蒙古沙冬青深色有隔内生真菌磷脂脂肪酸生物标记
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摘要
为探究深色有隔内生真菌(DSE)磷脂脂肪酸(PLFA)生物标记在DSE分类鉴定和土壤微生物检测上的应用,分别于宁夏银川、沙坡头、甘肃民勤和内蒙古乌海4个样地采集蒙古沙冬青(Ammopiptanthus mongolicus)根样,分离得到DSE,结合形态学和分子生物学进行分类鉴定,并利用Sherlock微生物鉴定系统检测DSE脂肪酸组成。4个样地共分离得到12株DSE,可划分为7属7种。通过对DSE脂肪酸组分进行检测,共得到91种已知脂肪酸和12种Summed Feature型。DSE脂肪酸以C_(14)酸、C_(16)酸和C_(17)酸为主,其中16∶0、16∶1Cis 9(ω7)、18∶2CIS 9,12/18∶0a、18∶0和Summed Feature 8为DSE共有脂肪酸,部分菌株中还具有特有脂肪酸组成,其可作为土壤微生物检测的生物标记。通过脂肪酸分析可将12株DSE分为4个类群,将3株DSE鉴定到种。结果表明,PLFAs生物标记可作为DSE分类鉴定指标,并为土壤微生物检测提供依据。
To explore application of phospholipid fatty acid(PLFA)biomarkers in dark septate endophytes(DSE)classification,identification and detection of soil microorganisms,root samples of Ammopiptanthus mongolicus were collected from Yinchuan,Shapotou,Minqin and Wuhai in northwestern China.DSE strains were isolated from roots and identified by morphology and ITS sequence analysis.DSE fatty acid composition was detect by Sherlock Microbial Identification System(Sherlock MIS).A total of 12 DSE strains were separated from 4sites,which it could be diveided into 7 genera and 7 species.The 91 known fatty acids and 12 Summed Feature were detected by the Sherlock MIS,and C_(14) acid,C_(16) acid and C_(17) acid were the major fatty acids of DSE.Of these,16∶0,16∶1Cis 9(ω7),18∶2CIS 9,12/18∶0a,18∶0 and Summed Feature 8 were the total fatty acids.Some of the strains also had the specific fatty acid composition,and specific fatty acids could be used as a biomarker for the detection of soil microorganisms.12 strains of DSE were divided into 4 groups by fatty acid analysis,and3 strains of DSE were identified to the species.This study suggested that PLFAs biomarkers could be used as DSE classification identification indicators,and provided a basis for the detection of soil microorganisms.
To explore application of phospholipid fatty acid(PLFA)biomarkers in dark septate endophytes(DSE)classification,identification and detection of soil microorganisms,root samples of Ammopiptanthus mongolicus were collected from Yinchuan,Shapotou,Minqin and Wuhai in northwestern China.DSE strains were isolated from roots and identified by morphology and ITS sequence analysis.DSE fatty acid composition was detect by Sherlock Microbial Identification System(Sherlock MIS).A total of 12 DSE strains were separated from 4sites,which it could be diveided into 7 genera and 7 species.The 91 known fatty acids and 12 Summed Feature were detected by the Sherlock MIS,and C_(14) acid,C_(16) acid and C_(17) acid were the major fatty acids of DSE.Of these,16∶0,16∶1Cis 9(ω7),18∶2CIS 9,12/18∶0a,18∶0 and Summed Feature 8 were the total fatty acids.Some of the strains also had the specific fatty acid composition,and specific fatty acids could be used as a biomarker for the detection of soil microorganisms.12 strains of DSE were divided into 4 groups by fatty acid analysis,and3 strains of DSE were identified to the species.This study suggested that PLFAs biomarkers could be used as DSE classification identification indicators,and provided a basis for the detection of soil microorganisms.
引文
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[2]MANDYAM K,JUMPPONEN A.Seeking the elusive function of the root-colonising dark septate endophytic fungi[J].Studies in Mycology,2005,53(53):173-189.
