STUDIES ON SWEET CLOVER FAILURES IN SOUTFIWESTERN ONTARIO1

Abstract 'The etiology of sweet clover f,~ilurcs tha t Ira\-c occurreti in reccilt years in

soutllrvestern Ontario Iva5 investigatctf further tllrough stueiies of the possit~le role of adverse ei1vironi~lerlta.l f a c~o r s , of insects, and of pathogenic h,tctei-in an(I fungi. 12 number of soil-ksorne pdrasitchs w i w r'ou~lcl. They cshibitecl ~~ i lueua l prlrasit is111 a t the. point of inil ial infection and as tlie (11-C.LSC occurretl i n thc field. Phytopkthorct carforum was fouiiti to cause tIic disease, and its p,lra?itibnl of sweet clovcr wn5 in\ estigaterl. Tlie susc-eptil~ility of the host v,\riecl .,l1ar1)Iy wit11 its 5t;kg-e of clevelopi~ient ant1 v\.a8i a t a rniniln1111l in the. scetlling stage ancl greatest early in the second season's gmwth. Ntrnrtsrc9us I(-nticels on the t ap roots provided natllr,tl 19rcaks in the peritlcrrn t hroupli ~+hic.h the fungiis prnttab1;- txleretl. Inva.;ic>n of thc tissr~e Biy the fuilgus mas inttat-c~li~1lar in tllc rclat ively srriall nu1riher of instances wlien first searon plants IVCTC a t t a c k ~ d ant1 was ;~ lways intercellular when seconrl sca5on 1)larits w trcJ at tnc-kvd. a ' h ~ fungus sporul*ltt.d when seecllinqi mere irlfccictl anti when extracts of seedlings wvre addccl to conlnltsn artificial ineclid. A4ycc~liurn of tlie fungus w a s found abtnnclant in cortical parcncl-~yni:~ c-olliitlcrably in atl\,anct> of lesions. Seconclary organisins q ~ ~ i ~ i i l y followed the initial infrr.tion. Epidemiological s t u d i e ~ o f sweet c-1ovt.r t:~ilures intlicated t f i ~ t oa~tl~realrs wcrt. c.I-laracterizetl iay relatively short periods of high disease incitle..n(.e ill the e:irly part of t lie secon(1 growiiag seascm in intlividual clovcr stantls. *An analy-i:3 of \ ~ c ~ ~ ~ t i l c i - clr-ata for tire pcriotl during wlaicli rccelat cI)itlcnlics oc.c.~rrrc.<t suggested that s~astairic-d high .;oil nroist nre in the carly p;ut of t l~t . gro\\fng si.,lson was ~,rerecj~~isi tc t o e1)itlcrriic. outl~reaLs of the diseasr, with higher than average soil te1n1,ernturc.s favora1,lc <is ~1.11. No plicnological conrlition, howt~ver, - c ~ n 1 c ~ 2 rt.sponsibl(. for confining the outl~rcaks t o the beginning of the second stiase,n of growth, mhic-11 appenrecl to he incidtxntal to soine physiological change ccsrrt~lat etl wit h thc. tlcve%opri~cntal rhythm of the host.

Introduction

Over 90% of Ont~irio's swcet clover crop is grown in southwestern Ontario. Centers of seed production lie in EIssex, 1 luron, ancf Siramcoe Counties. In districts where the soil is clayey and depletcri of nntnch organic matter by continuous cash cropping and mechanized farming, the growing of an increased acreage of sweet clover has hecn ~xt-ged by qualified agriculttmral workers. Consequently when failurcs occurred in recent years, attention Myas drawn to the importance of sweet clover diseases.

In this paper the designation ""s?cet clover failures" will be applied generally wiilmout regard to the cause of the trouble in specific instances. :Ilso, two distinct species, ilYe1ilolu.s qtEe;i~zalis (L.) I,anlask (the )rcllo\v-ilowering form) and A f . alba Desroussenux (the white-flowering form), are involvecl in observa- t ions and experiments. Throughout the paper ""sweet clover" will rcfer to

1 i$fan;i/.\cript received .S'e,tonbe~* 16, 1953. Joint confribution jrom the Ileficzrtw~ent qf fiotnny, l,i~t.~ersily cif Toronto, Torotcto,

Ofztario, c~nd the f?otc~tzy ( ~ n d PZa nt Pathology DizlY.sion, Science Seraire, Departnrent of '4 gricullure, Ottn;luu, Canuda. Contribz~fion iVo. 1.7 1.7.

Pascd on n thc..sis presented i n Ortoher, lQ52, to thc Ihiaersity of T'oronto in partic~l fulfill- n~crzt of the requireiltev,t.s,for the degree of Iloclor of Philosophy.

"It the time, i:rarimate sturlext (and dc~mo?tstrc~tur, I>eparfnzc?lt of Eotnny, I~?zia~rsi ly of ?'oronto; now r Z s ~ c ~ ~ i ( ~ t e P l ( ~ n t Patliolo,ai.st, Scienre Service Euborutot-y. P lant Ptmllzology Section, PIurrnw, Ontario.

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R E N E D I C T ~ SWEET CLOVER FAILURES 83

the yello\-2r-flowerlng form, unless special reference is made to the wlsite- flowering form.

The present stuclics were undertaken to include all of southwestern Ontario in an extension of investigations begun by Slykhuis (24) in 1047 concerning the nature of sweet clover failures locally in Essex County. Slykhuis found that Plzytophthorn root rot was the principal factor causing the failures. His investigations were made a t the peak of the root rot epidemic and have emphasized the destructiveness of the tlisease and some factors influencing root rot severity. The results of studies of recent recurrent sweet clover failures emphasize host-parasite interactions.

The history and geographic distribution of sweet clover has been fully reviewed elsewhere (18, 28). Fungus diseases ca~lsing root and crown rots of sweet clover in thc United States corn helt and in Canada h a w been reported variously in the literature for more than 25 years (8, 10, 11, 12, 13, 17, 22, 24, 25). K o t ~ i h t a i n this, sweet clover apparently has not been ravaged by root diseases throughout its wllole geographic range.

Symptoms

Compared with the healthy state, wilting and a soft water-soaked crown and root are major nlanifestations of the disease. Positive identification of the disease-producing agent is attendant upon isolations from diseased sweet clover roots and careful laboratory studies. 'The effects of the root rot causing sweet clover failures appear in the spring of the second season following resumption of growth of the tap root. In maily cases the disease appears in localized areas in the field conformiilg with the furrows and wide shallow depressions, and the plants die leaving bare patches. In other cases the disease is found in all parts of the field and thins the stand, or destroys it completely, after which the crop becomes weed infested and unprofitable.

