Evaluation of a crop rotation with biological inhibition potential to avoid N2O emissions in comparison with synthetic nitrification inhibition

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Evaluation of a crop rotation with biological inhibition potential to avoid N ₂ O emissions in comparison with synthetic nitrification inhibition

Adrián Bozal-Leorri

, Mario Corrochano-Monsalve

, Luis M. Arregui

, Pedro M. Aparicio-Tejo

, Carmen González-Murua

1DepartmentofPlantBiologyandEcology,UniversityoftheBasqueCountry(UPV/EHU),Apdo.644,E-48080,Bilbao 48940,Spain

2InstituteforInnovationandSustainableDevelopmentinFoodChain(ISFOOD),PublicUniversityofNavarre, Pamplona31006,Spain

3InstituteforMultidisciplinaryResearchinAppliedBiology(IMAB),PublicUniversityofNavarre,Pamplona31006, Spain

a r t i c l e i n f o

Articlehistory:

Received17January2022 Revised22April2022 Accepted25April2022 Availableonline4May2022

Keywords:

Fallow Sorghum Croprotation Nitrificationinhibitor N-cyclinggenes Soilmineralnitrogen

a bs t r a c t

Agriculture hasincreased the release of reactive nitrogen to the environment dueto crops’lownitrogen-useefficiency(NUE)aftertheapplicationofnitrogen-fertilisers.Prac- ticesliketheuseofstabilized-fertiliserswithnitrificationinhibitorssuchasDMPP(3,4- dimethylpyrazolephosphate)havebeenadoptedtoreducenitrogenlosses.Otherwise,cover cropscanbeusedincrop-rotation-strategiestoreducesoilnitrogenpollutionandbenefit thefollowingculture.Sorghum(Sorghumbicolor)couldbeagoodcandidateasitisdrought tolerantanditsculturecanreducenitrogenlossesderivedfromnitrificationbecauseitex- udatesbiologicalnitrificationinhibitors(BNIs).Thisworkaimedtoevaluatetheeffectof fallow-wheatandsorghumcovercrop-wheatrotationsonN2Oemissionsandthegrain yieldofwinterwheatcrop.Inaddition,thesuitabilityofDMPPadditionwasalsoanalyzed.

Theuseofsorghumasacovercropmightnotbeasuitableoptiontomitigatenitrogen lossesinthesubsequentcrop.Althoughsorghum–wheatrotationwasabletoreduce22%

theabundanceofamoA,itpresentedanincrementof77%incumulativeN2Oemissions comparedtofallow–wheatrotation,whichwasprobablyrelatedtoagreaterabundanceof heterotrophic-denitrificationgenes.Ontheotherhand,theapplicationofDMPPavoidedthe growthofammonia-oxidizingbacteriaandmaintainedtheN2Oemissionsatthelevelsof unfertilized-soilsinbothrotations.Asaconclusion,theuseofDMPPwouldberecommend- ableregardlessoftherotationsinceitmaintainsNH4+inthesoilforlongerandmitigates theimpactofthecropresiduesonnitrogensoildynamics.

© 2022TheResearchCenterforEco-EnvironmentalSciences,ChineseAcademyof Sciences.PublishedbyElsevierB.V.

ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/)

∗Correspondingauthor.

E-mail:[email protected](A.Bozal-Leorri).

https://doi.org/10.1016/j.jes.2022.04.035

1001-0742/© 2022TheResearchCenterforEco-EnvironmentalSciences,ChineseAcademyofSciences.PublishedbyElsevierB.V.Thisis anopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)

Introduction

Since the beginning of the green revolution, the applica- tion ofnitrogen (N)fertilisers to agriculturalcrops has in- creasedthelevelofreactivenitrogenpresentinthebiosphere (Subbaraoetal.,2017).Ammonium(NH4+)canbepresentin the soil afterbeing appliedwithfertilisers, but alsobe re- leasedfromorganicmatterthankstomicrobialactivityina processcalled“mineralisation” (Coskunetal.,2017).Inaerobic soils,NH4+isoxidisedbychemolithoautotrophicammonia- oxidisingbacteria(AOB)andarchaea(AOA)inwhatisknown asnitrification.Nonetheless,nitrificationismainlydrivenby AOB rather than AOA in soilsreceiving nitrogen-fertilisers (Dietal.,2009,2010).First,nitrifiersoxidiseNH4+tohydrox- ylamine(NH2OH)throughtheenzymeammoniummonooxy- genase(AMO)whichisencodedbytheamoAgene(Arpand Stein,2003).NH2OHisthenconvertedtonitrite(NO2−)and finally nitrite-oxidising bacteria (NOB) oxidise it to nitrate (NO3−)(Könnekeet al.,2005).Asananion,NO3− issuscep- tibletobelostthroughleachingbecauseitsnegativecharge isrepelledbynegativelychargedsoilcolloids(Fienckeetal., 2005).Inanoxicconditions,NO3−isthesubstrateforthedeni- trificationprocess.Duringdenitrification,NO3−issequentially reduced toNO2−,nitric oxide(NO)and nitrousoxide(N2O) byenzymes encodedbythegenesnarG,nirK,nirSand norB (HochsteinandTomlinson,1988).Inthisprocess,theemission ofN2OisaharmfulthreatfortheenvironmentsinceN2Oisa gaswithaglobalwarmingpotential(GWP)thatis265time greaterthanthatofCO2ina100-yearperiod(IPCC,2014).Fi- nally,bacteriaharbouringnosZIornosZIIgenescancarryout acompletereductionofN2OtoN2.Nonetheless,40%ofdeni- trifierslackofthegenestoperformthislaststep(Hallinetal., 2018).