[3]RODRIGUEZ R J,WHITE JR J F,ARNOLD A E,et al.Fungal endophytes:diversity and functional roles[J].New Phytologist,2009,182(2):314-330.
[4]PORRAS-ALFARO A,BAYMAN P.Hidden fungi,emergent properties:endophytes and microbiomes[J].Phytopathology,2011,49(1):291.
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[8]XU R,LI T,CUI H,et al.Diversity and characterization of Cd-tolerant dark septate endophytes(DSEs)associated with the roots of Nepal alder(Alnus nepalensis)in a metal mine tailing of southwest China[J].Applied Soil Ecology,2015,93:11-18.
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[10]徐风美,王春燕,褚洪龙,等.松萎蔫病发生区和未发生区油松根部真菌群落研究[J].西北植物学报,2014,34(8):1627-1634.XU F M,WANG CH Y,CHU H L,et al.Research on root fungal community of Pinus tabuliformis in pine wilt disease damaged and undamaged areas[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(8):1627-1634(in Chinese with English abstract).
[11]KAMPFER P.Limits and possibilities of total fatty acid analysis for classification and identification of Bacillus species[J].Systematic and Applied Microbiology,1994,17(1):86-98.
[12]FOSTER A L,MUNK L,KOSKI R A,et al.Relationships between microbial communities and environmental parameters at sites impacted by mining of volcanogenic massive sulfide deposits,Prince William Sound,Alaska[J].Applied Geochemistry,2008,23(2):279-307.
[13]ABEL K,PETERSON J I.Classification of microorganisms by analysis of chemical composition I.Feasibility of utilizing gas chromatography[J].Journal of Bacteriology,1963,85(5):1039-1044.
[14]MOSCA A,RUSSO F,MIRAGLIOTTA L,et al.Utility of gas chromatography for rapid identification of mycobacterial species frequently encountered in clinical laboratory[J].Journal of Microbiological Methods,2007,68(2):392-395.
[15]刘国红,刘波,林营志,等.基于脂肪酸生物标记与16S rRNA的芽胞杆菌系统发育分析比较[J].生物技术通报,2015,31(3):146-153.LIU G H,LIU B,LIN Y ZH,et al.The comparsion of Bacillus species classification based on fatty acid and 16S rRNA gene[J].Biotechnology Bulletin,2015,31(3):146-153(in Chinese with English abstract).
[16]曹艳峰,李彦,李晨华,等.荒漠灌木梭梭(Haloxylon ammodendron)周围土壤微生物的空间分布[J].生态学报,2016,36(6):1628-1635.CAO Y F,LI Y,LI CH H,et al.The spatial distribution of soil microbes around a desert shrub of Haloxylon ammodendron[J].Acta Ecologica Sinica,2016,36(6):1628-1635(in Chinese with English abstract).
[17]王秋红,陈亮,林营志,等.福建省青枯雷尔氏菌脂肪酸多态性研究[J].中国农业科学,2007,39(8):1675-1687.WANG Q H,CHEN L,LIN Y ZH,et al.Polymorphism of fatty acid of Ralstonia solanacearum in Fujian province[J].Scientia Agricultura Sinica,2007,39(8):1675-1687(in Chinese with English abstract).
[18]THOMPSON J D,GIBSON T J,PLEWNIAK F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(24):4876-4882.
[19]TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,24(8):1596-1599.
[20]TAMURA K,NEI M,KUMAR S.Prospects for inferring very large phylogenies by using the neighbor-joining method[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(30):11030-11035.
[21]张玉洁.植物深色有隔内生真菌(DSE)的研究进展[J].文山学院学报,2010,23(1):145-150.ZHANG Y J.Research advances on dark sepatate endophytes[J].Journal of Wenshan University,2010,23(1):145-150(in Chinese with English abstract).
