Additional file 2 of Deterministic colonization arises early during the transition of soil bacteria to the phyllosphere and is shaped by plant–microbe interactions
Creators: name: Mayer, Teresa
name: Teutloff, Erik
name: Unger, Kerstin
name: Lehenberger, Pamela
name: Agler, Matthew T.
Publisher: name: figshare
Year: 2025
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 2. Figure S1. Genus-level taxonomy and number of isolates of each genus recovered from in-planta isolation, where NG or PB-derived bacteria were inoculated onto NG, PB or Col-0 genotypes. Associated statistics can be found in Table S2. Figure S2. Early-inoculated plants (before the emergence of true leaves) develop distinct and diverse bacteriomes in mature leaves. Data is based on ASVs associated with the mature leaves of A. thaliana after inoculation at day 0, 7 or 14 or with heat-killed inoculum (D0, D7, D14, HK). A. RDA ordination of beta diversity (aitchison distance) of all samples including multiple sampling times. Results of a PERMANOVA test for significance of factors is in Table 1. B. RDA ordination of beta diversity (aitchison distance), including only live-inoculated samples collected 21 days post inoculation. Results of a PERMANOVA test for significance of factors is in Table 2. C. Alpha diversity (Chao1 estimates of total diversity and Shannon Index) of samples collected 21 days post inoculation. Figure S3. Early-inoculated plants (before the emergence of true leaves) develop distinct and diverse bacteriomes in mature leaves. Data is based on Order-level taxonomy of bacteria associated with the mature leaves of A. thaliana after inoculation at day 0, 7 or 14 or with heat-killed inoculum (D0, D7, D14, HK). A. RDA ordination of beta diversity (aitchison distance) of all samples including multiple sampling times. Results of a PERMANOVA test for significance of factors is in Table 1. B. RDA ordination of beta diversity (aitchison distance), including only live-inoculated samples collected 21 days post inoculation. Results of a PERMANOVA test for significance of factors is in Table 2. C. Alpha diversity (Chao1 estimates of total diversity and Shannon Index) of samples collected 21 days post inoculation. Figure S4. Between-sample beta diversity (Aitchison distances) comparing bacterial communities associated with mature A. thaliana leaf samples (21 DPI) at the ASV (left), Genus (middle) and Order (right) levels. Within-group distances (D0_D0, D7_D7 or D14_D14) show the variation in the bacterial community structure in a given treatment group. Between-group distances (D7_D0, D14_D0 and D14_D7) show how similar or different samples are between two treatments. In all cases, the stars show sinificant differences compared to the D0_D0 group. Figure S5. Bacterial taxa (ASVs) in mature leaf samples (combined SD21, 28, 35) that were differentially enriched between plants inoculated at D0 and D14. In the boxplots only the top 5 ASVs are shown (smallest fdr-adjusted p-values) because of space constraints. A. in Col-0, B. in NG-2, C. in PB. D. The total number of ASVs that were differentially enriched in each genotype and their overlap. Total detections are based on a DESeq2 analysis with p &lt; 0.001. Kruskall-wallis p-values are additionally shown for each plot. Figure S6. Bacterial taxa (Genera) in mature leaf samples (combined SD21, 28, 35) that were differentially enriched between plants inoculated at D0 and D14. In the boxplots only the top 5 genera are shown (smallest fdr-adjusted p-values) because of space constraints. A. in Col-0, B. in NG-2, C. in PB. D. The total number of genera that were differentially enriched in each genotype and their overlap. Total detections are based on a DESeq2 analysis with p &lt; 0.001. Kruskall-wallis p-values are additionally shown for each plot. Figure S7. Bacterial taxa (Orders) in mature leaf samples (combined SD21, 28, 35) that were differentially enriched between plants inoculated at D0 and D14. In the boxplots only the top 5 Orders are shown (smallest fdr-adjusted p-values) because of space constraints. A. in Col-0, B. in NG-2, C. in PB. D. The total number of taxa that were differentially enriched in each genotype and their overlap. Total detections are based on a DESeq2 analysis with p &lt; 0.001. Kruskall-wallis p-values are additionally shown for each plot. Figure S8. Total number of taxa correlated to ecotype in mature plants (combined SD21, 28, 35) that had been inoculated at D0, D7 or D14. A. ASV-level taxa, B. Genus-level taxa, and C. Order-level taxa. Detections were based on DESeq analysis with p &lt; 0.01. Figure S9. Bacterial taxa that were differentially enriched between ecotypes in mature leaf samples (combined SD21, 28, 35) in plants inoculated at D0. A. ASV-level taxa, B. Genus-level taxa, and C. Order-level taxa. Detections are based on DESeq analysis with p &lt; 0.01, but only the top 5 taxa are shown because of space constraints. The total number of differentially enriched taxa are shown in Figure S8. Kruskall-wallis p-values are additionally shown for each plot. Figure S10. Bacterial taxa that were differentially enriched between ecotypes in mature leaf samples (combined SD21, 28, 35) in plants inoculated at D14. A. ASV-level taxa, B. Genus-level taxa, and C. Order-level taxa. Detections are based on DESeq analysis with p &lt; 0.01, but only the top 5 taxa are shown because of space constraints. The total number of differentially enriched taxa are shown in Figure S8. Kruskall-wallis p-values are additionally shown for each plot. Figure S11. Processes shaping bacterial colonization of leaves of the A. thaliana genotypes Col-0, NG2 and PB depend on both inoculation time and genotype. A.