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Grazing by ungulate herbivores can greatly alter nitrogen ([N]) and phosphorus ([P]) concentrations in plants and soils. It is not clear, however, how grazing might affect N:P co‐limitation in grasslands depending on soil N and P availability. Here we selected 173 peer‐reviewed studies, which measured 12 key variables associated with changes in [N], [P] and N:P ratios (i.e. N:P stoichiometry) in soils and plants in the presence or absence of herbivore grazing. Subsequently, we addressed the magnitude and direction of grazing effects on these variables using a meta‐analysis approach. Grazing increased leaf [N] and [P] but decreased total and available soil [N] and [P]. Grazing also increased leaf N:P ratios while decreasing root and total soil N:P ratios. The response ratio (RR) of leaf N:P was negatively correlated with RR of plant‐available soil [P] and positively correlated with RR of available soil N:P ratio (rather than with RR of total soil N:P). Intensive grazing (e.g. heavy grazing or long‐term grazing) had in general more positive effects on plant N:P stoichiometry and negative effects on soil N:P stoichiometry than light grazing. Responses of plant‐soil N:P stoichiometry to grazing greatly varied depending on plant functional group identity, plant organizational level (i.e. species and community) and grassland type. Synthesis and applications. Our study suggests that understanding changes in available soil N:P stoichiometry (rather than total soil N:P) in response to grazing is crucial to predict nutrient co‐limitation in grassland biomes. Our findings show that P is more important for plant growth than generally thought due to greater reduction of plant‐available soil [P] under grazing. A better mechanistic understanding of the relationships between plant and available soil N:P stoichiometry under grazing will greatly help improve the sustainability of natural and semi‐natural grassland ecosystems.

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.

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.

In this study, we show that composition shifts of ammonia oxidizer communities imposed by a 22-year field fertilization regime could be well retained in both fresh and air-dried soils. The abundance and composition of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were measured in fresh soils, which received no fertilization (CK), chemical fertilization (NPK), and chemical plus organic matter fertilization (NPK/OM) for 22 years. The air-drying treatment of fresh soil was also conducted for pairwise analysis. We found that in fresh soils DGGE fingerprints of AOB showed significant changes under both NPK and NPK/OM treatments when compared with control (CK) and that microbial shift was almost identical in air-dried soils. Long-term nutrient fertilization did not affect AOA communities in either air-dried or fresh soils. Compared to CK treatment, real-time PCR indicated that AOB amoA genes increased significantly in fresh soils of NPK (59-fold) and NPK/OM (48-fold) plots and in air-dried NPK and NPK/OM soils by 22-fold and 19-fold respectively. Our results demonstrate that community shifts of AOB in fresh soils under chronic N fertilization could be well preserved in air-dried soils, despite the apparent decline in absolute abundance of ammonia oxidizers. These results suggest that air-dried soil could be a useful resource for deciphering the adaptive strategy of ammonia oxidizers under N enrichment when the significant changes of community composition occurred in fresh soils.

Soil organic carbon (SOC) sequestration across agroecosystems worldwide can contribute to mitigate the effects of climate change by reducing levels of atmospheric CO2. Stabilisation of organic carbon (OC) in the fine soil fraction (< 20 µm) is considered an important long-term store of SOC, and the saturation deficit (difference between measured OC and estimated maximum OC in the fine fraction) is frequently used to assess SOC sequestration potential following the linear regression equation developed by Hassink (1997). However, this approach is often taken without any assessment of the fit of the equation to the soils being studied. The statistical limitations of linear regression have previously been noted, giving rise to the proposed use of boundary line (BL) analysis and quantile regression (QR) to provide more robust estimates of maximum SOC stabilisation. The objectives of this work were to assess the suitability of the Hassink (1997) equation to estimate maximum fine-fraction OC in UK grassland soils of varying sward ages and to evaluate the linear regression, boundary line and quantile regression methods to estimate maximum fine-fraction OC. A chronosequence of 10 grasslands was sampled, in order to assess the relationship between sward age (time since the last reseeding event) and the measured and predicted maximum fine-fraction OC. Significantly different regression equations show that the Hassink (1997) equation does not accurately reflect maximum fine-fraction OC in UK grasslands when determined using the proportion of the fine soil fraction (< 20 µm, %) and measured fine-fraction OC (g C per kg soil). The QR estimate of maximum SOC stabilisation was almost double that of the linear regression and BL analysis (0.89 ± 0.074, 0.43 ± 0.017 and 0.57 ± 0.052 g C per kg soil, respectively). Sward age had an inconsistent effect on the measured variables and potential maximum fine-fraction OC. Fine-fraction OC across the grasslands made up 4.5 % to 55.9 % of total SOC, implying that there may be either high potential for additional C sequestration in the fine fraction of these soils or that protection in aggregates is predominant in these grassland soils. This work highlights the need to ensure that methods used to predict maximum fine-fraction OC reflect the soil in situ, resulting in more accurate assessments of carbon sequestration potential.

