Mingguang Chu

Clubroot caused by Plasmodiophora brassicae seriously threatens rapeseed production. Although metabolic networks are known to play important roles in plant defense, the precise mechanisms by which distinct pathways cooperate to confer resistance remain unclear. To address this, we performed a genome-wide association study (GWAS) on a resistant–susceptible segregating population (T13) and identified 51 resistance-associated single nucleotide polymorphisms (SNPs), four of which mapped to chromosome C04 and co-localized with known quantitative trait loci (QTLs)/genes. We next analyzed time-series root transcriptomes from resistant and susceptible genotypes after inoculation. Weighted gene co-expression network analysis (WGCNA) resolved six co-expression modules tightly linked to resistance and identified 29 hub genes among 2859 GWAS-annotated candidates. Functional enrichment and centrality analyses converged on a defense network centered on flavonoid biosynthesis and glucosinolate metabolism, with this subnetwork being the main contributor for clubroot resistance. Genome-wide identification of ADT, FLS, and upstream MYB families across A. thaliana and three Brassica species yielded 47, 494, and 497 members (divided into 6, 12, 12 subfamilies), and ADT, FLS, and MYB families expanded primarily through segmental duplication. Expression profiling and RT-qPCR confirmed that multiple members were significantly upregulated in resistant genotypes at late infection. Protein–protein interaction (PPI) networks showed that hub genes form a synergistic module with enzymes involved in phenylpropanoid, lignin, glucosinolate, and amino-acid metabolism, as well as in development. These findings link resistance loci and transcriptional architecture to a multi-layered synergistic model. Within this framework, flavonoid and glucosinolate pathways serve as core defenses, connected functionally by the MYB28–ADT5–FLS3 axis and key nodes such as PAL1, AOP3, and BCAT3/4; upstream primary metabolism, immune signaling, and development further shape a systemic network at late infection. This model offers a theoretical framework and concrete candidate genes for breeding.

Blackleg disease, a major threat to Brassica crops worldwide, is primarily caused by the pathogen Leptosphaeria biglobosa. Investigating the genetic variation of L. biglobosa is crucial for managing and preventing the disease in Brassica napus. To date, there is scarce genomic variation information available for populations of L. biglobosa in China. In this study, 73 L. biglobosa strains of canola stalks were collected from 12 provinces in China and subjected to re-sequencing. The 73 assemblies averaged 1340 contigs, 72,123 bp N50, and 30.17 Mb in size. In total, 9409 core orthogroups and 867 accessory orthogroups were identified. A total of 727,724 mutation loci were identified, including 695,230 SNPs and 32,494 indels. Principal component analysis (PCA) and population structure analysis showed that these strains could be divided into seven subgroups. The strains in most provinces were clustered into a single subgroup, suggesting a strong influence of the geographic environment on strain variation. The average nucleotide diversity (θπ) of all strains was 1.03 × 10⁻³, indicating important genetic diversity among strains from different regions of China. This study provides valuable resources for future comparative genomics, gives new insights into the population evolution of L. biglobosa, and supports the development of strategies for managing blackleg disease in canola.

Plasmodiophora brassicae, an obligate soilborne pathogen that causes clubroot on Brassica crops, is spreading rapidly in western Canada, threatening canola production in the region. Bioassays and molecular assays have been used to estimate the concentration of P. brassicae resting spores in soil, which can affect clubroot incidence and severity on crops. Droplet digital PCR (ddPCR) is a promising new approach for quantification of pathogen inoculum owing to its low sensitivity to inhibitors and consistency at low target concentrations. The objective of this study was to assess ddPCR against existing quantitative PCR (qPCR) for potential advantage and/or improvement in quantifying P. brassicae resting spores in soil. The new protocol enumerated resting spores accurately in spiked potting mix or soil samples ranging from 10² to 10⁷ spores per gram. At a spore concentration ≥10⁷ spores per gram, however, ddPCR became less accurate, with a tendency of overestimation. The protocol was validated by quantifying the resting spores in spiked brown, dark brown, and black soils using both ddPCR and qPCR simultaneously. These soil types are found commonly on the Canadian Prairies, and they vary in texture, pH, and organic content. ddPCR showed similar results among the different soil types, whereas qPCR often displayed lower counts for the same spore concentration, with the amplification of DNA inhibited completely in black soil samples. The inhibition can be removed by a 10-fold dilution of DNA samples. The results show that ddPCR can be a more versatile tool than qPCR for detection and quantification of P. brassicae resting spores in soil samples.

Clubroot disease is a serious threat to canola production in western Canada and many parts of the world. Rcr1 is a clubroot resistance (CR) gene identified recently and its molecular mechanisms in mediating CR have been studied using several omics approaches. The current study aimed to characterize the biochemical changes in the cell wall of canola roots connecting to key molecular mechanisms of this CR gene identified in prior studies using Fourier transform infrared (FTIR) spectroscopy. The expression of nine genes involved in phenylpropanoid metabolism was also studied using qPCR. Between susceptible (S) and resistance (R) samples, the most notable biochemical changes were related to an increased biosynthesis of lignin and phenolics. These results were supported by the transcription data on higher expression of BrPAL1. The up-regulation of PAL is indicative of an inducible defence response conferred by Rcr1; the activation of this basal defence gene via the phenylpropanoid pathway may contribute to clubroot resistance conferred by Rcr1. The data indicate that several cell-wall components, including lignin and pectin, may play a role in defence responses against clubroot. Principal components analysis of FTIR data separated non-inoculated samples from inoculated samples, but not so much between inoculated S and inoculated R samples. It is also shown that FTIR spectroscopy can be a useful tool in studying plant-pathogen interaction at cellular levels.