–C. are based on the phylogenetic bin-based null model analysis in iCAMP. A.-B. The relative importance of deterministic processes (homogeneous and heterogeneous selection, respectively) and C. the relative importance of of stochastic processes (the sum of Dispersal limitation, Drift, and Homogenizing dispersal) in colonization of the leaves of plants inoculated at ID0, ID7, ID14 or never inoculated. D. The phylogenetic normalized stochasticity ratio (phylogenetic NST) is also provided as an independent measure of stochasticity for comparison. The top row is Col-0, the second row is NG-2 and the third row is PB. Figure S12. Processes shaping bacterial colonization of leaves of the A. thaliana genotypes Col-0, NG2 and PB depend on both inoculation time and genotype. Assembly processes at the level of taxonomic bins. The trees are all three identical and are annotated with, from inside to outside: Colored dots representing the majority taxonomy of each bin, A barchart showing for plants inoculated at ID0 the relative contribution of each community assembly process for each bin (DL: Dispersal limitation, DR: Drift, HD: Homogenizing dispersal, HeS: Heterogeneous selection, HoS: Homogeneous selection), and A heatmap showing the change in relative importance of each assembly process for each bin between plants inoculated at ID0 and those inoculated at ID14 (yellow is higher at ID0 dark blue is higher at ID 14). Figure 3B in the main text also shows labeled the taxa that are mentioned in the text that were selected deterministically at ID0 and stochastically at ID14 (homologous selection increased by at least 20% to reach &gt;50% total contribution in D0). Figure S13. Correlation of the log mean abundance of ASVs to either the coefficient of variation (a measure of stochasticity of abundance) or the fraction of samples the taxa was detected in. Measurements are shown for ecotypes Col-0 (A), NG2 (B) and PB (C). The measures are calculated using data from leaf samples collected at SD 21, 28 and 35. Figure S14. Pseudomonas viridiflava 3D9 is an opportunistic pathogen of A. thaliana. Germinating A. thaliana Col-0 or NG2 seedlings were inoculated with very low levels of three bacteria (CFU/seedling is indicated). After 14 days, plant phenotypes were recorded. healthy = no symptoms, stressed = purple/discolored or chlorotic leaves, necrotic = spots of dead tissue on at least one leaf, or dead. N=10. Figure S15. Pseudomonas viridiflava 3D9 mixed into a natural soil promotes growth and alters the immune response of A. thaliana. A. The fresh weight of A. thaliana shoots 25-days post-germination. N= 5-9. B. The response of A. thaliana leaf tissue to bacterial flg22 peptide as measured in a luminescence assay for reactive oxygen species (ROS) production. The total ROS burst is presented as the area under the luminescence curve (N=6). P-values represent the results of a 1-sided t-test to evaluate if there was an increase in plant weight or ROS response of Peat+Soil vs 3D9 treatments. Figure S16. Pseudomonas viridiflava 3D9 transitions from soil to leaves based on fluorescent cell counts. Seeds were germinated in standard laboratory soil (LS) or LS amended with an extract of a natural garden soil (LS+GS). The three treatments in each soil are no Pseudomonas viridiflava 3D9, low 3D9 (~1000 cells/cm3) or moderate 3D9 (~1x106 cells/cm3). 14-d old plants were harvested and bacterial CFU counts were made on LB medium with nystatin (LB+N – total bacterial count), LB+N with gentamycin, chloramphenicol and bacitracin (LB+NGCB – selection against gram + bacteria) or LB+N with bacitracin and boric acid (LB+NBba – selection of Pseudomonads). The same plates were also observed for fluorescent colonies with a stereo microscope equipped with a GFP filter to estimate CFUs expressing mTurquoise2 (Pseudomonas viridiflava 3D9). Figure S17. Bacteria that deterministically transition from soil to mature leaves are influenced by P. viridiflava 3D9 soil amendment (ASV level analysis to complement Figure 5). (A. and B.) Alpha diversity of bacteria in mature leaf tissues (14 days post germination) assessed by Chao1 metric (total estimated diversity) and Shannon index (diversity considering evenness of distribution of taxa). A Kruskal-Wallis test was performed on the data overall and when this was significant a Wilcoxon test was used to evaluate