In d ia n P h y to p a th . edcbaZYXWVUTSRQPONMLKJIHGFEDCBA 5 6 (2 ) : 1 7 4 -1 7 9 (2 0 0 3 ) Natural occurrence of a yellow m osaic' disease on zucchini in India caused by a Potyvirus S .J. S IN G H , I.A . R .I. RAJ VERMA, R e g io n a l ABSTRACT: S ta tio n , A p o tyviru s P u n e , In d ia . T h e viru s co m m e rcia l fie ld s Y .S . A H LA W A T l, A g r ic u ltu r a l ca u sin g was m o ttle d , b y m e ch a n ica l sa p in o cu la tio n . T h e iso la te R e yn a n d a p re p a ra tio n s m e a su rin g a n tise ru m re ve a le d of th e T h e in fe cte d of pepo E le ctro n lo n g , m o s a ic (Z Y M V ). v ir u s a n d p a rticle m o rp h o lo g y in the w orld. It w as first reported et a l., and F rance in 1981 (Lecoq 1981) and subsequently (Lecoq et Lesem ann a l., 1983; e t a l., 1986; N am eth, fro m fro m o b se rva tio n s n o t e n ve lo p e d th ro u g h O n th e a n d b lo tch in g to th e m e m b e rs u su a lly b a sis of by M y z u s fle xu o u s re a cte d clo se ly and p e r s ic a e and viru s h o st p u rifie d p a rticle s stro n g ly sym p to m a to lo g y, iso la te w a s fo u n d o f fa m ily C o ste a n d e m e r e n tic o to r o f th e le a f d ip E L IS A , th e viru s w ith ra n g e , re la te d on tra n sm itte d th e a p h id to Z Y M V . and Y ilm az, e t a l., 1989; U llm an e t a l., 1986; W ickizer 1986; C rosslin et et.al., 1984; 1984; et aI., et aI., 1987; D avis a l., 1987; S am m ons 1991; W ong and Lee 1992; S tobbs et aI., 1990; G racia, 2000; P rieto e t 2001). B ecause of its aggressiveness virulence, Z Y M V is considered a P otyvirus serologically related to Z Y M V in zucchini crop in India. in Italy 1981, 1994; Lisa 1984; P row identi and M izuki, 1987; G reber of from other countries D avis H seu et aI., 1985; D avis, a l., id e n tifie d L .) co lle cte d le a ve s. T h e viru s w a s re a d ily L . T h e viru s w a s tra n sm itte d th e viru s Z ucchini yellow m osaic virus (Z Y M V ) is one of et a l., pepo P o tyviru s, a p h id th e m o s t a g g re s s iv e a n d d e s tru c tiv e v iru s e s squash ( C u c u r b ita w a s m a in ly co n fin e d m icro sco p ic te sts sq u a sh w a s re ce n tly o f zu cch in i re sp o n se o n C h e n o p o d iu m fila m e n to u s, n m . In se ro lo g ica l and R .P . P A N T 1 411 0 0 5 Pune p la n ts sh o w e d m o sa ic, ve in b a n d in g lo ca l le sio n s o n C u c u r b ita PRAKASH d a m a g e to zu cch in i sh a p e d b liste rs a n d filifo rm m a n n e r. p re se n ce y e llo w se ro lo g y S h iv a jin a g a r , le a ve s a n d fru its ch lo ro tic m o sa ic 700-750 Z u c c h in i tra n sm issib ility, K e y w o rd s: 2001. in a n o n -p e rsiste n t b e tw e e n P .o . T h e h o st ra n g e o f th e viru s in cite d se ve re syste m ic g o s s y p ii fro m irre g u la rly cu cu rb ita ce a e . R .K . S IN G H , S A T Y A E s ta te , se ve re e co n o m ic iso la te d n e a r P u n e d u rin g le a ve s a n d p ro d u ce d A p h is C o lle g e and a serious threat to M A T E R IA LS S a m p le co lle ctio n D uring survey of com m ercial