Identification and Correlation Analysis of Key Clostridia and LAB Species in Alfalfa Silages Prepared with Different Cultivars and Additives

Clostridial fermentation is the determining process causing the spoilage of direct-cut alfalfa silage, and the application of lactic acid bacteria (LAB) inoculant is considered as the most promising technology for inhibiting clostridial fermentation. In order to screen target-based LAB strains, identification and correlation analysis of key Clostridia and LAB species in alfalfa silage were conducted in this study. Three alfalfa cultivars (Sanditi, SD; Celsius, CE; SW5909, SW) were harvested at the early bloom stage and ensiled without (CK) or with LAB inoculant (LB) and sucrose (SC) for 60 d. Single-molecule real-time sequencing was used to identify dominant Clostridia and LAB species, and LAB with significant inhibitory effects on dominant Clostridia was screened via correlation network analysis. The results showed that silages CK and LB encountered severe clostridial fermentation as indicated by large amounts of butyric acid (BA) and ammoniacal nitrogen (NH3-N) production. Compared to silages CK and LB, SC treatment decreased (p < 0.05) BA and NH3-N concentrations, as well as decreasing (p < 0.05) the bacterial community indexes of Shannon and Chao1. Lactiplantibacillus pentosus was the first dominant LAB in silage CK of alfalfa SD and CE. The first dominant LAB in silage LB was also identified as L. pentosus, rather than Lentilactobacillus buchneri and Lactiplantibacillus plantarum in the used inoculant. L. buchneri became more abundant in silage SC of alfalfa SD and CE, accounting for the high fermentation quality of these silages. Clostridium tyrobutyricum, Clostridium luticellarii, Garciella sp._GK3, Clostridium sporogenes, Clostridium perfringens, and Clostridium sp._BTY5 were the most dominant Clostridia species in alfalfa silage. Furthermore, Enterococcus faecalis, L. buchneri, and L. pentosus exhibited significant (p < 0.05) inhibitory effects on C. tyrobutyricum, C. luticellarii, and Garciella sp._GK3, respectively, which were the top three Clostridia species associated with clostridial fermentation. In conclusion, E. faecalis, L. buchneri, and L. pentosus were screened and can be used as potential LAB inoculants for the targeted inhibition of clostridial fermentation.