Additional file 2 of Rhizosphere phage communities drive soil suppressiveness to bacterial wilt disease
Creators: name: Yang, Keming
name: Wang, Xiaofang
name: Hou, Rujiao
name: Lu, Chunxia
name: Fan, Zhe
name: Li, Jingxuan
name: Wang, Shuo
name: Xu, Yangchun
name: Shen, Qirong
name: Friman, Ville-Petri
name: Wei, Zhong
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 2: Table S1. Taxonomic classification of identified Ralstonia solanacearum phages. Table S2. Comparison of community network properties between healthy and diseased plant microbiomes. Table S3. Comparison of significance between different SEM model fits for healthy and diseased plant rhizosphere microbiome samples. Table S4. Detailed effects of different variables included in the first-step structural equation model for all five ‘functional groups’ based on Shannon alpha diversity index in healthy and diseased plant microbiome samples. Table S5. Detailed effects of different variables included in the second-step structural equation model based on Shannon alpha diversity index in healthy and diseased plant microbiome samples. Table S6. Linear model comparing the effect of ‘inhibitor-associated phage’ on ‘inhibitor bacteria’ in diseased plant microbiome samples. Formular: Inhibitor bacteria ~ Inhibitor-associated phage. Model R2 = 0.2909, F1,18 = 8.795, P = 0.00828. Table S7. The explanatory power of linear models for predicting Ralstonia solanacearum densities on each ‘functional group’ in both healthy and diseased microbiome samples. Table S8. Comparison of ‘inhibitory’ bacterial biomass in the presence and absence of their phages based on one-way ANOVA. Table S9. Comparison of Ralstonia solanacearum biomass between different treatments with each pair of isolated ‘inhibitor bacteria’ and ‘inhibitor-associated phage’ based on one-way ANOVA. Table S10. Comparison of tomato plant disease incidence between different treatments with each pair of isolated ‘inhibitor bacteria’ and ‘inhibitor-associated phage’ at the end of greenhouse experiment based on one-way ANOVA. Table S11. Comparison of R. solanacearum densities between different treatments with each pair of isolated ‘inhibitor bacteria’ and ‘inhibitor-associated phage’ at the end of greenhouse experiment based on one-way ANOVA. Table S12. Functions and packages used in R platform for statistical and bioinformatic analysis. Fig. S1. Comparison of bacterial community diversity and composition between healthy and diseased plant rhizosphere microbiome samples. a: Comparison of bacterial community diversity (Shannon index) between healthy (blue) and diseased (red) plant rhizosphere microbiome samples. Significances are shown as *: P &lt; 0.05, **: P &lt; 0.01, and ***: P &lt; 0.001 and n.s.: no significance, one-way ANOVA for each time point (see Supplementary Data S3 for details). b: Comparison of relative bacterial abundances at the phylum level between healthy (H) and diseased (D) plant rhizosphere microbiome samples. c-g: Comparison of bacterial community composition between healthy (blue circles) and diseased (red triangles) plant rhizosphere microbiome samples at different sampling time points at bacterial species level (PCA; pairwise comparisons based on PERMANOVA). h: Bray–Curtis distances of bacterial community in healthy and diseased plant rhizosphere microbiome samples at each time point (F4,75 = 76.5, P &lt; 0.001, Tukey’s multiple comparison after one-way ANOVA test). In panel a-g, n = 4 for all treatments per time point, while n = 16 in panel h. Fig. S2. Hierarchical composition of overall phage community based on predicted host bacterial lineages (all samples included) and distance between healthy and diseased plant microbiome. a. Circles indicate host bacterial taxonomic classifications from phylum (inner) to genus (outermost) level and percentage values show relative abundance of phages predicted to infect these bacterial taxa. b. Bray–Curtis distances of viral community in healthy and diseased plant rhizosphere microbiome samples at each time point (F4,75 = 22.59, P &lt; 0.001, Tukey’s multiple comparison after one-way ANOVA test) and the correlation with time (week 3-6, linear model). Fig. S3. Comparison of initial soil physicochemical properties between healthy and diseased plant samples. Significances are shown as *: P &lt; 