Lecomte P, Péros J P, Blancard D, Bastien N, Délye C
Unité Mixte de Recherches en Santé Végétale, Institut National de la Recherche Agronomique, Domaine de la Grande Ferrade, 33883 Villenave d'Ornon Cedex, France.
Appl Environ Microbiol. 2000 Oct;66(10):4475-80. doi: 10.1128/AEM.66.10.4475-4480.2000.
Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.
葡萄座腔菌是葡萄蔓割病的致病真菌，葡萄蔓割病是一种严重的葡萄坏死病。在培养基上特征性分生孢子出现不稳定且延迟（1至2个月），以及腐朽木材中存在大量微生物，使得在葡萄木材样本中鉴定或检测葡萄座腔菌变得困难。我们设计了六对用于诊断葡萄座腔菌的PCR引物。三对引物源自核糖体DNA内部转录间隔区序列，另外三对源自随机扩增多态性DNA片段。这六对引物可用于扩增从所有测试的葡萄座腔菌分离株中提取的DNA。它们不会扩增来自与葡萄相关的50多种不同微生物种类的真菌和细菌的DNA。我们开发了一种简单的方案，可快速释放DNA，这使我们能够从纯培养物或混合培养物以及葡萄木材样本中鉴定葡萄座腔菌。在木材中鉴定葡萄座腔菌只需几个小时，而不是在琼脂培养基上进行传统培养所需的几周时间。我们相信这里描述的程序可以适用于检测涉及木本植物病害的其他微生物。
