeaux mixture, but, of course, our interest in artificial hyper- trophies and hyperplasias centers chiefly around the question of their production with substances which are the by-products of parasites. In my first cases, as already stated, I did not detect any killing when vapors or large fluid dilutions were used, but with MacCarty’s findings in mind (Mayo Laboratories'), that in very early stages of breast cancers he was able always to detect a trace of cell-injury preceding the development of the malignant cells, I repeated some of my experiments, studying 1 For list of Dr. MacCarty’s suggestive papers on cancer see references at, end of the second one I have cited under “Literature.” carbonate by Caroline Rumbold. (See her paper in Jour. Am. Phil. Soc.) The wood occupies the upper one-half of the field and is bedded against the outer face of a row of hard bast fibers, the original location of the cambium from which it developed. Photographed by the writer from one of Dr. Rumbold’s sections. 484 BACTERIAL DISEASES OF PLANTS Fig. 362.—Outer part of same wood-island in bast as Fig. 361. The dark curved line in the middle is the cambium, the dark parallel lines below it are MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 485 earlier (2- to 3-day) stages of the tumor development (hyperplasias on cauliflower leaves resulting from acetic acid sprays) and al- ways found, judging by differences in staining, indications of at least a few killed cells under the stoma through which the acid penetrated (Fig. 378). In 1918, Harvey, of the U. S. Department of Agriculture, showed that cabbage leaves, exposed to —3°C., freeze at first Fie. 363.—Cabbage pith showing a hard woody cylinder which developed after injecting a solution of sodium bicarbonate. Needle track at N. Under X a little of the normal wood cylinder. irregularly in small spots and if the freezing be interrupted at the right moment, say at the end of 14 hour, and the plants re- turned to proper conditions, overgrowths (Fig. 379), which judging from his sections, may be either hypertrophies or hyper- plasias, develop from such chilled or frozen spots (Fig. 380) These frozen spots are visible at once because there is extrusion xylem medullary rays. The white islands in the upper part of the figure are groups of hard bast fibers in cross-section. Photographed by the writer from one of Miss Rumbold’s slides. 486 BACTERIAL DISEASES OF PLANTS Fic. 364.—Ricinus stem in longitudinal section showing tumors produced by exposure to the vapors from 0.2 ce. of a 20 per cent solution of monobasic ammonium phosphate held in a small serum tube resting on the next septum below. The unopened internode above shows its base also tumefied. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 487 Fig. 365.—Small tumors on under-surface of a cauliflower leaf produced by vapor of ammonia water. Exposed for 15 minutes in 10 cubic feet ot air space to 0.5 cc. of 0.90 sp. gr. water of ammonia. Photographed after,9 days. 4. 488 BACTERIAL DISEASES OF PLANTS Fig. 366.—Under-surface of a cauliflower leaf showing small tumors produced by exposure for 1 hour to vapors of heated Carnoy solution (acetic acid + ethyl alcohol) in 10 cubic feet of air space (temperature 20°C.). Exposure begun September 21, 1916. Photographed September 28, 1916. > 10. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 489 of water from the injured cells and this leads to a change in color, but this color difference soon disappears and the leaves appear to be normal until the growth has advanced to the second or third day. As I have recorded elsewhere (‘‘Mechanism of Tumor Growth in Crown gall’), the same transient spotting precedes the development of tumors on cauliflower leaves exposed to dilute vapors of ammonia (Fig. 381) and is undoubtedly to be explained in the same way, as due to loss of water from injured cells into the surrounding intercellular spaces. Earlier in the same year (1918) Wolf showed that intumes- cences may be produced on cabbage leaves by means of a sand- blast and ascribes intumescences occurring naturally on cabbage plants in the field to sand driven by the wind. In this he is unquestionably right. They may also be produced on cauli- flower leaves in the hothouse by sandpapering the leaves, and on bean plants by varicus woundings. All of Wolf’s figures are hypertrophies (Fig. 382). Some years ago (1892-93) George F. Atkinson experimented with the oedema of the tomato, which is an intumescence (Fig. 383) and reached the conclusion that on susceptible varieties (Fig. 384) oedema may be induced by insufficient light and bad ventilation coupled with too much water in the soil, and a soil temperature too near that of the air, leading to the accumu- lation of acids in the plant and to weak cell-walls, easily stretched as water is imbibed. He says: ‘‘When there is an abundance of water in the plant these acids draw large quantities into the cells, causing the cells to swell, resulting many times in oedema.” . . . ‘‘Ordinarily there is no increase in the number of cells.”’ He claims to have produced oedema by forcing an excess of water into the plants, but his experiments were few, 1n a place where oedema was naturally very prevalent, and they should be repeated. If he made any experiments to determine increased acidity they are not mentioned. Sorauer, who, following earlier writers, gave the name of intumescences to these wart-like growths which occur at times on plants of many species, thought they were ‘‘caused by an excess of water during a period of low assimilation.” In another place he speaks of these formations as due to ‘an BACTERIAL DISEASES OF PLANTS sbjccihictil se of oe bE = ee ee se i i ic oe sgn Fig. 367.—Under-surface of a cauliflower leaf 4 days after spraying with acetic acid in water (1490). Photographed August 5, 1918. x 4. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 491 abnormal elevation of temperature and excessive water sup- ply,” combined with weak illumination. With this view Atkinson agrees. In the last edition of his book Sorauer Fig. 368.—Cross-section, well above the leaf surface, of acetic acid tumor on cauliflower leaf 7 days after exposure. Surface covered by an epidermis. Block 1289. Fixed September 28, 1916. The exposure was for 45 hour in 10 cu. ft. of air space to vapor from 10 ce. of Carnoy’s fluid on a warm bath (about 65°C.). 8 mm. obj., 4 oc., bellows at 50, and enlarged 14 by engraver. Photo- micrograph by the writer. places intumescences under diseases due to ‘‘excessive moisture of the air.” Other observers regard strong light as favorable and specifi- cally on grape leaves (Viala and Pacottet, . c.) “‘excess of light in 492 BACTERIAL DISEASES OF PLANTS Fic. 369.—Cross-section of acetic acid cauliflower tumor at level of leaf surface (stomata of normal leaf surface above and below). oo acetic acid water sprayed April 10, 1917. Fixed in Carnoy, April 17. Slide 1336B2. tumor C. EE MISCELLANEOUS: TUMORS. IN ABSENCE OF PARASITES 493 Fic. 370.—Same series as Fig. 369, but tumor D and from the middle level of the leaf. In the center compact tumor tissue, surrounded by normal vessels and loose mesophyll of leaf. Slide 1336D3. Top row, 2d section from left. 8 mm. obj., 4 oc., bellows at 45. Photomicrograph by the writer. 494 BACTERIAL DISEASES OF PLANTS Fic. 371.—Middle part of Fig. 370 further enlarged. Normal mesophyll cells with large intercellular spaces, above, below and at the left. Tumor tissue very compact, like normal embryonic tissue. Photomicrograph by the writer. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 495 Fig. 372.—Longitudinal section of acetic acid tumor on cauliflower Jeaf. Tissue dead at X. Time, 7 days. 8 mm., 4 oc., bellows at 35. 496 BACTERIAL DISEASES OF PLANTS a humid atmosphere.’’ They say, ‘‘It is only during periods of the most brilliant illumination and directly under the glass of the houses, that the intumescences form in quantity. They do not occur in the same greenhouse on leaves which are in a diffuse light, or in the shade.”’ In commenting on this statement Dr. Hermann von Schrenk says, ‘‘Observations made in the greenhouse of the Missouri Botanical Garden during the present season [1904] on grape vines which were covered with these intumescences, fully bear out the observations made by Viala and Pacottet. The intumescences were formed only on the leaves immediately under the glass, while all the leaves in the shade were free from them.” Judging from my own observations and experiments, made on the potato, neither “‘insufficient light” nor ‘‘brilliant illu- mination” has anything to do with the formation of intu- mescences, at least with those which are hyperplasias. Also they may appear in the absence of any excess of moisture and when the ventilation is good. Not satisfied with the explanation of intumescences above given I made experiments of my own. After some thinking as to how best to begin, it appeared to me that I might imitate defective greenhouse conditions on a small scale by enclosing vegetation in sealed glass tubes. For this purpose I took unshriveled, carefully washed, sound potato tubers of several varieties, soaked them for 30 minutes in 1:1000 mercuric chlorid water to discourage surface organisms, pared away the poisoned surface with sterile knives and cut the remainder into rectangular blocks. These blocks were then dropped with sterile forceps into sterile cotton-plugged test tubes about an inch in diameter and containing at the bottom a wad of cotton, wet with 1 to 3 ec. of distilled water. The cotton plugs were then shoved down a half inch and the top filled with melted sealing wax. In these tubes I obtained the results detailed below. In 1910, the Russian botanist, P. Wisniewski, called attention to the production of intumescences on stems by obstruction of the lenticels with vaselin. Five years later (1915) the German, MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 497 «ee ie Fic. 373.—(1), (2) and (3). Smal] tumors on under surface of cauliflower leaves produced by formaldehyde (gas), exposure of February 21, 1917. Time, Gand 7 days. X 5. (4, Cross-section, showing nature of the overgrowth. The dark green palisade tissue is only slightly involved. At top, normal thickness of leaf tissue. From an unstained free-hand section. 16 mm., 4 oc., bellows at 35. 32 498 BACTERIAL DISEASES OF PLANTS ss Fig. 374.—Under-surface of a cauliflower leaf sprayed with formic. acid water (1700) on March 24, 1917. Photographed March 31. Natural size. Shows small tumors and white (killed) areas. See pl. 55 J. Ag. Res. Jan. 29, 1917. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 499 Fig. 375.—Under-surface of a cauliflower leaf in same series as Fig. 374, showing tumors due to formic acid (1 pt. to 100 pts. of water). plant. Time, 7 days. X 4, nearly. Second sprayed 500 BACTERIAL DISEASES OF PLANTS Fic. 376.—Under-surface of cauliflower leaf showing overgrowths due to formic acid water (100). Leaf sprayed March 24, 1917. Tumors not so well- developed as on plants sprayed with 4,99 formic acid water, 7.e., not raised so high. The very small round tumors are undoubtedly due to entrance of the acid in a Minimum quantity through a single stoma. Photographed March 31, 1917. X 4, nearly. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 50] ‘a ‘ £m ee ei ~ WHE gy gy, ae we es Si Q rey gee a a tg * a cs dee P lane, Bri 502 BACTERIAL DISEASES OF PLANTS E. Schilling, published on the same subject, having repeated and expanded Wisniewski’s experiments with similar results. The writer has also experimented on a number of plants, fig, mul- berry, olive, begonia, ginkgo, ete. (Figs. 385A, 386) using Squibb’s petrolatum. As soon as the lenticels are obstructed, gas interchange, 7.e., inflow of air and outflow of carbon diox- ide and vapor of water, ceases, or at least is greatly re- stricted, and following this (in susceptible species, but not in ginkgo) the cells under the lenticels at once begin to divide and a considerable cushion of cells (hyperplasia) may develop (Figs. 385B, and 387 to 389). Earlier than this it was known that the lenticels of potato tubers frequently proliferate in very moist earth (Sorauer), and also that the cut surface of potato tubers may proliferate. Fig. 390 shows proliferations that appeared on a pared sterile block of raw potato sealed into a test tube some months earlier by Mr. Shapovalov, who gave it to me thinking it might be crown gall. His experiments were for another purpose and these growths occurred in one of his check tubes. We could find no organisms in the tissues, either with the microscope or by means of agar-poured plates. Their internal structure (Fig. 391) is a twisted vascular hyperplasia not unlike crown gall. I tried to duplicate this in an atmosphere of nitrogen but only succeeded in asphyxiating the tissues. It would, I believe, be easy to get it with sensitive tubers and exactly the right reduction of air space or of oxygen. Possibly it is an abortive effort on the part of the flesh of the potato to reproduce the whole plant. Since the preceding paragraph was written I have repeated the experiments (spring of 1919) with pared blocks of raw potatoes sealed into a moist, confined air-space and have verified my prediction, as may be seen from Fig. 392. The blocks, which were some of those already referred to as in cotton- Fig. 377.—Structure of small and large tumor on cauliflower leaf sprayed with 4400 formic acid water. From unstained water mounts: (1) Weaker stimulus (stomata less open). (2) Edge of a large outgrowth. The whole tumor is about four times the length of the part hereshown. Both X 130 cirea (8 mm., 4 oc., and bellows at 35). MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 503 Fie. 378.—A. Cauliflower leaf showing slight sub-stomatal injury preceding acetic acid tumors. Time, 48 hours. Slide 1416, No. 23. Acid fuchsin stain. es 0. B. Same as A but after 6 days. Here a minimum of acid entered judging from small size of tumor. Slide 1420, No. 1. Acid fuchsin stain. 504 BACTERIAL DISEASES OF PLANTS Fig. 379.—Cabbage leaf showing tumors which followed a slight freezing. Leaf thawed in air. Time, about 7 days. X 2 circa. (After Harvey.) 505 ABSENCE OF PARASITES MISCELLANEOUS: TUMORS IN Fig. 380.—Varying structure (hypertrophy and hyperplasia) of frost tumors 0” (After Harvey.) cabbage leaves. 506 BACTERIAL DISEASES OF PLANTS plugged test tubes sealed in with sealing wax and resting on wet cotton, developed glistening ridges or hummocks of rounded cells and long hair-like cells (callous tissue) freely, especially in the cambium region of the tubers, also occasionally sprouts, and over most of their surface in course of a few weeks a well- defined cork-layer under which small hyperplasial tumors developed, pushing up the cork and frequently cracking it open; Fig. 381.—Cauliflower leaf three-fourths hour after exposure te vapor from 0.5 ec. strong ammonia water (0.90 sp. gr.) in 10 cu. ft. of air space to show fugitive mottling. sometimes also abortive buds developed from these tumors or in their vicinity (Figs. 3938, 394). The sealed tube experiments were continued for several months and many small tumors were obtained (Figs. 395 to 399). The nutrient substances in these small blocks of potato flesh are soon exhausted and the tumors cease to grow, but, if one could feed them, it would seem as if growth should continue for a considerable period, and that some of the tumors would become large. When the pared blocks of potato in the sealed tubes de- MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 507 Fic. 382.—From Wolf’s paper showing structure of intumescences (hypertrophies) produced on cabbage Jeaves by means of a sand blast. 508 BACTERIAL DISEASES OF PLANTS veloped shoots, the latter regularly produced, under stomata, which were always wide open, small intumescences (hyperpla- slas) in large numbers both on the stems and on the leaves (Figs. 400 to 404). ‘These intumescences were more abundant or rather Fre. 383.—Cross- section of a Vermont tomato leaf showing marked natural cedema. Upper surface at left. A few of the palisade cells unchanged. (After Atkinson.) more conspicuous at 28° to 35°C. (Fig. 405) than at 23° to 2o-C) but they, occurred also at the latter temperature (Figs. 406), although on some parts of the shoots at this temperature a microscopic examina- tion was necessary to determine their pres- ence (Fig. 407). The former were in bright light, and the latter in the dark or rather in very feeble diffused hight, packed away in quinine cans. The other conditions were the same, viz., a wet base (the block stood on very wet cotton or in water), saturated air, diminishing oxygen and in- creasing carbon dioxide. I am inclined to think, in this case, therefore, that the intumescences were due to excessive ab- sorption of water, coupled with acid stimuli liberated by a disturbed transpira- tion, due to a saturated or nearly saturated atmosphere. Of course, with temperatures near the optimum for growth (as would be the case in the top of a hothouse in bright light) there would be a more conspicuous development of such intumescences than at lower temperatures in which growth is much slower and in the latter it might re- quire examination with the microscope to demonstrate the beginnings of intumes- cences. On the pared sterile blocks of potato one of the most striking of these tumors, which was narrowly pediceled and covered with membrane, developed as a teratoma (Fig. 408). In these various examples it will be observed that the cells exhibit all grades of development from simple hypertrophy (Figs. MISCELLANEOUS: TUMORS IN ABSENCE OF PARASITES 509 Fic. 384.—Cross-section of intumescences on tomato produced, it is said, by forcing water into the stems: A. Variety No. 18. Normal tissue at the right. B. Variety Lorillard. Normal tissue at the right. (After George F. Atkinson.) 510 BACTERIAL DISEASES OF PLANTS 380, sub. 1, and 382 to 384) to marked hyperplasia (Figs. 368 to 372 and 377). They have one element, however, in common and in this they differ from all overgrowths due to active parasites, that is, corresponding to the fleeting nature of the stimulus, their growth is usually of short duration, whereas tumors due to para- sites, because supplied with a continuous stimulating exudate from the foreign organism, may continue their development indefinitely. The physico-chemical stimuli are, however, I believe, much the same in all cases where genuine hyperplasias occur. To these let us now turn our attention. IV. SPECULATIONS ON THE CHEMICAL AND PHYSICAL STIMULI UNDERLYING TUMOR-FORMATION Classed according to the number and size of their component elements, tumors are of three kinds: (1) simple hypertrophies (cell enlargements); (2) hyperplasias (cell multiplications); and (3) mixtures of the two, that is hyperplasias containing giant- cells. The size and shape of the cells forming the hypertrophy or the hyperplasia differ from tumor to tumor even in the same tissue, thus in animals we have round-celled, oat-celled and spin- dle-celled, large-celled and small-celled connective tissue tumors (sarcomas). The nature of tumors varies also, of course, greatly according to the nature of the tissue in which they originate, since the cells of each organ have a histology and an inheritance of their own. For this reason, connective tissue yields one type of tumor, glandular tissue another type, vascular tissue a third type and so on. An enormous amount of data has been accumulated on tumor differences, that is on the gross and minute anatomy of tumors, especially of human and animal tumors, because this has been the easiest method of approach, but it is not the most interesting side of the problem. That lies in quite another field, viz., in the field of hypothesis and experiment dealing with their etiology. All overgrowths, without reference to whether they are due to parasites or have developed independently of them, appear to me to be singularly alike in their chemical and physical origin and physiological requirements, their diverse appearances being LS —————_ MISCELLANEOUS: STIMULI UNDERLYING TUMOR-FORMATION 511 attributable to slight variations in the direction or force of the stimuli and to the diverse cell-inheritances, each and every tissue responding according to its own specific nature. All tumors begin, so far as we know, in injured places! and, fundamentally, I believe all may be regarded as excessive and continually modi- fied wound-repair reactions. In this chapter I shall deal with the secondary causes of tumors and shall endeavor to present my ideas as briefly as may be, premising that they are based on experiments and that where they pass beyond experiment into the field of hypo- thesis, no one need be led astray, if he keeps my title in mind. The best of any iconoclastic writing in science is not so much the new facts it has to offer as the changed outlook it gives, which new perspective often leads to renewed important experi- ments and to general discussion by many workers. I may claim to have contributed, at least, this much toward the elucidation of the complex and important problems involved in the origin of tumors. I may state at the outset that my conception of tumor forma- tion involves a loss of water and a change of chemical reaction in the cells which are to become tumor cells. This change which is toward cell-sap concentration and increased acidity must occur, I believe, to give the necessary stimulus to tumor forma- tion. Thestimulus may be long-continued or fleeting and may be brought about, as we shall see, in various ways. I will develop the subject as I proceed, and only add here at the beginning two other hypothetical postulates, first, that hyperplasias appear to me to represent a reponse of cells to oxygen-hunger or semi-asphyxiation, and, second, that the type of cell-response in the tumor, that is, whether a simple cell-enlargement with mitotic or amitotic nuclear multiplication (a giant-cell), or a full karyokinetic nuclear division with a corresponding hyperplasia, appears to depend on whether the partial proto- plasmic cell-paralysis involves both nucleus and hyaloplasm, or is confined to the latter, leaving the nucleus free to divide by 1 Of 850 breast cancers studied by MacCarty in the Mayo Laboratories every one showed evidences of having been preceded by inflammatory injuries (chronic mastitis). by BACTERIAL DISEASES OF PLANTS Fic. 385.—A. Under-surface of a young green branch of the common orna- mental rubber tree (Ficus elastica) showing outgrowths from lenticels 8 days after painting it with Squibb’s petrolatum. On the 5th day the treatment was re- peated. Nat. size, nearly. For section at X see B. B. Proliferation (hyperplasia) of semi-asphyxiated Jenticel of Ficus elastica. MISCELLANEOUS: STIMULI UNDERLYING TUMOR-FORMATION 913 mitosis and to form cell-walls. The extent of the cell-enlarge- ment will then depend on the amount of water imbibed and that in turn will depend on the acidity of the cell sap and on the corresponding extent to which the physiological control of imbi- bition, exerted by the hyaloplasm, is upset. But cell division will be rapid if the paralysis involves only the hyaloplasm. Let us take the simplest case first, that of hyperplasias developed under obturated lenticels (Figs. 385 to 389). Here we may suppose that some air still enters and that some vapor of water and gas still escapes, but the gas-exchange is demon- strably reduced to a very small fraction of what it was, that is, vapor of water and carbon dioxid cannot now escape through these openings as before, and air cannot now enter freely. In other words, there is a sfasis in the tissues under these openings, less entrance of air and less movement of aérated water, with more or less oxygen-hunger and with increase in cell-acidity (due to products of incomplete oxidation); also, owing to root- absorption, with increase of turgor pressure and, corresponding to these changes, a hyperplasia develops. Subjected to these conditions, many plants develop small tumors under the lenti- cels. The character of the hyperplasia, whether few-celled or many-celled, slow-growing or active, will depend on the nature of the tissues, and on the extent to which the lenticels are closed and the gas-exchange is interfered with. If there is less and less gas-exchange, the acid condition and the oxygen-hunger will be proportionately increased and the hyperplasia will be very small-celled and active. If there is still considerable en- trance of air and exit of aérated water and of gas either through imperfect closure of the lenticels or directly through the surface of the stem, the hyperplasia will be large-celled and slow-grow- ing, and this seems to correspond to the facts observed. Every growing cell is in constant need of oxygen—must have it at once or die. This is absorbed, it is now believed, through its whole periphery, either directly from the air or in- directly out of the aérated fluid which bathes its surface. If the Painted with petrolatum March 18, 1918. Photographed from a free-hand, unstained section in water, March 26. Upper part of proliferation torn away in making the section. 16 mm., 4 oc., bellows at 45. 33 oe BACTERIAL DISEASES OF PLANTS anaes SIE a sens Ro ss orm Se i k i Fig. 386.—Young vigorous shoot of Morus alba showing lenticel proliferations due to closure of the lenticels by Squibb’s petrolatum: (1) and (2). Two treated portions—2 was taken a foot above 1. (Time 13 days; 2d treatment on 11th day but not necessary.) Exposure begun March 5, 1918. Cut and photographed, March 18. X 5. (3) Untreated part, above 2. (4) Untreated part, below 1. MISCELLANEOUS: STIMULI UNDERLYING TUMOR-FORMATION 9515 volume of oxygen offered to such a cell (I am not here thinking of anaérobes) is reduced by the abstraction either of water or of air, 1t is plain that the only way the cell has of compensating for this reduction of an absolutely necessary substance is by cell-division, that is by increasing the area of its oxygen-absorb- ing surface, or to put it in other words, by increased respiration through the development of a hyperplasia.! If the amount of oxygen offered to the tissues is much below the amount required, then the hyperplasia will be fine-celled and active, if it is only a little below the needs of the tissues, the hyperplasia will be coarse-celled and slow-growing. All the evidence we have, enzymic and other, points to increased respiration in tumors of all kinds, and their feeble vascularization and correspondingly slow and uncontrolled circulation leads to Just the stasis neces- sary to produce more or less oxygen-hunger. I do not mean that there is complete absence of oxygen because in ordinary plants and animals that would mean prompt asphyxiation and death of tissues. Asphyxiation also occurs often in tumors but it is an end term that need not concern us here. What I mean is just sufficient reduction of the normal supply of oxygen to bring about cell-division for compensatory purposes, 1.€., to afford a larger oxygen absorbing surface. Two factors, at least, may be supposed to enter into this semi-asphyxiation hyperplasia under obturated lenticels: (1) oxygen-hunger, the cells being no longer bathed freely by air or by aérated water in movement toward the lenticel; (2) increased acidity of the cell- ap (from incomplete combustion of carbon compounds , leading to more or less paralysis of the proto- plasmic membrane (the hyaloplasm which governs intake and outflow) with correspondingly increased cell-permeability, allow- ing water to escape, and water, sugar and other food-stuffs to be brought back into the cells in increased quantities from 1The reason the bacterial cell accomplishes work out of all proportion to its size is just this, that its oxygen absorbing surface is enormously greater in propor- tion to volume of protoplasm than that of any other known organism. The surface of the rods in a cubic centimeter of bacterial slime, such as we frequently obtain in a test tube on our solid media and observe in the plant, represents an oxygen absorbing area equal to the surface of an ox. Indeed, we might say that the smallest bacteria are almost all surface. 516 BACTERIAL DISEASES OF PLANTS Fig. 387.—Cross-section of one of the mulberry tumors shown in Fig. 386. It is a hyperplasia: A, normal part; B, chlorophyll band; C, lenticel proliferation. Photographed from an unstained section mounted in water. 16 mm. obj., 4 oc. and bellows at 45. ~ MISCELLANEOUS: STIMULI UNDERLYING TUMOR-FORMATION 17 Fic. 388.—A. Hyperplasia in a lenticel en a young olive shoot due to treat- ment (March 4, and several times after that) with petrolatum. Response rather slow. From an unstained free-hand section. Photegraphed March 28, 1918. 8 mm. obj., 4 oc., bellows at 45. 8B. Cross-section showing a normal lenticel. Olive shoot of same age as A. The dark color under the lenticel to chlorophyll. is due 518 BACTERIAL DISEASES OF PLANTS ye 20 Veg j he / > i ; ~ ° < => » “ ‘ x ee? mee . Fig. 389.—Experimental intumescence on a silver spotted begonia (Begonia corallina lucerna): S . S08 eS A. Cross-section of internode of a young shoot, six days after painting with MISCELLANEOUS: STIMULI UNDERLYING: TUMOR-FORMATION 919 the surrounding tissues to compensate for the substances re- moved by growth as the tumors develop, and especially as they rupture to the surface and are not protected against irregular loss of water. Possibly there may be a third factor involved, viz., excessive turgor, due to the pressure of too much absorbed water. That there is an excessive movement of food-stuffs center- ing in all active tumors is shown not only by the rapid growth of such tumors, but also by chemical and microscopic analyses, by the overgrowth of neighboring tissues not actually involved in the tumor itself but affected by it (see Fig. 319, subs. 5 and 6), and, finally, by the starvation of remoter normal tissues. What proportion the air dissolved in the circulating fluids of the plant bears to the direct intake of air through the lenticels or the stomata in furnishing oxygen to the cells cannot be stated. It is conceivable that in many cases the first or indirect source would furnish more oxygen to many cells, especially in very young organs, the stomata on which are usually closed and the intercellular passages ‘n which are undeveloped, while in other cases the second or direct source would be most drawn upon. Certainly all the water that enters the transpiring plant through its root-system (in the aggregate, an enormous amount), as well as all the fluid that circulates in animals, is well aérated and bathes all the normal living tissues continuously, but there is not much active circulation in tumors, and consequently their cells will receive less oxygen from this source than normal cells. The hyperplasias produced from the flesh of raw potatoes in sealed tubes and those developed under stomata on young shoots in such sealed tubes, I would explain in the same way. As factors in the production of hyperplasias under the stomata, I believe we may eliminate both the decreasing external oxygen and the increasing external carbon dioxid in the sealed tubes, Squibb’s petrolatum. The cells of the lenticel have multiplied and pushed up the epidermis but have not yet ruptured it. Photographed March 11, 1918, from a water mount of an unstained free-hand section. 16 mm. obj., 4 oc., bellows at 52. For well developed intumescences see Fig. 394°. B. Same as A, but passing through a normal lenticel. From a free-hand unstained section in a water mount. The dark color is due to chlorophyll. Photo- graphed March 11, 1918. 16mm. obj., 4 oc., bellows at 52 (small upright stand). BACTERIAL DISEASES OF PLANTS Fic. 390.— Photographs showing small tumors developing under the cut surface (cork-layer) of a raw potato which was sealed into a very limited air space: (A) Frent view; (6) upper surface of A; (C) lower surface of A. The cork-layer is ruptured in places. Block enclosed in a test tube May 2, 1917, and sealed in with Photo. Sept. 25, 1917 sealing wax. At X one of the tumors has developed a bud. 21 5) TUMOR-FORMATION Y 7 RLYIN( = 4 4 TIMULI UNDI S MISCELLANEOUS: 390A, showing At left, below, a few normal cells. At left, above, some cells of the newly formed cork layer. o 5 7 Section of the outgrowth X on cut flesh of I 391. Fie distorted vy the tumor. structure of ascular € « 522 BACTERIAL DISEASES OF PLANTS Fig. 392.—Block of raw potato showing experimental production of small tumors like the accidental ones of Fig. 390. The pared sterile potato flesh, resting in wet cotton, was sealed into a test tube February 7, 1919, kept in the dark at room temperature, 22° to 25°C., and photographed April 17. xX 5. Small shoots were torn away at XY, which was the bottom of the block bedded in the wet cotton. MISCELLANEOUS: STIMULI UNDERLYING TUMOR-FORMATION 9523 Fic. 393.—Top and bottom (right side) of a pared block of sterile raw potato sealed into a test tube on wet cotton February 18, 1919, and kept in dull light at room temperatures (22° to 25°C.). The photos show top snd side views of the two tumors, both of which have ruptured the cork layer that covered them. At X, shoots are pushing. The lower left, block freshly pared to show the thickness of the cork-layer. Photographed March 22. x 5. OF PLANTS ES DISEAS RIAL uu BACTE iw potato sealed into test tubes on wet « sterile r Blocks of iry 18, 1919, kept in the d 394.1, ) oO Fie. cotton atures of temper at irk « Febru EL —™ MISCELLANEOUS: STIMULI UNDERLYING TUMOR-FORMATION 925 Le Fic. 394*.—Well developed intumescences which formed under lenticels on a silver spotted begonia that was painted with Squibb’s petrolatum April 19 and 22, 1920, Photo. May 20.. x 2: photographed March 24, x 5: (1C) A ridge of callous tissue from the cambium rezion, also small nodules. (2) At S stunted shoots pushing from a cambium, and at either side hyperplasias rupturing through the cork. (3) Small tumor at X bursting through the cork, at Y a naked small nodule. (4) Same as 1-3 but sealed in February 8, 1919, unpared and photographed March 29. Small hyper- plasias rupturing through the original tough cork layer. 526 BACTERIAL DISEASES OF PLANTS Fig. 395.—Pared, sterile block of an Early Rose potato tuber sealed into a test tube on wet cotton March 22, 1919, showing development of small tumors. Tube kept in the laboratory in dull light at 23° to 25°C. In these sealed tubes there was a saturated atmosphere and, owing to continued respiration of the tuber, reduction of oxygen and increase of carbon dioxide with probably some anaérobic respiration leading to the production of alcohol and acids. Photographed June 14ST ONOS eo: MISCELLANEOUS: STIMULI Fic. 396.—From the same series as Fig. 395. 7. 618 BACTERIAL DISEASES OF PLANTS Fic. 451.—A. Cross-section of a leaf from No. 18, second series, showing It arises from the colorless tissue (epidermis) above superficial origin of a shoot. As such a shoot grows it displaces P the palisade tissue (p) which is unbroken. MISCELLANEOUS: EXPERIMENTAL TERATOSIS 619 dance on other green plants. On the upper surface of the leaves I have not found any stomata and on the lower surface the aver- age is only about 20 per square millimeter, but in this respect it is not different from a half dozen other begonias I have examined. 