Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed.     

长期不同施肥制度下几种土壤微生物学特征变化

 为阐明土壤微生物对土壤健康的生物指示功能, 以国家褐潮土肥力与肥料效益监测基地的长期肥料试验为平台, 应用BIOLOG ECO微平板培养法与常规分析法研究了长期施肥15年后不同施肥制度对土壤微生物生物量、活性、群落代谢功能多样性及土壤肥力的影响。研究结果表明, 与对照(CK)相比, 长期化肥与有机肥配施土壤中土壤有机质(SOM)、全氮(STN)、全磷(STP)含量升高, 土壤C/N与pH值降低, 土壤微生物量碳(Soil microbial biomass carbon, SMBC)、生物微生物量氮(Soil microbial biomass nitrogen, SMBN)、微生物商(qMB)及脲酶(Urease)活性升高, BIOLOG ECO微平板平均颜色变化率(Average well color development, AWCD)、土壤微生物代谢功能多样性指数变化不明显。和长期单施化肥处理(NPK)相比, 长期化肥与有机肥配施处理中上述几种微生物学特征(SMBC、SMBN、qMB、Urease及AWCD、代谢功能多样性指数)均呈极显著增加。NPK处理与CK相比虽然SOM、STN、STP含量稍有升高, 土壤C/N与pH值降低, SMBC、SMBN、qMB及Urease活性增高, 但是AWCD、土壤微生物代谢功能多样性指数却显著下降。过氧化氢酶活性(Catalase)在各处理土壤中的差异不显著。土壤微生物碳源利用的主成分分析表明, 长期不同施肥各处理在土壤微生物利用碳源的种类和能力上有差异。此试验说明, 土壤微生物受农业管理措施和多种环境因素的影响, 土壤微生物学特征可作为土壤质量的敏感指标, 为提高作物产量、增强肥力提供理论参考。     

Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities.

We compared the abilities of Biolog's GN and ECO plates to distinguish among aerobic and heterotrophic bacterial communities in samples from six aquatic environments. The Biolog system is based on interpreting patterns of sole-carbon substrate utilization indicated by color development in a 96-well microtiter plate. Whether of fresh or saltwater origin, bacterial communities utilized > 95% of substrates in both types of plates. Samples from any one environment exhibited similar time courses of average well color development (AWCD) in both GN and ECO plates. Principal component analysis was performed on data sets resulting from combinations of algorithms (AWCD and curve-integration methods) and levels of color development (end-point and set-point approaches). In all cases, the two types of plates demonstrated an equal capacity to discriminate among the heterotrophic expressions of the six microbial communities. Substantial deviation from an anticipated 1:1 correspondence occurred when color development of 25 substrates common to both types of plates was compared. The discrepancies likely are related to the different formulations of low-nutrient media in GN and ECO plates.     

Heterotrophic microbial communities use ancient carbon following glacial retreat

When glaciers retreat they expose barren substrates that become colonized by organisms, beginning the process of primary succession. Recent studies reveal that heterotrophic microbial communities occur in newly exposed glacial substrates before autotrophic succession begins. This raises questions about how heterotrophic microbial communities function in the absence of carbon inputs from autotrophs. We measured patterns of soil organic matter development and changes in microbial community composition and carbon use along a 150-year chronosequence of a retreating glacier in the Austrian Alps. We found that soil microbial communities of recently deglaciated terrain differed markedly from those of later successional stages, being of lower biomass and higher abundance of bacteria relative to fungi. Moreover, we found that these initial microbial communities used ancient and recalcitrant carbon as an energy source, along with modern carbon. Only after more than 50 years of organic matter accumulation did the soil microbial community change to one supported primarily by modern carbon, most likely from recent plant production. Our findings suggest the existence of an initial stage of heterotrophic microbial community development that precedes autotrophic community assembly and is sustained, in part, by ancient carbon.     

