Microbial N Transformations and N2OEmission after Simulated GrasslandCultivation: Effects of the NitrificationInhibitor 3,4-DimethylpyrazolePhosphate (DMPP)

Yun-Feng Duan,a,b Xian-Wang Kong,b Andreas Schramm,a Rodrigo Labouriau,c

Jørgen Eriksen,b Søren O. Petersenb

Section of Microbiology, Department of Bioscience, Aarhus University, Aarhus, Denmarka; Department ofAgroecology, Aarhus University, Tjele, Denmarkb; Applied Statistics Laboratory, Department of Mathematics,Aarhus University, Aarhus, Denmarkc

ABSTRACT Grassland cultivation can mobilize large pools of N in the soil, with thepotential for N leaching and N2O emissions. Spraying with the nitrification inhibitor3,4-dimethylpyrazole phosphate (DMPP) before cultivation was simulated by use ofsoil columns in which the residue distribution corresponded to plowing or rotova-tion to study the effects of soil-residue contact on N transformations. DMPP wassprayed on aboveground parts of ryegrass and white clover plants before incorpora-tion. During a 42-day incubation, soil mineral N dynamics, potential ammonia oxida-tion (PAO), denitrifying enzyme activity (DEA), nitrifier and denitrifier populations,and N2O emissions were investigated. The soil NO3

� pool was enriched with 15N totrace sources of N2O. Ammonium was rapidly released from decomposing residues,and PAO was stimulated in soil near residues. DMPP effectively reduced NH4

� trans-formation irrespective of residue distribution. Ammonia-oxidizing archaea (AOA) andbacteria (AOB) were both present, but only the AOB amoA transcript abundance cor-related with PAO. DMPP inhibited the transcription of AOB amoA genes. Denitrifiergenes and transcripts (nirK, nirS, and clades I and II of nosZ) were recovered, and acorrelation was found between nirS mRNA and DEA. DMPP showed no adverse ef-fects on the abundance or activity of denitrifiers. The 15N enrichment of N2Oshowed that denitrification was responsible for 80 to 90% of emissions. With sup-port from a control experiment without NO3

� amendment, it was concluded thatDMPP will generally reduce the potential for leaching of residue-derived N, whereasthe effect of DMPP on N2O emissions will be significant only when soil NO3

� avail-ability is limiting.

IMPORTANCE Residue incorporation following grassland cultivation can lead to mo-bilization of large pools of N and potentially to significant N losses via leaching andN2O emissions. This study proposed a mitigation strategy of applying 3,4-dimethylpyrazole phosphate (DMPP) prior to grassland cultivation and investigatedits efficacy in a laboratory incubation study. DMPP inhibited the growth and activityof ammonia-oxidizing bacteria but had no adverse effects on ammonia-oxidizing ar-chaea and denitrifiers. DMPP can effectively reduce the potential for leaching ofNO3

� derived from residue decomposition, while the effect on reducing N2O emis-sions will be significant only when soil NO3

� availability is limiting. Our findings pro-vide insight into how DMPP affects soil nitrifier and denitrifier populations and havedirect implications for improving N use efficiency and reducing environmental im-pacts during grassland cultivation.

Received 5 July 2016 Accepted 7 October2016

Accepted manuscript posted online 14October 2016

Citation Duan Y-F, Kong X-W, Schramm A,Labouriau R, Eriksen J, Petersen SO. 2017.Microbial N transformations and N2O emissionafter simulated grassland cultivation: effects ofthe nitrification inhibitor 3,4-dimethylpyrazolephosphate (DMPP). Appl Environ Microbiol83:e02019-16. https://doi.org/10.1128/AEM.02019-16.

Editor Claire Vieille, Michigan State University

Copyright © 2016 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Yun-Feng Duan,Kevin.YF.Duan@gmail.com.

Y.-F.D. and X.-W.K. contributed equally to thisarticle.

ENVIRONMENTAL MICROBIOLOGY

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KEYWORDS grassland soil, 3,4-dimethylpyrazole phosphate (DMPP), plant residues,nitrifiers, denitrifiers

Temporary grasslands cover ca. 10% of the total arable land in the European Union(1), and even larger proportions in some regions (2). Temporary grasslands are

typically cultivated every few years as part of crop rotations (3), whereby plant residuesare incorporated into soil during tillage. Subsequent decomposition of residues con-taining up to 400 kg N ha�1 (4) will release significant quantities of inorganic N into thesoil, which is further transformed via nitrification and denitrification. As much as 100 to256 kg N ha�1 year�1 may be lost via leaching from grassland cultivation, dependingon the site and management (5–7). Atmospheric losses of N2O are relatively low (8), butthe global-warming and ozone-depleting potential of N2O is substantial (9, 10). Man-agement strategies are needed to reduce environmental impacts and to improve N useefficiency following grassland cultivation.

One strategy is to delay the oxidation of residue-derived NH4�, which is less prone

to losses than NO3� (11), thereby shifting the N balance toward uptake by a new crop

(12). This can be achieved with nitrification inhibitors (NIs). NIs are typically appliedtogether with mineral fertilizers and manures to increase N use efficiency (13), but theyhave also been applied to grazed pastures to reduce NO3

� leaching and N2O emissionsfrom excretal returns (14, 15). NIs can also manipulate N turnover associated withresidue decomposition. For example, Chaves et al. (16) observed reduced NO3

� accu-mulation during decomposition of cauliflower residues treated with two NIs, 3,4-dimethylpyrazole phosphate (DMPP) and dicyandiamide (DCD), and Francis et al. (6)found that application of DCD reduced NO3

� leaching following plowing of a grass-land. However, to date, no studies have investigated in detail the soil N dynamics ortheir microbiological basis following NI application to grassland before cultivation.

In the management strategy proposed here, DMPP is sprayed on aboveground partsof the vegetation, which are then incorporated during soil tillage. Two tillage methods,conventional plowing and shallower rotary tillage (rotovation), are common and resultin distinctly different patterns of residue distribution in the soil. Residue distributionmay itself have implications for the residue decomposition rate (17), N turnover (4), andN2O emissions (18, 19). In studying the effects of DMPP application, it is thereforenecessary to also examine interactions with residue distribution with respect to soil Ntransformations and N2O emissions.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) and denitrifying bacteria arekey components of inorganic N transformations in soil and are the major source of N2Oemissions (20). Understanding their response to NIs and residue incorporation is key todevising effective management strategies. NIs have been shown to reduce potentialammonia oxidation (PAO) and to inhibit the growth of AOB more than that of AOA(21–23). A recent study further revealed that DMPP inhibits the transcription of AOBamoA genes (24). Few studies have examined the effects of NIs on denitrifiers, butbased on the limited information available, DMPP appears to have little effect on theabundance of denitrifiers (23). Also, NIs do not seem to affect denitrifying enzymeactivity (DEA) (25, 26).

The present study was conducted to investigate how treating (aboveground partsof) grass-clover with an NI prior to incorporation will affect mineral N dynamics,microbial activities, and population dynamics of nitrifiers and denitrifiers. Also, theimplications for N2O emissions were examined by monitoring fluxes during incubation.We hypothesized that application of an NI before residue incorporation would delaynitrification of residue-derived N and that the effects on growth and activity ofdenitrifiers would be indirect, by reducing the availability of NO3

�. Soil columns wereconstructed with contrasting plant residue distributions in the soil, representing plow-ing and rotovation (Fig. 1). The soil N pool was labeled with 15NO3

� to trace thecontribution of denitrification to N2O emissions. DMPP was chosen as the NI for thisstudy because of its high efficiency, low mobility in soil (11, 27), and longer duration of

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activity than that of other NIs (16). Soil mineral N, PAO, DEA, and N2O fluxes weremonitored during a 42-day incubation experiment, and functional genes involved innitrification and denitrification (amoA, nirK, nirS, and clades I and II of nosZ) wereanalyzed for gene and mRNA copies on selected days (Fig. 1).

RESULTSDynamics of soil mineral N. Changes in soil ammonium (NH4

�) and nitrate (NO3�)

concentrations during the 42-day incubation are presented in Fig. 2. Ammonium

FIG 1 Overview of the experimental setup. Soil columns were constructed for five treatments (CT, PL,PLD, RO, and ROD). Soil columns for PL, PLD, RO, and ROD were compartmentalized into residue-associated soil (RS) and bulk soil (BS) compartments. DMPP was applied to the aboveground parts ofplant residues before incorporation into soil columns. In the sampling strategy, a solid circle indicatesthat N2O measurement or soil sampling took place on that specific day. An open circle denotes that N2Ofluxes were monitored on that day, but soil was not sampled and was kept for incubation until thesubsequent sampling (indicated by an arrow). See Materials and Methods for a detailed description of theexperimental setup.

FIG 2 Dynamics of soil ammonium (NH4�) (a1 and a2) and nitrate (NO3

�) (b1 and b2) concentrations insoil compartments during the 42-day incubation. Data for treatments simulating plowing (PL and PLD)and rotovation (RO and ROD) are shown in the left and right plots, respectively. Each point representsthe mean for triplicates, and error bars show standard errors (n � 3). Note that in panels a1 and a2, datapoints for CT overlap and are hidden underneath the data points for other samples.

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concentrations in the control (CT) and all bulk soil (BS) compartments were low (�0.5mg N kg�1 dry soil in most cases) and showed little change throughout the incubation.In the residue-associated soil (RS) compartments, a rapid increase in NH4

� content wasseen from day 1 to day 7 for treatments with DMPP (plowing [PLDRS] and rotovation[RODRS]) and, to a lesser extent, treatments without DMPP (PLRS and RORS). From thesecond week on, NH4

� concentrations in soil receiving DMPP showed either a gradualdecline (PLDRS) or a slow increase (RODRS) for the remainder of the incubation. Incontrast, the NH4

� content in PLRS and RORS samples took a rapid decline during thesecond week and then remained around the background (CT) level. Except for those atday 1, NH4

� concentrations of PLDRS and RODRS remained significantly higher thanthose of PLRS and RORS on all sampling days. Nitrate concentrations in all soilsconsistently increased during the 42-day incubation. From day 7 on, treatments with-out DMPP (PL and RO) generally gave higher NO3

� concentrations than correspondingtreatments with DMPP (PLD and ROD) for both RS and BS compartments. RS compart-ments generally had more NO3

� accumulation than BS compartments, except forPLDRS versus PLDBS. By day 42, all residue treatments had significantly higher NO3

�

concentrations than the CT level.Net N release (NH4

�-N � NO3�-N) from residues for each soil column (see Fig. S1 in

the supplemental material) was estimated by subtracting the background N in the CTsample and adjusting for the mass ratio of soil compartments (RS/BS ratio � 1:3 forPL/PLD and 1:1 for RO/ROD). Residue incorporation had a significant effect on net Nrelease, with RO and ROD showing significantly more net N release than PL and PLDbetween days 7 and 28. In contrast, DMPP had no effect on net N release within thesame residue incorporation type. Despite the different temporal dynamics, all treat-ments had similar net N releases on day 42, with an average of 34.2 mg N kg�1 dry soil,corresponding to ca. 33% of total N in residues.

The atom fraction 15N in soil [x(15N)] was monitored to trace the dilution of the15N-labeled NO3

� pool due to nitrification of residue-derived N (see Fig. S2 in thesupplemental material). The decline of x(15N) was faster with residue-amended treat-ments than with the CT treatment. Also, the decline of x(15N) was faster in RS than inBS compartments and in soils without DMPP than in the corresponding compartmentsreceiving DMPP. Hence, these data provided evidence that nitrification activity wasgreater in RS than in BS compartments and was more extensive without DMPP thanwith DMPP-treated residues.

Potential ammonia oxidation and denitrifying enzyme activity. Plant residueand DMPP application had clear effects on PAO activity (Fig. 3a1 and -2). PAO in thecontrol (CT) did not change during the 42-day incubation, and similar patterns wereseen for PAO in BS compartments, but with more fluctuations. For treatments withoutDMPP (both PL and RO), the presence of plant residues significantly stimulated PAO inPLRS and RORS samples between day 1 and day 14, and PAO remained at this high leveluntil the end of the experiment. In contrast, PAO in PLDRS and RODRS samples declined78% and 57%, respectively, during the first 2 weeks and remained inhibited until theend of the experiment.

