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土壤微生物分离新技术的研究进展 总被引：1，自引：0，他引：1
土壤是环境微生物学研究中最具挑战性的环境,也是生物分子、遗传资源开发的热点区域。在"宏-组学"方法快速发展的今天,土壤微生物分离培养技术在微生物环境功能研究、代谢途径的阐明、特定功能的验证及基础实验和生产实践的应用等方面仍然发挥着重要作用。本文首先分析了大多数环境微生物不能被分离培养的几个原因。然后重点综述了近年来在土壤微生物分离培养技术方面的探索,主要包括生长培养基的优化和重新设计如培养基稀释分离、添加生长限制因子、更换凝固剂等,在延长培养时间、改善含氧量、降低培养温度等方面对生长条件进行改进,采用"扩散盒"、"原位培养陷阱"等技术对微生物进行天然环境原位培养,对环境微生物群体培养和共培养,利用"宏基因组学""宏蛋白质组学"等非培养技术辅助分离等。最后,作者对未来土壤微生物分离研究的方向提出了自己的建议。 相似文献
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土壤微生物多样性研究的DGGE/TGGE技术 总被引：1，自引：0，他引：1
《核农学报》2009,23(4):721-727
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。 相似文献
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土壤微生物多样性研究的DGGE/TGGE技术进展 总被引：2，自引：0，他引：2
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。 相似文献
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磷脂脂肪酸分析方法及其在土壤微生物多样性研究中的应用 总被引：28，自引：1，他引：28
磷脂脂肪酸（PLFA）是活体微生物细胞膜的重要组分，不同类群的微生物可通过不同的生化途径合成不同的PLFA。一些PLFA可作为分析微生物量和微生物群落结构变化的“生物标记”。在土壤微生物分析中，越来越多地采用了PLFA方法。本文介绍了表征微生物的一些PLFA、从土壤中提取PLFA的方法以及用GC-MS分析PLFA的原理。本文还将常用的研究微生物多样性的几种方法进行了比较；传统的分析土壤微生物群落的方法依赖于培养技术，只能培养和分离出一小部分微生物群落；Biolog方法可用于研究土壤微生物群落生理多样性，即可反映微生物群落如何利用各种碳源底物，但对快速生长和适合在Biolog实验条件下生长的小部分群落成员有强烈的选择性；核酸分析方法的主要缺点是不能对土壤微生物进行定量分析；而PLFA方法相对于上述几种方法有诸多优势。本文通过一些实例证明PLFA方法的特色或独到之处，也分析了其缺点。采用PLFA方法并结合其他方法有助于获取土壤微生物群落多样性的更多和更全面而完整的信息。 相似文献
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土壤宏基因组学研究方法与进展 总被引：3，自引：0，他引：3
土壤微生物驱动着土壤中的物质循环和养分转化。在土壤学的研究中,长期将土壤作为一个黑箱系统来对待,对其中的生物组成及其参与的生化过程知之甚少。土壤中绝大部分微生物目前尚难以分离培养,因此基于传统的培养方法对于认识土壤微生物群落组成和功能有其局限性。宏基因组学直接从环境样品中提取全部微生物的DNA,或通过测序探究环境中微生物的群落结构和功能(序列驱动),或构建宏基因组文库,筛选新的基因或生物活性物质(功能驱动),克服了传统培养方法的缺陷,极大地丰富了对土壤微生物多样性及其功能的认知。本文在综述土壤宏基因组学研究基本流程的基础上,重点介绍了日益重要的第二代测序平台在土壤宏基因组学研究中的应用及其产生的海量数据的分析处理方法,并简要探讨了宏基因组学在土壤微生物生态学中的应用。最后,作者建议在国家层面上展开相关土壤宏基因组学研究,调查微生物群落及其变化,为生物资源开发、农业生产和环境保护作出应有的贡献。 相似文献
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土壤微生物生态学研究方法进展 总被引：28，自引：0，他引：28
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。 相似文献
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新近发展起来的稳定性同位素探针技术（SIP）与分子生物学方法结合,能够定向发掘复杂环境中参与特定生态过程的微生物资源,是土壤功能微生物原位鉴定的有效手段,具有广阔的应用前景。其原理是环境样品中的功能微生物代谢同化同位素标记的底物,通过对其生物标志物（即DNA、RNA、PLFA等）进行提取、分离、鉴定和比对分析,以此获取介导土壤物质转化和循环过程的功能微生物的直接信息。本文在分别介绍SIP技术在土壤功能微生物原位鉴定过程中的各种前处理方法、生物标志物选择及后续鉴定方法的基础上,综述了SIP技术在研究驱动土壤有机污染物生物降解、碳氮循环等过程的功能微生物原位鉴定的应用进展,展望了SIP技术在土壤微生物基因组学方面的应用前景。 相似文献
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污染土壤的微生物多样性研究 总被引：10，自引：0，他引：10
土壤微生物多样性是土壤微生物生态学的重要研究内容，目前已成为国际上生态学发展的崭新方向之一。土壤微生物多样性包括微生物群落功能多样性、结构多样性及分子遗传多样性，是指示土壤生态系统稳定性及其功能的重要传感器。本文基于分离培养以及生物标志分子方法。从不同生态层次上认识微生物多样性，较全面、系统地综合评述国内外污染土壤环境的微生物群落功能、结构及分子遗传多样性的研究进展，并针对新形势下土壤污染所面临的新问题，探讨了近期土壤微生物生态学过程研究的重要手段与科学问题。 相似文献
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自然界约99%的微生物不能通过传统的分离筛选途径进行培养(即未培养微生物),为获得新的基因资源,更全面地认识微生物多样性和微生物在自然环境和生物圈中的重要作用,近年来随着分子生物学的快速发展及其在微生物研究中的广泛运用,以环境中未培养微生物为研究对象的新兴学科--环境微生物基因组学成为国内外学者的研究热点.本文介绍了环境微生物基因组学的分析研究策略及生态作用:从环境样品中直接提取所有微生物的总DNA,采用适宜的载体克隆到宿主细胞中构建宏基因组文库,再筛选新的活性物质或基因;从生态学角度分析微生物不可培养的原因,可利用环境微生物基因组技术进行土壤污染修复、畜禽养殖除臭、鉴定新物种以及确定特定生态环境体系中未培养微生物种群与群落的结构组成及物种的进化模式. 相似文献
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PLFAs稳定同位素技术及其在土壤微生物学中的应用 总被引：1，自引：0，他引：1
磷脂脂肪酸(PLFAs)是微生物细胞膜的重要组分,可作为活体微生物的生物标记物。稳定同位素技术与生物标记物相结合为揭示微生物种群结构及其功能提供了一种有效的方法,可用来阐明复杂土壤生态系统中微生物源有机质代谢途径以及个别微生物种群特征,将特定微生物种群与相应生物化学过程相联系。介绍了PLFAs稳定同位素分析技术,包括气相色谱-燃烧-同位素比例质谱(GC-c-IRMS),气相色谱-质谱联机(GC-MS)以及核磁共振(NMR),并描述上述方法在土壤微生物学中的应用以及其优缺点。 相似文献
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Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods 总被引：6，自引：0，他引：6
B. K. Singh S. Munro E. Reid B. Ord J. M. Potts E. Paterson & P. Millard 《European Journal of Soil Science》2006,57(1):72-82
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. 相似文献
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Soil management practices, including the use of cover crops, affect soil and plant health through varied mechanisms. Impacts on microbial communities are known to be important, but are not well understood. Various techniques are used to measure the effect of treatments on microbial communities, but rarely are the results of more than one technique compared. This field study examined the impacts of a single-season application of cover crops on detection of pathogen species in the tomato crop rhizosphere. The study took place in Maryland, New York and Ohio (MD, NY and OH) in the summers of 2010 and 2011, with a total of 260 plots tested using both macroarray and T-RFLP analyses. The macroarray used in this study was specifically designed to detect thirty-one pathogens of solanaceous crops and had not previously been used for such a field study. The results of T-RFLP analysis, which is a common tool for examining microbial communities, were compared to the macroarray results and the limitations and benefits of each are presented. While not a quantitative measure, the macroarray was able to detect certain fungi with much greater sensitivity than T-RFLP. Our findings suggest that the results of PCR-based techniques used for microbial community studies should be compared to other methods to verify sensitivity. 相似文献
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Assessing bacterial diversity in soil 总被引：1，自引：1，他引：0
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核酸分析方法在土壤微生物多样性研究中的应用 总被引：3，自引：0，他引：3
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。 相似文献
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Microbial diversity and soil functions 总被引：43，自引：0，他引：43
P. Nannipieri J. Ascher M. T. Ceccherini L. Landi G. Pietramellara & G. Renella 《European Journal of Soil Science》2003,54(4):655-670
Soil is a complex and dynamic biological system, and still in 2003 it is difficult to determine the composition of microbial communities in soil. We are also limited in the determination of microbially mediated reactions because present assays for determining the overall rate of entire metabolic processes (such as respiration) or specific enzyme activities (such as urease, protease and phosphomonoesterase activity) do not allow any identification of the microbial species directly involved in the measured processes. The central problem posed by the link between microbial diversity and soil function is to understand the relations between genetic diversity and community structure and between community structure and function. A better understanding of the relations between microbial diversity and soil functions requires not only the use of more accurate assays for taxonomically and functionally characterizing DNA and RNA extracted from soil, but also high‐resolution techniques with which to detect inactive and active microbial cells in the soil matrix. Soil seems to be characterized by a redundancy of functions; for example, no relationship has been shown to exist between microbial diversity and decomposition of organic matter. Generally, a reduction in any group of species has little effect on overall processes in soil because other microorganisms can take on its function. The determination of the composition of microbial communities in soil is not necessary for a better quantification of nutrient transformations. The holistic approach, based on the division of the systems in pools and the measurement of fluxes linking these pools, is the most efficient. The determination of microbial C, N, P and S contents by fumigation techniques has allowed a better quantification of nutrient dynamics in soil. However, further advances require determining new pools, such as active microbial biomass, also with molecular techniques. Recently investigators have separated 13C‐ and 12C‐DNA, both extracted from soil treated with a 13C source, by density‐gradient centrifugation. This technique should allow us to calculate the active microbial C pool by multiplying the ratio between labelled and total DNA by the microbial biomass C content of soil. In addition, the taxonomic and functional characterization of 13C‐DNA allows us to understand more precisely the changes in the composition of microbial communities affected by the C‐substrate added to soil. 相似文献
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微生物功能多样性信息对于明确不同环境中微生物群落的作用具有重要意义 ,而微生物群落的定量描述一直是微生物学家面临的最艰巨的任务之一。目前 ,以群落水平碳源利用类型为基础的BIOLOG氧化还原技术为研究土壤微生物群落功能多样性提供了一种简单、快速的方法 ,并得以广泛应用。但它仍然是一种以培养为基础的方法 ,显示的代谢多样性类型也不一定反映整个土壤微生物群落的功能多样性。因此 ,这种方法优点明显 ,缺陷也存在 ,并且在应用过程中还有很多关键的操作要点与技巧。本文综述了BIOLOG研究土壤微生物群落功能多样性的原理、BIOLOG研究土壤微生物群落功能多样性的方法与技巧、应用过程中容易产生的问题及可能克服的办法 ,同时还提出了值得进一步研究的问题。旨在促进对BIOLOG测定土壤微生物群落功能多样性的了解 ,为正确运用这种方法开展土壤微生物群落功能多样性研究提供科学依据和理论指导。 相似文献
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Salinization of soil is recognised as one of the most pressing environmental challenges to resolve for the next century. We here conduct a synoptic review of the available research on how salt affects decomposer microbial communities and carbon (C) cycling in soil. After summarizing known physiological responses of microorganisms to salinity, we provide a brief overview and qualification of a selection of widely applied methods to assess microorganisms in soil to date. The dominant approaches to characterise microbial responses to salt exposure have so far been microbial biomass and respiration measurements. We compile datasets from a selection of studies and find that (1) microbial biomass-carbon (C) per C held in soil organic matter shows no consistent pattern with long-term (field gradients) or short-term (laboratory additions) soil salinity level, and (2) respiration per soil organic C is substantially inhibited by higher salt concentrations in soil, and consistently so for both short-term and long-term salinity levels. Patterns that emerge from extra-cellular enzyme assessments are more difficult to generalize, and appear to vary with the enzyme studied, and its context. Growth based assessments of microbial responses to salinization are largely lacking. Relating the established responses of microbial respiration to that of growth could provide an estimate for how the microbial C-use efficiency would be affected by salt exposure. This would be a valuable predictor for changes in soil C sequestration. A few studies have investigated the connection between microbial tolerance to salt and the soil salinity levels, but so far results have not been conclusive. We predict that more systematic inquiries including