ABSTRACT The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.

The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients.

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.

In this study, the structure and activity of the nitrate-reducers community were analysed in bulk and rhizospheric soils from three different non-isogenic transgenic cultivars of maize (two Bacillus thuringiensis maize and one glyphosate-resistant maize) in a long-term field experiment. DNA was extracted from both rhizospheric and non-rhizospheric soil sampled at three different development stages of the plants and amplified using primers targeting the genes encoding the␣membrane-bound nitrate reductase (narG). Nitrate-reducers community structure was analysed by generating fingerprints and sequencing of narG clone libraries. The season seems to be the most important factor controlling the genetic structure of the nitrate-reducers community. Smaller differences in the narG fingerprints were also observed between bulk and rhizospheric soils suggesting that presence of maize roots was the second important factor affecting the structure of this functional community. Similarly, a rhizosphere effect was observed on the nitrate reductase activity with a 2–3-fold increased in the rhizospheric soil compared to the non-rhizospheric soil. However, for both structure and activity of the nitrate-reducers community, no effect of the maize cultivar was observed. This study suggests that the effect of the cultivar and/or of the agricultural practices associated with the cultivation of transgenic maize is not significant compared to the effect of other environmental factors.

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.

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.

Emissions of N2O, a greenhouse gas, were measured in field conditions on a Rendzic Leptosol, an Eutric Leptosol, a Haplic Calcisol, a Haplic Luvisol and two Gleyic Luvisols under cultivation, and on a Haplic Fluvisol and two Gleyic Cambisols under cultivation and grassland conditions. The kinetics of N2O production and consumption during denitrification in these soils were studied during anaerobic incubations in the laboratory, with NO3- and N2O addition in the presence or absence of acetylene. The soils with the highest "in situ" levels of N2O emission, i.e. the Gleyic Luvisols under cultivation and the grassland soils, exhibited considerable transient accumulation of N2O during denitrification studied in the laboratory. We therefore propose an empirical indicator, measured in the laboratory, of the soil's potential to emit N2O on the field scale. Carbon addition to one of the Gleyic Luvisols promoted N2O reduction and limited the transient accumulation of N2O during laboratory denitrification.

Bacteria capable of denitrification are spread among phylogenetically diverse groups. In the present investigation, molecular methods (amplified ribosomal DNA restriction analysis (ARDRA) and partial 16S rDNA gene sequencing) were used to determine the genetic diversity of culturable denitrifying soil bacteria. The purpose of this work was to study the microbial density and diversity of denitrifying communities isolated from two luvisols and a rendosol. The denitrifying bacterial density was significantly higher in the two luvisols (3x10(6) and 4x10(6) bacteria g(-1) dry soil) than in the rendosol (4x10(5) bacteria g(-1) dry soil). Denitrifying isolates from soils were grouped according to the similarity of their restriction patterns into 26 ARDRA types. Interestingly ARDRA analysis suggests that some denitrifying isolates are specific to a soil type while others seem to be geographically widespread. The number of individual isolates found in each ARDRA type appeared to be highly variable between the two sampling dates but some denitrifying types were capable of persisting in soil. The tree obtained from the partial sequences revealed five major branches exhibiting highest identity to the following genera: (i) Burkholderia-Ralstonia, (ii) Pseudomonas, (iii) Xanthomonas-Frateuria, (iv) Bacillus and (v) Streptomyces. Our 16S rDNA-based analysis clearly reveals broad diversity exceeding that previously described in the literature.

Statistical relationships were established between the fate of C and N from 47 types of crop residues and their biochemical characteristics during a soil incubation at 15°C. The incubations were carried out under nonlimiting N in order to differentiate the effects of biochemical characteristics of residues from those of soil N availability. Depending on the residue, the apparent mineralization of residue C after 168 d varied from 330 to 670 g kg−1 of added C. Mineralization kinetics were described using a two‐compartment decomposition model that decomposes according to first‐order kinetics. Amounts of C mineralized after 7 d and the decomposition rate coefficient of the labile fraction were related mainly to the soluble C forms of the residue. No statistical relationship was established between the N concentration of residues and their decomposition in the soil. The incorporation of crop residues into soil led to various soil mineral N dynamics. Two residues caused net N mineralization from the time of their incorporation, whereas all the others induced net N immobilization (1–33 g N kg−1 of added C). After 168 d, only residues with a C/N ratio <24 induced a surplus of mineral N compared with the control soil. The mineral N dynamics were related mainly to the organic N concentration of the residues and to their C/N ratio. At the start of incubation, these dynamics were also influenced by the presence of polyphenols in the plant tissues. Finally, this study showed the need to include the biochemical quality of crop residues in any C and N transformation models that describe decomposition. In contrast, the N concentration or C/N ratio of the residues are sufficient to predict the net effects of crop residues on soil mineral N dynamics.