Becauseofthesenitrogenleaksandtransformations,agri- culture presents a low nitrogen-use-efficiency (NUE), since crops only assimilate anaverage of 30%–50% of the nitro- gen applied with fertilisers (Wendeborn, 2020). This leads field-crop agriculture, suchas wheat,to beresponsible for morethan61%oftotalglobalanthropogenicN2Oemissions (Montzkaetal.,2011).Therefore,wemustguideagricultural systemstowardssustainabilityinordertomaintainadequate productionlevelswhilereducingtheamountofreactivenitro- genlosttotheenvironment.Someoftheoptionstoachieve thisgoal andreducenitrogen-oxideemissionsaretheopti- misation ofnitrogensupply andsynchronisationwithcrop demandor the application ofstabilised nitrogenfertilisers with synthetic nitrification inhibitors (SNIs) (Thapa et al., 2016).SNIsinhibitthe AMOenzymedelayingthe oxidation of NH4+ to NO2−, giving plants more time to absorb the NH4+(Keeney,1983;RuserandSchulz,2015).Severalchemi- calcompoundswithnitrificationinhibitionactivityhavebeen developed(Subbaraoet al.,2006).The3,4-dimethylpyrazole phosphate (DMPP)isoneofthe mostworldwideusedSNIs (Gilsanz et al., 2016).In a 10 times lower application rate, DMPPpresentssimilarefficiencytoanotherworldwideused SNI, dicyandiamide(DCD) (Ruser and Schulz, 2015). In mi- crocosm experiments,DMPP ability todecrease N2O emis- sionsincreaseupto90%(Bozal-Leorrietal.,2021;Corrochano- Monsalveetal.,2021a).Infieldconditions,reductionsbetween

35% and50% inN2Oemissions are reportedwitha higher maintenanceofsoilNH4+contentforlongerperiod,without anydeleteriouseffectsontheyieldsofdifferentcropssuchas wheat(Huérfanoetal.,2015;Duncanetal.,2017),pastureand corn(Huérfanoetal.,2018;Nairetal.,2020).

Nevertheless, recent studies have shown an impact of dimethylpyrazole-based inhibitorson non-targetorganisms (Corrochano-Monsalveetal.,2020,2021a).Therefore,thepo- tential risksfor soilhealth ofusing SNIsinthe long term should be considered. As an alternative,the use of cover crops, in a crop rotation, with the ability to modify the soilnitrogen cycle through the release ofroot exudates is alsoconsideredagoodstrategytoreducenitrogenpollution (Subbaraoetal.,2013).Thisallelopathy,which isknownas biologicalnitrification inhibition(BNI),ishighlighted inthe frameworkofsustainableagriculturebasedontheuseofen- vironmentallyfriendlyagronomicpractices(Subbaraoetal., 2013;Zhangetal.,2015).Someofthesebiologicalnitrification inhibitors(BNIs)havethepotentialtoinhibitnotjustAOBbut alsoAOA(Byrnesetal.,2017),andthusimprovethenitrogen retentioninsoils,influencingpositivelycropNUE andmiti- gateGHGemissions.Amongcrops,sorghum(Sorghumbicolor L.)hasthe greatestBNI-releasing capacity(Subbaraoet al., 2017),whichmakesitadvisabletouseitincroprotationsasa covercrop.Thus,becauseofawakenednitrification,Npol- lutioninthefollowingcropshouldbereduced.Sorghumis droughttolerant(Hadebeetal.,2017),soitcandevelopdespite thedrysummerclimateofareaswithhumidMediterranean conditions.Moreover,ithasashortgrowthcycle,whichpro- vides winter crops enough time to settle. In addition, the use ofcover cropsalso bringmultiple environmentalben- efitssuchas animproved soilstructureand fertility,weed controlandareductioninnutrientleachingandsoilerosion (Muhammadetal.,2019; Garlandet al.,2021).Furthermore, the use ofa cover cropis avery efficient toolinreducing theamountofleachableNO3−insoil(Constantinetal.,2010; Plaza-Bonillaetal.,2015).

Thepresentworkaimedtoevaluatetheeffectoftwodif- ferentno-tillcroprotations:(1)asorghum-wheatrotation,in whichthepossiblebenefitsofacovercropwithBNIpotential willbeanalysedintermsofsoilmineralnitrogencontent,N2O emissionsandgrainyieldofwinterwheat;incomparisonto (2)aconventionalfallow-wheatrotation.Inthisstudy,theap- plicationofasyntheticnitrificationinhibitor(DMPP)willbe alsoconsideredas(1)acontrolofnitrificationinhibitionto comparewiththepotentialBNIactivityofsorghum,and(2)a complementforincreasingthesustainabilityofthesewheat rotationsintermsofN2Oemissions.

1. Materials and methods

This work was conducted in Pamplona, northern Spain (42°47’N,1°37’W,450mabovesealevel)duringthe2019/2020 growing season.Thesoilcharacteristics ofthe upperhori- zonbeforethestartoftheexperimentaregiveninTable1, whiledailyprecipitationandmeantemperaturesareshown

Table1– Physicalandchemicalpropertiesofthesoilcol- lectedin0– 30cmdepthlayerinPamplona beforethe startoftheexperiment(42°47^N,1°37^W,450mabovesea level,Navarre,Spain).

Soiltexture Soilchemicalproperties

Sand 38.6% pH^a 8.3

Silt 31.8 C:Nratio 8.9

Clay 29.6 N^b(g/kg) 1.4

Organic matter^c(g/kg)

21.5

CaCO3d(g/kg) 20.3

Mg^d(mg/kg) 53.5

K^d(mg/kg) 270.0

Ca^d(mg/kg) 2735.7

P^e(mg/kg) 11.5

a 1:2.5(m/V)soil:water

b Kjeldahldigestion(Keeney,1983);

cWalkleyandBlack,1934

dNH4AcO,MAPA,1994;

e WatanabeandOlsen,1965.

