Detection of grapevine leafroll associated viruses belonging to a distinct lineage within the ampelovirus genus
2009
Maliogka, V., Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki (Greece) | Dovas, Ch., Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine. Aristotle University of Thessaloniki (Greece) | Katis, N., Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki (Greece)
Leafroll disease (GLRD) is one of the most important grapevine diseases worldwide. In this study, a nested RT-PCR was developed, that allows the generic detection of a subgroup of GLRD-related ampeloviruses with a distinct evolutionary history within the genus Ampelovirus. Members of this lineage are GLRaV-4,- 5,-6,-9 and two isolates (GLRaV-De and GLRaV-Pr) that have been recently characterised and represent new species. The method involves a one step RT-PCR for the generic detection of Closteroviridae species using degenerate primers that target the HSP70h gene followed by a nested PCR, which detects all virusesmembers of the lineage and differentiates them from the other grapevine closteroviruses. The 490 bp nested PCR amplicons, corresponding to a phylogenetically informative region, can be sequenced directly to obtain initial genetic information for their partial characterization and rapid classification. Additional primers were designed, using a large dataset of partial HSP70h sequences, and successfully applied for the specific detection of GLRaV-4,-5,-6,-Pr and -De on respective single oras: multiplex nested PCR assays. The application of a ramped annealing thermal profile allowed all amplifications to run in parallel. A number of field-grown grapevine plants were tested using the generic coupled with the specific assays and the results confirmed their specificity and broad detection range. The proposed detection scheme can also be used for the enrichment of sequence information of known and novel ampeloviruses, classified within this lineage, enabling their selective amplification in mixed Closteroviridae virus infections.
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