The ozone-depleting gas N2O is an intermediate in denitrification, the biological reduction of NO3– to the gaseous products N2O and N2 gas. The molar ratio of N2O produced (N2O/N2O+N2) varies temporally and spatially, and in some soils N2O may be the dominant end product of denitrification. The fraction of NO3–-N emitted as N2O may be due at least in part to the abundance and activity of denitrifying bacteria which possess N2O reductase. In this study, we enumerated NO3–-reducing and denitrifying bacteria, and compared and contrasted collections of denitrifying bacteria isolated from two agricultural soils, one (Auxonne, soil A) with N2O as the dominant product of denitrification, the other (Châlons, soil C) with N2 gas as the dominant product. Isolates were tested for the ability to reduce N2O, and the presence of the N2O reductase (nosZ)-like gene was evaluated by polymerase chain reaction (PCR) using specific primers coupled with DNA hybridization using a specific probe. The diversity and phylogenetic relationships of members of the collections were established by PCR/restriction fragment length polymorphism of 16s rDNA. The two soils had similar numbers of bacteria which used NO3– as a terminal electron acceptor anaerobically. However, the soil A had many more denitrifiers which reduced NO3– to gaseous products (N2O or N2) than did soil C. Collections of 258 and 281 bacteria able to grow anaerobically in the presence of NO3– were isolated from soil A and soil C, respectively. These two collections contained 66 and 12 denitrifying isolates, respectively, the others reducing NO3– only as far as NO2–. The presence of nosZ sequences was generally a poor predictor of N2O reducing ability: there was agreement between the occurrence of nosZ sequences and the N2O reducing ability for only 42% of the isolates; 35% of the isolates (found exclusively in soil A) without detectable nosZ sequences reduced N2O whereas 21% of the isolates carrying nosZ sequences did not reduce this gas under our assay conditions. Twenty-eight different 16S rDNA restriction patterns (using two restriction endonucleases) were distinguished among the 78 denitrifying isolates. Two types of patterns appeared to be common to both soils. Twenty-three and three types of patterns were found exclusively among bacteria isolated from soils A and C, respectively. The specific composition of denitrifying communities appeared to be different between the two soils studied. This may partly explain the differences in the behaviour of the soils concerning N2O reduction during denitrification.

An experiment under glasshouse conditions with a sandy soil was conducted to evaluate the effect of the inoculation of wheat (Triticum aestivum L.) roots with Bacillus polymyxa on N uptake by plants and to investigate the effect of N fertilization on the dynamics of this strain. Different treatments were considered: (i) amended (100 kg N ha−1) or not with 15N labelled ammonium nitrate; (ii) inoculated (2.5 107 spores per seed and 0.5 109 spores kg−1 soil) or not with B. polymyxa CF43. No promoting effect on wheat growth (dry yield) or N uptake was observed after inoculation under these experimental conditions ; 62 per cent of the N fertilizer was assimilated by plants during the first 33 days. Inoculation had no effect on the quantity of soil adhering to the roots. Soluble organic C and inorganic N concentrations were higher in the soil adhering to the roots than in bulk soil. However, the nitrate concentration in soil adhering to the roots was influenced by inoculation at the beginning of plant growth, nitrate supply to the root system being delayed by its probable retention in the rhizosphere. Furthermore, total bacteria in roots, soil adhering to the roots and bulk soil was not affected by N fertilization. The number of B. polymyxa cells measured by an AB-ELISA method increased first in the bulk soil and later in the soil adhering to the roots after inoculation. Fertilization with N decreased the B. polymyxa population in adhering soil, competitive suppression of diazotrophs by non-fixing bacteria probably being induced by the presence of large amounts of nutrients in the rhizosphere when N was added.

Summary The decomposition of 15 N‐labelled catch‐crop materials (rape, radish and rye), obtained from field experiments, was studied in a chalky Champagne soil during a 60‐week incubation at 28°C. Mineralized N was assumed to come from either labile or recalcitrant fractions of plant residues. The labile fraction represented about one‐third of the catch‐crop N; its mineralization rate constant varied from 0.06 to 0.12 d −1 . The decomposition rate of the recalcitrant N fraction ranged from 0.03 × 10 −2 to 0.06 × 10 −2 d −1 . Catch‐crop species and rate of incorporation had no effect on N residue mineralized at the end of incubation. The decomposition of labelled rye was monitored in the same soil during a 5‐month pot experiment to determine the N availability to an Italian ryegrass crop and the effect of plants on the decomposition processes. The 15 N‐rye decomposed rapidly both in the presence or absence of Italian ryegrass, but the amounts of N mineralized were influenced by the presence of living roots: 42% of the 15 N in labelled rye was present as inorganic N in the pots without plants after 5 months, compared with only 32% in the ryegrass crop. Comparison of microbial‐biomass dynamics in both treatments suggested that there had been preferential utilization by soil micro‐organisms of materials released from the living roots than the labelled plant residues.

