农业土壤微生物基因与群落多样性研究进展

介绍了群落基因组多样性、结构多样性与功能多样性相互关系的研究方法 ,重点论述了近年来农业土壤微生物群落遗传、结构与功能多样性的研究进展。同时总结了耕作措施和养分管理对农业土壤微生物群落多样性的影响 ,提出微生物序列分析、比较基因组学和微生物芯片技术与传统研究技术结合将有助于对微生物群落结构与功能和生物与环境因素对土壤微生物群落影响的深刻理解     

转基因作物对土壤微生物多样性影响的研究方法

土壤微生物是土壤生态系统的一个重要组成部分,对土壤中的生物化学循环起着不可替代的驱动作用。转基因作物在生长过程中会不可避免地与土壤微生物发生交流,开展转基因作物对土壤微生物群落影响的研究对于科学评价转基因作物的潜在风险具有重要意义。随着现代生物技术的不断发展,土壤微生物多样性及其分析方法已经从传统的分离培养发展到从种群角度去研究整个土壤微生态系统内的微生物。但是由于土壤微生物的各种特性(如大部分不可培养、体积微小及群体效应等)和仪器设备检测性能的局限性,单一的研究方法会存在一些弊端,还需要结合其他的手段共同研究土壤生态系统中的微生物多样性。目前,人们对土壤微生物多样性的研究主要包括物种多样性、功能多样性、结构多样性及遗传多样性等4个方面,国内外关于转基因作物对土壤微生物多样性及其群落结构影响的研究方法很多,对常见的研究方法进行了总结,并提出了今后转基因作物对土壤微生物多样性及群落结构影响的研究策略。     

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.     

砷矿区农田土壤微生物群落碳源代谢多样性

采用Biolog方法研究了砷(As)矿区农田土壤微生物碳源利用多样性及其与土壤化学性质的关系.结果表明： 3种土壤的N、P、K、有机质(OM)、Cu和Zn全量依次为中As > 高As > 低As土壤.中As和高As土壤微生物平均吸光度以及多样性指数(H′、D、U)显著高于低As土壤.主成分分析与生理碳代谢指纹图谱分析表明,中As和高As土壤微生物对糖类、氨基酸类等碳源的利用程度显著高于低As土壤.典范对应分析显示,影响土壤微生物群落碳源代谢的主要有全P、OM、全Pb、全Zn、全N和pH,全As并不是最主要的影响因子.可见,养分是影响长期污染土壤微生物群落结构和功能多样性的主要因素.     

长期围封和自由放牧对高寒草甸土壤微生物群落结构及碳源代谢多样性的影响

土壤微生物群落结构和代谢多样性对于维持草地生态系统的功能和稳定性具有重要意义.本研究运用磷脂脂肪酸法(PLFA法)和Biolog微平板技术,研究了青藏高原长期围封和自由放牧2种草地利用方式下的土壤微生物群落结构和群落碳代谢多样性的差异以及影响机制.结果表明: 1)长期围封和自由放牧草地上的土壤微生物群落结构和碳代谢多样性存在显著差异;2)长期围封显著增加了土壤中总PLFA、细菌、真菌和放线菌生物量;3)长期围封草地土壤微生物的碳代谢活性、多样性和丰富度高于自由放牧草地,而均匀度低于放牧草地;4)与自由放牧相比,长期围封显著增加了土壤微生物对聚合物类、糖类、羧酸类、胺类碳源的利用;5)冗余分析表明,植被盖度显著影响了微生物的群落结构和碳代谢多样性.长期围封草地的土壤微生物量、碳代谢多样性指数和丰富度均高于自由放牧土壤,表明长期围封有利于提高草地生态系统中土壤微生物群落及其对碳源代谢的多样性.     

砷矿区农田土壤微生物群落碳源代谢多样性

采用Biolog方法研究了砷(As)矿区农田土壤微生物碳源利用多样性及其与土壤化学性质的关系.结果表明:3种土壤的N、P、K、有机质(OM)、Cu和Zn全量依次为中As＞高As＞低As土壤.中As和高As土壤微生物平均吸光度以及多样性指数(H'、D、U)显著高于低As土壤.主成分分析与生理碳代谢指纹图谱分析表明,中As和高As土壤微生物对糖类、氨基酸类等碳源的利用程度显著高于低As土壤.典范对应分析显示,影响土壤微生物群落碳源代谢的主要有全P、OM、全Pb、全Zn、全N和pH,全As并不是最主要的影响因子.可见,养分是影响长期污染土壤微生物群落结构和功能多样性的主要因素.     

