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Verticillium stripe, caused by Verticillium longisporum, presents an emerging threat to Canadian canola (Brassica napus). Initially detected in Manitoba in 2014, the presence of this pathogen has since been confirmed across western Canada. Infections by V. longisporum can result in yield losses of up to 50%, which is a cause for concern given the susceptibility of most commercial Canadian canola cultivars. The objective of this study was to screen a collection of 211 Brassica genotypes for their reactions to V. longisporum, and to use genome-wide association study (GWAS) to identify single nucleotide polymorphism (SNP) markers for resistance. The plant material consisted of 110 rutabaga (B. napus ssp. napobrassica), 35 canola, 40 Brassica rapa, and 15 Brassica oleracea accessions or cultivars, alongside 11 hosts of the European Clubroot Differential (ECD) set. These materials were screened for resistance under greenhouse conditions and were genotyped using a 19K Brassica SNP array. Three general linear models (GLM), four mixed linear models (MLM), and three GWAS methods were employed to evaluate the markers. Eleven non-commercial Brassica accessions and 9 out of 35 commercial canola cultivars displayed a low normalized area under the disease progress curve (AUDPCnorm.). The non-commercial accessions could prove valuable as potential sources of resistance against V. longisporum. Forty-five SNP markers were identified to be significantly associated with V. longisporum resistance using single-SNP based GWAS analysis. In comparison, haplotype-based GWAS analyses identified 10 to 25 haplotype blocks to be significantly associated with V. longisporum resistance. Between 20% and 56% of QTLs identified by the more conventional single-SNP based GWAS analysis were also detected by the haplotype-based GWAS analysis. The overlapping genomic regions identified by the two GWAS methods present promising hotspots for marker-assisted selection in the future development of Verticillium stripe-resistant canola.

The root systems of Brassica species are complex. Eight root system architecture (RSA) traits, including total root length, total root surface area, root average diameter, number of tips, total primary root length, total lateral root length, total tertiary root length, and basal link length, were phenotyped across 379 accessions representing six Brassica species (B. napus, B. juncea, B. carinata, B. oleracea, B. nigra, and B. rapa) using a semi-hydroponic system and image analysis software. The results suggest that, among the assessed species, B. napus and B. oleracea had the most intricate and largest root systems, while B. nigra exhibited the smallest roots. The two species B. juncea and B. carinata shared comparable root system complexity and had root systems with larger root diameters. In addition, 313 of the Brassica accessions were genotyped using a 19K Brassica single nucleotide polymorphism (SNP) array. After filtering by TASSEL 5.0, 6,213 SNP markers, comprising 5,103 markers on the A-genome (covering 302,504 kb) and 1,110 markers on the C-genome (covering 452,764 kb), were selected for genome-wide association studies (GWAS). Two general linear models were tested to identify the genomic regions and SNPs associated with the RSA traits. GWAS identified 79 significant SNP markers associated with the eight RSA traits investigated. These markers were distributed across the 18 chromosomes of B. napus, except for chromosome C06. Sixty-five markers were located on the A-genome, and 14 on the C-genome. Furthermore, the major marker-trait associations (MTAs)/quantitative trait loci (QTLs) associated with root traits were located on chromosomes A02, A03, and A06. Brassica accessions with distinct RSA traits were identified, which could hold functional, adaptive, evolutionary, environmental, pathological, and breeding significance.

The sustainable cultivation of canola is under threat from clubroot disease (Plasmodiophora brassicae). The pathogen’s resting spores can survive in the soil for extended periods, complicating disease management. Therefore, effective clubroot control requires a combination of tactics that provide multiple layers of protection. Management strategies have focused on pathogen avoidance and reducing disease levels in infested fields. The sanitation of machinery and field equipment remains the most effective method for preventing the pathogen’s introduction into non-infested fields. For disease reduction, crop rotation, liming, chemical control, and host resistance are commonly employed, with the use of clubroot-resistant cultivars being the most effective to date. However, resistance breakdown has been observed within four years of the introduction of new cultivars, jeopardizing the long-term effectiveness of this approach. A promising yet underexplored strategy is the use of cultivar mixtures. This approach leverages mechanisms such as the dilution effect, the barrier effect, induced resistance, disruptive selection, and the compensatory effect to control the disease. Cultivar mixtures have the potential to reduce the impact of clubroot on canola production while preserving pathogen population structure, thereby minimizing the likelihood of resistance breakdown. Given its potential, further research into cultivar mixtures as a management strategy for clubroot disease is warranted.

Clubroot is a soilborne disease of canola (Brassica napus) and other crucifers caused by the obligate parasite Plasmodiophora brassicae. In western Canada, clubroot is usually managed by planting-resistant cultivars, but the emergence of resistance-breaking pathotypes of P. brassicae represents a major threat to sustainable canola production. The rhizosphere and root contain beneficial microorganisms that can improve plant health. In this study, we evaluated the effect of two P. brassicae isolates (termed A and B) with different levels of virulence on the root and rhizosphere microbiomes of clubroot-resistant and clubroot-susceptible canola. Additionally, potential biocontrol microorganisms were identified based on taxa antagonistic to clubroot. Although both P. brassicae isolates were classified as pathotype 3A, isolate A caused a higher disease severity index in the resistant canola genotype compared with isolate B. Metabarcoding analysis indicated a shift in the bacterial and fungal communities in response to inoculation with either field isolate. Root endophytic bacterial and fungal communities responded to changes in inoculation, isolate type, sampling time, and canola genotype. In contrast, fungal communities associated with the rhizosphere exhibited significant differences between sampling times, while bacterial communities associated with the rhizosphere exhibited low variability.

Root rot disease poses a significant threat to canola (Brassica napus), underscoring the need for a comprehensive understanding of its causal agents for more effective disease mitigation. The composition and diversity of fungal pathogens associated with root rot of canola in Alberta, Canada, were evaluated from plant tissue samples collected in 2021 and 2022. The study revealed Fusarium spp. as the predominant pathogens found in almost all surveyed fields. Fusarium avenaceum, F. redolens, and F. solani were among the most frequently recovered species. Greenhouse trials confirmed their pathogenicity, with F. avenaceum and F. sporotrichioides found to be particularly aggressive. Additionally, F. sporotrichioides and F. commune were identified for the first time as canola root rot pathogens. Inoculation with isolates of most species resulted in significant reductions in seedling emergence, plant height, and shoot and root dry weights. Analysis of translation elongation factor 1-α (TEF-1α) and internal transcribed spacer (ITS) sequences confirmed the identity of the Fusarium spp., while concatenating the ITS and TEF-1α sequences enabled improved species differentiation. Geographic and year effects did not influence fungal diversity or aggressiveness, as determined by principal component analysis. This study emphasized the high diversity and impact of Fusarium spp. in causing canola root rot.

Verticillium stripe, caused by Verticillium longisporum, is an emerging disease of canola (Brassica napus) in Canada. Studies were conducted to assess the impact of pH on both the growth of V. longisporum and its virulence on the canola host. Fungal growth was assessed by measuring the colony diameter following 14 and 21 days of incubation on potato dextrose agar at varying pH levels (4.7, 5.5, 6.5, 7.4, or 8.6). The results indicated that colonies of V. longisporum were approximately 16% greater in diameter at pH 7.4 and 8.6 compared with those at pH 5.5. The impact of pH on disease development at the seedling stage was investigated using a semi-hydroponic system with different pH levels of 4.4, 5.4, 6.3, 7.5, and 8.4 in half-strength Hoagland’s solution. Verticillium stripe was most severe at pH 7.5 and 8.4 after a 10-day period in the semi-hydroponic system. In a second inoculation experiment, canola seedlings previously inoculated with the fungus were transplanted into potting mix amended to four pH levels (5.6, 6.4, 7.2, and 7.8). The transplants were cultivated under greenhouse conditions and evaluated for Verticillium stripe severity at plant maturity. Disease severity was greatest at pH 7.8. This is the first study on the effects of pH on V. longisporum in canola. It suggests a substantial risk of increased disease severity and yield losses due to Verticillium stripe in regions with neutral to slightly alkaline soils.

