Quantitative Isolation of Microbial DNA from Different Types of Soils of Natural and Agricultural Ecosystems

A novel procedure was developed for direct quantitative isolation of microbial DNA from soil. This technique was used to evaluate microbial DNA pools in soils of contrasting types (chernozems and brown forest soils) under different anthropogenic loads. A strong correlation was found between microbial biomass and DNA contents in soils of different types (R ²= 0.799). The ratio of soil CO₂ emission rate to the amount of extractable DNA in the soil was shown to reflect the physiological state of the soil microbial community; this ratio can be used as an ecophysiological parameter similarly to the metabolic quotient qCO₂.     

设施菜田与棚外粮田土壤菌群和反硝化气体产生的比较分析

【目的】对比设施菜田与棚外粮田土壤菌群以及N2O产生模式的差异。【方法】采用变性梯度凝胶电泳(DGGE)和反硝化功能基因(nirS,nosZ)方法分别比较两种土壤细菌群落以及功能基因类群丰度的差异,利用自动连续在线培养监测体系(Robot系统)测定两种土壤在好氧、厌氧阶段N2O等反硝化相关气态产物产生模式,分析N2O/(N2+N2O+NO)产物比。【结果】设施菜田与棚外粮田具有不同的土壤细菌群落结构,并且土壤细菌总量得到了显著的提升,然而两种反硝化功能基因(nirS,nosZ)丰度并没有显著变化。与设施菜田相比,棚外粮田有相对低的N2O积累量以及产物比,并且在厌氧初期气体产生模式有所不同。培养后铵态氮和亚硝态氮含量上升。【结论】设施菜田长期有别于棚外粮田的管理方式造成了土壤细菌群落的显著改变,增大了活跃微生物总量,造成土壤酸化,并导致N2O在气态产物中的比例升高。设施菜田土壤微生物进行了与棚外粮田不同的硝酸盐呼吸过程,异化硝酸盐还原成铵(DNRA)过程有可能贡献了两种土壤的部分厌氧N2O产生量。     

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent–bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.     

Extractable microbial DNA pool and microbial activity in paleosols of Southern Ural

An evaluation of microbial DNA pools was performed using direct quantitative isolation of DNA from contemporary soils of Southern Urals and paleosols sealed under burial mounds of early Bronze Age more than 5000 years B.P. Significant regression dependence was found between the biomass and DNA contents in these soils (R2 = 0.97). Activity and dominant ecological strategies of microbial communities of paleosols and contemporary southern black soil were compared from growth parameters obtained by analysis of respiratory curves. The ratio of maximum specific growth rates of soil microorganisms on glucose and on yeast extract was shown to provide an auxotrophy index for soil microbial communities.     

Microbial resource allocation for phosphatase synthesis reflects the availability of inorganic phosphorus across various soils

According to the resource allocation model for extracellular enzyme synthesis, microorganisms should preferentially allocate their resources to phosphorus (P)-acquiring enzyme synthesis when P availability is low in soils. However, the validity of this model across different soil types and soils differing in their microbial community composition has not been well demonstrated. Here we investigated whether the resource allocation model for phosphatase synthesis is applicable across different soil types (Andosols, Acrisols, Cambisols, and Fluvisols) and land uses (arable and forest), and we examined which soil test P and/or P fraction microorganisms responded to when investing their resources in phosphatase synthesis in the soils. The ratio of alkaline phosphatase (ALP) to β-d-glucosidase (BG) activities in the arable soils and the ratio of acid phosphatase (ACP) to BG activities in the forest soils were significantly negatively related with the available inorganic P concentration. We also observed significant effects of available inorganic P, pH, soil types, and land uses on the (ACP + ALP)/BG ratio when the data for the arable and forest soils were combined and used in a stepwise multiple regression analysis. These results suggest that microbial resource allocation for phosphatase synthesis is primarily controlled by available inorganic P concentration and soil pH, but the effects of soil types and land uses are also significant.     

Soil Microbial Biomass,Basal Respiration and Enzyme Activity of Main Forest Types in the Qinling Mountains