Microbial Diversity and Community Structure of Postdisturbance Forest Soils as Determined by Sole-Carbon-Source Utilization Patterns

Abstract The impact of clear-cutting, scarification, and prescribed burning on forest soil microbial community structure was assessed using sole-carbon-source utilization (SCSU). Organic and mineral soil samples were collected on two dates from Pinus banksiana plots that had been clear-cut, clear-cut followed by prescribed burning, clear-cut followed by scarification, or had not been harvested. Microorganisms were extracted from the soil samples and used to inoculate Gram-negative Biolog? plates. Patterns of substrate metabolism were used to calculate Shannon, Simpson, McIntosh, and related evenness indices. Principal component analysis (PCA) resolved organic and mineral soils. Organic soil exhibited higher metabolic diversity than mineral soil. Scarified plots showed lower diversity on one date, when diversity indices were calculated using all carbon sources, and on both dates when calculated using carboxylic acids, only. The results suggest that SCSU may be used to assess the impact of forestry practices on microbial diversity and community structure by using a subset of carbon substrates. Received: 30 July 1996; Accepted 18 November 1996     

Investigating biological control over soil carbon temperature sensitivity

Understanding the temperature sensitivity of soil respiration is critical for predicting the response of ecosystems to climate change, yet the microbial communities responsible are rarely considered explicitly in studies or models. In this study, we assessed total microbial community composition, quantified bacterial respiration temperature response, and investigated the temperature dependence of bacterial carbon substrate utilization in tropical, temperate, and taiga soils (from Puerto Rico, California, and Alaska). Microbial community composition was characterized using phospholipid fatty acid analysis. Bacterial community respiration on a standardized set of substrates was ascertained using the BiOLOG^™ substrate utilization assay incubated at four temperatures: 4, 12, 28, and 40 °C. First, we found that microbial communities from the three latitudes were compositionally distinct and that the bacterial component of the three communities had markedly different respiration temperature–response curves corresponding with their experienced temperature regimes. We use these data to highlight limitations of widely used temperature–response equations and investigate temperature-dependent patterns of substrate utilization. We found that temperature response, in terms of both respiration rates and substrate use, varied for these bacterial communities independent of substrate quality or quantity interactions such as labile depletion. In contrast to the common assumption of heterotrophic microbial ubiquity, we found that bacterial community differences from these diverse systems appeared to determine both rates of respiration and patterns of carbon substrate usage. We suggest that microbial community composition-specific responses to changing climate may be important in predicting the long-term role of ecosystems in atmospheric CO₂ dynamics.     

长期不同施肥对稻田土壤微生物群落功能多样性的影响

微生物群落功能多样性对土壤管理具有重要指示作用,施肥措施对土壤微生物群落功能多样性产生重要的影响,从而影响土壤有机质的含量,引起土地碳贮量变化,进而影响到陆地生态系统的碳汇.以位于湖南省桃江县国家级稻田肥力长期定位试验点的土样为研究对象,采用BIOLOG测试板对不同施肥处理下土壤微生物功能多样性进行了研究.研究结果显示,土壤微生物的碳源利用率因长期不同的施肥处理而发生分异.Shannon和Simpson指数的结果显示所有施肥处理均有利于维持微生物群落多样性,但秸秆还田和习惯施肥使群落均匀度(McIntosh指数)降低.主成分分析表明,试验点土壤微生物群落利用的主要碳源为氨基酸类和糖类,但不同施肥处理碳源利用类型有差异.结果可以得出,不同的施肥对土壤微生物功能多样性产生了不同影响,从而影响土壤有机质中碳、氮含量.这些信息可以为深入研究施肥对全球碳氮循环以及全球气候变迁提供依据.     

不同施肥处理与地膜覆盖对土壤微生物群落功能多样性的影响

微生物多样性是表征土壤质量变化的敏感指标。应用Biolog技术探讨了不同施肥处理与地膜覆盖对土壤微生物功能多样性的影响,从微生物功能多样性的角度评价施肥与地膜覆盖对土壤质量的影响。试验结果表明：裸地条件下,肥料合理配施可以增强微生物对碳源的利用程度（AWCD）,显著增加微生物功能多样性（Shannon指数）。地膜覆盖和施肥的交互作用降低了微生物对碳源的利用率,降低微生物的丰富度,改变其均匀度。土壤微生物碳源利用的聚类和主成分分析表明,各施肥处理在碳源的利用上存在较大差异,覆膜加剧了各处理之间的分异程度。糖类和氨基酸类碳源是微生物利用的主要碳源。土壤微生物对碳源利用受到土壤pH、速效钾的显著影响。此外,有机碳、速效氮含量和土壤碳氮比与土壤微生物群落功能多样性密切相关。     

扎龙季节性湿草甸土壤养分和土壤微生物特性

以扎龙自然湿地典型季节性湿草甸为试验对象,研究了土壤微生物特性和土壤养分空间分布规律及其主要影响因子.结果表明:土壤养分、微生物群落数量、微生物量碳、氮均呈现明显的垂直分布特征;土壤酶活性的空间分布因受多种因素影响而表现复杂.逐步线性回归分析表明:土壤微生物生物量碳、氮均分别与β-葡萄糖苷酶、脲酶、磷酸酶显著相关(P<0.05);有机碳与放线菌数量、过氧化氢酶活性显著相关(P<0.05);速效钾、全氮、水解性氮、C/N分别与放线菌数量、细菌数量、β-葡萄糖苷酶活性、土壤微生物生物量氮显著相关(P<0.05);全P、pH与土壤微生物活性无显著相关关系(P>0.05).采用主成分分析构建了湿地土壤养分的评价模型及微生物学预测模型.     

