The incorporation of honeysuckle as a silage additive in alfalfa production has yielded promising results; however, the underlying microbial mechanisms during fermentation remain poorly understood. This study leveraged high-throughput sequencing and nutrient profiling to elucidate microbial population dynamics over 45 days of anaerobic fermentation within a vacuum-assisted co-culture system comprising varying fresh weight ratios of honeysuckle branches, Lactobacillus, and alfalfa. The experimental design encompassed a Lactobacillus-supplemented treatment (M group) and an unsupplemented control (R group), each with five honeysuckle percentages (5%, 10%, 15%, 20%, 25%) and respective negative controls. Our findings reveal that the combined use of honeysuckle branches and Lactobacillus significantly impacts microbial community composition and diversity during alfalfa silage fermentation. The Mantel test underscores Lactobacillus's absence-dependent correlation of bacterial communities with pH and lactic acid, whereas its presence redirects these correlations towards total nitrogen, NH4+-N, neutral detergent fiber, and crude protein as key bacterial population drivers. Fungal populations exhibited analogous trends. Moreover, the combined additives reshaped microbial populations during anaerobic fermentation, altering interaction networks and intensifying microbial interplay. Notably, Lactobacillus introduction on a honeysuckle branch base diminished fungal core OTUs, thereby mitigating fungal contamination risks. This study also identified biomarkers unique to each treatment condition. Collectively, our research provides a molecular framework for employing honeysuckle branches and Lactobacillus as innovative silage additives in alfalfa production, fostering the development of eco-friendly and hygienic feed and farming industries.
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