芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定

Methylene Blue Combined Agarose Assay for Single-spore Isolation of Plasmodiophora brassicae and Pathotype Differentiation

对传统芸薹根肿菌(Plasmodiophora brassicae)单孢分离的水琼脂法进行改进,确定了亚甲基蓝和琼脂糖的最佳浓度,将0.01%亚甲基蓝染色液与1.5%琼脂糖溶液按体积比1︰300混匀,制成亚甲基蓝琼脂糖凝层,在显微镜下可成功分离芸薹根肿菌单孢。大白菜单孢接种最佳苗龄为催芽24 h,接种后暗培养24 h,再移栽至育苗钵,发病率最高。利用该方法分离获得中国11个省十字花科(大白菜、甘蓝、油菜)芸薹根肿菌单孢系菌株79株,采用Williams鉴别系统鉴定出P2、P3、P4、P5、P6、P7、P8、P9、P10、P12、P13和P14等12个不同的生理小种类型,其中P4为优势生理小种,出现频率为49.37%。明确了中国11个省份根肿菌的生理小种类型及其分布,为十字花科蔬菜抗病育种提供重要资源。

The traditional water agar method for single-spore isolation of Plasmodiophora brassicae was improved,and the optimum concentrations of methylene blue and agarose were determined.Methylene blue agarose gel layers were prepared by mixing 0.01%methylene blue and 1.5%agarose solution with a volume ratio of 1︰300,on which single-spore of P.brassicae could be successfully isolated under the microscope.The best seedling age for single-spore inoculation was germinating for 24 h.After inoculation,the seedlings were then cultured in darkness for 24 h before transplanted to bowls.A total of 79 single-spore strains of P.brassicae were isolated from 11 provinces in China by using methylene blue combined agarose assay developed in this study.And 12 pathotypes such as P2,P3,P4,P5,P6,P7,P8,P9,P10,P12,P13 were identified using the Williams identification system.Among which,P4 was the dominant pathotype,with a frequency of 49.37%.The pathotype differentiates and distributions of P.brassicae from 11 provinces of China were clarified,and the results may provide important resources for breeding disease-resistant varieties of crucifer vegetables.