Ff C) 2 RMP any re, rai a 7 fd a iN 4 a a | THE BLACKLE« | aT C f : aa a La ”~ wed esis for D } ni [= Soo) THESIS INVESTYGATIONS ON THE BLACKLEG DISEASE OF THE POTATO Thesis for the Degree of Master of Science. Michigan Agricultural College. | . y > yoo v toto qe EB Kotilsa , i June 1920. THESIS TABLE OF CONTENTS : Page INTRODUCTION - - - - - - = - - = - 1 SIGNS OF THE DISEASE - = = - = - - - = 1 PREVIOUS HISTRY- - - - - - -=- = = = 8 MICHIGAN OBSERVATIONS AND EXPERIMENTS Occurrence - - - = - - - - - - 6 Economig Losses - - - - = - = = = 9 Wintering Over of the P*rasite In Tubers - - - - = = fF = = = 8 In the Soil - = = = = = = = = 18 Other Hosts for the Parasite - - - - - - 16 Relation of Host and Paresite Uninjured Roots - - - - - - - - 17 Amount of Inoculum -- - - - - = - - 20 Enzyme Studies - - - - - - - - - 23 Toxin Studies - - - - - - - = = &§ Resistance and Susceptibility of Varieties - - 27 GENERAL DISCUSSION - - - - - - = = = gg BIBLIOGRAPHY = - - - = = - 2f© =e = = 82 PLATES - * - = 25S fF = = = = = = 88 102109 TaveetiGacIos O TES ELaCKLSG DIssass OF Tia POTAL Tate QUUCTION “or gex.rs serious lowses from poteto tuber rots, both in the field &nd in stora&fe, have been reported from verious sections of the Upper reninsula of wtichigsn. The diagnoses of srecimens sent to various plant patholorical leboretories resulted in considerable difference of opin- fon 6s to the causcél organism, «nd it was to clear up this mtter, @s well &s to tm%ke & more thorough study of the dicease, with espnecié1 refereace to its mnner of dis- semingetion, end hoct rels tions, the better to recomend control measures, thet the present investigations were undertsren, ICIS JO Tus vISuiew ane disease in micnigen presents a picture Similar to tne bi&cxleg disease of potato as described ty 1” E lor se in wine, Herrison in OG. neda, Fethybridge snd 2 4 murphy in Ireland, ven nell in tollend, &nd apnel in cn Germeny. This disease, €s the nime indie tes, is chéré&ecr at terized by an inky blsecxening of the stem usurlly et the ground region. ‘his cherecter is not, however, ¢ primery symptom of the diseese, tut on the other nand, is the * tthe numbers follo: ‘ing némes of authors mentioned refer to the nerers licted in the biblio.réephy «t the close of this never. mY characteristic which confirms the digegnosis. Tne Tirst Signs ére the greduel feding of the deen green color of the foliése into yellow-green, snd later, a yellow color. wnis sickly arrearence is ususlly eccompanied by an up- turning of the leaflets and a tendency of tne branches to grow upvard forming & more or less compact top. The plent gradually dies as the base of tne stem is rotted away by the bacteria. The diegnosis of tiese abnormel pleats is e&sily confirmed by vulling them up. cuch stems alreédy naving been considerstly rotted offer no resistance to being pulled up and show an inky bléck ap- pearénce, the extent of which devends unon the stege of the disease. (Plate I.) Under vensther conditions more favorable to renid bacterial growth, the disease presents some different asnects. ‘shen the disesse progresses révid- ly the stems &re rotted in a very short time énd the plaxts fall over before tne leaves heve had tiie to ascume a lignt green or yellow color. In these cases the stems may show & water-soaxed Epnearsnce un to eirsht or ten incnes above the surfece of the ground. This condition is imnediately followed by bleckening. Under less favoreble conditions when the digease nrogresses very slowly aerinl tubers rey be formed es in the case of Ahizoctonia disease of potstoes. Tne Blackleg ferm mixes repid progress in the pith urea of the stem and this in meny cases results in hollov. stalks. In tubers the disease is usually chtracterized by rotting vhich proceeds from the stem end, the infection heving spread from the diseased seed piece to the plant stem, £14 from the letter to the stolen tnd finally to the new tuber. ‘he writer hes observed but one cease of laterel infection of c& tuber wnile in tle soil and this wes brought ebout by the imuiediete contact vith a dis- ef.sed stem. ithe skin of tie tubers whien ere being rot- ted in the soil presents & gun-metsl ence rasce over those portioas wreich ere rotting. Under fsvoreble con- ditions new tubers sre very quicrly rotted, vwherees if unfavorable conditioas follow, the rotted portions dry un and sive the tubers a truncated Envearszice. in this condition the dise&se reméins dorment until more favor- able conditions alloy further rotting to progress. A Tubers rotting from plicileg when cut open show @ white, creamy becteriz1l growth currounded by & blécx line or border. There is &lso « tendency toward esnal formetion. ww spe Ne 7“ TL mT , -_ ae. 4 v¥ 1 wi u! iw) { oe lr vtem Gnd tuber rots of potetogc have heen in- vestiseted since & comreretively early d:.te. «mons the TT, eurlier investi;etors were Tellier (1878), weinke ¢ud Eertholi (1879), Prillicux end velecrois (1890), Mremer (1691) end Laureat (1699), but from their published eeccounts we esnnot be certzsin of tie exect n&ture or tre rots dealt with tec&use of the mesxrerness o. tnreir revorts und beesuse most of their expetiments, due to ti.¢e 9 faulty methods of petnolofsicul techniyue oo. their time, must of necessity h&éve been verformed with mixed cultures or, &t least, poorly described orpanismes. 6 Trek (1699) wesc the first to essociete the stem dise&ce of pot:.toes which he desipnated cs “schwerz- beinigkeit" ead the ecsomminying rot of tubers vith a definite beécteri:.1 orgeaism. 