Unlike PAO, DEA was not negatively affected by DMPP (Fig. 3b1 and -2). For BScompartments, treatments with (PLDBS and RODBS) and without (PLBS and ROBS) DMPPdid not differ in DEA compared to the control (CT). For the RS compartments, soilstreated with DMPP (PLDRS and RODRS) even showed a higher DEA than those of BS andCT soils in general. Overall, there was a trend of a decline in DEA from day 14 (forPL/PLD) or 21 (for RO/ROD) toward the end of incubation for all treatments.

Abundances of nitrifier and denitrifier genes. Figure 4 shows the abundances ofeach individual gene on days 1, 14, and 42. The results of a principal componentanalysis (PCA) of the abundances of the six genes are shown in Fig. S3 in thesupplemental material. For AOA amoA genes, there were no statistical differencesbetween the abundances on days 1, 14, and 42 for any treatment. For AOB, amoA genesremained unchanged between days 1 and 14. However, by day 42, there was a

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significant increase in bacterial amoA for PLRS and RORS, whereas a significant declinewas observed for PLDRS.

The abundances of denitrifier genes (nirK, nirS, and clades I and II of nosZ) eitherremained unchanged during the entire incubation or increased significantly by day 42.

FIG 4 Abundances of denitrifier (nirK, nirS, and clades I and II of nosZ) and nitrifier (archaeal and bacterial amoA) genes on days 1, 14, and 42. In each panel,for each soil compartment, data for samples from days 1, 14, and 42 with the same letter are not statistically different at an � level of 0.05.

FIG 3 Potential ammonia oxidation (a1 and a2) and denitrifying enzyme activity (b1 and b2) rates in soilcompartments during the 42-day incubation. Data for treatments simulating plowing (PL and PLD) androtovation (RO and ROD) are shown in the left and right plots, respectively. Each point represents themean for triplicates, and error bars show standard errors (n � 3).

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Overall, there was a clear trend of increase for the collective denitrifier genes. However,there was no clear effect of plant residue or DMPP on the abundances of denitrifiergenes.

Abundances of nitrifier and denitrifier mRNA transcripts. Figure 5 shows theabundances of nitrifier and denitrifier mRNA transcripts on day 14, representing asnapshot of gene transcription when microbial activities were most affected by treat-ments. The results of PCA of the mRNA abundances of the six genes are shown in Fig.S4 in the supplemental material. There were no significant differences in the abun-dances of archaeal amoA mRNA among treatments. In contrast, RS compartments withDMPP treatment (PLDRS and RODRS) had significantly smaller numbers of bacterialamoA mRNA than corresponding compartments without DMPP (PLRS and RORS). ThePLRS and RORS compartments had slightly larger numbers of bacterial amoA mRNA thanthose in BS compartments and the CT, although this difference was not alwayssignificant. There was high variability in the abundances of individual denitrifier mRNAs,without clear effects of residue distribution or DMPP. However, PCA did reveal aseparation between PLDRS and RODRS, where PLDRS appeared to have a larger numberof nosZ clade I mRNA copies than that for RODRS, while mRNAs for nirK and nirS werehigher in RODRS than in PLDRS (Fig. S4).

Regression analyses were performed to examine the relationships betweenamoA mRNA and PAO and between nirK/nirS mRNA and DEA (Fig. 6). A significantpositive correlation was found between the abundance of bacterial amoA mRNAand PAO, while no correlation was found between archaeal amoA mRNA and PAO.Also, there was a significant positive correlation between nirS, but not nirK, mRNAtranscripts and DEA.

FIG 5 Abundances of denitrifier (nirK, nirS, and clades I and II of nosZ) and nitrifier (archaeal and bacterial amoA) mRNA transcriptson day 14. In each panel, data for samples with the same letter are not statistically different at an � level of 0.05.

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Dynamics and sources of N2O. Nitrous oxide emissions were monitored in boththe main experiment with 15NO3

� amendment (Fig. 7a1 and -2) and a control exper-iment without NO3

� amendment (Fig. 7b1 and -2). Cumulative N2O emissions and therespective contributions from nitrification and denitrification are summarized in Fig. 8.In both experiments, residue incorporation greatly increased cumulative N2O emissionscompared to those in the CT (Fig. 8), with most of the emissions occurring during thefirst 2 to 3 weeks (Fig. 7).

In the main experiment, the PLD treatment gave a lower maximum N2O flux thanthat with PL, whereas the maximum N2O flux was higher for ROD than for RO. AlthoughRO and ROD gave slightly lower cumulative N2O emissions than PL and PLD, respec-tively, there were no significant differences between the two residue distributions.DMPP reduced the average cumulative N2O emission by 10% and 5% for PLD and ROD

FIG 6 Relationships of AOA/AOB amoA mRNA transcripts and PAO (a1 and a2) and of nirK/nirS mRNA transcripts and DEA(b1 and b2).

FIG 7 N2O fluxes during the 42-day incubation for the main experiment with 15NO3� amendment (a1

and a2) and during the 28-day incubation for the control experiment without NO3� amendment (b1 and

b2). Data for treatments simulating plowing (PL and PLD) and rotovation (RO and ROD) are shown in theleft and right plots, respectively. Each point represents the mean for triplicates, and error bars showstandard errors (n � 3).

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compared to PL and RO, respectively, but again, the effect was not significant. Nitrousoxide emission factors, expressed as the percentage of N in residues emitted as N2O-N,ranged from 1.46% (ROD) to 1.96% (PL).

The atom fraction 15N of soil NO3� [x(15N)] was determined for both the RS and BS

compartments of the main experiment (Fig. S2), but only the results for the RScompartments were used to estimate N2O emissions from denitrification. In residue-amended soil columns compared to the CT, the contribution to N2O emissions fromnitrification was ca. 3 to 9 times higher, but the contribution from denitrification was20 to 23 times higher. Accordingly, denitrification was responsible for 80 to 90% of N2Oemissions in residue-amended columns.

In the control experiment without NO3� amendment, DMPP reduced the maximum

N2O flux for treatment PLD significantly compared to that for PL, and there was a 58%reduction of cumulative N2O emissions. A reduction was also indicated for treatmentROD compared to RO, but this difference was not significant. Nitrous oxide emissionfactors ranged from 0.49% (PLD) to 1.21% (PL).

DISCUSSION

This incubation study with soil from a long-term grass-arable rotation representedconditions and practices typical for temperate grasslands in Northern Europe. While theeffects of DMPP may vary from soil to soil, depending on, e.g., sand content, H�

concentrations, and catalase activities (28), the observations described in this studyconcern a novel application of DMPP in which aboveground parts of the vegetation aretreated, and therefore subsequent transformations of residue-derived N in the soil maybe less sensitive to soil properties than other applications of nitrification inhibitors.

