To determine to which extent root-derived carbon contributes to the effects of plants on nitrate reducers and denitrifiers, four solutions containing different proportions of sugar, organic acids and amino acids mimicking maize root exudates were added daily to soil microcosms at a concentration of 150 microg C g(-1) of soil. Water-amended soils were used as controls. After 1 month, the size and structure of the nitrate reducer and denitrifier communities were analysed using the narG and napA, and the nirK, nirS and nosZ genes as molecular markers respectively. Addition of artificial root exudates (ARE) did not strongly affect the structure or the density of nitrate reducer and denitrifier communities whereas potential nitrate reductase and denitrification activities were stimulated by the addition of root exudates. An effect of ARE composition was also observed on N(2)O production with an N(2)O:(N(2)O + N(2)) ratio of 0.3 in microcosms amended with ARE containing 80% of sugar and of 1 in microcosms amended with ARE containing 40% of sugar. Our study indicated that ARE stimulated nitrate reduction or denitrification activity with increases in the range of those observed with the whole plant. Furthermore, we demonstrated that the composition of the ARE affected the nature of the end-product of denitrification and could thus have a putative impact on greenhouse gas emissions.

ABSTRACT Nitrous oxide (N2O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N2O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 101 and 102 target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 105 to 107 target copies g−1 of dry soil, whereas genes for 16S rRNA were found at 108 to 109 target copies g−1 of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.

One alternative to mineral fertilization is to use organic fertilizers. Our aim was to compare the impacts of 7-year applications of composted pig manure and ammonium nitrate on the structure and activity of the denitrifying community. Mineralization and organization of N, denitrification rates and N2O/N2 ratio were also investigated. Fourteen months after the last application, the potential denitrifying activity (+319%), N mineralization (+110%) and organization (+112%) were higher under pig compost than under ammonium nitrate fertilization. On the other hand, the N2O/(N2O+N2) ratio was lower (P<0.05, n=5) under organic fertilization. These effects of organic fertilization were in accordance with its higher total carbon content and microbial biomass. Fingerprints and clone library analyses showed that the structure of the denitrifying community was affected by the fertilization regime. Our results reveal that organic or mineral fertilizer applications could affect both structure and activity of the denitrifying community, with a possible influence on in situ N2O fluxes. These effects of the fertilization regime persisted for at least 14 months after the last application.

Nitrate reduction is performed by phylogenetically diverse bacteria. Analysis of narG (alpha subunit of the membrane bound nitrate reductase) trees constructed using environmental sequences revealed a new cluster that is not related to narG gene from known nitrate-reducing bacteria. In this study, primers targeting this as yet uncultivated nitrate-reducing group were designed and used to develop a real-time SYBR® Green PCR assay. The assay was tested with clones from distinct nitrate-reducing groups and applied to various environmental samples. narG copy number was high ranging between 5.08×108 and 1.12×1011 copies per gram of dry weight of environmental sample. Environmental real-time PCR products were cloned and sequenced. Data was used to generate a phylogenetic tree showing that all environmental products belonged to the target group. Moreover, 16S rDNA copy number was quantified in the different environments by real-time PCR using universal primers for Eubacteria. 16S rDNA copy number was similar or slightly higher than that of narG, between 7.12×109 and 1.14×1011 copies per gram of dry weight of environmental sample. Therefore, the yet uncultivated nitrate-reducing group targeted in this study seems to be numerically important in the environment, as revealed by narG high absolute and relative densities across various environments. Further analysis of the density of the nitrate-reducing community as a whole by real-time PCR may provide insights into the correlation between microbial density, diversity and activity.

In order to understand the effect of the maize rhizosphere on denitrification, the diversity and the activity of the denitrifying community were studied in soil amended with maize mucilage. Diversity of the denitrifying community was investigated by polymerase chain reaction (PCR) amplification of total community DNA extracted from soils using gene fragments, encoding the nitrate reductase (narG) and the nitrous oxide reductase (nosZ), as molecular markers. To assess the underlying diversity, PCR products were cloned and 10 gene libraries were obtained for each targeted gene. Libraries containing 738 and 713 narG and nosZ clones, respectively, were screened by restriction fragment analysis, and grouped based on their RFLP (restriction fragment length polymorphism) patterns. In all, 117 and 171 different clone families have been identified for narG and nosZ and representatives of RFLP families containing at least two clones were sequenced. Rarefaction curves of both genes did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity. Recovered NarG sequences were related to NarG from Actinomycetales and from Proteobacteria but most of them are not related to NarG from known bacteria. In contrast, most of the NosZ sequences were related to NosZ from alpha, beta, and gammaProteobacteria. Denitrifying activity was monitored by incubating the control and amended soils anaerobically in presence of acetylene. The N2O production rates revealed denitrifying activity to be greater in amended soil than in control soil. Altogether, our results revealed that mucilage addition to the soil results in a strong impact on the activity of the denitrifying community and minor changes on its diversity.

