Indian Journal of Experimental Biology Vol. 37, October 1999, pp. 1034-1036 Association of an unknown potyvirus isolate with a severe mosaic disease of Narcissus tazetta L. Aminuddin, J A Khan & S K Raj Plant Virus Laboratory, National Botanical Research Institute, Lucknow 226 00 I , Indi a Received 5 April 1999; revised 21 lilli e 1999 An isolate of potyvirus was found to be associated with severe mosaic disease in Narcisslls lazel/{! L. Electron microscopy, SDS -polyac rylamide gel el ec trophoresis of coat protein, serological relationship and RT-P CR were carried out to identify the virus isolate. Electron micro sco py of purified virus preparation revealed the prese ncc of tlexuous pa rti cles of 730 x 13 nm. One band with a molecular weight of 33 kDa was obt ained by SDS -po lyacrylamide ge l elec trophoresis. Serological relationship of the virus isolate with bean yellow mosaic virus was es tablished in cl ectrob lot immunoa ssay. Employing potyvirus specitic primers in RT-PCR, a band of about 300 bp was obtained from the core reg ion o f the coat protein, thus confirming the association of a potyvirus with the mosaic disease of N. /a ze lla. Narciss us tazetta L. cv. Paper White is an important ornamental plant cultivated in India for floriculture industry. Vegetative propagation through bulbs, leads to large sca le dissemination of systemic viru ses in Narcissu s spp. stocks. Several reports have on th e natural occ urrence of fi lamentous . I? d . 1 N' I po ty vlru scs '- an a potexv trl.l s·. arCISS ll S atent (Carlaviru s) was found on Narcissus,4 .5 Nerine 6 and bulbous iri s 7 . Other sli ghtl y fl exuous Carlaviruses li ke Nerine latent virus was iso lat ed from Nerine b I ··16 8 d H' I ' f owe e l7lf" an Ipp eastrum at e nt viru s Tom Hip peastrum hy bridum 9 . However, th ere is no report of potyvi ru s infecting Narcissus in India. We report here natural occurrence of a potyvirus on Nar cis sus tazetta L. N. taze tta cv. Paper White plants growing in th e nursery of the National Botanical Research In stitut e, Lucknow, exhibited severe mo sa ic symptoms accompanied with light-green to ye llow stripes on leaves. Plants showed extreme reduction in growth and flow e rs as compared to apparently healt hy plant s. In view of identifying putative virus associated with the di sease, viru s purificati on and electron microscopy were und ertaken followed by determination of molecular weight of th e coat prote in subunits and serological relationship analysis and RT- PCR. • Crude sap and purified virus preparations in phosphate buffer (0.1 M, pH 7. 2) were mechanically inoculated on the pl a nt speci es viz . Beta vulgaris, Che nopodium album, C. amarallti co lor, C. qUlllOa, Cucurbita pepo, Cucumis sativus, Datura stramonium, Go mphre m, g lohosa , Nicotiall{/ benfhamiana , N. r us fi ca, N. tabac ul11 cv. Samsun N N, Petunia hy brida, Phas eollls vLllg([ris cv. Th e Princ e, Solanum lfI elO l1g ella, Vig ll a mdiata and V. mung o. Virus was purified by clarifica ti on of buffered (phosphate buffer, 0.1 M, pH 7.2) NarcisslIs leaves extract with 15 % n-butanol fo ll owed by precipitation with 6% polyethylene glyco l (PEG) and se dimentation through 15 % sucrose co nt aining 0.1 % Triton X-IOO . Purifi ed preparations we re observed un der trans mi ssion elec tr on mi crosco pe (Philips 410) us in g 2% uranyl acetate (pH 4.2 ) as negative stain . Average size of th e viru s wa ca lcul ated by measuring 90 particles wit a ma gniri er, direc tly from electron mi crograph nega ti ve. 'The calibration was done us in g tobacco mosa ic vi rus as ex ternal standard . Virus infected crude sa mples we re mix ed ·with an equal volume of denaturing so luti on (0.04 M, Tris- HCI; pH 6.8 , 10% (v/v) glycerol, 2% (w/v) SDS. 5 lk (w/v) and O. OS o/c bro mop he nol blue) and heated in a boi lin g water ba th for 3 min . Electrophoresi s wa s ca rri ed out in I (separatin g) polyacrylamide ge ls acco rdi ng to th e meth od desc ribed by Laemmli 10 us in g Bio- Ra d Prote in 1I min i gel apparatu s. The marker prote in s ( Pharm<l cia . Sweden) used were phosphorylase b (94 kDa), bo vin e serum album in (67 kDa), ovalalbumin (43 kDa ), carbonic anh yd rase (30 kDa ), ,>oy bean trypsin inhibitor (2 0 .2 kDa) and (a- la ctalbumin ( 14.4 kDa ). Sample (20 J.l.1 ) was loaded to th e siot of th e gel.