Ali I, Malik A H, Mansoor S
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
Plant Dis. 2010 Feb;94(2):276. doi: 10.1094/PDIS-94-2-0276A.
Bitter gourd (Momordica charantia L.) is widely grown and consumed as a vegetable in Pakistan and other countries in the region. In 2007, a severe disease appeared on bitter gourd that reduced yield significantly. Symptoms of the disease included chlorosis, leaf crumpling, vein thickening, and stunting of plants that were suggestive of a virus infection. Symptomatic leaf samples were collected from fields in the vicinity of Faisalabad, Pakistan (Thikriwala, 12 km from Faisalabad, 31°22'0″N, 72°53'0″E). Seven infected samples were tested for the presence of Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus, Papaya ringspot virus, Melon necrotic spot virus, and Squash mosaic virus by double-antibody sandwich-ELISA according to the manufacturer's instructions (Bio-Rad, Hercules, CA). All samples of bitter gourd were found to be negative for all five RNA viruses, whereas melon samples collected from the same area (Thikriwala) were infected by ZYMV as reported earlier (3). Samples were also screened for begomoviruses by molecular tests. Total DNA was extracted with the cetyltrimethylammoniumbromide method (4). All seven symptomatic samples were positive for a begomovirus when DNA A of Tomato leaf curl New Delhi virus (ToLCNDV) was used as a general probe by Southern hybridization. A probe of the movement protein (MP) gene of ToLCNDV was also positive by Southern hybridization, suggesting the infection of a bipartite begomovirus. The presence of a begomovirus was confirmed by PCR with universal primers designed for amplification of begomoviruses (BegomoRe F 5'ACGCGT GCCGTGCTGCTGCCCCCATTGTCC3' and BegomoRe R 5'ACGCGT ATGGGCTGYCGAAGTTSAGACG3'). A fragment of the expected length (approximately 2.8 kb) was cloned in a T/A cloning vector (ptz57R/t; Fermentas, Burlington, Ontario, Canada) and partially sequenced. Sequence analysis of partial sequences (925 bp, GenBank Accession No. FN555137; 719 bp, GenBank Accession No. FN555138) showed maximum identity (97%) with Tomato leaf curl Palampur virus (ToLCPaV) recently reported from India and Iran (1,2). To our knowledge, this is the first report of ToLCPaV in Pakistan and the first report of the virus on bitter gourd. References: (1) J. Heydarnejad et al. Arch. Virol. 154:1015, 2009. (2) Y. Kumar et al. Virus Genes 38:193, 2009. (3) A. H. Malik et al. Plant Pathol. 55:285, 2006. (4) M. G. Murray and W. F. Thompson. Nucleic Acids Res.8:4321, 1980.
苦瓜（Momordica charantia L.）在巴基斯坦及该地区的其他国家作为一种蔬菜被广泛种植和食用。2007年，苦瓜上出现了一种严重病害，导致产量大幅下降。该病症状包括褪绿、叶片皱缩、叶脉增厚以及植株发育不良，提示可能是病毒感染。有症状的叶片样本采自巴基斯坦费萨拉巴德附近的田地（蒂克里瓦拉，距费萨拉巴德12公里，北纬31°22'0″，东经72°53'0″）。按照制造商的说明（Bio-Rad，美国加利福尼亚州赫拉克勒斯市），采用双抗体夹心酶联免疫吸附测定法（ELISA）对7个受感染样本进行西葫芦黄花叶病毒（ZYMV）、黄瓜花叶病毒、番木瓜环斑病毒、甜瓜坏死斑点病毒和南瓜花叶病毒检测。所有苦瓜样本对这5种RNA病毒均呈阴性，而从同一地区（蒂克里瓦拉）采集的甜瓜样本如先前报道（3）一样感染了ZYMV。还通过分子检测对双生病毒进行了筛查。采用十六烷基三甲基溴化铵法（4）提取总DNA。当用番茄曲叶新德里病毒（ToLCNDV）的DNA A作为通用探针进行Southern杂交时，所有7个有症状的样本对双生病毒均呈阳性。用ToLCNDV的运动蛋白（MP）基因探针进行Southern杂交也呈阳性，表明感染了双分体双生病毒。通过使用为扩增双生病毒而设计的通用引物进行PCR（BegomoRe F 5'ACGCGT GCCGTGCTGCTGCCCCCATTGTCC3'和BegomoRe R 5'ACGCGT ATGGGCTGYCGAAGTTSAGACG3'）证实了双生病毒的存在。将预期长度（约2.8 kb）的片段克隆到T/A克隆载体（ptz57R/t；加拿大安大略省伯灵顿市Fermentas公司）中并进行部分测序。部分序列（925 bp，GenBank登录号FN555137；719 bp，GenBank登录号FN555138）的序列分析显示，与最近在印度和伊朗报道的番茄曲叶帕拉姆布尔病毒（ToLCPaV）具有最高同源性（97%）（1,2）。据我们所知，这是ToLCPaV在巴基斯坦的首次报道，也是该病毒在苦瓜上的首次报道。参考文献：（1）J. Heydarnejad等人，《病毒学档案》154:1015，2009年。（2）Y. Kumar等人，《病毒基因》38:193，2009年。（3）A. H. Malik等人，《植物病理学》55:285，2006年。（4）M. G. Murray和W. F. Thompson，《核酸研究》8:4321，1980年。