in Appendix A Fig. S1. A bifactorial experimental design (crop rotation and type of wheat fertilisation) was imple- mented.Thecroprotations were:(1)fallow–wheat rotation (fallow–wheat);and(2)sorghumcovercropwithoutnitrogen fertilisation–wheatrotation(sorghum–wheat).Thesoiltreat- mentswerearrangedintwoblocks.Inthefirstblock,adventi- tiousplantsweredesiccatedonMay20,2019usingRoundUp (aglyphosate-basedherbicide)(36%W/V,Fertiberia,Spain)at arateof1.5L/ha,dosethatisroutinelyappliedinno-tillsys- temsfromthisregion.Inthesecondone,sorghum(Sorghum bicolorL.var.PR88P68PioneerCortevaAgriscience^R,USA)was sownunderno-tillconditionsatarateof15kg/haonMay20, 2019.ThesorghumcovercropwascrushedonOctober14,2019 andleftonthesoilsurface.Onemonthbeforesorghumtermi- nation,soilNH4+-Ncontentfromfallowandsorghumplots were 2.0and1.7kgN/ha,respectively,whilethesoilNO3−- Ncontentwas43.7and7.0kgN/haforfallowandsorghum plots.Winterwheat(TriticumaestivumL.cv.MarcopoloRAGT^R, Spain)wassownunderno-tillconditionsandoversorghum stoveratarateof220kg/haonOctober31,2019.Withineach block ofcrop rotation (fallow–wheat and sorghum–wheat), threewheatfertilisertreatmentswereappliedinfourrandom plotsreplicationsofindividualsizeof25m²(5m× 5m):(1) controlwithoutfertilisation(Control);(2)fertilisedwitham- monium sulphate21%-Nitrogen (AS);and (3)fertilisedwith ammonium 21%-Nitrogen sulphate combined with DMPP (AS+DMPP).Thefertilisationratewas90kgN/haappliedin asingledoseonFebruary28,2020atthebeginningofstem elongation(Z30)accordingtotheZadoksscale(Zadoksetal., 1974).TheAStreatmentusedammoniumsulphate(99%,Agro IberiaS.L.,Spain)asfertiliser,andAS+DMPPtreatmentused ENTEC^R (AgroIberiaS.L.,Spain),whichcontainsammonium sulphate(99%),andDMPP(99%)atarateof0.8%oftheNH4+- NpresentintheENTEC^R.ThewheatwasharvestedonJuly 24,2020.

1.2. Wheatsoilgeochemicalanalysisandwatercontent

SoilNH4+andNO3−contentswerefirstdeterminedtheday beforeapplyingthetreatments.Sampleswerethentaken10, 30and60dayspost-fertilisation.Threesoilsubsamples(3cm diameter × 0.3 m deep) were taken from each plot, rocks andstoneswereremovedandfinallytheywerehomogenised.

Next,100gfreshweightofthehomogenisedsubsampleswere mixedwith200 mLof1mol/L KCl(99%,PanReac Química, Spain)andshakenfor1hrat165r/min.Thesoilsolutionwas filteredthroughWhatmanNo.1filterpaper(GEHealthcare, Spain)toremoveparticlesandthenthroughSep-PakClassic C18125A˚ cartridges(Waters,USA)toeliminateorganicmat- ter.TheNH4+contentofthefilteredsolutionwasdetermined usingtheBerthelotmethod(PattonandCrouch,1977)andthe NO3−contentasdescribedbyCawse(1967).

Thesoilwatercontentwasalsomeasuredeachtimethe soiland/orGHGweresampled.Twosubsamples(3cmdiam- eter× 0.3mdeep)weretakenrandomlyfromthefield.Rocks were removed and the soilsubsampleswere driedat 80°C inacirculationovenfor72hruntiltheyreachedaconstant dryweight.Watercontentwasexpressedasthepercentageof water-filledporespace(WFPS,%)asperLinnandDoran(1984), Eq.(1):

WFPS=(C× Db)× (1− (D_b/Dp))⁻¹ (1) where,C(g)isthesoilgravimetricwatercontent,D_b(Mg/m³) isthebulkdensity;Dp(Mg/m³)istheparticledensity.

Dpwastakenas2.65Mg/m³.D_bwasmeasuredatthebe- ginningoftheexperimentandwasfoundtobe1.0Mg/m³. 1.3. MeasurementofN2Oemissionsfromwheatsoils

N2O soilemissions weremeasured usingthe closedcham- bermethod(Chadwicket al.,2014).Sampleswerecollected 3 times/week for 2 weeks after wheat fertilisation, then 2 times/week for the next 2 weeks and 1 times/week up to day 60. Considering the diurnal variation of emissions (Baggsand Blum, 2004), sampling was performed between 10:00a.m.and1:00p.m.Toaccountforsoilheterogeneity,four chambers(20cmdiameter× 16cmhighonceinsertedinthe soil)wereplacedineachplotandtwoweresampledonalter- natedays.Linearitywascheckedandgassamplesweretaken justafterclosingthechambersandthen45minlater.20mLof gaswastakenfromeachchamberandstoredatoverpressure inpre-evacuated 12mLglassvials.Sampleswere analysed inagaschromatograph(GC)(7890A,Agilent,USA)equipped withanelectroncapturedetectorforN2Odetection.Acapil- larycolumn(IAKRCIAES6017:240°C,30m× 320μm)wasused andthesampleswereinjectedusingaheadspaceautosam- pler(HT3,TeledyneTekmar,USA).N2Ostandardswereanal- ysedatthesametimeasthesamples.Gasemissionrateswere calculatedbyconsideringthevariationingasconcentration fromthebeginningtotheendofthe45min(Menéndezetal., 2008).CumulativeN2Oemissionsduringthesamplingperiod wereestimatedbyaveragingtherateoflossbetweentwosuc- cessivedeterminations,multiplyingthataverageratebythe lengthoftheperiodbetweenthemeasurements,andadding thatamounttothepreviouscumulativetotal(Menéndezetal., 2008).Soiltemperature(atadepthof10cm)wasmeasured

beforetakingthegassamples.Theairtemperaturewasmea- sured3timesduringthe45mingassamplingperiodtogetthe average.

1.4. AbundanceofN-cyclerelatedmicroorganismsin wheatsoils

Quantitative polymerasechainreaction(qPCR)wasusedto quantifytheabundanceofnitrifyinganddenitrifyinggenes.

SoilDNAwasisolatedfrom0to30cmsoilsamples(threesub- samplesperplot)collectedat10dayspost-fertilisation.DNA was extractedfrom0.25 gofdrysoilusingthe PowerSoil^R DNAIsolationKit(Qiagen,Germany) withthemodifications describedinHarteretal.(2014).Theconcentrationandqual- ityofDNAextractsweredeterminedbymeansofspectropho- tometry(NanoDrop^R 1000,ThermoScientific,USA).