Soil organic carbon (SOC) sequestration across agroecosystems worldwide can contribute to mitigate the effects of climate change by reducing levels of atmospheric CO2. Mineral associated organic carbon (MAOC) is considered an important long-term store of SOC and the saturation deficit (difference between measured MAOC and estimated maximum MAOC) is frequently used to assess SOC sequestration potential following the linear regression equation developed by Hassink (1997). However, this approach is often taken without any assessment of the fit of the equation to the soils being studied. The statistical limitations of linear regression have previously been noted, giving rise to the proposed use of boundary line (BL) analysis and quantile regression (QR) to provide more robust estimates of maximum SOC stabilisation. The objectives of this work were to assess the suitability of the Hassink (1997) equation to estimate maximum MAOC in UK grassland soils of varying sward ages and to evaluate the linear regression, BL and QR methods to estimate maximum MAOC. A chronosequence of 10 grasslands was sampled, in order to assess the relationship between sward age (time since last reseeding event) and current and predicted maximum MAOC. Significantly different regression equations show that the Hassink (1997) equation does not accurately reflect maximum MAOC in UK grasslands when determined using the proportion of fine soil fraction and current MAOC. The QR estimate of maximum SOC stabilisation was almost double that of linear regression and BL analysis (0.89 ± 0.074, 0.43 ± 0.017 and 0.57 ± 0.052 g C kg−1 soil, respectively). Sward age had an inconsistent effect on the measured variables and potential maximum MAOC. MAOC across the grasslands made up 4.5 to 55.9 % of total SOC, implying that there may be either high potential for additional C sequestration in the mineral fraction of these soils, or stabilisation in aggregates is predominant in these grassland soils. This work highlights the need to ensure that methods used to predict maximum MAOC reflect the soil in situ, resulting in more accurate assessments of carbon sequestration potential.

Plant roots can significantly alter soil pH and the chemical concentration and distribution of different elements in the rhizosphere environment. Here we ask whether cadmium (Cd) bioavailability in the rhizosphere of Cd-hyperaccumulator Sedum plumbizincicola can be influenced by root-induced effects on soil pH. The Cd-hyperaccumulator S. plumbizincicola and the Cd non-hyperaccumulator ecotype Sedum alfredii were both grown in four different Cd-contaminated soils. We used the planar optode imaging technique to produce two-dimensional and high-resolution measurements of soil pH. Shoot excess cation concentration, root architecture and Cd concentrations ([Cd]) in soil pore water were also measured. Spatial analyses based on kernel density estimate of roots (KDE) and a Moran’s I correlogram were performed to assess spatial patterns and potential relationships among root distribution, soil pH and [Cd]. Both Sedum species showed root-induced increases in soil acidification (i.e. soil pH decreases of 0.1 to 0.62 units), which were clearly associated with greater root density of these plants. Remarkable excess cation uptakes by both Sedum species were detected and likely a driving factor for the root-induced acidification. The presence of the roots of S. plumbizincicola were then related to higher [Cd] in the rhizosphere than in bulk soil in Orthic Acrisol (+342%) and in Hydragric Antrosol soils (+296%). The hyperaccumulator S. plumbizincicola had larger root systems, higher acidification ability, and was associated with greater soil [Cd] than S. alfredii. Spatial patterns of root distribution and soil pH were similar between Sedum plants, however, spatial patterns of [Cd] differed across polluted soils. Rhizosphere acidification induced by S. plumbizincicola plants can play an important role on soil Cd mobilization, but overall effects on soil Cd bioavailability will depend on intrinsic soil biogeochemical properties.

