Comprehensive Grapevine Virus Screening

During the fall of 2017, a total of 2,415 plant samples (S1 Table) were collected from grapevine populations with a historically high incidence of GLRaV-3 or with observable GLD symptoms such as interveinal reddening and downward rolling of leaf margins in red wine cultivars, and interveinal chlorosis and downward rolling of leaf margins in white cultivars [1 ]. These grapevine populations included: 1) the USDA National Clonal Germplasm Repository (NCGR) in Winters, CA, in which a previous study [18 (link)] identified plants infected with GLRaV-3 and originating from 12 different countries (1,206 samples); 2) the Davis Virus Collection (DVC) at University of California-Davis [21 ], which primarily consists of domestic GLRaV-3 isolates and is also the source of an isolate which is 99% identical to the GH24 isolate (109 samples); 3) the FPS pipeline of foreign and domestic introductions (417 samples); and 4) 89 vineyards in the main grape-growing areas of California including 77 samples from Napa Valley, 14 samples from Sonoma, 44 samples from San Luis Obispo, 39 samples from Monterey, 156 samples from Central Coast, 10 samples from Coachella Valley, 70 samples from the North Coast, 164 samples from the San Joaquin Valley and 109 samples from the Central Sierra region. Additionally, 45 samples from different GLRaV-3 collections outside of the USA were included in this study. These included 23 South African grapevines from eight different selections that represent the different GLRaV-3 variant groups present in that country; six samples originated from New Zealand, where several new GLRaV-3 isolates have been reported recently [8 (link)]; seven GLRaV-3 positive plants originated from Australia that showed mild leaf roll symptoms [22 ]; an asymptomatic GLRaV-3 infected ‘Pinot noir’ vine originating from Spain; and lastly, eight grapevine samples determined positive for GLRaV-3 by end-point RT-PCR and HTS during a study validating this technology for virus detection in Canada, including a sample (ON936) from a ‘Vidal Blanc’ vine infected with a putative new GLRaV-3 variant based on preliminary sequence analysis. Positive controls (described above) were included during the testing process, as well as a grapevine that tested negative by HTS for viruses and virus-like pathogens.
All the above-mentioned samples (leaf petioles or bark scrapings) were subjected to TNA extraction: 0.2 g plant tissue was homogenized in 2 ml of guanidine isothiocyanate lysis buffer (4 M guanidine isothiocyanate; 0.2 M sodium acetate, pH 5.0; 2 mM EDTA; 2.5% (w/v) PVP-40) and TNA extracts were prepared using a MagMAX-96 viral RNA isolation kit (Ambion, Austin, TX, USA) as per the manufacturer’s protocol. Subsequently, the integrity of RNA was verified using an 18S rRNA assay [18 (link)].