Rhizosphere priming effect on soil organic carbon decomposition under plant species differing in soil acidification and root exudation

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1 Reserh Rhizosphere priming effet on soil orgni ron eomposition uner plnt speies iffering in soil iifition n root exution Xiojun Wng 1, Cixin Tng 1, Juli Severi 1, Clyton R. Butterly 1 n Jeff A. Blok 2 1 Deprtment of Animl, Plnt n Soil Sienes, Centre for AgriBiosiene, L Troe University, Melourne Cmpus, Bunoor, VIC 386, Austrli; 2 CSIRO Agriulture, Glen Osmon, SA 564, Austrli Author for orresponene: Cixin Tng Tel: Emil: C.Tng@ltroe.eu.u Reeive: 3 Jnury 216 Aepte: 9 Mrh 216 oi: /nph Key wors: 13 C nturl unne, Cier rietinum,co 2 -trpping system, Lupinus lus, Pisum stivum, rhizosphere iifition, speies vrition, Tritium estivum. Summry Effets of rhizosphere properties on the rhizosphere priming effet (RPE) re unknown. This stuy ime to link speies vrition in RPE with plnt trits n rhizosphere properties. Four C 3 speies (hikpe, Cier rietinum; fiel pe, Pisum stivum; whet, Tritium estivum; n white lupin, Lupinus lus) iffering in soil iifition n root exution, were grown in C 4 soil. The CO 2 relese from soil ws trppe using newly evelope NOH-trpping system. White lupin n whet showe greter positive RPEs, in ontrst to the negtive RPE proue y hikpe. The gretest RPE of white lupin ws in line with its pity to relese root exutes, wheres the negtive RPE of hikpe ws ttriute to its gret ility to iify rhizosphere soil. The enhne RPE of fiel pe t mturity might result from high nitrogen eposition n relese of struturl root ron omponents following root senesene. Root iomss n length plye minor role in the speies vrition in RPE. Rhizosphere iifition ws shown to e n importnt ftor ffeting the mgnitue n iretion of RPE. Future stuies on RPE moelling n mehnisti unerstning of the proesses tht regulte RPE shoul onsier the effet of rhizosphere ph. Introution In eomposition stuies, priming effet is efine s strong short-term hnge in miroil minerliztion of soil orgni ron (SOC) in response to lile ron (C) inputs (Kuzykov et l., 2). When the soure of lile orgni C is rhizoeposits erive from living roots n hnge in eomposition of SOC in the iret viinity of roots is inue, it is terme rhizosphere priming effet (RPE). Rhizoeposits onsist of solule n insolule forms of orgni C suh s sugrs, orgni is, muilge, sloughe ell wlls n root hirs, ut n lso inlue nitrogen (N)-ontining orgni ompouns suh s mino is (H utsh et l., 22). Owing to the ongoing supply of lile root-erive rhizoeposit ron (RDC), the rhizosphere is hrterize y surplus of RDC whih n inrese, erese or not ffet the eomposition of SOC leing to positive, negtive or neutrl RPE, respetively (Kuzykov et l., 2). Mesure RPEs hve rnge from 7% to 38% (Zhu & Cheng, 211; Cheng et l., 214). Severl mehnisms hve een propose to explin ifferent RPEs. Positive RPEs re elieve to e riven y o-metolism of SOC with RDC (Kuzykov et l., 2), the tivtion of miroes le to ess n egre SOC (Fontine et l., 23) or N mining from N-rih SOC when N is limite (Murphy et l., 215). By ontrst, preferentil utiliztion of RDC involving swith from SOC to RDC y miroes, n N onstrints on SOC eomposition resulting from ompetition for N etween plnts n miroorgnisms, hve een propose to ount for negtive RPEs (Shimel et l., 1989). The miroil tivtion theory for positive RPE is supporte y the ft tht positive RPEs re often ssoite with inrese miroil iomss (Kuzykov, 21). The N mining n N ompetition theories knowlege the si N requirement of miroes to llow iomss umultion n exoenzyme synthesis in response to the ontinuous supply of RDC. Eh of these mehnisms my t lone or together, or the ominnt proess my hnge from one to nother over time, ounting for either positive or negtive RPEs (Blgotsky & Kuzykov, 28). The swith etween these mehnisms is possily triggere y hnges in the size n ommunity omposition of miroil ommunity (Tlot et l., 28; Doronikov et l., 29) or in nutrient vilility, espeilly N (Dijkstr et l., 213). Plnt speies hs een wiely reognize for its importne in regulting the mgnitue of RPE. For exmple, leguminous rop speies onsistently show greter RPEs thn nonlegumes (Fu & Cheng, 22; Cheng et l., 23; Cheng & Kuzykov, 25), n some tree speies suh s Poneros pine proue more prominent RPE thn other speies suh s Fremont ottonwoo 864

2 New Phytologist Reserh 865 (Dijkstr & Cheng, 27). Speies vritions in RPEs re frequently ttriute to ifferenes in the quntity n qulity of RDC (Cheng et l., 214). Other plnt trits suh s shoot n root iomss, n plnt phenology n lso ffet RPEs, ut the propose mehnisms re inevitly referre to plnt growthinue hnge in RDC (Fu & Cheng, 22; Cheng et l., 23; Dijkstr et l., 26). Positive reltionships etween the mount of RDC n RPEs hve een oserve in some tree speies (Dijkstr & Cheng, 27; Phillips et l., 211; Bengtson et l., 212). Also, greter RPEs of soyen thn whet or sunflower re elieve to e ttriute to its N-rih RDC (Fu & Cheng, 22; Cheng et l., 23; Cheng & Kuzykov, 25). Plnt speies lso iffer gretly in their pity to hnge rhizosphere properties suh s ph. Some speies suh s hikpe re ple of iifying their rhizosphere to greter extent thn others suh s white lupin n fiel pe, ue to greter exess uptke of tions over nions (Tng et l., 1997, 1999). There is mple eviene suggesting tht soil ph n regulte SOC trnsformtion iretly through its effet on SOC soluility or iniretly y ltering miroil tivity (Anersson et l., 2; Kemmitt et l., 26; Rousk et l., 29). The gretest priming effets re etete in the ph rnge of 6 8 (Blgotsky & Kuzykov, 28). Nevertheless, the