Accepted 1 June, 2012

Transcription

1 Afrin Journl of Biotehnology Vol. 11(58), pp , 19 July, 212 Aville online t DOI: /AJB12.63 ISSN Ademi Journls Full Length Reserh Pper Phosphorus use effiieny in ommon en (Phseolus vulgris L.) s relted to omptiility of ssoition mong rusulr myorrhizl fungi nd rhizoi Ftm Tjini 1,2 * nd Jen-Jques Drevon 1 1 INRA, UMR Eo nd Sols, Ple Vil, 346 Montpellier Cedex 1, Frne. 2 Fulté des Sienes de Gfs, 2112 Sidi Ahmed Zrroug, Tunisie. Aepted 1 June, 212 The triprtite symiosis of ommon en (Phseolus vulgris L.) reominnt inred line (RIL) 147 with rhizoi nd rusulr myorrhizl fungi (AMF) ws ssessed in snd ulture y ompring the effets of three AMF speies on the myorrhizl root oloniztion, rhizoil nodultion, plnt growth nd phosphorus use effiieny for symioti nitrogen fixtion. Although Glomus intrrdies well olonized roots of RIL147, Gigspor rose nd Aulospor melle wekly olonized roots-plnts. Higher oloniztion y Glomus ws lso otined y ontt with myorrhized Stylosnthes guinensis. Signifint differenes mong oloniztion nd nodultion of the roots nd growth were found etween AMF speies. Although the results showed tht the doule inoultion, espeilly with Glomus, indued signifint inrese in ll prmeters whtever the AMF tretments in omprison to the ontrol. In ddition, the omined inoultion of Glomus nd CIAT899 strins resulted in signifintly higher nitrogen nd phosphorus umultion of ommon en plnts nd improved phosphorus use effiieny ompred with their ontrols whih were not dully inoulted. It ws onluded tht phosphorus use effiieny for plnt growth nd nitrogen fixtion ould e improved y omptiility with rusulr myorrhizl fungi, rhizoil strin nd ommon en genotype, sme under P limited. Key words: Aulospor melle, rusulr myorrhizl fungi, Glomus intrrdies, Gigspor rose, nitrogen fixtion, Phseolus vulgris, phosphorus, rhizoi, Stylosnthes guinensis, symiosis. INTRODUCTION Bens ply n importnt role in the enhnement of the level of nourishment due to the ft tht this plnt is rih in elements like potssium, lium, iron nd phosphorus (P) nd it ontins 18 to 32% of protein in every grin vergely (Numn nd Nuri, 25). The plnt is lso rih in A, B nd D vitmins in developing ountries. Despite this interest of the food legume nutritionl, severl onditions limit the growth nd prodution of this legume, suh s phosphorus nd nitrogen whih onstitute the most limited nutriment for vegettive growth (Vne et l., 23; Tjini et l., 29, 211). However, nitrogen (N) nd P vilility is generlly low in rle soils, thus *Corresponding uthor. E-mil: ftmtjini@yhoo.fr. Tel: mking them mjor limiting ftors for rop growth nd yield, espeilly in tropil nd sutropil res (Hrdrson nd Atkins, 23). Xiurong et l. (211) reported tht during the pst 5 yers, the widespred use of hemil fertilizers to supply N nd P hs hd sustntil impt on food prodution, nd hs eome mjor input in rop prodution round the world. However, further inreses in N nd P pplition re unlikely to e s effetive t inresing yields. Tody, only 3 to 5% of pplied N fertilizer nd 1 to 45% of P fertilizer re tken up y rops (Adesemoye nd Kloeppe, 29). In ddition, the hevy use of hemil fertilizers in griulture uses frequent deleterious environmentl onsequenes, nd hs eome glol onern (Tilmn et l., 22). Therefore, developing nutrient-effiient vrieties will likely ply mjor role in inresing rop yield nd would e in

2 12174 Afr. J. Biotehnol. more sustinle nd eonomil pproh to griulture (Xiurong et l., 211). The pity of plnt to quire nd utilize nutrients hs enourged reserhers to study how nutrient effiieny is influened y oth nutrient sorption in roots nd utiliztion in plnts (Sepehr et l., 29). In order to ssess the pity of plnt to quire nutrients, rusulr myorrhizl fungi (AMF) nd rhizoi re two of the most importnt plnt symionts. They ply key role in nturl eosystems nd influene plnt produtivity, plnt nutrition nd improved inhiition of fungl plnt pthogens (Demir nd Akköpru, 27; Wehner et l., 21; Aohtem et l., 211). Myorrhiz enefits the host through moiliztion of phosphorus from non-lile soures, wheres rhizoi fix N 2 (Sheulin nd Vnder Heijden, 26). Previous works on the triprtite symiosis legume-myorrhiz-rhizoi hve shown stimultory (Demir nd Akköpru, 27; Xio et l., 21) or inhiitory (Sheulin nd Vnder Heijden 26; Frnzini et l., 21) effets on eh other or on the growth of plnts. A