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To develop a specific method for distinguishing and detecting Pythium species.Twenty PCR primers were designed from the sequences of the rDNA internal transcribed spacer 1 (ITS1) region from 34 Pythium species. The specificity of these forward primers paired with ITS2 or ITS4 and reverse universal primers was tested. Five species-specific primers were obtained, other primers showed different specificity to Pythium species. The specific amplifications enabled nine Pythium species to be differentiated. Specific detection of Pythium aphanidermatum from infested plants and P. dimorphum from soil was demonstrated.A method for identifying nine Pythium species using specific PCR amplification was achieved.Because of its rapidness and ease, the results of PCR amplified with different primers can be a powerful method for identifying Pythium species and detecting or monitoring the target fungus directly from plant material, soil and water samples.