基于高通量测序研究施肥对连作植烟土壤环境及微生物的影响
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摘要
为了研究不同施肥处理对连作植烟土壤环境因子及微生物群落多样性的影响,了解施肥后土壤微生物群落与环境因子的联系,采用Illumina Mi Seq高通量测序技术,对不同施肥处理下连作植烟土壤进行了细菌16S rRNAV4-V5区、真菌ITS rRNAV4-V5区测序,并通过主成分分析及冗余分析研究了连作植烟土壤的8种环境因子(有机质、碱解氮、速效磷、速效钾、蛋白酶、过氧化氢酶、磷酸酶、蔗糖酶)及微生物之间的关系。结果表明:测序质控后获得有效序列264 500个,获得22 035个操作分类单元(OTUs);菌落热图显示,施肥处理与对照相比增加了土壤中细菌与真菌的群落组成。施肥处理的细菌及真菌多样性与CK均呈较大差异,不同施肥处理的细菌或真菌结构聚集较紧密,而CK在排序轴中与其余处理较为分散,这说明施肥可以明显改变连作土壤细菌与真菌的结构;速效钾含量在一定范围内会影响土壤细菌群落的多样性和丰度,而8种环境因子对真菌的多样性和丰度均有不同程度的影响。运用高通量测序技术获取的烟草根际土壤微生物信息较为丰富,可为植烟土壤环境因子与微生物群落多样性研究提供理论参考。
In order to study the effects of different fertilization treatments on soil environmental factors and microbial community diversity in continuous cropping tobacco plants,and understand the relationship between soil microbial community and environmental factors,this paper adopted Illumina Mi Seq high-throughput sequencing technique to sequence bacteria 16 S rRNAV4-V5 district and fungi ITS rRNAV4-V5 district in soil of continuous cropping tobacco plants under different fertilization treatments and studied the relationship between 8 environmental factors(organic matter,available nitrogen,available phosphorus,available K,protease,catalase,phosphatase,sucrase) and the microbes by principal component analysis and redundancy analysis. The results showed that after sequencing,264 500 effective sequences and 22 035 operation classification units(OTUs) were obtained. Colonies showed that the fertilization treatment increased the community composition of bacteria and fungi in the soil compared with that of the contrast. There were bigger differences in bacterial and fungal diversity between fertilization treatment and the CK.The results showed that fertilization could significantly change the structure of bacteria and fungi in soil. The content of available K could affect the diversity and abundance of soil bacterial community in a certain range,and the 8 environmental factors could affect the diversity and abundance of fungi by different degree. The soil microbialinformation of tobacco rhizosphere obtained by high-throughput sequencing technology was rich,and provided theoretical references for different fertilization treatments affecting soil environmental factors and microbial community diversity.
In order to study the effects of different fertilization treatments on soil environmental factors and microbial community diversity in continuous cropping tobacco plants,and understand the relationship between soil microbial community and environmental factors,this paper adopted Illumina Mi Seq high-throughput sequencing technique to sequence bacteria 16 S rRNAV4-V5 district and fungi ITS rRNAV4-V5 district in soil of continuous cropping tobacco plants under different fertilization treatments and studied the relationship between 8 environmental factors(organic matter,available nitrogen,available phosphorus,available K,protease,catalase,phosphatase,sucrase) and the microbes by principal component analysis and redundancy analysis. The results showed that after sequencing,264 500 effective sequences and 22 035 operation classification units(OTUs) were obtained. Colonies showed that the fertilization treatment increased the community composition of bacteria and fungi in the soil compared with that of the contrast. There were bigger differences in bacterial and fungal diversity between fertilization treatment and the CK.The results showed that fertilization could significantly change the structure of bacteria and fungi in soil. The content of available K could affect the diversity and abundance of soil bacterial community in a certain range,and the 8 environmental factors could affect the diversity and abundance of fungi by different degree. The soil microbialinformation of tobacco rhizosphere obtained by high-throughput sequencing technology was rich,and provided theoretical references for different fertilization treatments affecting soil environmental factors and microbial community diversity.
引文
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[7]郭海燕,程国虎,李拥军,等.高通量测序技术及其在生物学中的应用[J].当代畜牧,2016(12):61-65.Guo H Y,Cheng G H,Li Y J,et al..The high throughput sequencing technology and its application in biology[J].Contemporary Anim.Husbandry,2016(12):61-65.
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[14]岳冰冰.烤烟连作改变了根际土壤微生物的多样性[D].哈尔滨:东北林业大学,硕士学位论文,2012.Yue B B.The rhizosphere soil microbial diversity is changed by flue-cured tobacco continuous cropping[D].Harbin:Northeast Forestry University,Master Dissertation,2012.
[15]顾美英,刘洪亮,李志强,等.新疆连作棉田施用生物炭对土壤养分及微生物群落多样性的影响[J].中国农业科学,2014,47(20):4128-4138.Gu M Y,Liu H L,Li Z Q,et al..Impact of biochar application on soil nutrients and microbial diversities in continuous cultivated cotton fields in Xinjiang[J].Sci.Agric.Sin.,2014,47(20):4128-4138.
