核酸分析方法在土壤微生物多样性研究中的应用

土壤微生物多样性一般包括微生物分类群的多样性、遗传(基因)多样性、生态特征多样性和功能多样性。传统的分离培养方法和土壤微生物的生化研究手段具有一定的局限性,核酸分析方法为土壤微生物多样性研究注入了新活力。本文主要综述了近年来国内外研究土壤微生物多样性所采用的核酸提取方法及核酸分析方法。重点阐述了基于PCR的分子指纹技术、核酸杂交技术、基因芯片等核酸分析方法的原理、优缺点和应用。各种土壤微生物多样性研究方法的综合应用可扬长避短,起到相互补充的作用,从而能够提供更加丰富而准确的土壤微生物群落结构及种群丰度变化等方面的信息,也将成为这一领域今后的发展趋势。     

长期施肥对黑土农田土壤微生物群落的影响

基于中国科学院海伦农业生态试验站长期定位试验区,应用实时荧光定量PCR(Real-time PCR)和变性梯度凝胶电泳(DGGE)技术研究了无施肥(NF)、单施N、P化肥(NP)以及化肥配施有机猪粪肥(NPM)等3种长期施肥措施对黑土区玉米田土壤微生物群落密度和结构的影响.Real-time PCR方法定量NF、NP及NPM措施土壤细菌群落基因组DNA质量分别为381、1 351和1 773 ng g-1干土,真菌群落基因组DNA质量分别113.3、127.3和20.6 ng g-1干土,真菌与细菌的比率分别为0.31、0.09和0.01,NPM措施显著低于另两种施肥方式(p＜0.05).DGGE方法研究表明,NP和NPM措施不能改善土壤细菌和真菌群落的多样性、均匀性及优势菌优势程度;但主成分分析结果显示NP和NPM措施均可改变土壤细菌和真菌群落的构成,且真菌群落的变化更为显著;聚类分析结果显示NP和NPM措施下细菌群落结构较相近,其相似系数为0.89,真菌群落中NP措施与NF措施相近,相似系数为0.63,高于NP与NPM措施的相似系数0.51.上述结果表明有机猪粪肥的长期施用可以显著降低黑土农田土壤真菌与细菌的比率,且明显地改变土壤细菌和真菌群落的结构.     

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     

土壤微生物多样性研究的新方法

传统的分离培养和鉴定土壤微生物方法所具有的困难性和局限性 ,是造成难以深入了解土壤微生物生态学特性和多样性组成方面的主要障碍。本文运用分子生物学技术 ,以澳大利亚两种主要森林类型的土壤微生物多样性研究为实例 ,介绍了从土壤中直接提取土壤微生物DNA的方法以及末端限制性酶切片段长度多态性 (T RFLP)分析的基本原理和方法。作者认为 ,用该方法提取的土壤真菌DNA的纯度高 ,完全适合PCR扩增和T RFLP分析的要求。T RFLP已成为国外深入研究土壤微生物多样性的理想方法之一     

Interactions between nematodes and microbial communities in a tropical soil following manipulation of the soil food web

The carrying capacity for microflora and nematofauna was manipulated (using a bactericide, a fungicide, manure or a growing millet plant) in a poor tropical soil, in order to identify relationships between the soil microbes and nematodes and to assess the influences of these organisms on nitrogen flux. The experiment was conducted for 4 months in containers under greenhouse conditions, with analyses of soil, nematofauna and microbial characteristics at regular intervals. Manure input and initial bactericide application led to a significant increase in bacterial-feeding and fungal-feeding nematodes of coloniser-persister classes 1 and 2, respectively, whereas high manure input stimulated omnivorous nematodes (i.e. Microdorylaimus rapsus) which became the dominant trophic group. Changes in abundance of the different bacterial-feeding nematode taxa between treatments seemed to be more related to changes in the structure of the microbial communities than to the total amount of micro-organisms, as suggested by the RISA fingerprint analysis of the bacterial communities. Canonical analysis of nematode feeding guilds, combined with soil microbial and mineral nitrogen parameters as well as multiple regression showed that the bacterial-feeding nematodes influenced the inorganic N content in the soil whereas microbial biomass was determined by total nematode abundance and not by any specific trophic group.     

