Detection and molecular characterization of Italian <i>Grapevine rupestris stem pitting‐associated virus</i> isolates

Abstract

The efficiencies of two published primer pairs (RSP13‐RSP14 and RSP2‐RSP21) were compared in reverse transcription‐polymerase chain reaction (RT‐PCR) to improve the diagnosis of Grapevine rupestris stem pitting‐associated virus (GRSPaV), which has consistently been found associated with rupestris stem pitting (RSP), one of the most common infectious diseases of grapevine. Fifty‐six grapevine accessions from different locations in Northern Italy were tested. Of these, 48 were positive with primers RSP13‐RSP14 and 37 with primers RSP2‐RSP21, suggesting that primer pair RSP13‐RSP14 has a broader detection spectrum than primers RSP2‐RSP21. The coat protein (CP) gene was also amplified by RT‐PCR and the amplicons analysed by single‐strand conformation polymorphism (SSCP) and sequencing. The results support the existence of wide variability within this virus, independent of cultivar or geographic origin. Sequence analysis of the 17 Italian GRSPaV isolates examined showed a molecular identity ranging from 78·8 to 99·7%, within which, four groups of sequence variants were recognized. Flexuous rod‐shaped particles, approximately 800 nm in length, were detected in plant extracts from infected grapevines and were positively decorated with the specific antiserum As7‐276 in immunosorbent electron microscopy (ISEM). This is the first report of GRSPaV particles in grapevines observed by transmission electron microscopy (TEM) in Italy.