Mekuria T A, Karasev A V, Martin R R, Naidu R A
Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser 99350.
Department of Plant, Soil, and Entomological Sciences, University of Idaho, Moscow 83844.
Plant Dis. 2009 Nov;93(11):1218. doi: 10.1094/PDIS-93-11-1218C.
In recent years, wine grape (Vitis vinifera) acreage in Idaho has expanded because of favorable climatic conditions for premium wine production. Nearly 95% of the 491.7 ha (1,215 acres) of wine grapes are in the Snake River Valley with Canyon County accounting for 81% of the vines. Previous studies have shown that grapevine leafroll disease (GLD) is the most widespread and economically significant virus disease in wine grapes in Washington and Oregon (1,2). However, little is known about the incidence and economic impact of GLD on wine grapes in Idaho. During the 2008 growing season, leaf samples were collected from approximately 25 individual grapevines of red-berried cultivars (Cabernet Sauvignon, Merlot, Syrah, and Petit Syrah) showing GLD symptoms and white-berried (Chardonnay) cultivars with suspected GLD symptoms growing in 10 geographically separate vineyards in Canyon County. An additional five samples were collected from a Lemberger block in Elmore County. Petiole extracts from these samples were tested by single-tube reverse transcription (RT)-PCR with primers LC 1 (5'-CGC TAG GGC TGT GGA AGT ATT-3') and LC 2 (5'-GTT GTC CCG GGT ACC AGA TAT-3') specific for the heat shock protein 70 homologue (HSP-70 gene) of Grapevine leafroll-associated virus-3 (GLRaV-3) (3). All samples, except the Petit Syrah, produced a single band of the expected size of 546 bp. ELISA with GLRaV-3-specific antibodies (BIOREBA AG, Reinach, Switzerland) confirmed the presence of the virus in samples that were positive in RT-PCR. GLRaV-3-specific amplicons were cloned in pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA) and 2 to 3 independent clones per isolate were sequenced in both orientations. A pairwise comparison of 22 sequences, six from Chardonnay (GenBank Accessions GQ344810, GQ344811, GQ344823, GQ344824, GQ344825, and GQ344826), five from Cabernet Sauvignon (GQ344807, GQ344808, GQ344809, GQ344827, and GQ344828), four each from Merlot (GQ344815, GQ344816, GQ344817, and GQ344818) and Syrah (GQ344819, GQ344820, GQ344821, and GQ344822), and three from Lemberger (GQ344812, GQ344813, and GQ344814) showed 87 to 100% identity at the nucleotide level and 92 to 100% identity at the amino acid level. A pairwise comparison of HSP-70 sequences of GLRaV-3 isolates from Idaho with corresponding sequences of GLRaV-3 isolates from GenBank showed nucleotide sequence identities between 88% (AJ748519) and 100% (DQ780885). Phylogenetic analysis of HSP-70 sequences from Idaho and GenBank showed clustering of Idaho sequences into five groups, with 12 sequences clustering with a Washington isolate (DQ780885), six sequences in a second group clustering with an isolate from Tunisia (AJ748522), two sequences in a third group clustering with an isolate from Austria (AJ748513), and one sequence each in groups four and five clustering with isolates from Italy (AJ748520) and Washington (DQ780889), respectively. The clustering was not cultivar- or vineyard-specific, suggesting separate introductions of different GLRaV-3 isolates in planting materials. To our knowledge, this is the first report of GLRaV-3 in grapevines grown in Idaho. These and previous results (1,2), indicate the wide distribution of GLRaV-3 in several grapevine cultivars in the Pacific Northwest Region. References: (1) R. R. Martin et al. Plant Dis. 89:763, 2005. (2) R. A. Naidu et al. (Abstr.) Phytopathology 96(suppl.):S83, 2006. (3) M. J. Soule et al. Plant Dis. 90:1461, 2006.
近年来，由于爱达荷州的气候条件有利于优质葡萄酒的生产，酿酒葡萄（欧亚种葡萄）的种植面积不断扩大。该州491.7公顷（1215英亩）的酿酒葡萄中，近95%位于蛇河谷，其中峡谷县的葡萄藤占81%。此前的研究表明，葡萄卷叶病（GLD）是华盛顿州和俄勒冈州酿酒葡萄中分布最广、经济影响最大的病毒病（1,2）。然而，关于GLD在爱达荷州酿酒葡萄中的发病率和经济影响，人们了解甚少。在2008年生长季节，从峡谷县10个地理位置分散的葡萄园里，采集了约25株表现出GLD症状的红浆果品种（赤霞珠、梅洛、西拉和小西拉）以及疑似感染GLD症状的白浆果品种（霞多丽）的单株葡萄的叶片样本。另外还从埃尔莫尔县的一个蓝贝格葡萄园采集了5个样本。用针对葡萄卷叶相关病毒3（GLRaV-3）热休克蛋白70同源物（HSP-70基因）的引物LC 1（5'-CGC TAG GGC TGT GGA AGT ATT-3'）和LC 2（5'-GTT GTC CCG GGT ACC AGA TAT-3'），通过单管逆转录（RT）-PCR对这些样本的叶柄提取物进行检测（3）。除小西拉外，所有样本均产生了一条预期大小为546 bp的条带。用GLRaV-3特异性抗体（瑞士雷纳赫的BIOREBA AG公司）进行的酶联免疫吸附测定（ELISA）证实，在RT-PCR呈阳性的样本中存在该病毒。将GLRaV-3特异性扩增子克隆到pCR2.1质粒（加利福尼亚州卡尔斯巴德的Invitrogen公司）中，每个分离株的2至3个独立克隆进行双向测序。对22个序列进行两两比较，其中6个来自霞多丽（GenBank登录号GQ344810、GQ344811、GQ344823、GQ344824、GQ344825和GQ344826），5个来自赤霞珠（GQ344807、GQ344808、GQ344809、GQ344827和GQ344828），梅洛（GQ344815、GQ344816、GQ344817和GQ344818）和西拉（GQ344819、GQ344820、GQ344821和GQ344822）各4个，蓝贝格3个（GQ344812、GQ344813和GQ344814），结果显示核苷酸水平上的同一性为87%至100%，氨基酸水平上的同一性为92%至100%。将爱达荷州GLRaV-3分离株的HSP-70序列与GenBank中GLRaV-3分离株的相应序列进行两两比较，结果显示核苷酸序列同一性在88%（AJ748519）至100%（DQ780885）之间。对爱达荷州和GenBank中HSP-70序列进行系统发育分析，结果表明爱达荷州的序列分为五组，其中12个序列与华盛顿州的一个分离株（DQ780885）聚类，第二组的6个序列与突尼斯的一个分离株（AJ748522）聚类，第三组的2个序列与奥地利的一个分离株（AJ748513）聚类，第四组和第五组各有一个序列分别与意大利（AJ748520）和华盛顿州（DQ780889）的分离株聚类。这种聚类不是品种或葡萄园特异性的，表明在种植材料中不同的GLRaV-3分离株是分别引入的。据我们所知，这是GLRaV-3在爱达荷州种植的葡萄藤中的首次报道。这些结果以及之前的结果（1,2）表明，GLRaV-3在太平洋西北地区的几个葡萄品种中广泛分布。参考文献：（1）R. R. Martin等人，《植物病害》89:763，2005年。（2）R. A. Naidu等人（摘要），《植物病理学》96（增刊）:S83，2006年。（3）M. J. Soule等人，《植物病害》90:1461，2006年。
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