Plant-microbe interactions are vital for ecosystem health and sustainable agriculture. The phyllosphere, the largest biological surface on Earth, harbors diverse microbial communities, including epiphytes (on leaf surfaces) and endophytes (within plant tissues). These communities influence plant productivity, food safety, and quality, influencing processes like disease resistance and nutrient cycling. Silage, a crucial animal feed, relies on microbial fermentation for preservation. China faces a significant gap between silage demand and production, underscoring the need for improved silage production. Alfalfa (*Medicago sativa* L.), a high-quality forage, is widely cultivated. Ensiling alfalfa involves complex microbial interactions, primarily driven by phyllosphere microorganisms including LAB, yeasts, molds, bacilli, and enterobacteria. While epiphytic bacteria's role in silage production is well-studied, the understanding of phyllosphere endophytes, particularly endophytic LAB, in silage fermentation remains limited. This study hypothesized that these endophytes, especially endophytic LAB, are important microbiome sources that influence forage silage fermentation quality. Endophytes, having co-evolved with plants, exhibit tolerance to plant-produced antibacterial substances and possess potential benefits in improving fermentation quality through metabolite production (e.g., polyphenols, polysaccharides, flavonoids). This research aimed to characterize the phyllosphere endophytic microbiota of alfalfa, its dynamics during ensiling, and its impact on silage fermentation quality, thereby exploring the potential of phyllosphere endophytes as efficient microbial inoculants.

Existing literature highlights the importance of plant-microbe interactions in agricultural systems and the significant role of phyllosphere microorganisms in plant health and productivity. Studies demonstrate the ability of phyllosphere microbes to enhance disease resistance and influence nutrient cycles. Conversely, undesirable microorganisms can compromise the safety and quality of plant products. In silage production, the focus has largely been on epiphytic bacteria, with studies investigating their assembly influenced by factors like host plant, environment, temperature, moisture, and radiation. There's a growing interest in employing LAB as inoculants to improve silage quality by accelerating acidification and suppressing undesirable microbes. However, exogenous LAB may struggle with environmental adaptability, leading to inconsistent effects. The role of endophytic LAB in promoting plant growth and inhibiting pathogens has been explored, but their influence on silage fermentation quality remains underexplored.

This study used a multi-faceted approach combining conventional microbial culture, high-throughput sequencing, and genomic comparative analysis to characterize the phyllosphere endophytic microbiota of alfalfa and its impact on silage fermentation. **Sample Collection:** 58 healthy alfalfa samples were collected from six major alfalfa-producing regions in China. **Phyllosphere Bacteria Isolation and Identification:** 795 bacterial strains were isolated using four different media (GS, MRS, TSA, LA). Epiphytic and endophytic bacteria were differentiated through surface sterilization. 16S rRNA gene sequencing identified the strains, revealing their taxonomic classifications. Phylogenetic analysis helped understand the relationships among the different strains. **LAB Screening and Characterization:** LAB strains were screened based on growth rate and acid production in MRS medium. Physiological and biochemical characteristics, as well as soluble sugar utilization rates (using Biolog AN MicroPlate), were determined for selected LAB strains. **Silage Preparation and Fermentation Quality Analysis:** Alfalfa was prepared in two ways: a phyllosphere bacteria group (PB) using fresh alfalfa and an endophyte group (EN) where epiphytes were removed. Chopped alfalfa was ensiled and analyzed at 9, 30, and 60 days. Analyses included pH measurement, organic acid (lactic acid, acetic acid, propionic acid, butyric acid) determination using HPLC, ammonia nitrogen quantification, dry matter measurement, and nutrient analysis. Microbial counts of LAB, molds, yeasts, and *E. coli* were determined using plate count methods. **PacBio Sequencing for Microbial Community Analysis:** DNA was extracted from silage samples at different time points. PacBio Sequel II sequencing was employed for 16S rRNA gene amplicon sequencing, generating high-accuracy CCS reads. Bioinformatic analysis included OTU clustering, taxonomic classification, and functional gene prediction using Tax4Fun and KEGG annotation. Alpha and beta diversity were assessed, and co-occurrence networks were generated to study the interactions within the microbial communities. **Genome Assembly and Comparative Genomics:** The genomes of four selected LAB strains (*L. lactis* EP2, *L. pentosus* EP3, *P. pentosaceus* EN5, and *L. plantarum* EN6) were sequenced and assembled using Oxford Nanopore Technology. Genomic features, including gene numbers, transport proteins, and CAZyme profiles were analyzed. Comparative genomic analysis, using downloaded *P. pentosaceus* genomes from NCBI, explored the pan-genome and core-genome, providing insights into gene diversity and evolution in different environments.

