长三角地区有机农药污染地下水的微生物群落分析

选择长江三角洲地区一处长期受到有机农药污染的浅层含水层,采集水样,采用不依赖于培养的16SrDNA序列分析方法,对该污染地下水中的微生物群落特征进行了分析和鉴定。结果表明,这种分析方法完全可行,该有机污染含水层具有较好的微生物多样性,且可能有一种未经鉴定的新菌种。     

长江口崇明东滩湿地微生物群落结构研究

以长江口发育最完善的河口型滩涂湿地-崇明东滩湿地为对象,采用高通量测序平台和定量PCR技术对光滩区和植被区沉积物的微生物群落结构进行研究,考察滩涂湿地自然演替过程中微生物群落变化规律及其环境驱动机制。结果表明,植被区沉积物表层的细菌16S rRNA基因拷贝数为光滩区表层的6.4倍,其Chao1指数与土壤有机碳、总氮和铵态氮呈显著负相关。植被区和光滩区沉积物中相对丰度大于1%的微生物门类有12个,占到总微生物群落的87%～90%。在属水平上,与光滩湿地相比,植被区湿地中相对丰度发生显著变化的优势属( 0.1%)为25个,占到所有属的8.3%和所有优势属的64.1%;显著增加的优势属( 0.2%)为10个,其增幅范围为0.22%～1.39%;其中,植被区沉积物中以脱硫球菌属和硫杆菌属微生物增加占主导,Cylindrospermopsis属为特有属。冗余分析表明,绿弯菌门微生物对土壤TOC、TN和NO3-等较为敏感,而酸杆菌门和拟杆菌门微生物对SO2-4、EC值和pH值等敏感。     

黄河三角洲盐生植被演替与土壤细菌群落结构的关系

研究黄河三角洲光板地和4种盐生植被(翅碱蓬、獐茅、白茅和罗布麻)下土壤细菌群落组成,揭示其与盐生植被演替的关系。利用细菌16S r DNA基因文库方法,构建系统发育树,在5个文库中各挑选180个阳性克隆子进行序列测定,并对数据进行统计分析。从光板地、翅碱蓬、獐茅、白茅和罗布麻5个文库中分别得到121、132、150、159、155条有效序列。重盐土壤(翅碱蓬土壤、獐茅土壤)细菌有着最高的Shannon指数和最低的Simpson优势度指数。土壤中检测到变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、酸杆菌门(Acidobacteria)等,共8门。其中变形菌门在5种土样中相对丰度为21.06%,拟杆菌门为11.16%,其他细菌相对丰度皆未超过10%。5种覆被类型下土壤细菌在种群组成上差异明显,但均匀度与丰富度差异较小,变形菌为所研究5种土壤中的优势菌群。当盐生植被处于相同演替阶段时,土壤细菌群落结构具有更大相似性;在不同演替阶段时,土壤细菌群落结构差别较大。     

应用T-RFLP技术分析不同土壤温度和施肥方式下设施土壤细菌群落结构

采用末端限制性片段长度多态性分析(T-RFLP)和荧光定量PCR技术,研究了两种土壤温度(对照不增温,10℃±2℃;增温,20℃±2℃)和两种施肥方式(对照不施肥;鸡粪有机肥底肥+尿素追肥)对土壤细菌群落结构及其多样性和数量特征的影响。结果表明:相同施肥方式下,土壤增温增加土壤速效养分含量,尤其在施肥时增加幅度更明显。分析显示施肥对土壤细菌群落结构的影响在不同土壤温度条件下表现不同。土壤不增温条件下,施肥增加细菌Pielou均匀度指数和Shannon-Weiner多样性指数,Simpson优势度指数降低;土壤增温条件下,则效果相反。与不增温比,增温使土壤细菌Pielou均匀度指数和Shannon-Weiner多样性指数降低,但物种个体数和Simpson优势度指数增加明显。增温改变了土壤细菌群落结构,62 bp和93 bp两种限制性末端片段在土壤增温条件下优势度提高,主要包括共生菌和杆菌。     

