Jarugula S, Soule M J, Rowhani A, Naidu R A
Department of Plant Pathology, Washington State University-Irrigated Agriculture Research and Extension Center, Prosser 99350.
Department of Plant Pathology, University of California, Davis 95616.
Plant Dis. 2008 Mar;92(3):485. doi: 10.1094/PDIS-92-3-0485C.
Grapevine leafroll disease (GLD) has been recognized as one of the major constraints to the production of wine grapes in Washington State. At least nine distinct Grapevine leafroll-associated viruses (GLRaV-1 to -9) have been detected in grapevines showing GLD symptoms in grape-growing areas of several countries. Previous studies documented the presence of GLRaV-1, -2, and -3 in Washington State (3). We initiated a program to test grapevine cultivars with GLD symptoms for the occurrence of the other GLRaVs. Leaf samples were collected from individual grapevines of red-berried grapevine cultivars showing typical GLD symptoms and tested by single-tube reverse transcription (RT)-PCR. Of nearly 300 samples from 13 cultivars in 19 vineyards, 14 samples from 5 cultivars (Cabernet Sauvignon, Merlot, Pinot Noir, Mourvedre, and Lagrein) in different vineyards tested positive for GLRaV-9 using primers LR9 F/F (5'-CGG CAT AAG AAA AGA TGG CAC-3') and LR9 R/R (5'-TCA TTC ACC ACT GCT TGA AC-3'), specific for the HSP-70h gene of GLRaV-9 (1). To confirm the identity of the RT-PCR products, the 393-bp amplicons obtained from each of these five cultivars were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparisons of these sequences (GenBank Accession Nos. EF101737, EF101738, EF101739, EF101740, and EU252530) with corresponding sequences of other GLRaVs in GenBank showed 94 to 100 and 96 to 100% identity at the nucleotide and amino acid level, respectively, with the sequence of HSP-70h gene of GLRaV-9 (GenBank Accession No. AY297819). Antiserum specific to GLRaV-9 was not accessible, therefore, an additional 540-nucleotide fragment specific to the coat protein (CP) gene of GLRaV-9 was amplified from cv. Lagrein using primers LR9-CP-F (5' TAC CGT CGA CAC TTT CGA AGC ACT 3') and LR9-CP-R (5' TGA GGC GTC GTA ACC GAA CAA TCT 3'). PCR amplified fragments were cloned and sequenced. A comparison of this sequence (GenBank Accession No. EU251512) with corresponding nucleotide sequences of other GLRaVs in GenBank showed 96% identity with CP of GLRaV-9 (GenBank Accession No. AY297819), further confirming the presence of GLRaV-9. Previously, GLRaV-9 was reported in grapevines in California (1), Tunisia (2), and Western Australia (4). To our knowledge, our results are the first evidence for the occurrence of GLRaV-9 in Washington State vineyards. Results from our study and previous reports (1,2,4) indicate the wide distribution of GLRaV-9 in several Vitis vinifera cultivars. The economic impact of GLRaV-9 on wine grape cultivars, however, remains to be determined. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) N. Mahfoudhi et al. Plant Dis. 91:1359, 2007. (3) R. R. Martin et al. Plant Dis. 89:763, 2005. (4) B. K. Peake et al. Aust. Plant Pathol. 33:445, 2004.
葡萄卷叶病（GLD）已被公认为是华盛顿州酿酒葡萄生产的主要限制因素之一。在几个国家葡萄种植区表现出GLD症状的葡萄藤中，至少检测到9种不同的葡萄卷叶相关病毒（GLRaV-1至-9）。先前的研究记录了华盛顿州存在GLRaV-1、-2和-3（3）。我们启动了一个项目，对有GLD症状的葡萄品种进行检测，以确定是否存在其他GLRaV。从表现出典型GLD症状的红浆果葡萄品种的单株葡萄藤上采集叶片样本，并通过单管逆转录（RT）-PCR进行检测。在来自19个葡萄园13个品种的近300个样本中，使用针对GLRaV-9的HSP-70h基因的引物LR9 F/F（5'-CGG CAT AAG AAA AGA TGG CAC-3'）和LR9 R/R（5'-TCA TTC ACC ACT GCT TGA AC-3'），对来自不同葡萄园5个品种（赤霞珠、梅洛、黑皮诺、慕合怀特和拉格瑞）的14个样本进行检测，结果显示GLRaV-9呈阳性（1）。为了确认RT-PCR产物的身份，将从这五个品种中每个品种获得的393-bp扩增子分别克隆到pCR2.1质粒（Invitrogen公司，卡尔斯巴德，加利福尼亚州）中。每个扩增子从两个方向对两个独立克隆进行测序。将这些序列（GenBank登录号EF101737、EF101738、EF101739、EF101740和EU252530）与GenBank中其他GLRaV的相应序列进行成对比较，结果显示在核苷酸和氨基酸水平上，分别与GLRaV-9的HSP-70h基因序列（GenBank登录号AY297819）有94%至100%和96%至100% 的同一性。由于无法获得针对GLRaV-9的抗血清，因此，使用引物LR9-CP-F（5' TAC CGT CGA CAC TTT CGA AGC ACT 3'）和LR9-CP-R（5' TGA GGC GTC GTA ACC GAA CAA TCT 3'）从拉格瑞品种中扩增出一段540个核苷酸的、特定于GLRaV-9外壳蛋白（CP）基因的片段。PCR扩增片段被克隆并测序。将该序列（GenBank登录号EU251512）与GenBank中其他GLRaV的相应核苷酸序列进行比较，结果显示与GLRaV-9的CP（GenBank登录号AY297819）有96%的同一性，进一步证实了GLRaV-9的存在。此前，在加利福尼亚州（1）、突尼斯（2）和西澳大利亚（4）的葡萄藤中报道过GLRaV-9。据我们所知，我们的结果是GLRaV-9在华盛顿州葡萄园出现的首个证据。我们的研究结果和先前的报告（1、2、4）表明GLRaV-9在几个酿酒葡萄品种中广泛分布。然而，GLRaV-9对酿酒葡萄品种的经济影响仍有待确定。参考文献：（1）R. Alkowni等人，《植物病理学杂志》86:123，2004年。（2）N. Mahfoudhi等人，《植物病害》91:1359，2007年。（3）R. R. Martin等人，《植物病害》89:763，2005年。（4）B. K. Peake等人，《澳大利亚植物病理学》33:445，2004年。
