Molecular and biological analysis of two Rupestris stem pitting-associated virus (RSPaV) isolates obtained from diseased Syrah and Pinot Noir grapevines

Rupestris stem pitting disease (RSP) is a graft-transmissible disorder of grapevines and one of the components of the Rugose Wood Complex (RW). Rupestris stem pitting-associated virus, which is a single-stranded positive sense polyadenylated RNA, has been associated with RSP. This virus belongs to the genus Foveavirus in the family Flexiviridae. Surveys in California vineyards revealed that some Syrah and Pinot Noir vines grafted on certain rootstocks were performing poorly. These plants showed red canopy, stunting, bark-splitting, union swellings and stem lesions on the rootstock starting at the graft union. Analysis of double-stranded RNA patterns obtained from these declining vines revealed the presence of dsRNAs of ca. 8.7 kbp, which were the template for making cDNA libraries. Two new RSPaVs were found in declining Syrah (RSPaV-SY) and Pinot Noir (RSPaV-PN) grapevines. The genome sequences of both isolates revealed six open reading frames, similar to other RSPaV isolates that have been previously characterized. The complete nucleotide sequences of RSPaV-SY and RSPaV-PN shared percent identities from 76% to 78% with RSPaV sequences in GenBank. The replicase gene was found to be the most variable gene and the coat protein gene the most conserved in the genome. Phylogenetic analysis of partial coat protein and replicase nucleotide sequences of 26 isolates showed that RSPaV-SY and RSPaV-PN clustered independently from the majority of RSPaV isolates. Strain-specific PCR primers designed from the replicase gene for specific detection of RSPaV-SY (SY9F/8R) or RSPaV-PN (PN1F/2R) and used in a survey of commercial vineyards indicated that SY9F/SR and PN1F/2R primers were specific in detecting these two variants. Biological indexing on St. George, LN33 and Kober 5BB indicator hosts, showed typical RSP symptoms, pitting and/or grooving developed under the point of inoculation or around the wood cylinder of the rootstock St. George, confirming the association of these two viruses with RSP disease. No symptoms were observed on healthy controls and on LN33 and Kober 5BB rootstocks. We also proved that RSPaV was seed-transmitted to seedlings. RSPaV was detected in intact seeds and seedlings at 18 days, 4.5 and at 11 months after transplanting.