20 this orgs nism he geve the aane Micrococcus rhytonhthorus but did aot publish a complete dese apnel (19C3) in his monogrrvrhic rerort upon the Lléczles disexse of the rotato geve in consideréble detail results of his investisations «-s to tne caussl organism, its nost relétions, its loss of virulence, re- Sistéuce and susceptibility of vérieties. “Nis work wes & continuation of thst berun ty frank. In this work, it was found tist tre ceusel orseniem is s btecillus and because Srenk failed to give s comrlete descrintion of the organism “ith which he worxed end left no cultures, the former rensimed the cousal org: nism cecillus vhytorh- 7 thorus. widith heving reecived = eulture of the orfen- weet AY te t 5 ism from -.npel gives the moet complete descrirntion of Pacillus shytorhthorusc aveiléble today. This descrip- be *- e eccorsin: to buectertiel struderds. Fearrison ya tion wag Le (1906) reported in * complete mounner the results of nis two ,ears' study of the Sleckles disesse of Can:da. In this report he comfirmed the txetcrial neture of the ro but escigned tne diserse to bicillus Loleniseprus. ne @lso took up the efvects won viericeties o potstoes and inodeulszted otrner plente. aw wet i 1 worse (1917) in sumtrizing nis nine yeore' ! ~wOrk on tne Sléckhlog diseese in +: im@geve ¢ review of the litereture, the geopraphnice:l1 distribution &¢s well es ecomomic a&srects of tne diset se, control nec sures, Ee. @ compérctive study of the causal orginicms. +e ¢lso re- ported és « recult of his observetions on the sources of infection and winterinse over of the orfrinism, thet dis- eccsed tubers cid not soil ves the cvring cource of in- fection. 8 Tosenbewn end Ramsey (1916) in investigating Cf the influence of temer ture tnd precipitxtion on the bleckleg disease of poteto, Tounn tuct taa severity of 7 V4 4 ~ L 4 soy? a. ae. . res ~) aS yt tT we ce ry Y S 4. ‘ - 2 MMA tee 8 , € ew. ° - - . . t - om | , “ Cc 3 qd Fl.a wt c ~e oO te le 71 Oters 1 (30 a oF 6 7. 3 toh Gd trae ‘. oo (3 Gs il -? a - 7 precipitation and ves devendent woon then. dvhese investi- getors consnucted exneriments on wintering over of the dis- both in Leine aud Virginia, coufirming the crevious views of uorce. g namcsey (1915) renvorted ctudies on the viclility of tre potato L’éckleg orgeonism efter being subjected to rN various climatic Eni environmentel conditions. nese ex- periments surplemeuted in &@ minor véy the more exteasive experiments of hogenb&um snd reimsey. his work consisted pe in subjecting infected tubers to low temmeratures, which invériebly proved fetal. se sleo obtvined nerz-&tive re- sults from éttempts to inoculate plitats by the vetering method. ine L Gri iGo. Oo. mw. Vee! TOuwi nd ie ..! oa a, dab cay ak J Occurrence wison us & result of Pield obcervetion end sub- sequent lebor:tor, work was led to believe thet the "DBlackleg" diseise in wiehifii differed from the dicesse as seen in isine £nd other rerts of the vnited stctes. In his opinion,the diseése fouid in the Upper genineuia of wichipgand more nearly resembled & “uséirium diseese of notse- toes vhich he observed in in.esots, and ne explceined tire preseiuce of bucterit« in tne tis tn cues by considering them of the soft rot tyre wnichn were werely second: ry iuveders. In order to determine definitely wheter the C rs rluckleg diseisce of the “iver *#eninsula of Michigan wes a Ssi:rium trouble or vwnether it was due to a bacterisl paré- site ¢s is the céese of Li&cxlegp diseases revorted fron other states of the United ctztes and from other countries, tre vriter poured dilution plétes from dise:.sed me terial obtéeined from two different fields in alger County locéted ebout eignteen miles enart. From these plates nine bac terial colonies were isole ted, five of whicn, wnen inoc- ulated into rew tubers end into heelthy,¢roving plants, produced chéréecteristic symptous of the digeise. In no se were &ny of the molds which develoned upou the pletes m2 C found to ke -ucerium. ‘Tre writer hes renestedly isoleteda bacteria from diseased plents from u frect many urrvrer re.sia- Suiw poiats which when tested out with cuthentie strvins of “Cillus phytopnthLoras obteined through the courtesy of the ‘ wigconsin sxveriment -tction proluced similer vi&ecuxles effects. sere seeus no chunce for question &gs to the etiolo;,7y of the olackleg disease of the Unrer seninsula of wichigan, and certeinly «custrium is not concerned, The orgenicm is & bacillus vhiciu is similer,if not iaen- tTicél, with that described es the causel orgenism of the Dlackleg disexse of potato in other sections of sericé and wurope. wconomic Losces Tic louses resulting from bléeckleg vary with the seesons. Juring se&sons which ere cool and wet conditions cre very fuvoréble for the disease &nd mucn erester losses follow thsn is the cise during dry sc&sons of little reinfell. as to the importence of tre dise&se in this and different countries, tle verious investi ;&tors report as follows: Gera 6 5 Frank ead s«prel ; The dise&se is comaon, wide- sere:.d ead often destructive. France: 9 Delacroix ; fernérs wider srread then tris in- vestigetor sunrosed because of the confusion between this disoase éndi "brunissure". ey Irel&énd: rethybridge :; 945 of diseased plants produced by selecting «ound seed tubers from €n in- fected crop of the previous season. Caneda: & iarrison : sastinctes totel loss through rot 1c-75. of the crop in irovince of Untsrio in 1305. Csnede: LO | murphy : to réerts of the leritime Frovinces of Ceneda 10, of total crop in 1915. én Some céses percentsce of tlackleg pleiuts higher then tnat, and total misses were és high es 6O,. End more. United wtites: morse’: sxtensive obcervations in Aroostook County, me., covering & period of Tive years revesled in rény fields only scattered tlickles vnlants wad not infreguently tlese éEmoundt to l or &€ nercent, 5 percent of dis- eased pnlunts being considered by the growers as representing & severe attack. wuosses of 1O or 15 vercent or more are by no inetns un- known, however. ft Coons: seports tiiat losses of from £5 to 75 percent were not uncommon in the Jpper Penin- Sula of wichifgan in 1915. tne writer studying the disease during the 1919 season, which vas very