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首页> 美国卫生研究院文献>Physiology and Molecular Biology of Plants >Molecular characterization of ‘Clover proliferation’ phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India
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Molecular characterization of ‘Clover proliferation’ phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India

机译：印度蔬菜作物相关的三叶草增殖植物质体D亚组（16SrVI-D）的分子特征

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Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches’ broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of ‘clover proliferation’ (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of ‘clover proliferation’ subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.
机译：2015年12月至2016年7月，在新德里印度农业研究所的实验田中观察到9种蔬菜植物物种，这些植物物种表现出可疑的白色/紫色叶，小叶，扁茎，女巫的扫帚，叶状体和叶黄等症状。从所有9种蔬菜的3种健康叶子和3个有症状叶子中提取的叶绿素经过植物质体特异性引物P1 / P7，R16F2n / R16R2和3Far / 3Rev的PCR检测，以扩增16S rDNA片段。在第一轮PCR分析中，未从任何有症状样品中引物对P1 / P7观察到DNA扩增。但是，从芸苔属油菜（Apiumgravolens L。）（两个分离株）扩增了约1.3kb的植物原质DNA特异性片段。通过使用3Far / 3Rev引物对对capitata L.（一种分离株）和对茄（Solanum melongena L。）（一种分离株）进行筛选，并使用R16F2n / R16R2引物对从莴苣（对一株）扩增1.2kb片段。在其他有症状的番茄，胡萝卜，葫芦，苦瓜和A菜的有症状蔬菜样品中未发现DNA扩增，使用P1 / P7引物对，随后使用3Far / 3Rev或R16F2n / R16R2引物对。在从有症状蔬菜田中收集的三种叶蝉物种中，仅发现Hishimonus phycitis对植物质体相关呈阳性。在健康植物样品和从无症状田野中收集的昆虫中未观察到DNA扩增。对发现的阳性植物质朴菌株的16S rDNA进行的序列比较分析表明，彼此之间以及与“三叶草繁殖”（16SrVI）组的质朴菌株之间具有100％的序列同一性。植原体序列，虚拟RFLP和16S rDNA序列的系统发育分析比较证实了在4种蔬菜和1种叶蝉（HP）物种中鉴定了16SrVI D亚型的植原体菌株。对蔬菜植原体菌株的16S rDNA序列进行的进一步虚拟RFLP分析证实了它们与“三叶草增殖”亚组D菌株的分类学分类。由于，研究中以有症状蔬菜为食的H. phycitis对16SrVI植原体亚组也呈阳性反应， D如蔬菜；它可能是感染植物和其他重要农作物的植物原质的16SrVI-D亚组的有效天然贮藏库。

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