Molecular biology techniques were carried out as described elsewhere [13 (link)]. The B28 strain was cultivated on a Czapek medium at 30°C under continuous agitation at 200 rpm. Mycelia from 48-h cultures were harvested and DNA was extracted.
The internal transcribed spacer regions (ITS) were submitted to PCR amplification using fungus-specific primers, namely ITSl (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT G-3′). The amplification was initiated by incubating the PCR reaction mixture at 95°C for 5 min, followed by 35 cycles of denaturation for 30 s at 94°C. The reaction was annealed at 50°C for 30 s and terminated with extension consisting of 1 min at 72°C and final steps of 10 min at 72°C. The PCR products were analyzed on an agarose gel (1%) and purified by the Polyethylene Glycerol- (PEG-) NaCl method. The nucleotide sequences were determined using the Big-Dye Terminator v3.1 Cycle Sequencing Kit and the automated ABI Prism1 3100-Avant Genetic Analyser (Applied Biosystems). The sequence obtained for the intergenic region rRNA. The BLAST search program (http://www.ncbi.nlm.nih.gov/BLAST/ ) was used to look for nucleotide sequence homology.
The internal transcribed spacer regions (ITS) were submitted to PCR amplification using fungus-specific primers, namely ITSl (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT G-3′). The amplification was initiated by incubating the PCR reaction mixture at 95°C for 5 min, followed by 35 cycles of denaturation for 30 s at 94°C. The reaction was annealed at 50°C for 30 s and terminated with extension consisting of 1 min at 72°C and final steps of 10 min at 72°C. The PCR products were analyzed on an agarose gel (1%) and purified by the Polyethylene Glycerol- (PEG-) NaCl method. The nucleotide sequences were determined using the Big-Dye Terminator v3.1 Cycle Sequencing Kit and the automated ABI Prism1 3100-Avant Genetic Analyser (Applied Biosystems). The sequence obtained for the intergenic region rRNA. The BLAST search program (