Micronutrient uptake in common bean (Phaseolus vulgaris L.) as affected by Rhizobium inoculation, and the supply of molybdenum and lime

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1 POJ 4(1):40-52 (2011) ISSN: Mironutrient uptke in ommon en (Phseolus vulgris L.) s ffeted y Rhizoium inoultion, nd the supply of molydenum nd lime Ptrik A. Ndkidemi, Sylvi Bmr nd Johim H.J.R. Mkoi Fulty of Applied Siene, Cpe Peninsul University of Tehnology, Cpe Town Cmpus, Keizersgrht, P.O. Box 652, Cpe Town 8000, South Afri * Corresponding uthor: ndkidemi56@yhoo.o.uk Astrt Pot nd field experiments were done to estlish the effet of Rhizoium inoultion, Molydenum nd lime supply on the vilility of mironutrients (Cu, Zn, Mn, Fe, B nd Mo) in Phseolus vulgris L. In oth field nd pot experiments, Rhizoium inoultion signifintly improved uptke of Mn, Fe, Cu, Zn, B nd Mo in ll orgns (roots, shoots, pods nd whole plnts) exept the Mo uptke in roots. Supplying Mo t 6 nd 12 g.kg -1 of seeds signifintly inresed Mo uptke ut redued tht of Mn, Fe, Cu, Zn nd B in the roots of P vulgris ompred with the zero ontrol. In the pods hrvested from the glsshouse nd field, nd whole plnt from glsshouse experiments, the uptke of Mn nd Fe were signifintly redued in tretments supplied with Mo t 6 nd 12 g.kg -1 of seeds reltive to the zero ontrol. Compred with zero-lime ontrol, the pplition of 2 nd 3 t.h -1 in the glsshouse signifintly redued the uptke of Mn, Fe, Cu, Zn nd B in the root, shoot nd whole plnt nd inresed tht of Mo. In the field onditions lime signifintly redued the uptke of Fe, Zn nd B in the roots, Fe nd Cu in the shoot nd whole plnt, nd Mn in pods nd whole plnts. Lime supply etween 2-3 t.h -1 inresed the uptke of Mo in ll orgns oth in the greenhouse nd field experiment. There ws signifint intertive effet etween Rhizoium inoultion nd Mo, Rhizoium x lime nd lime nd Mo supply. Intertively, the est uptke of the mironutrients ws reorded in tretments involving highest rte of Mo (12 g.kg -1 of seeds) nd tht of lime (3 t.h -1 ). Keywords: Nutrient uptke, Boron, Copper, Iron, Mngnese, Zin Introdution Phseolus vulgris L. is n importnt leguminous rop of greter nutritionl sttus to poor ommunities in Afrin ountries. It is niknmed s poor mn s protein due its potentil role in the dily diet settings of the poor where expensive niml protein nnot e fforded. From this kground, ny tehnologil or gronomil ttempt to improve the nutritionl qulity suh s the mironutrient ontent will e positive move in sustining helth of poor ommunities relying on this rop. The produtivity of P. vulgris in mny prts of developing world is onstrined y soil relted ftors. To mention the few, they inlude: idi soils with low ph (Wheln nd Alexnder, 1986; Rihrdson et l., 1988; Peoples et l., 1995); indequte nitrogen levels in the soil (Woomer et l., 1997 nd 1999) nd low levels of molydenum in idi soils, n importnt nutrient in the N 2 fixtion proess in legumes (Hfner et l., 1992; Crdoso et l., 1998; Vieir et l., 1998). Its pplition in most legume nutritionl progrms is mostly negleted lthough positive results on the vilility of nutrients nd yield of legumes is widely knowledged (López et l., 2007; Bmr nd Ndkidemi 2010). Being leguminous rop, P. vulgris hs unique property of symiotilly ssoiting with Rhizoium leguminosrum nd onvert tmospheri nitrogen into usle form to the plnts nd some my e leked to the soil nd therefore influening other iologil proess in the soil. Reserh evidene suggests tht Rhizoium inoultion of legumes nd the susequent N 2 fixtion proess ould hve positive effets on plnt growth nd finlly the mironutrient vilility nd hene improving the nutritionl qulity of different plnt omponents (Rodels et l., 1999; Rengel et l., 1999). It is estlished tht ertin groups of Rhizoium my produe siderophores (Wittenerg et l., 1996; Berrho et l., 1997; Duhn et l., 1998; Aror et l., 2001; Sridevi et l., 2008) whih my filitte the vilility of nutrients suh s Fe (Fino et l., 1994), Zn (Wni et l., 2008), P (Ad-All 1994; Sridevi et l., 2007) nd render them more ville