Station de Pathologie Végétale, INRA, Domaine Saint Maurice, BP 94, 84143 Montfavet cedex, France1
Department of Virology, Agricultural Research Organization, The Volcani Center, POB 6, Bet Dagan 50250, Israel2
Horticulture and Plant Protection Department, Faculty of Agriculture, University of Jordan, Amman, Jordan3
Author for correspondence: Hervé Lecoq. Fax +33 4 32 72 28 42. e-mail Herve.Lecoq{at}avignon.inra.fr
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In order to investigate the taxonomic position of CVYV further, cytopathological, serological and molecular studies were conducted. Cucumber (Cucumis sativus cv. Beit Alfa) and melon (Cucumis melo cv. Védrantais) plants were inoculated mechanically with the CVYV type strain from Israel (CVYV-Isr) or with a CVYV isolate from Jordan (CVYV-Jor) and were kept subsequently in independent screened cages maintained in an insect-proof greenhouse. The two strains induced similar vein-clearing symptoms in cucumber and melon, but CVYV-Jor caused a more severe stunting in cucumber.
For cytological studies, leaf pieces 1 mm across were collected from symptomatic young leaves 34 weeks after inoculation and similar samples from healthy plants were used as controls. Samples were fixed with glutaraldehyde, post-fixed with osmium tetroxide and embedded in araldite CY212 (Agar Scientific) (Delécolle, 1978 ). Thin sections were cut with a diamond knife by using an Ultracut E ultramicrotome (Reichert-Jung). Thin sections were stained in 5% uranyl acetate and lead acetate, pH 12, before observations.
Numerous cylindrical cytoplasmic inclusions were observed in CVYV-Isr- and CVYV-Jor-infected melon and cucumber cells. Depending on the section orientation, inclusions appeared as pinwheels (Fig. 1a, c
) or as bundles (Fig. 1b
, c
). Cylindrical inclusions were occasionally observed at the cell periphery, one end being associated with the plasmalemma perpendicular to the cell wall (Fig. 1b
). Cylindrical cytoplasmic inclusions are associated specifically with infections by virus species belonging to the family Potyviridae (Shukla et al., 1994
; Edwardson & Christie, 1996
). Numerous vesicles were also observed in the cytoplasm, sometimes associated with the cylindrical inclusions (Fig. 1c
). Increased vesiculation is also a feature commonly associated with infections by potyvirids (Shukla et al., 1994
). Only a few scattered virus particles were observed, generally close to the cylindrical inclusions. Neither cylindrical cytoplasmic inclusions nor numerous vesicles were observed in healthy plant samples.
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Further experiments were conducted with the potyvirid primers designed by Gibbs & Mackenzie (1997) , which are able to amplify genome fragments of aphid-, mite-, fungus- and whitefly-transmitted potyvirids. Total RNA was extracted from 50 mg infected, symptomatic young cucumber or melon leaves with TRI-Reagent (Molecular Research Center), resuspended in 20 µl DEPC-treated H2O and heated for 5 min at 65 °C prior to reverse transcription. Reverse transcription and PCR were performed following a procedure derived from Gibbs & Mackenzie (1997)
. cDNA was synthesized from 15% of the total RNA in a 20 µl reaction mixture using the Promega cDNA synthesis protocol and 1 µM of a primer derived from potyvirid primer 1 of Gibbs & Mackenzie (1997)
, Poty(dT)3' (5' CACGGATCCTTTTTTTTTTTTTTTTTV 3'). DNA encoding the 3' part of the viral genome was amplified in 25 µl of the PCR mixture described by Gibbs & Mackenzie (1997)
with 2 µl of the cDNA and Taq polymerase instead of Tth polymerase. Primers Poty(dT)3' and Poty5' (5' CCACGGATCCGGBAAYAAYAGYGGDCARCC 3') were used for amplification. PCR cycles were performed as described by Gibbs & Mackenzie (1997)
in a PEC2400 thermocycler (Perkin Elmer). A 1·9 kb fragment was amplified from CVYV-Isr- and CVYV-Jor-infected cucumber or melon extracts but not from extracts of healthy cucumber or melon plants.
The 1·9 kb fragment amplified from CVYV-Isr-infected plant tissue was ligated into a linearized and T-tailed pBlueScript plasmid (Stratagene). Plasmid DNA was purified from Escherichia coli DH10B and sent for double-strand sequencing to Genome Express (Grenoble, France). At least two sequence readings were obtained for each nucleotide. The deduced amino acid sequence of CYVY-Isr was compared to sequences in the GenBank database by using the FASTA procedure of Pearson & Lipman (1988) . Sequence alignments and distance matrices were obtained with CLUSTAL W (Thompson et al., 1994
). A PAM Dayhoff matrix was used to calculate distance trees with the NEIGHBOR algorithm of the PHYLIP package (Felsenstein, 1989
). Graphics were generated for the unrooted distance tree with the TreeView application (Page, 1996
).
The deduced amino acid sequence of CVYV was aligned with sequences from other potyvirids belonging to the whitefly-transmitted genus Ipomovirus (Sweet potato mild mottle virus, SPMMV), the aphid-transmitted genera Potyvirus [Potato virus Y (PVY) and ZYMV] and Macluravirus (Maclura mosaic virus, MacMV), the fungus-transmitted genus Bymovirus (Barley yellow mosaic virus, BaYMV) and the mite-transmitted genera Rymovirus (Agropyron mosaic virus, AgMV) and Tritimovirus (Wheat streak mosaic virus, WSMV). An unrooted tree was obtained using the amino acid sequences of the core of the CP of these potyvirids and those of Barley mild mosaic virus (BaMMV), Brome streak mosaic virus (BSMV), Narcissus latent virus (NLV), PRSV, RGMV, Sweet potato feathery mottle virus (SPFMV), Turnip mosaic virus (TuMV) and WMV.
The amplified fragment contained the 3' part of the CVYV genome, including the C-terminal part of the polymerase (NIb) coding region, the CP coding region and the 3' non-coding extremity. Sequence comparison with virus databases showed that CVYV-Isr exhibited 43% amino acid sequence identity to the Ipomovirus SPMMV, while the identity to other potyvirids did not exceed 32% for tritimoviruses and 27% for potyviruses. No significant sequence identity was observed outside the family Potyviridae. Fig. 2 presents the CLUSTAL W alignment of the C-terminal 559 amino acids of CVYV-Isr with the corresponding parts of seven potyvirids representing the six genera included in this family. The NIbCP cleavage site and the limit of the variable N-terminal part of CP were determined approximately by sequence comparisons and are underlined in Fig. 2
. The DAG triplet conserved in the N-terminal part of the CP of most aphid-transmitted potyviruses was not present in the sequence of the putative CVYV CP. However, two amino acids that are highly conserved among potyvirids in the core of the CP and involved in virion assembly (Jacquet et al., 1998
), R449 and D494, were present in the sequence of CVYV-Isr.
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Footnotes |
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References |
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Brunt, A., Crabtree, K., Dallwitz, M., Gibbs, A. & Watson, L. (1996). Viruses of Plants. Description and Lists from the VIDE Database. Wallingford, UK: CAB International.
Cohen, S. & Nitzany, F. E. (1960). A whitefly transmitted virus of cucurbits in Israel.Phytopathologia Mediterranea 1, 44-46.
Delécolle, B. (1978). Essais de rationalisation des méthodes de préparation déchantillons végétaux pour la microscopie électronique: problème des précipités parasites.Cellular and Molecular Biology 23, 431-436.[Medline]
Edwardson, J. R. & Christie, R. G. (1996). Cylindrical Inclusions. Bulletin 894. Gainesville, FL: University of Florida Agricultural Experiment Station.
Felsenstein, J. (1989). PHYLIP Phylogeny Inference Package (version 3.2). Cladistics 5, 164-166.
Gibbs, A. & Mackenzie, A. (1997). A primer pair for amplifying part of the genome of all potyvirids by RTPCR.Journal of Virological Methods 63, 9-16.[Medline]
Harpaz, I. & Cohen, S. (1965). Semipersistent relationship between cucumber vein yellowing virus (CVYV) and its vector, the tobacco whitefly (Bemisia tabaci Gennadius).Phytopathologische Zeitschrift 54, 240-248.
Jacquet, C., Delecolle, B., Raccah, B., Lecoq, H., Dunez, J. & Ravelonandro, M. (1998). Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.Journal of General Virology 79, 1509-1517.[Abstract]
Jordan, R. (1992). Potyviruses, monoclonal antibodies, and antigenic sites.Archives of Virology Supplementum 5, 81-95.[Medline]
Lecoq, H., Wisler, G. & Pitrat, M. (1998). Cucurbit viruses: the classics and the emerging. In Cucurbitaceae 98. Evaluation and Enhancement of Cucurbit Germplasm, pp. 126-142. Edited by J. D. McCreight. Alexandria, VA: ASHS Press.
Mansour, A. & Al-Musa, A. (1993). Cucumber vein yellowing virus; host range and virus vector relationships.Journal of Phytopathology 137, 73-78.
Mansour, A. N. & Hadidi, N. (1999). Cucumber vein yellowing virus: purification and serological studies.Dirasat Agricultural Sciences 26, 8-14.
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers.Computer Applications in the Biosciences 12, 357-358.[Medline]
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison.Proceedings of the National Academy of Sciences, USA 85, 2444-2448.[Abstract]
Pringle, C. R. (1999). Virus Taxonomy 1999. The universal system of virus taxonomy, updated to include the new proposals ratified by the International Committee on Taxonomy of Viruses during 1998. Archives of Virology 144, 421-429.[Medline]
Richter, J., Rabenstein, F., Proll, E. & Vetten, H. J. (1995). Use of cross-reactive antibodies to detect members of the Potyviridae. Journal of Phytopathology 143, 459-464.
Sela, I., Assouline, I., Tanne, E., Cohen, S. & Marco, S. (1980). Isolation and characterization of a rod-shaped, whitefly-transmissible, DNA- containing plant virus.Phytopathology 70, 226-228.
Shukla, D. D., Ward, C. W. & Brunt, A. A. (1994). The Potyviridae. Wallingford, UK: CAB International.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research 22, 4673-4680.[Abstract]
Yilmaz, M. A., Ozaslan, M. & Ozaslan, D. (1989). Cucumber vein yellowing virus in Cucurbitaceae in Turkey.Plant Disease 73, 610.
Zouba, A. A., Lopez, M. V. & Anger, H. (1998). Squash yellow leaf curl virus: a new whitefly-transmitted poty-like virus.Plant Disease 82, 475-478.
Received 24 February 2000;
accepted 6 June 2000.
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