A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping

Abstract

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases affecting the canola/oilseed rape (Brassica napus) industry worldwide. Currently, the planting of clubroot-resistant (CR) cultivars is the most effective strategy used to restrict the spread and the economic losses linked to the disease. However, virulent P. brassicae isolates have been able to infect many of the currently available CR cultivars, and the options to manage the disease are becoming limited. Another challenge has been achieving consistency in evaluating host reactions to P. brassicae infection, with most bioassays conducted in soil and/or potting medium, which requires significant space and can be labor intensive. Visual scoring of clubroot symptom development can also be influenced by user bias. Here, we have developed a hydroponic bioassay using well-characterized P. brassicae single-spore isolates representative of clubroot virulence in Canada, as well as field isolates from three Canadian provinces, in combination with canola inbred homozygous lines carrying resistance genetics representative of CR cultivars available to growers in Canada. To improve the efficiency and consistency of disease assessment, symptom severity scores were compared with clubroot evaluations based on the scanned root area. According to the results, this bioassay offers a reliable, less expensive, and reproducible option to evaluate P. brassicae virulence, as well as to identify which canola resistance profile(s) may be effective against particular isolates. This bioassay will contribute to the breeding of new CR canola cultivars and the identification of virulence genes in P. brassicae that could trigger resistance and have been very elusive to this day.

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

“…Phenotyping P. brassicae isolates serves the purpose of studying and describing the response of different hosts to the pathogen, aiming to comprehend its biology (Buczacki et al, 1975;Pang et al, 2020;Salih et al, 2023;Somé et al, 1996;Strelkov et al, 2018;Williams, 1966). This gathered information aids in revealing differences in virulence and host range.…”

“…Moreover, phenotyping allows the identification of genetic or environmental factors that impact the pathogen's growth and development, contributing to the formulation of effective management strategies for mitigating infections in Brassica crops. In a recent study published by our group in collaboration with the canola industry, we characterized several inbred homozygous (IH) canola lines using a hydroponic-based phenotyping system and the classic substrate inoculation method (Salih et al, 2023). Here, we elaborate on the substrate inoculation method because it allows phenotyping of the response of different hosts to the clubroot pathogen and facilitates propagation of the inoculum for further analyses.…”

“…Among the various protocols available for extracting P. brassicae resting spores, the methodology employed in this article is one that has been repeated extensively, consistently yielding reliable results (Hossain et al, 2021;Muirhead & Pérez-López, 2022;Pérez-López et al, 2020;Salih et al, 2023). The resting spores are extracted from infected clubroot galls, as these galls contain the highest concentration of inoculum.…”

“…Additionally, filter sterilization is another method for spore extraction. Fresh clubroot samples are homogenized and filtered multiple times through layered cheesecloth until all debris is removed (Pérez-López et al, 2020;Salih et al, 2023;Zamani-Noor, 2016). The method outlined in this article encompasses all these steps, optimizing the yield of resting spores from clubbed roots (Fig.…”

“…This response laid the foundation for categorizing P. brassicae isolates as races (Ayers, 1957;Buczacki et al, 1975;Williams, 1966), which are now referred to as pathotypes (Somé et al, 1996;Strelkov et al, 2018). Pathotyping the physiological reactions of different brassica cultivars to various P. brassicae isolates, as well as molecular phenotyping of the isolates themselves, plays a pivotal role in breeding clubroot resistance and selecting suitable germplasm for growers (Pang et al, 2020;Salih et al, 2023;Somé et al, 1996;Strelkov et al, 2018;Williams, 1966). However, challenges remain within the clubroot community, including the phenotyping process itself and basic tasks like DNA extraction from resting spores.…”