Olive mill wastewater effects on the microbial communities as studied in the field of olive trees by analysis of fatty acid signatures

The aim of this work was to study the effects of spreading olive mill wastewater (OMW) on the soil surface of an olive grove on the soil microbial communities. Analyses of ester-linked fatty acid methyl esters (EL-FAME) were used to assess variations in the soil microbial community structure following land spreading of OMW. Our data provide evidence that agronomic application of OMW has important effects on soil microbial community. Bacteria were relatively more reduced by these treatments than fungi and actinomycetes as revealed by an increased index of fungal/bacterial FAME and actinomycetes/bacterial FAME. Specific FAME markers indicated a significant reduction in the Gram-positive bacteria. However, the relative proportion of the Gram-negative bacteria was not significantly different after agronomic application of OMW. The ratios of cyclopropyl/monoenoic precursors decreased and the total monounsaturated/total saturated fatty acids increased in the OMW amended soils, suggesting that the microbes inhabiting the control soil are more carbon limited than the OMW amended soils. The changes in the FAME pattern of the soil organisms possibly were related (i) to an altered substrate quantity, that is the availability of substrates after the treatments, (ii) the complex nature of OMW which also contains high molecular-mass recalcitrant polyphenols.     

Artificial neural network modeling of microbial community structures in the Atlantic Forest of Brazil

Microbial communities vary across the landscape in forest soils, but prediction of their biomass and composition is a difficult challenge due to the large numbers of variables that influence their community structures. Here we examine the use of artificial neural network (ANN) models for extraction of patterns among soil chemical variables and microbial community structures in forest soils from three regions of the Atlantic Forest of Brazil. At each location, variations in soil chemical properties and FAME profiles of microbial community structures were mapped at 20 × 20 m intervals within 10 ha parcels. Geostatistical analyses showed that spatial variability in soil physical and chemical variables could be mapped at scale distances of 20 m, but that FAME profiles representing the microbial communities were highly variable and had no spatial dependence at the same scale in most cases. RDA analysis showed that FAME signatures representing different microbial groups were positively associated with soil pH, OM, P and base cations concentrations, whereas microbial biomass was negatively associated with the same environmental factors. In contrast, ANN models revealed clear relationships between microbial community structures at each parcel location, and generated verifiable predictions of variations in FAME profiles in relation to soil pH, texture, and the relative abundances of base cations. The results suggest that ANN modeling provides a useful approach for describing the relationships between microbial community structures and soil properties in tropical forest soils that were not able to be captured using geostatistical and RDA analyses.     

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。     

Can denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16s rDNA of soil bacterial populations be used in forensic investigations?

In criminal investigations, information on the origin of soils may be crucial for solving cases. The biological complexity of soil may potentially be used for sorting and differentiating between soil samples. Nucleic-acid based analyses of soil microbial populations are powerful tools, routinely used in studies of this habitat. Application of such approaches in forensics implies that a standardized DNA extraction method has to be applied to all samples. In this study, several DNA extraction protocols were compared. An improvement on the method proposed by Tsai and Olson (1991) was found to be most suited to extract DNA from various soil types, including from small samples. A blind test on soils from a crime, an alibi scene and unrelated locations was conducted to evaluate the potential of environmental PCR and denaturating gradient gel electrophoresis for use in forensic science. In most cases, soil patterns clustered according to soil type and location.     

温度水分对秸秆降解微生物群落功能多样性影响

秸秆腐解微生物群落结构受环境因子(温度和水分)影响显著。本试验利用BIOLOG技术,以秸秆腐解微生物碳源利用的平均颜色变化率(averagewell colordevelopment,AWCD)为指标,研究了不同温度(15℃,25℃和35℃和水分(40%,70%和90%田间持水量)条件下小麦和玉米秸秆腐解过程中微生物碳源代谢多样性的差异试验结果表明不同处理的AWCD随培育温度升高而降低,即15℃25℃35℃。随着培育时间的增加,其降低的趋势更加明显。同样地,小麦玉米秸秆腐解微生物的物种丰富度指数Shannon-Wiener(H)和优势度指数Simpson(D)也呈现出随温度升高而降低的趋势。微生物主要利用糖类和脂类物质。主成分分析结果表明在不同腐解时间,腐解微生物代谢多样性在不同温度下差异显著,而在不同水分下差异不显著。     

A field method to store samples from temperate mountain grassland soils for analysis of phospholipid fatty acids

