First Report of Grapevine red blotch virus in Seven Vitis Species in a U.S. Vitis Germplasm Repository

Grapevine red blotch virus (GRBV), in the genus Grablovirus of the family Geminiviridae, was first identified on Cabernet sauvignon, Cabernet franc, and Zinfandel in California and in New York in 2012 (Al Rwahnih et al. 2013; Krenz et al. 2012). Subsequently the virus was found in an archival grape cultivar Abouriou that was collected in California in 1940, suggesting its presence in grapes for over 70 years (Al Rwahnih et al. 2015a). GRBV was also detected in 73 out of 156 table grape accessions in the National Clonal Germplasm Repository of table grapes in California (Al Rwahnih et al. 2015b). Currently, GRBV has spread widely among commercial vineyards, and was also found in free-living Vitis species and alternative hosts adjacent to vineyards. GRBV infection reduces grape quality and yield, and poses a threat to sustainable viticulture. The USDA National Plant Germplasm repositories in Geneva, NY, and Davis, CA, maintain Vitis germplasm belonging to 31 species within the Vitis genus. They were collected from primary native Vitis habitats of Eurasian, East Asian, and North American regions over decades from 1893 to 2000. These preserved resources are distributed to growers and researchers worldwide for breeding and genetic analysis of economically and agriculturally important traits of grapevine. Knowing the status of viruses in the repositories will aid the prevention of further spread of grapevine viruses. In this report, we applied the triplex PCR assay for detecting GRBV by using the two primer sets specific to the coat protein (CP) and replicase (Rep) region of GRBV that were designed and used in a well-developed protocol (Krenz et al. 2014). The grapevine 16S rRNA-specific DNA fragment was amplified in all samples, indicating the integrity of DNA extracted from the 380 samples. The DNA fragments of 257 bp for the GRBV CP gene and 318 bp for the Rep gene were detected in 16 of the 380 samples. The DNA fragments were purified and subjected to Sanger sequencing. The sequenced region shares 100% identical nucleotides with the CP region (nt 1,096 to 1,326) to 26 GRBV accessions (represented by KY426921.1), and 100% identical nucleotides in the Rep region (nt 2,570 to 2,880) to five GRBV accessions (represented by KY426922.1). The 16 GRBV-infected vines were identified from samples in the Davis germplasm collection and are of the species V. aestivalis, V. nesbittiana, V. biformis, V. monticola, V. blancoii, V. bloodworthiana, and V. amurensis. No Vitis species samples from the Geneva germplasm repository were infected with GRBV. Identification of GRBV in the Davis repository germplasm showed that GRBV is present in Vitis species other than V. vinifera. It is interesting to note that GRBV was found only in the Davis germplasm repository, suggesting that GRBV has spread into the repository germplasm most likely by the three-cornered alfalfa hopper vector (Spissistilus festinus) from vineyards or free-living Vitis species. It remains unknown if GRBV is present in wild Vitis species in their native habitats that are remote from commercial vineyards. Screening of GRBV in wild Vitis plants will help us to find crucial clues on the geographical origins and natural reservoirs of GRBV.