Feau Nicolas, Weiland Jerry E, Stanosz Glen R, Bernier Louis
Centre de Recherche en Biologie Forestière, Université Laval, Sainte-Foy, Quebec G1K 7P4, Canada.
Mycol Res. 2005 Sep;109(Pt 9):1015-28. doi: 10.1017/s0953756205003242.
The development of a PCR assay for the detection of the poplar pathogenic fungi Septoria musiva (teleomorph Mycosphaerella populorum), S. populicola (M. populicola) and S. populi (M. populi) is described. Three pairs of species-specific PCR primers were designed using interspecific polymorphisms in the internal transcribed spacer (ITS) of nuclear ribosomal RNA gene (rDNA) repeats. The specificity of the three primer pairs was successfully tested on a collection of 40 S. musiva, 39 S. populicola and six S. populi isolates. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assay was further confirmed with DNA extracted from 12 additional Septoria species and 17 other fungal species obtained from stems or leaves of poplars. Specific amplification of the fragments for S. musiva and S. populicola was sensitive relatively to the technique used, detecting as low as 1 pg template DNA, and 10 pg of DNA of the target species in a background of 1 ng of DNA of the other species. Moreover, using DNA purified directly from disrupted conidia, it was possible to detect with a probability of 90%, using one unique PCR assay, the DNA equivalent of 166 conidia per microl of S. musiva and 156 conidia per microl of S. populicola. The procedures developed in this work can thus be applied for rapid and accurate detection and identification of Septoria species from poplars.
本文描述了一种用于检测杨树致病真菌缪氏壳针孢（有性型为杨树球腔菌）、杨树生壳针孢（有性型为杨树球腔菌）和杨树壳针孢（有性型为杨树球腔菌）的PCR检测方法。利用核糖体RNA基因（rDNA）重复序列的内部转录间隔区（ITS）中的种间多态性设计了三对种特异性PCR引物。在40株缪氏壳针孢、39株杨树生壳针孢和6株杨树壳针孢分离株上成功测试了这三对引物的特异性。在严格的PCR条件下，未观察到与任何测试分离株的交叉反应。从另外12种壳针孢属物种和从杨树茎或叶中获得的17种其他真菌物种中提取的DNA进一步证实了该PCR检测方法的特异性。缪氏壳针孢和杨树生壳针孢片段的特异性扩增对所用技术相对敏感，可检测低至1 pg的模板DNA，以及在1 ng其他物种DNA背景下10 pg的目标物种DNA。此外，使用直接从破碎分生孢子中纯化的DNA，通过一种独特的PCR检测方法，有90%的概率检测到每微升相当于166个缪氏壳针孢分生孢子和156个杨树生壳针孢分生孢子的DNA。因此，本研究中开发的方法可用于快速、准确地检测和鉴定杨树中的壳针孢属物种。