Different forest types exert essential impacts on soil physical-chemical characteristics by dominant tree species producing diverse litters and root exudates, thereby further regulating size and activity of soil microbial communities. However, the study accuracy is usually restricted by differences in climate, soil type and forest age. Our objective is to precisely quantify soil microbial biomass, basal respiration and enzyme activity of five natural secondary forest (NSF) types with the same stand age and soil type in a small climate region and to evaluate relationship between soil microbial and physical-chemical characters. We determined soil physical-chemical indices and used the chloroform fumigation-extraction method, alkali absorption method and titration or colorimetry to obtain the microbial data. Our results showed that soil physical-chemical characters remarkably differed among the NSFs. Microbial biomass carbon (Cmic) was the highest in wilson spruce soils, while microbial biomass nitrogen (Nmic) was the highest in sharptooth oak soils. Moreover, the highest basal respiration was found in the spruce soils, but mixed, Chinese pine and spruce stands exhibited a higher soil qCO₂. The spruce soils had the highest Cmic/Nmic ratio, the greatest Nmic/TN and Cmic/Corg ratios were found in the oak soils. Additionally, the spruce soils had the maximum invertase activity and the minimum urease and catalase activities, but the maximum urease and catalase activities were found in the mixed stand. The Pearson correlation and principle component analyses revealed that the soils of spruce and oak stands obviously discriminated from other NSFs, whereas the others were similar. This suggested that the forest types affected soil microbial properties significantly due to differences in soil physical-chemical features.     

Extractable Microbial DNA Pool and Microbial Activity in Paleosols of Southern Urals

An evaluation of microbial DNA pools was performed using direct quantitative isolation of DNA from contemporary soils of Southern Urals and paleosols sealed under burial mounds early in the Bronze Age more than 5000 years B.P. Significant regression dependence was found between the biomass and DNA contents in these soils (R ²= 0.97). Activity and dominant ecological strategies of microbial communities of paleosols and contemporary southern black soil were compared from growth parameters obtained by analysis of respiratory curves. The ratio of maximum specific growth rates of soil microorganisms on glucose and on yeast extract was shown to provide an auxotrophy index for soil microbial communities.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Influence of Drying–Rewetting Frequency on Soil Bacterial Community Structure

Soil drying and rewetting represents a common physiological stress for the microbial communities residing in surface soils. A drying–rewetting cycle may induce lysis in a significant proportion of the microbial biomass and, for a number of reasons, may directly or indirectly influence microbial community composition. Few studies have explicitly examined the role of drying–rewetting frequency in shaping soil microbial community structure. In this experiment, we manipulated soil water stress in the laboratory by exposing two different soil types to 0, 1, 2, 4, 6, 9, or 15 drying–rewetting cycles over a 2-month period. The two soils used for the experiment were both collected from the Sedgwick Ranch Natural Reserve in Santa Ynez, CA, one from an annual grassland, the other from underneath an oak canopy. The average soil moisture content over the course of the incubation was the same for all samples, compensating for the number of drying–rewetting cycles. At the end of the 2-month incubation we extracted DNA from soil samples and characterized the soil bacterial communities using the terminal restriction fragment length polymorphism (T-RFLP) method. We found that drying–rewetting regimes can influence bacterial community composition in oak but not in grass soils. The two soils have inherently different bacterial communities; only the bacteria residing in the oak soil, which are less frequently exposed to moisture stress in their natural environment, were significantly affected by drying–rewetting cycles. The community indices of taxonomic diversity and richness were relatively insensitive to drying–rewetting frequency. We hypothesize that drying–rewetting induced shifts in bacterial community composition may partly explain the changes in C mineralization rates that are commonly observed following exposure to numerous drying–rewetting cycles. Microbial community composition may influence soil processes, particularly in soils exposed to a significant level of environmental stress.     

不同土地利用方式下赤红壤生物学性状及其与土壤肥力的关系

以我国华南地区典型坡地的赤红壤为对象,研究了不同土地利用方式(新垦旱地、灌木林、桉树林及果园)下土壤微生物性状、酶活性及其与土壤肥力的关系.结果表明: 不同土地利用方式下土壤生物学性质差异极显著.其中,果园土壤的微生物数量和酶活性显著增加;新垦旱地土壤的呼吸速率显著增加,微生物数量、酶活性显著下降;灌木林和桉树林则介于果园和新垦旱地之间,且两者具有高度的相似性.不同土地利用方式下的土壤微生物数量和酶活性与土壤有机碳、大部分养分之间存在显著正相关;高有机质含量和高肥力水平的土壤有利于微生物的生长和酶活性的提高.     

接种泡囊假单胞菌对土壤生物性质及A. corsicum吸收Ni的影响

采用室内模拟试验方法,研究了在水稻土、元江土和墨江土中添加泡囊假单胞菌（Pseulormanas vesicularis）后土壤中微生物种群数量、土壤酶活性和镍超积累植物Alyssum corsicum对土壤镍的富集效果.土壤接种泡囊假单胞菌70d后,水稻土中DTPA提取态镍较对照土中的明显减少、元江土和墨江土中的有所减少;土壤中细菌、真菌和放线菌数量增加,5种土壤酶活性提高.试验结果表明,水稻土、元江土、墨江土添加泡囊假单菌后植物地上部生物量较对照分别增加了29%、309%和43%,进而提高了A.corsicum自土壤中富集镍的效率：水稻土中增加54%,元江土中增加306%,墨江土中增加32%.泡囊假单胞菌这一新用途的发现,可为植物修复微生物制剂和基因工程菌的开发提供本土的微生物的菌种资源.     

Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment

A bacterium, isolated from contaminated soils around a chemical factory and named strain DSP3 was capable of biodegrading both chlorpyrifos and 3,5,6-trichloro-2-pyridinol. Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences, DNA G+C content, and DNA homology between strain DSP3 and reference strains, strain DSP3 was identified as Alcaligenes faecalis. Chlorpyrifos was utilized as the sole source of carbon and phosphorus by strain DSP3. We examined the role of strain DSP3 in the degradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol under different culture conditions. Parathion and diazinon could also be degraded by strain DSP3 when provided as the sole sources of carbon and phosphorus. An addition of strain DSP3 (10(8)cells g(-1)) to soil with chlorpyrifos (100 mg kg(-1)) resulted in a higher degradation rate than the one obtained from non-inoculated soils. Different degradation rates of chlorpyrifos in six types of treated soils suggested that soils used for cabbage growing in combination with inoculation of strain DSP3 showed enhanced microbial degradation of chlorpyrifos.     

冻融期去根处理对小兴安岭6种林型土壤微生物量的影响

春季冻融期,在小兴安岭的阔叶红松(Pinus koraiensis)林、谷地云冷杉(Picea koraiensis-Abies nephrolepis)林、阔叶红松择伐林、白桦(Betula platyphylla)次生林、红松人工林、兴安落叶松(Larix gmelinii)人工林的去根处理样地和对照样地进行野外取土实验,分析了根去除对上述林型土壤微生物量的影响以及与土壤环境因子的关系。结果表明:冻融循环期间对照样地和去根处理样地的林型、土壤层次、取样时间均显著地影响土壤微生物量碳(MBC)(P0.05),对照样地中各林型的土壤微生物量氮(MBN)差异显著,而去根处理样地中各林型的MBN没有显著差异(P0.05);冻融循环期间去根处理显著地减少了大部分林型及土层(谷地云冷杉林0—10 cm及择伐林外)的MBC,而去根处理对大部分林型及土层(阔叶红松林0—10 cm,谷地云冷杉林和择伐林的10—20 cm除外)的MBN没有显著影响。说明在小兴安岭春季冻融期根系对土壤微生物量的影响不可忽视。     

水热增加下黑土细菌群落共生网络特征

黑土是有机质含量高且肥沃的土壤类型之一,气候变化会显著改变黑土中微生物群落的结构,同时影响群落间的潜在相互作用关系。[目的] 揭示水热增加对黑土中的细菌群落结构及潜在互作关系的影响。[方法] 基于土壤移置试验,采用16S rRNA高通量测序解析农田黑土（原位黑土、水热增加1和水热增加2）中的细菌群落结构对水热增加的响应;使用CoNet构建微生物群落共生网络,识别共生网络中的枢纽微生物;利用结构方程模型、相关性分析探究水热条件变化下土壤性质、微生物交互作用、多样性之间的直接、间接关系。[结果] 黑土中的微生物以疣微菌、变形杆菌、酸性杆菌和放线菌为主。水热增加下土壤微生物共生网络的拓扑性质发生显著变化,网络中表征微生物潜在竞争关系的负连线随着水热增加而显著增加。气候因素通过改变微生物潜在相互作用影响了群落水平分类多样性。物种竞争增强可能直接导致了土壤有机碳含量的降低。[结论] 水热增加会显著改变黑土中微生物之间的潜在交互作用,枢纽微生物的响应更加敏感。     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2, 4-D

The responses of three different soil microbial communities to the experimental application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2, 4-D, and one had no prior direct application of any herbicide. Dominant 2, 4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2, 4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2, 4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

