Biological inhibition of soil nitrification by forest tree species affects Nitrobacter populations.

Some temperate tree species are associated with very low soil nitrification rates, with important implications for forest N dynamics, presumably due to their potential for biological nitrification inhibition (BNI). However, evidence for BNI in forest ecosystems is scarce so far and the nitrifier groups controlled by BNI-tree species have not been identified. Here, we evaluated how some tree species can control soil nitrification by providing direct evidence of BNI and identifying the nitrifier group(s) affected. First, by comparing 28 year-old monocultures of several tree species, we showed that nitrification rates correlated strongly with the abundance of the nitrite oxidizers Nitrobacter (50- to 1000-fold changes between tree monocultures) and only weakly with the abundance of ammonia oxidizing archaea (AOA). Second, using reciprocal transplantation of soil cores between low and high nitrification stands, we demonstrated that nitrification changed 16 months after transplantation and was correlated with changes in the abundance of Nitrobacter, not AOA. Third, extracts of litter or soil collected from the low nitrification stands of Picea abies and Abies nordmanniana inhibited the growth of Nitrobacter hamburgensis X14. Our results provide for the first time direct evidence of BNI by tree species directly affecting the abundance of Nitrobacter.

Denitrifying bacterial communities display different temporal fluctuation patterns across Dutch agricultural soils.

Considering the great agronomic and environmental importance of denitrification, the aim of the present study was to study the temporal and spatial factors controlling the abundance and activity of denitrifying bacterial communities in a range of eight agricultural soils over 2 years. Abundance was quantified by qPCR of the nirS, nirK and nosZ genes, and the potential denitrification enzyme activity (DEA) was estimated. Our data showed a significant temporal variation considerably high for the abundance of nirK-harboring communities, followed by nosZ and nirS communities. Regarding soil parameters, the abundances of nosZ, nirS and nirK were mostly influenced by organic material, pH, and slightly by NO3-, respectively. Soil texture was the most important factor regulating DEA, which could not be explained by the abundance of denitrifiers. Analyses of general patterns across lands to understand the soil functioning is not an easy task because the multiple factors influencing processes such as denitrification can skew the data. Careful analysis of atypical sites are necessary to classify the soils according to trait similarity and in this way reach a better predictability of the denitrifiers dynamics.

Assessing the dynamic changes of rhizosphere functionality of Zea mays plants grown in organochlorine contaminated soils.

The persistent organochlorine pesticides (OCPs) in soils are suspected to disturb soil biogeochemical cycles. This study addressed the dynamic changes in soil functionality under lindane and chlordecone exposures with or without maize plant. Decreases in soil ammonium concentration, potential nitrogen mineralization and microbial biomass were only OCP-influenced in bulk soils. OCPs appeared to inhibit the ammonification step. With plants, soil functionality under OCP stress was similar to controls demonstrating the plant influence to ensure the efficiency of C- and N-turnover in soils. Moreover, OCPs did not impact the microbial community physiological profile in all tested conditions. However, microbial community structure was OCP-modified only in the presence of plants. Abundances of gram-negative and saprophytic fungi increased (up to +93% and +55%, respectively) suggesting a plant stimulation of nutrient turnover and rhizodegradation processes. Nevertheless, intimate microbial/plant interactions appeared to be OCP-impacted with depletions in mycorrhizae and micro/meso-fauna abundances (up to -53% and -56%, respectively) which might have adverse effects on more long-term plant growth (3-4 months). In short-term experiment (28days), maize growth was similar to the control ones, indicating an enhanced plasticity of the soil functioning in the presence of plants, which could efficiently participate to the remediation of OCP-contaminated soils.

Effect of plant diversity on the diversity of soil organic compounds.

