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.     

Differential Reactions Between the Genus Brassica and Aggressive Single Spore Isolates of Leptosphaeria maculans

Based on sirodesmin production and pathogenicity tests with Brassica cotyledons, strains of Leptosphaeria maculans were classified as aggressive (pathotype group A), or non-aggressive (pathotype group NA). NA strains caused no differential reactions. However, the pathotype group A could be divided into 5 sub-groups. AO isolates caused non-sporulating lesions with dark margins while Al isolates sporulated on cotyledons of most Brassica hosts tested. Only the cv. Erfurter Zwerg (B. oleracea var. botrytis) reacted resistant against AO and Al strains. A2 isolates caused resistance reactions on cotyledons of the cvs. Quinta (B. napus var. oleifera) and Runde (B. rapa var. rapa). A3 and A4 isolates were not detectable in our material. Isolates of these pathotype groups, supplied by Dr. P. H. Williams, Madison, USA, caused differential reactions on the oilseed rape cvs. Glacier, Quinta and Jet Neuf. In glasshouse and field experiments strains of pathotype groups Al, A2 and NA were tested on true leaves and hypocotyls of different oilseed rape cultivars. The low aggressiveness of NA isolates was evident under all experimental conditions. A2 strains caused resistance reactions not only on cotyledons but also on true leaves and hypocotyls of Quinta. Moreover, compared with Al, pathotype group A2 was more aggressive on hypocotyls of Jet Neuf. The resistance of this cultivar against Al isolates was clearly visible on hypocotyls and true leaves but not on cotyledons.     

Stress-driven discovery of metabolites from the phytopathogenic fungus Leptosphaeria maculans: structure and activity of leptomaculins A-E

A search for stress inducing metabolites produced by the plant pathogenic fungus Leptosphaeria maculans led to the isolation and structure elucidation of eight new metabolites, the leptomaculins and deacetylleptomaculins A-E. The chemical structures and absolute stereochemistry of the new metabolites were deduced by detailed analysis of 1D and 2D NMR spectroscopic data and chemical degradation of the toxin sirodesmin PL. Leptomaculins A and B are the first examples of naturally occurring 2,3-oxopiperazinethione and 2,3-dioxopiperazine, respectively. Stress inducing activity was found in the fungal phytotoxins sirodesmin PL and deacetylsirodesmin PL but not in any of the new leptomaculins, phomalide or phomamide. A metabolic pathway for biosynthesis of the first 2,3-(di)oxopiperazine(thione) from sirodesmin PL is proposed.     

Pathogenicity of Sirodesmin-deficient Mutants of Phoma lingam

Sirodesmin-deficient mutants were produced from aggressive isolates of Phoma lingam by UV-mutagenesis. The mutation rates were 7.1 × 10^-3 for mutants expressing a 100-fold reduction of sirodesmin PL in liquid cultures and 1.8 × 10^-3 for mutants with a 1000-fold reduction. The low ability of the mutants to produce sirodesmins was also apparent in planta after infection of cotyledons or the stem basis of Brassica napus. Growth rates under in vitro conditions, colony morphology, mating types and the formation of pycnidia and asci were not affected by the mutation. The mutants had the same ability as the wild types to infect cotyledons of the susceptible B. napus cv. Lirabon and to cause a typical grey-green tissue collapse. On the stem basis of the cv. Cobra, however, mutants caused significantly smaller lesions. These results indicate that phytotoxic sirodesmins produced by P. lingam have no basic role in pathogenesis and apparently are not involved in the virulence on cotyledons. However, it cannot be excluded that the compounds act as virulence factors on the stem base of B. napus.     

Morphological and molecular identification of Leptosphaeria maculans in canola seeds and flowers collected from the North Iran

Blackleg disease, caused by Leptosphaeria maculans, is one of the most important diseases of rapeseed Brassica napus in Iran as in other regions of the world. The samples including canola petals and seeds were collected during 2014–2015 from canola field in North Iran. Isolates characteristics of fungus were assessed based on the colony growth rate and pycnidia in Potato Dextrose Agar. The pycnidia of the fungus were black, globose to subglobose in shape, the single-celled conidia, hyaline and fusiform with diameters of 4–5 × 1.5–2 μm. Most of the isolates were produced pigment in the liquid culture in variable color brown to black. Thirteen isolates were then separated into pathogenicity groups based on the interactions on B. napus differential cultivars. For the direct detection of seed contamination with L. maculans, PCR was developed using specific primers pair (LmacF, LmacR) which can amplify ITS1 and ITS2 along with the 5.8S rRNA region of L. maculans genome. Based on the followed information and sequence analysis, the fungal isolates from these samples were identified as L. maculans. The findings of this research showed that the disease was aggressive and highly distributed from infested seeds to oilseed rape fields.     

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.     

