BIOLOG在土壤微生物群落功能多样性研究中的应用

一直以来,微生物的研究人员都无法解决关于微生物群落实验中的一些技术问题。BIOLOG技术的出现,为研究人员提供了新的研究思路。为了更好地描述土壤的微生物群落所具有的多样性功能,生物学家致力于对该实验的研究。BIOLOG技术是一种比较简单且快捷的研究方法,能够为研究人员提供比较基础的培养方法,使微生物在实验中反映出微生物群落的代谢多样性。笔者从BIOLOG技术释义角度出发,深入研究了实验研究的相关原理,找出了实验中将会存在的问题,并给出了合理的解决措施,期望能够为土壤的微生物群落实验提供借鉴。     

土壤微生物群落多样性研究方法及进展

微生物多样性是指群落中的微生物种群类型和数量、种的丰度和均度以及种的分布情况。研究土壤微生物群落多样性的方法包括传统的以生化技术为基础的方法(直接平板计数、单碳源利用模式等)和以现代分子生物技术为基础的方法(从土壤中提取DNA,进行G C%含量的分析,或杂交分析,或进行PCR,产物再进行DGGE/TGGE等分析)。现代生物技术与传统微生物研究方法的结合使用,为更全面地理解土壤微生物群落的多样性和生态功能提供了良好的前景。     

长江三角洲地区污水污泥与健康安全风险研究V．污泥施用对土壤微生物群落功能多样性的影响

利用BIOLOG测试方法对长江三角洲地区施污泥土壤的微生物群落功能多样性进行了初步探讨。研究结果表明,施用污泥后土壤微生物群落代谢剖面、多样性指数、动力学参数均发生了变化,说明微生物对单一碳源底物的利用能力发生了改变。微生物群落功能多样性动力学研究结果发现,供试土壤的微生物群落代谢剖面（AWCD）与培养时间之间呈非线性关系,其变化过程符合微生物种群生长动态模型（S型）。除滩潮土的杭州新鲜消化污泥和杭州风干污泥处理外,其他污泥处理均使土壤微生物群落功能多样性动力学参数K和r值降低,S值增大。土壤微生物群落功能多样性特征的变化可以较好地显示施用污泥后土壤微生物群落对碳源利用能力和模式的差异,反映施用污泥特定生境土壤微生物群落功能多样性的变化。     

长江三角洲地区污水污泥与健康安全风险研究Ⅴ.污泥施用对土壤微生物群落功能多样性的影响

利用BIOLOG测试方法对长江三角洲地区施污泥土壤的微生物群落功能多样性进行了初步探讨。研究结果表明,施用污泥后土壤微生物群落代谢剖面、多样性指数、动力学参数均发生了变化,说明微生物对单一碳源底物的利用能力发生了改变。微生物群落功能多样性动力学研究结果发现,供试土壤的微生物群落代谢剖面(AWCD)与培养时间之间呈非线性关系,其变化过程符合微生物种群生长动态模型(S型)。除滩潮土的杭州新鲜消化污泥和杭州风干污泥处理外,其他污泥处理均使土壤微生物群落功能多样性动力学参数K和r值降低,S值增大。土壤微生物群落功能多样性特征的变化可以较好地显示施用污泥后土壤微生物群落对碳源利用能力和模式的差异,反映施用污泥特定生境土壤微生物群落功能多样性的变化。     

不同土地利用方式对红壤微生物多样性的影响

试验采用碳素利用(BIOLOG)及磷酸酯脂肪酸(PLFA)法,研究了8种供试红壤微生物群落的功能多样性和结构多样性。两种方法均表明土地利用方式能显著影响微生物的多样性。采用磷酸酯脂肪酸法无需培养就能鉴别土壤微生物的组成结构,试验表明总PLFA含量与微生物生物量呈显著相关。碳素利用法的研究结果显示,茶叶园土中微生物对各类碳源的利用能力均很低,呈现出非常独特的微生物功能多样性。     

