Mekuria T, Martin R R, Naidu R A
Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser 99350.
USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR 97330.
Plant Dis. 2008 Aug;92(8):1250. doi: 10.1094/PDIS-92-8-1250A.
Grapevine fanleaf virus (GFLV; genus Nepovirus, family Comoviridae), responsible for fanleaf degeneration disease, is one of the most important viruses of grapevines worldwide (1). During our reconnaissance studies during 2007, dormant wood cuttings from individual grapevines of wine grape cv. Chardonnay were collected randomly from two geographically separate vineyards in eastern Washington State. Extracts made from cambial scrapings of these cuttings were tested separately for different viruses by single-tube reverse transcription (RT)-PCR using virus-specific primers. Two of the thirty-one grapevines in one vineyard tested positive for GLFV as mixed infection with Grapevine leafroll-associated virus (GLRaV)-3. In another vineyard, six of the twenty-six grapevines tested positive for GFLV as mixed infection with GLRaV-1, GLRaV-3, and Grapevine virus A (GVA) A forward primer (5'-ACCGGATTGACGTGGGTGAT, corresponding to nucleotides [nt] 2231-2250) and reverse primer (5'-CCAAAGTTGGTTTCCCAAGA, complementary to nt 2533-2552) specific to RNA-2 of GFLV-F13 isolate (GenBank Accession No. X16907) were used in RT-PCR assays for the detection of GFLV (4). Primers used for RT-PCR detection of GLRaV-1, GLRaV-2, and GVA were described in Martin et al (2) and Minafra et al (3). The RT-PCR results indicated mixed infection of GFLV with GLRaV-1, GLRaV-3, and GVA. To confirm the presence of GFLV, the 322-bp sequence representing a portion of the coat protein encoded by RNA-2 genomic segment was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Amplicons obtained from six individual grapevines in the two vineyards were used for cloning. Three independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 99 to 100% nucleotide sequence identity among themselves, indicating that GFLV isolates from the two vineyards may be identical. A comparison of the consensus sequence (GenBank Accession No. EU573307) with corresponding sequences of other GFLVs deposited in GenBank showed 89 to 91% identity at the nucleotide level and 95 to 99% identity at the amino acid level. However, mixed infection of GFLV with different viruses in the two vineyards suggests separate introduction of the planting material. ELISA with GFLV-specific antibodies further confirmed the presence of the virus in samples that were positive in RT-PCR. To our knowledge, this is the first report of GFLV in grapevines grown in the Pacific Northwest states of the United States. Further investigations are being carried out on the distribution, symptoms, molecular variability, and nematode vector transmission of GFLV. References: (1) P. Andret-Link et al. J. Plant Pathol. 86:183, 2004. (2) R. R. Martin et al. Plant Dis. 89:763, 2005. (3) A. Minafra et al. Arch. Virol. 142:417, 1997 (4) A. Rowhani et al. Phytopathology 83:749, 1993.
葡萄扇叶病毒（GFLV；线虫传多面体病毒属，豇豆花叶病毒科）是导致扇叶退化病的病原体，是全球最重要的葡萄病毒之一（1）。在2007年的勘查研究中，从华盛顿州东部两个地理位置相隔的葡萄园随机采集了酿酒葡萄品种霞多丽单株葡萄的休眠硬枝插条。使用病毒特异性引物通过单管逆转录（RT）-PCR分别对这些插条形成层刮屑提取物进行不同病毒检测。其中一个葡萄园的31株葡萄中有2株检测出GFLV呈阳性，为与葡萄卷叶相关病毒（GLRaV）-3的混合感染。在另一个葡萄园，26株葡萄中有6株检测出GFLV呈阳性，为与GLRaV-1、GLRaV-3和葡萄病毒A（GVA）的混合感染。使用针对GFLV-F13分离株（GenBank登录号X16907）RNA-2的正向引物（5'-ACCGGATTGACGTGGGTGAT，对应核苷酸[nt]2231 - 2250）和反向引物（5'-CCAAAGTTGGTTTCCCAAGA，与nt 2533 - 2552互补）进行RT-PCR检测GFLV（4）。用于RT-PCR检测GLRaV-1、GLRaV-2和GVA的引物见Martin等人（2）以及Minafra等人（3）的报道。RT-PCR结果表明GFLV与GLRaV-1、GLRaV-3和GVA存在混合感染。为确认GFLV的存在，将代表RNA-2基因组片段编码的外壳蛋白一部分的322 bp序列克隆到pCR2.1（Invitrogen公司，加利福尼亚州卡尔斯巴德）中。从两个葡萄园的6株单株葡萄获得的扩增子用于克隆。每个扩增子从两个方向对三个独立克隆进行测序。这些序列的两两比较显示它们之间的核苷酸序列同一性为99%至100%，表明来自两个葡萄园的GFLV分离株可能相同。将一致序列（GenBank登录号EU573307）与GenBank中其他GFLV的相应序列进行比较，在核苷酸水平上同一性为89%至91%，在氨基酸水平上同一性为95%至99%。然而，两个葡萄园GFLV与不同病毒的混合感染表明种植材料是分别引入的。用GFLV特异性抗体进行的ELISA进一步证实了RT-PCR呈阳性的样品中存在该病毒。据我们所知，这是美国太平洋西北部各州种植的葡萄中首次报道GFLV。目前正在对GFLV的分布、症状、分子变异性和线虫介体传播进行进一步调查。参考文献：（1）P.Andret-Link等人，《植物病理学杂志》86:183，2004。（2）R.R.Martin等人，《植物病害》89:763，2005。（3）A.Minafra等人，《病毒学档案》142:417，1997。（4）A.Rowhani等人，《植物病理学》83:749，1993。
