Epiphytic microflora and mycotoxin content in meadows–Is plant biodiversity affecting fungal contamination?

Results There were no differences observed between TMC found in monocultures and species-rich mixtures, average values measured were3.02x109 CFU g -1 and1.72x109 CFU g -1 (Table 3). Significantly higher amounts of both Saccharomycotina (yeasts) and other fungi (OF)were found in mixtures (yeasts 5.39x106 CFU g -1 and OF 4.82x106 CFU g -1) in comparison to monocultures (yeasts 4.81x104CFU g -1 and OF 2.44x105CFU g -1). The highest TMC present on the above-ground biomass was observed in perennial ryegrass (1.37x1010 CFU g -1). Furthermore, species-rich mixtures and ryegrass were more colonized by microorganisms than monocultures of tall fescue (Table 4). Significantly higher values were measured in Mixture II and III, there were no differences between Mixture I and fescues. The lowest microbial colonization was observed in ’Aprilia’ (4.09x107 CFU g -1). Highest number of yeasts was found in Mixture II (9.26 CFU g -1), lowest in ryegrass (7 500 CFU g -1), there were no significant differences between Mixtures I, Mixture III and the mono- cultures. Moreover, significantly higher amount of OF was found in Mixture II (10 915 151 CFU g -1) in comparison to the other variants. Lowest OF contamination was found in ’Aprilia’ (5.80x107 CFU g -1). In mycotoxin testing, average DON concentration was below the limit of detection in our monoculture samples, in mixtures it was 1.76 μg kg-1. ZEN levels of monocultures were signifi- cantly lower in comparison to species-rich mixtures, 0.42 μg kg-1 and 0.87 μg kg-1 respectively.

However, AFB1 levels were significantly higher in monocultures (0.05 μg kg-1) in comparison to mixtures (0.03 μg kg-1). No significant differences were found between ZEN and AFB1 levels of each individual var- iant (Table 5). Highest average ZEN levels were found in Mixture II (0.91 μg g -1), lowest in ’Kora’ (0.30 μg g -1). AFB1 levels were significantly lower in Mixture II and III in comparison to Mixture I, ryegrass and fescues. The highest AFB1 contamination was found in ’Aprilia’ (18.33 μg g -1), the lowest in Mixture III (4 μg g -1). Discussion Effects of acute or chronic mycotoxin toxicity can cause significant economic losses by decreasing feed efficiency, immune competence, and overall performance [13]. World annual crop losses of 1 billion metric tons have been estimated by Food and Agriculture Organization of the United (FAO) [14]. Hassan and Zhou [15] estimate that annual losses due to fungal infections and crop contamination can be as high as US$ 5 billion in the North America. Fur- thermore, these authors state that costs connected to mycotoxin occurrence in both food and feed will continue to rise. Marasas et al. [13] states, that estimated mean annual cost related to food and feed contamination by DON, aflatoxin and fumonisin damage in the USA is US$ 946 million, with over US$ 100 million for aflatoxins alone [16]. Infection of feed crops with mycotoxigenic fungal pathogens is a worldwide problem. The strict limits of aflatoxin concentration are defined for food in Commission recommendation

2005/925/EC to protect human health. In feed, the Directive 2002/32/EC on undesirable sub- stances in animal feed regulates maximum limits of aflatoxins in feed [17]. However, there are no actual limits set by the European Commission (EC) for fresh forage contamination in gen- eral. In case of AFB1, it may be due to low incidence of aflatoxins in the EU, however for other mycotoxins (ochratoxin A, DON, fumonisins, T-2 toxin, HT-2 toxin, ZEN and patulin) there are no limits either. On the other hand, Council Directive 95/53/EC recommends monitoring their occurrence in feed [18]. In the newer EC recommendation from 2006, guidance for maxi- mum concentration of several mycotoxins has been established; however, hay or fresh forage PLOS ONE Biodiversity and plant fungal contamination PLOS ONE | https://doi.org/10.1371/journal.pone.0288397 September 14, 2023 5 / 11