effet of root-inue rhizosphere iifition on root-inue eomposition of SOC, nmely RPE, is unknown. Furthermore, little ttention hs een given to the integrtion of multiple ftors n their reltive importne in efining the resultnt iretion n mgnitue of RPE. The ojetives of this stuy were to: exmine RPEs of rop speies with ontrsting rhizosphere iifition n root exution, n link the ifferenes in RPE with rhizosphere properties inluing ph, miroil iomss C n N n plnt trits suh s root exutes, shoot n root iomss. We hypothesize tht rop speies woul iffer in RPE with the speies exuing greter mounts of RDC resulting in greter RPE, n mesure RPEs woul orrelte negtively with rhizosphere iifition. Mterils n Methos Soil The experimentl soil ws ollete from the top 1-m lyer of C 4 Kngroo grssln (Theme trinr) t property, 2 km north of Gulgong, New South Wles, Austrli (32 11 S, E). The soil ws grnite-erive sny lom, n ws ir-rie n psse through 2-mm sieve. The 2-mm soil h the following si properties: ph 5. (.1 M CCl 2 ), orgni ron (C) 27 g kg 1, totl nitrogen (N) 1.6 g kg 1, ly 13 g kg 1 n soil ph uffer pity 3 mmol kg 1 ph 1. The 13 C vlue of the soil ws 18&. The 13 C signture of ifferent soil orgni ron (SOC) pools ws lso nlyse. Briefly, 2 g of ry soil ws sieve n ivie into pools with ifferent prtile sizes: < 53, 53 25, 25 5, 5 1, 1 2 lm. To ollet prtiulte orgni ron, nother 2 g of soil ws e into 8 ml wter n the soil suspension ws psse through 53-lm sieve. The orse orgni mterils retine on the 53-lm sieve were rinse, seprte from sn n ir-rie. All SOC frtions were ll-mille efore eing nlyse for 13 C y isotope rtio mss spetrometry (IRMS) (Seron 2-22, Crewe, UK). Experimentl set up This olumn experiment use 13 C nturl unne pproh where C 3 plnts were grown in the C 4 soil, n the 13 C ws use s trer to seprte plnt-erive CO 2 -C from soil-erive CO 2 -C. Common rop speies, hikpe (Cier rietinum L. v Slsher), fiel pe (Pisum stivum L. v Kosp), whet (Tritium estivum L. v Yitpi) n white lupin (Lupinus lus L. v Kiev), selete for their ontrsting effet on soil iifition n root exution, were grown in PVC olumns (imeter 1 m, height 4 m) ontining 2.8 kg ir-rie C 4 soil with the following sl nutrients (mg kg 1 ): KH 2 PO 4, 18; K 2 SO 4, 12; CCl 2.2H 2 O, 18; MgSO 4.7H 2 O, 5; MnSO 4.H 2 O, 15; ZnSO 4.7H 2 O, 9; CuSO 4.5H 2 O, 6; N 2 MoO 4.2H 2 O,.4; FeEDTA, 5.5. Four replites of eh speies were inlue. Columns without plnts (+/ N) were lso inlue s ontrols. Plnts were estrutively hrveste t 35, 56 n 84, whih represent the erly vegettive, lte vegettive n flowering stges, respetively, for whet n white lupin, n lte vegettive, flowering n grin-filling/mturity stge, respetively, for hikpe n fiel pe. Beuse isotopi frtiontion etween root tissue n root-respire CO 2 hs een inresingly reognize, n itionl set of olumns fille with orse river sn were lso inlue for eh speies with two replites. These itionl olumns were set up s esrie previously exept tht the wshe river sn, free of orgni C, ws inoulte with 1% (w/w) of C 3 soil efore plnting eh speies. The sn-fille olumns were wtere with Hogln solution (Hogln & Arnon, 195) to replenish oth wter n nutrients. Plnting The experiment ws onute in ontrolle environment room, with tempertures set t 14 h 2 C : 1 h 18 C, y : night, light intensity t 4 lmol m 2 s 1. Twelve pre-germinte sees of whet n eight sees of hikpe, fiel pe n white lupin were sown in row into eh olumn. All of the legumes were inoulte with n pproprite rhizoium inoulnt (EsyRhiz, New- Ege Miroils, Alury, Austrli). For whet, ure ws pplie t rteof3mgnkg 1 weekly from Week 2. In orer to reue the potentil of eveloping neroi onitions, 25 g of plsti es sele in nylon gs (mesh size 5 lm) were ple t the ottom of eh olumn. After emergene, plnts were thinne to seven plnts per olumn for whet n four per olumn for other speies. The soil wter ontent ws monitore grvimetrilly n mintine ily t. 8% of fiel pity. CO 2 trpping The CO 2 -trpping system onsiste of n inlet t the top of eh olumn where CO 2 -free ir ws introue fter eing pumpe

3 866 Reserh New Phytologist CO 2 -free ir Wter inlet Blu-Tk sel Cler PVC plte Vinyl tuing I M NOH Vlve.5 M NOH Air pump Vuum pump Air stone Plsti es () Air stone through 1 M NOH vi n ir stone (Fig. 1). On the opposite sie of the soil olumn, t the se, the outlet ws fitte with vuum line pssing through two CO 2 trps ontining 15 ml of.5 M NOH. The simultneous opertion of the pump n vuum filitte irflow through the soil olumns, reue the pressure within the hespe n stoppe possile leking roun the plnt stems. Before trpping CO 2 -C, two piees of hlve PVC sheet with prerille holes for plnt stems were fitte roun the plnt se n sele using Blu-Tk (Bostik, Thomstown, Austrli) (Fig. 1). The system ws heke for possile ir leks y vuuming the CO 2 -free ir (no pumping) through the soil olumn, efore it ws onnete to the trp of.5 M NOH (Fig. 1). A omplete sel t plnt se ws inite y ules in the trp of 1 M NOH t moerte vuum fore, euse low pressure oul e esily uilt up in sele system. All previously umulte CO 2 ws then remove y irulting CO 2 -free ir for eh olumn for 3 min. Totl elowgroun CO 2 ws trppe for 3 min every 6 h for 2 (Fig. 1). The ir-flow rte ws ontrolle t 8 1 ml min 1 to mximize the trpping effiieny (Cheng & Colemn, 1989). Plnts were wtere every y immeitely fter 3-min CO 2 trpping y injeting wter through the prerille holes (sele with Blu-Tk) on the PVC sheet in mounts se on the plnt requirement efore trpping. Although two trps were inlue to gurntee tht ll CO 2 ws pture, no CO 2 ws etete in the seon trp. Only the first trp ws use for quntifying