Martini M, Musetti R, Grisan S, Polizzotto R, Borselli S, Pavan F, Osler R
Department of "Biologia e Protezione delle Piante", University of Udine, Via delle Scienze 208, 33100 Udine, Italy.
Plant Dis. 2009 Oct;93(10):993-998. doi: 10.1094/PDIS-93-10-0993.
Aureobasidium pullulans and Epicoccum nigrum are frequently reported as endophytes of various crops, including grapevine (Vitis vinifera). Because of their potential role as biological control agents against grapevine pathogens, we examined the occurrence of A. pullulans and E. nigrum in two grapevine varieties (Merlot and Prosecco) in Italian vineyards where spontaneous recovery from phytoplasma disease is recurrent. Species-specific primers for A. pullulans and two genetically distinct strains of E. nigrum were designed in variable regions of ITS1 and ITS2. Primer specificity was confirmed by polymerase chain reaction using purified DNA from other fungal endophytes that are usually encountered during isolation attempts from grapevine tissues and from several other strains of A. pullulans and E. nigrum isolated from other sources. In order to determine the occurrence of the two endophytes in grapevine plants, DNA was extracted from shoots of 44 grapevines collected in six vineyards from different localities of northeast Italy. Both endophytes were detected and their identity was confirmed by restriction fragment length polymorphism (RFLP) patterns obtained from reference strains. RFLP analyses confirmed the presence of two E. nigrum strains belonging to different RFLP groups in grapevine. The molecular methods described allowed a sensitive, specific, and reliable identification of the two endophytes in grapevine.
出芽短梗霉和黑附球菌常被报道为包括葡萄（欧亚葡萄）在内的各种作物的内生菌。鉴于它们作为葡萄病原菌生物防治剂的潜在作用，我们在意大利葡萄园的两个葡萄品种（梅洛和普罗塞克）中研究了出芽短梗霉和黑附球菌的存在情况，这些葡萄园经常出现从植原体病害中自发恢复的情况。针对出芽短梗霉和两种遗传上不同的黑附球菌菌株，在ITS1和ITS2的可变区域设计了物种特异性引物。通过聚合酶链反应，使用从葡萄组织分离尝试过程中通常遇到的其他真菌内生菌以及从其他来源分离的出芽短梗霉和黑附球菌的其他几个菌株的纯化DNA，证实了引物的特异性。为了确定这两种内生菌在葡萄植株中的存在情况，从意大利东北部不同地区的六个葡萄园收集的44株葡萄的嫩枝中提取了DNA。检测到了这两种内生菌，并通过从参考菌株获得的限制性片段长度多态性（RFLP）模式确认了它们的身份。RFLP分析证实了葡萄中存在属于不同RFLP组的两种黑附球菌菌株。所描述的分子方法能够灵敏、特异且可靠地鉴定葡萄中的这两种内生菌。