中国亚热带典型天然次生林土壤微生物碳源代谢功能影响因素

天然次生林地比人工林地不仅土壤肥力较高,且土壤碳代谢功能更强。然而维持天然次生林高碳代谢功能的原因尚不十分清楚。分析天然次生林中土壤微生物碳源代谢功能的影响因素对于调控土壤微生物的功能乃至天然次生林的保护具有重要意义。本文选择中国亚热带地区典型天然次生林,研究了土壤微生物碳源代谢功能与土壤化学和物理因素及植物因素的关系。结果表明,3类因素能显著解释土壤微生物碳源代谢功能54.4% 的变异。乔木层植物多样性、土壤碳氮比、pH值和含水量是导致天然次生林碳代谢功能差异的主要因素,分别显著解释了土壤微生物碳源代谢16.7%、12.4%、10.5%和10.5%的变异。天然次生林较低的土壤碳氮比、较高的土壤含水量和土壤pH值（酸性范围内）,有利于土壤微生物碳源代谢功能的提高,同时天然次生林较高的阔叶树种的比例也能增加土壤微生物碳源代谢功能。     

Analysis of Factors Affecting the Accuracy, Reproducibility, and Interpretation of Microbial Community Carbon Source Utilization Patterns

We determined factors that affect responses of bacterial isolates and model bacterial communities to the 95 carbon substrates in Biolog microtiter plates. For isolates and communities of three to six bacterial strains, substrate oxidation rates were typically nonlinear and were delayed by dilution of the inoculum. When inoculum density was controlled, patterns of positive and negative responses exhibited by microbial communities to each of the carbon sources were reproducible. Rates and extents of substrate oxidation by the communities were also reproducible but were not simply the sum of those exhibited by community members when tested separately. Replicates of the same model community clustered when analyzed by principal-components analysis (PCA), and model communities with different compositions were clearly separated on the first PCA axis, which accounted for >60% of the dataset variation. PCA discrimination among different model communities depended on the extent to which specific substrates were oxidized. However, the substrates interpreted by PCA to be most significant in distinguishing the communities changed with reading time, reflecting the nonlinearity of substrate oxidation rates. Although whole-community substrate utilization profiles were reproducible signatures for a given community, the extent of oxidation of specific substrates and the numbers or activities of microorganisms using those substrates in a given community were not correlated. Replicate soil samples varied significantly in the rate and extent of oxidation of seven tested substrates, suggesting microscale heterogeneity in composition of the soil microbial community.     

Functional diversity and community structure of micro-organisms in three arctic soils as determined by sole-carbon-source-utilization

Functional diversities of micro-organisms in arctic soils at three incubation temperatures were assessed using sole-carbon-source-utilization (SCSU). Soil samples were collected from an area of anthropogenic fertilization (mixed Dorset/Thule/Historic site), an area of animal enrichment (bird rock perches), and unaltered tundra (raised beach; control soil site). The micro-organisms were extracted from the soil samples and inoculated into Gram-negative (GN) Biolog plates incubated at 30°C, 10°C, and 4°C. Calculations of the Shannon index, substrate utilization richness, Shannon evenness, and the Jaccard coefficient of similarity were based upon substrate utilization on the Biolog plates. Principal component analysis distinguished microbial communities in enriched soils from unenriched soils. At 10°C and 4°C, Shannon indices of enriched soil microbial communities (10°C: soils influenced by wild animals=4.28, soils influenced by human activities=4.20; 4°C: soils influenced by wild animals=4.15, soils influenced by human activities=4.03) were significantly higher than unenriched soil microbial communities (10°C: 3.66; 4°C: 3.38). Substrate utilization richness and evenness displayed similar trends. Although Jaccard coefficients showed uniformity across the different soil samples, cluster analysis supported patterns demonstrated by PCA. Lower temperatures (4°C and 10°C) yielded greater resolution between soil microbial communities than 30°C based on Biolog colour development patterns.     

