Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm4‐7, two avirulence effector genes of L. maculans, are involved in an ...unusual relationship: AvrLm4‐7 suppresses the Rlm3‐mediated resistance. Here, we assessed AvrLm3 polymorphism in a collection of 235 L. maculans isolates. No field isolates exhibited deletion or inactivating mutations in AvrLm3, as observed for other L. maculans avirulence genes. Eleven isoforms of the AvrLm3 protein were found. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3‐mediated resistance response was due to two distinct mechanisms. First, when AvrLm4‐7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Second, when only point mutations were observed in AvrLm4‐7, a3a7 isolates still contained an avirulent allele of AvrLm3. Directed mutagenesis confirmed that some point mutations in AvrLm4‐7 were sufficient for the fungus to escape Rlm7‐mediated resistance while maintaining the suppression of the AvrLm3 phenotype. Signatures of positive selection were also identified in AvrLm3. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3‐mediated resistance while preserving AvrLm3 integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize the importance of this effector in pathogenicity towards B. napus. While the common response to resistance gene pressure is local selection of isolates depleted in the cognate avirulence gene, this example contributes to complexify the gene‐for‐gene concept of plant‐pathogen evolution with a ‘camouflaged’ model allowing retention of nondispensable avirulence effectors.
Pathogen introductions into novel areas can lead to the emergence of new fungal diseases of plants. Understanding the origin, introduction pathways, possible changes in reproductive system and ...population size of fungal pathogens is essential in devising an integrated strategy for the control of these diseases. We used minisatellite markers to infer the worldwide invasion history of the fungal plant pathogen Leptosphaeria maculans, which causes stem canker (blackleg) of oilseed and vegetable brassicas. Clustering analyses partitioned genotypes into distinct populations corresponding to major geographic regions, along with two differentiated populations in Western Canada. Comparison of invasion scenarios using Approximate Bayesian Computation suggested an origin of the pathogen in the USA, the region where epidemics were first recorded, and independent introductions from there over the last few decades into Eastern Canada (Ontario), Europe and Australia. The population in Western Canada appeared to be founded from a source in Ontario and the population in Chile resulted from an admixture between multiple sources. A bottleneck was inferred for the introduction into Western Canada but not into Europe, Ontario or Australia. Clonality appeared high in Western Canada, possibly because environmental conditions there were less conducive to sexual reproduction. Leptosphaeria maculans is a model invasive pathogen with contrasting features in different regions: shallow population structure, high genetic variability and regular sexual recombination in some regions, by comparison with reduced genetic variability, high rates of asexual multiplication, strong population structure or admixture in others.
Map-based cloning of avirulence genes of the AvrLm1-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 ...resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLm1, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5(prime) and 3(prime) sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes are highly overexpressed during primary leaf infection. Using RNA interference, decreasing expression of AvrLm6 was shown to result in virulence toward Rlm6 genotypes whenever the expression was reduced by more than 60% compared with the wild-type isolate.
SUMMARY
Leptosphaeria maculans is the most ubiquitous pathogen of Brassica crops, and mainly oilseed brassicas (oilseed rape, canola), causing the devastating ‘stem canker’ or ‘blackleg’. This review ...summarizes our current knowledge on the pathogen, from taxonomic issues to specific life traits. It mainly illustrates the importance of formal genetics approaches on the pathogen side to dissect the interaction with the host plants. In addition, this review presents the main current research topics on L. maculans and focuses on the L. maculans genome initiative recently begun, including its main research issues.
Taxonomy: Leptosphaeria maculans (Desm.) Ces. & de Not. (anamorph Phoma lingam Tode ex Fr.). Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes (Loculoascomycetes), Order Pleosporales, Genus Leptosphaeria, Species maculans.
Host range: cultivated Brassicas such as Brassica napus (oilseed rape, canola), B. rapa, B. juncea, B. oleracea, etc., along with numerous wild crucifers species. Arabidopsis thaliana was recently reported to be a potential host for L. maculans. Primary disease symptoms are greyish‐green collapse of cotyledon or leaf tissue, without a visible margin, bearing tiny black spots (pycnidia). The fungus then develops an endophytic symptomless growth for many months. Secondary symptoms, at the end of the growing season, are dry necroses of the crown tissues with occasional blackening (stem canker or blackleg) causing lodging of the plants. Pseudothecia differentiate on leftover residues. Seedling damping‐off and premature ripening are also reported under certain environmental conditions.
Useful websites: Leptosphaeria maculans sequencing project at Genoscope: http://www.genoscope.cns.fr/externe/English/Projets/Projet_DM/organisme_DM.html; the SECURE site: http://www.secure.rothamsted.ac.uk/ the ‘Blackleg’ group at the University of Melbourne: http://www.botany.unimelb.edu.au/blackleg/overview.htm
Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, develops gene-for-gene interactions with its hosts. To date, eight L. maculans avirulence (Avr) genes, AvrLm1 to AvrLm8, have ...been genetically characterized. An additional Avr gene, AvrLm9, that interacts with the resistance gene Rlm9, was genetically characterized here following in vitro crosses of the pathogen. A worldwide collection of 63 isolates, including the International Blackleg of Crucifers Network collection, was genotyped at these nine Avr loci. In a first step, isolates were classified into pathogenicity groups (PGs) using two published differential sets. This analysis revealed geographical disparities as regards the proportion of each PG. Genotyping of isolates at all Avr loci confirmed the disparities between continents, in terms of Avr allele frequencies, particularly for AvrLm2, AvrLm3, AvrLm7, AvrLm8, and AvrLm9, or in terms of race structure, diversity, and complexity. Twenty-six distinct races were identified in the collection. A larger number of races (n = 18) was found in Australia than in Europe (n = 8). Mean number of virulence alleles per isolate was also higher in Australia (5.11 virulence alleles) than in Europe (4.33) and Canada (3.46). Due to the diversity of populations of L. maculans evidenced here at the race level, a new, open terminology is proposed for L. maculans race designation, indicating all Avr loci for which the isolate is avirulent.
The oilseed rape (Brassica napus) stem canker disease, due to the fungal pathogen Leptosphaeria maculans, is mainly controlled by host genetic resistance. Since 2004, the specific resistance gene ...Rlm7 is widely used in France. Specific resistance is effective when fungal populations are mainly composed of avirulent isolates. The development of molecular tools for the identification of virulent isolates towards Rlm7 was needed to undertake large‐scale surveys and to monitor the emergence of virulent populations in fields. Previous studies have described a large diversity of molecular events leading to virulence towards Rlm7, rendering conventional polymerase chain reaction (PCR) methods inapplicable to identify virulent isolates. Interestingly, a very limited nucleotide polymorphism was observed for avirulent, AvrLm7, alleles. Such characteristics were exploited here to develop a diagnostic method based on high resolution melting (HRM) analysis of the AvrLm4‐7 gene. High resolution melting analysis of a collection of 206 reference isolates revealed only four different profiles within 100 avirulent isolates and 87% of virulent isolates showed either no amplification or HRM curves distinct from those of avirulent isolates. The reliability of the method was confirmed using a second set of 119 unknown isolates, for which biological phenotyping and HRM genotyping were in agreement for 93% of the isolates. HRM combined with the PCR amplification of a larger fragment encompassing AvrLm4‐7 led to a correct diagnostic for 97.5% of the isolates.
Stem canker of oilseed rape (canola, Brassica napus) is associated with a species complex of two closely related fungal species, Leptosphaeria maculans and L. biglobosa. Of these, L. maculans is the ...most damaging and develops gene-for-gene relationships with the host. Here, a wide scale analysis of the L. maculans - L. biglobosa species complex was performed throughout the American continent (23 locations from Chile to Canada) plus several locations in Western Australia for comparison purposes, based on a collection of 1132 isolates from infected tissues of a susceptible cultivar. Fungal species were discriminated on the basis of morphological, phytopathological and molecular criteria and showed that L. biglobosa was closely associated with L. maculans in most of the locations. Multiple gene phylogeny using sequences of ITS, actin and β-tubulin confirmed the prevalence of the L. biglobosa'canadensis' sub-clade in Canada, whereas up to three different sub-clades of L. biglobosa were found in Georgia (USA). Race structure of L. maculans was investigated using a combination of pathogenicity tests and PCR amplification of avirulence alleles AvrLm1, AvrLm4 and AvrLm6. Three contrasting situations were observed: (i) race structure in Ontario, Chile and Georgia was related to that of European and Western Australian populations, with a low race diversity; (ii) only one race was found in Mexico, and not found outside of this country; (iii) a large diversity of races was observed in central Canada (Manitoba, Alberta and Saskatchewan) with very specific features including maintenance of avirulence alleles absent from Europe, absence of the AvrLm7 allele common in Europe (or eastern Canada) and wide location-to-location variability.
The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is through the breeding of resistant cultivars. Race ...specific major genes that mediate resistance from the seedling stage have been identified in B. napus or have been introgressed from related species. Many race specific major genes have been described and some of them are probably identical in B. napus (allotetraploid AACC) and the parental species B. rapa (diploid AA). More work is needed using a set of well-characterised isolates to determine the number of different major resistance genes available. In some B. napus cultivars, there is resistance which is polygenic (mediated by Quantitative Trait Loci) and postulated to be race non-specific. Many of these major genes and Quantitative Trait Loci for resistance to L. maculans have been located on B. napus genetic maps. Genes involved in race specific and polygenic resistance are generally distinct.
ABSTRACT Two types of genetic resistance to Leptosphaeria maculans usually are distinguished in Brassica napus: qualitative, total resistance expressed at the seedling stage and quantitative, partial ...resistance expressed at the adult plant stage. The latter is under the control of many genetic factors that have been mapped through quantitative trait loci (QTL) studies using 'Darmor' resistance. The former usually is ascribed to race-specific resistance controlled by single resistance to L. maculans (Rlm) genes. Three B. napus-originating specific Rlm genes (Rlm1, Rlm2, and Rlm4) previously were characterized. Here, we report on the genetic identification of two novel resistance genes, Rlm3 and Rlm7, corresponding to the avirulence genes AvrLm3 and AvrLm7. The identification of a novel L. maculans- B. napus specific interaction allowed the detection of another putative new specific resistance gene, Rlm9. The resistance genes were mapped in two genomic regions on LG10 and LG16 linkage groups. A cluster of five resistance genes (Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9) was strongly suggested on LG10. The relation between all these specific resistance genes and their potential role in adult-plant field resistance is discussed. These two Rlm-carrying regions do not correspond to major QTL for Darmor quantitative resistance.
The race structure and genotypic diversity of four Leptosphaeria maculans populations isolated from Brassica oleracea (broccoli, cauliflower, cabbage, etc.) in central Mexico (Aguascalientes, ...Guanajuato and Zacatecas states) were analysed. Race structure was characterized by an unusually low diversity at three locations out of four. Fourteen minisatellite markers revealed a high proportion of repeated multilocus genotypes in these populations, combined with a significant linkage disequilibrium and strong clonal fraction (65–87%). The occurrence of the mating‐type idiomorphs always significantly departed from the 1:1 proportion expected in the case of random mating. Each population thus consists of a few (four to nine) multilocus genotypes which are specific to each location. These data strongly support the hypothesis of exclusive, or a high rate of, clonal multiplication. Comparison of cropping practices between B. oleracea and B. napus indicate that the shift in reproductive behaviour of the fungus is chiefly man‐mediated.
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