Soil properties impacting denitrifier community size, structure, and activity in New Zealand dairy-grazed pasture

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1 Author(s) This work is distributed under the Cretive Commons Attribution 3.0 License. Soil properties impcting denitrifier community size, structure, nd ctivity in New Zelnd diry-grzed psture Neh Jh 1,2, Surinder Sggr 2, Donn Giltrp 2, Russ Tillmn 1, nd Julie Deslippe 2, 1 Soil & Erth Sciences Group, Institute of Agriculture nd Environment, Mssey University, Plmerston North, 4442, New Zelnd 2 Mnki Whenu Lndcre Reserch, Plmerston North, 4442, New Zelnd now t: School of Biologicl Sciences, Victori University of Wellington, Wellington, 6012, New Zelnd Correspondence to: Neh Jh (neh_jh@msn.com) Received: 14 September 2016 Discussion strted: 12 October 2016 Revised: 7 August 2017 Accepted: 16 August 2017 Published: 22 September 2017 Abstrct. Denitrifiction is n nerobic respirtion process tht is the primry contributor of the nitrous oxide (N 2 O) produced from grsslnd soils. Our objective ws to gin insight into the reltionships between denitrifier community size, structure, nd ctivity for rnge of psture soils. We collected 10 diry psture soils with contrsting soil textures, dringe clsses, mngement strtegies (effluent irrigtion or non-irrigtion), nd geogrphic loctions in New Zelnd, nd mesured their physicochemicl chrcteristics. We mesured denitrifier bundnce by quntittive polymerse chin rection (qpcr) nd ssessed denitrifier diversity nd community structure by terminl restriction frgment length polymorphism (T-RFLP) of the nitrite reductse (nirs, nirk) nd N 2 O reductse (nosz) genes. We quntified denitrifier enzyme ctivity (DEA) using n cetylene inhibition technique. We investigted whether vried soil conditions led to different denitrifier communities in soils, nd if so, whether they re ssocited with different denitrifiction ctivities nd re likely to generte different N 2 O emissions. Differences in the physicochemicl chrcteristics of the soils were driven minly by soil minerlogy nd the mngement prctices of the frms. We found tht nirs nd nirk communities were strongly structured long grdients of soil wter nd phosphorus (P) contents. By contrst, the size nd structure of the nosz community ws unrelted to ny of the mesured soil chrcteristics. In soils with high wter content, the richnesses nd bundnces of nirs, nirk, nd nosz genes were ll significntly positively correlted with DEA. Our dt suggest tht mngement strtegies to limit N 2 O emissions through denitrifiction re likely to be most importnt for diry frms on fertile or llophnic soils during wetter periods. Finlly, our dt suggest tht new techniques tht would selectively trget nirs denitrifiers my be the most effective for limiting N 2 O emissions through denitrifiction cross wide rnge of soil types. 1 Introduction Nitrous oxide (N 2 O) is potent greenhouse gs tht is produced s n intermedite product of biologicl nitrogen conversions in soils (Stevens et l., 1997). Denitrifiction is the stepwise nerobic reduction of queous nitrte (NO 3 ) to nitrite (NO 2 ) nd into the gseous forms N 2O nd benign dinitrogen (N 2 ). It is the mjor globl contributor to N 2 O production in grsslnd soils (Sggr et l., 2013) nd is responsible for significnt frction of griculturl greenhouse gs emissions (IPCC, 2014). Denitrifiction is medited by the ction of four enzymes: NO 3 reductse (NAR), NO 2 reductse (NIR), nitric oxide (NO) reductse (NOR), nd N 2 O reductse (N 2 OR) (Zumft, 1997), which re encoded by the nr/np, nir, nor, nd nos genes, respectively. Txonomiclly diverse bcteri, rche (Philippot et l., 2007; Tiedje, 1994; Ishii et l., 2010), nd eukryotes (Zumft, 1997) re known to hrbour two or more denitrifiction enzymes. Denitrifying bcteri re prticulrly widely distributed in psture soils (Grhm et l., 2014), nd more thn 60 gener hve been identified (Chen et l., 2012). Denitrifiers with ll four reductses re cpble of emitting N 2 nd re sid to be complete denitrifiers. Those denitrifiers tht lck N 2 OR nd emit N 2 O Published by Copernicus Publictions on behlf of the Europen Geosciences Union.

