Adam Dawidziuk

Zearalenone lactonohydrolase activity in Hypocreales and its evolutionary relationships within the epoxide hydrolase subset of a/b-hydrolases

AbstractBackgroundZearalenone is a mycotoxin produced by several species of Fusarium genus, most notably Fusarium graminearum and Fusarium culmorum. This resorcylic acid lactone is one of the most important toxins causing serious animal and human diseases. For over two decades it has been known that the mycoparasitic fungus Clonostachys rosea (synonym: Gliocladium roseum, teleomorph: Bionectria ochroleuca) can detoxify zearalenone, however no such attributes have been described within the Trichoderma genus.ResultsWe screened for the presence of zearalenone lactonohydrolase homologs in isolates of Clonostachys and Trichoderma genera. We report first finding of expressed zearalenone lactonohydrolase in Trichoderma aggressivum. For three isolates (T. aggressivum, C. rosea and Clonostachys catenulatum isolates), we were able to reconstruct full coding sequence and verify the biotransformation ability potential. Additionally, we assessed progression of the detoxification process (in terms of transcript accumulation and mycotoxin decomposition in vitro). In silico, search for origins of zearalenone lactonohydrolase activity in model fungal and bacterial genomes has shown that zearalenone lactonohydrolase homologs form a monophyletic fungal clade among the a/b hydrolase superfamily representatives. We corroborated the finding of functional enzyme homologs by investigating the functional sites (active site pocket with postulated, noncanonical Ser-Glu-His catalytic triad) conserved in both multiple sequence alignment and in homology-based structural models.ConclusionsOur research shows the first finding of a functional zearalenone lactonohydrolase in mycoparasitic Trichoderma aggressivum (an activity earlier characterised in the Clonostachys rosea strains). The supporting evidence for presence and activity of functional enzyme homologs is based on the chemical analyses, gene expression patterns, homology models showing conservation of key structural features and marked reduction of zearalenone content in cultured samples (containing both medium and mycelium). Our findings also show divergent strategies of zearalenone biotransformation ability (rapid induced expression and detoxification vs. gradual detoxification) present in several members of Hypocreales order (Trichoderma and Clonostachys genera). The potential for lactonhydrolase activity directed towards zearalenone and/or similar compounds is likely ancient, with homologs present in several divergent filamentous fungi among both Sordariomycetes (Bionectria sp., Trichoderma sp., Apiospora montagnei) and Leotiomycetes (Marssonina brunnea f. sp. ‘multigermtubi’).

Influence of meteorological parameters on Leptosphaeria maculans and L. biglobosa spore release in central and eastern Poland

Abstract Leptosphaeria maculans and L. biglobosa are fungal pathogens able to cause allergic reactions in humans and infect plants of Brassica species. The rate of their development and subsequent spore release depend on weather conditions. The aim of this paper was to pinpoint the exact meteorological conditions triggering the release of L. maculans and L. biglobosa ascospores in central and eastern Poland. Multiple regressions indicated that the frequency and amount of rainfall over short periods were important in mediating spore release. The first ascospore event depended mainly on the number of rainy days during the first 10 days of July and the cumulative precipitation during July and September. The most important variables for maximum spore release were cumulative rainfall in the beginning of July and the end of September, as well as the number of days with precipitation events in the first 10 days of August. The results highlighted for the first time the importance of the days preceding the collection of oilseed rape plants from the field. Higher moisture content of senescing but still living stems play a crucial role in the early start of the ascospore season and the maximum release of ascospores. This was not yet considered to date.

Molecular diagnostics on the toxigenic potential of Fusarium spp. plant pathogens

We propose and test an efficient and rapid protocol for the detection of toxigenic Fusarium isolates producing three main types of Fusarium‐associated mycotoxins (fumonisins, trichothecenes and zearelanone).

The distant siblings – a phylogenomic roadmap illuminates the origins of extant diversity in fungal aromatic polyketide biosynthesis

