Additional file 3 of Balancing the scales: assessing the impact of irrigation and pathogen burden on potato blackleg disease and soil microbial communities
Creators: name: Keating, Ciara
name: Kilbride, Elizabeth
name: Stalham, Mark A.
name: Nellist, Charlotte
name: Milner, Joel
name: Humphris, Sonia
name: Toth, Ian
name: Mable, Barbara K.
name: Ijaz, Umer Zeeshan
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Plant Biology

description: Supplementary Material 3: Supplementary Figure S1. Schematic diagram showing the experimental design of this study. This includes the choice of potato seed stock with high, low (both Jelly varieties) and zero (Estima mini-tubers) starting levels of Pectobacterium species and irrigation regimes (Unirrigated [rainfed only], Irrigation 1 [SMD 40 mm], Irrigation 2 [SMD &lt; 15 mm during the common scab control period then unirrigated], and Irrigation 3 [SMD &lt; 15 mm during the common scab control period then unirrigated]. Comparisons in microbial communities across time were made using soil sampled from the ridge (near the top of the furrow) at 50% plant emergence (T_E Ridge) and harvest (T_H Ridge). In addition, to determine if there were differences in microbial communities sampled closer to the plant roots, samples were also taken at the bottom of the furrow at harvest only (T_H Root), to avoid damaging roots in the emerging plants. Details about the plot layout can be found in Supplementary Data File 2. Supplementary Figure S2. Rarefaction curves showing the number of reads from the 16S rRNA gene in DNA from the soil samples on the x-axis and the number of OTUs within a 97% percent sequence similarity threshold on the y-axis. Treatment groups are indicated by colour-coded lines. Supplementary Table S1. Percentage of blackleg disease symptoms in potato plants per plot over the course of the experimental field trial in 2020. Supplementary Table S2. Proportion of tubers with rotting (%) and weight of rotted tubers (tubers/hectare) at potato harvest. Table also shows the results of the statistical analysis of individual stocks and irrigation regimes. The respective degrees of freedom (D.F.) is given within the standard error (S.E.). Supplementary Table S3. Genus identifier list for Supplementary Fig. 2 and the average percentage prevalence in all samples. Supplementary Figure S3.The ‘core microbiome of the soil microbial communities from the field trial. The ‘core microbiome’ was set at 85% minimum prevalence of samples. Numbers referring to the individual genera are shown on the x-axis (defined in Supplementary Table 3), with colours highlighting the taxonomic phyla. The y-axis shows the detection threshold of absolute genera abundances and heatmap colours showing the average prevalence of each genus within all samples, ranging from 0–1. Supplementary Table S4. Genus identifier list for Supplementary Fig. 2 and the average percentage prevalence in all samples. Supplementary Figure S4. Taxonomic diversity of the soil microbiomes (ridge samples) at the 50% plant emergence (T_E) timepoint. Colours represent the irrigation regime (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3). Symbols represent the potato seed stock (EstimaZero, JellyLow, and JellyHigh). A) Shows alpha diversity based on the relative distribution of taxa (Pielou’s evenness) and the number of different ASVs (Richness) within the communities; there were no significant differences between the watering regimes and no obvious clustering based on potato stock. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of beta diversity dissimilarity between the communities; note that there was no distinct clustering in relation to irrigation regime, but there was clustering with respect to potato stock. Supplementary Figure S5. Functional diversity of the soil microbiomes (ridge samples) at the 50% plant emergence (T_E) timepoint. Colours represent the irrigation regime (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3). Symbols represent the potato seed stock (EstimaZero, JellyLow, and JellyHigh). A) Shows diversity based on the relative distribution of KEGG Orthologs (KOs; Pielou’s evenness) and number of KEGG Orthologs (KOs; Richness) within the communities; there were no significant differences in relation to irrigation treatment and no obvious clustering by potato stock. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of functional beta diversity dissimilarity between the communities as assessed through PiCrust2 and hierarchical metastorms analysis; note that there was no distinct clustering in relation to any of the variables. Supplementary Figure S6. Taxonomic diversity of the soil microbiomes at the harvest (T_H) timepoint. Colours represent the irrigation regime (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3) and whether the sample was from the ‘Ridge’ or ‘Root’. Symbols represent the potato seed stock (EstimaZero, JellyLow, and JellyHigh). A) Shows diversity based on the relative distribution of taxa (Pielou’s evenness) and the number of different ASVs (Richness) within the communities; which were similar across irrigation regimes. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of beta diversity dissimilarity between the communities; note that samples clustered according to potato stock, sample type (ridge or root) and Irrigation 2 samples showed some clustering. Supplementary Figure S7. Functional diversity of the soil microbiomes at the harvest (T_H) timepoint. Colours represent the irrigation regime (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3) and the sample was from the ‘Ridge’ or ‘Root’. Symbols represent the potato seed stock (EstimaZero, JellyLow, and JellyHigh). A) Shows diversity based on the relative distribution of KEGG Orthologs (KOs; Pielou’s evenness) and number of KEGG Orthologs (KOs; Richness) within the communities; there were no significant differences in relation to irrigation treatment and no obvious clustering by potato stock. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of functional beta diversity dissimilarity between the communities as assessed through PiCrust2 and hierarchical metastorms analysis; note that samples loosely cluster according to irrigation regime, sample type (root or ridge) and primarily cluster according to potato stock. Supplementary Figure S8. Taxonomic diversity of the soil microbiomes at the 50% plant emergence (T_E) and plant harvest (T_H) time points from ‘Ridge’ samples only. Colours represent the irrigation regime (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3). Symbols represent the potato seed stock (EstimaZero, JellyLow, and JellyHigh). A) Shows diversity based on the relative distribution of taxa (Pielou’s evenness) and the number of different ASVs (Richness) within the communities; which were similar across irrigation regimes. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of beta diversity dissimilarity between the communities; note that samples clustered according to potato stock. Supplementary Figure S9. Functional diversity of the soil microbiomes at 50% plant emergence (T_E). Colours represent potato stock (EstimaZero – E, JellyLow – JL, and JellyHigh – JH). Symbols represent the irrigation regimes (Unirrigated, Irrigation_1, Irrigation_2, and Irrigation 3). A) Shows diversity based on the relative distribution of KEGG Orthologs (KOs; Pielou’s evenness) and number of KEGG Orthologs (KOs; Richness) within the communities; there were no significant differences in relation to irrigation treatment and no obvious clustering by potato stock. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of functional beta diversity dissimilarity between the communities as assessed through PiCrust2 and hierarchical metastorms analysis; note that samples very loosely cluster according to potato stock. Supplementary Figure S10. Functional diversity of the soil microbiomes at plant harvest (T_H). Colours represent potato stock (EstimaZero – E, JellyLow – JL, and JellyHigh – JH), time-point (50% plant emergence – T_H), and soil sample type (Ridge – Ri, Root – Ro). Symbols represent the irrigation regimes (Unirrigated, Irrigation_1, Irrigation_2, and Irrigation 3). A) Shows diversity based on the relative distribution of KEGG Orthologs (KOs; Pielou’s evenness) and number of KEGG Orthologs (KOs; Richness) within the communities; there were no significant differences in relation to irrigation treatment and no obvious clustering by potato stock. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of functional beta diversity dissimilarity between the communities as assessed through PiCrust2 and hierarchical metastorms analysis; note that samples loosely cluster according to potato stock and sample type. Supplementary Figure S11. Functional diversity of the soil microbiomes at the 50% plant emergence (T_E) and plant harvest (T_H) time points from ‘Ridge’ samples only. Colours represent the irrigation regime (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3). Symbols represent the potato seed stock (EstimaZero, JellyLow, and JellyHigh). A) Shows diversity based on the relative distribution of KEGG Orthologs (KOs; Pielou’s evenness) and number of KEGG Orthologs (KOs; Richness) within the communities; there were no significant differences in relation to irrigation treatment and no obvious