Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils

Microorganisms that use nitrate as an alternative terminal electron acceptor play an important role in the global nitrogen cycle. The diversity of the nitrate-reducing community in soil and the influence of the maize roots on the structure of this community were studied. The narG gene encoding the membrane bound nitrate reductase was selected as a functional marker for the nitrate-reducing community. The use of narG is of special interest because the phylogeny of the narG gene closely reflects the 16S ribosomal DNA phylogeny. Therefore, targeting the narG gene provided for the first time a unique insight into the taxonomic composition of the nitrate-reducing community in planted and unplanted soils. The PCR-amplified narG fragments were cloned and analyzed by restriction fragment length polymorphism (RFLP). In all, 60 RFLP types represented by two or more clones were identified in addition to the 58 RFLP types represented by only one clone. At least one clone belonging to each RFLP type was then sequenced. Several of the obtained sequences were not related to the narG genes from cultivated bacteria, suggesting the existence of unidentified nitrate-reducing bacteria in the studied soil. However, environmental sequences were also related to NarG from many bacterial divisions, i.e., Actinobacteria and alpha, beta, and gamma proteobacteria. The presence of the plant roots resulted in a shift in the structure of the nitrate-reducing community between the unplanted and planted soils. Sequencing of RFLP types dominant in the rhizosphere or present only in the rhizosphere revealed that they are related to NarG from the Actinobacteria in an astonishingly high proportion.     

Nitrous oxide reductase (nosZ) gene fragments differ between native and cultivated Michigan soils

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.     

Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in alpha-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.     

Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO3)2, (NH4)2SO4, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH4)2SO4 and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO3)2 or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.     

Comparative genetic diversity of the narG,nosZ, and 16S rRNA genes in fluorescent pseudomonads

The diversity of the membrane-bound nitrate reductase (narG) and nitrous oxide reductase (nosZ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay. Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively. The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes. The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed. Overall, there was a good correspondence between the reductase activities and the presence of the corresponding genes. Distribution in the different ribotypes of strains harboring both the narG and nosZ genes and of strains missing both genes suggests that these two groups of strains had different evolutionary histories. Both dissimilatory genes showed high polymorphism, with similarity indexes (Jaccard) of between 0.04 and 0.8, whereas those of the 16S rRNA gene only varied from 0.77 to 0.99. No correlation between the similarity indexes of 16S rRNA and dissimilatory genes was seen, suggesting that the evolution rates of ribosomal and functional genes differ. Pairwise comparison of similarity indexes of the narG and nosZ genes led to the delineation of two types of strains. Within the first type, the similarity indexes of both genes varied in the same range, suggesting that these two genes have followed a similar evolution. Within the second type of strain, the range of variations was higher for the nosZ than for the narG gene, suggesting that these genes have had a different evolutionary rate.     

Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

硫化物抑制潮土反硝化过程中氧化亚氮还原的菌群机制

【背景】土壤中的反硝化作用形成气态产物N_2O和N_2,会导致氮素的气态损失,并造成温室效应。硫化物对土壤的N_2O还原具有抑制作用,但其对菌群和功能基因的影响机制还不清楚。【目的】研究有无外加碳源情况下,硫化物对反硝化作用中间产物(NO、N_2O)的积累、反硝化功能基因(narG、nirS、nirK和nosZ)表达量以及菌群结构的影响。【方法】分别设置不同量葡萄糖(0和1000mg-C/kg干重土壤)和硫化钠(0和150mg-S/kg干重土壤)添加的交叉处理,进行室内微宇宙培养实验,利用自动化培养与实时气体检测系统检测培养过程中NO、N_2O和N_2的积累量,通过反转录定量PCR测定反硝化功能基因表达量,利用MiSeq技术平台基于16S rRNA基因序列的高通量测序分析样品的菌群结构。【结果】硫化钠的添加显著抑制N_2O还原,但是其对于N_2O积累量没有显著影响,却显著降低了NO的积累量。硫化钠的添加短时间内在转录水平上显著抑制N_2O还原酶的活性,并且抑制固氮弧菌属(Azoarcus)、微枝形杆菌属(Microvirga)、剑菌属(Ensifer)、氮氢单胞菌属(Azohydromonas)、芽孢杆菌属(Bacillus)、斯科曼氏球菌属(Skermanella)、申氏杆菌属(Shinella)和西索恩氏菌属(Chthoniobacter)的基因转录,降低它们的转录本丰度,结合Kyoto Encyclopedia of Genes andGenomes(KEGG)数据库的查询结果,发现硫化钠的添加抑制了不产生N_2O的N_2O还原反硝化细菌的生长。【结论】堆肥或其他原因引起的土壤硫化物增加,导致反硝化过程N_2O还原被抑制的原因是由于其对氧化亚氮基因转录的抑制和对不同反硝化菌的选择作用,研究结果有助于认识硫化物对氮代谢影响的微生物机制。     

