Plant metabolism of T-2 and HT-2 toxin in wheat, barley and oats

T2 toxin (T2) and HT2 toxin (HT2) are type-A trichothecenes produced by F. sporotrichioides, F. langsethiae and F. poae contaminating kernels of oats, barley and wheat. These toxins are of high concern to human health. T2 is a potent protein synthesis inhibitor and is rapidly metabolized in vivo to HT2 that induces similar adverse effects. Maximum levels for the sum of T2 and HT2 in cereals for human consumption are under discussion in the EU. A combined provisional TDI was already set at 0.06 microgr/kg. Plant metabolism of T2/HT2 may complicate risk evaluation with respect to human nutrition. T2- and HT2-3-O-glucosides and HT2-4-O-glucoside were detected in wheat and oats. Preliminary results show that wheat lines with the Fhb1 quantitative trait locus (QTL) are able to protect wheat against the phytotoxic effect of T2 and HT2. Fhb1 governs detoxification of deoxynivalenol (DON) to DON-3-O-glucoside. The detoxification mechanism of T2/HT2 is unknown and will be investigated. T2- and HT2-conjugates such as their glucosides could be “masked mycotoxins”: they are not detectable by routine analysis but in the intestinal tract the native toxin could be released. Part of the mycotoxins may be bound to the plant matrix and can either be accessible by chemical extraction or be completely insoluble. These bound mycotoxins might be released in the digestive tract upon consumption of contaminated plant material. Knowledge on the metabolisation of T2 and HT2 by plants is very limited to date. In this project we want to 1) investigate the metabolic fate of T2/HT2 in planta, 2) develop suitable analytical methods for detection and quantification of possible new metabolites and 3) produce standards of such metabolites. To detect metabolites a 1:1 mixture of natural and U-[13C]-toxins will be applied on wheat/barley ears and oat panicles. At different time points after application both known (i.e. predicted) and unknown metabolites will be traced and identified by liquid chromatography – high resolution mass spectrometry (LC-HRMS). Similar tests will be done with 14C-labeled toxins to assess the insoluble amount, measured by scintillation counting. A solvolysis procedure will be established to access the extractable biopolymer-bound and the non-soluble toxin fractions from the plant matrix. A molar sum of all metabolites will be estimated and compared to the total amount of toxins applied. To demonstrate the significance of the detected T2/HT2 biotransformation products, we will confirm their production after artificial inoculation with T2/HT2 producing Fusarium spp. and we will study their presence in naturally infected samples in the scope of a small survey. The resistance mechanism to T2/HT2 in wheat carrying Fhb1 is investigated using near-isogenic wheat lines with and without this QTL. To this end differential formation of T2 and HT2 metabolisation products will be studied by LC-HRMS.