*2.4. Isolation of Fungal DNA and PCR Amplification*

Fungal DNA was isolated directly from grain with the Bead-Beat Micro AX Gravity Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol. The quantity and quality of the isolated DNA were tested by measuring absorbance at 260 and 280 nm (NanoDrop 2000, Thermo Scientific, Wilmington, DE, USA). A metagenomic analysis of the fungal community was carried out in the ITS2 hypervariable region. The selected region was amplified and the library was prepared with the use of three specific primer sequences: fITS7 (GTGARTCATCGAATCTTTG), ITS4 (TCCTCCGCTTATTGATATGC) and an additional adapter sequence at the 5' end. PCR was conducted with the Q5 Hot Start High-Fidelity 2X Master Mix under the conditions recommended by the manufacturer. The Nextera Index Kit was used to add specific index adapter sequences to both ends of the analyzed DNA fragment.

## *2.5. Illumina MiSeq Sequencing*

The samples were sequenced in the Illumina MiSeq system (Poland) in paired-end (PE) mode, 2 × 250 nt, with the Illumina v2 kit (Genomed S.A., Warsaw, Poland). A preliminary analysis of the results was performed automatically in the MiSeq system with MiSeq Reporter (MSR) v2.6 software (Illumina, USA). The analysis was conducted in two steps: (1) automatic demultiplexing of samples, and (2) generation of fastq files with raw read data. A bioinformatics analysis with operational taxonomic unit (OTU) picking was conducted in the QIIME (Quantitative Insights Into Microbial Ecology) program based on the reference sequences in UNITE v7 [38]. The bioinformatics analysis was conducted in the following steps: (1) analysis of read quality and removal of low-quality sequences (quality < 20, minimal length—30)—cutadapt, (2) joining pair-ended sequences—fastq-join, (3) clustering based on a selected database of reference sequences—uclust, (4) removal of chimeric sequences with the usearch61 algorithm [39], and (5) taxonomic identification based on the UNITE-BLAST [40].
