棉秸秆自然腐解过程中细菌菌群多样性分析
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摘要
【目的】分析棉秸秆在自然腐解过程中细菌群落组成和多样性,获得与棉秸秆腐解有关的优势细菌菌属,为秸秆腐熟菌剂的制备奠定基础。【方法】采用高通量测序技术,对棉秸秆自然腐解过程、不同时间样品中的细菌16S rDNA基因的V4区测序,利用生物信息学方法分析测序结果。【结果】测序共得到354 067条有效序列,2 111条OTU序列,腐解初期有268个菌属,中期有300个菌属,末期有325个菌属。菌群α多样性指数分析显示,随着腐解时间的延长,7 d以后菌群多样性增加,与起始时相比差异显著(P<0.05),而7～28 d菌群多样性差异不显著(P>0.05)。在整个堆制过程中始终存在的优势菌属包括鞘氨醇杆菌属(Sphingobacterium)、橄榄球菌属(Olivibacter)、假黄单胞菌属(Pseudoxanthomonas)、德沃斯氏菌属(Devosia)、根瘤菌属(Rhizobium)。【结论】棉秸秆自然腐解过程中,在7 d时细菌群落多样性在属水平上与腐解起始阶段差异显著(P<0.05),随着腐解时间延长,菌落多样性趋于一致;在整个腐熟过程中始终存在五个优势属,功能预测为降解纤维素、木质素、果胶类物质以及提供氮素营养。
【Objective】 This project aims to analyze the composition and diversity of bacterial flora in the process of natural decomposition of cotton straw and obtain the dominant bacterial genera related to its decomposition, thus laying a foundation for the preparation of maturing fungicides. 【Methods】 High-through sequencing was used to sequence the V4 region in 16 S rDNA and based on this, the sequencing result was analyzed by bioinformatics methods. 【Results】 There were 354,067 effective tags, 2,111 OTUs, 268 bacterial genus at the beginning of degradation, 300 bacterial genera at the middle of degradation, 325 genera at the end. It was shown by α diversity index that the diversity of bacterial community was increased after 7 days. The difference was significant compared with that at the beginning(P<0.05). The diversity difference was not significant during 7 to 28 days(P>0.05). The dominant bacterial genera that existed during all the degradation included Sphingobacterium,Olivibacter,Pseudoxanthomonas,Devosia and Rhizobium. 【Conclusion】 During the natural decomposition of cotton straw, the diversity of bacterial community at the genus level was significantly different from that at the initial stage of decomposition at the 7 th days(P<0.05). With the prolongation of decomposing time, the diversity of colony tended to converge. There were five genus that existed as dominant genera throughout the decaying process. The function of these genera were predicted as degrading substance such as cellulose,lignin,pectic substance, which provided the nitrogen nutrition.
【Objective】 This project aims to analyze the composition and diversity of bacterial flora in the process of natural decomposition of cotton straw and obtain the dominant bacterial genera related to its decomposition, thus laying a foundation for the preparation of maturing fungicides. 【Methods】 High-through sequencing was used to sequence the V4 region in 16 S rDNA and based on this, the sequencing result was analyzed by bioinformatics methods. 【Results】 There were 354,067 effective tags, 2,111 OTUs, 268 bacterial genus at the beginning of degradation, 300 bacterial genera at the middle of degradation, 325 genera at the end. It was shown by α diversity index that the diversity of bacterial community was increased after 7 days. The difference was significant compared with that at the beginning(P<0.05). The diversity difference was not significant during 7 to 28 days(P>0.05). The dominant bacterial genera that existed during all the degradation included Sphingobacterium,Olivibacter,Pseudoxanthomonas,Devosia and Rhizobium. 【Conclusion】 During the natural decomposition of cotton straw, the diversity of bacterial community at the genus level was significantly different from that at the initial stage of decomposition at the 7 th days(P<0.05). With the prolongation of decomposing time, the diversity of colony tended to converge. There were five genus that existed as dominant genera throughout the decaying process. The function of these genera were predicted as degrading substance such as cellulose,lignin,pectic substance, which provided the nitrogen nutrition.