PCR-DGGE和FAMEs技术在土壤微生物多态性研究中的应用

将PCR-DGGE和FAMEs等生物技术新方法用于土壤微生物分析可有效的克服传统培养方法的局限性,为土壤微生物多样性、群落结构及动态变化的研究提供了更全面、可靠的方法,受到了国内外研究者的重视.本文综述了PCR-DGGE和FAMEs技术的原理及其在土壤微生物多样性、群落结构及动态变化方面的应用效果,指出了实际应用存在的问题.     

岩溶区植被和季节对土壤微生物遗传多样性的影响

基于土壤微生物遗传多样性随植被和季节的变化而变化的假设,运用变性梯度凝胶电泳技术(DGGE)检测岩溶区草丛(T)、灌丛(S)、次生林(SF)和原生林(PF)群落演替过程中土壤细菌和真菌群落的遗传多样性及其季节变化.随着地上植被的演替土壤细菌群落具有连续性但优势种群不明显,真菌群落没有连续性但优势种群明显.植被和季节对于细菌和真菌群落的Shannon多样性具有不同程度的显著影响,同时存在显著的植被和季节交互作用.草丛土壤中细菌和真菌群落的Shannon多样性有显著的季节变化(p<0.01);灌丛土壤中仅细菌群落多样性有显著季节变化(p<0.05);而森林土壤中细菌和真菌群落多样性没有显著的季节变化.土壤真菌和细菌多样性具有显著正相关关系.随着地上植被的正向演替,土壤微生物遗传结构逐渐稳定;植被恢复早期阶段,土壤中存在着丰富的微生物遗传多样性,但并不稳定.     

桑树-大豆间作对盐碱土碳代谢微生物多样性的影响

针对桑树-大豆间作可缓解盐碱土危害的特点,利用Biolog^TM技术研究了桑树-大豆间作对盐碱土作物根际碳代谢微生物多样性的影响.结果表明：表征土壤微生物代谢活性的平均颜色变化率(AWCD)在桑树-大豆间作下明显高于桑树单作和大豆单作,其中间作大豆的AWCD最高,单作桑树最低.桑树-大豆间作的土壤微生物均匀度指数高于单作,而土壤微生物的多样性指数和优势度指数在间作和单作之间差异不显著,说明桑树-大豆间作改变了盐碱土根际微生物群落结构组成,提高了根际微生物群落多样性.主成分分析表明,桑树-大豆间作和单作下土壤微生物的碳源利用模式出现分异,主要碳源为糖类、羧酸和聚合物类物质等.盐碱土pH和盐度是制约微生物群落多样性的主要因素,间作有效降低了土壤pH和盐度,促进了土壤微生物群落多样性的提高.     

土壤管理措施及环境因素对土壤微生物多样性影响研究进展

本文综述了土壤管理措施及环境因素对土壤微生物多样性影响的研究进展,并介绍了土壤微生物多样性的研究方法,土壤微生物多样性包括微生物物种多样性、遗传多样性和生态多样性。传统上,土壤微生物群落的分析依赖于培养技术,但使用该技术只能培养和分离出一部分土壤微生物群落。现在国际上普遍使用Biolog分析、磷脂脂肪酸(PLFA)分析和核酸分析等多种现代技术研究和表征土壤微生物多样性。土壤微生物多样性受土壤管理措施和多种环境因素的影响。农药可能使土壤微生物多样性减少或改变其结构和功能;施有机肥有利于维持土壤微生物的多样性及活性;但在施用无机肥的影响上目前的报道有矛盾之处。农业土壤减少耕作可能增加微生物多样性和生物量;轮作可能比单一栽培耕作更有利于维持土壤微生物的多样性及活性。土壤微生物多样性也受土壤有机质、植被、季节变化等因素的影响,且通常遭受干旱、过度放牧、营养缺乏等的胁迫作用。     

Influences of Plant Species Composition,Fertilisation and <Emphasis Type="Italic">Lolium perenne</Emphasis> Ingression on Soil Microbial Community Structure in Three Irish Grasslands

Semi-natural grassland soils are frequently fertilised for agricultural improvement. This practice often comes at a loss of the indigenous flora while fast-growing nitrogen-responsive species, such as Lolium perenne, take over. Since soil microbial communities depend on plant root exudates for carbon and nitrogen sources, this shift in vegetation is thought to influence soil microbial community structure. In this study, we investigated the influence of different plant species, fertilisation and L. perenne ingression on microbial communities in soils from three semi-natural Irish grasslands. Bacterial and fungal community compositions were determined by automated ribosomal intergenic spacer analysis, and community changes were linked to environmental factors by multivariate statistical analysis. Soil type had a strong effect on bacterial and fungal communities, mainly correlated to soil pH, as well as soil carbon and nitrogen status. Within each soil type, plant species composition was the main influencing factor followed by nitrogen fertilisation and finally Lolium ingression in the acidic upland and mesotrophic grassland. In the alkaline grassland, however, Lolium ingression had a stronger effect than fertilisation. Our results suggest that a change in plant species diversity strongly influences the microbial community structure, which may subsequently lead to significant changes in ecosystem functioning.     