dry «nd unfavorsble to the diseése, visited a number of fields which showed 10§. of infected plants. ‘he average for the entire Upver reninsula during that season, however, was considerably less and 1-3). would be & fir &verige. Yrom the aebove reports it can be concluded tnat Blackleg is indeed @ serious disease of tne potsto and thet in the Upper Peninsulé of michigen it is one of the limiting factors of the potato growing industry. f* wintering over or rarasite: In lubers: It hes been generally conceded by most invest ipa- tors thet dise:ised tubers kept in storase over winter sre the main source of infection. ‘Yrese tubers under favoreble conditions rot and céuse the rotting of adjoining healtay 14 tubers. ire bacterial slime m&y &lso spread over tne sur- face and into the eyes, cracks, or bruises of otherwise 1 norm] tubers. although ..orse and others have noted that tre orgsnism is resdily killed by dessice tion when it is dried on &@ smooth glass suffece, the writer has found that under other conditions the orgénism will re- tain its vitelity for @ much longer time. or instance, liorse found that the organism when dried on small egless discs et room tempers ture wes decd in less tran half an hour, whereas the writer has found that the orgenism will retain its vitelity for four days when dried on a Silk thre&d under similar conditions. (see Table I.) Téble I. Lbessication of the Blackler Yrgy nisin edium 3 wnere :iested on raw tubers o21 nutrient broth >; kept :l dv y:edeys :3d: ys: 4de7s:5days:6 dys: Cover Glass ; Desk ss: : ; : : . : s drswer: ded: dead : desd: deed : deta: de : ee TY Decric: : - , . : . - cator : ™" ::" 2. M4 2 -" =; -” : ; : ; : 5 ; : : Si¥k Thr8ed : H 5 oo st 2 oF fl oc o- s - : : : s wlive: &Slive alive: dead: dead ; 'T 1" e ° ° ° ° ° e ° e vesk e ° e e . e e e s dreveri&live: "7 > Fo; * 2 Me 3 ot : "rom tnese results it is seen thst the org: nism is able to live for &@ considerable period ander conditions of very little moisture and sufficient protection from dessica- tion to tide the org&nism over until more ft-vorable condi- tions arise. In regard to wintering over of the disease in 1 tubers which rem:in in the soil over winter, sorse basing his opinion upon field observations mekes tne following statement: "On fields which fre planted with potatoes tre second time in succession there is usuélly quite a percen- tage of volunteer plants which spring from tubers which remined in the soil over winter. ‘rese plants are fre- quently eesily recognized by their irregular occurrence on the sides of tre rows or between hills, The writer, (liorce) has never seen such plants affected by bleckleg. This ob- servation was cuite unexpected, for it seems not unreéson- &ble to sunpose that, if the tubers were sufficiently pro- tected from frost to te able to germinate, the bacteria causins the disease. Might live over in tne coth in such infected tubers &s well &s in storége." kamsey in studying the problem of over-wintering of the bacteris, inoculeted heslthy disinfected potato tubers with & pure culture of @ strain of B. atrosepticus van tall. After being left ina moist chember for 48 hours the tubers suoved marked rotting 7 ok Cu at the point of inmoculétion €1.d were tnen poeosk3ad iu Crosex 1.03 ine refrigerator. very twenty-four hours one of these tubers was taken out and planted. Out of the eleven tubers which were pleuted only two germineted but the sprouts were immediztely killed by Lleczleg. «ll other tubers decéjyed vithout showing eny sprouts ebove ground. Vlates which were poured from the tubers before planting showed thet the nun- ber of bacteri& vas reduced to @ minimum efter £64 consecu- tive nours. Tansey offers this es sn exnlanstion ts to the reason why sbleéckles is never found in volunteer vlints in iaine, aid stetes: "avidently the infected tubers which remain buried in the fieids ct diggins time, decay to such an extent thet they Gre unsble to germirmte, or if they do germiante, the sprouts are im.edistely Killed ty the orgéa- ism. Therefore, &11 volunteer rlents which maxe their Ennesrenace in the soring ere free from Eleckleg." 8 nosenbeumend russe; in more extensive experiments on ti.e wintering over of the Lla&ckle*s organism in naturally infected und ertificielly inoculeted tubers found that: "In no c&se wis the blecxleg orgenisn found to live over & winter in the-soil or in the tubers remaining in the soil." Tre experiments of Manuscy ond of Loseubéum end Ramsey mney be criticised in thet they were not sufficiently extensive to werrent definite conclusions to be drévn threre- from. ‘uen, “esin, it is very probable thet the wintering over might occur in tubers vhich in the fall show veryslight, ifeny outwerd indicetion of infection. In the experiments conducted ty the above investigsutors only vadly infected tubers were used, «nd these no doubt sccount for the results obtained. Soil conditions, or st least those of the Uprer Peninsuls of wichircan during the vinter ecannot be conrered to conditions represented by seusey's cracked-ice exmnerimeut, because in ordintry yer rs tre ground is not Prosen tefore it is covered with enovw End rermits the winterinpy over of pots- toes «t & comre ratively low but sot freezing teimpereture. oO on ~~ tle writer hes &1so observed from experiments on chilling érd Trost injury, tret notstoes :hich have becn exposed to Le - = ew freezing temneratures decay very rapidly through the in- veSion of secondary orgenisms, and it is possible thet these secondary bacteri& swamped end crowded out the Bleckleg organisms in Samsey's experiments. In order to deterinine the question of wintering Over, under conditions existing in the Unrer Teninsula of michigen, 2 plot of &pproximtely one-quarter of En acre was left undus in the fell of 1918. ctlackleg wes present in this plot during the 1916 frowing season. In the Svrine of 