Not only are Phytophthora, Pythium, and interesting Phytopythium species potentially devastating horticultural pathogens, they regularly present challenges in vitro. In this study, multiple established and novel methodologies were built upon to bolster researchers’ ability to quickly isolate, differentiate, and promote virulence of multiple oomycetes principally collected from symptomatic conifers and nearby water sources. These methods allow greater flexibility in generating clean mycelial cultures for genetic characterization, varying pathogen structures for use in novel bioassays (such as synchronized production of sporangia or zoospores), and ultimately inoculations to evaluate oomycide efficacy or make headway towards completion of Koch’s postulates for previously uncharacterized host: pathogen pairings. Phytopythium vexans (Pp. vexans) (n=8 isolates), three Phytophthora species / species complexes including P. cinnamomi (n=10), P. cryptogea / drechsleri complex (n=4) and P. humicola (n=2), as well as 5 tentative species of Pythium, were evaluated. Isolations took two forms, standard root sampling onto Phytophthora selective media (PARPH), or water-based sampling through a modified ‘baiting/trapping’ technique that utilized on-site collected water samples in the laboratory. The ‘baits’ were Cannabis sativa seed, Vigna radiata beans, and Rhododendron maximum leaves suspended in aerated water samples or slurry of silt/soil. Samples were evaluated on V8-agar (V8A), pea agar (PA), pea broth (PB), potato dextrose agar (PDA), cornmeal agar (CMA), and water agar (WA), each of which provided distinct morphological indicators and structures useful in diagnostic guides and in bioassays or inoculations. As is typical with all plant pathogens, the longer they remain in culture, the less virulent they may become. With oomycetes, this is compounded as the pathogen will often go into chlamydospore or oospore formation (long lived survival structures) which are not ideal for experimentation. Inclusion of germinated then surface sterilized (70% ethanol for 30s) Vigna radiata and Lupinus perennis sprouts into recently poured (still liquid) 1/8 clarified V8 juice agar (1/8 clV8A) provided a media capable of rejuvenating the pathogen due to presence of living roots and dynamic plant nutrients. This allows for more predictability of zoospore formation, especially if they are intended to be used with a time sensitive trials. In numerous incidences multiple species of Phytophthora and Phytopythium vexans isolates went into zoospore release simultaneously by utilizing these approaches in combination with resource starvation and culture washing with sterile distilled water. Taken together these approaches will greatly aid any researcher working with root disease oomycetes in culture.