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。     

土壤微生物生态学研究方法进展

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。     

菌剂与缓释肥配施对复垦土壤微生物生态的影响

在盆栽模拟复垦的条件下,利用传统平板计数法与BIOLOG方法相结合研究菌剂与缓释肥配施对土壤微生物生态的影响。结果表明,菌剂与缓释肥配施能显著提高土壤细菌、真菌和放线菌的数量。同时对土壤酶活性也具有有明显的影响。就脲酶而言,由于缓释肥与菌剂的配合施用,抑制了其活性,延长了缓释肥的肥效。通过对磷酸酶的研究发现,由于菌剂的加入促进了磷酸酶的活性,菌剂与缓释肥配施处理的土壤磷酸酶活性显著高于纯化肥处理的。通过多样性指数的测定表明菌剂与缓释肥配施处理土壤微生物群落的Shannon多样性指数、Simpson多样性指数和McIntosh均匀度等各项指标均显著高于其他处理的。说明菌剂与缓释肥配施不但可以提高土壤微生物功能多样性,而且还可以提高土壤微生物群落均匀度,从而可促进土壤微生物生态系统的稳定,提高土壤微生物群落的生态功能。     

BIOLOG微平板技术在土壤微生态研究中的应用

BIOLOG微平板技术是一种新型的鉴定物种群落结果多样性和均匀度的方法。随着土壤微生态研究的发展,这种方法在土壤微生态的研究上同样能够起到很好的效果。通过实验表明,应用BIOLOG技术可以明确反映土壤微生态的变化情况。与传统的平板计数法相比,可以更加有效地反映土壤微生物活性。     

秸秆施用下接种蚯蚓对农田土壤微生物特性的影响

在连续6年稻麦轮作系统中，研究不同秸秆施用方式下接种蚯蚓对土壤微生物生物量、活性（基础呼吸）及群落功能多样性（BIOLOG单一碳源利用指纹方法）的影响，试验设5个处理：对照（CK）、秸秆表施（M）、秸秆混施（I）、秸秆表施且接种蚯蚓（ME）、秸秆混施且接种蚯蚓（IE）。不同秸秆施用下接种蚯蚓均对土壤微生物生物量、微生物生物活性和群落碳源利用能力产生显著影响：两种秸秆施用方式下接种蚯蚓均增加微生物生物量；秸秆表施并接种蚯蚓导致微生物活性、碳源利用丰富度和多样性指数均降低，而在秸秆混施下则均升高；BIOLOG碳源利用分析结果表明在秸秆施用下接种蚯蚓后土壤的微生物群落组成发生明显变化。     

Changes of the functional diversity of soil microbial community during the colonization of abandoned grassland by a forest

The impact of secondary succession of grassland communities towards a Norway spruce forest on soil microbial community was studied on a belt transect established in the Pol’ana Mts., Central Europe. Data on understory vegetation, light availability, soil properties and microbial activity were collected on 147 plots distributed over regular grid. Moreover, distributions of functional groups of microorganisms were assessed using BIOLOG analysis on a subset of 27 plots. Mantel partial correlations between microbial community indicators and environmental variables showed that microbial activity generally decreased with increasing tree density and size, whereas it increased with increasing radiation at the soil surface, soil temperature, and cover and diversity of understory vegetation. Functional richness and diversity of microorganisms were positively correlated with solar radiation, but also with plant species richness and diversity. Abundance of several functional groups correlated closely with succession-related variables. Redundance analysis of microbial data provided slightly different outcomes. Forward selection yielded only two environmental variables significantly influencing the composition of the microbial community: tree influence potential and organic carbon content. Abundances of several functional microbial groups correlated with tree influence, documenting that microbial community changes are at least partially driven by the colonization of grassland by trees. Nevertheless, the relative importance of abiotic environment change and plant community succession on microbial community dynamics remains unresolved.     

