Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2006
Publication Date: 6/20/2006
Citation: Schroeder, K.L., Okubara, P.A., Tambong, J.T., Paulitz, T.C. 2006. Identification and quantification of pathogenic pythium spp. from soils in eastern washington using real-time pcr.. Phytopathology. Vol. 96: 637-647. Interpretive Summary: At present, total numbers of all Pythium spp. can be estimated with dilution plating, but techniques for accurately quantifying particular species of Pythium in the soil are non-existent. We developed species-specific primers for the 10 most abundant Pythium spp. in eastern Washington, using sequences from the ITS region. These primers can be used with quantitative real-time capillary PCR to measure the amount of DNA of each species from natural soil. This method has been tested in natural soils, and has detected up to 6 different species from a single sample. This technique will be useful for ecological and epidemiological studies; and will also provide a tool for growers to assess the risk of Pythium disease in fields.
Technical Abstract: A new technique for identification and quantification of Pythium spp. was developed. Current methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, there is a high level of variability inherent with dilution plating on agar media and counts may not accurately estimate populations. Both pathogenic and non-pathogenic species grow on selective media and some pathogenic species grow too slowly to be included in counts. In addition, methods for identification to species are labor intensive and difficult. Therefore, species-specific primers were designed from variable portions of the internal transcribed spacers of the rDNA of ten pathogenic species of Pythium commonly found in eastern Washington soils. These primers were used in combination with real-time PCR to develop a sensitive and specific technique for identification and quantification. Primers were screened for specificity using a collection of 90 Pythium isolates representing each of the ten species. Each primer set was found to be highly specific for only that species for which the primer was designed. In addition, a commercially available soil DNA extraction kit was modified for use with this system to quantify Pythium spp. directly from soil. Standard curves for quantification of P. debaryanum, P. irregulare and P. ultimum were developed for estimating populations of these species in soil based on the level of DNA amplification with real-time PCR, and preliminary tests in natural field soils were conducted.