Mineral N dynamics. The mineralization of residue-derived N began shortly afterincorporation, with a rapid net release of NH4

� during the first week (Fig. 2a1 and -2),but net N (NH4

� � NO3�) accumulation continued throughout the experiment for all

treatments (see Fig. S1 in the supplemental material). In a field study, Eriksen andJensen (4) also reported fast net N mineralization after cultivation, with the major partof N being released within 4 weeks. Nitrogen mineralization from plant residues willalso occur at low temperatures (2 to 14°C) (4, 16), which are typical for temperateregions in early spring, when grasslands are cultivated. Since the temperature sensi-tivities of N mineralization and nitrification are similar (29), a fast release of N fromincorporated residues will be accompanied by rapid conversion of ammoniacal N toNO3

�, which highlights the potential value of treating grasslands with a nitrificationinhibitor prior to cultivation.

The treatment of aboveground parts of grass-clover with DMPP prior to incorpora-tion clearly inhibited ammonia oxidation for at least 42 days (Fig. 3a1 and -2) at 20°C

FIG 8 Cumulative N2O emissions from the main experiment with 15NO3� amendment and the control

experiment without NO3� amendment. For the main experiment, contributions of denitrification and

nitrification to N2O emissions are indicated by different types of shading. Bars represent means fortriplicates. Error bars show propagated errors for nitrification and denitrification and standard errors forthe control experiment (n � 3). Data for treatments with the same letter are not statistically different atan � level of 0.05.

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in this study. At an average soil temperature of 7°C, which is more similar to fieldconditions in the spring, inhibition of nitrification by DMPP has been observed to lastfor up to 95 days (16), whereas the uptake of residue-derived N by a new crop can becomplete within 60 days (30). Therefore, treatment of grass-clover with DMPP beforegrassland cultivation in early spring may help to ensure that residue-derived N meetsthe demand of a new crop, reducing the need for supplementary fertilization as well asthe potential for N losses (11). While some studies have suggested that the activity ofDMPP applied to soil is reduced to 2 to 3 weeks at temperatures of �20°C (11, 31), theminimum 42-day life span of DMPP at 20°C observed in this study indicates that itwould be relevant to evaluate effects of treating grasslands with DMPP prior tocultivation in regions with warmer climates as well. However, DMPP applied in thespring will have little effect on autumn and winter N losses, which may be substantial(32), as DMPP will most likely be degraded by that time.

Effects of plant residues on NH4� release (Fig. 2a1 and -2) and PAO (Fig. 3a1 and -2)

were observed in RS but not BS compartments, indicating that the supply and trans-formations of residue-derived N were restricted to soil volumes close to plant residues.This is consistent with the work of Gaillard et al. (33), who observed that stimulation ofmicrobial responses was confined to a distance of a few millimeters from decomposingstraw. Such a localized stimulation of microbial activity near residue particles impliesthat soil contact, and hence the distribution of residues in soil, is important todecomposition and associated N turnover. When residues are concentrated in a layer,as in plowed soil, the limited residue-soil contact may restrict microbial colonizationand slow down decomposition (17). A stratified distribution of residues to simulateincorporation by plowing has been used in several studies (17, 34, 35). By this approach,the contact between plant residues and soil was restricted, although with a residuelayer of ca. 3 mm the distance to the surrounding soil was still relatively short. Inversiontillage only partly breaks up the vegetation cover, and therefore aboveground plantmaterial treated with DMPP will be distributed unevenly in the soil after incorporation.Residues more evenly mixed with soil, e.g., in RO treatments, which represent near-maximum contact, would have a larger surface area available for microbial attack, andthus a higher N turnover rate. Indeed, we observed a faster accumulation of net N inRO than in PL samples in this study (Fig. S1). In accordance with this, Eriksen and Jensen(4) reported for their field study that compared to plowing, rotovation enhanced Navailability and improved synchrony between N mineralization and plant N uptake,resulting in larger crop yields.

Soil nitrifier populations and activities. Similar to those in many other grasslandsoils (36, 37), AOA numerically dominated over AOB in the sandy loam soil used in thisstudy. However, PAO activity was correlated with the abundance of AOB but not AOAamoA mRNA (Fig. 6a2), indicating that AOB rather than AOA were responsible for theobserved nitrification activity. Although PAO rates represent activity in an environmentwithout substrate limitation, which is very different from the bulk soil environment,presumably the stimulation of nitrifier populations in residue-amended soil will belocalized around soil-residue interfaces with net N mineralization. Plant residues in-creased both the abundance (Fig. 4) and activity (in terms of mRNA copies) (Fig. 5) ofAOB but not AOA. Since the decomposing residues would have increased nutrient andN availability for microorganisms in RS compartments (33, 38), these observationssupport the contention that activity and growth of AOB are favored in nutrient-rich,high-N soil, whereas AOA may rather proliferate under nutrient-poor, low-N conditions(39, 40).

DMPP inhibited ammonia oxidation, targeting mainly AOB rather than AOA (Fig. 6),which is consistent with the observations of Kleineidam et al. (21). The inhibitionmechanism of DMPP remains unclear. Chaves et al. (16) speculated that DMPP inhibitsnitrification by indiscriminately binding to membrane-bound proteins, including am-monia monooxygenase, but this hypothesis cannot explain DMPP’s lack of nontargettoxicity (41). In addition, Florio et al. (24) and the present study both found that DMPP

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reduced the abundance of amoA mRNA, suggesting an unknown mechanism throughwhich DMPP downregulates the transcription of amoA genes. The transcription of AOBamoA genes was already inhibited by DMPP on day 14 (Fig. 5), but a reduction in AOBpopulation size was not observed until day 42 (Fig. 4); this may reflect the course ofnatural mortality in a situation where reduced ammonia oxidation prevented de novogrowth. The inhibition by DMPP may persist for several months (11, 16), but at leastsome AOB may survive NH4

� starvation for months or even a year (42, 43), and thusnitrification is expected to recover after the eventual degradation of DMPP.