Antibacterial Activity of Sirodesmin PL Phytotoxin: Application to the Selection of Phytotoxin-Deficient Mutants

Sirodesmin PL, a phytotoxin and mycotoxin produced by Leptosphaeria maculans, the causal agent of stem-canker disease of crucifers, exhibited antibacterial activity against gram-positive bacteria and particularly Bacillus subtilis. The importance of the disulfide bridge of the molecule in antibacterial activity was demonstrated. A simple and reliable bioassay based on the antibacterial activity of the toxin was performed for screening sirodesmin PL-deficient mutants when grown on solid culture medium. A mutant was selected and found to produce 3,700-fold less toxin than did the wild-type strain. A sensitive procedure for quantification of the toxin by high-pressure liquid chromatography was developed. Levels of product as low as 100 ng could be detected by this procedure.     

Biochemical and Molecular Tools for the Differentiation of Aggressive and Non-Aggressive Isolates of the Oilseed Rape Pathogen, Phoma lingam

A number of aggressive and non-aggressive isolates of the rape seed (Brassica napus) pathogen Phoma lingam (teleomorph: Leptosphaeria maculans) were analyzed by three different biochemical approaches: the quantitative measurement of extracellular enzymes, which are believed to be involved in plant attack, the electrophoretic analysis of fungal surface proteins, and the analysis of restriction fragment length polymorphisms (RFLPs) in Southern blot experiments. Aggressive strains show elevated activity levels for cellulase, α- and β-glucanase as well as for polygalacturonase. Furthermore, aggressive and non-aggressive isolates can clearly be characterized by their pattern of surface associated proteins and by RFLP analysis in Southern type experiments, using molecular probes for actin, ubiquitin, ribosomal RNA and the gene for elongation factor EFl (TEF). Seven out of nine aggressive isolates but only one out of eleven non-aggressive strains contained plasmid-like DNA.     

Pathogenicity studies on isolates of Leptosphaeria maculans from brassica seed production crops in south-east England

Out of 117 isolates of Leptosphaeria maculans collected from a range of brassica seed crops in south-east England, 26 were virulent when tested on cabbage cv. January King. Of these, 24 were isolated from oil-seed rape (Brassica napus) and the remainder from a swede (B. napus) and a cabbage (B. oleracea) seed crop. Virulent strains were derived from infected stems and from single ascospores of the sexual state present on diseased stubble. In glasshouse tests virulent isolates were not host-specific and caused severe cankers on cabbage, kale, swede, turnip and oil-seed rape; under field conditions, ascospores originating from diseased oil-seed rape stubble produced severe cankers and high levels of seed-borne infection in eight horticultural and vegetable brassica hosts (B. napus, B. campestris and B. oleracea). Virulent and non-virulent types could be distinguished by certain cultural characteristics. On nutrient agar virulent types grew slowly, irregularly and quickly staled whereas non-virulent types grew rapidly, regularly and did not stale. The latter also produced a yellow-brown pigment in liquid culture. The potential for cross-infection of virulent strains originating from oil-seed rape to other brassica seed crops has serious implications for disease incidence on forage and vegetable brassica seed crops.     

Survival of A-group and B-group Leptosphaeria maculans (phoma stem canker) ascospores in air and mycelium on oilseed rape stem debris

Mycelium of Leptosphaeria maculans survived on oilseed rape stem base debris buried in sand for 2,4, 6, 8,10 or 12 months and produced pseudothecia after subsequent exposure on the surface of the ground under natural conditions for 2–4 months, but did not survive on upper stem debris buried for 2 months. Only A‐group L. maculans ascospores were produced on the stem base debris which had been buried; no B‐group ascospores were produced. Mycelium of L. maculans survived on both stem base and upper stem debris exposed on the sand surface for 2, 4, 6, 8, 10 or 12 months and pseudothecia with viable ascospores were observed at the time of sampling. Both A‐group L. maculans (predominant on stem bases) and B‐group L. maculans (predominant on upper stems) ascospores were produced on unburied stem base and upper stem debris. Thus B‐group L. maculans survived longer on unburied debris than on buried debris. A‐group ascospores which were exposed in dry air in darkness at 5–20°C survived longer than B‐group ascospores; 10–37% of A‐group ascospores, compared with 2–31% of B‐group ascospores, survived after 35 days.     

The Use of Ergosterol as a Quantitative Measure of the Resistance of Cultured Tissues of Brassica napus ssp. oleifera to Leptosphaeria maculans

A simplified method for the quantitative assessment of the fungal lipid ergosterol was used to assess the levels of infection in tissue cultures of oilseed rape (Brassica napus ssp. oleifera) inoculated with Leptosphaeria maculans. The growth of L. maculans in liquid culture throughout a 36-day period correlated well (r = 0·92) with the amount of ergosterol extracted from the mycelium. There were significant differences (P < 0·05) in the amount of ergosterol extracted from infected thin cell layer (TCL) explants and callus tissue of two resistant and three susceptible cultivars of oilseed rape. Amounts of ergosterol extracted from resistant cultivars were < 100 (g and from susceptible > 100 (g. The mean amounts of ergosterol extracted from shoot cultures of two resistant and four susceptible cultivars were similar to those for TCL explants and callus tissue, although the values obtained were variable. This technique can be used in in vitro breeding programmes to accurately assess the resistance of tissue cultures of B. napus to L. maculans and could also have value in conventional breeding programmes.     

