**3. Results**

### *3.1. Fusarium Metabolites*/*Mycotoxins in Fusarium-Inoculated and Naturally-Infected Samples*

Deoxynivalenol (DON), DON-3-glucoside (D3G), 3-acetyldeoxynivalenol (3-ADON), culmorin (CULM), 15-hydroxyculmorin, 5-hydroxyculmorin, aurofusarin, rubrofusarin, enniatin (Enn) A1, B, B1 (Figure 1a–k) were found in all tested samples, although sometimes in ppb concentrations, while enniatin (Enn) B2 was present in at least half of the tested samples (Figure 1) Chrysogin was also found in all tested samples (Figure 1m), while zearalenone (ZEN), moniliformin (MON), nivalenol (NIV), siccanol, and equisetin were present in some of the tested samples (Figure 1n–r).

The concentrations of *Fusarium* mycotoxins DON, CULM, 3-ADON, 15-hydroxyculmorin, D3G, and 5-hydroxyculmorin were significantly elevated in the inoculated samples compared to naturally-infected samples of all tested winter wheat varieties (Supplementary Figure S1, Figure 1a–f). For other detected *Fusarium* metabolites, i.e., aurofusarin, rubrofusarin, and chrysogin, significant increases also occurred in inoculated samples compared to those that were naturally infected (Supplementary Figure S2, Figure 2c,d,f). The levels of DON measured in four FHB-inoculated samples were 17,586 μg/kg in Ficko, 22,337 μg/kg in Pepeljuga, 14,946 μg/kg in Mv Karizma, and 21,622 μg/kg in Mv Kolompos. Although FHB-inoculated samples were far more contaminated with DON, naturally-infected samples also contained higher levels of DON, as well as several other *Fusarium* mycotoxins, than expected (Figure 1a). Moreover, in Mv Karizma and Mv Kolompos, recorded DON concentrations were 1764 μg/kg and 2682 μg/kg, respectively, which is above the maximal allowed concentration for human consumption. As expected, D3G and 3-ADON were found in all samples that contained DON (Figure 1b,c). The highest concentration of D3G was recorded in Ficko in the inoculated samples (393 μg/kg), while the lowest concentration was in naturally-infected samples of Pepeljuga (31 μg/kg). 3-ADON concentration ranged from 9 μg/kg in naturally-infected samples to 3399 μg/kg in *Fusarium*-inoculated samples. FHB-inoculated samples of Mv Karizma had at least 2 times lower concentrations of 3-ADON compared to inoculated samples of other varieties.

**Figure 2.** *Alternaria* and *Aspergillus* mycotoxins in *Fusarium*-inoculated and naturally-infected samples: alternariol (**a**), alternariolmethylether (**b**), altersetin (**c**), infectopyron (**d**), tentoxin (**e**), tenuazonic acid (**f**), and kojic acid (**g**). The asterisk (\*) indicates < LOD.

The highest concentration of CULM (6551 μg/kg) was recorded in FHB-inoculated samples of Ficko, and the lowest was obtained in naturally-infected samples of Pepeljuga (443 μg/kg) (Figure 1d). The highest concentrations of 15-hydroxyculmorin and 5-hydroxyculmorin were found in FHB-inoculated samples of Mv Kolompos—63,329 and 23,864 μg/kg, respectively—similar to the levels detected in inoculated samples of Ficko and Pepeljuga, but lower levels were detected in inoculated samples of Mv Karizma (Figure 1e,f).

In this study, pigments were also detected; aurofusarin was detected in the range of 735 to 63,098 μg/kg and rubrofusarin from 65 to 5136 μg/kg. Although aurofusarin and rubrofusarin levels in all inoculated samples increased compared to naturally-infected samples, inoculated samples of Mv Karizma had at least three times lower concentrations of these mycotoxins compared to other varieties (Figure 1g,h).

The highest level of enniatins A1, B, B1, and B2 (8, 94, 57, and 4 μg/kg, respectively) were found in naturally-infected samples of Mv Kolompos. Overall, these mycotoxins were shown to be present in lower concentrations in FHB-inoculated samples compared to naturally-infected samples or were not detected at all in the inoculated samples (Figure 1i–l). The various enniatins were ranked as follows, in descending order of incidence and mean concentration: enniatin B > enniatin B1 > enniatin A1 > enniatin B2 > enniatin A > enniatin B3. Total enniatin levels varied from traces (0.02 μg/kg, enniantin A) to 94 μg/kg (enniantin B).

Chrysogin, in the inoculated samples compared to those that were naturally infected, decreased by 97, 97, 94, and 94%, respectively, in Ficko, Pepeljuga, Mv Karizma, and Mv Kolompos (Figure 1m).

All inoculated and two naturally-infected samples were contaminated with ZEN, and the highest concentration of this mycotoxin was detected in FHB-inoculated samples (26 μg/kg). No ZEN was detected in naturally-infected samples of Ficko and Pepeljuga, and all detected concentrations were below legal limits set in the EU by the European Commission regulation 1881/2006 (Figure 1n).

In the case of MON, the concentration in the inoculated sample of Ficko was 480 μg/kg, which is a 99% increase in relation to naturally-infected grain, while other varieties were far less contaminated with MON, and its concentration in other tested samples ranged from 3 (Pepeljuga, inoculated sample) to 86 μg/kg (Mv Kolompos, inoculated sample) (Figure 1o).

Nivalenol (NIV) was found in seven out of eight samples. Here, the inoculated samples had increased NIV levels compared to the naturally-infected grains, although the di fference between naturally-infected and inoculated samples was the least pronounced for Mv Kolompos, in which high values of NIV were detected in both treatments (Figure 1p). The di fferences between treatments were not statistically significantly di fferent (data not shown).

Inoculated sample of Ficko and naturally-infected samples of Mv Karizma and Mv Kolompos had no siccanol. The highest amount was recorded in naturally-infected samples of Ficko (300 μg/kg) (Figure 1q). The presence of siccanol is not specific for *Fusarium* strains used in this research, and such a high concentration in naturally-infected sample likely came from natural co-contamination by other *Fusarium* species.

Equisetin was detected only in samples of Mv Karizma, both FHB-inoculated and naturally-infected, as well as in naturally-infected samples of Pepeljuga and inoculated samples of Mv Kolompos (Figure 1r).

In addition to the mycotoxins presented in Figure 1, beauvericin, enniatin A, fumonisin B1, fumonisin B2, and antibiotic Y were also detected in the same samples, while enniatin B3, which was tested, was not detected in any of the analyzed samples. Concentrations of beauvericin and enniatin A were very low: 0.5 μg/kg of beauvericin was detected in inoculated samples of Ficko and enniatin A was detected in naturally-infected samples of Pepeljuga, Mv Karizma, and Mv Kolompos at concentrations of 0.5 μg/kg, 0.1 μg/kg, and 0.2 μg/kg, respectively. The inoculated sample of Pepeljuga was the only variety in which fumonisin B1 and B2 were detected at concentrations of 54 μg/kg and 15 μg/kg, respectively, while antibiotic Y was only detected in inoculated samples of Mv Karizma at a concentration of 73 μg/kg. The presence of those metabolites was not specific for *Fusarium* strains used in this research and probably resulted from natural co-contamination by other *Fusarium* species. T2 and HT2 toxins were absent in those tested wheat varieties in both treatments.
