Pak. J. Agri. Sci., Vol. 53(4), ; 2016 ISSN (Print) , ISSN (Online) DOI: /PAKJAS/

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1 Pk. J. gri. Sci., Vol. 53(4), ; 216 ISSN (Print) , ISSN (Online) DOI: /PKJS/ EFFECT OF INOCULTION Y MOROCCN ROCK PHOSPHTE- SOLUILIZING RHIZOI, VERSUS PHOSPHORUS FERTILIZTION, ON THE GROWTH ND THE PHOSPHORUS UPTKE Y Vici f Tsnime Mghroui 1,2, Nour echtoui 1, ntoine Glin 2, Sn Whi 2,3, Roin Duponnois 2, Mohmed Hfidi 3, Khlid Doui 4, Zin El idine Ftemi 4, Philippe de Ljudie 2 nd Khlid Oufdou 1,* 1 Lortory of iology nd iotechnology of Microorgnisms, Fculty of Sciences Semlli, Cdi yyd University, PO ox 239, Mrrkech, Morocco; 2 IRD, CIRD, LSTM, Cmpus de illrguet T 82/J Montpellier, Frnce; 3 Lortory of Ecology nd Environment (CNRST, URC32), Fculty of Sciences Semlli, Cdi yyd University, PO ox 239, Mrrkech, Morocco; 4 Centre Régionl de l Recherche gronomique de Meknès, INR, Morocco. * Corresponding uthor s e-mil: oufdou@uc.m Low Phosphorus vilility in soil is worldwide frequent mjor constrint for crops. Rhizoil strins, eneficil N 2-fixing symiotic prtners of legumes, were reported to soluilize oth orgnic nd inorgnic complex phosphtes. Thus they my ply significnt role s plnt growth-promoting rhizocteri (PGPR) in the iofertiliztion of crops. Nturl rock phosphte my ply key role in sustinle griculture, resulting in economic nd environmentl enefits. This work ims to evidence the cpcity of rhizoil strins nodulting Vici f L. plnts in Mrrkech-Houz region (Morocco) to soluilize Moroccn rock phosphte in vitro in either gr or liquid medium nd to evlute nd compre the effect of these strins on growth nd phosphorus uptke y V. f plnts of two Moroccn vrieties (Defes nd gudulce). Four rhizoil strins, contrsting for phosphte soluiliztion on gr medium, were further studied on liquid medium. RHOF147 nd RHOF174 strins formed cler hlos round colonies (1.37 cm nd 1.2 respectively fter 15 dys); opposite RHOF17 nd RHOF171 strins did not produce hlo. The four strins were le to moilize rock phosphte for growth in liquid medium (up to.59 mg/l of ssimilted phosphorus). Lower ph vlues nd most moilized phosphorus production were registered for RHOF147 nd RHOF174 strins. y screening the four rhizoil strins for growth on Chrome zurol S (CS) medium, only RHOF171 strin formed ornge hlo indicting its cpcity to produce siderophores. Greenhouse experiments were undertken including comprison of inoculted plnts with these four rhizoil strins in the presence of rock phosphte s sole P source versus noninoculted plnts growing on KH 2PO 4 s sole P source (P-fertilized plnts). Results show tht the root length, the root dry weight nd the rtio root /shoot dry weights were generlly higher in the inoculted plnts thn in the P-fertilized plnts, indicting the dpttion of the inoculted plnts to phosphorus deficiency conditions. The effect of rock phosphtesoluilizing cteri on plnt growth depends on the symiotic comintion of the inoculted strin nd the plnt genotype. s for phosphorus uptke, rhizoil strins mde phosphorus ville to the plnt t significnt incresed concentrtions in some symiotic comintions (e.g. gudulce-rhof147). The use of selected symiotic comintions hving the cpcity to soluilize rock phosphte my improve soil fertility nd phosphorus vilility to plnts. Keywords: Moroccn rock phosphte, rhizoi, Vici f, phosphorus soluiliztion. INTRODUCTION Morocco hrors the lrgest reserves of rock phosphte worldwide. The nturl rock phosphte my constitute vlule lterntive source of phosphorus fertilizer, when iotechnologicl process could e pplied to promote its soluiliztion nd mke phosphorus ville to the plnts (Hmdli et l., 28; Chng nd Yng, 29; Prk et l., 211). ioinoculums re most promising fertilizers for soil fertility, s lterntives for chemicl fertilizer use ffecting humn helth nd cusing environmentl pollution (Khn et l., 27; Svci, 212). Vlorizing the legume-rhizoi symiotic ssocitions my constitute friendly iotechnologicl pthwy to mintin the ecologicl lnce under severl environmentl stress conditions (Dulormne et l., 21; Fghire et l., 213; Oufdou et l., 214). fter nitrogen, phosphorus is n essentil plnt nutrient whose deficiency severely restricts plnt growth (ollnd et l., 2; Hinsinger, 21; Mjeed et l., 214). Low soil phosphorus vilility is mjor limiting fctor of crop yields, especilly when crop relies on symiotic nitrogen

