The Associative Effect of VA Mycorrhizae with Bradyrhizobium as Biofertilizers on Growth and Nutrient Uptake of Arachis hypogaea.

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1 Reserch Journl of Agriculture nd Biologicl Sciences, 4(2): , , INSInet Publiction The Associtive Effect of VA Mycorrhize with Brdyrhizobium s Biofertilizers on Growth nd Nutrient Uptke of Archis hypoge. Imn M. El-Azouni, Ysser Hussein nd Lmis D. Shbn Botny Deprtment, Fculty of Science, Zgzig University, Zgzig, Egypt. Abstrct: Pot experiments were crried out in virgin sndy soil mended with either rock phosphte or super phosphte. The experiment imed to investigte the influence of single or dul inocultion of N 2 fixer Brdyrhizobium nd the vesiculr-rbusculr mycorrhizl fungi VAM, s biofertilizers, on the growth nd nutrient uptke of penut plnts (Archis hypoge L.). The pots were received hlf of the recommended dose of P fertilizer in the rte of 20 mg P/kg soil, wheres, mmonium nitrte ws dded in the rte of 10 mg N/kg soil. The growth prmeters of the plnt were studied, the results reveled tht the biomss nd grin yield were significntly improved by using the dul biopreprtions of AM fungi with Brdyrhizobium. Dt lso reveled tht bcteril mycorrhizl legume symbiosis increse nodule number, nitrogense ctivity, totl pigments, crbohydrte, protein nd lipid content. Also, the nitrogen, phosphorus nd potssium (NPK) uptke were significntly incresed due to the single or dul inocultion. Generlly, inocultion with VA mycorrhize nd/or Brdyrhizobium cn, synergisticlly, remove the deficient effect of N nd P on N2-fixtion nd plnt growth in the soil of low nutrient content. In ll experimentl interctions, rock-p mended soil gve higher vlues thn tht obtined from super-p pplicble soil. Key words: Penut plnts, Brdyrhizobium, VA mycorrhize, nitrogen, phosphorus potssium INTRODUCTION About one-third of the lnd re of the world comprises rid nd semirid climtes. Arid desert soils were previously considered economiclly unimportnt; however, during the pst three decdes, the economic nd griculturl utiliztion of rid lnds hs emerged s criticl element in mintining nd improving the [73] world's food supply. The penut, or groundnut (Archis hypoge) is species in the legume fmily. It is n importnt oil nd protein source nd is grown widely in the semi-rid tropics. Legume crops re generlly cultivted in poor environments, even recently bred cultivrs re selected to grow in such poor environment nd ssocited with its rhizobium n ssocited microflor. Legume crops hve high phosphorus requirement for nodule [71] formtion, nitrogen fixtion nd optimum growth. Mycorrhizl condition of legume crops found to increse its vegettive growth nd seed yield in [41,47] ddition to improve nodultion on it's root system. Legume nitrogen-fixing bcteri nd rbusculr mycorrhizl symbiosis were studied by some [13,15,53] uthors who reported tht the nlysis of rbusculr mycorrhiz nd nitrogen-fixing nodules suggested tht the plnt hve universl system for monitoring their microbil ffinities tht my be either mutulistic or ntgonistic depending on the form of symbiosis, environmentl conditions nd individul genetic chrcters of intercting orgnisms. These regultory systems were not only pprently importnt for evolution of the beneficil microbil interctions tht contribute generlly to the dptive potentil of terrestril plnts, but lso, crete more fvourble environment for the development of ecosystems processes. Becuse of the importnce of penut s food legume in Egypt, the new reclimed soils were brought bout cultivtion for enhncing the product. One of the principle production constrints is poor fertility. This problem is gretly solved by the ddition of minerl fertilizers, which re