Abstract
Bacteriophages have been proposed as an alternative to pesticides to kill bacterial pathogens of crops. However, the efficacy of phage biocontrol is variable and poorly understood in natural rhizosphere microbiomes. We studied biocontrol efficacy of different phage combinations on Ralstonia solanacearum infection in tomato. Increasing the number of phages in combinations decreased the incidence of disease by up to 80% in greenhouse and field experiments during a single crop season. The decreased incidence of disease was explained by a reduction in pathogen density and the selection for phage-resistant but slow-growing pathogen strains, together with enrichment for bacterial species that were antagonistic toward R. solanacearum. Phage treatment did not affect the existing rhizosphere microbiota. Specific phage combinations have potential as precision tools to control plant pathogenic bacteria.
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Data availability
All sequence data generated in this study has been deposited in NCBI SRA database and the accession numbers are reported in the Methods. Phage sequence accession numbers are: SRR8402465 (NJ-P3), SRR8403229 (NB-P21), SRR8403928 (NC-P34) and SRR8410130 (NN-P42). The rhizosphere microbiome data determined at the end of the greenhouse experiment have accession numbers from SRR8417955 to SRR8417999 (45 samples, paired end sequencing). The rhizosphere microbiome data obtained in a separate laboratory experiment have accession numbers running from SRR8470488 to SRR8501098 (30 samples, paired end reads). All other data has been deposited to Dryad Digital Repository with the following digital identifier: https://doi.org/10.5061/dryad.02v6wwpzq.
Code availability
All code or algorithms used in this study are published and referenced in the Methods.
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Acknowledgements
This research was financially supported by the National Natural Science Foundation of China (41922053, 41671248; Z.W.), the National Key Basic Research Program of China (2015CB150503; Q.S.), the National Key Research and Development Program of China (2018YFD1000800; Z.W.) and the 111 project (B12009; Q.S.). A.J. is supported by the Netherlands Organization for Scientific Research (NWO) project ALW.870.15.050. V.P.F. is supported by the Wellcome Trust (ref. 105624) through the Center for Chronic Diseases and Disorders (C2D2) and Royal Society Research Grants (RSG\R1\180213 and CHL\R1\180031) at the University of York.
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V.P.F., X.F.W. and Z.W. developed the ideas and designed the experimental plans. X.F.W., J.N.W. and Z.W. performed the experiments. X.F.W., K.E.Y., A.J., Z.W. and V.P.F. analyzed the data. All the authors wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 A neighbor-joining phylogenetic tree showing the relatedness of the four phage types used in the experiments with similar phages publicly available in NCBI.
A neighbor-joining phylogenetic tree based on phage head-tail connector protein similarity between the phages used in this experiment (NJ-P3, NB-P21, NC-P34, NN-P42; red colour) and other similar phages that are publicly available at NCBI (black colour). All four phages belonged to order Caudovirales and the family Podoviridae, were highly similar with each other and a Ralstonia phage DU_RP_II. The scale bar represents 0.2 amino acid substitutions per site and values next to the nodes show bootstrap values based on 500 samples.
Supplementary Figure 2 Annotated genome maps for the four phage types used in the experiments.
In all circular genome maps, the outermost black circle represents the full length of the genome, the second outermost multicolored circle represents the forward and reverse reading frames, the third outermost green circle represents GC content and the innermost purple circle represents GC skew.
Supplementary Figure 3 Genetic distance between the four phage types used in our experiment and similar phages publicly available in NCBI with reported information on lytic and lysogenic activity.
Phylogenetic tree showing the relatedness of lytic and lysogenic phages based on genomic similarity. Each genome was first compared to another and to themselves to account and normalize for differences in phage genomes sizes and a neighbor joining tree was then constructed based on the complete distance matrix based on 500 bootstrap sample values as described previously (Mizuno, C.M., Rodriguez-Valera, F., Kimes, N.E. & Ghai, R. Expanding the Marine Virosphere Using Metagenomics. PLoS Genetics. 9 (2013)). Branch colours show different phage families and known lytic and lysogenic phages are shown in black and red, respectively. The phage types used in the experiments (NJ-P3, NB-P21, NC-P34, NN-P42) were most closely related to a lytic phage DU_RP_II.
Supplementary Figure 4 The mean infectivity of phage types to Ralstonia solanacearum clones isolated from China.
The average infectivity of four phage types against 96 R. solanacearum bacterial isolates originating from the tomato rhizosphere of four independent tomato fields in China (24 independent R. solanacearum isolates from each field: Nanjing, Nanchang, Ningbo and Nanning). Bars represent standard deviation between four different fields.
Supplementary Figure 5 Correlation between phage and pathogen densities in tomato rhizosphere.
The R2 and P value refer to the most parsimonious model fitted based on linear regression analysis (n=3 for ancestral and control treatment, n=12 for one- and three-phage treatments and n=18 for two-phage treatment; all n are biologically independent samples).
Supplementary Figure 6 Relationships between pathogen growth rate, phage combination treatment and phage resistance at the end of the greenhouse experiment.
