Microbial diversity in alpine tundra wet meadow soil: novel Chloroflexi from a cold, water-saturated environment

Cold, water-saturated soils play important biogeochemical roles, yet almost nothing is known about the identity and habitat of microbes active under such conditions. We investigated the year-round microenvironment of an alpine tundra wet meadow soil in the Colorado Rocky Mountains, focusing on the biogeochemistry and microbial diversity of spring snowmelt--a dynamic time for alpine ecosystems. In situ measurements revealed spring and autumn periods of long-term temperature stability near 0 degrees C, and that deeper soil (30 cm) was more stable than surface soil, with more moderate summers and winters, and longer isothermal phases. The soil was saturated and water availability was limited by freezing rather than drying. Analyses of bioavailable redox species showed a shift from Mn reduction to net Fe reduction at 2-3 cm depth, elevated SO4(2-) and decreased soluble Zn at spring snowmelt. Terminal restriction fragment length polymorphism profiles detected a correlated shift in bacterial community composition at the surface to subsurface transition. Bacterial and archaeal small-subunit rRNA genes were amplified from saturated spring soil DNA pooled along a depth profile. The most remarkable feature of these subsurface-biased libraries was the high relative abundance of novel, uncultivated Chloroflexi-related sequences comprising the third largest bacterial division sampled, and representing seven new Chloroflexi subdivisions, thereby dramatically expanding the known diversity of this bacterial division. We suggest that these novel Chloroflexi are active at near -0 degrees C temperatures, under likely anoxic conditions, and utilize geochemical inputs such as sulfide from upslope weathering.     

土壤中甲烷厌氧氧化菌多样性的分子检测

甲烷厌氧氧化作用是减少海洋底泥甲烷释放的重要生物地球化学过程,然而在陆地生态系统中甲烷厌氧氧化作用及其功能菌群的生态功能仍然不确定。对甲烷厌氧氧化菌多样性的研究可为减少甲烷排放提供重要科学依据。与传统的分离培养方法比较,分子检测方法是一种更为快速和高效的研究手段,可直接和全面的反映参与甲烷厌氧氧化作用的功能微生物。以DNA分子标记物为研究对象,重点探讨三类主要的分子标记基因,即16S rRNA,mcr A和pmo A,所采用的相关探针和引物信息,同时从定性和定量两个角度综述土壤甲烷厌氧氧化菌的多样性研究的主要进展,最后提出厌氧甲烷氧化菌多样性研究中存在的一些问题和相应的解决思路。     

自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

自然湿地是CH4排放的重要来源之一。产甲烷菌和甲烷氧化菌是介导自然湿地甲烷循环的重要功能菌群。开展产甲烷菌和甲烷氧化菌多样性的检测研究有助于揭示微生物介导的甲烷循环以及自然湿地甲烷排放的时空异质性。传统基于培养的检测方法已被证实无法充分描述产甲烷菌和甲烷氧化菌的多样性,而分子检测方法为自然湿地土壤产甲烷菌和甲烷氧化菌的多样性检测提供了一种更准确和科学的工具。本文综述了自然湿地土壤产甲烷菌和甲烷氧化菌的定性和定量分子检测方法,包括末端限制性片段长度多态性（T-RFLP）、变性梯度凝胶电泳（DGGE）、荧光原位杂交（FISH）和实时定量PCR（real-time qPCR）,重点分析了分子检测中两类重要的标记基因,总结了不同类型自然湿地产甲烷菌和甲烷氧化菌群落多样性的最新成果,提出了我国在该领域今后应深入研究探讨的一些问题及建议。     

Endogenous methanogenesis stimulates oxidation of atmospheric CH(4) in alpine tundra soil

