Direct colony PCR-SSCP for detection of multiple pythiaceous oomycetes in environmental samples

Colony PCR was developed for detection of pythiaceous species recovered on selective agar plates without DNA extraction. A minute amount of mycelia from a single colony was picked up with a pipette tip and added directly to the PCR mix as template for DNA amplification. Successful amplification was achieved in over 95% of the colonies recovered from plant tissues, irrigation water and soil with species-specific primers or oomycete ITS-1 primers. PCR was inhibited in the case of colonies emerging from unwashed pine bark potting mix plates. Direct colony PCR with ITS-1 primers combined with single-strand conformation polymorphism analysis (SSCP) was used to determine population levels of single and multiple species in plant and environmental samples. Application of this technique for disease diagnosis and monitoring pathogen sources was explored, and the potential for studying diversity and population dynamics of other cultivated microbial communities in the environment is discussed.