Influences of growth stage and ensiling time on fermentation profile, bacterial community compositions and their predicted functionality during ensiling of Italian ryegrass

The study evaluated the effects of growth stage and ensiling time on silage fermentation characteristics, bacterial communities and their predicted functional potential in Italian ryegrass (Lolium multiflorum Lam.) silage. Italian ryegrass (IR) was harvested at heading (Earing rate >50 %, IR1) and blooming stages (>50% bloom, IR2), respectively. The harvested IR was ensiled in 15-L plastic silos. The experiment had a completely randomized design. Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of fermentation, respectively. The fermentation products were analyzed on each sampling day. The bacterial communities and their functional potential and phenotype on day 3 and 60 were analyzed by high-throughput sequencing technique and PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) method. With the advancing maturity of IR, the neutral detergent fiber and dry matter (DM) contents, lactic acid bacteria, yeast, Enterobacteriaceae and aerobic bacteria populations increased, while water soluble carbohydrate contents and buffering capacity decreased. Larger lactic acid contents were accumulated in IR1 than IR2 at the early stage of fermentation. Higher contents of ethanol and ammonia nitrogen (NH3-N) were found in IR1 compared to IR2 at the late stage of ensiling. After 60 days, IR1 and IR2 both had good fermentation quality with low pH values (∼4.08), and acceptable levels of NH3-N (∼67.6 g/kg total nitrogen) and butyric acid (∼1.08 g/kg DM). The IR2 had lower abundance of Lactobacillus on day 3, and higher abundances of Kosakonia and Pantoea on day 60 compared to IR1. The bacterial community in IR2 had higher activity in metabolic pathway. Overall, high throughput sequencing technique combined with PICRUSt2 method can improve our understanding of the silage microbiology to further regulate the fermentation products.