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Long-term nitrogen (N) fertilization imposes strong selection on nitrifying communities in agricultural soil, but how a progressively changing niche affects potentially active nitrifiers in the field remains poorly understood. Using a 44-year grassland fertilization experiment, we investigated community shifts of active nitrifiers by DNA-based stable isotope probing (SIP) of field soils that received no fertilization (CK), high levels of organic cattle manure (HC), and chemical N fertilization (CF). Incubation of DNA-SIP microcosms showed significant nitrification activities in CF and HC soils, whereas no activity occurred in CK soils. The 44 years of inorganic N fertilization selected only 13C-ammonia-oxidizing bacteria (AOB), whereas cattle slurry applications created a niche in which both ammonia-oxidizing archaea (AOA) and AOB could be actively 13C-labeled. Phylogenetic analysis indicated that Nitrosospira sp. 62-like AOB dominated inorganically fertilized CF soils, while Nitrosospira sp. 41-like AOB were abundant in organically fertilized HC soils. The 13C-AOA in HC soils were affiliated with the 29i4 lineage. The 13C-nitrite-oxidizing bacteria (NOB) were dominated by both Nitrospira- and Nitrobacter-like communities in CF soils, and the latter was overwhelmingly abundant in HC soils. The 13C-labeled nitrifying communities in SIP microcosms of CF and HC soils were largely similar to those predominant under field conditions. These results provide direct evidence for a strong selection of distinctly active nitrifiers after 44 years of different fertilization regimes in the field. Our findings imply that niche differentiation of nitrifying communities could be assessed as a net result of microbial adaption over 44 years to inorganic and organic N fertilization in the field, where distinct nitrifiers have been shaped by intensified anthropogenic N input.

Plant litter decomposition is not only the major source of soil carbon and macronutrients, but also an important process for the biogeochemical cycling of trace elements such as iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu). The concentrations of plant litter trace elements can influence litter decomposition and element cycling across the plant and soil systems. Yet, a global perspective of the patterns and driving factors of trace elements in plant litter is missing. To bridge this knowledge gap, we quantitatively assessed the concentrations of four common trace elements, namely Fe, Mn, Zn, and Cu, of freshly fallen plant litter with 1411 observations extracted from 175 publications across the globe. Results showed that (1) the median of the average concentrations of litter Fe, Mn, Zn, and Cu were 0.200, 0.555, 0.032, and 0.006 g/kg, respectively, across litter types; (2) litter concentrations of Fe, Zn, and Cu were generally stable regardless of variations in multiple biotic and abiotic factors (e.g., plant taxonomy, climate, and soil properties); and (3) litter Mn concentration was more sensitive to environmental conditions and influenced by multiple factors, but mycorrhizal association and soil pH and nitrogen concentration were the most important ones. Overall, our study provides a clear global picture of plant litter Fe, Mn, Zn, and Cu concentrations and their driving factors, which is important for improving our understanding on their biogeochemical cycling along with litter decomposition processes.

Grassland management intensification can greatly influence nitrogen (N) dynamics between aboveground and belowground compartments mainly because of the large amount of available N forms, which are repeatedly added to soils. A better understanding of how chronic fertilisation might affect N use efficiency (NUE) in plants can contribute to reducing N losses from soils and improve the sustainability of managed grasslands. Here we address how NUE might be affected by (1) the addition of key nutrients (e.g. N, P, K, Mg) in different combinations, (2) grazing by rabbits, and (3) liming (i.e. CaCO3 applications) in a 22-year-old permanent grassland experiment established in Berkshire, UK, in 1991. We first calculate seven different NUE indexes, which are known to respond either to changes in soil N availability (i.e. endogenous N inputs from soil N mineralization processes) or to exogenous N inputs (i.e. synthetic N fertiliser). We found that plant NUE calculated as plant biomass produced per unit of N acquired significantly decreased under the chronic addition of multiple nutrients (NPKMg) and was even lower under N-only applications. Most NUE indexes significantly decreased under grazing but greatly increased under liming applications. We found evidence that NUE indexes, which accounted for endogenous N sources decreased at increased rates of soil N mineralization. Finally, we found no significant relationships between any of the NUE indexes and estimates of soil N losses (Mg N ha−1) or N retention in soils (i.e. units of soil N retained per unit of N added) calculated from changes in net soil N budget over 22 years. Our study carried out on relatively acidic sandy soils suggests how liming applications in combination with low levels of multi-nutrient additions (NPKMg) can significantly improve plant biomass production per unit of N added thus contributing to enhance the sustainability of managed grassland ecosystems.

