Growth stage vs. phyllosphere microbiota: dissecting their contributions to fermentation quality and bacterial community of fermented alfalfa.

A critical knowledge gap remains in disentangling the independent roles of growth stage and phyllosphere microbiota in driving fermentation profiles and bacterial community dynamics in fermented alfalfa. This study aimed to evaluate the respective contributions of alfalfa (Medicago sativa L.; AL) growth stage and phyllosphere microbiota to fermentation products, bacterial community diversity, co-occurrence networks, and functional potential using high-energy electron beam irradiation and microbiota transplantation methods. Alfalfa was harvested at the initial-flowering and full-flowering stages. The irradiated initial-flowering (AL1) and full-flowering (AL2) alfalfa were inoculated with 2 mL of phyllosphere microbiota inoculum eluted from the initial-flowering (AL1_IF, AL2_IF) and full-flowering (AL1_FF, AL2_FF) alfalfa, respectively. Chopped alfalfa (200 g fresh weight) was fermented in laboratory-scale plastic bags. Triplicate samples from each treatment were collected after 3 and 60 days of fermentation. After 60 days, growth stage exerted stronger effects on fermentation products, while phyllosphere microbiota significantly influenced bacterial community structure and co-occurrence network patterns. On day 60, AL1 groups exhibited higher (P < 0.05) acetic acid concentrations, pH, and proportions of potentially pathogenic bacteria than AL2 groups. Lactobacillus and Weissella dominated the bacterial community, with Pediococcus remarkably enriched in AL2 groups on day 60. Functional prediction indicated that glycolysis and lactate dehydrogenase were obviously upregulated during fermentation, and lactic acid was synergistically produced via both homolactic and heterolactic pathways. For practical production, initial-flowering alfalfa can be optimized by wilting or substrate supplementation, while full-flowering alfalfa benefits from inoculation with Lactobacillus and Pediococcus. These findings clarify the independent roles of growth stage and eluted phyllosphere microbiota and provide precise strategies for high-quality fermented alfalfa production.