First Report of Grapevine Red Blotch Virus in Idaho Grapevines

Wine grape production in Idaho occurs on approximately 1,300 acres, predominately in Canyon County in the southwest and Nez Perce County in the northwest (Idaho Wine Commission 2019). Two viruses were previously reported to affect wine grapes in the state, grapevine leafroll-associated virus 3 (Mekuria et al. 2009; Thompson et al. 2019) and grapevine fleck virus (Kanuya et al. 2012). Grapevine red blotch virus (GRBV) causes the red blotch disease in wine grapes (Sudarshana et al. 2015; Yepes et al. 2018) and belongs to the genus Grablovirus, family Geminiviridae, comprising single-stranded DNA viruses with a 3.2-kb genome (Zerbini et al. 2017). In the United States, GRBV was reported to occur in California and several other wine grape growing states (Krenz et al. 2014; Poojari et al. 2013; Sudarshana et al. 2015). In September of 2014 and 2015, a survey of wine grapes was conducted in Canyon and Nez Perce Counties of Idaho for the presence of GRBV. A total of 58 samples of red wine grape cultivars were collected in 2014 and again in 2015, along with an additional 46 collected in 2015 for a total of 104 vines sampled, based on visual symptoms of leaf reddening, and tested by PCR using two newly designed GRBV-specific primers, GRLBV-f5 (5′-TGCAAGTGGACATACGTTTA-3′) and GRBLV-R9 (5′-GGGATCCCATCAATTGTTCT-3′). Three grapevines were found positive by PCR producing the DNA fragment of expected size, 720-bp; all three positive samples came from a single vineyard in Canyon county, from the same wine grape cultivar, Syrah. The three PCR products were cloned into the pGEM-T Easy plasmid vector (Promega), sequenced, and confirmed to represent a fragment of the GRBV CP gene between positions 1,313 and 2,031 in the genome of GRBV, 99.8% identical to isolate GiGV-WA-MR (GenBank accession no. KC427995). To expand the GRBV survey, 434 random grapevine samples collected in 2009 to 2011 in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce Counties (Kanuya et al. 2012) were reanalyzed for the presence of GRBV; six additional GRBV-positive samples coming from two additional vineyards (Canyon County) and two additional grapevine cultivars, Merlot and Petite Sirah, were identified by PCR. To analyze the full-length sequences for GRBV, circular DNA present in the grapevine samples was enriched with rolling circle amplification (RCA) using the Illustra TempliPhi kit (GE Healthcare Life Sciences, U.K.). Based on the GRBV-specific sequence obtained, two abutting primers were synthesized, and the entire genome of GRBV was amplified from five GRBV-positive, RCA-enriched samples representing two grapevine cultivars, Syrah (one sample collected in 2011 and three in 2014) and Merlot (one sample collected in 2011), from two different vineyards about 50 km apart. The five whole genomes for these GRBV isolates were sequenced in DNA plasmids by Sanger methodology and subjected to phylogenetic analysis. All whole genomes sequenced (GenBank accession nos. MK928382 to MK928386) were assigned to clade 2 of GRBV (Krenz et al. 2014), most closely related to a group of GRBV isolates from Washington State (Adiputra et al. 2018). This phylogeny of the Idaho GRBV isolates suggests the introduction of GRBV to Idaho from the same infected source, probably through infected planting material. Visual observations and PCR tests for the virus in several vines adjacent to GRBV-positive Syrah plants in a single vineyard conducted in 2014 to 2017 suggested limited if any spread of the virus outside of the original three GRBV-positive plants. Idaho grape growers need to be aware of the presence of GRBV in the state. This is the first report of GRBV in wine grapes in Idaho.