The Influence of Rhizobium and Arbuscular Mycorrhizal Fungi on Nitrogen and Phosphorus Accumulation by Vicia faba

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1 Annls of Botny 94: , 24 oi:1.193/o/mh135, vill onlin t Th Influn of Rhizoium n Arusulr Myorrhizl Fungi on Nitrogn n Phosphorus Aumultion y Vii f YINSUO JIA, VINCENT MYLES GRAY* n COLIN JOHN STRAKER Shool of Molulr n Cll Biology, Univrsity of th Witwtrsrn, Privt g 3, Wits 25, Johnnsurg, South Afri Riv: 1 Sptmr 23 Rturn for rvision: 23 Jnury 24 Apt: 8 April 24 Pulish ltronilly: 17 Jun 24 Bkgroun n Aims Th im of this stuy ws to invstigt th ffts of th intrtions twn th miroil symionts, Rhizoium n rusulr myorrhizl fungi (AMF) on N n P umultion y ro n (Vii f) n how inrs N n P ontnt influn iomss proution, lf r n nt photosynthti rt. Mthos A multi-ftoril xprimnt onsisting of four iffrnt lgum miroil symioti ssoitions n two nitrogn trtmnts ws us to invstigt th influn of th iffrnt miroil symioti ssoitions on P umultion, totl N umultion, iomss, lf r n nt photosynthsis in ro n grown unr low P onitions. Ky Rsults AMF promot iomss proution n photosynthti rts y inrsing th rtio of P to N umultion. An inrs in P ws onsistntly ssoit with n inrs in N umultion n N proutivity, xprss in trms of iomss n lf r. Photosynthti N us ffiiny, irrsptiv of th inorgni sour of N (.g. NO 3 or N 2 ), ws nhn y inrs P supply u to AMF. Th prsn of Rhizoium rsult in signifint lin in AMF oloniztion lvls irrsptiv of N supply. Without Rhizoium, AMF oloniztion lvls wr highr in low N trtmnts. Prsn or sn of AMF i not hv signifint fft on noul mss ut high N with or without AMF l to signifint lin in noul iomss. Plnts with th Rhizoium n AMF symioti ssoitions h highr photosynthti rts pr unit lf r. Conlusions Th rsults init tht th synrgisti or itiv intrtions mong th omponnts of th triprtit symioti ssoition (Rhizoium AMF ro n) inrs plnt proutivity. ª 24 Annls of Botny Compny Ky wors: Arusulr myorrhizl fungi (AMF), nitrogn, phosphorus, Rhizoium, Vii f. INTRODUCTION Unr low soil P onntrtions, most plntspis r pnnt on symioti ssoition with rusulr myorrhizl fungi (AMF) for th quisition of P (Smith n R, 1997). Unr low N frtilizr inputs, soil P vilility is usully th mjor ftor limiting th rt of N 2 -fixtion in lgum rops (Toro t l., 1998) n, in th sn of AMF inftion, supplmntry P frtiliztion is gnrlly nssry for th mintnn of N 2 -fixtion rts y Rhizoium t th lvls rquir for onomilly vil rop proution (Anr t l., 1998). In lgums th positiv synrgisti intrtions mong th mmrs of th triprtit symioti ssoition (Rhizoium AMF lgum) rsult in improv rts of P uptk N 2 -fixtion n rop iomss proution unr onitionsofrunnpfrtilizrinputs(azóntl.,1991; Xvir n Grmi, 22, 23). Howvr, thr is littl informtion on th influn of P on N proutivity or photosynthti N us ffiiny. Nitrogn proutivity hs n fin s th rt of iomss proution pr unit iomss N ontnt (Ågrn, 1985), whrs th photosynthti N us ffiiny is th mount of CO 2 fix (mol CO 2 m 2 s 1 ) pr unit iomss N ontnt. In gnrl, photosynthti n spifi growth rts inrs with inrsing plnt tissu N onntrtion or N supply