Fusarium proliferatum is associated with the root rot of many plant species, but knowledge of its impact on western Canadian field crops is limited. This study assessed the host range of this fungus and its effect on plant emergence, plant height, and shoot and root dry weights in repeated greenhouse experiments with wheat, barley, faba beans, peas, lentils, canola, lupine, and soybeans. Infection was confirmed via PCR, and principal component analysis determined the utility of different parameters in assessing host responses. All crops were at least partly susceptible, developing mild to severe disease at the seedling and adult stages, and showing significant reductions in growth. In general, the barley and wheat demonstrated higher tolerances to infection, followed by the faba bean and the pea. The soybean, canola, lupine, and lentil were most susceptible. The canola and the soybean were particularly vulnerable to F. proliferatum at the pre-emergence stage, while infection greatly reduced the lentil’s biomass. Reductions in the barley’s emergence and other growth parameters, however, occurred only under a high inoculum concentration. Variability in root rot severity among cultivars of the same crop indicated some diversity in host reactions within species. Nonetheless, the absence of fully-resistant crops may pose challenges in managing F. proliferatum in western Canadian cropping systems.

Fusarium spp. are commonly associated with the root rot complex of soybean (Glycine max). Previous surveys identified six common Fusarium species from Manitoba, including F. oxysporum, F. redolens, F. graminearum, F. solani, F. avenaceum, and F. acuminatum. This study aimed to determine their pathogenicity, assess host resistance, and evaluate the genetic diversity of Fusarium spp. isolated from Canada. The pathogenicity of these species was tested on two soybean cultivars, ‘Akras’ (moderately resistant) and ‘B150Y1′ (susceptible), under greenhouse conditions. The aggressiveness of the fungal isolates varied, with root rot severities ranging from 1.5 to 3.3 on a 0–4 scale. Subsequently, the six species were used to screen a panel of 20 Canadian soybean cultivars for resistance in a greenhouse. Cluster and principal component analyses were conducted based on the same traits used in the pathogenicity study. Two cultivars, ‘P15T46R2′ and ‘B150Y1′, were consistently found to be tolerant to F. oxysporum, F. redolens, F. graminearum, and F. solani. To investigate the incidence and prevalence of Fusarium spp. in Canada, fungi were isolated from 106 soybean fields surveyed across Manitoba, Saskatchewan, Ontario, and Quebec. Eighty-three Fusarium isolates were evaluated based on morphology and with multiple PCR primers, and phylogenetic analyses indicated their diversity across the major soybean production regions of Canada. Overall, this study contributes valuable insights into host resistance and the pathogenicity and genetic diversity of Fusarium spp. in Canadian soybean fields.

In this study, a rutabaga (Brassica napus ssp. napobrassica) donor parent FGRA106, which exhibited broad-spectrum resistance to 17 isolates representing 16 pathotypes of Plasmodiophora brassicae, was used in genetic crosses with the susceptible spring-type canola (B. napus ssp. napus) accession FG769. The F2 plants derived from a clubroot-resistant F1 plant were screened against three P. brassicae isolates representing pathotypes 3A, 3D, and 3H. Chi-square (χ2) goodness-of-fit tests indicated that the F2 plants inherited two major clubroot resistance genes from the CR donor FGRA106. The total RNA from plants resistant (R) and susceptible (S) to each pathotype were pooled and subjected to bulked segregant RNA-sequencing (BSR-Seq). The analysis of gene expression profiles identified 431, 67, and 98 differentially expressed genes (DEGs) between the R and S bulks. The variant calling method indicated a total of 12 (7 major + 5 minor) QTLs across seven chromosomes. The seven major QTLs included: BnaA5P3A.CRX1.1, BnaC1P3H.CRX1.2, and BnaC7P3A.CRX1.1 on chromosomes A05, C01, and C07, respectively; and BnaA8P3D.CRX1.1, BnaA8P3D.RCr91.2/BnaA8P3H.RCr91.2, BnaA8P3H.Crr11.3/BnaA8P3D.Crr11.3, and BnaA8P3D.qBrCR381.4 on chromosome A08. A total of 16 of the DEGs were located in the major QTL regions, 13 of which were on chromosome C07. The molecular data suggested that clubroot resistance in FGRA106 may be controlled by major and minor genes on both the A and C genomes, which are deployed in different combinations to confer resistance to the different isolates. This study provides valuable germplasm for the breeding of clubroot-resistant B. napus cultivars in Western Canada.

Background: Tunisia harbors a rich collection of unexploited durum wheat landraces (Triticum durum ssp. durum) that have been gradually replaced by elite cultivars since the 1970s. These landraces represent an important potential source for broadening the genetic background of elite durum wheat cultivars and for the introgression of novel genes for key traits, including disease resistance, into these cultivars.Methods: In this study, single nucleotide polymorphism (SNP) markers were used to investigate the genetic diversity and population structure of a core collection of 235 durum wheat accessions consisting mainly of landraces. The high phenotypic and genetic diversity of the fungal pathogen Pyrenophora tritici-repentis (cause of tan spot disease of wheat) in Tunisia allowed the assessment of the accessions for tan spot resistance at the adult plant stage under field conditions over three cropping seasons. A genome-wide association study (GWAS) was performed using a 90k SNP array.Results: Bayesian population structure analysis with 9191 polymorphic SNP markers classified the accessions into two groups, where groups 1 and 2 included 49.79% and 31.49% of the accessions, respectively, while the remaining 18.72% were admixtures. Principal coordinate analysis, the unweighted pair group method with arithmetic mean and the neighbor-joining method clustered the accessions into three to five groups. Analysis of molecular variance indicated that 76% of the genetic variation was among individuals and 23% was between individuals. Genome-wide association analyses identified 26 SNPs associated with tan spot resistance and explained between 8.1% to 20.2% of the phenotypic variation. The SNPs were located on chromosomes 1B (1 SNP), 2B (4 SNPs), 3A (2 SNPs), 3B (2 SNPs), 4A (2 SNPs), 4B (1 SNP), 5A (2 SNPs), 5B (4 SNPs), 6A (5 SNPs), 6B (2 SNPs), and 7B (1 SNP). Four markers, one on each of chromosomes 1B, and 5A, and two on 5B, coincided with previously reported SNPs for tan spot resistance, while the remaining SNPs were either novel markers or closely related to previously reported SNPs. Eight durum wheat accessions were identified as possible novel sources of tan spot resistance that could be introgressed into elite cultivars.Conclusion: The results highlighted the significance of chromosomes 2B, 5B, and 6A as genomic regions associated with tan spot resistance.

