1 l

;!uh Ii ttcd i~o t.,,m facu11.~,f of t,/;:,f~ C1•adu.nl;.o bchool of the Oklahc,na Ar:rieultura.l nno neehm::i5.cal Collor;e

in port,inl :J.'"1:tliilLrat1nt o.f t,h.1 r-cicp.stl.rernents £or the c';et;ree of Ht,b'.i:lJ1 01·

1956

i.

- lMIM (iiltll TIUl l Wft:HjH«!Al ,OOtlESl

L.lRRARY

JUL1.6195 6

THE. rOJACGO RI JGS 'O VIRJS 01 A'illF ~LO IN OKLAH0.1 ~

ROBhRT J . ~HEPHERD

HASTER OF SCTu.lCE

1956

Thesi s and Abstract Approved:

Thesis Adviser

Dean of the J raciua te School

361532 ii

manuscr:i.;ut, to Dr. Oran D. Steffes· :for h:Ls helpfulness in preparation

of -the photo:;,:raphlc datQ, and to others on the staff of ti-1.e l:epcrt:.1~nt

of not,t.il\l nnd Plruct Pn.tbolog:;:f who t,hrow:;h their peda:t[ocical efi'orts

tion possible.

iii

••••:•••·••• • • •

SL!f'.i'Ot:15 .ru"\11: HOB'I' Pi.ANGE OF TUE liN.i.'E.Rt:.iE_l,ON VIRDS Illi GREENHOUSli: S'.ftlDIES • "' • • .. • • • • • • •

. ... . . ·• . 4

. ·• ·• ... 6

Sjr:mpt.o:ras on tobacco • • • • • • • • • •. • • • • • • • • • 6 S;y-mptorns on watermelon • • • • • • • •. • • • • • • • • • 9 Ro.action of other hosts to the water.m.clon virus ••••• 12

. •· . . . 20

• • . .. . 25

. . .. ·• . . . .. 29

... ·• a • ,a * • • 33

. . . .... 38

IllV·l';S'.i'ICA'iIONS OH IJATURII.L ?EAIIIS OF 'l:FLt..l~Sf<f.(SS:tGN 011 'l'Illi: tJA'rERiJE!J}i.t VIIWS • • ,. • • ,. .. • • • .. • • • • • • 46

I.nsec·t transrtl.ss:to:n trials · • • • • .. • • • • • • • • • • 46 Sood tra..D.Smissi-on ·t..ricils • • • • • • • • • • • • • • • • 50

.. . . . .. . 53

DISC{!SSION • • • • • .. -· . . . . • ,a • • • • •• .. •· . . .. 58a

• • • . . . ·• . . . . . . . ,. . . .. . . . .. ·•· . 61

• • .• • :ti ., .............. . • • • 63

iv

Sy:nptmrm on. torx~cco 2fter :tnoculD,tion with the wr:i;;crm:::>lor;. v.Lrus • • • • • • •

SJJ:mpta,ns on ot,her hc,str: &,:ft,0r ir•.oculation

• • •

• • . ll

tbe c;r,)0:.111.o~J,se • • • • • • • • • • • • • • 18

Fi;;:.n·e li ..., RE.JsnJJ:,s o:i? cro::.rn ... prot-eet:tmi t1:1t3tB on tob8oCCO plttnl:,s !'0<.;0'11er·ed fro:m. tobacco ringspot vi.rus :t:nfoct:torw • • • • • " • •

13nTc ) ... S:y111pt,m,is on natu.reJJ.y inf ectod wr13.:,(::ir­molon fruits •••••••••••• •

6 - Histological nature o;f tobacco ringspot virus losion.s on Dl,=-1.ck Dit;1nond wator­mel 011 fru.it,s • • • • • • • • • • • • •

Eri'ect, oJ; dilu:t.ion on the i:nhib:i:bor of

• •

" •

• • • •

1:·2))J,.e 1 --- Host .rar1r}:J an,,i s,y1nJJi.\o,-: iS o~t1 \~roe}1f1ot1ae plrn-ri;s ~,2t<:ir. irwculation. wh,l, the water ...

• • • • • • • • • • • • • • •

2 -· 'R.on1I·l t,G ofy 1-:{1,i·s~tcr<L tl10 w"£rt~r1-:1c:1lo:n. \r:l.r.1-1s •

• .. 26

• • 31

•• 42

•• 58

• 13

• • 23

To:bl,:;, J - 1~f.:f:Hr:rc of d1lut,ion on t,he of rn::.te11·1ulo[\ sa.p • • • . ·• • • • • • • • • • 57

~~.lie 1iir:~_:t.~c1\;~{;iv.: (CJ.·t,1··i~1lli: .. Li ,,.\~~; .. :._ttr-.'~.-Z r:clirti.ti.) .if.J 0110 of' ()1'~nJ1a;11.r:: 1s r101.~c ~ ·--D-~H-0~,~~~-

Collc:1tot.r:Lchu;·1 l,:1{,CrlDr'.l.urr-1 were co:J'J.on ._ ..... -...--.-~-

A sim:U.ar vir,,rn was .f'ounci. to

naso in

to

bn tnfccteG w:1.th the tobacco r:Lngspot virus. These iwrkcirs al.so r(;port.cid

mrturrtlly- infected squauh (Cucu.rbita. ~nruc.l..ma. ruchosne). ~Tohnson ( 25.) found

ti:ie v:Lr,:w occurrin.r,: nat,u:rally on cucuniber ( C,101mis na.tl\1u~; L.) and Husk-··- ,- -- , .... __ -----

,rieloa iu Kentucky, and Vallcau (hfa) re}JOrtol\ it cot1 (only

!nvesti.c~tions b~l(~lm in the SlVllI;;.er of' 19Slt in Oklahoma likowlne

iridict. tet.1 a vlrus a.llied with tho pim.ple disease. Inoculat-i on.s of pri­

ma.r.t leaven o:f cowpen \>r:ith sap trom infected f'rui.ts resulted in the pro­

duction o.f marr,1 redd.i5h-'b:r·o1;n 1,,sions {t:";.r;. 6). 'i.1'.lais S:lf~J;estod tho 1.1rc ...

sen.ce of c mochanically tr.ans,:li~sible virus.. 1:nocuJ.utiuns fr011 2h · oi'

these irirl ts from several fields ovor the stt:,t-0, :ta every case unw! the

srune characteristic losiona on eowpoa. In.oculations uu:i..nr; cJ~i}r,Js5utl saJJ

fro:) wat.err.t(!lon foliage fai.led. to reveal virus. 'tav.o·1.n:,i. tobacco :t)l.a;rl;.s•

sbrilrirly inoeillated with sap from di.sea.sod :fruits, I;:rod\.:,:c.:~d SYdFi,Otl~

characteristic of a rine;DL::ot d::.seiu.,e. Synpto,;15 were vor·y s:L;1ilr,r on

both cov-rf;ea and tobacco with all v:trils isolates obt,a.lned fra,,r t;.:e s.0·1.rorlil

fields.

'1'~10 disearna has 'been olwcrveo. in ,::1_11 plnn.t:ln[;s of the Black Diti.:aond

varict.;l \'isi te,1 in akla.hav1a. In i:,oiru? i':i0lcis alflost 100 percent :i.nf'cct:ion.

wat> :i:'0UJ.1ti. I;1 the lir;t.tt of thene observations it seemed ap_G,roprlate thti.t

the iut.c:tre of t,he disease be imrcst:tgatrni and i-ts imvortanco ev,2.lw:;.ted.

'.the wt~ wr~iielon var:ioty of ranking i;,1portanco, us judt;etl b;v acreec;o,

is l)lack I:ia;mn<l; thoreforc, i.rwotrM1;atim:1 wa.s, ill the mrd.z1, co11.i:i:omi to

the d:iseaoo on th-5.s variety. rlorida c:::a.:rrt~ Te:;w.s G:innt, C:.1nn<::m B:.111 and

Claru Lee are other vurietal oonms for this nelon.

acsay plants in tests whero eottmation;;;; of virus acti vtty were nee(icd.

r;ots.

plar.rts hi ,:.Ge were observod

'

the virw:; were rolled tn:t:.o a snall squar(c:i of non-ab:-.mrbent, cotto:1 .,1:nd

2'.i.11.0 tobacco mosaic virJ.u was i'rou nnk1rtd.ly infta)C'i.,od tt);11a.to plants at ttillwator, OklMhona. :Dr. ,J. P. :Fulton of t:.he Uni vm·Q.ity oi' 1Lrlv0xwas r;enerously supplied hLs A,.G, , anci F t;trsiris (16) of the c1.10u?il,.r,r mosaic vir..i.s. A green st.rain of the tobacco rincspot Yir::;s 0.nd t:1c yellow strain fron watornelon worH obl'tr:Lnf}lJT proviiJed by }Jr,. Ci:lom1 S.. I'cu.nd of ·the Uni­versity of Niscons'in.

4

mascerated ld th tllo finger tips. tJhen t,he co·t·ton becanc soaked with sap,

it w2s rubbed over. leaves 1:i.r;lrr,1.;y dust.cd Hi th 600 :r1er,,h carborunctun. Hlmre

1:-:rrzcr rm.:rib,irs of ·oost :plants we:i:•e, to te 1noffuli:J.:tcct, f'~ ~,10rta:r anB pestle

vjore usod to nascerate viruo-).rtl:'0ctcd ti~F110 211i: t.he juice w&,s tc:.. .. ko:n ·ap in

a cc.r(,tou pat. ~Ded in inoc,,J1.1tion. S1:;~.J.l,. Drou.:nd t:l~s£J spatulas were em­

ployed in mak:t:n~~ inoculations durin~ ph:fsical prop,arty ami ir.:11:i.hit.io11 t,est.s.

Plants were rinsed ·t-tl.·i,h tap wcrtor after i.noculation to remove excess inocu­

lun.