Abstract In recent years, the influx of newly sequenced fungal genomes has enabled sampling of secondary metabolite biosynthesis on an unprecedented scale. However, explanations of extant diversity which take into account both large-scale phylogeny reconstructions and knowledge gained from multiple genome projects are still lacking. We analyzed the evolutionary sources of genetic diversity in aromatic polyketide biosynthesis in over 100 model fungal genomes. By reconciling the history of over 400 nonreducing polyketide synthases (NR-PKSs) with corresponding species history, we demonstrate that extant fungal NR-PKSs are clades of distant siblings, originating from a burst of duplications in early Pezizomycotina and thinned by extensive losses. The capability of higher fungi to biosynthesize the simplest precursor molecule (orsellinic acid) is highlighted as an ancestral trait underlying biosynthesis of aromatic compounds. This base activity was modified during early evolution of filamentous fungi, toward divergent reaction schemes associated with biosynthesis of, for example, aflatoxins and fusarubins (C4–C9 cyclization) or various anthraquinone derivatives (C6–C11 cyclization). The functional plasticity is further shown to have been supplemented by modularization of domain architecture into discrete pieces (conserved splice junctions within product template domain), as well as tight linkage of key accessory enzyme families and divergence in employed transcriptional factors. Although the majority of discord between species and gene history is explained by ancient duplications, this landscape has been altered by more recent duplications, as well as multiple horizontal gene transfers. The 25 detected transfers include previously undescribed events leading to emergence of, for example, fusarubin biosynthesis in Fusarium genus. Both the underlying data and the results of present analysis (including alternative scenarios revealed by sampling multiple reconciliation optima) are maintained as a freely available web-based resource: http://cropnet.pl/metasites/sekmet/nrpks_2014.

The effect of winter weather conditions on the ability of pseudothecia of Leptosphaeria maculans and L. biglobosa to release ascospores

Leptosphaeria maculans and L. biglobosa are damaging pathogens of oilseed rape. The infection of plants occurs predominantly in early autumn or spring by spores produced in pseudothecia. The aim of this study was to investigate whether pseudothecia formed in the autumn are still viable in the spring and to what extend they are destroyed by winter frosts. The studies presented here demonstrated that winter frosts can render pseudothecia unable to release spores. Nevertheless, ascospores present in pseudothecia unable to discharge ascospores, were fully capable of germination, regardless of the incubation temperature. No significant differences were found between the studied Leptosphaeria species in their response to frost. A multiple regression equation has been elaborated to forecast the ability of pseudothecia to release ascospores, based on winter temperatures. Considerable correlation was found between the ascospore release in the autumn and the ability of pseudothecia to release ascospores over the winter period and the subsequent symptoms of stem canker before harvest. We have demonstrated that the potential and the survival of inoculum can have a large impact on the success of the pathogen. This may be particularly important in the light of forecasted climate change. Higher winter temperatures may increase the ability of pseudothecia to release ascospores and the discharge of ascospores of L. maculans and L. biglobosa into the air, and cause early plant infections. This in turn will increase the number of infected plants, the disease incidence at harvest, and reduce the yield of oilseed rape.

Optimal Trichoderma strains for control of stem canker of brassicas: molecular basis of biocontrol properties and azole resistance

Genus Trichoderma contains the most powerful agents used in biocontrol. Our study demonstrated that T. harzianum, T. hamatum and T. longibrachiatum can effectively control phytopathogenic fungi Leptosphaeria maculans and L. biglobosa. The effects of Trichoderma on pathogens’ growth (in dual cultures on agar media) and on disease severity (on seedlings in controlled conditions) were also confirmed by field experiments. Additionally, spraying with conidiospores in the autumn was helpful in reducing the incidence and severity of phoma leaf spots. It has also accelerated the degradation of plant stubble and the decomposition of pathogens’ fruiting bodies. Furthermore, all Trichoderma isolates showed higher cellulolytic activity and enhanced resistance to flusilazole treatments as compared to Leptosphaeria spp., which coincided with upregulation of 14α-sterol demethylases and an AbcG5 transporter. The effects we observed justify the use of Trichoderma to enhance the resistance of oilseed rape against pathogens, which in turn may lead to a decrease in the use of pesticides.

Adaptation and response to mycotoxin presence in pathogen-pathogen interactions within the Fusarium genus

The ability of fungal plant pathogens to exude bioactive compounds is an important element of competition in a changing environment. The filamentous fungi usually retain a number of adaptations related not only to the production of toxic compounds by themselves but also to the mitigation of exogenous influences by toxins present in the environment. We examined a distinct effect of toxins on morphology, growth patterns and gene expression after stimulation in mycotoxin-producing and nonproducing isolates representing four evolutionarily divergent species (and chemotypes) within the Fusarium genus (Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum and Fusarium verticillioides). The aim of our work was to investigate the influence of mycotoxins present in the environment on fungal isolates belonging to evolutionarily divergent complexes within Fusarium genus. The results point to retention of resistance mechanisms in non-producer isolates (F. oxysporum) and specific dose-dependent differences i...