Phenotypic and Genomic Analysis of Enterobacter ludwigii Strains: Insights into Mechanisms Enhancing Plant Growth Both Under Normal Conditions and in Response to Supplementation with Mineral Fertilizers and Exposure to Stress Factors

Abstract

In this research study, we investigated four strains of Enterobacter ludwigii that showed promising properties for plant growth. These strains were tested for their ability to mobilize phosphorus and produce ammonium, siderophores, and phytohormones. The strains exhibited different values of PGP traits; however, the analysis of the complete genomes failed to reveal any significant differences in known genes associated with the expression of beneficial plant traits. One of the strains, GMG_278, demonstrated the best potential for promoting wheat growth in pot experiments. All morphological parameters of wheat were improved, both when GMG_278 was applied alone and when combined with mineral fertilizer. The combined effect we observed may suggest various mechanisms through which these treatments influence plants. The amount of pigments and proline suggests that bacterial introduction operates through pathways likely related to stress resilience. A study on the genetic mechanisms behind plant resilience to stress has revealed a significant upregulation of genes related to reactive oxygen species (ROS) defense after bacterial exposure. It is important to note that, in the initial experiments, the strain showed a significant production of salicylic acid, which is a potent inducer of oxidative stress. In addition, the synthesis of some phytohormones has been restructured, which may affect root growth and the architecture of root hairs. When combined with additional mineral fertilizers, these changes result in a significant increase in plant biomass.

Keywords: Enterobacter ludwigii; Triticum aestivum; drought; mineral fertilizer; plant growth-promoting bacteria; reactive oxygen species; salinization.

Figure 1

Diagrams of agronomic indicators of plants after treatment with bacteria and mineral fertilizer. (A) the weight of the aboveground part of the plant, (B) the weight of the underground part of the plant, (C) the height of the plant, (D) the dry weight of the aboveground part of the plant, (E) the dry weight of the underground part of the plant. *—p (p values) < 0.05, **—p < 0.01, ****—p < 0.0001.

Figure 2

Alignment to the complete genomes of Enterobacter sp. by OrtoANI analysis. OrthoANI measures the overall similarity between two genome sequences. The threshold for species discrimination is 95~96%. The genomes for comparison were downloaded from the online accessible database https://www.ncbi.nlm.nih.gov/datasets/genome/ (accessed on 6 October 2024).

Figure 3

The Venn diagram shows the number of common genes in the studied strains.

Figure 4

Diagrams of morphological and physiological parameters of plants under stress conditions in experiments. Legend (MF—mineral fertilizer, 278—microbial fertilizer, NaCl—watering with 1% NaCl solution, PEG—watering with 10% PEG solution). (A,B) the weight of the wet (A) and dry (B) aboveground part of the plant; (C,D) the wet weight (C) and dry weight (D) of the underground part of the plant; (E) height of the plant; (I–K) the amount of pigment: chlorophyll A (I), chlorophyll B (J), carotenoids (K); (H) the amount of proline; (F,G) quantity ratio ChlA/ChlB (F), ChlA+B/car (G). *—p (p values) < 0.05, **—p < 0.01, ***—p < 0.001, ****—p < 0.0001.

Figure 5

Principal component analysis of the effect of strain GMG_278 on growth and physiological indices of wheat under stress conditions and at supplementing with mineral nutrition. Legend (MN—mineral nutrition, 278—microbial fertilizer, NaCl—watering with 1% NaCl solution, PEG—watering with 10% PEG solution). (A) PCA plot, (B) the contribution of indicators to the main component, (C) Redundancy analysis (RDA).

Figure 6

Heat map of changes in the morphological and physiological characteristics of wheat grown under stress conditions and at supplementing plants with mineral nutrition and the GMG_278 strain. Legend (MN—mineral nutrition, 278—microbial fertilizer, NaCl—watering with 1% NaCl solution, PEG—watering with 10% PEG solution).

Figure 7

Schematic representation of the effects of stress factors on plants. Most of the mechanisms for sensing and initiating adaptive responses to various abiotic stresses involve changes in proteins and lipids in biological membranes (1). Unfavorable conditions entail ultrastructural changes in biomolecules, which are sensed by receptors or specialized proteins, leading to the augmentation of the Ca²⁺ level in the cytosol (2), as well as REDOX imbalance (3). These signals activate kinase cascades and other secondary events that stimulate phosphorylation/dephosphorylation consecutive events (4), reaching a culmination in TF activation (5), and remodeling of gene expression (6). Moreover, the activation of certain enzymes stimulates the biosynthesis of osmolytes, pigments, thermoprotectants (7), and other secondary metabolites (8) in addition to ROS detoxifying enzymes (9), which are aimed to restore redox homeostasis in stressed cells. At the systemic level, various transformations are shown. They concern morphological changes, which are detected in the root and leaves and affect the proliferation of lateral roots in response to drought; biochemical changes associated with the secretion of phytochelants in response to heavy metals; and also the reclusion and closure of stomata in addition to a decrease in leaf area and abscission (10). The figure is based on the previous study [21].

Figure 8

Diagrams of the dependence of gene expression involved in response to stress factors in wheat grown under drought and salinity conditions on introducing GMG_278 strain. Legend (MF—mineral fertilizer, NaCl—watering with 1% NaCl solution, PEG—watering with 10% PEG solution). (A) gene ABARE, (B) gene WKY26, (C) gene MAPK, (D) gene CKX10, (E) gene DREB, (F) gene WKY71, (G) gene ARF2, (H) gene CTR, (I) gene POD, (J) gene CAT, and (K) gene LPX. *—p (p values) < 0.05, **—p < 0.01, ***—p < 0.001, ****—p < 0.0001.

Figure 9

Principal component analysis of data on the expression of genes involved in response to stress factors in wheat grown under drought and salinity conditions at introducing GMG_278 strains. Legend (MN—mineral nutrition, 278—microbial fertilizer, NaCl—watering with 1% NaCl solution, PEG—watering with 10% PEG solution). (A) PCA plot, (B) the contribution of indicators to the main component. The color indicates the maximum absolute coefficients for each PSA vector. For PSA1—green, for PSA 2 two colors are used: pink for conditions that shift the gene expression level up along the PSA2 axis, yellow—down along the PSA2 axis. (C) Redundancy analysis (RDA).