适于变性梯度凝胶电泳（DGGE）分析的草莓根际土壤微生物的DNA模板制备

根际土壤中微生物DNA的有效提取是土壤微生物变性梯度凝胶电泳(DGGE)分析的基础.研究对比了十二烷基硫酸钠(SDS)高盐抽提法、十六烷基三甲基溴化铵(CTAB)法和预洗处理的聚乙烯吡咯烷酮(PVP)法3种DNA提取方法对4个栽培地块草莓(Fragaria ananassa Duch.)根际土壤微生物总DNA提取的效果.结果表明,SDS高盐抽提法和预洗处理的PVP法从不同土样中提取的微生物总DNA片段长度大于23 kb,DNA均量分别达到32.94和43.06 靏/g;其中预洗处理的PVP法提取DNA的A260/A280和A260/A230比值平均为1.1623和0.8135,比SDS高盐抽提法(1.1238和0.5901)分别高出0.0385和0.2234,对土壤样品中蛋白质和腐植酸的去除效果较好;CTAB法提取的总DNA非常少.纯化后的DNA,分别采用细菌F341/R534和真菌FR1/FF390引物进行PCR扩增,成功获得细菌16S rDNAV3区和真菌18S rDNA目的片段,对其PCR扩增产物进行DGGE分析,4个土壤样品均得到清晰的DGGE图谱.草莓根际土壤微生物DGGE分析中DNA模板制备采用预洗处理的PVP法和SDS高盐抽提法均可成功获得,预洗处理的PVP法效果最好,而CTAB法制备效果较差.     

土壤微生物总DNA提取方法的比较

采用4种土壤DNA提取方法提取了5种类型土壤的微生物总DNA，并对4种提取方法的DNA提取效果进行综合分析，进一步通过PCR—DGGE扩增提取DNA中的细菌16S rDNA片段，并对扩增产物进行DGGE电泳分析。结果表明，SDS-高盐缓冲液法抽提的DNA得率最高，SDS-酚氯仿抽提法的DNA得率最低。DNA的纯度，以BIO-101 DNA Extraction Kit试剂盒法最高，改进试剂盒法纯度最低。通过PCR-DGGE分析土壤微生物多样性结果表明，BIO-101 DNA Extraction Kit试剂盒法提取的DNA代表的微生物多样性最全，而SDS酚氯仿法抽提的DNA代表性最差。     

土壤微生物多样性研究的DGGE/TGGE技术

分子生物学技术比传统的培养方法可得到土壤微生物种群多样性更全面的信息。变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)和温度梯度凝胶电泳(Temperature gradient gel electrophoresis, TGGE)可分离PCR扩增的DNA片段,已成为研究土壤微生物群落多样性的重要手段。本文综述了DGGE/TGGE技术在土壤微生物多样性研究中的应用进展,分析了该方法的主要影响因素及其优点和存在的问题。     

土壤微生物多样性研究的DGGE/TGGE技术进展

分子生物学技术比传统的培养方法可得到土壤微生物种群多样性更全面的信息。变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)和温度梯度凝胶电泳(Temperature gradient gel electrophoresis,TGGE)可分离PCR扩增的DNA片段,已成为研究土壤微生物群落多样性的重要手段。本文综述了DGGE/TGGE技术在土壤微生物多样性研究中的应用进展,分析了该方法的主要影响因素及其优点和存在的问题。     

三种土壤微生物总DNA提取方法的比较

本文对3种常用的土壤微生物总DNA提取方法Martin法、高盐改进法及试剂盒法进行了比较,并通过DNA得率、纯度及16S rDNA V3可变区的PCR扩增结合DGGE法（denatumg gradient gel electrophoresis）,分别对3种方法进行评价。结果表明,3种方法提取的DNA均能满足土壤微生物多样性分析的要求。其中试剂盒方法操作简单,提取的DNA质量较高,但DNA得率较低且成本昂贵。Martin法和高盐改进法用时较长,DNA得率较高,纯度较低,但对后续PCR扩增和DGGE分析没有明显影响,且成本低廉。     

