Development of a total nucleic acid extraction method for direct detection of potato pathogens in soil (P-14)

Soil-borne plant pathogens present a considerable risk to potato production due to their ability to cause both qualitative and quantitative losses. Detection and quantification of these pathogens in a field prior to planting can help ensure successful disease management strategies are used. Key criteria for a successful detection assay are its ability to extract from a biologically relevant sample size, ease of use by the operator and overall cost to the end user. Here we present a method to extract total nucleic acid from 300 g of soil in under two hours to produce material suitable for analysis with real-time PCR (TaqMan). This method was advantageous as it recovered both RNA and DNA from a soil sample enabling viral targets (PMTV and TRV) as well as eukaryotic targets (Rhizoctonia, Spongospora, Streptomyces, Colletotrichum and Verticillium) to be quantified from the same nucleic acid extract. It was determined that the pH of the lysis buffer was a key determinant in nucleic acid yield. The method was used to test 157 soil samples from Idaho potato fields prior to planting for Spongospora, Streptomyces (txtA gene) and PMTV. Spongospora was detected in 80% of fields tested, Streptomyces in 72% and PMTV in 40% of fields. Coupled with accurate thresholds for disease development this could be a useful decision support tool for growers.