2 Mghroui, echtoui, Glin, Whi, Duponnois, Hfidi, Doui, Ftemi, de Ljudie & Oufdou fixtion (Scervino et l., 211; Srinivsn et l., 212). Phosphorus is often present in soil s insolule complexes unville to plnt nutrition. Phosphorus is complexed with luminium, iron, silicium, nd other metllic ions in cidic soil (Whitelw, 2) or with clcium cronte in lkline soil (Gyneshwr et l., 22). Liu et l. (1994) reported tht the phosphorus deficiency is the most common nutritionl stress for plnts, ffecting 42% of the cultivted lnd over the world. Some rhizoil strins, eneficil N 2-fixing symiotic prtners of legumes, were reported to soluilize oth orgnic nd inorgnic phosphte complexes. These symiotic microorgnisms were shown to ffect positively plnt growth under phosphorus-deficient conditions y mking complex phosphorus sources ccessile to the plnt (Richrdson, 21; likhni et l., 26; Srinivsn et l., 212). Thus, using such rhizoi, selected for their phosphte-soluilizing cpcity s ioinoculnts to crops would result in the dul eneficil nutritionl effect from oth P moiliztion nd N 2- fixtion (Peix et l., 21). Vici f L. (f en) is considered s multipurpose crop over the world (Doui et l., 211, Mululem et l. 212; El- Komy et l., 215). V. f is the most importnt legume crop grown in Morocco over 4% of the totl re dedicted to legumes (MDRPM/DERD, 26). The f en is rich in proteins, minerl elements nd vitmins, nd hence used for oth humn nd niml nutrition (ond et l., 1985). V. f, due to its symiotic ssocition with rhizoi, plys gret role in griculturl production nd contriutes to food security nd economic development (Grhm nd Vnce, 23; Khn et l., 21). Few reports re ville on the role of rock phosphtesoluilizing rhizoi on f en plnt growth nd phosphorus uptke (Demissie et l., 213). During this study, our focus ws to evlute the cpcity of V. f-nodulting rhizoil strins to soluilize Moroccn rock phosphte, determine their ility to soluilize rock phosphte on gr medium nd in liquid roth of sme composition nd, ssess nd compre the inocultion effect of these rhizoil strins on growth nd phosphorus uptke y two Moroccn vrieties of V. f plnts (Defes nd gudulce) in the presence of rock phosphte s sole P source versus non-inoculted plnts growing on KH 2PO 4 s sole P source (P-fertilized plnts). MTERILS ND METHODS Isoltion nd purifiction of rhizoil strins: Rhizoil strins were isolted from field-grown V. f plnts locted in the Mrrkech-Houz region (Morocco). Nodules of V. f plnts were previously disinfected with sodium hypochlorite (4 ) nd wshed severl times with sterile physiologicl wter. The nodule ws crushed in sterile tue. The suspension ws streked on Petri dishes contining YEM medium gr with Congo Red. fter incution for 48 h to h t 28 C, colonies of rhizoi, chrcterized y gluey spect nd not uptking Congo red, were isolted on YEM medium (Vincent, 197). The rhizoi strins were purified y repeted streking on Yest-Extrct-Mnnitol gr (YEM gr) with Congo red. Pure isoltes were checked for their nodultion of septic V. f seedlings nd stored t -25 C in glycerol 3% v/v. Phosphte soluiliztion test on gr medium: Rhizoil strins were grown in Erlenmeyer flsks (25 ml) contining 1 ml YEM roth medium t 28 C during 48 h on rotry shker t 18 rpm. fter incution, the cultures were centrifuged t 6rpm for 5 min. The superntnt ws discrded, nd the pellet ws suspended in 1 ml of sterile physiologicl wter nd centrifuged gin. This opertion ws repeted 3 times to eliminte trces of phosphorus from the rhizoil cells. The finl pellet ws suspended in volume of sterile physiologicl wter such s to rech finl OD of.8. This constituted the inoculum. Using the drop plte method (likhni et l., 26), ech prt ws inoculted with 7 µl of the inoculum on TCPNH 4Cl medium gr contining 5g/L rock phosphte. The TCPNH 4Cl medium consists of: Glucose 1 g/l, MgSO 4.7H 2O 1 g/l, NH 4Cl 5 g/l nd NCl 1 g/l. Inoculted pltes were incuted t 28 C in drk. When occurring, the dimeter of the clering zone (hlo) surrounding the cteril colony, corresponding to P soluiliztion, ws mesured fter 3, 1 nd 15 dys. ll ssys were replicted three times. The Moroccn rock phosphte is hydroxyptite, nturlly occurring minerl form of clcium ptite, C 5(PO 4) 3(OH), usully written C 1(PO 4) 6(OH) 2 to denote tht the crystl unit cell comprises two entities. It consists of 56.53% O, 16.35% C, 9.37% P, 2.42% F, 2.3% l, 1.94% Mg, 1.81% N,.77% S,.6% Fe, nd.12% Sn (Hmdli et l., 28). Phosphte soluiliztion test in liquid medium: mong 8 rhizoil strins of our collection, we chose four strins with contrsting cpcities to soluilize rock phosphte in gr medium: RHOF147 nd RHOF174 strins form cler hlos round colonies wheres RHOF17 nd RHOF171 strins do not form hlo. Erlenmeyer flsks (25 ml) contining 1 ml of liquid TCPNH 4Cl medium contining 5 g/l of rock phosphte, were inoculted with 2 µl of ech rhizoil inoculum wshed three times s descried ove in the previous prgrph. The initil opticl density (OD) in the flsks ws.1. oth inoculted nd non-inoculted control flsks hd n initil ph of 7. The erlenmeyer flsks were incuted in drk on rotry shker (18 rpm) t 28 C. fter 48h, 96 h nd 168 h of incution, liquots of cultures were septiclly tken from ech flsk, centrifuged (6 rpm for 5 min) nd superntnt ws filtered on Millipore filter (.2 µm of porosity). ph vlues nd solule phosphorus relesed into the solution were mesured. Control flsk ws used s the seline (zero sornce) for determining the solule phosphorus relesed y ech rhizoil strin in culture medium. The dosge of the solule

