Interacting climate change environmental factors effects on Fusarium langsethiae growth, expression of Tri genes and T-2/HT-2 mycotoxin production on oat-based media and in stored oats.

This study examined the effect of climate change (CC) abiotic factors of temperature (20, 25, 30 °C), water activity (aw; 0.995, 0.98) and CO2 exposure (400, 1000 ppm) may have on (a) growth, (b) gene expression of biosynthetic toxin genes (Tri5, Tri6, Tri16), and (c) T-2/HT-2 toxins and associated metabolites by Fusarium langsethiae on oat-based media and in stored oats. Lag phases and growth were optimum at 25 °C with freely available water. This was significantly reduced at 30 °C, at 0.98 aw and 1000 ppm CO2 exposure. In oat-based media and stored oats, Tri5 gene expression was reduced in all conditions except 30 °C, 0.98 aw, elevated CO2 where there was a significant (5.3-fold) increase. The Tri6 and Tri16 genes were upregulated, especially in elevated CO2 conditions. Toxin production was higher at 25 °C than 30 °C. In stored oats, at 0.98 aw, elevated CO2 led to a significant increase (73-fold) increase in T2/HT-2 toxin, especially at 30 °C. Nine T-2 and HT-2 related metabolites were detected, including a new dehydro T-2 toxin (which correlated with T-2 production) and the conjugate, HT-2 toxin, glucuronide. This shows that CC factors may have a significant impact on growth and mycotoxin production by F. langsethiae.

Preparation of T-2-glucoronide with Rat Hepatic Microsomes and Its Use along with T-2 for Activation of the JAK/STAT Signaling Pathway in RAW264.7 Cells.

T-2 toxin (T-2), one of the most toxic trichothecene A-type mycotoxins, is biotransformed in animal tissues to modified T-2s (mT-2s) including T-2-glucuronide (T-2-GlcA). In this study, the optimal conditions for T-2-GlcA synthesis were established, and the JAK/STAT pathway in RAW264.7 cells was used to study the toxicity of T-2-GlcA. Because many mT-2 standards are not readily available, optimal conditions for T-2-GlcA synthesis in vitro were established by incubating T-2 with rat liver microsomes, UDPGA, and 0.2% Triton X-100 for 90 min. qRT-PCR and Western blot results showed 21- and 760-fold increases in IL-6 mRNA expression induced by T-2-GlcA and T-2, respectively. Similar differences were observed in JAK3, SOCS2/3, and CIS mRNA expression. T-2-GlcA induced a dose-responsive decrease in STAT1 mRNA expression, whereas the result with T-2 was the opposite. Moreover, the phosphorylation of STAT3 induced by T-2-GlcA was higher than that by T-2, whereas the phosphorylation of STAT1 was to the contrary. Overall, the results show that T-2-GlcA was somewhat toxic, but activation of the JAK/STAT pathway in RAW264.7 was higher by T-2.

T-2 mycotoxin: toxicological effects and decontamination strategies.

Mycotoxins are highly diverse secondary metabolites produced in nature by a wide variety of fungus which causes food contamination, resulting in mycotoxicosis in animals and humans. In particular, trichothecenes mycotoxin produced by genus fusarium is agriculturally more important worldwide due to the potential health hazards they pose. It is mainly metabolized and eliminated after ingestion, yielding more than 20 metabolites with the hydroxy trichothecenes-2 toxin being the major metabolite. Trichothecene is hazardously intoxicating due to their additional potential to be topically absorbed, and their metabolites affect the gastrointestinal tract, skin, kidney, liver, and immune and hematopoietic progenitor cellular systems. Sensitivity to this type of toxin varying from dairy cattle to pigs, with the most sensitive endpoints being neural, reproductive, immunological and hematological effects. The mechanism of action mainly consists of the inhibition of protein synthesis and oxidative damage to cells followed by the disruption of nucleic acid synthesis and ensuing apoptosis. In this review, the possible hazards, historical significance, toxicokinetics, and the genotoxic and cytotoxic effects along with regulatory guidelines and recommendations pertaining to the trichothecene mycotoxin are discussed. Furthermore, various techniques utilized for toxin determination, pathophysiology, prophylaxis and treatment using herbal antioxidant compounds and regulatory guidelines and recommendations are reviewed. The prospects of the trichothecene as potential hazardous agents, decontamination strategies and future perspectives along with plausible therapeutic uses are comprehensively described.

Influence of Selenium on the Production of T-2 Toxin by Fusarium poae.