Human-managed grasslands receive significant inputs of fertilizing materials, which can greatly influence soil biological processes associated with the cycling of nitrogen (N), including microbial nitrification. Here we specifically address how soil ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) respond to 23 years of different management practices in a permanent grassland experiment. We found that AOB amoA gene copy numbers were significantly higher in limed soils (associated with greater pH values) whereas AOA amoA gene copy numbers were higher in grazed grasslands. The chronic addition of inorganic N fertilizer in the form of ammonium nitrate (either applied alone or in combination with other macro-nutrients) greatly contributed to increase AOB abundance. Our study brings evidence of how soil-nitrifying communities can differently respond to the long-term effect of animal (i.e. rabbit) grazing and to repeated applications of nutrient (e.g. NH4NO3) and non-nutrient (i.e. CaCO3) fertilizing materials.

Common grassland management practices include animal grazing and the repeated addition of lime and nutrient fertilizers to soils. These practices can greatly influence the size and distribution of different soil aggregate fractions, thus altering the cycling and storage of carbon (C) and nitrogen (N) in grassland soils. So far, very few studies have simultaneously addressed the potential long-term effect that multiple management practices might have on soil physical aggregation. Here we specifically ask whether and how grazing, liming and nutrient fertilization might influence C and N content (%) as well as C and N pools of different soil aggregate fractions in a long-term grassland experiment established in 1991 at Silwood Park, Berkshire, UK. We found that repeated liming applications over 23years significantly decreased the C pool (i.e. gCKg-1 soil) of Large Macro Aggregate (LMA>2mm) fractions and increased C pools within three smaller soil aggregate fractions: Small Macro Aggregate (SMA, 250μm-2mm), Micro Aggregate (MiA, 53-250μm), and Silt Clay Aggregate (SCA

Important biochemical reactions in soils are catalyzed by extracellular enzymes, which are synthesized by microbes and plant roots. Although enzyme activities can significantly affect the decomposition of soil organic matter and thus influence the storage and cycling of carbon (C) and nitrogen (N), it is not clear how enzyme activities relate to changes in the C and N content of different grassland soils. Here we address whether the activity of C-acquiring (b-1,4-glucosidase, BG) and N-acquiring (L-leucine amino-peptidase (LAP) and b-1,4-N-acetyl-glucosaminidase (NAG)) enzymes is linked to changes in the C and N content of a variety of human-managed grassland soils. We selected soils which have a well-documented management history going back at least 19 years in relation to changes in land use (grazing, mowing, ploughing), nutrient fertilization and lime (CaCO 3) applications. Overall we found a positive relationship between BG activity and soil C content as well as between LAP þ NAG activity and soil N. These positive relationships occurred across grasslands with very different soil pH and management history but not in intensively managed grasslands where increases in soil bulk density (i.e. high soil compaction) negatively affected enzyme activity. We also found evidence that chronic nutrient fertilization contributed to increases in soil C content and this was associated with a significant increase in BG activity when compared to unfertilized soils. Our study suggests that while the activities of C-and N-acquiring soil enzymes are positively related to soil C and N content, these activities respond significantly to changes in management (i.e. soil compaction and nutrient fertilization). In particular, the link between BG activity and the C content of long-term fertilized soils deserves further investigation if we wish to improve our understanding of the C sequestration potential of human-managed grassland soils.

