Detoxification of the phytoalexin brassinin by isolates of Leptosphaeria maculans pathogenic on brown mustard involves an inducible hydrolase

Brassinin is a phytoalexin produced by plants from the family Brassicaceae that displays antifungal activity against a number of pathogens of Brassica species, including Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] and L. biglobosa. The interaction of a group of isolates of L. maculans virulent on brown mustard (Brassica juncea) with brassinin was investigated. The metabolic pathway for degradation of brassinin, the substrate selectivity of the putative detoxifying hydrolase, as well as the antifungal activity of metabolites and analogs of brassinin are reported. Brassinin hydrolase activity was detectable only in cell-free homogenates resulting from cultures induced with brassinin, N'-methylbrassinin, or camalexin. The phytoalexin camalexin was a substantially stronger inhibitor of these isolates than brassinin, causing complete growth inhibition at 0.5mM.     

Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: structures, syntheses and antifungal activity

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-methoxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.     

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.     

Camalexin induces detoxification of the phytoalexin brassinin in the plant pathogen Leptosphaeria maculans

The impact of the phytoalexins camalexin and spirobrassinin on brassinin detoxification by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], a pathogenic fungus prevalent on crucifers, was investigated. Brassinin is a plant metabolite of great significance due to its dual role both as an effective phytoalexin and as an early biosynthetic precursor of the majority of the phytoalexins produced by plants of the family Brassicaceae (Cruciferae). The rate of detoxification of brassinin in cultures of L. maculans increased substantially in the presence of camalexin, whereas spirobrassinin did not appear to have a detectable effect. In addition, the brassinin detoxifying activity of cell-free extracts obtained from cultures incubated with camalexin was substantially higher than that of control cell-free extracts or cultures incubated with spirobrassinin, and correlated positively with brassinin oxidase activity. The discovery of a potent synthetic modulator of brassinin oxidase activity, 3-phenylindole, and comparison with the commercial fungicide thiabendazole is also reported. The overall results indicate that brassinin oxidase production is induced by camalexin and 3-phenylindole but not by spirobrassinin or thiabendazole. Importantly, our work suggests that introduction of the camalexin pathway into plants that produce brassinin might make these plants more susceptible to L. maculans.     

Phylogenetic relationships between members of the crucifer pathogenic Leptosphaeria maculans species complex as shown by mating type (MAT1-2), actin, and beta-tubulin sequences

The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species differing in specificity and pathogenicity on Brassica napus. Twenty-eight isolates were investigated and compared to 20 other species of the Pleosporales order. Sequences of the mating type MAT1-2 (23), fragments of actin (48) and beta-tubulin (45) genes were determined and used for phylogenetic analyses inferred by maximum parsimony, distance, maximum likelihood, and Bayesian approaches. These different approaches using single genes essentially confirmed findings using concatanated sequences. L. maculans formed a monophyletic group separate from Leptosphaeria biglobosa. The L. biglobosa clade encompasses five sub-clades; this finding is consistent with classification made previously on the basis of internal-transcribed sequences of the ribosomal DNA repeat. The propensity for purifying and neutral evolution of the three genes was determined using sliding window analysis, a technique not previously applied to genes of filamentous fungi. For members of the L. maculans species complex, this approach showed that in comparison to actin and beta-tubulin, exonic sequences of MAT1-2 were more diverse and appeared to evolve at a faster rate. However, different regions of MAT1-2 displayed different degrees of sequence conservation. The more conserved upstream region (including the High Mobility Group domain) may be better suited for interspecies differentiation, while the more diverse downstream region is more appropriate for intraspecies comparisons.     

Wasalexins A and B, new phytoalexins from wasabi: isolation, synthesis, and antifungal activity

The chemical structure determination of two phytoalexins from wasabi (Wasabia japonica, syn. Eutrema wasabi), a plant resistant to virulent isolates of the blackleg fungus [Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.], as well as their synthesis and antifungal activity towards isolates of P. lingam and P. wasabiae is reported.     

Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp

Four filamentous ascomycetes, Leptosphaeria maculans, L. biglobosa, Oculimacula yallundae and O. acuformis, were transformed via Agrobacterium tumefaciens-mediated transformation with the genes encoding DsRed and GFP. Using vectors pCAMDsRed and pCAMBgfp, either germinated conidia of Leptosphaeria spp. and O. yallundae or physically fragmented cultures of Oculimacula spp. were transformed. In vitro, the expression of the two reporter proteins in mycelium of both Oculimacula and both Leptosphaeria species was sufficient to distinguish each species in co-inoculated cultures. In planta, transformants of L. maculans or L. biglobosa expressing DsRed or GFP could be observed together in leaves of Brassica napus. Either reporter protein could be used to view the colonization of leaf petioles by both Leptosphaeria spp. and growth in the xylem vessels could be clearly observed. With the generation of these transformants, further studies on interactions between pathogen species involved in disease complexes on various host species and between opposite mating types of the same species are now possible.     

Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phomalingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.     

Characterization and cross‐species amplification of microsatellite loci in the plant pathogenic fungus Leptosphaeria maculans

Seven polymorphic microsatellite markers suitable for population genetic studies and genetic mapping were developed for Leptosphaeria maculans, a fungal pathogen of canola (Brassica napus). Polymorphism was evaluated using 14 isolates from diverse geographical locations. Each locus had either two or three alleles. Cross‐species amplification was observed for almost all loci in L. biglobosa ‘brassicae’ and L. maculans ‘lepidii’.     

New sesquiterpenic phytotoxins establish unprecedented relationship between different groups of blackleg fungal isolates

A comprehensive search for sesquiterpenic metabolites produced by isolates of the blackleg fungus [Leptosphaeria maculans (Desm.)] Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] revealed that an isolate pathogenic on both canola and brown mustard (IBCN 18) and two isolates pathogenic on brown mustard (Laird 2 and Mayfair 2) produced similar sesquiterpenes. The isolation, chemical structure elucidation, and phytotoxicity of these new sesquiterpenes with silphinene and selinene type skeletons is reported. This is the first time that an isolate virulent on canola and brown mustard is found to produce metabolites characteristic of both virulent (sirodesmins) and avirulent (phomalairdenones) L. maculans/P. lingam. In the context of grouping the various isolates of L. maculans/P. lingam, this work suggests an additional pathogenicity group comprising isolates that produce both sirodesmins and phomalairdenones and are virulent on both canola and brown mustard.     

Isolation, structure determination, and phytotoxicity of unusual dioxopiperazines from the phytopathogenic fungus Phoma lingam.

The isolation, chemical structure elucidation, and bioactivity of polanrazines B-F, five new dioxopiperazines produced by isolates of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.] originating from Poland, are reported. Polanrazines C and E showed moderate but selective toxicity, causing necrotic and chlorotic lesions (1-3 mm diameter) on brown mustard leaves.     

Phomapyrones from blackleg causing phytopathogenic fungi: isolation, structure determination, biosyntheses and biological activity

The isolation and structure determination of phomapyrones D-G, three 2-pyrones and a coumarin, from a group of isolates of the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm, is reported. As well, phomenin B, infectopyrone, and polanrazines B and C were also obtained for the first time from these isolates. In addition, based on results of incorporations of 13C-labeled acetate and malonate, and deuterated methionine, a polyketide pathway is proposed for the biosyntheses of phomapyrones.     

Phomalairdenone: a new host-selective phytotoxin from a virulent type of the blackleg fungus Phoma lingam

The chemical structure and bioactivity of phomalairdenone (7), a new sesquiterpenic host-selective phytotoxin produced by an unusual virulent type isolate of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.] are reported.     

Characterization of an opsin gene from the ascomycete Leptosphaeria maculans.

An opsin gene (ops) has been characterized from Leptosphaeria maculans, the ascomycete that causes black-leg disease of Brassica species. This is the second opsin identified outside the archaeal and animal kingdoms. The gene encodes a predicted protein with high similarity (70.3%) and identity (53.3%) to the nop-1 opsin of another ascomycete Neurospora crassa. The L. maculans opsin also has identical amino acid residues in 20 of the 22 residues in the retinal-binding pocket of archaeal opsins. Opsin, on the fourth largest chromosome of L. maculans and 22 cM from the mating type locus, is the first cloned gene to be mapped in L. maculans. Opsin is transcribed at high levels in mycelia grown in the presence and absence of light; this pattern is in contrast with that of the N. crassa opsin, which is transcribed only in the light.     

Genetic variability and distribution of mating type alleles in field populations of Leptosphaeria maculans from France

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m2 field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity (H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m2 field plots. Population differentiation among the four field populations was low (GST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.     

Pathways of infection of Brassica napus roots by Leptosphaeria maculans

Infection of Brassica napus cotyledons and leaves by germinating ascospores of Leptosphaeria maculans leads to production of leaf lesions followed by stem cankers (blackleg). Leptosphaeria maculans also causes root rot but the pathway of infection has not been described. An L. maculans isolate expressing green fluorescent protein (GFP) was applied to the petiole of B. napus plants. Hyphal growth was followed by fluorescence microscopy and by culturing of sections of plant tissue on growth media. Leptosphaeria maculans grew within stem and hypocotyl tissue during the vegetative stages of plant growth, and proliferated into the roots within xylem vessels at the onset of flowering. Hyphae grew in all tissues in the stem and hypocotyl, but were restricted mainly to xylem tissue in the root. Leptosphaeria maculans also infected intact roots when inoculum was applied directly to them and hyphae entered at sites of lateral root emergence. Hyphal entry may occur at other sites but the mechanism is uncertain as penetration structures were not observed. Infection of B. napus roots by L. maculans can occur via above- and below-ground sources of inoculum, but the relative importance of the infection pathways under field conditions is unknown.     

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.     

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.     

Sterol composition of mycelia of the plant pathogenic ascomycete Leptosphaeria maculans

Analysis of sterols in mycelia of the ascomycete, Leptosphaeria maculans by gas chromatography-mass spectrometry revealed that ergosterol comprised 95% of the total sterols, with eight other sterols comprising the remaining 5%. Six of these latter sterols were putative precursors of ergosterol and their presence suggested a pathway for ergosterol biosynthesis in this fungus. Ergosterol biosynthesis in fungi is inhibited by the triazole antifungal agent flutriafol. When L. maculans was grown in the presence of flutriafol, ergosterol content decreased while two 14 alpha-methylated sterols, 24-methylene dihydrolanosterol and obtusifoliol, accumulated.