2.2 Purification and identification of fungi
The isolated colonies were transferred to tubes containing Sabouraud agar plus chloranphenicol. The isolated colonies were purified on Petri plates containing potato dextrose agar (PDA) incubated at 28 ºC±2 for 7 days. Pure cultures were transferred into tubes containing PDA. These pure cultures were stored in a refrigerator at 4 °C, with mineral oil and sterile distilled water. The isolated fungi were identified to the genus level by assessing the characteristics micro morphological characteristics using slide cultures [19].
The species of interest were identified via sequencing of the rDNA ITS region. Fungal DNA was extracted from mycelium the phenol:chloroform:isoamyl-alcohol method [20]. PCR reactions (3 µL of buffer (1X), 1.2 µL of MgCl2 (2.5 mM), 3 µL of DNTPs (200 µM), 1.5 µL ITS1 (5’- TCCGTAGGTGAACCTGCGG- 3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC- 3’) [21] and ultrapure water to a final volume of 30 μL) were carried out in a thermocycler (SuperCyclerTM SC-200, Kyratec) using the following amplification conditions: 94 ºC/5 min, 30 cycles of 94 ºC/30 s, 53 ºC/30 s, 72 ºC/1 min and 72 ºC/10 min. PCR products (8 µL) were visualized under UV light after electrophoresis (3 μL of SYBR® Safe (Invitrogen), 2 μL of Orange 6X (Fermentas), 10 µL 100-bp DNA Ladder, 40 min/100 V/100 A) on a 1.5% agarose gel in 1x TBE buffer. PCR products were purified via precipitation with polyethylene glycol (20% w/v PEG, 2.5 M NaCl). The sequencing reaction was performed using a commercial kit (BigColorant®, Applied Biosystems). Sequencing was carried out in a genetic analyzer (AB3130, Applied Biosystems). The sequences obtained were analyzed using BioEdit Sequence Alignment Editor and compared to those in the GenBank database. A phylogenetic tree was constructed using the program MEGA X v10.2.4 to verify the genetic and evolutionary relationship among the sequences.