1919 this plot vés dug and & nortion of tie crop was spreéd out upon €& cleén barn floor to greensprout. tne writer carefully selected six bushels of this green- sprouted stock, discerding s1l1 tubers wnich snowed evidence of rotting. Two bushels were disinfected with mercuric chloride, and two bushels with form&ldehyde in the usual manner, while the reméining two busLels were left untreated as a@ check. the potetoes vere pléiuted with e machine plént- er, the Corrosive suolinéte treated tubers being plénted first, followed by the formeldenyde trested snd untreeted tubers, respectively. ‘To avoid the possibility of e.il infection, the tubers were planted on virgia soil which had borne no previous crop. Careful examination res made of these plots twice a week throughout the entire growing Ssesson und eich diseased hill wes staked @s found in order to avoid dupliestion in recording the number of dise&sed hills in each plot. Tne results obteined from this ex- periment are shown in the following table: Table II. Cvervintering of Zlacklec in Takers remaining in the soil at the U. Pp. -ixn. ota., Chathnem, lich., 191€-1919. eno. disersed Treatment shills found > Percent ; sduring se&son ;: : Corrosive wutlim.te, 4 oz ; . : to 50 gt1l., ol min. ° & > 0.4 : formeldehyde, 1 vinat to . ° 30 gal., 15 min. : 7 : 1.4 : Untreated : 35 - 762 : rletes were poured from diseased plants, the causél orgs nicm isolated, and its pathogenicity proved b/ inoculation into raw tubers and into healthy groving plents. Murther evidence of Lilgéckleg was found in three other plots of the seme snring-dug seed stock which were plented in tnother field. The above expsriment and obser- vations indicate trat the Llackleg disease c&n winter over in tubers which remiin in the soil under conditions exist- ing in the Uprer reninsula of wichigan. The reletion of treatment to rercentaufe of Elacklesg &prearing is ulso sig- nificant. In the soil: as in the cease of wintering over of tie orré.nism in tubers, there is also difference of opinion &s to the 5 wintering over of the orygenicm directly in the soil. appnel i fJ an { C bo Bud Narrison botn believed in the orgenism remsining in a fue soil snd in their control measures recomend rotation 4. L 1 ferons. worse , dreving conclusions from otservetions meade of fields planted to potetoes tyvo yesars in succesion which during the second year snowed eitner not Sny or re- duced numbers depending upon, in his opinion, the thoroush- ness of seed treatment, stetes: "--infected, seed potutoes are the sole source of infection end distribution nd tret the disezse does not live over the vinter in the soil." 8 hosenbeum tnd weusey arrived «t the stme conclusion efter etteupting to isolete tne organism from soil in vérious perts of their winterin;g over test plcets. These investi- ators poured many series of plates ¢nd sade & great nun- Cy ber of isoletions, all of wrich vroved to be non-pithorenic vhen inoculited into rew tuber clices eud into frowing plants. The difficulty experienced in ende&voringe to sick up colonies of the Lleckleg orgénism from nlates poured from coil samples is, of course, obvious and militetes ainst the force of tneir conclusions. ‘WTnere is such a greet number of soil organisms wiicn develop upon agar plates very similer to the blacklee organism in apvearance, thet the chances of picking up the right org&nism ure very small. Tie folloving exneriment is notewortiy in view of the positive statements of other workers: in this ex- periment tiree kinds of soils vere used, Uley, vend, tad mucke)4 wll Ticient soil of essen kind wes used to fill two large wooden pé&ils and one .,¢eries wos kent in the green- house, while the other ves allowed to winter over outdoors. wien of these wee thoroushly mixed ufter receiving two we £ a liters of a decoction made up of severely rotted tubers and we.ter, the coils being literally satureted with the orpsaisn. In tie spring e&ch soil was thorouchly mixed tind ¢.n &verfeg¢e 7 axen according to the methods followed in soil ane- =) re jt D cr Lysis. Instesd of ett © isoleting tine orgenism from the soil, rav-tuber slices were Lnoculeted vith portions of the coil, tnus éllowing thre tuber slices, the neatursl host, to do the selecting. one hundred «nd eight rew tuber slices in soyce disnes were trea inoculited vith smell quantities of soil, .eig.iieen Sess > + - +. ‘ my qe Te, ra. Mg dee teing used for e&cl. of the Six semoles texen. tne ete D obtained from the emmeriment Ere re rs ©) orded in tne folloving teble: Giclee Ill. winterin;: over of tne Fléeckles Orgenism in verioue woils. LOLYA1TOEC, > Vind : ‘er sailaler 5 onuarer > Total : wOe. : Of 3; Kept sclices sslicses not: Slices : ; .Oil : shlackened: pltercned: rotted _: 1 ; Clay : Greenhouse ; 7 : 9 : L6 : ; | : : : 2 - vend : " : 9 . 7 : 16 . 3 luuelr : " : 11 6 : 17 : 4 . lay 2 Sutdoors : LO : 5 : LE : 5 e woud $ " : E ° 3 . 11 : 6 > Lmek : " : Lé ° © : Le. . e ° : : e ‘ : : : -l16- It chould te noted thet the soils wnich were ued &@S inoculum in tre ekove esmeriment were mixed cultures of bacteria end fucri, end not all of tie rotting which fol- 4 og. ej} ore aececy Jot oo root - Wk rib, OVee. wa ke ane @ wee aw tet bee td lowed cén be éttributed to the of tne tuber slices snowed discoloretion not entirely dis- Similar to the bi&ackening which Grnetre on tubers rotting Trom blackles. Direct inoculéitiou from one of these dis- colored tuber slices into & Le. lthy groving poteto plant resuited in @ brovn black discoloration cimiler to tne dis- cOlorétion of potito stems following inoculation with rure cultures of tie plackleg organism. Isoletions ére now in progress from tunis idaoculited ster. alti wer tre securlisg oF & pure culture of tie Biackleg orgenis: from the soil ses not attemted directly, there is sufficient evidence brought forwerd in the over- wintering experiment rerorted ebove to leéd tre writer to believe tict it is possible for the orgenicm to withustend the raveges of micnicén vinters. If in severe] cases tud "ith several tyres of soil,jorgsnisems cuneble of producing tyrical f£leckies rot of potsto elices ere Found, ©8 smitva nolds this cere city &s © decisive proof of identity, it is evident treat under the multinlicity of contitions rrossnted eree&t detl om overnvintering dosas tuxc rlece, e or cone Little lurorteuce in coutrol recou- - evye 4 4. 