few studies hve shown tht some teril speies respond to the presene of ertin AMF (Andrde et l., 1997; Artursson et l., 26), suggesting high degree of speifiity etween teri ssoited with AMF. Thus, the speifi teri together with AMF my rete more indiret synergism for plnt growth (Bre, 1997); inluding nutrient quisition (Bre et l., 22) nd enhnement of root rnhing (Gmlero et l., 24). In ddition, the AMF themselves hve lso een shown to hve n impt on the omposition of teril ommunities in their myelium environment (Artursson et l., 26). The rhizoi en symiosis when in ssoition with rusulr myorrhizl fungi (AMF) is known to enefit from etter supply of phosphorus (Snging et l., 2). The AMF is lso le to quire phosphorus in orgni form tht is not diretly ssimilted y plnts (Buher et l., 21). The mehnisms ffeting the effiieny of sorption nd utiliztion of phosphorus in plnts re relted to oloniztion y myorrhize (Ji et l., 24). Furthermore, Jin et l. (21) found tht dul inoultion with AMF nd rhizoi deresed the hrmful influene of sulphte slinity on plnt growth nd nutrient umultion (P, N nd proline) in Lthyrus stivus, ompred with the ontrol tretments. Both symioses shre prts of signlling pthwys, inditing intimte intertions etween ll three prtners during o-evolution (Demir nd Akköpru, 27; Xio et l., 21). On the other hnd Aysn nd Demir (29) reported tht the informtion on the mehnisms ontrolling intertions of teri with AMF nd plnt roots in the myorrhizosphere nd their tivities re very diffiult to generlize euse the intertions involving rusulr myorrhiz, root rot fungi nd Rhizoium vry with the miroil speies nd plnt ultivrs. In order to ssess the effet of inoultion with rusulr myorrhizl fungi or y ontt with myorrhized Stylosnthes guinensis on phosphorus use effiieny for plnt growth nd to investigte whether sensitivity of symioti nitrogen fixtion to phosphorus defiieny ws restored y symiosis with rusulr myorrhizl fungi, ommon en genotype from the ross of BAT477 y DOR364 ws grown in glsshouse with oth symionts. MATERIALS AND METHODS Two ses of triprtite symiosis were relized in this study. In the first, the ommon en genotype ws inoulted y myorrhizl inoulnt with AMF spp. diversity; ut in the seond, the ommon en genotype ws inoulted y ontt with myorrhized Stylosnthes guinensis or not (ontrol). In ll ses reeived similr rhizoil inoultion. The experimentl design onsisted of rndomized lok with 3 replitions. The SAS softwre (1997) ws used to perform the sttistil nlyses, results were sumitted to ANOVA, nd omprison of mens ws hieved y the Dunn's multiple rnge tests (P.5). Biologil mteril The ommon en genotype used in this study ws reominnt inred line (RIL) 147, from the ross of BAT477 nd DOR364 (CIAT-INRA oopertion). Seeds were surfe-sterilized with 1.3% lium hypohlorite for 15 min with onstnt stirring, nd then wshed with sterile distilled wter. They were germinted on.8% sterile gr pltes for 3 dys t 28 C in the drk, nd hd germintion rte of 8%. Rhizoil inoultion ws performed y soking the seedlings for 45 min in freshly prepred suspension of Rhizoium tropii CIAT899 ontining 1 8 teri ml -1. Seedlings were grown in 1 ml pots filled with n utolved snd-soil mixture (9:1 v:v) reolonized with soil teri ording to Jns et l. (22). Fifty grm (5 g) of one of three AMF inoulums, nmely, Glomus intrrdies BEG157 (Shenk nd Smith), Gigspor rose BEG9 (Niolson nd Shenk) or Aulospor melle NM54 (Spin nd Shenk), were pled elow the seedlings. The inoulum used for the pots onsisted of hopped roots of pot ultures plnted with leek (Allium porrum) nd grown for 18 months in glsshouse. Fifty grm (5 g) of AMF inoulum is pproximtely equl to 1 spores of the AMF speie ontined t lest 2 infetive propgules of AMF per grm of hopped root. In order to ssess the myorrhiztion y ontt, lso some surfe-sterile seeds were sown nd grown in soil-snd ulture in ontt with myorrhized Stylosnthes guinensis y Glomus intrrdies BEG157. The mount of myorrhize sustrte ws hrterized y low ville N (.7%) nd P (.1%). In non myorrhizl tretments, eh pot filled with sme mount of myorrhize free sustrte. Growth onditions Trils were performed in temperture-ontrolled glsshouse with night/dy tempertures of 25/35 C, nd 16 h photoperiod with omplementry illumintion of 4 µmol photons m -2 s -1. Inoulted seedlings with R. tropii CIAT899 nd AMF inoulnts or y ontt with myorrhized Stylosnthes or without AMF were grown in soilsnd sustrte. Pots were wtered with distilled wter every 2 dys until hrvest, nd reeived one week using the Vdez et l. (1996) nutrient solution: mroelements: K 2SO 4 (1.25 mm), MgSO 4.7H 2O (2.5 mm), CCl 2 (3.3 mm); miroelements: Fe EDDHA (8.5 µm Fe s sequestrene), H 3BO 3 (4. µm), MnSO 4 (6. µm), ZnSO 4 (.9 µm), CuSO 4 (1. µm), NMoO 4 (.1 µm). The nutrient solution ws supplemented with 2 mmol ure plnt -1 during the first two weeks, 1 mmol ure per plnt during the next two