不同作物间作对黄瓜病害及土壤微生物群落多样性的影响

分别采用RAPD和T-RFLP技术,研究了小麦、毛苕子和三叶草分别与黄瓜间作对黄瓜病害、黄瓜根际土壤微生物群落多样性和黄瓜产量的影响。结果表明,小麦、毛苕子与黄瓜间作均能提高黄瓜根际土壤微生物群落多样性,其中,小麦-黄瓜间作对黄瓜根际土壤微生物群落多样性的影响最为突出;3种作物分别与黄瓜间作均显著提高了黄瓜产量(p<0.01),其中小麦-黄瓜间作的产量优势最强;同时,3种作物分别与黄瓜间作均降低了黄瓜角斑病、白粉病、霜霉病和枯萎病的病情指数和尖孢镰刀菌的数量。间作有利于提高土壤微生物群落的多样性、减轻病害、提高黄瓜产量。     

土壤微生物群落结构的化学估价方法

由于认识到微生物在整个土壤生态系统中的重要功能，就促使人们不断地用最新垂头丧气估价土壤微生物群落结构和多样性。目前除了使用标志化合物外，利用生物细胞膜中磷酯类化合物的脂肪酸组成与土壤中可提取脱氧核糖核酸组成的复杂性作为指纹来估价土壤中微生物群落结构和多样性是两种最常用的方法。     

含油污泥的堆肥处理对微生物群落结构的影响

采用堆肥方法处理含油污泥,评价堆肥处理对含油污泥中石油烃的去除效果,并采用Biolog方法和构建16SrRNA基因克隆文库的方法对处理过程中微生物碳源利用特征和微生物群落结构进行了研究。结果表明,含油污泥经过90d的堆肥处理,石油烃降解率达53.3%±9.5%,显著高于对照处理。堆肥处理可以显著促进石油烃降解,是一种处理含油污泥的有效措施。Biolog分析结果表明,堆肥处理的孔的平均颜色变化率（AWCD）显著高于对照处理,堆肥处理提高了土壤微生物代谢活性。主成分分析结果表明,对照处理和堆肥处理的微生物碳源利用特征明显不同,堆肥处理改变了含油污泥中微生物的代谢功能特征。对照处理和堆肥处理的16SrRNA基因克隆文库之间存在显著差异,对照处理的优势类群是γ-Proteobacteria,堆肥处理的优势类群是Bacteroidetes,堆肥处理显著改变了含油污泥中的微生物群落结构。Marinobacter和Alcanivorax是对照处理中的优势菌,可能与石油烃的自然降解过程有关,而Pusillimonas和Agrobacterium可能对堆肥处理中石油烃的降解起一定作用。     

红壤荒草地氨氧化细菌富集液16SrDNA文库的RFLP分析

分析红壤荒草地富集液中氨氧化细菌的种群组成,选取氨氧化细菌16S rDNA特异性引物序列,利用PCR技术对从富集液中抽提的细菌总DNA进行扩增,并建立了氨氧化细菌特异性的16S rDNA文库。用酶HhaⅠ和RsaⅠ对该文库特异性片段进行了限制性酶切片断长度多态性分析(Restriction fragment lengthpolymorphism,RFLP),随机挑选的35个特异性克隆片段被分成3个不同的RFLP类型,其中优势型占了所有分析克隆子的94%,另两个型各占3%。从每个RFLP类型中挑取一定的转化子进行测序,测序结果经GenBank检索,发现在该富集液体系文库中存在大量亚硝化单胞菌属(Nitrosomonas)细菌序列,由此推测红壤荒草地中存在氨氧化细菌,Nitrosomonas属细菌能在富集条件下成为优势菌。     