U7hen a healitlly plant growing in the field under natural conditioils becomes diseased Gollowing dormancy, the first sign of the trouble in tlmc above-ground part of the plant is the drooping of leaf petioles (Figs. 1-3). One shoot may be corilpletely flaccid before other shoots wilt, or sorrietimes there is general and sudderl wilting of the whole plant. In the region of the cronTn the below- ground parts of tlie diseased plants exhibit a water-soaked, discolored, and downurard-advancing lesion that is at first turgid and is later wriilkled and rotted (1;ig-s. 4 and 5). Lateral roots are invaded by extension of the root- rot from the main root, or are attacked separately.

The problem of finding a prinsary organism capable of causing sweet clover failures was undertaken through investigations of such factors as environment, insects, bacteria, and fungi. Winter irajury due to heaving did not seem to cause any serious setback to tlae plants and winter killing was negligible in the region under study. The role that insects played in the sweet clover

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84 C'A V A D I A N JOUR.V.46, OF AOTt lSY . 1'01,. 32

failure ~sict~nre was more important. Larvae of the ('lovei- root curculio (201, Sr'to~ra hisPldul~ (Fab.), were foulat1 prevalent in 9 out of 10 sweet c-lover Gelds in June in Essex County. ?'he small gramt)s sc-ored tlle tap roots at- poir~ts someti~~~cxs sever:al incllcs k~elow the soil surface (1;igs. 6 and 7) and i t was cornrnon tts find root rot lesions ad jaccrlt to t hrbsie woui-ads. Thc sweet clover weevil (2), Sjio"r?i~ cylind~ii;oljZs (Fahr.), caused much loss of foliage in sweet clover stands in the S ~ B - I I I ~ whag~l the ndul t wvevils comt out of hiha-tatttion. ('11aractc.i-istically the wreevi!~ I-emovct <:a-escent-sln;i~~e:cI l)ic.{-cs inma 111e leaf margins (["iff. 8). It was i l ~ ~ a uncoa-rmrnon to find sweet clover seeflling-s virt~aa%%y delolia~cd by naitl-July (Fig. 9). 'The us(. o f DPI'I' has hcen rccornrnencled for the t:onf rol of this insect i r a order to secure a s t ;~ ldo f J ~ O I H ~ ~ Q ~ ~ L I ~ I S . Ke~la~~todes , sonic o f wllic*l1 1,clongcd to the genus Prntyie;izcizzc.s ( 3 5 ) , exart::rssively r ~ ~ ~ a t i l a t e ~ l ;trlci sloughed corticc~I tissalc of new rocsilhtts. This causcd a mucBa rvtai-ded i3cvcleprncnt of boih seedling arlci J-ear-old stwet c-love]- stands 011 sorllht sandy soils in the Hi~rr-ow district. Often tIlc nematodes were lo~lnel i r a roots along CT. it h the c.ntlog)hj-tic pIlycorn:~ca:tous nnycorlilizal furagus R h i z o p l r ~ ~ g u s (Fig. f 8). Rilites (1) I)eIongir~y: to the genus Ihrsomel-~zzcs \xTcl-rl fi~rrtlel irlnnelling thro~igh tissue cat the stem I):i:.s:- of seaieral young plants but s u r h mit:. i~aj t l r j? to sweet e:lovei- avas consitlered insignificant. I%athogenic bac.tcr-ia wcrc nor fo;ancl in sweet clovc.1- plants durin:< the present investigation,

Funlgi :~pparen tiy werc I-esponsible for the tlis~,~sc.. Iluring- the spring and

cloves- seedlings ga-own ir, "root rot" soil. This svil \\.;is Uro~lcst~11 rlay t~dterl in I'sses Count). frona ficdds of year-old sweet clover that llatl suffercd severely $1-on] S ~ I - ~ I B ~ failure of B he ca-op. At first a gro1a.t %I c.B-s:a~.;mt,e.r nritIa constarrt

Frr;. I . S v ~ ~ r ! t clover plan! s transplaalteci froni ! he fieltf Apri! 2 1, 1952, siio;ving stages 'of tile tliscasc: vc:ry cxilrrly in tlle growing season. H;ronl I-iglit tct left i r ) thct :jilc;~ogr:rl~l~ : progress frolu thc healthy con,litlon to tleatl pI;ll~ts. Ipot Xo. 1 sllows t!ie \vic!e spr;x:~tllng of the slloots and :L ~ C ' W ~'tlrooping. leaf petioles. Pots No. 3 ;~nrI 2 sltow wi!tccl pI.!nts.

I . 2 . iP h e ; ~ P t h ~ sweet. ciovcr plant as it npptbars a t the h:.ginnllfg of it.; s~r.orl;l siason of grc~v,.th. Note nuaiitrrous lvnticels which are associated with Iati-r:l! rcjot ~)ro:lu:.ticin. '1'Elv tlur~unnt crawl l )u( ls c.lnergc itbove ground and protincc t h c i ~ ; l f y s'llciots t11:lt ill:ar fjowc:rs and scetl.

I;[<;. 3- Firsb syl-mpto~us oi ciisense i11 the I~(:u- shoots- the. (In-ciopir~g of thc longc.1- I w f o . This plant was tr:ins;;jsl;rnte(l from tkr~ field :~t IlTootl>;lct> I:, t-hc iAiiscurlsin soil- ccm~p~ratrrre tank can in 'i':,ronto Fch. 1 1 , 10.52. 'The i)ll(~togr;lj)ll wrls taken ollc \vcek folio~virng tr:~nsplnnting anbl inc,c.~alrttiug; th(: plant was grotvn a t 20' ('.

1 ; . 4. A ciiso;ise(I pl:~nt infected 1)y Phyioijhthorn cuctov7c!n :in.;l cuIlibiting ;t water- scpaked, discolored, doxvnward-atjvancing lesion tha t is n t first turgitl 1~tit lattxr ~ri.inkled an(] rott ccl.

t i . 5 A cruwr1-rottirig Icsion a swect clovcr plant root forrnd lrltcr in !ht: set-onti season of gronrtlt.

Frc;. 6. Injury c311-id by I<lrv'~(: of S i t m u I z i ~ f i t d u f a to .n t ~ ~ t clrsv~r Intcr iai the sc.concl st~nsoil of grow-th. Larvae \tTcrr nuiraerous on tIae root,, ,~nt l in the. soil. Oltl ic.eding wounds are rallarest the soil surface. June, 19.51.

FIG. 9. Sweet clover plani infectert by the. root rot orgnnisnl. Xote water-~,oakc.d Icsion below old fet.tling wourads of t h e clover root curcuiio &lay, 1052.

