Kostadinovska E, Mitrev S, Bianco P A, Casati P, Bulgari D
Goce Delcev University, Faculty of Agriculture, Department for Plant and Environmental Protection, Krste Misirkov bb, 2000 Stip, Republic of Macedonia.
Dipartimento di Scienze Agrarie e Ambientali, Produzione, Territorio, Agroenergia, Università degli Studi, 20133 Milan, Italy.
Plant Dis. 2014 Dec;98(12):1747. doi: 10.1094/PDIS-05-14-0518-PDN.
Grapevine (Vitis vinifera L.) is an economically important crop and can host several different viruses, including those that have to be excluded from certified propagating material in Europe. Among these, the Vitivirus Grapevine virus A (GVA) and the Maculavirus Grapevine fleck virus (GFkV) are phloem-limited viruses that are associated with two different grapevine diseases, Kober stem grooving, belonging to the rugose wood complex, and fleck diseases, respectively. During a survey conducted in 2012 in the former Republic of Macedonia, symptomatic plants with reddening of leaves were collected for laboratory analyses. In this study, grapevine red varieties (Vranec, Francovka, and Pinot noir) from four different localities (Stip, Kavadarci, Valandovo, and Gevgelija) in Macedonia were examined. Thirty-four samples were analyzed by DAS-ELISA using commercially antibodies against Grapevine leafroll associated virus-3 (GLRaV-3). Ten selected samples were processed through DAS-ELISA and molecular assays also for the presence of GVA and GFkV. Total RNA was extracted as previously described (2) and retro-transcribed (RT) using random primers followed by PCR assay with primers GVA-MP (5'-GCCAGAGGTGTTTGAGACAAT-3') and GVA-CPdt (5'-TTTTGTCTTCGTGTGACAACCT-3') (1), which amplified a GVA-specific fragment of 986 bp, and with primers GFkV-U279 (5'-TGGTCCTCGGCCCAGTGAAAAAGTA-3') and GFkV-L630 (5'-GGCCAGGTTGTAGTCGGTGTTGTC-3') (3), which amplified a GFkV-specific region of 315 bp. Results from DAS-ELISA test showed the presence of GLRaV-3 in 21 tested samples and of GVA and GFkV in six and three out of 10 selected samples, respectively. GVA was found in Vranec and Francovka vines sampled in all the locations mentioned before, while GFkV was detected in Vranec and Pinot noir vines, in Stip, Kavadarci, and Gevgelija. These latter results were confirmed by RT-PCR assays; then, four GVA-specific and three GFkV-specific amplicons were sequenced from both directions to get a 3× coverage. For GVA fragment, a primer pair designed in the internal part of the sequence was also used. BLASTn analyses showed that (i) PCR products amplified with GVA-specific primers shared best nucleotide sequence identities, ranging from 91.7 to 93.7%, with GVA isolate at GenBank Accession No. X75433; (ii) PCR products amplified with GFkV-specific primers shared best nucleotide sequence identities, from 92.5 to 94.7%, with GFkV isolate at AJ309022. These evidences reinforced the serological and PCR results indicating that GVA and GFkV were identified in examined grapevine plants in this study. Nucleotide sequences of GVA (KF594432 to 35) and GFkV (KF594429 to 31) were submitted to GenBank. To our knowledge, this is the first report of GVA and GFkV grapevine viruses in the Former Yugoslav Republic of Macedonia. References: (1) J. De Meyer et al. Page 138 in: Extended Abstracts, 13th Meeting of ICVG, Adelaide, 12-17 March 2000. (2) D. J. MacKenzie et al. Plant Dis. 81:222, 1997. (3) B. J. Shi et al. Ann. Appl. Biol. 142:349, 2003.
葡萄（Vitis vinifera L.）是一种具有重要经济价值的作物，可携带多种不同病毒，包括欧洲认证繁殖材料中必须排除的病毒。其中，葡萄病毒A（GVA）和葡萄斑点病毒（GFkV）是韧皮部限制型病毒，分别与两种不同的葡萄病害有关，即属于皱纹木复合体的柯贝茎沟病和斑点病。在2012年对前马其顿共和国进行的一项调查中，采集了叶片发红的有症状植株进行实验室分析。在本研究中，对来自马其顿四个不同地区（斯科普里、卡瓦达尔奇、瓦兰多沃和盖夫盖利亚）的葡萄红色品种（弗拉内茨、弗朗科夫卡和黑皮诺）进行了检测。使用针对葡萄卷叶相关病毒3（GLRaV-3）的商业抗体，通过双抗夹心酶联免疫吸附测定（DAS-ELISA）对34个样品进行了分析。对10个选定样品也进行了DAS-ELISA和分子检测，以检测GVA和GFkV的存在。按照之前描述的方法提取总RNA（2），使用随机引物进行反转录（RT），然后用引物GVA-MP（5'-GCCAGAGGTGTTTGAGACAAT-3'）和GVA-CPdt（5'-TTTTGTCTTCGTGTGACAACCT-3'）（1）进行PCR检测，扩增出一个986 bp的GVA特异性片段，并用引物GFkV-U279（5'-TGGTCCTCGGCCCAGTGAAAAAGTA-3'）和GFkV-L630（5'-GGCCAGGTTGTAGTCGGTGTTGTC-3'）（3）进行PCR检测，扩增出一个315 bp的GFkV特异性区域。DAS-ELISA检测结果显示，在21个测试样品中存在GLRaV-3，在10个选定样品中，分别有6个和3个样品存在GVA和GFkV。在上述所有地点采集的弗拉内茨和弗朗科夫卡葡萄藤中均发现了GVA，而在斯科普里、卡瓦达尔奇和盖夫盖利亚的弗拉内茨和黑皮诺葡萄藤中检测到了GFkV。RT-PCR检测证实了后一结果；然后，从正反两个方向对四个GVA特异性和三个GFkV特异性扩增子进行测序，以获得3倍覆盖率。对于GVA片段，还使用了在序列内部设计的一对引物。BLASTn分析表明：（i）用GVA特异性引物扩增的PCR产物与GenBank登录号为X75433的GVA分离株的核苷酸序列一致性最高，范围为91.7%至93.7%；（ii）用GFkV特异性引物扩增的PCR产物与AJ309022的GFkV分离株的核苷酸序列一致性最高，范围为92.5%至94.7%。这些证据强化了血清学和PCR结果，表明在本研究中检测的葡萄植株中鉴定出了GVA和GFkV。GVA（KF594432至35）和GFkV（KF594429至31）的核苷酸序列已提交至GenBank。据我们所知，这是前南斯拉夫的马其顿共和国首次报道GVA和GFkV葡萄病毒。参考文献：（1）J. De Meyer等人。见：《扩展摘要》，第13届国际葡萄病毒学会议，阿德莱德，2000年3月12日至17日，第138页。（2）D. J. MacKenzie等人。《植物病害》81:222，1997年。（3）B. J. Shi等人。《应用生物学年鉴》142:349，2003年。