The objective of this study was to investigate the effects of selenium on the production of T-2 toxin by a Fusarium poae strain cultured in a synthetic medium containing different concentrations of selenium. The T-2 toxin contents in fermentative products were evaluated by a high performance liquid chromatography (HPLC). The results showed that the production of T-2 toxin was correlated with the concentration of selenium added to the medium. In all three treatments, the addition of 1 mg/L selenium to the medium resulted in a lower toxin yield than the control (0 mg/L); the yield of the toxin began to increase when selenium concentration was 10 mg/L, while it decreased again at 20 mg/L. In summary, T-2 toxin yield in the fermentative product was affected by the addition of selenium to the medium, and a selenium concentration of 20 mg/L produced the maximum inhibitory effect of T-2 toxin yield in the fermentative product of F. poae.

Effects of Phenolic Acids on the Growth and Production of T-2 and HT-2 Toxins by Fusarium langsethiae and F. sporotrichioides.

The effect of natural phenolic acids was tested on the growth and production of T-2 and HT-2 toxins by Fusarium langsethiae and F. sporotrichioides, on Mycotoxin Synthetic medium. Plates treated with 0.5 mM of each phenolic acid (caffeic, chlorogenic, ferulic and p-coumaric) and controls without phenolic acid were incubated for 14 days at 25 °C. Fungal biomass of F. langsethiae and F. sporotrichioides was not reduced by the phenolic acids. However, biosynthesis of T-2 toxin by F. langsethiae was significantly reduced by chlorogenic (23.1%) and ferulic (26.5%) acids. Production of T-2 by F. sporotrichioides also decreased with ferulic acid by 23% (p < 0.05). In contrast, p-coumaric acid significantly stimulated the production of T-2 and HT-2 toxins for both strains. A kinetic study of F. langsethiae with 1 mM ferulic acid showed a significant decrease in fungal biomass, whereas T-2 production increased after 10 days of incubation. The study of gene expression in ferulic supplemented cultures of F. langsethiae revealed a significant inhibition for Tri5, Tri6 and Tri12 genes, while for Tri16 the decrease in gene expression was not statistically significant. Overall, results indicated that phenolic acids had a variable effect on fungal growth and mycotoxin production, depending on the strain and the concentration and type of phenolic acid assayed.

Relationship between mycoparasites lifestyles and biocontrol behaviors against Fusarium spp. and mycotoxins production.

Global food security research is seeking eco-friendly solutions to control mycotoxins in grain infected by fungi (molds). In particular, mycotoxigenic Fusarium spp. outbreak is a chronic threat for cereal grain production, human, and animal health. In this review paper, we discuss up-to-date biological control strategies in applying mycoparasites as biological control agents (BCA) to prevent plant diseases in crops and mycotoxins in grain, food, and feed. The aim is to increase food safety and to minimize economic losses due to the reduced grain yield and quality. However, recent papers indicate that the study of the BCA specialists with biotrophic lifestyle lags behind our understanding of the BCA generalists with necrotrophic lifestyle. We examine critical behavioral traits of the two BCA groups of mycoparasites. The goal is to highlight their major characteristics in the context of future research towards an efficient biocontrol strategy against mycotoxin-producing Fusarium species. The emphasis is put on biocontrol of Fusarium graminearum, F. avenaceum, and F. culmorum causing Fusarium head blight (FHB) in cereals and their mycotoxins.

[Direct detection of T-2- and HT-2-mycotoxins producers of fungi the genus Fusarium in food grain by PCR (report 2)].

The improvement of the Fusarium DNA extraction method has been undertaken in order to reduce the error of PCR analysis for detection of toxigenic Fusarium species, including those contained in the grain in the uncultureted state, directly in the grain. The efficiency of Fusarium DNA extraction methods (nucleotides sorption and CTAB method) has been compared. The efficiency of CTAB method combined with 10-fold weight increase of milled grain sample has been demonstrated. This approach revealed a greater number of Fusarium species, than PCR analysis of combined Fusarium mycelium from the same samples. The uncultureted F. langsethiae was detected in the DNA extract from a sample of barley, which was not identified in the combined sample of the mycelium. This sample of the grain has the highest levels of T-2/NT-2-toxins--0,075/0,345 mg/kg (determined by HPLC) among positive samples. F. sporotrichioides--a potential producer of T-2- and HT-2-toxins has been revealed by PCR method in other grain samples both containing and not containing these toxins. The biosynthesis of T-2- and HT-2-toxins on the PSA-medium in vitro has been studied for 10 single-spores F. sporotrichioides isolates, allocated from grain. Synthesized T-2-toxin content (measured by ELISA) ranged from 0.4 to 184.5 mg per l of medium. Three strains showed very high levels from 117.2 to 184.4 mg/l, two of which have been isolated from barley which don't contain these toxins. The absence of the toxin in grain samples does not guarantee the absence of high-level producers of mycotoxins. The direct detection of Fusarium spp. in grain by PCR analysis with extraction of fungal DNA by CTAB method along with increased sample weight has been shown to make possible the detection of a more number of species of Fusarium (including uncultureol strains) compared with mycological method with PCR analysis of the combined sample of the mycelium.

