Validating methods to eradicate Select Agent and phylotype I Ralstonia solanacearum strains reveals that growth in planta increases bacterial stress tolerance

Abstract

Ralstonia solanacearum is a destructive pathogen that causes bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because R. solanacearum persists in soil, water, and plant material. Growers need practical methods to kill R. solanacearum in irrigation water, a common source of disease outbreaks. Additionally, the Race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated U.S. Select Agent. Plant protection officials and researchers need validated protocols to eradicate R. solanacearum for regulatory compliance. To meet these needs, we measured survival of four R3bv2 and three phylotype I R. solanacearum strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (HuwaSan), active chlorine, heat, ultraviolet radiation, and desiccation. No surviving R. solanacearum cells were detected after cultured bacteria were exposed for ten minutes to 400 ppm hydrogen peroxide, 50 ppm HuwaSan, 50 ppm active chlorine, or temperatures above 50{degrees}C. R. solanacearum cells on agar plates were eradicated by 30s UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. R. solanacearum did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with Select Agent research regulations and for management of a bacterial wilt outbreak in the field. ImportanceRalstonia solanacearum, a globally distributed wilt pathogen of many high-value crops, is spread via diseased plant material and contaminated soil, tools, and irrigation water. The Race 3 biovar 2 Select Agent subgroup of R. solanacearum is subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by: 1) growers seeking to disinfest water and manage bacterial wilt disease outbreaks; 2) researchers who must remain in compliance with regulations; and 3) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured R. solanacearum cells are sensitive to relatively low levels of oxidative chemicals, dessication, and heat, more aggressive treatment such as autoclaving or incineration is required to eradicate R. solanacearum cells growing inside plant material.