The amplification of the hypervariable ITS3–4 region of the ITS rRNA gene was carried out using fungal primers set ITS3F (GCATCGATGAAGAACGCAGC) and ITS4R (TCCTCCGCTTATTGATATGC) [76]. The PCR reactions were carried out in a 50 μL mixture with 1 mM dNTPs, 1× PCR buffer, 1 U Platinum Taq, 5 μM per primer, and 10 ng of template DNA. The PCR amplification included an initial denaturation at 94 °C for 3 min, denaturation (5 cycles at 94 °C) for 30 s, annealing at 45 °C for 20 s, extension at 65 °C for 30 s, denaturation (20 cycles at 94 °C) for 20 s, annealing at 55 °C for 20 s, extension at 72 °C for 30 s and a final extension at 72 °C for 5 min. After purification and quantification, the PCR product of the ITS3–4 region of the ITS rRNA gene was determined by pyrosequencing using an Illumina MiSeq sequencer (Sangon Biotech Shanghai Co., Ltd., China) [18, 77].

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