The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

Diagnosis and Management of Paroxysmal Supraventricular Tachycardia.

Importance: Paroxysmal supraventricular tachycardia (PSVT), defined as tachyarrhythmias that originate from or conduct through the atria or atrioventricular node with abrupt onset, affects 168 to 332 per 100 000 individuals. Untreated PSVT is associated with adverse outcomes including high symptom burden and tachycardia-mediated cardiomyopathy. Observations: Approximately 50% of patients with PSVT are aged 45 to 64 years and 67.5% are female. Most common symptoms include palpitations (86%), chest discomfort (47%), and dyspnea (38%). Patients may rarely develop tachycardia-mediated cardiomyopathy (1%) due to PSVT. Diagnosis is made on electrocardiogram during an arrhythmic event or using ambulatory monitoring. First-line acute therapy for hemodynamically stable patients includes vagal maneuvers such as the modified Valsalva maneuver (43% effective) and intravenous adenosine (91% effective). Emergent cardioversion is recommended for patients who are hemodynamically unstable. Catheter ablation is safe, highly effective, and recommended as first-line therapy to prevent recurrence of PSVT. Meta-analysis of observational studies shows single catheter ablation procedure success rates of 94.3% to 98.5%. Evidence is limited for the effectiveness of long-term pharmacotherapy to prevent PSVT. Nonetheless, guidelines recommend therapies including calcium channel blockers, ß-blockers, and antiarrhythmic agents as management options. Conclusion and Relevance: Paroxysmal SVT affects both adult and pediatric populations and is generally a benign condition. Catheter ablation is the most effective therapy to prevent recurrent PSVT. Pharmacotherapy is an important component of acute and long-term management of PSVT.

Systematic characterization of Brassica napus UBC13 genes involved in DNA-damage response and K63-linked polyubiquitination.

BACKGROUND: Ubc13 is the only known ubiquitin conjugating enzyme (Ubc/E2) dedicated to promoting Lys (K)63-linked polyubiquitination, and this process requires a Ubc/E2 variant (UEV). Unlike conventional K48-linked polyubiquitination that targets proteins for degradation, K63-linked polyubiquitination, which is involved in several cellular processes, does not target proteins for degradation but alter their activities. RESULTS: In this study we report the identification and functional characterization of 12 Brassica napus UBC13 genes. All the cloned UBC13 gene products were able to physically interact with AtUev1D, an Arabidopsis UEV, to form stable complexes that are capable of catalyzing K63-linked polyubiquitination in vitro. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects in spontaneous mutagenesis and DNA-damage responses, suggesting that BnUBC13s can replace yeast UBC13 in mediating K63-linked polyubiquitination and error-free DNA-damage tolerance. CONCLUSION: Collectively, this study provides convincing data to support notions that B. napus Ubc13s promote K63-linked polyubiquitination and are probably required for abiotic stress response. Since plant Ubc13-UEV are also implicated in other developmental and stress responses, this systematic study sets a milestone in exploring roles of K63-linked polyubiquitination in this agriculturally important crop.

Specific Recruitment of Phosphoinositide Species to the Plant-Pathogen Interfacial Membrane Underlies Arabidopsis Susceptibility to Fungal Infection.

Different phosphoinositides enriched at the membranes of specific subcellular compartments within plant cells contribute to organelle identity, ensuring appropriate cellular trafficking and function. During the infection of plant cells, biotrophic pathogens such as powdery mildews enter plant cells and differentiate into haustoria. Each haustorium is enveloped by an extrahaustorial membrane (EHM) derived from the host plasma membrane. Little is known about the EHM biogenesis and identity. Here, we demonstrate that among the two plasma membrane phosphoinositides in Arabidopsis (Arabidopsis thaliana), PI(4,5)P2 is dynamically up-regulated at powdery mildew infection sites and recruited to the EHM, whereas PI4P is absent in the EHM. Lateral transport of PI(4,5)P2 into the EHM occurs through a brefeldin A-insensitive but actin-dependent trafficking pathway. Furthermore, the lower levels of PI(4,5)P2 in pip5k1 pip5k2 mutants inhibit fungal pathogen development and cause disease resistance, independent of cell death-associated defenses and involving impaired host susceptibility. Our results reveal that plant biotrophic and hemibiotrophic pathogens modulate the subcellular distribution of host phosphoinositides and recruit PI(4,5)P2 as a susceptibility factor for plant disease.

Leptosphaeria maculans Isolates Reveal Their Allele Frequency in Western Canada.