I . 8. 1,eaf o f svcontf sc:iqon sweet clover plant. \!iu~ving fcedi~ig injury carlied t )y t h e sweet clover wecvil, Siiolzcz c y i i ~ s d ~ i t oLli.\, <:ornpnretl with unini~rrccl leaf.

FIG. 9. Plcfoliated an11 ~ tun tc t l swcet (-lover seedl i~~gs d11t. tip t h e ftlcdiilg of s\vc-c't clover weevils coanpareil x+9th ,L Ilealthy uninjured plant of the - ' L I I W ngc.

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-~empel-aturc of 20" C. and a lahotopcricsd of 14 hr. daily was utilized. Follow- ing emergence, whole scedlings were lilted, wdshcd free of r~tfiaering soil pal-tic-les, and placed in a snlall annount of distilled water in Petri dishes lor 21 to 38 hr. at room temperature. hiic-roscopic exana~inations of fungi tha t grcw out of 2 0 0 inlfccietd roois showed that I'ythiuan, ApJzanamyces, and Fusnriuvlr species were present. seco11(1 c'xperiment made use of LVisconsin soi 1-tempera t r~ re tanks in tkic green llouse. 1;our (-;ins of "1-oot ro tPP and four of '"Iacalth~7" soil were ~)lnccd TII each of five water baths regulated ;ti 1 0°, 12 . 5", 3 5", 17'. 5", ancl 20" ('. respec.ti\~elj~, I<x,arnin:rtion of anct isolaiion frona 843 aarnthrlfty seedlings fro111 both soil sa1n1zie.s showe(1 tha t several iul-agi were calaalslc of causing seedling blight of sweet clovct- (Table I). P ~ t h i u n a rlltz'il?zzd m (Fig. 1 1 ) ; ~ n d P. z'rreg zelnre (2 1 ) a . , ~ l~sc.d cl:~rraping-ofl ol soisle seed lings. Rl~izoclonz'cx solnlti liilled or retaa-tletB ~ l n e c%evelopimc~~t of many seedlings ( Fig. 3 2 ) and Thieltsviopsis basic-oia (Fig. 4 ,bi afiertcd rt few. The sarl-ath fungi loilncil i ia sectfll~~gs qrowaa in "root rot" soil also ;ti8;tclced scedlings growing in "llealt 11y" soil with thc t>xception of Ibhyfophkhora cactorum and 1'. basir:oia. 14~-~-c~7it~rgv~1e.e c.larnpil.ag off acc-ou~~retl for a l~ou t 367; of tlae seedlings at 21B0 C'. ancl a b o u t 607; ;el I 2 .5" C'. on "rm)t rot" soI1. I'TC'-CIII~S~CI~CC loss was bet wee-11 these cxtrt'n~es a t the otlaea- t e n ~ p ~ r a t11rt.s. IPost-ennergencc kill irng o f s:.cclli~lgs was sli;!ht. llndea- field conclitiorls lossrs of sweet clover seetilirag st an(1s cltri- to fungi n-erc., oil 110 occnsio11, al)~sraacinble (luring the years o f these studies. Also, the pr;~ctic;\l alifi7(1alt~ C X ~ I ~ T ~ C ' I I ( - ~ ~ % by growers has bee11 O I ~ C of 01,taininq :a scc'ontl seasola stan(1 of pl;ttlt s rat her than a seedling stand. K . solani and Fusa~iunz spp. ere the oaa8~- fulngi isolrated from sweet clover seed1in~:s growing i r t the field.

I . I . 31 i:.rophotogr,Lph of v( sicl<+ o h I'iki=ophngu r , tllc cradophytic phycc~~nyctltnrrs r l lyc 'orhi~~~i ~ L I I ~ ~ I I - . , ; I I ~ ~ I I ~ wit11 RC~II;L~O(!CS ill '1 nen lirnnch (tc-cdcr) root of a scc-ontl seasun iwLAt-t ('lover ~rl,lnt .

Frc,. 11. Oogorliuln all:[ ~~nt l lc~- i i I iu~i l 111 iPyli7iuw ?dttmzrm which attacked seetiling.; ot \15-~.clt clover.

E'rc,. l 2 t 1 . E l e ~ l t h l s\vc.c.t c-lovci- seetiling oncL month oI(1. A lanifoliate Bcaf is prex1ucetP 1)ef I V Y ( ~ I ~ tlic ( ~ ~ t ~ - i ( ~ , I o : ~ ; ~ r y It';lvi.> '111(1 the tr;folidtc' lea\re:;. Several bacterial nodulcs a re prescilt c~lrchdily. (O) \*c)rlng see(llings, the - a m . :rgck :rs the one in ( a ) attacltctB by KhZzoc r ~ ~ i t i a s:,lil n i.

Flc,. 13. '1'ypi~-nl ... c.lllanl>~tlos~)or(~i of 7'h ielcr.cl;oD\ 13 !)a > icolu. This fungus attacked some +\\re( t c1uvr.r 5ecrlling-.

I:rcy. 14. Nurnvro~;. zoosl~ol-angia oh P. c x r ~ ' o v ~ r a: ~) rod~rced tc.rminally on t he first flush of 11yl)llnt. enlvrging from ,in inftv.ted swc.~ ,~ i '!ov~r -.i'eJIi~ig hoot tha t was placerI 111 water.

Frc,, 1 T . %oosy)orarig~uin with zoospor,.s 1~11;ch i i ~ v e heconje inactive and spherical. Uotc characteristic- plug at the hd5e 01 t lle >mn-.lngium.

I 1 . Oogoniur~l 1% it11 p:rrngyno!ls ;~11t1>~'1-i(iitl~l: cllarncteristic of P. cmtnrum. I . 1 Rfyceli~~nl of P. cacforr4117 grow;i~g intrnccilul,~riy in a sccdlirag sweet clover

st cnl f : ~ c , . 18. I,o~igitu(linal st.ctia>n of a n i~lfertc:! >c. -onrl s ~ a s o n sweet clover root showing

ll>,l)ll:~e . ran~ifyiixg . intercellulnrly t l~roughout c.ora;i.;il y ) a r enchyn~~ in~mediately beneatla t h e l)cxl ldt>l-~ll.

Frc;. 19. 'I'r:~n.;vcr~c section ot infected secc~ntl ac'ison sweet clover root showing $egnlc.nts oi Ilyj)h.Ie lyin$ t)c,twecn thts (.ell?. Starch grains ;we abundant in this tissue.

Frc;. 20. Myceliunl oi P. c c ~ t f n ~ u n 2 grux ing throilgh tissue of a lenticel which may act ;1s an tli~trarace arid an exit in host-para-ite 1-elat ions.

