Reduction of deoxynivalenol, T-2 and HT-2 toxins and associated Fusarium species during commercial and laboratory de-hulling of milling oats.

Oats (Avena sativa L.) are well known for their nutritional properties but are susceptible to the growth of different Fusarium fungi resulting in mycotoxin contamination of harvested oats. In this study, oat samples from harvest years 2011 to 2017 were preselected for their suitability as milling oats for food purposes with DON contents below 1750 µg/kg. The reduction of DON, T-2 and HT-2 toxins during the commercial de-hulling process was analysed. While the average reduction for the sum of T-2 and HT-2 toxins in large oat kernels was 85%, the reduction for thin kernels was 66%. The reduction for DON was about 60% and did not differ for the two kernel fractions. In laboratory de-hulling experiments, milling oat samples and de-hulled oat kernels with known DON, T-2 and HT-2 toxin content were correlated with the associated DNA amount of Fusarium graminearum, Fusarium culmorum and Fusarium langsethiae. The reduction of the Fusarium DNA amount after de-hulling was comparable to the reduction of the associated mycotoxins. Notably, the correlation between F. langsethiae DNA amounts and the sum of T-2 and HT-2 toxin contents was R2 = 0.69 in milling oats and it rose to R2 = 0.85 in de-hulled oat kernels. In laboratory tests, at least one third of the initial levels of DON and the sum of T-2 and HT-2 toxins could be removed by polishing off the first parts of the outer layers; two thirds remained in the polished oat kernels. These observations indicate that de-hulling alone may not be completely sufficient to remove mycotoxin contamination in oats. These findings are of high importance in the discussion of determining legal maximum levels for DON or the sum of T-2 and HT-2 toxins in intermediate and final products.

Occurrence of Fusarium Mycotoxins and Their Modified Forms in Forage Maize Cultivars.

Forage maize is often infected by mycotoxin-producing Fusarium fungi during plant growth, which represent a serious health risk to exposed animals. Deoxynivalenol (DON) and zearalenone (ZEN) are among the most important Fusarium mycotoxins, but little is known about the occurrence of their modified forms in forage maize. To assess the mycotoxin contamination in Northern Germany, 120 natural contaminated forage maize samples of four cultivars from several locations were analysed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) for DON and ZEN and their modified forms deoxynivalenol-3-glucoside (DON3G), the sum of 3- and 15-acetyl-deoxynivalenol (3+15-AcDON), α- and ß-zearalenol (α-ZEL, ß-ZEL). DON and ZEN occurred with high incidences (100 and 96%) and a wide range of concentrations, reaching levels up to 10,972 and 3910 µg/kg, respectively. Almost half of the samples (46%) exceeded the guidance value in complementary and complete feeding stuffs for ZEN (500 µg/kg), and 9% for DON (5000 µg/kg). The DON related mycotoxins DON3G and 3+15-AcDON were also present in almost all samples (100 and 97%) with amounts of up to 3038 and 2237 µg/kg and a wide range of concentrations. For the ZEN metabolites α- and ß-ZEL lower incidences were detected (59 and 32%) with concentrations of up to 423 and 203 µg/kg, respectively. Forage maize samples were contaminated with at least three co-occurring mycotoxins, whereby 95% of all samples contained four or more mycotoxins with DON, DON3G, 3+15-AcDON, and ZEN co-occurring in 93%, together with α-ZEL in 57% of all samples. Positive correlations were established between concentrations of the co-occurring mycotoxins, especially between DON and its modified forms. Averaged over all samples, ratios of DON3G/DON and 3+15-AcDON/DON were similar, 20.2 and 20.5 mol%; cultivar-specific mean ratios ranged from 14.6 to 24.3 mol% and 15.8 to 24.0 mol%, respectively. In total, 40.7 mol% of the measured DON concentration was present in the modified forms DON3G and 3+15-AcDON. The α-ZEL/ZEN ratio was 6.2 mol%, ranging from 5.2 to 8.6 mol% between cultivars. These results demonstrate that modified mycotoxins contribute substantially to the overall mycotoxin contamination in forage maize. To avoid a considerable underestimation, it is necessary to analyse modified mycotoxins in future mycotoxin monitoring programs together with their parent forms.

Composition and Predominance of Fusarium Species Causing Fusarium Head Blight in Winter Wheat Grain Depending on Cultivar Susceptibility and Meteorological Factors.

Fusarium head blight (FHB) is one of the most important diseases of wheat, causing yield losses and mycotoxin contamination of harvested grain. A complex of different toxigenic Fusarium species is responsible for FHB and the composition and predominance of species within the FHB complex are determined by meteorological and agronomic factors. In this study, grain of three different susceptible winter wheat cultivars from seven locations in northern Germany were analysed within a five-year survey from 2013 to 2017 by quantifying DNA amounts of different species within the Fusarium community as well as deoxynivalenol (DON) and zearalenone (ZEA) concentrations. Several Fusarium species co-occur in wheat grain samples in all years and cultivars. F. graminearum was the most prevalent species, followed by F. culmorum, F. avenaceum and F. poae, while F. tricinctum and F. langsethiae played only a subordinate role in the FHB complex in terms of DNA amounts. In all cultivars, a comparable year-specific quantitative occurrence of the six detected species and mycotoxin concentrations were found, but with decreased DNA amounts and mycotoxin concentrations in the more tolerant cultivars, especially in years with higher disease pressure. In all years, similar percentages of DNA amounts of the six species to the total Fusarium DNA amount of all detected species were found between the three cultivars for each species, with F. graminearum being the most dominant species. Differences in DNA amounts and DON and ZEA concentrations between growing seasons depended mainly on moisture factors during flowering of wheat, while high precipitation and relative humidity were the crucial meteorological factors for infection of wheat grain by Fusarium. Highly positive correlations were found between the meteorological variables precipitation and relative humidity and DNA amounts of F. graminearum, DON and ZEA concentrations during flowering, whereas the corresponding correlations were much weaker several days before (heading) and after flowering (early and late milk stage).

Effect of ensiling duration on the fate of deoxynivalenol, zearalenone and their derivatives in maize silage.

Fusarium mycotoxins and their derivatives are frequently detected in freshly harvested forage maize. This study assessed the time course effects during ensiling of forage maize on the fate of Fusarium mycotoxins, using laboratory-scale silos and artificially contaminated raw material. A multi-mycotoxin liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was used to determine the levels of deoxynivalenol (DON), zearalenone (ZEN) and their derivatives DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, deepoxy-DON, α-zearalenol and ß-zearalenol. A significant increase of DON was observed during ensiling, whereas the levels of DON-3-glucoside and its acetylated forms proportionally decreased. In contrast, levels of ZEN, α-zearalenol and ß-zearalenol were not affected by the ensiling process. Based on these findings, ensiling is not a practical method for reducing the total amount of Fusarium mycotoxins present at harvest.

Evaluation of High-Resolution Mass Spectrometry for the Quantitative Analysis of Mycotoxins in Complex Feed Matrices.

The selective and sensitive analysis of mycotoxins in highly complex feed matrices is a great challenge. In this study, the suitability of OrbitrapTM-based high-resolution mass spectrometry (HRMS) for routine mycotoxin analysis in complex feeds was demonstrated by the successful validation of a full MS/data-dependent MS/MS acquisition method for the quantitative determination of eight Fusarium mycotoxins in forage maize and maize silage according to the Commission Decision 2002/657/EC. The required resolving power for accurate mass assignments (<5 ppm) was determined as 35,000 full width at half maximum (FWHM) and 70,000 FWHM for forage maize and maize silage, respectively. The recovery (RA), intra-day precision (RSDr), and inter-day precision (RSDR) of measurements were in the range of 94 to 108%, 2 to 16%, and 2 to 12%, whereas the decision limit (CCα) and the detection capability (CCß) varied from 11 to 88 µg/kg and 20 to 141 µg/kg, respectively. A set of naturally contaminated forage maize and maize silage samples collected in northern Germany in 2017 was analyzed to confirm the applicability of the HRMS method to real samples. At least four Fusarium mycotoxins were quantified in each sample, highlighting the frequent co-occurrence of mycotoxins in feed.