3 Tjini nd Drevon weeks nd no more ure during the lst two weeks. The pots were distriuted in omplete rndomized lok design with 3 replitions nd one plnt only per pot. Assessment of AMF oloniztion The plnts growing in pots were hrvested t 5 dys fter sowing (DAS), orresponding to flowering stge, hlf of the root system ws used for estimtion of the extent of root oloniztion y the AMF s follows: roots were lered in KOH 1% (w:v) t 8 C for 3 min followed y rinsing with wter nd two rinses with 1% HCl of 1 h eh. Therefter, the roots were immersed t 8 C for 1.5 h in stining solution onsisting of lti id: glyerol: wter (1:1:1 v:v:v) nd.1% of eh Trypn Blue nd Methylene Blue. After wshing wy the stin, the roots were de-stined in tp wter for 3 min t room temperture. The roots were exmined under ompound mirosope for quntittive oloniztion ssessment y mgnified-intersetion (MGonigle et l., 199). Biomss nd perentges of P nd N t hrvest At hrvest, shoot, nodules nd roots were seprted nd dried t 7 C for 2 dys, nd dry weight of eh frtion ws lulted. The perentge of P ws mesured in smples of ground tissues following wet digestion with nitri-perhlori id (6:1, v:v) t 25 C for 6 h, using the phosphovndo-molydte method (Tussky nd Shorr, 1953). The P use effiieny (PUE) ws lulted s the rtio of iomss (shoot + root) g -1 /men plnt P ontent mg -1. Totl nitrogen perentge (TN) ws mesured y the Kjeldhl proedure on plnts hrvested for iomss determintions. Beuse no signifint differene in most of the results of Gigspor nd Aulospor nd ontrol plnts, only plnts inoulted either with Glomus or y ontt with myorrhized Stylosnthes were nlyzed for this prmeter. RESULTS Myorrhizl oloniztion Dt in Figure 1A show tht the prmeters of rootoloniztion were ffeted y AMF tretments (P.1). The AMF effet ws predominnt in plnts inoulted y Glomus, nd signifint differene etween oth tretments (y ontt nd y myorrhizl inoulnt). Thus, rtes of oloniztion in plnts inoulted y Glomus were nerly two-fold higher for ll strutures in omprison to tht in plnt grown with myorrhized Stylosnthes. On the other hnd, higher vesiles nd hyphe levels were found in plnts inoulted with Glomus thn Gigspor, nd rusules were only enountered in roots of plnts inoulted with Glomus y myorrhizl inoulnt or y ontt with myorrhized Stylosnthes, ut oloniztion remined very low with Aulospor (Figure 1A). Figure 1B shows root oloniztion y vesiles nd hyphe of Glomus. Nodultion Nodultion ws strongly ffeted y AMF tretments (P.1), signifintly more nodules were enountered for plnts inoulted y Glomus thn other AMF speies (Figure 2A). In omprison to ontrol plnts, inoultion with AMF indued signifint inrese in nodule numer of 86 nd 77% in plnts inoulted with Glomus nd for plnts grown in ontt with myorrhized Stylosnthes, respetively, ut no signifint differene etween plnts inoulted with other AMF speies nd ontrols plnts (Figure 2A). On the other hnd, plnts inoulted with Gigspor nd Aulospor show low nodule numer in this experiment (Figure 2A). Also, AMF ffeted signifintly the nodule mss per plnt (P.5). Dt in Figure 2B show higher iomss of nodules in plnts grown in ontt with myorrhized Stylosnthes thn other AMF tretments. Thus, in omprison with ontrol plnts, AMF indued signifint inrese of 85, 78, 52 nd 27%, respetively y ontt with myorrhized Stylosnthes nd with Glomus, Gigspor nd Aulospor inoulnts (Figure 2B). Shoot nd root growth Plnt growth of RIL147 ws signifintly inresed y AMF tretments (P.5) sine higher dry weight ws oserved for plnts inoulted with AMF thn for ontrol plnts whtever the AMF tretments, nd the highest shoot dry weight ws oserved for plnts inoulted with Glomus thn other tretments (Figure 3A). AMF tretments indued signifint inrese in shoot dry weight of 46, 21 nd 26%, in plnts inoulted with Glomus, Gigspor nd for plnts grown