n smpling CO 2 -C. Hrvest n mesurements Plnts were hrveste immeitely fter eh CO 2 olletion. Plnt shoots were ut off t the soil surfe one olumn t time, in orer to minimize the eomposition of root exutes. The rhizosphere soil ws ollete y quikly shking off soil here to the roots n psse through 2-mm sieve to remove roken roots. Before moving on to the next olumn, the ollete rhizosphere soil ws immeitely store t 4 C for lter etermintion of soil miroil iomss ron (MBC) n soil respirtion. After hrvest, plnt roots were wshe refully with tp wter () Fig. 1 Trpping system of CO 2 relese from elowgroun; () lek heking, (, ) CO 2 trpping. n root length n imeter were quntifie using WinRHIZO Pro 23 (Regent Instruments, Quee City, Cn) n n EPSON EU-35 snner (Seiko Epson Corp., Suw, Jpn). All plnt mterils were oven-rie t 7 C n weighe. After the shoot n root smples were groun using ll mill, the 13 Cin the smples ws nlyse using IRMS. A susmple of homogenize rhizosphere soil ws ir-rie for the etermintion of soil ph, n totl orgni C n N ontent. Soil ph ws mesure in.1 M CCl 2 (1 : 5 soil : solution rtio, 1-h en-over-en shking, entrifuging t 7 g for 1 min). The C n N ontents of shoots, roots n soil smples were etermine y ry omustion using CHNS Anlyser (PerkinElmer EA24, Shelton, CT, USA). Soil respirtion ws etermine uring 12-h inution of rhizosphere soil t 25 C (Rukshn et l., 212). Respire CO 2 - C ws use s mesure of the miroil utiliztion of root exutes se on oservtions tht root exutes require only few hours to e respire in ontrst to few ys for other RDC suh s sloughe-off ell wlls n root hirs (Gregory, 26; Fisher et l., 21). Briefly, 1 g of fresh soil ws ple in glss inution jr n the CO 2 onentrtion within the hespe of inution jrs ws mesure using n infrre gs nlyser (421 Inustril Gs Anlyser; Servomex, Coworough, UK). The MBC ws etermine using hloroform fumigtionextrtion oring to Vne et l. (1987). Totl orgni C in the extrts (.5 M K 2 SO 4 ) ws etermine olorimetrilly fter wet igestion with K 2 Cr 2 O 7 n onentrte H 2 SO 4 t 135 C for 3 min (Ci et l., 211). Totl solule N ws mesure using QuikChem 85 flow injetion nlyser (Lht Instruments, Loveln, CO, USA) fter persulfte oxition of oth fumigte n nonfumigte extrts (Crer & Bere, 1993). The MBC n miroil iomss N (MBN) were lulte s the ifferene in totl orgni C n N onentrtion etween fumigte n nonfumigte soils, juste y proportionlity oeffiient (.45) for oth C n N (Jenkinson et l., 24). Soil inorgni N (NH NO 3 ) ws lso mesure y flow injetion nlyser (Lht Instruments) on nonfumigte.5 M K 2 SO 4 extrts. The mount of CO 2 in the lkli trps ws etermine y titrting 1 ml of.5 M NOH solution with.5 M HCl fter

4 New Phytologist Reserh 867 ing 5 ml 1 M BCl 2. To form pure SrCO 3 preipittes to quntify the 13 C of the trppe CO 2, 5 ml of 1 M SrCl 2 ws e to nother 1 ml of the.5 M NOH solution. The suspension ph ws juste to 7. y ing.3 M HCl t n mount se on the titrtion. The 13 C of the SrCO 3 ws nlyse y IRMS. Clultions The 13 C vlues mesure in the CO 2 trps were orrete for ontmintion from ir using the equtions of mss onservtion (Mry et l., 1992): C t ¼ C 1 þ C 2 t C t ¼ 1 C 1 þ 2 C 2 1 ¼ð t C t 2 C 2 Þ=ðC t C 2 Þ (C t, totl mount of C in the smple solution inluing C 2 ;C 1, mount of C in the smple solution without C 2 ;C 2, mount of C in lnk solutions; t, 13 C vlue of smple efore orretion; 1, 13 C vlue of smple fter orretion; 2, 13 C vlue of the ir ( 8&)). The totl CO 2 efflux from plnte soil ws prtitione using the following equtions (Mry et l., 1992): C 1 ¼ C t ð t 2 Þ=ð 1 2 Þ (C t, totl C from elowgroun CO 2 ; t, 13 C vlue of the C t ; C 1, mount of C erive from the C 4 soil; 1, 13 C vlue of the C 4 soil C ( 13 C vlue of CO 2 -C relese from the ontrol soil, i.e. no plnt); 2, 13 C vlue of the C 3 root-erive C (CO 2 trppe from the sn olumn)). Sttistil nlysis A two-wy ANOVA ws onute to ssess the effets of speies n hrvest time on plnt iomss, plnt N onentrtions, root length n 13 C unne of root n root-erive CO 2 (Tle 1). Signifint (P =.5) ifferenes etween mens were ientifie using Tukey s HSD test. One-wy ANOVA (Tukey s test) ws use to ompre soil-erive CO 2, rhizosphere ph, soil respirtion n miroil iomss C n N etween tretments t eh hrvest. The reltionship etween the rhizosphere priming effet (RPE) n selete vriles ws quntifie y onuting multiple regression nlyses, with the most signifint vriles in regulting RPE etermine y forwr stepwise regression proeure using GENSTAT (v.11; VSN Interntionl, Hemel Hempste, UK). Results Plnt growth Chikpe proue the gretest shoot iomss t Dy 84, followe y fiel pe, whet n white lupin. The root ry weight Tle 1 Shoot n root iomss, nitrogen (N) onentrtion, root length n 13 C unne of hikpe (Cier rietinum), fiel pe (Pisum stivum), whet (Tritium estivum) n white lupin (Lupinus lus) grown for 38, 56 n 84 Biomss (g per olumn) N onentrtion (mg g 1 ) 13 C unne (&) Speies Growth stge Shoot Root Shoot Root Root length (m per olumn) Root Root-erive CO 2 Dy 38 Chikpe Lte vegettion e e Fiel pe Lte vegettion ef e 28. Whet Erly vegettion g White lupin Erly vegettion g 3.9f Dy 56 Chikpe Flowering f ef Fiel pe Flowering Whet Lte vegettion f e White lupin Lte vegettion f 29.3ef Dy 84 Chikpe Poing 19.5f 4.6g f 333.7e Fiel pe Mturity 16.39e Whet Lte flowering 15.51e 2.29e f White lupin Lte flowering e 31.1f Two-wy ANOVA Speies *** *** *** ** *** *** *** Hrvest time *** *** *** * *** *** *** Speies 9 Hrvest time *** *** *** *** *** *** ** *, P <.5; **, P <.1; ***, P <.1. For eh olumn, ifferent letters inite signifint ifferenes etween mens (two-wy ANOVA, Tukey s test, P <.5).

