16-15(3)-07(최장경).fm
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1 Res. Plant Dis. 15(3) : (2009) Research in Plant Disease The Korean Society of Plant Pathology t (Lagenaria leucantha var. gourda) w IB m Cucumber mosaic virus Áy 1 Á»x 1 Á t 2 Á * w w, 1 w y w, 2 w w A Subgroup IB Isolate of Cucumber mosaic virus Isolated from Lagenaria leucantha var. gourda Sun Mi Oh, Jin Sung Hong 1, Ki Hyun Ryu 1, Gung Pyo Lee 2 and Jang Kyung Choi* Department of Applied Biology, Kangwon National University, Chuncheon , Korea 1 Division of Environment & Life Sciences, Seoul Women's University, Seoul , Korea 2 Division of Applied Plant Sciences, Chungang University, Ansung , Korea (Received on November 17, 2009; Accepted on December 7, 2009) An isolate of Cucumber mosaic virus (CMV), called as Lag-CMV, was identified from Lagenaria leucantha var. gourda showing mosaic symptom, and its properties was compared to Fny-CMV (subgroup IA) and As- CMV (subgroup IB) by host reaction in several indicator plants, dsrna analysis, RT-PCR analysis, restriction enzyme profile of the PCR products and nucleotide sequence of coat protein gene. Lag-CMV was similar to As-CMV used as a control CMV by the induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves of N. tabacum. cv. Xanthi nc. In the cucumber and zucchini squash, Lag-CMV and As-CMV induced a mild mosaic symptoms than that of Fny-CMV. Size and shapes of local lesions on Chenophodium amaranticolor and Vigna unguiculata induced by Lag-CMV was similar those by Fny-CMV or As-CMV. In experiments of dsrna profiles and RT-PCR analysis of coat protein gene, Lag-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using EcoRI, SalI, MspI, XhoI, and HindIII of the RT- PCR products and nucleotide sequence analysis of the coat protein gene showed that Lag-CMV belong to a member of CMV subgroup IB of the same to As-CMV. Keywords : Cucumber mosaic virus, Lagenaria leucantha var. gourda., Host reaction, Restriction enzyme profile, Subgroup IB Bromoviridae Cucumovirus w Cucumber mosaic virus(cmv), swš» ƒ vw š, ù y{ ƒ w š (Tomlinson, 1987). CMV RNA1, RNA2 RNA3 3 (+) ƒ RNA RNA3 3' w w RNA(RNA4) swwš (Palukaitis, 1992). ¾ š m CMV j I II š (Owen Palukaitis, 1988),» *Corresponding author Phone) , Fax) ) jkchoi@kangwon.ac.kr p x w (Kaper Waterworth, 1981), v rp ELISA(enzyme-linked immunosorbent assay) (Edwards Gonsalves, 1983), RNase protection w heterogenecity (Owen Palukaitis, 1988) RT-PCR(reverse transcription-polymerase chain reaction) w v wz (Rizos, 1992) w š. I w RNA3 5'» p k IA IB wš (Roossinck, 1999). t (Lagenaria leucantha var. gourda) (Lagenaria leucantha) w y. x
