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

⚬ Mixture 3: 90% grasses + 3% legumes + 7% forbs Botanical composition of the mixture was species-wise the same as the botanical composi- tion of experimental plots after two years after the establishment (during the sampling). Weed species that were found on experimental variants can be found in Table 2. The overall weed species’ percentage was <1% in monocultures and Mixture 1. The weed species’ percentage was <5% in Mixtures 2 and 3. Therefore their effect on the experiment can be considered negligible. All analyses were done in specialised laboratory at Mendel University in Brno (49˚21’ N, 16˚61’ E). For microbial analysis 10 g of fresh biomass were used. The sample was homoge- nized in sterile polyethylene bags on STOMACHER (Interscience, France) with 90 ml of sterile saline for 1 minute. From the inoculum a series of 10-fold dilutions were prepared. Following groups of microorganisms were cultivated: i) total microbial count (TMC) on Plate Count Agar at 30˚C for 72 h; ii) fungi (yeasts and other fungi) on Chloramphenicol Glucose Agar at 25˚C for 120 h. Cultivation media were prepared according to the producers’ instructions (BiokarDiagnostics, Allone, France). ColonyStarcolony counter (Funke Gerber, Berlin, Ger- many)withpressure-sensitive automatic counter and illuminated counting platewas used for countingof CFUs(colony forming units).The result was expressed asa number ofCFUsper gram offreshsample. Biomass samples for mycotoxin analysis were prepared by drying fresh biomass at 60˚C.

Pulverisette laboratory cutting mill (Fritsch, Idar-Oberstein, Germany) for preparation of sam- ples (<1 mm particles) was used. ELISA was used for the quantitative analysis of mycotoxins. Samples were prepared according to the test kits’ instructions (MyBioSource, San Diego, CA, USA). Kits’ detection limits were 0.03 μg kg-1 for deoxynivalenol (DON), 0.18 μg kg-1 for zear- alenone (ZEN) and 0.03 μg kg-1 for aflatoxin B1 (AFB1). Wavelenght in Synergy HTX Multi- Mode Microplate Reader (BioTek, Winooski, VT, USA) was adjusted to 450 nm. Concentra- tion of DON, ZEN and AFB1 was measured. Fig 1. Average monthly temperature and precipitation in the experimental location. https://doi.org/10.1371/journal.pone.0288397.g001 PLOS ONE Biodiversity and plant fungal contamination PLOS ONE | https://doi.org/10.1371/journal.pone.0288397 September 14, 2023 3 / 11