Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality

The naturally attached phyllosphere microbiota play a crucial role in plant-derived fermentation, but the structure and function of phyllosphere endophytes remain largely unidentified. Here, we reveal the diversity, specificity, and functionality of phyllosphere endophytes in alfalfa ( Medicago sativa L.) through combining typical microbial culture, high-throughput sequencing, and genomic comparative analysis. In comparison to phyllosphere bacteria (PB), the fermentation of alfalfa solely with endophytes (EN) enhances the fermentation characteristics, primarily due to the dominance of specific lactic acid bacteria (LAB) such as Lactiplantibacillus , Weissella , and Pediococcus . The inoculant with selected endophytic LAB strains also enhances the fermentation quality compared to epiphytic LAB treatment. Especially, one key endophytic LAB named Pediococcus pentosaceus EN5 shows enrichment of genes related to the mannose phosphotransferase system (Man-PTS) and carbohydrate-metabolizing enzymes and higher utilization of carbohydrates. Representing phyllosphere, endophytic LAB shows great potential of promoting ensiling and provides a novel direction for developing microbial inoculant. The study reveals the diversity, specificity and functionality of phyllosphere endophytes in alfalfa silage fermentation and finds a unique microbe resource in phyllosphere endophytes which tends to enrich genes related to carbohydrate utilization.