Q5 pcr master mix

For species assignment, we extracted genomic DNA from 40 mg of mycelium freshly scraped from an agar plate, using the Nucleospin Microbial DNA kit (Bioké, Leiden, the Netherlands) in combination with NucleoSpin Bead Tubes Type C (Bioké, Leiden, the Netherlands) for tissue disruption in a MM 301 vibratory mill (Retsch GmbH, Haan, Germany). The extraction was carried out according to manufacturer’s instructions except for the disruption time, where 2 cycles of 1 min each were used. Next, a PCR reaction was performed with 1 µL of extracted gDNA and the primers ITSF1 and ITS4, in a thermal cycler with a 2 × Q5 PCR master mix (New England Biolabs, Ipswich, MA, USA). The following program was used: 1 min at 98 °C, 30 cycles of 10 s at 98 °C, 15 s at 55 °C, 20 s at 72 °C, 5 min at 72 °C. The PCR product was visualized on agarose gel, purified, sequenced, and analyzed with BLAST as described above. The 25 best hits and the ITS sequences of other 18 Anthostomella strains obtained from the NCBI nucleotide database were compared by constructing a maximum-likelihood phylogenetic tree using MEGA 11 [90 (link)]. First, the sequences were aligned with the MUSCLE algorithm with default settings [91 (link)], then the tree was built using standard settings and number of bootstrap replications of 100.

For standard PCR amplification, a 25 μl reaction containing 1–10 ng genomic DNA, 2× Q5 PCR master mix (M0543L, New England Biolabs, USA), 0.4 µmol/l of appropriate forward and reverse primers and nuclease-free water were run on the BioRad C1000 Touch Thermal Cycler (BioRad, USA) using optimised cycling conditions with an initial step of 98°C for 30 s, followed by 30–35 cycles of 98°C for 10 s, 64–68°C (depending on the primer pairs) for 15 s and 72°C for 30 s to 1 min, with a final extension at 72°C for 2 min.