Jones T J, Westover F, Nita M
AHS Jr. Agricultural Research and Extension Center, Virginia Polytechnic Institute and State University, Winchester, VA.
Texas A&M AgriLife Extension Service, Houston, TX.
Plant Dis. 2014 Nov;98(11):1592. doi: 10.1094/PDIS-06-14-0619-PDN.
Grapevine leafroll disease (GLD), caused by the grapevine leafroll-associated viruses (GLRaVs, family Closteroviridae) is an important disease in all grapevine-growing regions of the world (2). It negatively affects vine vigor, fruit yield, and grape quality (e.g., sugar accumulation) (3). Typical disease symptoms include downward rolling of grape leaves accompanied by interveinal reddening in red-fruited varieties and interveinal chlorosis in white-fruited varieties (2). The state of Texas currently has over 275 commercial vineyards and acreage under grape production is expanding. Currently, there is limited information on the presence of either GLRaV-2 (genus Closterovirus) or GLRaV-3 (genus Ampelovirus) in this state. During the 2012 season, 19 individual, symptomatic grapevines (13 cv. Lenoir and 6 cv. Blanc du Bois) were sampled (14 petioles per vine) from one vineyard site in Richards, TX. Total nucleic acid was extracted from the samples as described before (5) and tested by RT-PCR using species specific primers to amplify a 334-bp fragment of the HSP70h gene of GLRaV-2 (L2 F: 5'-ATAATTCGGCGTACATCCCCACTT-3' and U2 R: 5'-GCCCTCCGCGCAACTAATGACAG-3') (1) and a 541-bp fragment of the HSP70h gene of GLRaV-3 (LC1 F: 5'-CGCTAGGGCTGTGGAAGTATT-3' and LC2 R: 5'-GTTGTCCCGGGTACCAGATAT-3') (4). Samples were also subjected to triple (TAS) and double (DAS) antibody sandwich ELISA for GLRaV-2 and GLRaV-3 using commercially available antibody test kits (AC Diagnostics, Fayetteville, AR). Five samples tested positive for GLRaV-2 and one for GLRaV-3, all from the variety Lenoir with no incidences of mixed infection. In addition to the RT-PCR and ELISA, the presence of GLRaV-2 and GLRaV-3 was confirmed by direct sequencing of select RT-PCR products, which was purified using the QIAquick PCR Purification kit (Qiagen Inc., CA). The sequencing took place at the Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA. GLRaV-2 isolate TX12 (GenBank Accession No. KF417612) and GLRaV-3 isolate TX9 (KJ545571) shared 85 to 100% and 94 to 100% nucleotide identity and 74 to 100% and 82 to 100% amino acid identity, respectively, with previously reported isolates from around the world. All samples tested negative for GLRaV-1, -4, -4 strain 5, and -4 strain 9 (4), suggesting that some of the symptomatic vines may have a different disease or abiotic disorder, such as a nutrient deficiency. To our knowledge, this is the first report of GLRaV-2 and GLRaV-3 in the state of Texas. References: (1) N. Bertazzon and E. Angelini. J. Plant Pathol. 86:283, 2004. (2) M. Fuchs et al. Plant Dis. 93:395, 2009. (3) L. Kovacs et al. Am. J. Enol. Vitic. 52:254, 2001. (4) F. Osman et al. J. Virol. Methods. 141:22, 2007. (5) A. Rowhani et al. Proc. ICVG (Adelaide). 13:82, 2000.
葡萄卷叶病（GLD）由葡萄卷叶相关病毒（GLRaVs，长线形病毒科）引起，是世界上所有葡萄种植区的一种重要病害（2）。它对葡萄树活力、果实产量和葡萄品质（如糖分积累）产生负面影响（3）。典型的病害症状包括葡萄叶片向下卷曲，红色果实品种伴有叶脉间变红，白色果实品种伴有叶脉间黄化（2）。得克萨斯州目前有超过275个商业葡萄园，葡萄种植面积正在扩大。目前，关于该州是否存在GLRaV - 2（Closterovirus属）或GLRaV - 3（Ampelovirus属）的信息有限。在2012年生长季，从得克萨斯州理查兹的一个葡萄园地点采集了19株有症状葡萄树（13株Lenoir品种和6株Blanc du Bois品种）（每株葡萄树采集14个叶柄）。按照之前描述的方法（5）从样品中提取总核酸，并使用种特异性引物通过RT - PCR进行检测，以扩增GLRaV - 2的HSP70h基因的334 bp片段（L2 F：5'-ATAATTCGGCGTACATCCCCACTT - 3'和U2 R：5'-GCCCTCCGCGCAACTAATGACAG - 3'）（1）以及GLRaV - 3的HSP70h基因的541 bp片段（LC1 F：5'-CGCTAGGGCTGTGGAAGTATT - 3'和LC2 R：5'-GTTGTCCCGGGTACCAGATAT - 3'）（4）。还使用市售抗体检测试剂盒（AC Diagnostics，阿肯色州费耶特维尔）对样品进行了针对GLRaV - 2和GLRaV - 3的三重（TAS）和双重（DAS）抗体夹心ELISA检测。5个样品检测出GLRaV - 2呈阳性，1个样品检测出GLRaV - 3呈阳性，所有阳性样品均来自Lenoir品种，未发现混合感染情况。除了RT - PCR和ELISA检测外，还通过对选定的RT - PCR产物进行直接测序确认了GLRaV - 2和GLRaV - 3的存在，这些产物使用QIAquick PCR纯化试剂盒（Qiagen公司，加利福尼亚州）进行了纯化。测序在弗吉尼亚生物信息学研究所进行，该研究所位于弗吉尼亚理工学院暨州立大学，弗吉尼亚州布莱克斯堡市。GLRaV - 2分离株TX12（GenBank登录号KF417612）和GLRaV - 3分离株TX9（KJ545571）与之前报道的来自世界各地的分离株相比，核苷酸同一性分别为85%至100%和94%至100%，氨基酸同一性分别为74%至100%和82%至100%。所有样品检测GLRaV - 1、- 4、- 4菌株5和- 4菌株9均为阴性（4），这表明一些有症状的葡萄树可能患有不同的病害或非生物失调，如营养缺乏。据我们所知，这是得克萨斯州首次报道GLRaV - 2和GLRaV - 3。参考文献：（1）N. Bertazzon和E. Angelini。《植物病理学杂志》86：283，2004。（2）M. Fuchs等人。《植物病害》93：395，2009。（3）L. Kovacs等人。《美国酿酒与葡萄栽培杂志》52：254，2001。（4）F. Osman等人。《病毒学方法杂志》141：22，2007。（5）A. Rowhani等人。《国际葡萄病毒学大会会议录》（阿德莱德）13：82，2000。
