Molecular characterization of Pythium species based on RFLP analysis of the internal transcribed spacer region of ribosomal DNA

The extent of variation in restriction sites within the spacers of the ribosomal RNA gene was determined for 36 important plant pathogenicPythiumspecies. The extent of intraspecific variation of twenty-two isolates representing threePythiumspecies collected from diverse hosts and geographical origins was also investigated. The ribosomal DNA internal transcribed spacer (ITS) regions were amplified by the polymerase chain reaction and digested with seven restriction enzymes. Length variations detected in this region ranged from 701 to 1103 base pairs. With a small number of exceptions, species could reliably be distinguished by restriction fragment length polymorphism (RFLP) analysis using four enzymes:CfoI,HinfI,MboI, andTaqI. Intraspecific variability was low. Data presented do not support the maintenance ofPythium aphanidermatumandPythium butlerias distinct species. Inferred relationships based on RFLP analysis of ITS regions were compared with those based on other characters; species with filamentous sporangia and those with globose sporangia were divided into two main branches. Sporangial form appears to be an important character. The correlation between molecular clusters and subgroups defined on the basis of sporangial form suggests an ancient differentiation of this characteristic.

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