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。     

指纹图谱技术在动物肠道微生物多样性研究中的应用

随着分子生物技术的发展,不可培养微生物多样性研究的难题得到了解决。肠道微生物处于特殊的生态环境条件下,分子生物学技术的应用使得肠道微生物多样性的研究进入了一个崭新的阶段。本文主要介绍了基于16S rRNA基因片段的一些肠道微生物研究工作中常用的分子生物学分析方法,主要包括变性梯度凝胶电泳(DGGE),温度梯度凝胶电泳(TGGE),单链构象多态性(SSCP),限制性片段长度多态性(RFLP),放大片断长度多态性(AFLP)和随机扩增多态性DNA(RAPD)等指纹图谱技术。     

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。     

变性梯度凝胶电泳(DGGE)技术在土壤微生物多样性研究中的应用

应用变性梯度凝胶电泳(DGGE)谱图分析技术对土壤微生物多样性进行描述,可以克服传统微生物分离纯化培养方法和显微技术的局限性.本文介绍了变性梯度凝胶电泳指纹图谱分析方法及其在土壤微生物多样性中的应用,包括对土壤特殊微生物生理类群,自然环境条件下微生物多样性变化,污染土壤微生物的多样性,不同种植制度下的土壤微生物多样性,转基因植物、微生物介入的土壤微生物多样性等方面的研究.     

不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。     

土壤微生物群落多样性研究方法及进展

微生物多样性是指群落中的微生物种群类型和数量、种的丰度和均度以及种的分布情况。研究土壤微生物群落多样性的方法包括传统的以生化技术为基础的方法(直接平板计数、单碳源利用模式等)和以现代分子生物技术为基础的方法(从土壤中提取DNA,进行G C%含量的分析,或杂交分析,或进行PCR,产物再进行DGGE/TGGE等分析)。现代生物技术与传统微生物研究方法的结合使用,为更全面地理解土壤微生物群落的多样性和生态功能提供了良好的前景。     

等碳投入的有机肥和生物炭对红壤微生物多样性和土壤呼吸的影响

施用有机肥是快速培育瘠薄土壤的一个重要措施。针对中亚热带第四纪红黏土发育的红壤旱地,建立了玉米和花生单作系统等碳量投入有机肥和生物炭的田间试验,利用聚合酶链式反应—变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)方法研究了土壤细菌和真菌群落组成和多样性的变化,分析了土壤呼吸速率(CO2通量)的变化及其与微生物多样性的关系。两年的试验表明,不同施肥方式导致微生物群落结构显著分异,施用有机肥和生物炭显著增加了细菌多样性,但施肥第二年真菌多样性有下降趋势。秸秆和猪粪配施显著增加了土壤呼吸速率,土壤呼吸速率与细菌和真菌多样性呈显著正相关,细菌多样性对土壤呼吸的影响(相对贡献率为71%)显著高于真菌(29%)。土壤磷素(全磷和速效磷)含量的变化是驱动红壤微生物多样性变化的主导因素,其对细菌和真菌多样性的相对贡献率分别为44.8%和47.4%。因此,合理配施秸秆和猪粪可以快速提高瘠薄红壤的生物功能。     

我国中亚热带毛竹林土壤微生物群落的空间分布特征及其影响因素

针对我国中亚热带毛竹林主要分布区,在福建、浙江、湖南、江西沿经度和纬度设置2个采样带,从5个县(市)采集了15个表层(0~20 cm)土样和15个土壤剖面(0~60 cm),利用磷脂脂肪酸(phospholipid fatty acids,PLFAs)和聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)方法研究毛竹林土壤微生物群落空间分布特征与剖面分布特征。结果表明:毛竹林表层土壤微生物生物量和细菌α多样性指数的地带性变化趋势不显著,但不同地点的土壤微生物群落结构存在显著差异;气候因子和土壤理化性质共同影响了土壤微生物的群落结构,但气候因子的影响随土壤剖面深度增加而减弱。毛竹林土壤细菌β多样性与距离之间存在显著的衰减关系,表层(0~20 cm)土壤细菌群落结构相似度(β多样性)随空间距离的衰减速率低于亚表层(20~40 cm)土壤,这可能与毛竹林根系的影响有关。总体上,环境选择和扩散限制共同影响了毛竹林土壤微生物的空间分布状况。     

