Abstract
In the current study, we describe a DNA isolation method that is based on an easy, quick polyvinylpolypyrrolidone-precipitation to release phytofungi from the soil, combined with lysozyme- RNase and -SDS lysis of the fungal population. DNA extracts were subjected to different techniques, including gel electrophoresis, restriction enzyme digestion, RAPD and ITS-PCR amplification. The proposed method yielded high-quality DNA, which was transparent, non-viscous and lacked visible contamination of RNA. Isolated DNA was efficiently digested with restriction enzymes. DNA extracted from soil was pure enough to be utilized at high concentrations for PCR amplifications. The extracted DNA was of high quality and allowed direct detection of specific genes by the polymerase chain reaction (PCR). The amplicon length of the fragment ITS4/ITS5, ranged in size from 550 to 680 bp. A polymerase chain reaction method used to detect soil-borne plant pathogens such as Fusarium spp, Rhizoctonia solani and Macrophomina phaseolina in the soil was developed and used with a range of soil textures. A direct method for the extraction of DNA from soil samples, which can be used for PCR-mediated diagnostics without a need for further DNA purification, was developed. The developed protocol seemed adequate to the range of soil textures that were artificially infested by a variety of soil-borne pathogens.