植物源有机物料对果园土壤微生物群落多样性的影响

【目的】苹果园土壤肥力持续下降,禽畜废弃物肥源严重不足,施用植物源有机肥成为生产中改善果园土壤状况的重要措施之一。本试验利用BIOLOG微平板技术研究了盆栽条件下添加植物源有机物料及其腐殖化过程驱动因子对果园土壤微生物群落多样性的影响,探讨葡萄糖、 尿素和蚯蚓在植物源有机物料向土壤碳库转化中的作用,为揭示果园土壤质量的演变机制提供参考。【方法】取苹果园020 cm土层土壤,与苹果枝条、 玉米秸秆和果园杂草粉碎物混合,栽植2a生苹果砧木山定子幼苗,分别添加尿素、 葡萄糖和蚯蚓,利用BIOLOG微平板技术进行土壤微生物群落多样性分析。不同处理的土壤浸提液在 BIOLOG生态测试板中培养,取培养96 h时微平板光密度值进行多样性指数计算,分别用丰富度指数S 表示被微生物群落利用的基质数量,多样性指数表示反应孔与对照孔光密度值之差和整块板总差的比值,均匀度指数 E表示可培养微生物的种类均匀度,优势度指数Ds用于评估某些最常见种的优势度。对土壤微生物群落利用BIOLOG微平板中六类碳源（碳水化合物类、 氨基酸类、 羧酸类、 多聚物类、 芳香族类和胺类）的情况进行主成分分析,明确不同处理微生物对碳源利用能力的差异。【结果】有机物料种类、 小分子有机物和蚯蚓数量均对平均吸光值（AWCD值）有显著影响,在培养0~24 h,玉米秸秆+葡萄糖+12条蚯蚓（T4）和果园杂草+葡萄糖（T9）处理的AWCD值明显高于其他处理,微生物群落的活性较强,碳源开始利用较早。2496 h时,AWCD呈指数增长,120 h后趋于平缓,以玉米秸秆+葡萄糖+12条蚯蚓（T4）、 苹果枝条+葡萄糖+6条蚯蚓（T2）、 果园杂草+葡萄糖（T9）处理斜率最大,其次为玉米秸秆+尿素+6条蚯蚓（T6）和苹果枝条+尿素（T1）处理; 小分子有机物种类对微生物群落丰富度指数（S）和优势度指数（Ds）的影响显著,丰富度指数（S）以苹果枝条+葡萄糖+6条蚯蚓（T2）最大,优势度指数（Ds）以玉米秸秆+葡萄糖+12条蚯蚓（T4）最大,各处理的多样性指数（H）和均一度指数（E）差异不显著; 对碳源利用主成分起分异作用的主要是碳水化合物类和多聚物类。【结论】与秸秆和杂草处理相比,苹果枝条处理土壤微生物群落多样性较丰富,加入葡萄糖为土壤微生物提供可迅速利用的碳源,微生物功能多样性也显著增加; 蚯蚓活动对微生物群落多样性的影响比葡萄糖小,尿素对微生物群落多样性的影响也较小,但同时添加尿素和葡萄糖有助于微生物多样性的增加。     

种植方式对玉米不同生长期土壤微生物群落功能多样性的影响

为探讨玉米不同种植方式下土壤微生物群落功能多样性的差异,进行田间定点试验,采用Biolog方法分别研究了4行轮作、4行连作、8行轮作和8行连作的种植方式对玉米种植前、拔节期、抽穗期和收获期土壤微生物功能多样性的影响。结果表明:4种种植方式的土壤微生物均在种植前代谢活性最弱、功能多样性最低,在玉米抽穗期土壤微生物代谢活性最强,功能多样性最高。在种植玉米前,轮作的土壤微生物代谢活性和功能多样性高于连作,8行轮作和4行轮作土壤微生物的物种多样性指数分别比相应的连作高22.93%和11.42%;4行轮作的土壤微生物物种多样性指数比8行轮作低3.17%,而4行连作比8行连作高6.83%。在玉米拔节期、抽穗期及收获期连作土壤微生物功能多样性略高于轮作,且有4行连作大于8行连作的趋势,但差异均未达显著水平。种植前,4种种植模式的土壤微生物对6大类碳源的利用程度整体上都较低,降解碳水化合物类、羧酸类和聚合物类碳源的微生物是种植方式影响的主要土壤微生物类群;随着玉米的生长,土壤微生物对6大类碳源的利用都逐渐增强,玉米拔节期、抽穗期和收获期之间土壤微生物特征碳源没有较大差异,4种种植方式的土壤微生物对聚合物类碳源利用程度差异都不显著。PLS-EDA分析结果表明种植方式对土壤微生物产生较大影响,种植前8行轮作和4行连作的土壤微生物碳源利用模式具有相似性;种植玉米后4种种植方式的土壤微生物对碳源的利用模式存在较大差异,其中4行连作的土壤微生物在玉米拔节期和收获期对碳源的利用模式与其他3种种植方式差异最大。试验说明作物长期连作栽培会影响土壤微生物群落功能,降低土壤微生物物种多样性,引起土壤微生物群落结构与功能的失调。     