4 a de 4. , 4. 4. ren : 4 at 6, ._ 3 wee: ~ 7 e aovel wes tie firct to attemtto aescertéin whetie2 ~17- the organism which was responsible for "¥8chwartzbeinigkeit" in Germany was parasitic on any other hosts. This investi- gator succeeded in getting infection from inoculations of B. phytophthorus on Cucumber, Winsor Bean, Carrot, Teltower Beet, Lupine and Tomto, but was not successful in inoculs- tions on Sugar Beets, iiangels, Pelargonium, and the grains. inrrison” Also reports successful results from in- Oculating other hosts with his B. Solanisaprus. Positive re- Sults were obtained from inoculating tomatoes, Capsicum “nnun, and cucumbers, while negative results were obtained from inoculations into egg plant, 5S. Melongens, Physalis, Datura stramonium, Schizanthus pinneétus, Collinsia verna, Salpifglos- Sis sinusta, and Impomoea purpurea. In a similsr experiment the writer obtained posi- tive results from inoculations into tomato end Nicotiana rustica, negative results being obtained from inoculations into Petunia, Bittersweet, Datura stramonium, and Physaloides. (Plate III.) From the above experiments it is evident that the causal organism of Blackleg of potato is also parasitic on a number of other hosts and under favorable conditions might Gause serious damge. This fact is also of importance in recommendation of control measures. Appel states that po- tatoes should not follow in rotation such crops as cucumbers, carrots, lupines, or tomatoes, nor should these immediately follow potatoes. Relation of Host and Parasite: Uninjured Roots: - 18 - There has been in the past some auestion &s to whether or not the Blackleg organism, in the event that it is able to live over winter in the soil, is able to pene- trate healthy roots. Hamsey conducted Several experiments to determine this question and secured negative results from ettempts to inoculate healthy growing plants by a watering method. As & result of his studies he concludes that unless the seed piece is infected at planting time, there is little chance that uninjured plants. will contract the disease even though the causal organism is washed about the stem and root system. The writer also has oar- ried on expetiments in this regard. Inoculation of healthy roots of potato plants growing in water cultures. has been attempted. MTubers were allowed to sprout in wet excelsior and when the roots were approximately 3 cm. long, © piece of the tuber about l cubic centimeter in size contsining the sprout end roots wes cut and pléced in a quart Mason jar containing & nutrient solution made up of the following: Magnesium Sulphate 60.0 cc Acid Potassium PhospAte 72.0 cc Calcium Nitrate 20.8 cc Water 1000.0 ac The stock solutions of the salts used were of one-fourth 13 molecular concentrations. Shives found tcat this nutrient solution produced optimum growth of wheat ®nd it was thought probable that it would also prove a good medium for potato- Two such cultures were prepared. growth. -/ About two months after the oultures were started, they were about 10 cm tall and had about seven leaves each. -~ 19 - One WAS inoculated by pouring about 5 cc of a 4-day-old broth culture of B. phytophthorus into the solution. ine deys after inoculetion it was noted that the plant had mde no further growth end that the lesves presented a lighter color than the check. The check hsd a good root system, healthy green leaves, &nd had begun to form ‘new tubers, the largest of which at this time was about the Size of a mrble. A month after the addition of the broth containing the Blackleg organism, the water culture potato wes dead. Contrary to what occurs in Bleckleg infected plents in the soil, there was no blackening of the roots or stem of the weter culture potato either ebove or below the surface of the solution. It *ppears that the organism was not able to penetrate the uninjured roots, but the toxins produced by the organism, being absorbed by the roots resulted in the death of the plant. The check remined nor- mal throughout the experiment. (Plste IV.) The water culture experiment, therefore confirms Ramsey's results in that it shows that the bacteria are un- able to penetrate uninjured roots . It my be well to point out that too general a conclusion must not be drawn from this fact. In nature the roots are often broken by culti- vation, or are injured by other diseases as Rhizoctonia or Fussrium and thus emple opportunities for the entrance of bacteria are presented. It would not seem justifiable, therefore, to give the significance as Ramsey h*s done, from exveriments conducted on uninjured roots and to deny all effects from washings of bacteria to the roots of plants in the field. FPurther field experimentation is necessary before any definite conclusions can be drawn in regard to the transmission of Blackleg from hill to hill through the soil. Amount of Inoculum: 7 It has been noted by Smith " -- that in culture- media not exactly adapted to the needs of an organism, a scenty inoculation my not give any growth - not even after & long time - whereas a copious one will lead to 6s growth which gradually clouds the fluid or covers the solid. -- fhe only explanation I can think of is that a multitude of bacteria is stronger than a few, and thus by union are able to overcome obstAcles too great for the few. The same fact comes repeatedly to the attention of the animal