Frc;. 21 . Y noc~~ lun i placer1 in wouilrlt~~t swcct clover plants in cac-h ISJisconsirm t ank can tfuririg infcc-tion eupr.rirnents. 'Fhe left c-ultr~rc. of P. ct~ctorum is growing on potato- ~Icxtroie ~ lga r and the right o n v in sterile water (for zoo=pore proci~~ction).

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C A N A D I A N JOLiRNAL OF BOII'ANY'. VOL. 32

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BELVEDICI': SWEET CLOVER FAII,UIPL~:S 8 7

The fungus that was consistently isolated from infected year-old sweet clover planis in early spring was Pl~,ytsphtizoru cac to~um. Many latcr isolations contained Fz~stzrit~?n spp. Where P. cnctorunz was in little evidence in the fields, other pathogenic fungi seemed to Ele insignificant also which suggested that tlais fungus was the primary pathogen. Pathogenicity tcsts sltowed that P. cactorum, inoculated into year-old sweet clover roots soon after winter rlorrnancy, procl~xced a severs wilt and root rot identical with tha t found in natur,~lly infectcrl plants in early spring. P3yt?z?'z~nr spp. caused pre- and post- emergence darnping-off of seedlings but cliei not affect oldc.1- plants. Kone of the Khixoctoniu isolates killed older plants although i hey were most tlcstructive to seedlings. Fusra-rium spp. and 7'. br~sirol~z retarrlcd the develop~neni sf some seediings. E'usariurn isolates c;i~asetl a wilt of year-old plants in sonme cases. Other fungi isolated from seedliilgs grown on "root ro tvv soil secrracd to be ernimportant in the sweet clovcr failure picture.

F;n~nsually precise limits were prescilt for- successful isolation of P. c r ~ c t o ~ u m . I t was most difficult tc, isolate the fungus except in very early spring when the roots \\-ere just breaking rlormancy and irlcipitnt infections were in evidence.

Barasi tism

Since Pl~ytcafi?z~tlzorn cnctorun.z readily reproduced the sweet clover failure elisease under cxperirnental conditio~ls, its parasitism was intensively investi- gated. 'I'he phases o f the life cycle obscrved in these studies correspontled with the life history of this fungus reported by Miss Blackwell (3, 5) whose terrni1ac)logy in ihis genus was also used. B. cactorum is homotllallic, and the species comprises several distinct strains (26).

C t ~ l t u ~ e Studies Cor1siderat)le difliculty was experienced in obtaining fungus fruiting bodies

wlaerl ordinary lat~oratory methods were tried. T;i~nally, however, zoosporangia and zoospores (Figs. 14 and 65) were ~~rocluced when infected seedlings or pieces of the ntycetial mat of the fungus miere placed in distilleti water in a Petri dish ant1 kept a t about 20° C. for 36 to 48 hr. The fungus produred oospores best on tjatrneal agar (23) in greenhouse temperature and light contlitions that prevail during the u~inter months. Constant t empe ra t~~ re and a dark chamber were never satisfactory for oosporc production. The antheridiu~n is prcdominan tly paragynous (Fig. 1 6), being attached in a lateral position on the oogoniurn. The oospore apparently is a ~ n ~ o s t resistant, perennitting body, and the conditions tha t ensure germination of i t lie within aaarrow* limits of ttmperature and moisture (4). Other perennating organas produced by the fungus are clalaruydospores which are larger than sporangia and arc spherical; resting spol-a~agia; and resting mycelia.

Thc growtlt rate of the fungus a n common media ~ v a s important in the separation of species of Plzytophtl~ora by Tucker (26). During the p r c ~ e n t investigation the isolations of P . cacforum from sweet clover hat1 a minimum temperature for growth below 6" C. and a rnaxiniurn a t~ove 30" C. on several

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88 CANi'lUI,lN JOUIINAL 01; BOTANY. VOL. 32

extract agars. The optimum teinperat ure 88" to 22' C . agreed with t hc Bo-ca; op t imu~n of il/)'i)llt 16' (1. for other Ph_vtoplzthara isolates from su~c:ct clovclr in illlserta ; u~d BI)~lt;\rBo (24), but is coi~siderably lower than the opt ina~l~n of abollt 27" 6:. fountl by \Yelsh (27:) for isolatiorls o f this fungus fro~rl iipple trees in h'iritish Columbia.

During the cultural studics of the fungus i t was thought possible tha t thab ;r(l(litio11 to cornmoil culture media of sorne growth promoting subsinincc~s ;ind cxtn-acts of sweet clover roots naigint stin-aallate scxlmal reprocl~~ctic,n. Sullse- clist.rlr tly, it was ot' great in wrest to note that a i~~andan t oogonaia ;11ld ;~n th~ ' r i~ l i ; t had cl:.veloped on the pot:~to-dextrose agar to which the extracts of srctllirlg ; X I B ~ ix11natlll-e sweet (-lover roots had Iwen atldcd, whereas the fung~:us grew on'n)~ vc?ge!ati\rcly on the agar with extract from seconcl season a-oats. This rc!at,ior~shi~> 1,ctween lungus fruiting and age of the host was evidci~t also wlierm inft"('tcc1 pl;tnts were err;lmiiaccl.

?lI~.cellilim of P. (;nctorzk?~z coloarized p;~rericlijrrnatous tissuc of the host intcr-- a11r.l intr;~cellularly. This was tlct-c:-111ined 1vhg.n more than 200 &r:Lns\.ersp ,111d longitutlinal stria! sections, each 50 p jn tllick~lcss, sf roots infectecl by the B'rrr~gus \~~ci-e i~~dividual ly plnc-cd on cleCm glass slidas in drops oC iodir~c. - jntassi1lm iodide, Imtlated for about one ~alirlutc after tllc addition ot' acid Euchsir~ in B;lctopIaenol, cooled, clearetf with lactopher-~ol, covered wit11 :L coLrer- slip, itrmtl exarnined anaicroscopi~~~lly. Wihole inlectcd seedlings uTci-e sinlil:lrlj~ ti-e.:t tea.

