Morphological and molecular studies of pathogenicity, virulence and resistance in clubroot caused by plasmodiophora brassicae

Abstract

Plasmodiophora brassicae Woronin is an obligate biotrophic plant pathogen that causes the disease clubroot, leading to root galls and stunted host plant growth, in vegetable and oilseed crops in the Brassicaceae (cabbage family). Clubroot is one of the most devastating diseases of vegetable brassicas worldwide and significantly reduces crop yield. Clubroot is partially managed by using plant quarantine, integrated control methods and a few resistant varieties. Australian populations of the pathogen have high pathotype diversity compared with other countries but their genetic diversity was unknown. Breeding resistant cultivars is difficult if the pathogen is very diverse. Variation in the phenotypes (virulence) of ten field populations of P. brassicae was studied using the European clubroot differential set (ECD) of Brassica hosts and compared with their genotypes using random and microsatellite primers. High diversity was seen in both phenotype and genotype but there was no apparent relationship to geographic origin or collection date. One profile of a highly pathogenic population was distinctive but no genetic marker for high or low virulence was found. The stability of two genotypes with different levels of virulence was investigated through three generations of plant passage by repeatedly inoculating and recovering P. brassicae. PCR profiles varied with each generation. Inoculum comprising an equal mixture of low and high virulence populations produced genotype profiles that did not resemble their parent inocula or one another. This lack of stability through plant passage makes breeding for resistance to P. brassicae very difficult. Single-spore isolates (SSIs) should theoretically be genetically uniform but three generations of four SSIs from the ‘e’ series (e1, e2, e3 and e6) showed high polymorphism and, like field populations, showed unexpectedly high divergence from parent profiles. This casts doubt on the assumption that they are genetically homogeneous and so useful for the study of events in infection and gall development. To pinpoint the critical morphological stage where susceptible and resistant host plants differed in gall development, the infection process was compared microscopically in cabbage and Chinese cabbage grown in a hydroponic system and inoculated with two populations of P. brassicae with different degrees of virulence (low and high). Root hair infection showed no differences with pathogen virulence or plant resistance, but secondary cortical infection did. Greater pathogen virulence resulted in a greater proportion of cells infected in the roots. Greater host plant resistance resulted in a predominance of resting spores in infected cells, whereas plasmodia predominated in resistant or partially resistant plants, suggesting that resistance delays the completion of the life cycle of P. brassicae. An understanding of the genotypic and phenotypic diversity, genetic stability and infection processes of the pathogen populations are important for the development of Brassica cultivars with effective and durable resistance to this disease. This thesis provides new evidence of high genotype diversity in Australian P. brassicae that is not constant through plant infection and changes current understandings of this pathogen