0.05 and n.s.: no significance, one-way ANOVA for each pair (n = 4, see Supplementary Data S3 for details). Fig. S4. Comparison of R. solanacearum-specific phage abundances between healthy and diseased plant rhizosphere microbiome sample replicates at different time points. Each pair of panels (a-h) show phage density dynamics for diseased and healthy plant rhizosphere microbiome samples and different line colours show individual plant replicates. Line symbols denote for different viral families as denoted in the legend. Phages RSF1, RSL2 and P4282 had relatively higher abundances in early samples, while phage P4282 persistently decreased in abundance, phages RSF1 and RSL2 edged up towards the end of the experiment in both diseased and healthy plant microbiome samples, respectively. In contrast, phages RSB3 and RS138 showed steady increase in their relative abundances in part of healthy plant microbiome samples. In addition, phages RSL1, RSK1 and RSJ5 showed multiple peak stages in different plant rhizosphere in both healthy and diseased samples. Fig. S5. Comparisons of interactions between ‘functional groups’ and pathogen densities in healthy and diseased plant rhizosphere microbiomes. a-b: Difference in the proportion of significant correlations between ‘functional groups’ and pathogen densities in healthy (a) and diseased (b) plant rhizosphere microbiomes. Light and dark blue colours denote for ‘inhibitor bacteria’ and ‘inhibitor-associated phages’, while light and dark green colours denote for ‘facilitator bacteria’ and ‘facilitator-associated phages’. c-e: Structural equation models based on different community indexes (Shannon index, Simpson index and average abundance) illustrating primary-phage-effect (R. solanacearum-specific phage to R. solanacearum), primary-bacteria effects (‘inhibitor bacteria’ and ‘facilitator bacteria’ to R. solanacearum) as well as secondary-phage effects (‘inhibitor-associated phages’ and ‘facilitator-associated phages’ to ‘inhibitor bacteria’ and ‘facilitator bacteria’, respectively and to R. solanacearum) in both healthy and diseased plant microbiomes. Fig. S6. Comparisons of secondary metabolism synthesis related gene abundances between healthy (blue) and diseased (red) sample pairs in week 6. Statistical significance between treatments was determined by one-way ANOVA test with *: P &lt; 0.05, **: P &lt; 0.01, n = 4 for all treatments (see Supplementary Data S3 for details). Fig. S7. Taxonomic classification of isolated ‘inhibitor bacteria’ and transmission electron microscope photograph of their isolated phages. a-c: Phylogenetic tree of three inhibitor bacterial strains based on 16s rRNA sequences using neighbour-joining method (on bold). The evolutionary distances were computed using the Maximum Composite Likelihood method and the scale bar indicates the average number of amino acid substitutions per site. d-f: TEM of representative isolates performed by HC-1 Hitachi TEM system at 80 kV. Fig. S8. Host bacterium abundance examined with (light blue) or without (dark blue) associated phage after co-culturing 24 h. Statistical significance between treatments was determined by one-way ANOVA test with ***:P &lt; 0.001. Error bar: standard deviation. n = 8 for all treatments (see Supplementary Table S8 for details). Fig. S9. Photograph of tomato plants at the end of greenhouse experiment. a-c: Treatments with plants inoculated with R. solanacearum and different ‘inhibitor bacteria’ in the presence (right) and absence (left) of ‘inhibitor-associated phages’. d: ‘R. solanacearum-only’ control plants inoculated only with R. solanacearum. e: Blank control inoculated only with sterile water without any bacteria or phages. f-h: Treatments with plants inoculated with different ‘inhibitor bacteria’ only. In panels a-d, DI represents average disease incidence and diseased plants are highlighted with red flags. No disease symptoms were observed in e-h. Fig. S10. Comparison of the relative bacterial biomass reduction at different initial multiplicity of infections (M.O.I.). With all‘inhibitor-associated phages’, highest bacterial biomass reduction was observed with M.O.I = 10. Statistical significance between treatments was determined by one-way ANOVA test with *: P &lt; 0.05, **: P &lt; 0.01, ***: P &lt; 0.001, n = 8 for all treatments