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

genera Alternaria, Phytophthora, Sclerotinia, Bipolaris, Rhizoctonia and Fusarium, while Fusar- ium oxysporum being one of the most important pathogens of legumes [32, 33]. Accensi et al. [34] compared three types of feed samples and found that higher occurrence of Aspergillus spp., which also include the main producer of aflatoxins Aspergillus flavus, was detected in the legumes (94.4%) in comparison to mixed feed samples (89.8%) and cereals (57.3%) [35]. Their findings suggest that higher AFB1 concentration would potentially occur in species-rich mix- tures containing legumes than in grass monocultures. However, this was not in accordance with the results obtained in this experiment. AFB1 concentration were significantly higher in monocultures than in mixtures containing legumes. Furthermore, mixtures that contained lower percentage of legumes (Mixture 2 and Mixture 3) did not differ from high legume Mix- ture 1 in the concentration of AFB1. Crops have a different level of susceptibility to infection by mycotoxigenic fungi and conse- quently to mycotoxin contamination [36]. Presence of mycotoxins in key fodder crops such as maize as well as in small grain cereals and legumes e.g. pea and soy is well known [21, 37, 38]. Infection by toxicogenic fungi depends on many abiotic factors, such as weather conditions, nutrient deficiency or excess, and biotic factors, such as insect damage. All of these factors pre- dispose plant biomass for contamination in field [39]. Late forage harvest may lead to higher

contamination by microscopical fungi and thus to higher levels of mycotoxins [22, 39]. This is in accordance with the study of Schenck et al. [40], where OF counts were higher in samples with higher dry matter content. It is possible to assume that species-rich mixtures will be usu- ally harvested later than grass monocultures, so that they can fulfill their ecosystem services. This hypothesis may partially explain the higher concentration of DON and ZEN in mixtures in comparison to monocultures. However, it does not support this hypothesis in terms of AFB1 contamination. Plant genotype is a factor affecting the phyllospheric microbial communities differently, depending on the observed microbial group [41]. When evaluated, forage crops grown in agro- forestry systems showed similar levels of fungal colonization and subsequent mycotoxin con- tamination as traditional monocultures. This may indicate the importance of species-rich stands in terms of ecosystem services, without significant negative effects on the fungal con- tamination of the final product [9]. In our study, there were significant differences among spe- cies-rich mixtures in terms of microbial counts. Even between Mixture II and III, where the species composition was the same, some differences were found. The abundance of species in the mixtures differed, however. Due to this we hypothesize, that the reason is that one or mul- tiple components, that are highly colonized by yeasts and fungi and, might have caused high CFU counts in the experiment.

Plant defense mechanisms may be affected by amounts of specific lipids, such as sphingoli- pids in cell membrane [42] or cell wall [43]. Zachariasova et al. [21], however, state that maize leaves and corncobs contain higher amounts of protein and polysaccharides than grass, there- fore they provide suitable conditions for surviving and spreading of fungi and other microor- ganisms. Moreover, in the study of Venslovas et al. [38], negative correlation between crude protein and crude fiber levels as well as ZEN concentration in silage samples was found. Higher percentage of protein-rich components occur in legumes species. This may be one of the factors responsible for higher OF counts as well as DON and ZEN contamination in mix- tures, where legume component was present, in comparison to monocultures. This theory, however, does not explain why Mixture 2 has higher OF counts in comparison to Mixture 1, where the legume component has higher percentage overall. This points to other factors affect- ing mycotoxin production. Similarly to our experiment, Schenck et al. [40] did not find any correlation between organic nutrients and OF counts or mycotoxin concentrations. On the PLOS ONE Biodiversity and plant fungal contamination PLOS ONE | https://doi.org/10.1371/journal.pone.0288397 September 14, 2023 8 / 11