摘要
【目的】研究甘蓝尾菜与干水稻(Oryza sativa L.)秸秆混贮发酵过程中理化指标、微生物群落变化,并分析二者之间的相关性,旨在为甘蓝尾菜和干水稻秸秆饲料化利用提供参考。【方法】将甘蓝(Brassica oleracea L.)尾菜与干水稻秸秆在30℃条件下混合贮存30 d,分别在发酵的第0,3,7,15,30天取样,分析样品的发酵品质、营养成分,利用Illumina Miseq高通量测序研究发酵过程中微生物菌群结构,使用Mothur 1.30.2分析细菌和真菌的Alpha多样性指数(Coverage指数、Shannon指数、Chao1指数),结合BugBase和FUNGuild数据库分析细菌和真菌群落的表型功能,再利用共现性网络、冗余分析(RDA)分别研究菌群互作以及理化指标与优势物种相关性。【结果】与发酵前相比,甘蓝尾菜与干水稻秸秆混贮发酵结束后pH、干物质(DM)含量、水溶性碳水化合物(WSC)含量显著下降(P<0.05),有机酸(乳酸、乙酸、丁酸)和乙醇显著积累(P<0.05),其中铵态氮含量在贮存结束时低于10%,pH降低到4.0;粗脂肪(CF)、粗蛋白(CP)、酸性洗涤纤维素(ADF)含量无显著变化(P>0.05),中性洗涤纤维素(NDF)含量显著上升(P<0.05)。细菌和真菌群落的Coverage指数为0.99~1.00;细菌群落的Shannon指数、Chao1指数于发酵第3天之后均显著下降(P<0.05),真菌群落的Shannon指数和Chao1指数无显著变化(P>0.05)。发酵过程中,优势细菌(相对丰度(RA)>5%)由初始的非理想菌群转变为乳酸菌和肠杆菌类,真菌群落中致病型菌群相对丰度显著下降(P<0.05)。9种细菌表型和12种真菌表型被注释,其中需氧型(aerobic)、形成生物膜(forms biofilms)、革兰氏阴性(gram negative)、潜在致病型(potentially pathogenic)细菌和动物致病型(animal pathogenic)真菌相对丰度均显著下降(P<0.05),在30 d时相对丰度分别为8.0%,31.5%,31.8%,31.3%,15.46%。基于物种属水平相对丰度构建的微生物共现性网络由38个点(代表细菌属或真菌属)和91条无向的边(代表相连细菌属或真菌属之间的相关性)组成。与发酵前相比,乳酸菌(片球菌属Pediococcus、肠球菌属Enterococcus、乳杆菌属Lactobacillus、未分类的乳杆菌科unclassified_f__Lactobacillacea)和腐生型真菌(节担菌属Wallemia和unclassified_f__Dipodascaceae)作为关键物种抑制了病原菌富集;片球菌属Pediococcus、unclassified_f__Dipodascaceae是甘蓝尾菜和干水稻秸秆混贮发酵过程中改善青贮品质的关键物种。RDA结果显示,pH、WSC、CF是影响微生物优势物种演替的关键理化因子,其中pH、WSC与优势物种镰孢菌属、曲霉属、泛菌属、肠杆菌属呈正相关关系,与片球菌属、未分类的肠杆菌科呈负相关关系;CF与优势物种肠球菌属、节担菌属呈负相关关系。【结论】甘蓝尾菜与干水稻秸秆混贮发酵过程中,干物质含量被有效保存,真菌优势物种显著富集,有效抑制了病原菌菌群的积累,可知混贮发酵是资源化处理甘蓝尾菜与干水稻秸秆的有效方式。
【Objective】To study the change of physicochemical indexes and microbial community in the co-ensiling process of cabbage and dry rice straw,and to analyse the correlation between them in order to provide references for the feed utilization of cabbage waste and dry rice straw.【Method】Cabbage waste was co-ensiled with dried rice straw in 30℃for 30 days,and samples were taken on the 0th,3rd,7th,15th and 30th days of fermentation to analyze fermentation quality and nutrient components.The microbial community was investigated using the Illumina Miseq high-throughput sequencing during ensiling.The Alpha diversity indexes(Coverage index,Shannon index and Chao1 index)of bacteria and fungi were analyzed by Mothur 1.30.2.The functions and phenotypes of microflora were predicted via BugBase and FUNGuild database.The co-occurrence network and redundancy analysis(RDA)were used to study microorganic interactions and correlation between physicochemical indexes and dominant taxa for revealing the role of keystone taxa during ensiling.【Result】Compared with before fermentation,pH,dry matter(DM)content and water-soluble carbohydrate(WSC)content decreased significantly after mixed storage and fermentation of cabbage and dry rice straw(P<0.05),while organic acids(lactic acid,acetic acid and butyric acid)and ethanol significantly accumulated(P<0.05).The content of ammonium nitrogen was less than 10%at the end of fermentation and pH dropped to 4.0.Contents of crude fat(CF),crude protein(CP)and acid detergent cellulose(ADF)had no significant changes(P>0.05),while the content of neutral detergent cellulose(NDF)increased significantly(P<0.05).The Coverage index of bacterial and fungal communities was 0.99 to 1.00,the Shannon index and Chao1 index of bacterial community decreased significantly after the 3rd day of fermentation(P<0.05),while the Shannon index and Chao1 index of fungal community did not change significantly(P>0.05).During the fermentation process,the dominant bacteria with relative abundance of>5%changed from the initial non-ideal flora to lactic acid bacteria and Enterobacter,and the relative abundance of pathogenic fungi in fungal community decreased significantly(P<0.05).Nine bacterial phenotypes and 12 fungal phenotypes were annotated.Among them,aerobic,biofilm-forming,gram-negative,curative potential bacteria and animal pathogenic fungi were significantly inhibited(P<0.05),the 30 rd day of relative abundance were 8.0%,31.5%,31.8%,31.3%and 15.64%.The microbial co-occurrence network constructed based on the relative abundance of species consisted of 38 points representing bacterial genera or fungal genera and 91 undirected edges representing correlation between connected bacterial genera or fungal genera.Compared with before fermentation,lactic acid bacteria(Pediococcus,Enterococcus,Lactobacillus and unclassified_f__Lactobacillacea)and saprophytic fungi(Wallemia and unclassified_f__Dipodascaceae)were key taxa to inhibit pathogen enrichment.Pediococcus and unclassified_f__Dipodascaceae were the key taxa to improve the quality of silage.RDA results showed that pH,WSC and CF were the key physical and chemical factors affecting the succession of dominant microorganisms.Among them,pH and WSC were positively correlated with dominant species Fusarium,Aspergillus,Pantoea and Enterobacter,but negativity correlated with Pediococcus and unclassified_f__Enterobacteriaceae.CF was negatively correlated with the dominant species Enterococcus and Wallemia.【Conclusion】In the mixed storage and fermentation process of Brassica oleracea and dry rice straw,the dry matter content was effectively preserved,the dominant species of fungi were significantly enriched,and the accumulation of pathogenic bacteria was effectively inhibited.Therefore,the mixed storage and fermentation is an effective way for resource treatment of cabbage waste and dry rice straw.
作者
张静娴
杜桂林
史吉平
马治国
袁辰阳
刘相岑
张保国
ZHANG Jingxian;DU Guilin;SHI Jiping;MA Zhiguo;YUAN Chenyang;LIU Xiangcen;ZHANG Baoguo(Shanghai Advanced Research Institute,Chinese Academy of Sciences,Shanghai 201210,China;University of Chinese Academy of Sciences,Beijing 100049,China;School of Life Science and Technology,Shanghai Tech University,Shanghai 201210,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2022年第2期90-102,共13页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家重点研发计划项目(2018YFD0800403)。
关键词
混贮发酵
甘蓝尾菜
水稻秸秆
微生物群落演替
共现性网络
关键物种
mixed storage and fermentation
cabbage waste
dry rice straw
microbial community succession
co-occurrence network
keystone taxon
