Phenazines are involved in the antagonism of a novel subspecies of Pseudomonas chlororaphis strain S1Bt23 against Pythium ultimum

Abstract

Long-term use of chemical fungicides to control plant diseases caused by fungi and oomycetes has led to pathogen resistance and negative impacts on public health and environment. There is a global search for eco-friendly methods and antagonistic bacteria are emerging as alternatives. We isolated a potent antagonistic bacterial strain (S1Bt23) from woodland soil in Québec, Canada. Taxonomic characterization by 16S rRNA, multi-locus sequence analysis, pairwise whole-genome comparisons, phylogenomics and phenotypic data identified strain S1Bt23 as a novel subspecies within Pseudomonas chlororaphis. In dual culture studies, strain S1Bt23 exhibited potent mycelial growth inhibition (60.2-66.7%) against Pythium ultimum. Furthermore, strain S1Bt23 was able to significantly bioprotect potato tuber slices from the development of necrosis inducible by P. ultimum. Annotations of the whole genome sequence of S1Bt23 revealed the presence of an arsenal of secondary metabolites including the complete phenazine biosynthetic cluster (phzABCDEFG). Thin-layer (TLC) and high-performance liquid (HPLC) chromatographic analyses of S1Bt23 extracts confirmed the production of phenazines, potent antifungal compounds. CRISPR/Cas9-mediated deletion of phzB (S1Bt23ΔphzB) or phzF (S1Bt23ΔphzF) gene abrogated phenazine production based on TLC and HPLC analyses. Also, S1Bt23ΔphzB and S1Bt23ΔphzF mutants lost antagonistic activity and bioprotection ability of potato tubers against P. ultimum. This demonstrated that phenazines are involved in the antagonistic activity of S1Bt23 against P. ultimum. Finally, based on genotypic and phenotypic data, we taxonomically conclude that S1Bt23 represents a novel subspecies for which the name Pseudomonas chlororaphis subsp. phenazini is proposed.

Keywords: Pseudomonas chlororaphis; Pseudomonas chlororaphis subsp. phenazini subsp. nov.; Pythium ultimum; Biological control; CRISPR/cas9; Phenazine; Phenazine-1-carboxylic acid; Secondary metabolites.

Fig. 1

Neighbor-joining evolutionary tree of 16S rRNA-gyrB-rpoB-rpoD (2964 bp) concatenated nucleotide sequences showing strain S1Bt23 clustering distinctly within the Pseudomonas chlororaphis subgroup. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. SG, subgroup.

Fig. 2

Genome-based TYGS-generated tree showing distinct evolutionary clustering of Pseudomonas chlororaphis subsp. phenazini strains (blue rectangle) relative to strains of the other P. chlororaphis subspecies. The TYGS algorithm classified S1Bt23 (in bold) and 15 NCBI strains as novel subspecies based on the cut-off for subspecies of 79.0%. Branch lengths are scaled according to the GBDP distance formula d5. Numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replicates. The tree was rooted at the midpoint.

Fig. 3

(A) Overview of the different genomic regions of potential secondary metabolites in Pseudomonas chlororaphis subsp. phenazini strain S1Bt23; and (B) Four complete biosynthetic clusters (cluster 1, 4, 9, and 15) previously reported to inhibit fungal/oomycete plant pathogens. NI-siderophore, NRPS-independent, IucA/IucC-like siderophores; NRPS, Non-ribosomal peptide synthetase; NAGGN, N-acetylglutaminylglutamine amide; RiPP-like, Other unspecified ribosomally synthesized and post-translationally modified peptide product (RiPP) and Hcn, hydrogen cyanide; GTS, glutathione S-transferase. The exact coordinates of the regions can be found in Figure S5. Red stars indicate the genes deleted (phzB and phzF) in our study.

Fig. 4

(A) Thin layer chromatography silica60 plate, under ultra-violet light, showing the loss of phenazine production (*dark gray band) in the pAKanCRISPR–cas9 mutants of S1Bt23: 1, positive control (synthetic phenazine; 20 µl of 1 mg/ml stock); 2, S1Bt23 wild type; 3, S1Bt23ΔphzB; and 4, S1Bt23ΔphzF mutants (right); (B) TLC extracts from WT inhibited growth of Pythium ultimum (Py. u.) but not those from mutants (ΔB or ΔF); and (C) WT inhibited Py. u. growth in dual culture assay but not ΔphzB or ΔphzF mutants.

Fig. 5

(A) Pseudomonas chlororaphis subsp. phenazini strain S1Bt23 (WT) shows significant protection of potato tuber slices against Pythium ultimum (Py. u.) compared with ΔphzB or ΔphzF mutants. No necrosis was observed on potato tuber slices treated with bacteria alone. (B) Statistical analysis of area of lesions calculated using mean diameter of the lesion [area = π(d/2)². Statistical analysis using one-way ANOVA. ** indicates p value < 0.01.