of zucchini w ere representing collected enzym e linked im m unosorbent by usual m echanical of potyvirus group that causes sym ptom s resem bling preparations present com m unication (W ebb 1984). et a l., (C . p e p o ) a w ide C ollected assay (D A S -E LlS A ) by the m ethod of C lark and A dam (1977) and also those incited by P a p a y a r in g s p o t v ir u s (P R S V ) and e t a l., plants range of sym ptom s from m ild to severe. infected to W M V -2 from sam ples w ere tested by double antibody sandw ich- hosts. 1965; A dlrej, 1969; P urcifull fields in P une in 2001, leaf and fruit sam ples cucurbit production w orld w ide. Z Y M V is a m em ber is related serologically AND M ETHODS inoculation Im m uno-electron sam ples w as as onto herbaceous m icroscopy w ell as perform ed by (IS E M ) from the of purified m ethod of A hlaw at and V arm a (1997). In the w e report the presence of V iru s iso la tio n , h o st ra n g e a n d p h ysica l p ro p e rtie s 1 D ivisio n o f P la n t P a th o lo g y, In d ia n R e se a rch In stitu te , N e w D e lh i 1 1 0 0 1 2 A g ricu ltu ra l T he virus isolate w as transm itted m echanically f.Q f host range studies and routine m aintenance of [V o l. 5 6 (2 ) In d ia n : 2003] c u ltu re s . S a p in o c u la tio n s w e re d o n e b y tritu ra tin g le a f tis s u e s w ith 0 .0 1 M p o ta s s iu m (p H 7 .3 ), c o n ta in in g 0 .0 1 M p h o s p h a te s o d iu m s u lp h ite ru b b in g th e e x tra c t o n c a rb o ru n d u m o f d ia g n o s tic e n s u re th e s p e c ie s v iru s re p o rte d p u rity , th e th ro u g h th re e s u c c e s s iv e C h e n o p o d iu m is o la te is o la te T IP a n d L lV in v itr o w e re c a rrie d m e th o d o n C . a m a r a n tic o lo r . onA was w h ic h s e rv e d a s m a x im a P h y s ic a l p ro p e rtie s V iru s s y s te m ic a lly a fte r in o c u la tio n E le c tro n T h e p la n ts in o c u la te d p H -4 .5 ) was a n d w e re a llo w e d fo r a c q u is itio n 5 m in o n a n in fe c te d w ith M y z u s fa s tin g access s y m p to m a tic h e a lth y z u c c h in i w e re p la n ts 1 0 m in u te s a n d th e n p la c e d fo r 1 h le a f. on each o f th re e fe e d in g b y in s e c tic id a l fo r s p ra y . T h e in o c u la te d p la n ts w e re m a in ta in e d o v e r a p e rio d 3 w e e k s in a n in s e c t p ro o f g la s s h o u s e assayed by D A C -E L IS A fo r and and done w ith e x a m in e d JE O L- m ic ro s c o p e d e s c rib e d o f th e v iru s u ra n y l a c e ta te under e le c tro n as p re p a ra tio n s s ta in e d (2 % , 100- CX (T E M ). b y A h la w a t and II IS E M V a rm a RESULTS p ro b e s o f z u c c h in i fo r in o c u la tio n k ille d M oghal (1 9 9 7 ). S y m p to m a to lo g y H e a lth y z u c c h in i le a v e s w e re u s e d a s c o n tro l.T e n v iru life ro u s a p h id s of G lo v e r. T h e g o s s y p ii p re -a c q u is itio n m e th o d a n d p u rifie d n e g a tiv e ly tra n s m is s io n A p h id tra n s m is s io n a n d A p h is th e m ic ro s c o p y L e a f-d ip o f th e v iru s b y D A