2 4244 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture s the finl product of denitrifiction re clled incomplete denitrifiers. NirS, nirk, nd nosz genes hve been trgeted s functionl mrkers of both complete nd incomplete denitrifiers in soils (Stres et l., 2008; Throbäck et l., 2004; Morles et l., 2010; Enwll et l., 2010). The blnce of complete nd incomplete denitrifiers in soils cn determine the rtio of N 2 O : N 2 produced during denitrifiction (Philippot et l., 2011; Bkken et l., 2012). Thus, denitrifier community structure nd bundnce cn be importnt fctors in determining nitrogen (N) loss nd griculturl greenhouse gs emissions from soils. Indeed, recent synthesis of 82 dtsets relting bcteril community structure nd environmentl chrcteristics to vriety of crbon (C) nd nitrogen (N) cycling processes found tht microbil community structure dt improved the power of models to explin denitrifiction process rtes better thn for ny other ecosystem process (Grhm et l., 2016). Still, strong reltionships re not lwys observed between denitrifiction rtes nd denitrifier community structure nd bundnce (Cvigelli nd Robertson, 2000, 2001; Chèneby et l., 1998; Mergel et l., 2001). In prticulr, the structure of denitrifier communities in environmentl smples is often poorly correlted with soil or environmentl fctors tht re known to influence process rtes (Dndie et l., 2011; Enwll et l., 2010; Philippot et l., 2009), indicting tht our understnding of the fctors controlling the diversity nd function of denitrifying communities is still indequte. Moreover, there is need to identify the soil conditions in which the presence nd ctivity of denitrifiers re likely to led to substntil N 2 O emissions so tht pproprite strtegies for trgeted nd effective mngement cn be deployed or developed where they re lcking. Pstorl diry frming is preferred lnd use in mild nd wet climtes on reltively fertile soils nd flt sites tht occupy low-lying positions in the lndscpe, s these loctions support high rtes of psture production (Sggr et l., 2013). The combintion of periodiclly noxic soil conditions, high concentrtions of N in cttle excrement ptches, nd reltively high microbil biomss t these sites combine to fvour denitrifiction s mjor oxidtive metbolic pthwy. Despite this, denitrifiction rtes nd potentils s well s N 2 O emissions through denitrifiction vry widely mong psture soils (Cyuel et l., 2013; Giltrp et l., 2012; Groffmn et l., 2006). Soil mngement prctices, including the ddition of orgnic mendments such s plnt residues, compost, mnure, or effluent irrigtion, cn increse soil fertility nd microbil biomss nd my led to structurl shifts in soil microbil communities, which in turn lter soil biochemicl processes (Kennedy nd Smith, 1995). The ddition of crop residues to soils increses the bundnce of denitrifier genes nd leds to greter denitrifiction in soils (Brrett et l., 2016; Go et l., 2016; Henderson et l., 2010). Likewise, incresing soil wter content is ssocited with incresing denitrifier gene bundnces in soils (Liu et l., 2012; Mergel et l., 2001). Mngement prctices tht lter the size of the denitrifier community in soils re lso likely to ffect its denitrifiction enzyme ctivity (DEA), s the bundnce of denitrifier genes cn be strong determinnt of DEA (Čuhel et l., 2010; Deslippe et l., 2014; Enwll et l., 2010; Hllin et l., 2009). However, the geologic origins of soil cn determine its dominnt properties over rnge of soil C nd wter contents (Bronick nd Ll, 2005). Indeed, we previously found tht soil texture, dringe clss, nd ltitude were powerful regultors of denitrifiction end products (N 2 vs. N 2 O) nd totl emissions. Also, both the forms nd quntities of gses emitted could be predicted by the 16S rrna gene communities of soil smples (Morles et l., 2015). However, we still lck detiled knowledge of how vrition in soil properties ffects denitrifier popultions nd denitrifiction. Better informtion on the role of soil physicochemicl chrcteristics in determining the size nd ctivity of denitrifiers my llow for improved nd soil-specific mngement of N 2 O emissions from pstorl griculture. Here, we sought better understnding of the reltionships between the structure, bundnce, nd ctivity of denitrifiers over rnge of New Zelnd diry psture soils, which vried widely in soil properties nd hd different mngement conditions. We investigted whether the properties of these soils drove unique denitrifier communities tht supported different DEA or were likely to generte different N 2 O emissions. We expected to find tht the size nd structure of denitrifier communities would vry most strongly in ccordnce with soil wter content nd tht soil physicl properties or mngement prctices tht increse soil wter would enhnce the size nd ctivity of denitrifiers. 2 Mterils nd methods 2.1 Sites nd soils Our im ws to smple soils tht would encompss the rnge of physicochemicl conditions tht predominte on New Zelnd diry frms. We therefore trgeted soils on the bsis of their geogrphicl loction (North or South Islnd of New Zelnd) nd minerlogy (llophnic or non-llophnic soils). As soil wter content is key fctor ffecting the structure nd ctivity of soil denitrifier communities (Liu et l., 2012; Mergel et l., 2001), it ws lso importnt to smple in both wet nd dry sesons. We therefore smpled soils over 6-month period from winter to summer. Soil textures vried from stony silt lom to fine sndy lom, nd the sites rnged from poorly drined to well drined (Tble 1). We smpled soils expected to hve the gretest soil wter contents in winter nd those we expected to be driest in summer, with other soils smpled between these times (see the Supplement Tble S1 for soil smpling dtes). We collected soils from 10 different commercil diry frms (Fig. S1 in the Supplement). All were fenced from livestock nd none hd been grzed within 8 weeks of smpling. All sites were

