Plant pathogenic bacterium Ralstonia solanacearum can rapidly evolve tolerance to antimicrobials produced by Pseudomonas biocontrol bacteria.

Soil-borne plant pathogens significantly threaten crop production due to lack of effective control methods. One alternative to traditional agrochemicals is microbial biocontrol, where pathogen growth is suppressed by naturally occurring bacteria that produce antimicrobial chemicals. However, it is still unclear if pathogenic bacteria can evolve tolerance to biocontrol antimicrobials and if this could constrain the long-term efficacy of biocontrol strategies. Here we used an in vitro experimental evolution approach to investigate if the phytopathogenic Ralstonia solanacearum bacterium, which causes bacterial wilt disease, can evolve tolerance to antimicrobials produced by Pseudomonas bacteria. We further asked if tolerance was specific to pairs of R. solanacearum and Pseudomonas strains and certain antimicrobial compounds produced by Pseudomonas. We found that while all R. solanacearum strains could initially be inhibited by Pseudomonas strains, this inhibition decreased following successive subculturing with or without Pseudomonas supernatants. Using separate tolerance assays, we show that the majority of R. solanacearum strains evolved increased tolerance to multiple Pseudomonas strains. Mechanistically, evolved tolerance was most likely linked to reduced susceptibility to orfamide lipopeptide antimicrobials secreted by Pseudomonas strains in our experimental conditions. Some levels of tolerance also evolved in the control treatments, which was likely correlated response due to adaptations to the culture media. Together, these results suggest that plant-pathogenic bacteria can rapidly evolve increased tolerance to bacterial antimicrobial compounds, which could reduce the long-term efficacy of microbial biocontrol.

Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media.

Three Web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of Escherichia coli that is commonly used in molecular biology and biochemistry research, e.g. in the production of recombinant proteins. Metal availabilities have been examined for cells grown in Luria-Bertani (LB) medium aerobically, anaerobically, and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by quantitative polymerase chain reaction (qPCR). The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterized in related bacterial cells enabling gene expression to be read out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial that discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

Combining in vitro and in vivo screening to identify efficient Pseudomonas biocontrol strains against the phytopathogenic bacterium Ralstonia solanacearum.

Although plant pathogens are traditionally controlled using synthetic agrochemicals, the availability of commercial bactericides is still limited. One potential control strategy could be the use of plant growth-promoting bacteria (PGPB) to suppress pathogens via resource competition or the production of antimicrobial compounds. This study aimed to conduct in vitro and in vivo screening of eight Pseudomonas strains against Ralstonia solanacearum (the causative agent of bacterial wilt) and to investigate underlying mechanisms of potential pathogen suppression. We found that inhibitory effects were Pseudomonas strain-specific, with strain CHA0 showing the highest pathogen suppression. Genomic screening identified 2,4-diacetylphloroglucinol, pyoluteorin, and orfamides A and B secondary metabolite clusters in the genomes of the most inhibitory strains, which were investigated further. Although all these compounds suppressed R. solanacearum growth, only orfamide A was produced in the growth media based on mass spectrometry. Moreover, orfamide variants extracted from Pseudomonas cultures showed high pathogen suppression. Using the "Micro-Tom" tomato cultivar, it was found that CHA0 could reduce bacterial wilt disease incidence with one of the two tested pathogen strains. Together, these findings suggest that a better understanding of Pseudomonas-Ralstonia interactions in the rhizosphere is required to successfully translate in vitro findings into agricultural applications.