C -E L IS A . a p h id s w e re g iv e n by 2 -3 w e e k s F ra n c k i (1 9 7 6 ). w e re p e r s ic a e (S u lz a r) in fe c te d le a v e s o f p u m p k in v iz ., D E P , o u t b y s ta n d a rd w a s c o n d u c te d b u ffe r, p H p u rific a tio n in h o s t ra n g e s tu d ie s w e re te s te d fo r th e p re s e n c e A p h id tra n s m is s io n in s u b s tra te A v iru s is o la te fro m z u c c h in i w a s p u rifie d fro m passed tra n s fe r The o f 0 .6 m g /m l 9.8. 1. To was s in g le -le s io n m a in ta in e d o n C u c u r b ita s o u rc e o f in o c u lu m . T a b le c o n c e n tra tio n and d u s te d le a v e s in a m a r a n tic o lo r . b u ffe r 175 P h y to p a th o lo g y th e . a n d th e n p re s e n c e of P o ty v iru s . Z u c c h in i p la n ts is o la te s h o w e d by s e v e re th e p re s e n t p ro m in e n t v e in a l c h lo ro s is in o c u la te d fo llo w e d m o ttlin g , m a lfo rm a tio n such to (F ig .1 ). M a lfo rm a tio n , and m a d e th e m v e in -b a n d in g a s b lis te rin g s im ila r y e llo w th e w ith s y m p to m s b lis te rin g and shoe o b s e rv e d d is c o lo ra tio n o n fru its u n m a rk e ta b le and in fro m le a f s trin g s th e fie ld g re e n to o f in fe c te d p la n ts (F ig . 2 ). S e ro lo g y D ire c t a n tig e n c o a tin g p e rfo rm e d a s d e s c rib e d (1 9 9 6 ). C o m m e rc ia l W a te r m e lo n m o s a ic m o s a ic v ir u s (D A C )-E L IS A b y C h a k ra b o rty p o ly c lo n a l P o ta to p u rc h a s e d fro m A g d ia , v ir u s E lk h a rt, b u ffe r, Y (P V Y ) In d ia n a , o f Z Y M V fro m B io -ra d . S a m p le s (w /v ) in c o a tin g a n tib o d ie s D e n m a rk ) w e re c o a te d o v e rn ig h t w ith im m u n o g lo b u lin w e re used p la te s a n tig e n at 4 °C . (2 0 0 1 J 1 /w e ll) (2 0 0 1 J 1 /w e ll) a t 3 7 °C fo r 2 h o u rs . G o a t a n ti ra b b it Ig G a lk a lin e p h o s p h a ta s e M 0) has in c u b a te d fo r (S ig m a C o ., S t. L o u is , 1 :1 0 0 0 d ilu tio n and 1 :1 0 (N u re , V iru s -s p e c ific a t 1 :2 0 0 a n d th e p la te s w e re in c u b a te d w e re USA and w e re g ro u n d p o ly s ty re n e a n d in c u b a te d of (W M V -1 ), C u c u m b e r v ir u s - 1 (C M V ), was a n d J a in used at p h o s p h a te of z u c c h in i s h o w in g s e v e re H o s t ra n g e h a t 3 7 °C . R e a c tio n w a s o b s e rv e d 2 0 m in u te s a fte r a d d itio n o f s u b s tra te p a ra -n itro p h e n y l p la n ts m o s a ic , d is to rtio n , filifo rm y o f le a v e s c o n ju g a te been 2 F ig .1 . In fe c te d (P N P P ) a t a The host in o c u la tin g ra n g e o f th e v iru s was s tu d ie d by s e v e ra l in d ic a to r p la n ts in g la s s -h o u s e . 