百菌清对落叶松人工防护林土壤微生物群落的影响

利用稀释平板法和Biolog-Eco检测技术,分析了喷施百菌清对落叶松人工防护林土壤微生物群落的影响。结果表明,土壤微生物中细菌数量占据绝对优势,其数量直接影响落叶松人工防护林土壤微生物总量的变化趋势。总体上,百菌清对细菌的数量具有抑制作用。喷洒后0d,仅抑制表层与上层(0-10cm)土壤细菌的增殖;喷洒后2个月,对表层与上层土壤细菌的抑制作用减弱,而喷洒后4个月时,其抑制作用最强。喷洒后0d与2个月时,百菌清对表层与上层土壤真菌数量的增殖均具有促进作用。喷洒后2个月、4个月时均对下层土壤放线菌数量具有抑制作用。将土壤3大类群微生物数量、土壤理化指标及群落多样性指数进行相关分析表明,土壤中有效磷和速效钾与放线菌数量、水解氮与细菌数量存在显著正相关性,而与群落多样性无显著相关性。喷洒百菌清2个月后,对表层土壤微生物群落碳源利用率的影响不显著,而对上层(0-10cm)土壤微生物存在显著影响,下层(10-20cm)土壤微生物群落的碳源利用率在百菌清喷洒2个月与4个月后具有显著差异;而不同土层微生物群落对碳源利用率的差异不显著。微生物群落利用31种碳源的主成分分析表明,喷洒百菌清后,微生物群落利用单一碳源的能力显著提高,且喷洒2个月后微生物群落对某些碳源如肝糖与2-羟基苯甲酸等的利用率明显增加。方差分析表明,喷洒百菌清后,不同作用时间及不同处理,不同土层间土壤微生物对碳源的利用率具有显著差异。喷洒百菌清前(5月)土壤微生物对多聚物与氨基酸、碳水类、芳香类及胺类的利用率差异较大(P<0.05),且对多聚物的利用率最高;处理组的表层土壤微生物对碳源的利用率高于其它处理组;而喷洒2个月后,对胺类、芳香类的利用率与其它4类碳源具有显著差异。     

天台山不同林型土壤微生物区系及其商值(qMB,qCO2)

研究了天台山8种林型下土壤微生物数量、微生物量碳、微生物量氮、微呼吸速率和微生物商值(qMB,qCO2),结果表明,均以云锦杜鹃林、黄山松林、茶园和竹林土壤中较大,以柳杉林土壤最小。天然林土壤的上述指标的平均水平均高于人工林土壤,所产生的差异是不同林型的立地条件和土壤性状综合影响的结果qCO2值以柳杉林、日本花柏林和茶园土壤最高(4．52％,4．43％,4．26％),云锦杜鹃林、黄山松林和七子花林土壤相对较低(3．475％,3．82％,3．70％),比前者土壤质量较好,可持续利用潜力大。     

3 次连续重复提取DNA 能较好反映土壤微生物丰度

【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。     

The Dry Limit of Microbial Life in the Atacama Desert Revealed by Calorimetric Approaches

The Atacama desert in Chile is one of the driest and most lifeless environments on Earth. It rains possibly once a decade. NASA examined these soils as a model for the Martian environment by comparing their degradation activity with Martian soil and looking for “the dry limit of life”. The existence of heterotrophic bacteria in Atacama soil was demonstrated by DNA extraction and by the isolation of microorganisms. So far, however, no data have been available about the metabolic activities in these soils due to the limitations of the existing methodologies when applied to desert soils. Calorimetry was used to obtain information on the metabolic and thermal properties of eleven soil samples collected at different sites in the Atacama desert. Differential scanning calorimetry and isothermal calorimetry were employed to determine the pyrolysis properties of the carbon‐containing matter and to measure biomass and microbial metabolism. They were compared to other soil properties such as total carbon and nitrogen, carbon to nitrogen ratio and pH. There was measurable organic matter in nine of the eleven samples and the heat of pyrolysis of those soils was correlated to the carbon content. In five of the eleven samples no biomass could be detected and the existence of basal microbial metabolism could not be established because all samples showed endothermic activity, probably from inorganic reactions with water. Six samples showed microbial activation after the addition of glucose. Carbon content, nitrogen content and the microbial activity after glucose amendment were correlated to the altitude and to the average minimum temperature of the sampling sites calculated from meteorological data. The detectable microbial metabolism was more dissipative with increasing altitude and decreasing temperature.     

黄土丘陵区植被类型与土壤微生物区系及生物量的关系

以陕西延安黄土丘陵区5种不同植被类型(人工刺槐林、天然侧柏林、天然辽东栎林、灌丛和裸地)为研究对象,分析了土壤微生物生物量碳、氮含量、细菌和真菌的丰度变化规律及其与土壤基本化学性质的关系。结果表明:(1)4种植被类型的土壤质量较之裸地都有不同程度的改善,总体趋势:天然林地人工林地裸地;(2)土壤微生物生物量碳、氮的总体趋势:天然林地最高,人工林次之,裸地最低,且与土壤有机碳(SOC)、全氮(TN)和速效磷(AP)极显著正相关(P0.01);(3)裸地土壤的细菌丰度最低,人工刺槐林真菌含量显著低于天然辽东栎林。细菌丰度与土壤营养状况呈显著正相关(P0.05),而真菌与土壤营养无明显相关性,只与土壤pH负相关。说明在该研究区域,植被类型与土壤质量对微生物资源都具有不同程度的作用。