引文
[1]秦都林,王双磊,刘艳慧,等.滨海盐碱地棉花秸秆还田对土壤理化性质及棉花产量的影响[J].作物学报,2017,43(7):1 030-1 042.QIN Du-lin,WANG Shuang-lei,LIU Yan-hui,et al.(2017).Effects of Cotton Stalk Returning on Soil Physical and Chemical Properties and Cotton Yield in Coastal Saline-Alkali Soil [J].Acta Agronomica Sinica,43(7):1,030-1,042.(in Chinese)
[2]连祎雯.秸秆还田与化肥配合施用对土壤肥力的影响[J].南方农机,2018,49(16):95.LIAN Yi-wen.(2018).Effect of Matching Use of Straw And Chemical Fertilizer on Soil Fertility [J].Southern Agricultural Machinery,49(16):95.(in Chinese)
[3]孙美娜,张凡凡,王永强,等.棉花秸秆纤维素降解菌的筛选鉴定与降解棉秆效果研究[J].新疆农业科学,2018,55(1):16-23.SUN Mei-na,ZHANG Fan-fan,WANG Yong-qiang,et al.(2018).Screening and Identification of Cellulolytic Strains from Cotton Straw and Its Effect on Degradation of Cotton Stalk [J].Xinjiang Agricultural Sciences,55(1):16-23.(in Chinese)
[4]李秀,韩佳乐,吴文雪,等.秸秆还田方式对关中盆地土壤微生物量碳氮和冬小麦产量的影响[J].水土保持学报,2018,32(4):170-176.LI Xiu,HAN Jia-le,WU Wen-xue,et al.(2018) Effects of straw returning on soil microbial biomass carbon and nitrogen and winter wheat yield in Guanzhong Basin [J].Journal of Soil and Water Conservation,32(4):170-176.(in Chinese)
[5]戴志刚,鲁剑巍,李小坤,等.不同作物还田秸秆的养分释放特征试验[J].农业工程学报,2010,26(6):272-276.DAI Zhi-gang,LU Jian-wei,LI Xiao-kun,et al.(2010).Experimental study on nutrient release characteristics of straw returned from different crops [J].Transactions of the Chinese Society of Agricultural Engineering,26(6):272-276.(in Chinese)
[6]任海伟,刘菲菲,李梦玉,等.玉米秸秆和白菜废弃物在不同贮存条件下的微生物群落[J].应用与环境生物学报,2018,24(2):281-291.REN Hai-wei,LIU Fei-fei,LI Meng-yu,et al.(2018).Microbial communities of corn stover and Chinese cabbage waste under different storage conditions [J].Chinese Journal of Applied and Environmental Biology,24(2):281-291.(in Chinese)
[7]尉良.农牧业废弃物堆肥腐熟质量控制指标研究[D].上海:东华大学硕士论文,2009.WEI Liang.(2009).Research on quality control indicators of composting and composting of agricultural and livestock wastes [D].Master Dissertation.Donghua University,Shanghai.(in Chinese)
[8]慕永红,李军,解保胜,等.生物菌剂在寒地水稻秸秆还田上的应用[J].垦殖与稻作,1999,(3):31-32.MU Yong-hong,LI Jun,XIE Bao-sheng,et al.(1999).Application of Biological Bacteria in Returning Rice Straw to Field [J].Recommended Cultivation and Rice Cultivation,(3):31-32.(in Chinese)
[9]邹金环,杨益民,吴伟.酵素菌肥改良滨海盐化潮土作用的初探[J].土壤肥料,1998,(4):35-36,34.ZOU Jin-huan,YANG Yi-min,WU Wei.(1998).Study on the Effect of Enzyme Fertilizer on Improving Coastal Salinization Soil [J].Journal of Soil and Fertilizer,(4):35-36,34.(in Chinese)
[10]刘娣.秸秆纤维素高效降解真菌的筛选、鉴定及其纤维素酶基因克隆[D].北京:中国农业科学院硕士论文,2008.LIU Di.(2008).Screening and identification of high-efficiency degrading fungi from straw cellulose and cloning of cellulase genes [D].Master Dissertation.Chinese Academy of Agricultural Sciences,Beijing.(in Chinese)
[11]高静,席琳乔,马春晖.土壤中棉秆纤维素降解菌的筛选[J].塔里木大学学报,2010,22(4):6-10.GAO Jing,XI Lin-qiao,MA Chun-hui.(2010).Screening of Cellulose Degrading Bacteria from Cotton Stalk in Soil [J].Journal of Tarim University,22(4):6-10.(in Chinese)
[12]王璐,张海红,杨柳,等.3株纤维素分解真菌的分子鉴定[J].江苏农业科学,2013,41(8):51-53.WANG Lu,ZHANG Hai-hong,YANG Liu,et al.(2013).Molecular identification of 3 strains of cellulolytic fungi [J].Jiangsu Agricultural Sciences,41(8):51-53.(in Chinese)
[13]张红.植物秸秆腐解特性及微生物多样性的响应研究[D].杨凌:西北农林科技大学硕士论文,2017.ZHANG Hong.(2017).Responses of plant straw decomposing characteristics and microbial diversity [D].Master Dissetation.Northwest A&F University,Yangling.(in Chinese)
[14] Edgar,R.C.(2013).Uparse:highly accurate otu sequences from microbial amplicon reads.Nature Methods,10(10):996-998.
