土壤微生物群落多样性解析法:从培养到非培养

土壤微生物群落多样性是土壤微生物生态学和环境科学的重点研究内容之一.传统的土壤微生物群落多样性解析技术是指纯培养分离法(平板分离和形态分析法以及群落水平生理学指纹法).后来,研究者们建立了多样性评价较为客观的生物标记法(磷脂脂肪酸法和呼吸醌指纹法).随着土壤基因组提取技术和基因片段扩增(PCR)技术的发展,大量的现代分子生物学技术不断地涌现并极大地推动了土壤微生物群落多样性的研究进程.这些技术主要包括:G+C％含量、DNA复性动力学、核酸杂交法(FISH和DNA芯片技术)、土壤宏基因组学以及DNA指纹图谱技术等.综述了这些技术的基本原理、比较了各种技术的优缺点并且介绍了他们在土壤微生物群落多样性研究中的应用,展望了这些技术的发展方向.     

土壤宏基因组学技术及其应用

传统的基于培养的研究方法只能反映土壤中少数(0.1%～10 %)微生物的信息,而大部分微生物目前还不能培养,因而这部分微生物资源尚难以被有效地开发利用.宏基因组学是分子生物学技术应用于环境微生物生态学研究而形成的一个新概念,主要技术包括土壤DNA的提取、文库的构建和目标基因克隆的筛选.它可为揭示微生物生态功能及其分子基础提供更全面的遗传信息,并已在微生物新功能基因筛选、活性物质开发和微生物多样性研究等方面取得了显著成果.本文对土壤宏基因组学技术的方法和应用作了详细介绍.     

五大连池新期火山熔岩台地不同植被类型土壤微生物群落的功能多样性

土壤微生物群落是陆地生态系统的重要生物活性成分,其结构和功能多样性直接影响到系统的碳、氮等生态过程,微生物群落功能多样性与地上植被类型变化密切相关,开展植被类型对土壤微生物群落功能多样性的影响研究具有重要意义。以五大连池新期火山熔岩台地苔藓、草本、灌丛、矮曲林、针阔混交林5种典型植被类型为对象,利用BIOLOG微孔板法研究不同演替阶段植被类型土壤微生物群落功能多样性特征。结果表明：不同植被类型土壤微生物群落功能多样性存在显著差异。平均颜色变化率（AWCD）随培养时间延长而逐渐增加,大小顺序为：苔藓 > 针阔混交林 > 矮曲林 > 草本 > 灌丛。灌丛土壤微生物多样性指数与其他植被类型间差异显著。主成分分析结果表明,主成分1和主成分2分别能解释变量方差的56.24%和29.59%,不同植被类型下土壤微生物的碳源利用格局差异主要是由氨基酸类和带磷基糖类引起,二者合计解释总变异量的47.51%。冗余分析表明,速效磷、铵态氮、C：N和pH对微生物功能多样性具有显著的影响,羧酸类、氨基酸类、酯类和胺类的降解更易受到环境因素的影响。研究结果为进一步探讨植被类型与土壤微生物之间在植被演替过程中的关系提供参考。     

污染土壤微生物群落结构多样性及功能多样性测定方法

土壤微生物在促进土壤质量和植物健康方面发挥着重要的作用,土壤微生物群落结构和组成的多样性及其变化在一定程度上反映了土壤质量.为了更好地了解土壤健康状况,非常有必要发展有效的方法来研究污染土壤微生物的多样性、分布以及行为等.回顾了近年来国内外污染土壤微生物群落结构多样性及功能多样性的测定方法,包括生物化学技术和分子生物学技术,现将它们的原理、优缺点、实用性及其发展动态作一阐述,同时指出结合这两种技术可为微生物群落分析提供一个更全面的、精确的方法.     

Diagnostic microbial microarrays in soil ecology

Soil microbial communities are responsible for important physiological and metabolic processes. In the last decade soil microorganisms have been frequently analysed by cultivation-independent techniques because only a minority of the natural microbial communities are accessible by cultivation. Cultivation-independent community analyses have revolutionized our understanding of soil microbial diversity and population dynamics. Nevertheless, many methods are still laborious and time-consuming, and high-throughput methods have to be applied in order to understand population shifts at a finer level and to be better able to link microbial diversity with ecosystems functioning. Microbial diagnostic microarrays (MDMs) represent a powerful tool for the parallel, high-throughput identification of many microorganisms. Three categories of MDMs have been defined based on the nature of the probe and target molecules used: phylogenetic oligonucleotide microarrays with short oligonucleotides against a phylogenetic marker gene; functional gene arrays containing probes targeting genes encoding specific functions; and community genome arrays employing whole genomes as probes. In this review, important methodological developments relevant to the application of the different types of diagnostic microarrays in soil ecology will be addressed and new approaches, needs and future directions will be identified, which might lead to a better insight into the functional activities of soil microbial communities.     

