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In a field experiment, annual nitrous oxide (N 2 O) emissions and grassland yield were measured across different plant communities, comprising systematically varying combinations of monocultures and mixtures of three functional groups (FG): grasses (Lolium perenne, Phleum pratense), legumes (Trifolium pratense, Trifolium repens) and herbs (Cichorium intybus, Plantago lanceolata). Plots received 150 kg ha -1 year -1 nitrogen (N) (150 N), except L. perenne monocultures which received two N levels: 150 N and 300 N. The effect of plant diversity on N 2 O emissions was derived from linear combinations of species performances' in monoculture (species identity) and not from strong interactions between species in mixtures. Increasing from 150 N to 300 N in L. perenne resulted in a highly significant increase in cumulative N 2 O emissions from 1.39 to 3.18 kg N 2 O-N ha -1 year -1 . Higher N 2 O emissions were also associated with the legume FG. Emissions intensities (yield-scaled N 2 O emissions) from multi-species mixture communities around the equi-proportional mixture were lowered due to interactions among species. For N 2 O emissions scaled by nitrogen yield in forage, the 6-species mixture was significantly lower than L. perenne at both 300 N and 150 N. In comparison to 300 N L. perenne, the same N yield or DM yield could have been produced with the equi-proportional 6-species mixture (150 N) while reducing N 2 O losses by 63% and 58% respectively. Compared to 150 N L. perenne, the same N yield or DM yield could have been produced with the 6-species mixture while reducing N 2 O losses by 41% and 24% respectively. Overall, this study found that multi-species grasslands can potentially reduce both N 2 O emissions and emissions intensities, contributing to the sustainability of grassland production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nitrous oxide (N 2 O) is a potent greenhouse gas (GHG) emitted from agricultural soils and is influenced by nitrogen (N) fertiliser management and weather and soil conditions. Source partitioning N 2 O emissions related to management practices and soil conditions could suggest effective mitigation strategies. Multispecies swards can maintain herbage yields at reduced N fertiliser rates compared to grass monocultures and may reduce N losses to the wider environment. A restricted-simplex centroid experiment was used to measure daily N 2 O fluxes and associated isotopomers from eight experimental plots (7.8 m 2 ) post a urea-N fertiliser application (40 kg N ha -1 ). Experimental pastures consisted of differing proportions of grass, legume and forage herb represented by perennial ryegrass (Lolium perenne), white clover (Trifolium repens) and ribwort plantain (Plantago lanceolata), respectively. N 2 O isotopomers were measured using a cavity ring down spectroscopy (CRDS) instrument adapted with a small sample isotope module (SSIM) for the analysis of gas samples ≤20 mL. Site preference (SP = δ 15 N α - δ 15 N β ) and δ 15 N bulk ((δ 15 N α + δ 15 N β ) / 2) values were used to attribute N 2 O production to nitrification, denitrification or a mixture of both nitrification and denitrification over a range of soil WFPS (%). Daily N 2 O fluxes ranged from 8.26 to 86.86 g N 2 O-N ha -1 d -1 . Overall, 34.2% of daily N 2 O fluxes were attributed to nitrification, 29.0% to denitrification and 36.8% to a mixture of both. A significant diversity effect of white clover and ribwort plantain on predicted SP and δ 15 N bulk indicated that the inclusion of ribwort plantain may decrease N 2 O emission through biological nitrification inhibition under drier soil conditions (31%-75% WFPS). Likewise, a sharp decline in predicted SP indicates that increased white clover content could increase N 2 O emissions associated with denitrification under elevated soil moisture conditions (43%-77% WFPS). Biological nitrification inhibition from ribwort plantain inclusion in grassland swards and management of N fertiliser source and application timing to match soil moisture conditions could be useful N 2 O mitigation strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Nitrous oxide (N 2 O) is a strong greenhouse gas produced by biotic/abiotic processes directly linked to both fungal and prokaryotic communities that produce, consume or create conditions leading to its emission. In soils exposed to nitrogen (N) in the form of urea, an ecological succession is triggered resulting in a dynamic turnover of microbial populations. However, knowledge of the mechanisms controlling this succession and the repercussions for N 2 O emissions remain incomplete. Here, we monitored N 2 O production and fungal/prokaryotic community changes (via 16S and 18S amplicon sequencing) in soil microcosms exposed to urea. Contributions of microbes to emissions were determined using biological inhibitors. Results confirmed that urea leads to shifts in microbial community assemblages by selecting for certain microbial groups (fast growers) as dictated through life history strategies. Urea reduced overall community diversity by conferring dominance to specific groups at different stages in the succession. The diversity lost under urea was recovered with inhibitor addition through the removal of groups that were actively growing under urea indicating that species replacement is mediated in part by competition. Results also identified fungi as significant contributors to N 2 O emissions, and demonstrate that dominant fungal populations are consistently replaced at different stages of the succession. These successions were affected by addition of inhibitors which resulted in strong decreases in N 2 O emissions, suggesting that fungal contributions to N 2 O emissions are larger than that of prokaryotes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Increased emissions of N 2 O, a potent greenhouse gas (GHG), from agricultural soils is a major concern for the sustainability of grassland agriculture. Emissions of N 2 O are closely associated with the rates and forms of N fertilisers applied as well as prevailing weather and soil conditions. Evidence suggests that multispecies swards require less fertiliser N input, and may cycle N differently, thus reducing N loss to the environment. This study used a restricted simplex-centroid experimental design to investigate N 2 O emissions and soil N cycling following application of urea-N (40 kg N ha -1 ) to eight experimental swards (7.8 m 2 ) with differing proportions of three plant functional groups (grass, legume, herb) represented by perennial ryegrass (PRG, Lolium perenne), white clover (WC, Trifolium repens) and ribwort plantain (PLAN, Plantago lanceolata), respectively. Swards were maintained under two contrasting soil moisture conditions to examine the balance between nitrification and denitrification. Two N 2 O peaks coincided with fertiliser application and heavy rainfall events; 13.4 and 17.7 g N 2 O-N ha -1  day -1 (ambient soil moisture) and 39.8 and 86.9 g N 2 O-N ha -1  day -1 (wet soil moisture). Overall, cumulative N 2 O emissions post-fertiliser application were higher under wet soil conditions. Increasing legume (WC) proportions from 0% to 60% in multispecies swards resulted in model predicted N 2 O emissions increasing from 22.3 to 96.2 g N 2 O-N ha -1 (ambient soil conditions) and from 59.0 to 219.3 g N 2 O-N ha -1 (wet soil conditions), after a uniform N application rate. Soil N dynamics support denitrification as the dominant source of N 2 O especially under wet soil conditions. Significant interactions of PRG or WC with PLAN on soil mineral N concentrations indicated that multispecies swards containing PLAN potentially inhibit nitrification and could be a useful mitigation strategy for N loss to the environment from grassland agriculture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)