SY!:I.?f'Q;.1Jl.'£01a~Y AJm ans '11 BAl:2GE OF '.IHB WA'lBRv:TBLON VIRUS II.{ GF&El\HlOUS}t s:cur.I.GS

anc :.noculated L1 tTcenho':lsen tiurinc t.t.c wintor months. Plants were inoou-

plrurt.:.:; oi co.ell species i~ero r,it,nned un'.nocul&tec. to serve r-:s hnalth;/ con-

trols. Hecovory of ·tho vlrus wua aturri:,tod from all inoc\*lr~tod iAiants

re.c;i".rdloss of the presence. or absence -of }>j'mpt.or:w. Gucu.dbcr plmrt;s were

used in recovery inocu1fftions to o'boorvo if nult.iplicatio.n lY-'ld .occ;.i.rrecl. in

each species. Cucum.001' wtw chosen for th:la purpose as it is appi:rently

less a.Zfcctod by· inhibitors of inrection. ::'ro/1 hi;/hcr plants {lB) 1 a.ntl it

virus.

l'Lc reaction. o:C' m.wc.,1a tobacco plant:;; whm i..nocu.lr-.::tocl vd. th tho w,'.tcr-

react.ion cons:tstod or snru.l chlorot5..c ()!' n0crot.:lc rin;;;s or s.l:)Ota. i:Lh.5.s

6

7

tenJx,rature ..

oven occurred. on

ctlo:r.ot;Lc

yellow

a pro TLncnt chJ.orot:L c: mottle

necrotic o

tc ti1~lk;. 1:ccovory was

pc>.rccptibl;;r more r,ap'ld at

•C) ... ·, t3· ., } i cu.nc " · :> •

virus.

8

Fig. 1 . Local sy..,tc lie sy;.1pto 01 tobacco after :reenhou inocula.tio ith th wat, r. lon virus . A. erotic rincs an flecks as pr:i.. • y ,Ympto, • B. Soli necrotic s>ots on inoc ·t leaf. c. y mic chlorosi , • Oak- le f tte :1 on y te.ni al y imucte leaves .

9

(L4· .) .• t t ·•.. .. . , . . 0 t·• t " Valleau q. . was the t1.rs·· to :no e s11ch i. y(d .. 1.ow ::n;raJ.n 02. '.n.e ·oco..cco

fea:ture o:l.' tiri.s strr;;in. Price (Jh} stated that co1,1:p1et,e m<1oldnr; i:n old.or

leavos of recovored

Only s other

lation. were c:lrcular and :inu:l.st:i.ncuish-

an<S: necrotic (F::1..g. 2,B). Loc2,.l symptoms

u::;;ually a., ,pearect .r,hou.t ?2 hours es:;:·tcr 'L:noculation.

Inoculated cot;flodons

sensit,iYc to the wat,ornelon vlrus.

lO

;:;rowinr:; point.

syst.e~nic ,Jevelopment of t[J() virus DJ1d f::Lrst showed us s~.1all yellow s1,;ot.s

( . ., ·.· 2 r) J.1.J~.. '··' .. S'_}ots of this sort dcvclo,,:ed at ran.d0iI1 over :i:.ntcrveinal areas

of lenves and frequ::rd:.l;y were sui'fic:'Lent in number to ::1 ve the le,:1.i' a

chlorot:'i..c :1ottlcd apprJarance. Chlorotic lesions of this nature often lmd

a tiny, pnlo center ,ts oociatod 'W:L th t,heir develo,µment.

Ir:rerular chlorot:.tc ::treas of1~CH1 app<:;iarcd on ve.ry young leaves ;just

:fatal -to plants hav:i.ng 1ni t:i.ally rncr~ived a larc;e amount of il:1ocuL:wi. In

m:.:,riy cases vines were killed b~ck t,o the .f:lcrst or second bas1:l node.

At hlrJmr temperatures

Sy 1pto is were also noted on other selected lJlltE'rld.elon variet:tos.

·ti10so of Pound (33),

A

• 2.

12

and stlmt:tnr~ in ·1tinos system:lcally infected wlt,i.'1. t,he Oklahoma watar:r;clon

vir.is were notably siru.lar to tho sy:;t:t:.1torns deser1bed 'by Po-and {33).

Heact.ion of other hosts to tt:e w·;.:.termolon virus

Rosu'..l.ts of these tests are SU.'l'ltiiarlzed in r.eable 1. Both local an.d

systemic sy.npt.ons are recorded in the t2ohle. 'l'he vlru.s was 1"'ecovcred from

all plants unless noted otherwise ..

Host rru)bo and sy1;1ptorns are similar to those reported for ·tLe tobt..teco

rinc;spot virus (51). Sot10 variation in SJiLlpto,'ls is lli1ciersta;:,1duhlc on t'h.e

basis of dif ferenccs in varieties of' host species used rulci env:lron ;ental

conditions.

Rosberg (37) i'ound p;1tilllia not. &"U.Sceptible -to t.he virus he recovered

fro:m naturally infected wuter;uelon in '.It':xas and noted this as a cl:U'fer­

ence bet,voen the Texas v:i.rus a:.>1d tl.".'te one w:t th which .Pound (.33) worked.

In i:noculat:tons here, howevGr, petimia invariably produced very· pro,'limn1t

local and systemic sy:t;Jto:as upon ini'ec'l:,io:n with tho Oklaho,,ia watoi-·.rnelon

v:tru.s (:C:l g;.. 3 ,c 1D). Ewn retarded, senescent petunia plants :tn the green­

house showeu. ahur..ciant symptom,. I:lcact.d.cn o.f tbi.s species was observed on

several occasions. 0£ t.tlc many watermelon 'llirus isolates rocovc:rred frot;:i

na.t;.::.ral.ly i11f'ectad fruits fro:;1. wldely .sca:ttered locali tics in Oklahon.a.,

all Sf*7r,i.ed to co:nsti t:ute a rather constant, euti ty and appeared. to oo v"Ory

similar or identical to the ·u-lruses from wator.E:clon whieh Poun<i (33} and

Uosborg (37) hav-e described.

As shown in 'l'al>le 1 all cucurbitaceous plan:ts teste6 were found s;12 ..

ceptible to the v:Lrus. Int.erostins:ly m1011E,J1, react:.Lonu ~tere SOiilOt>Jhut

similar on all spec'l.es and varletios. Local synptolim ccMiJonly appeared

Tabl 1 . lation with th

o t r e ter. l on

• OrOD A EAE: Beta vule:aris L. -rearden t, v

Blood turnip).

C

•

•

L.

•

13

cnhou e pl nts after inocu

• • •

chlorotic 0.1.t n erotic

• • •

ral chlorosi s .

·one; recov

not

Zonate, bl ck cro- C tic le ·ons.

s orotic o ten

• • • on • • •

• • • 0 • • •

• • • one . • •

•

c .

C e chloroti c tlc 'With i

lar cro ... i .

ener but faint chloro i .

not

one; vi ., not co ore .

on ; virus no covere .

u-

able 1 . (con•t . ) - Host range and symptoms in eenhouse plants after inoculation with the watermelon virus.

HOST SPECIES

CUCUR BIT ACEAE : Citrullus vulgaris Schrad. (watermelon, var. Black Diamond, Klondike, Charlest on Gray, Congo, Dixie Queen, Fairfax) .

Cucurbita maxima L. {squash, var. Early Prolific Straight Neck, Giant Crook­neck, White Bush Scallop) .

C. pepo L. -( pumpkin, var . Neu

England Pie) •

Cucumis sativus L. (cucumber, var. Na ti onal Pickling, Boston Pickling, Improved Long Green) .

C. melo L. (musk­-melon, var. Hale's Best, Honey Dew !felon) .

PRIMARY SYMPTO 15

Chlorotic spots or circular black necrotic lesions enlarging and be­coming irre ar; masked at high tem­peratures.

Circular chlorotic or necrotic lesions

• •• ditto •••

Chlorotic lesions

••• ditto •••

SYS'I'EMIC SYMPTO 5

Small yellow spots appearing first on immature leaves; occasional necrosis of growing tip with subsequent outgrowth of axillary buds; shortened internodes giving compact, bunchy growth habit with stunted and often malformed leaves; symptoms masked at high temperatures .

Yellow spots followed by chlorotic mottle ; stunting.

• •• ditto •••

••• ditto •••

• •• ditto •••

lS

T ble 1 . (con•t . ) - Host range syinptoms in rcenhou plants ar r inocul t:on With the ate elon virus .

• cocci L. -{Scarlet Runn r

).

Pi. um sativum L. ( , var. Al ka, Dwarf rvel) .

Glycine . rr. (soyb an) .

ecrotic £leckv an rin s .

••• one •••

S 11 chlorotic lesions, later

c · c:, nocrotic .

Small chlorotic pot .

· ddi • h- rawn necrotic lesions .

Zon to, crilorot, · c s ts becomi necrotic.

• • • • • •

Black necro ic cankers on veins , tioles and st s; coarse chlorotic leaf ottle follow by irreguJ. necrosis; necrosis or

owing point follow-ed by ath o! entire plait •

0 ; reco

not

Irre ar necrosis of lea s an st ; occasional eath of ntirc p nt.

Coars chlorotic ot tl with <>0.

cros..:.s of ste. •

;

tiole

cillorot..i..c ai n erotic

one ; virus not r cov r •

16

Table 1 . (con1t.) - Host range and symptoms in greenhouse plants after inoculation with the watermelon virus .

HOST SPECIES

Nicotiana tabacum L. ( tobacco, var . Havana, White Burley) .

N. glutinosa L.

Petunia hybrida Vilni. (petunia) .

PRIMA.RY SYMPTOMS

Necrotic spots and etch-like necrotic and chlorotic rings; masked at high tem­peratures .

Occasional faint chlorotic spots; often masked .

Zonate , necrotic · spots and rings .

SYSTEMIC SYMPTOMS

Chlorotic spots and coarse yellow mottle ; often prominent chloro­tic, oak-leaf patterns frequently becoming necrotic; plants even­tually recover; masked at high temperatures .

None; virus recovered.

Faint chloroti c mottle; necrotic flecking of leaves; necrosis of stems .

17

as either chlorotic or necrotic lesions. Systemic symptoms usually showed

in the form of characteristic yellow spots on leave~ (Fig. 3 , B) . Syste. -

ically infected loaves later bee e slightly chlorotic before plants began

a slow recovery. Cucu.mber usually pro uced innmoorable tiny yellow rings

over sy.sto ically infected leaves. 1ost synpto s disappeared after a t ·

al though plants at lower temperat s never fully recovere but re;nained

slightly chlorotic an markedly stunte for an inde inite period.

Primary sympto on bean and pea, as with cowpea., w re largely

necrotic . ecrotic f lecks and occasional rings ere produce on inoculate

leave of bean. Systemic infection resulted in a characte ·stic blighting

of the browing point of both bean and cowpea with dark, necrotic cank r s

appearing on ster1s, petiole· and leaf' vein· . Infect· on was usually fatal

on both species. On occasion, a coarse, chlorotic mottle devoloood on tri­

foliate 1 aves of b an with systemic development of the virus . At tirn. s,

an irregular necrosis subsequently appeared on these leaves . This necrosis

was part'cularly evident on veins and veinlets.

'Ihe Oklal oma watermelon virus was compared with tre tiscon in water­

melon virus in the symptOJ each ca so en inoculated tobacco., cowpea

and c cumber. Reactions on these ho ts with each viru were aJ.most i en­

tical an indicated that the two viruses w re imilar or iao1tical ..

Wi ard (.51) has investigated the host r ange an :;;ympto tology of the

tobacco r · -spot virus . T ho t ran ,o of tho Oklah watc elon virus

corres onds rather well, altho h differences are evident. his may be

explaine to s e extent by differences in viru strains. For example•

pea ( Pi sum sati.vum L. ) was a!)parently not susceptible to Wi.Df,a:r ' s en

strain., ereas, the watermelon vir studie here and the one in 11.::co in

( 33) ere tho eni.c on this '1.ost.

Fig . 3. Reaction o.f other hosts to the vato lon vlrus . • Sys m·' o !liOttle on soybean. B. Systemic reaction on

,uc ,her . C and D. Systemic and local neorot· c reactions, r ie:;~ ct,i vely, on petunia.

18

19

In general, other investicators have found few cruciferous plants

susceptible to t,e tobacco ri Similarly, the watermelon virus

was not found to ,ultiply in any of these plants . On the whole , how ver,

differences , thou ;h not at, see ed u.f'~ici nt to warrant .further i enti-

fication of the watermelon virus by physical properties and mores cific

means such as cross-i.munization an serological tests .

PHYSICA PROP.iiRTIES OF IE WA'r Rlit!,LON VIRUS

thods used in deter ti.nation of phy ical pro rties w re sil1Ii.l to

those described by Walker, LeDeau and PoUI d (47) . Tests on tbemal-

inacti vation-point, tolerance- to · iution and aging ~ vitro, were repeated

three ti.mes.

All tes e were ado using expressed sap fro young Havana tobacco

plants inoculated 2 week previously w:i th the watermelon virus. Stanley

(43) and Price (34, 35) found t tobacco ringspot virus to reach its

highest concentration in t obacco such an interin after inoculation

when plants were showing a ,w.xir.nira amotmt of necrosis. Since preliminary

results had indicated a relationship to t1e tobacco ringspot virus, a

sir.rl.lar perio of multiplication wa assume optir.ru.m .for the wate lon

virus. This point of concentration is pos "bly more i portant in working

with the ringspot vi.ruses as their concentration a parently decreases in

t obacco a the plants recover (35,43}. rowever., ulton (15) has found

evidence that this r,e.y not always be the case . Leaves of systemically

infected tobacco uere ashed an allowe to air dry before ntincing in a

meat grinder and extracting t juice . nis juice was filtered thro h

severnl layers of non-absorbent cotton, pipetted into tubes and held in an

ice bath until used.

Boston Pickling cucumber eedlin ,s were use as test plant • Coty-

ledons of these plants were inoculated just previous to e ion of t

first true L aves . Small ground glass s tula were dipped in inocul

an rub lir..htly over carborundU!ll dusted cotyle ons . When the inoculum

contained active virus , temic pto of small chlorotic s ts appeared

20

21

on t,he first true leaves about seven da.rs later. Twenty-four cucumber

plants were inoculated on both cotyledons ·with each test preparation.

Test plants were observe· daily; tho e showing sy:r,iptoms were promptly

discarded and recorded as positives.. Spatulas used in inoculation were

cleaned anrl sterilize before use by washing in soapy water,. rinsing in

clear water and flaming With alcohol.

In t.osts determining thennal- inactivation- r.;0int, small, thin-walled

gla ss tubes l . 5-2mm in diameter and 10 C.."ll in length 'Were filled With

expressed sap leaving approxim.;, tely 2 cm of each end empty. Both ends

we.re sealed in a srw.l flai'!e . A water bath with a meehanically driven

stirri rod and therntostatically controlled to vary not more than ! 0.1°c

was u ed., Tubes containing the sap wre placed in a small wire basket

constructed to hold the tubes well. separated. The basket with tubes '1/<JaS

completely i.'ll.mersed in the water bath at the desir: d temperature and

allo ,red to remain for 10 minutes.. Upon removal, basket an tubes were,

returned to an ice bath until inoculations were made. l<'or inoculations

tube ends were removed and. contents blo m out onto a. small glass surface,

then glass spatulas were dipped previo s to robbing over cotyledons of test

seedlings .

Dilutions of plant sap with col' distil.led wa.t.er were us d in the

t olerance- to ilution tests. Serial lot,"lll'ithraic dilutions of sap-> ranging

fron l part sap per 10 parts water to l part.- sap per 1.000, 000 parts water,

were used as inoculum. Each higher dilution was ma.de by pipetting 1 ml o;f

the preVi.ous lower dilution into 9 ml of water. A new dilution was mixed

well and allowed to stand f.or several minutes be.fore re..wval of an aliquot

for t.he next.

22

A constant te. pcrature incubator set at 20°c erve as storage for

plant saps during lo evi ty in ~ tests .

pipette into tubes stop re "'· th cotton ru

Un ·1uted expressed sap was

placed int incubator.

A sin e tube was re. oved · 1y at about the sarn.e hour and e a"' inocu­

lum.

R sults oft physical property te~ts presente L., Table 2.

int,lc 2 .... ]·osu.lt:J. o:t vir,.w on cucu:1ber.

'I'lmrmul ina.cti vation (10 r:.rl11]itca <lur,-:.'t.ionh

{);,s1;catcd • • • • • • 6o0c • • • " .. . . .. 65°c .. • •· • • • • .. ?0°c ·• • • • • • • •

Tola ranee to uilution: Und:llut,ed .. • • ·• • 1-10 • • • • • • .. • 1-100 • :w '" .. • • • 1-1000 • .. • • • .. • 1-10,000 .. • • •· • • 1-100.,000 • .. • • •

A,:irv~ ,Jitro ( : .. ·~o, .~:~-,-:-;-t "o0 c~.- '· .. t u.1. ~.-· o~ - L J._

0 • • . . • • • . ·• 2li •• . ... •· . " ••• . . • • 72. 96. 120 lL;h 1(}e 192 216

. .. ... . . ,. .. . -. . . - . . ............. . . ~ . . . . . • •· • :fl •. • • ...

• • • ... •. • * • .. . . . . . ...

23

l 2 'l'OiAL

2' /rJ ")!i/2! 2)/':IJ 72/12 ti ~·+ t> • , i 4 t.,i 19/21+ '''J/'°l)..j 2L/24 63/72 (.'. I '"' . ""''4'

0/''I 1'9/?l.i 3/2h 22/12 c,..i 0/2h 0/24 ()hi 0/72 '·I (.4,,

"J/?1 21/24 24/2h "''/72 2 · G.Lt c,i;;,. . . 23/2!i r· i .i> -1 r>t, l')L 71/72 d.;,/GJ, L!J 0.4

22/24 20/2h 2l/2h 63/72 o/2h 0/2h 1·/"1 rj/'P . u 2.-t l) ,_

0/Zh 1/c!h 2/2h 3/'12 o1,,.1 0/2h O/?i, 0/TZ . I .::l,j. . ;;.4

(. p \ ~ ... ,:-.--, ·. .·. ' •1 ,., 1 .(·. ,,e :r.eru.nc ,_

, .. .J,. :' .. l' ·ti.tro ti 'e n;' l ~-l4 __..v..--

l ;: .. 0,. \ .. ~ ,(,,.

.: \,.,. ·'< -~· .... _; ~---

r <',,1 •·\" .'"• ~ j ·.; ~~ ..... , .. ~.: J.

2 •• ~

. •. '-~ :.: ..

,. 101·c

Fig. 4.

D

Ro~ ts of cross-protection t st on toba co leave~ r cov re fro~ tobacco rin spot ~:r i ection~ . A. Jccovere le

26

after inoculation with tho wat lon virus . B. 1 al th,y leaf' of tl o sa. :e a . inocul ... e with lihe wate olon viru • C and ,ecovered leaves inoculate with tobacco onaic cucun1ber o ·aic vir lses, respectively.

•

27

All inoc late healthy control plants inclu ng tho e inoculate with the

wate elon viras proauc d abun ant sympto.s ( ig. 4,B) . Recover plants

inoculate with th watern. lon virus rm ,aine symptoml s for

r w which produc d f int o aic- ottlc e sy"llpto. s on t e inoculate

leav s (Fig. 4, A) . Thes plants in le te either co,plcte cros - immuniza­

tion was not occurring, or that th s pants were ~till pr ucing so

chroni.c s , pto s (31, 45) of the green strain. Price (36) has foun that

neither the een tobacco sot virus or Valleau (44), nor the o of

Wincar (Sl), will otect co plet ly aga:in t Valleau's yellow strain of

th virus . S · · 1ar lack or cro..,s-protection betw n strains is !mown in

cases of the s ar-beet curly- top viruses (19) , · an to to- s tte - ·it

vi.ruses (6) .

As an ad itional ch ck on the above noted result.., , e xperint nt

was r pcated usin recovere an healthy plants grown fro,a cuttings .

This all d a lon r porio for th plants to attal.n tho acquire toler­

ance stage o infection an provl e rapidly gro ·ng young plants to b

inoculate .

S cuttings, consisting of one leaf with its axillary bud, r

a a fro, botl healthy an recovered plants . hese re place in oist

.. an until roo an a new shoot began growth. Cutti were trans-

pl3nte to soil an f rtilized. These plant, infecte with the een

strain, w re s owin~ no sympt an appeare rfectly healthy. Groups

of the~e plants wer inoculate as in the original experinl nt .

In thee t sts compl te cross- unization wa~ apparent betwc n the

watermelon and green tobacco ring"·pot ru e a recoverc plants remain d

sym omles after inoculation with the wate lon viru • H thy controls

28

and recovered pilmts i:nocu.k:ood with either cucu,,1ber mo5aie or tobacco

2nosuic viruses, disr;layod sy.:rptom.s oharact,or:l.st:':..c oi' the new -virds with

wh~.ch they wore iuoculcted.

:Gecau.se cor'i~ai:n sim.ilari ties between the wa.ternelon ·virus and the

eucaruher r.1osaic virus hnd btJen shown w:l.th :respe<rt, to thorm~l-inact:1. wtion­

points and host ra:ue~, acklitional cross-protection tests were nwde with

the cucuHiber mosaic v-irus using cawpca (16).. 'l'hese plants., variety Black,

were inocuJJ;;,ted with :F'-ul ton I s stra'ln 11' ( 16} of cuc.u..>nber mosaic. This

strain induces a cblorotic mottle on syste:nical.ly i:nfected tri.foliate

leaves of eowpea. Infected plm1t.s e.xhl 'b:l. t,:ing su.ch SJ>7tlpto:f3G af'tor inocu-­

lation and several h3alth.v control plant.s of the same ~iigo were inoculated

,n:th t.he water:'i10lon '1-..i.rus on their tr:ifoliate leaves. f;;avertl of the sa.me

cucu:,fuer i?losaic plants were retained m1.inoculated as control.s.

IJewly inoculated plant,s soon proct.1ced rcddish-b:z•own priTs1ar-J lesions

chnractel'istic of' the waterr.1elon virus. Lesions appea.rccl onJ..y on inocu. ...

lated h1a.:flets. Like s:y:ciptOiiW 1-mre shown on h;;;eltby control pla.nts inocu.­

llitcd ·w:i:~h the 1.:1.atermclon virus. No lesions were observed on the u:rdn­

ocul.ated :mosaic pla."lts. 'l'hu.s,, cross-im.nunization was not .shown between

·che two nru.scs; hence,, the two are not closely rel~ited ..

·- .. ·, .. ~ • .::: <"'.:'L Lfl.tJ(\

A S18.ll

bot.to :i oi t.Le tu.Le.

l,t;evns were then

29

JO

• · ' · - -. " · , • -,- -~ ·t ""·'"'(' , ...... ,..,~,,,-.,., -t..· (-, ."·'_,.,,:,: t,'. ,·f:'_._ J·!·,· __ .; "',~ ·.1'·.'i" lt~1r<10'. s_·_,; · ....... ,,,.,_,_"._-i-._-, Li,,}l;C'(,J .. ()l:, ,1.c,-.::vf,b .. u.,,~) '11'7,V<Q :,, !.it•~, ...... , ••. ,,·_,;,.; "1 a..... .. -.-,,,; "' ,_ - ... "'"'·-

C-CJ "L to:;1.

t,:Ln l,dst. tobacco

t 3-•_"Jf)_f_'' ,, __ ,_Pg· r tuo- ' - > -, ,[). - I V !. .. H()Ulb,

'Yu.be 1 - Oee fxcrt ~bsorLed normal se:t~E1 w;_Jt,rr{,t0lon v1ru.s.

OI1(2 p:1.rt f,l:,i::;orbcid i'Lllme sel:'ixm t,,.,'.l.:i.d.nc; tho WLl.tQrr:1elo11 viruJ:;.

31

toi;;acco

o:r to

con.-

'l'ube 4 - One part absorbod :tnmuno oerun plus h pr,rts tobacco s;;-rp con­tobocco ,,ios;;d.e virus.

vr _,.. • 't \ ;J - tJ:r1C pc1rr, ri'.bsoroocL i::;1uu110 L,orun1 plu.n n p,1rts t,cbacco ~:;ap con ..

cuctt:ibor ,iiose1.c virus.

Tube 6 ... One part absorbe im un serum p us 4 parts tobacco sap con­taininb a green strain of the tobacco ringspot virus.

'l'he ·e tabes ware hel at 37°c . It was £oun necessary to ob rvo

32

tubes 2 hours afte~ mixing sap an serum as spontaneous precipitation of

plant, sa would later obscure positive tests . In all tests ositive

results were noted in tubm; 3 and 6 . Precipitates in these tubes were

e,ranular, flakiJ masses which gradually settled upon standin • In gen-

eral, precipitates did not form in other tubes until so hours lat r,

a.nd when appearing were less gl:"anula.r or aggregated and of a arker

color.

In conclusion, it may be stated that tne vate lon virus is scro ...

logically related to the tobacco l · ngspot v.i.ru.s as the two for 1 recilxl.-

tation complexes when pr "ent t,i th th same specific antisera. However ,

as sympto s on tobacco have shown, the watermelon virus i probably a

yellow strain, and tterefore , v y similar or identical to the virus

de cribea by Pollil (33) from watermelon in ~iisconsin.

During swmoor months the rincspoc vtr;;.s :in waterJ1olcn a.;;:1peared pri­

marily as a frt1it disease. :Foliac€? a:ad vino s;/r11pt,o:as were less evident

than 011 plant,s ~own ,:i:t. cooler tm~ratur(~s 1.n tho t;rcenho~rne. However,

folia;w syr1ptoms were observable on. so.10 plants; 1:hrt:tcu.lc::rly evident

uero the irregular, necrotic losi.oxIB on i:oliute• 1Ihow lesicns 1i8re

very siroila.r to those reanltint frOia greenhoti.:Je inoculations (F:l.:s• 2,B)

wl th the rincspot virus.

neath .of foliae,e oot,r centers of hals i: as also noticod in ::1any

})lantin:.,~s. Leaves al.one basal /;icrtions of' vines we1->e deaci a:rni drted

evi.cent. 'l'bls c-ont'"tl.ticn -was wJ.desy .. reatL in :fieldu by 1Bte J,ilJ"- J..n Gkl.a-

seldom (mcou.ntered when Nany of these observations were r~e 6::;ring the

oxtro,11ely c:r:1 ,_ etitb.0r :i.n tho m.Etr;Br of 19,h .•.

A. s'i::.ort, portion oi' st;ur1.tt:;d ;_.:;rowtri :i.:1:rl:.erpo1:;ed u-~t\~w:m nor:nal r~ppc~ng

1:x>rM.ont> on ei t,h,,~ sife 11,:;.,1 unotlK:r :fe&.tu.1-e <;.C courn.m occurrencu on i.n-

i'cctcd vi;--K:s. '.filj_s :,-:;ortion r1'7.cl ff·o1•ter intcrn.<)~fm .$.L"J(i sev(r.rely ;;;t,u.:uted

foliat,:e• 1'his sym.ptoH wus 1n"obc1bly analoe,0~1.s to ·1,11.:;.,t in ;::reent1c·use

plants in wi:ich stw.1t-int occurred soon £:'.J.''oor systt~nrl.c (tevolopmc'.1t., .of

rrowth habi·t. I:n ;Jn-a:oral., hmievor, ioli.:-,.g:e and vl.:.1e sy,,i.fYr..o;c1s ,.;,:;:ire

sporocii.e in occ-:n"l'.'cn.ce i11 field plimt'lnt.u uurin~:: 1-m.riucr ';,KW..1,imr.

.,, r' ) l~. ·0 ,.·, - .

Jh

'1 >ero

;e ~ ·~r.-r·l-;:\·, ,!,., l.>:' ~,--.;~i

·r.··. J,.,1 ·. -· >~~

. ~L\ t,:.1.1.•0 t!U lo., '..13:,

-., ~-···"-

t;/ O.· t.- ,;~-1\. t,. I~ ~ (::; .tJ G ,~ , ,fi.,r'.:. )5. ·ttJ~".i}?· ·:~en t. ilrl!S of1

~···.~ - -..•. , it, ...: .. .t ·1:,,,) .

J(;

+

'

Fig . 5.

D. f ac-

37

c. lrLtit

(

(1\). ( '';' ' \ .:,, . . ) 1rl ;_.re

;_·,.,_:._:~ .. ·~-·-~:,·.1··> (_:::.)· ·.,, .•• 1,:-.· .• ,_:·, .;;.··,'..- •. •,•,.~ .... 1·.··_._'!_,_,'(".·1.···:_ ,r_j.'_.,,-,-~ -~~"'l.v··r·.,·.• ... :··-.1.' :, ... ·-. t:~'',·,··)1(~ .. _ .. ,,. (""t·'·' ·1·-~·-t;··'\.1~, ,,.;·,·-r:c--" ·,·· 1:··,,.,-' {-, • • - ~ ~ -" • -- ... -... -- ' • ..,.. • • !.. J_ • · ·""" ,,.; - .J J .;,L ... , .. .,,._ __ .'..a.;-..Jd,.. ~.-. .. t.-.,i.,_ u~ . .1 •

• 6,r:).

'.r \. i ',.

' .. t}

l.:--:...

'_( ;c·. Cf/ C •

-_-i\..'.!

·:.,o

• ',''"",,c;,·;'

'j ·-~-' --~ !, •

:_,,_·_

c·

\. l•

•

'• ' l•

er oi

'\,O

,·,•

).

1'C

or

· __ ·,.,,

:uccro-

j ·,

\!'

\'f" ·. (?

·.t to

( ·-, ?) ,. - i •

42

Fig. 6. Histolo _ical nature of tobacco rint,spot virus lt'!sions on Black Diamond watermelon fruits . A. ~oction throut;h a healthy fruit . :a . A lesion after <lovelo;::inent in warm. weather and shcu:i.ng necrosi s ana hypertrop ied cells in mcsocarp. C and D. Lesions after develo ,ant in late season and showini; hyper • plasia of cells near the necrotic area. tiote extensive necro­sis extendini; t hrour,h sclerenchymatous sheath an hypoderm in D.

lkmco, ttOse cc11D

o;''. co1lfi·•

:fruits and caused ;;10s-t

fru~. t lcaionr,

discer.nible in many

les:Lons sectio:r~2'cl and Sin1i•

lesions ~,~re o:f

Jt .se-m.:t--solid.

sterilization

tunas.-

l'hv .. s 1 t,he ri.ngspcrt:, virus was the sole inc:i. tant

of f:ruJ.t lcwions ..

ne lon fruit~, grow:n in the greenhouse

v:i.rus.

noticed a1:morrmli ti:es on ini'ected

hosts.

+"'.'• VC"....t,

t,o ind.uce lesions o:n water-

i:noculr1ted with the ringspot.

t,he t,obacco ringspot virus,

Nest ~

·rhe cuctu,iber :mosa:'t..c virus

is, also kr.KWll'l to <~a.1 .. ise in:tum.escences on frui t,s of cucurhi ts ( 12) 1 but

as was prov~n in the cross-immuniza:eion and serologic&1l tests, the

Pimpled :fruits, the t,ype 6.esc:r:l.bed a:bove with the to·baeco rinf:;-

11~1 n'.1-,:vt"l:t:i'l. -b.,..Ot''"'i YH,nl ...,. ·· V,'!,.,..,..,_ •·-~,.,. ""' ·•tk'"'"'"·'·b

occur

out corn-

f:eult,s a.s origi:nally descr5J:.ied hl

l'Pl,'i"' "'')·,~t''i t···l '"l'' J.J.J...o:, .. tJ V\:e_.tJ.· ,t,___ ..J!,,.U .t

water.melon growers.