Blackleg, caused by Leptosphaeria maculans, is a major disease of canola in Canada, Australia, and Europe. For effective deployment of resistant varieties and disease management, it is crucial to understand the population structure of L. maculans. In this study, we analyzed L. maculans isolates from commercial fields in western Canada from 2014 to 2016 for the presence and frequency of avirulence (Avr) genes. A total of 1,584 isolates were examined for the presence of Avr genes AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm6, AvrLm7, AvrLm9, AvrLepR1, AvrLepR2, and AvrLmS via a set of differential host genotypes carrying known resistance genes and a PCR assay. Several Avr genes showed a higher frequency in the pathogen population, such as AvrLm6 and AvrLm7, which were present in >90% of isolates, whereas AvrLm3, AvrLm9, and AvrLepR2 showed frequencies of <10%. A total of 189 races (different combinations of Avr genes) were detected, with Avr-2-4-6-7-S, Avr-1-4-6-7, and Avr-2-4-6-7 as the three predominant races. When the effect of crop rotation was assessed, only a 3-year rotation showed a significantly higher frequency of AvrLm2 relative to shorter rotations. This study provides the information for producers to select effective canola varieties for blackleg management and for breeders to deploy new R genes in disease resistance breeding in western Canada.

Fungicide Mitigates Fusarium Head Blight in Durum Wheat When Applied as Late as the End of Flowering in Western Canada.

Fusarium head blight (FHB) is one of the most important diseases of durum, spring, and winter wheat in Canada. Growers rely on an integrated strategy to manage the disease, including fungicide application at the current recommendation of early to 50% anthesis (BBCH61-65). This study evaluated the effect of fungicide application timing and seeding rates of durum wheat on FHB. Field trials were carried out from 2016 to 2018 at three locations in Saskatchewan. Eight treatments of the metconazole fungicide Caramba were applied to durum seeded at 75 and 400 seeds m-2. The fungicide treatments consisted of a nontreated check, a treated check, and applications at BBCH59, BBCH61, BBCH65, BBCH69, and BBCH73 and a dual application treatment (BBCH61 followed by BBCH73). FHB index, proportion of Fusarium-damaged kernels (%FDK), deoxynivalenol (DON), grain protein content (GPC, %), and yield were evaluated. Seeding rates influenced all parameters. The high seeding rate had a higher yield and FHB index but lower DON and GPC than did the lower seeding rate. All fungicide treatments resulted in lower FHB index, DON, and %FDK than the nontreated check. Under FHB conducive conditions, all anthesis applications from BBCH61 to BBCH69 had a similar effect on FHB index, %FDK, DON, and yield, whereas in years with low disease severity, the application at BBCH65 had lower FHB index, %FDK, and DON relative to other single applications. The dual application (BBCH61 + 73) treatment resulted in similar FHB index levels, %FDK, and DON content as the BBCH65 application at all site years. Our results indicate that the window of fungicide application can be extended to the end of flowering when FHB risk is high.

First report of Leptosphaeria biglobosa 'brassicae' causing Black Leg on Brassica rapa subsp. pekinensis in China.

