BIOLOGY AND SPREAD OF GRAPEVINE RED BLOTCH-ASSOCIATED VIRUS Principal Investigator: Co-Principal Investigator: Collaborator:

Grapevine red blotch-associated virus (GRBaV) was isolated from table and wine grapes, as well as rootstocks, affected by red blotch, a recently recognized viral disease. Phylogenetic analyses revealed the clustering of GRBaV isolates two groups (clades) of genetic variants (Krenz et al., 2014, Al Rwahnih et al., 2015). Producing and using a full-length infectious clone of a representative isolate of each of the two clades showed systemic GRBaV infection of healthy grapevines following agroinoculation and the manifestation of typical disease symptoms, i.e. interveinal reddening on red-berried cultivars and chlorotic and necrotic leaf areas on whiteberried cultivars, while infection was latent in rootstocks with the exception of SO4. This work demonstrated that GRBaV isolates of both clades cause red blotch disease. Analysis of the spatio-temporal incidence of GRBaV in a selected vineyard of Cabernet franc in California was consistent with the occurrence of virus spread. GRBaV isolates spreading in California corresponded to phylogenetic clade 2. A survey of alternate hosts in proximity to the diseased Cabernet franc vineyard showed that free-living grapevines in riparian areas are infected with GRBaV (Perry et al., 2016). The GRBaV isolates from free-living grapevines, some of which being fingerprinted as hybrids of Vitis californica x Vitis vinifera cv. Sauvignon blanc, belonged to phylogenetic clade II, as did most of the GRBaV-infected vines in adjacent Cabernet franc and Merlot vineyards. The presence of GRBaV in freeliving grapevines suggests the existence of a hemipteran vector. Insect sticky traps placed in the section of the California vineyard with extensive clustering of diseased vines in 2014 and 2015 showed a diversity of insect families, genera and species that visited the vineyard, among which, the majority of specimens of four species consistently tested positive for GRBaV in PCR. These four species are vector candidates and their potential to transmit GRBaV in controlled conditions in the greenhouse is investigated. Among the four vector candidates, Spissistilus festinus the three cornered alfalfa treehopper was shown to transmit GRBaV from infected to healthy vines in the greenhouse. This finding revealed the potential of this treehopper as a vector of epidemiological significance. LAYPERSON SUMMARY Red blotch is a newly recognized viral disease of grapevines that is widely distributed in U.S. vineyards. Limited information is available on spread of its associated virus called Grapevine red blotch-associated virus (GRBaV). Similarly, limited information is available on the association between virus variability and pathogenicity. We showed that GRBaV isolates cause red blotch disease, regardless of their genetic makeup and variability. Studying changes in virus prevalence over time in selected vineyards of Cabernet franc in California and New York revealed an increased virus incidence in the California but not in the New York vineyard. Free-living grapevines proximal to diseased vines in the California vineyard were found infected with GRBaV, suggesting their potential role as alternate host. Among insects visiting the California vineyard, four species were found to carry the virus, suggesting a potential role as vector. Subsequent work in the greenhouse showed that one of these vector candidates, the three cornered alfalfa treehopper (Spissistilus festinus) transmits GRBaV from infected to healthy vines, revealing this treehopper is likely a vector of epidemiological importance in vineyards. INTRODUCTION Red blotch is a recently recognized disease of grapevines (Calvi 2011; Sudarshana et al., 2015). It was described for the first time on Cabernet Sauvignon at the UC Oakville Research Field Station in 2007 (Calvi 2011). Leaves of GRBaV-infected vines of red wine grapes show red specks and blotches first on old leaves at the bottom of the canopy in late June or July. Symptoms progressively appear upward in the shoots over time. Veins underneath the leaf blade often turn partly or fully red. For white wine grapes, foliar symptoms are less conspicuous; they correspond to localized and generalized foliar discoloration or chlorosis, sometimes combined with necrotic areas 2 at the edge of leaf blades (Sudarshana et al., 2015). Diagnosis based on specific symptoms can be challenging because of several confounding factors, including striking similarities between foliar symptoms elicited by red blotch and leafroll. There are also similarities between foliar symptoms of red blotch and abiotic factors such as poor root health, or physical injuries due to trunk or shoot girdling, mite damage, mineral deficiencies, or even the presence of Xyllela fastidiosa or Agrobacterium tumefaciens in young vines. Because symptom variation makes visual diagnosis of GRBaV-infected vines difficult, only DNA-based assays such as PCR are reliable for accurate diagnosis (Sudarshana et al., 2015). GRBaV was isolated from grapevines affected by red blotch disease (Sudarshana et al., 2015). This virus is a putative member of a new genus in the family Geminiviridae (Varsani et al., 2014; Sudarshana et al., 2015). The new genus is tentatively named Grablovirus (Zerbini, personal communication). GRBaV has a single-stranded DNA genome that codes for seven open reading frames (Al Rwahnih et al., 2013; Krenz et al., 2012; Perry, unpublished; Poojary et al., 2013; Seguin et al., 2014). Efforts to investigate the role of GRBaV in the etiology of red blotch disease showed that GRBaV is the causal agent of red blotch disease (Fuchs and Perry, unpublished). GRBaV was documented in major grape-growing US States (Krenz et al., 2014). The virus was also reported in British Columbia and Ontario (Poojari et al., 2016) in Canada, and in a Vitis germplasm collection (Al Rwahnih et al., 2015a), indicating its widespread presence in North America. GRBaV was found in table grapes, wine grapes, French-American interspecific hybrids, and rootstocks (Al Rwahnih et al., 2015a; Sudarshana et al., 2015). The widespread occurrence of GRBaV and its wide geographic distribution in North America suggest that propagation material has played a significant role in its dissemination. The virus was also found in an archival sample (Al Rwahnih et al., 2015b). Analysis of the genetic diversity among isolates of GRBaV indicated the existence of two groups (clades) of genetic variants (Krenz et al., 2014). The majority of isolates belong to the predominant clade II and recombination is underlying some of the variation seen among GRBaV genomes within clade I. Most vineyard managers and vintners report ripening issues with GRBaV-infected wine grapes. Reductions of 16°Brix have been consistently documented in fruits of infected vines, as well as lower berry anthocyanin and skin tannins, particularly in red wine grapes such as Cabernet franc and Cabernet Sauvignon (Calvi 2011; Sudarshana et al., 2015). Based on the effect of GRBaV on fruit quality and ripening, several growers are culling infected vines and replacing them with clean, virus-tested ones. Free-living grapevines proximal to vineyards were found infected with GRBaV (Bahder et al, 2016a; Perry et al., 2016). The GRBaV isolates in free-living grapevines was genetically related to clade II isolates in proximal Cabernet franc and Merlot vineyards (Perry et al., 2016). The presence of the virus in a potential alternative host that is at least 150 ft away from the natural host suggested the existence of a hemipteran vector. The ziczac leafhopper (Virginia creeper; Erythroneura ziczac) was claimed to transmit GRBaV from vine to vine in the greenhouse (Poojari et al., 2013) but a vector of GRaBV of epidemiological significance in vineyards remains to be identified. OBJECTIVES The overarching goal of this project is to advance our understanding of red blotch disease and its causal agent, GRBaV, with a major emphasis on horizontal spread in vineyards and optimized detection methodologies. Our specific objectives are to: 1. Investigate spread of GRBaV in selected vineyards in California and New York 2. Improve diagnostics for GRBaV 3. Determine if either of the two groups of GRBaV isolates show greater virulence and pose an increased threat to vineyard production 4. Disseminate research results to farm advisors and the industry RESULTS AND DISCUSSION