3 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture 4245 dominted by perennil ryegrss (Lolium perenne) nd white clover (Trifolium repens). Fertilistion regimes vried mong the frms nd consisted of pplictions of kgnh 1 nnully. Detiled descriptions of the individul fertiliser pplictions t the 10 frms re described in the Supplement. 2.2 Smpling nd nlysis of soil properties Tble 1. Description of soils. Soil Loction of the diry frm Geogrphicl loction Soil bbrevition Soil clssifiction Minerlogy clss TeK Typic orthic gley Glssy volcnic, kolinitic Te Kowhi silt lom AgReserch Rukur, Wikto S E OH Typic orthic Allophnic Otorohng silt lom Toknui, Wikto S E HR Typic orthic llophnic Allophnic Horotiu silt lom AgReserch Rukur, Wikto S E TM Argillic-frgic perch-gley pllic Vermiculitic S E Tokomru silt lom Mssey University, Plmerston North MW Wethered fluvil recent Illitic Mnwtu fine sndy lom Longburn, Plmerston North S E MWEI Wethered fluvil recent Illitic Longburn, Plmerston North S E Mnwtu fine sndy lom (effluent irrigted) PS Wethered orthic recent Illitic Ppru silt lom (Springston) Springston, Christchurch S E PL Wethered orthic recent Illitic Ppru silt lom (Lincoln) Lincoln, Christchurch S E LM Pllic orthic brown Vermiculitic Lismore stony silt lom Ashburton, Cnterbury S E MF No dt No dt Myfield deep silt lom Methven, Cnterbury S E Adpted from Morles et l. (2015). At ech frm, we rndomly selected six blocks of 100 m 2 for the collection of soil smples. At rndomly selected loctions within ech block, 25 soil cores (25 mm dimeter 100 mm long) were obtined using steel corer. The 25 cores from ech block were pooled to form single composited smple per block (n = 6 composited soil smples per frm). All soil smples were collected between August nd December 2010 once from ech site. Soil smples were tken to the lbortory, individully homogenised, sieved to 2 mm, nd stored t 4 C in plstic bgs (10 sites 6 replictes = 60 smples). A subsmple of ech soil ws stored t 20 C for moleculr nlysis. We mesured ph, nitrte N (NO 3 ) nd mmonium N (NH + 4 ) (minerl N), totl nitrogen (TN), totl crbon (TC), Olsen phosphorus (P), nd soluble C on the field-moist sieved soils using stndrd protocols (for detils see Jh, 2015). All soils were nlysed for these prmeters within 2 weeks of smpling. DEA ws determined using the cetylene inhibition method described in Luo et l. (1999), with the exception tht we dded chlormphenicol to inhibit the de novo synthesis of enzymes. Thus the vlues we report represent only the existing enzyme ctivity in soils. DEA ws ssessed for ll soil smples within 2 dys of collection. DEA incubtion conditions nd the gs smpling methods re described elsewhere (Jh, 2015). We intended to mesure microbil biomss crbon (MBC) within 48 h of soil smpling, but technicl problem with our set-up delyed mesurements of MBC for nerly 3 months. To stndrdise this effect cross soil smples we monitored chnges in the size of the MBC pool in two soils over 7 months. We found tht no significnt chnges in MBC occurred between 3 nd 7 months for soils stored t 4 C (see Tble S1). We therefore report MBC dt for ll soils tht were stored t 4 C for 4 to 6 months. 2.3 DNA extrction from soils Within 6 months of soil smple collection, soil smples were thwed on ice nd 0.25 g liquot ws obtined. DNA ws extrcted from these soil smples using the Mobio PowerSoil DNA Isoltion Kit (Mobio, Soln Bech, CA, USA) following the mnufcturer instructions. The yield nd qulity of DNA extrcts were verified s described in Deslippe et l. (2014). DNA ws stored t 20 C until nlysed.

4 4246 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture 2.4 Terminl restriction frgment length polymorphism (T-RFLP) of denitrifier genes Terminl restriction frgment length polymorphism (T- RFLP) ws performed to nlyse the community structure nd diversity of nir nd nos genes in soil smples. T-RFLP for nirs nd nosz genes ws conducted s described in Deslippe et l. (2014) except tht PCR for nir genes occurred in totl volume of 25 µl rection mixture, which contined 2.5 µl of 10 PCR buffer (1 mm MgCl 2 ), 0.5 mmmgcl 2, 0.2 mm of ech deoxynucleotide triphosphte (dntp), 1.25 U of Tq polymerse (Thermofisher Scientific ), 0.8 mg ml 1 bovine serum lbumin (BSA), 1.0 µm of ech primer, nd 10 ng DNA templte per rection. The PCR mplifiction consisted of n initil denturtion of the DNA templte t 94 C for 30 s followed by 35 cycles of 20 s t 94 C, 20 s t 56 C, nd 20 s t 68 C. The rection ws completed by 10 min t 68 C. For T-RFLP of the nirk gene we used the primers Copper 583F nd 909R (Dndie et l., 2011). The mplifictions of nirk nd nosz genes were chieved under slightly different conditions thn the nirs gene ccording to the specifictions of the regents used for PCR. The PCR mplifiction ws performed in totl volume of 25 µl rection mixture contining 10 µl of 2 NEB Tq mster mixes (New Englnd Biolbs Inc.), 0.4 µm of ech primer, nd 10 ng DNA templte per rection. PCR consisted of n initil denturtion of the DNA templte t 94 C for 2 min followed by 35 cycles of 30 s t 94 C, 1 min t 56 C, nd 1 min t 72 C. The rection ws considered complete fter 10 min t 72 C. The T-RFLP profiles generted for the soil smples were nlysed using Pek Scnner v1.0 softwre (Life Technologies) nd s described in Deslippe et l. (2014). The totl number of terminl restriction frgments (T-RFs) per electropherogrmme ws tken to indicte genotype richness per smple. We then clculted the gene Shnnon s diversity index nd Pielou s evenness index (Mgurrn, 1988) per smple nd used one-wy nlysis of vrince (ANOVA) to determine if soils belonging to the three physicochemicl groups differed with respect to gene richness, evenness, nd diversity. 