Experiments were done to test the hypothesis that atmospheric CH(4) oxidizers in a well-drained alpine tundra soil are supported by CH(4) production from anaerobic microsites in the soil. Soil was subjected to 22 days of anaerobic conditions with elevated H(2) and CO(2) in order to stimulate methanogenesis. This treatment stimulated subsequent atmospheric CH(4) consumption, probably by increasing soil methanogenesis. After removal from anaerobic conditions, soils emitted CH(4) for up to 6 h, then oxidized atmospheric CH(4) at 111 (+/- 5.7) pmol (g dry weight)(-1) h(-1), which was more than 3 times the rate of control soils. Further supporting our hypothesis, additions of lumazine, a highly specific inhibitor of methanogenesis, prevented the stimulation of atmospheric CH(4) oxidation by the anaerobic treatment. The method used to create anaerobic conditions with elevated H(2) and CO(2) also elevated headspace CH(4) concentrations. However, elevated CH(4) concentrations under aerobic conditions did not stimulate CH(4) oxidation as much as preexposure to H(2) and CO(2) under anaerobic conditions. Anaerobic conditions created by N(2) flushing did not stimulate atmospheric CH4 oxidation, probably because N2 flushing inhibited methanogenesis by removing necessary precursors for methane production. We conclude that anaerobic conditions with elevated H(2) and CO(2) stimulate atmospheric CH(4) oxidation in this dry alpine tundra soil by increasing endogenous CH(4) production. This effect was prevented by inhibiting methanogenesis, indicating the importance of endogenous CH(4) production in a CH(4-) consuming soil.     

Landscape patterns of litter decomposition in alpine tundra

A two-year study of the decomposition of alpine avens (Acomastylis rossii) foliage in alpine tundra of the Front Range of Colorado demonstrated a strong landscape-mediated effect on decay rates. Litter on sites with intermediate amounts of snowpack decayed more rapidly than litter on sites with larger or smaller amounts of snow. Annual decay constants (k-values) of this foliage ranged from-0.33 in dry tundra to-0.52 in moist tundra to-0.47 in the wettest habitat. No site differences in mass loss of litter were detected until late winter-early spring of the first year of decomposition, when significantly faster decomposition was observed for litter beneath the snowpack. In spite of obvious landscape-related patterns in rates of litter decomposition, total microarthropod densities in the top 5 cm of soil did not differ among habitats. However, the relative abundance of the oribatid and prostigmatid mites did vary significantly across the landscape in relation to the moisture gradient. Oribatid mites comprised a greater proportion of total mites in wetter areas. Microarthropod densities and composition, as well as patterns of decomposition, were compared with previous alpine, as well as arctic tundra, studies. The effects of soil invertebrates on decomposition rates in the alpine were evaluated with a mushroom litterbag decomposition experiment. Naphthalene was used to exclude fauna from a subset of litterbags placed in mesic and xeric habitats. Mushrooms without naphthalene additions decayed significantly faster in the mesic sites. Densities of invertebrates were also greater on mushrooms in these mesic sites. Mushrooms placed in xeric sites generally lacked fauna. Thus, both the activities and the composition of the detritus-based food web appear to change substantially across the moisture gradient found in alpine tundra.     

Molecular and functional diversity in soil micro-organisms

Traditional approaches to the study of microbial diversity have relied on laboratory cultivation of isolates from natural environments and identification by classical techniques, including analysis of morphology, physiological characteristics and biochemical properties. These approaches provide information on fine-scale diversity but suffer from bias, resulting from the media and cultivation conditions employed, and from the inability to grow and isolate significant proportions of natural communities in the laboratory. An alternative approach is the amplification of ribosomal RNA and functional genes from nucleic acids extracted directly from environmental samples, with subsequent analysis by `fingerprinting' methods or by sequencing and phylogenetic analysis. This approach avoids the need for laboratory cultivation and has provided major insights into species and functional diversity of bacterial and archaeal populations. This article reviews molecular approaches to the characterisation of prokaryote diversity in natural environments, their more recent application to fungal diversity and the advantages and limitations of molecular analyses.     

