Comparative transcriptome analysis of canola carrying a single vs stacked resistance genes against clubroot.

Pyramiding resistance genes may expand the efficacy and scope of a canola variety against clubroot (Plasmodiophora brassicae), a serious threat to canola production in western Canada. However, the mechanism(s) of multigenic resistance, especially the potential interaction among clubroot resistance (CR) genes, are not well understood. In this study, transcriptome was compared over three canola (Brassica napus L.) inbred/hybrid lines carrying a single CR gene in chromosome A03 (CRaM, Line 16) or A08 (Crr1rutb, Line 20), and both genes (CRaM+Crr1rutb, Line 15) inoculated with a field population (L-G2) of P. brassicae pathotype X, a new variant found in western Canada recently. The line16 was susceptible, while lines 15 and 20 were partially resistant. Functional annotation identified differential expression of genes (DEGs) involved in biosynthetic processes responsive to stress and regulation of cellular process; The Venn diagram showed that the partially resistant lines 15 and 20 shared 1,896 differentially expressed genes relative to the susceptible line 16, and many of these DEGs are involved in defense responses, activation of innate immunity, hormone biosynthesis and programmed cell death. The transcription of genes involved in Pathogen-Associated Molecular Pattern (PAMP)-Triggered and Effector-Triggered Immunity (PTI and ETI) was particularly up-regulated, and the transcription level was higher in line 15 (CRaM + Crr1rutb) than in line 20 (Crr1rutb only) for most of the DEGs. These results indicated that the partial resistance to the pathotype X was likely conferred by the CR gene Crr1rutb for both lines 15 and 20 that functioned via the activation of both PTI and ETI signaling pathways. Additionally, these two CR genes might have synergistic effects against the pathotype X, based on the higher transcription levels of defense-related DEGs expressed by inoculated line 15, highlighting the benefit of gene stacking for improved canola resistance as opposed to a single CR gene alone.

Characterization of a virulence factor in Plasmodiophora brassicae, with molecular markers for identification.

Symptom severity on differential host lines is currently used to characterize and identify pathotypes of Plasmodiophora brassicae, which is an obligate, soil-borne chromist pathogen that causes clubroot disease on canola (Brassica napus) and other brassica crops. This process is slow, variable and resource intensive; development of molecular markers could make identification of important pathotypes faster and more consistent for deployment of cultivars with pathotype-specific resistance. In the current study, a variant of gene 9171 was identified in the whole-genome sequences of only the highly virulent pathotypes of P. brassicae from around the world, including the new cohort of virulent pathotypes in Canada; its presence was confirmed using three KASP marker pairs. The gene was not present in the initial cohort of pathotypes identified in Canada. The putative structure, domains, and gene ontogeny of the protein product of gene 9171 were assessed using on-line software resources. Structural analysis of the putative protein produced by gene 9171 indicated that it was localized in the cytosol, and likely involved in cellular processes and catalytic activity. Identification of gene 9171 represents a potentially useful step toward molecular identification of the pathotypes of P. brassicae.

Identification of a genomic region containing genes involved in resistance to four pathotypes of Plasmodiophora brassicae in Brassica rapa turnip ECD02.

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 BC1 S1 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.

Stemphylium Leaf Blight: A Re-Emerging Threat to Onion Production in Eastern North America.

Stemphylium leaf blight (SLB), caused by Stemphylium vesicarium, is a foliar disease of onion worldwide, and has recently become an important disease in the northeastern United States and Ontario, Canada. The symptoms begin as small, tan to brown lesions on the leaves that can progress to defoliate plants. Crop loss occurs through reduced photosynthetic area, resulting in smaller, lower-quality bulbs. Leaf necrosis caused by SLB also can compromise bulb storage, as green leaves are required for the uptake of sprout inhibitors applied prior to harvest. The pathogen can overwinter on infested onion residue and infected volunteer plants. Asymptomatic weedy hosts near onion fields may also be a source of inoculum. Production of ascospores of the teleomorph (Pleospora allii) peaks in early spring in northeastern North America, often before the crop is planted, and declines rapidly as daily mean air temperatures rise. Conidia are usually present throughout the growing season. Application of fungicides is a standard practice for management of the complex of fungi that can cause foliar diseases of onion in this region. Recent assessments have shown that populations of S. vesicarium in New York and Ontario are resistant to at least three single-site mode-of-action fungicides. Three disease prediction systems have been developed and evaluated that may enable growers to reduce the frequency and/or number of fungicide applications, but the loss of efficacious fungicides due to resistance development within S. vesicarium populations threatens sustainability. The lack of commercially acceptable onion cultivars with sufficient resistance to reduce the number of fungicides for SLB also limits the ability to manage SLB effectively. Integrated disease management strategies for SLB are essential to maintain profitable, sustainable onion production across eastern North America.

