First report of grapevine leafroll-associated virus 3 in Vitis vinifera in North Carolina

Authors: HOFFMANN, M - North Carolina State University; TALTON, W - North Carolina State University; NITA, M - Virginia Polytechnic Institution & State University; JONES, T - North Carolina State University; AL RWAHNIH, M - University Of California, Davis; Sudarshana, Mysore; ALMEYDA, C - Virginia Polytechnic Institution & State University

Submitted to: Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2020
Publication Date: 11/24/2020
Citation: Hoffmann, M., Talton, W., Nita, M., Jones, T., Al Rwahnih, M., Sudarshana, M.R., Almeyda, C. 2020. First report of grapevine leafroll-associated virus 3 in Vitis vinifera in North Carolina. Journal of Plant Pathology. 103:385-386. https://doi.org/10.1007/S42161-020-00710-3.
DOI: https://doi.org/10.1007/S42161-020-00710-3

Interpretive Summary:

Technical Abstract: Grapevine leafroll disease (GLD) is associated with infections by several viruses that belong to three genera of the family Closteroviridae (Martelli, 2017). Grapevine leafroll-associated virus 3 (GLRaV-3) belongs to the genus Ampelovirus and is considered the economically most important virus in the GLD complex (Maree et al., 2013). GLRaV-3 can be transmitted through grafting, soft scale insects (Hemiptera: Coccoidae) and mealybugs (Hemiptera: Pseudococcoidae). While GLD is known to be present in most major grape growing regions in the US, little is known about the incidence of GLRaV in North Carolina (NC). Initial observations over the second half of 2017 in NC vineyards revealed that red grape cultivars exhibited leaf symptoms similar to those associated with GLD. Observed symptoms encompassed curled leaf edges and reddening of leafs. In October 2018, leaf petiole samples were collected in the Yadkin Valley American Viticulture Area (AVA), Crest of the Blue Ridge Henderson County AVA (CBRH) and the Upper Hiwassee Highlands AVA, following Foundation Plant Services procedures (FPS, UC Davis; http://fps.ucdavis.edu/samplecollection.cfm). A total of 80 samples were collected from 8 blocks in a total of 6 vineyards. Blocks were selected based on cultivar and visibility of symptomatic leaves. 10 individual vines were randomly selected in each block. In total, 10 vines of Vitisvinifera cv. ‘Cabernet Franc’, ‘Nero D’Avola’ and ‘Malbec’, 40 vines of ‘Merlot’, and 10 French-American ‘Chambourcin’ were sampled. Three petiole samples per grapevine were collected, sampling both cordons. Samples were immediately stored at 4°C and shipped overnight to the Micropropagation and Repository Unit at NCSU for further processing. Total nucleic acid was extracted from petiole tissue using guanidine thiocyanate buffer and a modified RNeasy Mini Kit (Qiagen) protocol (Osman et al., 2007). GLRaV-3 was detected by RT-qPCR using TaqManTM Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) using primers and probe sequences as described in the FPST assay (Diaz-Lara et al., 2018). GLRaV-3 was detected in 20 out of 80 samples. The presence of GLRaV-3 was confirmed using end-point RT-PCR and two primer sets to detect the coat protein (CP) gene and the homologous heat-shock protein 70 (hHSP70) gene (Klaassen et al., 2011). Complimentary DNA was made using SuperScript II reverse transcriptase and random hexamers (Thermo Fisher Scientific). OneTaq HotStart Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs) was used to amplify a 612 bp (CP) and 546 bp (hHSP70) products. Thermal cycling conditions were an initial heating to 94' for 30 seconds followed by 35 cycles of 94' for 30 seconds, annealing at 56' (CP) or 54' (hHSP70) for 30 seconds, and 68' for 1 minute; and a final extension of 68' for 5 minutes. GLRaV-3 was identified in 10 samples of ‘Merlot’ and 10 samples of ‘Cabernet franc’, both samples collected in CBRH. All 20 RT-qPCR positive samples amplicons were Sanger sequenced for the CP gene (GenBank: MK947125) and the hHSP70 gene (GenBank: MK947126). Sequenced CP and hHSP70 gene regions matched 99.98% among samples, and shared 99.84 % and 99.82% identity respectively to 25 GLRaV-3 public sequences (Top hit: accession numbers MH521111.2 for CP; MH814489.1 for hHSP70). Both fragments shared 99% homology with Group I isolates of GLRaV-3 (Diaz-Lara et al., 2018). While mealybug populations were observed in the affected vineyards, to this date the source of the virus is unclear.