The effect of plant diversity on aboveground organisms and processes was largely studied but there is still a lack of knowledge regarding the link between plant diversity and soil characteristics. Here, we analyzed the effect of plant identity and diversity on the diversity of extractible soil organic compounds (ESOC) using 87 experimental grassland plots with different levels of plant diversity and based on a pool of over 50 plant species. Two pools of low molecular weight organic compounds, LMW1 and LMW2, were characterized by GC-MS and HPLC-DAD, respectively. These pools include specific organic acids, fatty acids and phenolics, with more organic acids in LMW1 and more phenolics in LMW2. Plant effect on the diversity of LMW1 and LMW2 compounds was strong and weak, respectively. LMW1 richness observed for bare soil was lower than that observed for all planted soils; and the richness of these soil compounds increased twofold when dominant plant species richness increased from 1 to 6. Comparing the richness of LMW1 compounds observed for a range of plant mixtures and for plant monocultures of species present in these mixtures, we showed that plant species richness increases the richness of these ESOC mainly through complementarity effects among plant species associated with contrasted spectra of soil compounds. This could explain previously reported effects of plant diversity on the diversity of soil heterotrophic microorganisms.

Size evolution in microorganisms masks trade-offs predicted by the growth rate hypothesis.

Adaptation to local resource availability depends on responses in growth rate and nutrient acquisition. The growth rate hypothesis (GRH) suggests that growing fast should impair competitive abilities for phosphorus and nitrogen due to high demand for biosynthesis. However, in microorganisms, size influences both growth and uptake rates, which may mask trade-offs and instead generate a positive relationship between these traits (size hypothesis, SH). Here, we evolved a gradient of maximum growth rate (µmax) from a single bacterium ancestor to test the relationship among µmax, competitive ability for nutrients and cell size, while controlling for evolutionary history. We found a strong positive correlation between µmax and competitive ability for phosphorus, associated with a trade-off between µmax and cell size: strains selected for high µmax were smaller and better competitors for phosphorus. Our results strongly support the SH, while the trade-offs expected under GRH were not apparent. Beyond plasticity, unicellular populations can respond rapidly to selection pressure through joint evolution of their size and maximum growth rate. Our study stresses that physiological links between these traits tightly shape the evolution of competitive strategies.

Predicting the Responses of Soil Nitrite-Oxidizers to Multi-Factorial Global Change: A Trait-Based Approach.

Soil microbial diversity is huge and a few grams of soil contain more bacterial taxa than there are bird species on Earth. This high diversity often makes predicting the responses of soil bacteria to environmental change intractable and restricts our capacity to predict the responses of soil functions to global change. Here, using a long-term field experiment in a California grassland, we studied the main and interactive effects of three global change factors (increased atmospheric CO2 concentration, precipitation and nitrogen addition, and all their factorial combinations, based on global change scenarios for central California) on the potential activity, abundance and dominant taxa of soil nitrite-oxidizing bacteria (NOB). Using a trait-based model, we then tested whether categorizing NOB into a few functional groups unified by physiological traits enables understanding and predicting how soil NOB respond to global environmental change. Contrasted responses to global change treatments were observed between three main NOB functional types. In particular, putatively mixotrophic Nitrobacter, rare under most treatments, became dominant under the 'High CO2+Nitrogen+Precipitation' treatment. The mechanistic trait-based model, which simulated ecological niches of NOB types consistent with previous ecophysiological reports, helped predicting the observed effects of global change on NOB and elucidating the underlying biotic and abiotic controls. Our results are a starting point for representing the overwhelming diversity of soil bacteria by a few functional types that can be incorporated into models of terrestrial ecosystems and biogeochemical processes.

Plant species diversity affects soil-atmosphere fluxes of methane and nitrous oxide.