毛竹入侵森林对固氮微生物群落结构和丰度的影响

【目的】 毛竹入侵能够对生态系统的各个方面产生影响,包括森林生态系统氮循环。生物固氮是森林生态系统氮循环的重要一环,研究毛竹入侵过程中土壤固氮微生物的变化,可为毛竹入侵对生态功能影响的评价提供理论依据。 【方法】 选取天目山自然保护区的三个毛竹入侵带 (青龙山、石门洞和进山门),分别在三个入侵带中沿毛竹入侵方向采集毛竹纯林、毛竹与原林混交林以及原林地的表层土壤,分析pH、有机碳、碱解氮、有效磷和速效钾等土壤化学性质,应用基于nifH功能基因的末端限制性片段长度多态性 (T-RFLP) 和荧光定量PCR (qPCR) 技术,分析土壤固氮微生物群落结构和丰度的变化。 【结果】 毛竹入侵后土壤化学性质呈现三种不同情况,在三个入侵带中总体上升的有pH、有机碳、碱解氮、有效磷,下降的为速效钾,而硝态氮的变化均不显著,总体上,土壤养分含量在毛竹入侵后有所上升。土壤固氮微生物的丰度随毛竹入侵过程降低,其中进山门入侵带的变化显著 (P < 0.05)。土壤固氮微生物T-RFLP的结果显示,48 bp在三个入侵带中均为优势片段;不同的T-RFs在毛竹入侵过程中变化各异,有的片段在毛竹入侵后消失,有的片段在毛竹入侵后出现,有的片段变化不显著。土壤固氮微生物群落的Shannon指数和均匀度指数随毛竹入侵降低,Simpson指数相反,这些指标只在石门洞入侵带的不同林分之间存在显著差异 ( P < 0.05);多响应置换过程分析 (MRPP) 显示,石门洞和进山门入侵带毛竹入侵前后土壤固氮微生物群落结构有显著变化 ( P < 0.05);冗余分析 (RDA) 结果表明,土壤性质对固氮微生物群落变化的解释率普遍较低 (低于30%),三个入侵带显著影响土壤固氮微生物群落变化的土壤化学性质各不相同,并且三个入侵带的样点在RDA坐标图中分布格局并不统一。 【结论】 即使同一自然保护区的三个毛竹入侵带,土壤固氮微生物群落随着入侵过程的变化并不一致,母岩和原有植被产生的综合作用导致土壤固氮微生物群落发生变化,但需结合更多的因素进行进一步地探讨。      

地膜覆盖对土壤微生物群落结构的影响

采用磷脂脂肪酸(PLFA)法测定了沈阳农业大学棕壤长期定位试验站地膜覆盖条件下土壤微生物磷脂脂肪酸图谱及群落结构。结果表明:在玉米苗期,长期施氮肥处理土壤覆膜后大部分脂肪酸含量都有所提高;施有机肥处理的土壤覆膜后脂肪酸的含量有降低的趋势;有机无机肥配合施用处理的土壤覆膜会增加真菌的含量,但却降低了其他脂肪酸的含量;在不施肥处理的土壤中,覆膜会使一些支链脂肪酸的含量降低。抽雄期,施有机肥的土壤覆膜后除单饱和脂肪酸含量有所下降外,其他脂肪酸都会提高;不施肥土壤覆膜处理双不饱和支链脂肪酸及放线菌标志性脂肪酸10Me18:0的含量会有所提高。成熟期,施氮肥的土壤覆膜处理大部分脂肪酸的含量降低;有机肥处理土壤中各种脂肪酸如:15∶0,a17∶0,i17∶0,i19∶0,18∶0,10Me18∶0含量覆膜高于裸地;有机无机配施处理土壤中17∶0,br17∶0,18∶0,18∶2w6,10Me18∶0含量覆膜处理高于裸地;对照土壤中i15∶0,16∶0,17∶0,a17∶0,18∶0,18∶2w6,19∶0含量覆膜高于裸地。另外,从脂肪酸的变化看出,覆膜处理土壤微生物群落整体结构发生了改变,没有表现出明显的种群优势。但是,尽管土壤的施肥处理不同,覆膜处理土壤微生物群落结构有一致化发展的趋势。     

Microbial activity and community structure in two drained fen soils in the Ljubljana Marsh

