*2.3. Isolation of Fungi from Grain*

Grain was harvested in the over-ripe stage (BBCH 92) with a plot harvester on 12 August 2016. Fungal colonization of grain was analyzed, and fungal DNA was isolated immediately after harvest. Grain samples of 10 g each were placed in 250 cm3 flasks containing 90 cm3 of sterile water and 0.01 cm3 of Tween ®40 (Merck, Darmstadt, Germany). The flasks were shaken for 60 min on an Elpin Plus 358 S table shaker (180 rpm, Elpin Plus, Lubawa, Poland) to remove microorganisms from grain. Using a pipette, 0.1 cm<sup>3</sup> of the propagule suspension was transferred to Petri plates with a diameter of 9 cm and flooded with selective Martin medium [35] cooled to 42 ◦C. The experiment was conducted in four replications. Yeasts and filamentous fungi cultured on the Martin medium were incubated at 24 ◦C in darkness for 7 days (En 120 Incubator, Nuve, Ancara, Turkey). Yeast and fungal colonies were counted on plates, and different colonies of filamentous fungi were transferred to Petri plates filled with potato dextrose agar (PDA, Merck, Warsaw, Poland) for species identification under a microscope. The number of colony forming units (CFUs) was log-transformed (CFU+1). One hundred disinfected and non-disinfected kernels from each treatment were placed on PDA. Kernels were disinfected by

immersion in 1% sodium hypochlorite (NaOCl, ABO, Gda ´nsk, Poland) solution for 5 min and they were then rinsed three times in sterile water and dried on blotting paper. Colonies of filamentous fungi were identified at the species level based on the sporulation characteristics described in the literature [36,37].