[Trichothecene mycotoxins of Fusarium poae from different habitats].

Comparative study of the ability of three strains of Fusarium poae for the synthesis of trichothecen mycotoxins has been carried out. Studied strains were isolated from different habitats: forest soil, wheat (plant pathogen) and cranberry root (endophytic strain). All three strains were able to synthesize T-2 toxin, HT-2 toxin and T-2 tetraol but they were in various amounts. The soil strain 50660 was characterized by high level of synthesis of both HT-2 toxin and T-2 tetraol; plant pathogenic 50674 and endophytic 50685 strains were characterized by high level of T-2 tetraol synthesis and lower level of HT-2 toxin synthesis. The main trichothecene mycotoxin of this group - T-2 toxin - was detected in trace amounts for all three strains of F. poae.

Fusariotoxins in asparagus - their biosynthesis and migration.

Asparagus is often infected by fungi of the Fusarium genus, a causal agent of crown and root rot, which decreases the quantity and quality of spears. Fusarium oxysporum and Fusarium proliferatum are the most severe asparagus pathogens, well known as mycotoxin producers, mainly fumonisins and moniliformin. The present study was undertaken to estimate fumonisin B1, moniliformin and ergosterol concentrations in asparagus tissue. Moreover, the possibility of toxin transport to the edible asparagus part during the inoculation by F. oxysporum and F. proliferatum of different plant parts (root, crown and stem base) and the potential risk for consumers were assessed. Our studies showed that the highest capability of producing fumonisin B1 and moniliformin was demonstrated from isolates of F. proliferatum. The highest level of fumonisin B1 in edible spears was detected when the asparagus crown was inoculated with F. oxysporum and F. proliferatum. The lowest concentration was found in the case of storage root inoculation, which corresponds with mycelium absence and the long distance from the roots to the stem. Similar results were demonstrated for moniliformin. The mycotoxin content was confirmed even in healthy spears (without disease symptoms and mycelium presence), which might indicate that the transport of mycotoxins is possible from the soil through the root system to the top part of the plants.

Natural co-occurrence of fungi and mycotoxins in poultry feeds from Entre Ríos, Argentina.

A total of 120 pelleted poultry feed samples from Entre Ríos Province, Argentina, were evaluated. The aims were to investigate (1) the presence of relevant toxigenic fungi, as well as to determine the ability to produce aflatoxins (AFs) by Aspergillus section Flavi isolated strains; and (2) the natural co-occurrence of AFs, fumonisins (FBs), gliotoxin, diacetoxyscirpenol (DAS), HT-2 and T-2 toxin by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Total fungal counts were below the established value (1 × 104 CFU g⁻¹). Aspergillus flavus and A. parasiticus were the only aflatoxigenic species isolated. Co-occurrence of fumonisin B1 (FB1), HT-2 and T-2 toxin was detected in 100% of the feeds, with mean levels from 4502 to 5813; 6.7 to 21.6 and 19.6 to 30.3 µg kg⁻¹, respectively. A large number of starter samples were co-contaminated with aflatoxin B1 (AFB1), FB1, HT-2 and T-2 toxins. Gliotoxin and DAS were not found in this survey.

Mycotoxin contamination of cereal grain commodities in relation to climate in North West Europe.

