Occurrence of Grapevine leafroll-associated virus 1 in Two Ornamental Grapevine Cultivars in Washington State

Roger's Red, an interspecific hybrid between wild grape (Vitis californica, native to northern California) and the V. vinifera cv. Alicante Bouschet (1), and Claret Vine (V. vinifera cv. Purpurea Nana) are grown for their ornamental value in home gardens and other settings. We collected potted grapevines of Roger's Red and Claret Vine showing dull green-to-scarlet red leaves from two different retail nurseries in the Richland-Kennewick area and Prosser, WA, respectively. Since these symptoms 'mimic' grapevine leafroll disease, we tested petiole samples from four grapevines per cultivar for a panel of grapevine-infecting viruses by single-tube one-step reverse transcription (RT)-PCR (4). All samples tested positive only for Grapevine leafroll-associated virus 1 (GLRaV-1; genus Ampelovirus, family Closteroviridae). To further confirm these results, total RNA was subjected to RT-PCR to amplify a portion of the heat shock protein 70 homolog (HSP70h), coat protein duplicate 2 (CPd2), and ORF 9 (p24) of GLRaV-1. RT was performed at 52°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 5 min final extension step at 72°C. Primers specific to HSP70h (HSP70h/416F: 5'-CAGGCGTCGTTTGTACTGTG and HSP70h/955R: 5'-TCGGACAGCGTTTAAGTTCC), CPd2 (CPd2/F: 5'-GTTACGGCCCTTTGTTTATTATGG and CPd2/R: 5'-CGACCCCTTTATTGTTTGAGTATG) and ORF 9 (p24/F: 5'-CGATGGCGTCACTTATACCTAAG and p24/R (5'-CACACCAAATTGCTAGCGATAGC) were designed based on GLRaV-1 sequence (GenBank Accession No. AF195822) to amplify 540, 398, and 633 base pair (bp) DNA fragments, respectively. To verify that the amplified products were specific to the genome of GLRaV-1, the amplicons were cloned into pCR2.1 vector (Invitrogen Corp, Carlsbad, CA) and three independent clones for each amplicon were sequenced in both directions. Pairwise comparison of HSP70h (Accession Nos. HQ833472 and HQ833473), CPd2 (Accession Nos. HQ833474 and HQ833475), and p24 (Accession Nos. HQ833476 and HQ833477) sequences from Roger's Red and Claret Vine showed 100, 96, and 99% identities, respectively, between them, and 86 to 100, 80 to 97, and 86 to 90% nucleotide sequence identities, respectively, with corresponding sequences of GLRaV-1 isolates deposited in GenBank. We further confirmed the presence of GLRaV-1 in these two ornamental grape cultivars by double antibody sandwich-ELISA using commercially available antibodies (Bioreba AG, Reinach, Germany). Previous studies have reported the presence of GLRaV-2 and -3 (1,3) and Grapevine virus A and B (2,3) in Roger's Red. To our knowledge, this study represents the first report of the occurrence of GLRaV-1 in two Vitis species distributed as ornamental grapes. It is important to prevent virus spread via the supply of virus-tested ornamental grapevines by commercial nurseries. References: (1) G. S. Dangl et al. Am. J. Enol. Vitic. 61:266, 2010. (2) D. A. Golino et al. Phytopathology (Abstr.) 99(suppl.):S44, 2009. (3) V. Klaassen et al. Online publication. doi:10.1094/PDIS-09-10-0621. Plant Dis., 2011. (4) T. A. Mekuria et al. Phytopathology (Abstr.) 99(suppl.):S83, 2009.