in urvilinr fshion (Hiros n Wrgr, 1987; Sinlir n Hori, 1989; Ji n Gry, 24). This stuy tsts th hypothsis tht th synrgisti intrtions mong th mmrs of th triprtit symioti ssoition improv lgum proutivity through positiv ffts on th rts of photosynthti CO 2 ssimiltion, N 2 -fixtion n P uptk. Furthrmor, it is propos tht ths thr prosss r intrpnnt or vn tightly oupl. For xmpl, th rt of photosynthti CO 2 ssimiltion is influn y th rts of N n P supply, n th rt of N 2 - fixtion is influn y th rts of photosyntht n P supply to th nouls. Thus, th min ojtivs of this stuy wr to invstigt: () how th intrtions twn th miroil symionts, Rhizoium n AMF fft th rt of N n P umultion in ro n; () how N proutivity, irrsptiv of th inorgni sour of N (.g. NO 3 or N 2 ) ws fft y inrs P supply u to AMF; n () how photosynthti N us ffiiny, irrsptiv of th inorgni sour of N (.g. NO 3 or N 2 ), ws fft y inrs P supply u to AMF oloniztion. MATERIALS AND METHODS Plnt mtril n growth onitions Th ol-hry Vii f L. Aquul Clui (Strthof S Group), rquiring for rop vlopmnt, ws us. Outoor pot trils wr rri out in spring from August to Otor (mn ily xtrtrrstril * For orrsponn. E-mil vin@gko.iol.wits..z Annls of Botny 94/2, ª Annls of Botny Compny 24; ll rights rsrv Downlo from y gust on 1 Otor 218

2 252 Ji t l. N n P Aumultion in Vii f glol insoltion MJ m 2 ; mn y lngth h; mn ily mximum tmprtur C; mn ily minimum tmprtur C n mn A-pn vportion 8 64 mm 1 ). Initilly thr ss wr plnt pr pot in sn in 1.6-L pots. Th sn (25 mm siv msh) us in ll th trtmnts ws first thoroughly wsh with running tp wtr n thn utolv for 3 h t 121 C n 134 kp in mtl ukts. Th initil ph of th sn ws 7. Aftr grmintion n shoot mrgn, on sling of uniform siz pr pot ws slt n th othrs rmov. Exprimntl sign Th xprimntl sign ws rnomiz omplt lok with four rplitions for h hrvst intrvl, with omintions of trtmnt ftors rnomly ssign to pots in th lok. Th onntrtion of nitrt-n for th low N (LN) trtmnts ws 71 mm KNO 3 (1 ppm N) n 1786 mm KNO 3 (25 ppm N) for th high N trtmnts (HN). All trtmnts riv phosphorus supply of 5 mg P L 1 (161 mm NH 2 PO 4 ). Th vrious trtmnts wr: () two iffrnt N supply rts without ny miroil symioti ssoitions, LN n HN; () two iffrnt N supply rts plus AMF ssoition, LNM n HNM; () two iffrnt N supply rts plus Rhizoium ssoition, LNR n HNR; n () two iffrnt N supply rts plus oth AMF n Rhizoium symioti ssoitions: LNMR n HNMR. Plnts wr hrvst t 54 (4 Otor, T 1 ) n 63 (13 Otor, T 2 ) ftr plnting. Nutrints A moifi Long Ashton nutrint mixtur ws us to supply th non-nitrogn miro- n mro-nutrints. For th mironutrints 1 stok solutions wr m up s follows: NH 2 PO 4 28 gl 1, MgSO 4 7H 2 O369 gl 1, MnSO 4 H 2 O.223 g L 1, CuSO 4 5H 2 O 24 g L 1, ZnSO 4 7H 2 O29 g L 1,H 3 BO g L 1, (NH 4 ) 6 Mo 7 O 24 4H 2 O4 g L 1, CoSO 4 7H 2 O3 g L 1, NCl 585 g L 1. For th mro-nutrints, 1 stok solutions wr m up s follows: CCl 2 5 g L 1, K 2 SO g L 1, FEDTA 3 g L 1. Aftr ing th pproprit quntity of KNO 3 (HN or LN), 1 ml of th stok solutions wr. Th ph of th nutrint solutions ws thn just to ph 7 n th solution ws m up to 1 ml with istill wtr for ing ppli to th pots. Bus of th low wtr-holing pity of th snfill pots, it ws nssry to wtr plnts vry 2 with 15 ml of th moifi Long Ashton nutrint solution. Inlusion of 218 g L 1 of K 2 SO 4 in ll trtmnts mnt tht potssium supply, whil unnt for th high nitrogn trtmnts ws not limiting in th low nitrogn trtmnts. AMF inoul For AMF inftion, th myorrhizl inoulum onsist of th roots of n Ergrostis sp. from th grssln los to th univrsity mpus. Th ph of th grssln soil ws 7. Th inoulum ws prpr y homognizing n mixing surrouning soil with th Ergrostis sp. root systms, forming mix inoulum in trms of spis n propguls. Th inoulum ws ppli s 1 m lyr to th surf of th soil, n ovr with 1 m lyr of sn. Non-myorrhizl trtmnts riv inoulum tht h n utolv t 121 C for 2 min. No oloniztion of non-myorrhizl trtmnts y xtrnl sours of AMF spors ws tt uring th ours of th xprimnt. Rhizoium inoul Th originl Rhizoium lguminosrum v. vii inoulum ws otin from th tiv nouls of plnts of Vii f L. tht h n inft prviously with this strin (STIMUPLANT CC: Rg. No. CK89/4756/23; PO Box 11446, Brooklyn, South Afri 11). Rhizoium olonis wr mintin on soli ultur mium onsisting of 5 g K 2 HPO 4,2 g MgSO 4 7H 2 O, 1 g NCl, 1 g mnnitol, 4 g yst xtrt, 8 g gr n 25 % Congo R, issolv in 1 ml istill wtr n utolv t 121 C for 2 min. Rhizoium inoulum ws prpr y ulturing slt olonis in liqui ultur mium. Th ulturs wr grown in 25 ml Erlnmyr flsks n inut t 25 C until th tril ll optil nsity msur 6 t 6 nm (whih orrspon to pprox olony forming units pr ml). Svn ys ftr plnting, h pot for th Rhizoium trtmnts ws inoult thr tims with 1 ml of tn-fol ilution of th Rhizoium inoulum. To mintin uniformity of nutrint supply, ll othr non-rhizoium trtmnts wr inoult with utolv Rhizoium inoulum (121 C for 2 min). No nouls form on plnts inoult with utolv inoulum. Biomss msurmnts At h hrvst, four rplits of h trtmnt wr slt rnomly. Th plnts wr sprt into lvs, stms, roots n nouls, n ovn-ri t 65 C for 3. At h hrvst, root su-smpl from h trtmnt ws tkn for ovn rying for stimtion of AMF inftion of th root tissu. Th frsh mss of th su-smpl ws ror so tht th ry mss of th su-smpl oul to th totl root ry mss. N n P nlysis Aftr th trmintion of ry mss, tissus wr mill n nlys for totl N n P onntrtions. Su-smpls (1 1 g) wr igst in hyrogn proxi sulfuri i igstion mixtur y th Kjlhl prour follow y stnr olorimtri ssys (Anrson n Ingrm, 1993). All N n P msurmnts rprsnt totl lmntl N n P (orgni plus inorgni) prsnt in plnt tissus. AMF ttion n quntifition AMF oloniztion ws vlut from rnom susmpl of pprox. 15 root sgmnts pr plnt. Roots Downlo from y gust on 1 Otor 218

3 Ji t l. N n P Aumultion in Vii f 253 wr lr in 25 % KOH (9 C) for 45 min, iifi in 1 % HCl for 15 min, stin with.5 % Trypn Blu in i glyrol (9 C) for 45 min, n thn stor in i glyrol oring to th prour of Kosk n Gmm (1989). Rnomly slt root frgmnts wr mount in prmnnt mounting mium on slis (Omr t l., 1979) without squshing th root pis. Prntg oloniztion y rusuls, vsils n totl hyph wr ror, t 25 mgnifition using th mgnifi intrstions mtho of MGonigl t l. (199). Photosynthti stuis Photosynthti gs