clustering by potato stock. B) Principal coordinate analysis (PCoA) based on Bray–Curtis distance of functional beta diversity dissimilarity between the communities as assessed through PiCrust2 and hierarchical metastorms analysis; note that samples loosely cluster according to potato stock and time point. Supplementary Figure S12. Analysis of rare taxa from the Estima, JellyLow and JellyHigh samples at T_E (50% plant emergence) and T_H (plant harvest) divided into ridge and root samples (Ri and Ro). These figures show the percentage contribution (%) to taxonomic beta-diversity dissimilarity of: A) ‘Persistently Rare’ taxa B) ‘Abundant’ taxa; C) ‘Conditionally Rare’ taxa; and D) ‘Other Rare’ taxa. Supplementary Figure S13. Taxonomic map for differential heat tree analysis. The branches show the taxonomic identification of all ASVs. Note that the same tree can be used for each differential heat tree analysis instance. Supplementary Figure S14. Heat tree analysis to visualise the differential taxa between irrigation regimes at 50% plant emergence (T_E). The full legend for the tree branches is shown in Supplementary Fig. 10. Each tree shows a pair-wise comparison between irrigation regimes (denoted by either purple or blue). The size of the branch nodes shows the number of ASVs, while the differential Log2 ratio median proportion is shown by the colours. Note that there are no Log2 fold differences between taxa across the irrigation regimes. Supplementary Figure S15. Heat tree analysis to visualise the differential taxa between irrigation regimes at potato harvest (T_H) in soil ridge samples. The full legend for the tree branches is shown in Supplementary Fig. 10. Each tree shows a pair-wise comparison between irrigation regimes (denoted by either purple or blue). The size of the branch nodes shows the number of ASVs, while the differential Log2 ratio median proportion is shown by the colours. Note that there are no Log2 fold differences between taxa across the irrigation regimes. Supplementary Figure S16. Heat tree analysis to visualise the differential taxa between irrigation regimes at potato harvest (T_H) in soil root samples. The full legend for the tree branches is shown in Supplementary Fig. 10. Each tree shows a pair-wise comparison between irrigation regimes (denoted by either purple or blue). The size of the branch nodes shows the number of ASVs, while the differential Log2 ratio median proportion is shown by the colours. Note that there are no Log2 fold differences between taxa across the irrigation regimes. Supplementary Figure S17. Plot highlighting the impact of ‘Irrigation Regime’ on the abundance of microbial taxa in the potato harvest (T_H) ridge soil samples, based on the GLVMM analysis. The microbial genera names are indicated on the y-axis. The environment covariates values are indicated on the x-axis which are the coefficient values against the environmental covariates (e.g. Unirrigated, Irrigation_2, and Irrigation_3 as compared to the Irrigation 1 treatment). The values range from positive, neutral to negative associations with the specific taxa on the y-axis. The red lines show significantly positive taxa associated with the indicated environmental covariate and the blue lines show significantly negative taxa; grey lines are not statistically significant. Note: Irrigation 1 treatment is not displayed as the analysis uses this treatment as the reference. Supplementary Figure S18. Plot highlighting the impact of ‘Irrigation Regime’ on the abundance of microbial taxa in the potato harvest (T_H) root soil samples, based on the GLVMM analysis. The microbial genera names are indicated on the y-axis. The environment covariates values are indicated on the x-axis which are the coefficient values against the environmental covariates (e.g. Irrigation_1, Irrigation_2, and Irrigation_3, as compared to the Unirrigated treatment). The values range from positive, neutral to negative associations with the specific taxa on the y-axis. The red lines show significantly positive taxa associated with the indicated environmental covariate and the blue lines show significantly negative taxa; grey lines are not statistically significant. Note: Unirrigated treatment is not displayed as the analysis uses this as the reference. Supplementary Figure S19. Plot highlighting the impact of ‘Potato Stock’ on the abundance of microbial taxa at 50% plant emergence (T_E), based on the GLVMM analysis. The microbial genera names are indicated on the y-axis. The environmental covariates values are indicated on the x-axis which are the coefficient values against the environmental covariates (e.g. ‘Potato Stock’ either