Plant diversity effects on ecosystem functioning can potentially interact with global climate by altering fluxes of the radiatively active trace gases nitrous oxide (N2O) and methane (CH4). We studied the effects of grassland species richness (1-16) in combination with application of fertilizer (nitrogen:phosphorus:potassium = 100:43.6:83 kg ha(-1) a(-1)) on N2O and CH4 fluxes in a long-term field experiment. Soil N2O emissions, measured over 2 years using static chambers, decreased with species richness unless fertilizer was added. N2O emissions increased with fertilization and the fraction of legumes in plant communities. Soil CH4 uptake, a process driven by methanotrophic bacteria, decreased with plant species numbers, irrespective of fertilization. Using structural equation models, we related trace gas fluxes to soil moisture, soil inorganic N concentrations, nitrifying and denitrifying enzyme activity, and the abundance of ammonia oxidizers, nitrite oxidizers, and denitrifiers (quantified by real-time PCR of gene fragments amplified from microbial DNA in soil). These analyses indicated that plant species richness increased soil moisture, which in turn increased N cycling-related activities. Enhanced N cycling increased N2O emission and soil CH4 uptake, with the latter possibly caused by removal of inhibitory ammonium by nitrification. The moisture-related indirect effects were surpassed by direct, moisture-independent effects opposite in direction. Microbial gene abundances responded positively to fertilizer but not to plant species richness. The response patterns we found were statistically robust and highlight the potential of plant biodiversity to interact with climatic change through mechanisms unrelated to carbon storage and associated carbon dioxide removal.

Physiological effects of major up-regulated Alnus glutinosa peptides on Frankia sp. ACN14a.

Alnus glutinosa has been shown previously to synthesize, in response to nodulation by Frankia sp. ACN14a, an array of peptides called Alnus symbiotic up-regulated peptides (ASUPs). In a previous study one peptide (Ag5) was shown to bind to Frankia nitrogen-fixing vesicles and to modify their porosity. Here we analyse four other ASUPs, alongside Ag5, to determine whether they have different physiological effects on in vitro grown Frankia sp. ACN14a. The five studied peptides were shown to have different effects on nitrogen fixation, respiration, growth, the release of ions and amino acids, as well as on cell clumping and cell lysis. The mRNA abundance for all five peptides was quantified in symbiotic nodules and one (Ag11) was found to be more abundant in the meristem part of the nodule. These findings point to some peptides having complementary effects on Frankia cells.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI.

Identification of B-type procyanidins in Fallopia spp. involved in biological denitrification inhibition.

Nitrogen (N) is considered as a main limiting factor in plant growth, and nitrogen losses through denitrification can be responsible for severe decreases in plant productivity. Recently, it was demonstrated that Fallopia spp. is responsible for biological denitrification inhibition (BDI) through the release of unknown secondary metabolites. Here, we investigate the secondary metabolites involved in the BDI of Fallopia spp. The antioxidant, protein precipitation capability of Fallopia spp. extracts was measured in relation to the aerobic respiration and denitrification of two bacteria (Gram positive and Gram negative). Proanthocyanidin concentrations were estimated. Proanthocyanidins in extracts were characterized by chromatographic analysis, purified and tested on the bacterial denitrification and aerobic respiration of two bacterial strains. The effect of commercial procyanidins on denitrification was tested on two different soil types. Denitrification and aerobic respiration inhibition were correlated with protein precipitation capacity and concentration of proanthocyanidins but not to antioxidant capacity. These proanthocyanidins were B-type procyanidins that inhibited denitrification more than the aerobic respiration of bacteria. In addition, procyanidins also inhibited soil microbial denitrification. We demonstrate that procyanidins are involved in the BDI of Fallopia spp. Our results pave the way to a better understanding of plant-microbe interactions and highlight future applications for a more sustainable agriculture.

Management of Microbial Communities through Transient Disturbances Enhances the Functional Resilience of Nitrifying Gas-Biofilters to Future Disturbances.

