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.     

Genetic diversity and population structure of Leptosphaeria maculans isolates in Western Canada

Leptosphaeria maculans is a serious concern for canola production worldwide. For effective disease management, knowledge of the pathogen's genetic variability and population structure is a prerequisite. In this study, whole-genome sequencing was performed for 162 of 1590 L. maculans isolates collected in the years 2007–2008 and 2012–2014 in Western Canada. DNA variants in genome-wide and specific regions including avirulence (Avr) genes were characterized. A total of 31,870 high-quality polymorphic DNA variants were used to study L. maculans genetic diversity and population structure. Cluster analysis showed that 150 isolates were clustered into 2 main groups and 4 subgroups by DNA variants located in either Avr or small secreted protein-encoding genes and into 2 main groups and 6 subgroups by genome-wide variants. The analysis of nucleotide diversity and differentiation also confirmed genetic variation within a population and among populations. Principal component analysis with genome-wide variants showed that the isolates collected in 2012–2014 were more genetically diverse than those collected in 2007–2008. Population structure analysis discovered three distinct sub-populations. Although isolates from Saskatchewan and Alberta were of similar genetic composition, Manitoba isolates were highly diverse. Genome-wide association study detected DNA variants in genes AvrLm4-7, Lema_T86300, and Lema_T86310 associated with the years of collection.     

Ascospore Discharge, Leaf Infestation and Variations in Pathogenicity as Criteria to Predict Impact of Leptosphaeria maculans on Oilseed Rape

The aim of this investigation was to evaluate criteria for the forecast and targeted control of basal stem canker (blackleg) caused by Leptosphaeria maculans on oilseed rape. Ascospore discharge, ratio of aggressive and non-aggressive isolates and leaf and stem infestations were determined during 1991/92–1993/94 at 6–10 sites in Northern Germany. On a 1–9 scale, blackleg intensity varied from 2.3 to 6.3 at BBCH 81 between different sites and years. Ascospore discharge started in September or October, and reached maxima 1 or 2 months later, without an apparent relationship to blackleg or leaf infestation. There was a positive relationship between leaf infestation and blackleg. However, correlation coefficients were too low to be used as a basis for forecasting. On plant residues from the stem base, aggressive isolates were dominant (>80%) on all sites. From higher parts of the stem and from leaves also, non-aggressive isolates were isolated with higher frequencies on some locations, but the proportion of aggressive isolates was not related to the blackleg intensity. Taken all together, the three criteria alone seem to be insufficient for the development of a system of blackleg forecasting and targeted control. Further factors (e.g. climatic factors, seed-and soilborne inoculum, cultural practices) have to be included in models for forecasting the impact of blackleg on oilseed rape.     

Chemical suppression of the sexual stage of Leptosphaeria maculans on oil-seed rape and turnip seed crop straw

A selection of fungicides, herbicides and surfactants and urea were tested for their effect on the production of pseudothecia and ascospore release of Leptosphaeria maculans present on oil-seed rape straw and turnip seed crop straw. The fungicides ethyl mercury phosphate, triarimol, fenarimol, carbendazim, tridemorph and benomyl, each at 1 g/litre, the herbicides dinoseb and diquat, each at 10 g/litre the surfactants Bradasol, Cetrimide, Deciquam 222, Hyamine 1622 and Maxonol N, each at 50 g product/litre, and urea at 150 g/litre, applied to straw before pseudothecia had formed were more than 90% effective in preventing further development. These chemicals were also effective in preventing further ascospore production when applied to straw bearing mature pseudothecia but only dinoseb and urea prevented the release of mature ascospores. The results suggest that it may be possible to break the life cycle of L. maculans by chemical treatment and thereby obviate the need for subsequent control measures.     

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.     

