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Validating Methods for Eradicating Select Agent and Phylotype I Strains of Ralstonia solanacearum

Madeline Hayes: UW-Madison

<div><em>Ralstonia solanacearum</em> <span>(<span><em>Rs</em></span>) </span>is a highly destructive plant pathogen that causes bacterial wilt disease in many diverse crop species, including potato, tomato, and ornamentals. <span><em>Rs</em></span> can survive for extended periods in soil, water, and plant material, complicating disease prevention and management. Cool-tolerant potato- and ornamental-infecting phylotype II (R3bv2)<span> <em>Rs</em> strains are</span> quarantine pests in many countries and are highly regulated Select Agents in the U.S., so plant protection officials and researchers need a range of practical, reliable, and validated eradication methods for R3bv2 management and regulatory compliance. Additionally, g<span>rowers need practical methods to kill <span><em>Rs</em></span> in </span>contaminated irrigation water, a common source of disease outbreaks. To meet these needs, we measured survival of four R3bv2 strains and two phylotype I strains following treatment with hydrogen peroxide, HuwaSan (chemically stabilized hydrogen peroxide), active chlorine (bleach), heat, ultraviolet radiation, desiccation, and cell lysis steps of two commercial nucleic acid extraction kits. No surviving <span><em>Rs</em></span> cells were detected following ten minutes’ exposure to 200 ppm hydrogen peroxide, 20 ppm HuwaSan, 50 ppm active chlorine, temperatures above 50ºC, and 30s UV irradiation. All <em>Rs </em>strains were susceptible to desiccation on most abiotic and all biotic surfaces tested. Both extraction kits yielded 100% lysis and no surviving cells. Detailed eradication protocols will be discussed in the context of 1) compliance with Select Agent research regulations and 2) management of a major bacterial wilt outbreak in a greenhouse tomato production facility in West Africa.</div>