to plnts. Nevertheless, miroorgnisms suh s Rhizoium inoulnts my signifintly ffet the hemistry of mironutrients in soils y enhning mironutrient uptke y plnts. Bsed on these fts, it is therefore importnt to estlish the possile role whih ould e plyed y en Rhizoium inoulnts on the vilility of mironutrients in P. vulgris. Soil idity is n importnt ioti ftor ffeting the vilility of mironutrients in the soil (Rengel, 1999; Mrshner, 1991). This onstrin is llevited y liming (Bmr nd Ndkidemi 2010). Reserh evidene suggest tht the liming prtie is ntgonisti on the vilility of mironutrients suh s: Mn, Fe, Cu, Zn nd B (Gupt, 1972,; Gupt, 1979,1992). Studies y (Prsd nd Sinh, 1982) showed tht liming the soil inresed the soil ph nd resulted in Mn, Fe, Cu, Zn nd B defiieny in legumes. However, other reserh reports suggests improved uptke of ertin 40

2 mironutrients suh s Mo with liming (Fleming, 1980; Gupt, 1997 López, 2007). To-dte, most Rhizoium inoulnts hve een developed nd re primrily used for supplying N to plnts. Little is known out their effet on supplying mironutrients in legumes suh s P.vulgris. Furthermore, limited studies in South Afri hve reported the influene of lime nd molydenum on the vilility of mironutrients in different plnt orgns of P.vulgris inluding the edile prts suh s pods nd hene this study. Mterils nd methods Plnt growth nd growth onditions Glsshouse nd field experiments were onduted t the Cpe Peninsul University of Tehnology, nd the Agriulturl Reserh Counil Nietvoorij site (33 54 S, E) in Stellenosh, South Afri respetively, during the 2008 nd 2009 summer sesons. The field experiment ws onduted under irrigtion. The experimentl site for field experiment lies in the winter rinfll region of South Afri t n elevtion of 146 m ove se level, with the men nnul rinfll of mm nd men nnul tempertures of 22.6 C t dy nd 11.6 C t night. The soil type of the experimentl site ws sndy lom (Glenros, Hutton form), whih ording to the Soil Clssifition Working Group (SCWG) is equivlent to skeleti leptosol (SCWG, 1991). Experimentl design The experimentl design followed rndomized omplete lok design in ftoril rrngement with 4 replitions per tretment. The experimentl tretments onsisted of 2 levels of Rhizoium inoultion (with nd without Rhizoium), 3 levels of dolomiti griulturl lime (0, 2 nd 3 t.h -1 ) nd 3 levels of Mo (mmonium molydte [(NH 4 ) 6 Mo 7 O 24 2H 2 O]) (0, 6 nd 12 g.kg -1 of seeds). The field plots mesured 2.5 m x 4 m with 5 rows 0.5 m prt from one nother. P. vulgris ws sown with inter-row plnting distne of 20 m. The plots were intersped y smll terres of 1 m to prevent ontmintion. The plnt popultion density ws 200,000 plnts per hetre. Plnting ws done fter ploughing nd hrrowing. Dolomti griulturl lime pplition (Dolomite: CCO 3 nd MgCO 3 ) ws done 2 weeks efore plnting. Twelve hours efore plnting, P. vulgris seeds were soked into Mo solution (mmonium molydte [(NH 4 ) 6 Mo 7 O 24 2H 2 O]. The zero Mo ontrol ws lso soked in wter solution ontining zero Mo. To void ontmintion, ll Rhizoium uninoulted tretments were sown first. Rhizoium inoultion ws done mnully y putting the inoulnt (Rhizoium leguminosrum iovr phseoli-kteriee registrsienr. L1795 wet 36/1947) in the plnting hole. The inoulnts used were otined from University of Pretori, South Afri. Weeding ws done mnully with hoe t 3 nd 8 weeks fter plnting. Plnt hrvest nd smple preprtion At 60 d fter plnting, P. vulgris plnts were smpled for nutrient nlysis. Aout 10 plnts were smpled respetively from the middle rows of eh plot. The order plnts within eh row were exluded. The plnts were refully dug out with their entire root system, wshed nd divided into roots, shoots, pods. The plnt orgns were oven-dried t 60 C for 48 h weighed nd ground into fine powder (0.85 mm) for the nlysis of miro nutrients. Mesurement of minerl nutrients in orgns Mesurements of mironutrients (Mn, Fe, Cu, Zn, B nd Mo) were determined y shing 1 g ground smple in porelin ruile t 500 C overnight. This ws followed y dissolving the sh in 5 ml of 6 M HCl nd pling it in n