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.     

内蒙古贝加尔针茅草原不同利用方式土壤微生物功能多样性

基于Biolog-ECO技术,研究了贝加尔针茅草原在自由放牧、刈割和围封3种不同利用方式下土壤微生物群落功能多样性变化。结果表明:不同的利用方式能显著改变土壤微生物群落代谢活性,反映微生物活性的平均颜色变化率表现为围封>自由放牧>刈割,围封时土壤微生物群落代谢活性最高;不同的利用方式改变了土壤微生物群落多样性指数,自由放牧土壤微生物群落丰富度指数、均匀度指数和优势度指数均最高,围封次之,刈割最低。主成分分析结果表明:在自由放牧和刈割2种利用方式下土壤微生物群落碳源利用模式及代谢功能相似,而围封土壤微生物群落具有不同的碳源利用模式和代谢功能;糖类、氨基酸类和代谢中间产物及次生代谢物为土壤微生物利用的主要碳源。不同的利用方式改变了贝加尔针茅草原土壤微生物群落功能多样性。     

Community-Level Physiological Profiles of Cloacal Microbes in Songbirds (Order: Passeriformes): Variation Due to Host Species, Host Diet, and Habitat

The relationship between microorganisms and birds has received increased attention recently. The state of knowledge of this relationship, however, is based largely on examination of sick or dead birds, and knowledge of the prevalence and community structure and function of microbes in healthy wild populations is limited. Using carbon substrate utilization profiles, microbial communities were examined in 91 cloacal samples from 14 species within apparently healthy summer and winter passerine populations. Within each season, gradient lengths and eigenvalues from ordination analyses suggested that many samples differed in their carbon substrate utilization and several had very different communities. Cloacal microbe carbon utilization profiles were distinguishable among host species, season-specific diet, and study site in the ordination analyses. However, these patterns were only observed for the analysis of the summer data set. The results of this study support the idea that the avian host’s microbial community, relative to carbon substrate utilization, is related to host diet. Previously, this pattern had only been reported for potential pathogens isolated from the avian cloaca. Study site–specific patterns in the ordination analysis suggest that environmental conditions at a particular study site may influence cloacal microbial communities in birds. Results of this study indicate that examination of community-level physiological profiles may be a useful technique for distinguishing among avian cloacal samples, similar to that already established for discriminating aqueous and soil samples. Future studies that correlate microbe physiological profiles to condition-based indices of avian hosts may be most useful for eventually using the profile as an indicator of environmental conditions experienced by hosts.     

小麦和大豆茬口对黄瓜土壤微生物生态特征的影响

采用常规稀释平板法和BIOLOG ECO微平板反应系统,研究了小麦和大豆茬口对黄瓜土壤微生物生态特征的影响.结果表明,两种茬口均显著提高了黄瓜土壤微生物真菌、细菌和放线菌的数量,显著降低了尖孢镰刀菌黄瓜专化型的数量(P<0.05);显著提高了土壤微生物群落的Shannon-Wiener指数、均匀度指数、Simpson指数和McIntosh指数(P<0.05)、以及土壤微生物生物量碳,降低了土壤基础呼吸和代谢商(P<0.05),改变了土壤微生物对单一碳源的利用能力.此外,两种茬口还显著提高了土壤中速效磷和速效钾含量,以及黄瓜产量.说明小麦和大豆茬口改善了土壤微生态环境.     

土壤样品长期保存对微生物群落代谢活性和功能类群的影响

【目的】评估土壤长期保存(4个月)对土壤微生物群落代谢活性的影响。【方法】采用Biolog? EcoPlateTM生态板研究4 °C风干保存和?20 °C低温冻存的农田土壤和森林土壤中微生物群落的碳源利用模式。【结果】与新鲜土壤样品相比,长期保存的土壤样品的微生物群落对碳源的利用能力大大降低,其多样性、均匀度和Simpson指数均降低;风干保存和低温冻存两者对土壤微生物的碳源利用的影响没有显著差异;除风干保存的土壤样品中利用多聚物类的微生物类群的代谢活性外,两种保存方法显著降低微生物群落的代谢活性,降低幅度为54.5%–99.8%。【结论】长期保存土壤可能会导致对微生物群落信息的低估,土壤微生物代谢活性研究的最佳样品为新鲜 土壤。     