生物有机肥对土壤微生物活性的影响

通过两次连续温室玉米盆栽试验,研究了施用具有调节微生物功能的生物有机肥对土壤微生物数量与活性的影响,并利用传统平板计数法与BIOLOGECO方法相结合研究生物有机肥对土壤微生物生态的影响。结果表明,与化肥相比,施用生物有机肥可显著提高土壤微生物中3大菌群的数量;AWCD值及微生物对不同碳底物利用水平的测定结果表明,施用生物有机肥可明显提高土壤微生物对碳源的利用率,尤其土壤中的羧酸、胺类和其他类碳源等。表明生物有机肥的施用能增加土壤微生物利用碳源能力,改善微生物营养条件,使微生物保持较高活性,提高土壤微生物多样性。     

Effects of Ant Mounds on the Plant and Soil Microbial Community in an Alpine Meadow of Qinghai–Tibet Plateau

Ants are important soil engineers, affecting the structure and function of ecosystems. To address the impacts of ants (Camponotus herculeanus ) on the properties of an alpine meadow ecosystem of Qinghai–Tibet Plateau, we investigated the effects of ant mounds on plant biomass, soil physicochemical properties, microbial diversity, and functions. We found that the total biomass of plant community was significantly greater in ant mound periphery. Plant species richness in ant mounds was reduced compared with that of control plots without ant mounds. Significant changes in physicochemical properties of soil were also observed. Soil organic matter, total nitrogen, available phosphorous, total potassium, and available potassium increased in ant mound soil due to the excavation activities by ants as well as the accumulation of organic matter and other nutrients during mound construction. For example, roots/soil contents (g/g) and soil moisture in ant mound soils were lower than those in controls. Microbial community composition and microbial biomass were clearly changed in ant mound soils. BIOLOG analysis further indicated that the functional diversity of the microbial community of ant mound soil increased and differed from that of controls. This study indicates that ant‐induced modification of soil properties indirectly influences plant biomass and species composition, and ant mounds have different microbial communities from those of control soil. Copyright © 2016 John Wiley & Sons, Ltd.     

Changes in Microbial Functional Diversity and Activity in Paddy Soils Irrigated with Industrial Wastewaters in Bandung, West Java Province, Indonesia

Characteristics, such as microbial biomass, basal respiration, and functional diversity of the microbial communities, were investigated in paddy soils located in Bandung, West Java Province, Indonesia, that have been heavily polluted by industrial effluents for 31 years. Paddy soil samples (10?C20 cm) were taken from two sites: polluted soils and unpolluted soils (as control sites). The polluted soils contained higher salinity, higher sodicity, higher nutrient contents, and elevated levels of heavy metals (Cr, Mn, Ni, Cu, and Zn) than unpolluted soils. Soil physicochemical properties, such as maximum water holding capacity, exchangeable sodium percentage, sodium adsorption ratio, and swelling factor, in polluted soils were much greater than those in unpolluted soils (P?<?0.05). Changes in the physical and chemical soil properties were reflected by changes in the microbial communities and their activities. BIOLOG analysis indicated that the functional diversity of the microbial community of polluted soils increased and differed from that of unpolluted soils. Likewise, the average rate of color development (average well color development), microbial biomass (measured as DNA concentration), and the soil CO₂ respiration were higher in polluted soils. These results indicate that major changes in the chemical and physical properties of paddy soils following the application of industrial wastewater effluents have had lasting impacts on the microbial communities of these soils. Thus, the increased activity, biomass, and functional diversity of the microbial communities in polluted soils with elevated salinity, sodicity, and heavy metal contents may be a key factor in enhancing the bioremediation process of these heavily polluted paddy soils.     