Fen peatlands are specific wetland ecosystems containing high soil organic carbon (SOC). There is a general lack of knowledge about the microbial communities that abound in these systems. We examined the microbial activity and community structure in two fen soils differing in SOC content sampled from the Ljubljana Marsh under different seasonal conditions. Substrate-induced respiration and dehydrogenase activity were used as indicators of total microbial activity. Both methods indicated higher microbial activities in the fen soil with the higher SOC content on all dates of sampling. To determine whether the differences in microbial activity were associated with differences in the microbial community structures, terminal restriction fragment length polymorphism (T-RFLP) of bacterial 16S rRNA genes was performed. Comparison of the T-RFLP profiles revealed very similar community structures in both fens and in the two seasonal extremes investigated. This suggested a stable community structure in the two fens, which is not affected by the SOC content or seasonal variation. In addition, a bacterial 16S ribosomal RNA gene based clone library was prepared from the fen soil with the higher SOC content. Out of 114 clones analysed, approximately 53% belonged to the Proteobacteria, 23% to the Acidobacteria, 21% to a variety of other taxa, and less than 3% were affiliated with the Firmicutes.     

Methyl-mercury affects microbial activity and biomass,bacterial community structure but rarely the fungal community structure

Monomethyl-mercury is one of the most toxic compounds. Methylation of Hg usually appears under anoxic conditions. In Swiss forest soils, methyl-Hg concentrations of up to 3 μg kg⁻¹ soil dw have been observed, but the impact of methyl-Hg on soil microorganisms have rarely been examined so far. In this study, we investigated the effect of increasing concentrations of methyl-Hg (0, 5, 20, 90 μg kg⁻¹ soil dw) on the microbial communities in various forest soils differing in their physico-chemical properties. Experiments were conducted in microcosms under controlled conditions and the basal respiration (BR), the microbial biomass carbon (MBC) and the bacterial and fungal community structures using T-RFLP-profiling were investigated. BR was significantly affected by methyl-Hg. In general, the BR increased with increasing methyl-Hg concentrations, whereas the MBC was significantly reduced. Bacterial communities were more sensitive to methyl-Hg than fungal communities. In five out of seven soils, the bacterial community structures differed significantly between the treatments whereas the fungal communities did not. The impact of methyl-Hg on the soil bacterial communities was site specific. In one soil, a methyl-Hg concentration of already 5 μg kg⁻¹ soil dw significantly affected the relative abundance of 13% bacterial operational taxonomic units (OTU), whereas in other soils concentrations of even 90 μg kg⁻¹ soil dw rarely affected the abundance of OTUs. In this study, for the first time, the impact of methyl-Hg on soil bacterial and fungal communities in forest soils was assessed. We showed that its impact strongly depends on the physico-chemical conditions of the soil and that bacterial communities were more sensitive to methyl-Hg than fungi.     

Microbial activity and community structure of a soil after heavy metal contamination in a model forest ecosystem

We assessed the effects of chronic heavy metal (HM) contamination on soil microbial communities in a newly established forest ecosystem. We hypothesized that HM would affect community function and alter the microbial community structure over time and that the effects are more pronounced in combination with acid rain (AR). These hypotheses were tested in a model forest ecosystem consisting of several tree species (Norway spruce, birch, willow, and poplar) maintained in open top chambers. HMs were added to the topsoil as filter dust from a secondary metal smelter and two types of irrigation water acidity (ambient rain vs. acidified rain) were applied during four vegetation periods. HM contamination strongly impacted the microbial biomass (measured with both fumigation-extraction and quantitative lipid biomarker analyses) and community function (measured as basal respiration and soil hydrolase activities) of the soil microbial communities. The most drastic effect was found in the combined treatment of HM and AR, although soil pH and bioavailable HM contents were comparable to those of treatments with HM alone. Analyses of phospholipid fatty acids (PLFAs) and terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S ribosomal DNA showed that HM treatment affected the structure of bacterial communities during the 4-year experimental period. Very likely, this is due to the still large bioavailable HM contents in the HM contaminated topsoils at the end of the experiment.     