Risk assessment of secondary metabolites produced by fungi in the genus Stemphylium.

The fungal genus Stemphylium (phylum Ascomycota, teleomorph Pleospora) includes plant pathogenic, endophytic, and saprophytic species with worldwide distributions. Stemphylium spp. produce prodigious numbers of airborne spores, so are a human health concern as allergens. Some species also produce secondary metabolites, such as glucosides, ferric chelates, aromatic polyketides, and others, that function as toxins that damage plants and other fungal species. Some of these compounds also exhibit a low level of mammalian toxicity. The high production of airborne spores by this genus can result in a high incidence of human exposure. Concern about toxin production appears to be the reason that Stemphylium vesicarium, which is a pathogen of several vegetable crops, was classified in Canada as a potential risk of harm to humans for many years. A detailed assessment of the risk of exposure was provided to the relevant regulatory body, the Public Health Agency of Canada, which then determined that Stemphylium spp. in nature or under laboratory conditions posed little to no risk to humans or animals, and the species was re-assigned as a basic (level 1) risk agent.

Identification of resistance loci against new pathotypes of Plasmodiophora brassicae in Brassica napus based on genome-wide association mapping.

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.

Identification of Two Major QTLs in Brassica napus Lines With Introgressed Clubroot Resistance From Turnip Cultivar ECD01.

Plasmodiophora brassicae causes clubroot disease in brassica crops worldwide. Brassica rapa, a progenitor of Brassica napus (canola), possesses important sources for resistance to clubroot. A doubled haploid (DH) population consisting of 84 DH lines were developed from a Backcross2 (BC2) plant through an interspecific cross of B. rapa turnip cv. ECD01 (resistant, R) with canola line DH16516 (susceptible, S) and then backcrossed with DH16516 as the recurrent parent. The DH lines and their parental lines were tested for resistance to four major pathotypes (3A, 3D, 3H, and 5X) of P. brassicae identified from canola. The R:S segregation ratio for pathotype 3A was 1:3, and 3:1 for pathotypes 3D, 3H, and 5X. From genotyping by sequencing (GBS), a total of 355.3 M short reads were obtained from the 84 DH lines, ranging from 0.81 to 11.67 M sequences per line. The short reads were aligned into the A-genome of B. napus "Darmor-bzh" version 4.1 with a total of 260 non-redundant single-nucleotide polymorphism (SNP) sites. Two quantitative trait loci (QTLs), Rcr10 ECD01 and Rcr9 ECD01 , were detected for the pathotypes in chromosomes A03 and A08, respectively. Rcr10 ECD01 and Rcr9 ECD01 were responsible for resistance to 3A, 3D, and 3H, while only one QTL, Rcr9 ECD01 , was responsible for resistance to pathotype 5X. The logarithm of the odds (LOD) values, phenotypic variation explained (PVE), additive (Add) values, and confidence interval (CI) from the estimated QTL position varied with QTL, with a range of 5.2-12.2 for LOD, 16.2-43.3% for PVE, 14.3-25.4 for Add, and 1.5-12.0 cM for CI. The presence of the QTLs on the chromosomes was confirmed through the identification of the percentage of polymorphic variants using bulked-segregant analysis. There was one gene encoding a disease resistance protein and 24 genes encoding proteins with function related to plant defense response in the Rcr10 ECD01 target region. In the Rcr9 ECD01 region, two genes encoded disease resistance proteins and 10 genes encoded with defense-related function. The target regions for Rcr10 ECD01 and Rcr9 ECD01 in B. napus were homologous to the 11.0-16.0 Mb interval of chromosome A03 and the 12.0-14.5 Mb interval of A08 in B. rapa "Chiifu" reference genome, respectively.

The Role of Ascospores and Conidia, in Relation to Weather Variables, in the Epidemiology of Stemphylium Leaf Blight of Onion.