176A In d ia n P h y to p a th o lo g y ZYXWVUTSRQPONMLKJIHGFEDCBA Z u c c h in i p la n ts u p o n in o c u la tio n m o ttlin g , m a lfo r m a tio n d e v e lo p e d m o s a ic a n d filifo r m y . T h e h o s t r a n g e o f th e v ir u s w a s m a in ly c o n fin e d to th e m e m b e r s of fa m ily [V o l. C u c u r b ita c e a e a m a r a n tic o lo r ( T a b le e x h ib ite d P h y s a lis flo r id a n a b a n d in g and B la c k m o s a ic : 2003] C h e n o p o d iu m 1 ). c h lo r o tic 5 6 (2 ) lo c a l le s io n s T u r tle and 2 d e v e lo p e d s y m p to m s . No v e in s y m p to m s w e r e o b s e r v e d o n D a tu r a s tr a m o n iu m , L y c o p e r s ic o n e s c u le n tu m , N . g lu tin o s a , N . ta b a c u m V ig n a N ic o tia n a W h ite b e n th a m ia n a , u n g u ic u la ta . The not show v u lg a r is in o c u la te d in d e x e d b y E L IS A ( T a b le d id P h a s e o lu s B u r le y , p la n ts and w e re 1 ) . T h e p la n t s p e c ie s th a t s y m p to m s w e re a ls o n e g a tiv e in E L IS A . Physical properties The th e r m a l d ilu tio n in a c tiv a tio n fr o m in fe c te d d is c o lo u r a tio n , Z u c c h in i p la n ts m a lfo r m a tio n and s h o w in g The v ir u s on m e c h a n ic a l b e tw e e n 1 0 . 3, was 5 0 -5 5 °C and 4 8 h o u rs . in o c u la tio n a n d in d e x in g in E L IS A w a s tr a n s m itte d A. by S y m p to m s r e a c tio n p o in t v ir u s Aphid transmission m anner H ost o f th e b lis te r in g s y m p to m s Table 1. p o in t in v itr o w a s fo u n d lo n g e v ity F ig .2 . F r u its end g o s s y p ii in d u c e d tr a n s m is s io n m e c h a n ic a l w e re in in a n o n - p e r s is te n t p la n ts s im ila r in o c u la tio n M yzus and of p e r s ic a e . in fe c te d to th o s e th e v ir u s by a p h id in d u c e d by on z u c c h in i and p u r ifie d p la n ts . S p e c ie s R e a c tio n R e s u lts o f E L IS A C h e n o p o d iu m Cll.. M ild M M ild M ,L D M o d e r a te VC M ild V C , M , lD S e v e re V C , M , M t, lD S e v e re a m a r a n tic o lo r C itr u J lu s v u lg a r is C u c u m is C. m e lo s a tiv u s L . C u c u r b ita C. pepo m a x im a l. N3 D a tu r a s tr a m o n iu m L a g e n a r ia s ic e r a r ia L y c o p e r s ic o n M N ic o tia n a M M ild C S , M , lD S e v e re b e n th a m ia n a N3 N3 N . ta b a c u m v a r . W h ite B u r le y N3 P h a s e o lu s N3 lu n a tu s a n tis e r u m in IS E M show ed m e a s u r in g The 7 0 0 -7 5 0 p re s e n t of ZYM V of le a f fle x u o u s v ir u s and d ip fila m e n to u s nm (b a s e d is o la te v ir u s on r e a c te d n o t w ith W M V - I 47 w ith and PVY d e c o r a tio n w ith ( F ig .3 ) . N3 v u lg a r is P h y s a lis flo r id a n a B la c k T u r tle 2 p a r tic le s p a r tic le s ) . m ic r o s c o p y M ild c h a r a n tia N . g lu tin o s a P. E le c tr o n p r e p a r a tio n s N3 e s c u le n tu m L u ffa a c u ta n g u la M o m o r d ic a Electron microscopy and Serology V B ,M V ig n a u n g u ic u la ta M o d e r a te N3 H e a lth yC o n tr o l C ll = C h lo r o ticlo c a l le s io n s ,C S = C h lo r o tics p o ts , lD = le a f d is to r tio n ,M = M o s a ic ,M t = M o ttlin g ,N S = N o s y m p to m s ,V B = V e in b a n d in g , V C = V e in c le a r in g . - = N o r e a c tio n F ig .3 . E le c tr o n m ic r o g r a p h s h o w in g Z Y M V a n tis e r u m . M a g . 