Combined Humic Acid and Lichen-Derived Endophytic Bacteria Application Alleviates Salt Stress-Induced Oxidative Damage in Alfalfa (Medicago sativa L.)

This study investigates the synergistic effects of leonardite-sourced humic acid (HA) and a lichen-derived endophytic bacterium (Bacillus wiedmannii) in mitigating salt stress in alfalfa (Medicago sativa L., cv. Konya). Alfalfa seeds were grown in a peat-perlite mix with different combinations of 100 mM NaCl, HA (500 or 1000 mg L-1), and B. wiedmannii. After 35 days, relative water content (RWC), ROS (H2O2, O₂.-), MDA, antioxidant enzyme activities, and photosynthetic pigments were measured. The salt stress alone reduced RWC, the photosynthetic pigment amount, and the antioxidant enzyme activities, while elevating H2O2 and O2.-, and MDA accumulation. HA or B. wiedmannii (Bw) individually partially ameliorated these effects, however, the combined HA+Bw treatment under saline conditions (especially 1000 mg L⁻¹ HA+Bw+NaCl) elevated RWC, and reduced ROS and MDA levels close to the control values. In addition, the antioxidant enzyme activities and the photosynthetic pigment contents were enhanced under the combined treatments compared to the salt alone. These results demonstrate a synergistic effect between HA and B. wiedmannii in alleviating salt stress in the alfalfa by enhancing water status, reducing oxidative damage, and activating antioxidant defenses, suggesting that the co‐application of HA and B. wiedmannii is a promising strategy for improving salt tolerance in forage crops.