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Abscisic acid (ABA) plays multiple and pivotal roles in plant defence against abiotic and biotic stresses. Blackleg, mainly caused by the fungal pathogen Leptosphaeria maculans, is one of the most devastating diseases in canola (Brassica napus) resulting in significant yield loss worldwide. Few studies have investigated the function of ABA biosynthesis genes in response to fungal pathogens. In this study, we identified and characterized BnNCED3 (9‐cis‐epoxycarotenoid dioxygenease) and BnTX1 (trithorax‐like factor) from B. napus. Transgenic B. napus ‘Westar’ plants overexpressing BnNCED3 showed enhanced adult plant resistance to blackleg infection. Chromatin immunoprecipitation‐quantitative PCR demonstrated that BnTX1 bound to the promoter region of the BnNCED3 gene. However, the overexpression of BnTX1 in transgenic plants decreased adult plant resistance to blackleg pathogen infection. The expression analysis confirmed the abundance of BnNCED3 transcripts was higher in BnTX1‐RNAi knockdown plants than in overexpressing plants. Thus, BnTX1 might play a negative role in regulating BnNCED3 gene expression and hence ABA biosynthesis, thereby affecting plant disease resistance. Our study provides a new model demonstrating how increased ABA levels can enhance plant disease resistance, which may lead to the characterization of minor/quantitative genes in the B. napus–L. maculans interaction. [ABSTRACT FROM AUTHOR]

Leptosphaeria maculans is a fungal pathogen that causes blackleg disease in canola (Brassica napus), resulting in significant yield and economic losses in Canada and many parts of the world. Plant NAC transcription factors play critical roles in plant development and response to biotic or abiotic stress. In this study, we identified and characterized a BnNAC19 gene from Brassica napus. The overexpression of BnNAC19 in transgenic canola plants contributed to the improvement of seedling resistance against L. maculans. The mycelial growth of a green fluorescent protein‐tagged strain of L. maculans and production of pycnidiospores were shown to be inhibited in the transgenic canola plants overexpressing BnNAC19. In addition, the canola transgenic line overexpressing BnNAC19 showed increased disease resistance in the adult plant, which was determined by quantitative resistance. Both increased seedling and adult plant resistance in transgenic canola plants overexpressing BnNAC19 indicate that the BnNAC19 gene plays a positive role against L. maculans. The expression pattern of genes BnNAC19 upstream and downstream of BnNAC19 that participate in plant defence pathways were investigated to elucidate the B. napus resistance mechanisms to L. maculans infection, and hence to aid the long‐term blackleg disease‐resistant breeding programmes. [ABSTRACT FROM AUTHOR]

Abstract: Leptosphaeria maculans causes blackleg disease on Brassica napus, an economically important oilseed crop. Brassica juncea has high resistance to blackleg and is a source for the development of resistant B. napus varieties. To transfer the Rlm6 resistance gene from B. juncea into B. napus, an interspecific cross between B. napus “Topas DH16516” and B. juncea “Forge” was produced, followed by the development of F2 and F3 generations. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers linked to the L. maculans resistance gene Rlm6 were developed. Segregation of SCAR and CAPS markers linked to Rlm6 were confirmed by genotyping of F2 and F3 progeny. Segregation of CAPS markers and phenotypes for blackleg disease severity in F2 plants had a Mendelian ratio of 3:1 in resistant vs. susceptible plants, respectively, supporting the assumption that genetic control of resistance was by a single dominant gene. The molecular markers developed in this study, which show linkage with the L. maculans resistance gene Rlm6, would facilitate marker‐assisted backcross breeding in a variety development programme. [ABSTRACT FROM AUTHOR]

Abstract: Leptosphaeria maculans is the most important fungal pathogen of canola (Brassica napus, oilseed rape) that causes the devastating stem canker in canola fields of western Canada. The population genetic structure of L. maculans, represented by nine subpopulations from a 6‐year period and three different provinces in western Canada, was determined using ten minisatellite markers. Isolates collected at different locations in six consecutive years had an even distribution of MAT1‐1 and MAT1‐2 across the nine subpopulations. All subpopulations of L. maculans exhibited a moderate gene diversity (H = 0.356–0.585). The majority of the genetic variation occurred within subpopulations. Approximately 8% and 4% of the variations were distributed between sampling year and location, respectively. Genetic distance (FST) results, using analysis of molecular variation (AMOVA), indicated that subpopulation pairing within isolates by year ranged from FST = 0.010 to 0.109, and the location subpopulation ranged from FST = 0.038 to 0.085. Bayesian clustering analyses of multiloci inferred two distinct clusters in all the subpopulations examined. This study indicates a relatively high degree of gene exchange between the different L. maculans isolates. Our results suggest that this can occur in the wide growing areas of canola fields in western Canada. This gene exchange produced different gene allele frequencies and divergence between populations. [ABSTRACT FROM AUTHOR]

Blackleg, caused by the fungus Leptosphaeria maculans, is an important disease of canola. Both ascospores and pycnidiospores of the fungus can infect intact leaves under conditions of extended leaf wetness, with infection then progressing further into the stem. In western Canada, spring is typically cool and dry and additional factors may be involved for successful infection. This study was designed to assess the effect of wounding and wound age on cotyledon infection, to evaluate the likelihood that flea beetle-feeding injuries could contribute to increased disease. Infection of canola cotyledons occurred readily via mechanical wounds in the absence of leaf wetness when seedlings were spray inoculated with pycnidiospores of L. maculans, whereas no infection occurred on intact cotyledons even under 6-h leaf wetness. Wound age also played a role in susceptibility; wounded tissues were less susceptible when plants were kept in a greenhouse for 8 h or longer before inoculation, with substantially reduced infection success relative to fresh wounds. This wound-age effect was similar on susceptible and resistant canola varieties. A high temperature (25°C) seemed to favour the healing of wounds, reducing susceptibility when compared with control (21/16°C) and low (10°C) temperatures. Leaf wetness post-wounding may hinder healing, and high post-inoculation humidity (80–90% relative humidity) increased infection via older wounds slightly, relative to lower (50–60%) relative humidity. These data provide a rationale for studying the effect of improved flea beetle control on early L. maculans infection of canola. [ABSTRACT FROM AUTHOR]

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and β-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa, and constitute a new subclade, herein proposed as L. biglobosa 'americensis'. [ABSTRACT FROM AUTHOR]

Callose plays a critical role in different biological processes including development as well as in the response to multiple biotic and abiotic stresses. In this study, we characterized the callose deposition in cotyledons of different Brassica napus varieties post-inoculated with different Leptosphaeria maculans isolates. Further, members of the callose synthase gene were identified from the whole genome of B. napus using the 12 Arabidopsis thaniana callose synthase protein sequences, and were then classified into three groups based on their phylogenetic relationships. Chromosomal location and duplication patterns indicated uneven distribution and segmental duplication patterns of BnCalS genes in the B. napus genome. Subsequently, gene structures, conserved domains analysis, and protein properties were analyzed for BnCalS genes. In addition, 12 B. napus orthologs of the AtCalS were selected for investigating the tissue expression pattern, indicating diverse expression patterns for these BnCalS genes. Responses of the selected 12 orthologs and all the BnCalS genes were characterized in the different types (AvrLm1-Rlm1, AvrLm4-Rlm4, AvrLepR1-LepR1) of B. napus–L. maculans interactions and B. napus-Leptosphaeria biglobosa interactions, implying their potential roles in response to Leptosphaeria infection. [ABSTRACT FROM AUTHOR]

• LmCBP1 is a secreted protein and can bind to chitin. • Knockout of LmCBP1 in Leptosphaeria maculans using CRISPR-Cas9 lowered the pathogenicity of this pathogen on Brassica napus. • The knockout mutants of LmCBP1 were more sensitive to hydrogen peroxide. Leptosphaeria maculan s is the causal agent of blackleg disease on Brassica napus. Determining the underlying functions of genes required for pathogenesis is essential for understanding the infection process. A chitin-binding protein (LmCBP1) was discovered as a pathogenicity factor for the infection of B. napus by L. maculans through gene knockout using the CRISPR-Cas9 system. Chitin-binding activity was demonstrated through a chitin-protein binding assay. A secreted signal peptide was detected using a yeast secreted-signal peptide trap assay. An increased expression level during the infection stage was also observed, suggesting that LmCBP1 is a secreted protein. The knockout mutants showed decreased infection on B. napus , with reduced pathogenicity on ten cultivars with/without diverse R genes. The mutants were more sensitive to H 2 O 2 compared to wild type L. maculans isolate JN3. This study provides evidence of the virulence of a novel chitin-binding protein LmCBP1 on B. napus through mutants created via the CRISPR-Cas9 system. [ABSTRACT FROM AUTHOR]