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.     

Presence of Leptosphaeria maculans Group A and Group B Isolates in Sweden

Leptosphaeria maculans isolates have been assigned to one of two groups, A or B, on the basis of differences in their characteristics. Group A can further be divided into pathogenicity groups (PG) 2, 3 and 4 and group B into PG1. To determine if isolates belonging to the aggressive canker forming group A are present in Sweden, physiological and genetic characterisation of 120 isolates collected in the year 2000 were performed. Thirty‐seven isolates were classified as belonging to pathogenicity group PG3 and 63 isolates as PG4, based on a cotyledon assay. Twenty isolates did not cause any symptoms at all, and were classified as PG1. When comparing two geographical regions, Skåne and Östergötland, equal numbers of PG3 and PG4 isolates were found. By analysing the isolates by PCR, the collection was further classified into 100 group A isolates and 20 group B isolates. A corresponding classification of the isolates was observed when the ability to produce pigments in Czapek Dox broth was examined. The results showed a clear predominance of group A. This was also the case for the isolate collection from 2001. In a detailed survey of disease development in a L. maculans infected winter oilseed rape field in southern Sweden (Skåne), basal stem canker was not observed until early June Although the disease index value increased from 8.4 in June to 18.0 in July, few severely damaged plants were observed before harvest in mid‐July, despite infection with group A isolates.     

Random Primer Dependent PCR Differentiates Aggressive from Non-Aggressive Isolates of the Oilseed Rape Pathogen Phoma lingam (Leptosphaeria maculans)

A number of isolates of the aggressive an d non-aggressive pathotype group of the rape seed (Brassica napus) pathogen Phoma lingam (teleomorph: Leptosphaeria maculans) were analyzed by means of a simple, random primer dependent PCR (polymerase chain reaction) approach. Using the four synthetic nona- and decamer oligonucleotides 5′-GGAGCCCAC-3′, 5″-ACGGTCTTGG-3′, 5′GAAACAGCGG-3′, and 5′-GGCATCGGCC-3′ informative bands for both of the pathotype groups could be obtained. These amplified bands were shown to originate not only from repeated but also from single and low copy target sequences. This is the first report on molecular diagnostics of a plant pathogenic fungus, based on random primer dependent PCR. The experimental system is fast and reliable, does not require cloning and sequencing of L. maculans DNA, and works without time-consuming blotting or hybridization steps.     

Molecular and phenotypic characterization of near isogenic lines at QTL for quantitative resistance to Leptosphaeria maculans in oilseed rape (Brassica napus L.)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is by breeding resistant cultivars. Specific resistance genes have been identified in B. napus and related species but in some B. napus cultivars resistance is polygenic [mediated by quantitative trait loci (QTL)], postulated to be race non-specific and durable. The genetic basis of quantitative resistance in the French winter oilseed rape ‘Darmor’, which was derived from ‘Jet Neuf’, was previously examined in two genetic backgrounds. Stable QTL involved in blackleg resistance across year and genetic backgrounds were identified. In this study, near isogenic lines (NILs) were produced in the susceptible background ‘Yudal’ for four of these QTL using marker-assisted selection. Various strategies were used to develop new molecular markers, which were mapped in these QTL regions. These were used to characterize the length and homozygosity of the ‘Darmor-bzh’ introgressed segment in the NILs. Individuals from each NIL were evaluated in blackleg disease field trials and assessed for their level of stem canker in comparison to the recurrent line ‘Yudal’. The effect of QTL LmA2 was clearly validated and to a lesser extent, QTL LmA9 also showed an effect on the disease level. This work provides valuable material that can be used to study the mode of action of genetic factors involved in L. maculans quantitative resistance.     

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans–Brassica napus pathosystem

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.     

Protection of Oilseed Rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) Toward Fungal Pathogens by Strains of Plant-associated <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

In this report, four Bacillus strains were tested for effects on plant fitness and disease protection of oilseed rape (Brassica napus). The strains belonged to newly discovered plant-associated Bacillus amyloliquefaciens and a recently proposed species, Bacillus endophyticus. The fungal pathogens tested represented different infection strategies and included Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. The B. amyloliquefaciens strains showed no or a weak plant growth promoting activity, whereas the B. endophyticus strain had negative effects on the plant as revealed by phenological analysis. On the other hand, two of the B. amyloliquefaciens strains conferred protection of oilseed rape toward all pathogens tested. In vitro experiments studying the effects of Bacillus exudates on fungal growth showed clear growth inhibition in several but not all cases. The protective effects of Bacillus can therefore, at least in part, be explained by production of antibiotic substances, but other mechanisms must also be involved probably as a result of intricate plant–bacteria interaction. The protective effects observed for certain Bacillus strains make them highly interesting for further studies as biocontrol agents in Brassica cultivation.     

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 .