土壤微生物群落结构研究方法进展

分离培养技术在土壤微生物的研究中有很大的局限性，因为土壤中的微生物大部分还处于不能被分离培养状态。随着微生物研究技术的发展尤其是分子生物学技术的发展，一系列不依赖于培养的技术在土壤微生物研究中得到广泛应用。本文介绍了生物标志物法、SCSU、DNA复性分析、DGGE、TGGE、ARDRA、T-RFLP、SSCP、PAPD和ERIC—PCR图谱分析等方法在土壤微生物群落结构研究中的应用。     

紫色水稻土水稳性团聚体土壤微生物基因组DNA提取方法探讨

为了找到提取土壤微生物总DNA的最佳方法,通过OD值检验、凝胶电泳、PCR和DGGE分析,比较了Reddy法、基于DNAout kit试剂盒改进的实验方法、以及Kuske修订法、Edgcomb改进法、SDS高盐提取法、Eichner调整法等常用的不同土壤微生物基因组的DNA提取方法在亚热带地区长期免耕紫色水稻土水稳性团聚体0.25～2.0 mm粒径上的提取效果.结果表明,6种方法都可以从团聚体中提取到长度大于23.1 kb的DNA片段,但不同方法提取的DNA的产量存在明显差异,土壤总DNA均不需纯化就可以用于PCR扩增,使用细菌16S rDNA基因V3区的通用引物可扩增得到相应的片段.研究表明,改进的DNAout kit试剂盒法是长期免耕紫色水稻土水稳性团聚体中微生物基因组DNA的最佳提取方法.     

贵溪冶炼厂周边农田土壤重金属污染特性及评价

在对贵溪冶炼厂周边区域的农田土壤及生产的稻谷进行采样调查和数据处理分析的基础上,对农田土壤重金属污染特性和现状进行了监测和初步评价,结果表明:农田土壤重金属污染可能来源于贵溪冶炼厂废水的排放及尾矿渣的堆放,与灌溉水源无关;用国家土壤环境质量二级标准进行评价可知,农田土壤重金属的综合污染指数较高,土壤污染已处于重度污染等级,单项重金属污染指数表明Cu、Cd的含量已严重超标,并处于重度污染等级,Zn、Pb和As的含量未构成污染;水泉村、竹山村与所生产的稻谷三者之间呈显著或极显著正相关,即农田生态系统呈复合污染的趋势。     

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响,为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取,该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA,并对这两种方法的提取DNA的效果进行了比较。结果表明,试剂盒法不能用于提取土壤中的微生物DNA;可以从猪粪中提取到DNA,PCR扩增能得到目的产物,但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低,纯化后纯度增加,但DNA有所损失,用于PCR扩增时结果不理想;处理猪粪样品,提取的DNA浓度较低但纯度较高,PCR扩增结果比较理想。由此可见,化学裂解法用来提取猪粪样品中的微生物DNA是可行的,但需寻求更好的土壤样品微生物DNA的提取方法。     

土壤微生物生态学研究方法进展

土壤微生物研究方法经历了微生物纯培养，土壤酶活性（BIOLOG微平板分析），微生物库（如微生物生物量）和流（C和N循环），微生物生物标记物（FAMEs)，微生物分子生物学技术（从土壤中提取DNA，进行PCR－DGGE，PCR－SSCP，RLFP分析等），揭示了土壤微生物群落丰富的多样性和生态功能；现代生物技术和传统微生物研究方法的配合将为土壤微生物学研究提供了的前景。     

