Development of simple method for diagnosis of apple virus diseases by using gene-manupulation technique

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The present study was undertaken to develop the method for rapid diagnosis of diseased apple trees by using complementary DNA (cDNA) of causal viruses of apple topworking disease, particularly apple chlorotic leaf spot virus (ACLSV) and apple stem grooving virus (ASGV). The results obtained are as follows: 1. ACLSV and ASGV contained a single RNA species with molecular weight of 2.48 x 10^6 and 2.30 x 10^6, respectively. The 3'termini of both RNA species were polyadenylated. 2. Cloning of virus RNA was made; Plasmid vector inserting first- and second-strand cDNA were transformed into Escherichia coli, then cDNA of them was removed from vector by restriction enzyme digestion. cDNA clones thus obtained were corresponded to full length for ACLSV-RNA and 96% length for ASGV-RNA. 3. Method for hybrid formation between ACLSV (or ASGV) and its cDNA was established. From dot hybridization using ^<32>P-labeled cDNA of each virus, it was evident that limitation for detection by cDNA probe method was 1.6ng per spot for ACLSV or ASGV and 5.12 pg per spot for ACLSV-RNA. 4. Application of cDNA probe method to routine apple virus-indexing was investigated. When young leaves and petals collected in May was tested, specific hybrid formation for both viruses was observed. However, in old leaves, barks or fruit skins collected in August and November, any virus could not be detected so far. This method allowed to apply the certification of virus-free apple seedlings which have been propagated after meristem-tip culture.The cDNA probe method takes 3-4 days to perform in vitro, and principally can apply to other virus diagnosis, such as apple mosaic disease.