\]'hen seetlling-s uTere invader1 by the fungus, hyphae g~cnct ratccl Blot il I.ayp<)cotyl ancl root, rar-tlificd tlarouglaout the tissuc iritrac-ellularlj- (E:ig,:. 171, rtnct bore sexrral rcprodarctivc bodics i l l the tissues. 'T'hen. ~-1leil t he in le.cti.(f qceldlir-rgs lvere placed 1i11 water, h y r ~ l ~ a e emergrld directly through c .p ic l~~rn~al c-ells and procluccd tcrrninal sporangia which gave a mass diechargv of zoosy~oa-es. i3The1-a second season roots were invatled by thc fungils. tlyphae %;r-cp,v irat crc~llrllarlj- through cart ical and raj- pal-enchy rna and rcac-her1 the c.=nar;~B pith in ;LS short a p~ric)d as three days a t 17' C . ailtI, furthermore, 11;id .idv;aillc-cld frorra 5 to 8 mrn. bcyond the margin of the wet and sun1;e.n lesion. hl~rcclial r;arnificrations in c.c)rtical ~sarena:hynaa were at no time observctl p:lnrtr;lting cells of periderrn (Figs. 18 and 19) but were ahui1ti;int in Jen ticel iissue. soinetinles rra;tlting a11 exit from the root (Fig. 20). 7'he fa l r th~st 1,ene- iratiorl of the fungus lreyond the liinits of the root rot Iesion 17~7~~5 in pericycle eiss~ne :1nd much growth of the hyphae was parallel with the 1 0 1 1 ~ axis of the root. ,P few hj-phae pmctrated xylt-111 elements. Son~ci c-clls arlj accn t to l lq 'ph:~~ \vc-re plasmolj-zed am1 devoid of starch. J~adgi~ig by t l ~ e il~anllrea- of starc-h gr:~irls seen microscopically and the in teiasitj- of the coloa- in the starch test in serial scctions of root it sccn~ed tha t the carbohydrate rescrvcs were t leplet~d r-raturally from the crown clo-c~n~v~lrd. No starc-11 or rr~yceliknrla was fount1 where the fungus invasion had c-aused a lesion in the cr014~~i or root. ('onsidering l)hycomycetaus infec-tion in general, i t is usas~ally expcctt.cl to find

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the most advaric.ed pa - t oi the Icsio11 pronortnced and rnj~ccIium abennd~~nt tht'rt', whereas in tlie casr of P. ct~ctoru71z in swc't't clover it was i~iteresting to fi~rcl II.IC riij~ccliurn in1 tlrc p:rrcnchynnatous tissue coll~iclcraksly k)ej~oncl t l ~ o limits oi thc- lesio~i.

I',LI-iaticsns in thc inlectic~ri capabiliiies of the fung~ls deli-ing ~>i-eliuii~larj~ teats at it;ir~-oiv in 10.50 shometl that t l ~ c pat1log~:nicity of (he ;-aus.ll or-g;lnisul iilig%l t i~ 1-c'l;it~cl to iht' (*on~litio~l of the 11051 ;it a c:ert;~in phase in ils oirtoge~ay. C'ons, c l u~ .n t I>~ , irm t hrcc co11s2c:u t ivc er;l~::rianellts, ( a ) four-~i~ont h-olcl slvcct cI~Iv(.I- i~l,tnts, (b) g ~ ~ i - ~ ~ l i ~ l ; k t ~ [ i s\%'c'Q'~ ('!OVLT- st!cd, a11d (c) routs tsf swcci clover pl,rn t s i 1 ~ 1 t Ba.ar1 ex:~:~ricnc-td ;I ~ la t i i r~ i l dorin:~nt-, pei-io(: ila the iii.ls1 \~~(.i-e trans- p l i~n~c~(1 ,inti grown for six weeks in BVisco~lsin soil - t cm~~ ;~ ra t~ r r e talllts in the I J;liaic.i sit g- of 'I'oro~~ 10 g r c e l ~ l l o ~ l s ~ ~ (Iurillg t l l ~ wi1"bter of 19.5 1-52. l'hc. soil usctl nf::s X1,i:-r-trw sand , ~ r l t l 13rool;ston (-lay frc~ri~ fielcls t h ~ t hiad raisccl 11e;-~1?111~7 s\vc.a.t c-ic;i-er tiurillg 1950 ;-~r!d 6 95 1 in 12sses ('olln t 5-. The soil terrjpci-ature i:)r hht bix t ; ( l ~ k s o f eight C;BIIS C;XC!B W;L$ i-egralrated nl lo0, 14', f so, 2 2 O , 26", ancl ,iOo ('. rcspc>c.tivc.',j-. The rllc>isture 11d~lt1'119q c;tp~icity of the s0i2s was kept t o 0 , . I ,o/-allv grown seed was sbtninccl in the Hari-ow dist i-i6.a and n-ns of rhp coialriicm avtiita? slzcc-ies of swect clover.

'Two ol tlie f'ight ( xnls of soil a t each tcnlycrat~r~-c, one of clay ::ncI orar oi santl, 12-6.i-P stcxc~:n-sa c.1-ilizetl :anal four we]-c urntt-catctl, two of clay ansl tn-o of sanztl. 'The plaiits in t%acsc siu cans wcrc irlocu1~~tt.d with cr~Yirlrc of P. cucto;zi:jt grc:aaring vr:gcltitivt>ly on pot:~to-clcstrczs~ agar plus 21 watc.1- c~rltua-c of t!lis fung~ls tlzat c.vnt,tinc.al zoospores (Fig. 2 1). The. remiiinirlg tn-o c-ails, oncL e ~ f c.lnj- a n d o n e of sa~i t l , w t r c santrcatc-d, ;lnd the plants 1ve1-e i n o r ~ l i i t ( ~ i with sttirilized prstaro-dvstrosc agar. The fungus was thl-ce wccks old at thc ~inl t . oi i11ocul;~tion : ~ r i c l was iriserted in a scaI1)el wound cut loilgitutlinally ina thcx outa:r cortex of the 1-001, or, with seedlings, he 11tycelia1 nl:tt was plac-r.el aroun(l e n t l in c'on t;~ct with tllv base of f lle %~y~)oc:otyl o f t%1(: J ~ O L I P I ~ plant. r . She inocuIatr;)cl regions oT the p8;ints ~ \ ~ c r c lightly ~ox~erecl witla moist soil. Bcfore bt.irag t ra~lspln~l ted ill the cans of soil, t lie ger-~nii~ated seetls armel ~ ~ l a ~ l i s nTere a ashecl clcan in running tcip water. ,4rtificial lighti~ag sktpplieel k3y 500 nratt h1aztla Iikt~ips :+111)1)1~1lll'iitCd I he daj-light to give rr. ligl-1 t pel-iotl of about 14 hr. tlailj.. 'I'ile air tk>~n~,crature of thc co~r~pclrtnler~t was about 21" ('. and huiniclit y ~-:Ls kept iiigll 11) f l -~(~ucr l t watc'ring of the cemsrlt floor.