scrcity nd high cost. The gret deep gp between supply nd demnd nd their dverse effect on environment hve led griculturl scientists to [70] look for lterntive strtegies. Orgnic wstes nd biofertilizers re considered to be the lterntive source to meet the nutrient requirement of crops [61] nd to bridge the future gps. The lck of indigenous soil popultions of VA mycorrhize nd rhizobi hs restricted potentil yields of penut cultivted in this re. Applying of the soil microorgnisms which cn fix tmospheric nitrogen, solubilize phosphorus or stimulte plnt growth will be environmentlly benign pproch for nutrient mngement of ecosystem. Corresponding Author: Imn M. El-Azouni, Botny Deprtment, Fculty of Science, Zgzig University, Zgzig, Egypt. 187

2 The im of the present work ws conducted to exmine the role of vesiculr-rbusculr mycorrhizl (VAM) fungi with Brdyrhizobium spp. inocultion on the growth, yield nd nitrogen, phosphorus nd potssium (NPK) uptke of penut plnts grown in soil mended with either super-p or rock-p s biofertilizers, nd their response to N fixtion by the plnt. 2 MATERIALS AND METHODS Soil Preprtion: An griculturl virgin sndy lom soil, from New Al-Slheyi Region, Shrki Governorte, Egypt, contins 78% snd, 10% silt, nd -1 12% cly, ph 7.3, 4.7 g kg totl orgnic mtter, g kg totl nitrogen, 25 mg kg phosphorus, 34 mg kg 1-1 potssium nd 1.9 mmoh m E.C.The soil ws ir dried, sieved to pss through < 2mm mesh nd pcked in plstic pots. Seed: Seeds of penut (Archis hypoge L.) cv. Giz 5 were obtined from Field Crops Res. Institute, Agric. Res. Centre, Giz, Egypt. Experiments: Three seeds of penut were sown in ech plstic pot, seprtely. Pots were filled with 10 kg/pot of sndy soil. The seedlings were thinned to one plnt fter 15 dys of emergence. The experiment ws rrnged s rndomized complete block design with three replictes for ech tretment. Phosphorus ws pplied either s super phosphte (7.2% P) t the rte 20 mg P/kg soil or fine [22] rock-p (11.7% P), t sme rte. Rock phosphte (26.4 % P2O 5) ws obtined from Abu Zbl phosphte fertilizer Co. All pots were received 10 mg N/kg soil dded s mmonium nitrte, t the rte of 20-1 kg fed (NH4NO 3, 33.5%). Potssium ws pplied s kg K2O fed (k2so 4, 48%). Wtering, weeding nd sprying ginst pests nd diseses were done when necessry. The replictes were crried out with the following tretments: uninoculted control, inocultion with Brdyrhizobium, inocultion with VA mycorrhize, nd inocultion with both Brdyrhizobium nd VA mycorrhize, ll tretments were fertilized with either rock-p or super-p. Inoculnts: Brdyrhizobium strin USDA 3456 ws obtined from the Agriculture Reserch Centre, Giz, Egypt, nd used for n pproprite pet-bsed inocul 8-1 (3.5 x 10 cells g pet) for seed inocultion. Gum Arbic ws used s sticker to ensure vible rhizobi per seed, before sowing. Mixed VA mycorrhizl inoculum's consisted of spores, mycelium nd root segments of Glomus clrum; G. mossee nd G. fscicultum isoltes propgted with onion (mycorrhizl infection = 70%) roots for four months, [24] using the wet sieving technique. Approximtely spores pot were injected t 6-8 cm below the seed bed before sowing to ensure good infection. Smpling: Penut plnts were hrvest fter one hundred twenty dys, seprted into shoots nd roots. Pods were seprted nd their number/plnt hve been recorded. Fresh weights of shoots nd roots were determined. The roots were dipped in wter to remove dhering soil prticles, nd wshed with distilled wter. Plnt height nd root nodule number were estimted. Totl dry weight of shoots nd roots were determined fter drying t 70 C till constnt weight. The oven dried plnts were grounded nd minerlized by sulfuricperchloric cids. Nitrogen (N) ws extrcted from [58] plnts with sulfuric cid using