Bacterial community structure in lake sediments of microcosms contaminated with nonylphenol

The impact of nonylphenol, an estrogenic degradation product of alkylphenol polyethoxylates, on the bacterial community structure in contaminated sediments of aquatic microcosms was investigated over a period of 20 weeks using a 16S rDNA-based molecular phylogenetic approach. All microcosms showeda strong seasonal fluctuation of the dominant as well as the active bacterial microflora independent of their degree of contamination with nonylphenol. These changes were correlated with the dynamic of the total organic carbon content (TOC), ranging from 4–39 g/kg sediment dry weight and the redox potential in the sediment. Even at the highest observed nonylphenol concentration (3.4 mg/kg sediment dry weight) the bacterial community structure was mostly unchanged.     

Overland flow systems for treatment of landfill leachates—Potential nitrification and structure of the ammonia-oxidising bacterial community during a growing season

Overland flow systems are useful for treating landfill leachates, because they provide favourable conditions for nitrification and they are easy to maintain. However, little is known about the microbial communities in such systems or the nitrification capacity of those microorganisms. In this study, seasonal variations in potential nitrification and in community composition of nitrifying bacteria were investigated in two overland flow areas receiving leachate from landfills at Korslöt and Hagby, Sweden. Samples were collected in the settling ponds sediment and at two depths in the overland flow areas (the macrophyte litter layer and the rhizosphere) in May, August and November 2003. A short-term incubation method was used to measure potential oxidation of ammonia and nitrite (designated PAO and PNO). The ammonia-oxidising bacterial (AOB) community was investigated using a 16S rRNA gene approach that included PCR amplification and analysis of PCR products by denaturing gradient gel electrophoresis (DGGE), followed by nucleotide sequencing and phylogenetic analysis.PAO was determined in the range 5-2700 (NO₂⁻+NO₃⁻)-N g⁻¹ dw d⁻¹ and PNO in the range 60-2000 μg NO₂⁻-N g⁻¹ dw d⁻¹. At Korslöt, PAO and PNO showed similar temporal variation in the different ecosystems, whereas no such relationship was noticed at Hagby. Considering both sites, there was no obvious change in the composition of the AOB community over the growing season. However, the composition did differ between the ecosystems: Nitrosomonas-like sequences were more common in the ponds, and in the litter layers they were found as often as Nitrosospira-like sequences, whereas Nitrosospira-like sequences were more common in the rhizospheres. Altogether, we found nine different AOB sequences, five Nitrosomonas-like and four Nitrosospira-like, which belonged to clusters 0, 2, 3b, 6a, 6b and 7. There was no apparent relationship between the number of AOB populations and the PAO in different soil layers and sediments.     

Microarray analysis of bacterial diversity and distribution in aggregates from a desert agricultural soil

Previous research has shown that soil structure can influence the distribution of bacteria in aggregates and, thereby, influence microbiological processes and diversity at small spatial scales. Here, we studied the microbial community structure of inner and outer fractions of microaggregates of a desert agricultural soil from the Imperial Valley of Southern California. To study the distribution of soil bacteria, 1,536 clones were identified using phylogenetic taxon probes to classify arrays of 16S rRNA genes. Among the predominant taxonomic groups were the α-Proteobacteria, Planctomycetes, and Acidobacteria. When compared across all phyla, the taxonomic compositions and distributions of bacterial taxa associated with the inner and outer fractions were nearly identical. Our results suggest that the ephemeral nature of soil aggregates in desert agricultural soils may reduce differences in the spatial distribution of bacterial populations as compared to that which occur in soils with more stable aggregates.     

Changes in microbial community structure in soil as a result of different amounts of nitrogen fertilization

The changes in size, activity and structure of soil microbial community caused by N fertilization were studied in a laboratory incubation experiment. The rates of N fertiliser applied (KNO₃) were 0 (control), 100 and 2,000 μg N g⁻¹ soil. Despite no extra C sources added, a high percentage of N was immobilized. Whereas no significant increase of microbial C was revealed during incubation period, microbial growth kinetics as determined by the substrate-induced growth-response method demonstrated a significant decrease in the specific growth rate of microbial community in soil treated with 2,000 μg N g⁻¹ soil. Additionally, a shift in microbial community structure resulting in an increase in fungal biomarkers, mainly in the treatment with 2,000 μg N g⁻¹ soil was visible.     