The storage of soil samples for PLFA analysis can lead to shifts in the microbial community composition. We show here that conserving samples in RNAlater, which is already widely used to store samples for DNA and RNA analysis, proved to be as sufficient as freezing at?-20?°C and preferable over storage at 4?°C for temperate mountain grassland soil. The total amount of extracted PLFAs was not changed by any storage treatment. Storage at 4?°C led to an alteration of seven out of thirty individual biomarkers, while freezing and storage in RNAlater caused changes in the amount of fungal biomarkers but had no effect on any other microbial group. We therefore suggest that RNAlater could be used to preserve soil samples for PLFA analysis when immediate extraction or freezing of samples is not possible, for example during sampling campaigns in remote areas or during transport and shipping.     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

RAPD markers in the analysis of genetic diversity among common bean germplasm from Central Himalaya

A set of 99 common bean germplasm collected from central Himalaya was investigated for their genetic variability using random amplified polymorphic DNA (RAPD) markers. Ten oligonucleotide primers, selected from 60 initially screened, generated 123 amplicon products. Of these, two amplicons were shared by all the accessions whereas 112 were polymorphic at least in two pair wise comparison. Nine unique bands identified were as low as 0.32 kb M.W. to as high as 3.5 kb and were confined to eight collections. All primers produced polymorphic amplicons though the extent of polymorphism varied with each primer. The primer OPF-17 was found to be most powerful and efficient as it generated a total of 17 bands of which 15 were polymorphic. RAPD markers data were analysed statistically using NTSYSpc.2.02e software and a dendrogram was generated using Jaccard’s similarity coefficient. The similarity coefficient values varied from 0.19 to 0.91. Grouping analysis revealed the categorization of 99 germplasm into 12 major branches with different level of similarity. Three branches namely branch 2, 3 and 5 out of 12 had only one accession. Branch 1 which consisted of three accessions was the most divergent as revealed by Jaccard’s similarity coefficient. Branching pattern of the accessions did not show any correlation with morphological data or altitudinal alignment of the accessions.     

茶园土壤微生物总DNA不同提取方法的比较

传统的微生物分离培养方法,在反映茶园土壤微生物基因信息上有很大的局限性,因此,目前逐步被分子生态学的方法替代,而获得高质量、大片段、无偏好的土壤微生物总DNA则是茶园土壤微生物分子生态学研究的基础。本文采用SDS高盐法、变性剂加SDS高盐法、脱腐SDS高盐法、CTAB法和Krsek改进法5种土壤微生物DNA提取方法分别从茶园土壤微生物中提取总DNA,并对5种方法提取的DNA的片段大小、质量和产量进行了综合评价。结果表明,Krsek改进法提取到的DNA片段最大（〉23kb）、纯度最高（OD2UOD280〉1．70;OD2UOD230〉1．35）、产量较高（〉34．50μg／gdrysoil）且不需纯化就可以用于PCR扩增和RFLP分析。因此,Krsek改进法是一种高效、可靠且适合于茶园土壤微生物分子生态学研究的DNA提取方法。     

不同基肥对黄瓜根际土壤微生物群落多样性的影响

分别以RAPD分子生物学方法和BIOLOG生理学方法,研究了不同基肥对黄瓜根际土壤微生物群落DNA序列多样性和群落功能多样性的影响。结果表明,在本试验条件下,基肥为75000 kg/hm2有机肥处理和75000 kg/hm2有机肥加300 kg/hm2复合肥处理最好;基肥为600 kg/hm2复合肥处理而使土壤微生物群落DNA序列丰富度指数和多样性指数显著下降,与对照的DNA序列相似系数最低;有机肥处理有利于土壤微生物群落DNA序列多样性、均匀度和黄瓜产量的提高。此外,不同基肥处理改变了土壤微生物对单一碳源的利用能力。     

退化喀斯特植被恢复对土壤微生物数量及群落功能多样性的影响

采集不同恢复阶段的土壤样品,采用微生物培养法研究了退化喀斯特植被恢复对土壤微生物数量、群落功能多样性的影响。研究结果表明随着退化喀斯特植被的恢复,土壤微生物数量增加,表现为乔木群落阶段灌木群落阶段草本群落阶段裸地阶段。土壤微生物群落代谢功能分析表明:植被恢复往往导致较高的平均颜色变化率、物种丰富度和功能多样性。乔木群落阶段的平均诱导底物利用率最高,明显地与其他3个恢复阶段不同。总之,植被恢复使得土壤微生物数量增加,碳源平均利用率增强。因此,创造了更好的土壤条件更有利于退化喀斯特植被的恢复。     

