长期施肥对稻田土壤细菌、古菌多样性和群落结构的影响

稻田土壤是“迷失碳”的重要吸纳场所之一,也是温室气体(CH4和N2O等)的重要排放源.大气温室气体的动态变化与土壤碳氮转化的微生物过程紧密相关.以湖南桃江国家级稻田肥力变化长期定位试验点为平台,采用PCR-克隆测序和实时荧光定量PCR技术,研究不施肥(CK)、施氮磷钾肥(NPK)和氮磷钾肥+秸秆还田(NPKS)3种长期施肥制度(＞25 a)对稻田土壤细菌和古菌群落结构及数量的影响.细菌和古菌16S rRNA基因文库分析结果表明:稻田土壤细菌主要类群为变形菌、酸杆菌、绿弯菌,而古菌主要为泉古菌和广古菌.长期施肥导致土壤细菌和古菌种群结构产生明显差异,与CK相比,NPK和NPKS处理稻田土壤的变形菌、酸杆菌和泉古菌相对丰度增加.LIBSHUFF软件分析结果也表明,16S rRNA基因文库在CK、NPK及NPKS处理间存在显著差异.3种施肥处理的稻田土壤细菌16S rRNA基因拷贝数为每克干土0.58× 1010～1.06×1010个,古菌为每克干土1.16×106 ～ 1.72×106个.施肥(NPK和NPKS)后,细菌和古菌的多样性和数量增加,且NPKS＞NPK.说明长期施肥显著影响土壤细菌和古菌群落结构、多样性及数量.     

西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。     

西藏米拉山土壤古茵16S rRNA及amoA基因多样性分析

[目的]本研究旨在了解西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况.[方法]采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA基因和氨氧化古菌amoA基因克隆文库.利用DOTUR软件将古菌和氨氧化古菌序列按照相似性97%的标准分成若干个可操作分类单元(OTUs).[结果]通过构建系统发育树,表明古菌16s rRNA基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲.氨氧化古菌amoA基因克隆文库中序列均为泉古菌.古菌16s rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和75个OTUs.[结论]西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用.     

中国和美国原始土壤中非高温泉古菌的发现和鉴定

近年来在非极端环境中已经发现有古菌(Archaea)的存在, 但在中国原始土壤中还未见报道。本研究的目的是调查古菌是否存在于两个分别取自中国新疆和广西的土壤及两个美国亚利桑那州南部地区的土壤中。我们分别构建了这四个原始土壤的古菌16S rDNA文库并对28个克隆的16S rDNA进行了鉴定。所有这些16S rDNA的序列都归类于古菌的泉古菌门(Crenarchaeota)。进化树分析表明, 这些泉古菌的16S rDNA属于非高温陆地环境中的泉古菌种群, 明显区别于海洋和淡水地带的泉古菌种群。这个泉古菌种群又有两个分支, 这两个分支在16S rDNA序列上和G C含量上有明显的区别。本研究在两个中国和两个美国原始土壤中鉴定了非高温泉古菌的存在, 由此证明泉古菌的存在范围不只局限于高温等极端环境。另外, 美国原始土壤中的泉古菌只属于一个进化分支, 这说明非高温泉古菌种群的类型和土壤的地理位置及土壤特性有关。     

腾冲两热泉泉古菌多样性及系统发育的初步分析

通过构建16S rRNA基因片段的克隆文库对腾冲热海两温泉中泉古菌的多样性和系统发育关系进行了初步的研究.一共得到18个泉古茵克隆序列,可分为12个OTUs,两温泉的克隆序列与已知GenBank上关系最近序列的平均相似性较低,无名泉为92.56%,热爆区为93%.从基于16S rRNA基因片段序列构建的系统发育树来看,74℃的无名泉样点中既有属于超高温环境类群的泉古菌,同时又有属于和常温环境较接近的泉古菌;45℃的热爆区样点的泉古茵,相对来说则更接近于常温类群.本次研究表明,腾冲热泉与世界其它同类热泉之间的泉古茵类群存在着一定的差异;而且两实验样点代表了超高温和高温环境泉古菌逐渐向常温过度的两个重要环境.     

