乌龙江湿地土壤真菌群落结构的PCR-DGGE分析
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摘要
为了解乌龙江湿地土壤真菌群落结构组成及动态变化,本研究通过稀释平板法对湿地土壤可培养真菌数进行测定,并采用变性梯度凝胶电泳(DGGE)技术分析乌龙江湿地土壤真菌群落结构。平板计数结果表明,不同时间不同采样点可培养真菌数的变化幅度较小,每克干土中可培养真菌数介于0.13×10~4~8.26×10~4 CFU。真菌18SrDNA的PCR-DGGE图谱显示,每个泳道条带的位置、数目和亮度各有不同,说明不同采样时间不同采样点土壤真菌群落结构存在着差异。DGGE图谱条带多样性分析结果表明,2009年3月、2009年11月和2010年1月所采集土壤样品的真菌群落多样性较其他时间更为丰富;从采样点来看,真菌群落丰富度则表现为样品L>样品M>样品R。对DGGE主要条带和差异性条带进行克隆测序后发现,乌龙江湿地土壤主要真菌类群为子囊菌Ascomycota和毛霉菌Mucoromycota。
Composition and dynamics of the fungal community in soil of Wulongjiang wetlands were studied.The classical plate count and the denatured gradient gel electrophoresis(DGGE)were applied to determine the cultivable population and diversity of the community.The plate counts of the samples averaged between 0.13×10~4 and 8.26×10~4 CFU per g of dry soil with no significant difference due to the locations or time of collection.On the other hand,the PCR-DGGE patterns of 18 SrDNA(V1+V2)fragments exhibited variations on the positions,number and lightness of the bands from different lanes,indicating compositional differences among the fungal populations.The DGGE bands digitally analyzed by Quantity One software and the derived Shannon-Wiener index,evenness and abundance showed greater diversities in the samples obtained in March 2009,September 2009 and January 2010 than other times of a year.They also unveiled the differences in abundance of Sample L> Sample M> Sample R.The dominant fungi in the soil were identified to be Ascomycota and Mucoromycota according to a sequence analysis on the DGGE bands.
Composition and dynamics of the fungal community in soil of Wulongjiang wetlands were studied.The classical plate count and the denatured gradient gel electrophoresis(DGGE)were applied to determine the cultivable population and diversity of the community.The plate counts of the samples averaged between 0.13×10~4 and 8.26×10~4 CFU per g of dry soil with no significant difference due to the locations or time of collection.On the other hand,the PCR-DGGE patterns of 18 SrDNA(V1+V2)fragments exhibited variations on the positions,number and lightness of the bands from different lanes,indicating compositional differences among the fungal populations.The DGGE bands digitally analyzed by Quantity One software and the derived Shannon-Wiener index,evenness and abundance showed greater diversities in the samples obtained in March 2009,September 2009 and January 2010 than other times of a year.They also unveiled the differences in abundance of Sample L> Sample M> Sample R.The dominant fungi in the soil were identified to be Ascomycota and Mucoromycota according to a sequence analysis on the DGGE bands.
引文
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[19]亢宗静,袁楠,王蓥燕,等.若尔盖高原湿地的真菌群落结构及低温纤维素降解真菌特征[J].土壤通报,2017,48(4):830-836.
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[22]邱权,陈雯莉.三峡库区小江流域消落区土壤微生物多样性[J].华中农业大学学报,2013,32(3):15-20.
[23]CHAN O C,YANG X D,FU Y,et al.16SrRNA gene analyses of bacterial community structures in the soils of evergreen broad-leaved forests in south-west China[J].FEMS Microbiology Ecology,2006,58:247-259.
[24]吕新,陈丽华,刘兰英,等.抗真菌转基因水稻根际土壤真菌群落结构的动态变化[J].中国生态农业学报,2012,20(10):1340-1346.
[25]徐严.若尔盖高原湿地土壤真菌的初步研究[D].成都:四川农业大学,2008.
[26]陈会海,许修宏,李洪涛.应用PCR-DGGE分析三江湿地土壤真菌群落结构的多样性[J].环境科学研究,2012,25(11):1272-1278.
[2]陈爱辉,李朝霞,梁慧星,等.DGGE技术在湿地微生物多样性研究中的应用[J].湖北农业科学,2010,49(4):981-984.
[3]张晶,陈书明,王小国.天鹅湖湿地微生物群落PCR-DGGE分析[J].基因组学与应用生物学,2016,35(9):2423-2428.
