This project is expected to contribute solutions the agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for plant pathogen collection, detection, diagnosis and discrimination in plant tissue or associated material suspected of harboring microorganisms of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. Both microbial forensics and agricultural biosecurity approaches are required to avoid and/or minimize regional, national or global movement of unwanted pathogens. <P>Goal or objectives are: To develop and/or validate methods for plant pathogen detection, discrimination, and disease diagnosis. The new methods will be target-specific and broad detection to include variants within the target. The new methods can include diagnostic keys or diagnostics pathways if required. Methods will be developed for detection and diagnostic of specimens from active or passive surveillance activities, farm fields, borders or transitional sites, valuable imported or exported plant genetic material, including suspected bio-crime scenes. <P>To develop and validate sampling devices and/or sampling procedures, for rapid screening. It will include the assessment of new biomaterial to streamline microbial forensic investigations, diagnostics and screening of plant material potentially carrying unwanted plant pathogens of agricultural biosecurity relevance. Attention will be given to requirements of the three described biosecurity zones: global, borders and pathways, and within Oklahoma and the U.S. <P>The duration of this project is anticipated to be five years.
Non-Technical Summary: Increasing global trade, passenger traffic and natural atmospheric disturbances associated with climate change favor the continental and intercontinental movement of insects, microbes and pathogens. New introductions of unwanted organisms associated with these pathways can eventually lead to complex incursions of emerging pests and diseases costly to control and/or eradicate. Moreover, during the last 7 years it has become evident that changes in world-geopolitical scenarios have also increased the risk of terrorism interventions in general and also bioterrorism against relevant agricultural commodities and the associated industries in particular. This new scenario poses risks to agriculture due to inadvertent, illegal or criminal introductions of exotic pathogens, giving rise to serious threats to the economy, environment, human health and cultural heritage. This project is expected to contribute solutions the agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for plant pathogen collection, detection, diagnosis and discrimination in plant tissue or associated material suspected of harboring microorganisms of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. <P> Approach: This project will assess the potential for sensitive detection of viruses, oomycetes and bacteria, cost-effectiveness, and ease of use of forward osmosis (FO) hydration bag or filtration devices developed first for military, recreational, and emergency relief situations in which clean sources of drinking water are not available. Sampling irrigation water for detection of microorganisms is expected to work by immersion of the bags in tanks, ponds, rivers or irrigation channels. The sample to be analyzed will consist of the extracted membrane and the residual material captured on its outer surface during the FO of a standard volume of irrigation water. Currently available FO devices (HTI) filter up to 3 L/day over a 3 day period (overall filtration 9 L (~ 20 lbs). Sample volumes of cultured waterborne pathogens E. coli, Tomato bushy stunt virus and Pythium aphanidermatum will be optimized by assaying serially diluted preparations, and the limits of threshold sensitivity of current DNA-based molecular diagnostic methods will be determined. Testing will be repeated under stringent field conditions using irrigation water as a model in eastern, western and two central sites of the Oklahoma State. The information obtained as described above is expected to facilitate the design, standardization and validation of new water sampling devices and protocols, to be cost-effective and easy to use by growers for capturing targeted microorganisms within the threshold of sensitivity of current DNA-based molecular diagnostic methods and new high resolution melt (HRM) technology. Regarding primer design and molecular diagnostics, sequences of interest from the NCBI Genbank will be retrieved and aligned and subsequently analyzed for primer design using validated thermodynamic parameters and the Web-interface pathway Primer3-mFOLD-BLASTn as reported. The information obtained during the development of multiplex RT-PCR detection method will facilitate stepping toward the development of a multi-pathogen, diagnostic-detection platform by using solid phase, heat stable polymer surfaces to which primers or alternatively PCR products are immobilized. Procedures will be followed according to preliminary results and from experience documented at the OSU Microarray Core facility, including a variety of procedures and strategies reported on this regard. For development of artificial positive controls, which will contribute minimizing the risk of using true pathogens as positive controls, and in downstream routine or large scale diagnostic analysis. Constructions will be made by (a) ligation of a number of PCR amplifiable sequences which are targeted by optimized sets of diagnostics oligonucleotide primers. These constructions will be inserted in commercially available plasmids through standardized procedures as show in figure. This part of the research will require all constructs or synthetic primer strings to be validated by extensive PCR assays including reference positive and blank controls, cloning and sequencing verification.