This multi-disciplinary project directly relates to strengthening the nations agricultural security through the development of tools for the early detection of microbial pathogens. As a model system we will use the fungal potato pathogen Synchytrium endobioticum. This project will represent a significant advancement of sensors for use in field situations, while eliminating labile reagents and reducing costly chemical components. Traditional DNA based probes are either not feasible or not sensitive enough for the proposed techniques and the intended applications. Further, this project will develop tools needed that can be used for in situ detection of plant pathogens allowing more time for action to protect the unaffected crops. We will develop rapid detection methods specifically for S. endobioticum using a sandwich hybridization probes. This project will develop species- and pathotype-specific probes directed to S. endobioticum, the fungal potato pathogen. These probes will be developed using a DNA mimic molecule, PNA. PNA has several qualities that make it an attractive alternative to DNA, including better discrimination of mismatches, not degraded by natural enzymes, and higher binding efficiencies. The PNA-based probes will be coupled with detection based on nanoparticle aggregation and direct electronic detection of binding. These probes will be incorporated into rapid mobile technologies with the long-term goal of application to more organisms after development.
NON-TECHNICAL SUMMARY: The potato wart pathogen (Synchytrium endobioticum) can cause great economic harm to US agriculture and rapid methods of detection do not yet exist for this organism. This project will develop rapid, easy to use, inexpensive, mobile technologies for detection of pathogenic microorganisms from field soil samples, using the potato wart pathogen (Synchytrium endobioticum) as a model system.<P>
APPROACH: Species and pathotype specific PNA probes will be based on sequence obtained from Synchytrium endobioticum genes from the ribosomal large subunit (LSU), and the gene COB. Each probe will be tested first on artificial linkers and then using S. endobioticum RNA prior to field testing with soil (both spiked and naturally infected). Trials will be run to determine the most effective method of extraction of suitable quantity and quality of RNA from infected soils. Methods for functionalizing the surfaces of nanoparticles to enhance binding of the PNA probes will be developed. Nanoparticle aggregation will be detected using micro-tubes and electronic gates. Prototypes of field portable device will be manufactured and tested on-site in infected and control areas.