5 868 Reserh New Phytologist of hikpe ws 2.2, 3.8 n 6.6 times greter thn whet, white lupin n fiel pe, respetively, when verge over three hrvests (Tle 1). Whet showe the gretest root length: 1.5 times greter thn hikpe n 1 times greter thn white lupin n fiel pe. The N onentrtion ws gretest in the shoot n root of white lupin, n lowest in fiel pe (Tle 1). Totl elowgroun CO 2 efflux n 13 C unne The 13 C vlue for the roots of hikpe, whet, white lupin n fiel pe rnge from 28.5 to 32.8&. On the one hn, the 13 C vlue of CO 2 trppe from sn-fille olumns ws slightly enrihe (.3 1.8&) ompre with tht in the root tissue, initing isotopi frtiontion etween root tissue n root-respire CO 2 (Tle 1). On the other, the 13 C vlue of the prtiulte orgni mtter in the soil ( 14.8&) ws higher thn those of soil prtiles <.5 mm ( 18.8&). This suggeste tht ifferent SOC pools h ifferent 13 C vlues, with the esily eomposle C more 13 C enrihe thn the more nonlile C frtions ssoite with soil minerls (t not shown). Totl CO 2 efflux from the nonplnte ontrol showe slight eline over time (Fig. 2), n the 13 C vlue of CO 2 evolve ws onstnt throughout the experiment ( 14.5 to 15.2&) (Fig. 2). Growing plnts inrese totl CO 2 efflux y 24 43% ompre with the ontrols. The 13 C vlue of totl CO 2 ollete from plnte olumns ( 23.2 to 26.6&) ws signifintly lower thn the ontrol, n eme more eplete over time ( 29.7 to 3.7&) exept for fiel pe (Fig. 2). The 13 C vlue of CO 2 trppe for fiel pe erese y 3.9& t Dy 56 ut inrese y 1.6& t Dy 84. Chikpe showe muh lower 13 C vlue in the totl evolve CO 2 thn other speies (Fig. 2) (P <.5). Soil-erive CO 2, prime C n rhizosphere soil C onentrtion White lupin showe the gretest positive RPE t ys 38 n 56 (Fig. 3), with ily soil-erive CO 2 inresing y 53% reltive to the ontrol, n followe y 4% for whet (Fig. 3) (P <.5). The positive RPE of whet n white lupin showe erese t Dy 84. By ontrst, no RPE ws etete for fiel pe t Dy 38, ut soil-erive CO 2 inrese y 95% t Dy 84, ompre with the ontrol (Fig. 3) (P <.5). Both hikpe n fiel pe showe negtive RPE t Dy 56, where soilerive CO 2 erese y 7% n 4%, respetively (Fig. 3,) () Totl elowgroun CO 2 efflux (mg C kg 1 soil 1 ) () 1 δ 13 C of CO 2 ( ) Time fter plnting () Chikpe Fiel pe Whet White lupin Control + Ure Control Fig. 2 () Totl elowgroun CO 2 efflux n () 13 C vlues of CO 2 evolve from soil olumns with hikpe (Cier rietinum), fiel pe (Pisum stivum), whet (Tritium estivum), white lupin (Lupinus lus) n nonplnte ontrols t ys 38, 56 n 84. Error rs represent SEM of four replites. () Soil-erive CO 2 -C (mg C kg 1 soil 1 ) () Prime soil C (mg C kg 1 soil 1 ) Chikpe Whet Control e Fiel pe White lupin 38 ys 56 ys 84 ys Time fter plnting () Fig. 3 () Soil-erive CO 2 C n () prime soil ron (C) from soil olumns uner hikpe (Cier rietinum), fiel pe (Pisum stivum), whet (Tritium estivum), white lupin (Lupinus lus) n nonplnte ontrols t ys 38, 56 n 84. Error rs represent SEM of four replites. For eh pnel, ifferent itli letters ove the rs inite signifint ifferenes mong speies t eh hrvest time (Tukey s test, P <.5).

