A decision tool and molecular typing technique for a plant pathogen forensic application, using wheat streak mosaic virus as a model pathogen

The vulnerabilities of US agriculture and the easy availability of naturally abundant plant pathogens create the threat of a biological attack on our cropping systems. Before a forensic investigation of a disease outbreak occurs, it must be determined that the introduction was likely to have been intentional. Successful attribution of the crime may be facilitated by a genetic analysis of the causal pathogen. Strict method validations must be conducted for the procedure to withstand the scrutiny of the courtroom. In this study, I developed a decision tool to assist first responders in assessing whether an outbreak may have involved human intent. Additionally, I validated a molecular typing technique using Wheat streak mosaic virus (WSMV) as a model and evaluated its use in a forensic investigation through field trials and isolate relationship studies. The Crop Bioagent Introduction Intent Assessment Tool (CBIIAT) proved to be an effective method for assessing the likelihood that a plant disease outbreak was the result of a deliberate effort. The tool's effectiveness was evaluated by several testers, individuals who practiced using the tool at both a natural and intentionally inoculated WSMV outbreak in Oklahoma. With easy access via the online program and a user-friendly design, the tool can be quickly incorporated into investigative procedures by non-scientists and used as a model for similar tools for other pathogens. The WSMV SNP typing method was shown to be reproducible, sensitive, specific, and capable of discriminating between known strains and field isolates of the virus. In a two year study I evaluated the effect of common wheat field treatments and conditions on the consistency of the single nucleotide polymorphism (SNP) profiles of WSMV isolates. No effects due to treatment were found. Minor changes found in the profiles were most likely a natural phenomenon. The SNP typing method was further evaluated using two geographically distinct WSMV outbreaks in 2008 to determine if the assay could be used to evaluate the pattern or pathway of spatial spread of the virus. No correlation of genotype to geographical location was found, but each isolate yielded a unique SNP profile which could be useful in a forensic investigation, particularly if a correlation were shown with another element, such as the origin of seed.