[15] Wang,Q.,Garrity,G.M.,Tiedje,J.M.,& Cole,J.R.(2007).Naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,73(16):5,261-5,267.
[16] Quast,C.,Pruesse,E.,Yilmaz,P.,Gerken,J.,Schweer,T.,& Yarza,P.,et al.(2013).The silva ribosomal rna gene database project:improved data processing and web-based tools.Nucleic Acids Research,41(D1):D590-D596.
[17]吴杰.新疆棉花秸秆利用现状分析和探讨[J].中国棉花,2006,(2):9-11.WU Jie.(2006).Analysis and Discussion on the Status Quo of Cotton Straw Utilization in Xinjiang [J].China Cotton,(2):9-11.(in Chinese)
[18]王益,李春越,韩雅娇,等.白腐真菌与嗜热性侧孢霉复配对棉花秸秆降解的影响[J].地球环境学报,2018,9(3):273-281.WANG Yi,LI Chun-yue,HAN Ya-jiao,et al.(2018).Effects of white rot fungi and thermophilic genus Fusarium on the degradation of cotton straw [J].Journal of Earth Environment,9(3):273-281.(in Chinese)
[19]侯敏,包慧芳,王宁,等.棉秸秆纤维素降解菌系构建及固体发酵条件优化[J].新疆农业科学,2018,55(5):936-948.Hou Min,Bao Hui-fang,WANG Ning,et al.(2018).Construction of Cellulose Degrading Bacteria in Cotton Straw and Optimization of Solid Fermentation Conditions [J].Xinjiang Agricultural Sciences,55(5):936-948.(in Chinese)
[20]詹发强,崔卫东,周亚飞,等.降解棉秸秆耐热菌株的鉴定及降解条件优化[J].微生物学通报,2012,39(1):44-54.ZHAN Fa-qiang,CUI Wei-dong,ZHOU Ya-fei,et al.(2012).Identification of heat-resistant strains for degradation of cotton straw and optimization of degradation conditions [J].Microbiology,39(1):44-54.(in Chinese)
[21]徐丽萍,姜喆,葛英亮,等.Illumina MiSeq高通量测序降解黑龙江绥化地区玉米秸秆细菌菌群的研究[J].食品工业科技,2018,(23):105-110.XU Li-ping,JIANG Zhe,GE Ying-liang,et al.(2018).Study on high-throughput sequencing of Illumina MiSeq for degradation of bacterial flora of corn stover in Suihua area of Heilongjiang Province [J].Science and Technology of Food Industry,(23):105-110.(in Chinese)
[22]令利军,何楠,白雪,等.基于高通量测序的玉米秸秆自然发酵过程中细菌菌群结构特征[J].兰州大学学报(自然科学版),2017,53(4):526-533.LING Li-jun,He Nan,BAI Xue,et al.(2017).Structural characteristics of bacterial flora during natural fermentation of corn stover based on high-throughput sequencing[J].Journal of Lanzhou University (Natural Science Ed.),53 (4):526-533.(in Chinese)
[23]张保平,郭美辰,刘运,等.木质素及其衍生物在提取冶金中的研究进展[J].生物加工过程,2018,16(6):80-87.ZHANG Bao-ping,GUO Mei-chen,LIU Yun,et al.(2018).Research Progress of Lignin and Its Derivatives in Extraction Metallurgy [J].Bioprocessing,16(6):80-87.