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.     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Transmission of Leptosphaeria maculans from a cropping season to the following one

Current modelling of inoculum transmission from a cropping season to the following one relies on the extrapolation of kernels estimated on data at short distances from punctual sources, because data collected at larger distances are scarce. We estimated the dispersal kernel of Leptosphaeria maculans ascospores from stubble left after harvest in the summer previous to newly sown oilseed rape fields, using phoma stem canker autumn disease severity. We built a dispersal model to analyse the data. Source strengths are described in the spatial domain covered by source fields by a log‐Gaussian spatial process. Infection potentials in the following season are described in the space consisting of the target fields, by a convolution of sources and a power‐exponential dispersal kernel. Data were collected on farmers' fields considered as sources in 2009 and 2011 (72 and 39 observation points) and as targets in 2010 and 2012 (172 and 200 points). We applied the Bayesian approach for model selection and parameter estimation. We obtained fat tail kernels for both data sets. This estimation is the first from data acquired over distances of 0 to 1000 m, using several non‐punctual inoculum sources. It opens the prospect of refining the existing simulators, or developing disease risk maps.     

Inheritance of chromosomal length polymorphisms in the ascomycete Leptosphaeria maculans

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included β-tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.     

Distribution of cabbage root flies in brassica crops

Changes in the distribution of adult cabbage root flies were determined in 0–05-0-4 ha brassica plots during 1970 and 1971 using yellow water-traps. The three generations of flies occurred at similar times in both years and the numbers caught generally decreased from the first to the third generation. In resident populations, the numbers of males and females declined by 70 and 40% respectively, between the second and third generations. When there was no decline, immigration presumably compensated for flies trapped or otherwise lost. The results confirmed that male cabbage root fly movement is trivial, that wind has little effect on distribution at a brassica plot and that females move into the crop independently of males. They failed to confirm that females aggregate markedly at hedgerows or fences, that they spend little time in the crop or that they regularly return to hedgerows. Differences in the dispersal behaviour of the male and female flies were reflected in the numbers caught. Some males dispersed actively during the first 2 weeks after emergence but many were displaced slowly downwind. Once at a plot, wind appeared to have little effect on distribution of young males, but older males tended to shelter just downwind of the hedge. Relatively few males were caught near hedges but they tended to aggregate along the crop-interfaces, particularly during the first generation. Plot size did not appear to influence the pattern of fly distribution greatly. Females dispersed more actively than males and were generally more uniformly distributed through the crops even during periods of immigration. Gravid females entered an isolated plot without accompanying males, inferring that they are migratory and mate near the site of emergence. Female distribution was not related to the stage of ovarial development except during maximum oviposition when those that had laid some eggs visited the hedgerow. Aged females became progressively easier to capture and usually died in the crop.     

Factors affecting development of phoma canker (Leptosphaeria maculans) on stems of winter oilseed rape (Brassica napus) in southern England

In winter oilseed rape experiments at Rothamsted in 1997/98 (cvs Lipton and Capitol), 1998/99 (cv. Apex) and 1999/2000 (cvs Apex, Lipton and Capitol), development of crown canker and phoma stem lesions in spring was related to development of phoma leaf spot in the previous autumn/winter. There were differences in thermal time (degree‐days) from the first appearance of phoma leaf spot (autumn) to the first appearance of crown canker (spring) between cultivars (cvs Lipton and Capitol, 1220–1240; cv. Apex, 1120–1140 degree‐days) but not between growing seasons. In 1998/99 and 1999/2000, fungicide (November) treatment delayed the start of crown canker development in the spring but did not affect the rate of increase in severity. In 1997/98, fungicide treatments did not delay the appearance of crown canker but decreased the rate of increase in crown canker severity. In all three seasons, fungicide treatments generally decreased the proportions of plants at harvest with crown canker severity scores 3 or 4 and increased the proportions with scores 0 or 1. There were differences between seasons in the distributions of crown canker severity scores at harvest. The severity of both crown canker and phoma stem lesions increased linearly with accumulated degree‐days in plots with or without fungicide treatment in 1997/98 (cv. Lipton), 1998/99 (cv. Apex) and 1999/2000 (cv. Apex). Regressions showed that severity of crown canker at harvest in July was related to severity in the spring in 1997/98 (early June, cv. Lipton), 1998/99 and 1999/2000 (April, cv. Apex).     

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.     