comprehensive ranges of soil salinities will substantiate a connection between soil salinity and microbial tolerance to salt. This would confirm that salinity has a direct effect on the composition of microbial communities. While salt has been identified as one of the most powerful environmental factors to structure microbial communities in aquatic environments, no up-to-date sequence based assessments currently exist from soil. Filling this gap should be a research priority. Moreover, linking sequencing based assessments of microbial communities to their tolerance to salt would have the potential to yield biomarker sets of microbial sequences. This could provide predictive power for, e.g., the sensitivity of agricultural soils to salt exposure, and, as such, a useful tool for soil resource management. We conclude that salt exposure has a powerful influence on soil microbial communities and processes. In addition to being one of the most pressing agricultural problems to solve, this influence could also be used as an experimental probe to better understand how microorganisms control the biogeochemistry in soil. 相似文献
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Shifting microbial community structure across a marine terrace grassland chronosequence, Santa Cruz, California 总被引：1，自引：0，他引：1
Changes in the biomass and structure of soil microbial communities have the potential to impact ecosystems via interactions with plants and weathering minerals. Previous studies of forested long-term (1000s - 100,000s of years) chronosequences suggest that surface microbial communities change with soil age. However, significant gaps remain in our understanding of long-term soil microbial community dynamics, especially for non-forested ecosystems and in subsurface soil horizons. We investigated soil chemistry, aboveground plant productivity, and soil microbial communities across a grassland chronosequence (65,000-226,000 yrs old) located near Santa Cruz, CA. Aboveground net primary productivity (ANPP) initially increased to a maximum and then decreased for the older soils. We used polar lipid fatty acids (PLFA) to investigate microbial communities including both surface (<0.1 m) and subsurface (≥0.2 m) soil horizons. PLFAs characteristic of Gram-positive bacteria and actinobacteria increased as a fraction of the microbial community with depth while the fungal fraction decreased relative to the surface. Differences among microbial communities from each chronosequence soil were found primarily in the subsurface where older subsurface soils had smaller microbial community biomass, a higher proportion of fungi, and a different community structure than the younger subsurface soil. Subsurface microbial community shifts in biomass and community structure correlated with, and were likely driven by, decreasing soil P availability and Ca concentrations, respectively. Trends in soil chemistry as a function of soil age led to the separation of the biological (≤1 m depth) and geochemical (>1 m) cycles in the old, slowly eroding landscape we investigated, indicating that this separation, commonly observed in tropical and subtropical ecosystems, can also occur in temperate climates. This study is the first to investigate subsurface microbial communities in a long-term chronosequence. Our results highlight connections between soil chemistry and both the aboveground and belowground parts of an ecosystem. 相似文献