First Report of Grapevine Red Blotch Virus Causing Grapevine Red Blotch Disease in New England Vineyards

Grapevine red blotch virus (GRBV), a member of the Grabovirus genus in the Geminiviridae family, is the causative agent of grapevine red blotch disease (Al Rwahnih et al. 2013). This disease affects the production and quality of grapevines (Vitis vinifera L.) (Krenz et al. 2023) and has been reported in several grape-producing states across the US (EPPO 2025), but not in the Southeastern New England American Viticultural Area (SNE-AVA). During an extensive survey conducted from 2018 to 2020 on various grapevine RNA viruses in SNE-AVA vineyards (Dumas et al. 2023), we noticed that 28 leaf samples from five vineyards (two from Connecticut, one from Massachusetts, and two from Rhode Island) from plants displaying symptoms similar to leafroll disease (redness, yellowing, leaf distortion, and chlorotic mottling) were negative for all RNA viruses tested. Total RNA was extracted from the five vineyard samples and pooled into a composite sample (0.5µg of each RNA sample). The composite sample was used for library preparation following the ribosomal RNA depletion protocol (NEB Waltham, MA) and sequencing on an Illumina NextSeq2000 platform (producing 150 bp paired-end reads). A total of 66,300,500 clean reads were obtained and assembled into contigs using Megahit version 1.1.2 (Li et al. 2015). Nine contigs demonstrated a nucleotide identity ranging from 84% to 97% with the GRBV isolate CF214-1 (NC_022002), while none were mapped to other viral or viroidal sequences in GenBank. Total DNA was extracted from all 28 samples and amplified using rolling circle amplification (RCA) with random primers. The RCA product was used as a template for the PCR primers 1402c/1404v and 2287v/604c (Ouro-Djobo et al. 2023) targeting the GRBV genome. PCR products were Sanger sequenced and assembled on SnapGene software (Dotmatics; version 7.2) using the GRBV isolate CF214-1 (Al Rwahnih et al. 2013) as the reference genome. Multiple sequence alignment was conducted using ClustalW, and phylogenetic trees were constructed using the Maximum likelihood method based on the Tamura-Nei model on MEGA-11 Software (Version 11.0.3). Eighteen of the 28 samples analyzed tested positive for GRBV by PCR and were confirmed by blasting analysis results using the sequences acquired. The whole genome sequence (3204 bp) was acquired for seven isolates (PQ601719-PQ601725) and partial genome sequences (~1409 bp) for eleven isolates (PQ601707-PQ601718). Phylogenetic analyses of the sequences acquired in this study separated the SNE-AVA isolates into two distinct phylogenetic clades: sixteen isolates grouped in clade 1. In contrast, only two isolates, one from CT and another from RI, were grouped in clade 2. To the best of our knowledge, this is the first report of GRBV in the SNE-AVA vineyards. This report highlights the need to instate a grapevine testing program in NE to improve the sanitary status of planting material.