[24]田智辉,王亚妮,邹超,等.牛粪和秸秆好氧发酵堆肥的初始条件研究[J].中国土壤与肥料,2018,(6):192-197.TIAN Zhi-hui,WANG Ya-ni,ZOU Chao,et al.(2018).Study on Initial Conditions of Aerobic Fermentation Compost of Cow Manure and Straw [J].China Soil and Fertilizer,(6):192-197.(in Chinese)
[25]董震.棉秆皮纤维的制备及其纺织加工的可行性研究[D].无锡:江南大学博士论文,2016.DONG Zhen.(2016).Preparation of cotton stalk fiber and feasibility study of textile processing [D].PhD Dissertation.Jiangnan University,Wuxi.(in Chinese)
[2]连祎雯.秸秆还田与化肥配合施用对土壤肥力的影响[J].南方农机,2018,49(16):95.LIAN Yi-wen.(2018).Effect of Matching Use of Straw And Chemical Fertilizer on Soil Fertility [J].Southern Agricultural Machinery,49(16):95.(in Chinese)
[3]孙美娜,张凡凡,王永强,等.棉花秸秆纤维素降解菌的筛选鉴定与降解棉秆效果研究[J].新疆农业科学,2018,55(1):16-23.SUN Mei-na,ZHANG Fan-fan,WANG Yong-qiang,et al.(2018).Screening and Identification of Cellulolytic Strains from Cotton Straw and Its Effect on Degradation of Cotton Stalk [J].Xinjiang Agricultural Sciences,55(1):16-23.(in Chinese)
[4]李秀,韩佳乐,吴文雪,等.秸秆还田方式对关中盆地土壤微生物量碳氮和冬小麦产量的影响[J].水土保持学报,2018,32(4):170-176.LI Xiu,HAN Jia-le,WU Wen-xue,et al.(2018) Effects of straw returning on soil microbial biomass carbon and nitrogen and winter wheat yield in Guanzhong Basin [J].Journal of Soil and Water Conservation,32(4):170-176.(in Chinese)
[5]戴志刚,鲁剑巍,李小坤,等.不同作物还田秸秆的养分释放特征试验[J].农业工程学报,2010,26(6):272-276.DAI Zhi-gang,LU Jian-wei,LI Xiao-kun,et al.(2010).Experimental study on nutrient release characteristics of straw returned from different crops [J].Transactions of the Chinese Society of Agricultural Engineering,26(6):272-276.(in Chinese)
[6]任海伟,刘菲菲,李梦玉,等.玉米秸秆和白菜废弃物在不同贮存条件下的微生物群落[J].应用与环境生物学报,2018,24(2):281-291.REN Hai-wei,LIU Fei-fei,LI Meng-yu,et al.(2018).Microbial communities of corn stover and Chinese cabbage waste under different storage conditions [J].Chinese Journal of Applied and Environmental Biology,24(2):281-291.(in Chinese)
[7]尉良.农牧业废弃物堆肥腐熟质量控制指标研究[D].上海:东华大学硕士论文,2009.WEI Liang.(2009).Research on quality control indicators of composting and composting of agricultural and livestock wastes [D].Master Dissertation.Donghua University,Shanghai.(in Chinese)
[8]慕永红,李军,解保胜,等.生物菌剂在寒地水稻秸秆还田上的应用[J].垦殖与稻作,1999,(3):31-32.MU Yong-hong,LI Jun,XIE Bao-sheng,et al.(1999).Application of Biological Bacteria in Returning Rice Straw to Field [J].Recommended Cultivation and Rice Cultivation,(3):31-32.(in Chinese)
[9]邹金环,杨益民,吴伟.酵素菌肥改良滨海盐化潮土作用的初探[J].土壤肥料,1998,(4):35-36,34.ZOU Jin-huan,YANG Yi-min,WU Wei.(1998).Study on the Effect of Enzyme Fertilizer on Improving Coastal Salinization Soil [J].Journal of Soil and Fertilizer,(4):35-36,34.(in Chinese)
[10]刘娣.秸秆纤维素高效降解真菌的筛选、鉴定及其纤维素酶基因克隆[D].北京:中国农业科学院硕士论文,2008.LIU Di.(2008).Screening and identification of high-efficiency degrading fungi from straw cellulose and cloning of cellulase genes [D].Master Dissertation.Chinese Academy of Agricultural Sciences,Beijing.(in Chinese)
[11]高静,席琳乔,马春晖.土壤中棉秆纤维素降解菌的筛选[J].塔里木大学学报,2010,22(4):6-10.GAO Jing,XI Lin-qiao,MA Chun-hui.(2010).Screening of Cellulose Degrading Bacteria from Cotton Stalk in Soil [J].Journal of Tarim University,22(4):6-10.(in Chinese)
[12]王璐,张海红,杨柳,等.3株纤维素分解真菌的分子鉴定[J].江苏农业科学,2013,41(8):51-53.WANG Lu,ZHANG Hai-hong,YANG Liu,et al.(2013).Molecular identification of 3 strains of cellulolytic fungi [J].Jiangsu Agricultural Sciences,41(8):51-53.(in Chinese)
[13]张红.植物秸秆腐解特性及微生物多样性的响应研究[D].杨凌:西北农林科技大学硕士论文,2017.ZHANG Hong.(2017).Responses of plant straw decomposing characteristics and microbial diversity [D].Master Dissetation.Northwest A&F University,Yangling.(in Chinese)
[14] Edgar,R.C.(2013).Uparse:highly accurate otu sequences from microbial amplicon reads.Nature Methods,10(10):996-998.