32. When new phenomena appear our first thought is to inquire whether there are any old and well-known phenomena which can be linked up with the new appearances and thus serve to explain them and also whether there are any other unsolved problems on which they themselves will serve to throw light. In this case one naturally thinks of root-pruning or bruising, sometimes used to throw sterile fruit trees into bearing; of the development of young plants from the leaf margins of Bryo- phyllum calycinum, which also occurs, so far as I have observed, only when the water-supply is interfered with, 7.e., when the leaves are severed or partly severed from the stem or when water is withheld from the roots, or is drawn away from lower leaves to more active upper leaves, but here, while the stimulus appears to be the same, we have to do not with pathological or semi- pathological phenomena, or with regeneration, as I understand the term, but only with the growth of preformed dormant buds located in unusual places but otherwise normal; of prolepsis and prolification in peach trees attacked by peach yellows and peach rosette, where I have satisfied myself that there is premature death of a great many feeding roots, so that loss of water might exceed the intake, although the cause of this root-injury remains to be determined; of regeneration in general in plants and animals where the response is directly from the wounded surface or the cells in its vicinity, as it is in this begonia when young leaves are wounded; of crown-gall embryomas, where the shock which causes the development of roots and shoots in the tumors is connected with the presence in the tissues of the products of Bacterium tumefaciens and the growth of the tumors in the vicinity of totipotent cells, which are also set growing; of intu- and occupies deeper parts of the leaf. From a stained serial section. For a much earlier stage see Fig. 453 at X. X 55 circa. B. Cross-section of an internode showing superficial origin of the proliferous shoot. The phloem-xylem is a long distance below the part here shown and no vessels extended into it. Section photographed unstained in water. X 75 cirea. 620 BACTERIAL DISEASES OF PLANTS Fig. 452.—Leaves from adventive shoots showing fusions and other abnor- malities referred to in the text. At a, y, are stem glands (enlarged) giving rise | to shoots at their base. | MISCELLANEOUS: EXPERIMENTAL TERATOSIS 621 Fic. 453.—Cross-section of a leaf of Begonia phyllomaniaca showing early stages of adventive shoots at x and y. The dark central band is the only part of the leaf producing chlorophyll. From series V at end of three weeks. Photo- graphed in water from: a thick free-hand section. 8S mm., 4 oc., bellows at 35. 622 BACTERIAL DISEASES OF PLANTS mescences due to chemical and mechanical injury or to frost where clearly there is always some initial loss of water and in some instances, at least, increase of acidity; and finally, of tera- tomas in animals where nothing is known as to cause but where the consensus of expert opinion appears to be that the totipotent or pluripotent cell or cells giving rise to the fetal fragments dates from embryonic time, and in case of the atypical forms although ‘out of place,’ would have continued dormant but for the shock of the developing cancer. 33. In this connection one thinks also of the various processes used by gardeners to hasten the pushing of dormant buds. In 1885 Dr. Hermann Miller-Thurgau showed conclusively that potato tubers of varieties which ordinarily do not sprout until spring can be made to germinate in autumn or early winter by exposing them on ice for a number of weeks. If they are then removed and placed under conditions suitable for growth the dormant buds immediately begin to grow. He showed that potato tubers placed under these conditions change a portion of their starch into sugar and he believed that this increase of sugar is the cause of the germination. He mentioned incident- ally that there is also an increase of acid but lays no stress on this increase which, however, I believe to be the actual cause. His chilled potatoes which had become sweet were twice as acid as the unchilled ones (3.14 pro mille reckoned as malic acid. as against 1.74). In 1900 Dr. W. Johannsen, the Danish physiologist, experi- menting at first with sulphuric ether discovered that a great variety of plants which ordinarily do not push their winter buds until spring can be induced to push them in late autumn or early winter by etherizing the plants or by chloroforming them. Lilacs, for example, by this method can be brought into blossom at Christmas time and willows ean be induced to push their catkins in autumn within a week or ten days of the time they have been anesthetized. This method of procedure, worked out in detail by Johannsen for a variety of plants, proved so dependable and profitable that it is now used by florists the world over. The dose, temperature and time for lilacs is as follows: 30 to 40 grams of ether per hectolitre of air space MISCELLANEOUS: EXPERIMENTAL TERATOSIS 623 (about 3154 cubic feet), a temperature of 9° to 20°C., and gener- ally an exposure of 48 hours. When chloroform is used the time and temperature may be the same but the dose is reduced to 6 to 9 grams per hectolitre. The soil must be fairly dry otherwise much ether will be absorbed and the results dis- appointing. Ether vapor being heavier than air and explosive in the presence of fire, it must be liberated in the top of the air- chamber and the work must be done out of door or in a room where there is no fire. Johannsen states that he derived much benefit from a perusal of Miiller-Thurgau’s writings and from the earlier work of the great French physiologist, Claude Bernard. In 1909 Dr. Hans Molisch of Vienna published a very inter- esting paper showing that the same results obtained with ether and chloroform can be obtained simply by dipping the tops of the plants into warm water (30° to 40°C.) for a short period (6 to 12 hours). They are then set on the greenhouse bench under suitable conditions and bloom prematurely just as if they had been etherized. This method he states had been used for a considerable time by Russian gardeners but Molisch was the first to make exact experiments and to bring it to the attention of the scientific world. Neither Miller-Thurgau, Johannsen nor Molisch have off- ered satisfactory explanations for the results obtained. Miiller- Thurgau’s explanation that the hasty pushing of the buds is due to the presence of sugar in the tissues cannot be held to be a proper explanation since, as Johannsen points out, plants in a dormant state sometimes contain sugar in quantity, for exam- ple, garlic bulbs, and there is also always a considerable quan- tity of sugar circulating during the summer season in a variety of deciduous plants while their winter buds are forming and yet these buds do not ordinarily push. Preceding Johannsen’s work Dr. Raphael Dubois, professor of physiology in Lyons, France, published a paper (1891) on the action of chloroform in which he claims that the anesthetic acts by dehydration. I was unaware of the existence of Dubois’ paper until November, 1919, when I read for the first time Johannsen’s paper, although I had already arrived theoretically at the same conclusion, as may be seen from the preceding pages which were then in type. 624 BACTERIAL DISEASES OF PLANTS Johannsen makes light of the work of Dubois, as does also Overton. Johannsen objects that the figure which Dubois shows (that of an Echeveria exposed to chloroform. and exuding drops of water from all of its leaves) is the figure of a dead plant, and this may well be, but clearly there must be all stages in the exudation of cell-water from its mere beginnings in the live plant to its end, when not only the intercellular spaces and the sub-stomatal chambers are filled with the exuded sap, but also a sufficient quantity has exuded to appear on the sur- face in the form of tiny drops and to have exhausted the cells beyond recovery. In fact, Overton admits that there is loss of water from narcotized muscle and that Dubois is not wholly wrong. Recently I have observed all stages of dehydration in chloroformed cabbages. If the anesthetization is moderate there is only a spotting of the leaves (innumerable tiny spots, water-soaked or darker green by reflected light and translucent by transmitted light) without surface exudate. If the chloro- forming is continued longer, the change of color overspreads the whole leaf, and, if it is an undeveloped leaf with small inter- cellular spaces, there is an exudate of clear fluid from hundreds of stomata mostly to the under surface of the leaf, but, if it is an older leaf, the large amount of intercellular space is sufficient to accommodate the exuded water and it seldom appears on the surface. What becomes of such leaves depends on how far the dehydration is pushed. If the experiment is stopped before the exudate or spotting appears the leaves recover, if pushed until exudate appears on their surface I have not seen them recover. Temporary loss of function on the part of the protoplasmic cell membrane whether brought about by chilling, by hot water, by anesthetics, by acids, or by alkalies would lead, I believe, to the same set of phenomena; viz., loss of water, disturbed respira- tion, more or less oxygen-hunger, and compensatory cell-division with movement of water, sugar and other foods back into the growing point. Once the dormancy is overcome buds will continue to grow if placed under growing conditions. I believe, therefore, that we have in these common forcing methods added evidence that the phyllomania in this begonia must be due to MISCELLANEOUS: EXPERIMENTAL TERATOSIS 625 the shock produced by excessive loss of water acting on dormant totipotent cells. 34. I am inclined to think there is nothing in Weissmann’s theory making a sharp distinction between somatic and germinal cells. I believe that cells in many young undifferentiated parts are totipotent and that what finally becomes of them, that is whether they avail themselves of their totipotency, or not, depends on circumstances. Under ordinary conditions we know how they behave (that is, physiologically) but under extraordinary conditions they may behave quite differently, that is may at- tempt to reproduce the whole organism. In this begonia, toti- potent cells are distributed in great numbers over its superficial, actively growing parts. They occur, I believe, similarly if not so abundantly in the epidermis of other plants and in the skins of animals, but in most cases require a much stronger shock to set them growing, that is, a tumor, or a parasitic stimulus of some sort. That pluripotent cells should occur in the skin of a man, let us say, seems a very strange thing, yet man has in- herited his skin and all the rest of his anatomy from the lower animals, some of which, for aught we know, may have germinal cells as widely distributed and as sensitive to shock as they are in this curious begonia. No one knows, for instance, what effect a shock of some sort, such as a severe blood-letting or a drastic purgation of the mother, might have on a fetus in the way of starting dermoid cysts or other monstrous growths. The subject is one calculated to provoke thought and lead to further experi- ments. The effects of root-injury on this plant seem to me very suggestive as to the origin of Sereh of sugar-cane, of rosette diseases, of orange wilt, of peach yellows, and of the somewhat similar East Indian spike disease of sandalwood. It has also various other interesting bearings. . 30. That this begonia is more subject to shocks leading to phyllomania than other plants may be conceived to be due to its watery nature and especially to its inheritance of very sensitive and easily permeable cell-membranes. That it is alone in the world, in such behavior, I do not for a moment believe. 40 626 BACTERIAL DISEASES OF PLANTS TABLE I SHOWING RESULT OF WOUNDING UNDEVELOPED LEAF BLADES OF Begonia phyll- omaniaca. AFTER A MONTH KNIFE WOUNDS MADE Marcu 30, 1918. EXAMINATIONS MADE eo uw ee Contrasts on wounded leaves Wounded parts Unwounded parts Average | : . eel: = = | | | Plants and | nee eee mu race | | Total | | | pra branches ae jnumber of - _|number| a | number o of plants | 84-12. 07 | shoots perl Fraction Shoots Shoots | shoots on unwounded sq. in. on of sur- shoots | P each er | Total any equal leaf blades.| $4: 1"- | face oc- | Y%sq.in.