oven t 50 C for 30 min; 35 ml of deionised wter were dded nd extrt filtered through Whtmn no. 1 filter pper. Nutrient onentrtions in plnt extrts were determined using n indutively-oupled plsm (ICP) emission spetrophotometer (IRIS/AP HR DUO Thermo Eletron Corportion, Frnklin, Msshusettes, USA) (Giron, 1973). Sttistil nlysis A 3-ftoril design (3-wy ANOVA) ws used to sttistilly nlyse for mionutrients in plnt orgns. The nlysis ws done using the softwre of STATISTICA progrm Fisher s lest signifint differene ws used to ompre tretment mens t P 5 (Steel nd Torrie, 1980). Results Effets of Rhizoium inoultion on mironutrient uptke in roots, shoots, pods nd whole plnts of P. vulgris Rhizoium inoultion in P. vulgris plnts signifintly inresed shoot uptke of Mn, Fe, Cu Zn B nd Mo in oth the glsshouse nd field experiment reltive to the ontrol tretments in whih inoulnts were not supplied (Tle 1). With roots, the provision of Rhizoium inoulnts lso signifintly inresed root uptke of ll mironutrients listed ove exept Mo in the glsshouse nd the field (Tle 2) ompred with the zero Rhizoium tretments. As shown in Tle 3, with pods, the effet of Rhizoium inoultion in P. vulgris lso showed signifint inreses in the uptke of Mn, Fe, Cu Zn B nd Mo in oth the glsshouse nd field. The mironutrient uptkes in pods were signifintly elevted in the inoulted tretments reltive to the uninoulted ontrols plots. At the whole-plnt level, ll nutrient uptkes (Mn, Fe, Cu Zn B nd Mo) in en plnts were signifintly inresed with Rhizoium inoultion reltive to the ontrol tretments (Tle 4). Effets of Molydenum pplition on mironutrient uptke in roots, shoots, pods nd whole plnts of P. vulgris The uptke of mironutrients Mn, Fe, Cu, Zn nd B were signifintly redued y supplying Mo in the glsshouse nd field experiment (Tle 2). Generlly, signifintly lower uptke ws found y supplying Mo t 6 nd 12 g.kg -1 of seeds reltive to the zero ontrol (Tle 2). Contrry to the ove results, supplying Mo t 6 nd 12 g.kg -1 of seeds signifintly inresed Mo uptke in the roots of P vulgris ompred with the zero ontrol. Best Mo uptke ws reorded in tretments whih reeived the highest Mo level of 12 g.kg -1 of seeds. 41

3 A: Root-Fe B: Root-Cu Mo1 Mo2 Mo3 Root-Fe uptke (mg.plnt -1 ) Root-Cu uptke (mg.plnt -1 ) Without Rhizoium With Rhizoium 00 Without Rhizoium With Rhizoium 5 4 C: Root-Zn 4 Root-Zn uptke (mg.plnt -1 ) Without Rhizoium With Rhizoium Fig.1: Intertive effets of Rhizoium nd Molydenum on the uptke of A) Root-Fe; B) Root-Cu, nd C) Root-Zn of P. vulgris grown in the glsshouse. Mo1 = No Molydenum pplied, Mo2 = Molydenum pplied t 6 g.kg -1, Mo3 = Molydenum pplied t 12 g.kg -1. Brs followed y dissimilr letter re signifintly different y Fisher Lest signifint differene (LSD) test t P = 5 Applying Mo to P. vulgris plnts only signifintly inresed the shoot uptke of Mo in the glsshouse nd field experiments. The shoot ontent of other mironutrients (Mn, Fe, Cu Zn nd B) were not signifintly ltered y supplying Mo t 0, 6 nd 12 g.kg -1 kg of seeds (Tle 1). As shown in Tle 3, the pod uptke of Mn, Fe nd Mo in the glsshouse nd field were signifintly ffeted y Mo pplition. The uptke of Mn nd Fe were signifintly redued in tretments supplied with Mo t 6 nd 12 g.kg -1 of seeds reltive to the zero ontrol. However, s expeted, pplying Mo t 6 nd 12 g.kg -1 of seeds signifintly inresed the Mo uptke in the pods. At the wholeplnt level, the tissue uptke of Fe nd Mo were the only mironutrients tht were signifintly influened y Mo pplition in the glsshouse (Tle 4). In this experiment, the uptke of Fe in the glsshouse ws signifintly deresed with inresing Mo levels. But the uptke of Mo in oth the glsshouse nd field were signifintly inresed y inresing the Mo rtes from 6 nd 12 g.kg -1 of seeds. Effets of lime on mironutrient uptke in roots, shoots, pods nd whole plnts of P. vulgris Results from the glsshouse experiment on the uptke of mironutrients in the roots of P vulgris showed tht Mn, Fe, Cu, Zn were ll signifintly lowered y supplying lime (Tle 2). The uptkes of these mironutrients