The 16S rRNA gene (for quantification oftotal bacterial abundance)and functionalmarkergenesinvolvedinbacte- rialnitrification(amoA)anddenitrification(nirK,nirS,nosZIand nosZII)were amplifiedbymeans ofqPCR usingSYBR^R Pre- mix Ex TaqTM II (Takara-Bio Inc., Japan)and gene-specific primers (AppendixA TableS1) inthe StepOnePlus^TM Real- TimePCRSystem.EachqPCRreactionwasperformedintrip- licate foreach sample. Data analysiswas carried out with StepOnePlus^TM Software2.3(ThermoScientific,USA).Stan- dardcurveswerepreparedfromserialdilutionsoflinearised plasmidswithinsertionsofthetargetgenerangingfrom10⁷to 10³genecopies/μL.Thenumberofcopiesoftargetgene/gram ofdrysoil(N)wascalculatedaccordingtoamodifiedequation (Eq.(2))detailedinBehrensetal.(2008):

N=(Nper reaction× VDNAextracted/VDNAused× W)/[DNAextracted] (2) where, N_perreaction is the number of target gene copies/reaction; V_DNAextracted is the volume of DNA ex- tracted; V_DNAused is the volumeofDNA used per reaction;

W(g)istheweightofdrysoilextracted.[DNAextracted]isthe extractedDNAconcentration.

1.5. Wheatcropyieldparameters

Grain yields were calculated based on a harvested area of 7.5m²(1.5m× 5m)perplotandadjustedforamoisturecon- tentof12%.Anareaof0.36m²perplotwasmeasuredtocal- culatethenumberofspikes/m²anddryweightof1000grains.

Thetotal grain nitrogencontent wasanalysed byapplying theKjeldhalprocedure(AOAC,1980)withaKjeltecAutosam- plerSystem1035(Tecator,Spain)aftergrindingthegrainand passingitthrougha1mmscreen.Grainproteincontentwas takenas5.7timesthetotalnitrogencontent(Teller,1932).The yield-scaledN2Oemissions(YSNE)wereexpressedasthera- tiobetweentheamountofNemittedasN2Oandtheabove- groundnitrogenuptake(vanGroenigenetal.,2010).Thenitro- genuseefficiency(NUE)(kgdrymatter/kgN)wasdetermined as,Eq.(3):

NUE=(WNx− WN0)/Wnitrogen (3)

where,W_Nx(kg)isthedrymatterobtainedwhen90kgN/ha were added; WN0 (kg) is the dry matter obtained with no fertiliser application.W_nitrogen (kg) isthe weight ofapplied nitrogen.

1.6. Statisticalanalysis

Theresultsfromsoilmineralnitrogencontentdeterminations weresubject toatwo-way (croprotation,“R”;andfertiliser treatment,“F”)analysisofvariancestatisticalanalysis.The resultsofN2Omeasurements, microbialquantificationand grainyieldparameterswereanalysedbyone-wayANOVAus- ingDuncan’smultiplerangetestforseparationofmeansbe- tweentreatmentsandtheMann–WhitneyUtestwasusedto comparethetwotreatmentswiththeSPSSstatisticalsoftware package(2016,IBMSPSSStatisticsforWindows,Version24.0.

Armonk,NY,IBMCorp,USA).p-Values<0.05wereconsidered tobestatisticallysignificantdifferences.

2. Results and discussion

2.1. Nitrifyingmicroorganismsarereducedin sorghum-wheatcroprotation

Fertilisationhadastrongeffectonsoilmineralnitrogencon- tent(p<0.01).Soilfrombothcroprotationsstartedwitha similarsoilNH4+contentbeforefertiliserapplicationtothe wheatcrop(Fig.1a).After theaddition ofthenitrogen fer- tiliser,soilNH4+ contentincreasedtothesamelevelinthe ASandAS+DMPPtreatmentsforbothcroprotations,butthere wasasignificantdecreaseintheAStreatmentat30dayspost- fertilisation(DPF).However,atthesametime,theuseofDMPP meantthesoilretainedtwicetheamountofNH4+compared totheAStreatment.AlthoughtheNH4+ contentfortheAS andAS+DMPPtreatmentsdroppedtotheleveloftheControl treatmentat60DPF,DMPPwasabletoprolongtheavailabil- ityofNH4+atleastuntil30DPF.Theseresultsarecomparable tootherstudiesthatappliedDMPPasaSNI(Huérfanoetal., 2015, 2016; Liu et al.,2020).On the contrary,AS+DMPP re- ducedNO3− contentto40%at10DPFandto30%at30DPF comparedto the AStreatment (Fig.1b). TheControltreat- mentalsomaintainedlowsoilNO3− valuesthroughoutthe experiment.AsNH4+contentremainedhighandNO3−con- tentwaslowduetothedelayinNH4+oxidation(Ruserand Schulz,2015),AS+DMPPpresented the highestNH4+/NO3−

ratio ofall the fertilised treatments (Fig.1c).AS treatment showedahigherNH4+/NO3−ratiothanControltreatmentbut itwasonlyabletomaintain53%and22%ofAS+DMPPratio at10and30DPF,respectively.Nevertheless,althoughtheuse ofDMPPtreatmentwasabletomaintainmoreNH4+inthe soil,it canpresent someundesiredeffects.Ahigherreten- tionofsoilNH4+contentcould leadtoanincreaseonNH3

volatilization.EventhoughwedidnotmeasureNH3volatiliza- tioninourexperiments,meta-analysisestimatethattheuse ofSNIscanincreaseNH3volatilizationaround20%(Qiaoetal., 2015).Therefore,despitethefactthatSNIsapplicationallevi- atesglobalwarmingpotentialderivedfromdirectN2Oemis- sions,theirpotentialnegativesideeffectsshouldbefullycon- sidered.Ontheotherhand,wedidnotfindanydifferences betweenthe twocroprotations inthemaintenanceofsoil mineralnitrogen,intermsofsoilNH4+ andsoilNO3− con- tent,duringwheatdevelopment(Fig.1aandb).Thismayin- dicatethat,eventhoughcovercropscarrybenefitsforthefol- lowingculturesuchasimprovedsoilphysicochemicalprop-

