Role and mechanisms of moisture regulation in enhancing alfalfa silage quality

Background Regulating raw material moisture is a key strategy for inhibiting undesirable microorganisms and improving the fermentation quality of alfalfa silage. However, the effects of post-wilting rehydration on the microbial community and its metabolic pathways remain unclear. This study therefore aimed to elucidate the microbial mechanisms by which this rehydration process enhances fermentation quality through the modulation of key microorganisms and metabolic pathways. Results This study investigated the effects of four moisture pretreatment treatments—ND50, ND65, PS50, and PS65—on microbial community dynamics and metabolomic changes after 90 days of silage fermentation. The results showed that the contents of DM, WSC, pH, AN/TN, AA, and BA in the PS65 treatment were significantly lower than those in ND65 ( P  < 0.05). In the PS50 treatment, the contents of DM, CP, WSC, and pH were significantly lower than those in ND50 ( P  < 0.05). Across all treatments, the relative abundance of lactic acid bacteria exceeded 97%, with Lactiplantibacillus and Lentilactobacillus being the dominant genera. Notably, the relative abundance of Lentilactobacillus in the spraying treatments was significantly higher than that in the corresponding wilting treatments. The yeast counts in the spraying treatments (PS65 and PS50) were significantly lower than those in the wilting treatments (ND65 and ND50) ( P  < 0.05). In addition, the spraying treatments reduced the relative abundance of certain pathogenic fungi, including Fusarium and Aspergillus . Metabolomic analysis showed that the water-sprayed treatment PS65 significantly reduced pH ( P  < 0.05) and increased lactic acid (LA) content ( P  < 0.05) by regulating multiple metabolic pathways, including ascorbate and aldarate metabolism and phenylpropanoid biosynthesis. Further pathway analysis indicated that the PS65 treatment may influence ascorbate and aldarate metabolism, phenylpropanoid biosynthesis, thereby promoting fiber degradation in plant tissues. Microbial traceability analysis revealed that the differential metabolites were mainly derived from Lactiplantibacillus , Lentilactobacillus , and Weissella . Conclusion Within an appropriate moisture range, water spraying after wilting modulates the microbial community of alfalfa silage. This practice increases the relative abundance of Lentilactobacillus , inhibits the proliferation of pathogenic fungi such as Fusarium and Aspergillus , and reduces mycotoxin accumulation. Consequently, the water spraying treatment significantly lowers silage pH and increases LA content by altering key metabolic pathways, including ascorbic acid and aldaric acid metabolism, phenylpropanoid biosynthesis, and phenylalanine metabolism.