2.5 Quntittive polymerse chin rection (qpcr) of totl bcteril nd denitrifier genes Quntifiction of bcteril nirs, nirk, nd nosz genes ws ccomplished using qpcr following the methodology of Deslippe et l. (2014). Amplifiction efficiencies of qpcr rections for smples were within the rnge of vlues (E = %) published previously (McPherson nd Moller, 2006). The rections were liner over 7 orders of mgnitude nd sensitive down to 10 2 copies. The rtio of bundnces of denitrifier genes in environmentl smples hs been interpreted previously s n index of the potentil for complete denitrifiction (Philippot et l., 2009). Here, we clculted the nosz : (nirs + nirk) of soil smples. 2.6 Sttisticl nlysis The normlity nd homoscedsticity of ll soil physicochemicl, gseous emission, nd biologicl dtsets were exmined using Anderson Drling (Stephens, 1986) nd Levene s tests, respectively, in Minitb 16 softwre (Minitb Inc.). Box Cox trnsformtions (Box nd Cox, 1964) were pplied to dtsets s required to conform to model. The differences in the mens of soil chrcteristics, such s ph, nitrte N (NO 3 N) nd mmonium N (NH+ 4 N) (minerl N), totl nitrogen (TN), totl crbon (TC), Olsen phosphorus (P), microbil biomss crbon (MBC), soluble C, DEA, number of gene T-RFs, nd gene copy numbers, were ssessed using one-wy nlysis of vrince (ANOVA) test with soil type s fctor. Tukey s studentised rnge test t n α = 0.05 significnce level ws used post hoc to revel significnt differences mong mens. The reltionships mong the soil chemicl chrcteristics ph, nitrte N (NO 3 N) nd mmonium N (NH + 4 N), TN, TC, Olsen P, MBC, DEA, number of denitrifier gene T-RFs, nd gene copy numbers were determined using Person correltion nlysis. In order to reduce the dimensionlity of the mny correlted soil physicochemicl chrcteristics, we performed principl component nlysis (PCA) nd included % soil wter content (SWC), % SWC t field cpcity (% FC SWC), ph, TN, TC, soluble C, Olsen P, nd nitrte N (NO 3 N) nd mmonium N (NH + 4 N) s fctors in the PCA. Soils grouped long the first nd second ordintion xes. We used multiple response permuttion procedure (MRPP) to ssess the sttisticl significnce of these groupings. MRPP clcultes the chnce-corrected within-group greement (A), mesure of within-group homogeneity compred with tht expected by chnce, where A = 1 corresponds to identicl members within ech given group (mximum effect of fctor) nd where A 0 corresponds to within-group heterogeneity equl to or lrger thn tht expected by chnce (no effect of fctor; McCune nd Medford, 1999). We lso clculted Person correltions mong soil microbil chrcteristics nd the ordintion xes nd plotted those tht were significntly correlted (τ > 0.2) with xis 1 nd 2 s vectors on the PCA. Anlysis of the nirs, nirk, nd nosz community structure ws bsed on threshold normlised pek heights of T- RFs from electropherogrmmes (Deslippe et l., 2014). Nonmetric multidimensionl scling (NMS) ordintions were performed using Bry Curtis distnce (Bry nd Curtis, 1957) in the progrmme PC-ORD (McCune nd Mefford, 1999). In order to illustrte how the structure of denitrifier communities vried with the physicochemicl chrcteristic of soils, we clculted Kendll s rnk correltions mong the physicochemicl nd biologicl chrcteristics of soils with the NMS ordintion xes in PC-ORD. The significnt corre-

5 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture b T-RF richness 35 b b NirS nirs NirK nirk NosZ nosz Genes Figure 2. T-RF richness by soil group. White, grey, nd blck brs denote groups 1, 2, nd 3 soils. Men vlues re reported ±1 SE (stndrd error) of the men. Mens with the sme letters do not differ significntly t α = 0.5. Figure 1. Principl component nlysis of the physicochemicl chrcteristics of soil smples collected t 10 diry frms nd results of multiple response permuttion procedure (MRPP) to ssess the significnce of soil origin nd group. Vectors indicte significnt correltes (τ > 0.2) with ordintion xes in the first nd second PC xes. the three groups of soils. Overll, we found tht xes 1 nd 3 of the PCA were not significntly correlted with DEA or MBC. However, xis 2 of the PCA, which describes grdient in minerl-n form, ws significntly positively correlted with DEA (r 2 = 0.214) nd MBC (r 2 = 0.303; Fig. 1). 