in ontt with myorrhized Stylosnthes, respetively in omprison to tht in the ontrol plnts. But, plnts inoulted with Aulospor show low shoot dry weight without signifint differene with the ontrol plnts (Figure 3A). Root iomss ws lso ffeted y inoultion with AMF (P=.43) (Figure 3B). In omprison to the ontrol plnts, AMF tretments indued signifint inrese in root-growth of 48 nd 32% in plnts inoulted with Glomus nd for plnts grown in ontt with myorrhized Stylosnthes, respetively. But no signifint differene etween root-growth of plnts inoulted with Gigspor, Aulospor nd ontrol plnts (Figure 3B). P umultion nd P use effiieny Dt in Figure 4A show tht shoot phosphorus ontent vried signifintly with AMF tretments (P=.4). The highest phosphorus perentge ws oserved in plnts inoulted with oth tretment of Glomus (y myorrhizl inoulnt nd y ontt), nd the lowest phosphorus ontent ws otined in plnts inoulted with Aulospor without signifint differene with the ontrol plnts (Figure 4A). With AMF, the perentge phosphorus ontent inresed signifintly of 3, 14 nd

4 root oloniztion (%) Afr. J. Biotehnol. 1 8 A Figure 1A Figure 1A Glomus Gigspor Aulospor myorrhized Stylosnthes B Figure 1. A- Effet of Glomus, Gigspor nd Aulospor y myorrhizl inoulnt, nd effet of Glomus y ontt with myorrhized Stylosnthes guinensis on root oloniztion y vesiles (grey rs), hyphe (lk rs), nd rusules (hthed rs) of ommon en genotype RIL147 grown in snd ulture. Dt re mens ± SD of 3 replites, plnts hrvested t 5 dys fter sowing. For eh struture, different letters indite signifint differenes etween tretment mens ording to Dunn's multiple rnge test (P.5). B- root oloniztion of ommon en genotype RIL147 y vesiles nd hyphe of Glomus intrrdies.

5 Tjini nd Drevon Nodule numer pl A B.1 g DW nodule pl -1.5 Figure 2 Glomus Gigspor Aulospor myorrhized Stylosnthes ontrol Figure 2. Effet of AMF on numer (A) nd dry weight (B) of nodules of ommon en genotype RIL147, grown in snd ulture, fter inoultion with R. tropii CIAT899, nd with Glomus, Gigspor nd Aulospor y myorrhizl inoulnt (lk rs) or y ontt with myorrhized Stylosnthes guinensis with Glomus (hthed rs) or not s ontrol(open rs). Dt re mens ± SD of 3 replites, plnts hrvested t 5 dys fter sowing; different letters indite signifint differenes etween tretment mens ording to Dunn's multiple rnge test (P.5). 23% in plnts inoulted with Glomus, Gigspor nd for plnts grown in ontt with myorrhized Stylosnthes, respetively in omprison to tht in the ontrol plnts (Figure 4A). P use effiieny (PUE) ws lso ffeted y AMF tretments (P.3) (Figure 4B). The highest P use effiieny ws oserved in plnts inoulted with Glomus or y ontt with myorrhized Stylosnthes with mens of.1 ±.14 nd.12 ±.28 g DW mg -1 P, respetively. But the lowest P utiliztion ws otined in plnts inoulted with Aulospor with men of.6 ±.16 g DW mg -1 P. The inoultion with AMF inresed signifintly the P utiliztion of 4, 15 nd 5% in plnts inoulted with Glomus, Gigspor nd in plnts grown in ontt with myorrhized Stylosnthes, respetively in omprison to ontrol plnts (Figure 4B). N umultion Dt in Figure 5 show tht shoot nitrogen ontent ws inresed y AMF tretments (P.5). Thus, Glomus, in omintion with CIAT899, indued signifint inrese in shoot nitrogen perentge in omprison to ontrol plnts, wheres there ws no signifint differene etween oth AMF tretments (y myorrhizl inoulnt nd y ontt). With AMF, the perentge of nitrogen inresed signifintly of 45 nd 38% in plnts inoulted with Glomus nd y ontt with myorrhized Stylosnthes respetively, in omprison to tht in the ontrol plnts (Figure 5). DISCUSSION In this study, we estlished triprtite symiosis of ommon en with rhizoi nd AMF y myorrhizl inoulnt or y ontt with myorrhized Stylosnthes. The interesting finding of this work is the signifint use effiieny of phosphorus for growth enefit of the triprtite symiosis with AMF tretments. The highest phosphorus

6 12178 Afr. J. Biotehnol. g DW shoot pl A B g DW root pl Figure 3 Glomus Gigspor Aulospor myorrhized ontrol Stylosnthes Figure 3. Effet of AMF on dry weight of shoot (A) nd root (B) of ommon en genotype RIL147, grown in snd ulture, fter inoultion with R. tropii CIAT899, nd with Glomus, Gigspor nd Aulospor y myorrhizl inoulnt (lk rs) or y ontt with myorrhized Stylosnthes guinensis with Glomus (hthed rs) or not s the ontrol (open rs). Dt re mens ± SD of 3 replites, plnts hrvested t 5 dys fter sowing; different letters indite signifint differenes etween tretment mens ording to Dunn's multiple rnge test (P.5). use effiieny in the present work ws otined in plnts inoulted y Glomus or in plnts grown with myorrhized Stylosnthes (Figure 4) demonstrtes tht the mehnisms ffeting the effiieny of sorption nd utiliztion of phosphorus in plnts re relted to oloniztion y AMF (Buher et l., 21; Ji et l., 24). Also nitrogen umultion ws higher with Glomus under oth tretments in omprison to tht in the ontrol plnts (Figure 5), illustrtes the reltionship etween P use effiieny nd symioti nitrogen fixtion (Tng et l., 21; Tjini et l., 211) nd it ws lso indited tht the shoot growth nd N 2 fixtion were determined minly y the effiieny in P utiliztion (Rodino et l., 29; Brgz et l., 211, ). In snd-soil ulture where P limited onditions re more pronouned, the expression of myorrhizl enefits ws more ovious in plnts with oth tretments of Glomus (Figure 1), it is elieved tht myorrhize espeilly enefit plnts grown in soils where P is likely to limit plnt growth y inresing the soil volume explored y AM hyphe reltive to tht of root hirs of non-am plnts. This would gree with previous studies showing highest myorrhizl enefits to plnt growth under moderte P defiieny (Mthimrn et l., 25; Tjini et l., 29, 211; Fghire et l., 21; Xiurong et l., 211), espeilly with leguminous plnts hrouring orser root system with less extension of root hirs thn grmineous (Isoe nd Tsuoki, 1998). Different omptiilities etween myorrhizl nd rhizoil my explin the differenes in oloniztion nd nodultion of RIL147 upon inoultion with different AMF speies. They my explin the differenes in P use effiieny for Glomus- ompred to Gigspor or Aulospor inoulted plnts. The higher nodultion

7 PUE (g/mg) PUE (g/mg) P (%) P (%) Tjini nd Drevon A.1.12 B.8.4 Glomus Gigspor Aulospor myorrhized ontrol Figure 4 Stylosnthes Figure 4. Effet of AMF on plnt phosphorus perentge (A) nd phosphorus use effiieny (B) of ommon en genotype RIL147, grown in snd ulture, fter inoultion with R. tropii CIAT899, nd with Glomus, Gigspor nd Aulospor y myorrhizl inoulnt (lk rs) or y ontt with myorrhized Stylosnthes guinensis with Glomus (hthed rs) or not s ontrol (open rs). Dt re mens ± SD of 3 replites, plnts hrvested t 5 dys fter sowing; different letters indite signifint differenes etween tretment mens ording to Dunn's multiple rnge test (P.5). with Glomus in oth tretments (y myorrhizl inoulnt plnts nd shre some prt of the signlling pthwy or y ontt), thn with the other AMF speies is most (Antunes et l., 26). likely due to improved P nutrition of the plnts sine Previous studies with ommon en found tht nitrogen Glomus intrrdies is very effiient in trnsporting lrge fixtion ws signifintly limited y P defiieny, nd quntities of P to the plnts from remote zones (Jns et plnts defiient in P show deresed nodule-numer nd l., 23). Also, the oloniztion y Glomus ws higher iomss when grown in soil, snd nd lkline solution, under low P. So tht mens tht there my e signl or hydroeroponis (Brgz et l., 211, ; Mndri et oming from the plnts, in reltion to their P defiieny, l., 211; Vdez nd Drevon, 21). This ws not the tht im t inresing the level of myorrhiztion. se of our study (Figures 2 nd 5) nd this ould e Alterntively, Gigspor is known to require lrge ttriuted to the effet of inoultion with oth rhizoi quntities of photosynthtes from the plnts during its nd AMF, espeilly with Glomus, whtever the oloniztion estlishment, sometimes leding to plnt tretments (y myorrhizl inoulnt or y ontt with growth derese (Figure 3B) (Smith nd Smith, 1996). myorrhized Stylosnthes). In ddition, AMF improve This high ron requirement y Gigspor might plnt growth prmeters nd nutrient uptke. It hs een ompete with nodultion under low vilility of sugrs demonstrted for inresed N 2 fixtion in myorrhizl in roots (O'Hr, 21). The lter my lso explin the plnts tht when oth nitrogen nd phosphorus re very low detetion of oloniztion y Aulospor, unless limiting, AMF n improve phosphorus uptke y the we filed to visulize the root oloniztion strutures tht plnt whih in turn would result in more energy ville PUE (g/mg) re known to e diffiult to stin (Boddington nd Dodd, 2). Both symioses depend on signl exhnge with for nitrogen fixtion y rhizoi (Fitter nd Grye, 1994).