pathologist as & result of his inoculations. The animal body, we must assume, is often able to overeome and destroy @ few hostile organisms, where it would not be able to defend itself egainst msny; otherwise whole races would be exterminated by natural infections. ‘The sane is undoubtedly true in plénts. the modus operandi in plants is not altogether clear. We mey advance several hypotheses:(1) The formation of a resistant cork-layer before the bacteria have multiplied to such sn extent es to prevent cellpdivision; (2) the des- tructive action of antiseptic plant-substances, e.g., acids, before these can be neutralized or otherwise destroyed by the substances produced by the multiplying bacteria. -21- In some instances, the introduction of & very considerable mass of bacteris seems to be necessary to in- duce disease; in other oases a very few are sufficient. It would be extremely interesting to know the minimum number capable in any given case of induoing disease." In ordexs to determine the effect of amount of in- oculum in the case of the Blackleg disease of potato, the following experiment was performed. Dilutions of 1:100, 1:10,000, 1:100,000, and 1:1,000,000 were mde from 8 virulent 72-hour broth culture of B. phytophthorus. Sliss Triumph tubers were disinfected with corrosive sublimate snd cut into smell pieces by means of a sterile knife. These Slices were then placed in Soyke dishes upon pieces of &ab- sorbent cotton saturated with distilled water. Ey means of sterile pipettes one drop of the broth, full strength, and one drop ef each dilution was placed upon each of ten tuber Slices. One drop of each dilution was «lso placed in tubes of melted nutrient agar ana plates poured. Data obtained from this exneriment «ure resorted ian the folloving to2>le: mows. or ~~. CL YP a e lff3at of --4ay21.5 97 Ineculum :i0.cols. :nouba~:Growth on individual slices . stion . : Dilution ; pistes iperiod > 1: 2: 3: 4: 5: 6: 7: 8: 9: 10: Broth, Full:very ; $s 3 $3: $3: 3: 3: ¢$ $: $3 8 : strength ‘numerous: 2 daw: +: 4: 4: 4: $: 4:3 $: 4: 4: f: 1:100 ——-:: 19,650: 2 da.s:4: 4: 4: 4: 4: 4: 4: 4: 4: 4: 1:10,000 : 556: 3S de.: $: -: $: $: $: $: $2: ts 4: 4: 1:100,000 : 96: 5dea., sc Fi mr m2 tit: ts: $s ts oo: Liisillion : 6; 5 d&.: -3 $2 -: Ht: -: cs es $2: 2:3 ~«- —_ oo —_ bua be the data tebuleted in tuble IV reveal severil interestins feets. “irst, the neriod of inoculation is ineressead as tre dilution is increased. «t.ereas, in the cases of full strength broth and 1:100 dilution » ix wth avvec.red after the usual incubetion period of é€ déys, growth in the 1:10,000, 1:100,000 aad 1:1000,C00 dilutions weg not evident until 3 dsys in the ace of the 1:10,000 dilution aad not until 5 deys in the last two. Thet the growth in the two greatest dilutions, thst ceuced by 96 and & becteria, resvectively, wes considerably weeler then in the otkers is «lso significunt. The 96 bicteria per drop out of a possible ten, rnroduced six successful inocul: tions, wnile tre & tecteria ver drop were €ble to overcome the resisténce of only three Slices which shove tiuat this number is ebout the smellest which ean successfully c&use tne rotting of po- teto tubers. Tie effect of various qurntities of inoculum on Slices of raw tubers was &lso tested out in another experi- ment. Rew-tuber slices of Lliss Yriumyh vseriety were inoou- lated by pluciag varying quentities of & virulent g-deg-old broth culture on the cut surfeces by meens of @ 3 mu pleti- num vire loop. ‘he tuber slices were not pricxed vith a needle *«s is the metnod ordinerily pricticed ia muxing inoau- létionus. «after inoculetion the slices were placed in :ioist cnémbers, and 46 nours later shoved .earked dirtferences. Tnose Slices which were inoculated with very thin films of broth 1. culture, instead of snowing cresmy becterial growth &s in the cases of inoculétions with one or more loonsful of the cultur6, Sroved the effect of tre inherent resistence of tubers, which was exermmlified by & wurt-lixe nrotuber&.ce, or én oedema fownstion. (r£late V.) It was first thought tuat this phenomenon migrt possibly be due to renid division of cells, the minute quantity of inoculum beins the exciting factor. isicrosconic ex€mines tion, however, revesled the fact tint these protuberances instead of being composed of greet numbers of minute cells were nade up of giant cells, tn aud tict these cells contgsined very few starch fréins a compared to the nunber found in normal cells. «aS a probable explanetion of tne phenomenon it is thought that as a result of the effect of the organism or its products unon the cells the starcn prains are che nged into suger, thus increfsing tlhe ogsmatie pressure wnich resulted in greeter hydration of t the colloids, hence the incretsed size of cells. These experiments comfirm the telief of omith that & multitude of bacteria is strouger than a few, and shows that vnereeas in the cases of minute quantities of inoculum poteto tubers &re able to resist the ettack of tne Llackleg bectorisz, Jerger auounts a@re able to bres.x down tnis resis- tence and cause tne rotting of tre tubers. “1zgme Studies: L Harrison vas able to produce typic&l Llackleg lesions by the use of the chemical products (enzymes) of the growth of his bucillus, aad states thet the solvent action of the enzyme on the cement substance of the cells was quite mirked. The writer wes able to c&use the soften- ing of raw-tuber slices by placing smell quéntities of 4 dry enzymic extrect, by meens of & sterile scelpel cud platinum needle - oO , upon the rresnly cut surfaces. Tnis enzymic ex- tréct was made by masning 1E tuber slices, which were $e severely rotted by Bléeckleg, in 50 cc. of weter, tie mix- ture being then filtered through filter parer é€nd precini- tated witn 90%. alcohol, énd the precipitéte dried on gléees plétes. In order to escert&in vnetner ti.e enzyne produced by clecklegr was similer to thet produced by zecillus curoto- 1S, vorus, Cs described by Jones , snd to connsre béecterial with enzymic action, the following experiment was rerformedg six tuber G:d six e&rrot slices were inoculéted with the dry en- Zymic extrect, as described in the preceeding partgruph; wuile one tuber &£nd one exrrot Slize e&ch were inoculéted with £4-hour brotn cultures of ©, phytonhthorus, Becrpickerueni, aud distilled vater, the inocul:tions teing made by placing one dron of inoculum on the surfs.ce of e&ch tuber énd cérrot Slice. ‘Tne deta obtxined are tebuleted below: affect of sigymic oxtrect on Rew rotets sud larrot o Un FPOteto -o Un Clrrot I ae. O50. 