3.2 ltes (τ > 0.2) were overlid s vectors on the NMS ordintion plots Results Vritions in soil chemicl chrcteristics The 10 soils differed significntly with regrd to ll mesured physicochemicl chrcteristics (Tble S2). The PCA of soil chrcteristics generted three significnt xes, of which the first two ccounted for 83.4 % of the totl vrince (Fig. 1). Axis 1, which ccounted for 53.4 % of the vrition in soil properties, primrily described difference in % FC SWC, lthough totl N, totl C, nd Olsen P lso weighed hevily in forming xis 1. Axis 2, which ccounted for 30.0 % of the vrition in soil properties, primrily described grdi+ ent in minerl-n form with NO 3 N incresing nd NH4 N decresing long xis 2. The 10 soils segregted into three groups with replictes of soil tending to cluster closely together in the PCA. Firstly, the two llophnic soils OH (Otorohng silt lom) nd HR (Horotiu silt lom; group 1) were seprted from ll other soils by their reltively high % FC SWC, their high totl N, C, nd llophne contents. Secondly, the effluent-irrigted soil, MWEI (Mnwtu effluentirrigted fine sndy lom), ws seprted from ll other soils (group 2) due to its high NO 3 N content. The seven remining soils formed loose cluster due to their reltively high NH+ 4 N nd low Olsen P contents (group 3). MRPP confirmed tht these groups differed significntly in soil physicochemicl chrcteristics (A = 0.379, P < 0.001; Fig. 1). Tble S3 summrises the physicochemicl chrcteristics for Bcteril denitrifiers in New Zelnd diry-grzed psture soils Diversity indices of denitrifier gene T-RF profiles Across ll soil smples, totl T-RF richnesses were nirs = 52, nirk = 53, nosz = 47, which re typicl vlues for TRFLP studies of functionl genes in soils (Deslippe et l., 2014; Rich nd Myrold, 2004; Rösch nd Bothe, 2005). While the minimum nd mximum numbers of T-RFs vried mong smples, the ptterns of richness, evenness, nd diversity mong soils belonging to the three groups of soils were similr (see Tble S4). For simplicity, we therefore present only the vlues for T-RF richness in Fig. 2. NirS communities mong the three groups of soils hd similr richnesses. By contrst, the llophnic soils of group 1 hd significntly lower nirk richness, while the effluent-irrigted soils of group 2 hd significntly greter richness thn ll other soils (Kruskl Wllis H = 13.84, P = ). NosZ richness ws significntly greter in the effluent-irrigted soils of group 1 compred with ll other soils (Kruskl Wllis H = 8.59, P = 0.014) Denitrifier community structure in soils Ordintion of the soil smples in nirs T-RF spce indicted significnt structuring of the nirs community ccording to vrition in the physicochemicl chrcteristics of the soils (Fig. 3). Interestingly, however, the vrition in nirs community structure ws not driven by the sme physicochemicl chrcteristics tht vried most widely mong soils nd formed the first nd second PCA xes. Consequently the nirs communities of soil smples did not cluster ccording to the

6 4248 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture Figure 3. Non-metric multidimensionl scling (NMS) ordintions illustrting the Bry Curtis dissimilrities of nirs, nirk, nd nosz communities. Vectors represent those fctors tht were significntly correlted with the first nd second ordintion xes t τ = 0.2. Symbol colours re the sme s in Fig. 1. Symbol shpes represent the three groups of soils s determined by the PCA of their physiochemicl chrcteristics (Fig. 1). Finl stress vlues for ordintions nirs 12.53, nirk 5.58, nd nosz three groups of soils in our PCA. Rther, xis 1, which ccounted for 30.0 % of the vrition in nirs community structure, ws significntly correlted with SWC nd the Olsen P contents of the soils (Fig. 3). This indictes tht nirs community structure in diry psture soils from cross New Zelnd responded most strongly to SWC nd P grdients. Likewise, 66.9 % of the vrition in nirk community structure (xis 1) ws significntly correlted with the SWC nd Olsen P contents of the soils. Axis 1 of the nirk ordintion primrily seprted two soils (Horotiu silt lom nd Ppru Lincoln silt lom) from ll other soils. However, even when these two soils were removed from the dtset, NMS ordintion reveled tht the nirk community ws primrily structured ccording to Olsen P nd soil wter vribles (both SWC nd % FC SWC; Supplement Fig. S2A). An dditionl 7.7 % of the vrition in nirk community structure ws most strongly correlted with nir gene bundnce (nirs + nirk) in soils, which ws higher in the HR soil, n llophnic soil, thn for ny other. Ordintion of the soil smples in nosz T-RF spce reveled little clustering of soil smples by origin or group. Likewise, we detected no significnt ptterns of correltion mong the first nd second ordintion xes nd the soil physicochemicl chrcteristics. However, xis 1 of the NMS ordintion ws lso most strongly correlted with SWC nd Olsen P (Supplement Fig. S2B) Denitrifier gene bundnce The number of nirs nd nirk gene copies vried widely mong the 10 soils; nirs gene copies rnged from to copies g 1 soil, while nirk gene copies vried from to g 1 soil. Overll soils, nir genes were on verge n order of mgnitude more bundnt thn those encoding the finl step of denitrifiction. NosZ gene copies vried most widely ( to g 1 soil) with much greter rnge thn for nirs + nirk gene copies. The sum of nir gene (nirs + K) copies ws significntly greter in the llophnic soils of group 1 thn in the soils of group 3 (P < 0.005; Fig. 4), with the effluent-irrigted (group 2) soil hving intermedite vlues. Despite lrge vribility, the group 2 soils hd significntly more nosz gene copies thn the soils in groups 1 nd 3, while the group 1 soils hd significntly fewer thn the other two groups (P < 0.005; Fig. 4). Consequently the rtio of nosz : (nirs + K) genes, which my indicte the reltive bundnce of complete denitrifiers, vried significntly mong the three groups of soils (Fig. 4b), with the effluent-irrigted group 2 soil hrbouring the highest, the llophnic soils of group 1 the lowest, nd the group 3 soils intermedite rtios of nos : nir genes.

