Mycelia were collected by vacuum filtration and the total DNA isolation was performed using 50 mg of ground fungal material, as previously described [70 (link)]. The isolated DNA samples were used for PCR amplification of the ITS1-5.8S-ITS2 region of the Internal transcribed spacer (ITS) using the previously published primers, 18ITS1 [5′-GTCCCTGCCCTTTGTA-3′] and 28ITS2 [5′-CCTGGTGGTTTCTTTTCC-3′] [71 (link)].
PCR amplification reactions were performed with a KAPA Taq PCR Kit (KAPA Biosystems, Wilmington, MA, USA) in a PTC-200 Gradient Peltier Thermal Cycler (MJ Research, Waltham, MA, USA), according to the manufacturer’s instructions. The amplification protocol for the ITS region was: 3 min at 95 °C; 35 cycles of 30 s at 95 °C, 60 s at 48 °C, 2 min at 72 °C; and a final extension 5-min incubation at 72 °C. PCR amplicons were purified and cleaned using the PCR cleanup kit (NEB, Monarch PCR and DNA Cleanup Kit). All ITS amplicons were sequenced in both directions and assessed using the program SeqMan of Lasergene Suite 11 (DNASTAR Inc., Madison, WI, USA) [72 (link)]. The final sequences were deposited into GenBank. (Acc. Nos. OM993297–OM993326).
PCR amplification reactions were performed with a KAPA Taq PCR Kit (KAPA Biosystems, Wilmington, MA, USA) in a PTC-200 Gradient Peltier Thermal Cycler (MJ Research, Waltham, MA, USA), according to the manufacturer’s instructions. The amplification protocol for the ITS region was: 3 min at 95 °C; 35 cycles of 30 s at 95 °C, 60 s at 48 °C, 2 min at 72 °C; and a final extension 5-min incubation at 72 °C. PCR amplicons were purified and cleaned using the PCR cleanup kit (NEB, Monarch PCR and DNA Cleanup Kit). All ITS amplicons were sequenced in both directions and assessed using the program SeqMan of Lasergene Suite 11 (DNASTAR Inc., Madison, WI, USA) [72 (link)]. The final sequences were deposited into GenBank. (Acc. Nos. OM993297–OM993326).