3 Moroccn rock phosphte-soluilizing rhizoi phosphorus ws performed using the colorimetric method sed on the formtion nd reduction of the complex of orthophosphoric cid nd molydic cid. The Phosphorusmolydte reduction is ccompnied y lue color whose intensity is proportionl to the mount of solule phosphorus present in the medium (Olsen nd Sommers, 1982). Siderophore production test: We used the sme protocol reported ove in the prgrph Phosphte soluiliztion test on gr medium. Using the drop plte method, ech prt ws inoculted with 7 µl of the inoculum on the lue Chrome zurole S gr (CS) descried y lexnder nd Zuerer (1991). Development of yellow-ornge hlo round the colony is considered s positive test for siderophore production. Effects of rock phosphte-soluilizing rhizoi on f en plnts: Preprtion of seeds: Plnt tests were performed using two Moroccn V. f mjor vrieties: Defes nd gudulce. The seeds of Defes vriety (length x width x height = 1.98 cm x 1.28 cm x.44 cm) re smller thn the seeds of gudulce vriety (length x width x height = 2.49 cm x 1.78 cm x.37 cm). V. f seeds were sterilized with sodium hypochlorite 4 for 1 min, followed y severl rinsing in physiologicl distilled wter. The seeds were then plced on wet filter ppers in the Petri dishes for germintion t 25 C for 48h to 72h in the drk. Inoculums preprtion: We used the sme protocol reported ove in the prgrph Phosphte soluiliztion test on gr medium. The inoculum ws prepred y growing the rhizoil strin in YEM liquid medium t 28 C for 2-3 dys to otin n opticl density (OD) of 1 t 6 nm (pproximtely 1 9 colonies forming units (CFU)/mL) s descried previously (Lhrouni et l., 212; Oufdou et l., 214). Inocultion nd trnsfer to pot: Uniformly-germinted seedlings were immersed for 3 min in individul rhizoil liquid culture of ech of the tested strins RHOF147, RHOF174, RHOF17 or RHOF171. The seedlings were then grown on perlite in 5 L pots. Rigud nd Puppo nutrient solution in distilled wter ws dded to the trys (Rigud nd Puppo, 1975). Plnts were grown either with rock phosphte mendment s sole P-source nd seprtely inoculted y individul rhizoil test strins (iofertilized plnts: Tretments 2 to 5) or with KH 2PO 4 (P-fertilized, non-inoculted plnts: Tretment 1) (Tle 1). We tested whether plnt inocultion with selected rhizoil strins my compenste phosphorusdeficient conditions (rock phosphte s sole P-source) for plnt growth. The experimentl design ws complete rndom lock ssy. We pplied.4 g rock phosphte under ech germinted seedling in the pot. The plnts were hrvested t the flowering stge (4 dys fter sowing). The roots were thoroughly rinsed with distilled wter. The length, iomss nd totl 819 phosphorus content ws determined in oth shoots nd roots of ech plnt. Tle 1. The protocol of the greenhouse experimenttion. Tretments Prmeters Rock phosphte Rhizoi inoculum T1 (+) KH 2PO 4 (-) Non-inoculted T2 (-) KH 2PO 4 (+) RHOF147 T3 (-) KH 2PO 4 (+) RHOF174 T4 (-) KH 2PO 4 (+) RHOF17 T5 (-) KH 2PO 4 (+) RHOF171 + : presence, - : sence The trils were performed in the greenhouse of the Fculty of Sciences Semlli, Mrrkech, under nturl climtic conditions. Sttisticl nlysis: We used complete rndom lock ssy design. Registered growth vlues were mens of three replictes per tretment for the in vitro phosphte soluiliztion y rhizoi (in solid nd liquid medi), nd were mens of six replictes per tretment for the plnt tests in greenhouse. ll results were sujected to two-wy nlysis of vrince (NOV), with Lest Significnt Difference (LSD) for the comprison of mens using COSTT softwre. Mens nd stndrd errors (SE) were lso clculted nd re presented in the grphs. Mens with different letters re significntly different t p <.5. RESULTS Phosphte soluiliztion of rhizoil strins in gr medium: RHOF147 nd RHOF174 strins formed the lrgest cler hlos round their colonies on gr medium contining rock phosphte s sole source of phosphorus, wheres there ws no clering zone round the colonies of the strins RHOF17 nd RHOF171 until the 15 th dy of incution (Figure 1). We note tht the dimeter of the hlos incresed significntly (p<.5) during the period of incution. For exmple, the dimeter of the hlos produced y RHOF147 ws.77 in 3 dys, 1.18 in 1 dys nd 1.37 in 15 dys. Similrly, the strin RHOF174 exhiited hlos of dimeters.83 cm, 1.15 cm nd 1.2 cm respectively fter 3, 1 nd 15 dys of incution (Fig. 1). Phosphte soluiliztion of rhizoil strins in liquid medium: In liquid medium, the four studied strins were le to moilize differently rock phosphte (Tle 2). They produced solule phosphorus from the rock phosphte used s the sole source of phosphorus. The strin RHOF174 relesed the mximum of solule phosphorus throughout incution followed y the strin RHOF147 (Tle 2). There is positive correltion etween the produced soluilized phosphorus nd the ph decrese in the liquid medium (Tles 2 nd 3). The ph in superntnt turned cidic (Tle 3), with lower vlues in the cse of the

