Map-based cloning and functional analysis of a major quantitative trait locus, BolC.Pb9.1, controlling clubroot resistance in a wild Brassica relative (Brassica macrocarpa).

KEY MESSAGE: A causal gene BoUGT76C2, conferring clubroot resistance in wild Brassica oleracea, was identified and functionally characterized. Clubroot is a devastating soil-borne disease caused by the obligate biotrophic pathogen Plasmodiophora brassica (P. brassicae), which poses a great threat to Brassica oleracea (B. oleracea) production. Although several QTLs associated with clubroot resistance (CR) have been mapped in cultivated B. oleracea, none have been cloned in B. oleracea. Previously, we found that the wild B. oleracea B2013 showed high resistance to clubroot. In this study, we constructed populations using B2013 and broccoli line 90196. CR in B2013 is quantitatively inherited, and a major QTL, BolC.Pb9.1, was identified on C09 using QTL-seq and linkage analysis. The BolC.Pb9.1 was finely mapped to a 56 kb genomic region using F2:3 populations. From the target region, the candidate BoUGT76C2 showed nucleotide variations between the parents, and was inducible in response to P. brassicae infection. We generated BoUGT76C2 overexpression lines in the 90196 background, which showed significantly enhanced resistance to P. brassicae compared to the WT line, suggesting that BoUGT76C2 corresponds to the resistance gene BolC.Pb.9.1. This is the first report on the CR gene map-based cloning and functional analysis from wild relatives, which provides a theoretical basis to the understanding of the molecular mechanism of CR, and lays a foundation to improve the CR of cultivated B. oleracea.

Starch Plays a Key Role in Sporosorus Formation by the Powdery Scab Pathogen Spongospora subterranea.

Powdery scab disease, caused by the soilborne protist Spongospora subterranea f. sp. subterranea, poses a major constraint to potato production worldwide. Disease symptoms include damage to the tuber skin and the formation of root galls. This study aimed to investigate the potential mechanism behind the formation of sporosori, which are aggregates of resting spores, within root galls. Scanning electron microscopy analysis revealed that the early stage of gall formation, characterized by a white color, involved the accumulation of starch grains, which later disappeared as the gall matured and turned brown. The mature brown galls were found to contain fully formed sporosori. Light microscopy examination of ultramicrotome sections of the root galls showed that the high-amylopectin starches were surrounded by a plasmodium, a precursor to sporosorus. These findings suggest that starch grains contribute to the formation of a sponge-like structure within the sporosori. A significant reduction in total starch levels in both the root galls and their associated roots was observed compared with healthy roots. These findings indicate starch consumption by sporosori during the maturation of root galls. Interestingly, analysis of the transcript levels of starch-related genes showed downregulation of genes encoding starch degrading enzymes and an amylopectin-debranching enzyme, whereas genes encoding a starch synthase and a protein facilitating starch synthesis were upregulated in the infected roots. Overall, our results demonstrate that starch is consumed during sporosorus formation, and the pathogen likely manipulates starch homeostasis to its advantage for sporosorus development within the root galls.

A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping.

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 that have been very elusive to this day.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for Detection of the Potato Powdery Scab Pathogen Spongospora subterranea.

Spongospora subterranea is the causal agent of powdery scab of potato (Solanum tuberosum), which can significantly reduce potato quality. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) method for the detection of S. subterranea. A set of LAMP primers named PS-LAMP was designed and tested for specificity and sensitivity. In the specificity test, in silico analysis using the NCBI Primer-BLAST tool indicated that PS-LAMP was specific to S. subterranea. The in vitro tests confirmed specificity, showing that PS-LAMP could produce positive signals from DNA isolated from each of three potato tubers with powdery scab symptoms but did not produce positive signals from DNA isolated from 38 nontarget plant pathogens. The sensitivity of PS-LAMP was tested on both gBlocks and DNA isolated from potato samples with powdery scab symptoms. On gBlocks, the lowest number of copies for a positive LAMP reaction was six, which was similar to results obtained via qPCR, but it was 10 times more sensitive than conventional PCR. On a DNA sample from S. subterranea-infected potato, the lowest amount of template DNA for a positive LAMP reaction was 2 pg, which was incomparable with the sensitivity of qPCR. Considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, this assay can be very useful for plant pathology practitioners and diagnostic labs interested in rapid, accurate, and routine detection of S. subterranea and confirmation of powdery scab disease.