[4]ZELLES L,BAI Q Y,BECK T,et al.Signature fatty acids in phospholipids and lip-opolysaccharides as indicators of microbial biomass and community structure in agricultural soils[J].Soil Biology and Biochemistry,1992,24(4):317-323.
[5]陈子熙,陈磊,张卫文.单细胞尺度下的微生物学研究:意义与方法[J].微生物学报,2017,57(6):920-931.
[6]刘银银,李峰,孙庆业,等.湿地生态系统土壤微生物研究进展[J].应用与环境生物学报,2013,19(3):547-552.
[7]ZHANG C P,WANG B,DAI X Y,et al.Structure and function of the bacterial communities during rhizoremediation of hexachlorobenzene in constructed wetlands[J].Environmental Science and Pollution Research,2017,24:11483-11492.
[8]MARK B,HANNELE A,FREDERIK V K,et al.Susceptibility of constructed wetland microbial communities to silver nanoparticles:A microcosm study[J].Ecological Engineering,2016,97:476-485.
[9]ZHONG F,WU J,DAI Y R,et al.Bacterial community analysis by PCR-DGGE and 454-pyrosequencing of horizontal subsurface flow constructed wetlands with front aeration[J].Applied Microbiology and Biotechnology,2015,99:1499-1512.
[10]佘晨兴,仝川.闽江口芦苇沼泽湿地土壤产甲烷菌群落结构的垂直分布[J].生态学报,2012,32(17):5299-5308.
[11]曾志华,杨民和,佘晨兴,等.闽江河口区淡水和半咸水潮汐沼泽湿地土壤产甲烷菌多样性[J].生态学报,2014,34(10):2674-2681.
[12]李振高,骆永明,滕应.土壤与环境微生物研究法[M].北京:科学出版社,2008:90-92.
[13]MAY L A,SMILEY B,SCHMIDT M G.Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage[J].Canadian Journal of Microbiology,2001,47(9):829-841.
[14]BOWATTE S,ISHIHARA R,ASAKAWA S,et al.Characterization of ammonia oxidizing bacteria associated with weeds in a Japanese paddy field using amoA gene fragments[J].Soil Science and Plant Nutrition,2006,52:593-600.
[15]赵兴青,杨柳燕,陈灿,等.PCR-DGGE技术用于湖泊沉积物中微生物群落结构多样性研究[J].生态学报,2006,26(11):3610-3616.
[16]RAVENSCHLAG K,SAHM K,PERUTHALER J,et al.High bacterial diversity in permanently cold marine sediments[J].Applied and Environmental Microbiology,1999,65(9):3982-3989.
[17]蒲洋,李文军,王凯,等.烟台市区河流入海口微生物群落的分析[J].海洋科学,2017,41(7):9-15.
[18]赵先丽,周广胜,周莉,等.盘锦芦苇湿地土壤微生物数量研究[J].土壤通报,2008,39(6):1376-1379.
[19]亢宗静,袁楠,王蓥燕,等.若尔盖高原湿地的真菌群落结构及低温纤维素降解真菌特征[J].土壤通报,2017,48(4):830-836.
[20]FENG S G,ZHANG H X,WANG Y F,et al.Analysis of fungal community structure in the soil of Zoige Alpine W etland[J].Acta Ecologica Sinica,2009,29:260-266.
[21]赵先丽,周广胜,周莉,等.盘锦芦苇湿地土壤微生物特征分析[J].气象与环境学报,2006,22(14):64-67.
[22]邱权,陈雯莉.三峡库区小江流域消落区土壤微生物多样性[J].华中农业大学学报,2013,32(3):15-20.
[23]CHAN O C,YANG X D,FU Y,et al.16SrRNA gene analyses of bacterial community structures in the soils of evergreen broad-leaved forests in south-west China[J].FEMS Microbiology Ecology,2006,58:247-259.
[24]吕新,陈丽华,刘兰英,等.抗真菌转基因水稻根际土壤真菌群落结构的动态变化[J].中国生态农业学报,2012,20(10):1340-1346.
[25]徐严.若尔盖高原湿地土壤真菌的初步研究[D].成都:四川农业大学,2008.
[26]陈会海,许修宏,李洪涛.应用PCR-DGGE分析三江湿地土壤真菌群落结构的多样性[J].环境科学研究,2012,25(11):1272-1278.