0 10 20 30 40

Soil NO

-N (kg N/ha)

0 3 6 9 12 15

0 20 40 60

NH

-N/NO

-N ratio

Days post-fertilization 0

40 80 120

Soil NH

-N (k g N/ha)

a

b

c

10 30 60

R * NS NS

F ** ** **

R × ^{F * NS NS}

10 30 60

R * NS NS

F ** ** NS

R × ^{F NS NS NS}

10 30 60

R NS NS NS

F ** ** NS

R × ^{F NS NS NS}

Fig.1– Wheatcropsoilmineralnitrogen(0– 30cm)evolutionduring60dayspost-fertilisationinformofNH4+(a),NO3−(b) andtheratioofNH4+-N/NO3−-N(c).Control:controlwithoutfertilization;AS:fertilisedwithammoniumsulphate;

AS+DMPP:fertilisedwithammoniumsulphate+3,4-dimethylpyrazolephosphate.Statisticalanalysiswasmadethrough analysisofvariance(two-wayANOVA)showingtheeffectofcroprotation(R),fertilizertreatment(F)andtheirinteraction (R× F).Significantdifferencesaremarkedwithanasterisk(^∗)whenp<0.05anddoubleasterisk(^∗∗)whenp<0.01.

0.0E+00 4.0E+06 8.0E+06 1.2E+07 1.6E+07

C AS AS+DMPP C AS AS+DMPP

Fallow Sorghum

e c n a d n u b a B O A (am oA g/ s ei p o c )li os y r d

0.0E+00 5.0E+08 1.0E+09 1.5E+09 2.0E+09 2.5E+09

l ai r et c a B e c n a d n u b a (16S rRN A g/ s ei p o c) li os y r d

Fallow-wheat Sorghum-wheat

Fig.2– Abundanceoftotalbacteria(a)andammonia oxidizingbacteria(AOB)(b)at10dayspost-fertilisation (DPF)onsoilofwheatcrop.Significantdifferencesbetween treatmentsof“Fallow-wheat” rotationaremarkedwith lowercaseletters.Significantdifferencesbetween

treatmentsof“Sorghum-wheat” rotationaremarkedwith capitalletters.ForbothANOVA,theDuncanTestwasused (p<0.05;n=4).TheMann-WhitneyUtestwasusedfor thecomparisonbetweencroprotationswithinthesame fertilizationtreatment.Significantdifferencesatp<0.05 aremarkedwithanasterisk(^∗).

erties(suchaswaterholdingcapacity,aggregatestabilityand Cstock)(Buyeretal.,2010;Lal,2015;PoeplauandDon,2015) orreducednitrogenlosses(KayeandQuemada,2017),inthis case,theuseofsorghumasasummercoverplantationdidnot affecttheevolutionofsoilmineralnitrogenduringthefollow- ingcrop.Nonetheless,theanalysisofsoilmineralnitrogenin thesubsequentculturemaynotbesufficientlysensitivetode- tecttheeffectsofusingsorghumasacovercrop.

Fertilisertreatmentdidnotaffectthetotalbacterialabun- dance(measuredas16SrRNAgeneabundance)(Fig.2a).How- ever,theAStreatmentgreatlyenhancednitrification(interms

ofbacterialamoAgeneabundance),especiallyinfallow–wheat rotation(Fig.2b).Eventhoughadventitiousplantsweredesic- catedwithglyphosate-basedherbicidetocreateafallowplot, the greatAOBgrowth in AStreatment infallow–wheat ro- tationindicatedthelackofdeleteriouseffectsofglyphosate onnitrifyingmicroorganisms.Our resultsagreewithprevi- ousstudies wherewas demonstrated that higher doses of glyphosateorrepeatedexposuredidnotaffectnitrifyingpop- ulations(Allegrinietal.,2017;Zabaloyetal.,2017).Theappli- cationoftheDMPPwasveryeffectiveatreducingAOBabun- danceinsoilsfrombothcroprotations,evendowntothelev- elsoftheunfertilisedControl,withreductionsof56%and40%

comparedtoASinfallow–wheat andsorghum–wheatrota- tions,respectively.Notwithstandingthatsomestudiesinmi- crocosmshavereachedanAOBinhibitionofover85%with theuseofDMP-basedinhibitors(Torralboetal.,2017;Bozal- Leorri et al., 2021; Corrochano-Monsalve et al.,2021b), our resultsare similartothose obtainedinfield studieswhere the AOB inhibition was efficient but at lower percentages (Kleineidamet al.,2011; Duncanet al.,2017). Althoughthe differentcroprotationsdidnotaffectthesoilnitrogencon- tent,itdidinfluencesoilmicrobialpopulations.Weobserved asignificantincreaseinthetotalbacterialabundanceinsoil ofsorghum–wheat rotation,as it was 24% and 34% higher comparedtofallow–wheatfortheControlandAStreatments, respectively(Fig.2a).Furthermore,thetypeofcroprotation alsoaffectedAOBabundance,asthelevelsfortheControland AStreatmentsforthesorghum–wheatrotationwere35and 22%lowerthanthefallow–wheatplots(Fig.2b).Thisreduc- tionindicates that,duringits development,sorghummight haveexudedBNIsthatcankeepnitrifiersinhibiteduntilthe nextcrop.Dayanetal.(2010)indicatedthattheinhibitoryef- fectofsorghumcouldpersistforatleast60daysafterthehar- vestwasremoved.Inourcase,theBNIsmayhadamoreen- duringeffect(140days)becausethesorghumwasnotelimi- natedfromthecultivationsoilandtheexperimentwascar- riedoutunderno-tillageconditions,whichretardsBNIdegra- dation(Rothetal.,2000).Thus,leavingthesorghumstoverin thesoilunderno-tillageconditionsensuresslowerrootdegra- dationandtheconsequentreleaseofexudateswithaBNIca- pacitybecausesuchcompoundsareproducedexclusivelyin theroots(Baersonetal.,2008).

