**1. Introduction**

Ensiling forage crops is a common way of ensuring a continuous and stable supply of feed throughout the year in dairy husbandry. These silages, mostly grass or maize [1], represent 50–80% of the diet of dairy cows during the winter [2]. Especially in North-Western Europe, fodder maize cultivation for on-farm use is an essential part of dairy husbandry [3]. In the region of Flanders, Belgium, more than 127,000 ha of silage maize was grown in 2018, making it the second most grown crop behind pasture [4].

Maize silages can be contaminated with mycotoxins, secondary metabolites produced by a variety of moldy fungi. Mycotoxins can cause several acute and chronic toxic e ffects to humans and animals when ingested.

In general, ruminants are less sensitive to mycotoxins than monogastrics due to the ability of the ruminal flora to degrade several mycotoxins to less toxic substances [5]. However, not all mycotoxins are degraded in this way. Some can be converted to molecules with a higher toxicity level (e.g., zearalenone (ZEN) to α-zearalenol ( α-ZEL)), while others are not even converted at all (e.g., fumonisins (FUMs)) [6–9]. Moreover, if both the rumen microflora and the pH are not stable (e.g., in calves, high-yielding cows or animals in the transition period), mycotoxin metabolism is reduced [10]. Therefore, dairy cattle are susceptible to mycotoxic e ffects as well, including gastroenteritis, reduced feed intake and reduced fertility [10–12], leading to economic losses [13].

Mycotoxins can be produced in the field (preharvest) as well as in the silage (postharvest). In preharvest field conditions within temperate regions, mycotoxins are mainly produced by *Fusarium* spp., causing maize stem and ear rot [1,14–16]. Two main types of maize ear rot can be distinguished: red ear rot (or *Gibberella* ear rot), primarily caused by *F. graminearum*, *F. culmorum* and *F. poae*, and pink ear rot (or *Fusarium* ear rot), primarily caused by *F. verticillioides, F. proliferatum* and *F. subglutinans* [17–20]. The distribution and prevalence of these *Fusarium* spp. is dependent upon geography and climate. In Europe, the most isolated *Fusarium* species are *F. graminearum* and *F. culmorum*, dominantly in the North, and *F. verticillioides*, mostly found in the South [19,21–23]. However, maize ear rot is always caused by a *Fusarium* complex, rather than by a single species [16,19,24]. Di fferent *Fusarium* spp. interact with each other, leading to possible synergistic e ffects for infection, although reports have been contradictory [25–29]. Di fferent pathways can be used by *Fusarium* spp. to infect maize plants, and while some *Fusarium* spp. prefer a primary infection via the silks (e.g., *F. graminearum*), others use a systemic transmission from root to kernel, or co-occur as a secondary infection when insects damage the kernels (e.g., *F. verticillioides*) [30–34]. This makes the prevention and control of *Fusarium* spp. in maize very di fficult and complex.

During infection, *Fusarium* spp. can produce a variety of mycotoxins. Some of the most well-known *Fusarium* mycotoxins include deoxynivalenol (DON), causing reduced feed intake and diarrhea; zearalenone (ZEN), causing fertility problems; and the fumonisins (FUMs), causing liver and kidney injuries. Other important *Fusarium* mycotoxins include nivalenol (NIV), T-2 toxin (T2), diacetoxyscirpenol (DAS) and enniatins (ENN), among others [11,12,35,36]. Mycotoxins produced by other fungal species, such as aflatoxins produced by *Aspergillus* spp., are rarely found in temperate climates [37–40]. However, climate change may influence the geographical spread of mycotoxin-producing fungi in Europe, causing more tropical fungi, such as *Aspergillus flavus* and *Fusarium verticillioides*, to migrate northward [23,41–46].

*Fusarium* spp. cannot survive postharvest silage conditions if the silage is firmly pressed and sealed hermetically, but *Fusarium* mycotoxins are stable molecules that may remain unchanged during the silage process [24,47–50]. If a silage is not pressed and sealed correctly and oxygen remains present, *Fusarium* spores may germinate and colonize the maize silage and produce additional mycotoxins [51–53]. Furthermore, other fungal species such as *Penicillium* spp. and *Aspergillus* spp. are well adapted to the silage conditions and may produce additional mycotoxins [47,54–57].

This cocktail of mycotoxins, coming from different fungal species, has led to the observation that almost every maize or maize silage sample is contaminated with at least one mycotoxin, and often multiple. Numerous surveys have been conducted in many regions in the world [22,24,37–40,48,54,58–62]. However, most of these surveys were conducted on samples of maize ears, rather than on the entire plant. Some reports state that the ear can be a representative sample for the entire plant [38], although the fungal species composition and mycotoxin concentrations can di ffer [16]. Furthermore, most surveys focused on a selection of mycotoxins, rather than screening the entire mycotoxin load. Severe multi-mycotoxin contaminations could hence be overlooked.

The European Union (EU) has set a maximum level for aflatoxin B1 (AFB1) [63] and guidance values for DON, ZEN, ochratoxin A (OTA), fumonisin B1 (FB1) and B2 (FB2), T-2 toxin (T2) and HT-2 toxin (HT2) in several food- and feedstu ffs, including maize [64,65]. No recommendations have been formed on lesser researched mycotoxins like NIV, modified mycotoxins like 3- or 15-acetyldeoxynivalenol (3-ADON and 15-ADON), or emerging mycotoxins like enniatins (ENN) [66,67].

Neither do these guidance values take into account any possible synergistic e ffects of multi-mycotoxin contamination [40,62,68–72]. As a result, one cannot assess whether a particular feed sample is safe, based on these guidance values alone [2]. A better strategy to safeguard livestock health would be to avoid fungal infection and the production of mycotoxins in the first place.

The aim of this research was to investigate the natural mycotoxin load in harvested maize plants intended for silage in the Northwestern European region of Flanders over the course of three years, and link these concentrations to the presence of certain mycotoxigenic *Fusarium* species. A total of 257 samples were taken from harvested maize fields across Flanders during 2016–2018. Samples were analyzed for 22 di fferent mycotoxins using a multi-mycotoxin liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Then, using a quantitative polymerase chain reaction (qPCR), the DNA of three of the most prevalent *Fusarium* spp. in Flanders, namely *F. graminearum, F. culmorum* and *F. verticillioides* [21,73,74], was quantified in the same maize samples. With these data, we were able to quantify the mycotoxin load of silage maize fields in practice, compare mycotoxin occurrence between di fferent years and weather conditions, and identify correlations between these mycotoxins and the corresponding *Fusarium* spp.
