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

limits were not included [19]. In comparison to these EC recommendations, limits for DON (0.9–12 μg kg-1) or ZEN (0.25–3 μg kg-1) were not exceeded in this experiment. Interaction between several factors, such as year and plant density, may also affect myco- toxin content [20]. These interactions may explain differences between mixtures and mono- cultures. This is supported by the work of Zachariasova et al. [21], where DON, ZEN and aflatoxins were not detected in clover-grass forage samples. However, DON and ZEN did occur in hay. No differences in concentration of DON, ZEN and aflatoxins were found between perennial ryegrass monoculture and fescue-based mixture by Skladanka et al. [22]. Our results showed no significant differences between monoculture species and therefore are in agreement with their study. However, Baholet et al. [23] found significantly higher DON and ZEN contamination in samples of tall fescue than perennial ryegrass. These conflicting results may be caused by interannual variability that occurs in phyllosphere as well as myco- toxin contamination [24, 25]. Table 3. Average microbial colonization and mycotoxin contamination depending on diversity of botanical species in the sample. Mixtures (n = 9) Monocultures (n = 15) Mean value SD Mean value SD Total amount of microorganisms (log CFU g -1) 9.24 a 9.00 9.48 a 9.74 Saccharomycotina (log CFU g -1) 6.73a 6.65 4.68 b 4.59 Other fungi (CFU g -1) 6.68 a 6.69 5.39 b 5.44 Deoxynivalenol (μg kg-1) 1.76 a 1.31 <LOQ <LOQ Aflatoxin B1(μg kg-1) 0.03a 0.03 0.05 b 0.01

Zearalenone (μg kg-1) 0.87a 0.34 0.42 b 0.20 Results are expressed as a mean and standard deviation (SD). Mycotoxin concentration marked as <LOQ were under the limit of quantification (DON < 0.03 μg kg-1). Mean values are statistically significant at p < 0.05, when values in columns are marked with a different letter in the upper index. https://doi.org/10.1371/journal.pone.0288397.t003 Table 4. Average microbial colonization depending on botanical species. Total amount of microorganisms (log CFU g -1) Saccharomycotina (log CFU g -1) Other fungi (log CFU g -1) Festuca arundinacea ’Prostela’ (n = 3) Mean 8.13 a 4.35 a 4.92 a SD 6.66 2.83 4.22 Festuca arundinacea ’Aprilia’ (n = 3) Mean 7.61 a 4.53 a 7.61 a SD 7.13 3.50 3.06 Festuca arundinacea ’Philia’ (n = 3) Mean 8.80 a 5.01 a 5.50 a SD 8.10 4.33 4.86 Festuca arundinacea ’Kora’ (n = 3) Mean 8.79 a 4.87 a 4.99 a SD 8.07 4.46 2.36 Lolium perenne ’Promed’ (n = 3) Mean 10.14 d 3.88 a 5.82 a SD 8.64 3.53 5.54 Mixture I (n = 3) Mean 8.86 ab 5.95 a 6.13 a SD 7.66 5.34 5.10 Mixture II (n = 3) Mean 9.18 b 6.97 b 7.04 b SD 8.45 6.51 6.51 Mixture III (n = 3) Mean 9.47 c 6.78 ab 6.34 a SD 8.59 6.58 4.96 Results are expressed as a mean and standard deviation (SD). Mean values are statistically significant at p < 0.05, when values in columns are marked with a different letter in the upper index. https://doi.org/10.1371/journal.pone.0288397.t004 PLOS ONE Biodiversity and plant fungal contamination

PLOS ONE | https://doi.org/10.1371/journal.pone.0288397 September 14, 2023 6 / 11