7 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture () (b) A AB B b c NosZ: nirs+k gene copies 800 NosZ NosZ gene million copies g-1 dry soil NirS+K NirS+K genes million copies g-1 dry soil 4249 A AB B Soil groups Soil groups Figure 4. () Denitrifier gene (nirs, nirk, nosz) copy numbers in different soil groups; error brs denote SEM. (b) Denitrifier gene bundnce rtio (nosz: nirs, nirk) in different soil groups. Men vlues re reported ±1 SE of the men. Columns with the sme letters re not significntly different. Letter vlues with sme cse or font denote one test (one test for ech of the groups). 3.3 Denitrifiction enzyme ctivity (DEA) DEA vried considerbly mong the psture soils but lso mong replictes within soil (Tble S3). DEA vried by fctor of 5 mong the soil groups, with the group 2 soil (effluent-irrigted MWEI) chieving significntly higher DEA thn other groups, while the soils of group 1 (HR nd OH) hd significntly lower DEA vlues thn other groups (H = 12.09, P = 0.02; Tble S3). DEA vried considerbly in soils belonging to group 3. Overll, DEA ws mostly strongly positively correlted with soil NO 3 N contents nd ws mostly strongly negtively correlted with soil NH+ 4 N contents, driving its significnt correltion with xis 2 of our PCA (Fig. 1). All significnt physicochemicl correltes with DEA re given in Tble S Reltionships mong denitrifiction nd denitrifier community size nd structure cross rnge of soil moistures Given tht the structure of nirs, nirk, nd nosz communities vried primrily in response to soil wter content nd Olsen P (Fig. 3), we wished to know if unique reltionships between the richness nd size of the denitrifier gene community nd DEA exist t different SWCs. To ddress this question, we ctegorised soils ccording to corse-scle SWC (high, moderte, nd low) nd exmined the Person correltions mong these vribles within soil SWC ctegories. For soils in the highest SWC ctegory (MWEI, OH, nd HR), we found tht strong nd significnt positive correltions existed between denitrifier gene copy numbers nd DEA (nosz, r = nd P = 0.049; nirk, r = nd P = 0.007). Likewise, strong nd significnt positive correltions existed between DEA nd the T-RF richness of denitrifier genes (nosz, r = nd P = 0.010; nirk, r = nd P = 0.013; nd nirs, r = nd P = 0.011). However, these ptterns of correltion were not present in the soils ctegorised s moderte or low SWC. 4 Discussion Despite its reltively smll totl lnd re, New Zelnd is geologiclly diverse, nd the 1.8 million hectres of lnd tht were mnged s diry psture in 2015 (Diry NZ, 2017) hve soils derived from wide rnge of prent mterils. Here we studied 10 diry psture soils tht vried widely in texture, dringe clss, nd mngement strtegies. We found tht the % FC SWC nd grdient in minerl-n form ccounted for the gretest vrition in soil physicochemicl chrcteristics nd tht key microbil prmeters for denitrifiction, such s MBC nd DEA, were significntly positively correlted with higher soil NO 3 N. In our study, these ptterns were driven primrily by only three soils: the two llophnic soils, which hd high % FC SWC (group 1), nd the effluent-irrigted soil, which hd very high NO 3 N (group 2). The effluent irrigted soil, which hd the highest MBC, likely hrboured lrger popultion of nitrifiers with ctivities tht generted the NO 3 required by denitrifiers nd supported the highest rtes of DEA we observed. Nonetheless, our results re consistent with previous reports tht soil microbil biomss is key indictor of denitrifiction process rtes (Drury et l., 1991). From this perspective, cross wide rnge of soil properties, the size of the MBC pool my be n importnt corse-scle indictor of soil N2 O

8 4250 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture emissions under both noxic (denitrifiction) nd oxic (nitrifiction) conditions. Allophnic soils hve high wter content t field cpcity, but they dsorb copper nd re therefore likely to select ginst nirk denitrifiers, the periplsmic nitrite reductse of which requires six copper toms to mintin its trimeric structure. This ws reflected in our dt by the very low richness, evenness, nd diversity of nirk T-RFs nd in the very low numbers of nirk gene copies reltive to the other soils. We expected this to lso reduce the overll number of genes encoding nitrite reductse in group 1 soils but did not observe this. Insted we found tht nirs denitrifiers replced nirk denitrifiers in llophnic soils so tht the totl number of nir gene copies ws equivlent to tht in the effluentirrigted soil nd significntly greter thn the number of nir copies in ll other soils. Interestingly, despite the lrge size of the nirs community, llophnic soils did not, on verge, hve more diverse nirs communities thn other soils. However, the size nd diversity of nirs communities in llophnic soils ws more vrible thn for other soils. These findings suggest tht llophnic soils support reltively few microsites where denitrifiction driven by nirs