1 2 0 ,O O O X ,B a r = 1 0 0 n m [V o l. 5 6 (2 ) : 2003] In d ia n DISCUSSION tra n s m is s io n v iz ., M o re th a n 2 5 v iru s e s , in c lu d in g a t le a s t s e v e n P o ty v iru s e s , a re k n o w n to in fe c t c u c u rb its (L o v is o lo , 1 9 8 0 ). In In d ia , P o ty v iru s e s re p o rte d o n s e v e ra l c u c u rb its T h e s e v iru s e s n a tu ra lly have been (S in g h , 1 9 7 9 , 1 9 8 3 ). a ls o o c c u r in m a n y o th e r a re a s o f th e w o rld (W e b b GFEDCBA e t a l . , 1 9 6 5 ; S in g h , g o s s y p ii, et M yzus a l., w ith re p o rts o f Z Y M V e t a l., (L e c o q 1 9 8 4 ; L is a th a t th e re is little v a ria tio n in am ong th e o n s q u a s h a n d th e C o n n e c tic u t s e v e re y e llo w in g s y m p to m s . is o la te th a t p ro d u c e B ased is o la te b u t u n u s u a l s y m p to m s d iffe re n t fro m ZYM V filifo rm y , d is to rtio n o n fru it re p o rte d m akes m ic ro s c o p y fro m o th e r h a v e b e e n re p o rte d to b u t d u rin g s a tiv u m not P. in fe c t v u lg a r is cv. by B ru n t et th e re la te d to Z Y M V a s (1 9 9 6 ) a l. (2 0 0 0 ). H o w e v e r, The p re s e n c e of ZYM V a and van e x a c t id e n tific a tio n o f th e v iru s n e e d s its m o le c u la r c h a ra c te riz a tio n . y e llo w re q u ire s is know n squash to be m o s a ic s tric t v iru s q u a ra n tin e s e e d -tra n s m is s ib le a t a v e ry in z u c c h in i lo w ra te (S c h rijn w e rk e rs e t a l., P in to . v u lg a r is c v . P in to th e s a tiv u m H ow ever is s tra in p re s e n t and th e s tu d ie s P h a s e o lu s in fe c tio n s p e c ific and of P. re m a in s REFERENCES A d le rz , W .C . (1 9 6 9 ). D is trib u tio n v iru s e s 82: S oc. A h la w a t, V .S . and V a rm a , of s tra in s (L e c o q c ro p p o s s ib le 1981; a l., P ro w id e n ti et a l., 1 9 8 4 ). and In d ia n Z Y M V re p o rte d fro m 1 9 9 2 ) in fe c te d C itr u llu s C u c u m is m e lo S in g a p o re T r ic h o s a n th e s a n g u in a (T h u n b .) M a ts u n & L ., s a tiv u s , C. B ru n t, R oxb, and M o m o r d ic a C u c u r b ita m o s a ic S ta te H o r tic . A ., C ra b tre e , L is ts fro m ( 1 9 9 7 ). in fe c tio n m e a s u re 5 0 (2 ): fo r V iru s e s V ID E O xon, its S e ro lo g ic a l o n io n seed m a n a g e m e n t. M ., G ib b s , o f P la n ts . D a ta b a s e . UK. in 1 3 7 -1 4 0 . K ., D a llw itz , L . (1 9 9 6 ). W a liin g fo rt, A. v ira l 1484 CAB A ., a n d D e s c rip tio n s In te rn a tio n a l pp. L . c y lin d r ic a c h a r a n tia L. or L . H o w e v e r, th e v iru s u n d e r fu rth e r s u g g e s tin g m ix e d P h y to p a fn . W a ts o n , and s tu d y p ro d u c e d s y s te m ic s y m p to m s T . a n g u in a . e t a l., lu n a tu s L u fa a c u ta n g u la ( L .) R o x b . b u t d id n o t in fe c t (W o n g P r o c . F la . 