冬季覆盖作物秸秆还田对双季稻田根际土壤微生物群落功能多样性的影响

微生物群落功能多样性是土壤质量变化重要的指标,不同作物类型的秸秆还田措施对土壤微生物群落功能多样性具有明显的影响。以位于双季稻主产区不同冬季覆盖作物-双季稻种植模式大田定位试验田为研究对象,以冬闲-双季稻种植模式为对照(CK),应用Biolog-GN技术开展黑麦草-双季稻(Ry)、紫云英-双季稻(Mv)、油菜-双季稻(Ra)和马铃薯-双季稻(Po)种植模式条件下不同冬季覆盖作物秸秆还田后对双季稻田根际土壤微生物功能多样性影响的研究。研究结果表明,早稻和晚稻成熟期,与CK处理相比,冬季覆盖作物秸秆还田处理增加了稻田土壤碳源平均颜色变化率(AWCD),以Po处理AWCD均为最高,均显著高于Ry和CK处理。不同冬季覆盖作物秸秆还田处理土壤微生物代谢多样性指数表现出明显的差异,早稻成熟期,Po处理的Richness、Shannon和McIntosh指数均为最高,其次为Ry、Mv和Ra处理,CK处理最低;晚稻成熟期,各处理的Richness、Shannon和McIntosh指数大小顺序均表现为PoRaMvRyCK。土壤微生物碳源利用的主成分分析结果表明,各冬季覆盖作物秸秆还田处理根际土壤微生物利用的主要碳源为氨基酸类和糖类物质,不同处理间碳源利用类型有差异。冬季覆盖作物秸秆还田措施有利于提高双季稻田根际土壤微生物对碳源的利用能力、物种丰富度和均匀度。     

Changes in soil heterotrophic respiration,carbon availability,and microbial function in seven forests along a climate gradient

Soil microbial communities play an essential role in soil carbon (C) emission and C sequestration in forest ecosystems. However, little information is available regarding the relationship between soil C dynamics and microbial substrate utilization at large scales. Along the North–South Transect of Eastern China (NSTEC), seven forests representative of boreal, temperate and tropical biomes were examined. Soil heterotrophic respiration (R_h), soil dissolved organic C (DOC), microbial biomass C (MBC), and microbial community-level physiological profiles (CLPPs) were investigated using biochemical measurements, static chamber-gas chromatography analysis, and Biolog-Eco microplates, respectively. We found that soil R_h rates were significantly higher in subtropical and boreal forests than in temperate forests. Conversely, the concentrations of soil DOC and MBC, as well as microbial metabolic activity and functional diversity, were consistently higher in temperate forests than in subtropical forests. There were considerably different substrate utilization profiles among the boreal, temperate, and subtropical forests. Soil microorganisms from the temperate and boreal forests mainly metabolized high-energy substrates, while those from the subtropical forests used all substrates equally. In addition, soil R_h rates were significantly and negatively related to soil labile C concentrations, total metabolic activity, and the intensity of individual substrate utilization, indicating that soil microbes assimilated more soil substrates, thereby reducing CO₂ emissions. Overall, our study suggests that climate factors, as well as substrate availability, dominate the activities and functions of soil microbes at large scales.     

16S rDNA analysis reveals low microbial diversity in community level physiological profile assays

The metabolic diversity of microbial communities is fundamental for the multiple soil functions mediated by microorganisms. Community level physiological profiles (CLPPs) based on sole C source oxidation have been used as a fast and reproducible tool to study soil microbial functional diversity because the utilisation of available carbon is the key factor governing microbial growth in soil. Our aim was to assess the phylogenetic affiliation of the microorganisms responsible for C consumption after inoculating Biolog plates. For this purpose, two semi-arid Mediterranean forest soils with significantly different patterns of C consumption and microbial community structure were used. Following the inoculation of the Biolog plates, suspensions from seven wells were sampled after 1, 2 and 7 d of incubation. DNA was extracted and the microbial communities analysed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing of excised bands. Despite major differences in the microbial communities of the soils studied, their DGGE banding patterns after incubation were similar for all the analysed C source suspensions. Microorganisms belonging to beta-Proteobacteria (Ralstonia sp. and Burkholderia sp.) and alpha-Proteobacteria (Rhizobium sp.) were dominant. These opportunists had a competitive advantage under the conditions at which the CLPPs were analysed. This study reveals that significantly different CLPP patterns can be generated on the basis of only 3-4 genera, as reflected by PCR-DGGE analysis. Also for this reason, CLPPs based on incubations of soil suspensions should just be used as a screening method and always be accompanied by other techniques for community analysis.