HPLC analyses of cultures of Phoma spp.: differentiation among groups and species through secondary metabolite profiles

The metabolite profiles of 26 isolates of the blackleg fungus (Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.), obtained from diverse parts of the world (part of the International Blackleg Crucifer Network collection), were studied utilizing specific culture conditions, HPLC analysis, and a set of chemical markers. This fungus is the causative agent of blackleg disease of brassica oilseeds; a virulent strain of the pathogen has caused significant rapeseed (Brassica napus L., and B. rapa L.) and canola (B. napus L., and B. rapa L.) losses in Canada, and is also considered a serious agricultural problem worldwide. Effective surveys of blackleg epidemics require simple and reliable analytical methodology to differentiate among the diverse groups of isolates. The chemical analysis of phytotoxins and related secondary metabolites is perhaps one of the most discriminating and the least ambiguous methods for differentiation of Phoma blackleg isolates. Following HPLC analyses, the 26 isolates could be placed in three main groups, irrespective of country of origin: isolates producing phomamide and sirodesmins, isolates producing indolyl dioxopiperazines, and isolates producing polyketides. Discussion of the implications of our findings and suggestions for species reclassification are provided.     

Identification of two novel genes for blackleg resistance in Brassica napus

Blackleg, caused by Leptosphaeria maculans, is a major disease of Brassica napus. Two populations of B. napus DH lines, DHP95 and DHP96, with resistance introgressed from B. rapa subsp. sylvestris, were genetically mapped for resistance to blackleg disease with restriction fragment length polymorphism markers. Examination of the DHP95 population indicated that a locus on linkage group N2, named LepR1, was associated with blackleg resistance. In the DHP96 population, a second locus on linkage group N10, designated LepR2, was associated with resistance. We developed BC₁ and F₂ populations, to study the inheritance of resistance controlled by the genes. Genetic analysis indicated that LepR1 was a dominant nuclear allele, while LepR2 was an incompletely dominant nuclear resistance allele. LepR1 and LepR2 cotyledon resistance was further evaluated by testing 30 isolates from Canada, Australia, Europe, and Mexico. The isolates were from B. napus, B. juncea, and B. oleracea and represented different pathogenicity groups of L. maculans. Results indicated that LepR1 generally conferred a higher level of cotyledon resistance than LepR2. Both genes exhibited race-specific interactions with pathogen isolates; virulence on LepR1 was observed with one isolate, pl87-41, and two isolates, Lifolle 5, and Lifolle 6, were virulent on LepR2. LepR1 prevented hyphal penetration, while LepR2 reduced hyphal growth and inhibited sporulation. Callose deposition was associated with resistance for both loci.     

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B.?napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B.?napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B.?napus. Molecular analyses of the candidate genes using B.?napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.     

Molecular mapping of qualitative and quantitative loci for resistance to Leptosphaeria maculans causing blackleg disease in canola (Brassica napus L.)

Blackleg, caused by Leptosphaeria maculans, is one of the most important diseases of oilseed and vegetable crucifiers worldwide. The present study describes (1) the construction of a genetic linkage map, comprising 255 markers, based upon simple sequence repeats (SSR), sequence-related amplified polymorphism, sequence tagged sites, and EST-SSRs and (2) the localization of qualitative (race-specific) and quantitative (race non-specific) trait loci controlling blackleg resistance in a doubled-haploid population derived from the Australian canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum using the whole-genome average interval mapping approach. Marker regression analyses revealed that at least 14 genomic regions with LOD ≥ 2.0 were associated with qualitative and quantitative blackleg resistance, explaining 4.6-88.9 % of genotypic variation. A major qualitative locus, designated RlmSkipton (Rlm4), was mapped on chromosome A7, within 0.8 cM of the SSR marker Xbrms075. Alignment of the molecular markers underlying this QTL region with the genome sequence data of B. rapa L. suggests that RlmSkipton is located approximately 80 kb from the Xbrms075 locus. Molecular marker-RlmSkipton linkage was further validated in an F(2) population from Skipton/Ag-Spectrum. Our results show that SSR markers linked to consistent genomic regions are suitable for enrichment of favourable alleles for blackleg resistance in canola breeding programs.     

Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants

Brassica napus (canola) cultivars and isolates of the blackleg fungus, Leptosphaeria maculans interact in a 'gene for gene' manner whereby plant resistance (R) genes are complementary to pathogen avirulence (Avr) genes. Avirulence genes encode proteins that belong to a class of pathogen molecules known as effectors, which includes small secreted proteins that play a role in disease. In Australia in 2003 canola cultivars with the Rlm1 resistance gene suffered a breakdown of disease resistance, resulting in severe yield losses. This was associated with a large increase in the frequency of virulence alleles of the complementary avirulence gene, AvrLm1, in fungal populations. Surprisingly, the frequency of virulence alleles of AvrLm6 (complementary to Rlm6) also increased dramatically, even though the cultivars did not contain Rlm6. In the L. maculans genome, AvrLm1 and AvrLm6 are linked along with five other genes in a region interspersed with transposable elements that have been degenerated by Repeat-Induced Point (RIP) mutations. Analyses of 295 Australian isolates showed deletions, RIP mutations and/or non-RIP derived amino acid substitutions in the predicted proteins encoded by these seven genes. The degree of RIP mutations within single copy sequences in this region was proportional to their proximity to the degenerated transposable elements. The RIP alleles were monophyletic and were present only in isolates collected after resistance conferred by Rlm1 broke down, whereas deletion alleles belonged to several polyphyletic lineages and were present before and after the resistance breakdown. Thus, genomic environment and exposure to resistance genes in B. napus has affected the evolution of these linked avirulence genes in L. maculans.     

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 BC₁ 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.     

Recurring challenges from a necrotrophic fungal plant pathogen: a case study with Leptosphaeria maculans (causal agent of blackleg disease in brassicas) in Western Australia

BACKGROUND: Blackleg disease of Brassica napus, caused by the necrotrophic fungus Leptosphaeria maculans, causes severe yield losses in Australia, Europe and Canada. In Western Australia, it nearly destroyed the oilseed rape industry in 1972 when host genotypes and conducive environmental conditions favoured severe epidemics. The introduction of cultivars with polygenic resistance and the adoption of sound cultural practices two decades later helped to manage the disease. These were abandoned by many farmers in recent years in favour of the effective but ephemeral resistance conferred by the single dominant gene-based resistance derived from B. rapa ssp. sylvestris. Recently, several cultivars carrying this gene have collapsed widely within a period of 3 years after their commercial release. An environment conducive to the disease and the association of the pathogen with susceptible hosts in Western Australia for over 80 years together have led to the proliferation of L. maculans races, amounting to half of all races delineated to date from Europe, including the United Kingdom, Canada and Australia. SCOPE: This review demonstrates the problems that emerge when traditional cultural practices employed, along with cultivars containing polygenic resistance to a serious necrotrophic pathogen, are discarded in preference to the exclusive deployment of effective but ephemeral single dominant gene-based resistance to the disease across Southern Australia. CONCLUSIONS: Single dominant gene-based resistance currently available, on its own, will not confer durable resistance to blackleg disease in oilseed rape. Return to earlier management practices, including reliance upon polygenic resistance and induced resistance, may be the best currently available options to maintain production in regions across Southern Australia predisposed to severe epidemics.     

Introgression of Brassica rapa subsp. sylvestris blackleg resistance into B. napus

Blackleg, caused by Leptosphaeria maculans, is one of the most economically important diseases of Brassica napus worldwide. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris (BRS) were previously identified. To transfer LepR1 and LepR2 from BRS into B. napus, interspecific hybridizations were made between the two species to form allotriploids. Analysis of microsatellite markers in two BC1 populations, WT3BC1 and WT4BC1, indicated that segregation fit a 1:1 ratio for BRS and non-BRS alleles on the A-genome linkage groups N2 and N10, the locations of LepR1 and LepR2, respectively. However, recombination frequencies in the allotriploid BC1 populations were at least twice those in the amphidiploid. The number of C-genome chromosomes in the BC1 plants was determined through marker analysis, which indicated averages of 5.9 and 5.0 per plant in the WT3BC1 and WT4BC1 populations, respectively. Two L. maculans isolates, WA51 and pl87-41, were used to differentiate plants carrying resistance genes LepR1 and LepR2. Surprisingly, only 4.0 and 16.6 % of the plants were resistant to isolates WA51 and pl87-41, respectively, in the WT3BC1 population, while 17.9 and 33.3 % of the plants were resistant to these isolates, respectively, in the WT4BC1 population. No association of resistance to isolate WA51 or pl87-41 with linkage group N2 or N10 was found. Based on cotyledon resistance and marker-assisted selection (MAS), BC1 plant WT4-4, which carried a resistance gene similar to LepR1, herein designated LepR1′, and BC2S1 plant WT3-21-25-9, which carried LepR2′, were identified. These plants were successively backcrossed with B. napus and MAS was employed in each generation to reduce non-resistance alleles associated with the BRS genome and to recover the full complement of C-genome chromosomes, resulting in highly blackleg-resistant B. napus lines.     

Enhanced pathogenicity of Leptosphaeria maculans Pycnidiospores from paired co-inoculation of Brassica napus cotyledons with ascospores

BACKGROUND AND AIMS: Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) worldwide, including Australia. In most cases, the severity of the disease in the field is related to infections caused by airborne ascospores. In contrast, pycnidiospores originating from leaf and stem lesions and stubble are widely assumed to play only a relatively minor role in the epidemiology of blackleg. It is not clear whether, under certain conditions, pycnidiospores can cause severe disease in the field. The aim of the work reported was to determine if the pathogenicity of pycnidiospores is enhanced by paired co-inoculation of B. napus cotyledons with ascospores. METHODS: Three investigations were carried out under controlled-environment conditions using various L. maculans isolates and B. napus cultivars with different levels of host resistance to blackleg. KEY RESULTS: In all three experiments, co-inoculation with ascospores increased the ability of pycnidiospores to cause more disease on B. napus than when inoculations consisted of pycnidiospores alone. This effect was significantly influenced by the host resistance of the cultivar, but overall was independent of the L. maculans isolate used in the different experiments. This effect was also independent of timing of inoculation with the ascospores, with increased disease from pycnidiospores occurring on the cotyledon of the seedling in situations where inoculations with ascospores were carried out 0, 1 or 2 d after pycnidiospore inoculation. This enhanced pathogenicity of pycnidiospores was evident even when low concentrations of pycnidiospores were applied to the other cotyledon of the same seedling. CONCLUSIONS: These results may explain continuing severe blackleg disease cycles throughout the cropping season even when ascospore fallout was low or constrained only to a brief period or phase of the cropping season, and suggest that disease epidemics may be polycyclic rather than monocyclic.     

Complexities of chromosome landing in a highly duplicated genome: toward map-based cloning of a gene controlling blackleg resistance in Brassica napus

The LmR1 locus, which controls seedling resistance to the blackleg fungus Leptosphaeria maculans in the Brassica napus cultivar Shiralee, was positioned on linkage group N7. Fine genetic mapping in a population of 2500 backcross lines identified three molecular markers that cosegregated with LmR1. Additional linkage mapping in a second population colocalized a seedling resistance gene, ClmR1, from the cultivar Cresor to the same genetic interval on N7 as LmR1. Both genes were located in a region that showed extensive inter- and intragenomic duplications as well as intrachromosomal tandem duplications. The tandem duplications seem to have occurred in the Brassica lineage before the divergence of B. rapa and B. oleracea but after the separation of Brassica and Arabidopsis from a common ancestor. Microsynteny was found between the region on N7 carrying the resistance gene and the end of Arabidopsis chromosome 1, interrupted by a single inversion close to the resistance locus. The collinear region in Arabidopsis was assayed for the presence of possible candidate genes for blackleg resistance. These data provided novel insights into the genomic structure and evolution of plant resistance loci and an evaluation of the candidate gene approach using comparative mapping with a model organism.     

Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans

Restriction enzyme mediated insertional mutagenesis using a plasmid, pUCATPH, that confers hygromycin resistance, generated loss-of-pathogenicity mutants of Leptosphaeria maculans, the fungus that causes blackleg disease of Brassica napus. Of 516 L. maculans transformants analysed, 12 were pathogenicity mutants. When eight of these mutants were crossed to an isolate that attacks B. napus, cosegregation of pUCATPH sequences and loss of pathogenicity was not observed, suggesting that these mutations were not linked to plasmid sequences. In seven of eight crosses analysed, progeny with the hygromycin resistance gene were hygromycin-sensitive. Sequence analysis of an amplified fragment of pUCATPH in six clones derived from one 'silenced' progeny showed mutation of GC to AT on one DNA strand, reminiscent of repeat-induced point mutation (RIP) in Neurospora crassa. One loss-of-pathogenicity mutant had pUCATPH inserted in the promoter of a gene with an open reading frame of 529 amino acids that had no database match. Reintroduction of a wild-type copy of the gene to this mutant restored the ability to form lesions on cotyledons of B. napus.     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Progressive introgression between Brassica napus (oilseed rape) and B. rapa

We have earlier shown extensive introgression between oilseed rape (Brassica napus) and B. rapa in a weedy population using AFLP markers specific for the nuclear genomes. In order to describe the progress of this introgression, we examined 117 offspring from 12 maternal plants from the introgressed population with the same AFLP-markers; AFLP data were supported by chromosome counting. We also analysed the offspring with a species-specific chloroplast marker and finally evaluated the reproductive system in selected maternal plants. Our results indicated a high outcrossing rate of the introgressed maternal plants. It seemed that B. rapa most often functioned as the maternal plant in the introgression process and that the amount of oilseed rape DNA was highly diminished in the offspring compared to their introgressed maternal plants. However, our analysis of plants from the weedy population indicated that introgression can lead to both (1) exchange of chloroplast DNA between species producing B. rapa-like plants with B. napus chloroplasts and (2) incorporation of B. napus C-genome DNA into the B. rapa genome. Therefore, we question whether it can be regarded as containment to position transgenes in the chloroplast or in specific parts of the nuclear genome of B. napus.     

Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea.

Blackleg disease of crucifers, caused by the fungus Leptosphaeria maculans, is a major concern to oilseed rape producers worldwide. Brassica species containing the B genome have high levels of resistance to blackleg. Brassica juncea F2 and first-backcross (B1) populations segregating for resistance to a PG2 isolate of L. maculans were created. Segregation for resistance to L. maculans in these populations suggested that resistance was controlled by two independent genes, one dominant and one recessive in nature. A map of the B. juncea genome was constructed using segregation in the F2 population of a combination of restriction fragment length polymorphism (RFLP) and microsatel lite markers. The B. juncea map consisted of 325 loci and was aligned with previous maps of the Brassica A and B genomes. The gene controlling dominant resistance to L. maculans was positioned on linkage group J13 based on segregation for resistance in the F2 population. This position was confirmed in the B1 population in which the resistance gene was definitively mapped in the interval flanked by pN199RV and sB31143F. The provisional location of the recessive gene controlling resistance to L. maculans on linkage group J18 was identified using a subset of informative F2 individuals.     

RGA- and RAPD-derived SCAR markers for a Brassica B-genome introgression conferring resistance to blackleg in oilseed rape

An introgression derived from the B genome of Brassica juncea in spring-type oilseed rape (B. napus) conferring recessively inherited cotyledon resistance against several pathotypes of the blackleg fungus Leptosphaeria maculans was mapped using PCR-based molecular markers. Resistance-associated B-genome-specific randomly amplified (RAPD) and resistance gene analog (RGA) DNA polymorphisms were converted into three sequence-specific markers (SCARs; B5-1520, C5-1000, RGALm). The flanking sequence of the RGALm locus was determined by genomic walking, leading to a 1,610-bp EcoRV fragment which showed extensive homology to known and putative resistance genes of a cluster on Arabidopsis chromosome 5. Partial sequence analysis of the genomic RAPD segment OPC-05-1700 revealed strong homology to the gibberellin 2-oxidase gene of Arabidopsis. The SCAR markers were analyzed in two segregating populations and were found to be linked in coupling to each other, and in repulsion to the resistance locus. In both populations, markers deviated significantly from a monogenic 3:1 segregation ratio, with plants lacking the markers being more frequent than expected. Although the mode of introgression is yet unknown, the recombinant individuals observed among susceptible progeny suggest homeology between the B-genome-specific segment and its B. napus counterpart. This would offer prospects for reducing the size of the introgression and further fine mapping of the resistance locus.     

Recognition of avirulence gene AvrLm1 from hemibiotrophic ascomycete Leptosphaeria maculans triggers salicylic acid and ethylene signaling in Brassica napus

Interaction of a plant with a fungal pathogen is an encounter with hundreds of molecules. In contrast to this, a single molecule often decides between the disease and resistance. In the present article, we describe the defense responses triggered by AvrLm1, an avirulence gene from a hemibiotrophic ascomycete, Leptosphaeria maculans, responsible for an incompatible interaction with Brassica napus. Using multiple hormone quantification and expression analysis of defense-related genes, we investigated signaling events in Rlm1 plants infected with two sister isolates of L. maculans differentiated by the presence or absence of AvrLm1. Infection with the isolate carrying AvrLm1 increased the biosynthesis of salicylic acid (SA) and induced expression of the SA-associated genes ICS1, WRKY70, and PR-1, a feature characteristic of responses to biotrophic pathogens and resistance gene-mediated resistance. In addition to SA-signaling elements, we also observed the induction of ASC2a, HEL, and CHI genes associated with ethylene (ET) signaling. Pharmacological experiments confirmed the positive roles of SA and ET in mediating resistance to L. maculans. The unusual cooperation of SA and ET signaling might be a response to the hemibiotrophic nature of L. maculans. Our results also demonstrate the profound difference between the natural host B. napus and the model plant Arabidopsis in their response to L. maculans infection.