The soil-borne pathogen Plasmodiophora brassicae is the causal agent of clubroot, a major disease in Chinese cabbage (Brassica rapa ssp. pekinensis). The host’s resistance genes often confer immunity to only specific pathotypes and may be rapidly overcome. Identification of novel clubroot resistance (CR) from germplasm sources is necessary. In this study, Bap246 was tested by being crossed with different highly susceptible B. rapa materials and showed recessive resistance to clubroot. An F2 population derived from Bap246 × Bac1344 was used to locate the resistance Quantitative Trait Loci (QTL) by Bulk Segregant Analysis Sequencing (BSA-Seq) and QTL mapping methods. Two QTL on chromosomes A01 (4.67–6.06 Mb) and A08 (10.42–11.43 Mb) were found and named Cr4Ba1.1 and Cr4Ba8.1, respectively. Fifteen and eleven SNP/InDel markers were used to narrow the target regions in the larger F2 population to 4.67–5.17 Mb (A01) and 10.70–10.84 Mb (A08), with 85 and 19 candidate genes, respectively. The phenotypic variation explained (PVE) of the two QTL were 30.97% and 8.65%, respectively. Combined with gene annotation, mutation site analysis, and real-time quantitative polymerase chain reaction (qRT-PCR) analysis, one candidate gene in A08 was identified, namely Bra020861. And an insertion and deletion (InDel) marker (co-segregated) named Crr1-196 was developed based on the gene sequence. Bra013275, Bra013299, Bra013336, Bra013339, Bra013341, and Bra013357 in A01 were the candidate genes that may confer clubroot resistance in Chinese cabbage. The resistance resource and the developed marker will be helpful in Brassica breeding programs.

Abstract Background In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements. Results A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr’s adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr’s effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb ‘Starship’ transposon (dubbed ‘Horizon’) with a clearly defined target site and target site duplications. ‘Horizon’ was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative ‘Starship’ (dubbed ‘Icarus’) in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and ‘Icarus’ were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as ‘one-compartment’ based on calculated gene distances and evolutionary rates. Conclusions These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

Abstract Background Clubroot of canola (Brassica napus), caused by the soilborne pathogen Plasmodiophora brassicae, has become a serious threat to canola production in Canada. The deployment of clubroot-resistant (CR) cultivars is the most commonly used management strategy; however, the widespread cultivation of CR canola has resulted in the emergence of new pathotypes of P. brassicae capable of overcoming resistance. Several host differential sets have been reported for pathotype identification, but such testing is time-consuming, labor-intensive, and based on phenotypic classifications. The development of rapid and objective methods that allow for efficient, cost-effective and convenient pathotyping would enable testing of a much larger number of samples in shorter times. The aim of this study was to develop two pathotyping assays, an RNase H2-dependent PCR (rhPCR) assay and a SNaPshot assay, which could quickly differentiate P. brassicae pathotypes. Results Both assays clearly distinguished between pathotype clusters in a collection of 38 single-spore isolates of P. brassicae. Additional isolates pathotyped from clubbed roots and samples from blind testing also were correctly clustered. The rhPCR assay generated clearly differentiating electrophoretic bands without non-specific amplification. The SNaPshot assay was able to detect down to a 10% relative allelic proportion in a 10:90 template mixture with both single-spore isolates and field isolates when evaluated in a relative abundance test. Conclusions This study describes the development of two rapid and sensitive technologies for P. brassicae pathotyping. The high-throughput potential and accuracy of both assays makes them promising as SNP-based pathotype identification tools for clubroot diagnostics. rhPCR is a highly sensitive approach that can be optimized into a quantitative assay, while the main advantages of SNaPshot are its ability to multiplex samples and alleles in a single reaction and the detection of up to four allelic variants per target site.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus). Amisulbrom, a quinone inside inhibitor (QiI), was evaluated for its effectiveness in clubroot management in Alberta, Canada. Resting spores of P. brassicae were treated in vitro with 0, 0.01, 0.1, 1, and 10% (w/v) amisulbrom to determine its effect on spore germination and viability. Amisulbrom inhibited resting spore germination by up to 79% and reduced viable spores by 31% relative to the control. Applications of a liquid solution (AL1000, 1000 g active ingredient (ai) ha−1) and granular formulations (AF700, 700 g ai ha−1; AF1000, 1000 g ai ha−1; AF1500, 1500 g ai ha−1) of amisulbrom were tested on the canola cultivars ‘45H31’ (clubroot-susceptible) and ‘CS2000’ (moderately resistant) under greenhouse conditions and in field experiments in 2019 and 2020. In the greenhouse, the treatments were evaluated at inoculum concentrations of 1 × 105 or 1 × 107 resting spores g−1 soil. A trend of decreasing clubroot severity with an increasing amisulbrom rate was observed. At the lower spore concentration, treatment with AF1500 resulted in a clubroot disease severity index (DSI)

Multiple species of Fusarium can contribute to the development of root rot in canola (Brassica napus), making disease management difficult. We conducted field and greenhouse experiments to investigate the impacts of Fusarium avenaceum and Fusarium proliferatum, and the interaction between Fusarium oxysporum and F. proliferatum on root rot severity and canola yields. Inoculation with any of the three Fusarium spp. resulted in significant disease severity and reduced seedling emergence compared with non-inoculated controls, leading to yield reductions of up to 35%. Notably, there was a strong correlation (r = 0.93) between root rot severity at the seedling stage and at maturity. Regression analysis indicated a linear decline in seedling emergence with increasing disease severity. Furthermore, disease severity at maturity adversely affected the pod number per plant and the seed weight per plant, with both parameters ultimately approaching zero at a severity of 4.0 on a 0–4 scale. Co-inoculation with F. oxysporum and F. proliferatum induced more severe root rot than inoculation with each species on its own, suggesting synergistic interactions between these fungi. Knowledge of these interactions and the relative virulence of Fusarium spp. will contribute to the improved management of root rot in canola.

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease of crucifers associated with the formation of large root galls. This root enlargement suggests modulation of plant hormonal networks by the pathogen, stimulating cell division and elongation and influencing host defense. We studied physiological changes in two Brassica napus cultivars, including plant hormone profiles—salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), the auxin indole-3-acetic acid (IAA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)—along with their selected derivatives following inoculation with virulent and avirulent P. brassicae pathotypes. In susceptible plants, water uptake declined from the initial appearance of root galls by 21 days after inoculation, but did not have a significant effect on photosynthetic rate, stomatal conductance, or leaf chlorophyll levels. Nonetheless, a strong increase in ABA levels indicated that hormonal mechanisms were triggered to cope with water stress due to the declining water uptake. The free SA level in the roots increased strongly in resistant interactions, compared with a relatively minor increase during susceptible interactions. The ratio of conjugated SA to free SA was higher in susceptible interactions, indicating that resistant interactions are linked to the plant’s ability to maintain higher levels of bioactive free SA. In contrast, JA and its biologically active form JA-Ile declined up to 7-fold in susceptible interactions, while they were maintained during resistant interactions. The ACC level increased in the roots of inoculated plants by 21 days, irrespective of clubroot susceptibility, indicating a role of ethylene in response to pathogen interactions that is independent of disease severity. IAA levels at early and later infection stages were lower only in susceptible plants, suggesting a modulation of auxin homeostasis by the pathogen relative to the host defense system.