Chinese cabbage [Brassica rapa L. subsp. pekinensis (Lour.) Hanelt] is a major leafy vegetable crop grown in China and eastern Asia (Fordham and Hadley 2003). In December 2018, black leg symptoms were observed on of "Qingza No.3" of Chinese cabbage during harvest, Chibi (29°46'37.38''N, 114°05'6.88''E), Hubei, China. Symptoms were first noted in late Nov. as black spots on leaf petioles and basal stems. Then, black spots enlarged as oval or irregular-shaped grayish lesions. Finally, lesions enlarged and coalesced with black pycnidia were observed, and some diseased leaves became blighted. The disease incidence was about 80% in three fields surveyed (~2 ha in total). Diseased plant tissues were surface-sterilized, and incubated on potato dextrose agar (PDA) plates at 20°C for 4 days. Three fungal isolates, namely EP9-19, EP9-22 and EP9-26, were obtained from five of the diseased samples; all produced fluffy, white aerial mycelia and a yellow pigment on PDA. After 14 days, black-brown and globose pycnidia were produced, approximately 150 µm in diameter (n = 50). In addition, pink pycnidiospore ooze was observed on the top of pycnidium after 20-day culturing on a V8-juice (20%) agar. Conidia were cylindrical and hyaline, with the mean size of 4.6 × 2.7 µm (n = 50). Two fungal species have been reported to cause blackleg on Brassica crops (Williams and Fitt 1999), i.e. Leptosphaeria maculans and L. biglobosa. The former is much more destructive, but is not present in China. These isolates had morphological characteristics matching those of L. biglobosa (Williams and Fitt 1999). The genomic DNA of isolate EP9-22 was extracted and sequenced for its actin, ß-tubulin and the internal transcribed spacer (ITS) region of ribosomal DNA as described by Vincenot et al. (2008). Sequences of ITS (GenBank accession no. MN238766), actin (MN242213) and ß-tubulin (MN242214) for isolate EP9-22 showed 100%, 99.67%, and 97.93% identity to the corresponding regions of L. biglobosa 'brassicae' strain IBCN89 (Vincenot et al. 2008). In addition, the phylogenetic analysis also indicated that isolate EP9-22 belonged to L. biglobosa 'brassicae'. The pathogenicity test was performed according to established protocols (Balesdent et al., 2005). Cotyledons of the 15-day-old Chinese cabbage seedlings (cultivars Xiaoza No.55 and Hualiangzao No.5) were wound inoculated with 10 µl pycnidiospore suspension (1 × 107 conidia/ml) of the three isolates, with 20 cotyledons per isolate, respectively, and 20 cotyledons wound inoculated with sterile water served as a control group. The treated seedlings were maintained at 20°C and 100% relative humidity with a 12-h photoperiod. The experiment was repeated twice. At 7 days after inoculation, necrotic lesions became visible surrounding inoculation sites for the three isolates, while the control group remained healthy. Fungal isolates showing a similar colony morphology to the originals were re-isolated from ten diseased cotyledons but not from the control cotyledons. Based on these results, L. biglobosa 'brassicae' was shown to be the causal agent of blackleg on Chinese cabbage in China. We believe that this disease has historically often been misidentified as 'anthracnose' by local famers. The threat from L. biglobosa to the production of Chinese cabbage has been assessed. This accurate identification of the causal pathogen is a critical first step towards the development of disease management strategies.

Identification of resistance loci in Chinese and Canadian canola/rapeseed varieties against Leptosphaeria maculans based on genome-wide association studies.

BACKGROUND: The fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS). RESULTS: Using the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10- 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance. CONCLUSION: This study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola.

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.

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.

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.

The Effect of Fhb1 and Fhb5 Quantitative Trait Loci in Hard Red Spring Wheat Does Not Depend on Fungicide Use for Managing Fusarium Head Blight in Wheat.

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most damaging diseases that affect wheat in Canada. The disease is best managed by integrating host resistance and fungicides, mainly demethylation inhibitors. Research has shown that the effect of fungicides may be dependent on the level of resistance of the cultivar. However, whether the performance of genotypes carrying specific Sumai 3-derived major FHB quantitative trait loci is dependent on fungicide application has not been explored. In our study, the performance of near-isogenic lines (NILs; <1.0% genome/alleles from the resistance donor), carrying Fhb1 and Fhb5 in a hard red spring wheat cultivar CDC Go background compared with a moderately susceptible (MS) genotype, was evaluated with and without one application of metconazole during full flowering. Field experiments were conducted at five site-years in Saskatchewan, Canada, between 2016 and 2017. In both the individual and combined analysis (all trials), we found that the effect of NILs and metconazole in suppressing FHB symptoms and deoxynivalenol (DON) accumulation in the grain was additive. FHB severity was generally low and fungicide efficacy levels, relative to the untreated control, were increased in the MS cultivar than in the NILs carrying Fhb1 and Fhb5, which were least affected by the disease. The results confirm the importance of integrating fungicides with cultivar resistance to reduce FHB and DON, regardless of the presence of those well-characterized resistant genes.

Evaluating Changes in Cell-Wall Components Associated with Clubroot Resistance Using Fourier Transform Infrared Spectroscopy and RT-PCR.

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

Synchrotron based phase contrast X-ray imaging combined with FTIR spectroscopy reveals structural and biomolecular differences in spikelets play a significant role in resistance to Fusarium in wheat.

