Clubroot is a major soil-borne plant disease infecting a broad range of Brassica crops leading to significant (10-15%) yield and quality loss internationally. Since the identification of the clubroot pathogen, the disease has been recognised as a worsening problem worldwide [1, 2]. In Australia, clubroot disease is a serious problem in brassica vegetable crops. A recent report showed that more than 70% of brassica properties in Victoria are affected [3], leading to a ~10% yield loss [4]. Historically, canola in Australia is grown separately from brassica vegetable crops, preventing the clubroot from spreading to canola fields, but it is highly likely that this transfer will occur, taking into account water movement due to increasing flooding [5].
The causal agent of clubroot is the protist organism Plasmodiophora brassicae and it infects a wide range of crops important for the Australian economy, including canola, cabbage, and mustard. P. brassicae is an obligate biotrophic pathogen and it cannot be cultured without the plant host, making it extremely difficult to study [1, 6]. Recently, the whole genome assembly of the Canadian P. brassicae isolate was generated, but comparative genomic studies are hindered due to the lack of other complete genomes of the isolates from a broad geographic range [7].
In the current study, we address the current lack of genomic and phylogenomic data for Australian clubroot isolates. We have generated the first haplotype-resolved telomere-to-telomere complete genome assemblies for Australian field-sampled clubroot isolates. We have used Hi-C technology to capture the different haplotypes from the heterogeneous field isolates. We further used RNA-sequencing technology to carry out the high-quality gene annotation. We performed whole-genome comparisons with the Canadian reference isolate and identified some large structural variations. Interestingly, most of them were the results of transposon movements and affected the genes coding for putative virulence factors and carbohydrate-active enzymes (CAZymes).
We have further used this genome assembly as a reference to gain insight into the diversity of the clubroot isolates present in Australia. We have sequenced a subset of 13 historical clubroot samples from the Plant Pathology & Mycology Herbarium of the NSW Department of Primary Industries. The samples were collected across a wide geographic range (including NSW, TAS, SA and VIC) and sampling years between 1958 and 2010. Using the whole-genome assemblies, we could build the high-confidence phylogenetic tree, placing the Australian isolates into an international context and suggesting the multiple incursion events from different origins. We further identified the unique clade of isolates collected from Iberis and Sinapis plants across NSW showing early divergence and significant phylogenetic distance to any of the currently sequenced isolates.
This reference-quality whole genome assemblies and the phylogenomic analysis are a valuable resource for further studies of clubroot evolutionary dynamics and the molecular interplay between this pathogen and its plant host.