Phenazine-Producing Rhizobacteria Promote Plant Growth and Reduce Redox and Osmotic Stress in Wheat Seedlings Under Saline Conditions

Abstract

Application of plant growth promoting bacteria may induce plant salt stress tolerance, however the underpinning microbial and plant mechanisms remain poorly understood. In the present study, the specific role of phenazine production by rhizosphere-colonizing Pseudomonas in mediating the inhibitory effects of salinity on wheat seed germination and seedling growth in four different varieties was investigated using Pseudomonas chlororaphis 30-84 (wild type) and isogenic derivatives deficient or enhanced in phenazine production. The results showed that varieties differed in how they responded to the salt stress treatment and the benefits derived from colonization by P. chlororaphis 30-84. In all varieties, the salt stress treatment significantly reduced seed germination, and in seedlings, reduced relative water content, increased reactive oxygen species (ROS) levels in leaves, and in three of four varieties, reduced shoot and root production compared to the no salt stress treatment. Inoculation of seeds with Pseudomonas chlororaphis 30-84 wild type or derivatives promoted salt-stress tolerance in seedlings of the four commercial winter wheat varieties tested, but the salt-stress tolerance phenotype was not entirely due to phenazine production. For example, all P. chlororaphis derivatives (including the phenazine-producing mutant) significantly improved relative water content in two varieties, Iba and CV 1, for which the salt stress treatment had a large impact. Importantly, all P. chlororaphis derivatives enabled the salt inhibited wheat varieties studied to maintain above ground productivity in saline conditions. However, only phenazine-producing derivatives enhanced the shoot or root growth of seedlings of all varieties under nonsaline conditions. Notably, ROS accumulation was reduced, and antioxidant enzyme (catalase) activity enhanced in the leaves of seedlings grown in saline conditions that were seed-treated with phenazine-producing P. chlororaphis derivatives as compared to noninoculated seedlings. The results demonstrate the capacity of P. chlororaphis to improve salt tolerance in wheat seedlings by promoting plant growth and reducing osmotic stress and a role for bacterial phenazine production in reducing redox stress.

Keywords: phenazine; plant growth promoting rhizobacteria; plant-microbe interaction; reactive oxygen species; salt stress; wheat.

Figure 1

The effect of no inoculum or inoculation with 30-84 ZN, 30-84 WT, or 30-84 Enh (top to bottom, NO, ZN, WT, Enh, respectively) on reactive oxygen species (ROS) accumulation in leaves obtained from seedlings of different wheat varieties grown under the salt stress condition. (A) Iba, (B) CV 1, (C) TAM 112, and (D) TAM 113. Seeds were treated with either no inoculum (methylcellulose) or 30-84 ZN, 30-84 WT or 30-84 Enh and grown one week without stress. Then plants were exposed to salt stress (200mM NaCl). DAB staining revealed ROS accumulation in fully expanded leaves (third leaves) from three separate wheat plants/variety/experiment in three separate experiments. Representative images are shown.

Figure 2

The effect of no inoculum or inoculation with 30-84 ZN, 30-84 WT, or 30-84 Enh on catalase enzyme activity in leaves obtained from seedlings of different wheat varieties in no salt and salt stress treatments. (A) Iba, (B) CV 1, (C) TAM 112, and (D) TAM 113. Seeds were treated with either no inoculum (methylcellulose) or 30-84 ZN, 30-84 WT, or 30-84 Enh and grown one week without stress and the received either a salt stress (200mM NaCl) or no salt stress. Catalase activity was measured in fully expanded leaves (third leaves) from three different wheat plants/variety/experiment in three separate experiments. Data are the means and standard errors. Data were analyzed by variety using a two-way ANOVA and Tukey’s test for multiple comparisons (P < 0.05, N=9) and letters indicate significant differences.

Figure 3

Growth and phenazine production of P. chlororaphis 30-84 WT and derivatives under increasing salt concentrations. Bacterial populations and phenazine production for 30-84WT, 30-84ZN, and 30-84 Enh were measured after 12 and 24 h in Luria-Bertani medium (LB) amended with different salt concentrations (0%, 0.5%, 1.25%, 2.5%, 5%, and 10% NaCl, w/v, e.g., 85, 210, 425, 850, and 1,700 mM NaCl). Bacterial populations and phenazine production were quantified spectrophotometrically (OD₆₂₀ and OD₃₆₇, respectively) and means and standard errors are shown. Data were analyzed using a two-way ANOVA and Tukey’s test for multiple comparisons (P < 0.05, N=3) and letters indicate significant differences.