| P | Total y eq Total wounded | cupied (com- each | sur- shoats fraction eurtae leaf blades IH | pare Perea ad 4g sa. | face 90tS| (For comp. 734 Bcc: ee Saat with 9° f Co ene with col- sq. in. | wounds |1,5¢ eol-| Surface | waranard! umn) | and 5) Dgeateon eee 2.0 7 8 Me | 215 a4 5550 | UMol 2 4.0 I CR ee 0.61 19.0 AON lSs 61 0.07 | *%4o 8 0.2 II; lower leaf Sie 9.8 | Ka 71 32 Poses | PSA) 8G 1.6 Il, upperleaf, 1.0 11.6 M45 117 43 0.125] 4445] 57 Tes Tossa 1.6 20.6 Wy 238 24 Ovae ez egal soil 5.0 1S eae oe 2.0 210 vant) 141 47 | 0.09 | 1849) 5 0.3 Dies eee 10 19.0 toe 246 49 (2 2165) 24 ical WTis see) es oe | O68 36.0 164 267 89 0.18 | 291) 11 0.55 TVty Meas ate | a it 15.0 Wo | 241 48 0.3 | 25%9| 42 | 1.5 TV2 (dwarf | | shoot).....| 0.0 0.27 a | 8 3 0.0 48 0 0.0 Vi lower leaf. 123 (7 Yo | 46 31 0.4 £369) 41 0.6 Vi upper leaf.) |... 14.0 Me | 145 2a 10) 24 ea cele 2h eats Vodice ashen (O88 8.0 Ms 58 39 | 0.04 | 4343) 3 0.07 Av. 5 plants | (11 branches) | 2 Sie: LGV ITS) ee eed eee ee | erearace 1.4 | | 1 There was also a leaf (I3) which received eleven wounds, but the total wounded surface was not recorded. This was on a dwarfed shoot from the axil of the branch I2 and there were no shoots either from the wounded or unwounded parts. MISCELLANEOUS: EXPERIMENTAL TERATOSIS 627 TABLE II SHOWING NUMBER OF SHOOTS ON INTERNODES STIMULATED JULY 24-25, 1918, and ON UNSTIMULATED INTERNODES IMMEDIATELY ABOVE AND BELOW THE SAMB, COUNTS MADE IN SEPTEMBER-—OcTOBER, 1918 - Number of adventive shoots on internodes Sar fea: J Bas FRG a : _ aD Part used | 142 mbes Internodes next below | Fnac mee above eRe ee = — as te ——— 1 To, pMain axis.0-..... \. (05 of 90) 44 | 265 1150 10 0 0 0) 0 Tiraae |e Vieng axis senses | 2) 1 5 | 235 18 3] O} | feibranche eee ce ly hl Goh “0 220 | 6| 3] 0| Oj 0} Iss IMT PShic ga ekee see | 0| 10 295 130 | 4) 2 I¢ IMEI Eb 0 l45 oe ogee Mies |16 inches practically free Not counted but | 0 0 0 | | | very proliferous | | | | Underground branch | | 0) 0 360 COD |) OO! | | Ig SOsbT arc heer pees ee ORO 20 300 | 0| O Olney hig a) | 'Sdibranth 5... 5. 2, 8:|*'|._ | 0 1 134 160. We [eet Ol 0) In | 6th branch.......... | * Ile 0% emilee 1S 280 | 5| 3] o| o| | | | (all at top) | | | Lig) |let branch... .... Weal KON i= = es isaneel= (160 130 | 9j 0 0| 0] O Sdsbrancharraia eee | | 0 1 156 195 1] (0) 5) 0 Tis Miaimeaxdse merit. seer 5] 9] 8) 7 25 | 850 40 | 0; 0} 0} O| O | | (all on one | | side) | Ist branch (arising Ki | | underground)...... | = | | 360 42 | 3) O} a Os 4th branch (K)...... Nese pel} 0 | 65 | 5/10) 4] 5) 0 6th branch.......... Wes lieelpeal it aeseOr |) 128 160 | 3 2] o| o MUnaDTaneHe ys ce) 1. esl Foc lh a 200 18 | 0} 0} O| o lhe IMiaimeaxas es ese nicks 2 2: | | 6 5 5 6 110 | 3] 2) 4) O} O} II; Manga saeese re 2 eee | 0 0 0 475 25 0} 0] 0 | | (free except | at base) | Branchel 9.5.) 2.1 ee SIKOl a Oa e126 105) | OO ig II IMHO B5Ioc on op A hee 0 0| 0 0 | 285 22 (base) | 0} 0] 0| Ist branch...........| | 0] 0| o| o| 0 480+ (0 00 0 IIs Zdybran cheat we awe 0} 0} O} 0 62 32 3] 0] 0} 0] O Ile MIT Beets hoaschane | 3 4 95 250 | 6} 9| 8| 4] 0} O Ii |) in TEN. 5 ce se oe oe Vee ea iat 0 ) 65 2) 0] 0] o| | Mis _|‘Main axis.........3....: WOR Sie 6S 215 28 |28] 1| 0] 1] 0| O ita) |eMainaxis:s 0. fw, | o| o| 0 o| 0 540 | 2| ol o| | | Aas | Maincaxie: 2.) ). 0: bez} | 2a, 21 180 15 5/12) 8] 0 Ils Miainvaxisean 4 oe. | 0 22 |= 140 160 | 0} O} 0} Oj, | Pdebrancht saya.) =. lt |feslt 5|-0|40]. 0 95 300 | 3| 5] 1| oj o| Sdtbranch: .ae ners | Wettig ley areas || 170 | 4| 1] o| oj O| Tis | Main axis........... tHe? NON ealGre’ oi 288 220 |10] 0| 0| oj O| o | | | | top | (eorky) {hale | | Ii | Main axis........... Boia Ziel esd 33 95 |36/ 2| 4|12] 8| 0 | top (very corky) | | | | | | | (one side)| | I Ilis IMiamnaxis eee fea ec 30 shoots in a length of | 675 30 |10 on 4 inter- | 8 in. (free except) nodes | Hee tte base) | 3d branch (F),.......| | | | 0| 0] 0 110 35 | 0} 1] o| o| | * No internodes below first recorded one. 7 When but a single number is given there was only one proliferous internode. t In cases like this the shock undoubtedly influenced more than two internodes. ‘squeid oy} Jo do} 9y} 4¥ Bursin900 saaKo] snosajsyoud A[peploop ATU 9y} oe AoYT, “seovyd pepunom ApYSYS wos Jt sv sdnoas a]}41] ul ore suowwssjyoid vsoyy, “doz oy} AvOU Vsey} JO 9oINT, , ee | ZZ 240% BL 41S 1 2106/8421 | 2491) 2161 |FS\0G|2122|%1S/6S 61 SZ) F1 11 Go) 249) OE |ES 81 BBICZIPBOLcaincleala yee a ee ee ener pnq peddvim jo di} 0} Stxe UIvUL JO SoyOUT UL Y4SUaT | a a LI| L9T| STlZT) ZT] 91) OT) ZESTIZT| GT) ATIGTIOL/ST) SLIST) STj6L/ST\STor HURST EVAL SLRS HUN = Me ORB ira et orca oes emt (ATIpBos JSOT ST JojBM O04 SooR]d) Ssivos o_nd14ys 9y4 jnoqge suoyViejljoid SurMoys sepou jo Jaquinn I CO) |KO We a 0 T sO) Ose 0 |T |o |t aie 1 HO KO WE AO TE EHO HO EO) yo oe ee eee (szavd [eseq poze] | | -NuIljs 94} Ul ][[¥) SepourezuT AYO Jo JequINN — | | a | fa | al wil } i Tr, a xt) ET) STON) ZE\OL) 9D) “SE) Stl) PLISEjOL) Sh) ve QT/FI\ST| SI\6T| ST/9T EL\E1|F1/9T STILT etal LAGI cet aa NE BNO (souo raddn pu op -plur ay} A]JSOU) SapourI}UT YJCOUIS Jo Jaquin NY lle letoe alee Fe wcm Gel hl paslemGee acm a teil ao Go| erase 20m Gin Gin Da2e On| SaiGei (@euor HeuqiITe)isepOuten ur eno rey OAc Omecuan Ny GE ee ve €z\1z| 62| 98] 62] Tzlse|ze| 68] selzh\seler) s8i\ze| 6&|0F ze/8z oe|ze LZ/GZ\TE|LT/9S) 1 99, 10938 Moris YOIy Jo TB (queyd Jo yred | ioddn put o[ppiw) seavel yyCows jo JaquInN | G97 SEO e coe Gh eeze O92 ON Ge OalOe jG IGS BSAA Pal ZI Ghn On Ogg O)r Sh) wenorosoxca (as [aweU)) Se Avey Vere Olas oan Ny wD (@) 9 KG Me Ne SEI | Gaed IIMO] UI [[B) SAABI] SNOJejT[OId Jo aquUINN [| | | | | 6z| szizz| ozlez| ¥e| €2| | Tz\cz6r | 2t|91 aa eller] Tt\otle |e |2 | fatale. 0) eee ena | feed! sale Ii i | SIXV NIVW GHL dO GSOHL XINO ‘AONTINAANOO UVINAVL YOd ‘AGATIONI SINQOD ACONUAL -NI GNV AGON GHL ‘SHHONVUM AHL NO SHAVAT ACA IONI SLNN09 AVI GH], ‘(LX4L AGS) SHONVAUALSIG-LOOU GNV DNIAUC HDAOUHL UALVM FO SASSOT TVOASAND O% GHLOMAAS AUAM SENVId AHL GOWaGd HOIHM JO ATVH LSuld AHL ONTUAC ‘(GT HOUVIN OL STGI ‘ZI ATAL) SHINOW § AO GNA LV ([]] SATUAS) VoDYUMUMODLiYd DLObag AO SPNILLNAO AALOOU NO SUTASAY ONIMOHG Til Wav Paw ee Fale ee siecle eo hie Bi a Iaquinu Uv] J nN D - Ko) is) ~ ise) ios | cal BACTERIAL DISEASES OF PLANTS | 628 MISCELLANEOUS: EXPERIMENTAL TERATOSIS 629 BEGONIA LITERATURE 1852. Begonia phyllomaniaca Martius. Hooker’s Journal of Botany, Vol. IV, pp. 206-207. 1853. Dr Martius, C. F. Px. Begonia phyllomaniaca in “Delectus Seminum in Horto R. Bot. Monacensi” Anno 1852 Collectorum. Annales des Sciences Naturelles. 3 Série, 19, 1853, p. 366. 1854. Von Martius. Flora Brasiliensis, Vol. IV, Pars I pp. 386-387. Plates XCIX and C. 1854. KuorzscH, Hrn. Begonia phyllomaniaca in “Begoniaceen-Gattungen und Arten,’ Abhandlungen der Koniglichen Akad. der Wissenschaften zu Berlin, 1854, p. 129. 1863. WVeRutot, J. B. Begonia. Bulletin de la Société Botanique de France. Tome X, p. 474. ? Eight lines from a letter in which he states that hairs on the leaves of a begonia (sp. called B. geranioides) are changed into buds which render the leaves in some sort viviparous. The letter and specimen were sent to M. de Schoenefeld who exhibited them at a meeting of the Société Botanique de France. 1863. PRILLEUX, Ep. Observations sur une feuille gem- mipare de Begonia. Bulletin de la Société Botanique de France, Tome X, pp. 492-494. At a following meeting of the Société Botanique Prillieux discussed Verlot’s discovery and fogged the whole subject, denying that the shoots were the out- growth of hairs. 1875. CaruseLt, T. Nota su di una trasformazione di peli in gemme. (Begonia phyllomaniaca.) Nuova Giornale Botanico Italiana, Vol. 7, pp. 292-294, Pisa, 1875. Note on plants seen at the Kew Gardens in London. His observations confirm Verlot’s statements that hairs are converted into buds. 1880. HaANseN, ApoupH. Vergleichende Untersuchungen uber Adventivbildungen bei den Pflanzen. Abhandlungen Herausgegeben von der Senckenbergischen Naturforschenden Gesellschaft. Vol. XII. Frankfurt, pp. 147-154. 1887. DucHaRTRE, P. Sur un Begonia phyllomane. Bul- letin Société Botanique de France. Tome XXXIV, Paris, 1887, pp. 182-184. Forty plants raised by Nodot from seeds showed phyllomania on their stems, 630 BACTERIAL DISEASES OF PLANTS One of these was studied by Duchartre, who says, ‘‘ How are these little supple- mentary leaves produced? I have had neither the time nor the material neces- sary for seeking their first origin. ... Each one of these growths constitutes in reality a leafy branch” As a simple hypothesis he thought they originated from the epidermis. 1894. Warsura, O. Begonia phyllomaniaca. Die natiir- lichen Pflanzenfamilien. Engler und Prantl. Teil III, Abt. 6 and 6¢. p. 124. Leipzig. 1908. GorBEL, K. Einleitung in die Experimentelle Morphologie der Pflanzen. Naturwissenschaft und Technik in Lehre und Forschung eine Sammlung von Lehr- und Hand- biichern. [Begonia phyllomaniaca on pp. 153—-155.| 1913. Bargeson, WiLLIAM. Problems of Genetics. [Begonia phyllomaniaca on pp. 50-53.) Yale University Press. 1917. Smita, Erwin F. Embryomas in Plants (Produced by Bacterial Inoculations). The Johns Hopkins Hospital Bulletin, Vol. XXVIII, September, 1917. Also a repaged separate. Foot note on page 279. 1919. Smira, ERwin F. The Cause of Proliferation in Begonia phyllomaniaca. Proc. Nat. Acad. Sciences, Vol. V, February, 1919, pp. 36-37. LITERATURE ON EFFECT OF COLD, HEAT AND ANESTHETICS 1885. Miiller-Thurgau, Hermann. Beitrag zur Erklérung der Ruheperioden der Pflanzen. Landw. Jahrbiicher. XIV Bd. Berlin. Paul Parey, 1885, pp. 851-907. 1891. Dubois, Raphael. Mécanisme de L’ Action des Anes- thésiques. Revue Générale des Sciences Pures et Appliquées. Tome 2, Paris, Sept. 15, 1891, pp. 561-567. 2 text Figs. 1900. Johannsen, W. Mein Aetherverfahren in der Praxis. Die Gartenwelt, 5. Jahrg., 1900-1901, p. 265. 1901. Overton, E. Studien tiber die Narkose zugleich ein Beitrag zur allgemeinen Pharmakologie, pp. 195. Jena. Ver- lag von Gustave Fischer, 1901. (Dr. E. Overton, Privatdocent der Biologie und Assistent der Botanik an der Universitat Ziirich. ) 1904. Johannsen, W. Frihtreibversuche mit Straiuchern nach erfolgter Aetherisierung oder Chloroformierung. Sitzgs- ber. der © Flora,’ 1902-1903. Dresden 1904, pp. 71-83. MISCELLANEOUS: EXPERIMENTAL TERATOSIS 631 1906. Johannsen, W. Das Aetherverfahren beim Friihtrei- ben, mit besonderer Beriicksichtigung der Fledertreiberei. 2 Aufl., pp. 65. Jena. Gustav Fischer, 1906. 1909. Molisch, Hans. Das Warmbad als Mittel zum Treiben der Pflanzen. Jena. Verlag von Gustav Fischer. 1909, pp. IV, 38. Mit 12 Figuren im Text. ADDITIONAL LITERATURE ON INTUMESCENCES (See page 572) 1898. TusBrur, C. von. Uber Lenticellen-Wucherungen (Aérenchym) an Holzewiichsen. Forstlich-naturwissenschaftliche Zeitschrift, VII Jahrgang, 12 Heft, December, 1898, pp. 405- 414, 7 Figs. This very interesting paper, which was read with fear and trembling, came to my knowledge too late to be mentioned in the body of the text (page 477 et seq.), 2.e.. not until the corrected proofs had been returned to the printer. It anticipates by twenty years some of my findings but not all. The author raises the question of reduced transpiration as a possible cause of the tumors and decides against it because he also obtained them in his closed vessels when projecting leaves were on the plant and transpiration was in progress, but if the fluid transpired possessed a limited oxygen-content as it must in closed tubes, the peripheral stem tissues would still receive insufficient oxygen; and they might even, if the water transpired by the leaves, had sufficient oxygen, 7.e., was ground water, since the moist air in contact with the lenticels in the closed chambers would soon become deficient in oxygen. Concerning increased acidity of such tissues it does not appear to have occurred to Dr. Tubeuf to make any inquiries. Von Tubeuf states that the proliferation may be so extreme as to be patho- logical and that it is not due to an excess of water in the tissues. He cites Goebel to the effect that it arises in consequence of an undetermined irritation and Schenk that “it is not very probable that simple contact of the epidermis with the water, as such, is a factor, it is much rather to be supposed that lack of oxygen in the inner tissues, the plasma of the phellogen cells, leads to the production of the aérenchym.” Dr. Tubeuf’s experiments were made with stems and roots of small trees— elms, ete. 1913. Wehmer, C. Ubergang 4lterer Vegetationem von Aspergillus fumigatus in “ Riesenzellen’’ unter Wirkung ange- haufter Saure. Berichte der Deutschen Botanischen Gesellschaft, Vol. XX XI, No. 5, 1913-1914, pp. 257-274, 7 text figures. Wehmer obtained great numbers of giant cells in cultures of Aspergillus and Penicillium, using ammonium sulphate. He attributes these giant cells to the action of free acid ions on the spores of the fungus. 