Rolshausen P E, Trouillas F, Gubler W D
Department of Plant Pathology, University of California, Davis 95616.
Plant Dis. 2004 Sep;88(9):925-929. doi: 10.1094/PDIS.2004.88.9.925.
Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.
阔叶树溃疡病菌是木本植物的一种维管束溃疡病菌，通常通过从受感染组织中分离病原菌来进行诊断。在加利福尼亚州的葡萄园里，还发现了来自同一科（座囊菌科）的相关真菌与葡萄树有关联。已开发出一种原位聚合酶链反应（PCR）方法用于检测受感染木材组织中的阔叶树溃疡病菌。然而，我们的结果表明，该方法也会扩增其他座囊菌科真菌，这可能会导致错误诊断。因此，我们开发了一种PCR-限制性片段长度多态性（PCR-RFLP）分析方法。使用通用引物通过PCR扩增内部转录间隔区（ITS）1/5.8S/ITS2核糖体DNA区域，并用AluI消化后确定RFLP模式。所获得的限制性图谱有助于将阔叶树溃疡病菌与葡萄树干病原菌（葡萄拟茎点霉、葡萄座腔菌属、嗜土暗色座节孢和拟茎点霉）、座囊菌科真菌（座囊菌属、黑座囊菌属、葡萄小穴壳菌和细弱阔叶树溃疡病菌）、在葡萄枯木上发现的隐壳孢属以及其他阔叶树溃疡病菌种（佩氏阔叶树溃疡病菌、星状阔叶树溃疡病菌、皱皮阔叶树溃疡病菌和细弱阔叶树溃疡病菌）区分开来，但亚美尼亚阔叶树溃疡病菌（我们将其视为阔叶树溃疡病菌的同义词）和葡萄树上未发现的光滑阔叶树溃疡病菌除外。