r 7 l he results of th(.sc e>;pcrirncnts arc given in Table IT. The inoculated seedlings arnci pl;lnhs that rc~nairled hetll~hy grew ;as viga:,rously ;is the c-hecks. ' There seerncil to be lit llc tliffer-cnc-e irl the irlfcction cag)ak)iIities of the falngus on stel-il ized and un:;tel-ili~~cl soils throughout the range of ten~pcrattsrcs used. r \ I hc striking tlliilgs in the 111rvc. ;~laovc experinlaerlts scc.rt~ed to be the low infec- tion in 11mc first SCLLSOI~ 12li~ilts ~o~nl-)inled wit11 the ~harac:tcristic,a11~y normal rapid cievclopnlclnt oC S U ~ % B i1lfeL-tio11~ as did occiir. In the case of seconci season plants, nr:, pattern of incidence of wilt related to the lrrlgth of the incubation pcriotl coulcl 1):. found. '12'hy there slllouIc1 bc such a range w%ierr

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REI,PITION OF ONTO(;FSNE- TO SI:SCI;I-TIIIII,ITY OF SWEET C ~ O V E R FOJ.LOIVING INOCULATBON \TITI1 P. C ~ C I O Y U I ? E I N (;KISI-NHlOT~SE EXPERIMENTS

Seedling (3 weeks)

First season (4 months) I

i 9 . 7 I 7 - 0 I

Second season (irnmetlia~ely after 7 2 . 3 ~1nrm:iracv)

* There 7a.s 7x0 c ~ ~ r r ~ l ( ~ t i o n bef7~1e~:)z susceptibility and the soil type ur temperahre.

the nlethod of worlndiilg and the inoculum were unifornl seems indeed a problem that can hardly be ignored and requires further investigation. This anomaly seems to suggest the individuality of sweet clover plants mrhich must differ gerletically, ontogenetically, morpBaologically, and chemically, and in their dormancy experience i~a any population of plants. 'The complexity of thc life history of the causal organism and of the 8-~icnnial developmeill of the sweet clover host scenled to account for the i ~ ~ c o ~ n p l e t e infection in spite of the use of large amounts of inoculr~m. The nature o f the disease was typical of root rots once infection occur-red in the tap root. However, the occuri-ence of disease as measured by the small rlusnber of infections made the first two groups, dealing with first season seedlings and young plants, atypical. Some histo1ogic:~l studies of sweet clover roots during their develo1)ment have bee11 ~nade: by hIartin (10) 2nd RIiss Rottrrm (6).

Epidemiology and Control

Sweet clo-\ler failures in southwestern Ontario reached epiclenlic proportions in 1945, 1947, and 1948 and have been present, pitrtic-ularly in Essex ('ounty, with co~~s i s t en t l j~ fairly heavy losses each year since. Pertinent weather data obtained a t the Dominion Experinlental Station, Harrow, for the period August 1, 1944, to June 5, 1942, were analyzed anti showed that S U S ~ ;tined high soil nroisture in the early part of the growing season was prerequisite to out- breaks of the disease, with higher than average soil temperatures fa-\rorable as well. No weather factor, however, sccmeci responsible for limiting- the out- breaks to the beginning of the second season of growth. The outbreaks of disease sccnled to be incidental t o some p&j~siologic.al change correlateci with the developn~ental rhythm of the biennial host.

Surveys of sweet clover fields were rncrtle several times during the growing season of 1950, 1951, and 1952, over an area that included about 90% of the acreage of this crop seeded in Ontario. Former surveys in the literature (7, 9, 16) served to help estimate losses due to diseases in this crop. The extensive survcj-s showed that P. cuctnrunz was present in and could be isolated

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BENEDICT: SWEET CLOVER FAILL,iRES 9 1

from roots of plants displaying typical symptoms of the disease. This occur-rcri shortljr after new shouts appeared from dormant crown buds in early spring. The disease was nlost evident in fieltls in Essex and Kent Counties but ~XJ;XS prevalent in l,anll,ton and Huron Counties arscil present in sorrie other cc}unkics.

Since, in southtvrstern Ontario, the value of sweet clo\-el- depcnds to it

large extent on its suitabilitjr in a definite cropping system, the ap~slic;~tiors of 21 longer rotat ion could not be rccommencleti ;as 11 control measure. Prclirni~iary field expcrinlcnts irrdicated tha t neither a ch;~nge in soil type nor tZlc use of certain c11emic;al addenda affected a starcc~ssful irldirect i~iological control of the dise:we. 'This is in agreement with i~lvcstigations by (;crdenlann(44) and others who havr. observed that swect clover root rot occui-s in cert lain arcas 1-egardless of the ferr ility level of the soil. Therefore, i t appears that the most cffvctis-c and economic rnetlrotl for cotlkrol o f swvet clowi- failure is the use o f selections resistant to its attack. Jones (1 7 ) ancl (3ornlaclc (13) obtained sonle plants resistant to P. cnctorztr~z infection under field conclit ions. The seed of

sur-h selections has not breaa ~~rocluced in quantity or sown tvidclj~ in arcas most severely affcc-ted hy root rots. IIcjwevcr, since losses due tcs Phytophtf~src~ root rot can be effec-tively retlrrced by using resist:tnt strair~s and these strains can bc impi-ovtd in any section where infection is conmonly severe, the use of home grown seccX may be encouraged as the most effective Ineans for controtling the disease locallj-.

Discussion

The preserat in%-estigation has yieldetl ncuT as well as aclditional information reg-arcltr-ag the nature. of sweet clover failures in southwestern Ontario. Wvcause of llle complex it^^ of the sweet clover failurps it was decidecl that: suclr agents as envirnnnlcnt, insects, bacteria, :and fungi shoultl he investigated. Soon, frost irijury and &inter killing, nematodes and mites, a i d bacteria m7cl-e found to be relatively un i~r~por tan t as c-ausal ~tgents. However, it was thought tha t i n j ua-ies carlsetf by weevil 1 ai-vae feeding on the roots probably ~vould c-ontribute to an tlpidemic of discrase. Of the fungi u~llich were isolated, Rhi~ocLo?~ii~ soluni, Pythiunz spp., Thie1avioj1si.s bnsicola, and PhyLo~~J,htBrora cactorum all provet1 c:tpable oi killinq or severely stunting seecllings on diseased soil. I-'. ct~rtorum, Fusuri~cm spp., and Cy1znd~oc;a~pon sp. proved to be consisrcntly associa te(1, on the other- liancl, with sccoamti season sweet clover ~ ~ l a n t s illat had wilted in the fielrl. One of the facts disclosed by the present investigation was the limitation of the f u ~ ~ g u s P. cnctorunz t o specific host tissues. I ts viru1enc.c in patllogcnicitjr tests and the occurrence of epidemics c ~ f discase in early spring of the second season swect clover crops from \vhicla this furlgus was consistently isolatetf indicated ahat P. cuctorunz was responsible for most of the stveet clover failures in Essex County, Ontario. This p rom~~te t l further intensive investigr4tions of this fungus.

i111 interesting fii~clir~g ira connection with rultural studies of P . cnctoruan shorn-ec1 tha t this strain of the fungus had an optimum temperature for growth

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92 CA-\-AUIAN JOLIRNAL OF BOI'Ahl I.'. I'OI,. 32

of about 18' C . and tha t potato-dextrose agar was most suitable for growing it vcgr.tative!y and for sul~culttti-ing. Also, oosporcs of the Eur-rgus were prociuced in in f~s ted scedli~igs L L I ~ ~ FJY idcling ;in e'xtract froa-s-n m;-ac.e~-ated roots of l-aealthy scedliligs or irriniiature sweet c1ovt.r plants to 1)otato-dextrosc agar.