[22]刘茂军张兴涛赵之伟.深色有隔内生真菌(DSE)研究进展[J].菌物学报,2009,28(6):888-894.LIU M J,ZHANG X T,ZHAO ZH W.Advances in the research of dark septate endophytes[J].Mycosystema,2009,28(6):888-894(in Chinese).
[23]陈剑山,郑服丛.ITS序列分析在真菌分类鉴定中的应用[J].安徽农业科学,2007,35(13):3785-3786.CHEN J SH,ZHENG F C.Application of ITS Sequences in fungi classification and identification[J].Journal of Anhui Agricultural Sciencis,2007,35(13):3785-3786(in Chinese with English abstract).
[24]WHITE D C,DAVIS W M,NICKELS J S,et al.Determination of the sedimentary microbial biomass by extractible lipid phosphate[J].Oecologia,1979,40(1):51-62.
[25]吴愉萍,徐建明,汪海珍,等.Sherlock MIS系统应用于土壤细菌鉴定的研究[J].土壤学报,2006,43(4):642-647.WU Y P,XU J M,WANG H ZH,et al.Application of sherlock mis in identification of soil bacteria[J].Acta Pedologica Sinica,2006,43(4):642-647(in Chinese with English abstract).
[26]黄朱梁,裘迪红.MIDI Sherlock微生物自动鉴定系统鉴定方法的建立-以贻贝中分离的蜡样芽胞杆菌为例[J].宁波大学学报(理工版),2011,24(2):8-13.HUANG ZH L,QIU D H.Identification of Bacillus cereus separated from mussels using MIDI sherlock microorganism auto identification system[J].Ournal of Ningbo University(Nsee),2011,24(2):8-13(in Chinese with English abstract).
[27]陈晓芬,李忠佩,刘明,等.长期施肥处理对红壤水稻土微生物群落结构和功能多样性的影响[J].生态学杂志,2015.34(7):1815-1822.CHEN X F,LI ZH P,LIU M,et al.Effect of long-term fertilizations on microbial community structure and functional diversity in paddy soil of subtropical China[J].Chinese Jounral of Ecology,2015,34(7):1815-1822(in Chinese with English abstract).
[28]CHANG E H,CHEN T H,TIAN G,et al.The effect of altitudinal gradient on soil microbial community activity and structure in moso bamboo plantations[J].Applied Soil Ecology,2016,98:213-220.
[29]秦巧玲,粟婉媛,吕均,等.GC-MS联用分析测定志贺菌全细胞脂肪酸组成[J].中国卫生检验杂志,2008,18(3):426-428.QIN Q L,SU W Y,LU J,et al.GC-MS analysis of fatty acid from whole cell of Shigella[J].Chinese Journal of Health Laboratory Technology,2008,18(3):426-428(in Chinese with English abstract).
[30]HILL T C J,MCPHERSON E F,HARRIS J A,et al.Microbial biomass estimated by phospholipid phosphate in soils with diverse microbial communities[J].Soil Biology and Biochemistry,1993,25(12):1779-1786.
[31]周莉娜,苏润华,马思佳,等.基于PLFA法分析亚硝氮,硝氮和氨氮对厌氧微生物细菌群落的影响[J].环境科学学报,2016,36(2):499-505.ZHOU L N,SU R H,MA S J,et al.Effects of nitrite,nitrate and ammonia nitrogen on anaerobic microbial community characterized by using phospholipid fatty acid PLFA method[J].Acta Scientiae Circumstantiae,2016,36(2):499-505.
[2]MANDYAM K,JUMPPONEN A.Seeking the elusive function of the root-colonising dark septate endophytic fungi[J].Studies in Mycology,2005,53(53):173-189.
[3]RODRIGUEZ R J,WHITE JR J F,ARNOLD A E,et al.Fungal endophytes:diversity and functional roles[J].New Phytologist,2009,182(2):314-330.
[4]PORRAS-ALFARO A,BAYMAN P.Hidden fungi,emergent properties:endophytes and microbiomes[J].Phytopathology,2011,49(1):291.
