PCR-denaturing Gradient Gel Electrophoresis as a Simple Identification Tool of Arbuscular Mycorrhizal Fungal Isolates

Abstract

Due to their obligate symbiotic nature and lack of long-term storage methods, the strain collection of arbuscular mycorrhizal (AM) fungi requires periodic proliferation using a pot culture with host plants. Therefore, a method to evaluate the purity of proliferated AM fungal cultures is critical for the quality control of their collection. In a simple evaluation of the purity and identity of a proliferated AM fungal culture, DNA extracted from the culture was amplified using AM fungi-specific PCR followed by an analysis with denaturing gradient gel electrophoresis (PCR-DGGE). The present results showed that the DGGE band patterns of AM fungal strains differed according to their phylogenetic positions, allowing for the rapid and easy identification of the proliferated AM fungal strains. When a culture was contaminated with another AM fungal strain, the DGGE pattern became a mixture of those strains. A contaminant strain was detectable even when its ratio was 1/9 of the main strain. It was also possible to confirm the purity of the culture by comparing whether the DGGE band pattern of the proliferated culture was identical to that obtained from single spores isolated from the culture. Therefore, PCR-DGGE is useful as a quality control tool for maintaining culture collections of AM fungi.

Rapid screening of UPB1 gene variations by high resolution melting curve analysis

“…Besides, molecular hybridization-based methods, such as dynamic allele-specific hybridization and oligonucleotide ligation assays, which also involve expensive sequence-specific probes, increases the cost of detection ( 18 , 19 , 26 , 27 ). Other PCR-combined molecular methods are also available, such as single-strand conformation polymorphism and denaturing gradient gel electrophoresis ( 28 , 29 ). These techniques have their own distinctiveness, but the experimental design is relatively complex.…”

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus

“…One day before inoculation, seedlings were transferred to long-day conditions (16 h of light and 8 h of dark). Sterilised soil (1:1:1 mixture of black soil: Akadama soil: river sand) for pot culture was prepared as described by Ohtomo et al 45 . Briefly, all soil was autoclaved at 100 ˚C for 60 min, only black soil was sterilised twice and air-dried for a month.…”

Arbuscular Mycorrhizal Fungi as an Important Factor Enabling the Adaptation of Anthyllis vulneraria L. to Zn-Pb-Polluted Tailings

“…The PCR-DGGE is a method that separates DNA fragments on sequence differences [39]. PCR-DGGE has been used to identify AMF or to assess fungal compositions in the soil environment [40][41][42][43][44][45]. The technique has also been useful as a quality assessment tool in the collection of beneficial AM fungi [40].…”