Agricultural grasslands comprise over 50% of the total land area of the UK and provide important ecosystem services that include livestock and forage production. These services are rarely measured against the effects that key management practices might have on the long-term ability of grassland soils to cycle and store carbon (C). The common management practice of re-seeding (i.e. the ploughing and seeding of grasslands with more productive grass cultivars) can cause significant soil disturbance; yet the net long-term effects of re-seeding on soil C gains and losses in permanent grasslands are poorly understood. Here, we selected a chronosequence of 45 permanent grasslands across Northern Ireland with a well-documented history of single re-seeding events over the last 50 years. Second, we asked whether and how soil C cycling and storage might differ between recently re-seeded ‘young’ grasslands and increasingly ‘older’ (or never re-seeded) grasslands. We measured (1) soil CO2 fluxes, (2) soil C stocks, (3) the C content of different soil aggregate fractions, and (4) root C stocks. We found that soil CO2 fluxes were significantly higher in recently re-seeded, ‘young’, grasslands. However, total soil C stocks (0–20 cm depth) did not increase in ‘older’ grasslands despite these grasslands showing greater root C stocks. Instead, soil C stocks significantly decreased with increases in soil bulk density. Higher soil bulk density was also associated with lower C content in smaller organo-mineral aggregate sizes (i.e. more recalcitrant C pools) regardless of grassland age (time since re-seeding). Overall, our results suggest that management-induced effects on key soil physical properties, i.e. bulk density, may have significantly greater implications for C sequestration in permanent grassland soils than high disturbance, but infrequent, re-seeding events.

Chronic nutrient addition to grassland soils can strongly influence the composition and abundance of nitrifying microbial communities. Despite the fact that nitrifying microbes play a crucial role in regulating ecosystem nitrogen (N) cycling, our understanding of how long-term N fertilization might influence nitrifying microbial groups is still limited. Here we used soil from a 44-year-old grassland fertilization experiment and performed high-throughput pyrosequencing analyses (and real-time quantitative PCR) to determine whether and how the identity and abundance of nitrifying microbes has changed in response to chronic inorganic (chemical fertilizer) and organic (cattle slurry) N additions. We found that the amoA genes of ammonia-oxidizing archaea (AOA) significantly increased under organic N additions, whereas ammonia-oxidizing bacteria (AOB) increased with the addition of inorganic N. Proportional changes of AOA, AOB and nitrite-oxidizing bacteria (NOB) demonstrate that nitrifying phylotypes are influenced by chronic N additions. We also found that AOA/AOB ratios increased with higher application rates of cattle slurry suggesting that AOA may affect N cycling more in soils receiving animal manures, whereas AOB are functionally more important in chemically fertilized soils. Phylogenetic analysis shows that shifts in AOA and AOB community structure occurred through time across N fertilization treatments. For example, (a) fosmid 29i4-like AOA, (b) Nitrosospira cluster 3-like AOB, and (c) Nitrospira-like NOB dominated nitrifying communities in fertilized soils. Finally, high-throughput pyrosequencing of 16S rRNA genes show that N fertilization (either inorganic or organic) increased the abundance of Actinobacteria in soils while it decreased the abundance of Proteobacteria. Our study is one of the first to show that long-term N additions to soils can greatly affect nitrifying communities, and that phylogenetically and functionally distinct nitrifiers have developed through time in response to chronic N fertilization.

Long-term changes in soil organic carbon (SOC) are difficult to quantify experimentally because of measurement errors and high spatial and temporal variability. Modelling can help to provide a more robust assessment by reducing these uncertainties and reproducing greenhouse gas (GHG) and C exchange processes in an ecosystem by identifying key drivers. In this study, the Denitrification-Decomposition (DNDC95) model was used to evaluate SOC density (SOCρ) and its annual changes (ΔSOCρ) in temperate grassland soils, which received different forms of nitrogen (N) (i.e. inorganic and organic) and at different application rates for 45 years. We found that simulated values for SOCρ (0–15 cm depth) in unfertilized (54 t C ha−1) and fertilized soils (55 t C ha−1) were lower than measured values (73 and 77 t C ha−1, respectively). Despite some variations, measured and simulated SOCρ was higher under cattle (88–99 vs. 66–116 t C ha−1) than pig slurry (75–78 vs. 55–69) applications, and increased with increasing rates of added C. Irrespective of nutrient treatment, overall mean sequestration rates were 0.46 ± 0.06 (observed) and 0.37 ± 0.01 (simulated) t C ha−1 yr−1. Simulated values explained 66% of the variability between years and treatments (slope: 1.41; intercept: −34.58 t C ha−1) with reasonably good prediction efficiency The variations in simulated values could be explained by differences in applied N (63%), which were linked to differences in C (62%), rainfall (15%) and air temperature (11%). The model (R2 0.77–0.99/-0.99; p

