Separating chemical and microbial effects on ensiling characteristics and microbial community assembly during Italian ryegrass silage fermentation

This study aimed to evaluate the respective contributions of chemical compositions and phyllosphere microbiota of Italian ryegrass ( Lolium multiflorum Lam.; IR) to fermentation quality and microbial community diversity. The IR was harvested at the flowering and filling stages. Using gamma-ray irradiation and microbiota transplantation techniques, irradiated flowering (IR1) and filling (IR2) IR samples were inoculated with 2 mL of phyllosphere microbiota inoculum eluted from fresh IR at the flowering (IR1_FL, IR2_FL) and filling (IR1_FI, IR2_FI) stages, respectively. Chopped IR (200 g) was ensiled in laboratory-scale plastic bags. Triplicate samples from each treatment were collected after 3 and 60 days of fermentation. Fermented IR1 exhibited higher ( P < 0.05) lactic acid, ethanol and ammonia nitrogen contents than fermented IR2 on day 60, regardless of phyllosphere microbiota inoculum. Lentilactobacillus rapi , Lacticaseibacillus paracasei , and Saccharomyces sp. predominated in the IR1/IR2_FL groups, whereas Lactiplantibacillus plantarum , Levilactobacillus brevis , and Candida glabrata accounted for the majority of microorganisms in the IR1/IR2_FI groups on day 60. The IR1/IR2_FL groups demonstrated higher abundances of “Amino acid metabolism” and “Carbohydrate metabolism” pathways, along with increased relative abundances of “Alcohol dehydrogenase (EC:1.1.1.1)”, “ L -lactate dehydrogenase (EC:1.1.1.27)”, and “ D -lactate dehydrogenase (EC:1.1.1.28)” on day 60. Overall, chemical composition exerts a stronger effect on fermentation metabolites of IR silage, while phyllosphere microbiota more significantly shapes its microbial community structure, with both factors jointly determining the final silage fermentation quality through distinct biological mechanisms; a growth stage-specific microbial inoculation strategy is thus proposed for high-quality IR silage production, providing an operable technical basis for its industrial application. • Chemical and microbial factors affecting fermentation quality were studied. • Combined gamma-ray irradiation and microbiota transplantation were employed. • Chemical composition modulates the fermentation metabolic products in fermented Italian ryegrass. • Phyllosphere microbiota shapes the microbial community structure in fermented Italian ryegrass. • Chemical and microbial factors both decide the final fermentation quality of Italian ryegrass.