**4. Discussion**

This study presents the first report on the occurrence of 36 fungal metabolites in the wheat grain of four different wheat varieties from Croatia and Hungary and their concentrations after artificial inoculation with *Fusarium* spp. compared to non-inoculated field-grown wheat plants.

The major mycotoxins occurring in wheat, at levels of potential concern for human and animal health, are *Fusarium* mycotoxins [2]. *F. graminearum* occurs worldwide as well as in Croatia [37] and is the most important producer of deoxynivalenol (DON), type B trichothecene. The most frequently detected mycotoxins in wheat grain are DON, FB, ZEN, produced by *Fusarium* species, and aflatoxins (AFs) and ochratoxin A (OTA) produced by *Aspergillus* and *Penicillium* species, respectively [38]. In temperate areas, DON is the most prevalent mycotoxin in wheat [39]. Field survey reports clearly indicate that the mycotoxins most frequently produced in cereal head blight by *F. graminearum* and *F. culmorum* in all European countries are DON, 3-acetyldeoxynivalenol (3-ADON), and zearalenone (ZEN) [40].

Several studies reported a high incidence of multi-mycotoxin contamination in cereals and agricultural commodities [41]. The current investigation showed that in 2019, wheat samples in eastern Croatia were co-contaminated by 24 *Fusarium* mycotoxins/metabolites. A study carried

out in Italy showed that at least 80% of wheat samples were contaminated with one mycotoxin, while two mycotoxins were found in 27% of contaminated samples; 38% of the analyzed samples were contaminated with three or more mycotoxins [42].

### *4.1. Deoxynivalenol (DON), DON-3-Glucoside (D3G), and 3-Acetyldeoxynivalenol (3-ADON)*

As regards the contamination levels, the obtained results showed that mycotoxins such as DON, D3G, and CULM, which are mainly produced by *F. culmorum* and *F. graminearum*, were the most abundant mycotoxins in the environmental conditions of the considered field experiments. Average levels of DON contamination did not exceed risk threshold levels for Ficko and Pepeljuga in naturally-infected samples, but as the content range was very wide, all *Fusarium*-inoculated and two naturally-infected (Mv Karizma and Mv Kolompos) samples exceeded the maximum levels for DON contamination. The mean and maximum DON concentrations of naturally-infected wheat samples are summarized in Figure 1 and are lower than the maximum DON concentrations reported in the European wheat summary—a concentration as high as 10,000 μg/kg DON was reported in the European results [8].

Wheat samples had a high incidence of 3-ADON, which was observed in inoculated wheat samples. The modified mycotoxin, D3G was detected and quantified in both inoculated and naturally-infected samples. D3G, one of the several masked mycotoxins, is a phase II plant metabolite of the *Fusarium* mycotoxin DON [43], which can be hydrolyzed in the digestive tract of mammals, thus contributing to the total dietary DON exposure of individuals [20].

### *4.2. Culmorin (CULM), 15-Hydroxyculmorin, and 5-Hydroxyculmorin*

The average value of one of the "emerging mycotoxins" culmorin (CULM) in investigated inoculated samples was 6109 μg/kg, and in naturally-infected samples, this value was 1010 μg/kg. In Norway, the concentration in naturally-infected wheat was lower at median concentrations of 100 μg/kg [44]. This concentration was much higher in durum wheat in central Italy, where it was found that *F. graminearum* produced CULM at concentrations that were very high (2500–14,000 μg/kg) [45]. It was found that naturally-contaminated Norwegian wheat, barley, and oat samples with high DON concentrations also contained CULM and hydroxyculmorins at relatively high levels [46]. In addition, CULM and various hydroxy-culmorins (5- and 15-hydroxy-culmorin) were present in the same concentration ranges as DON in naturally-contaminated grain. This was also concluded by other researchers where levels were typically positively correlated with the amount of DON [44,47]. In Croatian wheat samples in the brewing industry, DON ranged from 595 μg/kg [48] to 2723 μg/kg in wheat malt [49], while concentrations in barley were considerably lower (up to 17.6 μg/kg) [50].

In the current research along with CULM, 15-hydroxyculmorin and 5-hydroxyculmorin also occurred. According to one study [51] CULM, 15-hydroxyculmorin, 5-hydroxyculmorin, and 15-hydroxyculmoron were detected after inoculation with *F. graminearum*, showing enhanced DON toxicity to insects, impacting both growth and mortality, although according to some researchers, CULM and DON can have a synergistic effect on toxicity [52]. Recent findings indicate that CULM can suppress the activity of uridine diphosphate glucosyltransferases (UGTs) that catalyze the glycosylation of DON into the less toxic DON 3-glucoside (D3G) [15]. Compared to DON, in the current research, D3G was found in lower concentrations. All varieties had higher CULM production in inoculated samples compared to those that were naturally infected, which can result in synergistic phytotoxic effects of DON and CULM, when CULM is present at a higher concentration than DON [16], which was not the case in our research.
