Isolation and DNA Extraction of Phytophthora from Four Wildland Soil Types in Northern California, Humboldt County.

Abstract

    Phytophthora spp., one of which causes sudden oak death, and other Oomycete pathogens have had major impacts on forests on both the east and west coasts of the United States.  For most of the species of Phytophthora, it is not fully understood how these pathogens travel and spread long distances. They are known to travel by air, in water, nursery stock, and via inadvertent human transport.  Many species of Phytophthora reside in the soil.  The purpose of this study was to identify different species of Phytophthora and other Oomycota present in four different forest/wildland soil types.  Soils from franciscan complex parent material (riparian, mixed conifer, and oak woodland), and serpentine parent material (mixed conifer) were sampled in Humboldt County, Northern California.  Phytophthora was baited from each soil type with Port-Orford cedar and Rhododendron leaf disc baits and cultured on plates of clarified V8 agar mixed with ampicillin, rifampin, pentachloronitrobenzene (PCNB), hymexazol, nystatin, and pimaricin (PARPNH).  Phytophthora morphotypes were isolated and bacterial contamination was excluded using a modified Van Tieghem plate method. Hyphal DNA was extracted using both a NaOH extraction method and Qiagen Plant DNeasy kit to compare results.  DNA was then amplified using PCR with ITS (internal transcribed spacer) primer pairs ITS4-5, ITS 4-6, and the nested primer pairs Phyto1-4, and Phyto2-3.  DNA from both extraction methods were submitted for sequencing after PCR amplification. Initial sequencing produced weak and noisy results.  To improve results, better isolation of hyphae, when culturing, to insure there is only one organism needs to be further practiced and investigated.