DETECTION OF APPLE STEM GROOVING VIRUS IN DORMANT APPLE-TREES WITH CRUDE EXTRACTS AS TEMPLATES FOR ONE-STEP RT-PCR

Partial nucleotide sequences of amplification products obtained from f our European apple stem grooving virus (ASGV) isolates using degenerat e primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Base d on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were d esigned to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV i solates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detec ted by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to co nstitute good templates for reliable amplification of ASGV sequences f rom leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermor e, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark t issues of apple trees during both active growth and the dormant season .