Mycotoxins and other fungal metabolites represent the most insidious safety risks to cereal food and the feed chain. Optimising agronomic practices is one of the main strategies adopted to minimise the contents of these undesirable substances in grain-based commodities. The aim of this study was to investigate the effect of the combination of sowing times and hybrids on the occurrence of emerging mycotoxins and fungal metabolites in maize. Field experiments were carried out in 2 sowing times (early vs late) and 3 maize hybrids were compared in the 2014 and 2015 growing seasons. Overall, 37 fungal metabolites produced by Fusarium and Alternaria species were detected. Apart from fumonisins type B (FBs), other metabolites produced by Fusarium verticillioides and F. proliferatum, such as fumonisins type A, fusaric acid, bikaverin and fusaproliferin, were also detected in all of the samples. Fusarin C was found in 61% of the samples. Deoxynivalenol (DON), deoxynivalenol-3-glucoside, culmorin and zearalenone, all of which are produced prevalently by Fusarium graminearum and F. culmorum, were found in all the samples. Their contents were clearly affected by the meteorological trend: the highest contamination was detected in the 2014 growing season, which was characterised by abundant rainfall and lower temperatures from flowering to maize ripening. Among the mycotoxins produced by other Fusarium species, aurofusarin was found to clearly be associated with DON, while moniliformin and beauvericin followed the same behaviour as the FBs.A late sowing time significantly increased the FBs and fumonisin-associated mycotoxins in both growing seasons. The increase in contamination with the delay of sowing was more pronounced in the 2015 growing season, as the environmental conditions were less favourable to the infection of other Fusarium species. The effect of sowing time on DON and DON-associated mycotoxins produced conflicting results for the two growing seasons, because contamination by these metabolites depends more on the conditions that occur during maize flowering than those that occur during ripening. A clearer hybrid susceptibility was observed for these compounds. Other metabolites, such as enniatins, equisetin, T-2 and HT-2 toxins and Alternaria toxins, were always found in traces. The occurrence of these metabolites seems to be influenced less by the considered agronomic practices.The results, obtained under naturally-infected field conditions, underline the key role that the sowing date and hybrid susceptibility play in influencing, in a variable way, the contamination of mycotoxins produced by different Fusarium species in maize subjected to different meteorological conditions. The content of mycotoxins produced by Fusarium spp. section Liseola is more directly and steadily related to late sowing time, while the contamination of mycotoxins associated to Fusarium spp. section Discolor depends more strongly on the environmental conditions at maize flowering and on hybrid susceptibility.

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The European corn borer (ECB) plays an important role in promoting Fusarium verticillioides infections and in the consequent fumonisin contamination in maize grain in temperate areas. The objective of this study was to evaluate whether the ECB feeding activity could also affect the occurrence of emerging mycotoxins in maize kernels. During the 2008-10 period, natural infestation of the insect was compared, in field research, with the protection of infestation, which was obtained by using an entomological net. The ears collected in the protected plots were free from ECB attack, while those subject to natural insect attacks showed a damage severity that varied from 10% to 25%. The maize samples were analysed by means of an LC-MS/MS-based multi-mycotoxin method, which led to the detection of various metabolites: fumonisins (FUMs), fusaproliferin (FUS), moniliformin (MON), bikaverin (BIK), beauvericin (BEA), fusaric acid (FA), equisetin (EQU), deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-G), zearalenone (ZEA), culmorin (CULM), aurofusarin (AUR) and butenolide (BUT). The occurrence of mycotoxins produced by Fusarium spp. of Liseola section was affected significantly by the ECB feeding activity. The presence of ECB injuries increased the FUMs from 995 to 4694 µg kg(-1), FUS from 17 to 1089 µg kg(-1), MON from 22 to 673 µg kg(-1), BIK from 58 to 377 µg kg(-1), BEA from 6 to 177 µg kg(-1), and FA from 21 to 379 µg kg(-1). EQU, produced by F. equiseti section Gibbosum, was also increased by the ECB activity, by 1-30 µg kg(-1) on average. Instead, the content of mycotoxins produced by Fusarium spp. of Discolor and Roseum sections was not significantly affected by ECB activity. As for FUMs, the application of a strategy that can reduce ECB damage could also be the most effective solution to minimise the other mycotoxins produced by Fusarium spp. of Liseola section.

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Cereals can be contaminated by several mycotoxins, whose co-presence may represent an undervalued risk for humans and animals. Maize and wheat are the most contaminated cereals and in temperate areas could be affected in field conditions by several Fusarium and Aspergillus infections. To date, only B-fumonisins (FBs), aflatoxins (AFs), zearalenone (ZEA), deoxynivalenol (DON) and T-2 and HT-2 toxins have been regulated in cereals in European Union. The other fungal metabolites, are commonly referred to as “emerging” and “masked” mycotoxins, and more information on their occurrence in combination with the regulated mycotoxins, are needed to design combined toxicological and exposure assessments. This research intends to develop and compare two multiresidue HPLC-ESI-TQ-MS/MS methods for the simultaneous determination of the main regulated, emerging and masked mycotoxins in maize and wheat, among which: FB 1 , FB 2 , DON, ZEA, AFB 1 , AFB 2 , AFG 1 , AFG 2 , moniliformin (MON), deoxynivalenol-3-glucoside (DON-3-G), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), enniatins A, A 1 , B, B 1 (ENNA, ENNA 1 , ENNB, ENNB 1 ). The extraction was performed for both methods using a mixture of CH 3 CN/H 2 O/CH 3 COOH (79/20/1, v/v/v), while the dilution/purification was carried out through two different procedures: (1) by the “dilute-and-shoot” technique diluting 1:2 the filtered extract with CH 3 CN/H 2 O/CH 3 COOH (20/79/1, v/v/v) to reduce the matrix effect; (2) using the Oasis ® PRiME HLB clean-up columns. The analysis was carried out using CH 3 OH and H 2 O both acidified with 0.1% of CH 3 COOH as eluents. The injection volume was 20 μL and the flow rate 200 μL min -1 . The analysis of two reference material (maize and wheat), was performed to evaluate the trueness and precision of the two methods by matrix-matched calibration curves. For all the regulated mycotoxins analyzed by both methods, the range of recovery percentage established by the Regulation (EC) No. 401/2006 was respected, except for ZEA by using the Oasis ® PRiME HLB clean-up columns. Nevertheless, the results suggest that the Oasis ® PRiME HLB clean-up columns, could be a valid alternative to the dilute-and-shoot method, although an additional cost for the clean-up has to be considered. In conclusion, both two analytical methods considerably reduce the analytical time and costs and therefore result to be promising and applicable for high-th...