Metagenomics approach for Polymyxa betae genome assembly enables comparative analysis towards deciphering the intracellular parasitic lifestyle of the plasmodiophorids.

Genomic knowledge of the tree of life is biased to specific groups of organisms. For example, only six full genomes are currently available in the rhizaria clade. Here, we have applied metagenomic techniques enabling the assembly of the genome of Polymyxa betae (Rhizaria, Plasmodiophorida) RES F41 isolate from unpurified zoospore holobiont and comparison with the A26-41 isolate. Furthermore, the first P. betae mitochondrial genome was assembled. The two P. betae nuclear genomes were highly similar, each with just ~10.2 k predicted protein coding genes, ~3% of which were unique to each isolate. Extending genomic comparisons revealed a greater overlap with Spongospora subterranea than with Plasmodiophora brassicae, including orthologs of the mammalian cation channel sperm-associated proteins, raising some intriguing questions about zoospore physiology. This work validates our metagenomics pipeline for eukaryote genome assembly from unpurified samples and enriches plasmodiophorid genomics; providing the first full annotation of the P. betae genome.

Advances in Biological Control and Resistance Genes of Brassicaceae Clubroot Disease-The Study Case of China.

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae. It occurs in cruciferous crops exclusively, and causes serious damage to the economic value of cruciferous crops worldwide. Although different measures have been taken to prevent the spread of clubroot disease, the most fundamental and effective way is to explore and use disease-resistance genes to breed resistant varieties. However, the resistance level of plant hosts is influenced both by environment and pathogen race. In this work, we described clubroot disease in terms of discovery and current distribution, life cycle, and race identification systems; in particular, we summarized recent progress on clubroot control methods and breeding practices for resistant cultivars. With the knowledge of these identified resistance loci and R genes, we discussed feasible strategies for disease-resistance breeding in the future.

Genome-Wide Identification and Characterization of the Trehalose-6-Phosphate Synthetase Gene Family in Chinese Cabbage (Brassica rapa) and Plasmodiophora brassicae during Their Interaction.

Trehalose is a nonreducing disaccharide that is widely distributed in various organisms. Trehalose-6-phosphate synthase (TPS) is a critical enzyme responsible for the biosynthesis of trehalose, which serves important functions in growth and development, defense, and stress resistance. Although previous studies have found that the clubroot pathogen Plasmodiophora brassicae can lead to the accumulation of trehalose in infected Arabidopsis organs, it has been proposed that much of the accumulated trehalose is derived from the pathogen. At present, there is very little evidence to verify this view. In this study, a comprehensive analysis of the TPS gene family was conducted in Brassica rapa and Plasmodiophora brassicae. A total of 14 Brassica rapa TPS genes (BrTPSs) and 3 P. brassicae TPS genes (PbTPSs) were identified, and the evolutionary characteristics, functional classification, and expression patterns were analyzed. Fourteen BrTPS genes were classified into two distinct classes according to phylogeny and gene structure. Three PbTPSs showed no significant differences in gene structure and protein conserved motifs. However, evolutionary analysis showed that the PbTPS2 gene failed to cluster with PbTPS1 and PbTPS3. Furthermore, cis-acting elements related to growth and development, defense and stress responsiveness, and hormone responsiveness were predicted in the promoter region of the BrTPS genes. Expression analysis of most BrTPS genes at five stages after P. brassicae interaction found no significant induction. Instead, the expression of the PbTPS genes of P. brassicae was upregulated, which was consistent with the period of trehalose accumulation. This study deepens our understanding of the function and evolution of BrTPSs and PbTPSs. Simultaneously, clarifying the biosynthesis of trehalose in the interaction between Brassica rapa and P. brassicae is also of great significance.

Effects of Exogenous Ergothioneine on Brassica rapa Clubroot Development Revealed by Transcriptomic Analysis.

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae that leads to a serious yield reduction in cruciferous plants. In this study, ergothioneine (EGT) was used to culture P. brassicae resting spores, the germination of which was significantly inhibited. Further exogenous application of EGT and P. brassicae inoculation in Chinese cabbage showed that EGT promoted root growth and significantly reduced the incidence rate and disease index. To further explore the mechanism by which EGT improves the resistance of Chinese cabbage to clubroot, a Chinese cabbage inbred line BJN3-2 susceptible to clubroot treated with EGT was inoculated, and a transcriptome analysis was conducted. The transcriptome sequencing analysis showed that the differentially expressed genes induced by EGT were significantly enriched in the phenylpropanoid biosynthetic pathway, and the genes encoding related enzymes involved in lignin synthesis were upregulated. qRT-PCR, peroxidase activity, lignin and flavonoid content determination showed that EGT promoted the lignin and flavonoid synthesis of Chinese cabbage and improved its resistance to clubroot. This study provides a new insight for the comprehensive prevention and control of cruciferous clubroot and for further study of the effects of EGT on clubroot disease.

Changes in Diversity and Composition of Rhizosphere Bacterial and Fungal Community between Resistant and Susceptible Pakchoi under Plasmodiophora brassicae.

Plasmodiophora brassicae (P. brassicae) is a soil-born pathogen worldwide and can infect most cruciferous plants, which causes great yield decline and economic losses. It is not well known how microbial diversity and community composition change during P. brassicae infecting plant roots. Here, we employed a resistant and a susceptible pakchoi cultivar with and without inoculation with P. brassicae to analyze bacterial and fungal diversity using 16S rRNA V3-V4 and ITS_V1 regions, respectively. 16S rRNA V3-V4 and ITS_V1 regions were amplified and sequenced separately. Results revealed that both fungal and bacterial diversity increased, and composition was changed in the rhizosphere soil of the susceptible pakchoi compared with the resistant cultivar. In the four groups of R_mock, S_mock, R_10d, and S_10d, the most relatively abundant bacterium and fungus was Proteobacteria, accounting for 61.92%, 58.17%, 48.64%, and 50.00%, respectively, and Ascomycota, accounting for 75.11%, 63.69%, 72.10%, and 90.31%, respectively. A total of 9488 and 11,914 bacteria were observed uniquely in the rhizosphere soil of resistant and susceptible pakchoi, respectively, while only 80 and 103 fungi were observed uniquely in the correlated soil. LefSe analysis showed that 107 and 49 differentially abundant taxa were observed in bacteria and fungi. Overall, we concluded that different pakchoi cultivars affect microbial diversity and community composition, and microorganisms prefer to gather around the rhizosphere of susceptible pakchoi. These findings provide a new insight into plant-microorganism interactions.

Fine mapping and candidate gene analysis of CRA3.7 conferring clubroot resistance in Brassica rapa.

KEY MESSAGE: In this study, we fine-mapped a clubroot resistance gene CRA3.7 in Chinese cabbage and developed its closely linked marker syau-InDel3008 for marker-assisted selection in CR cultivars breeding. Chinese cabbage is an important leafy vegetable rich in many nutrients widely grown in China. Clubroot disease caused by an obligate biotrophic pathogen Plasmodiophora brassicae was rapidly spread and challenged to Chinese cabbage production. A clubroot resistance (CR) gene, CRA3.7, was mapped on chromosome A03 of Brassica rapa. A Chinese cabbage line 'CR510', which harbor homozygous resistance locus CRA3.7 was selected from a BC4F3 family. 'CR510' was crossed with a clubroot susceptible Chinese cabbage inbred line '59-1'. Total 51 recombinant plants were identified from an F2 population including 3000 individuals. These recombinants were selfed and the clubroot resistance of F2/3 families was evaluated. Finally, a clubroot resistance gene CRA3.7 was fine-mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008. According to the reference genome, total 54 genes including five encoding the TIR-NBS-LRR proteins was annotated in the fine-mapped region. Further, nine candidate's gene expression in parental lines at 7, 14 and 21 days after inoculation of P. brassicae were evaluated. Bra019376, Bra019401, Bra019403 and Bra019410 are highly expressed in 'CR510' than '59-1'. Gene sequence of Bra019410 from 'CR510' was cloned and identified different from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are the most likely candidates for CRA3.7. Our research provides a valuable germplasm resource against P. brassicae Pb3 and CRA3.7 closely linked marker for marker-assisted selection in CR cultivars breeding.

