A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries
Author:
Nita Mizuho12ORCID, Jones Taylor1, McHenry Diana1, Bush Elizabeth1, Oliver Charlotte1, Kawaguchi Akira3ORCID, Nita Akiko1, Katori Miyuki2
Affiliation:
1. Alson H. Smith Jr. Agricultural Research and Extension Center, Virginia Polytechnic Institute and State University (Virginia Tech), Winchester, VA 22602, USA 2. Department of Law and Economics, Shinshu University, Nagano 390-8621, Japan 3. National Agriculture and Food Research Organization (NARO), Western Region Agricultural Research Center, Hiroshima 721-8514, Japan
Abstract
We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020–2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.
Funder
Japan Grants-in-Aid for Scientific Research
Subject
Virology,Infectious Diseases
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