Our knowledge of root mass decomposition processes has greatly benefited from studies carried out in natural and semi‐natural ecosystems. It is less known, however, how root mass loss might occur in intensively managed ecosystems, such as agricultural grasslands, which receive chronic additions of nitrogen (N) fertilizers. Key questions remain about (1) how repeated N applications either in organic or inorganic forms might influence fine root mass decomposition, and (2) what implications root mass loss might have for soil carbon (C) storage. Here, we use a long‐term grassland experiment established in 1970 at Hillsborough, Northern Ireland (UK), to address how repeated additions of organic (i.e., liquid manures) and inorganic (i.e., NPK) fertilizers over >45 yr might influence fine root mass decomposition and how. We found that after a 10‐month incubation period under field conditions, highest root mass loss (~43%) was associated with highest additions of organic slurries, whereas lowest root mass loss (~29%) occurred in inorganically N‐fertilized and unfertilized soils. Root decomposition was significantly and negatively related to root C:N ratios and fungal‐to‐bacterial (F:B) ratios. Root decomposition, however, was not related to changes in soil C stocks or rates of soil C accumulation. N applications either maintained or increased (up to three times) soil C accumulation compared to unfertilized soils after five decades of intensive management. Our findings suggest that in the short‐term organic fertilization can enhance root detritus decomposition (thus promoting N release in soils for plant uptake) while contributing in the long‐term to soil C accumulation either through extra C inputs from manures or through N‐induced effects on microbial activity or both. Further studies need to address how this dual effect of organic fertilization could contribute making grassland intensification more sustainable by reducing the use of artificial NPK fertilizers.

Grassland management intensification can significantly affect the structure and composition of important soil microbial groups such as bacteria and fungi. Changes to these microbial communities can greatly influence carbon (C) and nitrogen (N) cycling in grassland soils. Here we specifically address how microbial abundances might shift under the effect of multiple management practices and how this in turn might relate to changes in soil C and N storage. Soil samples were collected from a 23-year-old grassland experiment and real-time quantitative Polymerase Chain Reaction (PCR) was performed to address whether and how (1) chronic nutrient additions, (2) liming (i.e., the addition of CaCO3 to soils), and (3) grazing by rabbits might affect archaeal, bacterial and fungal microbial groups. We found that liming additions significantly increased archaeal and bacterial abundance whilst strongly reducing fungal abundance. The addition of N-only (as NH4NO3) significantly reduced bacterial abundance while chronic grazing by rabbits resulted in positive effects on archaeal abundance. Despite long-term grassland management significantly affecting soil microbial abundances (and Fungal-to-Bacterial ratios), microbial changes were not related to either changes in soil C or N pools. Overall, our results suggest that (1) important microbial-‘soil functioning’ relationships may only be detected at lower taxonomic levels, and (2) liming-induced increases in soil pH determined significant shifts in soil microbial abundance, which could have important consequences for the delivery of multiple soil ecosystem services (i.e., nutrient regulation, C and N sequestration) from permanent grassland.

The addition of organic amendments to soils can significantly increase soil organic matter (SOM) content as well as the mass of large (e.g. 2000–250 μm) particulate organic matter (POM) fractions. Here we address whether the addition of organic amendments might affect both POM pools and the phytoextraction efficiency of Sedum plumbizincicola (S. plumbizincicola) in cadmium (Cd) polluted soils. We carried out a 442-days long pot experiment and measured Cd uptake by S. plumbizincicola for four successive plant crops in two polluted paddy soils subjected to rice straw (RS) amendment. We also measured soil pH, nutrient availability, pore water dissolved organic carbon (DOC) and Cd concentrations across different soil particle-size fractions. We found that RS amendments had no significant effects on soil pH, DOC or Cd concentrations in bulk soils during repeated phytoextraction. However, RS amendments were associated with significant (1) increases (i.e. 14.3 to 20.7%) in Cd uptake by S. plumbizincicola plants from the first crop harvest, (2) increases in available soil nutrients content, (3) increases in the percentage of oxidizable Cd fractions and the mass of soil POM fractions, and (4) decreases in POM Cd concentrations after repeated phytoextraction. These findings suggest that POM fractions may represent important accessible pools of Cd for uptake by S. plumbizincicola plants. RS amendments enhance both Cd bioavailability within soil POM pools and Cd uptake by the hyperaccumulator S. plumbizincicola thus providing an effective management practice to improve Cd removal from polluted soils through phytoextraction.