denitrifiers is the dominnt respirtory pthwy. New Zelnd s llophnic soils re porous nd free drining with reltively low bulk densities (Molloy, 1998). As such, noxic microsites conducive to denitrifiction re expected to be few. Likewise, fewer ctive microsites for denitrifiction fits with the low to moderte DEA we observed in the llophnic soils. Allophnic soils re known to dsorb P (Hshizume nd Theng, 2007), nd the binding of denosine by llophnes my hve limited DEA in these soils despite their reltively lrge nir popultions. Nonetheless, we lso found fr fewer copies of nosz genes, reltively low nosz diversities, nd the lowest nos : nir gene rtios in the llophnic soils, suggesting tht complete denitrifiers re reltively rre in these soils. Consequently, where nd when it occurs, denitrifiction in llophnic soils is likely to led to significnt N 2 O emissions. This result fits with other work from our group, which indictes tht llophnic soils emit greter N 2 O : (N 2 O + N 2 ) reltive to other soil types (McMilln et l., 2016). Tken together, these results suggest tht trgeted mngement of nirs denitrifiers in llophnic soils during wet sesons my be n effective strtegy to combt greenhouse gs emissions from pstorl griculture in volcnic regions. The effluent-irrigted soil (MWEI), with physicochemicl properties tht seprted it from ll other soils, ws chrcterised by very high NO 3 nd Olsen P concentrtions, reltively high ph (5.9), nd high MBC, which supported very high DEA. This modertely drined, fine sndy lom hd the highest SWC t the time of smpling. MWEI hd the lrgest number of nirk gene copies but only moderte numbers of nirs, leding to intermedite totl numbers of nir genes. Likewise, it hd the gretest diversity of nirk genotypes but only moderte diversity of nirs genotypes. These findings emphsise the potentil for effluent irrigtion to increse denitrifiction enzyme ctivity, likely through incresing both the size of the totl microbil community (MBC), SWC, nd NO 3 vilbility, which in turn selects for denitrifiers. However, MWEI supported significntly lrger popultion of nosz denitrifiers thn the other soils nd this led to the highest nos : nir of ny soil. Overll, these findings suggest tht MWEI is likely to support lrge nd ctive community of denitrifiers but tht complete denitrifiction my limit N 2 O emissions from this soil. Consequently, mngement of greenhouse gs emissions from highly fertile psture soils like MWEI my benefit from strtegies tht limit NO 3 vilbility in soils, such s the ppliction of nitrifiction inhibitors (e.g. DCD). When considered in isoltion, the seven soils of group 3 still vried significntly with regrd to physicochemicl chrcteristics. In prticulr MW, PS, nd PL, which rnked higher on xis 2 of the PCA, differed from the four remining soils (Fig. 1). For exmple, they hd on verge three times the NO 3 nd hlf the NH + 4 s the other group 3 soils. These soils lso hd the three highest ph vlues ( ), high MBC, nd reltively low SWCs. They supported reltively lrge numbers of nirs nd nosz denitrifiers but only verge numbers of nirk genes, leding to overll intermedite nir : nos. Likewise, these soils hd intermedite diversities of nirs, nirk, nd nosz genes nd ordintions of the nirs, nirk, nd nosz gene T-RFs filed to distinguish these soils from those in the other groups. Despite this, when incubted under non-limiting conditions, these three soils together with MWEI supported the highest DEAs. These findings indicte tht denitrifiction responds quickly to SWC in modertely fertile soils. Thus, creful mngement of NO 3 lods by limiting diry stock, using nitrifiction inhibitors, or both is lso likely to be useful in limiting greenhouse gs emissions from these soils during wetter periods of the yer. The four remining soils of group 3 were the lest fertile nd the most cidic (ph ) with the lowest MBC in our study. They were lso mong the driest. Despite modertely high totl numbers of nirs, nirk, nd nosz genes, the nos : nir rtios in these soils were equivlent to the other soils of group 3 nd intermedite overll. With the exception of the two llophnic soils, the four remining soils of group 3 hd the lowest DEA, which ws on verge bout one-qurter of tht mesured in the other group 3 soils. Tken together, these results suggest lower risk of N 2 O emissions from these soils, s oxic conditions nd low concentrtions of substrtes re likely to limit denitrifiction much of the time, nd modertely high numbers of nosz denitrifiers will fvour some complete denitrifiction of this smller totl N pool. Overll, ordintions of T-RFLP dt reveled no structuring of the nirs, nirk, or nosz communities ccording to the three groups tht defined the mjor physicochemicl chrcteristics of the soils, grdient in soil wter content t field cpcity, nd grdient in minerl-n form. Rther, SWC t the time of soil smple collection nd Olsen P were the primry drivers of the structure of denitrifier communities.