Iaafection experiments with P. cuctorunz rcvealccl several irltercsting phenornen:e c-o~~ccrni~ig tlic association of this i'u~ag~as with iks host. Tha t sn-eet c l o v ~ r lail~11-cs were not conncctetl wit11 the first season plaasos o f host desrelop~n~nt seemt.c% evicletit . 111 the secclnt-1 s,.asora ol growth, t l ~ c produt-tion by thc plant of a whale new filjr-ous root systetm with numerous lcrltic-cIs appvrzl-i11g on the tap roots provitled riiany natural breaks in ahe peritlera7-h n7llic11 itself seemed to 1~ a b;arrier to this fua-sgus. I t was also found t11;1t, although n wouaad resulting either by a scalpi.1 cut or thc Iccciing of insect larvae was utilized cffectivcly by P. cacto~um, :L ~ilechanic;~l injury was not essciit ial for inleetion of sweet clovers I ts ri~orpliological response to ~ntc11anic:el iiajui-y apparcnaly was not dissimilar to other pli~nts that effectively w~xll out injured areas by new tissue izi the ahs2nc.e of the fur~gus, but , n7ht.ra P. cticiorum iris-add worrncls, the walli~ig-off r)rocess seenlcd to be inhibitecl. 1h7hcrl infect ion d it1 occur, tissue irmvasion 1 ~ ; ~ s irl trac.cllular ira the case of seedling infection, 1)ut was ~ ~ U T ~ L Y S intercell~rlitr wl~en older ~ ~ l a n t s were par;~si- tizcd. H t ur:is also u~ausrual to obst:rve n~j-celi~irri ol P. c c r c t o r ? ~ ~ consitlerat~ly in adva~ic-c of I lle rot tctl tissue- of leraio~led roots. \Vl~e ~ h e s r;errniniat ion of the zoospore or of the pci-e~lnatinp oospore was rcsporisibla for i~i i t iatiogl of irlfection in tlie spring could not he determined definitely.

Tlac current ir-hvt~t;tig:ltion has estal~lished that the lnthogenlic-it?- (pi tlle causal 01-ganisrn was related to the ctdmlclitio~~ of the host a t a1 certain phase oj

its ontogclnq-. Previously, it was repartcci (24) that iaaoculattd plants of :ill ages from t lmrctb wct.l\-s to 10 ~non th s readily became infected with P h y ~ o p h t l z ~ ~ ~ root rot . f Ioivevcl-, the p r ~ s c n t investigations cliscloscal c-onsist en t l ~ ~ lon infection irk scct f lirig anti first season p%a~ i t s of tlie biennial sweet clovel-, c-omt~inecl with thc c.Ilr~r;~ctca-istic:~lly nosanal rapid dc-veloprner~t of sucli infect ioaas 3s ~ P C C B I T T ~ ( ~ . I 'hr fact tha t laigher than a17eragc air teairpt.rat 111-cs along with sustained high soil moisture in the early part of the gronring season seemt-(1 prerequisiae to epiclcrnic outbreaks of the ciisc~ase is not ill keeping with previous i~~vcst ig:~t ic)~~s ' . . Howevca-, 110 we;tthes fkxctor cotlld be found respon- sik~lt. for Iitnitii?g the outlrrenks to the k)egi~?l~irig of the seconcl season of gro\~~l i i . C:rop Er+ilemrc. appearect to bc ir~cident;kl to some physiological cliangc coa-relatecl with the de~c lopme l~ t i~ l rhythm o f the biennial host . The fact that i~s fectioil occurretI more reatlily aiid mortalitjr was fc~~rilti to be greatest when 21

I-riechanical injury was provided for the causal fungus to invade the host plant is in agrecil-aent with previous reports.

It is ilnportant to note that in greellhvtls.;c% experiments, with air temperature about 28" ('. ant1 ~ T ; ~ H ~ O U S constant soil tennkpcraturc:s rangiiig fror-nl 10° to 30° C., ~vilti~ag of tlie p1a1its following artificial Inoc~llation occurrvti in frona 6 to 2 6 days ~vithout a correlation hetween susceptibility of the host and the soil type or teinpernturc. illost rapid growth of the chec-l\- plants occ~arred at a soil

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BESI<bPdCT: SB,rEE:7' CLOF'ER Fi l ILURES 93

temperature of about 26" C. In the present invesilgatioi~ the cq~ti~nunr ternperati~re for infection appeared to bc aE)tsalt 22" C. whereas Sly!iIl~~is reported that root rot of sweet clover is most clcstri~ctivc when the soil. arouncl tl-ne roots is s~n~e 'm~herc between 6" and 86" (1. It is possible tha t tlic above- iridicatetl irlconsistcncy of findings corlcernirig tenaperature rcc-gkairernen ts of the fungus may I)(, explainable thc fact tha t different physiological races w-ere I~eing i~tilized b>- the different investig;ttom-s.

I t snay be conc$uclt:ci lronl prclinlitlary field cxperinwnts that P. cnctor;i~.m is c.st:tl~lishetf in 111uch of the 13rookston clay soil of the area 111lcler investigation, anti i l ~ a t no sat isfitctory soil a~ncnctazlen t has 1-et been i o ~ ~ n d for ctsratrol of this patlnogen.

l'hr. writer ac-l;tlom-leitges sincerest a~,prec.iaiion to I'rc3E. D. I,. Bailey for- valuc(l suggestions during the courses of ~ I I Y S C s tud i~s a n d in the ~rreparatiorl of tht" rnanus:-ript. An edrlcatio11:tf grant and scholarship was gratefully rc*cei\.ed from the Tlepartment of I'etcl-ans Affairs. Thanlis are due to Dr. I> . IV. Iicjcln for c-ontinned iritcrcst i11 t11c problem and for the L I S ~ of the. fari1itic.s of the SC~CH~CC Sc>rvice l,ahor;~tory, IPla~lt I'itthology Divisicm, Ef;lrr~\v, Orit. The \-\'riles also wishes to express his ;~pprea.iatic~n ttr otller professional workers lor reatling and helpful cn-iticisnl of the lanu nu script.

