DOI: 10.5061/dryad.dz08kps40
title: Phages enhance both phytopathogen density control and rhizosphere microbiome suppressiveness

description: Bacteriophages, viruses that specifically target plant pathogenic
 bacteria, have emerged as a promising alternative to traditional
 agrochemicals. However, it remains unclear how phages should be applied to
 achieve efficient pathogen biocontrol, and to what extent their efficacy
 is shaped by indirect interactions with the resident microbiota. Here we
 tested if the phage biocontrol efficacy of Ralstonia solanacearum
 phytopathogenic bacterium can be improved by increasing the phage cocktail
 application frequency, and if the phage efficacy is affected by
 pathogen-suppressing bacteria already present in the rhizosphere. We find
 that increasing phage application frequency improves R. solanacearum
 density control, leading to a clear reduction in bacterial wilt disease in
 both greenhouse and field experiments with tomatoes. The high phage
 application frequency also increased the diversity of resident rhizosphere
 microbiota and enriched several bacterial taxa that were associated with
 the reduction in pathogen densities. Interestingly, these taxa often
 belonged to Actinobacteria known for antibiotics production and
 soil suppressiveness. To test if they could have had secondary effects on
 R. solanacearum biocontrol, we isolated Actinobacteria from Nocardia and
 Streptomyces genera and tested their suppressiveness to the pathogen in
 vitro and in planta. We found that these taxa could clearly inhibit R.
 solanacearum growth and constrain bacterial wilt disease, especially when
 combined with the phage cocktail. Together, our findings unravel an
 undiscovered benefit of phage therapy, where phages trigger a second line
 of defense by the pathogen-suppressing bacteria that already exist in
 resident microbial communities.
description: # Phages enhance both phytopathogen density control and rhizosphere
 microbiome suppressiveness
 [https://doi.org/10.5061/dryad.dz08kps40](https://doi.org/10.5061/dryad.dz08kps40) ## Description of the data and file structure Datasets included: ### Figure 1 * In the row of “trt”, “0” represents no phage cocktail application, “1” represents applied once (day 2), “2” represents applied two times (days 2 and 9), “3” represents applied three times (days 2, 9 and 16) after pathogen inoculation. "trt" means treatment. * In the row of “rep”, “1” represents the first replicate, “2” represents the second replicate, “3” represents the third replicate, “4” represents the fourth replicate. * “index” is the mean disease incidence of 6 plants within each replicate. * “audpc” is the area under the disease index curve, calculated as the integral. ### Figure 2 * The alpha diversity metrics, Species Richness, and Simpson Index were calculated using the 'diversity' function within the R package 'vegan', considering all bacterial ASVs. ### Figure 3 * We conducted correlation-based indicator analysis using the 'multipatt' function in the R package 'indispecies'. This analysis determined the point-biserial general correlation coefficient (r.g.) for ASVs positively associated with phage cocktail application frequency, with statistical significance determined after 999 permutations and a cutoff line of p &lt; 0.05. * For the co-occurrence network analysis, we included the most common genera with an appearance frequency of ≥50%. We calculated pairwise Spearman correlation matrices using the 'Hmisc' package and adjusted p-values using the 'fdrtool' package. Only correlations with absolute Spearman R-values &gt; 0.90 and adjusted p-values &lt; 0.05 were retained in the final co-occurrence network. * To identify groups of taxa responding similarly to phage cocktail application frequency, we utilized the 'cluster_fast_greedy' function from the 'igraph' package, applying the Newman algorithm. * “s.0” represents no phage cocktail application, “s.1” represents applied once, “s.2” represents applied two times, “s.3” represents applied three times. * In the row of “trt”, 'I' signifies genera present in all phage cocktail treatments. 'II' signifies genera present in treatments with twice and thrice phage cocktail applications. 'III' signifies genera present in treatments with once and twice phage cocktail applications. 'IV' signifies genera present only in treatments with once phage cocktail application. 'V' signifies genera present only in treatments with twice phage cocktail application. 'VI' signifies genera present only in treatments with thrice phage cocktail application. 'O' signifies genera present only in the treatment without phage cocktail application. ### Figure 4 * Displayed here are the relative abundances of the top 8 phyla and the top 8 genera belonging to the Actinobacteriota phylum. We conducted a Random Forest analysis to identify key predictor taxa for pathogen suppression. ### Figure 5 * The suppressiveness of 10 Actinobacterial isolates for the growth of *R. solanacearum* based on the supernatant assay. “ie” represents the inhibition effect on *R. solanacearum*. * “Control”: pathogen only, “P”: phage alone, “N”: *Nocardioides* alone, “S”: *Streptomyces* alone, “PN”: phage and *Nocardioides*, and “PS”: phage and *Streptomyces*. ## Sharing/Access information The raw sequencing reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA819579) ## Code/Software NA