2 t (Lagenaria leucantha var. gourda) w IB m Cucumber mosaic virus 255 (KI /QUCKE U[ORVQOU KP PCVWTCNN[ KPHGEVGF.CIGPCTKC NGWECPVJCXCTIQWTFC wš w š. ¾ t š ù, l Cucumber mosaic virus(cmv) (Choi, 1999), Cucumber green mottle mosaic virus(cgmmv) (Lee, 1990; Kim Lee, 2000; Antignus, 2001), Zucchini yellow mosaic virus(zymv) (Lisa, 1981), Zucchini green mottle mosaic virus(zgmmv) (Ryu, 2000; Lee, 2003) y , w s x j t (Fig. 1) w g l CMV w m wš, ¾ t CMV m w p w. ¾. j t 0.01 M (ph 7.0) w z, 5-6» Nicotiana benthamiana w. 10 z N. benthamiana w t Cucumovirus p v (CPT-all primer) (Choi, 1999) w RT-PCR(reverse transcription-polymerase chain reaction)) w, 950 bp j» cdnaƒ. t j CMV w m w š Chenopodium amaranticolor w w. mw N. benthamiana C. amaranticolor w x 2z w, x 5-6» N. benthamiana w œ w š, t w CMV Lag- CMV w. wr CMV ¾ Fny-CMV( IA) As-CMV( IB) œ w. t. Lag-CMV» p w» w CMV p š N. tabacum cv. Xanthi nc, N, glutinosa, Cucubita pepo cv. Black beauty, Cucumis sativus cv. Suyo, Vigna unguiculata cv. Kurotanesanzaku Capsicum annuum cv. Cheongyang œ w. e 0.01 M w w, w t 25 o C-30 C 2 o š ùkù Fny-CMV As- CMV w t wì w. Lag-CMV Fny-CMV As-CMV» ùkù p Table 1 6CDNG4GCEVKQPQHKPFKECVQTRNCPVUD[OGEJCPKECNKPQEWNCVKQP QH.CI(P[CPF#U *QUVRNCPV.CI 5[ORVQOU C (P[ #U 0KEQVKCPCDGPVJCOKCPC / / / 0INWVKPQUC / / / 0VCDCEWOEX:CPVJKPE %5/ / %5/ %JGPQRQFKWOCOCTCPVKEQNQT... %CRUKEWOCPPWWOEX%JGQPI[CPI / O/ %WEWDKVCRGRQEX$NCEMDGCWV[ / %5/ / %WEWOKUUCVKXWUEX5W[Q / / / 8KIPCWPIWKEWNCVC... a Inoculated leaves/upper leaves, M: mosaic, mm: mild mosaic, CS: chlorotic spot, L: necrotic local lesion, -: symptom less or not infected. (KI5[ORVQOUQPKPFKECVQTRNCPVUD[OGEJCPKECNKPQEWNCVKQP QH.CI KUQNCVGF HTQO.CIGPCTKC NGWECPVJC XCT IQWTFC (P[ CPF #U YGTG KPQEWNCVGF CU C EQPVTQN U 2JQVQITCRJU CTG RTGUGPVGF QP KPQEWNCVGF NGCXGU QH 0KEQVKCPC VCDCEWOEX:CPVJKPECPFQPWRRGTNGCXGUQH%WEWOKUUCVKXWU EX 5W[Q %WEWTDKVC RGRQ EX $NCEM DGCWV[ CPF %CRUKEWO CPPWWOEX%JGQPI[CPITGURGEVKXGN[
3 256 Áy Á»xÁ tá w. t w Lag-CMV N. benthamiana x j ùkü œ w CMV w. ù N. tabacum. cv. Xanthi nc n x As-CMV w, ùkü Fny- CMV (Fig. 2). wr Fny-CMV j y w j ùkü, Lag-CMV As-CMV j x. š Fny-CMV As-CMVƒ 7~10 z j x ù, Lag-CMV y. ù Lag-CMV w š w Cucumovirus p v w RT-PCR w, cdnaƒ Lag-CMV š ƒ. wr C. amaranticolor V. unguiculata 2-3 z x CMV. DsRNA. Lag-CMV RNA j»» w Lag-CMV, Fny-CMV As-CMV N. benthamiana l Morris Dodds (1979) ƒ RNA(double stranded RNA; dsrna) w. w dsrna 6% polyacrylamide gel 1 TAE (40 mm Tris, 40 mm acetic acid, 2 mm EDTA, ph 7.8)» w z, ethidium bromide w w. Lag- CMV N. benthamiana l w dsrna» ql Fny-CMVù As-CMV dsrna j» ùkü (Fig. 3). dsrna wù CMV ƒ š, RNA satellite RNA w w (Dodds, 1993). w Wang (1988) m CMV l w dsrna», dsrna ƒ m x x(serotype) ew š šw. w š l w, Lag-CMV dsrna Fny-CMVù As-CMV x x q. RT-PCR wz. Lag-CMV N. benthamiana w total RNA wš, w RT-PCR w. Total RNA Choi (1998) w, RT-PCR v IR 3' sww Cucumovirus p v (Choi, 1999) w (KI 2QN[CET[NCOKFG IGN GNGEVTQRJQTGUKU QH FU40# KUQNCVGF HTQO0KEQVKCPCDGPVJCOKCPCKPHGEVGFYKVJ.CI#U CPF(P[TGURGEVKXGN[#NKSWQVQHFU40#RWTKHKGFD[%( EGNNWNQUGYCUGNGEVTQRJQTGUGFKPRQN[CET[NCOKFGIGN.» PCR 1.2% agarose gel 1 TAE» w, 3 CMV l 950 bp cdnaƒ (Fig. 4A). cdna wz EcoRI HindIII, MspI, SalI XhoI w z, wz ql(rflp) w Lag-CMV w. Lag-CMV RFLPql œ w 5 wz As-CMV ew Lag-CMVƒ IB (Fig. 4B). Singh (1995) CMV m w v cdna w EcoRI MspI w, I II w w š w. ù z w IA IB w. x HindIII IA IB CMV w» w wz ƒ. v p. Lag-CMV v w» w» w, CMV RNA3 v sww 950 bp cdna w. Lag-CMV CMV N. benthamiana l w total RNA x RT-PCR wwš, 1.2% agarose gel» w, ƒ CMV cdna w. w ƒ cdna pgem-t easy vector (Promega) ligation k z E. coli JM109 j w, GFX Micro Plasmid Prep kit (Amersham Parmacia) w w v w. w v wz EcoRI w y w, y v w» w. v» MacAlign(DNAstar 3.30C) w
4 t (Lagenaria leucantha var. gourda) w IB m Cucumber mosaic virus 257 (KI4GXGTUGVTCPUETKRVKQPRQN[OGTCUGEJCKPTGCEVKQP462%4CUUC[#CPFTGUVTKEVKQPGP\[OGCPCN[UKUQHVJGRTQFWEVU$HQTVJG JCNHTGIKQPQH40#EQXGTKPIHWNNNGPIVJEQCVRTQVGKPIGPGHTQO.CI#UCPF(P[TGURGEVKXGN[&KIGUVGFTGUVTKEVKQP HTCIOGPVUYGTGUGRCTCVGFQPCCITQUGIGNCPFUVCKPGFYKVJGVJKFKWODTQOKFG.CPG/MD&0#NCFFGTNCPG'EQ4+NCPG *KPF+++NCPG/UR+NCPG5CN+CPFNCPG:JQ+TGURGEVKXGN[ (KI%QORCTKUQPQHCOKPQCEKFUGSWGPEGQHEQCVRTQVGKPDGVYGGP.CI#UCPF(P[+FGPVKECNTGUKFWGUCTGKPFKECVGF D[J[RJGP w z ƒ w (Fig. 5).» Lag-CMV As-CMVƒ 99.1%, Lag-CMV Fny-CMV 94.5%,» w w Lag-CMV As-CMV 100%, Lag-CMV Fny-CMV 97.3%. Lag-CMV As- CMV 218 ew, Fny- CMV v 5 (25, 65, 71, 99, 205 ) ey ùkþ. t w Lag-CMV v As-CMV ew IB w CMV y. x j t (Lagenaria leucantha var. gourda) l CMV wš Lag-CMV w. t w Lag-CMV N. benthamiana x j ùkü œ w CMV w. ù N. tabacum. cv. Xanthi nc n x As-CMV w, ùkü Fny- CMV. w Fny-CMV j y w j ùkü, Lag-CMV As-CMV j x. š Fny-CMV As-CMV ƒ j x ù, Lag-CMV. Lag-CMV N. benthamiana l w dsrna» ql Fny- CMVù As-CMV dsrna j» ùkü. Lag-CMV N. benthamiana l w total RNA v IR 3' sww 950 bp cdnaw w v w RT-PCR w 950 bp cdnaƒ. cdna wz EcoRI, HindIII, MspI, SalI XhoI w z RFLP w, Lag-CMV RFLPql As-CMV ew. wr cdna w» w,» As-CMV 99.1%, Fny- CMV 94.5%, v As-CMV 100%, Fny-CMV 97.3% ùkþ. l t w Lag- CMV As-CMV IB w y.
