Grapevine rupestris stem pitting associated virus (GRSPaV) is a component of the rugose wood disease complex. The vast majority of collected samples through field surveys in Californian vineyards that showing rugose wood disease complex symptoms, were tested negative for viruses typically associated with graft union disorders, meanwhile 50% were found positive for GRSPaV using RT-PCR methodology. The dsRNA extracted from GRSPaV-positively tested samples were used as a template for cDNA construction. Molecular analysis of the several sequenced clones from different vines, using ClustalW bioinformatics program revealing significant variations in the virus genome. Two different GRSPaV-isolates named later as GRSPaV-SY and GRSPaV-PN had been identified, showing significant nucleotide sequence variability reached up to 29%. Specific pairs of primers were designed and optimized to detect each isolate specifically, using one step RT-PCR techniques. The use of these detection primers in studying the etiology of GRSPaV and correlation of the symptoms variation will be inevitable.
Declining cv. Pinot noir clone 23 plants on 3309 Couderc rootstock were observed in different vineyards in California exhibiting severe stunting, solid red leaves and rootstocks with stem necrosis-distortion. High molecular weight dsRNA of ca. 8.7 kbp was extracted from symptomatic vines, used as a template for cDNA libraries construction and sequencing and identified as a new strain of Grapevine rupestris stem pitting-associated virus designated as Pinot noir strain (GRSPaV-PN). Molecular characterization of GRSPaV-PN genome revealed that the complete nucleotide sequence was composed of 8,724 nt, excluding the poly-A tail. This strain shared nucleotide identity of 76% to 78% with other GRSPaV sequences present in the GenBank. Its genome organization included six open reading frames where its replicase gene had 76% and 85% nucleotide and deduced amino acid identities, respectively, to other GRSPaV sequences. The coat protein gene was the most conserved with nucleotide and amino acid identities of 81-88% and 91.8-95%, respectively. Limited field survey revealed that GRSPaV-PN was present mainly in Pinot noir clone 23. However, the causal nature of GRSPaVPN and 3309C stem necrosis-distortion is not known.