2.2. Fungi identification

Genomic DNA was extracted from two sample of fungal mycelium (200 mg) by the CTAB (Hexadecyltrimethylammonium bromide) method [19]. The integrity of DNA was observed on a 0.6 % (wt/vol) agarose gel. The DNA concentration was measured (Nano Vue, GE). Amplification of the ITS ribosomal region was performed following the protocol [20]. The primers used for PCR were ITS4 (reverse) and ITS5 (forward) described in [21]. In this experiment, a negative control was created using water instead of genomic DNA. PCR products were run on a 1.2 % agarose gel and stained with ethidium bromide for visualization on a UV transilluminator (Vilber Lourmat). The negative control was visualized without amplicons. Three samples were sequenced using an automated Sanger sequencer (AB1 3100, Applied Biosystems) and a Big Dye cycle sequence kit (Applied Biosystems). A consensus sequence was subjected to nBLAST to retrieve type material sequences. The related sequences were aligned with the consensus sequence in CLUSTAL Omega; Phylip format and the sequences’ global alignment were cut in Phylogeny FR, Gblocks. The Phylip archive was used to construct a distance tree using the Kimura algorithm (2000 replicates). The phylogenetic tree was visualized in FigTree 1.4.2.