A PCR-DGGE method for detecting arbuscular mycorrhizal fungi in cultivated soils

Abstract

Arbuscular mycorrhizal (AM) fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Molecular techniques are used to investigate fungal community composition 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 of seven isolates (representing five AMF species). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in the province of Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these cultures. The effectiveness of the procedure was tested by its application to soil samples from 38 farms. Prominent 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. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods.