Regulatory Mechanisms Underlying Flavonoid-Mediated Restructuring of the Rhizosphere Microbiome in Alfalfa (Medicago sativa L.) Under Salt Stress

Abstract

This study employed two Medicago sativa L. varieties with contrasting salt tolerance—Zangmu No.1 (ZM1, salt-tolerant) and Zangmu No.2 (ZM2, salt-sensitive)—to investigate root exudate composition, rhizosphere microbiome assembly, and their potential correlations under 0 mM, 100 mM, and 200 mM NaCl stresses. Under 200 mM NaCl, ZM1 exhibited specific activation of flavonoid-related metabolic pathways (including flavonoid degradation and tyrosine metabolism), resulting in significant upregulation of characteristic flavonoids such as naringin (a metabolite designated as NEG885 in our LC-MS library) and apigenin (POS148) in root exudates. Correlation analysis revealed that these metabolites were significantly and positively associated with the enrichment of salt-tolerant bacterial taxa, including Pseudarthrobacter and Adhaeribacter. In contrast, ZM2 under identical stress conditions showed significantly enhanced synthesis of p-hydroxyphenylacetic acid (NEG151, p < 0.05) which correlated with Limnobacter and Flavobacterium, indicating ZM2’s comparatively limited metabolic regulatory capacity. Notably, exogenous supplementation of salt-tolerant plant growth-promoting rhizobacteria (PGPR) from γ-Proteobacteria (Atlantibacter, Enterobacter) and Bacilli (Priestia) effectively alleviated growth inhibition in Medicago sativa (both ZM1 and ZM2) under 200 mM NaCl, primarily through promoting root/shoot elongation, leaf expansion, and photosynthetic efficiency while maintaining root “high K+/low Na+” ion homeostasis. These PGPR demonstrated multifunctional traits including indole-3-acetic acid biosynthesis, phosphate solubilization, and nitrogen fixation. qRT-PCR analysis confirmed their regulatory effects on salt-responsive genes such as CHI1 (chalcone isomerase 1) and CYP75A1 (flavonoid 3′,5′-hydroxylase), mediating root development and ion balance. This work elucidates the potential regulatory role of flavonoid metabolism in Medicago sativa’s recruitment of beneficial microbiota under salt stress, providing a theoretical foundation for developing salt-resistant cultivars through plant–microbe synergy strategies.