Soil denitrifier populations and activities. While both nirK- and nirS-type denitri-fiers were found in this study, a correlation between the number of mRNA copies andDEA was found only for nirS, which may suggest that nirS denitrifiers were primarilyresponsible for denitrification in these soils. However, Penton et al. (44) tested thecoverage of several commonly used nirK and nirS primer sets and found that they cancapture only a subset of known denitrifiers, and especially the coverage of nirK primerswas extremely low. Therefore, the abundance of nirK denitrifiers and their involvementin DEA may be underestimated in the present study. Moreover, Graf et al. (45) reportedthat ca. 35% of nirK denitrifiers lack the nor gene for converting NO to N2O, whereas noris almost always present in nirS denitrifiers. The inability of many nirK denitrifiers toproduce N2O may therefore also partly explain the lack of correlation between nirKmRNA abundance and DEA, since this assay relies on the production of N2O.

The nosZ genes encoding nitrous oxide reductase were recently separated into twogroups: clade I (nosZI) and the previously unaccounted clade II (nosZII) (46). An averageof 40% of the nosZ genes recovered in this study belonged to clade II, confirming thatnosZII-containing organisms are abundant and widespread in soils (46). However,dissociation curve analysis following quantitative real-time PCR (qPCR) showed thatthere was sometimes minor unspecific coamplification, suggesting that the abundanceof nosZII organisms may be overestimated in some cases. The nosZ N2O reducers arecritical in determining the soil N2O sink capacity (47), and conceivably the abundanceof nosZ genes and/or mRNA should be correlated negatively with N2O emissions (48).However, no such correlation was found in the present study, suggesting that thecorrelation between nosZ genes and N2O emissions is more complex. For example, Diet al. (49) reported increased nosZ abundance in response to elevated N2O production,and they proposed that this was due to an improved substrate supply.

In contrast to the inhibition of AOB, we did not find adverse effects of DMPP ongrowth or activity of denitrifiers in this study. Di et al. (49) concluded that DCD reducedthe abundance of the nirK gene by affecting AOB populations that also bear nirK. Whilethe size of AOB populations was reduced by DMPP in the present study, there was nocorresponding reduction in the abundance of nirK genes. Florio et al. (24) observedvariable inhibition of nosZ mRNA by DMPP during a 7-day incubation, and theyproposed that there may be a short-term effect of DMPP on nontarget microorganisms.However, a similar pattern was not observed with any denitrifier mRNA in the presentstudy, in which soil samples were analyzed on day 14. Also, Kong et al. (41) found noshort-term toxicity of DMPP to nontarget microorganisms or functions even at a dosage10 times higher than the normal application rate. Therefore, any effect of DMPP ondenitrifiers would have to be transient and possibly restricted to the first few days afterDMPP exposure. In addition, consistent with the work of Müller et al. (25), DMPP did notinhibit DEA in this study. Rather interestingly, we observed that RS compartments in soilwith DMPP-treated residues showed significantly higher DEA than compartmentswithout DMPP on most days (Fig. 3b). High NO3

� concentrations may inhibit nitratereductase activity (50) or even reduce the abundance of nitrate reductase genes (narG)(49). We therefore speculate that by inhibiting ammonia oxidation, DMPP resulted inless NO3

� accumulation than that for treatments without DMPP, and possibly in asmaller chance of inhibition of nitrate reduction by excess NO3

�.Although plant residues did not stimulate the growth of denitrifiers, the distribution

of residues in soil seemed to have influenced the transcription of denitrification genes.

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There is a separation between PL/PLD and RO/ROD soil samples in Fig. S4, especiallybetween PLDRS and RODRS, indicating a larger number of nosZI mRNA copies butsmaller numbers of nirK and nirS mRNAs in plowed than in rotovated soil. Thetranscription of both nir and nos genes could be induced at low O2 levels (51) that likelydeveloped around decomposing residues (52), and it remains unclear what determinedthis variation in nirK/nirS and nosZ transcripts with different residue distributions. It isalso unclear whether this was a transient situation or a treatment effect, as mRNA wasanalyzed at only one time point in this study.

N2O fluxes and sources of N2O. In both the main experiment and the controlexperiment, residue incorporation stimulated N2O emissions (Fig. 7), consistent withfield observations after grassland cultivation (53–55). The temporal dynamics weresimilar to those reported by Baggs et al. (53), with most of the N2O emissions occurringduring the first 2 to 3 weeks after incorporation, coinciding with the rapid N transfor-mations described above. Presumably the rapid residue decomposition provided Nsubstrates for nitrification and denitrification (56) and eventually promoted O2-limitedconditions around residues (52, 57), thereby increasing the potential for N2O emissions.

Denitrification contributed 80 to 90% of the N2O emitted in this study, irrespectiveof DMPP application and residue distribution (Fig. 8). This is consistent with the workof Li et al. (58), who identified denitrification as the major source of N2O emissions fromdifferent plant residues for a 40 to 60% water-filled pore space. The atom fraction 15Nof RS compartments was used to estimate the contribution of denitrification to N2Oemissions, rather than the average value for both compartments. This seemed moreappropriate because microbial N transformations were far more active in RS than in BScompartments (Fig. 2 and 3). Due to possible gradients in the distribution of 15NO3

�

within RS compartments, the contribution of denitrification may be somewhat under-estimated (58).

By inhibiting the activity and growth of nitrifiers, DMPP would be expected toalways reduce N2O emissions from nitrification and/or nitrifier denitrification. We foundno evidence for direct effects of DMPP on denitrifiers, and thus any effect on N2Oemissions from denitrification would be expected to be indirect, i.e., via changes insubstrate availability (59). In the main experiment, the absence of DMPP effects on N2Oemissions implied that NO3

� availability did not limit denitrification, as NO3� could be

obtained from soil (up to 32 mg N kg�1 dry soil) even though derivation from residueswas blocked. This was in contrast to the control experiment (with a background NO3

�

level of 7 mg N kg�1 dry soil) without an external NO3� source, where DMPP reduced

N2O emissions for the PLD treatment by 58% compared to the level for PL. Inaccordance with this, in an incubation experiment with layered plant residues similar tothe present study, Li et al. (58) found evidence for NO3

� limitation of denitrificationduring the first week of residue decomposition, which was later alleviated by nitrifica-tion of N released from residues. This inhibition of N2O emissions by DMPP is compa-rable to effects observed when nitrification inhibitors are used together with inorganicfertilizers or manure (59, 60). However, the effect of DMPP in the RO treatment wassmaller and not significant; presumably, the shallow and uniform distribution of plantresidues shifted decomposition toward more aerobic processes, while the supply ofNO3

� from soil and nitrification was sufficient to support the residual denitrification(19).