Ref ercnces I . 131\slcs, N. 'T'Ilc a~:arir~n (11- nnites. 1J.S. BBcpt. rlgr. Report No. 108. 1015. 2. I ~ I K D , 14. I b. The s wctbt c'Iover weevil, Sifonci. ryIindricollis (I;ahr.). C a n . l~:ntc:i-uoIogis~

79 : .?-Il* 19-47. 3, 1Sr21ri;w~~i.r, E. 7'11c B i f t b lli5tory of Yhyfoi>izthnrc~ cuctorz~ri~ (L,eh. ant1 Co i~n j Sr'Rroct.

ijrii. lIyc.01. Soc. Trans. 2 0 : 71-89. 1913. 9 13 i \C Kwr r,r,, E. Igre\itlt,tit ial atlt11-css. 0 1 1 gvi-llii~l'it ing tliv o o s ~ ) o i - t ~ ~ ) t 1T)l,yfr7l~/~tl~ol*u

cactnrrlw~. Hrit, RIycol. Sue. 'Trans. 2 0 : 93-100. 1 0 t 3 . 5. 3 C - I , ~ ?'eri?litloIc~g\. in P~cyfophi/zora. Co11111101r~vtieaith l lycol. Jtlst. Eol:i7cologicaI

T'<~pcrq, No= 3). 1949. 0. 13r~rirT~.r, F. R . Iiisio%ogical studies on the root of iVfe2ilotz~s ( L / ~ I ( I . 130I;i11. Gnz. 103 :

132-145. 194;. 7. 131-sc~r, I,. 1'. an,] ~;LI ,T 'ATRTCK, J . 1). A I C ~ ~ I I ~ I C - roo^ I-ot survt3y ~ I B w ~ ~ t l l ~ v e \ t ~ r r ~ Otlt'trio

i r a 1950. I'lant lliscasr ltq)tr. 35 : 296. 1058. F. C'rrr ; t<~.wrcrc , iV. J. Rl-rizoctonia root rot of sncct clovcr. Phytts~~athology, 31 : 673-674.

1041. 0 . C' : i i -, i I K, I<. S. I'Iant disease losscc,: their .~pl~l-ai+ai ant! ilatcrprctntiori. IPlanl Di~c~rtsc

Kcjlt:-. Sup1)ii~nkc~nt 143. j r~nc 15, 10.cO. 0 . . 1 ? O r r t hc inr.,i4ol~ of root- of Jiedirngo antP zllcIilnt~is by h C ~ I ~ ~ o t i n i a SIi.

rind Plr~:ocinw:~~r waliloti 1). an(l S . Can. J. Kcsc..~rch, 11 : 47 t-480. 19361. 1 I . ('oxi%f.zck-, 31. lV. F z t s n r ~ i m ~ .'p,r). as roo1 l9.ira~ites oh ,iLf,~ifn ant1 su-ect clover in iillscbrt,t.

C ' m . J . I~CSPA~CII , 15 : 493-2 10. 1037. 12. Co~iw;lcr;, >I . 1;. PiT~yfophthnra r c t t f o n ~ t ~ z as a ~ ' 1 1 1 ~ o f root rot of swcet elox-cr. Phyto-

~)<ltliology, 30 : 700-701. 1940. 13. Co~ i~ l i c .~ ; , 51. IV. Iynrit.tai rc$i-tnmce of nlf,llf:~ anti ~.v;c\et clovcr to root ant1 i-rown-rot ring

fungi i r r , I l b r ~ t ~ . Sci. Agr. 2 2 : 775-;80. 1042. 11. C'$r~snl;ar I Y N , J. i V Thc efiect of fertilizer arc3~tlncnt on s w c ~ t clovcr root lot. Plant

Tlisi~asr Ktxptr. 34 : 261-262. 10.50. (;oonr<\r, 'F. Sdil and fresh tvatcr nc-lilatotles. John Wilcy C% Sons, Inc., Ncrv Jrorli.

1950.

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91 CJ,IN.4 DI-4 A\' JOl JRNA L O F BOl'il N Y , VOL. 32

16. HORSF-ILI,, J. C;. A study of meadow-(-rop diseases in New York, K.Y. Cortiell Agr. Expt. Sta. Mem. 130. 1930.

17. J O N I C ~ , F. R. Evidence of resistance irn sweet clover to a l['hytophthora root rot. Pl~yto- pc~tho1~)gy, 29 : 909-010. 1639.

18. ?,a,oun, %ti. A. Sweet clo.i7er; (:1IeliEolus). Ohio Agr. Expt. Sta. Bull. 244. 1912. 10. ~ ~ L P R T I V , J. N. Tlle anatoiny of the crown and roots of the :rnnual ant1 bfetnnial white

sweet c1ovr.r compared. Proc. Iowa Acati. Sci. 37 : 209-210. 1930. 20. & ~ I L ~ ~ A I , F , C. 1,. and F L I N ~ , ?T. P. T)t.struc.tivc~ and useful insr,cts. 3rd etl. hIcGraw-

Hill Book Coenpany. TIIP., New York. 8351. 21. r 1 . . The taxonorn?;, host range and geographic ciisti-ibu~ic~n of the genus

Py:hiu.itz. Mem. Torrey riotan. Club, 20 : 1-71. 1943. 2 2 . L O t . I 1 , . . I.; the apparent winter k i l l i~~g of swcct clover and red

clover a r ~ i u l t oi disease iiijury ? Sci. Agr. 5 : 03-06, 1924. 33. KIRER, A. J. ant1 Ra~;r;it, K. S. Imtroiluc-tion to research on plant cliseases. Jo111i S.

Swift t o . , lnc.. New York. 1936. 24. Se ,u~r~rrr \ , J. T. I~ivcxstigntions on sweet clover fnilurc in southwcster~i Ontario. Sci.

Agr. 32 : 1-18. 19.52. 2.5. T ~ ~ O V A S , R. C. A n e w diqeasc of sweet clovcr-. Ohio ijgr. I3spt, Sta. 131111. 446 : 72-73.

1 930. 26. 'TL~CICFR, (2. R4. Tnxonnnriy of tlip genus Phytophtho~u de Rary. Missouri Agr. ISxpt.

Std. Resedrcll Bull. 153. 1031. 27. %~;EY.SII, hl. F. Studies on crown rot of apple treeq. Can. J. Rescarch, 20 : 4.:7-4!'0.

1942. 28. 1&'1~,r,a~n, C. J. An cxperinlrntal study of sweet c1ovc.r. Ollio Agr. H'xpt. Sta. Bull. 405.

1927.

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