2.2. N2Oemissionsareaffectedbythetypeofrotation

Daily N2O emissions ranged from 0.89 to 13.74 g N2O- N/(ha•day)infallow–wheatrotationandfrom0.38to24.58g N2O-N/(ha•day)insorghum–wheatrotation(Fig.3a).Thecu- mulative N2O emissions from the AS treatments were the highestofallthefertilisertreatmentswith382.9and678.3g N2O-N/hainsoilfromthefallow–wheatandsorghum–wheat rotations,respectively(Fig.3b).Asiswellsupportedelsewhere (RuserandSchulz,2015),DMPPreducedcumulativeN2Oemis- sionsto valuesakinto the Controltreatment, correspond- ingtoreductionsof79%and86%comparedtotheAStreat- mentforthefallow–wheatandsorghum–wheatrotations,re- spectively.SinceAOBpopulationswerereducedinthewheat crop(seeSection2.1),probablybecauseoftheBNIsreleased from sorghum,we also expected a decrease in N2O emis- sions(eitherduetoareductioninN2Oemittedbynitrifiers

Fig.3– Daily(a)andcumulative(b)N2Oemissionduring56dayspost-fertilisationonsoilofwheatcrop.Significant differencesbetweentreatmentsof“Fallow-wheat” rotationaremarkedwithlowercaseletters.Significantdifferences betweentreatmentsof“Sorghum-wheat” rotationaremarkedwithcapitalletters.ForbothANOVA,theDuncanTestwas used(p<0.05;n=4).TheMann-WhitneyUtestwasusedforthecomparisonbetweencroprotationswithinthesame fertilizationtreatment.Significantdifferencesatp<0.05aremarkedwithanasterisk(^∗).

or a decrease indenitrifying activity becauseofa delay in the transformationofNH4+into NO3−).However,asshown inFig.3a,N2OemissionsfromtheAStreatmentofsorghum–

wheatrotationwerehigherthanthoseforthefallow–wheat rotationthroughouttheentireexperiment,resultingina77%

increaseincumulativeN2Oemissions(Fig.3b).Itshouldbe remarkedthatsorghumstoverisanextracarbonsourcein the soil, and the main mechanism connecting carbon cy- cling withnitrogengasemissionsisthe carbonavailability inthesoilthatenhancesheterotrophicdenitrification,which isoneofthemainprocessesresponsibleforN2Oproduction (Davidsonetal.,2000).Furthermore,N2Oemissionsarerelated tosoilwatercontent(Davidson,1991),withathresholdof60%

WFPSbetweenwater-limitedandaeration-limitedmicrobial processes. Inthepresent work,duringN2Omeasurements, thesoilWFPSremainedbetween45%and60%(AppendixA Fig.S2),arangeinwhichdenitrifyingmicroorganismsbecome morerelevantforN2Orelease.Therefore,wearguethatthein- crementinN2Oemissionsfromsoilsinthesorghum–wheat rotationisduetoanenhancedheterotrophicdenitrification because ofa greater carbon availability, as larger portions oflabilecarbonsubstratespromotedenitrificationreactions (Sureyetal.,2020).Thisgreater abundanceofheterotrophic denitrifiersinsorghum–wheatrotationswasevidencedbythe increasedabundanceofnitritereductase(NIR)enzymecon- tainingdenitrifyingbacteria(Fig.4aandb).TheControlandAS treatmentsfromthesorghum–wheatrotationshoweda56%

and73%increaseinnirKabundancecomparedtotheequiva- lenttreatmentsonthefallow–wheatrotation(Fig.4a).Onthe otherhand,theabundanceofnirKwasnotaffectedbyanyof thetreatments(withorwithoutnitrogen)onthefallow–wheat rotation,buttheAS+DMPPtreatmentonthesorghum–wheat rotation had alower nirK abundancethan the Controland AStreatments.ComparingcroprotationsinthecaseofnirS

abundance,anincreaseof30%wasonlysignificantfortheAS treatment(Fig.4b).SimilarlytonirK,nirSabundancewasnot affectedbyfertilisertreatmentinthefallow–wheatrotation, buttheAS+DMPPtreatmentpresentedalowerlevelthanthe ControlandAStreatmentsforthesorghum–wheatrotation.

Inthiscase,regardingN2O-reducingbacteria,neitherthecrop rotationsnortheNtreatmentsaffectedbothnosZIandnosZII genesabundances(Fig.4candd).

In contrast tothe soilmineral nitrogen,the changes in the abundances of N-related microorganisms were sensi- tive enough to detect the effects of using a cover crop.

Insoilwithfallow–wheat rotation, AStreatmentpresented the highest amoA/nirK and amoA/nirK+nirS ratios of all three treatments (Table 2). This means that those soils weremorebalancedtowardsnitrificationsincetheaddition of N-fertilisation increases the level of nitrification genes (Ouyang et al., 2018). In the same manner, there was a higherratio betweenN2O productionindenitrificationand N2Oreduction(nirK+nirS)/(nosZI+nosZII).Ontheotherhand, the addition of DMPP decreased amoA abundance, which iswhy this treatment presentedthe lowest amoA/nirK and amoA/nirK+nirSratios.Fertilisertreatmentdidnotaffectthe nitrifying/denitrifyingratiosinthesorghum–wheatrotation.

