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Identification of Rathayibacter and other bacteria associated with gumming disease of grasses in Oregon, U.S.A.

Melodie Putnam: Oregon State University, Botany and Plant Pathology

<div><em>Rathayibacter toxicus</em> is toxin-producing bacterium that is designated a U.S. Select Agent. It is closely related to other, non-toxic bacteria associated with gumming disease of grass seed produced in the U.S. Pacific Northwest. Accurate, rapid identification of <em>Rathayibacter</em>-like bacteria is an essential need. <em>Rathayibacter</em> can be detected with species-level (SL) PCR primers, but there is no genus-level (GL) detection to distinguish <em>Rathayibacter</em> from other bacterial genera, nor is there a rapid means of discriminating infected seed. We used seed washes from 699 seed lots collected between 2012 and 2015 from 13 counties in Oregon to recover bacteria associated with gumming disease. We tested whether seed rinsate could be used for culture-free detection of <em>Rathayibacter</em> using SL PCR primers. To identify GL primers, we analyzed whole genome sequences from approximately 120 <em>Rathayibacter</em> isolates and identified regions predicted to distinguish it from other genera. We tested 93 <em>Rathayibacter</em> spp. and genetically distant bacteria with the primers. We found the intra-genus diversity of <em>Rathayibacter</em> at the nucleotide level too great for sufficiently specific GL primers. Preliminary results showed seed washes to be an effective way to directly detect <em>Rathayibacter</em> species using SL PCR without culturing, reducing detection time from 10-14 days to two days. Additionally, we identified a new species of <em>Rathayibacter</em> from four different grass species.</div>