Abstract Clubroot, caused by Plasmodiophora brassicae, is an important disease of brassica crops worldwide. Vegetable turnip (Brassica rapa L.) have proven to be a source of clubroot resistance genes effective against many pathotypes of P. brassicae. The F1 progeny from the cross B. rapa canola ACDC (susceptible, S) × B. rapa turnip ECD02 (resistant, R) were backcrossed with ACDC, then self‐pollinated to produce BC1S1 lines. All the F1 plants were resistant to four pathotypes (3A, 3D, 3H, and 5X) of P. brassicae. Segregation for R and S in BC1 to each pathotype was 1:1 and resistance reactions were highly correlated. From whole genome sequencing, 192.1 M sequences with 96% template coverage from ECD02, and 478.9 M sequences with 92% coverage from ACDC, were aligned with the reference genome of B. rapa. Genotyping‐by‐sequencing was performed on the BC1 population. The number of aligned short reads per plant in the BC1 ranged from 1.4 to 8.5 M sequences with 4–8% template coverage. We obtained 1,344 high‐quality single‐nucleotide polymorphism (SNP) loci with a mean missing rate at 0.27% and distributed them on 10 chromosomes. A single co‐localized quantitative trait loci (QTL), designated as Rcr9ECD02 on chromosome A08, conferred resistance to the four pathotypes. The QTL explained 68.9–74.4% of phenotypic variation with the logarithm of the odds values of 24.3 to 31.1. Bulked segregant analysis was performed, and 14 SNP markers linked to the gene were developed using the Kompetitive Allele Specific PCR. Rcr9ECD02 was mapped into an interval of 2.2 cM, flanked by CF_A08_10664692 and CF_A08_12230973, which spanned 1.51 Mb on the chromosome and included 219 B. rapa genes. Four of these genes (BraA08g012910.3C, BraA08g012920.3C, BraA08g013130.3C, and BraA08g013630.3C) encoded disease resistance proteins.

Abstract In this study, clubroot resistance in the resynthesized European winter Brassica napus cv. ‘Tosca’ was introgressed into a Canadian spring canola line ‘11SR0099’, which was then crossed with the clubroot susceptible spring line ‘12DH0001’ to produce F1 seeds. The F1 plants were used to develop a doubled haploid (DH) mapping population. The parents and the DH lines were screened against ‘old’ pathotypes 2F, 3H, 5I, 6M and 8N of the clubroot pathogen, Plasmodiophora brassicae, as well as against the ‘new’ pathotypes 5X, 5L, 2B, 3A, 3D, 5G, 8E, 5C, 8J, 5K, 3O and 8P. Genotyping was conducted using a Brassica 15K SNP array. The clubroot screening showed that ‘Tosca, ‘11SR0099’ and the resistant DH lines were resistant to three (2F, 3H and 5I) of the five ‘old’ pathotypes and four (2B, 3O, 8E and 8P) of the 12 ‘new’ pathotypes, while being moderately resistant to the ‘old’ pathotype 8N and the ‘new’ pathotypes 3D and 5G. ‘Tosca’ was susceptible to isolates representing pathotype 3A (the most common among the ‘new’ pathotypes) as well as pathotypes 6M, 5X, 5L, 5K and 8J. Linkage analysis and QTL mapping identified a ca. 0.88–0.95 Mb genomic region on the A03 chromosome of ‘Tosca’ as conferring resistance to pathotypes 2F, 3H, 5I, 2B, 3D, 5G, 8E, 3O and 8P. The identified QTL genomic region housed the CRk, Crr3 and CRd gene(s). However, the susceptibility of ‘Tosca’ to most of the common virulent pathotypes makes it unattractive as a sole CR donor in the breeding of commercial canola varieties in western Canada.

Abstract Background Rutabaga or swede (Brassica napus ssp. napobrassica (L.) Hanelt) varies in root and leaf shape and colour, flesh colour, foliage growth habits, maturity date, seed quality parameters, disease resistance and other traits. Despite these morphological differences, no in-depth molecular analyses of genetic diversity have been conducted in this crop. Understanding this diversity is important for conservation and broadening the use of this resource. Results This study investigated the genetic diversity within and among 124 rutabaga accessions from five Nordic countries (Norway, Sweden, Finland, Denmark and Iceland) using a 15 K single nucleotide polymorphism (SNP) Brassica array. After excluding markers that did not amplify genomic DNA, monomorphic and low coverage site markers, the accessions were analyzedwith 6861 SNP markers. Allelic frequency statistics, including polymorphism information content (PIC), minor allele frequency (MAF) and mean expected heterozygosity ( H ¯ $$ \overline{H} $$ e) and population differentiation statistics such as Wright’s F-statistics (F ST ) and analysis of molecular variance (AMOVA) indicated that the rutabaga accessions from Norway, Sweden, Finland and Denmark were not genetically different from each other. In contrast, accessions from these countries were significantly different from the accessions from Iceland (P

Abstract Genetic resistance is a successful strategy for management of clubroot (Plasmodiophora brassicae) of brassica crops, but resistance can break down quickly. Identification of novel sources of resistance is especially important when new pathotypes arise. In the current study, the reaction of 177 accessions of Brassica napus to four new, virulent pathotypes of P. brassicae was assessed. Each accession was genotyped using genotyping by sequencing to identify and map novel sources of clubroot resistance using mixed linear model (MLM) analysis. The majority of accessions were highly susceptible (70–100 DSI), but a few accessions exhibited strong resistance (0–20 DSI) to pathotypes 5X (21 accessions), 3A (8), 2B (7), and 3D (15), based on the Canadian Clubroot Differential system. In total, 301,753 SNPs were mapped to 19 chromosomes. Population structure analysis indicated that the 177 accessions belong to seven major populations. SNPs were associated with resistance to each pathotype using MLM. In total, 13 important SNP loci were identified, with 9 SNPs mapped to the A-genome and 4 to the C-genome. The SNPs were associated with resistance to pathotypes 5X (2 SNPs), 3A (4), 2B (5) and 3D (6). A Blast search of 1.6 Mb upstream and downstream from each SNP identified 13 disease-resistance genes or domains. The distance between a SNP locus and the nearest resistance gene ranged from 0.04 to 0.74 Mb. The resistant lines and SNP markers identified in this study can be used to breed for resistance to the most prevalent new pathotypes of P. brassicae in Canada.

Contamination of barley by deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum, causes considerable financial loss to the grain and malting industries. In this study, two atmospheric cold plasma (ACP) reactors were used to produce plasma-activated water (PAW) bubbles. The potential of PAW bubbles for the steeping of naturally infected barley (NIB) during the malting process was investigated. The PAW bubbles produced by treating water for 30 min using a bubble spark discharge (BSD) at low temperature resulted in the greatest concentration of oxygen-nitrogen reactive species (RONS). This treatment resulted in 57.3% DON degradation compared with 36.9% in the control sample; however, the same treatment reduced germination significantly (p < 0.05). Direct BSD ACP treatment for 20 min at low temperature and indirect treatment for 30 min increased the percentage of germinated rootlets of the seedlings compared with the control. Considering both the DON reduction and germination improvement of barley seeds, continuous jet ACP treatment for 30 min performed better than the other treatments used in this study. At higher temperature of PAW bubbles, the concentration of RONS was significantly (p < 0.05) reduced. Based on quantitative polymerase chain reaction (qPCR) analysis and fungal culture tests, the PAW bubble treatment did not significantly reduce infection of NIB. Nonetheless, this study provides useful information for the malting industry for PAW treatment optimization and its use in barley steeping for DON reduction and germination improvement.

