Novel Pseudomonas sp. SCA7 Promotes Plant Growth in Two Plant Families and Induces Systemic Resistance in Arabidopsis thaliana

Abstract

Pseudomonas sp. SCA7, characterized in this study, was isolated from roots of the bread wheat Triticum aestivum. Sequencing and annotation of the complete SCA7 genome revealed that it represents a potential new Pseudomonas sp. with a remarkable repertoire of plant beneficial functions. In vitro and in planta experiments with the reference dicot plant A. thaliana and the original monocot host T. aestivum were conducted to identify the functional properties of SCA7. The isolate was able to colonize roots, modify root architecture, and promote growth in A. thaliana. Moreover, the isolate increased plant fresh weight in T. aestivum under unchallenged conditions. Gene expression analysis of SCA7-inoculated A. thaliana indicated a role of SCA7 in nutrient uptake and priming of plants. Moreover, confrontational assays of SCA7 with fungal and bacterial plant pathogens revealed growth restriction of the pathogens by SCA7 in direct as well as indirect contact. The latter indicated involvement of microbial volatile organic compounds (mVOCs) in this interaction. Gas chromatography-mass spectrometry (GC-MS) analyses revealed 1-undecene as the major mVOC, and octanal and 1,4-undecadiene as minor abundant compounds in the emission pattern of SCA7. Additionally, SCA7 enhanced resistance of A. thaliana against infection with the plant pathogen Pseudomonas syringae pv. tomato DC3000. In line with these results, SA- and JA/ET-related gene expression in A. thaliana during infection with Pst DC3000 was upregulated upon treatment with SCA7, indicating the ability of SCA7 to induce systemic resistance. The thorough characterization of the novel Pseudomonas sp. SCA7 showed a remarkable genomic and functional potential of plant beneficial traits, rendering it a promising candidate for application as a biocontrol or a biostimulation agent.

Keywords: Arabidopsis thaliana; ISR; PGPB; Pseudomonas; Triticum aestivum L.; biocontrol; mVOCs; plant-microbe interactions.

Figure 1

The maximum-likelihood phylogenetic tree of Pseudomonas sp. SCA7 and 11 closely related type strains generated with FastTree 2.1 from 1,510 genes of the core genome. Values represent local support values based on the Shimodaira–Hasegawa test (1 SH = 100% bootstrap). The scale bar represents nucleotide substitutions per site (0.01 scale = 1% nucleotide substitutions per site).

Figure 2

Rhizosphere competence of SCA7. Swarming ability of SCA7 (A) and DH5α (B) on a semi-solid medium after 18 h. Ability to produce biofilm in vitro (C) and quantification of biofilm production compared to WCS417 (positive control) and DH5α (negative control), with averaged results of 12 replicates normalized to OD600 = 1 (E). Absorbance measured at 550 nm; error bars indicate standard deviation, **p = 0.001677. (D) In situ detection of root colonization of Arabidopsis thaliana roots by SCA7 on 2-week-old Arabidopsis thaliana roots grown in sterile quartz sand and visualized after fluorescence in situ hybridization (FISH) in a confocal laser scanning microscope (CLSM). Bacterial cells are identified by yellow color from overlayed channels of probes EUB (green) and gam42 (red). (F) An AHL biosensor assay with biosensor strain A136, positive control N35, and SCA7. Blue color indicates AHL production.

Figure 3

Plant growth-promoting traits of SCA7 in vitro. (A) IAA production after 24 h normalized to OD₆₀₀ = 1 with Herbaspirillum frisingense GSF30 as positive control. Error bars indicate standard deviation, N = 3, ***p = 0.00000012 (B) Siderophore production indicated by color change of the medium from blue to orange with RL1 as positive control and BG43 as negative control.

Figure 4

Ability of SCA7 to change root architecture and promote plant growth in Arabidopsis thaliana seedlings. Representative seedlings after 10-day growth on ½ MS agar (A) without and (B) with SCA7. (C) Primary root length difference of seedlings in absence or presence of SCA7. Number of (D) lateral roots and (E) fresh weight of seedlings treated with SCA7. Differential root hair formation on the terminal 1.5 cm of the primary root in absence or presence of SCA7 (F,G). Results averaged from three experiments each with 10 replicates. (H) Relative to control expression of marker genes after 24 h involved in auxin, nutrient uptake, and defense responses in A. thaliana seedling roots inoculated with SCA7. Mock represents control treatment with PBS, WCS417r (positive control), HK (heat-killed SCA7), and DH5α as negative control. Results were obtained from three pooled experiments. Asterisks indicate significant differences (p value * < 0.05, ** < 0.01, *** < 0.001). Error bars indicate standard deviation.

Figure 5

Influence of SCA7 on (A,C) fresh weight and (B,D) dry weight in (A,B) shoots and (C,D) roots in Triticum aestivum grown in sand-clay system for 3 weeks. Results averaged from two experiments each with 15 replicates. Asterisks indicate significant differences (p value* < 0.05, ** < 0.01). DW = dry weight, FW = fresh weight. Error bars indicate standard deviation.

Figure 6

A confrontation assay of SCA7 against plant pathogenic bacteria (A) Xanthomonas translucens, (B) Pseudomonas syringae pv. tomato DC3000 and plant beneficial, and (C) Bacillus velezensis FZB42 in direct (left) and indirect (right) contact. Antagonistic activity is indicated by smaller bacterial colonies. Control plates (= Mock) without SCA7.

Figure 7

A confrontation assay of SCA7 against plant pathogenic fungi (A) Fusarium oxysporum, (B) Fusarium culmorum, and (C) Rhizoctonia solani in direct (left) and indirect (right) contact grown for 7 days. Antagonistic activity is indicated by an inhibition zone around bacteria. Control plates (= Mock) without bacteria.

Figure 8

mVOCs detected in the headspace of Pseudomonas sp. SCA 7 cultures. The emission is reported as percentage (mean ± SD) of total emission for (1) 1-undecene, (2) octanal, (3) 1,4-undecadiene, and (4) an unknown compound. The chemical structures of the compounds 1 to 3 are given within the graph (drawn with Marvin JS by ChemAxon LtD; http://www.chemaxon.com). Further characteristics of the detected compounds, including retention time, Kovats retention index, and Chemical Abstracts Service (CAS)–registry numbers, are given in the Supplementary Table S7.

Figure 9

Effect of SCA7 on Pst DC3000 infection in A. thaliana. (A) Pst DC3000 proliferation (CFU/mg fresh weight) in A. thaliana leaf tissue. Plant roots were inoculated with SCA7 with 4-x-10⁷ CFU/ml and 2-x-10⁸ CFU/ml. Results averaged from three experiments (N = 4, N = 8, N = 8). Asterisks indicate significant differences (p value * < 0.05, ** < 0.01). (B) Effect of SCA7 on the relative expression of genes involved in common induced defense responses in A. thaliana shoots infected with Pst DC3000 and co-inoculated with SCA7 (or PBS, as control) at the roots. Three-week-old plants were inoculated with SCA7 and PBS at the roots, and then, after 24 h, the plants were spray infected with a suspension of Pst DC3000. Leaves were harvested after 24 h of infection with the pathogen, and expression of marker genes involved in defense responses mediated by SA and JA and was profiled. Error bars indicate standard deviation.