广谱抗真菌蛋白转基因水稻秸秆模拟还田对土壤真菌群落结构的影响

为探讨广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响,本文在室温条件下进行田间秸秆还田模拟试验,设不添加秸秆(S)、添加转基因水稻‘转品1’秸秆(S-Z1)、添加转基因水稻‘转品8’秸秆(S-Z8)、添加非转基因水稻‘七丝软粘’秸秆(S-CK)4个土壤处理,采用传统的平板计数法和变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术,分析广谱抗真菌蛋白转基因水稻秸秆模拟还田过程中土壤可培养真菌数和土壤真菌群落的变化情况。平板计数结果表明,在秸秆降解的第40 d,转基因水稻秸秆处理(S-Z1、S-Z8)与非转基因水稻秸秆处理(S-CK)土壤之间的可培养真菌数差异显著,但秸秆降解中后期(50~90 d),S-Z1、S-Z8和S-CK之间土壤可培养真菌数的差异均不显著。真菌18S r RNA的PCR-DGGE图谱显示,S-Z1、S-Z8和S-CK在秸秆降解过程中没有显著不同的条带出现,仅有个别条带在亮度上存在差异。DGGE图谱条带多样性分析结果表明,在秸秆降解的个别时间段,S-Z1、S-Z8和S-CK之间在丰富度和Shannon-Wiener多样性指数上存在显著差异,而在秸秆降解的整个过程均匀度指数差异均不显著。对DGGE主要条带和差异性条带进行克隆测序后发现,子囊菌占最大比重,其次为担子菌、壶菌,而在转基因和非转基因土壤处理间亮度上存在差异的条带属于子囊菌。以上研究结果表明,广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响是短暂的、不持续的。     

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。     

氯氰菊酯与铜复合污染对土壤微生物群落的影响

采用常规手段研究了土壤在受氯氰菊酯、铜及二者复合污染后土壤呼吸率及微生物量碳的变化,采用了分离效果较好的双变性梯度凝胶电泳（DG—DGGE）技术研究微生物群落的变化。结果表明,低浓度的铜与高浓度的氯氰菊酯复合污染更能促进微生物量碳及土壤呼吸率的增加,微生物的群落结构也会受到明显影响。而两种污染物分别单独作用时,铜对微生物的胁迫更大,有铜组和无铜组在DGGE条带上差异较大,Shannon指数上也有明显不同。当铜的浓度较高时,加入高浓度的氯氰菊酯在较长的时间后（60d）对土壤呼吸作用、微生物量碳有一定影响,可能高浓度氯氰菊酯的加入在一定程度上减弱了高浓度铜对微生物的胁迫,而微生物群落并无显著的变化。     

Bacterial communities are more dependent on soil type than fertilizer type, but the reverse is true for fungal communities

The soil microbial community is strongly influenced by a wide variety of factors, such as soil characteristics and field management systems. In order to use biological indicators based on microbial community structure, it is very important to know whether or not these factors can be controlled. The present study aimed to determine whether soil type or fertilization has a greater influence on the soil microbial community based on denaturing gradient gel electrophoresis (DGGE) analysis of 12 experimental field plots containing four different soil types, Cumulic Andosol, Low-humic Andosol, Yellow Soil and Gray Lowland Soil, kept under three different fertilizer management systems since 2001 (the application of chemical fertilizer, the application of rice husk and cow manure, and the application of pig manure). Bacterial DGGE analysis using 16S rRNA genes and fungal DGGE analysis using 18S rRNA genes revealed that the bacterial community was related to the soil type more than the fertilization; however, the fungal community was related to the fertilization more than the soil type. These results might suggest that the fungal community is easier to control by fertilization than the bacterial community. Thus, we propose that indicators based on the fungal community might be more suitable as microbial indicators for soil quality.     

Distribution of microbial communities in a forest soil profile investigated by microbial biomass, soil respiration and DGGE of total and extracellular DNA

We studied the distribution of the indigenous bacterial and fungal communities in a forest soil profile. The composition of bacterial and fungal communities was assessed by denaturing gradient gel electrophoresis (DGGE) of total and extracellular DNA extracted from all the soil horizons. Microbial biomass C and basal respiration were also measured to assess changes in both microbial biomass and activity throughout the soil profile. The 16S rDNA-DGGE revealed composite banding patterns reflecting the high bacterial diversity as expected for a forest soil, whereas 18S rDNA-DGGE analysis showed a certain stability and a lower diversity in the fungal communities. The banding patterns of the different horizons reflected changes in the microbial community structure with increasing depth. In particular, the DGGE analysis evidenced complex banding patterns for the upper A1 and A2 horizons, and a less diverse microflora in the deeper horizons. The low diversity and the presence of specific microbial communities in the B horizons, and in particular in the deeper ones, can be attributed to the selective environment represented by this portion of the soil profile. The eubacterial profiles obtained from the extracellular DNA revealed the presence of some bands not present in the total DNA patterns. This could be interpreted as the remainders of bacteria not any more present in the soil because of changes of edaphic conditions and consequent shifting in the microbial composition. These characteristic bands, present in all the horizons with the exception of the A1, should support the concept that the extracellular DNA is able to persist within the soil. Furthermore, the comparison between the total and extracellular 16S rDNA-DGGE profiles suggested a downwards movement of the extracellular DNA.     