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

Multi-functional silvopastoral systems provide a wide range of services to human society including the regulation of nutrients and water in soils and the sequestration of atmospheric carbon dioxide (CO2). Although silvopastoral systems significantly contribute to enhance aboveground carbon (C) sequestration (e.g. C accumulation in woody plant biomass), their long-term effects on soil C pools are less clear. In this study we performed soil physical fractionation analyses to quantify the C pool of different aggregate fractions across three land use types including (1) silvopastoral system with ash trees (Fraxinus excelsior L.), (2) planted woodland with ash trees, and (3) permanent grassland, which were established in 1989 at Loughgall, Northern Ireland, UK. Our results show that 26 years after the conversion of permanent grassland to either silvopastoral or woodland systems, soil C (and N) stocks (0–20 cm depth) did not significantly change between the three land use types. We found, however, that permanent grassland soils were associated with significantly higher C pools (g C kg−1 soil; P 2 mm) whereas soil C pools of the micro-aggregate (53–250 μm) and silt and clay (

Long-term nutrient fertilization of grassland soils greatly increases plant yields but also profoundly alters ecosystem phosphorus (P) dynamics. Here, we addressed how long-term P fertilization may affect ecosystem P budget, P use efficiency (PUE) and the abundance of arbuscular mycorrhizal fungi (AMF), which play a key role in the acquisition of P by plants. We found that 47 years of organic P applications increased soil P availability and total soil P stocks up to 1600% and 400%, respectively, compared to unfertilized-control soils. Grassland soils could retain up to 62% and 48% of P applied since 1970 in organic and inorganic forms, respectively. Nutrient treatments significantly affected rates of AMF root colonization (%), which were higher in control and NPK-fertilized plots when compared to soils receiving increasing applications of organic P. Plant PUE increased with greater AMF root colonization, which remained high (i.e. 50-to-75%) even after ~50 years of continuous ‘normal’ rates of agronomic P inputs (~30 kg P ha−1 year−1). AMF abundance, however, decreased under higher P applications and we found a negative relationship between soil P availability or soil P stocks and rates of AMF root colonization. Our study demonstrates that (1) AMF root colonization is still high in soils, which have received consistent but moderate P inputs for over four decades, and (2) moderate rates of P fertilization are related to a more conservative P ecosystem budget whereby the amount of P retained in soils and up-taken by plants on an annual basis is higher than the amount of P added through fertilization. This is possible only if extra P is ‘mined’ from the soil P ‘bank’ and made available to plant uptake. We suggest that AMF could play a significant role in intensively-managed grasslands contributing to increase P sustainability by reducing the need for extra P fertilizer.

Aim We sought to understand how the individual and combined effects of multiple environmental change drivers differentially influence terrestrial nitrogen (N) concentrations and N pools and whether the interactive effects of these drivers are mainly antagonistic, synergistic or additive. Location Worldwide. Time period Contemporary. Major taxa studied Plants, soil, and soil microbes in terrestrial ecosystems. Methods We synthesized data from manipulative field studies from 758 published articles to estimate the individual, combined and interactive effects of key environmental change drivers (elevated CO2, warming, N addition, phosphorus addition, increased rainfall and drought) on plant, soil, and soil microbe N concentrations and pools using meta‐analyses. We assessed the influences of moderator variables on these effects through structural equation modelling. Results We found that (a) N concentrations and N pools were significantly affected by the individual and combined effects of multiple drivers, with N addition (either alone or in combination with another driver) showing the strongest positive effects; (b) the individual and combined effects of these drivers differed significantly between N concentrations and N pools in plants, but seldom in soils and microbes; (c) additive effects of driver pairs on N concentrations and pools were much more common than synergistic or antagonistic effects across plants, soils and microbes; and (d) environmental and experimental factors were important moderators of the individual, combined and interactive effects of these drivers on terrestrial N. Main conclusions Our results indicate that terrestrial N concentrations and N pools, especially those of plants, can be significantly affected by the individual and combined effects of environmental change drivers, with the interactive effects of these drivers being mostly additive. Our findings are important because they contribute to the development of models to better predict how altered N availability affects ecosystem carbon cycling under future environmental changes. ispartof: GLOBAL ECOLOGY AND BIOGEOGRAPHY vol:28 issue:5 pages:1-35 status: Published online

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.