A'i''.l.l;m?i'S TO f'INI; HA '{URAL MttAN$ Oli' tRll.1'1$ifi8t1IOW (}fl' 'liPJ;. RINGS PO'i' vnws

reactions wore spo1 .. acdc or absent. Thus, insect transmission trials

were not availabl1:; i'or these tests unless reared or colonized.. For

this reason only difforentlal cruoshoppers (l\~lanopus. dif'ferentialis

'l'homas) and nelon or cotton aphio.s (Aphis go.ssyJ,-ii Glover) were used

r:wlun to waten1mlon.

bility is watermelon seed. This -was tested., althcrv.gh not exl1austively.

w.tth seed collected .f'rom nat,uralJ;r infected fruits.

Insect transmis;sion trl.-tls . .

their av"Uila'o1.lity and a.s !:!alters (h8) has found are potent,:lal vectors

be more e.fi'icient in transmission to watermelon; however, th:te insect

is not usual];r a prominent- feeder on tM.$ C'.l."Op.,

. This insect is found in all fields in t,hi.s &re~t e.nd often causes looses

46

47

with which they could be col.on:i.zed on watermelon plants in the labora­

tor.r.

tJhen possi"'ule, insects were kept caged in t,he labora..tor;t to p!'0Vtoint

accidental in:fest,atio:ns in greenhm1.ses. Insects were t.akcn into (;roen­

hou::;es only when used ln transmission t,rial~.

Diffe:rential crasshoppers were roarotl in the labo1~atory using the

:c1.et,hod of .Hnydak (21). l1'0:r1talos heavy wit,h eg~\s were collected in field

during early f-:tll and caged. Small contairters ·!.\.1:;;.h tl8d'll',i? ;:;a.:nc were

placed in t11e cag0s.. li'mJttlcs ovlpositeo. in t;10 sand an~;_ ci:'Led soon

af'·terward. Egg capsules were col'lecrood and plttce.:i in <.1a.H.P sand. at; room

t,e111poratu.res for about two weeks, and then stored in an icebox ~t a

few det·rees above fre,ezing for appro.:id.r,mtely one month. E.g;(;s i·Jere then

placed in cont,ai:.1ers at laboratory ternpor.;;,.tlU'es beneath a shallow 18;:ver

of (:amp sand unt.11 the young hoppe:rs emerged about three i-reel~s 1~:i,ter ..

Young hoppers ,:rere collecte0. and plf.iccc~ in ti, s:mll car,e in constant

lit)rt... A food 1nixtur0 consi.st,:i.ng of one part dri8d ba.kerts y1;)ast, two

pfl.rts dried skim nilk ru:16 t;wo perts dried alf oJ.i'a :meal wi1e kept in small

flat co:atainers in the cages at all t,i,;,ws.. Water was provided. by lay .....

ing large test tu.bes £rtO})pered with absorbent eot,ton on the floor of

ca.:j1s. liop:i;,ers grow rep:Ldly .lnei. matm•cd in e.bout thirty deys,

Systernically in:fected we.temelon plants served as virus source

plants in all tre.rwr-£ission trials. Youn6, heal thy watermelon plants

in vigorous gro .... 1:1..ng condition t:rere used as test plants.

Grasshoppers wo1·c stt:'it'ved approximately 21.i. hour-s previous to use

in tra:nsrllissior1 trials. 'th:l.s insured tha-t hoppers would .. feed readily

on test plants and po.ssibl;f increased the likelihood of" t,ransmission.

and

•

then tran;;;fcrrcd :Ln •

•

(

woro

OTJ c.ach_

fJl,rirtts .•

49

were held for about two wcelrn ·to allow an;;r inf\c,ctions to becO,ile sys­

te:mic in the plant. After t.i'd .. s period,, when virus would have l;i-tH.~n

present in sufficitmt quant,i

test plant~,; wt:re indc:xed on oucur:tbe.r plants. Cuca"\1.l,'er proved to be

t:he only rel:lable host for indexing wa'ber:m.elon plax1ts for- presence of

the tobacco ringspot, virus as cucu.mber W!lB apparently either :non ... mmsi­

tiw to the :tnhihitor in wa:te~n1elon foliagt:i (18) or more suscept:Lbl:e

to the ring5pot virus (31)_. Due to the OC(!asional mas1d.ng of infection

in water;ne1on, part,lcularly at warmer t.,11tperat,tll'BS;; symptons alone were

:not rel::Led upon to in.dic:ftte presence o:t vlras in thc.1 test plants!

the v1J:.''\rn with differential grasshoppers. This sinL,,le tr£1mmrlssi.on was

evidence that these insects im,cy· occasionally i1:ct fts vectors but, th~.t

their o.f.ficioncy is probably t,oo low to account for> the per-

centages of. infection in field plant:i.ngs,. Also, the relatively eiirl;T

spread of the disease ·the field and the nurrJoer of hoppers ordinarily

foJ.n.d in fields at this tirn.e do :not rtmem su.f:f:lcieu:t to i1;1plieate this

insect as an i:ntporta:nt vector.. l1'rom ·tohttcco to tobacco, lJaltierf:; ( )

reported only six percent transll1ission of the tobacco r'.i.ng:spot virus

vli. th this insuct.

Of the h5 ati;cm.pts to tra.,smi t t~he virus with melon a_phl.cts, all were

seeH to be a vector of

tobacco rin,;:spot v:'e.1:"<.m, Ht least rn:rl; ruroler the eirmllnstri::nces

t.lwse tests.

'.ro date., 11riU:.ers• (h8) data are the only .im,tance of insect trans-

vtrus ..

50

;for natural spread of the vlru{, are unknm·m. 'l'hese viruses do not

appear to be trunsmittecl to any extent by mechanical contact because

of their low concentration in host plants (42). In tho present stu<lies

not a si.ngle case of accidental transmission of the ringspot viru.s was

observed in the greenhou.se tests.

Sev"Cral mwJ.ccess.tul at,te.mpts at insect transmission of tobacco

r.ingspot virus have been l"el)orted. H:0,.,-ever:, existence of natural ve.c.­

tors is only circumstantial. Pound (33) obt,ai:n.ed oogativ-e results iiith

l{Yzus persicae Sulzer.,yet others have found cireu.m.stantial evidence

1,1tch points to this insect as a possfble vector.. S:rni th and Brierley

(u) have reported -on accid-ental infection of f;la.oiolus with tobaeeo

ringspot vll"US as explainable only i;,y the fact that plants had been

.exposed to i!• r..erslcae. Vallaau (46} believes that '.fhri.ps tabaci

Lindeman .may be a potenti.al vcotor but that it probably transmits the

virus in only a small percentage ot cases. He has .suggested that the

manner in which sul.fur du.stlng eontrol1S eggplant yellows, tobacco ring ...

spot on eggplan~, is probably eJ:plainable by the fact that sulfur acts

a.a a repellant. to 1'. t:.a.baci• :a veet,or of tobacco ringspot virus. ..... . ..

An attempt w:as made to hold field collected. twelve-spotted cucumber

boetles (Diabrot-ica u.ndecimpunctata howardi Barb.) at low temperatures

in an icebox for use in transmission trials as the insect is known to

overwinter as adul_t.s; however, all died be.fore transn1ission t.!"lals

could be :m.a.-0.e.

Seed trans.mission . trials

Tobacco ring;spct virus has been show to be seod tranami tted in

tobacco (44l, petunia (22) a.Tld soybean (ll). \Jith this fact in mind

51

i.t seemed possible that wat.err<Wlon seed :might. also herbor the 1;irus.

Seed were harvested during the summer from natu.ra.lly infected melons.

'l'hose melons t,R:,re all exhibiting numerou$ lesions uhet1 collected; however,,.

as an udo..i tional check a ar:iall ;µiecm of rind tissue fron each was frozen

for use later in reco1,-,ering the v.i.ru:s. 'fh:i..s woulc. provo that the melon

was infected.. This recovery o:f the virus was :made to cucu:mber du:riiig the

w::Lnter., Only the seed of fruits fror.1 which the virus was recovered were

used in these tests.

During winter mont,hs se0d from infected melons was planted in flats

in the greenhouse.. All restu t:i.ng seedlings 1:rere obs-0rved. dur"'lug the

first several weeks of grotnh. and aU. those apr:JOaring abnormal or stunted

were used to inoculate cucuaber. 'lb:is manner of ind-exi.J1.g syste:.Il.cally

infected wate:rmelon pl,9.:rrts hy tranii.1rlitt:Lng t,be virus to cacumber was

fow1ci rzl ta·ble in othe:r tests where symptoms were .:nasked. in watarrimlon.

Ap91,o:icimately 900 seedlings wore gro-wn from t-::1e set.'d of inXeo'tod melons

ru.1d. suspected plants ·m~re indexed.. Nona o.f these seedlings proved to

be infected..

As tllo above method sem,ied somewhat inadequate because of the like­

lihood of symptoms being entirely masked in seed..lin;:;s, more seed from

infected tr!lits were pla"lted and all resultir:g seedlings inc:iexed on cu­

cumber. Approxi'fi:1atel.y 300 seedlings were indexed in t.his manner w:i.th

negative :tiesul.ts.

~:evcral thousand w:atermolon seedli:ngs were used in othc'.?r phases of

thts investigation. 'lhene seedlings Tu>·i,re from certifi€ld seed .from.

growers in Oklahoma.. Du.ring harvest. of th:Ls seed many infected m.elons

are used. Several fields of' certified seed growers wt~re visited durii.1g

harvest and a h.tgh incidence of ir1fection was noteci. on the .mature fruits •.

Hence, many of the ecrt.::i.:fied seed iised in this study were undoubtedly

.?ro.n infected melons, ytJ"t., in no ini.;ta'noe were seodliri,gs fouuo. infected

during th~ ereeni:louse studies unless inoculated.