xhng rts wr trmin t h hrvst on qul-g ohorts. All plnts wr fully limtiz to full sunlight (2 mmol m 2 s 1 ). Nt photosynthsis unr light sturting onitions ws msur with th portl CIRAS-1, PP systms, infrr gs nlysr (Ji n Gry, 24), using ight iffrnt youngst fully xpn lvs for h trtmnt. Sttistil nlysis AMF% inftion t wr trnsform using rsin squr root to stisfy norml istriution n homognity of vrin ssumptions for thr-wy ANOVA. Dry mss n lmntl (N n P) onntrtions of lvs, stms, roots n nouls wr nlys y thr-wy ANOVA for min ffts n intrtionl ffts (Zr, 1984). Th signifin of th iffrns in vrils us of th intrtions twn ftors A (N, NM, NR n NRM), B (N supply LN n HN) n C (hrvst t T 1 n T 2 ) wr tst. For th multiftoril xprimnt, th sours of vrition wr suivi into thr min ffts: A (prsn or sn of miroil symiont(s); B (low n high nitrogn supplis); n C (hrvst ts); thr first-orr intrtions, A B, A C, n B C n on son-orr intrtion, A B C. Multipl omprisons of mns wr prform y th Tuky HSD tst (P < 5) ftr prforming thr-wy ANOVA with rsiul stimtion. Th STA- TISTICA (vrsion 6.) pkg ws us for sttistil nlysis. RESULTS AND DISCUSSION Effts of trtmnt ftors on plnt vrils Eh of th trtmnt ftors (A, B, C), whn onsir iniviully, h signifint ffts on ll of th iffrnt plnt vrils list Tl 1. In rltion to thir omin ffts, th thr-wy ANOVA rsults show tht th mjority of first-orr intrtions h signifint ffts on ll plnt vrils. Th omin ffts of th thr ftors on miroil oloniztion, plnt iomss, rts of N n P umultion, lf r n photosynthti rts r shown in Figs 1 5, n th omin ffts of th thr ftors on N n P onntrtions r shown in Tl 2. In ll instns, miroil oloniztion of plnts ws ssoit with signifint inrss in th mgnitus of plnt vrils suh s iomss, lf r, photosynthti rt, N n P ontnt. Th fft of hrvst t on th vrils suh s tissu N onntrtion n tissu T ABLE 1. Th signifin of th iffrns in plnt prmtrs rsulting from th intrtions mong ftors A (N, NM, NR n NRM), B (N supplis LN n HN) n C (hrvst ts T 1 n T 2 ) Sour of vrin A B C A B A C B C A B C Dgrs of from Vrils: Photosynthti rt (mmol CO 2 m 2 s 1 ) *** *** *** *** ** ** * AMF AC (%) *** *** *** * ns ns ns VC (%) *** *** * *** * ns ns HC (%) *** *** *** *** ns ns ns N, P ontnt Lf N (mg g 1 ) *** *** *** *** *** * ns Stm N (mg g 1 ) *** *** *** *** *** ns ns Root N (mg g 1 ) *** *** *** *** ** * ns Noul N (mg g 1 ) *** *** *** ns ns * ns Lf P (mg g 1 ) *** *** *** *** ** ** ns Stm P (mg g 1 ) *** *** *** *** *** *** ** Root P (mg g 1 ) *** *** *** *** *** *** ** Noul P (mg g 1 ) *** *** *** ns ns ** ns Biomss of plnt prt Lf r (m 2 plnt 1 ) *** *** *** *** *** ** * Lf. wt (g plnt 1 ) *** *** *** ** * ** ns Stm. wt (g plnt 1 ) *** *** *** ns * * ns Root. wt (g plnt 1 ) *** *** *** * * ** ns Noul. wt (g plnt 1 ) * *** *** ns ns *** ns Biomss (g plnt 1 ) *** *** *** *** *** *** ns Downlo from y gust on 1 Otor 218 Prntg AMF oloniztion, ry mss, lf r, lmntl N n P onntrtions of lvs, stms, roots n nouls wr nlys y thr-wy ANOVA with rsiul stimtion, for min ffts n intrtion ffts. *, **, *** Effts tht r signifint t P < 5, 1 n 1, rsptivly; ns, ffts tht r not signifint t P < 5.