Structural differences in the rhizosphere communities of legumes are not equally reflected in community-level physiological profiles

The relationship of structural diversity and differences in the functional potentials of rhizosphere communities of alfalfa, common bean and clover was investigated in microcosms. PCR-SSCP (single strand conformation polymorphism) analysis of 16S rRNA genes revealed significant differences in the composition of the leguminous rhizosphere communities at the shoot stage of plants grown in the same soil. Sequencing of dominant SSCP-bands indicated the presence of plant specific organisms. The partial rRNA gene sequences were related to members of the α- and γ-Proteobacteria, Bacteroidetes and Actinobacteria. Besides the plant species, the soil also affected the structural diversity in rhizospheres. The dominant bacterial populations of alfalfa grown in soils with different agricultural histories were assigned to different taxonomic groups. Addressing the functional potentials, community-level physiological profiles (CLPP) were generated using BIOLOG GN^®. The three leguminous rhizosphere communities could be differentiated by principle component analysis, though the overall analysis indicated that the metabolic potential of all rhizosphere samples was similar. The functional variation examined in rhizospheres of alfalfa was minor in response to the soil origin and was found not to be significant different at different growth stages. The results indicate that similar functional potentials may be provided by structurally different bacterial communities.     

Microbial community shifts in Pythium ultimum-inoculated suppressive substrates

In this study, the role of compost amendments for the biocontrol of Pythium ultimum was evaluated in bioassays with cucumber (Cucumis sativa L. variety “Chinesische Schlangen”). The addition of compost to the peat-based growing substrates resulted in a significant reduction of disease symptoms of cucumber plants in the presence of P. ultimum compared to pure substrate. Microbial community composition of compost-amended substrates and with different levels of P. ultimum inoculum (0, 5‰) was analyzed by polymerase-chain-reaction-based techniques. To detect and compare dominant bacterial and fungal representatives of suppressive substrate mixes with different pathogen inoculum, 16S and 18S rRNA clone libraries were established. Phylogenetic analysis of the 16S rRNA clones revealed Actinobacteria and α-Proteobacteria to be the prominent classes in the presence of P. ultimum, which are not part of the dominant microflora in the mixes without the pathogen. 18S rRNA sequences for the Pythium-inoculated compost supplemented samples were dominated by Chytridiomycota and Sordariomycetes, whereas in uncontaminated soil–compost mixes, a large part of the sequences were related to Homobasidiomycetes. Thus, it is assumed that the presence of P. ultimum induces distinct shifts in microbial communities favoring to groups known to comprise potential biocontrol agents.     

Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by geographic features

Here, we examine soil-borne microbial biogeography as a function of the features that define an American Viticultural Area (AVA), a geographically delimited American wine grape-growing region, defined for its distinguishing features of climate, geology, soils, physical features (topography and water), and elevation. In doing so, we lay a foundation upon which to link the terroir of wine back to the soil-borne microbial communities. The objective of this study is to elucidate the hierarchy of drivers of soil bacterial community structure in wine grape vineyards in Napa Valley, California. We measured differences in the soil bacterial and archaeal community composition and diversity by sequencing the fourth variable region of the small subunit ribosomal RNA gene (16S V4 rDNA). Soil bacterial communities were structured with respect to soil properties and AVA, demonstrating the complexity of soil microbial biogeography at the landscape scale and within the single land-use type. Location and edaphic variables that distinguish AVAs were the strongest explanatory factors for soil microbial community structure. Notably, the relationship with TC and TN of the <53 μm and 53–250 μm soil fractions offers support for the role of bacterial community structure rather than individual taxa on fine soil organic matter content. We reason that AVA, climate, and topography each affect soil microbial communities through their suite of impacts on soil properties. The identification of distinctive soil microbial communities associated with a given AVA lends support to the idea that soil microbial communities form a key in linking wine terroir back to the biotic components of the soil environment, suggesting that the relationship between soil microbial communities and wine terroir should be examined further.