differences between groups. Significant p-values are shown. (C.) Principal components analysis of Aitchison distance between samples, constrained for treatment, showing biplot arrows for the top 1% of ASVs that correlate to the two axes. (D.) Boxplots showing the abundance of the bacteria identified using the biplot analysis in (C.) with p-value result of a Kruskal-Wallis test for significant differences between groups. Figure S18. Bacteria that deterministically transition from soil to mature leaves are influenced by P. viridiflava 3D9 soil amendment (Order-level analysis to complement Figure 5). (A. and B.) Alpha diversity of bacteria in mature leaf tissues (14 days post germination) assessed by Chao1 metric (total estimated diversity) and Shannon index (diversity considering evenness of distribution of taxa). A Kruskal-Wallis test was performed on the data overall and when this was significant a Wilcoxon test was used to evaluate differences between groups. Significant p-values are shown. (C.) Principal components analysis of Aitchison distance between samples, constrained for treatment, showing biplot arrows for the top 3% of taxa that correlate to the two axes. (D.) Boxplots showing the abundance of the bacteria identified using the biplot analysis in (C.) with p-value result of a Kruskal-Wallis test for significant differences between groups. Figure S19. Preston’s Octave plots for the number of strains (y-axis) that fall within a range for a given number of cells per strain (x-axis). The three plots correspond to three orders of magnitude of N, the density of bacterial cells in soil (cells/mm3) and each plot is calculated for three levels of alpha, Fisher’s diversity index. The equations that the plots are based on can be found in the supplementary results. Figure S20. Results of test experiment to identify leaf bacteria that can reach and colonize leaves when only a few cells encounter a germinating seed. Bacteria collected from wild NG2 plants were diluted to near extinction (0-1 cells/plant) and inoculated onto liquid or solid R2 medium or onto germinating seedlings. The bar chart shows the taxonomy of randomly picked isolates from the medium and from mature leaves where colonization was detected by PCR. Table S1: Number of bacterial colonizers recovered by traditional medium-based isolation methods in comparison with our in-planta approach (2018) and comparison between cross-inoculations of leaf inocula onto different plant genotypes (2019). Table S2: fdr-adjusted p-values for genus-level taxonomy and number of isolates of each genus recovered from in-planta isolation, where NG or PB-derived bacteria were inoculated onto NG, PB or Col-0 genotypes. P-values are based on a X2 test to determine whether a genus significantly more often originated from PB or NG (origin) or was recovered significantly more often in PB, Col or NG. p-values are only shown for genera where there were at least two observations in the tested conditions. Table S3: P-values for the likelihood that the number of times a genus was observed co-colonizing with another taxa was by chance alone. P-values are based on a C2 test comparing the expected number of observations (87.5% of total observations alone and 12.5% of total observations with a co-colonizer – based on the totals in the experiment) vs. the actual number of times the genus was observed alone and with a co-colonizer. Genera in bold are those who were mentioned in the text because they were found more often paired with another bacterium than alone. Orange colored genera denote the Burkholderiales. Table S4: Significance of factors in describing the variation in bacterial communities between samples. Results are based on the Aitchison distance between samples collected at all time points at the level of ASVs. Analysis was carried out with PERMANOVA with 1000 permutations. Table S5: Significance of factors in describing the variation in bacterial communities between samples. Results are based on the Aitchison distance between samples collected at all time points at the Order level. Analysis was carried out with PERMANOVA with 1000 permutations. Table S6: Significance of factors in describing the variation in bacterial communities between samples. Results are based on the Aitchison distance between samples collected at 21 DPI at the ASV level. Analysis was carried out with PERMANOVA with 1000 permutations. Table S7: Significance of factors in describing the variation in bacterial communities between samples. Results are based on the Aitchison distance between samples collected at 21 DPI at the Order level. Analysis was carried out with PERMANOVA with 1000 permutations. Table S8: Counts and p-values for the likelihood that the number of times a genus was observed in-planta is the same as the expected  number of observations based on counts in R2A medium. P-values are based on a C2 test comparing the expected number of observations (calculated based on the frequency observed in R2A medium) vs. the actual number of observations.