6 New Phytologist Reserh 869 () Rhizosphere SOC (mg g 1 ) () Rhizosphere soil ph () Rhizosphere soil respir on (μg CO 2 g 1 soil) Chikpe Fiel pe Whet White lupin Control (P <.5). The erese in prime C y hikpe ws persistent t Dy 84, lthough to lesser extent. Consistently, when ompre with the ontrol, the presene of white lupin erese SOC onentrtion in the rhizosphere soil y 4% (P <.5) t the en of the experiment (Fig. 4). By ontrst, SOC onentrtion in the rhizosphere of hikpe ws equl to or greter thn the ontrol. There ws no hnge in SOC onentrtion in the rhizosphere of fiel pe n whet t ys 38 n 56, ut SOC erese y 4% t Dy 84 for fiel pe. Dy 38 Dy 56 Dy 84 Time fter plnting () Fig. 4 () Soil orgni ron (SOC), () ph n () respirtion of the rhizosphere soil of hikpe (Cier rietinum), fiel pe (Pisum stivum), whet (Tritium estivum), white lupin (Lupinus lus) n nonplnte ontrols t ys 38, 56 n 84. Error rs represent SEM of three replites. For eh pnel, ifferent itli letters ove the rs inite signifint ifferenes mong speies t eh hrvest time (Tukey s test, P <.5). Inorgni N (mg N kg 1 soil) Chikpe Whet Control Fiel pe White lupin Dy 38 Dy 56 Dy 84 Time fter plnting () Fig. 5 Inorgni nitrogen (N) (NH NO 3 ) onentrtions in the rhizosphere soil of hikpe (Cier rietinum), fiel pe (Pisum stivum), whet (Tritium estivum), white lupin (Lupinus lus) n nonplnte ontrols t ys 38, 56 n 84. Error rs represent SEM of three replites. Different itli letters ove the rs inite signifint ifferenes mong speies t eh hrvest time (Tukey s test, P <.5). Rhizosphere ph, soil respirtion n inorgni N onentrtion Among the four speies, only hikpe iifie its rhizosphere (y ph units) (Fig. 4) (P <.5). The rhizosphere ph of other speies ws the sme or higher thn the ontrol. For ll tretments, rhizosphere ph tene to eline with growth stge (Fig. 4). Regrless of growth stge, the rhizosphere soil respirtion uring the 12-h inution ws onsistently the gretest in the rhizosphere of white lupin, followe y hikpe, whet n fiel pe n the lowest in the ontrol (Fig. 4). Inorgni N (NH NO 3 ) onentrtions were lower in the rhizosphere soil of ll speies thn in the ontrol, espeilly for fiel pe n hikpe (Fig. 5). For fiel pe, greter inorgni N onentrtion ws etete in the rhizosphere soil t Dy 84 thn t ys 38 n 56. Rhizosphere soil miroil C n N When ompre with the ontrol, the presene of white lupin inrese MBC (Fig. 6) (P <.5). An inrese in MBC in the rhizosphere of fiel pe ws only etete t Dy 84. The MBN showe n pprent response to rhizosphere effet for most speies. For instne, MBN ws invrily greter in the rhizosphere of white lupin n whet thn in the ontrol, wheres it ws 2% lower in the rhizosphere of hikpe thn in the ontrol t ys 58 n 84 (P <.5). The hnge in MBN in the rhizosphere of fiel pe showe istint pttern: erese y 3% t Dy 58 ut inrese y 25% t Dy 84, in omprison with the ontrol (Fig. 6). Correltion etween RPE n selete ftors Rhizosphere ph n MBN orrelte positively with RPE (Tle 2). However, rhizosphere ph n MBN only ounte e

7 87 Reserh New Phytologist () 5 Miroil C (mg C kg 1 soil) () 35 Miroil N (mg N kg 1 soil) for 52% of the vrition in RPE. When multiple regression nlysis ws performe t eh smpling time, soil respirtion n soil ph ounte for 78% of vrition in RPE t Dy 38. However, MBN, rhizosphere ph n soil respirtion expline 96% of hnge in RPE t Dy 58. At Dy 84, only rhizosphere ph ws shown s signifint vrile, ounting for 54% of the vrition in RPE. Disussion Chikpe Fiel pe Whet White lupin Control Dy 38 Dy 56 Dy 84 Time fter plnting () Fig. 6 Miroil iomss () ron (C) n () nitrogen (N) in the rhizosphere soil of hikpe (Cier rietinum), fiel pe (Pisum stivum), whet (Tritium estivum), white lupin (Lupinus lus) n nonplnte ontrols t ys 38, 56 n 84. Error rs represent SEM of three replites. For eh pnel, ifferent itli letters ove the rs inite signifint ifferenes mong speies t eh hrvest time (Tukey s test, P <.5). Speies vrition in RPE The four speies iffere sustntilly in their rhizosphere priming effet (RPE). Exept t Dy 84, white lupin showe the gretest positive RPE of ll speies, in ontrst to the negtive RPE proue y hikpe. This ws further eviene y n pprent loss n umultion of soil orgni ron (SOC) in the rhizosphere of white lupin n hikpe, respetively. The positive RPE of whet i not use signifint erese in SOC, possily euse the mount of C loss ue to RPE ws equl to the retention of C input in the forms of rhizoeposition. Using oth Tle 2 Prmeters of multiple regressions of the rhizosphere priming effet (RPE) s funtion of soil ph, miroil nitrogen (N) n soil respirtion Moel Prmeter Vlues Signifine level (P-vlue) All t Interept <.1 Soil ph 3.65 <.1 44 Miroil N r 2.54 Hrvest I Interept Soil respirtion Soil ph r 2.78 Hrvest II Interept Miroil N.25 <.1 77 Soil ph 5.35 <.1 16 Soil respirtion r 2.96 Hrvest III Interept Soil ph r 2.54 Vrition expline (%) the C uget n 13 C trer metho, Cheng (29) foun tht the RPE of whet plnts resulte in fster C yling in soil, ut no net hnge in SOC. Different RPEs hve lso een reporte mong other rop speies suh s mize n soyen, n tree speies suh s Poneros pine n Fremont ottonwoo (Fu & Cheng, 22; Cheng et l., 23; Dijkstr & Cheng, 27; Bengtson et l., 212). Our stuy expne the rnge of plnt speies for whih RPEs hve een quntifie through the inlusion of three rop legumes, with the mgnitue of RPE rnging from 7% to 53%. These vlues n tht otine for whet fell within the rnge of 7% to 38% (Zhu & Cheng, 211; Cheng et l., 214). Unerstning the impt tht vritions in plnt speies n hve on SOC eomposition oul help to efine the mehnisms ounting for hnges in SOC stoks uner fiel onitions. Plnt speies vrition in RPE ws lso ffete y plnt growth stge. Given tht the speies rehe ifferent growth stges t given hrvesting time, the speies vrition in RPE t eh hrvest might e ue prtly to vritions in plnt phenology. For exmple, fiel pe, the only speies tht rehe mturity t the finl hrvest, showe greter RPEs t Dy 84 thn ys 34 n 56. A greter RPE t mturity thn other growth stges ws lso etete for C 4 plnt speies Amrnthus (Fu & Cheng, 22). For the speies tht i not reh mturity y Dy 84, RPE tene to pek t lte vegettive stge n eline therefter, in line with finings of other stuies (Cheng et l., 23; Cheng & Kuzykov, 25). Nevertheless, the RPE of white lupin n whet oul hve een unerestimte t Dy 84, ue to the use of 13 C vlue of soil-erive CO 2 for the no-plnt ontrol olumn (i.e. 14.8&). The persistent positive RPE of whet n white lupin woul e expete to use epletion of lile SOC n possily stle SOC pools. In orer to test the sensitivity of RPE lultion to hnges in 13 C vlues of soil-erive C, we hnge the 13 C vlue from 14.8 to 18.8& (i.e. ssuming ll of the CO 2 originte from the stle C pools) n foun the

8 New Phytologist Reserh 871 RPE of these two speies were still lower t Dy 86 thn tht t ys 56 n 36. Thus, the erese in RPE of whet n white lupin with time ws not n experimentl rteft. Consiering tht plnt trits suh s root exutes n root morphology oul vry ross plnt growth stges, hnges in the RPE with time were expete. However, without gining eep n omplete unerstning of ll ftors regulting RPE, it is iffiult to explin speies vrition in RPE in this stuy. The speies vrition in RPE oul not e expline y ifferenes in root morphology. Neither root iomss nor totl length orrelte with RPEs ross the speies n growth stges. Although the lrger RPE of whet, whih lke notiele relese of root exutes, oul e prtly ttriute to its extensive root system, the negtive/low RPE of hikpe resulte minly from the gret pity of its lrge n ense root system to iify the rhizosphere soil. White lupin, low in totl root length, hs istint luster-root struture relesing lrge mounts of root exutes (Neumnn & Mrtinoi, 22), whih likely ontriute to the lrger RPE note in this stuy. In nother stuy, Fu & Cheng (22) foun tht the mount of prime-c orrelte positively with root iomss of C 3 plnt speies suh s soyen n sunflower. Priniplly, the length n morphology of plnt roots re elieve to ffet RPEs through influening the rhizosphere volume. Nevertheless, given tht only zones immeitely ehin the root tips re generlly tive in exution (Bri & Vivno, 29), lrger root iomss or length might not neessrily result in more root exution in the rhizosphere. It ppere tht plnt root physiologil trits suh s root exute relese n rhizosphere iifition plye more preominnt role in ounting for mesure RPEs thn root morphologil trits. Root exutes The signifint (P =.2) orreltion etween rhizosphere soil respirtion n RPE t Dy 35 suggeste tht the quntity of root exutes te s n importnt ftor ffeting RPE uring erly stges of plnt growth. In this stuy, white lupin ws selete for its well-known high root exution pity in ontrst to whet n fiel pe (Ss et l., 21; Venekls et l., 23; Wng et l., 28). As expete, the gretest rhizosphere soil respirtion n RPE of white lupin were in line with its pity to generte root exutes. Previous stuies lso showe tht the mount of root exutes relese ws positively orrelte with miroil growth n SOC eomposition (Dijkstr & Cheng, 27; Phillips et l., 211; Bengtson et l., 212). Our stuy provies further eviene tht plnt speies with high root exution exhiit greter RPE t erly growth stges. The mgnitue of RPE oul not e simply expline y hnges in the quntity of root exutes, s reflete y the poor orreltion etween soil respirtion n RPE t lte growth stges. On the one hn, for fiel pe, ellulr root mteril oul ontriute sustntilly to rhizoeposits following senesene of roots t mturity, in ition to root exutes (Gvito et l., 21; Wihern et l., 27; Arn et l., 213), whih oul ount for n inrese miroil growth n enhne RPE t Dy 84. The inorgni N onentrtion in the rhizosphere of fiel pe t Dy 84 nerly oule tht t Dy 56 (Fig. 5), possily refleting greter N eposits from the root t mturity. Nitrogen-rih sustrtes oul stimulte miroil growth, inrese the proution of exoenzymes n use greter priming effets thn sustrtes of low or no N (Drke et l., 213). On the other hn, the erese in RPE of whet n white lupin with time ws possily ttriute to eline in the esily eomposle SOC pool, s inite y eresing totl CO 2 evolve from the ontrol olumns. In short-term inution experiments, Kuzykov et l. (2) showe tht priming effets originte minly from the minerliztion of lile SOC. Drke et l. (213) lso foun tht the relese of low-moleulr-weight ompouns y plnt roots enhne the proution of exoenzymes trgeting low-moleulr-weight SOC. Finlly, other ftors suh s N vilility n rhizosphere ph oul lso hve ounte for the lk of positive reltionship etween root exutes n RPE t lte growth stges. N vilility Miroil N, ut not miroil C, orrelte positively with mesure vlues of RPE. At the lter growth stges, miroil C : N rtios were generlly higher in the rhizosphere of ll N 2 - fixing legumes thn whet whih ws regulrly fe with fertiliser N. In ition, higher miroil C : N rtios n negtive RPEs of hikpe n fiel pe were in orne with the lowest inorgni N onentrtion in their rhizospheres t Dy 56. Thus, lower miroil N n higher miroil C : N rtios possily reflete miroil N limittion with time. Severl stuies foun tht RPEs were onstrine t extremely low N onitions (Cheng & Kuzykov, 25; Drke et l., 213). Alterntively, low miroil N might e