Blackleg, caused by Leptosphaeria maculans, is an important disease of canola (Brassica napus). The pathogen can attack stems, leaves and pods, but basal stem cankers are most damaging and can result in significant yield losses. In Canada, Verticillium stripe (Verticillium longisporum) has recently emerged as another disease threat to canola. Symptoms of Verticillium stripe can resemble those of blackleg, and the two diseases may occur together. The effect of blackleg on yield was explored in field experiments with two canola hybrids and by evaluating a wider variety of hybrids in commercial crops in central Alberta, Canada. The impact on yield of L. maculans/V. longisporum interactions was also assessed under field and greenhouse conditions. In most hybrids, the relationship between blackleg severity and yield components was best explained by second-degree quadratic equations, although a linear relationship was found for one variety sampled in commercial fields. When L. maculans was co-inoculated with V. longisporum, blackleg severity and yield losses increased. In some cases, Verticillium stripe caused greater yield losses than blackleg. The results suggest that the interaction between L. maculans/V. longisporum may cause more severe losses in canola, highlighting the need for proactive disease management strategies.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases of canola (Brassica napus) in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of clubroot, as these genes may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defense signaling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA. Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous atpla knockout/knockdown mutants of Arabidopsis, and tested their performance following inoculation with P. brassicae. Four mutants (pla1-iiα, pla1-iγ3, pla1-iii, ppla-iiiβ, ppla-iiiδ) developed more severe clubroot than the wild-type, suggesting increased susceptibility to P. brassicae. The homologs of these Arabidopsis PLAs (AtPLAs) in B. napus (BnPLAs) were identified through Blast searches and phylogenic analysis. Expression of the BnPLAs was subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae, and promising candidates for further characterization identified.

Clubroot is a serious soil-borne disease of crucifers caused by the obligate parasite Plasmodiophora brassicae. The genetic basis and histopathology of clubroot resistance in two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines Bap055 and Bap246, challenged with pathotype 4 of P. brassicae, was evaluated. The Chinese cabbage cultivar “Juxin” served as a susceptible check. The resistance in Bap055 was found to be controlled by the CRa gene, while resistance in Bap246 fit a model of control by unknown recessive gene. Infection of the roots by P. brassicae was examined by inverted microscopy. Despite their resistance, primary and secondary infection were observed to occur in Bap055 and Bap246. Primary infection was detected at 2 days post-inoculation (DPI) in “Juxin,” at 4 DPI in Bap055, and at 6 DPI in Bap246. Infection occurred most quickly on “Juxin,” with 60% of the root hairs infected at 10 DPI, followed by Bap055 (31% of the root hairs infected at 12 DPI) and Bap246 (20% of the root hairs infected at 14 DPI). Secondary infection of “Juxin” was first observed at 8 DPI, while in Bap055 and Bap246, secondary infection was first observed at 10 DPI. At 14 DPI, the percentage of cortical infection in “Juxin,” Bap055 and Bap246 was 93.3, 20.0, and 11.1%, respectively. Although cortical infection was more widespread in Bap055 than in Bap246, secondary infection in both of these hosts was restricted relative to the susceptible check, and the vascular system remained intact. A large number of binucleate secondary plasmodia were observed in “Juxin” and the vascular system was disrupted at 16 DPI; in Bap055 and Bap246, only a few secondary plasmodia were visible, with no binucleate secondary plasmodia. The defense mechanisms and expression of resistance appears to differ between Chinese cabbage cultivars carrying different sources of resistance.

Fusarium root rot, caused by a complex of Fusarium spp., is a major disease of field pea (Pisum sativum). The development of genetic resistance is the most promising approach to manage the disease, but no pea germplasm has been identified that is completely resistant to root rot. The aim of this study was to detect quantitative trait loci (QTL) conferring partial resistance to root rot and wilting, caused by five fungal isolates representing Fusarium solani, F. avenaceum, F. acuminatum, F. proliferatum, and F. graminearum. Evaluation of the root rot-tolerant cultivar “00-2067” and susceptible cultivar “Reward” was carried out with the five species. There was a significant difference (p < 0.001) between the mean root rot values of the two cultivars inoculated with the F. avenaceum (F4A) and F. graminearum (FG2) isolates. Therefore, in the QTL study, the F8 recombinant inbred line (RIL) population derived from “Reward” × “00-2067” was inoculated in the greenhouse (4 ×) with only F4A and FG2. The parents and F8 population were genotyped using 13.2K single nucleotide polymorphisms (SNPs) and 222 simple sequence repeat (SSR) markers. A significant genotypic effect (p < 0.05) and high heritability (79% to 92.1%) were observed for disease severity, vigor, and plant height following inoculation with F4A and FG2. Significant correlation coefficients were detected among and within all traits. This suggested that a high proportion of the genetic variance was transmitted from the parents to the progeny. However, no significant QTL (LOD > 3) were detected for the RILs inoculated with F4A. In the case of the RILs inoculated with FG2, 5 QTL for root rot severity and 3 QTL each for vigor and plant height were detected. The most stable QTL for plant height (Hgt-Ps3.1) was detected on Chrom5/LGIII. The two most stable QTL for partial resistance to FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in a 15.1-cM and 11.2-cM genomic region, respectively, on Chrom4/LGIV. The most stable QTL for vigor (Vig-Ps4.1) was found in the same region. Twenty-five major and moderate effect digenic epistatic interactions were detected. The identified region on chrom4/LGIV could be important for resistance breeding and marker development.

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of canola (Brassica napus) in Canada. Disease management relies heavily on planting clubroot resistant (CR) cultivars, but in recent years, new resistance-breaking pathotypes of P. brassicae have emerged. Current efforts against the disease are concentrated in developing host resistance using traditional genetic breeding, omics and molecular biology. However, because of its obligate biotrophic nature, limited resources have been dedicated to investigating molecular mechanisms of pathogenic infection. We previously performed a transcriptomic study with the cultivar resistance-breaking pathotype 5X on two B. napus hosts presenting contrasting resistance/susceptibility, where we evaluated the mechanisms of host response. Since cultivar-pathotype interactions are very specific, and pathotype 5X is one of the most relevant resistance-breaking pathotypes in Canada, in this study, we analyze the expression of genes encoding putative secreted proteins from this pathotype, predicted using a bioinformatics pipeline, protein modeling and orthologous comparisons with effectors from other pathosystems. While host responses were found to differ markedly in our previous study, many common effectors are found in the pathogen while infecting both hosts, and the gene response among biological pathogen replicates seems more consistent in the effectors associated with the susceptible interaction, especially at 21 days after inoculation. The predicted effectors indicate the predominance of proteins with interacting domains (e.g., ankyrin), and genes bearing kinase and NUDIX domains, but also proteins with protective action against reactive oxygen species from the host. Many of these genes confirm previous predictions from other clubroot studies. A benzoic acid/SA methyltransferase (BSMT), which methylates SA to render it inactive, showed high levels of expression in the interactions with both hosts. Interestingly, our data indicate that E3 ubiquitin proteasome elements are also potentially involved in pathogenesis. Finally, a gene with similarity to indole-3-acetaldehyde dehydrogenase is a promising candidate effector because of its involvement in indole acetic acid synthesis, since auxin is one of the major players in clubroot development.

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.

Clubroot, caused by Plasmodiophora brassicae, is a major disease of cruciferous crops in Colombia. Limited information is available, however, regarding its distribution or epidemiology in this country. The objectives of this study were to determine the occurrence of clubroot in the main regions of Colombia where cruciferous crops are grown, and to examine the relationship between pathogen inoculum density and the likelihood of field infestation with crop management practices and climatic information. In total, 127 fields were surveyed across eight departments, the pathogen inoculum density was estimated, climatic information was obtained, and farmers were surveyed on their crop management practices. More than half (53.7%) of the fields visited were found to be clubroot-infested and pathogen DNA was detected in 91.3% of the surveyed fields. The only department where clubroot symptoms were not observed was Nariño. In infested fields, P. brassicae inoculum density varied between 3 × 102 and 1 × 106 resting spores per gram of soil, with the highest inoculum density observed in Norte de Santander. All other departments had comparable spore loads. Inoculum density positively affected the likelihood of infestation of a field, and both spore loads and infestations were positively affected by the average temperature.

Aphanomyces root rot, caused by Aphanomyces euteiches, causes severe yield loss in field pea (Pisum sativum). The identification of a pea germplasm resistant to this disease is an important breeding objective. Polygenetic resistance has been reported in the field pea cultivar ‘00-2067’. To facilitate marker-assisted selection (MAS), bulked segregant RNA-seq (BSR-seq) analysis was conducted using an F8 RIL population derived from the cross of ‘Carman’ × ‘00-2067’. Root rot development was assessed under controlled conditions in replicated experiments. Resistant (R) and susceptible (S) bulks were constructed based on the root rot severity in a greenhouse study. The BSR-seq analysis of the R bulks generated 44,595,510~51,658,688 reads, of which the aligned sequences were linked to 44,757 genes in a reference genome. In total, 2356 differentially expressed genes were identified, of which 44 were used for gene annotation, including defense-related pathways (jasmonate, ethylene and salicylate) and the GO biological process. A total of 344.1 K SNPs were identified between the R and S bulks, of which 395 variants were located in 31 candidate genes. The identification of novel genes associated with partial resistance to Aphanomyces root rot in field pea by BSR-seq may facilitate efforts to improve management of this important disease.

Aphanomyces root rot (ARR) of field pea (Pisum sativum), caused by Aphanomyces euteiches, can cause severe root damage, wilting, and large yield losses under wet soil conditions. To identify ways to manage this disease, the effect of A. euteiches inoculum density on field pea was studied under greenhouse and field conditions in 2015 and 2016. Increases in inoculum density reduced seedling emergence, root nodulation, and plant vigor, and resulted in increased root rot severity in both field and greenhouse tests. Seed treatments with the fungicides Apron Advance (thiabendazole + fludioxonil + metalaxyl) + Vibrance (difenoconazole + metalaxyl-M + sedaxane), INTEGO Solo (ethaboxam), BAS 516F (boscalid + pyraclostrobin), BAS 720F (metalaxyl + pyraclostrobin + fluxapyroxad), and BAS 516F + BAS 720F (3:1) were evaluated for their efficacy against ARR. All seed treatments except Apron Advance + Vibrance reduced root rot severity under controlled conditions. BAS 516F, BAS 720F and INTEGO Solo improved plant vigor and all treatments reduced seedling blight to varying degrees under greenhouse conditions, but not in the field. A collection of 22 pea genotypes was evaluated for resistance to root rot in field plot experiments. Line 00–2067 showed the least severe root rot symptoms, whereas ‘Spring D’ showed the lowest reduction in yield. The results suggest that there may be an opportunity to combine partial host resistance and fungicidal seed treatments to adequately manage ARR of field pea. Keywords: Aphanomyces euteiches, Field pea, Fungicides, Inoculum density, Resistance

Clubroot, caused by Plasmodiophora brassicae Woronin, is one of the most important diseases of oilseed rape (Brassica napus L.). The rapid erosion of monogenic resistance in clubroot-resistant (CR) varieties underscores the need to diversify resistance sources controlling disease severity and traits related to pathogen fitness, such as resting spore production. The genetic control of disease index (DI) and resting spores per plant (RSP) was evaluated in a doubled haploid (DH) population consisting of 114 winter oilseed rape lines, obtained from the cross ‘Aviso’ × ‘Montego,’ inoculated with P. brassicae isolate “eH.” Linkage analysis allowed the identification of three quantitative trait loci (QTLs) controlling DI (PbBn_di_A02, PbBn_di_A04, and PbBn_di_C03). A significant decrease in DI was observed when combining effects of the three resistance alleles at these QTLs. Only one QTL, PbBn_rsp_C03, was found to control RSP, reducing resting spore production by 40%. PbBn_rsp_C03 partially overlapped with PbBn_di_C03 in a nucleotide-binding leucine-rich repeat (NLR) gene-containing region. Consideration of both DI and RSP in breeding for clubroot resistance is recommended for the long-term management of this disease.

Plasmodiophora brassicae Wor. is a soilborne parasite causing clubroot of canola (Brassica napus L.), a serious disease managed mostly by planting clubroot-resistant (CR) cultivars. Recently, new pathotypes of P. brassicae have emerged that overcome resistance, highlighting the need for a greater understanding of resistance stewardship and an integrated approach to clubroot management. Replicated field experiments were conducted in Edmonton, AB, in 2018 and 2019, to evaluate the effect of lime application and weed management on clubroot severity, crop yield and various growth parameters in clubroot-susceptible (CS) and CR canola cultivars. P. brassicae resting spore densities were also monitored by quantitative PCR for each treatment. When hydrated lime was applied to increase the soil pH from initial values of 5.2–5.5 to 7.2, clubroot severity decreased by 34–36% in the CS canola cultivar, while seed yield increased by 70–98%. The application of hydrated lime also resulted in resting spore densities that were 48–80% lower, relative to untreated controls, in plots where the CS cultivar was grown. Lime application or management of weeds did not significantly affect spore densities in plots with the CR cultivar. The results from the field trial suggest that the application of hydrated lime may be a useful strategy to manage clubroot, when used in combination with genetic resistance to reduce disease pressure.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is an important disease of the Brassicaceae and poses a significant threat to the $26.7 billion canola/oilseed rape (Brassica napus) industry in western Canada. While clubroot is managed most effectively by planting resistant host varieties, new pathotypes of P. brassicae have emerged recently that can overcome this resistance. Whole genome analyses provide both a toolbox and a systemic view of molecular mechanisms in host-pathogen interactions, which can be used to design new breeding strategies to increase P. brassicae resistance. We used RNA-seq to evaluate differential gene expression at 7, 14 and 21 days after inoculation (dai) of two B. napus genotypes with differential responses to P. brassicae pathotype 5X. Gall development was evident at 14 dai in the susceptible genotype (the oilseed rape ‘Brutor’), while gall development in the resistant genotype (the rutabaga (B. napus) ‘Laurentian’) was limited and not visible until 21 dai. Immune responses were better sustained through the time-course in ‘Laurentian’, and numerous genes from immune-related functional categories were associated with salicylic acid (SA)-mediated responses. Jasmonic acid (JA)-mediated responses seemed to be mostly inhibited, especially in the resistant genotype. The upregulation of standard defense-related proteins, like chitinases and thaumatins, was evident in ‘Laurentian’. The enrichment, in both host genotypes, of functional categories for syncytium formation and response to nematodes indicated that cell enlargement during P. brassicae infection, and the metabolic processes therein, share similarities with the response to infection by nematodes that produce similar anatomical symptoms. An analysis of shared genes between the two genotypes at different time-points, confirmed that the nematode-like responses occurred earlier for ‘Brutor’, along with cell metabolism and growth changes. Additionally, the susceptible cultivar turned off defense mechanisms earlier than ‘Laurentian’. Collectively, this study showed the importance of SA in triggering immune responses and suggested some key resistance and susceptibility factors that can be used in future studies for resistance breeding through gene-editing approaches.

