Trkulja V, Jošić Kovačić D, Mihić Salapura J, Stanković I, Vučurović A, Bulajić A, Krstić B
Department of Plant Protection, Agricultural Institute of Republic of Srpska, Knjaza Miloša 17, 78000 Banja Luka, Bosnia and Herzegovina.
Institute of Phytomedicine, Department of Phytopathology, University of Belgrade-Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia. This research was partly supported by the grant III 43001 of the Ministry of Education, Science, and Technological Development, Republic of Serbia and FP7 Project Area 316004.
Plant Dis. 2014 Jun;98(6):858. doi: 10.1094/PDIS-11-13-1156-PDN.
Several potyvirus species cause severe economic losses in cucurbit crops in the Mediterranean region, but Zucchini yellow mosaic virus (ZYMV) is regarded as one of the most destructive (2,3). In June 2012, field-grown watermelon plants (Citrullus lanatus [Thunb.] Matsum and Nakai) showing mild to severe mosaic, mottling, and bubbling followed by leaf deformation with blistering were observed in the Kukulje locality (Region of Banja Luka) in Bosnia and Herzegovina. Incidence of virus infection in the field was visually estimated at 15%. Symptomatic watermelon plants were collected and tested for the presence of the most prevalent watermelon viruses including ZYMV, Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Papaya ringspot virus (PRSV), and Squash mosaic virus (SqMV) (1) using commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls were included in each assay. Of the 14 watermelon plants tested, all were positive for ZYMV and negative for WMV, CMV, PRSV, and SqMV. Sap prepared from an ELISA-positive sample (isolate 314-12) and healthy watermelon plants, using 0.01 M phosphate buffer (pH 7) was mechanically inoculated onto five carborundum-dusted plants of each Chenopodium quinoa and Citrullus lanatus 'Creamson sweet'. Mechanically inoculated C. quinoa plants exhibited chlorotic spots 5 days post-inoculation, while severe mosaic accompanied by crinkling and leaf deformation were observed on all inoculated watermelon plants 12 days post-inoculation. For further confirmation of the virus identity, total RNAs from all 14 naturally and 5 mechanically infected watermelon plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and subjected by reverse transcription (RT)-PCR. RT-PCR was carried out with One-Step RT-PCR Kit (Qiagen) using ZYMV-specific primer pair, CPfwd and CPrev (4), designed to amplify an 1,100-bp fragment covering the entire coat protein (CP) gene and part of the nuclear inclusion (NIb) and 3'-UTR. Total RNAs obtained from the Serbian ZYMV isolate from winter squash (GenBank Accession No. JN315861) and tissue sample from healthy watermelon leaves were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from each of the watermelon plants assayed confirming serological virus identification. One amplicon derived from isolate 314-12 was purified (QIAquick PCR Purification Kit, Qiagen) and sequenced directly (KF836440). Sequence analysis of the complete CP gene, conducted by MEGA5 software, revealed that watermelon isolate from Bosnia and Herzegovina showed the highest nucleotide identity of 99.8% (99.6% amino acid identity) with 14 ZYMV isolates originating from different hosts from Serbia (HM072431, JF308189 to 90, JN315856 to 57, JN315859 to 61) and Austria (AJ420012 to 17). To our knowledge, this is the first report of ZYMV in Bosnia and Herzegovina, which is an important discovery. It represents expansion of this virus to new geographical area. Considering that the ZYMV is among the most devastating pathogens of cucurbits (3), further survey is needed to determine its distribution in Bosnia and Herzegovina. References: (1) L. M. da Silveira et al. Trop. Plant Pathol. 34:123, 2009. (2) H. Lecoq et al. Virus Res. 141:190, 2009. (3) H. Lecoq and C. Desbiez. Adv. Virus Res. 84:67, 2012. (4) M. F. Pfosser and H. Baumann. Arch. Virol. 147:1599, 2002.
几种马铃薯Y病毒属病毒给地中海地区的葫芦科作物造成了严重的经济损失，而西葫芦黄花叶病毒（ZYMV）被认为是最具破坏力的病毒之一（2,3）。2012年6月，在波斯尼亚和黑塞哥维那巴尼亚卢卡地区的库库列村，观察到田间种植的西瓜植株（西瓜[Citrullus lanatus (Thunb.) Matsum and Nakai]）出现了从轻度到重度的花叶、斑驳和气泡，随后叶片变形并起泡。目测估计田间病毒感染率为15%｡采集了有症状的西瓜植株，使用商业双抗体夹心（DAS）-ELISA诊断试剂盒（瑞士雷纳赫的Bioreba AG公司）检测是否存在最常见的西瓜病毒，包括ZYMV、黄瓜花叶病毒（CMV）、西瓜花叶病毒（WMV）、番木瓜环斑病毒（PRSV）和南瓜花叶病毒（SqMV）（1）。每次检测都包括商业阳性和阴性对照。在检测的14株西瓜植株中，所有植株对ZYMV呈阳性，而对WMV、CMV、PRSV和SqMV呈阴性。用0.01 M磷酸盐缓冲液（pH 7）从ELISA阳性样品（分离株314 - 12）和健康西瓜植株中制备汁液，机械接种到藜麦和西瓜‘Creamson sweet’的各5株经金刚砂摩擦处理的植株上。机械接种的藜麦植株在接种后5天出现褪绿斑点，而接种的所有西瓜植株在接种后12天出现严重花叶，伴有皱缩和叶片变形。为进一步确认病毒的身份，用RNeasy植物小提试剂盒（德国希尔德的Qiagen公司）从所有14株自然感染和5株机械感染的西瓜植株中提取总RNA，并进行逆转录（RT）-PCR。使用一步RT-PCR试剂盒（Qiagen），用ZYMV特异性引物对CPfwd和CPrev（4）进行RT-PCR，该引物对设计用于扩增一个1100 bp的片段，覆盖整个外壳蛋白（CP）基因以及部分核内含体（NIb）和3'-UTR。分别以从塞尔维亚西葫芦中获得的ZYMV分离株（GenBank登录号：JN315861）的总RNA和健康西瓜叶片的组织样品作为阳性和阴性对照。从每个检测的西瓜植株中扩增出预期大小的RT-PCR产物，证实了血清学病毒鉴定结果。从分离株314 - 12获得的一个扩增子经纯化（QIAquick PCR纯化试剂盒，Qiagen）后直接测序（KF836440）。用MEGA5软件对完整CP基因进行序列分析，结果显示来自波斯尼亚和黑塞哥维那的西瓜分离株与来自塞尔维亚（HM072431、JF308189至90、JN315856至57、JN315859至61）和奥地利（AJ420012至17）不同寄主的14个ZYMV分离株的核苷酸同一性最高，为９９．８％（氨基酸同一性为９９．６％）。据我们所知，这是ZYMV在波斯尼亚和黑塞哥维那的首次报道，这是一项重要发现。它代表了这种病毒向新地理区域的扩散。鉴于ZYMV是葫芦科最具毁灭性的病原体之一（3），需要进一步调查以确定其在波斯尼亚和黑塞哥维那的分布情况。参考文献：（1）L. M. da Silveira等人，《热带植物病理学》34:123，2009年。（2）H. Lecoq等人，《病毒研究》141:190，2009年。（3）H. Lecoq和C. Desbiez，《病毒研究进展》84:67，2012年。（4）M. F. Pfosser和H. Baumann，《病毒学档案》147:1599，2002年。
