Identification and Characterization of Bacteria-Derived Antibiotics for the Biological Control of Pea Aphanomyces Root Rot

Abstract

Antibiosis has been proposed to contribute to the beneficial bacteria-mediated biocontrol against pea Aphanomyces root rot caused by the oomycete pathogen Aphanomyces euteiches. However, the antibiotics required for disease suppression remain unknown. In this study, we found that the wild type strains of Pseudomonas protegens Pf-5 and Pseudomonas fluorescens 2P24, but not their mutants that lack 2,4-diacetylphloroglucinol, strongly inhibited A. euteiches on culture plates. Purified 2,4-diacetylphloroglucinol compound caused extensive hyphal branching and stunted hyphal growth of A. euteiches. Using a GFP-based transcriptional reporter assay, we found that expression of the 2,4-diacetylphloroglucinol biosynthesis gene phlA_Pf-5 is activated by germinating pea seeds. The 2,4-diacetylphloroglucinol producing Pf-5 derivative, but not its 2,4-diacetylphloroglucinol non-producing mutant, reduced disease severity caused by A. euteiches on pea plants in greenhouse conditions. This is the first report that 2,4-diacetylphloroglucinol produced by strains of Pseudomonas species plays an important role in the biocontrol of pea Aphanomyces root rot.

Keywords: 2,4-DAPG; Aphanomyces euteiches; Pseudomonas spp.; antibiotics; biocontrol.

Figure 1

Bacterial antagonists inhibit the growth of A. euteiches.A. euteiches was cultured in the center of the plates and the bacterial cells were inoculated around the pathogen. All strains were cultured on ½ PDA plates at 28 °C for three days before the results of the inhibition were recorded (A) and the size of the inhibition zone was measured (B). The experiment was repeated three times with similar results. (C); the taxonomy of the bacterial antagonists that were isolated in this work was identified by 16S rRNA analysis. The phylogenetic tree was constructed using IQ-tree and visualized using Figtree v 1.4.4. Five different bacterial genera forming distinct clades are represented by different colors at the branches. The ten bacterial isolates highlighted with red color were isolated in this study. The GenBank accession number of each isolate was shown. The values at the node of the tree represent bootstrap support.

Figure 2

Inhibition of Pf-5 (A–D) and 2P24 (E) and their derivatives against A. euteiches on plates. A. euteiches was cultured in the center of the plates and the bacterial cells were inoculated around the pathogen. WT: wild type; 5-fold: Pf-5 mutant, ΔpltAΔpcnCΔofaAΔrzxBΔhcnB; 6-fold: Pf-5 mutant, ΔpltAΔpcnCΔofaAΔrzxBΔhcnBΔphlA; control has no bacteria inoculation. All strains were cultured on ½ PDA plates at 28 °C for two days before the results were recorded. Photos are representatives of three replicates and the experiment was repeated three times with similar results.

Figure 3

Impact of metabolites generated in the 2,4-DAPG biosynthesis pathway on hyphal growth of A. euteiches. (A), 2,4-DAPG gene cluster of Pf-5. Genes phlABCD encode four enzymes (PhlABCD) in the 2,4-DAPG biosynthesis pathway (B), and their expression is negatively regulated by the transcriptional repressor PhlF; PhlG degrades 2,4-DAPG to MAPG and its expression is controlled by PhlH; phlE encodes a putative permease of 2,4-DAPG. (C), Filter paper disks containing the indicated amount of the tested compounds were dried and placed around A. euteiches on ½ PDA plates. Representative results from three replicates were recorded two days after inoculation. (D), the oomycete hyphae were sampled at the A. euteiches growth margin on plates of (C) and examined under a microscope immediately after the inhibition was observed. Control indicates no compound treatment. Arrows show the excessive hyphal branching and stunted branches. Size bars indicate 15.9 μm. The experiments were repeated at least two times.

Figure 4

Expression of phlA by Pf-5 in the interactions with pea germinating seeds and A. euteiches. Wild type Pf-5 containing the GFP-based transcriptional reporter constructs pphlA_promoter-gfp (A), or the translational reporter construct pphlA_translation-gfp (B) was cultured with germinating pea seeds with or without the presence of A. euteiches hyphae. The expression of phlA was measured as relative GFP [fluorescence of GFP divided by (OD₆₀₀ × 1000)] recorded at 24 h post inoculation. * indicates treatments are significantly different (p < 0.05), as determined by Student t-test. Data are means and standard deviations of three replicates from a representative experiment repeated two times with similar results.

Figure 5

Biocontrol assays of pea Aphanomyces root rot by P. protegens Pf-5 in greenhouse. Pea seeds without inoculation served as negative control; pea seeds in positive control were planted in soil containing A. euteiches (50 oospores per gram soil) but not the Pf-5 strains. 5-fold mutant: ΔpltAΔpcnCΔofaAΔrzxBΔhcnB; 6-fold mutant: ΔpltAΔpcnCΔofaAΔrzxBΔhcnBΔphlA. Disease severity was evaluated on a 0–4 disease level scale. Scatter plot shows combined data from two independent experiments each had six replicates (i.e., six growth pots) per treatment and each replicate included five to six pea plants. * indicates treatments are significant different (p < 0.05), as determined by one-factor ANOVA analysis.