Alaniz Sandra, Armengol Josep, García-Jiménez José, Abad-Campos Paloma, León Maela
Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022-Valencia, Spain.
Plant Dis. 2009 Aug;93(8):821-825. doi: 10.1094/PDIS-93-8-0821.
Cylindrocarpon liriodendri and C. macrodidymum are the causal agents of grapevine black foot disease. Recently, a third species, C. pauciseptatum, has been isolated from roots of grapevine showing decline symptoms. Currently, reliable identification of isolates of these species through phenotypical characteristics has not been possible. The polymerase chain reaction (PCR)-based method developed in this study allows a quick and easy detection of Cylindrocarpon spp. associated with grapevine. Three primer pairs annealing to variable, partly species-specific sites of the internal transcribed spacer regions amplified species-specific PCR fragments of different sizes in C. liriodendri, C. macrodidymum, and C. pauciseptatum in a multiplex assay with DNA obtained with both quick and traditional extraction methods. They did not generate any PCR product in other fungal trunk pathogens or contaminants commonly associated with grapevines. When universal fungal ITS primers were used in a nested multiplex PCR, the three primer pairs also detected C. liriodendri, C. macrodidymum, and C. pauciseptatum in total DNA extracted from roots of inoculated grapevines. The designed methods can be used for the diagnosis of these fungi from pure culture or infected grapevines.
葡萄柱枝孢和巨孢柱枝孢是葡萄黑脚病的病原菌。最近，已从表现衰退症状的葡萄根部分离出第三种柱枝孢——少隔柱枝孢。目前，通过表型特征可靠鉴定这些物种的分离株是不可能的。本研究开发的基于聚合酶链反应（PCR）的方法能够快速、简便地检测与葡萄相关的柱枝孢属真菌。三对引物与内转录间隔区的可变、部分物种特异性位点退火，在多重分析中，利用快速和传统提取方法获得的DNA，扩增出葡萄柱枝孢、巨孢柱枝孢和少隔柱枝孢中不同大小的物种特异性PCR片段。它们在其他通常与葡萄相关的真菌树干病原菌或污染物中未产生任何PCR产物。当在巢式多重PCR中使用通用真菌ITS引物时，这三对引物也能在从接种葡萄根部提取的总DNA中检测到葡萄柱枝孢、巨孢柱枝孢和少隔柱枝孢。所设计的方法可用于从纯培养物或受感染葡萄中诊断这些真菌。