4 Shoot length (cm/ plnt) Shoot length (cm/ plnt) Mghroui, echtoui, Glin, Whi, Duponnois, Hfidi, Doui, Ftemi, de Ljudie & Oufdou two hlo-producing strins: RHOF147 nd RHOF174 ( ) compred to the non-producing ones RHOF17 nd RHOF171 ( ). slight decrese in ph ws lso registered in the control non-inoculted culture medium (.2-.8) used s the seline (zero sornce) for determining the solule phosphorus moilized y ech rhizoil strin in culture medium. Tle 2. Solule phosphorus in mg/l mesured in the superntnt of rhizoil test strin cultures grown in TCPNH4Cl liquid medium contining rock phosphte s sole P source. Strin Time of incution h 48h 96h 168h RhOF ±.1.22±.3.15±.3 RhOF ±.2.39±.3.59±.4 RhOF ±.1.7±.1.2±.3 RhOF ±.1.6±.1.17±.4 Tle 3. Differentil ph vlue in liquid TCP NH4Cl medium during rock phosphte soluiliztion tests y studied strins. Strin Time of incution h 48h 96h 168h RhOF RhOF RhOF RhOF Control Dimeter of the hlo (cm) 1,6 1,2,8,4 RhOF 147 RhOF Time (dys) Figure 1. Rhizoi rock phosphte soluiliztion test in gr TCPNH4Cl medium fter 3 dys, 1 dys nd 15 dys of incution. Results re expressed s dimeters of cler hlos oserved round colonies of the test strins. Mens (± stndrd errors) with different letters re significntly different t p <.5. Siderophore production test: The strin RHOF171 when tested for phosphte soluiliztion, did not produce ny hlo on gr medium nd low level of phosphorus ws soluilized from rock phosphte in liquid medium. This strin developed n ornge hlo on CS gr. RHOF171 strin hs een found to produce siderophores. Effects of rock phosphte-soluilizing rhizoi on f en plnts: The root length of Defes plnts ws generlly higher in the inoculted plnts especilly in the presence of RHOF147 (Fig. 2). The length of the plnts inoculted with RHOF147 (34.5 cm) ws significntly higher thn in the P- fertilized plnts (2 cm). c c 8 6 c Tretments Tretments Figure 2. Shoot lengths of V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: rock phosphte (RP)+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5. 82

5 Root dry weight (g / plnt) Root dry weight (g / plnt) Moroccn rock phosphte-soluilizing rhizoi s for the gudulce vriety, the P-fertiliztion did not increse significntly in either shoot or root lengths in comprison to the inoculted plnts (Fig. 3). Figure 4 presents the shoot nd the root dry weights of V. f plnts (Defes vriety) sumitted to different tretments. The highest shoot dry weights were noted in the P-fertilized plnts nd Defes-RHOF17 symiotic comintion (Figure 4). The P-fertiliztion with KH 2PO 4 did not increse significntly the root dry weight of Defes plnts in comprison to the ioinoculted plnts (Fig. 4). In the inoculted gudulce plnts, root dry weights were generlly incresed, especilly those nodulted y RHOF171 (.7 g/plnt) followed y the plnts nodulted y RHOF174 (.65 g/plnt), wheres the root dry weight ws only.49 g/plnt in the P-fertilized plnts (Fig. 5). Root length (cm/ plnt) Root length (cm/ plnt) Tretments Figure 3. Roots lengths of V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: RP+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5. Shoot dry weight (g / plnt) c c Tretments c Figure 4. Shoot dry weights of V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: RP+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5. 1,,5 Shoot dry weight (g / plnt) 1,, Tretments Tretments, Tretments Figure 5. Roots dry weights of V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: RP+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5., Tretments 821

6 Totl phosphorus (mg/g DW) Totl phosphorus (mg/g DW) Mghroui, echtoui, Glin, Whi, Duponnois, Hfidi, Doui, Ftemi, de Ljudie & Oufdou Roots DW / Shoots DW,6,4,2 Roots DW / Shoots DW,6,4,2 Tr1 Tr2 Tr3 Tr4 Tr5 Tretments Figure 6. Roots DW / Shoots DW rtio in V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: RP+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5. Tr1 Tr2 Tr3 Tr4 Tr5 Tretments Totl phosphorus (mg/g DW) 1,,8,6,4,2 c c c Totl phosphorus (mg/g DW),8,6,4,2 d c cd, Tretments Figure 7. Phosphorus content in shoots of V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: RP+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5., Tretments,4,2 c c c,2, Tretments Figure 8. Phosphorus content