A PCR-based 'molecular tool box' for in planta differential detection of Verticillium dahliae vegetative compatibility groups infecting artichoke
A PCR-based 'molecular tool box' for in planta differential detection of Verticillium dahliae vegetative compatibility groups infecting artichoke
复制标题
DOI：
10.1111/j.1365-3059.2008.01981.x
复制
发表时间：
2009-06-01
期刊：
影响因子：
2.7
通讯作者：
Mercado-Blanco, J.
中科院分区：
文献类型：
--
作者：
Collado-Romero, M.;Berbegal, M.;Mercado-Blanco, J.
建立了多重套式-聚合酶链式反应方法，用于对侵染朝鲜蓟的黄萎菌分离物进行现场检测和营养体亲和群(VCGS)的评价。从VCG1A或VCG2B(334)菌株中扩增出334bp的标记，从VCG2A或VCG4B菌株中扩增出688bp的标记，从VCG2B(824)菌株中扩增出688bp和964bp的标记。两轮扩增后，根据扩增标记的特异性图谱，在洋蓟组织中鉴定出侵染大丽弧菌的VCGS。首先使用从洋蓟植株中提取的DNA来优化基于PCR的分子工具箱，这些DNA被人工接种到具有已知VCGS代表的菌株中。此后，使用从表现出一系列症状严重程度的自然感染的朝鲜蓟植物以及从没有症状的植物中提取的DNA来测试分子程序的效率。与传统的分离方法相比，新的多重套式聚合酶链式反应方法在检测病原体方面具有明显的优势，此外，它还提供了关于VCGS的信息。此外，在没有症状但受感染的植物中，通过微生物分离程序检查产生假阴性的大丽弧菌，该方法可以检测和VCG鉴定大丽叶弧菌的感染。这一“分子工具箱”揭示了几种大丽弧菌VCGS感染瓦伦西亚纳地区同一种洋蓟植物的存在。此外，它还有助于研究侵染洋蓟的大丽弧菌种群的遗传多样性和致病力多样性，并可能有助于预测黄萎病流行的严重程度。
A multiplex-nested-PCR procedure was developed for in planta detection of Verticillium dahliae isolates infecting artichoke and assessment of their vegetative compatibility groups (VCGs). PCR markers were identified and assigned to V. dahliae VCGs, including: i) a 334 bp marker amplified from VCG1A or VCG2B(334) isolates; ii) a 688 bp marker amplified from VCG2A or VCG4B isolates; and iii) a 688 bp and a 964 bp PCR marker amplified from VCG2B(824) isolates. The infecting V. dahliae VCGs were identified in artichoke tissues according to specific patterns of amplified markers after two rounds of PCR. The PCR-based 'molecular tool box' was first optimized using DNA extracted from artichoke plants artificially inoculated with isolates representative of known VCGs. Thereafter, the efficiency of the molecular procedure was tested using DNA extracted from naturally-infected artichoke plants showing a range of symptom severity as well as from symptomless plants. The novel multiplex-nested-PCR assay was clearly superior in detecting the pathogen compared to conventional isolation procedures, and in addition was informative about the VCGs. Moreover, the PCR method allowed the detection and VCG identification of V. dahliae infections in symptomless but infected plants, which had yielded false negatives when checked by microbiological isolation procedures. This 'molecular tool box' has uncovered the presence of several V. dahliae VCGs infecting the same artichoke plants in the Comunidad Valenciana Region. In addition, it is useful for genetic and pathogenicity diversity studies of V. dahliae populations infecting artichoke, and may help in predicting the severity of verticillium wilt epidemics.