[16]陈广波.肥料和菌剂配施对红壤地区连作花生生育性状和土壤微生物多样性的影响[D].南京:南京农业大学,硕士学位论文,2012.Chen G B.Effects of fertilizers and microbial agents combined application on the biological characteristics of continuous cropping peanut and microbial diversities in red soil region[D].Nanjing:Nanjing Agricultural University,Master Dissertation,2012.
[17]卡着才让,德科加,徐成体.不同施肥时间及施氮水平对高寒草甸生物量和土壤养分的影响[J].草地学报,2015,23(4):726-732.Ka Z C R,De K J,Xu C T.The effect of different fertilization time and nitrogen levels on biomass and soil nutrients in alpine meadow[J].Acta Agrestia Sin.,2015,23(4):726-732.
[18]陈丹梅,段玉琪,杨宇虹,等.长期施肥对植烟土壤养分及微生物群落结构的影响[J].中国农业科学,2014,47(17):3424-3433.Chen D M,Duan Y Q,Yang Y H,et al..Effects of long-term fertilization on flue-cured tobacco soil nutrients and microorganisms community structure[J].Sci.Agric.Sin.,2014,47(17):3424-3433.
[19]常安然,李佳,张耸,等.基于宏基因组学16S r DNA测序对烟草根际土壤细菌群落组成分析[J].中国农业科技导报,2017,19(2):43-50.Chang A R,Li J,Zhang S,et al..Analysis of bacterial community structure in rhizosphere soil of tobacco based on the metagenomics 16S r DNA sequencing technology[J].J.Agric.Sci.Technol.,2017,19(2):43-50.
[20]焦晓光,高崇升,隋跃宇,等.不同有机质含量农田土壤微生物生态特征[J].中国农业科学,2011,44(18):3759-3767.Jiao X G,Gao C S,Sui Y Y,et al..Research on soil microbial ecology under different soil organic matter levels in farmland[J].Sci.Agric.Sin.,2011,44(18):3759-3767.
[21]隋跃宇,焦晓光,高崇生,等.土壤有机质含量与土壤微生物量及土壤酶活性关系的研究[J].土壤通报,2009,40(5):1036-1039.Sui Y Y,Jiao X G,Gao C S,et al..The relationship among organic matter content and soil microbial biomass and soil enzyme activities[J].Chin.J.Soil Sci.,2009,40(5):1036-1039.
[22]陈桂芬,刘忠,黄雁飞,等.不同施肥处理对连作蔗田土壤微生物量、土壤酶活性及相关养分的影响[J].南方农业学报,2015,46(12):2123-2128.Chen G F,Liu Z,Huang Y F,et al..Effects of different fertilizer treatments on microbial biomass,enzyme activity and related nutrients in soils of continuous cropping sugarcane field[J].J.Southern Agric.,2015,46(12):2123-2128.
[2]邓家军,张仕祥,张富生,等.烟草幼苗根系分泌自毒物质种类及PAEs对根系抗氧化性能的影响[J].生态学报,2017,37(2):495-504.Deng J J,Zhang S X,Zhang F S,et al..Autotoxins exuded from roots and the effects of PAEs on antioxidant capacity in roots of tobacco seedlings[J].Acta Ecol.Sin.,2017,37(2):495-504.
[3]邱梅.黄土坡地核桃林不同间作模式土壤养分及酶活性研究[D].陕西咸阳:西北农林科技大学,硕士学位论文,2014.Qiu M.Study on soil nutrient&enzyme activity under different intercropping patterns of walnut forest on loess olateau[D].Shaanxi Xianyang:Northwest A&F University,Master Dissertation,2014.
[4]曹仕明,廖浩,张翼,等.施用腐熟秸秆肥对烤烟根系土壤微生物和酶活性的影响[J].中国烟草学报,2014,20(2):75-79.Cao S M,Liao H,Zhang Y,et al..Effects of decayed straw manure on microbe and enzyme activities in soil around tobacco roots[J].Chin.J.Tob.,2014,20(2):75-79.
[5]杨宇虹,陈冬梅,晋艳,等.不同肥料种类对连作烟草根际土壤微生物功能多样性的影响[J].作物学报,2011,37(1):105-111.Yang Y H,Chen D M,Jin Y,et al..Effects of different fertilizers on functional diversities of microbial flora in rhizospheric soil of monoculture tobacco[J].Acta Agron.Sin.,2011,37(1):105-111.
[6]汪涵.增产煤层气与产氢菌种的筛选及群落结构研究[D].北京:北京科技大学,博士学位论文,2015.Wang H.Research on screening of microbial communities for bioenhancement of coal seam methane and biohydrogen production and microbial community structure[D].Beijing:Beijing University of Science and Technology,Doctor Dissertation,2015.
[7]郭海燕,程国虎,李拥军,等.高通量测序技术及其在生物学中的应用[J].当代畜牧,2016(12):61-65.Guo H Y,Cheng G H,Li Y J,et al..The high throughput sequencing technology and its application in biology[J].Contemporary Anim.Husbandry,2016(12):61-65.