The observations in this study thus point to an important difference in the efficacyof DMPP for mitigating N2O emissions and N leaching. Since N2O emissions mayoriginate from both soil and residue-derived NO3

�, DMPP will have the potential toreduce N2O emissions only when soil NO3

� availability is low. This implies that there ispotential for a DMPP-induced reduction of N2O emissions following grassland cultiva-tion, as soil NO3

� availability is usually low in early spring due to previous plant uptakeand leaching (5). On the other hand, N leaching is associated mainly with highly mobileNO3

�, and therefore DMPP would always reduce the risk for leaching of residue-derivedN to the extent that accumulation of NO3

� is prevented.

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Conclusions. This laboratory incubation experiment reproduced all the key ele-ments of grassland cultivation and thus provides a reliable prediction of DMPP effectsin the field. Our results indicate that treatment of grass-clover with DMPP prior tograssland cultivation can change the soil N dynamics as hypothesized. Residue distri-bution to simulate rotovation or plowing had no significant effect on the abundance oractivity of soil nitrifiers and denitrifiers, except that net N release was faster in rotovatedthan in plowed soil. Independent of residue distribution, DMPP lowered ammoniaoxidation by inhibiting the transcription of AOB amoA genes, but it had little effect onthe abundance or activity of AOA or denitrifying bacteria. This management strategythus appears to reduce the N-leaching potential following grassland cultivation. Deni-trification was the main source of N2O emissions associated with residue decomposi-tion, and the results showed that DMPP has the potential to reduce N2O emissions, butonly when the soil NO3

� availability is low and thus dependent on nitrification ofresidue N.

MATERIALS AND METHODSCollection of soils and plant materials. A sandy loam soil (0 to 25 cm) was collected from a

grass-clover pasture at Foulumgård Experimental Station, Denmark, in January 2014. The soil wasclassified as Typic Hapludults, with 7.4% clay, 10% silt, 81% sand, 1.6% C, and a pH of 5.0 (in 1 M KCl; 1:4[wt/vol]), and it contained 0.2 and 7.0 mg N kg�1 dry soil of NH4

� and NO3�, respectively. The cropping

history and other properties were previously described by Eriksen et al. (61). The soil was stored in plastictrays with a loose cover at 2°C after sieving and homogenization.

Ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) were collected from the same site.Intact soil blocks with vegetation were brought to the laboratory, where plants, including the roots, wereseparated from soil by rinsing with tap water and then separated into aboveground (leaves and stems,and stolons for clover) and belowground (roots) parts. Plant materials were stored in ziplock bags at 2°Cuntil needed for experiments. A subsample was used to determine total C and N levels by the Dumascombustion process (Flash 2000 NC analyzer; Thermo Scientific, Bergman Labora, Sweden) after ovendrying (80°C, 24 h) and ball milling by use of a Mixer MM 400 mill (Retsch GmbH, Haan, Germany).

Experimental setup. Five treatments were included in this experiment (Fig. 1). A control treatment(CT) was prepared without addition of plant residues or DMPP. For treatments with residues, thefollowing two methods of tillage were simulated: plowing (PL), where plant residues were placedbetween two layers of soils in a “sandwich” structure; and rotovation (RO), with plant residues mixedevenly into the upper part of soil columns. Aboveground plant parts were sprayed with DMPP (PLD andROD) or left untreated (PL and RO). Soil columns were further defined by two compartments. Theresidue-associated soil (RS) compartment refers to the combined 1-cm soil layers on both sides of theresidue layer in plowing treatments (PLRS and PLDRS) and to the soil in the upper 0 to 4 cm that wasmixed with residues in rotovation treatments (RORS and RODRS). Soils without contact to residues arereferred to as the bulk soil (BS) compartments (PLBS, PLDBS, ROBS, and RODBS).

To keep the number of samples manageable while allowing sufficient replicates, a total of 90 soilcolumns were prepared as described in the next section, with 18 replicates for each treatment, whichallowed destructive sampling on six occasions (days 1, 7, 14, 21, 28, and 42) for three replicates pertreatment. Measurement of N2O fluxes and sampling of soils for analyses took place on selected days asillustrated in Fig. 1. Samples from the first (day 1) and last (day 42) soil samplings were selected foranalyses of functional gene abundances to examine treatment effects on microbial growth. Additionally,to investigate the relationship between microbial abundances and activities, samples from day 14 wereanalyzed for functional genes and mRNA transcripts. Day 14 was selected because treatment effects onmicrobial activities were most prominent around this time, when N2O fluxes also peaked, while nitrifierand denitrifier population sizes had not yet changed significantly.

Packing and incubation of soil columns. One week before the experiment, soils were transferredto 20°C to allow microorganisms to adapt to the incubation temperature (62). Four days before residueincorporation, soils were packed into transparent acrylic tubes (10 cm tall and 6.4 cm in diameter), to adry bulk density of 1.3 g cm�3, by use of a custom-made piston. Soil was added in four portions of 2 cmof soil. After packing of each portion, 1.2 ml KNO3 solution (1.74 mg N ml�1; atom fraction 15N � 10%)(Icon Services Inc.) was evenly added dropwise on the surface. The total NO3

� amendment correspondedto 25 mg N kg�1 dry soil, and the soil had a water-filled pore space of 50%.

Plant materials were cut into ca. 5-mm pieces before incorporation. For aboveground parts, residuesof clover were mixed with a leaf-plus-stem/stolon ratio of 1:4, and then clover was mixed with ryegrassat a ratio of 3:7. Belowground parts (roots) of clover and ryegrass were also mixed at a ratio of 3:7. Fivemilliliters of DMPP (2.32 mg ml�1; EuroChem Agro, Mannheim, Germany) was then evenly sprayed onhalf of the aboveground mixture, while the other half was left without DMPP. Finally, the above- andbelowground mixtures were combined at a ratio of 1:4. The above mixing ratios were decided based onprevious characterizations of residue composition (63) and the availability of materials. The amount ofgrass-clover residues corresponded to an incorporation rate of 4 t dry matter ha�1. The DMPP treatmentcorresponded to an application rate of 1 kg DMPP ha�1, which is identical to the recommended rate forits application together with mineral fertilizers or manure (11).

