Dewberry, R. J. | Search bioRxiv

bioRxiv2024

Genotypic and Phenotypic Analyses Show Ralstonia solanacearum Cool Virulence is a Quantitative Trait Not Restricted to 'Race 3 biovar 2'

Most Ralstonia solanacearum species complex strains cause bacterial wilts in tropical or subtropical zones, but the group known as Race 3 biovar 2 (R3bv2) is cool virulent and causes potato brown rot at lower temperatures. R3bv2 has invaded potato-growing regions around the world but is not established in the United States. Phylogenetically, R3bv2 corresponds to a subset of the R. solanacearum phylotype IIB clade, but little is known about the distribution of the cool virulence phenotype within phylotype IIB. Therefore, genomes of 76 potentially cool virulent phylotype IIB strains and 30 public genomes were phylogenetically analyzed. A single clonal lineage within the sequevar 1 subclade of phylotype IIB that originated in South America has caused nearly all brown rot outbreaks worldwide. To correlate genotypes with relevant phenotypes, we quantified virulence of ten Ralstonia strains on tomato and potato at both 22{degrees}C and 28{degrees}C. Cool virulence on tomato did not predict cool virulence on potato. We found that cool virulence is a quantitative trait. Strains in the sequevar 1 pandemic clonal lineage caused the most disease, while other R3bv2 strains were only moderately cool virulent. However, some non-R3bv2 strains were highly cool virulent and aggressively colonized potato tubers. Thus, cool virulence is not consistently correlated with strains historically classified as R3bv2 group. To aid detection of sequevar 1 strains, this group was genomically delimited in the LINbase web server and a sequevar 1 diagnostic primer pair was developed and validated. We discuss implications of these results for the R3bv2 definition.

microbiology↗

bioRxiv2022

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

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.

microbiology↗