[15] Wang,Q.,Garrity,G.M.,Tiedje,J.M.,& Cole,J.R.(2007).Naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,73(16):5,261-5,267.
[16] Quast,C.,Pruesse,E.,Yilmaz,P.,Gerken,J.,Schweer,T.,& Yarza,P.,et al.(2013).The silva ribosomal rna gene database project:improved data processing and web-based tools.Nucleic Acids Research,41(D1):D590-D596.
[17]吴杰.新疆棉花秸秆利用现状分析和探讨[J].中国棉花,2006,(2):9-11.WU Jie.(2006).Analysis and Discussion on the Status Quo of Cotton Straw Utilization in Xinjiang [J].China Cotton,(2):9-11.(in Chinese)
[18]王益,李春越,韩雅娇,等.白腐真菌与嗜热性侧孢霉复配对棉花秸秆降解的影响[J].地球环境学报,2018,9(3):273-281.WANG Yi,LI Chun-yue,HAN Ya-jiao,et al.(2018).Effects of white rot fungi and thermophilic genus Fusarium on the degradation of cotton straw [J].Journal of Earth Environment,9(3):273-281.(in Chinese)
[19]侯敏,包慧芳,王宁,等.棉秸秆纤维素降解菌系构建及固体发酵条件优化[J].新疆农业科学,2018,55(5):936-948.Hou Min,Bao Hui-fang,WANG Ning,et al.(2018).Construction of Cellulose Degrading Bacteria in Cotton Straw and Optimization of Solid Fermentation Conditions [J].Xinjiang Agricultural Sciences,55(5):936-948.(in Chinese)
[20]詹发强,崔卫东,周亚飞,等.降解棉秸秆耐热菌株的鉴定及降解条件优化[J].微生物学通报,2012,39(1):44-54.ZHAN Fa-qiang,CUI Wei-dong,ZHOU Ya-fei,et al.(2012).Identification of heat-resistant strains for degradation of cotton straw and optimization of degradation conditions [J].Microbiology,39(1):44-54.(in Chinese)
[21]徐丽萍,姜喆,葛英亮,等.Illumina MiSeq高通量测序降解黑龙江绥化地区玉米秸秆细菌菌群的研究[J].食品工业科技,2018,(23):105-110.XU Li-ping,JIANG Zhe,GE Ying-liang,et al.(2018).Study on high-throughput sequencing of Illumina MiSeq for degradation of bacterial flora of corn stover in Suihua area of Heilongjiang Province [J].Science and Technology of Food Industry,(23):105-110.(in Chinese)
[22]令利军,何楠,白雪,等.基于高通量测序的玉米秸秆自然发酵过程中细菌菌群结构特征[J].兰州大学学报(自然科学版),2017,53(4):526-533.LING Li-jun,He Nan,BAI Xue,et al.(2017).Structural characteristics of bacterial flora during natural fermentation of corn stover based on high-throughput sequencing[J].Journal of Lanzhou University (Natural Science Ed.),53 (4):526-533.(in Chinese)
[23]张保平,郭美辰,刘运,等.木质素及其衍生物在提取冶金中的研究进展[J].生物加工过程,2018,16(6):80-87.ZHANG Bao-ping,GUO Mei-chen,LIU Yun,et al.(2018).Research Progress of Lignin and Its Derivatives in Extraction Metallurgy [J].Bioprocessing,16(6):80-87.
[24]田智辉,王亚妮,邹超,等.牛粪和秸秆好氧发酵堆肥的初始条件研究[J].中国土壤与肥料,2018,(6):192-197.TIAN Zhi-hui,WANG Ya-ni,ZOU Chao,et al.(2018).Study on Initial Conditions of Aerobic Fermentation Compost of Cow Manure and Straw [J].China Soil and Fertilizer,(6):192-197.(in Chinese)
[25]董震.棉秆皮纤维的制备及其纺织加工的可行性研究[D].无锡:江南大学博士论文,2016.DONG Zhen.(2016).Preparation of cotton stalk fiber and feasibility study of textile processing [D].PhD Dissertation.Jiangnan University,Wuxi.(in Chinese)