The regulatory effect of inoculation with dark septate endophyte on root exudates of Sophora alopecuroides L. under salt stress

Objectives The salt-tolerant plant Sophora alopecuroides L.holds immense potential in the restoration of saline-alkali soil. This study delved the regulatory effects of dark septate endophytes (DSE) inoculation on growth performance and metabolic responses in S. alopecuroides under salt stress conditions, offering theoretical support for the ecological restoration of plant-microbial interaction in saline-alkali land.

Methods A controlled hydroponic system was conducted using S. alopecuroides as the test material. Five distinct concentrations of Na₂SO₄ salts (0%, 0.15%, 0.30%, 0.45%, 1.00%) and two groups of treatments (inoculated with DSE and uninoculated control, CK) were set up, with a total of 30 samples (5 salt concentrations×2 treatments×3 replicates). After the plants have grown in the nutrient solution for 90 days, the root exudates of the plants were collected. and the plant biomass accumulation, ion (Na⁺, K⁺, Ca²⁺, Mg²⁺) content, and root morphology parameters (total length, surface area, tip number) were quantitatively assessed. In the detection of root exudates, ultra-high performance liquid chromatogre-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for non-targeted metabolomics analysis. Comparative analysis was conducted to evaluate the two-factor effects (salt concentration and inoculation) on plant growth, aiming to elucidate DSE-mediated regulation of biomass accumulation, ion homeostasis, and metabolite profiles in S. alopecuroides, and employ pathway analysis to establish response relationships among variables, ultimately revealing the mechanisms underlying DSE-enhanced salt tolerance.

Results Na₂SO₄ stress significantly reduced S. alopecuroides biomass. Inoculation with DSE had a significant biological stimulating effect on S. alopecuroides. Compared with the control (CK) at the identical salt concentration, biomass of the S. alopecuroides increased by 93%−254%, root length by 4%−181%. The promotional effects of DSE were the most pronounced under 0.30% Na₂SO₄ stress. The K⁺ content of inoculated S. alopecuroides increased by 53%−108%, the ratio of Na⁺/K⁺ decreased by 23%−33%. Metabolic analysis uncovered that DSE specifically triggered six main pathways such as glycerol phospholipid metabolism and carotenoid biosynthesis, and boosted the synthesis of metabolites such as lipids and carotenoids. It forms a salt-tolerant defense mechanism by enhancing membrane stability through phospholipid remodeling and carotenoid scavenging of ROS. Correlation analysis demonstrated that inoculation was significantly correlated with plant biomass, root morphology, ion content and metabolite content. Path analysis revealed that inoculation exhibited significant positive correlations with glycosylphosphatidylinositol (GPI) biosynthesis, glycerolipid metabolism, and carotenoid biosynthesis, while demonstrating significant negative correlations with fructose and mannose metabolism in S. alopecuroides. These four core metabolic pathways collectively regulated root growth and modulating Na⁺/K⁺ balance.

Conclusions DSE activates pathways for membrane stabilization and ROS scavenging by upregulating metabolites such as lipids and carotenoids. It also facilitates K⁺ uptake and maintaines Na⁺/K⁺ balance, thereby effectively promoting the growth and root development of S. alopecuroides and enhancing their salt resistance.