Strain typing of polish Leptosphaeria maculans isolates supports at the genomic level the multi-species concept of aggressive and non-aggressive strains

47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE. With two exceptions all Polish isolates do not form sirodesmins. grow rapidly without penetrating into the substrate and form in most cases yellow or brown pigments in Czapek-Dox liquid cultures. With respect to cellulase secretion and molecular fingerprinting Polish A strains (aggressive) fit into the general picture of the aggressive pathotype group, whereas the NA isolates (non-aggressive) display a higher degree of heterogeneity. This matches with inoculation tests on rape seedlings, which revealed a considerable number of isolates ranging in aggressivity between the conventional A and NA pathotype group. Molecular fingerprinting techniques unequivocally sorted intermediately aggressive isolates into the NA pathotype group. Isolate Ph Bial, which produces sirodesmin but groups within NA isolates according to molecular and physiological markers, may represent a novel third group besides A and NA strains with intermediate aggressivity (IA). We hybridized Southern blots of electrophoretically separated chromosomes with radioactively labelled PCR fragments used for differentiation between A and NA isolates. The specificity of diagnostic PCR amplicons is reflected at the genomic level. The A probe reveals a single hybridizing chromosome exclusively in A strains. The NA probe reveals several chromosomes and is specific for the NA pathotype group. Chromosomes from intermediately aggressive strains are equally well recognized by the NA probe as are Polish isolates with low aggressivity and give no signal with the A probe. Both diagnostic DNA sequences are highly specific for the pathotype group they were derived from. The lack of correspondence of both genetic elements between A and NA strains strongly supports the idea of ascribing the pathotype groups to different species. Whereas the A pathotype group is genetically homogeneous and congruent with the species Leptosphaeria maculans, the NA group needs to be revised taxonomically. NA isolates will presumably have to be split into several independent species.     

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 (G_ST < 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.     

Nonspecific Phytotoxic Effects of Sirodesmins on Host and Nonhost Plants of Leptosphaeria maculans

Sirodesmin PL, deacetylsirodesmin PL and sirodesmin C were isolated from cultures of an aggressive strain of Leptosphaeria maculans, purified by thin layer and column chromatography, and tested on leaves and roots of 14 host and non-host plants of the pathogen. The sirodesmins showed no host-specificity. However, various plant species developed symptoms of different severities. In both bioassay systems deacetylsirodesmin PL had a lower phytotoxicity than sirodesmin PL and sirodes-min C. Our results show that sirodesmins belong to the non-host-specific phytotoxins.     

Quantification of Leptosphaeria maculans Growth in Cotyledons of Brassica napus Using ELISA

The result of the Leptosphaeria maculans/Brassica napus interaction is usually assessed by symptom scoring following a cotyledon-inoculation test. However, an early evaluation of the interaction, and reliable quantitaive data of fungal growth inside plant tissues are needed to supplement the visual assessment of the symptoms. For this purpose, we developed a quantitatve double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using rabbit polyclonal antisera directed against soluble mycelial proteins. The specificity of the serum was first assessed by immunoblotting following isoelectric focusing of soluble proteins (Western blot) and by DAS-ELISA. Except for Alternaria brassicae, no cross-reactions were observed with my celial extracts of saprophytes or pathogens of B. napus following DAS-ELISA. Although Tox⁺ and Tox⁰ isolates of L. maculans were unequivocally discriminated by Western blot, they were quantitatively indistinguishable following ELISA, thus enabling us to analyse a wide range of L. maculans isolates in planta. The detection limit of the assay was less than 10 ng of fungal proteins per ml of plant extract. For a given isolate, time-course studies showed that fungal growth in cotyledons was correlated with symptom scoring. In the case of hypersensitive response, only 34% of the plants were ELISA-positive, and these plants never contained more than 10 ng of fungal protein per cotyledon. In contrast, in the cases, of susceptibility, 100% of the plants were ELISA-positive and fungal protein content was higher than 10 μg per cotyledon. Moreover, significant differences in ability to colonize the tissues were observed among Tox⁺ isolates. Finally, using the ELISA quantification, intermediate symptoms could be differentiated as lateresistance responses or susceptibility.