in roots of V. f plnts (: Defes vriety nd : gudulce vriety) sumitted to the five tretments. Tr 1: KH 2PO 4; Tr 2: RP+RHOF147; Tr 3: RP+RHOF174; Tr 4: RP+RHOF17; Tr 5: RP+RHOF171. Mens (±stndrd errors) within the sme grphic followed y different letters re significntly different t p <.5. To chrcterize the plnt orgn ehvior, we determined the rtio root dry mtter /shoot dry mtter (Fig. 6). We noticed tht the inoculted plnts presented generlly the higher rtio dry mtter /shoot dry mtter thn tht of the P-fertilized plnts. The inoculted plnts presented high development of the root system. Phosphorus uptke in f en plnts: Phosphorus content would e generlly higher in the plnts continuously irrigted, 822 Tretments with the solule form of phosphorus (KH 2PO 4). However, the rhizoi strins cn compenste the phosphorus deficiency conditions in the presence of rock phosphte. They mintined the phosphorus uptke higher or not significntly lower thn tht in the P-fertilized plnts continuously irrigted with solule phosphorus (KH 2PO 4) (Fig. 7 nd 8). In some symiotic comintions, for instnce, the comintion gudulce-rhof147 resulted in significnt higher

7 Moroccn rock phosphte-soluilizing rhizoi phosphorus uptke in the shoots in comprison to the P- fertilized plnts (Fig. 7). DISCUSSION Rhizospheric microorgnisms re importnt components in soils mediting soil processes such s decomposition, nutrient moiliztion nd minerliztion, storge relese of nutrients nd wter (Zhrn, 21; Duponnois et l., 26; Khn et l., 27). Specificlly, nitrogen-fixing rhizoi-legume symioses represent gret potentil to improve crop yields nd to reduce the use of fertilizers in griculture. In ddition, some rhizoi, together with Pseudomons nd cillus, were reported to hve phosphte-soluilizing cpcity (likhni et l., 26; Khn et l., 27; Demissie et l., 213; ouizgrne et l., 215). In the present work, we investigted in vitro Moroccn rock phosphte soluiliztion cpcity of selected rhizoil strins in oth liquid nd gr medium conditions, nd we exmined the effect of these strins on f en plnt growth nd phosphorus uptke. mong the four rhizoil strins tested two of them RHOF147 nd RHOF174 formed clering zones round their colonies, wheres there ws no cler hlo round the colonies of the strins RHOF17 nd RHOF171. The clering zone dimeter incresed significntly during the time of incution. These results re consistent with reports in literture of soluilizing-phosphte cteri selection y similr in vitro tests of cler hlo formtion round colonies on gr medium contining insolule phosphte (likhni et l., 26; Khn et l., 27; n et l., 213). The rhizoil strins were then tested for their ility to soluilize rock phosphte under liquid culture medium conditions, to further confirm their phosphte-soluilizing ctivity nd to quntify the moilized phosphorus y ech strin. The four studied rhizoi re le to moilize differently phosphorus from rock phosphte lthough two of them did not produce clering zones on gr medium. likhni et l. (26) hve demonstrted the need to crry out liquid medium tests to check the ility of the cteri to soluilize phosphte even it didn t not produce hlo on gr medium. This my e due to differentil diffusion rtes of the vrious orgnic cids secreted y n orgnism tht my e higher in liquid medium thn in gr medium (Johnson, 1959). Xie (28) reported tht the phosphte gr ssy is fst method useful for screening lrge numers of isoltes rpidly nd instntneously. Nevertheless, it my discount isoltes, lthough unle to produce hlo zone on gr pltes, le to soluilize vrious types of insolule inorgnic phosphtes in liquid medi. Therefore, concerning P soluiliztion, the phosphte gr ssy gives qulittive informtion wheres the liquid ssy results in quntittive dt. 823 Since strin le to grow on gr medium contining insolule phosphte s sole P-source without producing hlo round its colony, my soluilize rock phosphte y other processes, performing liquid medium phosphte soluiliztion tests. Hmdli et l. (28) hve reported tht some rhizospheric ctinomycetes strins re not le to produce hlos on gr medium contining rock phosphte, lthough they cn soluilize rock phosphte in liquid medium. These uthors showed tht the soluiliztion of rock phosphte is done y lterntive mechnisms such s the production of siderophores chelting phosphorus. Zidi et l. (29) discussed other mechnisms, which could e indirectly involved in phosphte soluiliztion, such s the secretion of siderophores nd the cheltion y exopolyschride (EPS) of the metl onds to the phosphte nd relese in the roth medium. In these cses, the cpility to reduce the ph did not usully correlte with the ility to soluilize minerl P. However, the study of kinetic production of phosphorus y cteri would offer cler imge of cellulr ehvior towrd P oserved y the vrition of