A polymerase chain reaction and denaturing gradient gel electrophoresis procedure for analysis of arbuscular mycorrhizal fungi in soil
Arbuscular mycorrhizal fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Traditional spore morphology-based identification of AMF in biodiversity studies is subjective and requires expertise and time. Researchers have used molecular techniques to investigate community composition of AMF in uncultivated, disturbed, or contaminated soils, but this approach to community analysis of AMF in agricultural soils has not been reported. In this study, a polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) procedure for the detection of fungal 18S ribosomal RNA gene was developed with reference cultures. Five AMF species were procured from the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these INVAM cultures. The method’s technical limitations were investigated. The optimized procedure’s effectiveness was tested by its application to soil samples from 38 farms. Bands from the PCR-DGGE profiles of these samples were excised for sequence analysis. The total number of species recovered was low in comparison to other AMF community surveys of temperate climate locations. The majority of the sequences recovered were Glomus species. Scutellospora calospora, a previously undetected AM fungus in Saskatchewan was found. A trend in AMF distribution in Saskatchewan was observed and it was relatable to their phylogenetic taxonomy. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods.
PCR, AMF, DGGE
Master of Science (M.Sc.)