In vitro culture of Plasmodiophora brassicae

Term: 2 years, beginning 2024
Status: Ongoing
Researcher(s): Mary Ruth McDonald, University of Guelph
SaskCanola Investment: $24,955
Total Project Cost: $99,820
Funding Partners: WGRF, ACPC

Objective

1. Develop a method for in vitro culture of P. brassicae 

2. Produce single spore cultures of different pathotypes of P. brassicae and submit for long read sequencing. 

Project Description

Plasmodiophora brassicae is an obligate pathogen so must have a host to complete its life cycles and, by definition, cannot be grown in pure culture. The pathogen lives within the cells of its host throughout most its life cycle. Resting spores are produced in infected roots and are released into the soil as the roots decay. These represent the only source of pure pathogen available. However, when trying to get ‘clean’ cells for sequencing and other research, the P. brassicae material is generally contaminated with genes from plants and soil microbes which causes problems. A method to grow pure cultures of cells of P. brassicae, outside of the host, would be very useful for many types of research, and especially as a tool for selecting clubroot resistant canola lines, and advancing breeding canola for clubroot resistance. 

Dual cultures of P. brassicae and brassica crops have produced the ‘cleanest’ cultures of the pathogen so far. Dual-culture techniques, where the pathogen grows within tissue culture callus of a Brassica host, permit the study of P. brassicae under controlled conditions and provide a cleaner genomic material (only the pathogen and a single host) for further studies. The McDonald lab group has used dual tissue cultures of P. brassicae and Brassica host to remove the soil microbial contaminations. However, producing dual cultures of P. brassicae and brassica crops is challenging and time-consuming. Brassica plants do not produce callus in tissue culture easily, and only survive for a month or two, even under ideal conditions. The success rate of dual cultures with Brassicas is very low. The isolation of the pathogen and growth of pure cultures under controlled conditions in the lab would have many benefits and would advance research about P. brassicae biology, genomics, mechanism of virulence, and consequently breeding for clubroot resistance in crops and the management of clubroot. The group in the McDonald lab were able to remove the cell walls of P. brassicae and keep the cells alive on culture media for several hours up to a few days, without the presence of the host. These cells were not zoospores, which have two flagella and swim in culture. It appeared that some of the cells were dividing, but the numbers declined over time, probably because cells were dying faster than others could divide. However, when a suspension of the cell wall-free cells was used to inoculate susceptible plants, there was more disease severity on plants inoculated with the amoebae-like cells, than with a similar concentration of the resting spores from the same club. This demonstrates that the amoebae-like cells were alive and capable of infecting plants and causing clubroot. It also suggests (as expected) that not all resting spores germinate when applied to a root. We postulate that an optimum growth medium can be developed that will mimic conditions in a Brassica root, and then the cells will divide, increase over time form small cell colonies. 

Obligate parasites such as Plasmodiophora and rust fungi obtain some essential nutrients for their growth from living hosts such as amino acids and sugars. The axenic culture of rust fungi was possible by providing Czapek's minerals, sucrose or glucose, and various combinations and concentrations of 19 amino acids and a tripeptide, glutathione. However, the nutritional requirement for P. brassicae has not yet been studied. Several reports have suggested that the organism may have a free-living saprotrophic amoebal stage. Some studies provided evidence of plasmodial growth on culture media in related organisms such as Spongospora subterranea and P. brassicae. Resting spores were germinated on Ringer's agar spread previously with a suspension of Escherichia coli. Each resting spore produced a single uninucleate amoeba. The amoebae encysted if the culture media was left to dry out. However, the cyst germinated new amoeba if transferred to a fresh media. Previous attempts to culture P. brassicae used either the resting spores of the pathogen or infected canola root tissues and were not successful. Many of the attempts were not published in the literature, as most scientists do not like to publish negative results. Our proposed research will examine the possibility that P. brassicae has a culturable amoebal-like growth stage, which is like the protoplast stage that develops in root hairs and root cells. The start will be resting spores without cell walls. There is no universal protocol for the culturing of cells without cell walls. Like everything in science, it depends on the organism and the tissue from which these cells are taken. Since P. brassicae cell wall removal has been successfully performed in our laboratory for the first time, we are only a few steps away from culturing P. brassicae. For this proposed project a high auxin/kinetin ratio will be used to induce cell division, similar to conditions in an infected root. The best growth media and conditions for the production of P. brassicae cell colonies will be tested. 

The in vitro culture of P. brassicae will facilitate the screening of resistance as well as studying the ability of the organism to infect and cause disease, the pathogenesis. In addition, these cells have the potential to reform the cell wall. Thus, they will also be an important tool for investigating the mechanism of cell wall formation, membrane biology, and macromolecular uptake as well, which is unknown.