Wv.tcr:melon seed a1)parently do not. trans:m.:U:. the virus or at least

have not been demonstrated to do so with the exper-"J.mmtal procedures

used in t!r~s investigation.

ov [

malies sugges'beti eit,her that an :i.1ihH:rltor of in.fection (4) was present,

or that the vl.I"t:iS was not systf-.:mic in f oUau) and pre.sent in only low

cowpen. LE,ter., :Lnoculations to tobacco from melon foliage rcsU:lJ:;ed in

trv,nsm:tssion in onl:f a £aw instances. Transmission of 'the virus from

tob,icco to cou1x1a usually rosul ted in abundant infec t:3.on. As a conse-

quence of the results Jtff.,t out,l:c.ned an tnvesti,r:c:atio:n was init.tat.ed to

Initial tests for inhibition were made by comparing the virus

.:icti vi t;1 of infec t:l. vc tobacco stips niixed wit.h dist:lllec. water on one

:,erve as control; sap from the srune source was diluted oqnally with

One tobacco control

53

54

.solution and tho other h2J.f•lo~f :',noculated wi.t..11 t.he tobacco sap-foliage

sap mixture. Several leaves wc,re inocul2,tecl. in this ma.nner in each o!

sever~l tests. In every case 1nan;r loslons R<;ip•.::ared on the control ho.lf­

le:.wes, while in no instance cid a s:l.nr;le lesion result on the half'•

leaves inoculated wi t,h t,hc prept:"iration. co:ntaininG '\"Jatermclon foliage· sap.

'£hus, it beca111e apparent th::1t melon foU.ar;c sap did contain an inhibitor

and cou.ld completely der:troj' nctivlty in a ,rery infective prep[;ration

of t.he virus. A sit1.ilnr b11t lctis marked inro.bitive action ~s .found

with sap from 1mteri:1clon fruits.

~ubsoqu.ent tests 'kKJ.re st.andaruized in order that more reliable

estinmtes of degro~ of inhibi·tion could be had. ?riuar-y leaves of cow­

pea, variety Black,. were used t.o assay prepBN!.t,ions ccntaini.ng t,he to­

l,acco riJ:11_1spot. virus or cacurnber mosaic v:Lru.s, and l\Sie-etiana fl'.lt~

14 wrts ui;:ed to ass&y prepa.rat:1.ons cont,ainLng tobacco mosaic virus. In

all tests equivalent preps.rations were cor.1pared with a stande.rd inoculum

cons:lst1.ng oi sap from systemically infectftd Havana toba.c(;o mix~d with

eqm:-:.1 :?iit101mts of ci..atillod watser. Virus activities of all te.st prep:.ira­

tiono were col.-.1pared ·with this cont,rol by· the half-leaf r:ieth.od. Inocu­

lations were made With a glass spatula which v.i;,1.s ·washE:d and fla11ed in

alcohol before uoo w:l. th each different te;:,t, inoculu.rn. This spatula. was

pass0d twice across each half-loaf prioviou.sly du.st0d as nverJ.y as pos ...

si.ble -wtt.h cn.rl:>orundum; hen.ce, each half-leaf received approx:i.nw.tely

the sa."11.e amount of inocU:lum. .Ji small pad of eot ton was held wrie;r t,he

leaf during rubbing to serve as supi)Ort s._nct absorb excess inoculmn.

1he effect of dilution upon watert~lon foliage sap was studied in

att.o:11pts to eve:luete I!lore acm.tt'ately the de1;.reo of inhibition.. Vnrious

dilutions of tollnge sap were made with distilled water 111 ·M1e same

:manner as when st-u.dying dilu. tio:n-end-point of the virus in the physical

property tests. k'l aliquot oi' ea.ch of these dillttions we.s then :r{d.xcd

Ni th equal axaou.nts of' infoc-t.i ve ·tobacco sap and c.or\"ro'ed with the con­

trol by inocalations of hulf-leaves. Sirnila.r tests were m.ade using

expressec:1. saps £r0111 the rind areas of waterm.elon ±'rui ts. Sample data

are given in Table 3 and Fig. 7. 'I'bree of these tests -were made; all

gave sinilar results.

Dilution-enc.'l.-point. of the irihi.1:xi:t:i.ve action of foliage sap was

between 10-2 and 10•3. Inhibition was, in all cases, still signi.fi­

cant at the 10-2 dilution. Similar results were had with expressed

sap from. waternelon .fruits; ho1,ie·ver, d.ilution-end-point was approxi­

mately t,on-fold lower. The inhibitor may have been present in lower

concentration in t.he t:ratecy fruit. Tohiht t,i vo action of fruit sap

was nuver sufficient to neu.tralize corn.pletely the h.ighly active virus

preparation fra..1 ·tobt>.coo.

\Jc.tt-0:rn1elon. foliage saps 1-qore found. si:failarly to inhibit infect.ion

m. th tobacco mosaic vlr-J.s preparations in transmissions to Nicotiana

glutinosa. Althoue}1 tho effect of dilution was not. st,ucli.ed., undiluted

foliae-e sa:p 1nixed w.i. th an equal volume of virus solution were found to

give 3.3.3 lesions ~r half-leaf as compared to 2h4.7 lesions per half­

leaf inoculated ·h1i th a mixt,ure of equal amo-unts of virtm soh.itton and

wu.ter. '.l.'his am.ov.nted to a reduction of 89. 7%. In no instance wns the

1rir-a.s ~~ct:i.:vit;:ir cortpletely neutralized., hou-ever, as it was with ringspot

-vi..rus. '11his rt'1,;;y he.vo been due ·to the unusw:> .. lly higher concentration

t.hat tobacco mosaic virus roaches in tobacco ss conp.::1-retl wlth the ring­

spot viruses. l!Jntor.;1.elon f olia.ge sap was si:milarly f'ou:nd to inhib;i:t

56

transrn.isslon of cucun1ber mosaic virus to cciWpea.. In this case inhibi­

tion SE!Oi't:.ed complete 1ihcn un.d:i.luted foliaEe sap ws 1;'.,ixed w:1. th equal

a."1.-om1tu of cucu:.~eor mosaic in tobacco sap ns no lesions Wt.',re · proGv.cod

on any of' the half-lemres inocu1;;.terl with tM.s miY:.ture. The ef'fll:JCt of

d:Uu.tion w2:s not st1ldied in this case.

\he 1.nb1bi tor contained in watermelon fol.i.nee was found to be des­

troyed by hoat:i..ng: the sap to ao0c for 10 m.inutHs. The inhib:l.tive action

of snp WHS pc'irt.ially dostroyed after heat:in3 at 75°C for a simi..lDX period;

however, L:,sion co·:.L-qts showed appro:rlma:t;ely 50,Z:; reduction eepacity still

rmnainetl tn the i'oliag;e juice.. Similar ef,:E,ets of heat arc imown with

ot::Hr in.hi bi tors ( 29 ,h:9) and Su,[{:7.GS'& that thene may be of a proteinaceous

n~ture i;imilar to t,he glycop:eotein isofatcd and described by Kassan:i.s and

Kleczkowski (28) from Phytolacca ,!:_Sculenta.

The inhib:i. tor frorn water.,1elon was f oune ineff'ccti ve in preventing

transmissions of tobacco r:i.nf~spot virus to cucumber. Gendron and Kassanis

( 13) f o,;J'ld infection of cucu.r.1ber less affected than any of several other

species tried when imresti0,-ating the im:-,ortance of host species to wh:Lch

·transmit.sion WD.S attEu1pte<l in pl'('H3f,nce of various inhibit.ors of infection

fron hlgher plan:ts. Bawden (h) has stated that an obvious GX~lanation

w-culc! be thnt cn:cu.mher also contf>.ina the inrdb:'i.tors, as :i:.t is c0i11monly

know-n that inh:ibtt,ors are ineffective in preYent,ing infection 011 the sar;ie

species frofi1 which the irmibitor. coma. How-erver, it Plft.Y l2e that cucwnber

:i.s simply more scnsj_t,ive to infection hy tobacco r:lngsr:,ot. virus. HoKinney

&nd Clayton ( 31) be-1:tev-e cucumber to he more susceptible than tobacco to

the r~l.ngspot v:Lrus.

Cu.cumber proved to be the only reliable h.ost f'or indexing watermelon

plants for infection 1:iith the ri:ngspot virus :ln these invostii:;e,tions.

'l'nblo 3. Zi'fuct 6£ <l:i.lution on the :lnh:'1.hitive action oi: wot.ermelon sap before mixing 1:1 'With sap o.f tohacc:o infected t·rl.th the rinf;spot virus and comi,firinc -with a. ~.t.nncia:rd :tnocul.11,nl by hru.f.-1(:'J;;_f inoculations o:n cowpea..

b'aterm.olon foliage sap:

l.ind.ilu:ted ••.•• S tanda:rd 5.noculura

. .. •· -~ ...... .. .

.. . . " ·• ... ..... 1:10 dilut,io:n • • • • • .. Stanuard inoculum .....

1.: 100 dilution ••• • • • •

.. .

....... ·• . . •· .... .. . ... •· .... -·

1:1000 dJ.lution • • St.anciard inoculm11 • • • . •· ·• ......

,_:;,. t"' ,...,..~,,, 1 ·0<1 -rr»-l t <' n '). ~~ ~ -ti.1.$,. U.h,J-=.. .. -- ~- . µ~J.'. ~

Und:Uuted ...... .stanc~ard 1noculum

1: 10 dilution .. •

•. •· .. . ........ ·• . ... . . .. , .. ·• ....

....... .. .. .. . .. ... ......... ... . . ... 1:100 dilu'l:,icn ... .,. , ... ·• ... ·• .. S ta:ruiard inoculur.1. • ......... . ·• .

Averai;e lesions per half-le~£ in 6 replications

No.

o.o 65.0

o.J 62.2

2.3 16.5

54.8 39.8

J .. 2 ,2.9

3~2 12.J.

170 .. 9 l79.1

Reduction hy

sap

Per cent

100.0

86.1

o.o

95.9

73.6

o.o

ls~·,-,n· (''"r ' i u1 . . .Ii,~" ~ •.• 1 -4' . 0:-~ ·1"" u _noc mn cons:i.Bv;::~ 0.1. ss,p o:r toi)acco iu.i:'ecterl ·with the

ringspot vlrus mixed l;l witb distilled water.