BACKGROUND: Fusarium head blight (FHB), a scab principally caused by Fusarium graminearum Schw., is a serious disease of wheat. The purpose of this study is to evaluate the potential of combining synchrotron based phase contrast X-ray imaging (PCI) with Fourier Transform mid infrared (FTIR) spectroscopy to understand the mechanisms of resistance to FHB by resistant wheat cultivars. Our hypothesis is that structural and biochemical differences between resistant and susceptible cultivars play a significant role in developing resistance to FHB. RESULTS: Synchrotron based PCI images and FTIR absorption spectra (4000-800 cm(-1)) of the floret and rachis from Fusarium-damaged and undamaged spikes of the resistant cultivar 'Sumai3', tolerant cultivar 'FL62R1', and susceptible cultivar 'Muchmore' were collected and analyzed. The PCI images show significant differences between infected and non-infected florets and rachises of different wheat cultivars. However, no pronounced difference between non-inoculated resistant and susceptible cultivar in terms of floret structures could be determined due to the complexity of the internal structures. The FTIR spectra showed significant variability between infected and non-infected floret and rachis of the wheat cultivars. The changes in absorption wavenumbers following pathogenic infection were mostly in the spectral range from 1800-800 cm(-1). The Principal Component Analysis (PCA) was also used to determine the significant chemical changes inside floret and rachis when exposed to the FHB disease stress to understand the plant response mechanism. In the floret and rachis samples, PCA of FTIR spectra revealed differences in cell wall related polysaccharides. In the florets, absorption peaks for Amide I, cellulose, hemicellulose and pectin were affected by the pathogenic fungus. In the rachis of the wheat cultivars, PCA underlines significant changes in pectin, cellulose, and hemicellulose characteristic absorption spectra. Amide II and lignin absorption peaks, persistent in the rachis of Sumai3, together with increased peak shift at 1245 cm(-1) after infection with FHB may be a marker for stress response in which the cell wall compounds related to pathways for lignification are increased. CONCLUSIONS: Synchrotron based PCI combined with FTIR spectroscopy show promising results related to FHB in wheat. The combined technique is a powerful new tool for internal visualisation and biomolecular monitoring before and during plant-microbe interactions to understand both the differences between cultivars and their different responses to disease stress.

Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae.

BACKGROUND: The protist Plasmodiophora brassicae is a biotrophic soil-borne pathogen that causes clubroot on Brassica crops worldwide. Clubroot disease is a serious threat to the 8 M ha of canola (Brassica napus) grown annually in western Canada. While host resistance is the key to clubroot management, sources of resistance are limited. RESULTS: To identify new sources of clubroot resistance (CR), we fine mapped a CR gene (Rcr1) from B. rapa ssp. chinensis to the region between 24.26 Mb and 24.50 Mb on the linkage group A03, with several closely linked markers identified. Transcriptome analysis was conducted using RNA sequencing on a segregating F1 population inoculated with P. brassicae, with 2,212 differentially expressed genes (DEGs) identified between plants carrying and not carrying Rcr1. Functional annotation of these DEGs showed that several defense-related biological processes, including signaling and metabolism of jasmonate and ethylene, defensive deposition of callose and biosynthesis of indole-containing compounds, were up-regulated significantly in plants carrying Rcr1 while genes involved in salicylic acid metabolic and signaling pathways were generally not elevated. Several DEGs involved in metabolism potentially related to clubroot symptom development, including auxin biosynthesis and cell growth/development, showed significantly lower expression in plants carrying Rcr1. CONCLUSION: The CR gene Rcr1 and closely linked markers will be highly useful for breeding new resistant canola cultivars. The identification of DEGs between inoculated plants carrying and not carrying Rcr1 is an important step towards understanding of specific metabolic/signaling pathways in clubroot resistance mediated by Rcr1. This information may help judicious use of CR genes with complementary resistance mechanisms for durable clubroot resistance.

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.

Resilience of Canola to Plasmodiophora brassicae (Clubroot) Pathotype 3H under Different Resistance Genes and Initial Inoculum Levels.

In this study, we explored the resilience of a clubroot resistance (CR) stacking model against a field population of Plasmodiophora brassicae pathotype 3H. This contrasts with our earlier work, where stacking CRaM and Crr1rutb proved only moderately resistant to pathotype X. Canola varieties carrying Rcr1/Crr1rutb and Rcr1 + Crr1rutb were repeatedly exposed to 3H at low (1 × 104/g soil) and high (1 × 107/g soil) initial resting spore concentrations over five planting cycles under controlled environments to mimic intensive canola production. Initially, all resistant varieties showed strong resistance. However, there was a gradual decline in resistance over time for varieties carrying only a single CR gene, particularly with Crr1rutb alone and at the high inoculum level, where the disease severity index (DSI) increased from 9% to 39% over five planting cycles. This suggests the presence of virulent pathotypes at initially low levels in the 3H inoculum. In contrast, the variety with stacked CR genes remained resilient, with DSI staying below 3% throughout, even at the high inoculum level. Furthermore, the use of resistant varieties, carrying either a single or stacked CR genes, reduced the total resting spore numbers in soil over time, while the inoculum level either increased or remained high in soils where susceptible Westar was continuously grown. Our study demonstrates greater resistance resilience for stacking Rcr1 and Crr1rutb against the field population of 3H. Additionally, the results suggest that resistance may persist even longer in fields with lower levels of inoculum, highlighting the value of extended crop rotation (reducing inoculum) alongside strategic CR-gene deployment to maximize resistance resilience.

Identification and mapping of a novel blackleg resistance locus LepR4 in the progenies from Brassica napus × B. rapa subsp. sylvestris.

Blackleg, caused by Leptosphaeria maculans, is one of the most economically important diseases of Brassica napus worldwide. Two blackleg-resistant lines, 16S and 61446, were developed through interspecific hybridization between B. napus and B. rapa subsp. sylvestris and backcrossing to B. napus. Classical genetic analysis demonstrated that a single recessive gene in both lines conferred resistance to L. maculans and that the resistance alleles were allelic. Using BC(1) progeny derived from each resistant plant, this locus was mapped to B. napus linkage group N6 and was flanked by microsatellite markers sN2189b and sORH72a in an interval of about 10 cM, in a region equivalent to about 6 Mb of B. rapa DNA sequence. This new resistance gene locus was designated as LepR4. The two lines were evaluated for resistance to a wide range of L. maculans isolates using cotyledon inoculation tests under controlled environment conditions, and for stem canker resistance in blackleg field nurseries. Results indicated that line 16S, carrying LepR4a, was highly resistant to all isolates tested on cotyledons and had a high level of stem canker resistance under field conditions. Line 61446, carrying LepR4b, was only resistant to some of the isolates tested on cotyledons and was weakly resistant to stem canker under field conditions.

Effects of nitrogen fertilization and a commercial arbuscular mycorrhizal fungal inoculant on root rot and agronomic production of pea and lentil crops.

In the Canadian prairies, pulse crops such as field pea (Pisum sativum L.) and lentil (Lens culinaris L.) are economically important and widely grown. However, in recent years, root rot, caused by a variety of fungal and oomycete pathogens, including Aphanomyces euteiches, has become a limiting factor on yield. In this study, we examined the impacts of nitrogen (N) fertilization and a commercial arbuscular mycorrhizal fungal (AMF) inoculant on pea and lentil plant health and agronomic production at three locations in Saskatchewan: Swift Current, Indian Head and Melfort. The AMF inoculation had no impact on root rot severity, and therefore is not considered a reliable method to manage root rot in pea and lentil. In contrast, N fertilization led to reductions in root rot in Swift Current, but not the other two sites. However, N fertilization did reduce nodulation. When both pea and lentil are considered, the abundance of A. euteiches in soil increased from pre-seeding to mid-bloom. A negative correlation between soil pH and disease severity was also observed. The high between-site variability highlights the importance of testing root rot mitigation strategies under multiple soil conditions to develop site-specific recommendations. Use of N fertilizer as a root rot management strategy merits further exploration, including investigation into its interactions with other management strategies, soil properties, and costs and benefits.

Canola with Stacked Genes Shows Moderate Resistance and Resilience against a Field Population of Plasmodiophora brassicae (Clubroot) Pathotype X.

Genetic resistance is a cornerstone for managing clubroot (Plasmodiophora brassicae). However, when used repeatedly, a clubroot resistance (CR) gene can be broken rapidly. In this study, canola inbred/hybrid lines carrying one or two CR genes (Rcr1/CRaM and Crr1rutb) were assessed against P. brassicae pathotype X by repeated exposure to the same inoculum source under a controlled environment. Lines carrying two CR genes, either Rcr1 + Crr1rutb or CRaM + Crr1rutb, showed partial resistance. Selected lines were inoculated with a field pathotype X population (L-G3) at 5 × 106 resting spores/g soil, and all clubs were returned to the soil they came from six weeks after inoculation. The planting was repeated for five cycles, with diseased roots being returned to the soil after each cycle. The soil inoculum was quantified using qPCR before each planting cycle. All lines with a single CR gene were consistently susceptible, maintaining high soil inoculum levels over time. The lines carrying two CR genes showed much lower clubroot severity, resulting in a 10-fold decline in soil inoculum. These results showed that the CR-gene stacking provided moderate resistance against P. brassicae pathotype X, which may also help reduce the pathogen inoculum buildup in soil.