若尔盖高原湿地泥炭沼泽土亚硝酸盐还原酶（nirK）反硝化细菌群落结构分析

若尔盖泥炭湿地是世界少有的低纬度永久冻土湿地,具有高海拔、高紫外辐射、高有机质的特点。该区域N2O的排放量对全球气候变暖有重要影响。对若尔盖高原湿地泥炭沼泽土中的亚硝酸盐还原酶(nir K)反硝化细菌群落结构多样性进行分析,以期揭示该区域N2O释放的微生物调控机制。基于若尔盖高原湿地泥炭沼泽土的理化性质和反硝化活性(PDA),结合限制性酶切片段长度多态性(Restriction fragment length polymorphism,RFLP)技术、克隆文库及分子测序对该生态系统中的nir K反硝化细菌群落结构及多样性进行分析。反硝化活性测定结果显示:阿西地区麦溪地区分区地区,反硝化活性与土壤有机碳、总氮和丰富度呈显著正相关(P0.05)。Shannon-wiener多样性指数以阿西最高、分区最低。3个样品中共测序15条nir K基因代表序列,系统发育表明若尔盖高原湿地优势nir K反硝化菌群为变形门菌群。其中,阿西地区主要为α-变形菌门,麦溪地区主要为β-变形菌门,分区地区无法确定优势种群。冗余分析(Redundancy analysis,RDA)显示:有效钾和有效磷是影响nir K反硝化细菌群落结构的关键环境因子。本论文显示,若尔盖高原湿地存在着明显的反硝化作用,调控这些反硝化作用的nir K反硝化细菌多样性较高,且与土壤有效钾和有效磷密切相关。     

Reduction in denitrification activity in field soils exposed to long term contamination by 2,4,6-trinitrotoluene (TNT)

Terrestrial sites contaminated with 2,4,6-trinitrotoluene (TNT) are a widespread and persistent problem and often contain non-vegetated areas with TNT concentrations well in excess of 1000 mg kg(-1). In this study, we examined the effect of TNT on denitrification activity in field soils, and compared the sensitivity of denitrifying enzymes to TNT. DNA probes assessed the prevalence of nirS, nirK and nosZ (encoding cd(1) or copper nitrite reductase and nitrous oxide reductase, respectively), denitrifying genotypes in the culturable and total microbial community. The nitrate (NaR), nitrite (NiR) and nitrous oxide (N(2)OR) reductase activities in field soil and in isolates were assessed by gas chromatography. The relative occurrence of the nirK, nirS or nosZ genotypes increased in the cultured community and in total uncultured community DNA as nitroaromatic concentrations increased. However, denitrifying activity decreased in response to increasing TNT concentrations, with an IC(50) for NaR+NiR+nitric oxide reductase (NOR) of 400 mg TNT kg(-1) soil and for N(2)OR of 26 mg TNT kg(-1) soil. The denitrifying activity of four soil isolates also decreased in response to TNT, with N(2)OR activity being three times more sensitive to TNT than NaR+NiR+NOR activity. Interestingly, there were 118 times more nirK isolates than nirS isolates in uncontaminated soil but only 1.5 times more in soil containing 17400 mg kg(-1) TNT. The results from this study indicated that TNT reduced denitrification activity in field soils, and N(2)OR was much more sensitive to TNT than NaR+NiR+NOR.     

过量施肥对设施菜田土壤菌群结构及N2O产生的影响

【背景】N_2O是一种很强的温室气体,其温室效应强度大约是CO_2的265倍。土壤氮肥施加量是影响N_2O排放的重要因素,而厌氧条件下微生物反硝化则是N_2O产生的重要途径。【目的】研究过量施肥条件下蔬菜大棚土壤菌群结构变化及其对N_2O气体排放的影响。【方法】利用自动化培养与实时气体检测系统(Robot)监测土壤厌氧培养过程中N_2O和N_2排放通量,比较过量施肥和减氮施肥模式下土壤N_2O排放模式的差异。通过Illumina二代测序平台对这2种不同施肥处理的土壤微生物群落进行高通量测序,研究不同施肥量对土壤菌群组成的影响。【结果】过量施肥土壤中硝酸盐的含量大约是减氮施肥土壤的2倍,通过添加硝酸盐使2种土壤的硝酸盐含量均为60 mg/kg或为200 mg/kg时,过量施肥土壤在厌氧培养前期N_2O气体的产生量及产生速度都明显高于减氮施肥土壤。另外,过量施肥导致土壤菌群结构发生显著改变,并且降低了土壤微生物的多样性。相对于减氮施肥,过量施肥方式富集了Rhodanobacter属的微生物。PICRUSt预测结果显示,传统施肥没有显著改变反硝化功能基因相对丰度。【结论】长期过量氮肥施用显著增加了土壤N_2O的排放,可能原因是施肥改变了包括氮转化相关微生物在内的土壤菌群组成,从而影响了土壤N_2O气体的形成与还原过程。     

Nitrous Oxide Reductase (nosZ) Gene Fragments Differ between Native and Cultivated Michigan Soils

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are α-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in γ-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D_mean = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D_mean = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.     