Higher plant diversity enhances soil stability in disturbed alpine ecosystems

Genotypic differences in acquiring immobile P exist among species or cultivars within one species. We investigated the P-efficiency mechanisms of rapeseed (Brassica napus L.) in low P soil by measuring plant growth, P acquisition and rhizosphere properties. Two genotypes with different P efficiencies were grown in a root-compartment experiment under low P (P15: 15 mg P kg^?1) and high P (P100: 100 mg P kg^?1) treatments. The P-efficient genotype produced more biomass, and had a high seed yield and high P acquisition efficiency under low P treatment. Under both P treatments, both genotypes decreased inorganic P (Pi) and organic P (Po) fractions in the rhizosphere soil. However there was no decrease in NaHCO₃-Po at P100. For the P15 treatment, the concentrations of NaHCO₃-Po and NaOH-Po were negatively correlated with soil acid phosphatase activity. The P-efficient genotype 102 differed from the P-inefficient genotype 105 in the following ways. In the rhizosphere the soil pH was lower, acid phosphatase activity was higher, and depletion of P was greater. Further the depletion zones were wider. These results suggested that improving P efficiency based on the character of P efficiency acquisition in P-efficient genotype would be a potential approach for maintaining rapeseed yield potential in soils with low P bioavailability.     

Plant-microbe competition for soil amino acids in the alpine tundra: effects of freeze-thaw and dry-rewet events

Amino acids have been shown to be a potentially significant N source for the alpine sedge, Kobresia myosuroides. We hypothesised that freeze-thaw and dry-rewet events allow this plant species increased access to amino acids by disrupting microbial cells, which decreases the size of competing microbial populations, but increases soil amino acid concentrations. To test this hypothesis, we characterized freeze-thaw and dry-rewet events in the field and simulated them in laboratory experiments on plant-soil microcosms. In one experiment, ¹⁵N,¹³C-[2]-glycine was added to microcosms that had previously been subjected to a freeze-thaw or dry-rewet event, and isotopic concentrations in the plant and microbial fractions were compared to non-stressed controls. Microbial biomass and uptake of the labeled glycine were unaffected by the freezing and drying treatments, but microbial uptake of ¹⁵N was lower in the two warmer treatments (dry-rewet and summer control) then in the two colder treatments (freeze-thaw and fall control). Plant uptake of glycine-¹⁵N was decreased by climatic disturbance, and uptake in plants that had been frozen appeared to be dependent on the severity of the freeze. The fact that intact glycine was absorbed by the plants was confirmed by near equal enrichment of plant tissues in ¹³C and ¹⁵N. Plants under optimal conditions recovered 3.5% of the added ¹⁵N and microbes recovered 5.0%. The majority of the ¹³C and ¹⁵N label remained in a non-extractable fraction in the bulk soil. To better understand the isolated influences of environmental perturbations on soil amino acid pools and population sizes of amino-acid utilizing microbes, separate experiments were performed in which soils, alone, were subjected to drying and rewetting or freezing and thawing. Potential respiration of glycine and glutamate (substrate-induced respiration; SIR) by the soil microbial communities was unaffected by a single freeze-thaw event. Glycine SIR was decreased slightly (∼10%) by the most extreme drying treatment, but glutamate SIR was not significantly affected. Freezing lowered the concentration of water-extractable amino acids while drying increased their concentration. We interpret the surprising former result as either a decrease in proteolytic activity in frozen soils relative to amino acid uptake, or a stimulation in microbial uptake by physical nutrient release from the soil. We conclude that climatic disturbance does not provide opportunities for increased amino acid uptake by K. myosuroides, but that this plant competes well for amino acid N under non-stressed conditions, especially when soils are warm. We also note that this alpine tundra microbial community's high resistance to freeze-thaw and dry-rewet events is novel and contrasts with studies in other ecosystems. Received: 24 February 1997 / Accepted: 28 August 1997     

