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.

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.

Fluctuations in Ammonia Oxidizing Communities Across Agricultural Soils are Driven by Soil Structure and pH.

The milieu in soil in which microorganisms dwell is never constant. Conditions such as temperature, water availability, pH and nutrients frequently change, impacting the overall functioning of the soil system. To understand the effects of such factors on soil functioning, proxies (indicators) of soil function are needed that, in a sensitive manner, reveal normal amplitude of variation. Thus, the so-called normal operating range (NOR) of soil can be defined. In this study we determined different components of nitrification by analyzing, in eight agricultural soils, how the community structures and sizes of ammonia oxidizing bacteria and archaea (AOB and AOA, respectively), and their activity, fluctuate over spatial and temporal scales. The results indicated that soil pH and soil type are the main factors that influence the size and structure of the AOA and AOB, as well as their function. The nitrification rates varied between 0.11 ± 0.03 µgN h(-1) gdw(-1) and 1.68 ± 0.11 µgN h(-1) gdw(-1), being higher in soils with higher clay content (1.09 ± 0.12 µgN h(-1) gdw(-1)) and lower in soils with lower clay percentages (0.27 ± 0.04 µgN h(-1) gdw(-1)). Nitrifying activity was driven by soil pH, mostly related to its effect on AOA but not on AOB abundance. Regarding the influence of soil parameters, clay content was the main soil factor shaping the structure of both the AOA and AOB communities. Overall, the potential nitrifying activities were higher and more variable over time in the clayey than in the sandy soils. Whereas the structure of AOB fluctuated more (62.7 ± 2.10%) the structure of AOA communities showed lower amplitude of variation (53.65 ± 3.37%). Similar trends were observed for the sizes of these communities. The present work represents a first step toward defining a NOR for soil nitrification. The sensitivity of the process and organisms to impacts from the milieu support their use as proxies in the NOR of agricultural soils. Moreover, the clear effect of soil texture established here suggests that the NOR should be defined in a soil type-specific manner.

Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance.

Analysing the consequences of the decrease in biodiversity for ecosystem functioning and stability has been a major concern in ecology. However, the impact of decline in soil microbial diversity on ecosystem sustainability remains largely unknown. This has been assessed for decomposition, which is insured by a large proportion of the soil microbial community, but not for more specialized and less diverse microbial groups. We determined the impact of a decrease in soil microbial diversity on the stability (i.e. resistance and resilience following disturbance) of two more specialized bacterial functional groups: denitrifiers and nitrite oxidizers. Soil microbial diversity was reduced using serial dilutions of a suspension obtained from a non-sterile soil that led to loss of species with low cell abundance, inoculation of microcosms of the same sterile soil with these serial dilutions, and subsequent incubation to enable establishment of similar cell abundances between treatments. The structure, cell abundance and activity of denitrifying and nitrite-oxidizing communities were characterized after incubation. Increasing dilution led to a progressive decrease in community diversity as assessed by the number of denaturating gradient gel electrophoresis (DGGE) bands, while community functioning was not impaired when cell abundance recovered after incubation. The microcosms were then subjected to a model disturbance: heating to 42 degrees C for 24 h. Abundance, structure and activity of each community were measured 3 h after completion of the disturbance to assess resistance, and after incubation of microcosms for 1 month to assess resilience. Resistance and resilience to the disturbance differed between the two communities, nitrite oxidizers being more affected. However, reducing the diversity of the two microbial functional groups did not impair either their resistance or their resilience following the disturbance. These results demonstrate the low sensitivity of the resistance and resilience of both microbial groups to diversity decline provided that cell abundance is similar between treatments.

Maintenance of soil functioning following erosion of microbial diversity.

The paradigm that soil microbial communities, being very diverse, have high functional redundancy levels, so that erosion of microbial diversity is less important for ecosystem functioning than erosion of plant or animal diversity, is often taken for granted. However, this has only been demonstrated for decomposition/respiration functions, performed by a large proportion of the total microbial community, but not for specialized microbial groups. Here, we determined the impact of a decrease in soil microbial diversity on soil ecosystem processes using a removal approach, in which less abundant species were removed preferentially. This was achieved by inoculation of sterile soil microcosms with serial dilutions of a suspension obtained from the same non-sterile soil and subsequent incubation, to enable recovery of community size. The sensitivity to diversity erosion was evaluated for three microbial functional groups with known contrasting taxonomic diversities (ammonia oxidizers < denitrifiers < heterotrophs). Diversity erosion within each functional group was characterized using molecular fingerprinting techniques: ribosomal intergenic spacer analysis (RISA) for the eubacterial community, denaturing gradient gel electrophoresis (DGGE) analysis of nirK genes for denitrifiers, and DGGE analysis of 16S rRNA genes for betaproteobacterial ammonia oxidizers. In addition, we simulated the impact of the removal approach by dilution on the number of soil bacterial species remaining in the inoculum using values of abundance distribution of bacterial species reported in the literature. The reduction of the diversity of the functional groups observed from genetic fingerprints did not impair the associated functioning of these groups, i.e. carbon mineralization, denitrification and nitrification. This was remarkable, because the amplitude of diversity erosion generated by the dilution approach was huge (level of bacterial species loss was estimated to be around 99.99% for the highest dilution). Our results demonstrate that the vast diversity of the soil microbiota makes soil ecosystem functioning largely insensitive to biodiversity erosion even for functions performed by specialized groups.

Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing, denitrifying and nitrifying bacterial communities in grassland soils.

Management by combined grazing and mowing events is commonly used in grasslands, which influences the activity and composition of soil bacterial communities. Whether observed effects are mediated by management-induced disturbances, or indirectly by changes in the identity of major plant species, is still unknown. To address this issue, we quantified substrate-induced respiration (SIR), and the nitrification, denitrification and free-living N(2)-fixation enzyme activities below grass tufts of three major plant species (Holcus lanatus, Arrhenatherum elatius and Dactylis glomerata) in extensively or intensively managed grasslands. The genetic structures of eubacterial, ammonia oxidizing, nitrate reducing, and free-living N(2)-fixing communities were also characterized by ribosomal intergenic spacer analysis, and denaturing gradient gel electrophoresis (DGGE) or restriction fragment length polymorphism (RFLP) targeting group-specific genes. SIR was not influenced by management and plant species, whereas denitrification enzyme activity was influenced only by plant species, and management-plant species interactions were observed for fixation and nitrification enzyme activities. Changes in nitrification enzyme activity were likely largely explained by the observed changes in ammonium concentration, whereas N availability was not a major factor explaining changes in denitrification and fixation enzyme activities. The structures of eubacterial and free-living N(2)-fixing communities were essentially controlled by management, whereas the diversity of nitrate reducers and ammonia oxidizers depended on both management and plant species. For each functional group, changes in enzyme activity were not correlated or were weakly correlated to overall changes in genetic structure, but around 60% of activity variance was correlated to changes in five RFLP or DGGE bands. Although our conclusions should be tested for other ecosystems and seasons, these results show that predicting microbial changes induced by management in grasslands requires consideration of management-plant species interactions.