Grasslands cover more than a third of the European agricultural area and are often intensively managed to support the livestock-farming sector. Despite grassland intensification can greatly influence soil carbon (C) cycling, changes in soil C stocks both across years and at increasing soil depths remain difficult to quantify simultaneously. Here we measured spatial and temporal changes in soil C stocks to assess C stocks’ response to intensive grassland management. We measured soil C stocks (a) in a long-term nutrient fertilization experiment on permanent grassland (established in 1970), and (b) in 126 grassland fields distributed across 11 lowland farms in Northern Ireland (UK), which are associated with different frequencies of soil tillage. Using 45 years of data from the plot-scale grassland experiment we found evidence that significant changes in soil C stocks mainly occurred in the soil top 20 cm (not in deeper soils) and only between ‘extreme’ nutrient treatments (i.e. unfertilized vs. highly fertilized soils). Soil physical fractionation, radiocarbon and stable nitrogen isotope analyses all suggest that new C has accumulated in these soils but perhaps not fast enough to affect C stocks in deeper soil layers. Results at the field-scale from intensively managed grasslands show how the frequency of soil tillage neither affected soil C stocks between 0–20, 20–40 or 40–60 cm depth layers nor the C pool of different soil physical fractions at increasing soil depths. Our findings demonstrate how the response of soil C stocks to grassland intensification (i.e. C stocks response to different rates of nutrient fertilization or frequency of soil tillage) can be very slow both in time and space under cool and humid climate conditions. We suggest how the persistence of these soil C stocks under intensive grassland management offers the unique opportunity to improve nutrient use efficiency and cycling thus promoting the delivery of multiple ecosystem processes.

Two primary approaches to perennial biofuel crop production studied so far are fertilized grassmonocultures and low-input high-diversity grasslands. While high-yielding perennial grass varieties arebeing developed in fertilized monocultures, breeding for yield in low-input high-diversity systemswould be difficult. Before initiating breeding for low-input systems, it is therefore important to know theminimum number of functional groups and species required for maximum biomass harvest from lowinputgrasslands. We controlled the number of perennial grassland species in 168 plots in Minnesota,USA. Species were selected at random from a pool of 18, and 1, 2, 4, 8, or 16 were planted in each plot.Aboveground biomass was measured annually, and the plots were burned each spring. We found astrongly positive log-linear relationship between average annual aboveground biomass and plantedspecies number, but a large proportion of plot-to-plot variability remained unexplained. Weperformed aconditional analysis of the aboveground biomass data to determine whether considering species identitywould reduce the minimum number of species necessary in order to achieve yields similar to the highestdiversity treatments. A model that accounted for the presence of legumes in general, and for the presenceof the legume species Lupinus perennis in particular, showed no increase in biomass yield with increasedspecies number. Over 11 years, average yields of L. perennis/C4 grass bicultures were similar to those of16-species (maximum diversity) plots, and both were >200% greater than the average of monocultures.Thus, under low-input conditions, the choice of the appropriate few perennial plant species for eachlocation might result in systems with biomass yields similar to those from high-diversity systems.Because breeding biofuel crops in diverse mixtures would introduce complexity that is unwarranted interms of maximum biomass yield, the first biofuel crop breeding programs for low-input systems arelikely to accelerate progress by focusing on grass–legume bicultures.

We studied the composition of a savanna woody plant community across a natural herbivory gradient maintained by both browsing and grazing ungulates in an arid part of the Kruger National Park, South Africa. We focused on (1) short-term browsing effects on reproductive and morphological traits of a dominant-palatable woody species, Acacia nigrescens, Miller, (2) the relationship between browsing–grazing intensity and soil parameters, (3) the effects of herbivore–soil interactions on woody species richness and composition, and (4) browser-induced effects on the representation of woody plant functional traits. We show that the number of pods carried by A. nigrescens trees as well as the internode length of external tree branches both decreased significantly at high browsing intensity. Moreover, we found that total soil nitrogen (N) and soil cation (Ca, Na, Mg, and K) concentrations varied significantly according to grazing rather than browsing intensity with soil nutrients decreasing at heavily grazed sites. Although herbivory and soil