JellyHigh, JellyLow as compared to the EstimaZero. The values range from positive, and neutral to negative associations with the specific taxa on the y-axis. The red lines show significantly positive taxa associated with the indicated environmental covariate and the blue lines show significantly negative taxa; grey lines are not statistically significant. Note: The EstimaZero group is not displayed as the analysis uses this as the reference. Supplementary Figure S20. Plot highlighting the impact of ‘Potato Stock’ on the abundance of microbial taxa in the root samples at potato harvest (T_H), based on the GLVMM analysis. The microbial genera names are indicated on the y-axis. The environment covariates values are indicated on the x-axis which are the coefficient values against the environmental covariates (e.g. ‘Potato Stock’ either JellyHigh, JellyLow as compared to the EstimaZero. The values range from positive, neutral to negative associations with the specific taxa on the y-axis. The red lines show significantly positive taxa associated with the indicated environmental covariate and the blue lines show significantly negative taxa; grey lines are not statistically significant. Note: The EstimaZero group is not displayed as the analysis uses this as the reference. Supplementary Figure S21. Plot highlighting the impact of ‘Potato Stock’ on the abundance of microbial taxa in the ridge samples at potato harvest (T_H), based on the GLVMM analysis. The microbial genera names are indicated on the y-axis. The environment covariates values are indicated on the x-axis which are the coefficient values against the environmental covariates (e.g. ‘Potato Stock’ either JellyHigh, EstimaZero as compared to the JellyLow. The values range from positive, neutral to negative associations with the specific taxa on the y-axis. The red lines show significantly positive taxa associated with the indicated environmental covariate and the blue lines show significantly negative taxa; grey lines are not statistically significant. Note: The JellyLow group is not displayed as the analysis uses this as the reference. Supplementary Figure S22. Plot highlighting the impact of time on the abundance of microbial taxa in the ridge soil samples, based on the GLVMM analysis. The microbial genera names are indicated on the y-axis. The environment covariates values are indicated on the x-axis which are the coefficient values against the environmental covariate (e.g. Harvest [T_H] time point, as compared to the 50% plant emergence [T_E] time point. The values range from positive, neutral to negative associations with the specific taxa on the y-axis. The red lines show significantly positive taxa associated with the indicated environmental covariate and the blue lines show significantly negative taxa; g grey lines are not statistically significant. Note: The 50% emergence time point (T_E) is not displayed, as the analysis uses this as the reference. Supplementary Figure S23. Minimum subset of amplicon sequencing variants (ASVs) associated with percentage blackleg symptoms during the field trial. Analysis using the Ensemble Quotient Optimisation (EQO) technique considering at most 20 microbial taxa and the root samples at harvest. The left y-axis shows the relative abundance of the microbial taxa, and the right y-axis shows the percentage blackleg symptoms observed. The x-axis shows the sample identity with a colour legend for potato stock (Green EstimaZero, Blue JellyLow, and Red JellyHigh) and the irrigation regimes are shown in text (Unirrigated, Irrigation 1, Irrigation 2, and Irrigation 3). Supplementary Figure S24. Continuous ensemble quotient analysis (EQO) of the harvest ridge samples. Plot shows a stacked bar chart showing the minimum subset of amplicon sequencing variants (ASVs) associated with common scab symptoms. The x-axis shows the sample details including the potato stock (EstimaZero – Green, JellyLow – Blue, and JellyHigh – Red) and the irrigation regime is indicated in text. The right y-axis shows the relative abundance of the ASVs and the right y-axis shows the percentage (%) of common scab symptoms. Supplementary Figure S25. Continuous ensemble quotient analysis (EQO) of the harvest root samples. Plot shows a stacked bar chart showing the minimum subset of amplicon sequencing variants (ASVs) associated with common scab symptoms. The x-axis shows the sample details including the potato stock (EstimaZero – Green, JellyLow – Blue, and JellyHigh – Red) and the irrigation regime is indicated in text. The right y-axis shows the relative abundance of the ASVs and the right y-axis shows the percentage (%) of common scab symptoms.