632 BACTERIAL DISEASES OF PLANTS 1919. Tayior, WILLIAM RANDOLPH. On the production of new cell formations in plants. Contributions from the Botani- cal Laboratory of the Univ. of Pennsylvania, Vol. IV, No. 2, pp. 271-299, 8 pls. 1920. RumpBoup, CARoLINE. The injection of chemicals into chestnut trees. American Journal of Botany, Vol. VII, No. 1, Jan., pp. 1-20, 7 text: figures. 1920. RumBoLp, CAROLINE. Causes for the production of pathological xylem in the injected trunks of chestnut trees. Phytopathology, Vol. X, No. 1, Jan., pp. 23-33, 2 pls. 1920. Harvey, R. B. Relation of Catalase, Oxidase, and H+ Concentration to the Formation of Overgrowths. Am. Journal of Botany, May, 1920, pp. 211-221. ese ard Re AY) GENERAL OBSERVATIONS How to make the most of one’s education, how to achieve the largest success, must ever be a matter of immediate concern to the student who has to win his own way. With such persons in view, and I am speaking to no others in these pages, I will here set down some observations that have grown out of my own experience. If occasionally they prove useful and help to smooth ways which are often hard in the beginning, I shall feel well repaid. I have expressed my individuality very de- cidedly on a variety of subjects in the following pages but I could not do otherwise. If anyone thinks these observations smack too much of “Thus spake Zarathustra” he has the remedy in his own hands. We are often compelled to listen to an in- dividual when we are bored, but never to a book. ‘‘ Si ce livre me fasche, ven prens un aultre.” ON SUBSIDIARY STUDIES I have spoken farther along about the need of modern lan- guages and may say a word here about the despised Latin and Greek. As cultural studies, there can be no doubt of their value. The student of Latin and Greek is generally a more discrimi- nating student and forceful writer of his own language than other men and this is a sufficient reason for their study. In the case of the naturalist there are other reasons: (1) the terminology of science is derived from these languages, and (2) all the oldest scientific writings and some of the modern ones are in Latin and Greek, and these, in some instances at least, must be read. Finally, Latin is the mother of all the great Romance languages, whose literatures will be to you a source of profit and delight for many other reasons than the purely pathological one. My advice to the pathologist therefore would be: study both Latin and Greek, or at least Latin, and get as much out of it as you can. 633 634 BACTERIAL DISEASES OF PLANTS Of the sciences, the higher mathematics would seem to be of least use to the experimental pathologist, and yet I may be wrong inthis judgment. Certainly the end of all experimenting is to be able to express one’s data in plots and curves, but biology is a very complex subject, too complex apparently for any mathe- matician to understand, and biologists, for the most part, are very far from being able to express themselves after the manner of mathematicians, however desirable it might be. Their language and ours are unlike almost to mutual exclusion. If, then, you are only an average biologist do not spend several years on the higher mathematics, because in the end you will be only an indifferent mathematician, a plodder and a grubber like the rest of us, not a member of the great race. When, as a student, I lamented to Harrington, the astronomer, my lack of proficiency in the higher mathematics, he said: ‘‘ You have not cut as much underbrush in this direction, that is all.’ But I am sure the defect lies deeper, viz., in a type of mind, and one very common among biologists. The case is quite different, however, if your liking for mathematics is second only to your love of biology. Then you may study it as long as you feel inclined. You will be a kind of a white blackbird among your fellow biologists but this need not disturb you, since you will be able to do some things which they cannot do. Of sciences which are closer to the pathologist I may mention experimental physics (especially those branches of it dealing with heat, electricity, hydrostatics, surface tension, viscosity, etc.) and chemistry, of which he cannot have too much. Bio-chemis- try in particular will be of service to him at every turn. He cannot do without it unless he can arrange to work jointly with some chemist and even then he should not be content simply to look over the fence. The type of chemistry the pathologist should cultivate is that which deals with organic compounds such as his parasites produce or attack, and the problems con- nected with which he will have to face. I mean the chemistry of starches, sugars, celluloses, pectoses, tannins, acids, aldehyds, amino acids, glucosides, enzymes, ethers, esters, and the like. The pathology of the future lies right in the midst of these things and more and more the pathologist must be a chemist if he would succeed in a large way. GENERAL OBSERVATIONS: ON SUBSIDIARY STUDIES 635 The student should also know something of meteorology and of surface geology and soil physics. He must have some knowledge of zoology and especially of entomology, both because insects act as carriers of disease and because he must know how to keep his experimental plants free from all sorts ef depreda- tors. He should certainly know all the common insect pests, and the broad general conditions under which all animal life develops and functions. To know these things will give him a much broader and firmer grasp of his own problems. In botany, the pathologist may be trusted to acquire as he goes along a knowledge of the morphology and structure of plants because all his life he will be making sections of various organs on a variety of plants, but plant physiology he should study thoroughly from the beginning, for how can one know the meaning of a disease if he does not know the functions and behavior of a normal plant! He should also understand garden- ing, that is the proper care and cultivation of plants in the open and under glass, and to this end he should affiliate with compe- tent gardeners. There is only one other group of studies I would touch upon. Human and animal pathology and modern medicine, with its stimulating outlook, are close neighbors, and the plant pathologist will be wise to make friends with the well-trained physician and the animal pathologist and to keep in touch as much as he can with the progress of these sister sciences. There is a large program laid out, I hear it said. So be it, but if you are not lazy nor wasteful of your time, but hew to the line through a series of years you can accomplish it all and much more, and must, because what I have mentioned is only subsidiary to the main task. ON SEEING THINGS The successful student of nature, and especially the successful scientific man, must not belong to that type against whom it was said of old ‘“‘ Having eyes they see not!” In him “that inner eye which,”’ according to the poet, ‘‘is the bliss of solitude”’ must be forever open to the faintest impressions from the 636 BACTERIAL DISEASES OF PLANTS natural world, if he would fathom its meaning. Seeing is not enough but it is the first step, the beginning of all the others. How to see with the eyes of a Darwin, a Pasteur, or an Asa Gray, that is the question! Poets are said to be born, not made, and inheritance must also play no inconsiderable part in the lives of all great men of science. Yet another saying has it that genius counts only for one-tenth while hard work is nine-tenths of every man’s success. These are extreme statements and the truth les somewhere between the two. Both environment and heredity are important. Certain it is, however, given some basis of good material to work upon, that patience and perse- verance will do much to cultivate and sharpen the seeing eye. This must be so, otherwise the amateur would be as efficient as the highly trained man and we know that this is not the case in any field of endeavor. As every teacher knows, it is hopeless to try to make students out of many persons because ‘‘It isn’t in them,” as the saying goes. They carry an insurmountable inheritance of dullness. On the contrary, long pondering on a subject with oft-repeated observations of the physical phe- nomena involved gradually enables the right sort of a person to see definite principles quickly and clearly in that which was at first only a maze of obscurity and uncertainty. The plainest things are often the hardest to see because all our seeing and all our thinking runs, or is apt to run, in stereotyped channels and the older we grow the greater the danger. Strive, then, to keep an open mind and to enlarge your horizon as you grow old. But the first inertia is the most difficult to overeome— cest le premier pas qui cotite. The new and strange are always hard to comprehend and interpret. For this reason the first foreign language, especially if it is Latin or some other much inflected tongue, is I believe, always hardest for English-speak- ing persons. A Chinese student once told me that Latin was easy for him (because inflected, I presume) but English ‘‘ very hard.’ For the same reason first impressions of a strange coun- try are always most vivid but generally very inaccurate, witness many books. Every one has heard the story of Agassiz’s stud- ent to whom a fish was given that he might point out its most conspicuous feature. The bilateral symmetry of the fish was GENERAL OBSERVATIONS: ON SEEING THINGS 637 what the master had in mind, but this was the very last thing the student thought of. Why? Learn then to see, and to think upon what you have seen! And look again and again lest you should miss something. By seeing I mean not loose general observations such as would enable you to distinguish a man from a tree (Smile, if you will, but this is the common way of seeing. I have exaggerated it only a little) but patient, long-continued discriminating observation. In this way, gradually, all the hidden details of an object become visible. When they are clear enough to be drawn or to be reflected upon as separate entities then only can you be said to know them. By thinking I mean prolonged log- ical reflection leading to clarity of view, not mere hap-hazard dreaming. “Learning without thought is labor lost; thought without learning is perilous.”’ ON EXPERIMENTATION Observations and reflections, however extensive and _ pro- found, are not sufficient guides in pathology. These might serve to make a statesman or a philosopher but not a scientist. Things observed are to be questioned—and this questioning is done by means of well-planned experiments. These experi- ments lead necessarily to many new observations and often to a materially changed point of view, so that the imperfect frame- work of a discovery, which may have been nine-tenths insight at first, is gradually filled in and worked over experimentally until it becomes a substantial and lasting structure. In path- ology, as in all subjects dealing with phenomena, experimental tests of the validity of one’s ideas are necessary at every step and the term ‘‘scientist’’ is a misnomer when applied to any one who does not try his hypotheses in the reducing fire of experiment. The world is full of shouting theorists who have never made an experiment in all their lives, certainly not one worthy of the name, and yet they are asking all men to follow them. This is why most politics, economics, socialism, spiritualism, psychic research, psychology, philosophy and 1 Doubtless, Louis Agassiz (1807-1873) tried this on many students, but Dr. W. J. Beal is the one who told it to me. 638 BACTERIAL DISEASES OF PLANTS theology are such bogs and quicksands of the human intellect. They have not been, and from their very nature, in many cases, cannot be subjected to rigid experiment, and, therefore, have not arrived at, and in many cases, never can arrive at cer- tainty. They belong on another plane, that of possibilities or probabilities and some are not even possibilities. The best advice I can give the young pathologist is this: If you would go far, experiment continually. Try out all of your theories and other men’s theories by experiment. Let no day pass without something done to verify the correctness of the various ideas you have formed from your observations. In this way you will be able to discard many specious but erroneous assumptions, and will be continually adding to your sum of exact information. The reason many men are only hewers of wood and drawers of water is because they are content with simple observations and reflections, often very superficial ones, and stop short of experiment which would show them where the truth lies.. They may lack the seeing eye and the inquiring mind, may have ‘“‘ hook worm,” be simply lazy, or perhaps only untaught. In too many children the eager questioning spirit is repressed by a hard and unsympa- thetic environment. Such persons are conspicuously weak in memory, and in a knowledge of the past. Consequently they are the natural and easy prey of the walking delegate, the political demagogue and the yellow journalist. ON BEGINNING WORK THOUGHTLESSLY The best advice I can give the ambitious student is this: As far as possible, think out carefully in advance all the main ramifications of your experiments. This is not easy, even for the advanced worker, and surely you will have overlooked something, however thoughtful you may be, but by such preliminary cogitation you will escape many pitfalls, and come at once into the only proper way of research. ‘‘But I cannot afford the time,’ I hear someone say. Well, time wisely spent in the beginning of an undertaking is often time saved in the end. The shrewd commander takes into account all possible contingen- GENERAL OBSERVATIONS: BEGINNING WORK THOUGHTLESSLY 639 cies, as nearly as may be, and thereby wins a battle or a cam- paign. The commander who cannot afford the time, or who lacks the foresight and the acumen, is beaten and disgraced. You have your choice. But what profit a student thinks he will derive from a blundering course of experimentation ending in some dead end or no thoroughfare, I cannot imagine. Your results, you may be sure, will not be commensurate with your labors. ‘‘Palma non sine pulvere.”’ Yes, but Seneca is careful to add “‘per viam rectam.”? You may flounder through the mud and dust desperately, but if you are on the wrong road all of your energy will not save you. Literature is full of examples of this sort of bungling, espe- cially Theses, which once printed have to be read but which really have no raison d’étre, since often they do not add materially either to human knowledge or to the reputation of the writers. Sit down, therefore, with your problem and think it over seriously in all its various aspects before you attempt a stroke of work. The more thought you put upon it in advance the more likely you will be to obtain convincing results when you actually begin to experiment. Here I cannot resist telling an old story. An Irishman invented a cumbrous cover to keep water out of a gig in case of sudden rains while on the road, which cover, he said, was to be stored away underneath the gig in clear weather. ‘‘But there is no room for it underneath,” said a critic. As this was only too evident, Pat was nonplussed for a moment and then replied, ‘‘ Well, you can leave it at home.” This is hke many a human cogitation! Tried out it does not work! I am not attacking any one. Some of my own experiments have been of this sort, but fewer I trust in recent years than earlier. Now I always spend more time, often very much more, thinking over my proposed experiments, than I do in the per- formance of them. And generally speaking, I know in advance, barring some unforeseen contingency, just how they are coming out. If they fail, I begin to search shamefacedly for that something which I have overlooked, and sometimes it turns out, when discovered, to be as plain as the nose on a man’s face, or as the bilateral symmetry of the fish. 640 BACTERIAL DISEASES OF PLANTS ON INTERPRETATION OF PHENOMENA First of all, you are to remember that very often things are not what they seem! Two sets of phenomena may resemble each other superficially but be of quite unlike origin. Herein lies many a pitfall for the unwary. Probably most blunders in science result from failure to distinguish between similarity and identity, between resemblances that are fundamental and must depend on community of origin, and those that are only superficial and consequently must have diverse origins. The student, and the older worker as well, should be on his guard continuously against the fallacy of mistaken identity. The difference between a careful worker and one who is sometimes careless, or habitually so, lies in just this, that the latter sees the superficial similarity and is content with an inference, while the former probes the inference, demonstrates its non-validity, and saves his reputation. Laziness, or inhibitions due to overwork, lie at the bottom of most such blunders, I think, but sometimes over-confidence. Usually it is quite easy to show that a given result corresponds exactly to another or differs from it in various particulars, if environmental conditions are duplicated, and if cultures are made and sections are cut and studied, but all this takes time and painstaking care, which some persons are loth to give. It also involves good judgment and good training. Especially must you demonstrate, if you have made inoculations and obtained results: (1) that the resulting lesions are identical with those occurring naturally on the plant; and (2) that the organism in the lesions is identical with the one isolated from the natural disease and used for the inoculations. Not to do these two things thoroughly well is to leave your whole paper a tissue of uncertainties. “Verify everything!” is the best advice I can offer. Then you will have no after regrets. Nearly every productive scientific man, however, has some regrets of this sort. ON REPETITION OF EXPERIMENTS—OTHER PEOPLE'S, ONE’S OWN There is a mistaken notion abroad that if someone has worked on a subject and published a book or paper, that settles GENERAL OBSERVATIONS: ON REPETITION OF EXPERIMENTS 641 it, and no one else need consider the problem farther, especially if that someone is a person of reputation. No supposition could be wider of the mark! Some reputations are founded on a rock, others are mere bubbles. Moreover, nature hides from us very securely her secret things, and the chances of going astray in their interpretation are many. The young scientific man, filled with his intellectual pride and knowing very little really, either about the complexities of nature or the history of science, which for the most part is the story of one long series of blunderings (toward the light, however, not into deeper darkness), is apt to judge the mistakes of his fellows and of older men harshly; the experienced honest man, on the contrary, knows that to err is human and judges all honest work leniently, since he knows that even the best work is certain to contain some erroneous observa- tions, or some errors of interpretation. Remember this, therefore, as a fundamental doctrine in science: Nothing is too sacred to investigate, and nothing can be regarded as indubitably established until various careful observers and experimenters have arrived independently at the same conclu- sion. Copy this out and stick it up where you can see it every day! Ifthe second man over a subject finds the first man correct in all essential observations and interpretations, the more credit to the former. The second man will, nevertheless, usually be able to extend the first man’s observations somewhat and should leave the field clearer than he found it and in any event his ob- servations will be useful, as confirmation. Unfortunately, often, as the history of science shows, the second man over a subject is only a bumptious fool, and, when he has finished, the subject is covered with a cloud of uncertainty, until some third man, of greater ability, goes once more methodically over the entire field, blows away the dust, and again sets matters in their true light. If you repeat a man’s experiments, try to be at least as painstaking and circumspect as he was, unless you wish to be intellectually pilloried for the contempt of oncoming genera- tions. Never think it a waste of time or a work of superer- ogation to repeat the experiments of another person. Do not eall it ‘‘duplication of work.” It is not that, because no two individuals ever bring to a problem just the same sort of train- 41 642 BACTERIAL DISEASES OF PLANTS ing or outlook, and consequently, very often, one man finds what many others have missed. To exactly ‘‘duplicate”’ another man’s work you would need to have exactly the other man’s type of mind. Only by the labors of many minds has modern science come to stand where it does, as the only trustworthy interpretation of the world. So much, about the repetition of other people’s experiments! It is still more important to repeat your own experiments. Most mistakes in science result from neglect of this simple and funda- mental precaution. If this practice were universal there would not be so many papers published, it is true, but those which did see the light would be much more worth reading, and would redound far more to the credit of the author and of the publisher. New species would not then be made from different shoots of the same root nor from different branches of the same tree. Remember: Rushing into print with some half-finished article may give you an ephemeral success, but not any lasting one! “Though the mills of God grind slowly, yet they grind exceed- ing small,’ and the clarified and final judgment of the world on any human performance is apt to be very near its true worth, certainly not in excess of it. Be careful then of what you publish. Repeat your experiments again and again, and only conclude that you have the truth when they advance each time to a definite result like clock-work. Then, rightly, you may be full of that joy of discovery than which there are few keener delights, and may publish as speedily as possible with the full assurance that confirmation and due credit will not fail to appear. I now endeavor to repeat all my own experiments several times over and in the end I have a rounded-out and better view than one series only could possibly give me. Incidentally, I usually succeed in eliminating some errors or half-truths, which appertained to the first experiment. I consider this subject so important that the whole chapter ought really to be printed in capitals! Pasteur’s two golden rules are worth remembering: N’a- vancez rien qui ne puisse étre prouvé d’une fagon simple et décisive, and in the presence of failure, Refaisons les mémes expériences, Vessentiel est de ne pas quitter le sujet. Se GENERAL OBSERVATIONS: ON PUBLICATION 643 ON PUBLICATION The object of publication is to let other persons know what we have discovered. We cannot reach everyone, nor is that the aim, but we should be able to reach those cultivating the same field. The choice of a place for publication is, therefore, not unimportant. Generally we should choose some journal de- voted to our