Microbial communities have a key role for the performance of engineered ecosystems such as waste gas biofilters. Maintaining constant performance despite fluctuating environmental conditions is of prime interest, but it is highly challenging because the mechanisms that drive the response of microbial communities to disturbances still have to be disentangled. Here we demonstrate that the bioprocess performance and stability can be improved and reinforced in the face of disturbances, through a rationally predefined strategy of microbial resource management (MRM). This strategy was experimentally validated in replicated pilot-scale nitrifying gas-biofilters, for the two steps of nitrification. The associated biological mechanisms were unraveled through analysis of functions, abundances and community compositions for the major actors of nitrification in these biofilters, that is, ammonia-oxidizing bacteria (AOB) and Nitrobacter-like nitrite-oxidizers (NOB). Our MRM strategy, based on the application of successive, transient perturbations of increasing intensity, enabled to steer the nitrifier community in a favorable way through the selection of more resistant AOB and NOB sharing functional gene sequences close to those of, respectively, Nitrosomonas eutropha and Nitrobacter hamburgensis that are well adapted to high N load. The induced community shifts resulted in significant enhancement of nitrification resilience capacity following the intense perturbation.

Functional and structural responses of soil N-cycling microbial communities to the herbicide mesotrione: a dose-effect microcosm approach.

Microbial communities driving the nitrogen cycle contribute to ecosystem services such as crop production and air, soil, and water quality. The responses to herbicide stress of ammonia-oxidizing and ammonia-denitrifying microbial communities were investigated by an analysis of changes in structure-function relationships. Their potential activities, abundances (quantitative PCR), and genetic structure (denaturing gradient gel electrophoresis) were assessed in a microcosm experiment. The application rate (1 × FR, 0.45 µg g(-1) soil) of the mesotrione herbicide did not strongly affect soil N-nutrient dynamics or microbial community structure and abundances. Doses of the commercial product Callisto® (10 × FR and 100 × FR) or pure mesotrione (100 × FR) exceeding field rates induced short-term inhibition of nitrification and a lasting stimulation of denitrification. These effects could play a part in the increase in soil ammonium content and decrease in nitrate contents observed in treated soils. These functional impacts were mainly correlated with abundance shifts of ammonia-oxidizing Bacteria (AOB) and Archaea (AOA) or denitrifying bacteria. The sustained restoration of nitrification activity, from day 42 in the 100 × FR-treated soils, was likely promoted by changes in the community size and composition of AOB, which suggests a leading role, rather than AOA, for soil nitrification restoration after herbicide stress. This ecotoxicological community approach provides a nonesuch multiparameter assessment of responses of N-cycling microbial guilds to pesticide stress.

Using plant traits to explain plant-microbe relationships involved in nitrogen acquisition.

It has long been recognized that plant species and soil microorganisms. are tightly linked, but understanding how different species vary in their effects on soil is currently limited. In this study, we identified those. plant characteristics (identity, specific functional traits, or resource acquisition strategy) that were the best predictors of nitrification and denitrification processes. Ten plant populations representing eight species collected from three European grassland sites were chosen for their contrasting plant trait values and resource acquisition strategies. For each individual plant, leaf and root traits and the associated potential microbial activities (i.e., potential denitrification rate [DEA], maximal nitrification rate [NEA], and NH4+ affinity of the microbial community [NHScom]) were measured at two fertilization levels under controlled growth conditions. Plant traits were powerful predictors of plant-microbe interactions, but relevant plant traits differed in relation to the microbial function studied. Whereas denitrification was linked to the relative growth rate of plants, nitrification was strongly correlated to root trait characteristics (specific root length, root nitrogen concentration, and plant affinity for NH4+) linked to plant N cycling. The leaf economics spectrum (LES) that commonly serves as an indicator of resource acquisition strategies was not correlated to microbial activity. These results suggest that the LES alone is not a good predictor of microbial activity, whereas root traits appeared critical in understanding plant-microbe interactions.

Resource pulses can alleviate the biodiversity-invasion relationship in soil microbial communities.