青藏高原高寒草甸土壤环境对线虫功能多样性的影响

土壤线虫是地下食物网的重要组成部分, 在生态系统能量流动和物质循环中起着至关重要的作用。大量研究报道了肥力等土壤环境对土壤线虫物种多样性及各功能群多度的影响, 而我们对土壤线虫功能多样性如何响应土壤环境变化依然知之甚少。本研究以群落水平个体大小和个体大小多样性表征土壤线虫功能多样性。在青藏高原高寒草甸选择3个研究点, 调查和分析了不同生境(沟底平地、阴坡、阳坡和山顶)土壤线虫物种多样性、各功能群多度和功能多样性及其与土壤理化因子和植物多样性的关系。结果表明: (1)土壤线虫个体多度和物种多样性在阳坡最高, 随土壤pH值和土壤总磷增加而升高; 而基于个体大小的土壤线虫功能多样性主要受土壤养分影响, 随土壤总氮和有机质增加而增加, 随土壤总磷含量增加而减少; (2)食细菌和食真菌线虫多度在沟底最高, 植食与捕食杂食线虫多度在山顶最低; 除捕食杂食线虫外, 各功能群多度也主要随土壤磷增加而升高; 除食真菌线虫外, 各功能群多度随植物物种丰富度的增加而减少。本研究强调了土壤线虫物种和功能多样性受不同土壤环境因子的影响, 丰富了土壤线虫多样性研究的内容, 为理解高寒草甸土壤动物多样性形成、维持和变化提供了更广阔的 视角。     

长白山高山冻原生态系统的碳储量

对长白山高山冻原生态系统碳在植被-凋落物-土壤中的分布进行了研究。结果表明,在长白山高山冻原植被亚系统中,碳储量分布规律是典型高山冻原(TT)>石质高山冻原(ST)>草甸高山冻原(MT)>沼泽高山冻原(WT)>石海高山冻原(RT),长白山高山冻原的植被碳储量为3.3457×104t。而生物量分布规律是WT>TT>MT>ST>RT。在长白山高山冻原凋落物亚系统中,平均凋落物量是0.991 kg.hm-2.年-1;凋落物碳储量为1.5043×104t,碳储量分布规律是TT>ST>MT>RT>WT。长白山高山冻原土壤(0~20 cm)亚系统中,有机碳储量为3.162×105t;每年约有1.4105×104t碳通过土壤呼吸释放到大气圈,长白山高山冻原植被-土壤系统现已成为3.6471×105t碳储库。     

长白山苔原带土壤动物群落结构及多样性

长白山苔原生态系统对环境变化非常敏感,一旦破坏很难恢复。土壤动物是陆地生态系统的重要组成部分,联系着地上和地下生态系统。为探讨长白山苔原带土壤动物群落的组成、结构和多样性特征,于2009年5月、7月和9月对上部和下部苔原带土壤动物进行了研究。研究表明:长白山整个苔原带土壤动物优势类群为甲螨亚目、节跳虫科和球角跳虫科。下部苔原亚带土壤动物个体数和类群数多,群落结构较为复杂。土壤动物的个体数随生境和时间变化有很大波动,类群数变化则较小。在7月份个体数最少,而类群数随着时间的变化有减少的趋势。土壤动物的垂直分布随时间和生境的不同而异。上部苔原亚带9月的甲螨亚目(P﹤0.01)、革螨亚目(P﹤0.05)及下部苔原亚带5月的甲螨亚目、革螨亚目、辐螨亚目(P=0.03,P=0.011,P=0.027)0—5 cm土层显著高于凋落物层;下部苔原亚带5月的球角跳虫科(P=0.001)及各月的节跳虫科和山跳虫科在各层之间存在显著差异(P=0.001,P=0.036,P=0.005,P=0.001,P=0.04,P=0.009)。两个亚带土壤动物多样性随时间的变化都有递减的趋势。土壤动物的多样性指数、丰富度指数和均匀度指数均是下部苔原亚带高于上部苔原亚带,而优势度指数则相反。相似性指数表明两个亚带土壤动物群落组成上存在一定的差异,各类群的相对数量差异在9月最大。下部苔原亚带的生境条件较适宜土壤动物生存,生物多样性较高。     

长白山高山冻原生态系统磷硫生物循环的研究

利用分室模型对长白山高山冻原生态系统磷硫生物循环进行了研究.结果表明,长白山高山冻原植被-土壤系统中总磷和总硫净储存分别为16 088.6 t和26 079.4 t,其中土壤库分别占99.2%和99.5%.磷硫在土壤库、凋落物库和植被库的分布极不平衡,储量分别是:植被库中磷46.14 t、硫64.82 t,其中地上部分储存磷21.88 t、硫44.21 t,地下根系储存磷24.28 t、硫20.61 t,在植物亚系统中47.4%的磷和68.2%的硫储存在地上部分;凋落物库中磷89.63 t、硫53.16 t;土壤库中磷15 952.8 t、硫26 014.6t.长白山高山冻原植被-土壤系统中,磷年吸收量和年凋落物归还量分别为24.52和31.59 t;硫在地上植物活体、地下根系中年转移量和年凋落物归还量分别为31.18、10.12和21.06 t,硫自然归还率为67.5%.     

Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.

Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains.     

围封对退化高寒草甸土壤微生物群落多样性及土壤化学计量特征的影响

围封对植被处于近自然恢复状态的退化草地有一定的修复作用,开展轻度退化草地围封过程中生物与非生物因素的协同互作研究是完整地认识草地生态系统结构和功能的基础.本试验对围栏封育10年的轻度退化草地的土壤化学计量特征进行了研究,同时采用高通量基因测序技术并结合Biolog-Eco方法,调查了土壤微生物多样性和功能的变化.结果表明:轻度退化草地实施围封后,土壤铵态氮含量显著升高,全钾含量显著降低,土壤有机碳、全氮、全磷、硝态氮、速效磷和速效钾则无明显变化.高寒草甸土壤微生物碳和氮在轻度退化和围栏封育草地间差异不显著;围栏封育后草地土壤微生物碳氮比显著高于轻度退化草地.随培养时间的延长,高寒草甸不同土层土壤微生物碳代谢强度均显著升高,土壤微生物碳代谢指数在轻度退化和围栏封育草地间差异不显著.高寒草甸土壤细菌OTUs显著高于真菌,轻度退化与围栏封育草地土壤微生物相似度为27.0%～32.7%.围封后,土壤真菌子囊菌门、接合菌门和球壶菌门相对丰富度显著升高,担子菌门显著降低,土壤细菌酸杆菌门显著低于轻度退化草地.土壤真菌和细菌群落组成在不同土层间差异较大,在轻度退化和围栏封育草地间仅有表层土壤真菌群落组成表现出较大差异.土壤细菌多样性受土壤全氮和速效钾影响较大,真菌多样性受地上生物量影响较大.土壤微生物对碳源利用能力主要受土壤速效钾影响.综上,长期围封禁牧对轻度退化草地土壤养分和土壤微生物无明显影响,且会造成牧草资源浪费,适度放牧可以保持草地资源的可持续利用.     

Molecular and metabolic characterization of cold-tolerant alpine soil Pseudomonas sensu stricto

Alpine soils undergo dramatic temporal changes in their microclimatic properties, suggesting that the bacteria there encounter uncommon shifting selection gradients. Pseudomonads constitute important members of the alpine soil community. In order to characterize the alpine Pseudomonas community and to assess the impact of shifting selection on this community, we examined the ability of cold-tolerant Pseudomonas isolates to grow on a variety of carbon sources, and we determined their phylogenetic relationships based on 16S ribosomal DNA sequencing. We found a high prevalence of Pseudomonas in our soil samples, and isolates from these soils exhibited extensive metabolic diversity. In addition, our data revealed that many of our isolates form a unique cold-adapted clade, representatives of which are also found in the Swedish tundra and Antarctica. Our data also show a lack of concordance between the metabolic properties and 16S phylogeny, indicating that the metabolic diversity of these organisms cannot be predicted by phylogeny.     

长白山高山冻原种子植物的调查

通过多次对长白山高山冻原的实地考察,统计得到冻原种子植物共计34科、94属、146种,有6种属的分布区类型(包括3个变型),北温带分布类型最多,占总属数的68.09%。在整个冻原上,草本有125种,占总数的85.62%,其中多年生草本124种。高山冻原植物中具有多种生态,生理适应方式,低矮平卧状植株、极矮小草本、密集丛生是植物的重要适应方式,细长密集须根是冻原上主要的适应方式,占植物总数的54.94%。果实中干果共计137种,其中蒴果56种。由于高山冻原上生态环境十分残酷,极易受人为因素的影响。