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

Abstract

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 β-zearalenol (α-ZEL, β-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 β-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.

Keywords: 15-acetyl-deoxynivalenol; 3-acetyl-deoxynivalenol; Fusarium; cultivar; deoxynivalenol; deoxynivalenol-3-glucoside; forage maize; modified mycotoxins; zearalenone; α-zearalenol.

Figure 1

Mean concentrations (±SD) of DON, DON3G, 3+15-AcDON, ZEN, and α-ZEL (µg/kg) in forage maize samples of four different cultivars (Cv I–IV) (a–d) at eight trial locations (L1–L8). DON = deoxynivalenol; DON3G = deoxynivalenol-3-glucoside; 3+15-AcDON = sum of 3- and 15-acetyl-deoxynivalenol; ZEN = zearalenone; α-ZEL = α-zearalenol. n = 120.

Figure 2

Distribution (%) of the number of Fusarium mycotoxins in forage maize samples of four different cultivars (Cv I–IV). n = 120.

Figure 3

Percentages of most co-occurring Fusarium mycotoxin combinations containing three, four, and five mycotoxins in forage maize samples of four different cultivars (Cv I–IV). DON = deoxynivalenol; DON3G = deoxynivalenol-3-glucoside; 3+15-AcDON = sum of 3- and 15-acetyl-deoxynivalenol; ZEN = zearalenone; α-ZEL = α-zearalenol. n = 120.

Figure 4

Boxplots and means (black rhombus) of ratios between (a) DON3G and DON, (b) 3+15-AcDON and DON, (c) 3+15-AcDON + DON3G and DON, (d) α-ZEL and ZEN (mol%) in forage maize samples of four different cultivars (Cv I–IV). Five statistics are represented in each boxplot from bottom to top: the smallest observation, lower quartile, median, upper quartile, and largest observation, respectively. Different letters describe significant differences between cultivars for each ratio (p ≤ 0.05). The p-values describe significant or insignificant differences of mean ratios from zero. DON = deoxynivalenol; DON3G = deoxynivalenol-3-glucoside; 3+15-AcDON = sum of 3- and 15-acetyl-deoxynivalenol; ZEN = zearalenone; α-ZEL = α-zearalenol. n = 120.

Figure 5

Total DON concentrations (µg/kg) in forage maize samples of four different cultivars (Cv I–IV) (a–d) by considering DON3G and 3+15-AcDON. The concentration of DON + DON3G + 3+15-AcDON of each sample was calculated by adding up all concentrations of the measured DON and DON arising from DON3G and 3+15-AcDON assuming a total conversion of DON3G and 3+15-AcDON into parent DON. For the determination of DON concentrations arising from DON3G and 3+15-AcDON concentrations of DON3G and 3+15-AcDON were normalised to the molecular mass of DON. Samples were sorted by increasing concentration of DON + DON3G + 3+15-AcDON for each cultivar separately. The guidance value for DON in complementary and complete feeding stuffs (5000 µg/kg) [27] is shown as red line. DON = deoxynivalenol; DON3G = deoxynivalenol-3-glucoside; 3+15-AcDON = sum of 3- and 15-acetyl-deoxynivalenol. n = 120.