58

A B C D

ig. 7. Eff .ct of 'lution on t e inhibitor of infection fr wat~r­elon f olia ... e in preventin infection with the t obacco r in s pot · r., on cowvea. All left hal f - l eave"' inoculate' itt "'tan

inoculum consL,tin& of sap of infect tobacco 1nixo 1:1 with ater . All r:i.- ht half-1 a-\les inoculated 'th sa.p o ' nfectcd

tobacco mixe 1:1 with watermelon folia esp f ter ilution: A. - Un il ted, B. - 1:10, c. - 1:100, D. - 1:1000.

The present :I.nv,zst,iLation has dern.onstraf.:it)d the coiw1on occurrence

of the tobacco ringspot v:irus in assoctation with t,he pinple riisaase of

watermelon in Oklal:1cma. The ident,1:f'ication of this virus from wat.ormelon.

substantiel.tes 'tlm work .oi' Pound (33) and Rosberg (.37) and prov:i.des fur-

ther evld.ence on the w:L<iespread occun~ence of the tobacco ringspot virus

on we.teraelon.

Several viruses causing rine;spot.-like diseases on tobacco are k;;1m:rn

and arc usually tHstinguisha1)1e on the basis of host ranf;e and physical

-p·rO"'.J.'""'r·t~,, sfndi E""~ ( "'6 112). r:iowever ... Jp some eases, more sponifi.c ,nr,thods " .f _ ...... .~ """· v.,,._ ··-" ,, '°" L _ J. ---,- - . f _ ~ .., . - J:j _ '-" .o...;,

some of tl'.:e viruses wi tbin tho r:Lni:;spot group ( 2).

it would

seem t,hf,t some ho~,t oth:sr thc1n water.11,.:lon is acting as cJ, ri?;servoir from

tm insect vector or vectors. Several possible m:i:t.u.ra1 hosts occur ubun-

dantly in and ~u·ou.nd Oklahc.na watermelon f:lelds. Ai,1ong t.herJe sweet

clover (13,.23)., J:1.mson weed (2h)i and horse :ne'l:,tle (2~,4l.i) have been

.founci naturally l:r1fected 1tl'th the tobacco r:5.ngspot vi.rus.. l~h:i.le soy-

bean (1) has been demonstrated as a co:r,1x:1on host for this virus it seems

unlikely that it serves as a virus ret,ervo:'Lr in Olrlahoma beca:use ot the

limited acreage of soybeans and. the fiic-t, tl:1at tho t't'JO crops are usually

:not grown :ln the samo areas., In the pres~n::ct :l.n:vestiga:l:.ion t!J!'1! r:Lngs;;ot

v:Lrus has not been demonstrated. .f1s ocmll'ril.'1g in nature in tests of sev-

eral possiblE:1 weed hosts. S:tnce these tests were l:im:i.ted and s:tnce

$Ba

di.f.ficultios were encountered in handllnis t.he virus., the present results

do not preclude the possibility that the vtrus does occur co!ltit0nly in

sorrie host other than t<m.termolon in Oklahoma .•

Only differential ttrasshoppers (i.i.8) and the green peach e.phid (.39)

have been repo~s.d as vectors for the tobacco ringspot virus. In the

present study the diff,erE:mtia.l grasshopper was found to trans:r11it this

virus .from watermelon to waterr;ie.lon in only <me of 48 trials. :Few of

the insects com1;1only encountered in Oklnhoma. wa.terfaelon plantings have

been tried as vectors. Halon aJ;Jtdd,s l'lBI'e tried in the present work with

neg;utive results. An insect wh:i.eh appears as a likely suspect is t,he

twelve-spotted cucumber heetle (Diabrotica undecimpu.nctata howardi 13~rb.} -------·--·-·-· . .

At this time the vector or vectors responsible £or introducing the virus

to waterraelon and responsible for spread from watermelon to waterI11elon

remains unknm-m.

The evicie:nce from present work is that th.a virus is not carried in

watermelon seed. Since, hot:1evor, only a ;te.w thousar!ri seed ·were tested

th:1.s evidence ls not conelusi ve. 'loo problem remalns tnen to explain

the wi.despreaci., un:i.f orm occurrenCfj of tMs disease in Oklahoma ,Jatermelons.

1'he only reasonable explanation see1,1s to be that a rather cod::1on host is

servlnc; as a Virus source and that one or nmre insects are involved as

vector~.

It shouJd be pointod out that the extent. a.nu thoroughness of the

st.ucLi.es presented have been limited by two 1:mµortru-.rt factors.. 'l'e.1nperature

has been a limiting factor ·throughout 'these stud:les. At Jo0 c and above

it is dii'ficult or impossible to obtain symptom expression with tob.Uceo

ringspot virus on the hosts t,ried. t.Jhet,hE;;r the effect of ter;1pera-ture ts

60

'l'he viru.s has

bt~en rccovorecl in a J\,w Instances :;.rom :1.:noculated hosts showing no syr,1p ...

toms at. high temperature. 'Int) other p:c::Lncifk'll liuiting factor has been

and fruits of an i:nhibit,or oz :Ln:tec-

t.ion. 1he demonstration here of thie inhi.b:i:t.or com:rl::.i tu:tes t.h€ firG;;t

known report .of wat,ernelon sap having thi.s propert,y .•

'Iha tobacGo r:tngspot virus

as tlcsertboc. herein, doos not

:t10 ;y1.eln ars t:rvai ·tb uhi c l1 to losses.. Al.Bo wor·tlry

v1ull occur ui th

cooler seasons in the.c:c sout.l:wrn areas as greenhouse tests hai1t'l shovm the

v:Lrus capable of caus:Lnl; let,hal infections on watermelon at cooleir tem­

peratures.

t:toned thEt due to the i:ndistingtr:Lshabl~ nfl:ture of the fruit lesions

infected fr"J.it,s are (;ulled by insp,jctors at rail std.pp:Lng pr1:Lnts ..

Anthracnosc is kr~own to be a::::;t:,ociated w:Lth a rap:i.,i;l deca_y of i.I'li.lt.s.

Fer t)t:'LS reason, all melons showing t,be ,.n~ry sir.ulr,r virus lesicns are

SUN:ciARY

A mechanically transmitted vi:nw was fouml constantly associated.

with a disease of Black Diamond wa.te:rm.elons in. Oklah:or:ia.. Th:ts virus

was ident.i.fied as a yellow strain of the, tobacco ringspot virus through

a study of symptomatology, host. range, phys"ical properties, cross-irm;nmi ...

zation, and scrolOGY•

Uost ranee and syn.ptoH1atology ivere, studied and described on water-

1nelon., tobacco, c:ucu.rri:ber;; bean., :petunia., end cowpea in addition 'to sev•

eral othor plants. Ph,/sieal properties as determ.ined on cuemllbor were

as follows: thermal inae·tivation, 10 minutes at 70°c; clilu.tiou, l to

100,000; lontl'lvity 1::! vit-ro, 8 days at 20°c. The watermelon virus

e:ffecti vely protect1-.1d tobacco plants against subsequent infection with

tobacco rine;spot vlrus but not against tobacco :mosaic viru.s or cucut~1r)er

mosaic vlrus. Confirmation of the identifieation of the wa:terr<1olon virus

as tobacco ringspot was obtained through precipi t1.n t-est,s .•

The ooture and development or the v.i.rus induced lesions on Black

Diamond watermelon fruits was investigated. Histolog$Lcally these lesions

were found to 1::ie gall-like.. Lesions. were alwa:1i"s in the mesocarp region

of the fruit and were compos~d of necrotic tissue with au area of hyper­

troph.1.ed eolls i,,"ll.mediatel;v oeneatl-1. ~the hypertrophy appeared as a re­

sponse to necrosis.

Attora>.;:>ts to tI·an&1u.t the tcibacco ringspot vtrus fron watermelon to

water.melon with differential grasshoppers or :melon aphids f'ai.led e:.;:cept

in o:ne ·trial in :;,1h5.ch grasshoppers successfully tra.11sr.1itted the virus.

'fransm:i.ssi.on of tobacco ri.ngspot vlrus through watermelon soed.s wa.a not

61

62

An :i.nl1~i..b:1.tor of vir;:1s infection wr:is demonstratE)d in watermelon

Infection w:;,s 5.nhi.bited by sap from watermelon

cowpea and with tobacco r,lo::;aic v:Lrus on i:licot,5-ana glut:Lnosa.

10 minutes., and b;r c:ilu-tion, l to 1,000.

1. Allinc;ton, H. J. 19Li.6. B~,idbli;;ht of so;ioean cauned by t.he tobacco ;.,;1 __ -.y-1,·r, __ ·,.',,"';',¥'_··,'O,l'~,~,--·_w_>' •6 ! ".l. l:7 _3:::..-:;. • rj .. 1] .. g-s:;Jot ,r:Lrue • .1"- \•_.,~u,~J." -..~?.ii J .. .J,,,. .,,,.c.

2. Anderson, c. 'd. 199.i. aster ringr:;pot vJ.ros:is of central :b'lor:i.da.

3.

P11,.yt,('>part,t10·1or;J:· 4l~.: l;?-92.

±3;d.:efJ0r .. , rtc1~te ii. certo.:1.n. s11:-Jc:l.es

1909. C crn1parati ve of Gucurb:i. taceae.

hi. si,ology of i'rui ts and Dot. Gaz .. 1/l: 261-310.

seeds

ti. Dawden, F. c. 195h. I:nhJ.bit,ors and µlant vtruses. Adv. in V:lrus Res. 2: 31-57.

~;. :3erk;.;;-ley, G. H. l9:,3. So;,JH vir.,J.sos affect:'i.n.g gladiolus. 2tyto­pat.hology :~3: 11.1-11$.

6. Dest, n. J. 1954. Gross protection st.rains of tomato s1Joti:,f;6.

of

wilt, virns and. a .new tht)ory to e:x-pla:il1 it.. kust. Jo1...:e. B:l<Jl.. •

~,. I •

7: t-15-J-i2)4.

Br:tc.;ymn, C. roservoir.

H., and J. C. l-S.:1tlker. 19S2. J?L·y~-(;<J~oa:t~Ltolot,;' h2:, 65-70.

Cheo • .D. G., and. ti. J .• za,w1eyor. 1952. rinF.-SDot isclat,eo. from. bean. u. S. 36: -Li59-tih:; • .

Glaci.iolus as a virus

A nsv¥ stra::.n of tobacco At;r .. :r,1. I'.:ls. Rept.r.

9. Ch~st(?r, K .. S. 1934. S9ec:Lfic quarrt:Lt.,rtivc m:rntral:Lu.ition of the v:i.ruses of tobacco .imse.ic, tobacco ri:nr; spot, and cuomaber 1,1osa:i.c by immune sera.. Phytopatholo;s:r 2b neo-1202.

