The purpose of this study was to clarify the genetic diversity of Phytopythium helicoides and to understand the transmission mode of the pathogen in Japan. In total, 232 P. helicoides isolates were collected from various host plants and geographic origins, including farms and natural environments. We developed 6 novel microsatellite markers for use in the study and found 90 alleles among the 6 markers in the 232 isolates. The analysis of molecular variance suggested that P. helicoides has high variance within individuals and low fixation indices between populations. A phylogenetic analysis revealed that isolates collected from the same hosts and/or geographic origins were often grouped together. For example, several isolates from natural environments were grouped with isolates from nearby agricultural areas. On the other hand, 2 geographically distant populations collected from the same host plant had similar genotypes. Our results suggested that migration of the pathogen could be facilitated naturally via drainage systems or by human activity in the transport of agricultural materials.

A strategy combining dual-suppression PCR and thermal asymmetric interlaced PCR was used to determine sequences flanking microsatellite regions in Pythium helicoides. The primer pairs were designed to amplify loci containing (AC)n, (GA)n, (AGC)n, (CAC)n(CAA)n, (TCA)n and (CTTT)n repeats from the P. helicoides nuclear genome. The PCR products of each primer pair, amplified from three representative isolates collected from different hosts and locations, were cloned and sequenced. Different degrees of polymorphism were detected among these microsatellite markers. The numbers of alleles were 6, 2, 4, 11, 4 and 4 in YL-AC, YL-AGC, YL-CAA, YL-CTTT, YL-GA and YL-TCA, respectively. Allele analysis of 30 P. helicoides isolates showed length polymorphisms in all loci, except for YL-AC, using capillary electrophoresis. Thus, we have developed a simple method for designing PCR primers to amplify microsatellite markers from P. helicoides.