非刚竹属5种竹子丛枝病病原菌的分离和鉴定

Isolation and Identification of the Pathogens Causing Witches’Broom Disease of Five Bamboo Species of Non-Phyllostachys

【目的】分离和鉴定非刚竹属5种竹子丛枝病病原菌,并分析其与麦角菌科其他真菌间的系统发育关系,为竹子丛枝病病原菌的鉴定、病害的防控和麦角菌科物种间亲缘关系的确定提供科学依据。【方法】采用组织分离法从非刚竹属5种竹子丛枝病样品上分离病原菌,利用显微镜观察病原菌的形态结构并测量分生孢子大小,结合形态学与分子生物学方法鉴定病原菌物种,基于ITS rDNA和LSU rDNA序列构建麦角菌科的系统发育树。【结果】中华业平竹、巴山木竹和薄箨茶竿竹丛枝病组织分离的病原菌形态结构与竹针孢座囊菌一致,分生孢子在PDA平板上形成表面凹凸不平的褶皱、略有光泽、边缘不整齐的浅黄色菌落,分生孢子针形,大小为(22.4~52.0)μm×(2.2~6.0)μm。黄麻竹和牡竹丛枝病组织分离的病原菌菌丝在PDA平板上形成表面平整、边缘整齐的圆形菌落,病原菌培养72天后产生短棒状分生孢子,大小为(5.4~10.5)μm×(1.2~2.8)μm。ITS rDNA和LSU rDNA序列分析和系统发育分析表明,分离自中华业平竹、巴山木竹和薄箨茶竿竹丛枝病样品的病原菌与针孢座囊菌聚为一支,支持率为99%;分离自黄麻竹和牡竹丛枝病样品的病原菌与竹异香柱菌聚为一支,支持率为100%。【结论】中华业平竹、巴山木竹和薄箨茶竿竹丛枝病的病原菌为竹针孢座囊菌,该菌与麦角菌属的亲缘关系近。黄麻竹和牡竹丛枝病的病原菌为箣竹异香柱菌,该菌与肉瘤座菌属的亲缘关系近,与香柱菌属的亲缘关系远。

【Objective】In this study,we isolated and characterized the causal agents of witches’broom disease on 5 bamboo species of non-Phyllostachys,and analyzed the morphological features and culture characteristics of the pathogens,which would provide a scientific basis for identifying of the pathogen,controlling the witches’broom disease of bamboo,and determining the phylogenetic relationship of Clavicepitaceae.【Method】The pathogens of witches’broom disease were isolated from the infected tissues of five bamboo species of non-Phyllostachys in Hangzhou of Zheijiang and Xishuangbanna,Yunnan through the conventional tissue isolation approach.The species identification was conducted based on morphological methods combined with molecular biology.The morphology and conidia size of the pathogens were determined by microscope,and the phylogenetic relationships of Clavicipitaceae were investigated based on the ITS rDNA and LSU rDNA sequences.【Result】The morphological structure of the pathogens isolated from Semiarundinaria sinica,Bashania fargesii and Pseudosasa amabilis var.tenuis were similar to Aciculosporium take.The conidia of pathogenic fungus formed a pale yellow colony with uneven fold,slightly glossy surface and irregular edge on PDA plate.The conidia of pathogenic fungus was needle shape,(22.4-52.0)μm×(2.2-6.0)μm in size.The mycelia of pathogenic fungi isolated from the infected tissues of Dendrocalamopsis stenoaurita and Dendrocalamus strictus formed round colony with smooth surface and regular edge on PDA plate.The pathogen produced short rod-like conidia after 72 days of culture,and the size of the conidia was(5.4-10.5)μm×(1.2-2.8)μm.The sequence analysis and phylogenetic analysis based on ITS rDNA and LSU rDNA showed that the pathogenic fungus isolated from S.sinica,B.fargesii and P.amabilis var.tenuis were clustered with A.take with 99%bootstrap support,and the pathogenic fungus isolated from D.stenoaurita and D.strictus were clustered with Heteroepichlo bambusae with 100%bootstrap support.【Conclusion】The causal agent of witches’broom disease of S.sinica,B.fargesii and P.amabilis var.tenuis is A.take,which is closely related to the genus Claviceps.The causal agent of witches’broom disease of D.stenoaurita and D.strictus.is H.bambusae,which is closely related to the genus Balansia,but distantly related to the genus Epichlo.