1 6 1 -1 6 5 . d e te c tio n et o f w a te rm e lo n 1 a n d 2 in F lo rid a . s y m p to m le s s w ith s o m e F re n c h a n d N o rth A m e ric a n and tra n s m is s io n , a n d s e ro lo g y 1 9 9 1 ). S o m e s tra in s o f Z Y M V l., but m e a s u re s w h ily in tro d u c in g Z u c c h in i s e e d a s Z Y M V it p ro d u c e s y s te m ic in fe c tio n w ith o u t v is ib le s y m p to m s pepo w ith W M V -2 . g ro u p s e ro lo g ic a lly P o ty v ir u s re s e m b lin g c o u n trie s . N a k a i, re a c tio n o n th e s y m p to m a to lo g y , R e g e n m o rte l T h e v iru s u n d e r s tu d y m o s a ic , a n d y e llo w in g o f th e le a v e s lik e th a t o f C o n n e c tic u t P is u m P V Y . S im ila r 1. h o s t ra n g e , e le c tro n d e s c rib e d b y th e F lo rid a is o la te th a t p ro d u c e m ild e r s y m p to m s in (1 9 9 0 ) g a v e w e a k n o t w ith W M V of 1 9 8 4 ) to fa ll in to b io ty p e s , th o s e re p re s e n te d v iru s d id w ith a n tis e ru m 1 and v iru s u n d e r s tu d y h a s b e e n id e n tifie d a s a m e m b e r Z Y M V is o la te s h a v e b e e n re p o rte d (P ro w id e n ti th e b u t n o t w ith W M V in th e h o s t ra n g e a m o n g d iffe re n tia tin g p ro d u c e d v e ry s e v e re has a n d L e c o q (1 9 9 7 ). Z Y M V is o la te re p o rte d b y S to b b s e t a l. P o ty v iru s e s th a t in fe c t c u c u rb its . e t a l., m anner re s u lts h a v e b e e n re p o rte d b y D a v is (1 9 8 6 ), D e s b ie z Z V M V is o la te s a ll o v e r th e w o rld a n d th e s e re a c tio n s u s e fu l and e r y s im i in a n o n -p e rs is te n t a ls o b e e n re p o rte d . et 1 9 8 1 a n d L is a a n d L e c o q , 1 9 8 4 ). T h is in d ic a te s be L ip a p h is c r a c c iv o r a , 1979, 1983; a n d h o s t ra n g e o f th e v iru s u n d e r 1983, 1981; Lesem ann c o u ld A. A. 1 9 8 1 ; L is a e t a l., T h e v iru s re a c te d s e ro lo g ic a lly s tu d y a re c o n s is te n t o f a p h id 1 9 8 4 ), a l., m id d le tr ii, p is u m , spp. U r o le u c o n of ZYM V e t a l., A p h is et (L e c o q p e r s ic a e 1 9 8 1 ), a l., b y o th e r s p e c ie s (P u rc ifu ll c itr ic o la A c h y r th o s ip h o n L o v is o lo , 1 9 8 0 ). S y m p to m s of ZYM V A p h is 177 P h y to p a th o lo g y in M . c h a r a n tia th e d is tin c tn e s s C h a k ra b o rty , N .K . a n d J a in , R .K . (1 9 9 6 ). E n z y m e L in k e d Im m u n o s o rb e n t A ssay. In : P a th o g e n s , (E d s ., P ., M a la th i, V .G . a n d V e rm a , P la n t P a th o lo g y , A h la w a t, IA R I, D e te c tio n V .S ., New of P la n t R a m a c h a n d ra n , J .P .) p . 3 -8 . C A S in D e lh i. o f th e p re s e n t is o la te fro m k n o w n is o la te s p re s e n t C la rk , e ls e w h e re.. M .F . a n d A d a m s , o f m ic ro T h e v iru s A p h is g o s s y p ii under s tu d y and M yzus was tra n s m itte d p e r s ic a e . by H o w e v e r, s o rb e n t G en. p la te assay V ir a l. m e th o d A .N . of (1 9 7 7 ). enzym e fo r th e d e te c tio n 3 4 : 4 7 5 -4 8 3 . C h a ra c te riz a tio n lin k e d im m u n o - o f p la n t v iru s e s . J. 178 CBA In d ia n C rosslin, J.M ., B row n, F irst [V ol. 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