Poster: Diseases of Plants: Disease Detection & Diagnosis
In-field detection of the select agent Rathayibacter toxicus using loop-mediated isothermal amplification
M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) (1) Kansas State University, U.S.A.; (2) Department of Plant Pathology, Kansas State University, U.S.A.; (3) Department of Economic Development, Jobs, Transport and Resources, Latrobe Uni
Early, accurate and sensitive detection of Rathayibacter toxicus by plant health officials would improve inspections of imported annual ryegrass hay and seed at ports of entry and enhance in-field detection by biosecurity surveillance personnel. The gram positive bacterium and select agent R. toxicus produces a toxin in annual ryegrass (Lolium rigidum). Livestock deaths in Australia due to toxin contaminated ryegrass have raised concerns about the global spread of R. toxicus in ryegrass hay and seed. Genomes from all available R. toxicus populations were explored to design target-specific loop-mediated isothermal amplification (LAMP) primers. Gene rpoD was selected for primers design. Internal LAMP primers targeting the ITS gene of host plant L. rigidum were also designed to enhance reliability and accuracy. Genie II, Sybr Green I and lateral flow device were used for LAMP assays. The developed assays were validated in silico against the NCBI GenBank nucleotide database and in vitro against the inclusivity and exclusivity panels for specificity. In-field assays were performed in South Australia; accurately detected R. toxicus from infected annual ryegrass samples. The developed LAMP assays are sensitive, accurate and easy to implement. The proposed LAMP method will be helpful in diagnostics, surveys, disease management, and biosecurity decisions to support export/import of annual ryegrass.