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.