Rampersad S N, Umaharan P
The University of the West Indies, Department of Life Sciences, St. Augustine, Republic of Trinidad and Tobago.
Plant Dis. 2003 May;87(5):602. doi: 10.1094/PDIS.2003.87.5.602A.
Severe symptoms of suspected geminivirus etiology were manifested as intense yellow or golden mottling or mosaic of the lamina accompanied by mild leaf margin distortion on dicotyledonous weed species, Sida rhombifolia (L.) and Rhynchosia minima (L.), collected from 1999 to 2002 from the northeastern and central regions of Trinidad. S. rhombifolia is a common roadside weed while R. minima may have been introduced through restricted cultivation as a forage legume for livestock. Potato yellow mosaic virus-Trinidad isolate (PYMV-TT) has been implicated as the primary causal agent of begomoviral disease in large-scale tomato cultivation in Trinidad (2). It has been suggested that these weeds may be alternative hosts to PYMV-TT. However, all samples tested negative for PYMV-TT in dot blot hybridization assays using a PYMV-TT-specific DNA-A probe under high stringency. These results excluded the presence of PYMV-TT in these weeds. Polymerase chain reaction (PCR) amplification using clarified leaf extracts with degenerate primers for DNA-A (MP16 and MP82, PAL1v1978 and PAR1c715, and prV324 and prC889) and for DNA-B (PBL1v2040 and PCRc1) was performed on the weed samples (S. N. Rampersad and P. Umaharan, unpublished). Degenerate primers MP16 and MP82 target the 5' terminal region of the coat protein (cp) (2); PAL1v1978 and PAR1c715 direct amplification of the replication-associated protein gene (rep) and part of the cp gene (1); prV324 and prC889 amplify the core cp sequence (3). Primers PBL1v2040 and PCRc1 target the intergenic region and the 5' terminal of the BL1 ORF (1). PCR fragments obtained through amplification using this primer pair confirmed the presence of a DNA-B component for the unknown viruses. PCR fragments were sequenced and alignments were performed using DNASTAR (DNASTAR Inc., Madison, WI) and BLASTN ( www.ncbi.nlm.nih.gov/blast/ ) programs. None of the partial nucleotide sequences obtained for the viruses produced significant alignments with each other (5' terminal cp: 74% identity; core cp sequence: 78% identity), suggesting the detection of two distinct viruses. In addition, the partial sequences obtained were aligned to sequences of homologous regions of 11 New World begomoviruses (from the major representative clusters). The nearest match for R. minima, using alignments with 5' terminal cp (GenBank Accession No. AY221124), core cp (GenBank Accession No. AY217344), and 5' terminal BL1 region (GenBank Accession No. AY220490) was obtained for Rhynchosia golden mosaic virus (RhGMV, GenBank Accession Nos. AF408199 and AF442117) with 84 and 88% identity. There were no significant similarities found for sequence comparisons of the BL1 ORF. For S. rhombifolia, the highest homology using the 5' terminal cp (GenBank Accession No. AY220489), core cp (GenBank Accession No. AY217345), rep/cp region (GenBank Accession No. AY220488), and the 5' terminal BL1 region (GenBank Accession No. AY221125) was obtained for Sida golden mosaic virus (SiGMV, GenBank Accession Nos. AF049336, AF070923, and Y11100), with 82, 89, 84, and 87% identity. To our knowledge, this is the first report of geminivirus infection in these weed species in Trinidad. This may have substantial implications to future geminivirus disease outbreaks especially if there is expansion of the host range of these viruses to include economically important crops. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) P. Umaharan et al. Phytopathology 88:1262, 1998. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.
1999年至2002年期间，从特立尼达岛东北部和中部地区采集的双子叶杂草物种——菱叶黄花稔（Sida rhombifolia (L.)）和小果灰叶豆（Rhynchosia minima (L.)）上，疑似双生病毒病因的严重症状表现为叶片出现强烈的黄色或金色斑驳或花叶，并伴有轻微的叶缘扭曲。菱叶黄花稔是一种常见的路边杂草，而小果灰叶豆可能是作为家畜饲料豆科植物通过有限种植引入的。马铃薯黄花叶病毒特立尼达分离株（PYMV-TT）被认为是特立尼达大规模番茄种植中番茄曲叶病毒病的主要病原体（2）。有人认为这些杂草可能是PYMV-TT的替代寄主。然而，在高严谨度条件下，使用PYMV-TT特异性DNA-A探针进行的斑点杂交试验中，所有样本对PYMV-TT检测均呈阴性。这些结果排除了这些杂草中存在PYMV-TT的可能性。使用针对DNA-A（MP16和MP82、PAL1v1978和PAR1c715、prV324和prC889）以及针对DNA-B（PBL1v2040和PCRc1）的简并引物，对杂草样本（S. N. Rampersad和P. Umaharan，未发表）的澄清叶提取物进行了聚合酶链反应（PCR）扩增。简并引物MP16和MP82靶向外壳蛋白（cp）的5'末端区域（2）；PAL1v1978和PAR1c715指导复制相关蛋白基因（rep）和部分cp基因的扩增（1）；prV324和prC889扩增核心cp序列（3）。引物PBL1v2040和PCRc1靶向基因间隔区和BL1 ORF的5'末端（1）。使用该引物对扩增获得的PCR片段证实了未知病毒存在DNA-B组分。对PCR片段进行了测序，并使用DNASTAR（DNASTAR公司，威斯康星州麦迪逊）和BLASTN（www.ncbi.nlm.nih.gov/blast/）程序进行了比对。所获得的病毒部分核苷酸序列彼此之间均未产生显著比对结果（5'末端cp：74%同一性；核心cp序列：78%同一性），这表明检测到了两种不同的病毒。此外，将所获得的部分序列与11种新大陆番茄曲叶病毒（来自主要代表性簇）同源区域的序列进行了比对。使用与5'末端cp（GenBank登录号AY221124）、核心cp（GenBank登录号AY217344）和5'末端BL1区域（GenBank登录号AY220490）的比对，小果灰叶豆与黄金花叶病毒（RhGMV，GenBank登录号AF408199和AF442117）的匹配度最高，同一性为84%和88%。在BL1 ORF的序列比较中未发现显著相似性。对于菱叶黄花稔，使用5'末端cp（GenBank登录号AY220489）、核心cp（GenBank登录号AY217345）、rep/cp区域（GenBank登录号AY220488）和5'末端BL1区域（GenBank登录号AY221125），与黄花稔金色花叶病毒（SiGMV，GenBank登录号AF049336、AF070923和Y11100）的同源性最高，同一性分别为82%、89%、84%和87%。据我们所知，这是特立尼达这些杂草物种中双生病毒感染的首次报道。这可能对未来双生病毒病的爆发具有重大影响，特别是如果这些病毒的寄主范围扩大到包括经济上重要的作物。参考文献：（1）M. R. Rojas等人，《植物病害》77:340，1993年。（2）P. Umaharan等人，《植物病理学》88:1262，1998年。（3）S. D. Wyatt和J. K. Brown，《植物病理学》86:1288，1996年。