were lowest in the highest lime tretment of 3 t.h -1 (Tle 2). However, Mo pplition inresed the root Mo uptke oth in the glsshouse nd in the field. Mironutrient uptke in roots followed the similr trend in the field experiment, ut signifint redutions in their uptke were only oserved in Fe, Zn nd B. There ws signifint response in the shoot mironutrient uptke of P. vulgris to exogenous supply of lime. In the glsshouse experiment, the ddition of lime signifintly redued the shoot uptke of Mn, Fe, Cu, Zn nd B, nd inresed tht of Mo. Compred with zero-lime ontrol, the pplition of 2 nd 3 t.h -1 signifintly redued the shoot uptke of Mn, Fe, Cu, Zn nd B nd inresed tht of Mo 42

4 Tle 1. Effet of with nd without Rhizoium, Mo nd lime supply on the miroelements uptke in roots of P. vulgris s mesured in the glsshouse nd field. Tretments GLASSHOUSE FIELD Mn Fe Cu Zn B Mo Mn Fe Cu Zn B Mo mg.plnt -1 Rhizoium R- 19± ± ±01 20±01 16±01 26± ± ± ± ± ± ±76 R+ 45± ± ±01 28±02 23±01 33± ± ± ± ± ± ±67 Molydenum (g.kg -1 ) 0 48± ± ±01 28±02 23±02 15± ± ± ± ± ± ± ± ± ±01 24±02 20±01 32± ± ± ± ± ± ± ± ± ±01 19±02 15±01 42± ± ± ± ± ± ±97 Lime (t.h -1 ) 0 46± ± ±01 29±02 23±02 13± ± ± ± ± ± ± ± ± ±01 24±02 20±01 27± ± ± ± ± ± ± ± ± ±01 19±02 15±01 49± ± ± ± ± ± ± Wy ANOVA (F- Sttisti) R 13.46*** 32.38*** 10.14** 27.22*** 29.89*** 3.02ns 11.4** 25.3*** 7.1** 20.6*** 19.6*** 0.9ns Mo 5.27** 12.83*** 6.18** 12.99*** 17.59*** 16.46*** 5.6** 9.9*** 4.4* 10.5*** 11.7*** 8.7*** L 4.68* 23.75*** 7.48** 11.26*** 15.44*** 29.76*** 3.0ns 9.8*** 2.6ns 3.5* 4.7* 19.8*** R*Mo 2.8ns 5.5** 3.2* 5.1** 3.0ns 0.1ns 3.1ns 4.7* 2.4ns 4.3* 2.8ns 0.3ns R*L 1.8ns 9.9*** 1.2ns 0.9ns 0.3ns 0.2ns 1.4ns 6.1** 1.0ns 0.7ns 0.5ns 0.2ns Mo*L 0.1ns 0.3ns 0.5ns 0.3ns 0.7ns 1.4ns 0.2ns 0.4ns 0.5ns 0.6ns 0.5ns 0.8ns -R: Without Rhizoium; +R; With Rhizoium, R; Rhizoium, Mo; Molydenum, L; Lime. Vlues presented re mens ± SE, n = 4. *; **; *** = signifint t P 5, P 1, P 01 respetively, ns = not signifint, SE = stndrd error. Mens followed y dissimilr letters in olumn re signifintly different from eh other t P=5 ording to Fisher lest signifine differene. 43

5 Shoot-Zn uptke (m g.plnt - 1 ) A: Shoot-Zn Shoot-B uptke (mg.plnt - 1 ) B: Shoot-B P o d -F e u p t k e (m g.p l n t - 1 ) C: Pod-Fe Mo1 Mo2 Mo Without Rhizoium With Rhizoium 0 Without Rhizoium With Rhizoium Without Rhizoium With Rhizoium D: Pod-Zn 25.0 E: Whole plnt-fe Pod -Zn uptke (mg.plnt - 1 ) W P -F e up t ke (m g.pln t-1 ) Without Rhizoium With Rhizoium Without Rhizoium With Rhizoium Fig.2: Intertive effets of Rhizoium nd Molydenum on the uptke of A) Shoot-Zn, B) Shoot-B, C) Pod-Fe, D) Pod-Zn nd E) Whole plnt-fe of P. vulgris grown in the glsshouse. Mo1 = No Molydenum pplied, Mo2 = Molydenum pplied t 6 g.kg -1, Mo3 = Molydenum pplied t 12 g.kg -1. Brs followed y dissimilr letter re signifintly different y Fisher Lest signifint differene (LSD) test t P = 5 (Tle 1). In the field onditions, supplying lime t 2 nd 3 t.h - 1 signifintly redued the shoot uptke of Fe nd Cu nd inresed tht of Mo reltive to ontrol (Tle 1). In pods, exept for Zn nd B in the glsshouse, other uptkes of mironutrients (Mn, Fe Cu) were signifintly redued in the pods supplied with lime t 2 nd 3 t.h -1 (Tle 3). Similr results were reported in the field experiment, ut in ddition to Zn nd B, Cu uptke in the pods ws lso not signifintly ffeted y lime tretments. Whether mesured in the pods olleted from the glsshouse or in the field, Mo uptke ws signifintly inresed with lime supply t 2 nd 3 t.h -1 ompred with the ontrol. At whole plnt level, lime pplition signifintly redued the uptke of Mn, Fe, Cu, Zn nd B in the glsshouse, nd tht of Fe, Cu nd Zn in the field (Tle 4). The uptke of these mironutrients ws lowest s lime rtes were inresed from 2 to 3 t.h -1 reltive to the lime zero ontrol. Intertive effets of Rhizoium inoultion, Mo nd lime supply on mironutrient uptke in roots, shoots, pods nd whole plnts of P. vulgris. There ws signifint intertive effet etween Rhizoium inoultion nd Mo supply to the P vulgris orgns grown in the glsshouse nd in the field (Figure 1-2). Generlly, Rhizoium inoultion resulted into signifintly more uptke of Zn nd B in shoots, Fe, Cu nd Zn in the roots nd pods nd Fe in the whole plnt. Interestingly, in ll tretments with no Rhizoium inoultion, the uptke of ll the ove mentioned nutrients ws lowest in the zero Mo tretment. However, supplying inoulnts nd with no Mo resulted into greter 44