Microbial Communities in Long-Term Heavy Metal Contaminated Ombrotrophic Peats

High concentrations of heavy metals are known to be toxic to many soil organisms. The effects of long-term exposure to lower levels of metals on the soil microbial community are, however, less well understood. The southern Pennines of the U.K. are characterised by expanses of ombrotrophic peat soils that have experienced deposition of high levels of heavy metals since the mid to late 1800s. Concentrations of metals in the peat remain high but the effect of the contamination on the in-situ microbial communities is unknown. Geochemical and molecular polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing techniques were used to derive new information on the metal chemistry and microbial populations in peat soils from six locations in the southern Pennines. All sites were highly acidic (pH 3.00–3.14) with high concentrations of potentially toxic heavy metals, particularly porewater Zn and particulate-associated Pb. The results also reveal a split in site characteristics between the most polluted sites with the highest levels of bioavailable metals (Bleaklow, FeatherBed Moss and White Hill) and those with much lower bioavailable metals (Cowms Moor, Holme Moss and Round Hill). There was no difference in the number of dominant bacterial species between the sites but there were significant differences in the species composition. At the three sites with the highest levels of bioavailable metals, bacterial species with a high similarity to acidophilic sulphur- and iron-oxidizing bacteria and those from high metal environments were detected. The transformations carried out by these metal mobilising and acid producing bacteria may make heavy metals more bioavailable and therefore more toxic to higher organisms. Bacteria with similarity to those typically found in forest and grassland soils were documented at the three sites with the lowest levels of bioavailable metals. The data highlight the need for further studies to elucidate the species diversity and functionality of bacteria in heavy metal contaminated peats in order to assess implications for moorland restoration.     

Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

 We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods, i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples. The profiles generated by all four methods were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community. However, the patterns of clustering also indicated that different populations of bacteria could be detected in the same soil using different soil DNA extraction methods. The application of two dilution levels of DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods. The indirect methods gave clustered profiles at both dilution levels. It is likely that these methods extracted DNA from a major, easily desorbed, bacterial fraction, consisting of low-density populations. PCR-DGGE was found to be a suitable technique with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination of microbial community diversity at the molecular level. Received: 22 June 1999     

Influence of different temperatures on metal tolerance measurements and growth response in bacterial communities from unpolluted and polluted soils

The effects of temperature on the growth rate and metal toxicity in soil bacterial communities extracted from unpolluted and polluted soils were investigated using the thymidine and leucine incorporation techniques. An agricultural soil, which was contaminated in the laboratory with Cu, Cd, Zn, Ni or Pb, and an uncontaminated forest soil were used. Measurements were made at 0°C and 20°C. Leucine incorporation was found to be as sensitive to heavy metals as thymidine incorporation in the short-term trial used to indicate heavy metal tolerance. Similar IC₅₀ values (the log of the metal concentration that reduced incorporation to 50%) were also obtained at 0 and 20°C, independently of the technique used. Metal tolerance could thus be measured using both techniques at any temperature in the range 0–20°C. In the long-term experiment different temperature-growth relationships were obtained on the basis of the rate of thymidine or leucine incorporation into bacterial assemblages from unpolluted and polluted soils, as judged from the minimum temperature values. This could not be attributed to the metal addition alone since different patterns were observed when different metals were added to the soil. Thus, the minimum temperature for thymidine incorporation was similar in Cu-polluted and unpolluted soil, while in soils polluted with Cd and Zn the minimum temperature increased by 2°C, and Ni and Pb additions increased the minimum temperature by 4°C compared to the unpolluted soil. This suggested that heavy metal pollution led to bacterial communities showing different temperature characteristics to those in the corresponding unpolluted soil. Similar observations were deduced from the minimum temperatures required for leucine incorporation. Three groups of bacterial communities were distinguished according to the growth response to temperature in polluted soils, one group in Cu-polluted soil, a second group in soil polluted with Zn and Cd, and a third group in soils polluted with Ni and Pb.     