9 N. Jh et l.: Soil properties impcting denitrifier community size in New Zelnd diry-grzed psture 4251 Given the overll high correltion of SWC nd Olsen P in soil smples, this result is likely to indicte considerble plsticity of the denitrifier community in response to mbient soil wter content. In the wettest soils, we found strong nd significnt positive correltions between the diversity of nirs, nirk, nd nosz genes nd DEA. We lso found strong nd significnt positive correltions between nirk nd nosz gene copy numbers nd DEA in those soils. However, these reltionships broke down for soils with moderte or low SWCs. While the im of our study ws to smple psture soils over wide rnge of physicochemicl chrcteristics in order to gin insight into the properties of the denitrifier communities they support, sesonl vrition in the structure of nirs nd nirk denitrifiers in cultivted nd psture soils is well estblished (Wertz et l., 2016; Ttti et l., 2017; Bent et l., 2016; Yu et l., 2016; Smith et l., 2010). The plsticity of the denitrifier community in response to mbient soil wter content together with the strong correltions between the size, diversity, nd ctivity of denitrifying communities in very wet soils suggests tht future work towrds chrcterising the denitrifier communities most likely to contribute to greenhouse gs emissions from pstorl soils should focus smpling efforts on the wettest times of the yer. Severl lines of evidence collected here suggest tht nirk denitrifiers were more sensitive to the rnge of physicochemicl chrcteristics in soils thn were nirs denitrifiers. For exmple, the gene copy numbers nd diversity metrics of nirs communities were firly uniform cross soil groups but vried significntly for nirk denitrifiers. Likewise, the ptterns of nirk diversity cross soil groups were mirrored by the ptterns of nos : nir, suggesting tht chnges in the size of the nirk community hd dominnt influence on the overll rtio of complete nd incomplete denitrifiers in soil groups. Independent shifts in nirs nd nirk community structures in response to common physicochemicl chrcteristics were recently observed in eutrophic reservoir (Zhou et l., 2016). In contrst, the structure of nosz communities did not correspond to ny physicochemicl property mesured. Together, these results my suggest tht nirs nd nosz genotypes re equivlently dpted to the physicochemicl conditions of wide rnge of diry psture soils, while nirk denitrifiers re more sensitive. Given tht our dt suggest greter N 2 O emissions in llophnic soils where nirk denitrifiers re few, it my be the microbil communities dominted by nirs denitrifiers tht should be the trget of efforts to reduce greenhouse gs emissions from psture soils. However, further work is necessry to confirm whether microbil communities dominted by nirs denitrifiers support greter N 2 O emissions thn nirk denitrifier communities of equivlent size. 5 Conclusions Here we chrcterise the size, structure, nd diversity of nirs, nirk, nd nosz genes in soils tht vried widely in physicochemicl chrcteristics to ddress the question of whether different denitrifier communities develop under these vried soil conditions, nd if so, whether they re ssocited with different denitrifiction ctivities nd likely to generte different N 2 O emissions. Overll, we found strong correltion between MBC nd DEA nd tht modertely high to highly fertile soils supported the lrgest popultions of denitrifiers. Given tht the more fertile soils were lso likely to hrbour significnt popultions of nitrifiers, MBC my be n importnt corse-scle indictor of totl potentil N 2 O emissions from such soils. However, our results for llophnic soils suggest tht even reltively low rtes of denitrifiction my led to significnt N 2 O emissions given their reltively low nos : nir. Consequently, we conclude tht mngement strtegies to limit N 2 O emissions through denitrifiction re likely to be most importnt for diry frms on fertile or llophnic soils during wetter periods. Finlly, our dt suggest tht new techniques tht would selectively trget nirs denitrifiers my be the most effective for limiting N 2 O emissions through denitrifiction cross wide rnge of soil types. Dt vilbility. All code nd dt used to produce the results of this pper cn be provided upon request by contcting the corresponding uthor. The Supplement relted to this rticle is vilble online t Competing interests. The uthors declre tht they hve no conflict of interest. Acknowledgements. We cknowledge funding nd support for this study from the New Zelnd Agriculturl Greenhouse Gs Reserch Centre (NZAGRC) through Lndcre Reserch Strtegic Science Investment Funding from the Ministry of Business, Innovtion nd Employment (MBIE). We lso cknowledge support from Peter Berben nd Thilk Plmd t Lndcre Reserch in Plmerston North, Jif Luo t AgReserch in Hmilton, nd Mohmmd Zmn t Blnce Agrinutrients for ssistnce with nd rrngements for the collection of soil smples from Mnwtu, Wikto, Christchurch, nd Ashburton. Edited by: Tin Treude Reviewed by: three nonymous referees

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