新疆沙湾冷泉沉积物中免培养古菌多样性初步研究

【目的】了解新疆沙湾冷泉沉积物的古菌组成及多样性。【方法】采用免培养法,液氮研磨提取冷泉沉积物总DNA,使用古菌通用引物进行16S rRNA基因扩增,构建16S rRNA基因文库。对阳性克隆进行HhaI限制性酶切分型,选出具有不同酶切图谱的序列进行测序,将所得序列与GenBank数据库中序列比对并构建16S rRNA基因系统发育树。【结果】从冷泉沉积物古菌16S rRNA基因文库中随机挑选了121个阳性克隆,共得到22个不同的可操作分类单元,BLAST结果表明全部克隆子归属于泉古菌门(Crenarchaeote)中免培养类群。系统发育分析归类为Soil-Freshwater-subsurface group和MarinegroupI,2个亚群并且各占整个文库的50%。其中40%左右的克隆子与具有无机碳和硝酸盐同化能力的泉古菌有高的相似性。此外还发现40%的克隆子与低温泉古菌类群具有很高的相似性。【结论】新疆沙湾冷泉沉积物中古菌类群多样性较低,但存有大量高度适应此低温、贫营养环境的泉古菌类群。     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

洋山深水港海域表层海水中古菌多样性特点

利用DGGE、定量PCR以及16S rRNA基因克隆文库技术对洋山深水港海域表层海水中的古菌多样性组成进行了调查和分析。通过Quantity One软件分析不同样品的DGGE图谱,发现洋山深水港3个不同海域(小洋山港口区、大洋山港口区、中间航道)的古菌群落多样性组成不存在明显的区别;利用定量PCR的方法对3个不同样品中的古菌16S rRNA基因拷贝进行计数,发现3个不同海域的古菌16S rRNA基因拷贝数的数量关系为:大洋山海域((1.69±0.19)×10~6copies/mL)中间航道((2.17±0.20)×10~6copies/mL)小洋山港口区((2.42±0.22)×10~6copies/mL),这说明洋山深水港3个不同海域的古菌群落组成是一致的,只是在数量上略有差异,为对该海域古菌的更深入调查研究提供了便利和数据基础。洋山深水港海域表层海水的古菌16S rRNA基因文库分析表明,古菌由三大门类组成:广古菌门(Euryarchaeota)、泉古菌门(Crenarchaeota)和奇古菌门(Thaumarchaeota)。广古菌门所占比例约为26.8%,泉古菌门约为33.0%,奇古菌门约为40.2%。广古菌门含有2个类群,分别是Mathanobacteriales(10.3%)和Marine GroupⅡ(16.5%);泉古菌门含有1个类群,为Unidentified Crenarchaeotic Group(33.0%);奇古菌门含有1个类群,为Marine GroupⅠ(40.2%)。结果显示,Mathanobacteriales类群和Marine GroupⅡ类群的比例失调,这可能预示着洋山深水港海域存在油污污染及外来微生物物种入侵的风险。     

不同利用方式对红壤坡地微生物多样性和硝化势的影响

采集了中国科学院桃源农业生态试验站红壤坡地农田、自然恢复林和茶园土壤样品,采用末端限制性酶切片段长度多态性分析(T.RFLP)技术分析土壤细菌、古菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的多样性,采用好气培养法测定不同土壤的硝化势,研究不同土地利用方式对微生物多样性和硝化势的影响.结果表明:土壤AOB和AOA多样性指数差异不显著,且在3种不同土地利用方式中呈现相同的趋势,均为农田=茶园>自然恢复林;通过RDA分析发现,不同利用方式造成土壤理化性状的改变是影响土壤AOA和AOB群落结构的主要原因;好气培养法测得不同土壤硝化势农田最高,茶园次之而自然恢复林最低;相关性分析显示,硝化势与细菌16S rRNA、AOA和AOB amoA基因多样性指数呈显著正相关,其中与AOA amoA基因关系最为密切;总体来说,红壤坡地不同利用方式改变了土壤细菌、古菌、AOA和AOB的多样性,土壤AOB和AOA积极参与了土壤的硝化过程,且AOA在氨氧化微生物群落生态功能中占有重要地位,AOA比AOB与硝化势的关系更为密切.     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

Metagenomic studies reveal the critical and wide-ranging ecological importance of uncultivated Archaea: the role of ammonia oxidizers

Microbial genome sequencing has entered a new phase, where DNA sequence information is gathered from entire microbial communities (metagenomics or environmental genomics) rather than from individual microorganisms. By providing access to the genetic material of vast numbers of organisms, most of which are organisms that have never been isolated or cultivated, a new level of insight is being gained into the diversity and extent of the microbial processes that are presently occuring in environmental communities. By extending metagenomic-based approaches to the study of very complex and methodologically recalcitrant soil environments, a recent study has found that ammonia-oxidizing archaea are more abundant in many soils than bacteria.1 These findings not only highlight the undoubtedly critical yet unknown roles that archaea play in global nutrient cycles but illustrate the importance of genomic studies for informing us about the functional capacity of life on Earth.     