The roles of species richness, resource use, and resource availability are central to many hypotheses explaining the diversity-invasion phenomenon but are generally not investigated together. Here, we created a large diversity gradient of soil microbial communities by either assembling communities of pure bacterial strains or removing the diversity of a natural soil. Using data on the resource-use capacities of the soil communities and an invader that were gathered from 71 carbon sources, we quantified the niches available to both constituents by using the metrics community niche and remaining niche available to the invader. A strong positive relationship between species richness and community niche across both experiments indicated the presence of resource complementarity. Moreover, community niche and the remaining niche available to the invader predicted invader abundance well. This suggested that increased competition in communities of higher diversity limits community invasibility and underscored the importance of resource availability as a key mechanism through which diversity hinders invasions. As a proof of principle, we subjected selected invaded communities to a resource pulse, which progressively uncoupled the link between soil microbial diversity and invasion and allowed the invader to rebound after nearly being eliminated in some communities. Our results thus show that (1) resource competition suppresses invasion, (2) biodiversity increases resource competition and decreases invasion through niche preemption, and (3) resource pulses that cannot be fully used, even by diverse communities, are favorable to invasion.

Coupling Between and Among Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms Submitted to Elevated CO2 and Nitrogen Supply.

Many studies have assessed the responses of soil microbial functional groups to increases in atmospheric CO2 or N deposition alone and more rarely in combination. However, the effects of elevated CO2 and N on the (de)coupling between different microbial functional groups (e.g., different groups of nitrifiers) have been barely studied, despite potential consequences for ecosystem functioning. Here, we investigated the short-term combined effects of elevated CO2 and N supply on the abundances of the four main microbial groups involved in soil nitrification: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (belonging to the genera Nitrobacter and Nitrospira) in grassland mesocosms. AOB and AOA abundances responded differently to the treatments: N addition increased AOB abundance, but did not alter AOA abundance. Nitrobacter and Nitrospira abundances also showed contrasted responses to the treatments: N addition increased Nitrobacter abundance, but decreased Nitrospira abundance. Our results support the idea of a niche differentiation between AOB and AOA, and between Nitrobacter and Nitrospira. AOB and Nitrobacter were both promoted at high N and C conditions (and low soil water content for Nitrobacter), while AOA and Nitrospira were favored at low N and C conditions (and high soil water content for Nitrospira). In addition, Nitrobacter abundance was positively correlated to AOB abundance and Nitrospira abundance to AOA abundance. Our results suggest that the couplings between ammonia and nitrite oxidizers are influenced by soil N availability. Multiple environmental changes may thus elicit rapid and contrasted responses between and among the soil ammonia and nitrite oxidizers due to their different ecological requirements.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites.

Previous studies on the effect of secondary metabolites on the functioning of rhizosphere microbial communities have often focused on aspects of the nitrogen (N) cycle but have overlooked biological denitrification inhibition (BDI), which can affect plant N-nutrition. Here, we investigated the BDI by the compounds of Fallopia spp., an invasive weed shown to be associated with a low potential denitrification of the soil. Fallopia spp. extracts were characterized by chromatographic analysis and were used to test the BDI effects on the metabolic and respiratory activities of denitrifying bacteria, under aerobic and anaerobic (denitrification) conditions. The BDI of Fallopia spp. extracts was tested on a complex soil community by measuring denitrification enzyme activity (DEA), substrate induced respiration (SIR), as well as abundances of denitrifiers and total bacteria. In 15 strains of denitrifying bacteria, extracts led to a greater BDI (92%) than respiration inhibition (50%). Anaerobic metabolic activity reduction was correlated with catechin concentrations and the BDI was dose dependent. In soil, extracts reduced the DEA/SIR ratio without affecting the denitrifiers: total bacteria ratio. We show that secondary metabolite(s) from Fallopia spp. inhibit denitrification. This provides new insight into plant-soil interactions and improves our understanding of a plant's ability to shape microbial soil functioning.

Are plant-soil feedback responses explained by plant traits?

