Detection and molecular characterization of Italian Grapevine rupestris stem pitting-associated virus isolates

Grapevine rupestris stem pitting-associated virus (GRSPaV) has been reported and found to be consistently associated with Rupestris stem pitting (RSP), one of the most common grapevine infectious disease. In this work, two published primer pairs (RSP13-RSP14 and RSP2-RSP21), using total RNA from plant samples, have been compared to improve the diagnosis of this virus by reverse transcription-polymerase chain reaction (RT-PCR) technology. The RT-PCR procedure has been successful in detecting GRSPaV. Fifty six grapevine accessions from different locations of Northern Italy were tested. Forty eight were positive with the primer pair RSP13-RSP14 and 37 with the primers RSP2-RSP21. This study show that oligonucleotide pair RSP13-RSP14 has a broad spectrum detection for the virus compared to the primer pair RSP2-RSP21. RT-PCR amplification of the coat protein (CP) gene was also performed. The CP amplicons were then subjected to single–strand conformation polymorphism (SSCP) and sequence analysis. The results have suggested the existence of wide variability within this virus, independently of the cultivar or geographic origin. Sequencing data of the seventeen Italian GRSPaV isolates examined showed a molecular identity ranging from 78.8% to 99.7%. Four groups of variants were recognized. Flexuous rod particles, about 800 nm in length, were detected in plant extracts from infected grapevines and positively decorated with the specific antiserum As7-276 by immunosorbent electron microscopy (ISEM). This is the first report on GRSPaV particles in grapevines observed at the transmission electron microscope (TEM) in Italy.