Interplay between rhizospheric Pseudomonas chlororaphis strains lays the basis for beneficial bacterial consortia

Abstract

Pseudomonas chlororaphis (Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the plant. In this study we described the interactions between the rhizospheric Pc strains PCL1601, PCL1606 and PCL1607 with a focus on their effects on root performance. Differences among the three rhizospheric Pc strains selected were first observed in phylogenetic studies and confirmed by genome analysis, which showed variation in the presence of genes related to antifungal compounds or siderophore production, among others. Observation of the interactions among these strains under lab conditions revealed that PCL1606 has a better adaptation to environments rich in nutrients, and forms biofilms. Interaction experiments on plant roots confirmed the role of the different phenotypes in their lifestyle. The PCL1606 strain was the best adapted to the habitat of avocado roots, and PCL1607 was the least, and disappeared from the plant root scenario after a few days of interaction. These results confirm that 2 out 3 rhizospheric Pc strains were fully compatible (PCL1601 and PCL1606), efficiently colonizing avocado roots and showing biocontrol activity against the fungal pathogen Rosellinia necatrix. The third strain (PCL1607) has colonizing abilities when it is alone on the root but displayed difficulties under the competition scenario, and did not cause deleterious effects on the other Pc competitors when they were present. These results suggest that strains PCL1601 and PCL1606 are very well adapted to the avocado root environment and could constitute a basis for constructing a more complex beneficial microbial synthetic community associated with avocado plant roots.

Keywords: Pseudomonas chlororaphis; avocado; competitive index; consortia; rhizosphere; root colonization; syncom.

Figure 1

Rhizospheric Pseudomonas spp. phylogeny including Pc strains PCL1601, PCL1606 and PCL1607, marked with a red point and bold letters. Sequences were aligned by MUSCLE. The tree was made with MEGA 11 by multilocus sequence typing using concatenated 16S and the housekeeping genes rpoD, gyrB and recA. The neighbour-joining method with 1000 bootstraps and the Juke-Cantor model were used. The numbers in the nodes are the bootstrap values. P. aeruginosa PAO1 was used as an outgroup.

Figure 2

Positive (filled square) and negative (empty square) matches from a BLASTp search of PGP-related activities in the genomes of the selected Pc strains. PCA, phenazine-1-carboxylic acid; 2-OH-PCA, 2-hydroxyphenazine-1-carboxylic acid; PCN, phenazine-1-carboxamide; 2,4-diacetyphloroglucinol HCN, hydrogen cyanide; HPR, 2-hexyl-5-propyl resorcinol; ACCD, 1-aminocyclopropane-1-carboxylate deaminase; 2,3-btd, 2,3-butanediol; IAA, indol-3-acetic acid; PAA, Phenylacetic acid; PQQ, pyrroloquinoline. *: presence of 7 of 8 genes considered for a functional T3SS by software analysis.

Figure 3

Competitive index (CI) in liquid media (TPG and M9+succ) calculated after 24 hours of growth at 25°C. All inocula were adjusted to an O.D. of 0.05 at 600 nm (10⁵ cfu/ml). Then, dual and triple inoculations were made at the same proportion of 1:1 (1:1:1) in (A) TPG and (B) M9+succ. P. chlororaphis PCL1601 (red); P. chlororaphis PCL1606 (blue); P. chlororaphis subsp. piscium PCL1607 (red); 3P: combination of three Pc strains. Average competitive index (bar) and standard error (error bars) of three independent experiments are presented.

Figure 4

Colony morphology and biofilm formation of the individual strains and dual and triple strain combinations. Colony morphology growing after 5 days of incubation in (A) TPG, in (B) TPG plates supplemented with Congo red (40 µg/ml) and Coomassie brilliant blue (20 µg/ml). (C) Details of colonies grown on TPG supplemented with Congo red and Coomassie brilliant blue captured with a stereomicroscope. (D) Air-liquid interface pellicle formed after 5 days of incubation in TPG liquid media at 25°C. Single numbers correspond to single strain inoculation, and two numbers and 3P correspond to the dual and triple combinations of strains in the same proportion of 1:1 or 1:1:1, respectively. Average quantification and standard error of four independent experiments of bacterial adhesion are presented by measuring crystal violet absorbance at 590 nm in (E) TPG and (F) M9+succ. 01: P. chlororaphis PcPCL1601; 06: P. chlororaphis PCL1606; 07: P. chlororaphis subsp. piscium PCL1607; 3P: combination of the three Pc strains. Different letters represent a statistically significant difference after one-way analysis of variance, followed by Fisher’s least significance test (P = 0.05).

Figure 5

Competitiveness of Pc strains in avocado plant root assays. The competitive index in plant roots was calculated after 24 h of incubation. P. chlororaphis PCL1601 (red); P. chlororaphis PCL1606 (blue); P. chlororaphis subsp. piscium PCL1607 (red); 3P: combination of three Pc strains. Average competitive index (bar) and standard error (error bars) of three independent experiments are presented.

Figure 6

Time course of the bacterial counts of Pc strains inoculated in avocado roots (A) individually and (B) in triple combination (3P). P. chlororaphis PCL1601 (red); P. chlororaphis PCL1606 (blue); P. chlororaphis subsp. piscium PCL1607 (green). Bacterial counts and standard error (error bars) of three independent experiments are presented.

Figure 7

Bacterial distribution pattern on avocado roots after 24 h visualized under confocal laser microscopy. Single-strain biofilms (A–C) are shown: P. chlororaphis PCL1606 (in blue); P. chlororaphis PCL1601 (in red); and P. chlororaphis subsp. piscium PCL1607 (in green), as well as the dual and 3P combinations (D–G). Yellow squares shown in the bottom right corner images correspond to areas magnified three times. Scale bars represent 100 µm. 3P: Three-strain combination.

Figure 8

Biocontrol ability of combined 3 rhizospheric Pc strains (3P) against white root rot of avocado plants caused by Rosellinia necatrix. P. chlororaphis PCL1606 was used as a positive biocontrol reference. The area under the disease progress curve was calculated and analysed for significance after arcsine square root transformation with analysis of variance, followed by Fisher’s least significant difference test (P = 0.05). Average AUDPC values and standard error (error bars) of three independent experiments are presented. Values with different letters represent a statistically significant difference. 3P: Three-strain combination (P. chlororaphis PCL1601, P. chlororaphis PCL1606 and P. chlororaphis subsp. piscium PCL1607).