A novel and efficient method for producing high-purity single protoplast-derived isolates of Plasmodiophora brassicae

Plasmodiophora brassicae, a root-infecting protist, causes the devastating clubroot disease of cruciferous crops worldwide. Field populations of this pathogen often consist of multiple strains, leading to inconsistent experimental results and unreliable resistance classification in breeding programs. The extremely low infection success rate associated with single resting spore inoculation has limited the generation of genetically uniform single spore-derived isolates (SSIs). In this study, we developed a simple and highly effective single-protoplast-derived isolate (SPI) method to obtain near-genetically pure isolates of P. brassicae. The method involves enzymatic digestion of clubroot-infected root tissue to remove plant cell wall and release individual plant protoplasts. Each released protoplast contains thousands of resting spores. Individual protoplasts are then isolated and purified from the digestion suspension. All resting spores contained within a single protoplast are subsequently released and used as the sole inoculum to generate one SPI on susceptible canola seedlings. Using soilless Sunshine Mix #3, SPI infection success rates ranged from 77.8 to 100%, depending on the strain. Genetic purity of 20 SPIs derived from strain AB11 was assessed using Kompetitive Allele-Specific PCR, revealing 95% homogeneity at informative SNP loci. Whole-genome sequencing further demonstrated a marked reduction in genomic heterogeneity, from 91.22 to 96.40% in the progenitor strains AB11 and AB16, respectively, to ≤ 1.0% in their corresponding SPIs, compared with 14.95 and 44.26% observed in two of nine published SSIs. Using a panel of Brassica napus lines, each carrying a single clubroot resistance gene (Rcr1, Rcr3, Rcr5, Rcr8, Rcr9, or Rcr10), we identified the corresponding avirulence (Avr) genes among 18 SPIs. The frequencies of Avr1, Avr3, Avr5, Avr8, Avr9, and Avr10 were 0.0, 11.1, 44.4, 33.3, 27.8, and 50.0%, respectively. Seven races were identified, including the highly aggressive race avr1-3-5-8-9-10, underscoring the urgent need for novel resistance genes. The SPI method provides a robust, efficient, and reproducible strategy for generating near-genetically pure P. brassicae isolates with high infection success, improving the reliability of resistance screening and virulence monitoring, and facilitating informed and durable deployment of clubroot resistance genes for long-term disease management.