Effect of organic mulches on soil bacterial communities one year after application

The application of organic mulches as a soil cover is effective in improving the quality of soil. However, very little information is available on the effect of mulches on the soil microbial community. In this study, we investigated the effect of various organic mulches on soil dehydrogenase activity (DHA) and microbial community structures in the top 1 cm and 5 cm below the soil surface 1 year after application of the mulches. DHA was stimulated at both depths in plots mulched with grass clippings (GC), but was not significantly different from the control for the other mulch treatments. Fatty acid methyl ester (FAME) analysis and denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rDNA fragments were used to assess changes in the soil microbial community structure. Cluster analysis and principle component analysis of FAME profiles showed that only soil mulched with pine chips distinctively clustered from the other treatments. At the soil surface, bacterial DGGE profiles revealed that distinct shifts in several bacterial populations occurred in soils mulched with GC and eucalyptus yardwaste (EY), while DGGE profiles from soil at the 5 cm depth revealed no distinct changes. Changes in bacterial diversity at the soil surface under different mulches were calculated based on the number of bands in the DGGE profile using the Shannon-Weaver index of diversity ( H). Compared to the control ( H =0.9), the GC- and EY-treated soils showed slightly increased bacterial diversity, with an H of 1.1 and 1.0, respectively. These results indicate that the long-term effect of organic mulches on the soil microbial activity and community structure is highly dependent upon the type of mulch and is mostly exerted in the top few centimeters of the soil profile.     

Diversity–function relationship of ammonia-oxidizing bacteria in soils among functional groups of grassland species under climate warming

Although warming and plant diversity losses have important effects on aboveground ecosystem functioning, their belowground effects remain largely unknown. We studied the impact of a 3 °C warming and of three plant functional groups (forbs, grasses, legumes) on ammonia-oxidizing bacteria (AOB) diversity (polymerase chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE) and their function (potential nitrification) in artificial grasslands. Warming did not influence AOB diversity and function. Sequencing of 16S rRNA gene fragments retrieved from DGGE gel revealed that they were all related to Nitrosospira-like sequences. Clustering analysis of DGGE profiles resulted in two nodes, separating AOB community structure under legumes from all other samples. Decreased AOB richness (number of DGGE bands) and concurrent increased potential nitrification were also observed under legumes. We hypothesized that ammonium availability was the driving force regulating the link between aboveground and belowground communities, as well as the AOB diversity and function link. The results document that the physiology of AOB might be an important regulator of AOB community structure and function under plant functional groups. This study highlights the major role of the microbial community composition in soil process responses to changes in the functional composition of plant communities.     

Molecular approaches to investigate herbicide-induced bacterial community changes in soil microcosms

Since biochemical and microbiological methods used to study microbial community changes induced by anthropogenic activities can be biased, the impact of two herbicides on soil microorganisms was investigated by culture-independent molecular techniques. The effect of three different amounts (the recommended field dose, tenfold, and 100-fold the dose) of propanil or prometryne on the bacterial community of a clay soil, two modalities of incubation (soil moisture at 70% of the field capacity and a soil-herbicide suspension, 1:10, w:v), and time of incubation were investigated by denaturing gradient gel electrophoresis (DGGE) and amplified rDNA restriction analysis (ARDRA). Two sets of primers for 16S rDNA were used to amplify total soil DNA. Sterile and non-sterile samples were used to determine, by HPLC, the amounts of herbicides adsorbed on soil and transformed by soil microorganisms. Prometryne persisted in soil longer than propanil. Propanil was removed significantly more by non-sterile than by sterile samples, while for prometryne, slight differences were observed. 3,4-Dichloroaniline, a product of propanil hydrolysis, was detected in non-sterile samples and increased with incubation time. Propanil did not affect soil bacteria significantly as indicated by DGGE and ARDRA, with the only exception being the soil-herbicide suspension. Despite a lower utilization of prometryne by soil microorganisms, DGGE analysis showed a more diverse banding than with propanil. Some bands were also detected in the DNA sample extracted from the soil-prometryne suspension, and could be representative of bacterial species utilizing the herbicide as a carbon source, in two very different soil microcosms.     

植物促生菌接种对中国芥蓝根际固有微生物群落结构的影响

Plant growth-promoting rhizobacteria (PGPR) have been widely recognized as an important agent,especially as a biofertilizer,in agricultural systems.The objectives of this study were to select efective PGPR for Chinese kale (Brassica oleracea var.alboglabra) cultivation and to investigate the efect of their inoculation on indigenous microbial community structure.The Bacillus sp.SUT1 and Pseudomonas sp.SUT19 were selected for determining the efficiency in promoting Chinese kale growth in both pot and field experiments.In the field experiment,PGPR amended with compost gave the highest yields among all treatments.The Chinese kale growth promotion may be directly afected by PGPR inoculation.The changes of microbial community structure in the rhizosphere of Chinese kale following PGPR inoculation were examined by denaturing gradient gel electrophoresis (DGGE) and principal coordinate analysis.The DGGE fingerprints of 16S rDNA amplified from total community DNA in the rhizosphere confirmed that our isolates were established in the rhizosphere throughout this study.The microbial community structures were slightly diferent among all the treatments,and the major changes depended on stages of plant growth.DNA sequencing of excised DGGE bands showed that the dominant species in microbial community structure in the rhizosphere were not mainly interfered by PGPR,but strongly influenced by plant development.The microbial diversity as revealed by diversity indices was not diferent between the PGPR-inoculated and uninoculated treatments.In addition,the rhizosphere soil had more influence on eubacterial diversity,whereas it did not afect archaebacterial and fungal diversities.