茶树炭疽菌分离鉴定与遗传转化体系建立

Isolation and Identification of Anthrax from Tea Plant and Establishment of Genetic Transformation System

【目的】炭疽菌属(Colletotrichum spp.)病原菌主要引起茶树叶部炭疽病害。为了降低茶树受到炭疽菌的侵害,从而防控茶树炭疽病,作者进行了病原菌的分离鉴定和遗传体系的建立。【方法】对福建省的政和县、福州市、福安市和宁德市不同茶产区的茶树炭疽病病叶采用单孢分离法分离病原菌,对分离得到的菌株采用形态学结合分子鉴定方法(rDNA-ITS序列)进行鉴定。为深入研究其致病性机理,将含有潮霉的抗性标记基因片段和融合有绿色荧光蛋白(Green fluorescent protein)GFP基因的Histone 1片段的质粒pCZ007,采用聚乙二醇PEG-CaCl_(2)介导法,转化茶树胶胞炭疽病原菌。【结果】从4个地区共分离得到致病炭疽菌12株,并进行观察和测定。发现引起茶树炭疽病的胶孢炭疽菌(Colletotrichum gloeosporioides)是优势菌株,并成功建立的该茶树胶孢炭疽菌的遗传转化体系,随机挑取的4个经分子验证的转化子均能观察到定位于细胞核的绿色荧光和具有抗潮霉素的特性,随后的多次传代均能够稳定观察到绿色荧光定位信号。【结论】本研究分离并经形态学结合分子检测鉴定了福建省4个茶产区的茶树炭疽病的主要病原菌为胶孢炭疽菌(C.gloeosporioides),成功建立能稳定表达目的基因的遗传转化体系,为后续深入研究茶树炭疽菌相关关键致病功能基因的分子机制提供新的线索,运用绿色荧光蛋白观察茶树胶孢炭疽菌对茶树叶片的侵染过程奠定基础。

【Objective】The disease of tea tree leaves was mainly caused by Colletotrichum spp..In order to control the process of anthrax infection on tea trees,and reduce the invasion of the impact on tea trees,isolation and identification were carried out and the genetic system was established.【Method】The pathgens were isolated from different tea production areas from Zhenghe,Fuzhou,Fu’an and Ningde in Fujian province.The isolates were isolated by single spore separation and identified by morphological combined molecular identification(rDNA-ITS sequence)method.For further research on pathogenic mechanism,the GFP system of C.gloeosporioides was established,containing hygromycin resistance marker gene and the green fluorescent protein gene,by using polyethylene glycol PEG-CaCl_(2) mediated transformation process.【Result】A total of 12 strains were obtained from tea tree diseases,and the representative strains from 4 regions were selected for observation and determination,showing that the C.gloeosporioides was the dominant strain,which caused the anthracnose of tea tree.The GFP system of anthrax fungus of tea tree has been successfully established.The randomly selected transformants have fluorescence phenomenon and adaptability against hygromycin.【Conclusion】In the study,C.gloeosporioides,isolated and identified by morphological and molecular methods,was the main pathogen of tea tree anthracnose in 4 tea producing regions of Fujian province.A genetic transformation system of C.gloeosporioides that can stably express the target gene was successfully established.It provides a new clue for further study on the molecular mechanism of key pathogenic genes related to anthrax in tea tree,and lays a foundation for observation of the infection process of anthrax in tea tree leaves by using green fluorescent protein.