外源砷胁迫对两种土壤中细菌和古菌群落的影响

采矿和冶炼等活动会导致土壤中砷的累积,给农产品质量安全和土壤微生物带来不利影响。本文研究了外源砷进入黄壤(YS)和紫色砂页岩发育土壤(RS)后有效砷含量随时间的变化,并采用MiSeq高通量测序研究土壤中细菌和古菌在未加砷和外源砷胁迫1、30、360 d后的群落变化,探讨砷胁迫下土壤细菌和古菌的适应机制。结果表明: 外源砷进入土壤后,土壤有效砷含量随时间推移逐渐降低,并显著影响土壤细菌和古菌的群落组成。土壤细菌的优势菌群丰度变化显著;而古菌中仅丰度较低的菌群发生显著改变,优势菌群丰度变化较小,推测古菌群落具有高耐砷性和稳定性。与砷胁迫时间相比,土壤砷的有效性对细菌和古菌群落结构的影响更大。研究结果可为砷污染农田的安全利用及微生物修复等提供参考。     

Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Selenoproteins and the metabolic features of the archaeal ancestor of eukaryotes

In all three branches of life, some organisms incorporate the rare amino acid selenocysteine. Selenoproteins are relevant to the controversy over the metabolic features of the archaeal ancestor of eukaryotes because among archaea, several known selenoproteins are involved in methanogenesis and autotrophic growth. Although the eukaryotic selenocysteine-specific translation apparatus and at least one selenoprotein appear to be of archaeal origin, selenoproteins have not been identified among sulfur-metabolizing crenarchaeotes. In this regard, both the phylogeny and function of archaeal selenoproteins are consistent with the argument that the archaeal ancestor was a methanogen. Selenium, however, is abundant in sulfur-rich environments, and some anaerobic bacteria reduce sulfur and have selenoproteins similar to those in archaea. As additional archaeal sequence data becomes available, it will be important to determine whether selenoproteins are present in nonmethanogenic archaea, especially the sulfur-metabolizing crenarchaeotes.     

Analysis of covalent flavinylation using thermostable succinate dehydrogenase from Thermus thermophilus and Sulfolobus tokodaii lacking SdhE homologs

Recent studies have indicated that post-translational flavinylation of succinate dehydrogenase subunit A (SdhA) in eukaryotes and bacteria require the chaperone-like proteins Sdh5 and SdhE, respectively. How does covalent flavinylation occur in prokaryotes, which lack SdhE homologs? In this study, I showed that covalent flavinylation in two hyperthermophilic bacteria/archaea lacking SdhE, Thermus thermophilus and Sulfolobus tokodaii, requires heat and dicarboxylic acid. These thermophilic bacteria/archaea inhabit hot environments and are said to be genetically far removed from mesophilic bacteria which possess SdhE. Since mesophilic bacteria have been effective at covalent bonding in temperate environments, they may have caused the evolution of SdhE.     

Archaeal abundance in relation to root and fungal exudation rates

Archaea are ubiquitous in forest soils, but little is known about the factors regulating their abundance and distribution. Low molecular weight organic compounds represent an important energy source for archaea in marine environments, and it is reasonable to suspect that archaeal abundance is dependent on such compounds in soils as well, represented by, for example, plant and fungal exudates. To test this hypothesis, we designed a microcosm experiment in which we grew ponderosa pine, sitka spruce, and western hemlock in forest soil. Root and mycorrhizal exudation rates were estimated in a 13C pulse-chase experiment, and the number of archaeal and bacterial 16S rRNA genes was determined by qPCR. Archaeal abundance differed among plant species, and the number of archaeal 16S rRNA genes was generally lower in soil receiving high concentration of exudates. The mycorrhizal fungi of ponderosa pine seemed to favor archaea, while no such effect was found for mycorrhized sitka spruce or western hemlock. The low abundance of archaea in the proximity of roots and mycorrhiza may be a result of slow growth rates and poor competitive ability of archaea vs. bacteria and does not necessarily reflect a lack of heterotrophic abilities of the archaeal community.     

Long-term effects of nitrogen and phosphorus fertilization on soil microbial community structure and function under continuous wheat production

Soil microorganisms play a critical role in the biosphere, and the influence of cropland fertilization on the evolution of soil as a living entity is being actively documented. In this study, we used a shotgun metagenomics approach to globally expose the effects of 50-year N and P fertilization of wheat on soil microbial community structure and function, and their potential involvement in overall N cycling. Nitrogen (N) fertilization increased alpha diversity in archaea and fungi while reducing it in bacteria. Beta diversity of archaea, bacteria and fungi, as well as soil function, were also mainly driven by N fertilization. The abundance of archaea was negatively impacted by N fertilization while bacterial and fungal abundance was increased. The responses of N metabolism-related genes to fertilization differed in archaea, bacteria and fungi. All archaeal N metabolic processes were decreased by N fertilization, while denitrification, assimilatory nitrate reduction and organic-N metabolism were highly increased by N fertilization in bacteria. Nitrate assimilation was the main contribution of fungi to N cycling. Thaumarchaeota and Halobacteria in archaea; Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria in bacteria; and Sordariomycetes in fungi participated dominantly and widely in soil N metabolic processes.