*Article* **Multiple Fungal Metabolites Including Mycotoxins in Naturally Infected and** *Fusarium***-Inoculated Wheat Samples**

**Valentina Spanic 1,\*, Zorana Katanic 2, Michael Sulyok 3, Rudolf Krska 3,4, Katalin Puskas 5, Gyula Vida 5, Georg Drezner 1 and Bojan Šarkanj 6**


Received: 21 March 2020; Accepted: 14 April 2020; Published: 17 April 2020

**Abstract:** In this study, the occurrence of multiple fungal metabolites including mycotoxins was determined in four different winter wheat varieties in a field experiment in Croatia. One group was naturally infected, while the second group was inoculated with a *Fusarium graminearum* and *F. culmorum* mixture to simulate a worst-case infection scenario. Data on the multiple fungal metabolites including mycotoxins were acquired with liquid chromatography with mass spectrometry (LC-MS/MS) multi-(myco)toxin method. In total, 36 different fungal metabolites were quantified in this study: the *Fusarium* mycotoxins deoxynivalenol (DON), DON-3-glucoside (D3G), 3-acetyldeoxynivalenol (3-ADON), culmorin (CULM), 15-hydroxyculmorin, 5-hydroxyculmorin, aurofusarin, rubrofusarin, enniatin (Enn) A, Enn A1, Enn B, Enn B1, Enn B2, Enn B3, fumonisin B1, fumonisin B2, chrysogin, zearalenone (ZEN), moniliformin (MON), nivalenol (NIV), siccanol, equisetin, beauvericin (BEA), and antibiotic Y; the *Alternaria* mycotoxins alternariol, alternariolmethylether, altersetin, infectopyron, tentoxin, tenuazonic acid; the *Aspergillus* mycotoxin kojic acid; unspecific metabolites butenolid, brevianamid F, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and tryptophol. The most abundant mycotoxins in the inoculated and naturally contaminated samples, respectively, were found to occur at the following average concentrations: DON (19,122/1504 μg/kg), CULM (6109/1010 μg/kg), 15-hydroxyculmorin (56,022/1301 μg/kg), 5-hydroxyculmorin (21,219/863 μg/kg), aurofusarin (43,496/1266 μg/kg). Compared to naturally-infected samples, *Fusarium* inoculations at the flowering stage increased the concentrations of all *Fusarium* mycotoxins, except enniatins and siccanol in Ficko, the *Aspergillus* metabolite kojic acid, the *Alternaria* mycotoxin altersetin, and unspecific metabolites brevianamid F, butenolid, cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In contrast to these findings, because of possible antagonistic actions, *Fusarium* inoculation decreased the concentrations of the *Alternaria* toxins alternariol, alternariolmethylether, infectopyron, tentoxin, tenuazonic acid, as well as the concentration of the nonspecific metabolite tryptophol.

**Keywords:** fusarium; LC-MS/MS; mycotoxin; occurrence; wheat
