First report of Grapevine red blotch virus, the causal agent of grapevine red blotch disease, in Vitis vinifera in North Carolina.

Grapevine red blotch virus (GRBV), genus Grablovirus, family Geminiviridae, is the causal agent for grapevine red blotch disease (GRBD) (Al Rwahnih et al. 2013; Yepes et al. 2018). GRBV has been found in several wine-grape growing regions in the United States (Krenz et al. 2014), as well as in germplasm repositories (Al Rwahnih et al. 2015). However, the incidence of GRBV in parts of the southeastern United States is unknown. Initial observations between August and October of 2017 in North Carolina (NC) vineyards revealed that the commonly grown red cultivars Vitis vinifera ‘Merlot’, ‘Malbec’, and ‘Cabernet franc’ frequently exhibited leaf symptoms similar to those associated with GRBD. The symptoms were seemingly unrelated to location, management, or age of the plants. A total of 80 grapevine samples were collected from eight blocks in six vineyards (10 vines per block) in three different wine-grape growing regions in NC (Upper Hiwassee Highlands American Viticultural Area [AVA], Crest of the Blue Ridge Henderson County AVA, and Yadkin Valley AVA), following the recommended sampling strategy from Foundation Plant Services (FPS, University of California–Davis) (http://fps.ucdavis.edu/samplecollection.cfm). All samples were collected in October 2018. Blocks were selected based on the cultivar and the visibility of symptomatic leaves. Ten vines with leaf symptoms were selected randomly within each block. Three samples per grapevine were collected, sampling both cordons. Samples were directly stored at 4°C and shipped overnight to the NC State University Micropropagation and Repository Unit for further processing. Following FPS protocols, total nucleic acid (TNA) was extracted from leaf petiole tissue using the RNeasy Mini Kit (Qiagen), and samples were analyzed by quantitative reverse transcription PCR (RT-qPCR) for a panel of viruses known to infect grapevine, including GRBV. GRBV was identified in 21 samples of cultivars Merlot and Malbec in Yadkin and Surry Counties, NC (both Yadkin Valley AVA). All positive vines showed GRBD symptoms. To confirm the RT-qPCR results, aliquots of TNA from GRBV-positive samples were run by end-point PCR for further sequencing. GRBV-specific primers as described by Al Rwahnih et al. (2013) were used. RT-PCR yielded products of the expected size (557 bp) for all positive samples, and they were directly sequenced. All the positive samples shared 100% identity with 22 GRBV GenBank accessions, with the top hit being an isolate from Washington state (MF795177), based on BLASTN analysis. Complete genomes of three representative GRBV samples, one from each positive vineyard, were amplified using Phusion Hot Start Flex 2× Master Mix (NEB) and overlapping primers, GRBV-OL-F (5′-ATTCCTGCAGTTCTAGTGAAAG-3′) and GRBV-OL-R (5′-TAGAACTGCAGGAATCGC-3′). Sequencing of the 3.2-kb amplicons was done and the sequences deposited in GenBank (accession nos. MN186403 to MN186405). The GRBV sequences from NC shared 97 to 99% complete genome identities with isolates from Washington State (MF795161 and MF795177). Phylogenetic analysis revealed the clustering of the three NC isolates into GRBV clade 2 (Krenz et al. 2014). This report is part of a comprehensive survey of NC vineyards to study grapevine virus incidence in the state. The source of the virus is unclear; however, studies are underway to determine source and potential vector capacity in NC. To our understanding, this is the first time GRBV is being reported in NC.