Location: Emerging Pests and Pathogens
Project Number: 8062-22000-019-09
Start Date: Sep 20, 2010
End Date: Sep 19, 2015
Objective 1. Specific primers and highly sensitive assays will be designed for known and new Pythium and Phytophthora spp. DNA sequences of target genes will be retrieved from the National Center for Biotechnology Information (NCBI) Genbank or newly generated by DNA sequencing, aligned, and analyzed. Primers will be designed by using validated thermodynamic parameters through a Web-interface pathway. Morphological characters will be used to validate molecular identifications where applicable. Studies under this objective will involve informal collaboration with M. Daughtrey of Cornell University. Objective 2. Sampling protocols will be developed to optimize pathogen detection according to facility size and substrate type (plant tissues, water, soil, or potting mixes). Oomycete isolates collected from greenhouses and nurseries in OK and NY will be identified to species level morphologically. Small portions of roots containing oospores will be plated onto selective media and grown at 23C for 3-7 days. When necessary, reproductive structures will be produced in water-grass cultures. Soil aliquot samples from pots will be plated on selective media. Collection of Pythium and Phytophthora strains from water samples will be obtained from each facility water source and fertilizer holding tank. Water samples will be processed using a novel reverse osmosis water filtration protocol. Preliminary tests have demonstrated the utility of this protocol for detection/recovery of Pythium aphanidermatum by growers (Garzon et al. unpubl.). Commercial kits for DNA purification from plant tissues, mycelium, and soil samples will be used as recommended by the manufacturer. Protocols will be modified or replaced by standard protocols as needed. Objective 3. Novel primers designed in objective 1 and DNA sequencing of the ITS region will be used to identify strains to species level using species-specific primers. Diversity of Pythium and Phytophthora spp. in each facility will be recorded. Strains with ambiguous identification will be characterized thoroughly by DNA sequencing of multiple loci (ITS, cox I-II, hsp 90, TigA, and Beta-tubulin) and compared to sequences available in the NCBI database. New species will be characterized and reported. Predominant species in each facility will be identified using three types of molecular markers: SSR, AFLP and ISSR. Data analyses will be conducted using population genetic software. Genetic structure and diversity of pathogen populations will be characterized, dominant genotypes will be identified, and their location in facilities will be mapped. The genetic profiles of dominant strains will be compared between facilities to identify common genotypes (lineages). Population genetic information will be combined with information on crop management in different facilities, and possible sources of inoculum and means of long distance movement will be identified. ARS Objective. Laboratory assays and small-scale greenhouse tests conducted by collaborating USDA-ARS researchers in Ithaca, NY will elucidate the nature of Bradysia fungus gnat/Phytophthora associations and the role of fungus gnats in Phytophthora disease outbreaks.