[5]KOVACS G M,SZIGETVARI C.Mycorrhizae and other root-associated fungal structures of the plants of a sandy grassland on the great hungarian plain[J].Phyton(Horn,Austria),2002,42(2):211-224.
[6]KHIDIR H H,EUDY D M,PORRAS-ALFARO A,et al.A general suite of fungal endophytes dominate the roots of two dominant grasses in a semiarid grassland[J].Journal of Arid Environments,2010,74(1):35-42.
[7]ZHAN F,HE Y,LI T,et al.Tolerance and antioxidant response of a dark septate endophyte(DSE),exophiala pisciphila,to cadmium stress[J].Bulletin of Environmental Contamination and Toxicology,2015,94(1):96-102.
[8]XU R,LI T,CUI H,et al.Diversity and characterization of Cd-tolerant dark septate endophytes(DSEs)associated with the roots of Nepal alder(Alnus nepalensis)in a metal mine tailing of southwest China[J].Applied Soil Ecology,2015,93:11-18.
[9]SIEBER T,GRNIG C.Biodiversity of fungal root-endophyte communities and populations,in particular of the dark septate endophyte Phialocephala fortinii s.l.[J].Microbial Root Endophytes,2006,9:107-132.
[10]徐风美,王春燕,褚洪龙,等.松萎蔫病发生区和未发生区油松根部真菌群落研究[J].西北植物学报,2014,34(8):1627-1634.XU F M,WANG CH Y,CHU H L,et al.Research on root fungal community of Pinus tabuliformis in pine wilt disease damaged and undamaged areas[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(8):1627-1634(in Chinese with English abstract).
[11]KAMPFER P.Limits and possibilities of total fatty acid analysis for classification and identification of Bacillus species[J].Systematic and Applied Microbiology,1994,17(1):86-98.
[12]FOSTER A L,MUNK L,KOSKI R A,et al.Relationships between microbial communities and environmental parameters at sites impacted by mining of volcanogenic massive sulfide deposits,Prince William Sound,Alaska[J].Applied Geochemistry,2008,23(2):279-307.
[13]ABEL K,PETERSON J I.Classification of microorganisms by analysis of chemical composition I.Feasibility of utilizing gas chromatography[J].Journal of Bacteriology,1963,85(5):1039-1044.
[14]MOSCA A,RUSSO F,MIRAGLIOTTA L,et al.Utility of gas chromatography for rapid identification of mycobacterial species frequently encountered in clinical laboratory[J].Journal of Microbiological Methods,2007,68(2):392-395.
[15]刘国红,刘波,林营志,等.基于脂肪酸生物标记与16S rRNA的芽胞杆菌系统发育分析比较[J].生物技术通报,2015,31(3):146-153.LIU G H,LIU B,LIN Y ZH,et al.The comparsion of Bacillus species classification based on fatty acid and 16S rRNA gene[J].Biotechnology Bulletin,2015,31(3):146-153(in Chinese with English abstract).
[16]曹艳峰,李彦,李晨华,等.荒漠灌木梭梭(Haloxylon ammodendron)周围土壤微生物的空间分布[J].生态学报,2016,36(6):1628-1635.CAO Y F,LI Y,LI CH H,et al.The spatial distribution of soil microbes around a desert shrub of Haloxylon ammodendron[J].Acta Ecologica Sinica,2016,36(6):1628-1635(in Chinese with English abstract).
[17]王秋红,陈亮,林营志,等.福建省青枯雷尔氏菌脂肪酸多态性研究[J].中国农业科学,2007,39(8):1675-1687.WANG Q H,CHEN L,LIN Y ZH,et al.Polymorphism of fatty acid of Ralstonia solanacearum in Fujian province[J].Scientia Agricultura Sinica,2007,39(8):1675-1687(in Chinese with English abstract).
[18]THOMPSON J D,GIBSON T J,PLEWNIAK F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(24):4876-4882.
[19]TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,24(8):1596-1599.