内蒙草原不同植物功能群及物种对土壤微生物组成的影响

为了分析不同植物群落组成对内蒙古典型草原土壤微生物群落组成的影响,本研究利用植物功能群剔除处理实验平台,采用荧光定量PCR(real-timePCR)和自动核糖体间隔区基因分析(automated ribosomal intergenic spacer analysis,ARISA)技术,对不同植物功能群组成的非根际土壤和常见物种的根际土壤中细菌和真菌的数量及群落结构进行了分析。结果表明,在非根际土壤中,不同植物功能群组成对细菌数量有显著影响,而对真菌数量及细菌和真菌的群落结构影响不明显;在根际土壤中,不同植物物种对细菌、真菌的数量都有显著影响。此外,聚类分析表明,不同物种的根际土中细菌和真菌的群落结构也有所不同,尤其以细菌的群落结构变化较为明显。研究结果表明不同植物物种可以通过根系影响土壤微生物群落组成。     

农用化学品污染对土壤微生物群落DNA序列多样性影响研究

采用ＲＡＰＤ分子遗传标记技术研究了农用化学品不同使用环境下的４种土壤微生物群落ＤＮＡ序列多样性的变化。结果表明,４种土壤微生物群落ＤＮＡ序列在其丰富度、多样性指数、均匀度等方面均存在差异;农用化学品的使用会对土壤微生物群落在ＤＮＡ分子水平上的多样性产生影响;而冰同的农用化学品对土壤微生物群落ＤＮＡ序列多样性影响各不相同：化肥污染会引起某些土壤微生物的富集和一些微生物物种的丧失;农药杂会引起土壤微生     

Rhizosphere Microbial Characterization in Petroleum-Contaminated Soil

Contamination of soil with petroleum compounds is of concern worldwide. Although there are a variety of physical and chemical technologies available to remediate petroleum waste sites, biological methods are often used due to lower cost and public acceptance. Growth and enhanced activity of microbial communities in contaminated soil is a key factor for the success of bioremediation. Establishing vegetation in petroleum-contaminated soil may enhance microbial activity and remediation success even further by providing root exudates to the rhizosphere microorganisms. In this study, microorganisms were characterized in petroleum-contaminated soils and sediments quantitatively and qualitatively based on enumeration and metabolic diversity assessments. Contaminated soils and sediments were obtained from a phytoremediation field demonstration project in California. Microbial numbers in the unvegetated soil, based on plate counts and most probable number of hydrocarbon degraders, were significantly lower than the vegetated soils. Metabolic microbial characterization using BIOLOG was also conducted and based on principle component analysis (PCA), there was a distinct difference between the metabolic diversity of microbial communities in vegetated and unvegetated soils. Results from this research indicate that the presence and type of plants, and level of contamination may greatly influence microbial communities in polluted soils.     

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.     

DNA extraction from soils: old bias for new microbial diversity analysis methods

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.     

盐城滨海滩涂湿地典型植物群落土壤微生物组成与结构特征

土壤微生物是生态系统维持正常结构与功能的重要组成部分,为探究盐城滩涂典型湿地土壤微生物群落结构特征,以江苏盐城滩涂互花米草、藨草、盐地碱蓬、芦苇及淤泥质光滩5种典型群落为对象,采用16S rRNA高通量测序技术分析0—10 cm(表层)、10—30 cm(中层)、30—60 cm(深层)土壤微生物多样性及群落结构。结果表明：(1)几种植物群落间,土壤微生物群落结构差异较大,主要体现在细菌群落结构的差异性,古菌群落结构差异相对较小。光滩与植物群落间,在土壤细菌种类及相对丰度上差异相对较大,互花米草群落与本土植物群落间,在微生物群落的细菌种类组成上存在较大差异;藨草群落土壤表层微生物群落结构与互花米草群落相似,深层与盐地碱蓬、芦苇群落相似。(2)同一群落不同层次土壤微生物群落结构相似,差异小于不同群落间土壤微生物群落的结构差异性;不同群落对应层次间,表深层土壤中五种群落土壤微生物多样性存在显著差异,中层土壤中五种群落微生物多样性差异不显著。总体上,植物群落类型对土壤微生物群落结构的影响大于土壤深度;与本土植物群落相比,互花米草群落土壤主要优势门微生物种类差异较小,但部分优势门微生物相对丰度...     

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.