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Cucumber mosaic virus (CMV), the type member of the cucumovirus group, contains four positive sense single-stranded(ss) RNA species designated RNA1-4. CMV has a very broad host range and is one of the most widespread and economically important viruses. Numerous isolates (strains) of CMV have been reported on the basis of host range and symptoms1), but it is difficult to classify and identify genuine differences among them. Replicative form double stranded RNAs (RF-dsRNAs), that are twice the size of viral genomic ssRNA have been isolated from plants infected with RNA viruses2). Analytical results of the dsRNA species have been used for diagnosis as well as for virus classification3-6). This report deals with the comparison of the CMV isolates by dsRNA analysis, demonstrating the effectiveness of the method in such identification, and suggests that the individual isolates may be distinguished by this means. Six CMV isolates were isolated from naturally infected plants collected in Tochigi, Ibaraki, and Shizuoka prefectures and designated PU, TU, KU, IU, EU and SU, respectively, for this study (Table 1). All isolates were propagated in Nicotiana tabacum cv. Xanthi. DsRNAs were extracted from plants infected by the six isolates as described by Dodds and Bar-Joseph (1983)3). Frozen leaf tissue (5g) which had been stored at -80C was ground with a mortar and pestle to a fine powder and homogenized with 10ml of 2•~STE buffer (1/STE= 0.1M NaCl, 0.05M Tris-HCl, 1mM EDTA, pH7.0) containing 1% SDS, 0.1ml of 2-mercapto ethanol, 10ml of 80% phenol containing 0.1% 8-hydroxyquinoline, 10ml of chloroform and 0.4ml of pentanol. After the mixture was centrifuged at 8,000•~g for 10min, ethanol was added to a final concentration of 15%. Following a second centrifugation, 1.2g of CF-11 cellulose

Comparison of cucumber mosaic virus isolates by double-stranded RNA analysis.