Fungal Diversity in Soils as Assessed by Direct Culture and Molecular Techniques

It has been consistently reported that bacterial diversity in a given environment as determined by culture techniques represents a small fraction of the total bacterial diversity detected by molecular methods. This may be attributable to a variety of related factors, such as: the limitations of the culture techniques employed; complex interdependencies between microorganisms in soil that cannot be replicated in the laboratory; and the presence of bacteria in dormant or “viable but non-culturable” states. We hypothesize that a similar situation exists with regard to soil fungi. Traditional culture methods were used to cultivate fungi from different soil samples. In brief, 10% (w/v) soil was dispersed in phosphate buffered saline containing 0.02 % sodium dodecylsulfate, agitated for ten minutes and the resulting liquid phase was used to prepare dilutions for cultivation of fungi. Concurrently, the DNA from each soil was extracted using the Qbiogene Soil DNA Extraction Kit and a portion of the rRNA gene was amplified by the polymerase chain reaction (PCR) using fungal specific primers. The PCR products were ligated into the pCR4Blunt-TOPO vector (Invitrogen) and cloned into E. coli. Individual clones were examined by restriction fragment length polymorphism (RFLP) analysis to identify unique patterns. The number of fungal species that could be cultured from various soils ranged from three to fifteen. However, the total fungal diversity of the sample, as assessed by the number of unique RFLP patterns, varied significantly from the number obtained by direct culture.