Effect of drying treatment on the respiratory quinone profile in soil

Recently, the analysis of the microbial community structure in soil has received a great deal of attention. Various analytical methods based on biomarkers have been developed: 16S-rDNA, total DNA, phospholipid fatty acids, ergosterols, muramic acids, etc. (Tunlid and White 1992; Carter and Lynch 1993). In a previous paper, we reported that respiratory quinones are useful biomarkers to characterize the microbial community structure in soil (Fujie et al. 1998). In these analyses, only fresh moist soils or frozen soils had been used as samples. However, the soil samples are generally stored in the dark after air drying in many research institutes and experimental stations. It was considered that the analysis of microbial communities in dried soil samples was not possible.

In this paper, we observed that the drying of soils did not affect the proportions of quinone species in soil although the treatment decreased the amount of extracted quinones. These findings suggest that the analysis of the respiratory quinone profile of dried soils reflects the micro biota present in fresh moist soils before drying.     

墙体材料及其组合对日光温室墙体保温性能的影响

堆肥是由群落结构演替非常迅速的多个微生物群体共同作用而实现固体废物资源化、无害化的动态过程。本文在综合国内外文献资料的基础上,结合本实验室的研究工作,从堆肥过程中微生物群落的演替、有机物降解菌的选育应用、变性梯度凝胶电泳(DGGE)在堆肥微生物研究中的应用等方面介绍了现代堆肥过程中微生物研究的进展及存在问题,并且指出堆肥过程中微生物菌系组成变化复杂和实验手段有限是限制本研究的主要因素,今后应重视利用分子生物学方法进行微生物的研究工作,并根据微生物之间的协同关系有目的地构建降解多种有机废弃物的高效稳定复合菌系,以适应复杂的堆肥环境。     

Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods

Several biochemical and molecular methods are used to investigate the microbial diversity and changes in microbial community structure in rhizospheres and bulk soils resulting from changes in management. We have compared the effects of plants on the microbial community, using several methods, in three different types of soils. Pots containing soil from three contrasting sites were planted with Lolium perenne (rye grass). Physiological (Biolog), biochemical (PLFA) and molecular (DGGE and TRFLP) fingerprinting methods were employed to study the change in soil microbial communities caused by the growth of rye grass. Different methods of DNA extraction and nested PCR on TRFLP profiles were examined to investigate whether they gave different views of community structure. Molecular methods were used for both fungal and bacterial diversity. Principal component analysis of Biolog data suggested a significant effect of the plants on the microbial community structure. We found significant effects of both soil type and plants on microbial communities in PLFA data. Data from TRFLP of soil bacterial communities showed large effects of soil type and smaller but significant effects of plants. Effects of plant growth on soil fungal communities were measured by TRFLP and DGGE. Multiple Procrustes analysis suggested that both methods gave similar results, with only soil types having a significant effect on fungal communities. However, TRFLP was more discriminatory as it generated more ribotype fragments for each sample than the number of bands detected by DGGE. Neither methods of DNA extraction nor the nested PCR had any effect on the evaluation of soil microbial community structure. In conclusion, the different methods of microbial fingerprinting gave qualitatively similar results when samples were processed consistently and compatible statistical methods used. However, the molecular methods were more discriminatory than the physiological and biochemical approaches. We believe results obtained from this experiment will have a major impact on soil microbial ecology in general and rhizosphere–microbial interaction studies in particular, as we showed that the different fingerprinting methods for microbial communities gave qualitatively similar results.     

Rapid PCR-based method for the direct analysis of fungal communities in complex environmental samples

Fungal community analysis using 18S rDNA primer pairs and denaturing gradient gel electrophoresis of PCR products (Vainio, E.J., Hantula, J., 2000. Mycological Research 104, 927–936) was applied to field studies of the forest ecosystem. We report a DNA extraction method producing high quality DNA allowing successful PCR amplification from problematic samples without use of nested polymerase chain reaction (PCR) procedures. The analysis was found to be applicable for samples from environments of varying fungal diversities and high organic matter content: wood samples from fallen branches of trees, laboratory mini-ecosystems and forest humus samples. When the method was tested using replicate forest soil samples, it was shown to be highly reproducible.