Plant-soil feedbacks can influence plant growth and community structure by modifying soil biota and nutrients. Because most research has been performed at the species level and in monoculture, our ability to predict responses across species and in mixed communities is limited. As plant traits have been linked to both soil properties and plant growth, they may provide a useful approach for an understanding of feedbacks at a generic level. We measured how monocultures and mixtures of grassland plant species with differing traits responded to soil that had been conditioned by model grassland plant communities dominated by either slow- or fast-growing species. Soils conditioned by the fast-growing community had higher nitrogen availability than those conditioned by the slow-growing community; these changes influenced future plant growth. Effects were stronger, and plant traits had greater predictive power, in mixtures than in monocultures. In monoculture, all species produced more above-ground biomass in soil conditioned by the fast-growing community. In mixtures, slow-growing species produced more above-ground biomass, and fast-growing species produced more below-ground biomass, in soils conditioned by species with similar traits. The use of a plant trait-based approach may therefore improve our understanding of differential plant species responses to plant-soil feedbacks, especially in a mixed-species environment.

Phytochemical analysis of mature tree root exudates in situ and their role in shaping soil microbial communities in relation to tree N-acquisition strategy.

Eperua falcata (Aublet), a late-successional species in tropical rainforest and one of the most abundant tree in French Guiana, has developed an original strategy concerning N-acquisition by largely preferring nitrate, rather than ammonium (H. Schimann, S. Ponton, S. Hättenschwiler, B. Ferry, R. Lensi, A.M. Domenach, J.C. Roggy, Differing nitrogen use strategies of two tropical rainforest tree species in French Guiana: evidence from (15)N natural abundance and microbial activities, Soil Biol. Biochem. 40 (2008) 487-494). Given the preference of this species for nitrate, we hypothesized that root exudates would promote nitrate availability by (a) enhancing nitrate production by stimulating ammonium oxidation or (b) minimizing nitrate losses by inhibiting denitrification. Root exudates were collected in situ in monospecific planted plots. The phytochemical analysis of these exudates and of several of their corresponding root extracts was achieved using UHPLC/DAD/ESI-QTOF and allowed the identification of diverse secondary metabolites belonging to the flavonoid family. Our results show that (i) the distinct exudation patterns observed are related to distinct root morphologies, and this was associated with a shift in the root flavonoid content, (ii) a root extract representative of the diverse compounds detected in roots showed a significant and selective metabolic inhibition of isolated denitrifiers in vitro, and (iii) in soil plots the abundance of nirK-type denitrifiers was negatively affected in rhizosphere soil compared to bulk. Altogether this led us to formulate hypothesis concerning the ecological role of the identified compounds in relation to N-acquisition strategy of this species.

Soil environmental conditions and microbial build-up mediate the effect of plant diversity on soil nitrifying and denitrifying enzyme activities in temperate grasslands.

Random reductions in plant diversity can affect ecosystem functioning, but it is still unclear which components of plant diversity (species number - namely richness, presence of particular plant functional groups, or particular combinations of these) and associated biotic and abiotic drivers explain the observed relationships, particularly for soil processes. We assembled grassland communities including 1 to 16 plant species with a factorial separation of the effects of richness and functional group composition to analyze how plant diversity components influence soil nitrifying and denitrifying enzyme activities (NEA and DEA, respectively), the abundance of nitrifiers (bacterial and archaeal amoA gene number) and denitrifiers (nirK, nirS and nosZ gene number), and key soil environmental conditions. Plant diversity effects were largely due to differences in functional group composition between communities of identical richness (number of sown species), though richness also had an effect per se. NEA was positively related to the percentage of legumes in terms of sown species number, the additional effect of richness at any given legume percentage being negative. DEA was higher in plots with legumes, decreased with increasing percentage of grasses, and increased with richness. No correlation was observed between DEA and denitrifier abundance. NEA increased with the abundance of ammonia oxidizing bacteria. The effect of richness on NEA was entirely due to the build-up of nitrifying organisms, while legume effect was partly linked to modified ammonium availability and nitrifier abundance. Richness effect on DEA was entirely due to changes in soil moisture, while the effects of legumes and grasses were partly due to modified nitrate availability, which influenced the specific activity of denitrifiers. These results suggest that plant diversity-induced changes in microbial specific activity are important for facultative activities such as denitrification, whereas changes in microbial abundance play a major role for non-facultative activities such as nitrification.