10. • 1937. ~;:orolog:tcal studies or 1Jlant v:trnses. Pi:wto--pathology 27: 903-912.

11. I:esj<irciino, ? .. R., R. L. Latterrd.l, and ,J. K. H'i.-tchell. 195k. S0ed transr;•insion oi' i;obacco ringspot in Li:ncoln varJ.ety oi soybEiari. Phytopatholor_;:-r L~: 86.

12. Doolittle, S. P. 1920. :B:1t: mosaic d:l.seaGe of cucurb:tts. u. s. rcept .. Agr .. Bu.I. 8?9.

lJ.. Fenne_, [j. D. 19.31. Field i;;tudies on the ring-spot, d:l.see.ce of Durley tobacco in U'ashi.:.i.gton County, Virg:inia. 1-'hytopat,holq:_;y 21: 891 .... e99.

Fos'lk1r, A. s.. 193/.i. 1lhe 1.i.~3e of tawric erl,ai.P..ing cell walls in I11eristemutic tis;:;ue.

auc;_ iron cbloricie for Stain '.Coch .. 10: 91-92.

l''ulton, H.. vJ. l.9h9. V1r;;,s concentration in p1r .. ntti acqtdring t.ol,;;;.r­ance ho tobacco streak. Phytopatl1olot;f 39: 231 ... 2'-3.

63

16.

17.

]/1.

64

F'ulton., J. sp:inach.

p. 19$0. StudiE,s on strains of cucumber v:Lrus l from Phyt,opatholory 729-736.

(;ardm1r, H. VJ. tomato fru.its.

1925. ,Jour.

1:ie ores.is, A(~~I'1J... Iit?!·S·

s~peci.,2~s ir1 dt;·t,cr!_:1-i.r11.r1(r Biol.. !~.l: 1B3-11l9.

hyperpla,:;;i,o;. and adhesicms in f/lOSttlc

(tl. s.) 30: 871-888.

'fhe tmz;ortance of tbe host

19;.. !}idd.ir1r;s, ;,,s. ,J·. i95(). t~,.tlf'.&r,.,..1):~ct c;1}rlJ' to.p.

sorn·.a 'int,errela.tionships of virus stra:Lns in to: 377 ... 31m.

20. Gilbert,, u .. "•, anc E .. ilrtschwn[;;er.. 192;,',, r;at;,,:n"L101.on internal 'brc.wnin\. Phyt.opath.olo:y 15: 119-121.

21. Haycak., i,z. ff. 19;.,.,2. r::rasshoppers uncmr laboratory cQnditions.

22.

23.

f:ci.enco 657-658.

Henderson, p,:r:;ttm:i.a.

H .• Ct. 1-931... '_f1,.a:nsrn:t.ss:Lor1 Pl1,ytopatholc1cy 21: 22~; .. 2~?:9.

tobacco

• in--s-w-·e-,,-;-.t_c_l_o_V_'r:_,,r. 193h. Occurrence oi' tobact:o ring-spot-like viruses

Ph;rtopatholce;y 2i.n 2h8-256.

,._ tiJ:1C. .S • ---r---~~_,, .... .,__.,"" .

ring spot in Vir;::::1.11ia. A.-.. lJJj:J~lftarrl.,

,Jour. Affr. 1931.. li'urt,hcr studies on tobacco

i:E,s. (u. s.) h3: 191-207.

25. Johnson, }~. 11. 19.30.. Virus diseasos oi' tobacco :ln Kentucky. Ky. Arr. Kx:pt. Sta. Dul. 306.·

26. , a.nd w., n. Val.lea.u. -a.2;:.easci's. cit··-plant~;. Ky. A{;J.~ •.

,Jones, s. E. 19h2. Cmrt.rol of $ta. Bul. 623.

2e. Ktu,t:o.nis, 13., r.nd A. Klect1l:ww1,k:L. 1948. l'ho isolation and propo:rties of a virus-:tnh::.'bit,ing :pt·ot,ein fron Phytolae:ca escu.1,mta. Sour. G~n .. rii.crcbi.o·lc. 2.:: l-.h2~J~53. '., C" ~

JO.

31.

Kuntz, ,l. 15;., and l .. c. 19h?. Vir~rn inS.ibi tion by ex"l",racts 561-579 .. spinach. Phyt,ovathf)lcgy 37:

lie Kinney, tiss•le.

H.. II. 191+7. $ tfl:bili'l~y of flt.y-t.opatho1oey .37 :. 139-1h2 ..

, and E. E. Clayton. ~,----,--~..--,-,~~ in tbe tobacco ririe;-spot disease •

.32. Peterson., Alvah. 1953. A rramml of £~ntomological 'I'ecb.1u.ques. 7th B:d.

3J. Poii:nd, G .. S. 194_9. A v:Lrus m.sca.se of wa:termelc:n :tn Uisco.nsi:n incited by ·i~ho t,obacco ri:n.gsr,ot v:irus. ,Jour .• Agr .. E0H3 .. (U •. S,.) 78: 6).~7-656.

36.

JD.

65

Pr:i,:e, W. G. 19J2. Acqulrf,d. ii,nunity to rinr3s9ot in Nicotiana .. In:st. Pl. Hes. ti: 3~;9 ... J-i.03. Sot."st,r1."b. L~cyct?

• 1936 • concent:rfct:t,:;n rclati:m to a<;qt:;;:.red s,,)ot. Pz1srt~;:rpaU1olo:!'tT 26: )GJ-~;29. _ ti - ~u ,

Hcc,r,.u:rr;,; j,,,I:rm1:t ty fron tobacco .. ~26: {S6:J-(/fS.

:1!t.Jr~Jl)t:~r·r;.;, Ii., spot v:I:rus rn:pt. !L ·r.

t,J. l?:-:;3. _:{: [30c:LfJ.tJ·i·on ol" t't strciin of tubae co r:ttlg_-wi.tti p.ir1tJl~1s o·l.· vz-atc:rr;1ol.on 2 .. 11 1\1xtts.. tr. &. .PJ. .i:-:2..~) .• rt~~·:pt.r. 37 -~ 392-396.

;:)ass, r1;J •. r,. l.S',.5.1 .• Press., A:,1es. PP• 228.

Aphic. t,ransmission cf tobticco r:ts. 1~eptr .• 39·: 35 •.

li(J. tt1itl1, r:. i'I. 1933. 'i}1e l)r'&f;e:n't r .. rt,at;us cf I1lrrn,t ~-.t:Lrt1~:; r~es<.~;;1.~c11.

48 .•

49.

$1.

Biol. Hr,v. o:l:' Prot~. Gm;lbric\:e Phil. Soc. t1: 136-178.-

S11ith, K. 2nd Ed.

::,1., 1951. lhcent, iu;;,1:;<.:rlcEJS in Blak:tston Co • ., I11iladelphia.

t"lai1t; 11'lrus E~csei1rct1. PP• JDO.

• ';O\H~;f;,O lll:fiClt-r-luf\! 0. l1.Gt'1 ~vit'l1S d:Lsea[30 • ?rnsZtolor:Y 37: 126-130.

St..a)-1],.ey, T,J,. tf .. :tro:M tolJe,cco :r~

1939. spot disease.

o~t ·v·i:rlis /:ro1n r,l:1r1ts 1:<ecovr2l"l'.~ti Jour. Biol. Ctte:11. 129: 1i29-h36 ..

Vallea,J.., t·J .. I:.. 1932. ;.',eed tra.nni,d.st;;:ton s;11d ster:L1:L't;{ studj_(,s of t\'w stra:L.1s oz tobc.cr.o r:1.r111;spot.. Ky .. A;r. Lxp .. Lit;,. Bul. 327: 1-80.

• 191;1. 2xp:::rinrmta1 -c&llecl recovi;>r0r, r:Ln.g-s:;-;ot tobacco

e,cquirf,6- L-r1;1uni ty.. Pl\rtopai;hology

prciiuct,ion of synpto:i:ls in and its bc,aring on

31: 522-533.

• _____ ,..,,,..,,,,..,•-··--11i..E· n~~ ~tf;.L.LOl•:jl:1,.

19.'.Jl. '/'ohacco r:Ln::,-spnt Yirns: Je}·1;/t10l)~tt.tolot:1r hl: 2t)~)-f212.

1iialkcr, J. c • ., J,. J. Lc1:!eau, <.1:LHtcci w-1.th c,:;.hba;_f) rriosaic.

t,.rr~ci C}., S •. f'utin(i. SoLtr. A";r. R6s.

l;,liS .. (U. S.)

Viruses eic:;6o-

70t 379-i:tOh.

,1,sTLc:cG., n. J. 19$1. ;~cisnce 113: 36-37 •

~~e:i.ntra1:1.b, i-1., ::1ri.c ,J. JJ. G:UpGtr:lck. 19;52. An l1ost, of to1)t:.<::co 1"'ir{gs1"io-~ v·5 .. 1~u:.:i. Gar1 •. Jo1.xr. Dcrt~.

1951.t. pp. 239.

t.or in a now 30~ Sh9-:,57.

W:ingard, s. A. 192f1.. Hosts and sympt,or,,:J of ring spot,, a virus disoase of plant;:;. Jour. A;:r. Ron. (U. S.) 37: 127-153.

Hohort Jar.1os Shepherd earl.did.ate :for the dotree oi'

!},faster of t,cienue

ItLoc~aphieal and Other Items:

Born: ,June 5, 19.30 at Clinton, Oklahomai

Undergr•ldxmte Study: thrbrorsity of Oklahoma., 1948-1950; o.1t.u.o ., 1952-19,h

l\Icnbcr of S":.s,;ma, 'I'hfJ OkldKr:1a Actu:k1m,y of Sc:Le:nce, Arlerican PlJytopath(:ilo;Jc<':l Society., a~1d Associate I~mbor of t.l1e Sod.et;v of S:t2~111a Xi.

Aw--"'ust · -l.9· r~r.;:."' CJ , .,,,,

66

67

AO'.i.'HOll: Robort ,J. Shepherd

THESIS AffVISim: Dr. F. Ben Struble

The content and form have been checked and approved by the author and thesis achr.i.ser. 'i'he Craduo.te School Office assum.es no responsi­bility for errors either in form or content. The copies are sent to the bindery just . as they a.re approved by the author and i"acul t.y adviser.

'l'Y PIS'l': Bernice Winter