A Basic Guide to the Propagation and Manipulation of the Clubroot Pathogen, Plasmodiophora brassicae

Abstract

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio‐economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagationSupport Protocol 1: Evans blue staining to identify resting spore viabilitySupport Protocol 2: Storage of Plasmodiophora brassicaeBasic Protocol 2: Generation of single spore isolates from P. brassicae field isolatesBasic Protocol 3: Phenotyping of Plasmodiophora brassicae isolatesBasic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting sporesBasic Protocol 5: Molecular detection of Plasmodiophora brassicae

Telomere-to-telomere Genome Assembly of the Clubroot Pathogen Plasmodiophora Brassicae

“…Pb3A resting spores propagated in Westar, a susceptible B. napus accession, were extracted as previously described ( Salih et al 2024 ). Using 3 to 5 mL of spores at a concentration ranging from 10 8 to 10 9 spores/mL, HMW DNA was extracted from the resting spores using a modified cetyltrimethylammonium bromide (CTAB) method to avoid DNA shearing, followed by a cleaning step using the Nanobind CBB kit (PacBio, USA).…”