Lyu M D, Li M J, Li J, Li X M, Cheng Y-Q
Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, Key Laboratory of Beijing Municipality, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China.
Plant Dis. 2013 Jan;97(1):150. doi: 10.1094/PDIS-08-12-0760-PDN.
Grapevine leafroll disease (GLD) is one of the most economically important diseases of cultivated grapevines (Vitis vinifera), causing decrease in yield, as well as decreasing the sugar levels and increasing the acidity of the berries (1). There are currently at least 10 serologically distinct viruses, referred to as grapevine leafroll-associated viruses (GLRaVs), from the family Closteroviridae that are associated with leafroll disease (4). China is one of the world's leading grape producers, and nearly 75% of the vineyards in China are located in Xinjiang Uygur Autonomous Region, and Hebei, Shandong, Gansu, Ningxia, and Yunnan provinces. Grapevine leafroll-associated virus 7 (GLRaV-7) isolates have been reported so far in Liaoning (GQ849392, GQ849393, and JF927943) and Henan (EF093187) provinces in China (3). The four Chinese isolates were isolated respectively from grape varieties, Cabernet Sauvignon (GQ849392, GQ849393), Centennial Seedless (JF927943), and Semillon (EF093187), and these grape varieties are introduced from abroad. Cow's Nipple and Dragon's Eye are old grape varieties native to China. Cow's nipple is extensively cultivated in Xinjiang Uygur Autonomous Region, while Dragon's Eye is widely planted in Heibei Province. To determine if GLRaV-7 was present in these two varieties, six samples (three per variety) were collected from six individual grapevines showing GLD-like symptoms in two vineyards in Xinjiang Uygur Autonomous Region and Hebei Province, respectively, in September 2011. Total RNA extracts obtained from phloem scrapings of samples using the RNeasy plant mini kit (QIAGEN) were tested by reverse transcription (RT)-PCR with primers F1 (5'-TATATCCCAACGGAGATGGC-3') and R1 (5'-ATGTTCCTCCACCAAAATCG-3') (2) specific to the heat shock protein 70 homologue (HSP-70 gene) of GLRaV-7. All samples produced a single band of the expected size of 502 bp. One GLRaV-7-specific amplicon per variety was cloned into pMD 18-T simple vector (TaKaRa). Plasmid DNA was purified using Column Plasmid DNA (TIANDZ, Beijing, China) from three individual clones and sequenced from both directions. The sequence of the two isolates (GenBank Accession Nos. JX494722 and JX494723) shared 97.81% identity at the nucleotide level and 100% identity at the amino acid level. A pairwise comparison of HSP-70 sequences of the two isolates from this report with nine corresponding sequences of GLRaV-7 isolates (including four previously reported Chinese isolates) showed nucleotide sequence identities ranging from 91.24% (EF093187) to 98.80% (GQ849392). These samples were further analyzed by double antibody sandwich (DAS)-ELISA using antibody specific to GLRaV-7 (NEOGEN Europe, Ayr, Scotland) according to the manufacturer's instructions, and the results confirmed the presence of the virus in these samples that were positive by RT-PCR. To our knowledge, this is the first report of GLRaV-7 occurring in native grape varieties in China. These results could be helpful in developing sound diagnostic systems for implementing efficient disease management strategies. References: (1) B. Akbas et al. Hort. Sci. 36:97, 2009. (2) E. Engel et al. Plant Dis. 92:1252, 2008. (3) X. Fan et al. Acta Hortic. Sinica 39:949, 2012. (4) G. P. Martelli. Extended Abstr. 16th Meet. International Council for the Study of Virus and Virus-like Diseases of Grapevines (ICVG). 15-23, 2009.
葡萄卷叶病（GLD）是栽培葡萄（欧亚种葡萄）最具经济重要性的病害之一，会导致产量下降，以及降低浆果的糖分水平并增加酸度（1）。目前至少有10种血清学上不同的病毒，称为葡萄卷叶相关病毒（GLRaVs），属于长线形病毒科，与卷叶病有关（4）。中国是世界主要葡萄生产国之一，中国近75%的葡萄园位于新疆维吾尔自治区以及河北、山东、甘肃、宁夏和云南等省份。到目前为止，在中国辽宁省（GQ849392、GQ849393和JF927943）和河南省（EF093187）已报道了葡萄卷叶相关病毒7（GLRaV-7）分离株（3）。这4个中国分离株分别从赤霞珠（GQ849392、GQ849393）、无核白百年（JF927943）和赛美蓉（EF093187）葡萄品种中分离得到，这些葡萄品种均从国外引进。牛奶葡萄和龙眼葡萄是中国本土的古老葡萄品种。牛奶葡萄在新疆维吾尔自治区广泛种植，而龙眼葡萄在河北省广泛种植。为了确定这两个品种中是否存在GLRaV-7，2011年9月，分别从新疆维吾尔自治区和河北省的两个葡萄园的6株表现出类似GLD症状的葡萄植株上采集了6个样本（每个品种3个）。使用RNeasy植物小提试剂盒（QIAGEN）从样本的韧皮部刮取物中提取的总RNA，通过逆转录（RT）-PCR，用针对GLRaV-7热休克蛋白70同源物（HSP-70基因）的引物F1（5'-TATATCCCAACGGAGATGGC-3'）和R1（5'-ATGTTCCTCCACCAAAATCG-3'）（2）进行检测。所有样本均产生了一条预期大小为502 bp的条带。每个品种的一个GLRaV-7特异性扩增子被克隆到pMD 18-T简易载体（TaKaRa）中。使用柱式质粒DNA（TIANDZ，中国北京）从3个独立克隆中纯化质粒DNA，并从两个方向进行测序。这两个分离株的序列（GenBank登录号JX494722和JX494723）在核苷酸水平上具有97.81%的同一性，在氨基酸水平上具有100%的同一性。将本报告中的两个分离株的HSP-70序列与9个GLRaV-7分离株的相应序列（包括4个先前报道的中国分离株）进行成对比较，结果显示核苷酸序列同一性范围为91.24%（EF093187）至98.80%（GQ849392）。根据制造商的说明，使用针对GLRaV-7的抗体（NEOGEN Europe，Ayr，苏格兰）通过双抗体夹心（DAS）-ELISA对这些样本进行进一步分析，结果证实了这些通过RT-PCR呈阳性的样本中存在该病毒。据我们所知，这是GLRaV-7在中国本土葡萄品种中出现的首次报道。这些结果可能有助于开发完善的诊断系统，以实施有效的病害管理策略。参考文献：（1）B. Akbas等人，《园艺科学》36:97，2009年。（2）E. Engel等人，《植物病害》92:1252，2008年。（3）X. Fan等人，《园艺学报》39:949，2012年。（4）G. P. Martelli，《第16届葡萄病毒和类病毒病害国际研究理事会会议扩展摘要》。15 - 23页，2009年。