Molecular data reallocates Sorosphaerula radicalis (Plasmodiophorida, Phytomyxea, Rhizaria) to the genus Hillenburgia.

This study reports the first record of Sorosphaerula radicalis (Phytomyxea, Rhizaria) in continental Europe (Tirol, Austria) and provides first molecular data for this species. An 18S rRNA phylogeny placed S. radicalis into the Plasmodiophorida, although distant from other members of the genus Sorosphaerula and close to the parasite of water cress Hillenburgia nasturtii. To resolve this polyphyly, we compare morphological data and life cycles of Sorosphaerula veronicae (the type species of the genus Sorosphaerula), Hillenburgia nasturtii, and Sorosphaerula radicalis. We conclude that Sorosphaerula radicalis belongs to the recently established genus Hillenburgia.

Plasmodiophora brassicae in Mexico: From Anecdote to Fact.

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been stated as a fact. However, an intensive search of the scientific literature in English and Spanish, as well as gray literature including theses and government reports, did not reveal any information about the actual detection of the pathogen, affected hosts, or areas with clubroot presence, or any information about clubroot (hernia de la col in Mexico). We followed a multistep process to confirm whether P. brassicae was indeed in Mexico. First, we identified agricultural communities with a history of cruciferous crop cultivation. Second, we asked growers if they had seen clubroot on their crops, using pictures of the characteristic root galls. Third, we collected soil from the locations where clubroot was reported and looked for clubroot/P. brassicae in the soil using several cruciferous bait plants. For the first time we confirm the presence of the clubroot pathogen P. brassicae in Mexico, through a bioassay, the presence of resting spores, and a P. brassicae-specific PCR assay. The identification of P. brassicae in Mexico will contribute to our understanding of the genetic diversity of this elusive and devastating plant pathogen in future studies.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

Application of the NanoString nCounter System as an Alternative Method to Investigate Molecular Mechanisms Involved in Host Plant Responses to Plasmodiophora brassicae.

Clubroot, caused by the soilborne pathogen Plasmodiophora brassicae, is an important disease of canola (Brassica napus) and other crucifers. The recent application of RNA sequencing (RNA-seq) technologies to study P. brassicae−host interactions has generated large amounts of gene expression data, improving knowledge of the molecular mechanisms of pathogenesis and host resistance. Quantitative PCR (qPCR) analysis has been widely applied to examine the expression of a limited number of genes and to validate the results of RNA-seq studies, but may not be ideal for analyzing larger suites of target genes or increased sample numbers. Moreover, the need for intermediate steps such as cDNA synthesis may introduce variability that could affect the accuracy of the data generated by qPCR. Here, we report the validation of gene expression data from a previous RNA-seq study of clubroot using the NanoString nCounter System, which achieves efficient gene expression quantification in a fast and simple manner. We first confirm the robustness of the NanoString system by comparing the results with those generated by qPCR and RNA-seq and then discuss the importance of some candidate genes for resistance or susceptibility to P. brassicae in the host. The results show that the expression of genes measured using NanoString have a high correlation with the values obtained using the other two technologies, with R > 0.90 and p < 0.01, and the same expression patterns for most genes. The three methods (qPCR, RNA-seq, and NanoString) were also compared in terms of laboratory procedures, time, and cost. We propose that the NanoString nCounter System is a robust, sensitive, highly reproducible, and simple technology for gene expression analysis. NanoString could become a common alternative to qPCR to validate RNA-seq data or to create panels of genes for use as markers of resistance/susceptibility when plants are challenged with different P. brassicae pathotypes.

What Can We Learn from -Omics Approaches to Understand Clubroot Disease?