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The packed 8-cm soil columns were separated into two equal parts, 0 to 4 and 4 to 8 cm, and plantresidues were either placed between the two parts as a ca. 3-mm layer (plowing treatments) or mixedwith the soil from 0 to 4 cm (rotovation treatments). Finally, packed tubes were covered with perforatedParafilm on both ends to prevent water loss but allow gas exchange and were placed at 20°C in a darkroom for incubation. Tubes were weighed every 2 to 3 days; the water loss was negligible throughoutthe experiment.

Fluxes and 15N labeling of N2O. For N2O flux measurements, soil columns were placed individuallyin 1-liter gastight jars with a rubber septum for gas sampling. Zero, 1, 2, and 3 h after closure, a 10-mlgas sample was taken from the headspace and immediately transferred to a 6-ml preevacuated Exetainer(Labco, High Wycombe, United Kingdom). Nitrous oxide concentrations were determined with an Agilent7890 gas chromatograph (GC) system with a CTC CombiPal autosampler (Agilent, Nærum, Denmark),configured as described by Petersen et al. (64). Fluxes of N2O were estimated with the HMR fluxestimation package in R (version 3.2.2; R Core Team), using either linear or nonlinear regression (65).

By the end of the N2O flux measurement (3 h after closure), an additional 75-ml gas sample was takenfor direct injection into an isotopic N2O analyzer (LGR model 914-0022; Los Gatos, Mountain View, CA)to determine �15N-N2O as described by Li et al. (58). Nitrous oxide derived from denitrification (N2Od) wascalculated according to the method of Stevens et al. (66). On days 3, 10, and 35, when soil columns werenot sampled for 15N analyses, N2Od was estimated as the average for N2Od values calculated for theprevious (days 1, 7, and 28, respectively) and subsequent (days 7, 14, and 42, respectively) samplings.

Measurement of soil N dynamics. On the days indicated in Fig. 1, N2O flux measurements werefollowed by destructive sampling of soil columns. Sections corresponding to the RS and BS compart-ments were isolated. Soil was carefully separated from plant residues, mixed, and subsampled. Soils tobe used for chemical and microbiological assays were stored at 2°C until the following day, whilesubsamples for molecular analyses were immediately stored at �80°C until needed.

Soil mineral N was extracted from ca. 10 g of soil by shaking in 40 ml 1 M KCl for 30 min. The extractwas filtered through a 1.6-�m-pore-size glass microfiber filter (VWR, France), and the filtrate was analyzedfor NH4

� and NO3� by continuous-flow analysis on an Autoanalyser III (Bran�Luebbe GmbH, Norder-

stedt, Germany), using standard colorimetric methods. The soil pH of this filtrate was also measured,while soil gravimetric water content was determined for separate subsamples by oven drying at 105°Cfor 24 h.

The 15N abundance of NO3� in soil extracts was determined by the sequential diffusion method

described by Sørensen and Jensen (67), using an elemental analyzer interfaced with a continuous-flowisotope ratio mass spectrometer (IRMS) at the Stable Isotope Facility, University of California, Davis.

PAO was determined for ca. 10 g fresh soil by the shaken soil-slurry method as described byElsgaard et al. (68). Soil DEA was determined with ca. 10 g fresh soil by using the acetylene inhibitionmethod (69).

Nucleic acid extraction and purification. Total nucleic acids (DNA and RNA) were extracted fromca. 0.5-g soil samples following a modular method described by Lever et al. (70). Briefly, samples wereweighed into 2-ml bead-beating lysis matrix E tubes (containing 1.4-mm ceramic spheres, 4-mm glassbeads, and 0.1-mm silica spheres; MP Biomedicals, Santa Ana, CA) and soaked with 100 �l of a solutioncontaining a 10 mM concentration of each deoxynucleoside triphosphate (dNTP). After being frozen solidat �80°C, samples were mixed by brief vortexing with 400 �l lysis solution I (70), followed by 400 �lphenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol/vol]). Cells were lysed on a TissueLyser LT instrument(Qiagen, Copenhagen, Denmark) 3 times for 45 s each at a speed of 50 Hz, with a 30-s incubation on icebetween each step to prevent overheating. This combined mechanical and chemical cell lysis has beenproven sufficient for DNA/RNA extraction from soil samples (70). After centrifugation (12,000 � g for 5min at 4°C), the upper, aqueous phase containing nucleic acids was transferred to new sterile Eppendorftubes and reextracted with an equal volume of chloroform-isoamyl alcohol (24:1 [vol/vol]). Nucleic acidswere recovered by isopropanol-NaCl precipitation with linear polyacrylamide (final concentration, 20 �gml�1) as a coprecipitant. Nucleic acid pellets were washed with 70% (vol/vol) ice-cold ethanol, moder-ately dried in a Savant DNA120 Speed-Vac concentrator (Thermo Scientific, Waltham, MA), and resus-pended in MilliQ water.

The crude nucleic acid extract was separated into two portions. One portion, used for DNA analysis,was purified using a Norgen CleanAll kit (Norgen Biotek, Ontario, Canada). The other portion, used forRNA analysis, was first treated with a Turbo DNA-free kit (Life Technologies, Carlsbad, CA) to remove DNAand then purified using a Norgen CleanAll kit. The absence of residual genomic DNA was confirmed by(a lack of) PCR amplification of target genes from these RNA extracts. Reverse transcription (cDNAsynthesis) was performed using an Omniscript reverse transcription kit (Qiagen, Copenhagen, Denmark)with random hexamer primers and Ambion Anti-RNase (Fisher Scientific, Slangerup, Denmark) as anRNase inhibitor, according to the manufacturers’ instructions. DNA and cDNA were stored at �20°C untilneeded for downstream analysis.

Quantification of nitrifying and denitrifying genes and mRNA transcripts. Quantitative real-timePCR (qPCR) was performed using a Stratagene Mx3005P instrument (Agilent, Santa Clara, CA). PotentialPCR inhibitors in DNA and cDNA samples were tested prior to quantification as described by Kong et al.(41), and no inhibition was observed in these initial tests.