DEHP对土壤脱氢酶活性及微生物功能多样性的影响

选用肥熟旱耕人为土(黄棕壤),设置了在土壤中施加100 mg kg-1 DEHP与不施加DEHP两个水平,盆栽试验研究了DEHP对土壤脱氢酶活性以及土壤微生物群落功能多样性的影响,以及植物在污染土壤中的修复作用。结果表明,施加DEHP显著抑制了土壤脱氢酶活性,30 d时与对照相比降低了约30%, 第60 d时尽管有缓慢的回升,但仍明显低于对照(p<0.05)。从BIOLOG反应的结果可以看出,DEHP也显著影响土壤微生物的功能多样性,土壤微生物群落的Shannon指数、Simpson指数、McIntosh指数和均度均显著低于无污染的对照,说明DEHP的污染导致了土壤微生物群落功能多样性的下降。种植植物对土壤脱氢酶和微生物活性有很明显的促进作用,并且在一定程度上缓解了DEHP的毒害作用,但并未消除DEHP对土壤微生物的影响。     

Does altered rainfall regime change pesticide effects in soil? A terrestrial model ecosystem study from Mediterranean Portugal on the effects of pyrimethanil to soil microbial communities under extremes in rainfall

In this century, agroecosystems are subjected to multiple global change stressors acting in concert such as alterations in rainfall regimes and pesticide use. Alterations in rainfall regimes, characterised by more extreme intra-annual rainfall regimes, have been forecasted for the Mediterranean region. At the same time, the use of pesticides continues to rise. Here, we report the responses of soil microbial community to a model pesticide, i.e., fungicide pyrimethanil (PYR) under altered rainfall regimes (i.e., drought and heavy rainfall) two and eight weeks after PYR application. We measured the functional responses as enzyme activities, potential nitrification and BIOLOG carbon substrate utilisation. We also characterised the soil bacterial communities using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) method. After two weeks, enzyme activities were mainly responsive to PYR and kinetic parameters, calculated from BIOLOG carbon substrate utilisation, indicated interaction effects from PYR and rain treatments. Bacterial band richness increased with PYR treatment under normal rain and drought regimes, but bacterial band richness was higher at 1X than 5X PYR under heavy rainfall. Bacterial community structure was also different with the PYR and rainfall treatments. By week eight, PYR treated soils remained functionally different from untreated soils. Bacterial band richness was consistent across PYR treatment regardless of rain regime. However, the bacterial community structure remained significantly different among the PYR treatments under different rain regimes. We conclude that rainfall extremes can alter the effect of PYR on the soil microbial community structure without altering PYR effects on soil functions (measured as enzyme activities, potential nitrification and BIOLOG carbon substrate utilisation).     

Plant biomass, soil water content and soil N:P ratio regulating soil microbial functional diversity in a temperate steppe: A regional scale study

Soil microorganisms are influenced by various abiotic and biotic factors at the field plot scale. Little is known, however, about the factors that determine soil microbial community functional diversity at a larger spatial scale. Here we conducted a regional scale study to assess the driving forces governing soil microbial community functional diversity in a temperate steppe of Hulunbeir, Inner Mongolia, northern China. Redundancy analysis and regression analysis were used to examine the relationships between soil microbial community properties and environmental variables. The results showed that the functional diversity of soil microbial communities was correlated with aboveground plant biomass, root biomass, soil water content and soil N: P ratio, suggesting that plant biomass, soil water availability and soil N availability were major determinants of soil microbial community functional diversity. Since plant biomass can indicate resource availability, which is mainly constrained by soil water availability and N availability in temperate steppes, we consider that soil microbial community functional diversity was mainly controlled by resource availability in temperate steppes at a regional scale.