DOI: 10.5061/dryad.1g1jwsv3g
title: Data from: Plant pathogenic bacterium Ralstonia solanacearum can rapidly evolve tolerance to antimicrobials produced by Pseudomonas biocontrol bacteria

description: 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 strain 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.
description: # Data from: Plant pathogenic bacterium Ralstonia solanacearum can rapidly
 evolve tolerance to antimicrobials produced by Pseudomonas biocontrol
 bacteria All the data
 produced by the experiments. ## Description of the data and file structure
 README file Author names: S.E. Clough (1,2), J.G. Elphinstone (3) and V-P.
 Friman (1,4) (1) University of York (Dept. of Biology, Wentworth Way,
 University of York, York, YO10 5DD, U.K) (2) Durham University (Dept. of
 Chemistry and Dept. of Biosciences, South Road, Durham University, Durham,
 DH1 3LE, U.K) (3) Fera Science Ltd (National Agri-Food Innovation Campus,
 Sand Hutton, York, U.K) (4) University of Helsinki (Department of
 Microbiology, University of Helsinki, 00014, Helsinki, Finland) Title of
 study: Plant pathogenic bacterium Ralstonia solanacearum can rapidly
 evolve tolerance to antimicrobials produced by Pseudomonas biocontrol
 bacteria Summary of study: Here, we used an in vitro experimental
 evolution approach to investigate the capability of phytopathogenic
 bacterium Ralstonia solanacearum, which is the causative agent of
 bacterial wilt, to evolve tolerance to antimicrobials produced by
 Pseudomonas bacteria. We first conducted a selection experiment followed
 by fitness assays in bacterial supernatant as well as in synthetic
 Pseudomonas metabolites. Most of the data is based on changes in bacterial
 densities, which was measured as optical density (OD) using
 spectrophotometer (600 nm wavelength). S.E. Clough is responsible for
 collecting data. Overview of files: 1\) Selection experiment dataset
 (Figure 1).csv Dataset used to produce Figure 1 with OD values – each row
 represents which Ralstonia culture (7 strains) was inoculated into which
 Pseudomonas supernatant (8 strains) and at which supernatant concentration
 (50% or 80%). What date that OD value was recorded (0-21 days), growth is
 displayed as the raw OD value and normalised OD value (these were
 normalised by subtracted the OD of LB broth alone). 2\) Selection
 experiment dataset as reductions (Figure1 analysis.csv Dataset used for
 Figure 1 analysis with OD data converted into growth reduction shown as a
 percentage - each row represents which Ralstonia culture (7 strains) was
 inoculated into which Pseudomonas supernatant (8 strains) and at which
 supernatant concentration (50% or 80%). What date that OD value was
 recorded (24-504 hours), the raw OD value and normalised OD value (these
 were normalised by subtracting the OD of LB broth alone). The additional
 column ‘OD of control’ is the average OD for the control sample at that
 timepoint. ‘OD reduction’ is the ‘OD of control’ column subtracted from
 the ‘Normalised OD’ column which is then converted to ‘Reduction %’. 3\)
 Fitness assays dataset (Figure 2).csv Dataset for initial analysis of
 Figure 2 of the growth data (OD values) - each row represents which
 Ralstonia culture (7 strains) was inoculated into which Pseudomonas
 supernatant (8 strains), the evolutionary origin of the Ralstonia sample
 (Ancestral, Evolved, Control) and which condition they were grown in
 during the fitness assay (LB broth ‘LB’ or Pseudomonas supernatant ‘SN’).
 What date that OD value was recorded (24 or 72h), the raw OD value and
 normalised OD value (these were normalised by subtracted the OD of LB
 broth alone). 4\) Fitness assays reduction dataset by Ralstonia strain
 (Figure 2 and 3 analysis).csv Dataset used to produce Figure 2 and 3 as
 well as the analysis with OD data converted into growth reduction shown as
 a percentage – each row represents the growth reduction of each Ralstonia
 culture (7 strains), the evolutionary origin of the Ralstonia sample
 (Ancestral, Evolved, Control), what date that OD value was recorded (24 or
 72h), ‘Reduction OD’ was calculated like previously described and
 converted to ‘Reduction %’. The column ‘Supernatant concentration in
 comparisons’ identifies the different high and low supernatant environment
 comparisons (e.g. 50LB = 50% LB broth and 100LB = 100% LB broth, 50SN =
 50% supernatant, 80SN = 80% supernatant). 5\) Fitness assays reduction
 dataset by Pseudomonas strain (Figure 4 analysis).csv Dataset used to
 produce Figure 4 as well as the analysis with OD data converted into
 growth reduction shown as a percentage – this is the same as the ‘fitness
 assays reduction dataset by Ralstonia strain (Figure 2 and 3
 analysis).csv’ above except it is averaged over the Ralstonia strains to
 focus on Pseudomonas identity effects. 6\) Fitness assay reduction dataset
 for DAPG exposure (Figure 5A analysis).csv Dataset used to produce Figure
 5A with normalised OD data and OD data converted into growth reduction
 shown as a percentage - each row represents the growth reduction of each
 Ralstonia culture (7 strains), the evolutionary origin of the Ralstonia
 sample (Ancestral, Evolved, Control), what date that OD value was recorded
 (24 or 72h), ‘OD Reduction’ was calculated like previously described and
 converted to ‘Reduction %’ and at what concetration of DAPG (50-1000uM).
 7\) Fitness assay dataset for orfamide exposure (Figure 5B-C).csv Dataset
 used to analyse Figure 5B-C with OD values- Dataset for initial analysis
 of Figure 2 of the growth data (OD values) - each row represents which
 Ralstonia culture (strain #1) was inoculated into which Pseudomonas
 supernatant during the selection experiment (2 strains), the evolutionary
 origin of the Ralstonia sample (Ancestral, Evolved, Control) and which
 metabolite they were exposed to during the fitness assay (orfamide A
 ‘OrfA’ or orfamide B ‘OrfB’). 8\) Fitness assay reduction dataset of
 orfamides (Figure 5B-C analysis).csv Dataset used to produce Figure 5B-C
 with OD values converted into growth reduction shown as a percentage. 9\)
 Fitness assay dataset of pyoluteorin exposure (Figure 5D).csv Dataset used
 to analyse Figure 5D with OD values - Dataset used to analyse Figure 5B-C
 with OD values- Dataset for initial analysis of Figure 2 of the growth
 data (OD values) - each row represents which Ralstonia culture (strain #1
 or strain #7) was inoculated into which Pseudomonas supernatant during the
 selection experiment (2 strains), the evolutionary origin of the Ralstonia
 sample (Ancestral, Evolved, Control) grown in one concentration of
 pyoluteorin. 10\) Fitness assay reduction dataset of pyoluteorin (Figure
 5D analysis).csv Dataset used to produce Figure 5D with OD values
 converted into growth reduction shown as a percentage. All statistical
 analyses and graphs were produced using R (R Foundation for Statistical
 Computing, R Studio Version.3.4.4. Packages included: ggplot (v3.4.3),
 dplyr (v1.1.2), magrittr (v2.0.3), multcomp (1.4-25), plyr (v1.8.8),
 RColorBrewer (v1.1-3), readr (v2.1.2), reshape2 (v1.4.4) and tidyr
 (v1.3.0). Colour blind friendly palettes were used in all figures
 (viridis, magma, plasma and inferno).