8 N(%) 1218 Afr. J. Biotehnol Figure 5 Glomus myorrhized Stylosnthes ontrol Figure 5. Effet of Glomus on shoot nitrogen perentge of ommon en genotype RIL147 grown in snd ulture, fter inoultion with R. tropii CIAT899, nd with Glomus y myorrhizl inoulnt (lk rs) or y ontt with myorrhized Stylosnthes guinensis (hthed rs) or not s ontrol (open rs). Dt re mens ± SD of 3 replites, plnts hrvested t 5 dys fter sowing; different letters indite signifint differenes etween tretment mens ording to Dunn's multiple rnge test (P.5). In the sme wy nd in order to ground one's rgument for our hypothesis, Krndshov nd Buher (25) found tht the enhned N 2 -fixing ility in myorrhizl plnts ompred with non-myorrhizl plnts, usully disppers if the non-myorrhizl plnts re supplied with redily ville P soure. The otined results indited tht AMF tretments, espeilly with Glomus, signifintly inresed nitrogen umultion in the shoot nd inresed the phosphorus ontent nd phosphorus use effiieny in plnts, ompred with their ontrols whih were not dully inoulted. These findings re in greement with tht of Aysn nd Demir (29), Askr nd Rshd (21) nd Xiurong et l. (211) with other AMF tretments, it is well known tht AMF n improve the nutrient sttus of their host plnts (Smith nd Red, 28; Tjini et l., 29; Kim et l., 21). It is lso thought tht the plnt-rhizoium system enefits from the presene of AM fungi euse the myorrhize meliorte not only P defiieny ut lso ny other nutrient defiienies tht might e limiting to rhizoium (Smith, 22). Similrly, Nutiyl et l. (21) found tht dul inoultion of Cier rietinum L. with rhizoi nd AMF signifintly enhned the numer of nodules nd the dry weight per plnt. Kim et l. (21) showed tht the omined inoultion of Methyloterium oryze strins nd AMF in soil ulture inresed plnt growth, resulted in signifintly higher nitrogen umultion in roots s well s shoots, nd inresed the phosphorus ontent of red pepper plnts ompred with uninoulted ontrols. However, under drought stress inoultion with AMF nd rhizoil strins inhiited of nodule development nd N 2 fixtion, nd used derese in plnt growth (Frnzini et l., 21, Bllesteros-Almnz et l., 21). On the other hnd, Lisette et l. (23) reported tht oinoultion with rhizoi nd omptile AMF ould drmtilly enhne pe growth, N nd P uptke. Therefore, the AMF (Glomus) we used for the present study re omptile with the rhizoil strin nd ommon en genotype, whih might hve potentil for griulturl pplition. Conlusion AM fungl speies used in this study hve different level of root oloniztion nd nodultion, ontriution to plnt growth nd nutrient uptke of ommon en plnts s ompred to the ontrol plnts. The results indite tht inoultion with myorrhizl speies, espeilly with Glomus, hd positive effets on the growth of ommon en RIL147. Myorrhize inoulted plnt hd higher yield (SDW nd RDW) nd inresed P plnt onentrtion nd P utiliztion for symioti nitrogen fixtion. Suitle omintions of AMF (y ontt or y inoultion) nd rhizoi nd the omptiility etween oth symionts, my derese sensitivity of symioti nitrogen fixtion to phosphorus defiieny. For the suessful pplition of these results in iotehnology, multi- lotionl field trils re needed to determine the most effiient tri-prtite symioses. These results show n interesting prtil pplition for griulturl

9 Tjini nd Drevon development in mrginl lnds tht re often defiient in P. The present study opens possiilities for in-situ nondestrutive studies of energy lne in terms of ron nd oxygen requirements for symioti respirtion, metoli monitoring (NMR), nd moleulr nlyses with in-situ hyridiztion nd rel time polymerse hin retion (RT-PCR) with prtiulrly len mteril. REFERENCES Aohtem M, Chkrfi F, Jiti F, Dihzi A, Bziz M (211). Arusulr Myorrhizl Fungi Limit Inidene of Fusrium oxysporum f.sp. ledinis on Dte Plm Seedlings y Inresing Nutrient Contents, Totl Phenols nd Peroxidse Ativities. Open Horti. J., 4:1-16. Adesemoye AO, Kloeppe JW (29). Plnt-miroes intertions in enhned fertilizer-use effiieny. Appl. Miroiol. Biotehnol., 85:1 12. Andrde G, Mihr KL, Lindermn RG, Bethlenflvy GJ (1997). Bteri from rhizosphere nd hyphosphere soils of different rusulr-myorrhizl fungi. Plnt Soil, 192: Antunes PM, Deville