6 Tle 2. Effet of with nd without Rhizoium, Mo nd lime supply on the miroelements uptke in shoots of P. vulgris s mesured in the glsshouse nd field. Tretments GLASSHOUSE FIELD Mn Fe Cu Zn B Mo Mn Fe Cu Zn B Mo mg.plnt -1 Rhizoium R- 8±1 3.23±0.31 2±0 8±1 8±1 5±1 2.0± ± ± 1.6± ± ±0.2 R+ 0.18±1 5.63±0.38 3±0 0.12±1 0.13±1 8±1 5.6± ± ± ± ± ±0.3 Molydenum (g.kg -1 ) ±2 4.57±0.53 2±0 0.10±1 0.10±1 3±1 3.5± ± ± ± ± ± ±2 4.64±0.58 2±0 0.10±1 0.10±1 6±1 4.0± ± ± ± ± ± ±2 4.07±0.32 3±0 0.11±1 0.11±1 0.11±2 3.8± ± ± ± ± ±0.4 Lime (t.h -1 ) ±2 5.66±0.57 3±0 0.11±1 0.12±1 3±1 4.9± ± ± ± ± ± ±2 4.48±0.40 2±0 0.10±1 0.11±1 6±1 3.5± ± ± ± ± ± ±2 3.15±0.32 2±0 8±1 9±1 0.11±2 2.9± ± ± ± ± ± Wy ANOVA (F- Sttisti) R 32.00*** 30.88*** 2.18ns 24.70*** 45.24*** 10.77** 15.9*** 28.1*** 22.0*** 36.1*** 37.7*** 22.2*** Mo 0.29ns 0.69ns 1.11ns 0.41ns 1.00ns 22.07*** 0.1ns 0.7ns 1.7ns 0.4ns 0.9ns 14.7*** L 3.39* 11.27*** 3.51* 3.69* 5.11** 19.61*** 1.8ns 3.9* 3.3* 2.6ns 2.0ns 12.4*** R*Mo 1.9ns 3.0ns 3.2* 4.1* 4.8* ns 0.2ns 0.2ns ns 0.1ns 0.1ns 1.3ns R*L 0.6ns 1.1ns 0.3ns 0.2ns 0.6ns 3.6ns 0.6ns 1.7ns 2.0ns 1.8ns 1.4ns 1.7ns Mo*L 0.1ns 0.2ns 0.1ns 0.1ns 0.2ns 1.6ns ns 0.1ns 0.2ns 0.2ns 0.2ns 0.4ns -R: Without Rhizoium; +R; With Rhizoium, R; Rhizoium, Mo; Molydenum, L; Lime. Vlues presented re mens ± SE, n = 4. *; **; *** = signifint t P 5, P 1, P 01 respetively, ns = not signifint, SE = stndrd error. Mens followed y dissimilr letters in olumn re signifintly different from eh other t P=5 ording to Fisher lest signifine differene. 45

7 Tle 3. Effet of with nd without Rhizoium, Mo nd lime supply on the miroelements uptke in pods of P. vulgris s mesured in the glsshouse nd field. Tretments GLASSHOUSE FIELD Mn Fe Cu Zn B Mo Mn Fe Cu Zn B Mo mg.plnt -1 Rhizoium R- 3±0 0.67±5 2±0 8±0 6±0 3±0 2±0 0.53±4 1±0 6±0 5±0 2±0 R+ 8±1 1.50±0.12 3±0 0.14±1 0.10±0 5±1 8±1 1.60±0.15 3±0 0.14±1 0.10±0 6±1 Molydenum (g.kg -1 ) 0 6±1 1.34±0.20 2±0 0.11±1 7±0 2±1 6±1 1.26±0.21 2±0 0.11±2 7±1 2±1 6 5±1 0.99±0.12 2±0 0.10±1 7±0 4±1 5±1 1.05±0.19 2±0 0.10±1 7±1 4±1 12 4±1 0.92±7 2±0 0.11±1 8±0 6±1 4±1 0.88±0.10 2±0 0.10±1 8±0 6±1 Lime (t.h -1 ) 0 6±1 1.50±0.17 3±0 0.11±1 8±0 2±0 6±1 1.51±0.23 2±0 0.11±1 7±1 2±0 2 5±1 1.06±0.12 3±0 0.11±1 8±0 5±1 5±1 1.01±0.13 2±0 0.11±1 7±1 4±1 3 4±1 0.68±8 2±0 0.10±1 7±0 6±1 4±1 0.68±8 2±0 9±1 7±1 6±1 3 - Wy ANOVA (F- Sttisti) R 78.13*** *** *** 49.19*** 89.01*** 36.68*** 113.4*** 84.9*** 183.6*** 74.1*** 190.3*** 55.6*** Mo 3.45* 10.68*** 0.26ns 0.85ns 2.31ns 33.83*** 3.2* 3.7* 0.5ns 0.3ns 2.0ns 25.1*** L 4.79* 34.71*** 3.17* 1.81ns 0.19ns 37.87*** 4.9* 17.7*** 1.7ns 0.9ns ns 27.2*** R*Mo 3.2* 12.8*** 7.0** 5.5** 4.1* 0.1ns 4.5* 6.6** 7.9** 5.1** 5.8** 0.3ns R*L 1.8ns 7.8** 1.3ns 1.3ns 0.5ns 5.1** 2.7ns 8.2*** 1.7ns 1.7ns 1.0ns 5.5** Mo*L 0.2ns ns 0.3ns 0.6ns 4.3** 0.1ns 0.3ns 0.3ns ns 0.5ns 2.1ns -R: Without Rhizoium; +R; With Rhizoium, R; Rhizoium, Mo; Molydenum, L; Lime. Vlues presented re mens ± SE, n = 4. *; **; *** = signifint t P 5, P 1, P 01 respetively, ns = not signifint, SE = stndrd error. Mens followed y dissimilr letters in olumn re signifintly different from eh other t P=5 ording to Fisher lest signifine differene. 46