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

Sustainable grassland intensification aims to increase plant yields while maintaining the ability of soil to act as a sink rather than sources of atmospheric CO2. High biomass yields from managed grasslands, however, can be only maintained through long-term nutrient fertilization, which can significantly affect soil carbon (C) storage and cycling. Key questions remain about (1) how long-term inorganic vs. organic fertilization influences soil C stocks, and (2) how soil C gains (or losses) contribute to the long-term C balance of managed grasslands. Using 43 years of data from a permanent grassland experiment, we show that soils not only act as significant C sinks but have not yet reached C saturation. Even unfertilized control soils showed C sequestration rates of 0.35 Mg C ha−1 yr−1 (i.e. 35 g C m−2 yr−1; 0–15 cm depth) between 1970 and 2013. High application rates of liquid manure (i.e. cattle slurry) further increased soil C sequestration to 0.86 Mg C ha−1 yr−1 (i.e. 86 g C m−2 yr−1) and a key cause of this C accrual was greater C inputs from cattle slurry. However, average coefficients of slurry-C retention in soils suggest that 85 % of C added yearly through liquid manure is lost possibly via CO2 fluxes and organic C leaching. Inorganically fertilized soils (i.e. NPK) had the lowest C-gain efficiency (i.e. unit of C gained per unit of N added) and lowest C sequestration (similar to control soils). Soils receiving cattle slurry showed higher C-gain and N-retention efficiencies compared to soils receiving NPK or pig slurry. We estimate that net rates of CO2-sequestration in the top 15 cm of the soil can offset 9–25 % of GHG (greenhouse gas) emissions from intensive management. However, because of multiple GHG sources associated with livestock farming, the net C balance of these grasslands remains positive (9–12 Mg CO2-equivalent ha−1 yr−1), thus contributing to climate change. Further C-gain efficiencies (e.g. reduced enteric fermentation and use of feed concentrates, better nutrient management) are required to make grassland intensification more sustainable.

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.

The functioning of human-managed grassland ecosystems strongly depends on how common management practices will affect grassland ‘belowground compartment’ including soil biogeochemistry and plant roots. Key questions remain about how animal grazing, liming (e.g. the addition of CaCO3 to soils), and nutrient fertilization might affect, in the long-term, soil nutrient cycling and multiple root traits. Here we focus on a mesotrophic grassland located in Berkshire, UK, where contrasting levels of rabbit grazing, liming and different inorganic fertilizers have been applied since 1991. We ask how (1) soil nitrogen (N) availability and cycling, (2) total root mass, (3) root mass decomposition, and (4) arbuscular mycorrhizal fungal (AMF) root colonisation might respond to 22 years of very different management. We found that liming strongly affected total root mass, root decomposition, root AMF colonisation as well as soil N availability and cycling and that these effects were mainly driven by liming-induced increases in soil pH. Increases in soil pH were associated with significant (1) decreases in root mass, (2) increases in root mass decomposability and in the mineralization of N in decomposing root detritus, and (3) increases in AMF infection. Soil pH was also significantly related to greater N availability (i.e. soil NO3 levels) and to lower δ15N natural abundance, which suggests more efficient N uptake by plants in limed soils as we found in our study. The application of multiple nutrients (NPKMg) also reduced total root mass, while N-only fertilization was associated with greater AMF infection. Surprisingly the long-term impact of grazing was generally weak and not significant on most plant and soil parameters. Despite soil pH affecting most belowground variables, changes in soil pH were not associated with any change in soil C and N stocks. Because liming can improve nutrient cycling (and benefits soil pH and grass yields) without negatively affecting soil C sequestration, we suggest that regular liming applications may provide management solutions for increasing the long-term sustainability of permanent grassland. This article is protected by copyright. All rights reserved.

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.