8 6 6 648 33 O63 64 ct 4B Cg Te: oocuLum sThoesshours:Yours::Inoe: tours: Hours : Hours: ° ° § sake maces shLigquid pulivht it cuorced ; ° Soy te ~£97 ~ ey UC Ve ecsoften -:soften: ye mi¢ ° 6 5uO0,t- ° 3 “se @ 6 ° SN ' a Ext te ot Pacing eae: ; :Shhe ging ting bevuytovi-: ; seliguts: L: —- ssligut : ; thorus ; Od 5 7 8 Or euyi yn: : pai-¢GQ1l-; divto; : : go es : -Ore tion. - Be. grigue.: $3 : sc dtent, rt aL ee =: J]: ~ - " 2OPo ial: : : : 2: : : Seo! vistilled ; : : 3 : : : : Wever > lo: |= 3: -:: 1: - : - ; - : oa iicroscopic exztminm tion oF ti.c softened tuber and c&rrot slices revealed many free cells, showinz thet the enzyne acts on pectin of tne middle lgmells, which would sugvest treet tne enzume ves pectéese. The fect tnat cévrots were also softened, trough not :s repidly as were potetoes, signifies tnet the enzyme produced by 5. thorus, if not identical, is very closely related to that mnroduced by b. curotovorus, Tne deta recorded in suble V also show tiri:t tire enzymic activity wes much more vigorous then in the céce of trie becterisl inoculztions. wnereés, in £4 nours, in the former there wos marred evidence of activity, in tne latter there v&aS none. Tnere were also severél other points of difference between enzymic and bscterizl activity. ue tube slices softened by the former were not blacxened €s is the case when tubers are rotted by the BSlacklerz germ. Tnen cf: in, neitner tne softening of the tuber slices nor those of tne carrot wes accompenied by the disaprree&étle odor which is pro- duced when tuber slices ére inoculéted with the live orgenisn. On the other hant, the odor ws réther nlecsent, and esrecizll; z so in the case of tre e€rrot slices. If was &lso observed from the above e:izyue exeri- ments thet tiere wes no discoloratioa of tubers as a result of the enzymic activity, and ss blackening both in tubers end on the stems of infected plants is such & fixed charac- ter of blackleg, the cuestion was raised as to what caused the discoloration. «As the enzyme was unable to produce dis- - 26 - coloration, it was thought very probable that the organism must in addition produce a toxin substance which causes the blackening. This theory was readily confirmed by plécing & virulent 24 hour broth culture of Bacillus phytophthorus into a thin sterile collodion sack about & mm. in dismeter and 4 cm. long and then placing the sack on sterile raw potato plug ina sterile test tube. vcubstances of colloi- dal nature, the enzyme, etc., which are produced dy. eul- tures were thus kept inside the sack while the orystalloidal substances were able to diffusethrough to the surface of the potato plug. This effect produced a brownish discoloration resembling typical Blackleg staining in its first stages of development. There is therefore produced & crystalloid toxic substénce. The presence of a toxic substance was &lso shown by placing leaves from healthy vigorously-growing plants in beef and potato broth. That part of the petioles which was kept in the broth wes enveloped in a thin collodion sack. In trree days these leaves vere wilted while the check, which wes enveloped in a simibar sack and kept in distilled water, was still turgid. Plate VI. From the above studies of the reletions of tie host and parasite, it seems extremely likely that two agencies operate in the mechanism of the attack by the Blackleg organ- ism upon potato. The Blackleg germ produces a pectin-dis- solving enzyme and also a toxic substance which causes dis- coloration, and the two working in conjunction, under favor=- able conditions, produce the symptoms by which the disease is recognized. Viewed in the light of these two factors, the rotting effects of heavy dosage and the oedema formation with light dosage are very clear. The difference in patho- Benicity between B- carotovorus and B- phytophthorus is thus found to rest upon differences in toxic substances. ' Resistance and Susceptibility of Varieties: Practically all of the investigators who have studied the Blackleg problem have noted the difference in resistance and susceptibility of varieties of potatoes. Appe1® was the first to note this difference, and Harrison® also found that some varieties seemed to be more susceptible than others. The latter investigator, however, in variety tests found out that many varieties whach showed comparative resistance in the field were comparatively susceptible when subjected to artificial inoculation. Morse? in studying the disease in Maine observed that the Irish Cobbler, an early maturing variety, was more susceptible to Blackleg than the Green Mountain which is a late maturing variety, and goes on to state that: "As long as Msine potato growers planted this variety ajmost exclusively, Blackleg was of minor conseauence." Field observations of the writer during the 1919 season, supplemented by laboratory and greenhouse tests has led the writer to conclude that the Green Mountein is not as resis- tant as one would be apt to believe after reeding the obser- -~ 28 - vations of Morse. JLaAboratory tests on tubers of eight warieties were conducted and from the data obtained * dif- ference in resistance and susceptibility was noted. These showed