The study revealed significant differences in microbial community composition and function between the phyllosphere bacteria (PB) group and the endophyte (EN) group. The EN group exhibited enhanced fermentation characteristics, including significantly higher lactic acid content and lower pH, acetic acid, and ammonia nitrogen levels compared to the PB group. Specific LAB, notably *Lactiplantibacillus*, *Weissella*, and *Pediococcus*, were dominant in the EN group. The inoculant containing selected endophytic LAB strains also showed improved fermentation quality compared to epiphytic LAB treatment. *Pediococcus pentosaceus* EN5, a key endophytic LAB, exhibited high carbohydrate utilization efficiency due to gene enrichment related to the mannose phosphotransferase system (Man-PTS) and carbohydrate-metabolizing enzymes. Comparative genomic analysis of *P. pentosaceus* EN5 revealed the presence of two IIB genes in the Man-PTS, along with specific active enzymes (CBM35, CBM4, GT32, GT8) correlated with glucose and mannose utilization, not found in other tested strains. Phylogenetic analysis of *P. pentosaceus* strains indicated a diverse distribution across plant and animal habitats. The study also revealed differences in the co-occurrence networks of microbial communities, with *Pediococcus* showing negative correlations with many microorganisms, suggesting a stable microbial network structure. The EN5 strain showed superior adaptability to the silage environment, maintaining dominance even when its initial growth and acid production rates were lower than other strains in laboratory settings. This superior adaptability was linked to EN5's high utilization of alfalfa sugars, particularly D-mannose, α-D-glucose, and D-cellobiose. The presence of the SecB protein in EN5, absent in other *P. pentosaceus* strains, was also noted, potentially contributing to its stress tolerance in the silage environment.

The findings address the research question by demonstrating the superior performance of phyllosphere endophytic LAB, particularly *P. pentosaceus* EN5, in enhancing silage fermentation quality compared to naturally occurring phyllosphere bacteria. The improved fermentation characteristics, including higher lactic acid content and lower pH, are directly linked to the dominance of specific LAB within the endophytic community. The superior carbohydrate utilization efficiency of *P. pentosaceus* EN5, supported by its genomic characteristics, explains its successful competition and domination in the silage environment. The lower diversity and more stable structure of the microbial communities in the EN group, reflected in the Shannon index and co-occurrence networks, contrast with the PB group's higher diversity and more dynamic community shifts. These results highlight the potential of endophytic LAB as effective inoculants for silage production. The identification of specific genes and enzymes involved in carbohydrate metabolism, along with the unique presence of the SecB protein in EN5, provides valuable insights into the mechanisms underlying the enhanced fermentation efficiency. This work provides strong evidence for a novel approach to enhancing silage fermentation quality, potentially addressing the growing demand for efficient silage production while also advancing the understanding of plant-microbe interactions and the ecological roles of endophytic bacteria.

This research provides compelling evidence that phyllosphere endophytic lactic acid bacteria, especially *Pediococcus pentosaceus* EN5, offer a promising strategy for improving alfalfa silage fermentation. The superior performance of EN5 is attributed to its efficient carbohydrate metabolism and high adaptability to the silage environment, supported by its genomic characteristics. This study underscores the significance of exploring endophytic microbial communities as a potential source for developing effective microbial inoculants for enhancing silage fermentation quality. Future research should focus on investigating the specific mechanisms underlying the unique properties of EN5, optimizing inoculation strategies, and evaluating the long-term effects on animal health and environmental sustainability. Further studies are needed to address potential concerns regarding antibiotic resistance genes found in EN5.

The study primarily focused on alfalfa silage, and the findings may not be directly generalizable to other forage crops. The sample size, while substantial, might be further expanded to increase statistical power and enhance the generalizability of the results. The study primarily relied on 16S rRNA gene sequencing to characterize the microbial community, which may not fully capture the functional diversity within the communities. Although the genomes of several key LAB strains were sequenced and analyzed, a more comprehensive metagenomic analysis of the entire silage microbial community would provide a more holistic understanding of the functional interactions involved. The potential risk of antibiotic resistance gene transfer from EN5 to other microorganisms in the silage environment requires further investigation.