Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions

Assessment of fungal diversity in environmental samples is currently a challenge. Several recently developed molecular methods offer new avenues for determining the presence and diversity of fungi in complex microbial communities. Terminal restriction fragment (TRF) pattern analysis was tested as a method for assessing the fungal molecular diversity of a terrestrial microbial community. Community DNA was isolated from sand samples taken from a pilot-scale petroleum-contaminated land treatment unit. PCR amplification was carried out using primers, one of which was fluorescently labeled, designed to hybridize to conserved sequences in the fungal ribosomal small subunit (l8S) or the internal transcribed spacer ITSI-5.8S-ITS2 (ITS) ribosomal region. Amplicons were then digested separately with HpaII or HaeIII; fluorescently labeled TRFs were detected by capillary gel electrophoresis. ITS region TRF patterns were predicted and observed to generate a greater richness than 18S TRF patterns. Unique TRF patterns were also observed for each community examined. Finally, the ITS region showed a higher degree of specificity in matching observed TRF profiles to those generated from GenBank sequence data for species identification. These data suggest that ITS rDNA TRF pattern analysis has great potential as a rapid and specific method for fungal community analysis and species identification