Biodiversity Of The Clostridial Community And Mechanism Of The Resulting Clostridial Fermentation In Alfalfa Silage

Alfalfa is viewed as a difficult crop to ensile,primarily because of its high moisture content,low concentration of water soluble carbohydrates,high buffering capacity,and vulnerability to the undesirable secondary clostridial fermentation.The objective of this study is to describe the clostridial community dynamics during the ensiling of alfalfa,and to elucidate the mechanism by which the clostridial fermentation develops.The main contents and conclusions of this paper are as follows:1.Four alfalfa cultivars[Sanditi(Al),AC Caribou(A2),WL319HQ(A3)and 4030(A4)]were ensiled immediately after harvest(wilting for 0-h)or after wilting(2-h and 4-h)for 14,28 and 56 days,and changes in the clostridial community were assessed by DGGE analysis.The results indicated that Cl.sporogenes resulted in the enhanced clostridial fermentation of direct-cut A4 silage,while Cl.perfringens was associated with the clostridial fermentation of direct-cut silages of other 3 cultivars.Cl.ghonii and Cl.sartagoforme were involved in the restricted clostridial fermentation of 2-h wilted silage.The clostridial fermentation was completely inhibited in 4-h wilted silage.2.Dynamics of clostridial community during ensiling direct-cut alfalfa were determined by high-throughput sequencing.Alfalfa silages were prepared with or without the addition of a lactic acid bacterial inoculant(105 cfu/g FM)and sucrose(20 g/kg FM),and were labeled as LS and CK respectively.Silages were sampled for analysis at 0,3,7,14,28 and 56 days after ensiling.The results indicated that Romboutsia sp.,Cl.tyrobutyricum,Cl.perfringens,Garciella sp.and Terrisporobacter mayombei were the prevalent Clostridia species epiphytic on alfalfa.For LS silage which was well preserved,a similar clostridial community was observed between pre-ensiled alfalfa and alfalfa silage.For CK silage which was subjected to the clostridial fermentation,the relative abundance of Cl.perfringens,Garciella sp.and Cl.baratii increased rapidly once the herbage was ensiled at 3 days.Cl.perfringens and Cl.baratii were the predominant Clostridia species at 7 days.With prolonged ensiling,Cl.tyrobutyricum increased rapidly and became the most abundant Clostridia species during the remaining storage period.Therefore,it suggested that Cl.perfringens、Garciella sp.and Cl.baratii started the clostridial fermentation and that Cl.tyrobutyricum was important promoter of the clostridial fermentation for a longer fermentation period.3.Composition and biochemical characteristics of the dominant clostridial community in alfalfa silage were investigated by conventional culture method.65 isolates were divided into eight groups on the basis of their morphological and biochemical characteristics,and were indentified based on 16S ribosomal DNA analysis:Cl.tyrobutyricum(24.6%),Cl.sordellii(7.7%),Cl.sporogenes(18.5%),Cl.subterminale(7.7%)and Cl.perfringens(41.5%).Cl.perfringens was the most abundant member of the clostridial community in alfalfa silage.Compared to the other four strains,Cl.perfringens fermented more carbohydrates and was found to grow well at 45 ℃ and 50 ℃.Therefore,it is necessary to confirm the role of Cl.perfringens in the clostridial fermentation.4.Effect of Cl.perfringens on dynamics of clostridial community during ensiling direct-cut alfalfa was determined by high-throughput sequencing.Alfalfa silages were prepared with or without the addition of a lactic acid bacterial inoculant(105 cfu/g FM)and sucrose(20 g/kg FM),and were labeled as LS and CK respectively.Cl.perfringens was inoculated at 14,28 and 42 days of ensiling.Silages were sampled for analysis at 56 days after ensiling based on the time origin of silage preparation and Clostridia inoculation,respectively.The results indicated that inoculation with Cl.perfringens had no significant effect on the fermentation process of LS silage,while enhanced the clostridial fermentation of CK silage.Compared to Clostridium,Garciella had a greater effect on the clostridial fermentation of CK silage with prolonged ensiling.In CK silage,the more enhanced clostridial fermentation was observed when Cl.perfringens was inoculated at 28 days compared to silages inoculated with Cl.perfringens at 14 and 42 days,this might also be related to the activity of Garciella.Compared to Cl.perfringens,Garciella sp.and Cl.tyrobutyricum were the predominant clostridia in CK silage.Therefore,it suggested that the activities of Garciella sp.and Cl.tyrobutyricum have a greater effect on the promotion of the clostridial fermentation than Cl.perfringens.