Human-induced increases in nitrogen (N) deposition are common across many terrestrial ecosystems worldwide. Greater N availability not only reduces biological diversity, but also affects the biogeochemical coupling of carbon (C) and N cycles in soil ecosystems. Soils are the largest active terrestrial C pool and N deposition effects on soil C sequestration or release could have global importance. Here, we show that 27 years of chronic N additions to prairie grasslands increased C sequestration in mineral soils and that a potential mechanism responsible for this C accrual was an N-induced increase in root mass. Greater soil C sequestration followed a dramatic shift in plant community composition from native-species-rich C4 grasslands to naturalized-species-rich C3 grasslands, which, despite lower soil C gains per unit of N added, still acted as soil C sinks. Since both high plant diversity and elevated N deposition may increase soil C sequestration, but N deposition also decreases plant diversity, more research is needed to address the long-term implications for soil C storage of these two factors. Finally, because exotic C3 grasses often come to dominate N-enriched grasslands, it is important to determine if such N-dependent soil C sequestration occurs across C3 grasslands in other regions worldwide.

The application of calcium- and magnesium-rich materials to soil, known as liming, has long been a foundation of many agro-ecosystems worldwide because of its role in counteracting soil acidity. Although liming contributes to increased rates of respiration from soil thereby potentially reducing soils ability to act as a CO2 sink, the long-term effects of liming on soil organic carbon (Corg) sequestration are largely unknown. Here, using data spanning 129 years of the Park Grass Experiment at Rothamsted (UK), we show net Corg sequestration measured in the 0–23cm layer at different time intervals since 1876 was 2–20 times greater in limed than in unlimed soils. The main cause of this large Corg accrual was greater biological activity in limed soils, which despite increasing soil respiration rates, led to plant C inputs being processed and incorporated into resistant soil organo-mineral pools. Limed organo-mineral soils showed: (1) greater Corg content for similar plant productivity levels (i.e. hay yields); (2) higher 14C incorporation after 1950s atomic bomb testing and (3) lower C:N ratios than unlimed organo-mineral soils, which also indicate higher microbial processing of plant C. Our results show that greater Corg sequestration in limed soils strongly reduced the global warming potential of long-term liming to permanent grassland suggesting the net contribution of agricultural liming to global warming could be lower than previously estimated. Our study demonstrates that liming might prove to be an effective mitigation strategy, especially because liming applications can be associated with a reduced use of nitrogen fertilizer which is a key cause for increased greenhouse gas emissions from agro-ecosystems.

In a 13-year grassland biodiversity experiment in Minnesota, USA, we addressed two main questions: What set of ecological mechanisms caused aboveground productivity to become approximately 340% greater in highly diverse plant mixtures than in the average monoculture? Why did the effect of diversity on productivity become so much stronger through time? Because our grassland system is N limited, we simultaneously measured critical variables associated with the storage and cycling of this element, such as plant and soil N pools, soil N availability, soil N mineralization rates, and plant N-use efficiency, as well as the initial soil N concentration of each diversity plot when the experiment was established in 1994. We used linear and multiple regression analyses to test for potential effects of these variables on aboveground productivity and to address whether and how such variables were in turn affected by plant species diversity and functional composition across years and also at different time intervals within the same year. We found that seven variables simultaneously controlled productivity: (1) initial total soil nitrogen (N) of each plot, (2) diversity-dependent increases in total soil N through time, (3) soil N mineralization rates, (4) soil nitrate (NO3-) utilization, (5) increases in plant N-use efficiency at greater plant diversity, (6) legume presence, and (7) higher species numbers. The surprising continued significance of higher plant diversity may occur because of its effects on seasonal capture of soil NO3- and moisture and on the accumulation of root-N pools, all of which may have also increased productivity through time at higher species numbers.

