Development of a Large-Scale Soil DNA Extraction Method for Molecular Quantification of Fusarium oxysporum f. sp. fragariae in Soil.

Abstract

The most common soilborne diseases affecting the strawberry industry in California include Verticillium wilt due to Verticillium dahliae, charcoal root rot due to Macrophomina phaseolina, and Fusarium wilt due to Fusarium oxysporum f. sp. fragariae. Detection of these pathogens in soil is an important facet of disease management and fumigation recommendations. Whereas the soil populations of both M. phaseolina and V. dahliae can be readily quantified with quantitative PCR (qPCR) assays using DNA extractions with 500 mg of soil, the single-cell nature of the F. oxysporum chlamydospore does not provide enough pathogen DNA from 500-mg extractions to be reliably quantified. Here, we describe an improved DNA extraction protocol from 10 to 15 g of soil that allows for the quantification of F. oxysporum f. sp. fragariae populations below 10CFU/g. The relationship between results from the TaqMan qPCR assay and pathogen population density in soil was determined by using this extraction method in pathogen-free soils artificially infested with a hygromycin-resistant strain of F. oxysporum f. sp. fragariae to facilitate accurate colony counts when plated on a selective medium. Although the protocol was developed for F. oxysporum f. sp. fragariae, it is applicable for detection and quantification of other soilborne pathogens.