5 258 Áy Á»xÁ tá ( y: ) w,. š x Antignus, Y., Wang, Y., Perlsman, M., Lachman, O., Lavi, N. and Gal-On, A Biological and molecular characterization of a new cucurbit-infecting Tobamovirus. Phtopathoiogy 91: Choi, J. K., Kim, H. J., Hong, J. S., Kim, D. W. and Lee, S. Y Identification and differentiation of cucumber mosaic virus isolates in Korea. Korean J. Plant Pathol. 14: Choi, S. K., Choi, J. K., Park, W. M. and Ryu, K. H RT- PCR detection and identification of three species of cucumoviruses with a genus-specific single pair of primers. J. Virol. Methods 83: Dodds, J. A DsRNA in diagnosis. In : Diagnosis of Plant Virus Diseases, ed. by R.E.F. Matthews. pp CRC Press, Boca Raton, USA. Edwards, M. C. and Gonsalves, D Grouping of seven biologically defined isolates of cucumber mosaic virus by peptide mapping. Phytopathology 73: Kim, D. H. and Lee, J. M Seed treatment for Cucumber green mottle mosaic virus (CGMMV) in gourd (Lagenaria siceraria) seeds and its detection. J. Kor. Soc. Hort. Sci. 44: 1-6. Kaper, J. M. and Waterworth, H. E Cucumoviruses. In : Handbook of Plant Virus Infections, ed. by E. Kurstak, pp Elsevier/Holland Biolmedical, New York, USA. Lee, G. P., Min, B.E., Kim, C. S., Choi, S. H., Ham, C. H., Kim, S. U. and Ryu, K. H Plant virus cdna chip hybridization for detection and differentiation of four cucubit-infecting tobamovirus. J. Virol. Methods 110: Lee, K. W., Lee, B. C., Park, H. C. and Lee, Y. S Occurrence of cucumber green mottle mosaic virus disease of watermelon in Korea. Korean J. Plant Pathol. 6: Lisa, V., Boccardo, G., Dgostino, G. and Dellavlle, G Characterization of a potyvirus causing zucchini yeiiow mosaic virus. Phytopathology 71: Morris, T. J. and Dodds, J. A Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69: Owen, J. and Palukaitis, P Characterization of cucumber mosaic virus. I. Molecular heterogenecity mapping of RNA3 in eight CMV strains. Virology 166: Palukaitis, P., Roossinck, M. J., Dietzgen, R. G. and Francki, R. I. B Cucumber mosaic virus. Adv. Virus. Res. 41: Rizos, H., Gunn, L. V., Pares, R. D. and Gillings, M. R Differentiation of cucumber mosaic virus isolates using the polymerase chain reaction. J. Gen. Virol. 73: Roossinck, M. J., Zhang, L. and Hellwald, K. H Rearrangement in the 5' nontranslated region and phylogenetic analysis of cucumber mosaic virus RNA3 indicated radial evolution of three subgroups. J. Virol. 73: Ryu, K. H., Min, B. E., Choi, G. S., Choi, S. H., Kwon, S. B., Noh, G. M., Yoon, J. Y., Choi, Y. M., Jang, S. H., Lee, G. P., Cho, K. H. and Park, W. M Zucchini green mottle mosaic virus is a new Tabamovirus; Comparison of its coat protein gene with that of Kyuri green mottle mosaic virus. Arch. Virol. 145: Singh, Z., Jones, R. A. C. and Jones, M. G. K Identification of Cucumber mosaic virus subgroup I isolates from banana plants affected by infectious chlorosis disease using RT-PCR. Plant Dis. 79: Tomlinson, J. A Epidemiology and control of virus diseases of vegetables. Ann. Appl. Biol. 110: Wang, W. Q., Natsuaki, T., Okuda, S. and Teranaka, M Comparison of cucumber mosaic virus isolate by duoblestrand RNA analysis. Ann Phytopath. Soc. Japan 54:
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