Summary 1. Plant functional composition may indirectly affect fine root processes both qualitatively (e.g. by influencing root chemistry) and quantitatively (e.g. by influencing root biomass and thus soil carbon (C) inputs and the soil environment). Despite the potential implications for ecosystem nitrogen (N) cycling, few studies have addressed the linkages between plant functional composition, root decay, root detritus N dynamics and soil N mineralization rates. 2. Here, using data from a large grassland biodiversity experiment, we first show that plant functional composition affected fine root mass loss, root detritus N dynamics and net soil N mineralization rates through its effects on root chemistry rather than on the environment of decomposition. In particular, the presence of legumes and non-leguminous forbs contributed to greater fine root decomposition which in turn enhanced root N release and net soil N mineralization rates compared with C3 and C4 grasses. 3. Second, we show that all fine roots released N immediately during decomposition and showed very little N immobilization regardless of plant composition. As a consequence, there was no evidence of increased root or soil N immobilization rates with increased below-ground plant biomass (i.e. increased soil C inputs) even though root biomass negatively affected root decay. 4. Our results suggest that fine roots represent an active soil N pool that may sustain plant uptake while other soil N forms are being immobilized in microbial biomass and/or sequestered into soil organic matter. However, fine roots may also represent a source of recalcitrant plant detritus that is returned to the soil (i.e. fine roots of C4 and C3 grasses) and that can contribute to an increase in the soil organic matter pool. 5. Synthesis . An important implication of our study is that the simultaneous presence of different plant functional groups (in plant mixtures) with opposite effects on root mass loss, root N release and soil N mineralization rates may be crucial for sustaining multiple ecosystem services such as productivity and soil C and N sequestration in many N-limited grassland systems.

Summary 1. The mechanisms controlling soil carbon (C) and nitrogen (N) accumulation are crucial for explaining why soils are major terrestrial C sinks. Such mechanisms have been mainly addressed by imposing short-term, step-changes in CO 2 , temperature and N fertilization rates on either monocultures or low-diversity plant assemblages. No studies have addressed the long-term effects of plant functional diversity (i.e. plant functional composition) on rates of soil C accumulation in N-limited grasslands where fixation is the main source of N for plants. 2. Here we measure net soil C and N accumulation to 1 m soil-depth during a 12-year-long grassland biodiversity experiment established on agriculturally degraded soils at Cedar Creek, Minnesota, USA. 3. We show that high-diversity mixtures of perennial grassland plant species stored 500% and 600% more soil C and N than, on average, did monoculture plots of the same species. Moreover, the presence of C4 grasses and legumes increased soil C accumulation by 193% and 522%, respectively. Higher soil C and N accrual resulted both from increased C and N inputs via (i) higher root biomass, and (ii) from greater root biomass accumulation to 60 cm soil depth resulting from the presence of highly complementary functional groups (i.e. C4 grasses and legumes). 4. Our results suggest that the joint presence of C4 grass and legume species is a key cause of greater soil C and N accumulation in both higher and lower diversity plant assemblages. This is because legumes have unique access to N, and C4 grasses take up and use N efficiently, increasing below-ground biomass and thus soil C and N inputs. 5. Synthesis. We demonstrate that plant functional complementarity is a key reason why higher plant diversity leads to greater soil C and N accumulation on agriculturally degraded soils. We suggest the combination of key C4 grass‐legume species may greatly increase ecosystem services such as soil C accumulation and biomass (biofuel) production in both high- and low-diversity N-limited grassland systems.

Chronic nitrogen (N) fertilization can greatly affect soil carbon (C) sequestration by altering biochemical interactions between plant detritus and soil microbes. In lignin-rich forest soils, chronic N additions tend to increase soil C content partly by decreasing the activity of lignin-degrading enzymes. In cellulose-rich grassland soils it is not clear whether cellulose-degrading enzymes are also inhibited by N additions and what consequences this might have on changes in soil C content. Here we address whether chronic N fertilization has affected (1) the C content of light versus heavier soil fractions, and (2) the activity of four extracellular enzymes including the C-acquiring enzyme β-1,4-glucosidase (BG; necessary for cellulose hydrolysis). We found that 19 years of chronic N-only addition to permanent grassland have significantly increased soil C sequestration in heavy but not in light soil density fractions, and this C accrual was associated with a significant increase (and not decrease) of BG activity. Chronic N fertilization may increase BG activity because greater N availability reduces root C:N ratios thus increasing microbial demand for C, which is met by C inputs from enhanced root C pools in N-only fertilized soils. However, BG activity and total root mass strongly decreased in high pH soils under the application of lime (i.e. CaCO3), which reduced the ability of these organo-mineral soils to gain more C per units of N added. Our study is the first to show a potential ‘enzyme link’ between (1) long-term additions of inorganic N to grassland soils, and (2) the greater C content of organo-mineral soil fractions. Our new hypothesis is that the ‘enzyme link’ occurs because (a) BG activity is stimulated by increased microbial C demand relative to N under chronic fertilization, and (b) increased BG activity causes more C from roots and from microbial metabolites to accumulate and stabilize into organo-mineral C fractions. We suggest that any combination of management practices that can influence the BG ‘enzyme link’ will have far reaching implications for long-term C sequestration in grassland soils.