[20]TAMURA K,NEI M,KUMAR S.Prospects for inferring very large phylogenies by using the neighbor-joining method[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(30):11030-11035.
[21]张玉洁.植物深色有隔内生真菌(DSE)的研究进展[J].文山学院学报,2010,23(1):145-150.ZHANG Y J.Research advances on dark sepatate endophytes[J].Journal of Wenshan University,2010,23(1):145-150(in Chinese with English abstract).
[22]刘茂军张兴涛赵之伟.深色有隔内生真菌(DSE)研究进展[J].菌物学报,2009,28(6):888-894.LIU M J,ZHANG X T,ZHAO ZH W.Advances in the research of dark septate endophytes[J].Mycosystema,2009,28(6):888-894(in Chinese).
[23]陈剑山,郑服丛.ITS序列分析在真菌分类鉴定中的应用[J].安徽农业科学,2007,35(13):3785-3786.CHEN J SH,ZHENG F C.Application of ITS Sequences in fungi classification and identification[J].Journal of Anhui Agricultural Sciencis,2007,35(13):3785-3786(in Chinese with English abstract).
[24]WHITE D C,DAVIS W M,NICKELS J S,et al.Determination of the sedimentary microbial biomass by extractible lipid phosphate[J].Oecologia,1979,40(1):51-62.
[25]吴愉萍,徐建明,汪海珍,等.Sherlock MIS系统应用于土壤细菌鉴定的研究[J].土壤学报,2006,43(4):642-647.WU Y P,XU J M,WANG H ZH,et al.Application of sherlock mis in identification of soil bacteria[J].Acta Pedologica Sinica,2006,43(4):642-647(in Chinese with English abstract).
[26]黄朱梁,裘迪红.MIDI Sherlock微生物自动鉴定系统鉴定方法的建立-以贻贝中分离的蜡样芽胞杆菌为例[J].宁波大学学报(理工版),2011,24(2):8-13.HUANG ZH L,QIU D H.Identification of Bacillus cereus separated from mussels using MIDI sherlock microorganism auto identification system[J].Ournal of Ningbo University(Nsee),2011,24(2):8-13(in Chinese with English abstract).
[27]陈晓芬,李忠佩,刘明,等.长期施肥处理对红壤水稻土微生物群落结构和功能多样性的影响[J].生态学杂志,2015.34(7):1815-1822.CHEN X F,LI ZH P,LIU M,et al.Effect of long-term fertilizations on microbial community structure and functional diversity in paddy soil of subtropical China[J].Chinese Jounral of Ecology,2015,34(7):1815-1822(in Chinese with English abstract).
[28]CHANG E H,CHEN T H,TIAN G,et al.The effect of altitudinal gradient on soil microbial community activity and structure in moso bamboo plantations[J].Applied Soil Ecology,2016,98:213-220.
[29]秦巧玲,粟婉媛,吕均,等.GC-MS联用分析测定志贺菌全细胞脂肪酸组成[J].中国卫生检验杂志,2008,18(3):426-428.QIN Q L,SU W Y,LU J,et al.GC-MS analysis of fatty acid from whole cell of Shigella[J].Chinese Journal of Health Laboratory Technology,2008,18(3):426-428(in Chinese with English abstract).
[30]HILL T C J,MCPHERSON E F,HARRIS J A,et al.Microbial biomass estimated by phospholipid phosphate in soils with diverse microbial communities[J].Soil Biology and Biochemistry,1993,25(12):1779-1786.
[31]周莉娜,苏润华,马思佳,等.基于PLFA法分析亚硝氮,硝氮和氨氮对厌氧微生物细菌群落的影响[J].环境科学学报,2016,36(2):499-505.ZHOU L N,SU R H,MA S J,et al.Effects of nitrite,nitrate and ammonia nitrogen on anaerobic microbial community characterized by using phospholipid fatty acid PLFA method[J].Acta Scientiae Circumstantiae,2016,36(2):499-505.