Nevertheless,thetypeofcroprotationinfluencedthesera- tios,asthereweresignificantdifferencesbetweenthem.As hasbeenmentionedbefore,thehigheravailabilityofsoilcar- bonduetosorghumcovercropresiduesmightberesponsi- ble foran increaseindenitrification reactions(Palmer and Horn,2015;Sureyetal.,2020).Then,theallegedincreaseof denitrifyingmicroorganismscontainingNIRenzymebecause of sorghum stover (Fig.4a and b),balanced the amoA/nirK andamoA/nirK+nirSratios towardsdenitrification.Thispro- duced a 60% reduction in the amoA/nirK ratio and 55% in the amoA/nirK+nirS ratio for the Control treatment on the

Fig.4– AbundanceofdenitrifyingbacteriameasuredastheabundanceofnirK(a),nirS(b),nosZI(c)andnosZII(d)genesat10 dayspost-fertilisation(DPF)onsoilofwheatcrop.Significantdifferencesbetweentreatmentsof“Fallow-wheat” rotationare markedwithlowercaseletters.Significantdifferencesbetweentreatmentsof“Sorghum-wheat” rotationaremarkedwith capitalletters.ForbothANOVA,theDuncanTestwasused(p<0.05;n=4).TheMann-WhitneyUtestwasusedforthe comparisonbetweencroprotationswithinthesamefertilizationtreatment.Significantdifferencesatp<0.05aremarked withanasterisk(^∗).

Table2– TheamoA/nirK,amoA/(nirK+nirS)and(nirK+nirS)/(nosZI+nosZII)ratioonsoilofwheatcrop.

amoA/nirK amoA/(nirK+nirS) (nirK+nirS)/(nosZI+nosZII)

Fallow- wheat

Control 2.76± 0.44 ab 1.77± 0.24 ab 1.49± 0.25 b

AS 3.37± 0.45 a 2.79± 0.61 a 2.27± 0.15 a

AS+DMPP 1.86± 0.29 b 1.25± 0.19 b 2.36± 0.18 a

Sorghum- wheat

Control 1.11± 0.14 A^∗ 0.78± 0.14 A^∗ 2.45± 0.14 B^∗

AS 1.64± 0.09 A^∗ 1.26± 0.05 A^∗ 3.00± 0.24 A^∗

AS+DMPP 1.82± 0.32 A 1.29± 0.21 A 2.32± 0.13 B

Significantdifferencesbetweentreatmentsof“Fallow-wheat” rotationaremarkedwithlowercaseletters.Significantdifferencesbetween treatmentsof“Sorghum-wheat” rotationaremarkedwithcapitalletters.ForbothANOVA,theDuncanTestwasused(p<0.05;n=4).The Mann-WhitneyUtestwasusedforthecomparisonbetweencroprotationswithinthesamefertilizationtreatment.Significantdifferencesat p<0.05aremarkedwithanasterisk(^∗).

sorghum–wheatrotationcomparedtofallow–wheat.Further- more,thereductionofthe AOBpopulationinthe AStreat- ment (Fig. 2b) due to the potential release of BNIs from sorghumrootsalsocontributedtothedecreaseinamoA/nirK andamoA/nirK+nirSratioswitha51%and54%reduction,re- spectively,comparedtothefallow–wheatrotation.Moreover, althoughtheabundancesofnirKandnirSmightincreasedue totheextracarbon,noeffectscouldbeobservedontheabun- danceofnosZIandnosZIIgenes.Wetheorizethat,somehow, theuseofsorghumasacovercropfavouredascenarioofin- completedenitrification,whichincreasedtheemissionofN2O duetothelackofincreaseofthemicroorganismsthatcould reduceitcompletelytoN2.

Consequently,focusingonthecapacitytomodifytheni- trifying/denitrifyingratioofthedifferentcroprotations,we might developa betterunderstanding ofhow soilNemis- sionsrespond,suchasthe differentN2O emissionsforthe AStreatment.However,sincetheAS+DMPPtreatmentsignif- icantlyinhibitedAOBgrowth(Fig.2b),soilNO3−formationdi- minishedcomparedtothe AStreatmentand therewasno increaseinnirKandnirSgenes(Fig.4aandb),ultimatelyre- sultinginsimilarnitrifying/denitrifyingratiosbetweencrop rotations.Inaddition,AS+DMPPtreatmentshowedthelower (nirK+nirS)/(nosZI+nosZII)ratio, resultingin abetterbalance betweenN2Oproduction/N2Oreduction.Wethereforesuggest the useofsyntheticNIssuchasDMPPtoreducethepollu- tionderivedfromtheuseofsorghumasacovercrop.More- over,Menéndezetal.(2012)reportedthatthereductioninN2O emissionsinducedbyDMPPisconditionedbythemagnitude ofthelossesfromthefertiliserwithoutNIs.Thus,DMPPcan counteracthigher N2Oemissionswithgreater efficiency,as canbeobservedinourexperiment,witha79%reductionof N2OemissionswithrespecttoASinthefallow–wheatrota- tionversus86%inthesorghum–wheatrotation(Fig.3b).

2.3. Yieldparametersarenotaffectedbythetypeof rotation

Planting winterwheat after asorghum cropis a common practice, yet it can affect the settlement of wheat seed.

Guenzi et al.(1967)demonstrated that water extractsfrom sorghum residues could inhibit corn and wheat seed ger- mination. Thiseffectis duetothe allelopathicsubstances, suchassorgoleone,thatsorghumreleasesthroughitsroots.

Rothetal.(2000)suggestedthatsoilmanagementisthekeyto counteractingtheseeffects.Insoilswithconventionaltillage management,sorghumexudatesarequicklysolubilisedand degraded.Inno-tillagesoils,bycontrast,sorghumremainsre- leasingtheirdegradationcompoundsgraduallyandtherefore affectcropyield.Evenso,theeffectsofsorghumresidueson wheatseedgerminationcanbemitigatedbyincreasingthe seedingrateordelayingtheplantingofsubsequentcropsun- tiltheresidueshavedecomposedorweathered(Westonetal., 2013). In our experiment, the wheat sowing density was 220kg/ha,whichseemshighenoughtopalliatetheeffectsof theprevioussorghumcropsincethetypeofcroprotationdid notaffectthe wheatgrainyieldoffertilised treatments.As expected,wheatgrainyieldwasmuchhigherfortheASand AS+DMPPtreatmentswith6616and6133kg/ha,respectively, for thefallow–wheat rotation and 6230and 6543kg/ha for

thesorghum–wheatrotation(AppendixATableS2),surpass- ingtheaverageforthatregionin2019,whichwas5000kg/ha (MAGRAMA,2019). Similarly,the use ofSNIsdidnot affect grainyield,whichwasinlinewithotherworkswhereDMPP maintainedthegrainyieldofrainfedwintercerealscompared tofertilisertreatmentswithout inhibitor(Arregui and Que- mada,2008;Huérfanoetal.,2016).Furthermore,thenumber ofspikes/m²wasalsohigherintheASandAS+DMPPtreat- mentsinbothcroprotationsthanintheControltreatments.