Fresh farmyard manure (C-to-N ratio = 20.3) was composted in the presence of 15N-labelled (NH4)2SO4 using a bench-scale reactor under temperature conditions which reproduce the evolution of compost in a pile. C and N mineralization of composted farmyard manure (C-to-N ratio = 12.5) was monitored in two silty soils during an aerobic incubation at 28-degrees-C and 100% of WHC for 76 weeks. C mineralization of compost was assumed to come from a labile and a recalcitrant fraction which decomposed according to 1 and 0 order kinetic reactions, respectively [Cm = Clabile.(1 - e(-klabile.t)) + kresistant.t]. Tle size of the labile fraction (Clabile) represented 31.3 and 19.9% of compost-C for the two soils, and its mineralization rate constant (klabile) ranged from 0.03 to 0.09 day-1. N-mineralization was assumed to come from only one labile fraction [Nm = Nlabile.(1 - e(-klabile.t))]. This fraction ranged from 34 to 25% of compost-N for the two soils; its mineralization mte constant did not vary with soils (0.006 day-1). Composted farmyard manure produced under these laboratory conditions may not have been completely mature when added to soil and totally representative of comparable material composted in a pile. Nevertheless, it was considered as fairly mineralizable and its mineralization was greatly influenced by soil type.

C and N cycling were examined in a soil incubated at 4, 12, 20 or 28-degrees-C for 140 days. Before incubation the soil was amended with (KNO3)-N-15, and either glucose-C-14 or holocellulose-C-14. The kinetics of tracer and non-tracer C and N from the biomass, mineralized-C and inorganic-N were measured.C and N behaviours in soil were influenced by temperature, substrate and substrate-temperature interactions. Labelled-C mineralization rates after 140 days ranged from 41 to 58% for glucose and from 34 to 65% for holocellulose. Maximal immobilization was 21.8-31.6 mg N kg-1 soil for holocellulose and 24.3-33.5 mg N kg-1 soil for glucose. Re-mineralization began earlier with glucose and at higher temperatures: 6-23% of immobilized-N were re-mineralized for glucose and 0-19% for holocellulose. More labelled C and N were incorporated into the microbial biomass from both carbon sources at lower temperatures. The biomass turnover was highly influenced by temperature: 40-46% of labelled C or N incorporated in the biomass remained in this compartment at 20-28-degrees-C, while corresponding values at 4-12-degrees-C were only 0-40%.Organic-C-14 mineralization and immobilization rate constants were influenced by temperature, the different trends depending on the carbon source. Thus an overall temperature coefficient (Q-10) could not be determined for these complex transformations. Variations in the rate constant with temperature were described using polynomial regressions.

The behaviour and availability of soil organic-C and -N were studied by means of soil particle-size fractionation techniques. In a first experiment, three soils were incubated with 14C-glucose or 14C-cellulose and K15NO3 for 104 weeks at 28°C and were fractionated at different sampling dates. The radioactivity was preferentially incorporated in the sand and coarse clay fractions for the cellulose treatment, and in the coarse clay, light silt and soluble fractions for the glucose treatment. All fractions for the glucose treatment and the sand fraction from the cellulose treatment were responsible for the mineralization of organic-14C during incubation. With the glucose and cellulose treatments, organic-15N was incorporated in the coarse clay, light silt and soluble fractions, and inorganic-15N produced during incubation was derived essentially from mineralization of these fractions. In a second experiment, 15N labelled soil materials (0-2, 2-20, 20-50, 50-200 μm fractions and whole soil) were then separately incubated for 224 days at 28 °C in shaken liquid media. For the calcic cambisoil, the labelled-N contained in the clay and sand fraction was more available than in the silt fraction. For the loamy soil, all fractions exhibited comparable 15N mineralization rates, except for the 20-50 μm fraction. At the same time, the non-labelled-N present in all fractions appeared to be less mineralizable than recently immobilized 15N.

, IRD UR179 SeqBio, LRI Université d'AntananarivoBP 434, 101 Antananarivo, Madagascar Iydie.lardy@ird.fr 21RD UR179 SeqBio, LEMSA T BP 1386, Dakar, Sénégal 3 INRA, CMSE, 21065 Dijon, France • CNRS UMR5175, CEFE, 1919 route de Mende, 34293 Montpellier, France 5 ISTOM, 32 boulevard du Port, 95094 Cergy-Pontoise, Cedex 5, France e FOFIFA URP SCRiD cio FOFIFA B.P. 1690, Antananarivo, Madagascar 7 LRI-SRA Université d'Antananarivo, BP 3383, 101 Antananarivo, Madagascar e IRD UR179 SeqBio, 2 place Viala, bâtiment 12, 34060 Montpellier cedex 1, France