Panel A shows the growth rate for evolved pathogen clones isolated from different phage combination treatments at the end of the greenhouse experiment. Lowercase letters above boxplots denote for significant differences between phage combination treatments (multiple comparisons were conducted by Tukey test, FDR adjusted P<0.05) and R2 and red dashed line represents the maximum growth rate of ancestral pathogen strain. Box-plots show interquartile range (25 to 75% of the data), the median as lines and outliers as dots. Panel B shows the correlation between pathogen maximum growth rate and mean phage resistance to all ancestral phages at the end of the greenhouse experiment. The R2 and P values refer to the most parsimonious model, black line shows the mean regression based on all the data points and the small inset in top right corner shows the change in regression coefficient between phage combination treatments (n=3 for ancestral and control treatment, n=12 for one- and three-phage treatments and n=18 for two-phage treatment; all n are biologically independent samples). N0 to N3 denote for no-phage, single-phage, two-phage and three-phage treatments, respectively.
Supplementary Figure 7 Relationship between pathogen competitiveness and phage combination treatment at the end of the greenhouse experiment.
Pathogen competitive ability was measured as the relative growth against the phage-susceptible ancestral pathogen in terms of reduction in the ancestral pathogen abundance. All box-plots show interquartile range (25 to 75% of the data) and median as line. Lowercase letters above boxplots denote for significant differences between phage combination treatments (multiple comparisons were conducted by Tukey test, FDR adjusted P<0.05, n=3 for ancestral and control treatment, n=12 for one- and three-phage treatments and n=18 for two-phage treatment; all n are biologically independent samples).
Supplementary Figure 8 Effects of phage combination treatments on microbial OTU richness at the end of the greenhouse experiment.
Microbial OTU richness correlated positively with the number of phages present in the phage combination treatment. The R2 and P value refer to the most parsimonious model fitted based on linear regression analysis and red dashed line shows the OTU richness of no-phage control treatment (n=3 for control treatment, n=12 for one- and three-phage treatments and n=18 for two-phage treatment; all n are biologically independent samples).
Supplementary Figure 9 Effects of phage combination treatments on relative abundance of different bacterial phyla at the end of the greenhouse experiment.
Correlations between relative abundances of different bacterial phyla and the number of phages present in the phage combinations at the end of the greenhouse experiment. Panels A-H show the correlation for Ralstonia, Proteobacteria, Bacteroidetes, Chloroflexi, Acidobacteria, Planctomycetes, Firmicutes and Actinobacteria, respectively. Red dashed lines in all panels show the no-phage control treatment. The R2 and P value refer to the most parsimonious model fitted based on linear regression analysis (n=3 for control treatment, n=12 for one- and three-phage treatments and n=18 for two-phage treatment; all n are biologically independent samples).
Supplementary Figure 10 Effects of phages on bacterial co-occurrence networks at the end of the greenhouse experiment.
The bacterial co-occurrence networks associated with single-phage and three-phage combinations treatments. Each node represents a bacterial OTU, and each edge represents a Spearman correlation with negative (blue) and positive correlations (red). The node colours represent taxa classification at the phylum level (n=12 biologically independent samples for one- and three-phage treatments).
Supplementary Figure 11 The resistance of pathogenic and non-pathogenic bacterial isolates to phage types used in the experiments.
The average infectivity of four phage types against 96 R. solanacearum bacterial isolates originating from the tomato rhizosphere of four independent tomato fields in China (24 independent R. solanacearum isolates from each field: Nanjing, Nanchang, Ningbo and Nanning) and 400 non-pathogenic bacterial isolates from the soil used in the greenhouse experiment. Bars represent standard deviation between four different field for pathogen isolates.
Supplementary Figure 12 Effects of phage on the abundance of bacterial genera.
Panel A shows significant positive (red) and negative (blue) correlation coefficients between bacterial genera abundances and number of phages present in the phage combinations. Correlations were determined using Spearman method and all correlations were FDR adjusted P<0.05. Panel B shows to which phyla positively (top pie chart) and negatively (bottom pie chart) correlated bacteria belonged to.
Supplementary Figure 13 Simplified structural equation models explaining pathogen density, microbiome diversity and disease incidence.
Structural equation model path diagrams showing the phage combination-mediated effects on the pathogen density (a), microbiome diversity (b) and the disease incidence (c). Red, blue and grey arrows denote for positive, negative and non-significant pathways, respectively, and the numbers beside arrows denote for the magnitude of these effects. Numbers within the circles show how much of the variance of each variable was explained by the other variables and χ2 and NFI values denote for the fit of the models.
Supplementary Figure 14 Phylogenetic relationship between Ralstonia solanacearum clones isolated from four different tomato fields.
Phylogenetic neighbour-joining tree based on partial endoglucanase (egl) gene sequences of Chinese Ralstonia solanacearum strains and most closely related reference strains (red circles), that were used to test the phage type infectivity ranges (Supplementary Fig. 4). The tips of the tree show 96 Chinese R. solanacearum isolates with sample location abbreviation and sample ID. Chinese isolates are assigned to specific sequevar types based on their closest relative reference strains and sequevar type numbers are shown in boxes over each clade.
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Wang, X., Wei, Z., Yang, K. et al. Phage combination therapies for bacterial wilt disease in tomato. Nat Biotechnol 37, 1513–1520 (2019). https://doi.org/10.1038/s41587-019-0328-3
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DOI: https://doi.org/10.1038/s41587-019-0328-3
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