specialty or, at least, concerned with kindred topics, some publication in which one would naturally look for papers on plant pathology. Journals are better than Transactions because they are issued more regularly and frequently, and are read more widely. Among journals select that recognized as a leading journal. If you print in a Transactions or ina Report be careful to select one that is published on time, not a year or two after going to press. Remember: printing is not publica- tion, but distribution by sale or otherwise is. By no means bury your contribution in a newspaper or other ephemeral sheet, nor publish it in a journal, or Transactions, that seldom prints pathological papers, lest it should be overlooked and _ perish still-born! Do not print it in the middle of some other man’s paper, nor in the middle of one of your own papers devoted to some other subject. Iam referring to actual cases! Print your paper, don’t send it out mimeographed. Yet such copies are better than none. Finally, always secure and distribute several hundred separates, so that no one will have an excuse for neglect- ing it. In this distribution include all the leading journals and workers both at home and abroad. This is the more essential, in many cases, because certain journals have only a limited circulation outside of their own country, whereas science is in- ternational. Pathological problems also are international. I approve of patriotism, but that sort which has no international outlook is a narrow and vicious kind, fit only for barbarians. ON CLEARNESS IN PRESENTATION Having selected a place for publication, the serious question arises how best to present the subject matter. This is compara- tively easy only when the subject is a simple one and the contri- 644 BACTERIAL DISEASES OF PLANTS bution is but a note. It is grave if the subject is complex, and the writing extensive. Moreover, I have observed that the difficulty increases in proportion to the ignorance of the writer. Many a big book could have been boiled down to a few chapters, and in some cases to a few sentences, or to nothing at all, had its author been possessed of clear ideas. As a means of com- pression, learn to think. This is too much to expect of every one, but not too much to insist upon for the man of science. Whatever is worth doing at all is worth doing well. Clarity is the soul of truth, and especially in science there should be an idea behind every expression, and this idea should be stated as clearly as language permits. To read the dictionary is usually considered in the light of a joke, but I doubt if any student could do better, and that, too, through a long series of years. If he does not continually thumb grammar and dictionary, and per- sistently read the best authors, he will seldom acquire a luminous and persuasive style, than which, exclusive of a single-minded devotion to the truth, nothing is more to be desired. There are various Ways of saying things, but only one best way. Never- theless, to read the contributions of many scientific men one would suppose they must think any method of expression suffi- cient, even the most clumsy and ambiguous. Yet such is not the case. In spite of this motley array of bad writers, it is best that subject and predicate should agree, that one should avoid split infinitives and especially that each statement should be susceptible of but one interpretation! Every paragraph and every sentence in your paper should receive careful and repeated consideration, first, as to whether it tells the exact truth; second, as to whether it is absolutely clear, i.e., will convey the same meaning to all as to yourself (try it on your friends, if they will submit to it); third, as to whether it is complete, or requires various additions or qualifications— science is an eternal qualification; fourth, as to whether the sentences in it are entirely logical and move convincingly toward your final conclusions. These things can be determined only by repeated readings and much pondering. It helps greatly, when one has finished a paper, judging from my own experience, to turn back and re-write the whole of it. During this laborious GENERAL OBSERVATIONS: ON CLEARNESS IN PRESENTATION 645 and more or less irksome process, many new ideas occur to me, and better ways of stating ideas already expressed. It helps also, I find, to put aside the completed paper and come back to it months later, as to a new subject, or to one by another author. Occasionally there is a person who can write a thing as it should be the first time trying, but I have known only one or two such persons. Generally, easy writing is hard reading. Dar- win sometimes recast his paragraphs a dozen times, and most of us may expect to reach a good style, if at all, only by dint of much labor and repeated re-writing. Yet who can doubt that it is an end worth all it may cost? You publish to convince your readers and advance your own branch of science, and incidentally to enhance your own reputation. Look to it, then, that your writings are not only permeated with a love of the truth, but are forceful and impid as a mountain stream. To this end, avoid technical terms when common words will serve, even if you must do so at the expense of some conciseness. Nothing is more discouraging to the general reader than a book or paper bristling with a newly in- vented terminology, or full of mathematical formulae. ON COMPLETENESS OF PRESENTATION If you wait for absolute completeness, you will never publish anything but be always following up some one of the many side paths ramifying entrancingly in every direction from the great central subject under consideration. Nature is boundless and our own working lives are very short. There must, then, be some compromises. The investi- gation must be broken off somewhere. The question is, where? This is solved, partly, but not altogether satisfactorily, by not undertaking very complex problems. All I can say is—Do each piece of work as thoroughly as time permits, but publish, otherwise, especially on the assumption that you have something really worth publishing, your generation is more or less defrauded. Granted that you intend full publication, how complete the first paper should be, whether it should include all, or only 646 BACTERIAL DISEASES OF PLANTS a synopsis, or only some particular features of what is to follow, is a matter depending on various contingencies. If you have time, and are not likely to be forestalled, put it all into one com- plete and convincing paper, and illustrate it as thoroughly as possible. If, on the other hand, various other workers are in the field and you have reason to believe that their eyes are quite as sharp as your own, then it is important that you should get your discoveries into print as quickly as possible, if you are to receive due credit. You may then publish only a preliminary note, stating clearly what you have found and referring your readers to your later full paper for details and supporting proofs. Be sure of your facts, however, if you do this, since it is much better to let the credit of a discovery go to another than to rush into print only to discover later that you are wrong in places’ where you might have been right by taking a little more time for verification. In this connection it is well to recall the remark of the great zoologist of Johns Hopkins University, the late William Keith Brooks, when some one, alluding to an unpub- lished research of his, asked him if he did not fear anticipation. ‘“‘T long since ceased to be troubled by such thoughts, for if another should publish on this or any other subject before I do, his work would probably be better or worse than mine. If it was better, I should be glad to be saved the mortification of having published poorer work; if worse, it would only afford additional material for my paper.” This, I should say, is better advice for a mature worker with a well-established reputation than for a young man with his reputation to make, and yet it is worth the young man’s pondering. By complete presentation I do not mean extensive and tedious presentation. Far from it! Many scientific papers, especially in Germany, are spun out to great length simply, it would seem, to increase the size of the honorarium. By all means avoid such doings. I shall deal more at length with this in the next section. ON BREVITY OF STATEMENT—WHEN BREVITY IS NOT DESIRABLE A good rule is never to use two pages for a subject that can be compressed by a little thinking into one. The generality GENERAL OBSERVATIONS: ON BREVITY OF STATEMENT 647 of men use more words to express an idea than are actually necessary, if the best words had been chosen. Study the mean- ing of words, their shades of meaning, and re-write a subject twenty times, if necessary, to state it cogently and with brevity. Remember: nearly everybody will read a brief statement on an interesting subject, while only the most phlegmatic and deter- mined will hold themselves to a long-winded one. You will more than treble the number of your readers by halving your paper! Moreover, for the necessity of those who can’t spend even the minimum of time necessary to read a short paper, and for the convenience of everybody, especially of the foreigner, it is your solemn duty to sum up the substance of your contribu- tion in a series of brief conclusions which everyone will read, and which, if well put, may induce many to turn back and read your whole paper. No little thing vexes me more than to take up a paper two hundred pages long, let us say, often in a foreign language, and find no summary. I dip into it here and there trying to find what it is all about, without actually reading it word for word, and if I cannot do this the chances are that I throw it aside. Other people beside an author have some rights! Once I might have read it verbatim, but I have read too many such without profit, and now Iam wary. It may be nearly all ambrosia, but how is one to know if its author has not respected it enough to provide a summary of its contents, as an appetizer? Study then with all your might how to be brief, how to say much in little, and do not use a word more than is requisite! Yet at the same time, use all the space that is necessary to follow your subject into all its various ramifications, and to present each and every feature of it clearly. Brevity is never desirable when it leads to obscurity. Often, especially in abstracts of papers read at scientific meetings, a few words more, especially if well chosen ones, would have converted a glittering generality which tells nothing, nothing exactly and usefully, and therefore is worth nothing, into a helpful note. There is a great opportunity for reform in this particular. Either journals should publish no abstracts whatever, or else exact, useful ones. Not every one can make a good abstract, in fact, very few can; and in general you should consult original papers rather than abstracts 648 BACTERIAL DISEASES OF PLANTS if you would be well-served and master of your subject. Often it is some slight side remark of an author, sure to be missed by the reviewer, that will prove suggestive to you and fruitful. Another prevalent sin is neglect to provide long papers and books with a table of contents and with a suitable index. It is too much for any author to expect the reader to make an index to his book, unless he is a very guileless individual. My own opinion is that such authors are lazy, rather than unsophisti- cated. Any way, they deserve to be put into a pillory because sometimes unfortunately it is necessary to use their books, and to read much in order to get a little. Publishers are also to blame for accepting and printing unindexed works. That a second volume with a general index is contemplated is no proper excuse for neglecting to index the first volume, because the second volume may be long delayed or never published. I recall several such cases. Ebermayer’s © Physiologische Chemie der Pflan- zen’ is a capital example.