However,eventhough croprotations didnotinfluence the grainyieldoffertilisedtreatments,thiswasnotthecasefor theControltreatment,whichpresentedadecreaseinwheat grainyieldinthesorghum–wheatrotation(AppendixATable S2).Thisdecreasemaybeduetothecompetitionfornutrients betweenplantsandsoilmicrobesinsoilswithalownitrogen content.Itisassumedthatheterotrophicsoilmicroorganisms arestrongercompetitorsforinorganicNthanplants(Kayeand Hart,1997).Moreover,thegrowthofthesemicroorganismsis carbon-limited.Thus,whensoilmineralnitrogenincreasedin theControltreatments,suchasbetween0and10DPFwhen mineralisationcanbeobserved(Fig.1aandb),theadditional Cfromsorghumstoverledtoanincreaseinthetotalbacterial abundancecomparedtothe fallow–wheatrotation(Fig.2a), andthereforetoagreatercompetitionagainsttheplantsfor soilnitrogenuptake.In addition,atthesestagesforwheat plantsgrowninasorghum–wheatrotation,thereductionin Nuptakewasevidentasthenumberofspikes/m²intheCon- troltreatmentofthesorghum–wheatrotationwaslowerthan thoseforthefallow–wheatrotation.Despitethis,therewere nosignificantdifferencesbetweenfertilisertreatmentsorbe- tweencroprotationsonthenumberofgrains/spikeandthe percentageofgrain protein(AppendixATableS2).Further- more,attendingto thenitrogen use efficiency (NUE),there werealsonodifferencesbetweenfertilisationtreatmentsor croprotations.Finally,toevaluatetheN2Oefficiencyofcrop- pingsystemsanddevelopstrategiesforoptimalcropproduc- tivity,andhenceminimiseenvironmentalcontamination,it maybemoreinformativetoexpressN2Oemissionsinrela- tiontocropproductivity (YSNE)(vanGroenigenetal.,2010; Schwenke and Haigh, 2016). The AS treatment applied to bothcroprotationspresentedanincreasedYSNEcompared totheControlandAS+DMPPtreatments,whichwereequally low.Thesetreatmentsdidnotshowanydifferencesbetween thetwocroprotations,butfertilising wheatwithASinthe sorghum–wheatrotationhada93%higherYSNEthanwheat fertilisedwithASinthefallow–wheat rotation.Thus,when sorghum was used as a cover crop, it produced twice the N2Oemissions/kgofnitrogenuptakebecauseitincreasedthe gaseousnitrogenlosses.

3. Conclusions

Theuse ofsorghuminacroprotationmightnotbeasuit- ableoptiontomitigatethenitrogenlosses,suchasN2Oemis- sions,derivedfromthenitrogenfertiliserapplicationinthe subsequentculture.Sorghum–wheatrotationdidnotpresent anyeffectonthemaintenanceofsoilNH4+ contentduring wheatcropdevelopment,asthelevelswerethesameasthe fallow–wheatrotation.AlthoughthepotentialreleaseofBNIs

fromsorghumrootsproduceda22%decreaseinthegrowth ofAOBintheAStreatmentcomparedtothefallow–wheatro- tation,thisdidnotleadtolowerthenitrogenlossesthrough N2Oemissions.Wetheorizethatthe77%increaseincumu- lative N2O emissions for the AS treatment applied to the sorghum–wheatrotationwastheresultoftheincreaseofhet- erotrophicdenitrificationduetoahighercarbonavailability fromsorghumstover,sincenirKandnirSgeneswere73%and 30%moreabundantcomparedtothefallow–wheatrotation.

Moreover,whilethetypeofcroprotationdidnotaffectwheat grainyields,thehighercumulativeN2OemissionsoftheAS treatmentonthesorghum–wheatrotationproduceda93%in- crease inthe emissionsofN2O/kgofnitrogenuptakecom- paredtothefallow–wheatrotation.However,wesuggestthe useofsyntheticNIssuchasDMPPtoavoidthegreaterN2O releasederivedfromtheuseofsorghumasacovercrop.The applicationofDMPPmaintainedAOBgrowthatthelevelsof theunfertilisedsoils,therebydelayingsoilNH4+oxidation.As moresoilNH4+contentwasmaintained,soilNO3−formation wasdiminishedcomparedtoAS,thusmitigatingtheincrease ofnirKandnirSgenesresultingfromthehighercarbonavail- abilityinthesorghum–wheatrotation.ThecumulativeN2O emissions were alsomaintainedatthe levelsofthe unfer- tilisedsoils.Therefore,asgrainyieldwasnotaffectedbythe useofthesyntheticNI,theAS+DMPPtreatmentyieldedre- ductionsof73%and86%intheemissionsofN2O/kgofnitro- genuptakecomparedtotheAStreatmentinthefallow–wheat andsorghum–wheatrotations,respectively.

Declaration of Competing Interest

Theauthorsdeclarethattheyhavenoknowncompetingfi- nancialinterestsorpersonalrelationshipsthatcouldhaveap- pearedtoinfluencetheworkreportedinthispaper.

Acknowledgments

This work was supported by the Spanish Government (RTI2018-094623-B-C21andC22MCIU/AEI/FEDER,UE)andthe BasqueGovernment(IT-932-16).Dr.AdriánBozal-Leorriheld agrantfrom theBasqueGovernment(PRE-2020-2-0142).Dr.

MarioCorrochano-MonsalveheldagrantfromtheMinistryof EconomyandBusinessoftheSpanishGovernment(BES-2016- 076725).

Appendix A Supplementary data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversionatdoi:10.1016/j.jes.2022.04.035.

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