Clubroot is one of the most economically significant diseases worldwide. As a result, many investigations focus on both curing the disease and in-depth molecular studies. Although the first transcriptome dataset for the clubroot disease describing the clubroot disease was published in 2006, many different pathogen-host plant combinations have only recently been investigated and published. Articles presenting -omics data and the clubroot pathogen Plasmodiophora brassicae as well as different host plants were analyzed to summarize the findings in the richness of these datasets. Although genome data for the protist have only recently become available, many effector candidates have been identified, but their functional characterization is incomplete. A better understanding of the life cycle is clearly required to comprehend its function. While only a few proteome studies and metabolome analyses were performed, the majority of studies used microarrays and RNAseq approaches to study transcriptomes. Metabolites, comprising chemical groups like hormones were generally studied in a more targeted manner. Furthermore, functional approaches based on such datasets have been carried out employing mutants, transgenic lines, or ecotypes/cultivars of either Arabidopsis thaliana or other economically important host plants of the Brassica family. This has led to new discoveries of potential genes involved in disease development or in (partial) resistance or tolerance to P. brassicae. The overall contribution of individual experimental setups to a larger picture will be discussed in this review.

Comparative Proteomic Analysis of Potato Roots from Resistant and Susceptible Cultivars to Spongospora subterranea Zoospore Root Attachment In Vitro.

Potato (Solanum tuberosum L.) exhibits broad variations in cultivar resistance to tuber and root infections by the soilborne, obligate biotrophic pathogen Spongospora subterranea. Host resistance has been recognised as an important approach in potato disease management, whereas zoospore root attachment has been identified as an effective indicator for the host resistance to Spongospora root infection. However, the mechanism of host resistance to zoospore root attachment is currently not well understood. To identify the potential basis for host resistance to S. subterranea at the molecular level, twelve potato cultivars differing in host resistance to zoospore root attachment were used for comparative proteomic analysis. In total, 3723 proteins were quantified from root samples across the twelve cultivars using a data-independent acquisition mass spectrometry approach. Statistical analysis identified 454 proteins that were significantly more abundant in the resistant cultivars; 626 proteins were more abundant in the susceptible cultivars. In resistant cultivars, functional annotation of the proteomic data indicated that Gene Ontology terms related to the oxidative stress and metabolic processes were significantly over-represented. KEGG pathway analysis identified that the phenylpropanoid biosynthesis pathway was associated with the resistant cultivars, suggesting the potential role of lignin biosynthesis in the host resistance to S. subterranea. Several enzymes involved in pectin biosynthesis and remodelling, such as pectinesterase and pectin acetylesterase, were more abundant in the resistant cultivars. Further investigation of the potential role of root cell wall pectin revealed that the pectinase treatment of roots resulted in a significant reduction in zoospore root attachment in both resistant and susceptible cultivars. This study provides a comprehensive proteome-level overview of resistance to S. subterranea zoospore root attachment across twelve potato cultivars and has identified a potential role for cell wall pectin in regulating zoospore root attachment.

Identification of lncRNAs in response to infection by Plasmodiophora brassicae in Brassica napus and development of lncRNA-based SSR markers.

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), is yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot-resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with Plasmodiophora brassicae pathotype 3. Pathway enrichment analysis showed that most of the target genes regulated by these DE lncRNAs belonged to plant-pathogen interaction and hormone signaling, as well as primary and secondary metabolic pathways. Comparative analysis of these lncRNAs with 530 previously reported DE lncRNAs, identified using resistance located on BnaA08, detected 12 lncRNAs that showed a similar trend of upregulation in both types of resistant lines; these lncRNAs probably play a fundamental role in clubroot resistance. We identified SSR markers within 196 DE lncRNAs. Genotyping of two DH populations carrying resistance on BnaA03 identified a marker capable of detecting the resistance in 98% of the DH lines. To our knowledge, this is the first report of the identification of SSRs within lncRNAs responsive to P. brassicae infection, demonstrating the potential use of lncRNAs in the breeding of Brassica crops.

