Phylogeny of the genus Pythium is analyzed based on sequences of the large subunit ribosomal DNA D1/D2 region and cytochrome oxidase II gene region of Pythium isolates and comprehensive species of related taxa belonging to the Oomycetes. The phylogenetic trees show that the genus Pythium is a highly divergent group and divided into five well-or moderately supported monophyletic clades. Each clade is characterized by sporangial morphology such as globose, ovoid, elongated, or filamentous shapes. Based on phylogeny and morphology, the genus Pythium (s. str.) is emended, and four new genera, Ovatisporangium, Globisporangium, Elongisporangium, and Pilasporangium, are described and segregated from Pythium s. lato.

We investigated virus infection in the oomycete Pythium polare from the Arctic. From 39 isolates investigated, 14 contained virus-like double-stranded RNA (dsRNA). Next generation sequencing revealed that the P. polare isolate OPU1176 contained three different virus-like sequences. We determined the full-length genome sequence of one of them. The 5397 nt-length genome had two overlapped open reading frames (ORFs) consistent with a toti and toti-like viruses, that we named Pythium polare RNA virus 1 (PpRV1). The ORF2 encoded an RNA-dependent RNA polymerase (RdRp). The shifty heptamer motif and RNA pseudoknot were predicted near the stop codon of ORF1, implying that the RdRp could be translated as a fusion protein with the ORF1 protein. Phylogenetic analysis with deduced RdRp amino acid sequences indicated that oomycete virus PpRV1 was closely related to the unclassified arthropod toti-like viruses. The comparison of PpRV1-free and -infected lines suggested that PpRV1 infected in a symptomless manner.

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It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.

The standard Pythium selective medium PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar), was modified by replacing PCNB and pimaricin with other antifungal agents. Several antifungal agents such as fluazinam, miconazole, 2,4,5,6-tetrachloroisophthalonitrile (TPN), iminoctadine triacetate, tolclofos-methyl, captan, and nystatin, initially were screened for effects on Pythium growth. Based on these results, the following three media were developed: PARF (pimaricin + ampicillin + rifampicin + fluazinam agar), NARF (nystatin + ampicillin + rifampicin + fluazinam agar), and NARM (nystatin + ampicillin + rifampicin + miconazole agar). New media were comparable with PARP on yield of naturally occurring Pythium spp. from two different types of soil using the soil-dilution plating technique. PARF and NARF were significantly better than PARP on inhibition of non-pythiaceous microbes on the soil-dilution plates, but were significantly lower than PARP on the rate of mycelial growth of six of eight isolates belonging to seven species of Pythium. NARM was equivalent to PARP on inhibition of non-pythiaceous microbes except for Fusarium oxysporum, and was significantly better than PARP on rate of mycelial growth of five of eight isolates of Pythium.