D, Goss MJ (26). Effet of two AMF life strtegies on the triprtite symiosis with Brdyrhizoium jponium nd soyen. Myorrhiz, 16: Artursson V, Finly RD, Jnsson JK (26). Intertions etween rusulr myorrhizl fungi nd teri nd their potentil for stimulting plnt growth. Environ. Miroiol., 8: 1-1. Askr AA, Rshd YM (21). Arusulr myorrhizl fungi: A ioontrol gent ginst ommon en Fusrium root rot disese. J. Plnt Pthol., 9: Aysn E, Demir S (29). Using rusulr myorrhizl fungi nd Rhizoium leguminosrum, Biovr phseoli Aginst Slerotini slerotiorum (Li.) de ry in the ommon en (Phseolus vulgris L.). J. Plnt Pthol., 8: Bllesteros-Almnz L, Altmirno-Hernndez J, Pen-Criles JJ, Sntoyo G, Snhez-Ynez JM, Vleni-Cntero E, Mis- Rodriguez L, Lopez-Buio J, Crdens-Nvrro R, Fris-Rodriguez R (21). Effet of o-inoultion with myorrhiz nd rhizoi on the nodule trehlose ontent of different en genotypes. Open Miroiol. J., 4: Bre JM (1997). Myorrhiz-teri intertions on plnt growth promotion. In: Ogoshi K et l. (eds) Plnt Growth Promoting Rhizoteri, Pris, OECD Press, pp: Bre JM, Azon R, Azon-Aguilr C (22). Myorrhizosphere intertions to improve plnt fitness nd soil qulity. Anton. Vn. Leeuwen, 81: Brgz A, Drevon JJ, Oufdou K, Mndri B, Fghire M, Ghoulm C (211). Nodule phosphorus requirement nd O2 uptke in ommon en genotypes under phosphorus defiieny. At Agri. Sn. Setion B-Soil Plnt Si., 61: Brgz A, Ghoulm C, Fghire M, Asln Attr H, Drevon JJ (211). The nodule ondutne to O2 diffusion inreses with high phosphorus ontent in the Phseolus vulgris-rhizoi symiosis. Symiosis, 53(6): Boddington CL, Dodd JC (2). The effet of griulturl prties on the development of indigenous rusulr myorrhizl fungi. II. Studies in experimentl miroosms. Plnt Soil, 218: Buher M, Rush C, Drm P (21). Moleulr nd iohemil mehnisms of phosphorus uptke into plnts. J. Plnt Nutr. Soil Si., 164: Demir S, Akkopru A (27). Using of rusulr myorrhizl fungi (AMF) for ioontrol of soil-orne fungl plnt pthogens. In: Chinholkr SB, Mukerji KG (eds) Biologil Control of Plnt Diseses, Hworth Press, USA, ISBN: , pp: Fghire M, Smri S, Meddih A, Bslm M, Goioehe N, Qddoury A (21). Positive effets of rusulr myorrhizl fungi on iomss prodution, nutrient sttus nd wter reltions in dte plm seedlings under wter defiieny. At Hort., (ISHS) 882: Fitter AH, Grye J (1994). Intertions etween myorrhizl fungi nd other soil orgnisms. Plnt Soil, 159: FrnziniVI, Azón R, Ltnze-Mendes F, Aro R (21). Intertion etween Glomus speies nd Rhizoium strins ffet the nutritionl physiology of drought stressed legume hosts. J. Plnt Physiol., 167: Gmlero E, Mrtinotti MG, Trott A, Lemneu P, Bert G (24). Morphogeneti modifitions indued y Pseudomons fluoresens A6RI nd Glomus mossee BEG12 in the root system of tomto differ ording to plnt growth onditions. New Phytol., 155: Hrdrson G, Atkins C (23). Optimising iologil N2 fixtion y legumes in frming systems. Plnt Soil, 252: Isoe K, Tsuoki Y (1998). The reltionship etween growth promotion y rusulr myorrhizl fungi nd root morphology nd phosphorus sorption in grmineous nd leguminous rops. Crop Si., 67: Jns J, Mozfr A, Anken T, Ruh R, Snders IR, Frossrd E (22). Diversity nd struture of AMF ommunities s ffeted y tillge in temperte soil. Myorrhiz, 12: Jns J, Mozfr A, Frossrd E (23). Long-distne trnsport of P nd Zn through the hyphe of n rusulr myorrhizl fungus in symiosis with mize. Agronomie, 23: Ji Y, Gry VM, Strker CJ (24). The influene of rhizoium nd rusulr myorrhizl fungi on nitrogen nd phosphorus umultion y Vii f. Ann. Bot., 94: Jin L, Sun XW, Wng XJ, Shen YY, Hou FJ, Chng SH, Wng C (21). Synergisti intertions of rusulr myorrhizl fungi nd rhizoi promoted the growth of Lthyrus stivus under sulphte slt stress. Symiosis, 5: Krndshov V, Buher M (25). Symioti phosphte trnsport in rusulr myorrhizs. Tren. Plnt Si., 1: Kim K, Yim W, Trivedi P, Mdhiyn M, Hri P Boruh D, Rshedul Islm Md, Lee G, S T (21). Synergisti effets of inoulting rusulr myorrhizl fungi nd Methyloterium oryze strins on growth nd nutrient uptke of red pepper (Cpsium nnuum L.). Plnt Soil, 327: Lisette J, Xvier C, Germid JJ (23). Seletive intertions etween rusulr myorrhizl fungi nd Rhizoium leguminosrum v. viee enhne pe