Svoboda J, Leisova-Svobodova L
Crop Research Institute, Drnovská 507, 161 06 Prague, Czech Republic. This work was supported by Project NAZV QH71229 of the Ministry of Agriculture, Czech Republic.
Plant Dis. 2011 Oct;95(10):1321. doi: 10.1094/PDIS-05-11-0444.
In August 2010, yellow mosaic and leaf deformation were observed on leaves of field planted ornamental pumpkin (Cucurbita pepo L. convar. microcarpina Grebenščikov) plants near Tasovice Village in the Znojmo District of the Czech Republic. These symptoms were typical of a virus infection. Nine leaf samples were collected and examined for the presence of commonly occurring cucurbit viruses by double-antibody sandwich (DAS)-ELISA. Eight samples were infected with Zucchini yellow mosaic virus (ZYMV), seven with Watermelon mosaic virus-2 (WMV-2), five with Cucumber mosaic virus (CMV), and two with Squash mosaic virus (SqMV). Some aphid species were observed on inspected plants but we did not carried out a search for vectors. SqMV isolates, Tas-1 and Tas-5, were also infected with ZYMV and WMV-2. SqMV was separated from the other viruses by mechanical inoculation onto the resistant plant species Cucumis sativus L. 'Taichung Mou Gua-1', which is resistant to ZYMV and WMV-2 (3). The virus was transmitted back to C. pepo plants, and DAS-ELISA used for testing for multiple viruses proved the presence of SqMV alone. A C. pepo leaf sample was examined by electron microscopy to validate the presence of the virus. Isometric particles of approximately 30 nm in diameter, corresponding in size and shape to the described particles of SqMV (2), were observed. The presence of SqMV was verified by reverse transcription (RT)-PCR using specific primers designed on the sequence for coat proteins of SqMV found in NCBI and EMBL databases (SqMV-F: TGTGTACAAGATTGGTGGAGATGC; SqMV-R: AGGCTTCTAAAGCGAACTGGG). The obtained amplicons of approximately 1,900 bp were sequenced (GenBank Accession No. JF922966), and by using nucleotide blast analysis (4), identified as a part of RNA-2 genome. Blast analysis showed that the Czech SqMV isolates, Tas-1 and Tas-5, were identical and similar to published SqMV sequences from the United States, Japan, and China. The highest similarity was found between the Czech isolates and one isolate from the United States (Accession No. M96148; E = 0, nucleotide sequence identity = 90%) and one from China (Accession No. AF059533; E = 0, nucleotide sequence identity = 90%). To our knowledge, this is the first report of a natural occurrence of SqMV in the Czech Republic. SqMV can be transmitted via infected seeds by as much as 35% (1); therefore, protection against epidemics should be based on clean seed programs. References: (1) M. Alvarez et al. Phytopathology 68:257, 1978. (2) H. M. Mazzone et al. Biochim. Biophys. Acta 55:164, 1962. (3) T. Wai et al. J. Hered. 88:454, 1997. (4) Z. Zhang et al. J. Comput. Biol. 7:203, 2000.
2010年8月，在捷克共和国兹诺伊莫区塔索维采村附近种植于田间的观赏南瓜（西葫芦变种小果南瓜，Cucurbita pepo L. convar. microcarpina Grebenščikov）植株的叶片上，观察到黄斑花叶病和叶片变形症状。这些症状是病毒感染的典型症状。采集了9片叶片样本，通过双抗体夹心（DAS）-酶联免疫吸附测定法检测常见葫芦科病毒的存在情况。8个样本感染了小西葫芦黄花叶病毒（ZYMV），7个感染了西瓜花叶病毒2型（WMV-2），5个感染了黄瓜花叶病毒（CMV），2个感染了南瓜花叶病毒（SqMV）。在受检植株上观察到了一些蚜虫种类，但我们未对传毒介体进行调查。SqMV分离株Tas-1和Tas-5还感染了ZYMV和WMV-2。通过机械接种到对ZYMV和WMV-2具有抗性的黄瓜品种‘台中牟瓜-1’（Cucumis sativus L. 'Taichung Mou Gua-1'）上，将SqMV与其他病毒分离（3）。该病毒被回传到西葫芦植株上，用于检测多种病毒的DAS-酶联免疫吸附测定法证实仅存在SqMV。通过电子显微镜检查一个西葫芦叶片样本，以验证该病毒的存在。观察到直径约30 nm的等轴颗粒，其大小和形状与已描述的SqMV颗粒一致（2）。使用根据美国国立生物技术信息中心（NCBI）和欧洲分子生物学实验室（EMBL）数据库中SqMV外壳蛋白序列设计的特异性引物，通过逆转录（RT）-聚合酶链反应（PCR）验证了SqMV的存在（SqMV-F：TGTGTACAAGATTGGTGGAGATGC；SqMV-R：AGGCTTCTAAAGCGAACTGGG）。对获得的约1900 bp扩增子进行测序（GenBank登录号：JF922966），并通过核苷酸比对分析（4），鉴定为RNA-2基因组的一部分。比对分析表明，捷克的SqMV分离株Tas-1和Tas-5相同，且与来自美国、日本和中国的已发表SqMV序列相似。在捷克分离株与来自美国的一个分离株（登录号：M96148；E = 0，核苷酸序列同一性 = 90%）以及来自中国的一个分离株（登录号：AF059533；E = 0，核苷酸序列同一性 = 90%）之间发现了最高的相似性。据我们所知，这是SqMV在捷克共和国自然发生的首次报道。SqMV可通过受感染种子传播高达35%（1）；因此，预防疫情应基于清洁种子计划。参考文献：（1）M. Alvarez等人，《植物病理学》68:257，1978年。（2）H. M. Mazzone等人，《生物化学与生物物理学报》55:164，1962年。（3）T. Wai等人，《遗传学杂志》88:454，1997年。（4）Z. Zhang等人，《计算生物学杂志》7:203，2000年。
