基于ITS序列的丹参连作根际土壤真菌群落组成及多样性分析
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摘要
目的:该文研究了丹参连作根际土壤真菌群落组成及多样性变化,有利于阐述丹参-土壤-真菌根际微环境的变化,为揭示连作障碍形成的原因提供理论基础。方法:基于高通量测序技术,分别对未种植丹参(CK),连作1年(Y1)及连作两年(Y2)的根际土壤进行转录间隔区(ITS)测序,3种处理中分别得到14 153,19 024及7 622条有效序列,注释为1 027,1 095及712种真菌可操作分类单元(OTU)。结果:随着连作年限的增加,真菌群落多样性呈现出先下降后上升的变化趋势。在纲分类水平上,优势菌群分别为伞菌纲(Agaricomycetes),锤舌菌纲(Leotiomycetes)及银耳纲(Tremellomycetes),在3种处理中所占比例大小分别为Y1>CK>Y2,Y2>Y1>CK,CK>Y2>Y1;在属分类水平上,CK中优势菌属为耐冷酵母(Guehomyces),Y1及Y2较CK相比分别下降96.13%和90.21%,异常威克汉姆酵母(Wickerhamomyces)在Y2中所占比例增幅明显(6.64%),较CK及Y1相比分别增加290.59%及564.00%。结论:CK及Y1样品中根际土壤真菌在属水平上群落结构上具有更高的相似性,Y2样品群落结构分布热图显示炭疽菌属(Cryptosporiopsis)等致病真菌成为优势菌属,推测与连作丹参病害率高有一定相关性。研究结果为阐述丹参连作障碍形成、发展绿色种植提供理论支撑。
Objective: To study rhizosphere soil fungal community composition and diversity of Salvia miltiorrhiza,in order to explain the changes of the rhizosphere microenvironment of S. miltiorrhiza,and provide theoretical basis for revealing the reasons for the formation of continuous obstacles. Method: Based on the highthroughput sequencing technology,three kinds of soil samples were collected,namely soil of non-plant( CK),soil for cultivating for one year( Y1) and soil for cultivating for two years( Y2),respectively. According to the sequencing of the internal transcribed space of nuclear ribosomal DNA( ITS) region of fungal rRNA gene,14153,19 024 and 7 622 valid sequences were obtained and annotated as 1 027,1 095 and 712 varieties of OTUs.The diversity of fungal communities showed a trend of decreasing first and then increasing. Agaricomycetes,Leotiomycetes and Tremellomycetes were the dominant bacterial communities at the class level, their orders in three treatments were Y1 > CK > Y2, Y2 > Y1 > CK, CK > Y2 > Y1. At the genus level, Guehomyces was dominant species in CK, and both Y1 and Y2 had a decrease of 96. 13% and 90. 21%, respectively.Wickerhamomyces had a significantly increase in Y2( 6. 64%),250. 59% and 564. 00% compared with CK and Y1,respectively. Result: The results of cluster analysis showed that CK and Y1 had a higher similarity at the genus level in soil fungal community composition compared with Y2. Conclusion: Cryptosporiopsis and other pathogenic fungal communities were dominant genuses according to the heatmap analysis in Y2, suggesting a correlation with the incidence of S. miltiorrhiza. The findings provide theoretical basis for the formation of continuous cropping obstacle.
Objective: To study rhizosphere soil fungal community composition and diversity of Salvia miltiorrhiza,in order to explain the changes of the rhizosphere microenvironment of S. miltiorrhiza,and provide theoretical basis for revealing the reasons for the formation of continuous obstacles. Method: Based on the highthroughput sequencing technology,three kinds of soil samples were collected,namely soil of non-plant( CK),soil for cultivating for one year( Y1) and soil for cultivating for two years( Y2),respectively. According to the sequencing of the internal transcribed space of nuclear ribosomal DNA( ITS) region of fungal rRNA gene,14153,19 024 and 7 622 valid sequences were obtained and annotated as 1 027,1 095 and 712 varieties of OTUs.The diversity of fungal communities showed a trend of decreasing first and then increasing. Agaricomycetes,Leotiomycetes and Tremellomycetes were the dominant bacterial communities at the class level, their orders in three treatments were Y1 > CK > Y2, Y2 > Y1 > CK, CK > Y2 > Y1. At the genus level, Guehomyces was dominant species in CK, and both Y1 and Y2 had a decrease of 96. 13% and 90. 21%, respectively.Wickerhamomyces had a significantly increase in Y2( 6. 64%),250. 59% and 564. 00% compared with CK and Y1,respectively. Result: The results of cluster analysis showed that CK and Y1 had a higher similarity at the genus level in soil fungal community composition compared with Y2. Conclusion: Cryptosporiopsis and other pathogenic fungal communities were dominant genuses according to the heatmap analysis in Y2, suggesting a correlation with the incidence of S. miltiorrhiza. The findings provide theoretical basis for the formation of continuous cropping obstacle.
引文
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[5]郭俊霞,李青苗,杨玉霞,等.丹参连作土壤水提液对其幼苗生长的影响[J].甘肃中医学院学报,2013,30(3):76-79.
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[9]董林林,牛玮浩,王瑞,等.人参根际真菌群落多样性及组成的变化[J].中国中药杂志,2017,42(3):443-449.
[10]张重义,陈慧,杨艳会,等.连作对地黄根际土壤细菌群落多样性的影响[J].应用生态学报,2010,21(11):2843-2848.
[11]刘伟,张琳,章云云,等.不同连作年限对白花丹参生长及其活性成分含量的影响[J].中国中药杂志,2013,38(24):4252-4256.
[12]姚佳,尹海波,赵容,等.基于连作障碍条件下穿山龙无机元素吸收规律及总皂苷含量分析[J].中国实验方剂学杂志,2015,21(24):61-64.
[13]刘伟,章云云,魏莹莹,等.耕作模式对白花丹参连作土壤理化性质及酶活性的影响[J].作物杂志,2015(1):72-77.
[14]章云云,朱端卫,王晓,等.植物根系分泌物的作用及其与药用植物连作障碍的关系[J].湖北农业科学,2014,53(6):1241-1245.
[15]Bokulich N A,Sathish S,Faith J J,et al.Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nat Methods,2012,10(1):57-59.
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[17]Jay L G,Aaron L M.Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization[J].Appl Environ Microbiol,1991,57(8):2351-2359.
[18]孟品品,刘星,邱慧珍,等.连作马铃薯根际土壤真菌种群结构及其生物效应[J].应用生态学报,2012,23(11):3079-3086.
[19]张子龙,王文全.植物连作障碍的形成机制及其调控技术研究进展[J].生物学杂志,2010,27(5):69-72.
[20]李坤,郭修武,孙英妮,等.葡萄连作对土壤细菌和真菌种群的影响[J].应用生态学报,2009,20(12):3109-3114.
[21]严铸云,王海,何彪,等.中药连作障碍防治的微生态研究模式探讨[J].中药与临床,2012,3(2):5-9.
[22]徐红梅,倪方方,宋腾蛟,等.高通量测序揭示杭白芍根际真菌群落年际变化[J].浙江中医药大学学报,2016,40(12):942-947.
[2]王雪,陈美兰,杨光,等.丛枝菌根真菌与哈茨木霉菌合用对连作丹参生长及质量的影响[J].中国中药杂志,2014,39(9):1574-1578.
[3]刘伟,魏莹莹,孙鹏,等.须根自然腐解对白花丹参生长及其活性成分含量的影响[J].中国中药杂志,2015,40(13):2548-2552.
[4]赵磊,余弦,宋玉丹,等.川牛膝不同种植模式比较[J].中国实验方剂学杂志,2015,21(8):86-88.
[5]郭俊霞,李青苗,杨玉霞,等.丹参连作土壤水提液对其幼苗生长的影响[J].甘肃中医学院学报,2013,30(3):76-79.
[6]张辰露,孙群,叶青.连作对丹参生长的障碍效应[J].西北植物学报,2005,25(5):1029-1034.
[7]李俊仁,陈秀珍,曾湘达,等.连作对穿心莲基因组DNA甲基化的影响[J].中国实验方剂学杂志,2018,24(15):65-71.
[8]谭勇,崔尹赡,季秀玲,等.三七连作的根际、根内微生物变化与生态学研究进展[J].中草药,2017,48(2):391-399.
[9]董林林,牛玮浩,王瑞,等.人参根际真菌群落多样性及组成的变化[J].中国中药杂志,2017,42(3):443-449.
[10]张重义,陈慧,杨艳会,等.连作对地黄根际土壤细菌群落多样性的影响[J].应用生态学报,2010,21(11):2843-2848.
[11]刘伟,张琳,章云云,等.不同连作年限对白花丹参生长及其活性成分含量的影响[J].中国中药杂志,2013,38(24):4252-4256.
[12]姚佳,尹海波,赵容,等.基于连作障碍条件下穿山龙无机元素吸收规律及总皂苷含量分析[J].中国实验方剂学杂志,2015,21(24):61-64.
[13]刘伟,章云云,魏莹莹,等.耕作模式对白花丹参连作土壤理化性质及酶活性的影响[J].作物杂志,2015(1):72-77.
[14]章云云,朱端卫,王晓,等.植物根系分泌物的作用及其与药用植物连作障碍的关系[J].湖北农业科学,2014,53(6):1241-1245.
[15]Bokulich N A,Sathish S,Faith J J,et al.Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nat Methods,2012,10(1):57-59.
[16]Quast C,Pruesse E,Yilmaz P,et al.The SILVA ribosomal RNA gene database project:improved data processing and web-based tools[J].Nucleic Acids Res,2013,41(Database issue):590-596.
[17]Jay L G,Aaron L M.Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization[J].Appl Environ Microbiol,1991,57(8):2351-2359.
[18]孟品品,刘星,邱慧珍,等.连作马铃薯根际土壤真菌种群结构及其生物效应[J].应用生态学报,2012,23(11):3079-3086.
[19]张子龙,王文全.植物连作障碍的形成机制及其调控技术研究进展[J].生物学杂志,2010,27(5):69-72.
[20]李坤,郭修武,孙英妮,等.葡萄连作对土壤细菌和真菌种群的影响[J].应用生态学报,2009,20(12):3109-3114.
[21]严铸云,王海,何彪,等.中药连作障碍防治的微生态研究模式探讨[J].中药与临床,2012,3(2):5-9.
[22]徐红梅,倪方方,宋腾蛟,等.高通量测序揭示杭白芍根际真菌群落年际变化[J].浙江中医药大学学报,2016,40(12):942-947.