yield nd nutrition. Biol. Fertil. Soil, 37: Mthimrn N, Ruh R, Vullioud P, Frossrd E, Jns J (25). Glomus intrrdies domintes rusulr myorrhizl ommunities in hevy textured griulturl soil. Myorrhiz, 16: Mndri B, Drevon JJ, Brgz A, Oufdou K, Fghire M, Plssrd C, Pyre H, Ghoulm C (211). Intertions etween ommon en (Phseolus vulgris) genotypes nd rhizoi strins isolted from Moron soils for growth, phosphtse nd phytse tivities under phosphorus defiieny onditions. J. Plnt Nutr., 34: (rtile in press). MGonigle TP, Miller MH, Evn DG, Fihild GL, Swn JA (199). A new method whih gives n ojetive mesure of oloniztion of roots y vesiulr-rusulr myorrhizl fung. New Phytol., 115: Nutiyl CS, Chuhn PS, DsGupt SM, Seem K, Vrm A, Stddon WJ (21). Triprtite intertions mong Peniillus lentimorus NRRL B-3488, Piriformospor indi DSM 11827, nd Cier rietinum L. W. J. Miroiol. Biotehnol., 26: Numn B, Nuri Y (25). The Effets of Different Nitrogen nd Phosphorus Doses nd Bteri Inoultion (Rhizoium phseoli) on the Yield nd Yield Components of Field Ben (Phseolus vulgris L.). J. Agron., 4: O'Hr GW (21). Nutritionl onstrints on root nodule teri ffeting symioti nitrogen fixtion: review. Austr. J. Exp. Agri., 41: Rodino AP, Metre R, Guglielmi S, Drevon JJ (29). Vrition mong ommon-en essions (Phseolus vulgris L.) from the Ierin Peninsul for N 2-dependent growth nd phosphorus requirement. Symiosis, 47: Snging N, Lysse O, Singh BB (2). Phosphorus use effiieny nd nitrogen lne of owpe reeding lines in low P soil of the derived svnn zone in West Afri. Plnt Soil, 22: Sheulin TR, Vnder Heijden MGA (26). Arusulr myorrhizl fungi olonize nonfixing root nodules of severl legume speies. New Phytol., 172: Sepehr, E., M.J. Mlkouti, B. Kholderin, A. Smdi, nd N. Krimin

10 12182 Afr. J. Biotehnol. (29). Genotypi vrition in P effiieny of seleted Irnin erels in greenhouse experiment. Int. J. Plnt Prod., 3(3): Smith FA, Smith SE (1996). Mutulism nd prsitism: diveristy in funtion nd struture in the "rusulr" (VA) myorrhizl symiosis. Adv. Bot. Res., 22: Smith SE (22). Soil miroes nd plnts-rising interest, mutul gins. New Phytol., 156: Smith SE, Red DJ (28). Myorrhizl Symioses. London, UK; Ademi Press. Tjini F, Suriykup P, Vilhe H, Jns J, Drevon JJ (29). Assess suitility of hydroeroponi ulture to estlish triprtite symiosis etween different AMF speies, ens, nd rhizoi. B.M.C. Plnt Biol. 9: Tjini F, Trelsi M Drevon JJ (211). Co-inoultion with Glomus intrrdies nd Rhizoium tropii CIAT899 inreses P use effiieny for N2 fixtion in ommon en under P defiieny in hydroeroponi ulture. Symiosis, 53: Tjini F, Suriykup P, Jns J, Drevon JJ (211). Assessing hydroeroponi ulture for the triprtite symiosis of mungen (Vign rdit L.) with rusulr myorrhizl fungi nd rhizoi. Afr. J. Biotehnol., 1: Tng C, Hinsinger P, Jillrd B, Rengel Z, Drevon JJ (21). Effet of phosphorus defiieny on growth, symioti N 2 fixtion nd proton relese y two en (Phseolus vulgris L.) genotypes. Agronomie, 21: Tussky HH, Shorr E (1953). Miroolorimetri method for the determintion of inorgni phosphorus. Biol. Chem., 22: Tilmn D, Cssmn KG, Mtson PA, Nylor R, Polsky S (22). Agriulturl sustinility nd intensive prodution prties. Nture, 418: Vdez V, Rodier F, Pyre H, Drevon JJ (1996). Nodule permeility to O 2 nd nitrogense-linked respirtion in en genotypes vrying in the tolerne of N 2 fixtion to P defiieny. Plnt Physiol. Biohem., 34: Vdez V, Drevon JJ (21). Genotypi vriility in P use effiieny for symioti N2 fixtion in ommon-en (Phseolus vulgris L.). Agronomie, 21: Vne CP, Uhde-Stone C, Alln DL (23). Phosphorus quisition nd use: ritil dpttions y plnts for seuring nonrenewle resoure. New Phytol., 157: XioTJ, Yng QS, Rn W, Xu GH, Shen QR (21). Effet of inoultion with rusulr myorrhizl fungus on nitrogen nd phosphorus utiliztion in uplnd rie-mungen interropping system. Agri Si., 9: Xiurong W, Qing P, Fengxin C, Xiolong Y, Hong L (211). Effets of o-inoultion with rusulr myorrhizl fungi nd rhizoi on soyen growth s relted to root rhiteture nd vilility of N nd P. Myorrhiz, 21: Wehner J, Antunes PM, Powell JR, Mzuktow J, Rillig MC (21). Plnt pthogen protetion y rusulr myorrhizs: A role for fungl diversity?. Pedoiologi, 53: