The phytopathogenic fungi Leptosphaeria maculans and Leptosphaeria biglobosa: chemotaxonomical characterization of isolates and metabolite production in different culture media

Previous molecular chemotaxonomic analyses of isolates of the plant pathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. (asexual stage Phoma lingam (Tode ex Fr.) Desm.) in a chemically defined medium suggested that this species complex was composed of at least three distinct groups. Subsequently, a group within L. maculans was classified as Leptosphaeria biglobosa, on the basis of morphologic characteristics and the lack of sexual crossing. To obtain clarification regarding the metabolite profiles of the various groups or species of blackleg fungi, the objectives of this work were (i) to determine the chemical structures of metabolites produced by Canadian V isolates and Polish-type isolates in potato dextrose broth (PDB) and (ii) to determine the chemotaxonomic relationship among French isolates of L. biglobosa and among Canadian W isolates and Thlaspi isolates of L. maculans. Here, we report for the first time that Canadian V isolates grown in PDB produced 2,4-dihydroxy-3,6-dimethylbenzaldehyde, a metabolite never reported from L. maculans, but none of the usual phytotoxins (sirodesmins). In addition, we report a new metabolite, 2-[2-(5-hydroxybenzofuranyl)]-3-(4-hydroxyphenyl)propanenitrile, from Polish-type isolates of L. maculans grown in PDB and the metabolite profiles of 16 Thlaspi isolates. The metabolite profiles of Thlaspi isolates indicate that these are part of two distinct groups, the Polish W group and the Canadian W group, i.e., L. biglobosa. Finally, we demonstrate that the metabolite profiles of the French isolates classified as L. biglobosa are similar to those of Canadian W isolates.     

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.     

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.     

Effect of eyespot caused by Oculimacula yallundae and O. acuformis, assessed visually and by competitive PCR, on stem strength associated with lodging resistance and yield of winter wheat

Winter wheat, (cv. Consort) was inoculated with three isolates of either Oculimacula yallundae or O. acuformis to determine the effect of eyespot caused by each species on yield and lodging resistance of winter wheat. Plants were visually assessed for disease incidence and severity, and pathogen DNA was quantified at GS 33 and GS 60. At early milk development of the crop (GS 72), 900 main shoots were also visually assessed for the disease and subjected to mechanical tests for stem strength. Pathogen DNA was extracted from each shoot and quantified using competitive polymerase chain reaction (PCR). Although slight and moderate eyespot lesions caused by either species had no effect on ear weight, severe lesions caused by O. acuformis and O. yallundae reduced ear weight by 3% and 7%, respectively. Stem lodging failed to occur at the site; however, yield losses of 11% for O. acuformis and 6% for O. yallundae were observed. Visual assessment failed to reveal differences between species in their effect on plant characteristics, stem bending strength, or stem safety factor. PCR data, however, showed that the two species had similar effects determined by different DNA concentrations. Both species reduced lodging resistance (stem safety factor) compared with the control. In contrast to healthy plants, where reductions were related predominantly to the height and weight distribution of the plants, the observed reductions of stem lodging resistance in infected plants with Oculimacula spp. were associated primarily with reductions in stem bending strength.     

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’.     

Polyubiquitin-regulated DsRed marker for transgenic insects.

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP The DsRed fluorescentprotein has spectral characteristics that are most distinct from GFP and GFP variants, and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitin-regulated DsRed1 were detected throughout development at a relatively high frequency, and they exhibited brighter fluorescence than transformants marked with EGFP. The use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressedfrom a reporter construct could be unambiguously detected when co-expressed with EGFP DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.     

Brassinin oxidase, a fungal detoxifying enzyme to overcome a plant defense -- purification, characterization and inhibition

Blackleg fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and Leptosphaeria biglobosa] are devastating plant pathogens with well-established stratagems to invade crucifers, including the production of enzymes that detoxify plant defenses such as phytoalexins. The significant roles of brassinin, both as a potent crucifer phytoalexin and a biosynthetic precursor of several other plant defenses, make it critical to plant fitness. Brassinin oxidase, a detoxifying enzyme produced by L. maculans both in vitro and in planta, catalyzes the detoxification of brassinin by the unusual oxidative transformation of a dithiocarbamate to an aldehyde. Purified brassinin oxidase has an apparent molecular mass of 57 kDa, is approximately 20% glycosylated, and accepts a wide range of cofactors, including quinones and flavins. Purified brassinin oxidase was used to screen a library of brassinin analogues and crucifer phytoalexins for potential inhibitory activity. Unexpectedly, it was determined that the crucifer phytoalexins camalexin and cyclobrassinin are competitive inhibitors of brassinin oxidase. This discovery suggests that camalexin could protect crucifers from attacks by L. maculans because camalexin is not metabolized by this pathogen and is a strong mycelial growth inhibitor.     

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.     

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.     

A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans

We evaluated the usefulness and robustness of Agrobacterium tumefaciens-mediated transformation (ATMT) as a high-throughput transformation tool for pathogenicity gene discovery in the filamentous phytopathogen Leptosphaeria maculans. Thermal asymmetric interlaced polymerase chain reaction allowed us to amplify the left border (LB) flanking sequence in 135 of 400 transformants analysed, and indicated a high level of preservation of the T-DNA LB. In addition, T-DNA preferentially integrated as a single copy in gene-rich regions of the fungal genome, with a probable bias towards intergenic and/or regulatory regions. A total of 53 transformants out of 1388 (3.8%) showed reproducible pathogenicity defects when inoculated on cotyledons of Brassica napus, with diverse altered phenotypes. Co-segregation of the altered phenotype with the T-DNA integration was observed for 6 of 12 transformants crossed. If extrapolated to the whole collection, this indicates that 1.9% of the collection actually corresponds to tagged pathogenicity mutants. The preferential insertion into gene-rich regions along with the high ratio of tagged mutants renders ATMT a tool of choice for large-scale gene discovery in L. maculans.     

Leptosphaeria maculans, the causal agent of blackleg disease of Brassicas.

The loculoascomycete Leptosphaeria maculans (anamorph: Phoma lingam) causes blackleg of Brassicas, including Brassica napus (canola or rapeseed). This fungus probably comprises several morphologically similar species; taxonomic relationships between them are being clarified and nomenclature is being revised. The pathotype ("A" group) responsible for major economic losses to canola has been studied in more detail than other members of this species complex and is the focus of this review. L. maculans is haploid, outcrossing, can be transformed, and has a genome size of about 34 Mb. Preliminary genetic and physical maps have been developed and three genes involved in host specificity have been mapped. As yet, few genes have been characterized. Chemical analysis of fungal secondary metabolites has aided understanding of taxonomic relationships and of the host-fungal interaction by the unraveling of pathways for detoxification of antimicrobial phytoalexins. Several phytotoxins (host and nonhost specific) have been identified and a complex pattern of regulation of their synthesis by fungal and host metabolites has been discovered.     

Establishment of an efficient RNA silencing system in Trichoderma koningii using DsRed as a reporter

We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.     

Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.     

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.     

B-genome derived resistance to Leptosphaeria maculans in near isogenic Brassica napus lines is independent of glucosinolate profile

The role of the glucosinolate-myrosinase system in resistance to Leptosphaeria maculans was studied by monitoring changes in glucosinolate profiles in leaf tissue surrounding the site of inoculation. Susceptible Brassica napus cv. Hanna, resistant B. nigra and near isogenic lines derived from interspecific hybrids between the two species were compared. Expression of myrosinase binding protein and presence of genetic markers were also assayed. No correlation between degree of resistance and amount of sinigrin or other aliphatic glucosinolates was found. However, in time course experiments the glucosinolate profile of the L. maculans inoculated plants differed significantly from the water-inoculated control plants in the amount of 4-methoxy-glucobrassicin observed. Five to eight days post-inoculation an increased level of 4-methoxy glucobrassicin, ranging from 30% to 47% on average, was found in the inoculated plants, whilst controls varied between 7.6% and 9.2%. This increase was seen both in susceptible and resistant material. Other changes observed could mainly be assigned as effects of wounding. Although inoculation with L. maculans elicited changes in the leaf indolyl glucosinolate profiles, in the material studied, these changes could not be correlated to resistance against the fungus.     

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or beta-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.