This study aimed to investigate mycotoxin contamination of cereal grain commodities for feed and food production in North Western Europe during the last two decades, including trends over time and co-occurrence between toxins, and to assess possible effects of climate on the presence of mycotoxins. For these aims, analytical results related to mycotoxin contamination of cereal grain commodities, collected in the course of national monitoring programmes in Finland, Sweden, Norway and the Netherlands during a 20-year period, were gathered. Historical observational weather data, including daily relative humidity, rainfall and temperature, were obtained from each of these four countries. In total 6382 records, referring to individual sample results for mycotoxin concentrations (one or more toxins) in cereal grains were available. Most records referred to wheat, barley, maize and oats. The most frequently analysed mycotoxins were deoxynivalenol, 3-acetyl-deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and zearalenone. Deoxynivalenol had the highest overall incidence of 46%, and was mainly found in wheat, maize and oats. Mycotoxins that showed co-occurrence were: deoxynivalenol and 3-acetyl-deoxynivalenol in oats; deoxynivalenol and zearalenone in maize and wheat; and T-2 toxin and HT-2 toxin in oats. The presence of both deoxynivalenol and zearalenone in wheat increased with higher temperatures, relative humidity and rainfall during cultivation, but the presence of nivalenol was negatively associated with most of these climatic factors. The same holds for both nivalenol and deoxynivalenol in oats. This implies that climatic conditions that are conducive for one toxin may have a decreasing effect on the other. The presence of HT-2 toxin in oats showed a slight decreasing trends over time, but significant trends for other toxins showed an increasing presence during the last two decades. It is therefore useful to continue monitoring of mycotoxins. Obtained results can be used for development of predictive models for presence of mycotoxins in cereal grains.

Control of T-2 toxin in Fusarium langsethiae and Geotrichum candidum co-culture.

Due to contamination of barley grains by Fusarium langsethiae, T-2 toxin can be present in the brewing process. It has been observed that the presence of the yeast Geotrichum candidum during malting can reduce the final concentration of this mycotoxin in beer. In this work, a co-culture method was carried out for both microorganisms in order to evaluate the effect on T-2 mycotoxin concentration in comparison with the pure culture of F. langsethiae in the same conditions. The microbial growth of both microorganisms was assessed using three different methods: dry weight, DOPE-FISH, and DNA quantification. In coculture, both microorganisms globally developed less than in pure cultures but G. candidum showed a better growth than F. langsethiae. The concentration of T-2 was reduced by 93 % compared to the pure culture. Hence, the interaction between G. candidum and F. langsethiae led to a drastic mycotoxin reduction despite the only partial inhibition of fungal growth.

Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium.

Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a(w) at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED50 and ED90 values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a(w) and the influence of temperature on growth rates was 25>20>15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED50 and ED90 values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a(w), type of fungicide and dose. Levels of T-2 were usually higher than those of HT-2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a(w) T-2 levels increased in cultures containing fenpropimorph while at 0.96 a(w) the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a(w) and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a(w). This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxins in oat-based media.

Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae.

Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups.

Observation of T-2 toxin and HT-2 toxin glucosides from Fusarium sporotrichioides by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).

The trichothecenes produced by solid and liquid cultures of Fusarium sporotrichioides were evaluated with high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Along with the expected T-2 toxin HT-2 toxin and neosolaniol, two additional compounds were detected, which had ions 162 m/z higher than those in the mass spectra of T-2 toxin or HT-2 toxin. Fragmentation behavior of these two compounds was similar to that of T-2 toxin and HT-2 toxin. Based on LC-MS/MS behavior, it is proposed that the two compounds are T-2 toxin 3-O-glucoside and HT-2 toxin 3-O-glucoside. Production of the two glucosides was measured in kernels from wheat and oat inoculated with F. sporotrichiodes, as well as in cultures grown in liquid media and on cracked corn or rice. Production of glucosides in wheat and oats suggest that they may also be present in naturally contaminated cereals.

Influence of barley malting operating parameters on T-2 and HT-2 toxinogenesis of Fusarium langsethiae, a worrying contaminant of malting barley in Europe.

The fungus Fusarium langsethiae, exclusively described in Europe at present, seems to have taken the place of other Fusarium species in barley fields over the last 5 years. It has proved to be a highly toxic type-A trichothecene producer (T-2 and HT-2 toxins). The aim of this work was to study the ecotoxinogenesis of this fungus the better to identify and manage the health risk it may pose during the beer manufacturing process. The influence of temperature and water activity on its growth rate and production of toxins are particularly assessed from a macroscopic point of view. Different cultures were grown on sterilized rehydrated barley with a water activity between 0.630 and 0.997 and a temperature ranging from 5 to 35 degrees C. Biomass specific to F. langsethiae and T-2 and HT-2 toxins were quantified by real-time polymerase chain reaction and liquid chromatography-mass spectrometry, respectively. It appears that the optimal temperature and water activity for F. langsethiae toxinogenesis are 28 degrees C and 0.997. This fungus was able to produce 2.22 g kg(-1) of these toxins in 16 days on barley in optimal production conditions. The malting process seems to be a critical step because, in its temperature range, specific production was six times higher than under optimal temperatures for fungus growth. In the short-term, this work will help redefine the process conditions for malting. In the medium-term, the results will contribute to the development of a molecular tool to diagnose the presence of this contaminant and the detection of the toxins in barley, from fields to the end product.