4 254 Ji t l. N n P Aumultion in Vii f Coloniztion (%) Coloniztion (%) Coloniztion (%) f g Arusulr Vsiulr Hyph P onntrtion is prsnt in Tl 2. Expt for lf r, stm P ontnt n root P ontnt, thr wr no signifint son-orr intrtions (Tl 1). Effts of N supply n Rhizoium on myorrhizl oloniztion Tl 1 shows tht first-orr intrtions (A B) h signifint ffts on AMF oloniztion. Coloniztion lvls of AMF wr highr in low N trtmnts (Fig. 1). With rgr to rusulr oloniztion, two kins of myorrhizl root oloniztion rspons wr vint (Fig. 1). Th prsn of Rhizoium inftion rsult in signifint lin in %AC, %VC n %HC t low n high onntrtions of N supply. Low onntrtions of N in th prsn of Rhizoium inftion (LNMR) rsult in %AC vlus similr to thos ssoit with high rts of N supply in th sn of Rhizoium inftion (HNM). At low N supply, th prsn of Rhizoium inftion (LNMR) rsult in out 5 % rution in %VC (Fig. 1). Howvr, t high onntrtions of N thr ws no fft of Rhizoium inftion on %VC. Th prsn of Rhizoium inftion n/or high onntrtions of N supply wr h ssoit with 5 % or lowr %VC ompr with th T1 T2 LNM LNMR HNM HNMR F IG. 1. Prntg of rusulr (AC%), vsiulr (VC%) n hyph oloniztion (HC%) in rspons to Rhizoium inoultion, low nitrogn (LN = 1 ppm) n high nitrogn (HN = 25 ppm N) supply t two hrvsts (T 1 n T 2 ). Vrtil rs rprsnt s.. (n = 4) of th mns. Diffrnt lttrs init signifint iffrns ssss y th Tuky HSD tst (P < 5) ftr prforming thr-wy ANOVA with rsiul stimtion. Totl iomss (g pr plnt) Lf ry wight (g pr plnt) Stm ry wight (g pr plnt) Root ry wight (g pr plnt) Noul ry wight (g pr plnt) T1 Biomss j T2 i hi i fg fg gh f LN Lf h fg fgh gh f f Stm Root f f f f f Noul LNM LNR LNMR HN HNM HNR HNMR roots of LNM plnts. In th s of hyphl oloniztion (%HC), th prsn of Rhizoium inftion ws ssoit with signifint lin in %HC (Fig. 1). Whnvr N supply ws not limiting signifint rution in %HC ourr. For xmpl, unr high N Rhizoium inftion rsult in furthr signifint lin in %HC (Fig. 1). A similr pttrn ws osrv for %AC. In ll trtmnts, th prsn of myorrhizl ssoition h positiv impt on nutrint uptk n iomss proution. For xmpl, whn th trtmnts for HN, HNM n HNMR r ompr, th plnts h similr f f f F IG. 2. Lf, stm, root, noul ry wight proution n totl iomss s influn y nitrogn supply (LN n HN) n miroil symioti ssoition (N, NM, NR n NMR) t two hrvsts (T 1 n T 2 ). Vrtil rs rprsnt s.. (n = 4) of th mns. Diffrnt lttrs init signifint iffrns ssss y Tuky HSD tst (P < 5) ftr prforming thrwy ANOVA with rsiul stimtion. f Downlo from y gust on 1 Otor 218

5 Ji t l. N n P Aumultion in Vii f 255 P uptk rt (mg plnt 1 1 ) P uptk rt (mg plnt 1 1 ) Lf r (m 2 plnt 1 ) LN Noul Root Stm Lf T1 T2 LNM totl root iomss (Fig. 2), ut, irrsptiv of th lvl of prntg AMF oloniztion (Fig. 1), th prsn of n AMF ssoition lwys rsult in nhn nutrint uptk n totl iomss proution (Figs 2 n 3). Effts of N supply n AMF on Rhizoium inftion fg f Tl 1 shows tht first orr intrtions (A B) h signifint ffts on noul ry mss. Figur 2 shows th impt of AMF n N supply on noul mss. Unlik th Rhizoium ffts on AMF oloniztion, AMF inftion i not hv signifint fft on noul mss. Howvr, high fg LN LNM LNR LNMR Totl N Totl P g f HN HNM HNR HNMR F IG. 4. Lf r (m 2 plnt 1 ) proution s influn y nitrogn supply (LN n HN) n miroil symioti ssoition (N, NM, NR n NMR) t two hrvst intrvls (T 1 n T 2 ). Vrtil rs rprsnt s.. (n = 4) of th mns. Diffrnt lttrs init signifint iffrns ssss y Tuky HSD tst (P < 5) ftr prforming thr-wy ANOVA with rsiul stimtion. LNR LNMR HN HNM HNR