First Report of Grapevine Red Blotch Virus in Mexico

Abstract

Grapevine red blotch virus (GRBV) (Al Rwahnih et al. 2013) is a member of the recently recognized genus Grablovirus in the family Geminiviridae (Varsani et al. 2017). GRBV is the causal agent of grapevine red blotch disease (Yepes et al. 2018), affecting viticulture in North America and some other regions around the world (reviewed in Sudarshana et al. [2015]). In this study, we sampled grapevine plants displaying red blotch symptoms in the major wine producing region of Mexico: Ensenada, Baja California, in 2016 and 2017. A total of 46 samples from cultivars Vitis vinifera L. ‘Pinot Noir’ and ‘Merlot’ were tested in 2016 by end-point polymerase chain reaction (PCR) using primers GRBV-V2F (5′-ATGGGTTAGGGGATGAGGCT-3′) and GRBV-V1R (5′-CGGCAATGACTCCTGCGGCT-3′). Results showed amplification of the expected 798-bp product from 31 samples. One of these amplicons was cloned, sequenced, and compared with reported sequences through a BLASTn search. The fragment had high nucleotide sequence identity (98%) with GRBV isolate BCRB5 from Canada (GenBank accession no. KX234092). The presence of GRBV was corroborated using the AmplifyRP Acceler8 kit (Agdia, Elkhart, IN), which detects the GRBV genome by isothermal recombinase polymerase amplification (RPA). In 2017 symptomatic V. vinifera ‘Nebbiolo’ plants were tested by multiplex PCR to amplify a region of the replicase-associated protein (Rep) gene with primers Repfor/Reprev (expected product 318 bp) and a fragment from the coat protein (CP) gene with primers CPfor/CPrev (expected product 257 bp) (Krenz et al. 2014). Amplicons from a positive sample were cleaned using a GenJet PCR purification kit (Thermo Fisher Scientific, Waltham, MA) and analyzed by Sanger sequencing. Both Rep and CP fragments shared >99% nucleotide sequence identity with GRBV isolates ONRB7 (KY316025) and ONRB4 (KY316022) from Canada (Poojari et al. 2017). To gain a deeper insight into the phylogeny of local GRBV isolates, we sequenced the full-length viral genome of 2016 and 2017 representative isolates. Each genome was amplified by high-fidelity PCR in two fragments, combining primers CPfor/Reprev (expected product 1.8 kb) and Repfor/CPrev (expected product 1.9 kb). After Sanger sequencing, the genomes were assembled in the GeneStudio sequence analysis software version 2.2.0.0 (GeneStudio, Suwanee, GA). A phylogenetic tree constructed after comparing the local isolates GRBV-Gpe-JGB (MH557096) and GRBV-Gpe-JCT (MH557095) to GRBV isolates from Canada and the United States grouped the Baja Californian isolates with clade 1 isolates. To our best knowledge, this is the first report of GRBV in Mexico.