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Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (6): 1290-1300.doi: 10.16420/j.issn.0513-353x.2021-0421

• Research Papers • Previous Articles     Next Articles

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

RUI Tingting1,2, GAO Qingyun1,3, LI Xiaojing1, SHI Yanxia1, XIE Xuewen1, LI Lei1, ZHANG Hongjie3, XU Wenjuan2, CHAI Ali1,**(), LI Baoju1,**()   

  1. 1The Institute of Vegetable and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China
    2Anhui Agricultural University,Hefei 230031,China
    3Hebei North University,Zhangjiakou,Hebei 075061,China
  • Received:2021-12-20 Revised:2022-02-21 Online:2022-06-25 Published:2022-07-05
  • Contact: CHAI Ali,LI Baoju E-mail:chaiali@caas.cn;libaojuivf@163.com

Abstract:

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 1300,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.

Key words: Crucifer vegetable, clubroot disease, Plasmodiophora brassicae, single-spore isolation, pathotype differentiation

CLC Number: