The Foliar Application of Rice Phyllosphere Bacteria induces Drought-Stress Tolerance in Oryza sativa (L.)

Abstract

This study assessed the potential of Bacillus endophyticus PB3, Bacillus altitudinis PB46, and Bacillus megaterium PB50 to induce drought tolerance in a susceptible rice cultivar. The leaves of the potted rice plants subjected to physical drought stress for 10 days during the flowering stage were inoculated with single-strain suspensions. Control pots of irrigated and drought-stressed plants were included in the experiment for comparison. In all treatments, the plant stress-related physiochemical and biochemical changes were examined and the expression of six stress-responsive genes in rice leaves was evaluated. The colonization potential on the surface of the rice leaves and stomata of the most successful strain in terms of induced tolerance was confirmed in the gnotobiotic experiment. The plants sprayed with B. megaterium PB50 showed an elevated stress tolerance based on their higher relative water content and increased contents of total sugars, proteins, proline, phenolics, potassium, calcium, abscisic acid, and indole acetic acid, as well as a high expression of stress-related genes (LEA, RAB16B, HSP70, SNAC1, and bZIP23). Moreover, this strain improved yield parameters compared to other treatments and also confirmed its leaf surface colonization. Overall, this study indicates that the foliar application of B. megaterium PB50 can induce tolerance to drought stress in rice.

Keywords: Bacillus megaterium PB50; drought stress; induced systemic tolerance; phyllosphere; plant-growth-promoting bacteria.

Figure 1

Plot of principal component analysis (PCA) showing ordination of the samples based on the physiochemical and biochemical properties of the plant leaves. Treatment are indicated by 95% confidence ellipses. RWC, relative water content; APX, ascorbate peroxidase activity; CAT, catalase activity; GPX, glutathione peroxidase activity; MDA, malondialdehyde; IAA, indole acetic acid; ABA, abscisic acid.

Figure 2

Bar graph of the relative gene expression of LEA, HSP70, RAB16B, AP2/ERF, bZIP23, and SNAC1 in 70-day-old rice plants under different treatment conditions. Shown are means and standard deviation values (n = 4). Values with different letters are significantly different according to Duncan’s test (p ≤ 0.05).

Figure 3

Heatmap showing the fold-change expression of genes in different treatments and clustering of samples according to the relative expression of the set of analyzed genes.

Figure 4

Co-inertia analysis (CIA) results based on two datasets (plant physico- and biochemical variables and gene expression data). (A) Projections of the principal axes of the two datasets onto the axes of the co-inertia analysis. X axes: Plant physico- and biochemical variables; Y axes: Drought-responsive gene expression data. (B) Scree plot of eigenvalues. (C) Correlation of gene expression data with the first two axes of the co-inertia analysis. (D) Correlation of plant physico- and biochemical variables with the first two axes of the co-inertia analysis. (E) Plot of the first two components in the sample space. Each sample is represented by a square, where the two datasets for each sample are connected by lines to a center point (global score).

Figure 5

Bar graphs representing means and SD of the yield parameters: Panicle length (a), weight (b), and grain weight (c) of the rice plants of different treatments. PB3, drought stress with B. endophyticus PB3 foliar spray; PB46, drought stress with B. altitudinis PB46 foliar spray; PB50, drought stress with B. megaterium PB50 foliar spray; Cws, drought stress control; and CI, irrigated control. Values with different letters (above the bars) are significantly different according to Duncan’s test (p ≤ 0.05).

Figure 6

Bar graph showing means and SD of the stomatal aperture of the rice leaves of treatments under gnotobiotic conditions. T₁—PBS spray; T₂—PEG6000 (32.6%) + PBS spray; T₃—PB50 spray inoculation; and T₄—PEG6000 (32.6%) + PB50 spray inoculation. PBS, phosphate buffer solution. Values with different letters are significantly different according to Duncan’s test (p ≤ 0.05).

Figure 7

Representative SEM images showing the change in stomatal aperture in control rice plants under normal (A) and osmotic stress (B) conditions, and B. megaterium PB50 spray inoculated rice plants under normal (C) and osmotic stress (D) conditions.

Figure 8

SEM images of Bacillus megaterium PB50 cells on the leaf surface of rice plants (T₄). PB50 strain colonization in the ridges of rice leaf surface (A), on trichomes (B), near silica bodies (C), and near stomata produced with exopolysaccharides (EPS) (D). S, silica body; T, trichome; and EPS, exopolysaccharides.

Figure 9

The figure presents schemes of two different mechanisms of rice plants under moderate drought stress conditions (−1.20 to −1.40 MPa) with and without foliar application of B. megaterium PB50 during the flowering period of rice plants (variety CO51). In control stress plants where the expression of the bZIP23 gene is low due to the low inheritance potential to ABA production, excessive ethylene can be produced that induces the expression of AP2/ERF genes and causes leaf senescence. This stress-responsive mechanism is insufficient to protect plant cells, resulting in the excessive release of MDA from damaged cells due to reactive oxygen species. In the case of rice plants with leaves treated with a suspension of Bacillus megaterium PB50, the scenario can be just the opposite. The release of 1-Aminocyclopropane-1-carboxylate (ACC) deaminase by the PB50 strain reduces the expression of the AP2/ERF gene by breaking down the ethylene precursor molecule, and the other exogenous PB50 strain metabolites could induce ABA-dependent mediated stress-responsive mechanisms to reduce cell damage and induce stomatal closure to avoid evapotranspiration during drought stress.

Figure 10

Photo of illustrating experimental setup. The rice plants during the flowering stage (70 days after sowing (DAS)) after 10 days of drought stress are shown. From left to right, the treatments are as follows: PB3, drought stress with B. endophyticus PB3 foliar spray; PB46, drought stress with B. altitudinis PB46 foliar spray; PB50, drought stress with B. megaterium PB50 foliar spray; Cws, drought stress control; and CI, irrigated control. R₃ is the replicate 3 of each treatment.