Refined PCR protocol for detection of plant pathogens in soil

Kageyama, Koji; Komatsu, Tsutomu; Suga, Haruhisa
June 2003
Journal of General Plant Pathology;Jun2003, Vol. 69 Issue 3, p153
Academic Journal
A polymerase chain reaction method to detect soil-borne plant pathogens such as Pythium ultimum, Plasmodiophora brassicae, and Verticillium dahliae in soil was developed and used with a range of soil textures. DNA extraction was based on lyses with alkali buffer (pH 9.0) containing sodium dodecyl sulfate, extraction with benzyl chloride, and collection by ethanol precipitation. Four modified protocols � purification of extracted DNA from soil, independent addition of glass beads or skim milk to the extraction buffer, and addition of bovine serum albumin (BSA) to the PCR reaction mixture � were evaluated to enhance the sensitivity of detection. The effectiveness varied for three tested pathogens, but none of the four protocols negatively affected PCR detection. DNA purification was essential for detecting the three tested pathogens. Physical disruption with glass beads and the addition of BSA to the PCR reaction mixture were necessary for detecting V. dahliae, and the addition of skim milk was needed for Pl. brassicae. Additions of BSA and skim milk enhanced the detection of P. ultimum. The developed protocol seems applicable to the range of soil textures that are naturally inhabited by these three pathogens. By integrating multiple protocols to enhance sensitivity, PCR can be used to detect various soil microorganisms.


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