8 R o o t- F e u p t k e ( m g. p l n t - 1 ) A: Root-Fe P o d - F e u p t k e ( m g. p l n t - 1 ) B: Pod-Fe W P - F e u p t k e ( m g. p l n t - 1 ) C: Whole plnt-fe L1 L2 L3 1.0 Without Rhizoium With Rhizoium Without Rhizoium With Rhizoium Without Rhizoium With Rhizoium D: Pod-Mo E: Whole plnt-mo P o d - M o u p t k e ( m g. p l n t - 1 ) W P - M o u p t k e ( m g. p l n t - 1 ) Without Rhizoium With Rhizoium 0 Without Rhizoium With Rhizoium Fig.3: Intertive effets of Rhizoium nd Lime on the uptke of A) Root-Fe, B) Pod-Fe, C) whole plnt-fe D) Pod-Mo, nd E) Whole plnt-mo of P. vulgris grown in the glsshouse. L1 = No lime pplied, L2 = Lime pplied t 2 t.h -1, L3 = Lime pplied t 3 t.h -1. Brs followed y dissimilr letter re signifintly different y Fisher Lest signifint differene (LSD) test t P = 5. uptke of ll nutrients in ll orgns (Figure 1-2). In most ses, inresing the supply of Mo from 6 nd 12 g.kg -1 of seeds in omintion with Rhizoium inoultion signifintly redued the uptke of mironutrients (Figure 1-2). The Rhizoium x lime intertion ws signifint for Fe uptke in the roots, pod nd whole plnt (Figure 3A - C). In generl, the uptkes of these mironutrients were etter in the plots supplied with Rhizoium. However, the est uptke of Fe ws reorded in the zero lime ontrol tretments supplied with Rhizoium nd the uptke of this element ws signifintly redued s lime rtes were inresed from 2-3 t.h -1. The Rhizoium x lime intertion ws lso signifint for Mo uptke in the pods nd whole plnt (Figure 3D - E). Generlly, Rhizoium inoulted tretments exhiited the gretest uptke of Mo. Applying lime t 2-3 t.h -1, signifintly elevted the uptke of Mo reltive to the zero lime tretment. In this study, liming nd Mo pplition interted positively nd influened the uptke of Mo nd B in pods nd Mo in the whole plnts grown in the glsshouse (Figure 4). Generlly, the uptke of these elements in their orgns inresed stedily nd signifintly y inresing the pplition rtes of Mo from 0-12 g.kg -1 of seeds nd tht of lime from 0-3 t.h -1 respetively. The est uptke of the mironutrients ws reorded in tretments involving highest rte of (12 g.kg -1 of seeds) nd tht of lime (3 t.h -1 ). 47

9 Tle 4. Effet of with nd without Rhizoium, Mo nd lime supply on the miroelements uptke in whole plnt in P. vulgris s mesured in the glsshouse nd field. Tretments GLASSHOUSE FIELD Mn Fe Cu Zn B Mo Mn Fe Cu Zn B Mo mg.plnt -1 Rhizoium R- 0.12±2 6.35±0.42 5±0 0.18±1 0.15±1 0.10±2 2.3± ± ± 1.9± ± ±0.2 R+ 0.30± ±0.73 7±0 0.29±1 0.25±1 0.17±2 6.4± ± ± ± ± ±0.4 Molydenum (g.kg -1 ) ±4 9.63±1.06 6±0 0.24±2 0.19±2 6±2 4.4± ± ± ± ± ± ±3 9.02±0.90 6±0 0.22±2 0.19±2 0.13±2 4.5± ± ± ± ± ± ±2 7.29±0.50 6±0 0.23±1 0.21±1 0.22±3 4.1± ± ± ± ± ±0.4 Lime (t.h -1 ) ± ±1.04 6±0 0.25±2 0.22±2 6±1 5.7± ± ± ± ± ± ±3 8.59±0.61 6±0 0.24±2 0.20±1 0.14±2 4.1± ± ± ± ± ± ±2 6.00±0.49 5±0 0.20±1 0.18±1 0.22±3 3.2± ± ± ± ± ± Wy ANOVA (F- Sttisti) R 42.0*** 73.1*** 45.2*** 63.8*** 86.4*** 24.7*** 18.8* 41.6*** 36.6*** 45.0*** 45.9* 22.6*** Mo 1.8ns 6.8** ns 0.4ns 0.8ns 43.6*** 0.1ns 1.5ns 0.5ns 0.2ns 0.6ns 19.2*** L 4.9* 33.0*** 8.3*** 5.4** 4.6* 47.5*** 2.4ns 7.3** 6.1** 3.3* 2.6ns 20.1*** R*Mo 2.8ns 8.5*** 8.1*** 8.4*** 6.5 ns 0.3ns 0.5 ns 0.6 ns 0.3 ns 0.1ns 0.8ns R*L 0.9ns 6.5** 0.5ns 1.0ns 0.7ns 4.9* 0.8 ns 3.6* 3.2* 2.2 ns 1.6ns 1.3ns Mo*L 0.1ns 0.4ns 0.3ns 0.1ns 0.2ns 3.2* ns ns 0.1 ns 0.2 ns 0.2ns 0.5ns -R: Without Rhizoium; +R; With Rhizoium, R; Rhizoium, Mo; Molydenum, L; Lime. Vlues presented re mens ± SE, n = 4. *; **; *** = signifint t P 5, P 1, P 01 respetively, ns = not signifint, SE = stndrd error. Mens followed y dissimilr letters in olumn re signifintly different from eh other t P=5 ording to Fisher lest signifine differene 48

10 R o o t- F e u p t k e ( m g.p l n t - 1 ) A: Root-Fe P o d -M o u p t k e (m g.p l n t - 1 ) B: Pod-Mo L1 L2 L3 Without Rhizoium With Rhizoium 0 Without Rhizoium With Rhizoium C: Whole plnt-fe D: Whole plnt-cu W P -F e u p t k e ( m g.p l n t - 1 ) W P - C u u p t k e (m g.p l n t - 1 ) Without Rhizoium With Rhizoium Without Rhizoium With Rhizoium Fig.4: Intertive effets of Rhizoium nd Lime on the uptke of A) Root-Fe, B) Pod-Mo, C) whole plnt-fe nd D) Whole plnt- Cu of P. vulgris grown in the field experiment. L1 = No lime pplied, L2 = Lime pplied t 2 t.h -1, L3 = Lime pplied t 3 t.h -1. Brs followed y dissimilr letter re signifintly different y Fisher Lest signifint differene (LSD) test t P = 5. Disussion Supplying plnts of P vulgris with Rhizoium inoulnts signifintly ltered the uptke of Mn, Fe, Cu Zn, B