P concentrtion in the liquid medium (Rodriguez nd Frg, 1999). decrese in ph ws noticed in liquid medium especilly in the cse of the two strins RHOF147 nd RHOF174 lso forming cler hlos on gr medium. The most cidic ph vlues nd the mximum moilized phosphorus, were registered for these strins. The non-producing hlo RHOF171 strin, with low phosphorus relesed from rock phosphte in the liquid medium, hs een found to produce siderophores. This is consistent with previous works reporting tht orgnic cids production is mjor mechnism for higher phosphte soluiliztion (likhni et l., 26; Khn et l., 27). cteri cn dissolve phosphtes y the production of orgnic cids for the secretion of H + ions. The predominnt orgnic cids produced y the phosphte-soluilizing cteri include oxlic cid, citric cid, lctic cid, gluconic cid, mlic cid, succinic cid, fumric cid etc (Vzquez et l., 2; Mlih et l., 24). Orgnic cids cn lso dissolve the insolule phosphte s result of nion exchnge of PO 4 3- y cid nion nd/or cn chelte oth iron nd luminum ions ssocited with phosphte (Omr, 1998). Inorgnic cids cn lso soluilize phosphte ut they hve less ctivity thn tht of orgnic cids t the sme ph (Kim et l., 1997). We further investigted the effect of the rhizoil strins on the growth nd the phosphorus uptke y two vrieties of V. f (Defes nd guducle). We compred the effect of ioinocultion y rock phosphte-soluilizing rhizoi versus the chemicl phosphorus fertiliztion with K 2HPO 4. The effect of rock phosphte-soluilizing cteri on plnt growth depends on the inoculted strin nd on the genotype used for symiotic comintion. In some comintions, the root length, the roots dry weight nd the rtio roots dry weight/shoots dry weight were generlly higher in the

8 Mghroui, echtoui, Glin, Whi, Duponnois, Hfidi, Doui, Ftemi, de Ljudie & Oufdou inoculted plnts. It indicted the development nd the dpttion of roots exposed directly to phosphorus deficiency conditions in the inoculted plnts in comprison to the P- fertilized continuously irrigted with solule phosphorus. When compred to the P-fertilized plnts, the effect of phosphorus deficiency conditions ws more pronounced on shoot dry weight of Defes plnts thn tht of gudulce plnts. s for phosphorus uptke y V. f plnts, some rhizoil strins mde phosphorus ville to the plnt t no significntly different levels to those registered for the plnts continuously fertilized with KH 2PO 4 nd sometimes t significnt incresed levels (eg. gudulce-rhof147). These results re in ccordnce with the finding of Dhnkhr et l. (213) who hve reported tht the phosphtesoluilizing cteri used s ioinoculnts simultneously increses crop yield nd phosphorus uptke y the plnt. Demissie et l. (213) hve demonstrted tht most of V. f plnt iomss such s shoot nd root dry weight were significntly incresed s result of inocultion y two rhizoi strins with rock phosphte. Yseen et l. (213) hve reported tht rock phosphorus fertiliztion with the ppliction of Rhizoium nd rusculr mycorrhiz inocultion incresed significntly the growth prmeters of the legume plnt Mcroptilum rctetum. Mndri et l. (212) hve reported tht phosphorus uptke y Phseolus vulgris my e improved y selection of oth the rhizoil strin nd the plnt genotype most dpted to phosphorus limittion in soil. Otherwise, Vlverde et l. (27) hve found tht the cteril in vitro phosphte-soluiliztion ility is not lwys correlted with the plnt phosphorus uptke. In our study, we noticed tht different symiotic comintions result in contrsted levels of totl phosphorus, whtever the ility of the rhizoil isolte to soluilize phosphorus. This result is consistent with the oservtions of Ormeno et l. (23) reporting tht Rhizoium nd Sinorhizoium meliloti strins isolted from nodules of the legume plnt Phseolus luntus, exhiited good cpcity to soluilize phosphorus in vitro ut only Sinorhizoium could moilize it in the plnt. Conclusions: The four studied rhizoil strins were differently le to moilize rock phosphte in liquid medium of sme composition lthough two of them not produced hlos on gr medium. The most cidic ph vlues nd the mximum of moilized phosphorus production were noted for the hlo-producing strins RHOF147 nd RHOF174. The effect of rock phosphte-soluilizing cteri on plnt growth depends oth on the inoculted strin nd on the plnt genotype for symiotic comintion. In some symiotic comintions, the root length, the root dry weight nd the rtio roots dry weight/shoots dry weight were higher in the inoculted plnts thn in the P-fertilized plnts. The development of root system my e n dpttion to 824 phosphorus deficiency conditions in the inoculted plnts in comprison to the P-fertilized continuously irrigted with solule phosphorus. Rhizoi strins mde phosphorus ville to the plnt t significnt incresed concentrtions in some symiotic comintions (e.g. gudulce-rhof147). The use of selected symiotic comintions hving the cpcity to soluilize rock phosphte my improve soil fertility y mking phosphorus more ville to the plnts. The ppliction of efficient phosphte-soluilizing rhizoilegume symioses is promising iotechnology for chemicl fertilizer use limittion. The use of rhizoi symioticlly efficient with the est legume genotype comintion, nd hving the cpcity to soluilize rock phosphte represents fscinting lterntive for frmers over the world nd especilly in Morocco, holding the lrgest reserves of nturl rock phosphte worldwide. cknowledgments: This work ws supported y the Gret Federtive Project FTROPIMED finnced y gropolis Foundtion under the reference ID 11-9 nd VERROES scholrship progrm provided y the EU for Tsnime MGHROUI PhD work. REFERENCES lexnder, D.. nd D.. Zuerer Use of Chrome zurol S regents to evlute siderophore production y rhizosphere cteri. iol. Fertil. Soils 12: likhni, H.., N. Sleh-Rstin nd H. ntoum. 26. Phosphte soluilizing ctivity of rhizoi ntive to Irnin soils. Plnt Soil 287: n,.h.,.h. Dicko, K. Mïg nd D. Troré Chrcteriztion of rock phosphte-soluilizing microorgnisms isolted from whet (Triticum estivum L.) rhizosphere in Mli. J. Microiol. Microil Res. 1:1-6. ollnd, M.D.., K.H.M. Siddique nd R.F. rennn. 2. Grin yield responses of f en (Vici f L.) to pplictions of fertilizer phosphorus nd zinc. ust. J. Exp. gr. 4: ouizgrne,., K. Oufdou nd Y. Ouhdouch ctinorhizl nd rhizoil-legume symioses for llevition of iotic stresses, pp In: N.K. ror (ed.), Plnt Microes Symiosis: pplied Fcets. Springer, Indi. ond, D.., D.. Lwes, G.C. Hwtin, M.C. Sxen, nd J.S. Stephens F en (Vici f L.). pp In: R.J. Summerfield nd E.H. Roerts (eds.), Grin Legume Crops. Willim Collins Sons Co. Ltd. 8 Grfton Street, London, WIX 3L, UK. Chng, C.H. nd S.S. Yng. 29. Thermo-tolernt phosphte-soluilizing microes for multi-functionl iofertilizer preprtion. ioresource Technol. 1:

9 Moroccn rock phosphte-soluilizing rhizoi Doui, K., M. Krrou, R. Mret, Z. Ftemi, X. Drye nd J.F. Ledent Genotypic vrition of phosphorus use efficiency mong Moroccn f en vrieties (Vici f mjor) under rinfed conditions. J. Plnt. Nutr. 35: Demissie, S., D. Mulet nd G. erech Effect of phosphte soluilizing cteri on seed germintion nd seedling growth of f en (Vici f L.). Int. J. gric. Res. 8: Dhnkhr, R., S. Sheorn,. Dhk nd R. Soni The role of phosphorus soluilizing cteri (PS) in soil mngement- n overview. Int. J. Dev. Res. 3: Dulormne, M., O. Musseu, F. Muller,. Toriio nd. â. 21. Effects of NCl on growth, wter sttus, N 2 fixtion, nd ion distriution in Pterocrpus officinlis seedlings. Plnt Soil 327: Duponnois, R., M. Kis nd C. Plenchette. 26. Phosphte soluilizing potentil of the nemtofungus rthrootrys oligospor. J. Plnt. Nutr. Soil Sci. 169: El-Komy, M.H.,.. Sleh nd Y.Y. Moln Resistnce/susceptiility of f en to otrytis fe: the cusl gent of chocolte spot with respect to lef position. Int. J. gric. iol. 17: Fghire, M., M. Frissi, K. Toufiq, R. Fghire,. rgz,. Mndri, K. Oufdou,. Lury, J.J. Drevon nd C. Ghoulm Genotypic vrition of nodules enzymtic ctivities in symiotic nitrogen fixtion mong common en (Phseolus vulgris L.) genotypes grown under slinity constrint. Symiosis 6: Grhm, P.H. nd C.P. Vnce. 23. Legumes: Importnce nd constrints to greter use - Updte on legume utiliztion. Plnt Physiol. 131: Gyneshwr, P., G.N. Kumr, L.J. Prekh nd P.S. Poole. 22. Role of soil microorgnisms in improving P nutrition of plnts. Plnt Soil 245: Hmdli, H., M. Hfidi, M.J. Virolle nd Y. Ouhdouch. 28. Growth promotion nd protection ginst dmping-off of whet y two rock phosphte soluilizing ctinomycetes in P-deficient soil under greenhouse conditions. ppl. Soil Ecol. 4: Hmdli, H.,. ouizgrne, M. Hfidi,. Lerihi, M. Virolle nd Y. Ouhdouch. 28. Screening for rock phosphte soluilizing ctinomycetes from Moroccn phosphte mines. ppl. Soil Ecol. 38: Hinsinger, P. 21. iovilility of soil inorgnic P in the rhizosphere s ffected y root-induced chemicl chnges: review. Plnt Soil 237: Johnson, H.W The soluiliztion of insolule phosphte IV the rection etween orgnic cids nd triclcium phosphte. New Zel. J. Sci. 2: Khn, H.R., J.G. Pull, K.H.M. Siddique nd F.L. Stoddrd. 21. F en reeding for drought-ffected environments: physiologicl nd gronomic perspective. Field Crop Res. 115: Khn, M.S.,. Zidi nd P.. Wni. 27. Role of phosphte-soluilizing microorgnisms in sustinle griculture- review. gron. Sustin. Dev. 27: Kim, K.Y., D. Jordn nd G.. Mc Donld Soluiliztion of hydroxyptite y Enterocter gglomerns nd cloned Escherichi coli in culture medium. iol. Fert. Soils 24: Lhrouni, M., K. Oufdou, M. Fghire,. Peix, F. El Khlloufi, V. Vsconcelos nd. Oudr Cynocteril extrcts contining microcystins ffect the growth, nodultion process nd nitrogen uptke of f en (Vici f L., Fcee). Ecotoxicology 21: Liu, J.Z., Z.S. Li nd J.Y. Li Utiliztion of plnt potentilities to enhnce the ioefficiency of phosphorus in soil. Eco-gric. Res. 2: MDRPM/DERD. 26. Sttistiques du Ministère de l griculture, du Développement Rurl et des Pêches Mritimes, 26, Morocco Mjeed,., S. Jvid, Z.. hmd,. Muhmood nd. Niz Fertigtion helps in incresing phosphorus use efficiency of whet (Triticum estivum L.) compred to conventionl method. Pk.J. gri. Sci. 51: Mlih, R., K. Smin,. Njm,. Sdi nd L. Frooq. 