Stemphylium leaf blight (SLB), caused by Stemphylium vesicarium, is an important foliar disease of onion in northeastern North America. The pathogen produces conidia and ascospores, but the relative contributions of these spore types to epidemics in onion is not known. Determining the abundance of ascospores and conidia during the growing season could contribute to a disease forecasting model. Airborne ascospores and conidia of S. vesicarium were trapped during the growing season of 2015 and 2016 at an onion trial in southern Ontario, Canada, using a Burkard 7-day volumetric sampler. Meteorological data were recorded hourly. Ascospore numbers peaked before the crop was planted and declined rapidly with time and at daily mean air temperatures >15°C. Conidia were present throughout the growing season and appear to be closely related to the development of SLB on onion. Daily spore concentrations were variable, but 59 to 73% of ascospores and approximately 60% of conidia were captured between 0600 and 1200 h. Spore concentrations increased 24 to 72 h after rainfall and precipitation and leaf wetness duration were consistently and positively associated with increases in numbers of conidia and subsequent SLB incidence. The first symptoms of SLB coincided with high numbers of conidia, rainfall, leaf wetness duration ≥8 h, and days with average daily temperature ≥18°C. The number of airborne ascospores was very low by the time SLB symptoms were observed. Ascospores may initiate infection on alternative hosts in early spring, while conidia are the most important inoculum for the epidemic on onions.

Transcriptome analysis identified the mechanism of synergy between sethoxydim herbicide and a mycoherbicide on green foxtail.

Certain synthetic herbicides can act synergistically with specific bioherbicides. In this study, a sethoxydim herbicide at 0.1× label rate improved biocontrol of herbicide-sensitive green foxtail (Setaria viridis, GFT) by Pyricularia setariae (a fungal bioherbicide agent), but did not change the efficacy on a herbicide-resistant GFT biotype. Reference transcriptomes were constructed for both GFT biotypes via de novo assembly of RNA-seq data. GFT plants treated with herbicide alone, fungus alone and herbicide + fungus were compared for weed-control efficacy and differences in transcriptomes. On herbicide-sensitive GFT, sethoxydim at the reduced rate induced ABA-activated signaling pathways and a bZIP transcription factor 60 (TF bZIP60), while improved the efficacy of biocontrol. The herbicide treatment did not increase these activities or improve biocontrol efficacy on herbicide-resistant plants. An exogenous application of ABA to herbicide-sensitive plants also enhanced bZIP60 expression and improved biocontrol efficacy, which supported the results of transcriptome analysis that identified the involvement of ABA and bZIP60 in impaired plant defense against P. setariae. It is novel to use transcriptome analysis to decipher the molecular basis for synergy between a synthetic herbicide and a bioherbicide agent. A better understanding of the mechanism underlining the synergy may facilitate the development of weed biocontrol.

Two Clubroot-Resistance Genes, Rcr3 and Rcr9wa, Mapped in Brassica rapa Using Bulk Segregant RNA Sequencing.

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.

Quantification of Plasmodiophora brassicae Resting Spores in Soils Using Droplet Digital PCR (ddPCR).

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

Whole-genome DNA similarity and population structure of Plasmodiophora brassicae strains from Canada.

BACKGROUND: Clubroot is an important disease of brassica crops world-wide. The causal agent, Plasmodiophora brassicae, has been present in Canada for over a century but was first identified on canola (Brassica napus) in Alberta, Canada in 2003. Genetic resistance to clubroot in an adapted canola cultivar has been available since 2009, but resistance breakdown was detected in 2013 and new pathotypes are increasing rapidly. Information on genetic similarity among pathogen populations across Canada could be useful in estimating the genetic variation in pathogen populations, predicting the effect of subsequent selection pressure on changes in the pathogen population over time, and even in identifying the origin of the initial pathogen introduction to canola in Alberta. RESULTS: The genomic sequences of 43 strains (34 field collections, 9 single-spore isolates) of P. brassicae from Canada, the United States, and China clustered into five clades based on SNP similarity. The strains from Canada separated into four clades, with two containing mostly strains from the Prairies (provinces of Alberta, Saskatchewan, and Manitoba) and two that were mostly from the rest of Canada or the USA. Several strains from China formed a separate clade. More than one pathotype and host were present in all four Canadian clades. The initial pathotypes from canola on the Prairies clustered separately from the pathotypes on canola that could overcome resistance to the initial pathotypes. Similarly, at one site in central Canada where resistance had broken down, about half of the genes differed (based on SNPs) between strains before and after the breakdown. CONCLUSION: Clustering based on genome-wide DNA sequencing demonstrated that the initial pathotypes on canola on the Prairies clustered separately from the new virulent pathotypes on the Prairies. Analysis indicated that these 'new' pathotypes were likely present in the pathogen population at very low frequency, maintained through balancing selection, and increased rapidly in response to selection from repeated exposure to host resistance.

Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa.

BACKGROUND: Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC1 and 3R:1S in the F2, which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. RESULTS: Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8-15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1-6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819. CONCLUSION: Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study.

Analysis of genome-wide variants through bulked segregant RNA sequencing reveals a major gene for resistance to Plasmodiophora brassicae in Brassica oleracea.

Two cabbage (Brassica oleracea) cultivars 'Tekila' and 'Kilaherb' were identified as resistant to several pathotypes of Plasmodiophora brassicae. In this study, we identified a clubroot resistance gene (Rcr7) in 'Tekila' for resistance to pathotype 3 of P. brassicae from a segregating population derived from 'Tekila' crossed with the susceptible line T010000DH3. Genetic mapping was performed by identifying the percentage of polymorphic variants (PPV), a new method proposed in this study, through bulked segregant RNA sequencing. Chromosome C7 carried the highest PPV (42%) compared to the 30-34% in the remaining chromosomes. A peak with PPV (56-73%) was found within the physical interval 41-44 Mb, which indicated that Rcr7 might be located in this region. Kompetitive Allele-Specific PCR was used to confirm the association of Rcr7 with SNPs in the region. Rcr7 was flanked by two SNP markers and co-segregated with three SNP markers in the segregating population of 465 plants. Seven genes encoding TIR-NBS-LRR disease resistance proteins were identified in the target region, but only two genes, Bo7g108760 and Bo7g109000, were expressed. Resistance to pathotype 5X was also mapped to the same region as Rcr7. B. oleracea lines including 'Kilaherb' were tested with five SNP markers for Rcr7 and for resistance to pathotype 3; 11 of 25 lines were resistant, but 'Kilaherb' was the only line that carried the SNP alleles associated with Rcr7. The presence of Rcr7 in 'Kilaherb' for resistance to both pathotypes 3 and 5X was confirmed through linkage analysis.

Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) was identified and fine mapped in Chinese cabbage cv. "Jazz" using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene Rcr2. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2.

Genotyping-by-sequencing reveals three QTL for clubroot resistance to six pathotypes of Plasmodiophora brassicae in Brassica rapa.

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.

Propidium Monoazide Improves Quantification of Resting Spores of Plasmodiophora brassicae with qPCR.

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) analysis is often used to quantify resting spores but does not distinguish between DNA of viable and nonviable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from nonviable microorganisms, was assessed in several experiments. Spore suspensions from immature and mature clubs were heat treated; then, PMA-PCR analyses and bioassays were performed to assess spore viability. Prior to heat treatment, assessments comparing PMA-PCR to qPCR for mature spores were similar, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified in PMA-PCR. Bioassays demonstrated that clubroot severity was much higher in plants inoculated with mature spores than with immature spores. Heat treatment produced little or no change in estimates of mature spores from qPCR but spore estimates from PMA-PCR and clubroot severity in bioassays were both substantially reduced. Estimates of spore concentration with PMA-PCR were less consistent for immature spores. To facilitate use of PMA-PCR on infested soil, a protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard methods.

Identification of Genome-Wide Variants and Discovery of Variants Associated with Brassica rapa Clubroot Resistance Gene Rcr1 through Bulked Segregant RNA Sequencing.