利用Illumina高通量测序对复合重金属污染下根际和非根际稻田土壤细菌的研究

There is an increasing concern about rice (Oryza sativa L.) soil microbiomes under the influence of mixed heavy metal contamination.We used the high-throughput Illumina MiSeq sequencing approach to explore the bacterial diversity and community composition of soils in four paddy fields,exhibiting four degrees of mixed heavy metal (Cd,Pb and Zn) pollution,and examined the effects of these metals on the bacterial communities.Our results showed that up to 2 104 to 4 359 bacterial operational taxonomic units (OTUs) were found in the bulk and rhizosphere soils of the paddy fields,with the dominant bacterial phyla (greater than 1％ of the overall community) including Proteobacteria,Actinobacteria,Firmicutes,Acidobacteria,Gemmatimonadetes,Chloroflexi,Bacteroidetes and Nitrospirae.A number of rare and candidate bacterial groups were also detected,and Saprospirales,HOC36,SC-I-84 and Anaerospora were rarely detected in rice paddy soils.Venn diagram analysis showed that 174 bacterial OTUs were shared among the bulk soils with four pollution degrees.Rice rhizosphere soils displayed higher bacterial diversity indices (ACE and Chao 1) and more unique OTUs than bulk soils.Total Cd and Zn in the soils were significantly negatively correlated with ACE and Chao 1,respectively,and the Mantel test suggested that total Pb,total Zn,pH,total nitrogen and total phosphorus significantly affected the community structure.Overall,these results provided baseline data for the bacterial communities in bulk and rhizosphere soils of paddy fields contaminated with mixed heavy metals.     

贵州万山汞矿区某农田土壤重金属污染特征及来源解析

研究采集万山汞矿区典型农田土壤样品,分析测试其Hg、As、Cd、Cr、Pb、Cu、Zn、Ni含量,利用综合污染指数法、地累积指数法和潜在生态危害指数法评估农田土壤的污染状况及生态风险,结合相关分析和主成分分析解析农田土壤中重金属的来源。结果表明,该农田土壤Hg、As、Cd、Cr、Pb、Cu、Zn、Ni的平均含量分别为4.29、117.6、0.43、59.06、48.99、43.77、29.13、18.80 mg kg~(-1)。土壤重金属综合污染指数为7.16,表明该农田土壤重金属重度污染,其中100%的位点Hg、As重度污染,66.7%的位点Pb轻度污染,25%的位点Cd轻度污染。土壤重金属的综合潜在生态危害指数为469.0,生态风险强,Hg对综合潜在生态危害指数的贡献率为78.30%,是该农田土壤生态风险的主要来源。该农田中重金属的来源包括:交通运输源、矿业污染源、农业污染源和自然活动源,主要污染物Hg来源于矿业活动,As来源于交通运输和矿业活动,Cd来源于农业活动,Pb来源于交通运输。     

Changes in Microbial Functional Diversity and Activity in Paddy Soils Irrigated with Industrial Wastewaters in Bandung, West Java Province, Indonesia

Characteristics, such as microbial biomass, basal respiration, and functional diversity of the microbial communities, were investigated in paddy soils located in Bandung, West Java Province, Indonesia, that have been heavily polluted by industrial effluents for 31 years. Paddy soil samples (10?C20 cm) were taken from two sites: polluted soils and unpolluted soils (as control sites). The polluted soils contained higher salinity, higher sodicity, higher nutrient contents, and elevated levels of heavy metals (Cr, Mn, Ni, Cu, and Zn) than unpolluted soils. Soil physicochemical properties, such as maximum water holding capacity, exchangeable sodium percentage, sodium adsorption ratio, and swelling factor, in polluted soils were much greater than those in unpolluted soils (P?<?0.05). Changes in the physical and chemical soil properties were reflected by changes in the microbial communities and their activities. BIOLOG analysis indicated that the functional diversity of the microbial community of polluted soils increased and differed from that of unpolluted soils. Likewise, the average rate of color development (average well color development), microbial biomass (measured as DNA concentration), and the soil CO₂ respiration were higher in polluted soils. These results indicate that major changes in the chemical and physical properties of paddy soils following the application of industrial wastewater effluents have had lasting impacts on the microbial communities of these soils. Thus, the increased activity, biomass, and functional diversity of the microbial communities in polluted soils with elevated salinity, sodicity, and heavy metal contents may be a key factor in enhancing the bioremediation process of these heavily polluted paddy soils.     

重金属复合污染下土壤微生物群落功能多样性动力学特征

用碳素利用法对浙江省天台铅锌银尾矿区重金属复合污染土壤微生物群落功能多样性动力学特征进行了初步探讨。研究结果表明 ,矿区重金属复合污染降低了供试土壤的微生物群落代谢剖面 ,且群落代谢剖面值与培养时间之间呈非线性关系 ,其变化过程符合微生物种群生长动态模型 (S形 )。随着重金属复合污染程度的加剧 ,土壤微生物群落功能多样性动力学参数K和r值越低 ,参数s值所需的时间则越长。上述动力学参数与群落代谢剖面各自的主成分分析结果显示 ,微生物群落功能多样性的动力学参数K值和s值能够很好地区分矿区土壤重金属污染程度 ,并且其区分效果比微生物群落代谢剖面值好。土壤微生物群落功能多样性动力学特征的变化可以较好地显示矿区重金属复合污染土壤微生物群落对碳源利用模式的差异 ,反映矿区特定生境土壤微生物群落功能多样性的变化 ,在一定程度上揭示重金属胁迫下环境微生物种群作用机理。     

铅锌银尾矿区土壤微生物活性及其群落功能多样性研究

通过对浙江省天台铅锌银尾矿区土壤微生物活性指标以及微生物群落功能多样性研究 ,结果表明 ,尾矿污染区土壤几种重金属含量比非矿区土壤有明显的增加。尾矿区土壤微生物特征发生了显著的变化 ,微生物生物量和可培养细菌数量显著降低 ,但土壤基础呼吸和微生物代谢商 (qCO2 )值却明显升高。Bi olog测试结果显示 ,随着重金属污染程度的加剧其土壤微生物群落结构发生了相应变化 ,尾矿区土壤微生物群落代谢剖面 (AWCD)及群落丰富度、多样性指数均显著低于非矿区土壤 ,且供试土壤间均达极显著水平差异 (p <0 .0 1) ,表明尾矿区重金属污染引起了土壤微生物群落功能多样性的下降 ,减少了能利用有关碳源底物的微生物数量、降低了微生物对单一碳源底物的利用能力     

Occurrence and infectivity of arbuscular mycorrhizal fungi in some norwegian soils influenced by heavy metals and soil properties

Mycorrhizae are ubiquitous symbiosis which can mediate uptake of some plant nutrients. In polluted soils they could be of great importance in heavy metal availability and toxicity to plants. Mycorrhizae have also been reported to protect plants against toxic metals. We investigated the occurrence and infectivity of arbuscular mycorrhizal (AM) spores as affected by heavy metal levels and other soil properties in Norwegian soils collected from heavy metal polluted, high natural background and non-polluted areas. Spore numbers, mycorrhizal infectivity and spore germination of indigenous mycorrhizal fungi and of a reference strain (Glomus mosseae) in soils showed lower values in two soils with high metal concentrations and in one soil with a low pH. Mycorrhizal infectivity was negatively correlated with extractable metals. Spore number and mycorrhizal infectivity in a soil with naturally high heavy metal content were not different to in non-polluted soils, and indigenous AM fungi appeared more tolerant to metals than those in non-polluted soils. Mycorrhizal infectivity, expressed as MSI₅₀ values, was significantly correlated (r′=0.89, P< 0.05) with the percentage of germinating G. mosseae spores in the soils. However, the number of spores per volume of soil was not significantly correlated with infectivity or spore germination of the reference strain. The spore germination method is discussed as a bioassay of heavy metal toxicity in soil.