the semi-micro Kjeldhl [30] method. Phosphorus (P) ws extrcted by nitricperchloric cid digestion nd mesured using the [29] vndono-molybdophosphoric colorimetric method. Potssium (K) ws ssyed using flme [6] spectrophotometer. The sugr nd protein content of [51] plnt tissues were estimted ccording to Nguib nd [14] Brdford. Lipids were extrcted with petroleum ether [10] using Soxhlet pprtus ccording to AOAC. Nitrogense ctivity of plnt ws mesured by using [25] cetylene reduction ssy. Vlues of nitrogense -1-1 ctivity were recorded s nmoles C2H 4 gm plnt h. The method used for the quntittive determintion of photosynthetic pigments chlorophyll nd b ws tht [49] of Metzner et l.,. A subsmple of wshed roots ws clered in 10% KOH (w/v) nd fungus stined with 0.03% (v/v) trypn blue in lctoglycerol ccording to [57] the method of Philips nd Hymn, percentge of mycorrhizl infection ws determined ccording to [24] Gerdemnn nd Nicolson. Sttisticl Anlysis: The dt obtined in the present study were expressed s men of tretment vribles nd the sttisticl nlysis done by using SPSS progrm (Sttisticl Pckge for the Sciences System). P-vlue <0.05 ws considered sttisticlly significnt. RESULTS AND DISCUSSION Fresh Biomss Yield: Effect of inocultion with VA mycorrhizl fungi nd /or Brdyrhizobium on shoot nd root fresh weights were presented in Tble 1. Dt indicted tht the dependence of penut growth on inocultion with Brdyrhizobium increse the fresh biomss yield of eril nd ground prts of penut plnts thn the uninoculted plnts in both P-mended [2] soils. Abdel-Ghffr reported tht, inoculting fbben with Rhizobium incresed the yield nd the qulity of grin compred to uninoculted tretments. Also dt reveled tht single inocultion with 188

3 Tble 1: Shoot nd root fresh weight of penut plnts, inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium, grown in soil mended with rock-p or super-p. Inocultion Fresh Fresh Totl Fresh Fresh Totl weight of weight Fresh weight of weight of Fresh shoot (g) of root(g) weight shoot (g) root (g) weight Uninoculted Plnt+Rh ** Plnt+VAM ** 26.62*** Plnt+VAM+Rh *** 38.44*** ** 38.40*** Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P mycorrhize in presence of either rock-p or super-p significntly enhnced the totl fresh shoot nd root weights of penut plnts over the non-mycorrhizl plnts by bout 39% nd 29%, respectively. With this [37] respect, Khnizdeh et l., reported tht vesiculrrbusculr mycorrhiz inocultion in combintion with phosphorus incresed dry nd fresh shoot weight, lef re nd lef number of strwberry compred to ppliction of phosphorus lone. Lmbert nd [41] [47] Weidensul nd Mthur nd Vys reported tht mycorrhizl condition of legume crops found to increse its vegettive growth nd seed yield in ddition to improve nodultion on it s root system. Highly significnt improvement were reported with the inocultion of VA mycorrhizl fungi in combintion with Brdyrhizobium, either in rock-p or super-p, where the totl fresh weight content reched up to 88% nd 90% for both soils, respectively, s compred with the uninoculted plnts. This my ttribute to the potentil benefit of this mutulistic ssocition on growth biomss. For ll experimentl interction, shoot nd root fresh weights were higher in cse of soil mended with rock-p thn super-p, but the increse ws not significnt. These results were in greement [23] with those obtined by El-Ghndour et l.,. Dry Weight Yield: Shoot nd root dry weights, s ffected by inocultion of VA mycorrhizl nd/or Brdyrhizobium, grown in soil mended with either rock-p or super-p, were shown in Tble 2. Dt indicted tht inocultion with Brdyrhizobium incresed the dry mtter yield of shoot nd root penut plnts, over the uninoculted plnts cultivted in soil mended with rock-p