24. Orgnic cids production nd phosphte soluiliztion y phosphte soluilizing microorgnisms under in vitro conditions. Pk. J. iol. Sci. 7: Mndri,., J. Drevon,. rgz, K. Oufdou, M. Fghire, C. Plssrd, H. Pyer nd C. Goulmn Interctions etween common en genotypes nd rhizoi strins isolted from Moroccn soils for growth, phosphtse nd phytse ctivities under phosphorus deficiency conditions. J. Plnt Nutr. 35: Mululem, T., T. Desslegn nd Y. Desslegn Prticiptory vrietl selection of f en (Vici f L.) for yield nd yield components in Dt district, Ethiopi. Wudpecker J. gric. Res. 7: Olsen, S.R. nd L.E. Sommers Phosphorus, pp In:.L. Pge nd R.H. Miller (eds.), Methods of Soil nlysis; Prt 2, 2 nd Ed. gronomy Monogrph 9, S nd SSS, Mdison, WI. Omr, S The role of rock phosphte soluilizing fungi nd vesiculr rusculr mycorrhiz (VM) in growth of whet plnts fertilized with rock phosphte. World J. Micro. iotechnol. 14: Ormeno, E., R. Toress, R. Myo, R. Rivs,. Peix, E. Velzquez nd D. Zunig. 23. Phseolus luntus is nodulted y phosphte soluilizing strin of Sinorhizoium meliloti in Peruvin soil, pp In: E. Velzquez nd C. Rodriguez-rrueco (eds.), First Interntionl Meeting on Microil Phosphte Soluiliztion, Springer. Oufdou, K., L. enidire, L. Lyuenov, K. Doui, Z.. Ftemi nd P. Schröder Enzymes of the

10 Mghroui, echtoui, Glin, Whi, Duponnois, Hfidi, Doui, Ftemi, de Ljudie & Oufdou glutthione-scorte cycle in leves nd roots of rhizoi-inoculted f en plnts (Vici f L.) under slinity stress. Eur. J. Soil iol. 6: Prk, J.H., N. oln, M. Meghrj nd R. Nidu Concomitnt rock phosphte dissolution nd led immoiliztion y phosphte soluilizing cteri (Enterocter sp.). J. Environ. Mnge. 92: Peix,.,.. Rivs-oyero, P.F. Mteos, C. Rodriguez- rrueco, E. Mrtinez-Molin nd E. Velzquez. 21. Growth promotion of chickpe nd rley y phosphte soluilizing strin of Mesorhizoium mediterrneum under growth chmer conditions. Soil iol. iochem. 33: Richrdson,.E. 21. Prospects for using soil microorgnisms to improve the cquisition of phosphorus y plnts. ust. J. Plnt Physiol. 28: Rigud, J. nd. Puppo Indole-3-cetic cid ctolism y soyen cteroids. J. Gen. Micro. 88: Rodriguez, H. nd R. Frg Phosphte soluilizing cteri nd their role in plnt growth promotion. iotechnol. dv. 17: Svci, S n griculturl pollutnt: Chemicl fertilizer. Intern. J. Env. Sci. Dev. 3:77-8. Scervino, J.M., V.L. Ppinutti, M.S. Godoy, J.M. Rodriguez, I.D. Monic, M. Recchi, M.J. Pettinri nd.m. Godes Medium ph, cron nd nitrogen concentrtions modulte the phosphte soluiliztion efficiency of Penicillium purpurogenum through orgnic cid production. J. ppl. Microiol. 11: Srinivsn, R.,.R. lgwdi, S. Mhesh, K.K. Meen nd.k. Sxen Chrcteriztion of phosphte soluilizing microorgnisms from slt-ffected soils of Indi nd their effect on growth of sorghum plnts [Sorghum icolor (L.). Moench]. nn. Microiol. 62: Vlverde,., J.M. Igul nd E. Cervntes. 27. Polyphsic chrcteriztion of phosphte soluilisting cteri isolted from rhizospheric soil of the North-Estern region of Portugl, pp In: E. Velzquez nd C. Rodriguez-rrueco (eds.), First Interntionl Meeting on Microil Phosphte Soluiliztion, Springer. Vzquez, P., G. Holguin, M.E. Puente,. Lopez Cortes nd Y. shn. 2. Phosphte soluilizing microorgnisms ssocited with the rhizosphere of mngroves in semirid costl lgoon. iol. Fert. Soils 3: Vincent, J.M The cultivtion isoltion nd mintennce of rhizoi, pp In: J.M. Vincent (ed.), Mnul for the Prcticl Study of Root-nodule. lckwell Scientific Pulictions, Oxford. Whitelw, M.. 2. Growth promotion of plnts inoculted with phosphte soluilizing fungi. dv. gron. 69: Xie, J. 28. Screening for clcium phosphte soluilizing Rhizoium leguminosrum. Thesis of Mster Science. University of Ssktchewn Ssktoon, SK, Cnd; p.97. Yseen, T., F. Hussin, H.U. Rhmn nd M. Noor Chnge in growth nd productivity of urgundy due to rock phosphte, VM nd Rhizoium inocultion. Srhd J. gric. 29: Zhrn, H.H. 21. Rhizoi from wild legumes: diversity, txonomy, ecology, nitrogen fixtion nd iotechnology. J. iotechnol. 91: Zidi,., M.S. Khn, P.. Wni, M. hemd nd M. Oves. 29. Recent dvnces in plnt growth promotion y phosphte-soluilizing microes, pp In: M.S. Khn,. Zidi nd J. Musrrt (eds.), Microil Strtegies for Crop Improvement. Springer erlin, Heidelerg. 826