烤烟蛙眼病病原菌的分离鉴定与生物学特性

【目的】明确贵州烤烟蛙眼病病原菌种类及生物学特性，为烤烟蛙眼病的综合防治提供科学依据。【方法】以采集自贵州烟区的烤烟蛙眼病病叶为材料，采用常规组织分离法和离体叶片接种法进行病原菌分离及致病性测定，利用形态学特征和分子生物学方法对病原菌进行种类鉴定，并对典型菌株进行生物学特性测定。【结果】从采集的病叶样本中共分离获得11株菌株，选取具有代表性的3株菌株YC1110、YC1111和YC1112接种到健康烤烟叶片上，接种6 d后出现典型症状，与田间症状一致，证明3株菌株为烤烟蛙眼病病原菌。ITS序列及系统发育分析表明，供试3株菌株与烟草尾孢（Cercospora nicotianae）的遗传关系很近，且菌落及孢子等形态特征亦符合烟草尾孢。生物学特性测定结果显示，病原菌在25℃、pH 7、光照条件为24 h全黑暗、碳氮源分别为果糖和牛肉浸粉、培养基为番茄琼脂培养基（TA2）时产孢量最多；在25℃、pH 7、碳氮源分别为甘露醇和蛋白胨、培养基为胡萝卜琼脂培养基（CA）和烟叶煎汁琼脂培养基（TA1）时生长最快。病原菌菌丝致死温度为54℃水浴10 min。【结论】引起贵州烤烟蛙眼病的病原菌为烟草尾孢。

Isolation,identification and biological characteristics of pathogens causing frog eye disease in flue-cured tobacco

【Objective】To clarify species and biological characteristics of frog eye disease pathogen from flue-cured tobacco，so as to provide scientific basis for controlling flue-cured tobacco frog eye disease.【Method】 Flue-cured tobacco leaves with frog eye disease in Guizhou tobacco planting areas were taken as material.Pathogens were isolated and their pathogenicity was determined by conventional tissue isolation method and in vitro leaf inoculation method.Pathogens were classified and identified according to morphological characteristics and molecular biology methods.And the biological characteristics of typical strains were determined.【Result】Eleven strains of bacteria were isolated from collected diseased tobacco leaves，and 3 typical strains of YC1110，YC1111 and YC1112 were inoculated into healthy flue-cured tobacco.After six d of inoculation，tobacco leaves showed typical symptoms as the symptoms in field，proving the 4 strains were frog eye disease pathogen.ITS sequence and phylogenetic analysis showed that the 3 tested strains had close genetic relationship with Cercospora nicotianae，and the morphological characteristics of colonies and spores were also consistent with those of C.nicotianae.Biological characteristic determination showed that the highest spore yield required a temperature with was 25℃，pH of 7，24 h darkness of light condition，carbon and nitrogen sources of fructose and beef extract powder，the medium of tomato agar medium（TA2）.The optimum condition for the fastest growth were temperature of 25℃，pH of 7，carbon and nitrogen sources of mannitol and peptone，medium of carrot agar（CA）and tobacco leaf juice agar medium（TA1）.The lethal temperature of pathogen mycelia was 54℃ water bath for 10 min.【Conclusion】 The pathogen causing frog eye disease of flue-cured tobacco in Guizhou is identified as C.nicotiana.