Clubroot, caused by Plasmodiophora brassicae, is an important disease on Brassica species worldwide. A clubroot resistance gene, Rcr1, with efficacy against pathotype 3 of P. brassicae, was previously mapped to chromosome A03 of B. rapa in pak choy cultivar "Flower Nabana". In the current study, resistance to pathotypes 2, 5 and 6 was shown to be associated with Rcr1 region on chromosome A03. Bulked segregant RNA sequencing was performed and short read sequences were assembled into 10 chromosomes of the B. rapa reference genome v1.5. For the resistant (R) bulks, a total of 351.8 million (M) sequences, 30,836.5 million bases (Mb) in length, produced 120-fold coverage of the reference genome. For the susceptible (S) bulks, 322.9 M sequences, 28,216.6 Mb in length, produced 109-fold coverage. In total, 776.2 K single nucleotide polymorphisms (SNPs) and 122.2 K insertion / deletion (InDels) in R bulks and 762.8 K SNPs and 118.7 K InDels in S bulks were identified; each chromosome had about 87% SNPs and 13% InDels, with 78% monomorphic and 22% polymorphic variants between the R and S bulks. Polymorphic variants on each chromosome were usually below 23%, but made up 34% of the variants on chromosome A03. There were 35 genes annotated in the Rcr1 target region and variants were identified in 21 genes. The numbers of poly variants differed significantly among the genes. Four out of them encode Toll-Interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat proteins; Bra019409 and Bra019410 harbored the higher numbers of polymorphic variants, which indicates that they are more likely candidates of Rcr1. Fourteen SNP markers in the target region were genotyped using the Kompetitive Allele Specific PCR method and were confirmed to associate with Rcr1. Selected SNP markers were analyzed with 26 recombinants obtained from a segregating population consisting of 1587 plants, indicating that they were completely linked to Rcr1. Nine SNP markers were used for marker-assisted introgression of Rcr1 into B. napus canola from B. rapa, with 100% accuracy in this study.

Identifying and Managing Root Rot of Pulses on the Northern Great Plains.

Pulse crops (annual grain legumes such as field pea, lentil, dry bean, and chickpea) have become an important component of the cropping system in the northern Great Plains of North America over the last three decades. In many areas, the intensity of damping-off, seedling blight, root rot, and premature ripening of pulse crops is increasing, resulting in reduction in stand establishment and yield. This review provides a brief description of the important pathogens that make up the root rot complex and summarizes root rot management on pulses in the region. Initially, several specific Fusarium spp., a range of Pythium spp., and Rhizoctonia solani were identified as important components of the root rot disease complex. Molecular approaches have recently been used to identify the importance of Aphanomyces euteiches on pulses, and to demonstrate that year-to-year changes in precipitation and temperature have an important effect on pathogen prevalence. Progress has been made on management of root rot, but more IPM tools are required to provide effective disease management. Seed-treatment fungicides can reduce damping-off and seedling blight for many of the pathogens in this disease complex, but complex cocktails of active ingredients are required to protect seedlings from the pathogen complex present in most commercial fields. Partial resistance against many of the pathogens in the complex has been identified, but is not yet available in commercial cultivars. Cultural practices, especially diversified cropping rotations and early, shallow seeding, have been shown to have an important role in root rot management. Biocontrol agents may also have potential over the long term. Improved methods being developed to identify and quantify the pathogen inoculum in individual fields may help producers avoid high-risk fields and select IPM packages that enhance yield stability.

Sensitivity of Mycosphaerella pinodes to Pyraclostrobin Fungicide.

Mycosphaerella blight, caused by Mycosphaerella pinodes, is a destructive disease of field pea that is managed using foliar fungicides. Strobilurin fungicides have been used in western Canada for disease management since 2003. To assess the baseline sensitivities of M. pinodes isolates to the strobilurin fungicide pyraclostrobin, the effective concentration to reduce mycelial growth by 50% (EC50) was determined for 70 isolates collected prior to 2003 from Alberta, Saskatchewan, North Dakota, and Washington State. Each of these isolates was sensitive to pyraclostrobin, with EC50 values ranging from 0.03 to 0.29 mg liter-1. The pyraclostrobin concentrations required to reduce conidia germination by 50% was lower, ranging from 0.008 to 0.041 mg liter-1. In all, 324 isolates collected in 2010 and 2011 were tested for high levels of insensitivity by examining mycelial growth using a discriminatory dose of 5 mg liter-1. Nineteen isolates were highly insensitive to pyraclostrobin, with EC50 values of 80 to 216 mg liter-1. Conidia of these isolates germinated when exposed to a discriminatory dose of 0.1 mg liter-1. Insensitive isolates infected field pea plants treated with pyraclostrobin but sensitive isolates did not. The identification of insensitive isolates indicates that insensitivity may be emerging in the pathogen population.