The aim of this study was to determine the effect of the rhizosphere of maize on the diversity of denitrifying bacteria. Community structure comparison was performed by constructing a collection of isolates recovered from bulk and maize planted soil. A total of 3240 nitrate-reducing isolates were obtained and 188 of these isolates were identified as denitrifiers based on their ability to reduce nitrate to N2O or N-2. 16S rDNA fragments amplified from the denitrifying isolates were analysed by restriction fragment length polymorphism. Isolates were grouped according to their restriction patterns, and 16S rDNA of representatives from each group were sequenced. A plant dependent enrichment of Agrobacterium-related denitrifiers has been observed resulting in a modification of the structure of the denitrifying community between planted and bulk soil. In addition, the predominant isolates in the rhizosphere soil were not able to reduce N2O while dominant isolates in the bulk soil evolve N-2 as a denitrification product.

We report the development of quantitative competitive (QC) PCR assays for quantifying the 16S, 18S ribosomal and atzC genes in nucleic acids directly extracted from soil. QC-PCR assays were standardised, calibrated and evaluated with an experimental study aiming to evaluate the impact of atrazine application on soil microflora. Comparison of QC-PCR 16S and 18S results with those of soil microbial biomass showed that, following atrazine application, the microbial biomass was not affected and that the amount of 16S rDNA gene representing 'bacteria' increased transitorily, while the amount of 18S rDNA gene representing fungi decreased in soil. In addition, comparison of atzC QC-PCR results with those of atrazine mineralisation revealed that, in response to atrazine treatment, the amount of atzC gene increased transitorily in soil pre-treated with atrazine, suggesting that accelerated atrazine biodegradation in soil could be due to a transient increase in the size of the atrazine mineralising community.

The ability of facultative anerobes to respire nitrate has been ascribed mainly to the activity of a membrane-bound nitrate reductase encoded by the narGHJI operon. Respiratory nitrate reduction is the first step of the denitrification pathway, which is considered as an important soil process since it contributes to the global cycling of nitrogen. In this study, we employed direct PCR, cloning, and sequencing of narG gene fragments to determine the diversity of nitrate-reducing bacteria occurring in soil and in the maize rhizosphere. Libraries containing 727 clones in total were screened by restriction fragment analysis. Phylogenetic analysis of 128 narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria. Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch. All together, our results revealed a more complex nitrate-reducing community than did previous culture-based studies. A significant and consistent shift in the relative abundance of the nitrate-reducing groups within this functional community was detected in the maize rhizosphere. Thus a substantially higher abundance of the dominant clone family and a lower diversity index were observed in the rhizosphere compared to the unplanted soil, suggesting that a bacterial group has been specifically selected within the nitrate-reducing community. Furthermore, restriction fragment length polymorphism analysis of cloned narG gene fragments proved to be a powerful tool in evaluating the structure and the diversity of the nitrate-reducing community and community shifts therein.

The mineralisation rate of atrazine measured in soil pre-treated with this herbicide, was significantly higher in the maize rhizosphere than in bulk soil. Maize rhizosphere was also shown to significantly increase microbial biomass C as compared with bulk soil. Ribosomal intergenic spacer analysis conducted on nucleic acids extracted directly from soil samples revealed that the structure of microbial communities observed in the rhizosphere was slightly different from that of bulk soil. The quantification of the relative amount of the gene atzC, which encodes an enzyme involved in atrazine mineralisation, was carried out on soil nucleic acids by using quantitative–competitive PCR assays. It revealed that atzC was present at a higher level in the rhizosphere than in bulk soil. In addition, the amount of atzC was transiently enhanced in both rhizosphere and bulk soils following atrazine treatment. These results suggest that the stimulation of atrazine mineralisation in the maize rhizosphere depends on the abundance of atrazine-degrading communities.

ABSTRACT 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 α, β, and γ 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.

ABSTRACT The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.