onsequene of negtive RPEs. In ontrst to positive RPEs, whih oul enhne miroil N immoiliztion (Kuzykov & Xu, 213), erese RPEs were inite to erese miroil uptke of soil-erive N n hene miroil iomss N. Further stuy is require to ssign the uses n effets etween miroil N n RPEs. Rhizosphere ph The speies vrition in RPE oul e prtilly ttriute to the plnt speies effet on rhizosphere ph. The greter pity of hikpe to iify the rhizosphere oul e expline y its pprent exess uptke of tions over nions uring N 2 fixtion (MLy et l., 1997; Tng et l., 1999). Low ph woul reue miroil iomss, miroil tivity n/or sustrte vilility (Anersson et l., 2; Kemmitt et l., 26; Rousk et l., 29), n hene RPE. In the present stuy, the rhizosphere ph of hikpe reue from 5 to 4.3, t whih miroil tivity oul e suppresse (Leifel et l., 28; Rousk et l., 21). Quiqumpoix (2) foun tht soil priming effets erese t low soil ph ue to the sorption of enzymes on surfes of ly prtiles. The gretest priming effets hve often een etete ner neutrl ph s isusse erlier (Blgotsky & Kuzykov, 28). Moreover, hikpe relese greter quntity of root

9 872 Reserh New Phytologist exutes thn whet or fiel pe in this stuy, or the sme mount s white lupin y others (Venekls et l., 23). The negtive/low RPEs of this speies suggeste tht low ph ws the preominnt ftor ffeting RPE. Rhizosphere iifition oul therefore e n importnt mehnism ontriuting to negtive RPE n rhizosphere ph shoul e reognize for its importnt role in regulting RPE. Further stuies re require to ientify the ounry ph vlue triggering the funtioning n reltive ontriutions of other mehnisms. Conlusion This stuy provie simplifie, low-ost n relile CO 2 trpping system for exmining RPEs of ifferent speies. Speies with ifferent pity to relese root exutes h quite ifferent RPEs. For the first time, rhizosphere ph ws revele s n importnt ftor ffeting the mgnitue n iretion of RPE, n oul prtly ount for speies vrition in RPE long with root exution, N vilility n lile ron pool size. Uner fiel onitions, option of legumes speies in the ropping system nees to onsier oth soil iifition n C loss or gin ue to RPE. For speies suh s white lupin with gret RPEs, mximizing plnt resiue return might e essentil to ompenste for the totl SOC loss from the system. Future stuies on either RPE moelling or mehnisti unerstning of RPE shoul lso onsier the effet of rhizosphere ph on SOC stility n longterm C storge. Aknowlegements This reserh ws supporte uner Austrlin Reserh Counil s Disovery Projets funing sheme (projet DP12141). Author ontriutions X.W. n C.T. plnne n esigne the reserh. X.W. n J.S. performe experiments n nlyse t. X.W., C.T., J.S., C.R.B. n J.A.B. wrote the mnusript. Referenes Anersson S, Nilsson SI, Setre P. 2. Lehing of issolve orgni ron (DOC) n issolve orgni nitrogen (DON) in mor humus s ffete y temperture n ph. Soil Biology n Biohemistry 32:1 1. Arn MM, Knight JD, Frrell RE Temporl ynmis of nitrogen rhizoeposition in fiel pe s etermine y 15 N lelling. Cnin Journl of Plnt Siene 93: Bri DV, Vivno JM. 29. Regultion n funtion of root exutes. Plnt, Cell & Environment 32: Bengtson P, Brker J, Gryston SJ Eviene of strong oupling etween root exution, C n N vilility, n stimulte SOM eomposition use y rhizosphere priming effets. Eology n Evolution 2: Blgotsky E, Kuzykov Y. 28. Mehnisms of rel n pprent priming effets n their epenene on soil miroil iomss n ommunity struture: ritil review. Biology n Fertility of Soils 45: Crer ML, Bere MH Alkline persulfte oxition for etermining totl nitrogen in miroil iomss extrts. Soil Siene Soiety of Ameri Journl 57: Ci Y, Peng C, Qiu S, Li Y, Go Y Dihromte igestionspetrophotometri proeure for etermintion of soil miroil iomss ron in ssoition with fumigtion-extrtion. Communitions in Soil Siene n Plnt Anlysis 42: Cheng W. 29. Rhizosphere priming effet: its funtionl reltionships with miroil turnover, evpotrnspirtion, n C-N ugets. Soil Biology n Biohemistry 41: Cheng W, Colemn DC A simple metho for mesuring CO 2 in ontinuous ir-flow system: moifitions to the sustrte-inue respirtion tehnique. Soil Biology n Biohemistry 21: Cheng W, Johnson DW, Fu SL. 23. Rhizosphere effets on eomposition: ontrols of plnt speies, phenology, n fertiliztion. Soil Siene Soiety of Ameri Journl 67: Cheng W, Kuzykov Y. 25. Root effets on soil orgni mtter eomposition. In: Wright S, Zoel R, es. Roots n soil mngement: intertions etween roots n the soil. Agronomy monogrph no. 48. Mison, WI, USA: Amerin Soiety of Agronomy, Cheng W, Prton WJ, Gonzlez-Meler MA, Phillips R, Aso S, MNikle GG, Brzostek E, Jstrow JD Synthesis n moelling perspetives of rhizosphere priming. New Phytologist 21: Dijkstr FA, Crrillo Y, Penll E, Morgn JA Rhizosphere priming: nutrient perspetive. Frontiers in Miroiology 4: 1 8. Dijkstr FA, Cheng WX. 27. Intertions etween soil n tree roots elerte long-term soil ron eomposition. Eology Letters 1: Dijkstr FA, Cheng WX. 27. Moisture moultes rhizosphere effets on C eomposition in two ifferent soil types. Soil Biology n Biohemistry 39: Dijkstr FA, Cheng WX, Johnson DW. 26. Plnt iomss influenes rhizosphere priming effets on soil orgni mtter eomposition in two ifferently mnge soils. Soil Biology n Biohemistry 38: Doronikov M, Blgotsky E, Blgotsky S, Fngmeier A, Kuzykov Y. 29. Stimultion of r- vs. K-selete miroorgnisms y elevte tmospheri CO 2 epens on soil ggregte size: reserh rtile. FEMS Miroiology Eology 69: Drke JE, Dry BA, Gisson MA, Krmer MA, Phillips RP, Finzi AC Stoihiometry onstrints miroil response to root exution insights from moel n fiel experiment in temperte forest. Biogeosienes 1: Fisher H, Ingwersen