Location: Emerging Pests and Pathogens Research2012 Annual Report
1a. Objectives (from AD-416):
Ornamental growers face diverse plant diseases that represent major management challenges. Companies produce multiple species and often multiple varieties of each crop, which may differ in susceptibility to plant pathogens and respond differently to agricultural practices. Furthermore, ornamental crops are exposed to pests in unnatural ways. Although environmental conditions inside greenhouses may be adjusted to encourage optimum growth of the crops, the same conditions also may be favorable, if not optimum, for pathogens. In the absence of natural competitors/inhibitors, pathogens may thrive under greenhouse conditions. Seed rots, damping-off of seedlings, black leg, and root rots caused by Pythium and Phytophthora spp. are among the most devastating, recurrent problems affecting ornamental crops. Initial studies on the optimization of management strategies for Pythium and Phytophthora diseases in ornamental crops were first conducted in the early 1900s. More recent studies have detailed environmental conditions favorable for infection in ornamental production facilities, and robust tools for pathogen identification and genetic characterization have been developed. Many protocols for detection of inoculum and management of diseases in ornamental crops have been developed. Selected species associated with ornamental crops have been surveyed and their genetic diversity and fungicide sensitivities have been characterized. However, it is necessary to synthesize the available information, fill in the vast information gaps that still exist, and deliver updated protocols, providing growers with tools and strategies for efficient management of plant diseases. This project will focus on some of the most common diseases affecting floriculture crops, those due to Pythium and Phytophthora species that infect root systems. It is important to develop information to help growers choose effective management strategies tailored to the specific pathogens that threaten the plants they grow. Learning how to use molecular tools effectively to pinpoint the sources of pathogen outbreaks within production pathways is essential for developing effective disease management strategies. This project will build on previous USDA FNRI-funded research on root rots and will make use of the latest scientific methods to develop better disease management options for successful integrated pest management programs for floriculture crops. Specific objectives of the proposed research include 1) development of improved molecular methods to accurately identify oomycete (Pythium and Phytophthora) species that cause diseases on many important floriculture crops, 2) design of standardized oomycete sampling techniques from plants, growing media, and irrigation water for routine and forensic applications, and 3) development of improved detection and identification technologies for Pythium and Phytophthora species and use of these technologies to track populations of a species within individual greenhouses or nurseries (especially to track introductions of new strains, insect vectoring of strains, and development of fungicide-resistant strains).
1b. Approach (from AD-416):
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.
3. Progress Report:
DNA extraction and Polymerase chain reaction: A total of 119 additional isolates of Pythium and Globisporangium species isolated from Floricultural Crops from Long Island, New York, by collaborator Margery Daughtrey were barcoded by sequencing of the ITS region. DNA was purified from lyophilized mycelia using Qiagen DNeasy Plant Mini kits according to manufacturer's instructions. PCR of the ITS region was performed. Molecular identification: Isolates identified to species based on morphology were verified by DNA sequence analysis of the ITS region (including the 5.8S subunit). PCR products were treated with ExoSAP-IT (USB, Cleveland) and sequenced directly. The resulting ITS sequences were compared using BLAST with sequences available at the National Center for Biotechnology Information (NCBI). Analysis of the sequences obtained since 2010 (264 total) identified 14 species in the genera Pythium (n=5) and Globisporangium (n=9; formerly Pythium). Most of the isolates (n=198) belonged to the Globisporangium irregulare species complex, with sequences matching available reference sequences (NCBI, blastn) of G. irregulare sensu stricto, G. cryptoirregulare, G. cylindrosporum, and G. irregulare sensu lato. The phylogenetic relationships among these four groups are not well defined (morphology and ITS sequences overlap between them) which complicates diagnostics and identification of inoculum sources and making management recommendations. Hence population genetics analyses are being conducted to define species boundaries. SSR DNA fingerprinting: A subset of 22 isolates that represented the diversity of the G. irregulare species complex in Long Island greenhouses was analyzed using 9 microsatellite (SSR) loci. Analyses of molecular variance (AMOVA), principal coordinate analysis (PCO), genetic distance analysis (UPGMA) and population genetics analyses (GST, Nm) were conducted. Our results identified four populations distributed across greenhouses without correlation to location. Phylogenetic and population genetics analyses indicate that the G. irregulare isolates collected belonged to main lineages, of which the first included G. irregulare sensu stricto (including the type strain) only, while the second lineage contained three major genetic groups, including G. cryptoirregulare, G. cylindrosporum and G. irregulare sensu lato isolates. The differences between the four groups are large, potentially representing a speciation process; however, we also found evidence of potential hybridization. Current efforts are focused on comparing these strains with strains from other agricultural systems to define species boundaries, characterization of nursery populations using SSRs, and validating diagnostic primers for G. sylvaticum, G. irregulare s.s. and G. cryptoirregulare.