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Summary The ubiquitous filamentous fungus F usarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F . graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX‐2) was characterized by liquid chromatography‐tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX‐2 as 3α‐acetoxy‐7α,15‐dihydroxy‐12,13‐epoxytrichothec‐9‐ene. Compared with the well‐known 3‐acetyl‐deoxynivalenol (3‐ADON), it lacks the keto group at C‐8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10‐fold higher contamination with its deacetylated form, named NX‐3, (up to 540 mg kg−1) compared with NX‐2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga C hlamydomonas reinhardtii. NX‐3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX‐2 is far less toxic, similar to 3‐ADON. Genetic analysis revealed a different TRI 1 allele in the N‐isolates, which was verified to be responsible for the difference in hydroxylation at C‐8.

Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB1 (pHFB1 and HFB1) and thermal treatment in the presence of reducing sugars leads to formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially. We produced high purity FB1, pHFB1a+b, HFB1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC-MS/MS based methods. While the latter was significantly increased in the FB1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans.

This paper reports the identification of a barley UDP-glucosyltransferase, HvUGT14077, which is able to convert the estrogenic Fusarium mycotoxin zearalenone into a near-equimolar mixture of the known masked mycotoxin zearalenone-14-O-β-glucoside and a new glucose conjugate, zearalenone-16-O-β-glucoside. Biocatalytical production using engineered yeast expressing the HvUGT14077 gene allowed structural elucidation of this compound. The purified zearalenone-16-O-β-glucoside was used as an analytical calibrant in zearalenone metabolization experiments. This study confirmed the formation of this new masked mycotoxin in barley seedlings as well as in wheat and Brachypodium distachyon cell suspension cultures. In barley roots, up to 18-fold higher levels of zearalenone-16-O-β-glucoside compared to the known zearalenone-14-O-β-glucoside were found. Incubation of zearalenone-16-O-β-glucoside with human fecal slurry showed that this conjugate can also be hydrolyzed rapidly by intestinal bacteria, converting the glucoside back to the parental mycotoxin. Consequently, it should be considered as an additional masked form of zearalenone with potential relevance for food safety.

Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination.

Pentahydroxyscirpene, a novel trichothecene-type compound, was isolated from Fusarium-inoculated rice. The structure of pentahydroxyscirpene was elucidated by 1D and 2D NMR spectroscopy and X-ray single-crystal diffraction. The conformation in solution was determined by NOESY experiments supported by quantum chemical calculations. In vitro toxicity tests showed that pentahydroxyscirpene inhibits protein synthesis as do other trichothecenes.

An improved and reproducible procedure for the preparation of four different glycosides of the mycotoxins α- and β-zearalenol (α,β-ZEL), both metabolites of the Fusarium toxin zearalenone (ZEN), is reported. These conjugated or masked mycotoxins are formed during phase II metabolism in plants (glucosides) or animals and humans (glucuronides). Improved regioselective Konigs–Knorr glucuronidation was applied to ZEN followed by reduction of the keto group of the mycotoxin, leading to α- and β-configuration of ZEL and also to a partial reduction of the glucuronic acid methyl ester to obtain the corresponding glucosides. After deprotection of the sugar moiety, α- and β-zearalenol-14-β,d-glucuronide as well as the corresponding glucosides were isolated at once using preparative HPLC. The reduction step was studied under different reaction conditions to finally develop an optimized and also tunable procedure for the first simultaneous preparation of both, glucosides and glucuronides of a xenobiotic substance in reasonable amounts to be used as reference materials for bioanalytical and toxicological investigations.

Beer is one of the most popular beverages worldwide. Malted cereal grains are among the basic ingredients and hence mycotoxin contamination might occur. Previous studies reported the presence of the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3ADON), as well as of the masked mycotoxin deoxynivalenol-3-glucoside (D3G) in beer. In the present survey, 374 beer samples from 38 countries with a focus on Austrian (156) and German (64) beers were analysed for the presence of D3G, DON and 3ADON. Beers were assigned to the following six categories: pale (217), wheat (46), dark (47), bock (20), nonalcoholic beers (19) and shandies (25). In total, 348 and 289 beers (93 and 77%, respectively) contained D3G and DON at the levels above the limit of detection, whereas 3ADON was not detected in any of the samples. Average concentrations of all beers were 6.9 µg L(-1) for D3G and 8.4 µg L(-1) in the case of DON. Nonalcoholic beers and shandies showed the lowest contaminations, 1.5 and 3.2 µg L(-1) for D3G and 2.7 and 4.4 µg L(-1) for DON, respectively. In bock beers characterised by a higher gravity, a significant trichothecene load of 14.8 µg L(-1) D3G and 12.4 µg L(-1) DON was found. The highest contamination (81 µg L(-1) D3G, 89 µg L(-1) DON) was detected in a pale beer from Austria, underlining the importance of this study for food safety. The molar D3G to DON ratio ranged between 0.11 and 1.25 and was 0.56 on average. Concluding, the average contamination of beer is not of toxicological concern for moderate beer drinkers. However, in the case of heavy beer drinkers, beer consumption may considerably contribute to the overall intake of DON, which might even lead to exceeding the maximum tolerable limits established for this Fusarium toxin.

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B(2) and B(4) by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25-30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.

Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism.