Damayanti T A, Alabi O J, Rauf A, Naidu R A
Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University, Bogor 16680, Indonesia.
Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, 24106 N. Bunn Road Prosser 99350.
Plant Dis. 2010 Apr;94(4):478. doi: 10.1094/PDIS-94-4-0478B.
Yardlong bean (Vigna unguiculata subsp. sesquipedalis) is extensively cultivated in Indonesia for consumption as a green vegetable. During the 2008 season, a severe outbreak of a virus-like disease occurred in yardlong beans grown in farmers' fields in Bogor, Bekasi, Subang, Indramayu, and Cirebon of West Java, Tanggerang of Banten, and Pekalongan and Muntilan of Central Java. Leaves of infected plants showed severe mosaic to bright yellow mosaic and vein-clearing symptoms, and pods were deformed and also showed mosaic symptoms on the surface. In cv. 777, vein-clearing was observed, resulting in a netting pattern on symptomatic leaves followed by death of the plants as the season advanced. Disease incidence in the Bogor region was approximately 80%, resulting in 100% yield loss. Symptomatic leaf samples from five representative plants tested positive in antigen-coated plate-ELISA with potyvirus group-specific antibodies (AS-573/1; DSMZ, German Resource Center for Biological Material, Braunschweig, Germany) and antibodies to Cucumber mosaic virus (CMV; AS-0929). To confirm these results, viral nucleic acids eluted from FTA classic cards (FTA Classic Card, Whatman International Ltd., Maidstone, UK) were subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (CIFor: 5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3' and CIRev: 5'-ACICCRTTYTCDATDATRTTIGTIGC-3') (3) and degenerate primers (CMV-1F: 5'-ACCGCGGGTCTTATTATGGT-3' and CMV-1R: 5' ACGGATTCAAACTGGGAGCA-3') specific for CMV subgroup I (1). A single DNA product of approximately 683 base pairs (bp) with the potyvirus-specific primers and a 382-bp fragment with the CMV-specific primers were amplified from ELISA-positive samples. These results indicated the presence of a potyvirus and CMV as mixed infections in all five samples. The amplified fragments specific to potyvirus (four samples) and CMV (three samples) were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 93 to 100% identity among the cloned amplicons produced using the potyvirus-specific primers (GenBank Accessions Nos. FJ653916, FJ653917, FJ653918, FJ653919, FJ653920, FJ653921, FJ653922, FJ653923, FJ653924, FJ653925, and FJ653926) and 92 to 97% with a corresponding nucleotide sequence of Bean common mosaic virus (BCMV) from Taiwan (No. AY575773) and 88 to 90% with BCMV sequences from China (No. AJ312438) and the United States (No. AY863025). The sequence analysis indicated that BCMV isolates from yardlong bean are more closely related to an isolate from Taiwan than with isolates from China and the United States. The CMV isolates (GenBank No. FJ687054) each were 100% identical and 96% identical with corresponding sequences of CMV subgroup I isolates from Thailand (No. AJ810264) and Malaysia (No. DQ195082). Both BCMV and CMV have been documented in soybean, mungbean, and peanut in East Java of Indonesia (2). Previously, BCMV, but not CMV, was documented on yardlong beans in Guam (4). To our knowledge, this study represents the first confirmed report of CMV in yardlong bean in Indonesia and is further evidence that BCMV is becoming established in Indonesia. References: (1) J. Aramburu et al. J. Phytopathol. 155:513, 2007. (2) S. K. Green et al. Plant Dis. 72:994, 1988. (3) C. Ha et al. Arch. Virol. 153:25, 2008. (4) G. C. Wall et al. Micronesica 29:101, 1996.
长豇豆（Vigna unguiculata subsp. sesquipedalis）在印度尼西亚被广泛种植，作为绿色蔬菜供人食用。在2008年生长季，西爪哇省茂物、勿加泗、苏邦、印德拉马尤和井里汶、万丹省丹格朗以及中爪哇省北加浪岸和蒙蒂兰等地农民田间种植的长豇豆爆发了一种类似病毒的严重病害。受感染植株的叶片出现严重花叶至亮黄色花叶以及叶脉失绿症状，豆荚变形，表面也出现花叶症状。在品种777中，观察到叶脉失绿，症状叶片上形成网状图案，随着生长季推进，植株死亡。茂物地区的发病率约为80%，导致产量损失100%。用马铃薯Y病毒属特异性抗体（AS - 573/1；德国不伦瑞克生物材料德国资源中心DSMZ）和黄瓜花叶病毒（CMV；AS - 0929）抗体对五株代表性植株的有症状叶片样本进行抗原包被板酶联免疫吸附测定（ELISA），结果呈阳性。为证实这些结果，从FTA经典卡（FTA Classic Card，英国Whatman International Ltd.公司，梅德斯通）洗脱的病毒核酸，使用马铃薯Y病毒简并引物（CIFor：5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3'和CIRev：5'-ACICCRTTYTCDATDATRTTIGTIGC-3'）（3）和CMV亚组I特异性简并引物（CMV - 1F：5'-ACCGCGGGTCTTATTATGGT-3'和CMV - 1R：5' ACGGATTCAAACTGGGAGCA-3'）（1）进行逆转录（RT）-PCR。用马铃薯Y病毒特异性引物从ELISA阳性样本中扩增出一个约683个碱基对（bp）的单一DNA产物，用CMV特异性引物扩增出一个382 bp的片段。这些结果表明，所有五个样本中均存在马铃薯Y病毒和CMV混合感染。将扩增得到的马铃薯Y病毒特异性片段（四个样本）和CMV特异性片段（三个样本）分别克隆到pCR2.1（美国加利福尼亚州卡尔斯巴德市Invitrogen公司）中。每个扩增子从两个方向对两个独立克隆进行测序。这些序列的成对比较显示，使用马铃薯Y病毒特异性引物产生的克隆扩增子之间的同一性为93%至100%（GenBank登录号FJ653916、FJ653917、FJ653918、FJ653919、FJ653920、FJ653921、FJ653922、FJ653923、FJ653924、FJ653925和FJ653926），与来自台湾的菜豆普通花叶病毒（BCMV）相应核苷酸序列的同一性为92%至97%（登录号AY575773），与来自中国（登录号AJ312438）和美国（登录号AY863025）的BCMV序列的同一性为88%至90%。序列分析表明，来自长豇豆的BCMV分离株与来自台湾的分离株的亲缘关系比与来自中国和美国的分离株更近。CMV分离株（GenBank登录号FJ687054）彼此间100%相同，与来自泰国（登录号AJ810264）和马来西亚（登录号DQ195082）的CMV亚组I分离株的相应序列同一性为96%。BCMV和CMV在印度尼西亚东爪哇省的大豆、绿豆和花生中均有记录（2）。此前，在关岛的长豇豆上记录到了BCMV，但未记录到CMV（4）。据我们所知，本研究是印度尼西亚长豇豆中CMV首次确诊报告，进一步证明BCMV正在印度尼西亚定殖。参考文献：（1）J. Aramburu等人，《植物病理学杂志》155:513，2007年。（2）S. K. Green等人，《植物病害》72:994，1988年。（3）C. Ha等人，《病毒学档案》153:25，2008年。（4）G. C. Wall等人，《密克罗尼西亚》29:101，1996年。
