Improved DNA extraction from field soils for a highly sensitive quantification of Phomopsis sclerotioides by real-time PCR. Jpn. J. For highly sensitive quantification of Phomopsis sclerotioides DNA from naturally infested watermelon field soils by real-time PCR, we improved a DNA extraction method and evaluated the extraction efficiency and the inhibitory effect of the soil extracts on the PCR. We used a FastDNA Spin Kit for Soil (MP Biomedicals) to examine the effect of pretreatment of

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... soils, conditions for soil beating, and purification of soil extracts to obtain better quantitative values for the DNA of the fungus. Pretreatment with shaking and grinding the soil increased values to twice those obtained without the pretreatment, and optimal conditions for bead-beating of fungal cells in soil and subsequent purification to obtain higher values were determined. Use of this optimal extraction method increased quantification sensitivity over the previously reported method; the fungus was quantifiable from soils containing over 1 cfu/g fresh soil. For field soils, DNA quantities were adjusted based on extraction efficiencies of internal standard DNA because the extraction efficiency differed depending on the soil samples. These adjusted values were highly positively correlated with disease severities in cucumber seedling tests. Such DNA quantification of a pathogenic fungus from soil using real-time PCR and this optimal extraction method with an internal standard DNA should be useful for assessing the disease potential of field soils.