Exploring the ensiling characteristics and bacterial community of red clover inoculated with the epiphytic bacteria from temperate gramineous grasses.

Abstract

To explore the microbiological factors that cause the difference in silage fermentation characteristics between temperate gramineous (Italian ryegrass and oat) grass and legume forage. Through γ-ray irradiation sterilization and bacterial transplantation technology, the sterilized red clover was inoculated as follows: (i) aseptic water (STRC); (ii) epiphytic bacteria on red clover (RCRC); (iii) epiphytic bacteria on oat (RCOT); and (iv) epiphytic bacteria on Italian ryegrass (RCIR). Red clover was ensiled in laboratory-scale silos made of vacuum-packed plastic bags for 1, 3, 7, 14, 30 and 60days. STRC remained unfermented state based on similar chemical components with fresh red clover. Compared with RCRC and RCIR, higher lactic acid content and ratio of lactic acid to acetic acid, and lower pH, acetic acid and ammonia nitrogen contents were observed in RCOT after 60days of ensiling. Using next-generation sequencing, higher abundances of Methylobacterium and Sphingomonas were observed in the epiphytic bacteria on red clover. Pediococcus was dominant in RCRC after 3days of ensiling. Lactobacillus was the most predominant in each group after 60days of fermentation. RCRC and RCIR had a more hetero-fermentative process, while RCOT possessed a more homo-fermentative pathway. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was accelerated by the epiphytic bacteria from red clover. Inoculating the epiphytic bacteria from oat could alter the fermentation characteristics and bacterial compositions of red clover silage. The relative abundance and activity of Methylobacterium, Sphingomonas, Enterobacteriaceae and hetero-fermentative Lactobacillus in red clover silage could be changed by the epiphytic bacteria on temperate gramineous grass. The exogenous micro-organisms inhibiting the metabolism of amino acids can be a good potential source to improve the silage quality of legume forage.