Fungal Identification: Phenotypic and Molecular Protocols

Firstly, the obtained fungi were identified using classic phenotypic methods according to available monographs and articles [33 (link),34 (link),35 (link),36 (link),37 ,38 (link),39 (link),40 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 ]. In the next step, a molecular analysis of the obtained fungal cultures was performed. For this purpose, DNA was extracted from a 28-day-old culture on PDA using Bead-Beat Micro AX Gravity (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s instructions. Fungal rDNA was amplified using two fungal-specific PCR primers: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [47 ]. PCR was performed in a T100 Thermal Cycler for 35 cycles (Bio-Rad, Berkeley, CA, USA) according to Ogórek et al. [48 (link)]: after initial denaturation for 5 min at 94 °C, each cycle comprised 30 s denaturation at 94 °C, 30 s annealing at 55 °C, 45 s extension at 72 °C with a final extension for 7 min at 72 °C at the end of 35 cycles. The fungal internal transcribed spacer regions were verified by electrophoretic separation on a 1.2% agarose gel, subsequently purified using Clean-Up Kit (A&A Biotechnology, Gdańsk, Poland), and sequenced at Macrogen Europe (The Netherlands, http://dna.macrogen.com/eng/, accessed on 12 May 2021).