短期落干对水稻土反硝化微生物丰度和N2O释放的影响

为了探明水稻土落干过程对温室气体排放和反硝化微生物的影响,通过模拟水稻土淹水落干过程,系统监测了落干开始后24 h内N2O的释放和氧化还原电位(Eh)的变化,并利用实时PCR(qPCR)方法测定了反硝化功能基因narG和nosZ的丰度.结果表明：落干开始后4 h N₂O释放量就明显增加,在24 h时N₂O的释放量比淹水对照增加了5倍多;narG和nosZ基因丰度也随着落干过程的推移而快速增加;而且N₂O排放通量与narG基因呈极显著相关(P<0.01).表明水稻土短期淹水落干过程中,含narG基因反硝化微生物是驱动N₂O释放的主要功能微生物.     

短期落干对水稻土反硝化微生物丰度和N2O释放的影响

为了探明水稻土落干过程对温室气体排放和反硝化微生物的影响,通过模拟水稻土淹水落干过程,系统监测了落干开始后24 h内N2O的释放和氧化还原电位(Eh)的变化,并利用实时PCR(qPCR)方法测定了反硝化功能基因narG和nosZ的丰度.结果表明：落干开始后4 h N₂O释放量就明显增加,在24 h时N₂O的释放量比淹水对照增加了5倍多;narG和nosZ基因丰度也随着落干过程的推移而快速增加;而且N₂O排放通量与narG基因呈极显著相关(P<0.01).表明水稻土短期淹水落干过程中,含narG基因反硝化微生物是驱动N₂O释放的主要功能微生物.     

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.     

恩诺沙星残留对土壤中反硝化细菌氧化二氮还原酶基因(nosZ)多样性的影响

为了解恩诺沙星在环境中残留对土壤微生物的影响,通过PCR扩增、基因克隆、RFLP分析法对恩诺沙星影响下的土壤反硝化细菌氧化二氮还原酶nosZ基因的分子多样性进行了研究。结果表明,恩诺沙星作用于土壤后第35天,ⅠⅥ组(Ⅰ组0μg/g、Ⅱ组0.01μg/g、Ⅲ组0.1μg/g、Ⅳ组1μg/g、Ⅴ组10μg/g、Ⅵ组50μg/g)的OTUs与克隆子的百分比分别为:48.30%、41.88%、34.78%、33.62%、25.42%、23.81%;第70天,ⅠⅥ组的OTUs与克隆子的百分比分别为:29.66%、24.24%、18.10%、16.67%、15.83%、14.39%。对照组多样性指数均高于添加药物组,第35天,对照组的M argalef指数与添加药物各组差异均显著(P0.05),第70天,仅与10μg/g和50μg/g两组差异显著;第35天,除了0.01μg/g组,对照组的Shannon-W iener指数与其他添加药物组差异均显著,第70天,仅与50μg/g组差异显著。由此可见,随着药物作用的时间延长,药物含量0.0110μg/g组土壤反硝化细菌的多样性与对照组之间的差异变小。     

Abundance of narG, nirS, nirK, and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 x 10(5) to 8.9 x 10(5) copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 x 10(3) to 2.6 x 10(4), 7.4 x 10(2) to 1.4 x 10(3), 2.5 x 10(2) to 6.4 x 10(3), and 1.2 x 10(3) to 5.5 x 10(3), respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Microbial succession of nitrate-reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central Alps

Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (>2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community.     

化肥对稻田土壤细菌多样性及硝化、反硝化功能菌组成的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,采用聚合酶链式反应(polymerase chain reaction,PCR)和DNA序列测定技术分析研究了3种长期施肥制度(对照不施肥-CK,单施氮肥-N,氮磷钾肥-NPK)对土壤细菌群落以及硝化、反硝化微生物种群的影响.通过系统分析细菌16S rDNA、细菌的硝化基因氨单加氧酶(ammonia monooxygenase,amoA)和反硝化基因氧化亚氮还原酶(nitrous oxide reductase,nosZ)等基因文库发现,长期单施氮肥导致细菌16S rDNA和amoA的多样性明显低于CK和NPK处理,而nosZ的多样性与之相反,即单施氮肥处理明显高于CK和NPK处理.LUBSHUFF软件统计分析显示:16S rDNA和amoA基因文库在CK与N,CK与NPK,NPK与N处理间均存在显著性差异.而对于nosZ基因文库,N和NPK与CK处理相比呈现出了显著性差异,N与NPK之间的差异没有达到显著水平.上述结果表明长期施用化肥对水稻土细菌的群落结构及硝化和反硝化细菌组成产生了明显的影响,但这种影响因基因类型而异.