J, Kuzykov Y. 21. Miroil uptke of low-moleulr weight orgni sustnes out-ompetes sorption in soil. Europen Journl of Soil Siene 61: Fontine S, Mriotti A, Aie L. 23. The priming effet of orgni mtter: question of miroil ompetition? Soil Biology n Biohemistry 35: Fu S, Cheng W. 22. Rhizosphere priming effets on the eomposition of soil orgni mtter in C 4 n C 3 grssln soils. Plnt n Soil 238: Gvito ME, Curtis PS, Jkosen I. 21. Neither myorrhizl inoultion nor tmospheri CO 2 onentrtion hs strong effets on pe root proution n root loss. New Phytologist 149: Gregory PJ. 26. Plnt root growth, tivity n intertion with soil. Oxfor, UK: Blkwell Pulishing. Hogln DR, Arnon DI The wter-ulture metho for growing plnts without soil. Berkeley, CA, USA: University of Cliforni, College of Agriulture, Agriulturl Experiment Sttion. H utsh BW, Augustin J, Merh W. 22. Plnt rhizoeposition n importnt soure for ron turnover in soils. Journl of Plnt Nutrition n Soil Siene 165: Jenkinson DS, Brookes PC, Powlson DS. 24. Mesuring soil miroil iomss. Soil Biology n Biohemistry 36: 5 7. Kemmitt SJ, Wright D, Gouling KWT, Jones DL. 26. ph regultion of ron n nitrogen ynmis in two griulturl soils. Soil Biology n Biohemistry 38: Kuzykov Y. 21. Priming effets: intertions etween living n e orgni mtter. Soil Biology n Biohemistry 42: Kuzykov Y, Frieel JK, Sthr K. 2. Review of mehnisms n quntifition of priming effets. Soil Biology n Biohemistry 32: Kuzykov Y, Xu X Competition etween roots n miroorgnisms for nitrogen: mehnisms n eologil relevne. New Phytologist 198:

10 New Phytologist Reserh 873 Leifel J, Zimmermnn M, Fuhrer J. 28. Simulting eomposition of lile soil orgni ron: effets of ph. Soil Biology n Biohemistry 4: Mry B, Mriotti A, Morel JL Use of 13 C vritions t nturl unne for stuying the ioegrtion of root muilge, roots n gluose in soil. Soil Biology n Biohemistry 24: MLy CDA, Brton L, Tng C Aiifition potentil of ten grin legume speies grown in nutrient solution. Austrlin Journl of Agriulturl Reserh 48: Murphy CJ, Bggs EM, Morley N, Wll DP, Pterson E Rhizosphere priming n promote moilistion of N-rih ompouns from soil orgni mtter. Soil Biology n Biohemistry 81: Neumnn G, Mrtinoi E. 22. Cluster roots: n unergroun pttion for survivl in extreme environments. Trens in Plnt Siene 7: Phillips RP, Finzi AC, Bernhrt ES Enhne root exution inues miroil feeks to N yling in pine forest uner long-term CO 2 fumigtion. Eology Letters 14: Quiqumpoix H. 2. Mehnisms of protein sorption on surfes n onsequenes for extrellulr enzyme tivity in soil. In: Bollg JM, Stotzky G, es. Soil iohemistry, vol. 1. New York, NY, USA: Mrel Dekker, Rousk J, Bth E, Brookes PC, Luer CL, Lozupone C, Cporso JG, Knight R, Fierer N. 21. Soil teril n fungl ommunities ross ph grient in n rle soil. ISME Journl 4: Rousk J, Brookes PC, Bth E. 29. Contrsting soil ph effets on fungl n teril growth suggests funtionl reunny in ron minerlistion. Applie n Environmentl Miroiology 75: Rukshn F, Butterly CR, Blok JA, Xu JM, Tng C Moel orgni ompouns iffer in priming effets on lklinity relese in soils through ron n nitrogen minerlistion. Soil Biology n Biohemistry 51: Ss L, Rengel Z, Tng C. 21. Root morphology, exess tion uptke, n extrusion of proton n orgni i nions in Lupinus lus L. uner phosphorus efiieny. Plnt Siene 16: Shimel JP, Jkson LE, Firestone MK Sptil n temporl effets on plnt miroil ompetition for inorgni nitrogen in Cliforni nnul grssln. Soil Biology n Biohemistry 21: Tlot JM, Allison SD, Treseer KK. 28. Deomposers in isguise: myorrhizl fungi s regultors of soil C ynmis in eosystems uner glol hnge. Funtionl Eology 22: Tng C, MLy CDA, Brton L A omprison of the potentil proton exretion of twelve psture legumes grown in nutrient solution. Austrlin Journl of Experimentl Agriulture 37: Tng C, Unkovih MJ, Bowen JW Ftors ffeting soil iifition uner legumes. III. Ai proution y N 2 -fixing legumes s influene y nitrte supply. New Phytologist 143: Vne ED, Brookes PC, Jenkinson DJ An extrtion metho for mesuring soil miroil iomss C. Soil Biology n Biohemistry 19: Venekls EJ, Stevens J, Cwthry GR, Turner S, Grigg AM, Lmers H. 23. Chikpe n white lupin rhizosphere roxyltes vry with soil properties n enhne phosphorus uptke. Plnt n Soil 248: Wng X, Tng C, Guppy CN, Sle PWG. 28. Phosphorus quisition hrteristis of otton (Gossypium hirsutum L.), whet (Tritium estivum L.) n white lupin (Lupinus lus L.) uner P efiient onitions. Plnt n Soil 312: Wihern F, Myer J, Joergensen RG, M uller T. 27. Rhizoeposition of C n N in pes n ots fter 13 C 15 N oule lelling uner fiel onitions. Soil Biology n Biohemistry 39: Zhu B, Cheng W Rhizosphere priming effet inreses the temperture sensitivity of soil orgni mtter eomposition. Glol Chnge Biology 17: New Phytologist is n eletroni (online-only) journl owne y the New Phytologist Trust, not-for-profit orgniztion eite to the promotion of plnt siene, filitting projets from symposi to free ess for our Tnsley reviews. Regulr ppers, Letters, Reserh reviews, Rpi reports n oth Moelling/Theory n Methos ppers re enourge. We re ommitte to rpi proessing, from online sumission through to pulition s rey vi Erly View our verge time to eision is <28 ys. There re no pge or olour hrges n PDF version will e provie for eh rtile. The journl is ville online t Wiley Online Lirry. Visit to serh the rtiles n register for tle of ontents emil lerts. If you hve ny questions, o get in touh with Centrl Offie (np-entrloffie@lnster..uk) or, if it is more onvenient, our USA Offie (np-usoffie@lnster..uk) For sumission instrutions, susription n ll the ltest informtion visit