The functioning of human-managed grassland ecosystems strongly depends on how common management practices (e.g., animal grazing and the chronic addition of fertilizing materials to soils) interact to influence plant and soil element stoichiometry. Here we use data from a 22-yr-long grassland experiment to address whether and how plant element stoichiometry (i.e., carbon [C], nitrogen [N], phosphorus [P] ratios) might respond to (1) animal grazing, (2) agricultural liming (i.e., CaCO3) applications, and (3) nutrient fertilization. We also ask whether plant C:N:P stoichiometry could predict changes in soil N and P availability and in soil C, N, and P stocks. We found that grassland management significantly affected plant C:N:P ratios as predicted by ecological stoichiometry theory. For example, plant aboveground and belowground C:N and C:P ratios decreased under chronic N and P fertilization, respectively. Plant C:N and C:P ratios were significantly greater in unfertilized (control) soils. Also plant C:N ratios were highest under P-only additions, whereas plant C:P ratios were highest under N-only additions. However, unpredictable changes in C:N:P ratios also occurred, suggesting that plant tissue chemistry may not be a simple reflection of soil nutrient availability. Changes in plant C:nutrient ratios well predicted variation in soil nutrient availability, but not in soil C, N, and P stocks. Contrary to expectations, soil C stocks significantly increased with decreasing plant C:N ratios in the nutrient-fertilized grasslands and not with increasing plant C:N ratios in the unfertilized grasslands. We suggest that a better mechanistic understanding of the negative relationship between plant C:N stoichiometry and soil C accrual will greatly help in improving the sustainability of human-managed grasslands.

Human activities have greatly increased the availability of biologically active forms of nutrients [e.g., nitrogen (N), phosphorous (P), potassium (K), magnesium (Mg)] in many soil ecosystems worldwide. Multi-nutrient fertilization strongly increases plant productivity but may also alter the storage of carbon (C) in soil, which represents the largest terrestrial pool of organic C. Despite this issue is important from a global change perspective, key questions remain on how the single addition of N or the combination of N with other nutrients might affect C sequestration in human-managed soils. Here, we use a 19-year old nutrient addition experiment on a permanent grassland to test for nutrient-induced effects on soil C sequestration. We show that combined NPKMg additions to permanent grassland have 'constrained' soil C sequestration to levels similar to unfertilized plots whereas the single addition of N significantly enhanced soil C stocks (N-only fertilized soils store, on average, 11 t C ha(-1) more than unfertilized soils). These results were consistent across grazing and liming treatments suggesting that whilst multi-nutrient additions increase plant productivity, soil C sequestration is increased by N-only additions. The positive N-only effect on soil C content was not related to changes in plant species diversity or to the functional composition of the plant community. N-only fertilized grasslands show, however, increases in total root mass and the accumulation of organic matter detritus in topsoils. Finally, soils receiving any N addition (N only or N in combination with other nutrients) were associated with high N losses. Overall, our results demonstrate that nutrient fertilization remains an important global change driver of ecosystem functioning, which can strongly affect the long-term sustainability of grassland soil ecosystems (e.g., soils ability to deliver multiple ecosystem services).

Biomass allocation to plant aboveground and belowground compartments may change in response to changes in soil nutrient fertility (e.g., nitrogen (N) and phosphorous (P) availability). It is not clear however whether and how changes in soil fertility could influence carbon(C):nutrient stoichiometry of wild plants distributed along environmental gradients. Here we use ecological stoichiometric theory to test two hypotheses: (1) C:N (or C:P) ratios of different plant compartments (i.e., roots, stems and leaves) increase when soil N (or P) availability decreases, (2) the relative availability of N compared to P in soils predictably influences C:nutrient ratios (e.g., high soil N:P ratios are associated with high plant C:P ratios). Data from 72 wild plant species were collected along a gradient of soil development determined by the temporal progression of four primary ecological successions across Europe. We found significant changes in soil N and P availability and content along the four vegetation successio...