Glucose influence on the production of T-2 toxin by Fusarium sporotrichioides.

Toxigenic isolate of Fusarium sporotrichioides was tested for the T-2 toxin production on PDA plates during 10 days under various glucose concentrations. T-2 toxin was determined by LC-MS and confirmed with LC-MS/MS. This analytical method has been applied, for the first time, to an extensive study of T-2 accumulation. Results showed that the production of this mycotoxin is directly correlated to the concentration of glucose present in the medium. Concentrations of T-2 toxin produced by the strain of F. sporotrichioides ranged from 0 to 1.45 mg/kg. The better T-2 production was evidenced in the fermentation operated with 20% of glucose.

The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation.

The fungal proteins of the White Collar photoreceptor family, represented by WC-1 from Neurospora crassa, mediate the control by light of different biochemical and developmental processes, such as carotenogenesis or sporulation. Carotenoid biosynthesis is induced by light in the gibberellin-producing fungus Fusarium fujikuroi. In an attempt to identify the photoreceptor for this response, we cloned the only WC-1-like gene present in the available Fusarium genomes, that we called wcoA. The predicted WcoA polypeptide is highly similar to WC-1 and contains the relevant functional domains of this protein. In contrast to the Neurospora counterpart, wcoA expression is not affected by light. Unexpectedly, targeted wcoA disruptant strains maintain the light-induced carotenogenesis. Furthermore, the wcoA mutants show a drastic reduction of fusarin production in the light, and produce less gibberellins and more bikaverins than the parental strain under nitrogen-limiting conditions. The changes in the production of the different products indicate a key regulatory role for WcoA in secondary metabolism of this fungus. Additionally, the mutants are severely affected in conidiation rates under different culture conditions, indicating a more general regulatory role for this protein.

Fusarium toxins of the scirpentriol subgroup: a review.

Scirpentriol and its seven acetylated derivatives comprise a family of type-A trichothecene toxins produced by several species of Fusarium fungi. Out of this group 4,15-diacetoxyscirpenol has attracted most attention. It elicits toxic responses in several species and was detected in a variety of substrates. Out of the three possible monoacetylated derivatives 15-monoacetoxyscirpenol and the parent alcohol scirpentriol received some attention, whereas the remaining members of the family were mentioned in few reports. The present review deals with the structure, biosynthesis, analysis and toxicity of scirpentriol toxins. Formation by Fusarium species as well as culture conditions used for toxigenicity studies are reviewed; data about the natural occurrence of scirpentriol toxins in different cereal types, cereal associated products as well as in non-grain matrices including potato and soya bean are reported. Basing on literature reports about the toxicity of scirpentriol toxins an attempt is made to summarise the state of knowledge for risk evaluation for human and animal health.

Fusarium mycotoxins in Lithuanian cereals from the 2004-2005 harvests.

Fusarium mycotoxins deoxynivalenol (DON), T-2 toxin, and zearalenone (ZEN) contamination in 5 kinds of cereal grain harvested in 2004 and 2005 in different regions of Lithuania was examined for their occurrence frequency and level. In all cereal species DON was the most frequently detected mycotoxin with an incidence rate of 98.0-100% and range in positive samples from traces to 691 microg kg(-1) in 2004 and 62.5-94.0%, range from traces to 1,121 microg kg(-1) in 2005, respectively. All the tested oat samples collected in 2004-2005 were found to be contaminated with the T-2 toxin. In one sample from the year 2004 the level of T-2 toxin (121.5 microg kg(-1)) exceeded the allowable level. In 2004, ZEN contamination was more frequent in spring wheat, barley and oats grain, whereas in 2005 this toxin was identified at higher levels only in barley grain (68.0%). In one barley grain sample from 2004, ZEN content (193.4 microg kg(-1)) exceeded the allowable level. Variation in the relative air-humidity exerted some effect on the incidence of Fusarium spp. fungi and mycotoxin content in wheat grain. The weather conditions at harvesting contributed to an increase in the contents of Fusarium fungi and DON and ZEN mycotoxins produced by them in winter wheat grain. This risk factor increases the threat to human and animal health.