Inhibition of Three Potato Pathogens by Phenazine-Producing Pseudomonas spp. Is Associated with Multiple Biocontrol-Related Traits

Abstract

Phenazine-producing Pseudomonas spp. are effective biocontrol agents that aggressively colonize the rhizosphere and suppress numerous plant diseases. In this study, we compared the ability of 63 plant-beneficial phenazine-producing Pseudomonas strains representative of the worldwide diversity to inhibit the growth of three major potato pathogens: the oomycete Phytophthora infestans, the Gram-positive bacterium Streptomyces scabies, and the ascomycete Verticillium dahliae. The 63 Pseudomonas strains are distributed among four different subgroups within the P. fluorescens species complex and produce different phenazine compounds, namely, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxphenazine. Overall, the 63 strains exhibited contrasted levels of pathogen inhibition. Strains from the P. chlororaphis subgroup inhibited the growth of P. infestans more effectively than strains from the P. fluorescens subgroup. Higher inhibition was not associated with differential levels of phenazine production nor with specific phenazine compounds. The presence of additional biocontrol-related traits found in P. chlororaphis was instead associated with higher P. infestans inhibition. Inhibition of S. scabies by the 63 strains was more variable, with no clear taxonomic segregation pattern. Inhibition values did not correlate with phenazine production nor with specific phenazine compounds. No additional synergistic biocontrol-related traits were found. Against V. dahliae, PCN producers from the P. chlororaphis subgroup and PCA producers from the P. fluorescens subgroup exhibited greater inhibition. Additional biocontrol-related traits potentially involved in V. dahliae inhibition were identified. This study represents a first step toward harnessing the vast genomic diversity of phenazine-producing Pseudomonas spp. to achieve better biological control of potato pathogens. IMPORTANCE Plant-beneficial phenazine-producing Pseudomonas spp. are effective biocontrol agents, thanks to the broad-spectrum antibiotic activity of the phenazine antibiotics they produce. These bacteria have received considerable attention over the last 20 years, but most studies have focused only on the ability of a few genotypes to inhibit the growth of a limited number of plant pathogens. In this study, we investigated the ability of 63 phenazine-producing strains, isolated from a wide diversity of host plants on four continents, to inhibit the growth of three major potato pathogens: Phytophthora infestans, Streptomyces scabies, and Verticillium dahliae. We found that the 63 strains differentially inhibited the three potato pathogens. These differences are in part associated with the nature and the quantity of the phenazine compounds being produced but also with the presence of additional biocontrol-related traits. These results will facilitate the selection of versatile biocontrol agents against pathogens.

Keywords: Phytophthora infestans; Pseudomonas; Solanum tuberosum; Streptomyces scabies; Verticillium dahliae; biocontrol; phenazine.

FIG 1

In vitro antagonism of plant-beneficial phenazine-producing Pseudomonas spp. (A) Inhibition of Phytophthora infestans. (B) Inhibition of Streptomyces scabies. (C) Inhibition of Verticillium dahliae. For the 63 strains under study, the inhibition zone, defined as the distance between the edges of the bacterial colonies and the pathogen vegetative tissues, was measured. The colors used for each strain correspond to the following phylogenetic groups: P. fluorescens subgroup (orange), P. gessardii subgroup (yellow), CMR12a/CMR5c subgroup (green), and P. chlororaphis subgroup (light blue for the phzH⁺ strain, blue for the phzO⁺ strain, and dark blue for B25, which does not harbor phzH nor phzO). When it was not possible to display the phylogenetic affiliation of the strain on the histogram bar, a colored symbol was added next to its name. Statistical analyses (Kruskal-Wallis test, followed by post hoc tests) discriminated two groups of strains significantly different from each other (P < 0.05). Error bars represent the standard errors.

FIG 2

Phenazine production in King’s B broth by the 63 strains under study. Three phenazine compounds (PCA, PCN, and 2-OH-PHZ) were quantified from 5-day-old KB broth cultures using HPLC. The colors correspond to PCA (white), PCN (gray), and 2-OH-PHZ (black). The symbol “–” indicates the absence of phenazine detection. Phenazine production by the different strains was compared between each other using Kruskal-Wallis and post hoc tests. Two groups of strains were statistically discriminated, one group encompassing the strains with high phenazine production (H) and the other encompassing the strains with low phenazine production (L). Error bars represent the standard errors. The colors used for each strain correspond to the following phylogenetic groups: P. fluorescens subgroup (orange), P. gessardii subgroup (yellow), CMR12a/CMR5c subgroup (green), and P. chlororaphis subgroup (blue).

FIG 3

Phenazine production in three different agar-solidified growth media. Three phenazine compounds (PCA, PCN, and 2-OH-PHZ) were quantified in V8 agar, OBA, and PDA in the presence of the three potato pathogens. The histogram bars correspond to the amount of PCA (white), PCN (gray), and 2-OH-PHZ (black) being produced. For each medium, strains with different letters are significantly different (P < 0.05). Phenazine production was not compared across the different media. Error bars represent the standard errors. For each strain, a symbol indicates the phenazine compounds likely to be produced based on the presence or absence of the two accessory phenazine biosynthetic genes in their genome: PCA producer (rectangle), PCN (and PCA) producer (triangle), and 2-OH-PHZ (and PCA) producer (circle). The color of each symbol specifies the phylogenetic group to which the strain belongs: P. fluorescens subgroup (orange), P. gessardii subgroup (yellow), CMR12a/CMR5c subgroup (green), and P. chlororaphis subgroup (blue).

FIG 4

Correlation between pathogen inhibition and total phenazine production by Pseudomonas spp. (A) Phytophthora infestans. (B) Streptomyces scabies. (C) Verticillium dahliae. For each pathogen, correlations between the width of the inhibition zones and phenazine production in the confrontation medium was examined using Kendall rank tests.

FIG 5

Mean inhibition of the three studied potato pathogens by phenazine-producing Pseudomonas spp. The 13 strains were grouped according to the phenazine compounds they produce in the three media used for the confrontation assays. The colors correspond to strains producing only PCA (white), strains producing PCN (gray), strains producing 2-OH-PHZ (black), and strains producing no phenazine compound of any kind (dotted). For each pathogen, groups with different lowercase letters are significantly different (P < 0.05). Groups were not compared across pathogens. Error bars represent the standard errors.

FIG 6

Antibiotic activities of PCA, PCN, and 2-OH-PHZ against three potato pathogens. Phenazine compounds dissolved in DMSO were added to culture media inoculated with one of the three potato pathogens: Phytophthora infestans (A), Streptomyces scabies (B), and Verticillium dahliae (C). The colors correspond to PCA (white), PCN (gray), and 2-OH-PHZ (black). Statistical analyses (Kruskal-Wallis, followed by post hoc tests) were conducted to compare each concentration, and significant differences (P < 0.05) are indicated with different lowercase letters. Relative inhibitions were not compared across different concentrations or pathogens. Error bars represent the standard errors.

FIG 7

Mean pathogen inhibition ratios associated with the presence or absence of phytobeneficial traits. Ratios for each phytobeneficial trait were calculated by dividing the mean inhibition achieved by Pseudomonas strains harboring it by the mean inhibition achieved by strains not harboring it. For example, if a gene/cluster has a ratio of 1.5 for a given pathogen, it means that strains harboring this gene/cluster inhibit the pathogen 50% more than strains not harboring it. Only phytobeneficial traits that significantly correlate (P < 0.05) with higher or lower pathogen inhibition and are present in five strains or more are presented.