Maize Endophytic Plant Growth-Promoting Bacteria Peribacillus simplex Can Alleviate Plant Saline and Alkaline Stress

Abstract

Soil salinization is currently one of the main abiotic stresses that restrict plant growth. Plant endophytic bacteria can alleviate abiotic stress. The aim of the current study was to isolate, characterize, and assess the plant growth-promoting and saline and alkaline stress-alleviating traits of Peribacillus simplex M1 (P. simplex M1) isolates from maize. One endophytic bacterial isolate, named P. simplex M1, was selected from the roots of maize grown in saline-alkali soil. The P. simplex M1 genome sequence analysis of the bacteria with a length of 5.8 Mbp includes about 700 genes that promote growth and 16 antioxidant activity genes that alleviate saline and alkaline stress. P. simplex M1 can grow below 400 mM NaHCO₃ on the LB culture medium; The isolate displayed multiple plant growth-stimulating features, such as nitrogen fixation, produced indole-3-acetic acid (IAA), and siderophore production. This isolate had a positive effect on the resistance to salt of maize in addition to the growth. P. simplex M1 significantly promoted seed germination by enhancing seed vigor in maize whether under normal growth or NaHCO₃ stress conditions. The seeds with NaHCO₃ treatment exhibited higher reactive oxygen species (ROS) levels than the maize in P. simplex M1 inoculant on maize. P. simplex M1 can colonize the roots of maize. The P. simplex M1 inoculant plant increased chlorophyll in leaves, stimulated root and leaf growth, increased the number of lateral roots and root dry weight, increased the length and width of the blades, and dry weight of the blades. The application of inoculants can significantly reduce the content of malondialdehyde (MDA) and increase the activity of plant antioxidant enzymes (Catalase (CAT), Superoxide Dismutase (SOD), and Peroxidase (POD)), which may thereby improve maize resistance to saline and alkaline stress. Conclusion: P. simplex M1 isolate belongs to plant growth-promoting bacteria by having high nitrogen concentration, indoleacetic acid (IAA), and siderophore, and reducing the content of ROS through the antioxidant system to alleviate salt alkali stress. This study presents the potential application of P. simplex M1 as a biological inoculant to promote plant growth and mitigate the saline and alkaline effects of maize and other crops.

Keywords: Peribacillus simplex; endophytic bacteria; maize; plant growth promote; saline and alkaline stress.

Figure 1

Colonies grown in the NA medium added with 400 mM NaHCO₃ and blasted with standard bacterial strain in the BERGEY’S MANUAL. (A) Colonies grown in the NA medium added with 400 mM NaHCO₃ (B). Blast with standard bacterial strain in BERGEY’S MANUAL.

Figure 2

Physiological and biochemical reactions of bacteria. (A) Catalase characteristic experiment for catalase positive or negative. (B) hydrogen sulfide test for H₂S production. (C) Voges–Proskauer (VP) test for producing acid reaction. (D) methyl red test for producing acid reaction.

Figure 3

Schematic illustration showing the various tests performed on plates (Petri dishes 9 cm in diameter) for the assessment of the PGP traits of P. simplex M1. (A) Nitrogen fixation ability on the Nfb culture medium; (B) phosphorus solubilization ability on the NBRIP culture medium; (C) Siderophore production on the CAS culture medium; (D) produce IAA grown on no added tryptophan.

Figure 4

Growth of P. simplex M1 under different concentrations of NaHCO₃ stress. A total of 5 μL P. simplex M1 (OD₆₀₀ = 0.5) were spotted on solid LB media supplemented with the indicated stresses and grew at 30 °C for 3 d. No treatment is a control (CK).

Figure 5

Growth of P. simplex M1 under different pH conditions. A total of 200 μL P. simplex M1 (OD₆₀₀ = 0.5) liquid was added to 1 mL sterilized LB liquid medium with different pH values (3–10) at 30 °C with constant shaking at 1300 rpm. The OD₆₀₀ value was measured using an ELISA reader every hour and measured continuously for 24 h. * p < 0.05, *** p < 0.001, standard error of three biological replicates.

Figure 6

The effect of P. simplex M1 on maize seed germination. (A) Left: the sterile maize seeds were planted on the 1/2 MS; right: the sterile maize seeds in the inoculation with P. simplex M1 were planted on the 1/2 MS; (B) left: the sterile maize seeds were planted on the 1/2 MS + 10 mM NaHCO₃; right: the sterile maize seeds in the inoculation with P. simplex M1 were planted on the 1/2 MS + 10 mM NaHCO₃; (C) the leaf length of maize seed germination; (D) the root length of maize seed germination for 10 d. The germination percentage of the maize seeds was recorded during 10 d of NaHCO₃ treatment. Data show the means ± SE of three replicates. At least 50 seeds in each treatment were measured in each repeat. * p < 0.05, ** p < 0.01, standard error of three biological replicates.

Figure 7

TTC, DAB, and NBT staining of maize seed. (A) Each group represents the sterile maize seeds under free salt stress (control); (B) the sterile maize seeds in the inoculation with P. simplex M1 under free salt stress; (C) the sterile maize seeds under the 10 mM NaHCO₃ stress; (D) the sterile maize seeds in the inoculation with P. simplex M1 under the 10 mM NaHCO₃.

Figure 8

Colonization of P. simplex M1 on maize seedling root. (A) The morphology of GFP-expressing P. simplex M1; (B) the maize root as a control; (C) the colonization of root with GFP- expressing P. simplex M1. The white arrow points to a bacterial cluster, scale Bar = 50 µm.

Figure 9

Effect of P. simplex M1 strain on maize growth parameters after 7 days of cultivation under saline conditions. (A) The growth of maize seedling, from leaf to right: uninoculated maize seedling growth under free salt, inoculated maize seedling with P. simplex M1, uninoculated maize seedling growth under NaHCO₃ stress, and inoculated maize seedling with P. simplex M1 under NaHCO₃ stress; (B) chlorophyll content index; (C) leaf length; (D) leaf depth; (E) leaf dry weight; (F) root number; (G) root length; (H) root dry weight. E- represents uninoculated maize seedlings, E+ represents inoculated maize seedlings, E-ASS represents uninoculated maize seedlings irrigated with 400 mM NaHCO₃, and E + ASS represents inoculated maize seedlings irrigated with 400 mM NaHCO₃. The values represent the means of replicates (n = 4) ± standard deviations. Asterisks in superscript indicate a significant difference from the control at 95% between treatments. Each data point is the average of five replicates, and error bars represent ± SE. Error bars indicate ± SD. * Significance at p < 0.05, ** Significance p < 0.01, *** Significance p < 0.001.

Figure 10

Antioxidant enzyme activity determination in maize seeding. (A) MDA content; (B) POD activity; (C) SOD activity; (D) CAT activity; (E) superoxide anion; (F) H₂O₂ content. (G) CAT relative expression; (H) POD relative expression; (I) SOD relative expression. * Significance at p < 0.05, ** Significance p < 0.01, *** Significance p < 0.001.

Figure 11

A model for the mechanism underlying P. simplex M1 promoted maize development. The P. simplex M1 has the ability to fix atmospheric nitrogen, produce IAA, produce siderophore, and enhance the antioxidant enzyme activity. When the maize is inoculated with P. simplex M1, P. simplex M1 can increase seed vigor and seedling development (root length, leave length, and so on), thereby promoting maize growth. When the maize was treated with NaHCO₃, with the increase in ROS in plants, the activity of antioxidant enzymes caused by P. simplex M1 also enhanced, which alleviated the saline–alkaline stress on plants.