In the present study, we reinvestigate the diversity of Trichoderma in Poland utilizing a combination of morphological and molecular/phylogenetic methods. A total of 170 isolates were collected from six different substrata at 49 sites in Poland. These were divided among 14 taxa as follows: 110 of 170 Trichoderma isolates were identified to the species level by the analysis of their ITS1, ITS2 rDNA sequences as: T. harzianum (43 isolates), T. aggressivum (35), T. citrinoviride (11), T. hamatum (9), T. virens (6), T. longibrachiatum (4), T. polysporum (1), and T. tomentosum (1); 60 isolates belonging to the Viride clade were identified based on a fragment of the translation-elongation factor 1-alpha (tef1) gene as: T. atroviride (20 isolates), T. gamsii (2), T. koningii (17), T. viridescens (13), T. viride (7), and T. koningiopsis (1). Identifications were made using the BLAST interface in TrichOKEY and TrichoBLAST (http://www.isth.info). The most diverse substrata were soil (nine species per 22 isolates) and decaying wood (nine species per 75 isolates). The most abundant species (25%) isolated from all substrata was T. harzianum.

Three Fusarium species: F. graminearum, F. culmorum and F. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identify Fusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates of F. graminearum, the 15Ac-DON chemotype dominated, and among those where F. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates of F. graminearum tested, displayed the NIV chemotype. An increase in the frequency of F. graminearum and a decrease in the frequency of F. culmorum were found during 1998 to 2006.

BackgroundZearalenone 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’).

Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted antagonistic compounds by yeast cells is one of the prominent examples. Although this killer behavior has been thoroughly studied in laboratory yeast strains, our knowledge of the antagonistic specificity of killer effects in nature remains limited. In this study, yeast strains were collected from various niches and screened for antagonistic activity against one toxin-sensitive strain of Saccharomyces cerevisiae and three pathogenic fungi. We demonstrate that some strains with antagonistic activity against these pathogenic fungi can be found in antagonist culture tests. These yeasts were identified as members of Trichosporon asahii, Candida stellimalicola, Wickerhamomyces anomalus, Ustilago esculenta, Aureobasidium pullulans, and Pichia kluyveri. The results indicated that the antagonistic activity of these killer yeasts has a narrow optimal pH range. Furthermore, we found that the antagonistic activity of some species is strain-dependent.

AimsWe 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).Methods and ResultsThe novel approach utilizes partially multiplexed markers based on genes essential for mycotoxin biosynthesis (fumonisin—fum6, fum8; trichothecenes—tri5, tri6; zearalenone, zea2) in Fusarium spp. The protocol has been verified by screening a collection of 96 isolates representing diverse species of filamentous fungi. Each Fusarium isolate was taxonomically identified through both molecular and morphological techniques. The results demonstrate a reliable detection of toxigenic potential for trichothecenes (sensitivity 100%, specificity 95%), zearalenone (sensitivity 100%, specificity 100%) and fumonisins (sensitivity 94%, specificity 88%). Both presence and identity of toxin biosynthetic genes were further confirmed by direct sequencing of amplification products.ConclusionsThe cross-species-specific PCR markers for key biosynthetic genes provide a sensitive detection of toxigenic fungal isolates, contaminating biological material derived from agricultural fields.Significance and Impact of the StudyThe conducted study shows that a PCR-based assay of biosynthetic genes is a reliable, cost-effective, early warning system against Fusarium contamination. Its future use as a high-throughput detection strategy complementing chemical assays enables effective targeted application of crop protection products.

wojska polskiego 75, pL-60-625 poznań popiel D., Kwaśna H., Chełkowski J., stępień ł., Laskowska M.: Impact of selected antagonistic fungi on Fusarium species -toxigenic cereal pathogens. Acta Mycol. 43 (1): 29-40, 2008.Fusarium-ear blight is a destructive disease in various cereal-growing regions and leads to significant yield and quality losses for farmers and to contamination of cereal grains with mycotoxins, mainly deoxynivalenol and derivatives, zearalenone and moniliformin. Fusarium pathogens grow well and produce significant inoculum on crop resiudues. Reduction of mycotoxins production and pathogen sporulation may be influenced by saprophytic fungi, exhibiting antagonistic effect.Dual culture bioassays were used to examine the impact of 92 isolates (belonging to 29 fungal species) against three toxigenic species, i.e. Fusarium avenaceum (Corda) saccardo, F. culmorum (w.G.smith) saccardo and F. graminearum schwabe. Both F.culmorum and F. graminearum isolates produce trichothecene mycotoxins and mycohormone zearalenone and are considered to be the most important cereal pathogens worldwide. infection with those pathogens leads to accumulation of mycotoxins: deoxynivalenol (DoN) and zearalenone (ZEA) in grains. Fusarium avenaceum isolates are producers of moniliformin (MoN) and enniatins. isolates of Trichoderma sp. were found to be the most effective ones to control the growth of examined Fusarium species. the response of Fusarium isolates to antagonistic activity of Trichoderma isolates varied and also the isolates of Trichoderma differed in their antagonistic activity against Fusarium isolates. the production of MoN by two isolates of F. avenaceum in dual culture on rice was reduced by 95% to 100% by T. atroviride isolate AN 35. the same antagonist reduced the amount of moniliformin from 100 μg/g to 6.5 μg/g when inoculated to rice culture contaminated with MoN, which suggests the possible decomposition of this mycotoxin.

Fusarium head blight and maize ear rot are destructive diseases in various cereal production regions, leading to significant yield losses and to the contamination of cereal grain with Fusarium mycotoxins. The mycotoxin zearalenone belongs to the three most important mycotoxins produced by Fusarium species worldwide; moreover, its hormonal oestrogenic activity is higher than its toxicity. The compound also exhibits fungitoxic activity. Toxigenic Fusarium species sporulate on cereal crop residues and produce inoculum to infect the next crop. Reduction of mycotoxin production and pathogen sporulation may be influenced by saprophytic fungal antagonists. Selected Trichoderma and Clonostachys isolates in dual culture bioassays on rice reduced zearalenone, α-zearalenol and zearalenone sulphate production by two isolates of Fusarium graminearum Schwabe and two isolates of F. culmorum (W.G. Smith) Saccardo belonging to three different chemotypes, by over 97%. Two Trichoderma isolates reduced the amount of zearalenone produced by the same four Fusarium isolates by 5-83% in solid substrate bioassays, whereas several Clonostachys isolates reduced the amount of zearalenone by over 96%. Examination of the dynamics of zearalenone reduction showed that the reduction occurred at the fastest rate during the first four days of incubation. Selected non-toxigenic Trichoderma and Clonostachys isolates are candidates for biocontrol agents against toxigenic Fusarium pathogens of cereals and may be useful for decomposition of zearalenone in contaminated cereal grain and cereal products.

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