Virulence Spectrum of Single-Spore and Field Isolates of Plasmodiophora brassicae Able to Overcome Resistance in Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important disease of canola (Brassica napus L.) that is managed mainly by planting clubroot-resistant (CR) cultivars. Field isolates of P. brassicae can be heterogeneous mixtures of various pathotypes, making assessments of the genetics of host-pathogen interactions challenging. Thirty-four single-spore isolates were obtained from nine field isolates of the pathogen collected from CR canola cultivars. The virulence patterns of the single-spore and field isolates were assessed on the 13 host genotypes of the Canadian Clubroot Differential (CCD) set, which includes the differentials of Williams and Somé et al. Indices of disease (IDs) severity of 25, 33, and 50% (±95% confidence interval) were compared as potential thresholds to distinguish between resistant and susceptible reactions, with an ID of 50% giving the most consistent responses for pathotype classification purposes. With this threshold, 13 pathotypes could be distinguished based on the CCD system, 7 on the differentials of Williams, and 3 on the hosts of Somé et al. The highest correlations were observed among virulence matrices generated using the three threshold IDs on the CCD set. Genetically homogeneous single-spore isolates gave a clearer profile of the P. brassicae pathotype structure. Novel pathotypes, not reported in Canada previously, were identified among the isolates. This large collection of single-spore isolates can serve as a reference in screening and breeding for clubroot resistance.

Genetic Structure of Plasmodiophora brassicae Populations Virulent on Clubroot Resistant Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae Woronin, is a significant threat to the canola (Brassica napus L.) industry in Canada. Clubroot resistance has been overcome in more than 200 fields since 2013, representing one of the biggest challenges to sustainable canola production. The genetic structure of 36 single-spore isolates derived from 12 field isolates of P. brassicae collected before and after the introduction of clubroot resistant (CR) canola cultivars (2005-2014) was evaluated by simple sequence repeat (SSR) marker analysis. Polymorphisms were detected in 32 loci with the identification of 93 distinct alleles. A low level of genetic diversity was found among the single-spore isolates. Haploid linkage disequilibrium and number of migrants suggested that recombination and migration were rare or almost absent in the tested P. brassicae population. A relatively clear relationship was found between the genetic structure and virulence phenotypes of the pathogen as defined on the differential hosts of Somé et al., Williams, and the Canadian Clubroot Differential (CCD) set. Although genetic variability within each pathotype group, as classified on each differential system, was low, significant genetic differentiation was observed among the pathotypes. The highest correlation between genetic structure and virulence was found among matrices produced with genetic data and the hosts of the CCD set, with a threshold index of disease of 50% to distinguish susceptible from resistant reactions. Genetically homogeneous single-spore isolates provided a more complete and clearer picture of the population genetic structure of P. brassicae, and the results suggest some promise for the development of pathotype-specific primers.

Revised Taxonomy and Expanded Biodiversity of the Phytomyxea (Rhizaria, Endomyxa).

Phytomyxea (phytomyxids) is a group of obligate biotrophic pathogens belonging to the Rhizaria. Some phytomyxids are well studied and include known plant pathogens such as Plasmodiophora brassicae, the causal agent of clubroot disease. Despite this economic importance, the taxonomy and biodiversity of this group are largely cryptic, with many species described in the premolecular area. Some of these species were key for establishing the morphotaxonomic concepts that define most genera to this day, but systematic efforts to include and integrate those species into molecular studies are still lacking. The aim of this study was to expand our understanding of phytomyxid biodiversity in terrestrial environments. Thirty-eight environmental samples from habitats in which novel and known diversity of Phytomyxea was expected were analysed. We were able to generate 18S rRNA sequences from Ligniera verrucosa, a species which is well defined based on ultrastructure. Phylogenetic analyses of the collected sequences rendered the genera Lignera, Plasmodiophora and Spongospora polyphyletic, and identified two novel and apparently diverse lineages (clade 17, clade 18). Based on these findings and on data from previous studies, we formally establish the new genera Pseudoligniera n. gen. for L. verrucosa,Hillenburgia n. gen. for Spongospora nasturtii and revert Plasmodiophora diplantherae to its original name Ostenfeldiella diplantherae.