Grapevine Rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the family Flexiviridae. GRSPaV is closely associated with the disease Rupestris stem pitting and is frequently detected in grapevines worldwide. Previous research in several laboratories suggests that GRSPaV consists of a family of sequence variants. However, the genetic composition of GRSPaV variants in viral isolates from scion and rootstock varieties has not been studied extensively. In this report, the genetic diversity and population structure of GRSPaV isolates from scion and rootstock varieties were analysed using two pairs of primers targeting two different genomic regions encoding the helicase domain of the replicase and the capsid protein. In total, 190 cDNA clones derived from 24 isolates were sequenced and analysed. At least four major groups of GRSPaV variants were found to exist in grapevines. Interestingly, the majority of the scion varieties (9/10) that were analysed, regardless of their genetic background and geographical origin, harboured complex viral populations composed of two to four distinct viral variants. In contrast, the viral populations in isolates from rootstock varieties were homogeneous and comprised a single variant. The practice of grafting between scion and rootstock varieties commonly used in modern viticulture, coupled with the frequent regional and international exchange of propagating materials, may have played a major role in the ubiquitous distribution and mixed infections of distinct GRSPaV variants among scion varieties. The possible origin and evolution of GRSPaV are also discussed.

Isolation of pure RNA from woody perennials, especially fruit crops such as grapevine rich in complex secondary metabolites, has remained very challenging. Lack of effective RNA isolation technology has resulted in difficulties in viral diagnosis and discovery as well as studies on many biological processes of these highly important woody plants. It is imperative to develop and refine methodologies with which large amounts of pure nucleic acids can be readily isolated from woody perennials. We compared five commonly used RNA isolation kits in isolating total RNA from twelve species of woody perennials. We made modifications to select RNA isolation systems to simplify and improve their efficiency in RNA isolation. The yield and quality of isolated RNAs were assessed via gel electrophoresis and spectrophotometric measurement. We also performed RT-PCR and RT-qPCR to detect several major viruses from grapevines. Two of the kits were shown to be the best in both the yield and quality of the isolated RNA from all twelve woody species. Using disposable extraction bags for tissue homogenization not only improved the yield without affecting quality, but also made the RNA isolation technology simpler, less costly, and suitable for adoption by many potential users with facility limitations. This system was successfully applied to a wide range of woody plants, including fruit crops, ornamentals and timber trees. Inclusion of polyvinylpyrrolidone in the extraction buffer drastically improved the performance of the system in isolating total RNA from old grapevine leaves collected later in the season. This modification made our system highly effective in isolating quality RNA from grapevine leaves throughout the entire growing season. We further demonstrated that the resulting nucleic acid preparations are suitable for detection of several major grapevine viruses with RNA or DNA genomes using PCR, RT-PCR and qPCR as well as for assays on plant microRNAs. This improved RNA isolation system would have wide applications in viral diagnostics and discovery, studies on gene expression and regulation, transcriptomics, and small RNA biology in grapevines. We believe this system will also be useful in diverse applications pertaining to research on many other woody perennials and recalcitrant plant species.

In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario. A total of 657 composite leaf samples representing 3285 vines collected from 137 vine blocks of 33 vineyards from three appellations: Niagara Peninsula, Lake Erie North Shore and Prince Edward County. These samples covered six major red cultivars and five major white grape cultivars. Using a multiplex RT-PCR format, we tested these samples for 17 viruses including those involved in all major viral diseases of the grapevine, such as five grapevine leafroll-associated viruses (GLRaV-1, 2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine Pinot gris virus (GPGV), grapevine rupestris stem sitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine fleck virus (GFkV), arabis mosaic virus (ArMV), tomato ringspot virus (ToRSV), trapevine fanleaf virus (GFLV), among others. Fourteen of the 17 viruses were detected from these samples and the predominant viruses are GRSPaV, GLRaV-3, GFkV, GPGV and GRBaV with an incidence of 84.0, 47.9, 21.8, 21.6 and 18.3%, respectively. As expected, mixed infections with multiple viruses are common. 95.6% of the samples included in the survey were infected with at least one virus; 67% of the samples with 2–4 viruses and 4.7% of the samples with 5–6 viruses. The major grape cultivars all tested positive for these major viruses. The results also suggested that the use of infected planting material may have been one of the chief factors responsible for the recent outbreaks of viral diseases across the province. This is the first such comprehensive survey for grapevine viruses in Ontario and one of the most extensive surveys ever conducted in Canada. The recent outbreaks of viral diseases in Ontario vineyards were likely caused by GLRaV-3, GRBV and GPGV. Findings from this survey provides a baseline for the grape and wine industry in developing strategies for managing grapevine viral diseases in Ontario vineyards.

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

Grapevine rupestris stem pitting-associated virus (GRSPaV), a member of the genus Foveavirus within the family Flexiviridae, is the putative causal agent of the disease Rupestris stem pitting (RSP) of grapevines. GRSPaV comprises a family of variants whose pathological characteristics have not been determined. Recently, many of the indicator ‘St George’ plants (Vitis rupestris) used throughout the world to index RSP tested positive for GRSPaV. This finding questions the validity of past biological indexing results. In this work, a representative genomic region of GRSPaV was first sequenced from ten ‘St George’ plants from two sources and it was demonstrated that nine of them carried a new variant, GRSPaV-SG1. The genomes of GRSPaV-SG1 and GRSPaV-BS from ‘Bertille Seyve 5563’ plants were sequenced, revealing a genome structure identical to that of GRSPaV-1. It was demonstrated experimentally that infection of ‘St George’ plants with GRSPaV-SG1 is asymptomatic and thus it is proposed that GRSPaV-SG1 infection should not have interfered with the outcome of past indicator indexing. This represents the first attempt to link a GRSPaV variant with pathological properties.