Standards ranging from 107 down to 101 gene copies �l�1 were prepared from linearizedplasmids with insertions of target gene fragments. Standards, samples, and nontemplate controlswere prepared in triplicate. Primers, PCR mixture compositions, and PCR procedures for amplificationof archaeal and bacterial amoA, nirK, nirS, and nosZ clade I and II genes are listed in Table 1.Following amplification, the specificity of products was confirmed by dissociation curve analysis and

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(for a random subset of samples) gel electrophoresis. Nontemplate controls did not show anyamplification. Results were processed using MxPro v4.10 software (Agilent Technologies Inc., SantaClara, CA) with default parameters.

Effect of nitrate availability (control experiment). Soil columns in the main experiment wereamended with 15N-labeled NO3

� to assess the contributions from nitrification and denitrification to N2Oemissions, which would increase the potential for N2O emissions via denitrification. For reference, acontrol experiment without NO3

� amendment was conducted with the same five treatments (CT, PL,PLD, RO, and ROD) in three replicates. These columns were incubated for 28 days, with measurement ofN2O fluxes on days 1, 3, 7, 10, 14, 21, and 28 of incubation. By the end of the incubation, soil columnswere sectioned and subsampled for chemical analyses as described above.

Statistical analyses. All statistical analyses, except for principal component analysis (PCA), wereperformed with the statistical software R (version 3.2.2; R Core Team). The effects of DMPP and residueincorporation were tested using two-way analysis of variance (ANOVA), and pairwise differences, with an� level of 0.05, were identified by the lsmeans package, using the Tukey multiple-comparison test. TheNH4

�-N and NO3�-N data from the two soil compartments in the main experiment were ln(n � 1) and

ln transformed prior to analysis. The relationships between mRNA abundances and microbial activitieswere analyzed by linear regression. A PCA of the abundances of nitrifier and denitrifier genes and mRNAswas performed using OriginPro 9.0 (OriginLab, Northampton, MA); abundance data were log transformedbefore analysis.

Due to the destructive sampling, cumulative N2O emissions and treatment effects on cumulativeemissions were not readily calculated. The N2O data were modeled using a generalized linear mixedmodel assuming gamma-distributed responses, identity linking, and a linear predictor containing aninteraction of day and treatment (inference was performed using the glmer function of the lme4package). In the model described above, the dependency of repeated measurements was taken intoaccount by introducing a Gaussian random component, taking the same value for all observations fromthe same tube. Cumulative N2O emissions were estimated by a trapezoidal approximation of the areaunder the flux curve; to do so, contrasts (i.e., linear combinations of the predicted emissions on each ofthe measurement days) were constructed in such a way that they coincided with the sum of the areasof the trapezoidal approximation of the integral of the flux curve (see the supplemental material for thetheoretical background). The estimates, variances or standard errors of estimates, tests comparingtreatment levels, and confidence intervals were computed using the standard theory of inference forcontrasts under generalized linear mixed models. The P values for simultaneous tests were adjusted formultiple comparisons by the false discovery rate (FDR) method; P values of �0.05 were consideredstatistically significant.

TABLE 1 Primers, compositions of PCR mixtures, and PCR procedures for amplification of nitrifier and denitrifier genes

GenePrimername Primer sequence Contents of PCR mixture (20 �l) PCR procedure Reference

Archaeal amoA Arch-amoAFArch-amoAR

STAATGGTCTGGCTTAGACGGCGGCCATCCATCTGTATGT

1� LightCycler 480 SYBR green I mastermix (Roche, Mannheim, Germany), 5�g bovine serum albumin (BSA), 0.5�M (each) primers, 2 �l template

Initial denaturation at 95°C for 10 min;40 cycles of 95°C for 30 s, 57°C for60 s, 72°C for 30 s, 82°C for 15 s,plate reading

71

Bacterial amoA amoA-1FamoA-2R

GGGGTTTCTACTGGTGGTCCCCTCKGSAAAGCCTTCTTC

1� LightCycler 480 SYBR green I mastermix, 5 �g BSA, 0.8 �M (each) primers,2 �l template

Same as that for archaeal amoA 72

nirK F1aCuR3Cu

ATCATGGTSCTGCCGCGGCCTCGATCAGRTTRTGGTT

1� LightCycler 480 SYBR green I mastermix, 5 �g BSA, 0.3 �M (each) primers,2 �l template

Initial denaturation at 95°C for 10 min;5 cycles of 95°C for 15 s, 63°C for30 s (decreased by 1°C per cycle),and 72°C for 30 s; 40 cycles of 95°Cfor 15 s, 58°C for 30 s, 72°C for 30 s,83°C for 15 s, plate reading

73

nirS Cd3aFR3cd

AACGYSAAGGARACSGGGASTTCGGRTGSGTCTTSAYGAA

1� LightCycler 480 SYBR green I mastermix, 5 �g BSA, 0.8 �M (each) primers,2 �l template

Same as that for nosZ clade I genes 74

nosZ clade I nosZ_1FnosZ_1R

CGYTGTTCMTCGACAGCCAGCGSACCTTSTTGCCSTYGCG

1� LightCycler 480 SYBR green I mastermix, 5 �g BSA, 0.8 �M (each) primers,2 �l template

Initial denaturation at 95°C for 10 min;5 cycles of 95°C for 15 s, 65°C for30 s (decreased by 1°C per cycle),72°C for 30 s; 40 cycles of 95°C for15 s, 60°C for 30 s, 72°C for 30 s,83°C for 15 s, plate reading

75

nosZ clade II nosZ-II_FnosZ-II_R

CTXGGXCCXYTKCAYACGCXGARCARAAXTCBGTRC

1� LightCycler 480 SYBR green I mastermix, 5 �g BSA, 0.8 �M (each) primers,2 �l template

Initial denaturation at 95°C for 10 min;40 cycles of 95°C for 30 s, 54°C for60 s, 72°C for 60 s, 82°C for 15 s,plate reading

46

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02019-16.

TEXT S1, PDF file, 1.2 MB.

ACKNOWLEDGMENTSThis study was part of the project “Mitigating Nitrous Oxide Emissions following

Grassland Cultivation,” funded by the Danish Council for Independent Research (grant12-127378).

We greatly appreciate the skilled technical assistance of Bodil Steensgaard Jensen,Susanne Nielsen, Trine Bech Søgaard, and Britta Poulsen. We also thank Sara Hallin andMaria Hellman from the Swedish University of Agricultural Sciences, who kindly pro-vided plasmids carrying denitrifier genes.

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