nd Mo in orgns of P. vulgris (shoot, root, pod nd whole plnt) grown in the glsshouse nd verified in the field (Tle 1-4). There ws signifint inrese in mironutrient ontent in ll orgns for the Rhizoium inoulted tretments. Therefore, the greter levels of Mn, Fe, Cu Zn, B nd Mo in shoots, roots, pods, nd whole plnts of P vulgris with Rhizoium inoultion is n dditionl dvntge prt from the si funtion of fixing the tmospheri nitrogen into usle forms in legumes. The mehnisms involved re not well doumented. However, reserh evidene suggests tht different nitrogen fixing orgnisms my produe siderophores (Wittenerg et l., 1996; Berrho et l., 1997; Duhn et l., 1998; Aror et l., 2001; Sridevi et l. 2008) whih my filitte the soluility of nutrients suh s Fe (Fino et l., 1994) Zn (Wni et l., 2008) P (Young et l., 1990; Ad-All 1994; Chot et l., 1996; Sridevi et l., 2007) from different soures, phenomenon similr to the oservtions mnifested in our study. The simultive effet of Rhizoium on the uptke ould e due to their tivities on the soluiliztion of the mironutrients, phenomenon whih requires quntifition. Similr to our study, Howell (1987) reported tht superior rhizoil strins enhned the uptke of other minerls nd lned the nutritionl requirements of penut plnts. In our study, supplying Mo t fro 0-12 g.kg -1 of seeds signifintly inresed the uptke of this element in ll orgns t the glsshouse nd in the field (Tle 1-4). Molydenum is 49

11 known to e reltively moile nutrient in the plnt system nd redily onentrted y plnts (Clrkson nd Hnson, 1980). However, Mo supply led to the signifintly redued uptke of Mn, Fe, Cu Zn nd B in roots (Tle 1), Mn nd Fe in pods (Tle 3) nd Fe in the whole plnt (Tle 4). The derese in minerl ontent of plnt prts ws more drmti with inresing the supply rtes of Mo. A seprte report from this study (Bmr nd Ndkidemi, 2010) showed signifint inreses in soil ph from 6.1 to 6.3 nd 6.5 y supplying Mo t 0, 6 nd 12 g.kg -1 of seeds respetively. It is widely doumented tht, Mo is the only tre minerl whose vilility to plnts inreses with n inrese in soil ph under norml soil onditions (Fleming, 1980; Gupt, 1997) sitution similr to the oservtions reported in this study. Furthermore, the redution in the uptke of Mn, Fe, Cu Zn nd B my lso e strongly relted to similr hnges in soil ph s it is widely epted tht the vilility nd uptke of these mironutrients is ph dependent (Brdy nd Weil, 2008). Similr to our oservtion, Fleming (1980) showed tht high Mo onentrtions in iologil systems n indue Fe-defiieny. The intertions etween Mo nd the iovilility of Fe, Cu Zn nd B nd re well doumented in nimls (Dowdy nd Mtrone, 1968; Smith nd Wright, 1975; Bremner, 1979; Suttle et l., 1984; Gengelh et l., 1994), ut little informtion is ville for plnts. Studies involving different niml types hve shown tht high Mo intkes my interfere with the iovilility of these mironutrients, senrio whih ws lso onfirmed in P. vulgris in our study. These hnges in mironutrient ontent of P. vulgris shoots with Mo supply ould hve implitions in the dietry use of the lef omponent s vegetles in Afri. Therefore, this lls for detiled studies to estlish other possile mehnism of Mo deresing the uptke nd iovilility of Mn, Fe, Cu Zn nd B in P. vulgris orgns. Results from our study showed tht liming hd ntgonisti effet on the iovilility nd the uptke of Mn, Fe, Cu, Zn nd B. Although in some orgns the vilility ws not signifintly redued (Tles 1-4), ut in ll instnes, the mounts of Mn, Fe, Cu, Zn nd B were signifintly redued with the ddition of lime. The levels of these mironutrients were onsistently lower y inresing lime rtes from 2-3 t.h - 1 reltive to the zero lime tretment. The results re onsistent with reports y Chen et l. (1982); Rengel et l. (1999); Bsu et l. (2008). As expeted, lime in this study signifintly inresed the soil ph (Bmr nd Ndkidemi 2010) nd thus inresing the uptke nd iovilility of the Mo in orgns of P vulgris. Similr to our report, López et l. (2007) lso reported n inrese in Mo in leguminous plnt Trifolium Prtense L. with the ddition of