that the Bliss Triumph, ® very early méturing variety, was more resistant to Blackleg than was the Green Mountain, which proved to be only slightly more resistant than the Russet Rural, another late variety. To determine ‘whether resistance ®nd susceptibility as expressed by tuber inoqulations was correlated with simi- lar resistance and susceptibility of stems, healthy succu- lent plants of the same eight varieties were inoculated with the same auantity of a virulent 24-hour broth culture of B. phytophthorus, the inoculatibns being made on the stems just above the surface of the soil. The plants were of the same &ge and were grown in the greenhouse. After inoculation the plants were pl*ced in 4 Wardian chamber where & very humid condition was kept. Ten days after inoculation the plants were examined and,as in the case of tuber inoculations, a difference in resistance and susceptibility was spparent. The Green Mountain and Bliss Triumph varieties, instead of being resistant:, however, were the first varieties to suc- cumb to the attack of the disesse, while the Irish Cobbler and Rural New Yorker types proved to be more resistant. A comparison of the resistance and susceptibility of varieties as expressed by tuber and stem inoculations may be made from the following table: - 29 - Table VI. Resistance and Susceptibility of varieties to Blackleg ee Tubers Growing Plants Resistant Resistant 1. Bliss Triumph 1. Sir Walter Raleigh 2. Green Mountain 2. Carmen #3 5. Russet Rural 3. Irish Cobbler 4. Irish Cobbler .4. Russet Rural 5. Early Ohio 5. Barly Vhio 6. Early Rose 6. Barly Rose 7. Sir Walter halel gi 7.. Bliss Triumph 8. Carmen #3. 8. Green Mountain. Susceptible Susceptible From the above experimentation it is evident that there is some difference in resistance and suscepti- bility of varieties of potatoes to the Blackleg disease, but the contradictory evidence presented also mikes it Glear that conclusions in regard to such resistance or susceptibility cannot be drawn from tuber or stem inoc- ulations alone. Extensive trials in the field and green» house as well as in the laboratory must be made before definite conclusions in regard to varietal resistance can be drawn. GEAERAL DISCUSSION These investigations heving been undertaken to aettempt to clear up the question of the causal organism of the blackleg disease of potato in Michigan as well as to m&ke & more thorough study of the trouble, have brought forward the following facts: 1. The Blackleg disease of the potato as found in the Upper Peninsuls of Michigan is caused by a bacteriel organism, identical so fer as behévior is concerned, with B. phytophthorus. - £. The organism is able to winter over in tubers which remain in the soil under conditions existing in the Upper Peninsule of Michigan. 3. It is also able to winter over directly in the soil. 4. The disease is not limited to the potato. Tomato, Nicotianeé, «nd other plants ere susceptible to attack. 5. The organism is unable to attack healthy uninjured roots, but injures them by its soluble toxic substances. 6. The rapidity of the progress of the rotting of tubers is dependent upon the amount of inoculum, and the type of reaction of the host varies with the mss applied. 7. Two products are formed abundantly by the organism in rotting tubers and in’ pure cultures, an en- zyme which causes softening, and » toxic substancé “which causes discoloration. 8. There is some difference in resistance and susceptibility of varieties. 9, The disease is readily controlled by careful selection and disinfection of seed tubers in the spring hefore planting. L. Se 5. 7. 10. ll. 12. 13. 14. ~3]< BIBLIOGRAPHY Morse, W. J. Blackleg disease of potato. Jour.Agr. Res. §: 79-126, 1917. Harrison, F. C. A bacterial rot of potato caused by Bacillus solanisaprus. Centralkbl. Bakt. . Abt. 2; BA 17: 34-39, 120-128, 166-174, 384-395. Pigs. 17. 1 Pethybridge, G. H. and Murphy, P. A. A bacteriak disease of the potato in Irelend. Proc. Roy. Irish Acad. 9; 1-37. Pls. 1-3. 1911. van Hall, C. J. J. Bijdragen tot de Kennis der bakterieele Plentenziekten. 198 p. Amsterdam Inaug. Diss. 1902. Appel, Otto. Untersuchungen tber die Schwarzbeinig- keit und die durch Bakterien hervorgerufene Knollenf@ule der Kartoffeln. Arb. Biol. Abt. K. Gsndhtsamt. Bd 3: 365-432. Figs. 15. Pls. 1.(001). 1903. Frenk, A. B. Die Bakterienkrankheiten der Kartoffeln. Centralb. Bakt. Abt. 2. Ba 5: 96-102, 134-139. Figs. 2. 1898. Smith, Erwin F. Bacillus phytophthorus Appel. Science $1:748-749. 1910. Beoteria in relation to plant diseases. 2: 70471. Figs. 2. 1911. Rosenbaum, J» and Ramsey, G. Bb. Influence of tempere- ture and precipitation on the Blackleg of potato. Jour. Agr. Res. B: 507-5136. 1918. Ramsey, G 3B. Studies on the viebility of the potato blackleg organism. Phytopath. $:285-2688, 1919. Murphy, P. A. The blackleg disease of potatoes. Circ. x1, Dept. of Agr. Dom. of Canada. Pl. 1. 1916. Coons, G. H. Michigan potato diseases. Spec. Bul. 85. Mich. Agr. Exp. Sta. Figs. 2. 1918. Kotila, J. E. Frost injury to potato tubers. Mich. Acad. Soi. Rept. 1918: 4651-460. Fls. &. 1918. medeae Shi tolane: yyw Nudvaendt- SA LAG d Mgore oF ape’ 191¢. Jones, L. R. Eectinace the eplelfes mnpymepodeccl ly fr bnetly rey. ag. ap SMa, Teh, £, ft pow 2 igen PLATES Plate I. Characteristic blackening of stem. =-33- Plate II. Rotting tuber, showing creamy growth, blackening, and canal formation. ates Plate III. Nicotiana rustica L. inoculated with Bacillus phytophthorus. -35- Plete IV. Effect produced by addition of 5 cc. of broth culture of B. phytophthorus to uninjured roots of potato plant growing in water culture. «36= Plete V. Oedema formation, produced when tuber Slices were inoculated with small amounts of inoculum. =37% Plate VI. Effect of toxic substance on potato leaves. an « ~a& & yes re we ' ". “e Fer sca si ‘ A “ - , + S4 j TMT EE OE Tiana 733