A cost-effective protocol for molecular detection of fungal pathogens in soil
DNA EXTRACTION FROM FUNGI IN SOIL often fails because of humic substances that are co-extracted with the DNA and subsequently inhibit PCR analyses. Moreover, it is difficult to release the fungal DNA because of the diverse fungal structures residing in soil. Since available DNA extraction protocols and commercial kits are expensive or time-consuming, we have devised a superior method by testing different components of these procedures on grapevine nursery soils. The best DNA yield and sensitivity were obtained by a short and easy extraction method with sodium dodecyl sulphate buffer using the FastPrep homogenizer. An easy-to-prepare spin column with polyvinylpoly-pyrrolidone was developed to remove PCR inhibitors. In the presence of bovine serum albumin, PCR reactions were possible without further dilutions of the DNA. Our method was more sensitive for detecting Phaeomoniella chlamydospora, the organism responsible for Petri grapevine decline, and Cylindrocarpon black-foot pathogens in grapevine nursery soils than the FastDNA SPIN Kit for Soil and enabled us to perform 25 extractions for the price of one with the kit. This is the first report of molecular detection of Cylindrocarpon macrodidymum from soil and the first account of Pa. chlamydospora from soils in South Africa.