lime. The intertive effets etween Rhizoium, nd Mo pplition were oserved in the glsshouse for the uptke of Fe, Cu nd Zn in roots (Fig. 1A - C), Zn nd B in shoots, Fe nd Zn in pods nd Fe in the whole plnt (Fig 2A - E). Generlly, more mironutrient uptke ourred in the tretments involving Rhizoium inoultion. Interestingly, the est signifint mironutrients uptke ws found in tretments involving Rhizoium inoultion with zero Mo (Figs. 1A-C nd 2A - E). Supplying inoulnts nd pplying Mo t 6 nd 12 g.kg -1 of seeds hd signifint negtive effets on the uptke of mironutrients in different orgns nd the whole plnt of P. vulgris. The mehnism involved is not well estlished, thus, wrrnting further investigtion. The intertive effet etween Rhizoium nd lime were lso reorded in the glsshouse study on the uptke of Fe in roots nd pods, nd whole plnt (Fig. 3A, B nd C nd 5C), nd Mo in pods nd whole plnt (Figs 3C nd D nd 5C). Plnts reeiving lime nd rhizoil inoulnts hd signifintly higher uptke of Fe nd Mo reltive to the uninoulted tretments. Signifint redution in the uptke of Fe in root, pods nd whole plnt were mostly reorded in inoulted tretments nd were ggrvted more y supplying lime t 2-3 t.h -1 reltive to the zero lime tretment. It is well estlished tht lime hs positive effets on inresing the soil ph (Bmr nd Ndkidemi, 2010) nd ultimtely deresing the uptke of Fe (Ptr nd Mohnty, 1994). In our study, Rhizoium inoultion in omintion with lime inresed the soil ph (Bmr nd Ndkidemi, 2010), thus, reduing the soluility of Fe in the soil solution (Römheld, 1987; Ptr nd Mohnty, 1994), nd then, redued the vilility of Fe to plnts nd hene its uptke nd trnslotion into roots, pods nd whole plnt s oserved in our study. Signifint inreses in Mo levels were oserved in pods nd whole plnts of P. vulgris supplied with omintion of lime nd Rhizoium inoultion. The tretments supplied with Rhizoium nd lime t 2-3 t.h -1 hd signifintly more Mo thn the uninoulted tretments (Fig. 3C nd E). In this study it is evident tht Rhizoium inoultion in omintion with lime inresed the Mo in pods, whole plnt nd roots supporting the ft tht the miroorgnism hd mehnism whih filitted the uptke (Figs 3C & E 5A & B). This is due to the ft tht Mo levels in the uninoulted tretments supplied with lime t 2-3 t.h -1 were lower, nd y introduing the inoulnts they were signifintly douled (Figs. 3C & E 5A & B). Looking t the mens of two ontrol tretments (0 t.h -1 of lime) in the inoulted nd uninoulted groups (Figs. 3C & E 5A & B), the response in Mo uptke in inoulted tretment ws indued y Rhizoium inoultion. Therefore, prt from lime, inresed Mo uptke in pods nd whole plnts of P vulgris reported in this study ws due to Rhizoium inoultion. In their studies, other workers hve lso reported tht lime pplition inresed the soil ph; promoting the molydte desorption nd hene mking it more ville to leguminous plnt T. Prtense (López, 2007). Inresed uptke of Mo in pods oserved in this study (Fig. 4A) with the supply of lime nd Molydenum in plnts is similr to results reported y other reserhers (During, 1984; Wheeler, 1998). The inresed uptke of Mo with Mo pplition ws expeted. However, the improved uptke of Mo ttriuted to lime ould e ssoited with inresed ph (Bmr nd Ndkidemi, 2010) whih mde Mo more ville into the pods (López et l., 2007). The uptke of B in the pods followed the similr trend to tht of Mo (Figure 4B). In onlusion, the provision Rhizoium inoulnts to P. vulgris pper to promote greter nutrient uptke (Mn, Fe, Cu, Zn B nd Mo) nd umultion in their tissues. The supply of externl nutrients suh s Mo nd lime lso hd some ntgonisti influenes y reduing the vilility of Mn, Fe, Cu, Zn nd B. The findings otined in this study lerly suggest the need for further experimenttion on the effets of miroorgnisms nd other fertilizer inputs on the nutrient qulity nd quntity of different legumes used in the food industry. Aknowledgements This study ws supported y the Cpe Peninsul University of Tehnology through University Reserh Fund (RP 03). 50

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