An improved technique for isolation and characterization of single-spore isolates of Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae, is a soil-borne disease that occurs in cruciferous crops worldwide. P. brassicae usually exists as a mixture of several pathotypes, which has hampered the research on resistance mechanisms of cruciferous crops against P. brassicae. In this study, clubroot galls were collected from a field in Shenyang, China, as a pathogen source to develop an efficient protocol for a single-spore isolation system of P. brassicae by optimizing the seedling age for inoculation, host inoculation method, and plant culture method. The operational steps of the single-spore isolation method were optimized as follows: the use of two-day-old seedlings for inoculation, substituting a cryobox (100 × 2.0 mL vials) for culture dishes, the addition of nutrient solution culture, and microscopic observations of single spores. The rate of infection success was substantially improved, and single-spore isolates of four pathotypes: 4, 8, 9, and 11, were acquired in this system. Subsequently, the optimized system was used to isolate and characterize the pathotypes of single-spore isolates of P. brassicae collected from five fields in regions in China. Approximately 4 to 9 pathotypes were isolated from each region. Among these, pathotype 4 was the most prevalent. This study provides a source of valuable information that can eventually be used for the genetic analysis of host–P. brassicae interaction.