HNMR F IG. 3. N n P umultion rt n prtitioning into lvs, stms, roots n nouls s influn y nitrogn supply (LN n HN) n miroil symioti ssoition (N, NM, NR n NMR). Vrtil rs rprsnt s.. (n = 4) of th mns. Diffrnt lttrs init signifint iffrns ssss y th Tuky HSD tst (P < 5) ftr prforming two-wy ANOVA with rsiul stimtion. N n P umultion rt (mg plnt 1 1 ) rprsnts n vrg rt of umultion lult s (T 2 mount T 1 mount)/9. A mx (µmol CO 2 m 2 s 1 ) LN T1 T2 LNM h f fgh gh h f f fg LNR LNMR HN HNM HNR HNMR F IG. 5. Light sturt (2 mmol qunt m 2 s 1 ) photosynthti rt s influn y nitrogn supply (LN n HN) n miroil symioti ssoition (N, NM, NR n NMR) t two hrvst intrvls (T 1 n T 2 ). Vrtil rs rprsnt s.. (n = 8) of th mns. Diffrnt lttrs init signifint iffrns ssss y Tuky HSD tst (P < 5) ftr prforming thr-wy ANOVA with rsiul stimtion. N supply ws ssoit with lrg rution in noul ry mss rltiv to low N plnts (Fig. 2). At low N thr ws ouling in noul ry mss twn T 1 n T 2 ut only slight inrs ovr th sm prio t high N, initing tht high onntrtions of nitrt inhiit noul growth. A signifintly lrgr quntity of P umult in LNR n LNMR nouls twn hrvsts ompr with th HNR n HNMR nouls (Tl 2 n Fig. 3). Th onntrtion of P in th LNR n LNMR nouls ws lso signifintly highr thn in th HNR n HNMR nouls (Tl 2). In th s of low N trtmnts, P onntrtion ws signifintly highr in LNMR nouls thn in LNR nouls (Tl 2). Th fft of th N : P rtio on iomss Th lvl of P supply h signifint fft on plnt iomss (Fig. 2), root noultion (Fig. 2), N umultion rt (Fig. 3), lf r (Fig. 4), nt photosynthti rt (Fig. 5) n tissu N onntrtion (Tl 2). Inrs in P supply s onsqun of AMF oloniztion ws onsistntly ssoit with signifint inrs in N umultion n iomss proution (Figs 2 n 3). For th LNMR plnts th N : P rtio ws 11 mg N 1 :1mgP 1, n 14 mg N 1 :1 mg P 1 for th HNMR plnts. For HNM n HN th N : P rtios wr 18 mgn:1mgp 1 n 217mgN 1 :1mgP 1, rsptivly. Ths rsults init tht th rt of iomss proution inrs s th proportion of totl P umult inrs rltiv to th totl N umult. AMF ontriut to n inrs in N proutivity in trms of iomss proution y inrsing th supply of P. Efft of P on totl N umultion Th mounts of xtrnl hyph in th pots wr not trmin, nor th mtolilly tiv proportion of th intrnl oloniztion. In LNM th %HC ws lmost 1 %, whrs for HNMR it ws pprox. 6 %, lthough th HNMR plnts umult signifintly mor P thn i th LNM plnts. Plnts with oth Rhizoium n AMF ssoitions (LNMR n HNMR) umult th Downlo from y gust on 1 Otor 218

6 256 Ji t l. N n P Aumultion in Vii f T ABLE 2. () N n () P ontnt in lf, stm, root n noul of Vii f L. s influn y th ftors A (N, NM, NR n NRM), B (N supplis LN n HN) n C (hrvst intrvls T 1 n T 2 ) () Vril vlu = N onntrtion (mg g 1 ry mss) Ftors Vril Vril Vril Vril A B C Lf N Tuky-t Stm N Tuky-t Root N Tuky-t Noul N Tuky-t N LN T N LN T N HN T N HN T NM LN T NM LN T NM HN T NM HN T f f NR LN T f NR LN T f gh NR HN T f f fg NR HN T fg f fgh NMR LN T f f fg NMR LN T g g h NMR HN T g f fg NMR HN T h g gh () Vril vlu = P onntrtion (mg g 1 ry mttr) Ftors Vril Vril Vril Vril A B C Lf P Tuky-t Stm P Tuky-t Root P Tuky-t Noul P Tuky-t N LN T N LN T N HN T N HN T NM LN T gh 22 9 NM LN T gh i f NM HN T f NM HN T f fg NR LN T NR LN T NR HN T NR HN T NMR LN T fg h NMR LN T h j f NMR HN T fg NMR HN T fg gh Vlus r mns s., n = 8. Diffrnt lttrs init signifint iffrns ssss y Tuky HSD tst (P < 5) ftr prforming thr-wy ANOVA with rsiul stimtion. most P. In low N trtmnts AMF oloniztion h signifint positiv impt on th rt of nitrt-n umultion (Fig. 3), n in high N trtmnts, non-amf plnts suh s th HNR plnts umult signifintly lss N thn i HNMR plnts. Ths rsults r onsistnt with th fining of Tor t l. (1994, ) tht AMF oloniztion inrs plnt nitrt-n uptk. P-limittion of N 2 -fixtion rts pprs to th rson for th lowr rts of N umultion in LNR rltiv to LNMR plnts (Fig. 3). Nitrt ws th mjor N sour for HNMR plnts minly us of thir lowr noul mss ompr with th LNR n LNMR plnts (Figs 2 n 3). Th N 2 -fixing LNMR plnts iffr only slightly from th nitrt ssimilting HNMR plnts in rt of totl lmntl N umultion. For xmpl, t T 1, HNMR h umult 7. % mor N thn LNMR n, t T 2, HNMR h umult 5.3 % mor N. It is not lr whthr th iffrns in nitrt umultion rts mong th LN, HN, LNM, HNM n HNMR plnts wr irt onsqun of inrs uptk n moiliztion of nitrt-n y th xtrnl hyph, or inirt onsqun of n inrs in th plnt P ontnt. If th lttr hl, thn plnt N proutivity in trms of lf r proution woul hv n nhn y n inrs in P umultion. Inrs in th plnt s totl trnspirtionl surf r woul tn to inrs th flux of wtr through th soil plnt tmosphr ontinuum n inrs th flux of nitrt to th plnt root systm. This oul possil xplntion for inirt ffts of AMF oloniztion on plnt nitrt uptk. Downlo from y gust on 1 Otor 218

7 Ji t l. N n P Aumultion in Vii f 257 T ABLE 3. Th influn of N n P supply rts on lf N n P onntrtion, totl P umult, lf r proution, light sturt photosynthti rt (Amx), n quntum yil ffiiny (QYE) Ftors A B SLN (g N m 2 ) SLP (g P m 2 ) Totl P (g plnt ) Lf r (m 2 plnt 1 ) A mx (mmol CO 2 m 2 s 1 ) QYE (mmol CO 2 mmol Q ) 1 ppm N LP ppm N LP ppm N LP ppm N LP ppm N LP f f ppm N LP f fg ppm N HP f ppm N HP f 12 9 f f 5 ppm N HP fg f g 1 ppm N HP g f g h 25 ppm N HP h g h i 5 ppm N HP i h h i n Low P (LP) plnts wr suppli with 5 mm P n high P (HP) plnts wr suppli with 16 mm P. Vlus r mns s.. Diffrnt lttrs init signifint iffrns ssss y Tuky HSD tst (P <.5) ftr prforming thr-wy ANOVA with rsiul stimtion. Downlo from y gust on 1 Otor 218

8 258 Ji t l. N n P Aumultion in Vii f Efft of N on totl P umultion Root iomss i not ppr to mjor ftor in ithr P or N uptk. Sin, prt from th LN plnts, root iomss i not iffr signifintly in th othr trtmnts (Fig. 2) it lon oul not ount for th vrition in th quntity of P umult in th iffrnt trtmnts (Fig. 3). Plnts with roots oloniz y AMF h P umultion rts tht wr tims th rts for th non-miroil low N trtmnts (LN vrsus LNM n LN vrsus LNMR in Fig. 3). At high N, ll plnts with AMF oloniz roots h P umultion rts tht wr tims th rts for th nonmiroil high N trtmnts; s HN vrsus HNM n HN vrsus HNMR in Fig. 3. Howvr, roots inft only with Rhizoium lso show n inrs in P umultion rltiv to th low N or high N non-myorrhizl ontrol plnts. For xmpl, LNR plnts h 66-fol inrs in th lvl of P umultion ompr with LN plnts, n thr ws 18-fol inrs in th lvl of P umultion whn HN plnts wr inft with Rhizoium (Fig. 3). Hr th inrs in P umultion ppr to influn inirtly y th ssoition with N 2 -fixing Rhizoium in th low N plnts, or y th inrs nitrt N supply in th high N trtmnt plnts. Inrs N umultion u to N 2 -fixtion in LNR n HNR plnts (Fig. 3) ws ssoit with n inrs in lf r xpnsion (Fig. 4). Thus, it ws possil tht th AMFinpnnt nhn rts of P umultion in LNR n HNR plnts wr u to th ffts tht N umultion h on lf r proution. Th influn of N, P n miroil symionts on photosynthsis Figurs 3 n 5 show tht photosynthti rts inrs s th rtio of P to N supply inrs (not lso th rltionship twn th N : P trn in Tl 2 n th trn in photosynthti rts in Fig. 5). Th impt tht th miroil symionts h on photosynthti rts ppr to mit y thir ffts on th plnt N : P rtio. Sprt xprimnts to tst th fft of P supply on th photosynthti N us ffiiny n th quntum yil ffiiny of lf N show tht n inrs in th supply of P inrss th photosynthti N us ffiiny n th quntum yil ffiiny of lf N (Tl 3). Th rsults in Tl 2 n Fig. 5 r onsistnt with th hypothsis tht inrsing P supply nhns photosynthti N us ffiiny. CONCLUSIONS Th finings of this xprimnt r onsistnt with othr osrvtions (Xvir n Grmi, 22, 23) of th positiv impt of th synrgisti intrtions twn AMF n Rhizoium on lgums. Th mgnitu of th inrss in oth lf r n iomss proution with h inrmnt in N supply ws pnnt on th lvl of P supply. Inrsing P supply s irt onsqun of AMF oloniztion or s n inirt onsqun of Rhizoium inftion h positiv ffts on N umultion, lf r proution n iomss proution. Inrsing P umultion h positiv influn on photosynthti N us ffiiny. Thus, it is rsonl to onlu tht, with rspt to th lgum triprtit symioti ssoition xmin hr, th uppr limits of nitrogn proutivity or photosynthti N us ffiiny n pn on th lvl of P supply. ACKNOWLEDGEMENTS Th uthors thnk Prof. M. Shols for ssisting with N n P nlysis n Prof. J. S. Glpin for hlping with th sttistil nlysis. Th rsrh ws fun y th Univrsity Snt Rsrh Grnt. LITERATURE CITED Ågrn GI Thory for growth of plnts riv from th nitrogn proutivity onpt. Physiologi Plntrum 64: Anrson JM, Ingrm JSI Tropil soil iology n frtility hnook of mthos, 2n n. Wllingfor, UK: CAB Intrntionl, Anr G, D Lij F, Lynh JM Plnt mit intrtions twn Psuomons fluorsns, Rhizoium lguminosrum n rusulr myorrhiz on p. Lttrs in Appli Miroiology 26: Azón R, Ruio R, Br JM Sltiv intrtions twn iffrnt spis of myorrhizl fungi n Rhizoium mliloti strins, n thir ffts on growth, N 2 -fixtion ( 15 N) n nutrition of Migo stiv L. Nw Phytologist 117: Hiros T, Wrgr MJA Nitrogn us ffiiny in instntnous n ily photosynthsis of lvs in th nopy of Soligo ltissim stn. Physiologi Plntrum 7: Ji YS, Gry VM. 24. Intrrltionships twn nitrogn supply n photosynthti prmtrs in Vii f L. Photosynthti 41: Kosk RE, Gmm NJ A moifi prour for stining roots to tt VA mykorrhiz. Myologil Rsrh 92: MGonigl TP, Millr ML, Evns DG A nw mtho whih givs n ojtiv msur of oloniztion of roots y vsiulr-myorrhizl fungi. Nw Phytologist 115: Omr MB, Bolln L, Hthr WA A prmnnt mounting mium for fungi. Bulltin of th British Myologil Soity 13: Sinlir TR, Hori T Lf nitrogn, photosynthsis n rop rition us ffiiny: rviw. Crop Sin 29: Smith SE, R DJ Myorrhizl symiosis, 2n n. Sn Digo, CA: Ami Prss. Tor RM, Azón R, Br JM Improv nitrogn uptk n trnsport from 15 N-lll nitrt y xtrnl hyph of rusulr myorrhiz unr wtr-strss onitions. Nw Phytologist 126: Tor RM, Azón R, Br JM Th improvmnt of plnt N quisitionfrom n mmonium-trt, rought-strss soil y th fungl symiont in rusulr myorrhiz. Myorrhiz, 4: Toro M, Azón R, Br JM Th us of isotopi ilution thniqus to vlut th intrtiv ffts of Rhizoium gnotyp, myorrhiz fungi, phospht-soluilizing Rhizotri n rok phospht on nitrogn n phosphorus quisition y Migo stiv. Nw Phytologist 138: Xvir LJC, Grmi JJ. 22. Rspons of lntil unr ontroll onitions to o-inoultion with rusulr myorrhizl fungi n rhizoi vrying in ffiy. Soil Biology & Biohmistry 34: Xvir LJC, Grmi JJ. 23. Sltiv intrtions twn rusulr myorrhizl fungi n Rhizoium lguminosrum v. vi nhn p yil n nutrition. Biology n Frtility of Soils 37: Zr JH Biosttistil nlysis. Englwoo Cliffs, NJ: Prnti-Hll. Downlo from y gust on 1 Otor 218