Oral Technical Session: Epidemiology and Disease Management
54-O
Genome-informed diagnostics to discriminate geographically isolated populations of the select agent Rathayibacter toxicus
M. ARIF (1), G. Y. Busot (1), R. Mann (2), B. Rodoni (2), S. Liu (3), J. P. Stack (1)
(1) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (2) Department of Primary Industries, La Trobe University, Bundoora, Australia; (3) Department of Plant Pathology, Kansas State University, Manhattan, Australia
Rathayibacter toxicus is a toxin-producing, gram-positive bacterial pathogen of annual ryegrass (Lolium rigidum) and a select agent in the United States. Livestock deaths in Australia due to consumption of contaminated ryegrass have raised concerns about the global spread of R. toxicus in ryegrass hay and seed. A method for early and accurate detection of R. toxicus is needed. Whole genome sequences (Illumina; MiSeq) were generated for representative isolates from different geographic areas. The genome assemblies of different isolates were used to design target-specific primers and probes. A sensitive qPCR assay based on an rpoD region was developed to detect all R. toxicus isolates. A multiplex PCR based on genome-informed strategically identified genes was developed to discriminate among three genetically-distinct populations of R. toxicus. Each amplification assay included a multi-target artificial internal control to enhance reliability and accuracy. The detection assays were validated in silico and in vitro with 64 DNAs of R. toxicus, other Rathayibacter species and Dietzia cinnamea for specificity; they accurately detected R. toxicus from infected annual ryegrass and successfully identified the R. toxicus population type. These sensitive detection methods are easy to implement and will be appropriate for routine diagnostics, pathogen and population specific surveys, disease management, and biosecurity decisions to support export/import of annual ryegrass.