[8]鲍士旦.土壤农化分析[M].北京:中国农业出版社,2000.Bao S D.Soil Agrochemical Analysis[M].Beijing:China Agriculture Press,2000.
[9]关松荫.土壤酶及其研究法[M].北京:中国农业出版社,1986.Guan S Y.Soil Enzyme and Its Research Method[M].Beijing:China Agriculture Press,1986.
[10]杨兰芳,曾巧,李海波,等.紫外分光光度法测定土壤过氧化氢酶活性[J].土壤通报,2011,42(1):207-210.Yang L F,Zeng Q,Li H B,et al..Measurement of catalase activity in soil by ultraviolet spectrophotometry[J].Chin.J.Soil Sci.,2011,42(1):207-210.
[11]Li W Z,Godzik A.Cd-hit:A fast program for clustering and comparing large sets of protein or nucleotide sequences[J].Bioinformatics,2006,22(13):1658-1659.
[12]Quast C,Pruesse E,Yilmaz P,et al..The SILVA ribosomal RNA gene database project:Improved data processing and webbased tools[J].Nucl.Acids Res.,2013,41:590-596.
[13]史志华,朱华德,陈佳,等.小流域土壤水分空间异质性及其与环境因子的关系[J].应用生态学报,2012,23(4):889-895.Shi Z H,Zhu H D,Chen J,et al..Spatial heterogeneity of soil moisture and its relationships with environmental factors at small catchment level[J].Chin.J.Appl.Ecol.,2012,23(4):889-895.
[14]岳冰冰.烤烟连作改变了根际土壤微生物的多样性[D].哈尔滨:东北林业大学,硕士学位论文,2012.Yue B B.The rhizosphere soil microbial diversity is changed by flue-cured tobacco continuous cropping[D].Harbin:Northeast Forestry University,Master Dissertation,2012.
[15]顾美英,刘洪亮,李志强,等.新疆连作棉田施用生物炭对土壤养分及微生物群落多样性的影响[J].中国农业科学,2014,47(20):4128-4138.Gu M Y,Liu H L,Li Z Q,et al..Impact of biochar application on soil nutrients and microbial diversities in continuous cultivated cotton fields in Xinjiang[J].Sci.Agric.Sin.,2014,47(20):4128-4138.
[16]陈广波.肥料和菌剂配施对红壤地区连作花生生育性状和土壤微生物多样性的影响[D].南京:南京农业大学,硕士学位论文,2012.Chen G B.Effects of fertilizers and microbial agents combined application on the biological characteristics of continuous cropping peanut and microbial diversities in red soil region[D].Nanjing:Nanjing Agricultural University,Master Dissertation,2012.
[17]卡着才让,德科加,徐成体.不同施肥时间及施氮水平对高寒草甸生物量和土壤养分的影响[J].草地学报,2015,23(4):726-732.Ka Z C R,De K J,Xu C T.The effect of different fertilization time and nitrogen levels on biomass and soil nutrients in alpine meadow[J].Acta Agrestia Sin.,2015,23(4):726-732.
[18]陈丹梅,段玉琪,杨宇虹,等.长期施肥对植烟土壤养分及微生物群落结构的影响[J].中国农业科学,2014,47(17):3424-3433.Chen D M,Duan Y Q,Yang Y H,et al..Effects of long-term fertilization on flue-cured tobacco soil nutrients and microorganisms community structure[J].Sci.Agric.Sin.,2014,47(17):3424-3433.
[19]常安然,李佳,张耸,等.基于宏基因组学16S r DNA测序对烟草根际土壤细菌群落组成分析[J].中国农业科技导报,2017,19(2):43-50.Chang A R,Li J,Zhang S,et al..Analysis of bacterial community structure in rhizosphere soil of tobacco based on the metagenomics 16S r DNA sequencing technology[J].J.Agric.Sci.Technol.,2017,19(2):43-50.
[20]焦晓光,高崇升,隋跃宇,等.不同有机质含量农田土壤微生物生态特征[J].中国农业科学,2011,44(18):3759-3767.Jiao X G,Gao C S,Sui Y Y,et al..Research on soil microbial ecology under different soil organic matter levels in farmland[J].Sci.Agric.Sin.,2011,44(18):3759-3767.
[21]隋跃宇,焦晓光,高崇生,等.土壤有机质含量与土壤微生物量及土壤酶活性关系的研究[J].土壤通报,2009,40(5):1036-1039.Sui Y Y,Jiao X G,Gao C S,et al..The relationship among organic matter content and soil microbial biomass and soil enzyme activities[J].Chin.J.Soil Sci.,2009,40(5):1036-1039.
[22]陈桂芬,刘忠,黄雁飞,等.不同施肥处理对连作蔗田土壤微生物量、土壤酶活性及相关养分的影响[J].南方农业学报,2015,46(12):2123-2128.Chen G F,Liu Z,Huang Y F,et al..Effects of different fertilizer treatments on microbial biomass,enzyme activity and related nutrients in soils of continuous cropping sugarcane field[J].J.Southern Agric.,2015,46(12):2123-2128.