In this study, Brassica rapa subsp. rapifera (ECD 02) which exhibits broad-spectrum resistance to many Canadian Plasmodiophora brassicae isolates was crossed with two clubroot-susceptible B. rapa accessions to produce two F2 populations. The F2 plants were screened against P. brassicae pathotypes 3H, 5X, and 5G. The Chi-square goodness of fit test showed that the vast majority (≈75%) of the crosses that produced the F2 populations showed segregation ratios of 9R:7S, 7R:9S, 13R:3S, 3R:13S, 5R:11S, 11R:5S, and 1R:15S. These were modifications of the 15R:1S ratio expected for the inheritance of two dominant major clubroot resistance (CR) genes from ECD 02. The distorted segregation ratios suggest that the two resistance genes are on different chromosomes and that two genes interact in an epistatic manner to confer resistance. Genotyping was conducted with 144 PCR-based markers in the two F2 populations. Linkage and QTL analysis with the polymorphic markers identified two QTLs on chromosome A03 to be associated with resistance to P. brassicae pathotypes 5X and 5G in Popl#1 while only the second QTL on chromosome A03 was associated with resistance to pathotypes 5X and 5G in Popl#2. The QTLs clustered in genomic regions on the A03 chromosome of B. rapa where the CRa/CRbKato gene(s) are mapped. In addition, the Crr1 gene on the A08 chromosome of B. rapa was detected in the two F2 populations. Therefore, the phenotypic and molecular data confirm the existence of two CR genes in ECD 02. This is the first study that shows that major dominant genes in Brassica interact in a non-additive manner to confer resistance to different P. brassicae pathotypes.Key Message: This study provides knowledge on the inheritance and type of gene action for clubroot resistance derived from Brassica rapa subsp. rapifera (ECD 02). The results indicated that duplicate recessive and recessive suppression epistatic interactions, digenic additivity and complementary gene action between the CRa/CRbKato gene(s) on the A03 and the Crr1 gene on the A08 chromosome of B. rapa controlled clubroot resistance to P. brassicae pathotypes 3H, 5X and 5G.

Rutabaga [Brassica napus ssp. napobrassica (L.) Hanelt] is reported to be an excellent source of clubroot (Plasmodiophora brassicae) resistance genes. In this study, 124 rutabaga accessions from the Nordic countries (Norway, Sweden, Finland, Denmark, and Iceland) were evaluated for their reaction to five single-spore isolates representing P. brassicae pathotypes 2F, 3H, 5I, 6M, and 8N and 12 field isolates representing pathotypes 2B, 3A, 3O, 5C, 5G, 5K, 5L, 5X (two isolates, L-G2 and L-G3), 8E, 8J, and 8P. The accessions were also genotyped using a 15K Brassica SNP array and 60 PCR-based primers linked to previously identified clubroot resistance genes. Six thousand eight hundred sixty-one SNP markers were retained after filtering with TASSEL 5.0, and used to evaluate four general linear models (GLM) and four mixed linear models (MLM). The PCA + K and Q + K MLM models gave the minimal deviance of the observed from the expected distribution in quantile-quantile plots, and hence were used for SNP-clubroot association analyses. In addition, 108 alleles derived from the PCR-based markers and the phenotypic data were analyzed with the PCA + K model. Forty-five SNPs and four PCR-based markers were identified to be associated strongly with resistance to isolates representing 13 pathotypes (2F, 3H, 5I, 6M, 8N, 2B, 3A, 3O, 5C, 5G, 5K, 5L, and 8P). These markers revealed the top and bottom segments of rutabaga chromosome A03 and the middle segment of chromosome A08 as genomic hotspots associated with resistance to the different P. brassicae pathotypes.

Abstract Background Plasmodiophora brassicae is a soilborne pathogen of the family Brassicaceae and the causal agent of clubroot disease. In Canada, P. brassicae is now one of the most important constraints to canola (Brassica napus) production, and is managed mainly by the deployment of resistant cultivars. In recent years, however, new strains of the pathogen have emerged that are capable of overcoming host resistance, posing new challenges for disease management. Despite its economic significance, molecular studies of P. brassicae are rare, mainly because this microorganism cannot be cultured outside of its host. Results Restriction site-associated DNA sequencing (RADseq) was used to examine the genetic diversity within P. brassicae single-spore and field isolates collected from across Canada. The isolates included individuals that were either capable or incapable of causing disease on clubroot resistant canola cultivars. Over 8750 variants were identified through RADseq. Population analysis indicated that most isolates belonged to one of two distinct populations, corresponding with the ability of isolates to cause disease on resistant cultivars. Within each population, there were low levels of genetic diversity. One thousand and fifty of the genetic variants that distinguished the two populations were nonsynonymous, altering the coding sequences of genes. Conclusion The application of RADseq revealed two distinct populations of P. brassicae in Canada, suggesting multiple introductions of the pathogen into the country. The genetic variation found here will be important for future research and monitoring of the pathogen.

Clubroot, caused by Plasmodiophora brassicae, is one of the most detrimental threats to crucifers worldwide and has emerged as an important disease of canola (Brassica napus) in Canada. At present, pathotypes are distinguished phenotypically by their virulence patterns on host differential sets, including the systems of Williams, Somé et al., the European Clubroot Differential set, and most recently the Canadian Clubroot Differential set and the Sinitic Clubroot Differential set. Although these are frequently used because of their simplicity of application, they are time-consuming, labor-intensive, and can lack sensitivity. Early, preventative pathotype detection is imperative to maximize productivity and promote sustainable crop production. The decreased turnaround time and increased sensitivity and specificity of genotypic pathotyping will be valuable for the development of integrated clubroot management plans, and interest in molecular techniques to complement phenotypic methods is increasing. This review provides a synopsis of current and future molecular pathotyping platforms for P. brassicae and aims to provide information on techniques that may be most suitable for the development of rapid, reliable, and cost-effective pathotyping assays.

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of the Brassicaceae. Knowledge of the spatial dynamics of P. brassicae at the field level and the influence of soil properties on pathogen spatial patterns can improve understanding of clubroot epidemiology and management. To study the spatial patterns of P. brassicae inoculum density and their relationship to different soil properties, four clubroot-infested fields in central Alberta, Canada, were sampled in 2017 and 2019, and P. brassicae inoculum density, soil pH, and boron, calcium, and magnesium concentrations were quantified. Spatial autocorrelation of the inoculum density was estimated for each of the fields in both years with the Moran’s I and semi-variograms. A Bayesian hierarchical spatial approach was used to model the relationship between P. brassicae inoculum density and the soil parameters. Patchiness of the pathogen was detected, with most patches located at the field edges and adjacent to the entrance. Infested patches grew in size from 2017 to 2019, with an average increase in diameter of 221.3 m and with this growth determined by the maximum inoculum density and active dispersal methods such as movement by machinery and wind. Soil pH, boron, calcium, and magnesium concentrations were not found to have an important effect on the inoculum density of P. brassicae.

Abstract Clubroot, caused by Plasmodiophora brassicae, is an important disease of Brassica crops worldwide. F1 progeny from the Brassica rapa lines T19 (resistant) × ACDC (susceptible) were backcrossed with ACDC, then self-pollinated to produce BC1S1 lines, From genotyping-by-sequencing (GBS) of the parental lines and BC1 plants, about 1.32 M sequences from T19 were aligned into the reference genome of B. rapa with 0.4-fold coverage, and 1.77 M sequences with 0.5-fold coverage in ACDC. The number of aligned short reads per plant in the BC1 ranged from 0.07 to 1.41 M sequences with 0.1-fold coverage. A total of 1584 high quality SNP loci were obtained, distributed on 10 chromosomes. A single co-localized QTL, designated as Rcr4 on chromosome A03, conferred resistance to pathotypes 2, 3, 5, 6 and 8. The peak was at SNP locus A03_23710236, where LOD values were 30.3 to 38.8, with phenotypic variation explained (PVE) of 85–95%. Two QTLs for resistance to a novel P. brassicae pathotype 5x, designated Rcr8 on chromosome A02 and Rcr9 on A08, were detected with 15.0 LOD and 15.8 LOD, and PVE of 36% and 39%, respectively. Bulked segregant analysis was performed to examine TIR-NBS-LRR proteins in the regions harboring the QTL.

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important soilborne disease of Brassica napus L. and other crucifers. To improve understanding of the mechanisms of resistance and pathogenesis in the clubroot pathosystem, the rutabaga (B. napus subsp. rapifera Metzg) cultivars ‘Wilhelmsburger’ (resistant) and ‘Laurentian’ (susceptible) were inoculated with P. brassicae pathotype 3A and their transcriptomes were analyzed at 7, 14, and 21 days after inoculation (dai) by RNA sequencing (RNA-seq). Thousands of transcripts with significant changes in expression were identified in each host at each time-point in inoculated vs. non-inoculated plants. Molecular responses at 7 and 14 dai supported clear differences in the clubroot response mechanisms of the two genotypes. Both the resistant and the susceptible cultivars activated receptor-like protein (RLP) genes, resistance (R) genes, and genes involved in salicylic acid (SA) signaling as clubroot defense mechanisms. In addition, genes related to calcium signaling and genes encoding leucine-rich repeat (LRR) receptor kinases, the respiratory burst oxidase homolog (RBOH) protein, and transcription factors such as WRKYs, ethylene responsive factors, and basic leucine zippers (bZIPs), appeared to be upregulated in ‘Wilhelmsburger’ to restrict P. brassicae development. Some of these genes are essential components of molecular defenses, including ethylene (ET) signaling and the oxidative burst. Our study highlights the importance of activation of genes associated with SA- and ET-mediated responses in the resistant cultivar. A set of candidate genes showing contrasting patterns of expression between the resistant and susceptible cultivars was identified and includes potential targets for further study and validation through approaches such as gene editing.

The fungus Pyrenophora tritici-repentis (Died.) Drechs. infects the leaves and kernels of wheat, causing tan spot and red smudge, respectively. Isolates of P. tritici-repentis have been reported to be both phytopathogenic and mycotoxigenic. This research investigates the influence of nitrogen sources on growth and production of mycotoxins by eight different isolates of P. tritici-repentis. A synthetic agar medium (SAM) was used with different nitrogen sources, both inorganic [(NH4Cl, NH4NO3 and (NH4)2SO4)] and organic (l-alanine, l-histidine, and l-lysine), at a concentration of 37.5 mmol L− 1. Individual isolates exhibited different growth rates that varied according to the nitrogen source added to the medium. The choice of nitrogen source also had a major effect on production of the mycotoxins emodin, catenarin and islandicin. The highest concentrations of emodin, 54.40 ± 4.46 μg g− 1, 43.07 ± 23.39 μg g− 1 and 28.91 ± 4.64 μg g− 1 of growth medium, were produced on the complex medium (V8-potato dextrose agar) by the isolates Alg-H2, 331-2 and TS93-71B, respectively. A relatively high concentration of emodin also was produced by isolates Az35-5 (28.29 ± 4.71 μg g− 1 of medium) and TS93-71B (27.03 ± 4.09 μg g− 1 of medium) on synthetic medium supplemented with l-alanine. The highest concentrations of catenarin (174.54 ± 14.46 μg g− 1 and 104.87 ± 6.13 μg g− 1 of medium) were recorded for isolates TS93-71B and Alg-H2 on synthetic medium supplemented with l-alanine and NH4Cl, respectively. The highest concentration of islandicin (4.64 ± 0.36 μg g− 1 medium) was observed for isolate 331-2 in the presence of l-lysine. There was not a close relationship between mycelial growth and mycotoxin production by the fungal isolates. This is the first report on the influence of nitrogen sources on the production of mycotoxins by P. tritici-repentis.

Genetic resistance is widely used to manage clubroot (Plasmodiophora brassicae) in brassica crops, but new pathotypes have recently been identified on canola (Brassica napus) on the Canadian prairies. Resistance effective against both the most prevalent pathotype (3H, based on the Canadian Clubroot Differential system) and the new pathotypes is needed. BC1 plants of Brassica rapa from a cross of line 96-6990-2 (clubroot resistance originating from turnip cultivar ‘Waaslander’) and a susceptible doubled-haploid line, ACDC, exhibited a 1:1 segregation for resistance against pathotypes 3H and 5X. A resistance gene designated as Rcr3 was mapped initially based on the percentage of polymorphic variants using bulked segregant RNA sequencing (BSR-Seq) and further mapped using Kompetitive Allele Specific PCR. DNA variants were identified by assembling short reads against a reference genome of B. rapa. Rcr3 was mapped into chromosome A08. It was flanked by single nucleotide polymorphisms (SNP) markers (A90_A08_SNP_M12 and M16) between 10.00 and 10.23 Mb, in an interval of 231.6 Kb. There were 32 genes in the Rcr3 interval. Three genes (Bra020951, Bra020974, and Bra020979) were annotated with disease resistance mechanisms, which are potential candidates for Rcr3. Another resistance gene, designated as Rcr9wa, for resistance to pathotype 5X was mapped, with the flanking markers (A90_A08_SNP_M28 and M79) between 10.85 and 11.17 Mb using the SNP sites identified through BSR-Seq for Rcr3. There were 44 genes in the Rcr9wa interval, three of which (Bra020827, Bra020828, Bra020814) were annotated as immune-system-process related genes, which are potential candidates for Rcr9wa.

Pyrenophora tritici-repentis, the causal agent of tan spot of wheat, differentially induces tan necrosis and/or chlorosis in wheat. A chlorosis-inducing, host-specific toxin, termed Ptr ToxB (formerly Ptr chlorosis toxin), was purified from the culture filtrates of a race 5 isolate of P. tritici-repentis. Partial purification was performed by 25 to 80% ammonium sulfate precipitation and passage through a carboxy-methyl-Sephadex C-25 cation exchange column. Final purification was performed by fast performance liquid chromatography, with a Mono S HR 5/5 cation exchanger, followed by size fractionation on a Superose 12 HR 10/30 column. The toxin was shown to be proteinaceous in nature, and purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of Ptr ToxB was determined to be 6.61 kDa. The amino acid composition and partial N terminus amino acid sequence of the toxin were also obtained. Ptr ToxB was found to be heat stable, maintaining full toxic activity after 1 h at 55°C. Infiltration of toxin concentrations as low as 14 nM produced chlorosis on susceptible cultivars.

Blackleg, caused by Leptosphaeria maculans, is an important disease of oilseed rape (Brassica napus L.) in Canada and throughout the world. Severe epidemics of blackleg can result in significant yield losses. Understanding disease-yield relationships is a prerequisite for measuring the agronomic efficacy and economic benefits of control methods. Field experiments were conducted in 2013, 2014, and 2015 to determine the relationship between blackleg disease severity and yield in a susceptible cultivar and in moderately resistant to resistant canola hybrids. Disease severity was lower, and seed yield was 120%–128% greater, in the moderately resistant to resistant hybrids compared with the susceptible cultivar. Regression analysis showed that pod number and seed yield declined linearly as blackleg severity increased. Seed yield per plant decreased by 1.8 g for each unit increase in disease severity, corresponding to a decline in yield of 17.2% for each unit increase in disease severity. Pyraclostrobin fungicide reduced disease severity in all site-years and increased yield. These results show that the reduction of blackleg in canola crops substantially improves yields.