Changes in functional diversity of soil microbial community with addition of antibiotics sulfamethoxazole and chlortetracycline

Potential effects of antibiotics on agricultural soil microflora have recently become increasing concerns with antibiotic-contaminated biosolid now being used in agricultural land. However, changes of soil microbial community function caused by the antibiotic-associated disturbance are less addressed. This paper investigated the changes in microbial functional diversity by spiking sulfamethoxazole (SMX) and chlortetracycline (CTC) in a loam paddy soil and then incubating for 21 days. The dose-effect and time-dependent changes of antibiotic-associated disturbance on soil microbial community were analyzed with the soils sampled at 7 and 21 days using Biolog EcoPlate. At day 7 following treatment, SMX decreased functional diversity of soil microbial community, and the treatment of 100 mg SMX kg?1 dry soil had a significant inhibition of average well color development (AWCD) and Shannon index as compared to the control (p?相似文献   

车八岭山地常绿阔叶林冰灾后土壤微生物群落功能多样性

研究了我国南方冰灾后常绿阔叶林林冠开度及土壤养分的空间异质性对土壤微生物功能多样性的影响.在受冰灾影响的粤北车八岭山地常绿阔叶林2 hm2固定样地中按照冠层受损程度选取16个20 m×20 m的样方,用半球面影像技术获取林冠开度,并取0～20 cm的表层土壤混合样品分析土壤的理化性质,同时应用Biolog技术分析微生物功能多样性.按林冠开度梯度对各样方土壤微生物群落利用单一碳源的分析发现,林冠开度大的样方土壤微生物的活性、丰富度、多样性和均匀度都较低,反之则较高.聚类分析的结果与林冠开度的梯度有高度的一致性.主成分分析表明各样方土壤微生物功能多样性具有显著差异(第一轴p<0.005;第二轴p<0.001),其结果与聚类结果基本吻合.冗余分析揭示了土壤全磷、全钾、全氮、速效氮、有机碳、容重、总孔隙度和林冠开度的综合作用对土壤微生物功能多样性有显著影响(p<0.005),其中林冠开度与土壤微生物群落功能多样性的关系最密切.土壤微生物功能多样性受土壤养分的影响,具体表现为:与土壤有机碳呈明显的正相关;与全氮正相关;与速效氮、全磷负相关.研究说明冰灾所造成林冠开度和土壤养分的空间异质性会影响到土壤微生物功能多样性,而土壤微生物功能多样性可用于对生境恢复的指示和评价.     

冬季火对川西亚高山草地土壤微生物功能多样性及其强度的短期影响

为可持续管理川西亚高山草地生态系统, 全面地了解火后土壤微生物功能的多样性和强度的变化及其恢复状况, 基于2010年“12·5”冬草场的火烧事件, 以川西亚高山草地为研究对象, 对比了火烧和未火烧区域0-20 cm土层土壤中7种酶(β-葡糖苷酶、酸性磷酸酶、碱性磷酸酶、脲酶、蔗糖酶、蛋白酶和过氧化氢酶)的活性变化, 分析了土壤微生物功能多样性及其强度对火处理的响应。结果发现, 7种酶的潜在活性在0-5 cm土层中皆有所增加, 但对火处理、土层深度和两者交互作用的响应有所差异; 其中, 碱性磷酸酶在指示该区域火后微生物功能多样性和强度短期内的变化时具有较好的灵敏性和指示性。火在一定程度上促进了表土层微生物功能的发挥, 但是土壤微生物功能多样性及其强度对火和土层深度(0-20 cm)的响应并不显著。因此, 为能更好地揭示干扰行为对微生物生物多样性的影响机制, 未来应加强土壤微生物群落功能稳定性的研究。     

环境DNA技术在地下生态学中的应用

地下生态过程是生态系统结构、功能和过程研究中最不确定的因素。由于技术和方法的限制,作为"黑箱"的地下生态系统已经成为限制生态学发展的瓶颈,也是未来生态学发展的主要方向。环境DNA技术,是指从土壤等环境样品中直接提取DNA片段,然后通过DNA测序技术来定性或定量化目标生物,以确定目标生物在生态系统中的分布及功能特征。环境DNA技术已成功用于地下生态过程的研究。目前,环境DNA技术在土壤微生物多样性及其功能方面的研究相对成熟,克服了土壤微生物研究中不能培养的问题,可以有效地分析土壤微生物的群落组成、多样性及空间分布,尤其是宏基因组学技术的发展,使得微生物生态功能方面的研究成为可能;而且,环境DNA技术已经在土壤动物生态学的研究中得到了初步应用,可快速分析土壤动物的多样性及其分布特征,更有效地鉴定出未知的或稀少的物种,鉴定土壤动物类群的幅度较宽;部分研究者通过提取分析土壤中DNA片段信息对生态系统植物多样性及植物分类进行了研究,其结果比传统的植物分类及物种多样性测定更精确,改变了以往对植物群落物种多样性模式的理解。同时,环境DNA技术克服传统根系研究方法中需要洗根、分根、只能测定单物种根系的局限,降低根系研究中细根区分的误差,并探索性地用于细根生物量的研究。主要综述了基于环境DNA技术的分子生物学方法在土壤微生物多样性及功能、土壤动物多样性、地下植物多样性及根系生态等地下生态过程研究中的应用进展。环境DNA技术对于以土壤微生物、土壤动物及地下植物根系为主体的地下生态学过程的研究具有革命性意义,并展现出良好的应用前景。可以预期,分子生物学技术与传统的生态学研究相结合将成为未来地下生态学研究的一个发展趋势。     

土壤微生物生态过程与微生物功能基因多样性

土壤微生物在陆地生态系统中具有重要的生态功能,包括参与地球化学物质循环、污染物降解、环境剧烈变化的缓冲等.土壤微生物的生态功能与土壤功能联系密切,微生物群落结构与组成变化会直接影响土壤功能的发挥.土壤微生物通过具有生物活性的酶参与一系列的代谢活动,编码酶的功能基因成为微生物功能标记物.近10年中,以功能基因多样性为核心的分子生态学研究迅速发展,为从功能基因角度了解土壤微生物的生态功能提供了一个新的切入点.本文综述了与土壤微生物生态功能相关的功能基因多样性研究进展,并对该领域的发展前景提出展望.     

Short-term effects of a winter wildfire on diversity and intensity of soil microbial function in the subalpine grassland of western Sichuan,China

为可持续管理川西亚高山草地生态系统, 全面地了解火后土壤微生物功能的多样性和强度的变化及其恢复状况, 基于2010年“12·5”冬草场的火烧事件, 以川西亚高山草地为研究对象, 对比了火烧和未火烧区域0-20 cm土层土壤中7种酶(β-葡糖苷酶、酸性磷酸酶、碱性磷酸酶、脲酶、蔗糖酶、蛋白酶和过氧化氢酶)的活性变化, 分析了土壤微生物功能多样性及其强度对火处理的响应。结果发现, 7种酶的潜在活性在0-5 cm土层中皆有所增加, 但对火处理、土层深度和两者交互作用的响应有所差异; 其中, 碱性磷酸酶在指示该区域火后微生物功能多样性和强度短期内的变化时具有较好的灵敏性和指示性。火在一定程度上促进了表土层微生物功能的发挥, 但是土壤微生物功能多样性及其强度对火和土层深度(0-20 cm)的响应并不显著。因此, 为能更好地揭示干扰行为对微生物生物多样性的影响机制, 未来应加强土壤微生物群落功能稳定性的研究。     

金沙土遗址土壤细菌群落结构和推测的代谢特征

【背景】金沙土遗址被认为是3 200年前商周时期大型祭祀场所,具有重要的古文化和历史意义,目前金沙土遗址在物理、化学、生物等因素的影响下已出现不同程度的劣化,物理、化学因素的影响已有报道,而生物因素尚鲜有关注。【目的】研究金沙土遗址中微生物群落结构组成,解析微生物菌群活性及代谢特征,为金沙土遗址的科学保护提供依据。【方法】根据遗址目前的保存状况,从金沙土遗址采集具有代表性的土壤样品,所采区域样品的劣化程度依次为J4J3J2J1,应用Biolog平板法与PCR-DGGE技术对各样品中的微生物群落结构组成和代谢功能多样性进行研究。【结果】Biolog结果显示,随着金沙土遗址的劣化,各土壤样品中的微生物功能多样性存在较大差异,各样品的微生物群落代谢活性依次为:J2J3J4J1,表明随着土壤的劣化,微生物的代谢活性表现出增大的趋势。主成分分析结果反映出4个样品中的微生物碳代谢方式具有显著的变化,样品J2中的细菌群落对底物碳源利用种类最多,且偏好的碳源类型与其它样品存在明显的不同。PCR-DGGE图谱显示,金沙土遗址不同土壤样品中的细菌多样性和种群组成存在明显差异,在DNA和RNA水平上,各样品的微生物组成多样性依次为:J2J4J3J1。主成分分析结果显示,除了样品J1,劣化样品的细菌群落结构和活性细菌群落结构具有较高的一致性,表明随着金沙土遗址的劣化,土壤中微生物多样性呈现出上升趋势。DGGE图谱主要条带的测序结果表明,样品中主要的细菌类群归属于放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、变形菌门(Proteobacteria)和异常球菌-栖热菌门(Deinococcus-Thermus),分属于7个属,在DNA和RNA水平上均能检测到红色杆菌属(Rubrobacter)和短波单胞菌属(Brevundimonas)。【结论】首次对金沙土遗址的细菌群落结构组成和功能进行分析,结果表明随着金沙土遗址劣化程度的增加,土壤微生物类群及功能多样性都随之增大,其中仅在劣化土壤样品中检出或者具有高表达活性的红色杆菌属、Tellurimicrobium、短状杆菌属细菌可能参与了土壤劣化过程,这为今后土遗址的科学保护提供了理论依据。     

Dispersal alters soil microbial community response to drought

Microbial communities will experience novel climates in the future. Dispersal is now recognized as a driver of microbial diversity and function, but our understanding of how dispersal influences responses to novel climates is limited. We experimentally tested how the exclusion of aerially dispersed fungi and bacteria altered the compositional and functional response of soil microbial communities to drought. We manipulated dispersal and drought by collecting aerially deposited microbes after precipitation events and subjecting soil mesocosms to either filter-sterilized rain (no dispersal) or unfiltered rain (dispersal) and to either drought (25% ambient) or ambient rainfall for 6 months. We characterized community composition by sequencing 16S and ITS rRNA regions and function using community-level physiological profiles. Treatments without dispersal had lower soil microbial biomass and metabolic diversity but higher bacterial and fungal species richness. Dispersal also altered soil community response to drought; drought had a stronger effect on bacterial (but not fungal) community composition, and induced greater functional loss, when dispersal was present. Surprisingly, neither immigrants nor drought-tolerant taxa had higher abundance in dispersal treatments. We show experimentally that natural aerial dispersal rate alters soil microbial responses to disturbance. Changes in dispersal rates should be considered when predicting microbial responses to climate change.     

元蛋白质组学在微生物功能研究中的应用

后基因组时代,仅依靠基因组方法来研究原位微生物群落的功能已远远不够,在这种背景下元蛋白质组学研究逐渐兴起。应用元蛋白质组学技术可大规模研究原位微生物群落的蛋白质表达,分析生态系统中微生物的功能,寻找新的功能基因和代谢通路,为微生物群体的基因和功能多样性研究提供数据。同时,还可鉴定与微生物功能相关的蛋白质,这些蛋白质未来可以作为生物标记物为环境可持续发展铺路。综述了元蛋白质组学的发展概况及其在微生物功能研究中的重大作用,强调了元蛋白质组学方法在分析新功能基因及其相关基因,揭示微生物多样性与微生物群体功能之间的关系等方面起到的作用,并对其应用前景进行了展望。     

黄土丘陵沟壑区地形变化对土壤微生物群落功能多样性的影响

研究黄土高原丘陵沟壑区破碎地形对土壤微生物功能多样性的影响,对于理解复杂地形区生态过程与系统功能的空间变化具有重要意义。选择陕西省安塞县陈家洼为研究区,依据坡面地形变化选择不同坡位土壤,采用Biolog微平板培养法探究地形变化对土壤微生物群落功能多样性的影响。实验发现,土壤微生物群落培养的平均颜色变化率(AWCD)增长曲线总的呈现出坡下部坡中部坡上部的规律,且坡下部AWCD值与坡中部、坡上部间差异显著(P0.05);坡下部土壤微生物群落功能多样性显著高于坡中部和坡上部,但不同土层深度(0—10 cm、10—20 cm)间无显著性差异(P0.05);对土壤微生物群落功能多样性差异贡献较大的碳源是糖类、羧酸类和多酚化合物类碳源;土壤含水率高低是不同坡位土壤微生物群落功能多样性差异显著的主要原因;微生物群落丰富度(H)和均一度(D)与土壤全氮含量正相关,优势度(U)反之,土壤全碳、全磷和p H对土壤微生物群落结构和功能多样性差异作用不显著。     

Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches

Until recently, our understanding of microbial community development in soil ecosystems exposed to different inorganic and organic pollutants has been limited to culturable microorganisms because of the techniques available. The discovery that most soil microorganisms are non-culturable but potentially viable and metabolically active accelerated the application of different culture-independent methods for structural diversity assessments of the microbial community. This review examines the results of recent studies on the impact of heavy metals and organic pollutants on the diversity of the microflora obtained with methods based on analyses of signature biomarkers such as nucleic acids and fatty acids. The application of these techniques allowed researchers to pinpoint reduction of microbial diversity in contaminated soil, and significant shifts in the community structure, leading to the dominance of only a few populations (species) and the disappearance of others, some of which were never isolated by conventional methods (e.g. an increase in Acidobacterium or a decrease in terrestrial non-thermophilic Crenarchaeota). Although the new techniques are not free from limitations, they allow the monitoring of the virtual impact of stressors on soil microorganisms and the direction of resuscitation of the microbial community during natural or induced bioremediation, especially when using combined approaches.     

Remarkable Recovery and Colonization Behaviour of Methane Oxidizing Bacteria in Soil After Disturbance Is Controlled by Methane Source Only

Little is understood about the relationship between microbial assemblage history, the composition and function of specific functional guilds and the ecosystem functions they provide. To learn more about this relationship we used methane oxidizing bacteria (MOB) as model organisms and performed soil microcosm experiments comprised of identical soil substrates, hosting distinct overall microbial diversities (i.e., full, reduced and zero total microbial and MOB diversities). After inoculation with undisturbed soil, the recovery of MOB activity, MOB diversity and total bacterial diversity were followed over 3 months by methane oxidation potential measurements and analyses targeting pmoA and 16S rRNA genes. Measurement of methane oxidation potential demonstrated different recovery rates across the different treatments. Despite different starting microbial diversities, the recovery and succession of the MOB communities followed a similar pattern across the different treatment microcosms. In this study we found that edaphic parameters were the dominant factor shaping microbial communities over time and that the starting microbial community played only a minor role in shaping MOB microbial community     

基于BIOLOG技术分析氮沉降和降水对土壤微生物功能多样性的影响

土壤微生物是有机物分解和养分循环的主要介质,因此在维持土壤的功能多样性和持续性方面发挥着关键作用。气候变化驱动因素会影响土壤微生物的生理活动,引起其群落结构和功能多样性的改变,并对生物地球化学循环和气候―生态系统反馈产生连锁效应,其中氮沉降和降水是全球气候变化的研究热点。土壤氮(N)的有效性有可能通过改变微生物的群落组成以调节微生物对降水变化的响应,但目前关于N沉降和降水及其交互作用对土壤微生物群落功能多样性的影响机制仍不清楚。为了准确预测未来气候条件下生态系统的功能状况,需要更好地了解土壤微生物对环境变化的响应。基于BIOLOG技术综述了氮沉降和降水变化及其交互作用对土壤微生物功能多样性影响的相关研究进展,可以为进一步研究全球气候变化背景下地下生态学的发展提供参考。另外,分析阐述了当前工作中存在的一些主要瓶颈,并对未来的研究热点进行了探讨和展望。     

Culture-independent discovery of natural products from soil metagenomes

Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or “metagenomic,” methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth’s microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.     

Accessing the soil metagenome for studies of microbial diversity

Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.