or super-p. El-Ghndour et l., [22] reported tht fungl infection nd rhizobil inocultion either lone or in combintion incresed dry mtter yield s compred to uninoculted plnts. Also, dt showed tht VA mycorrhizl coloniztion with penut plnt roots significntly increse dry weight of shoot nd root, tht the totl weight exceeded tht of uninoculted plnts by bout more thn 2-fold in both rock nd super phosphte pplied soil. This result ws [40] in correspond to Krishn nd Bgyrj who reported tht inocultion with the mycorrhizl fungus Glomus fscicultum enhnced penut growth nd incresed its dry mtter more thn 2-fold compred with the noninoculted control, in both sterilized nd non-sterilized [4] soil. Also Al-Krki et l., indicted tht shoot dry mtter, shoot phosphorus nd root dry mtter were higher for mycorrhizl infected whet (Triticum estivum L.) plnts thn for non infected plnts. Chuln [17] nd Mrtin reported significnt shoot dry weight increment when Theobrom cco ws inoculted with [3] VA-mycorrhiz. Aggngn nd Del Cruz reported dry mtter yield increment of up to 631% when L. leucocephl ws inoculted with vesiculr-rbusculr [16] mycorrhiz. Chien et l., mrked tht dry mtter yield of different soyben plnt prts showed n increse in dry mtter yield with different P sources s compred with check. The highest significnt yields were recorded with the inocultion of both VA mycorrhizl fungi nd Brdyrhizobium either in soil mended with rock-p or super-p. The totl dry weights of shoot nd root, for both soils, reched pproximtely more thn 4-folds over the uninoculted plnts. The ppliction of bioinoculnts like Am fungi nd one of the plnt growth-promoting rhizobcteri is n environment-friendly. The beneficil effects of these bcteri in combintion with Am fungi hve been [42,55] reported by number of workers. It hs been reported tht these bcteri my ffect Am fungi nd their plnt host through vriety of mechnisms tht include (1) effects on the receptivity of the root; (2) effects on the root-fungus recognition; (3) effects on the fungl growth; (4) modifiction of the chemistry of the rhizospheric soil; nd (5) effects on the germintion of the fungl propgules. Moreover, Am fungi nd nitrifying bcteri often ct synergisticlly on infection rte, minerl nutrition nd plnt growth. Dt showed tht soil mended with rock-p significntly yielded 189

4 Tble 2: Shoot nd root dry weight of penut plnts, inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium, grown in soil mended with rock-p or super-p. Inocultion Dry weight Dry weight Totl Dry Dry weight Dry weight Totl Dry of shoot (g) of root(g) weight of shoot (g) of root (g) weight Uninoculted Plnt+Rh *** 7.50*** *** 6.02** Plnt+VAM *** 10.19** *** 9.61*** Plnt+VAM+Rh *** 16.82*** *** 16.07*** Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P shoot nd root dry weights higher thn tht of super-p, [23] this ws in greement with El-Ghndour et l., who reported tht shoots nd roots dry weight ws higher in cse of rock thn super-p wheres the men of shoots were 33.5 when rock-p ws pplied while it ws 31.4 in cse of super-p. Height nd Root Length: Tble 3 showed tht inoculting penut plnts with Brdyrhizobium significntly incresed the shoot height nd root length in either rock-p or super-p mended soil. Dt lso reveled n enhnced height increment in penut plnts with mycorrhizl symbiosis up to 51% in rock-p, nd 45% in super-p mended soil, respectively, s compred to the non inoculted plnts. Mrschner nd [48] Dell reported tht mycorrhiz infection is known to enhnce plnt growth by incresing nutrients uptke. The higher height increment registered with inoculted plnts could be s result of enhnced inorgnic [19] nutrient bsorption nd greter rtes of [5] photosynthesis, which obviously could hve given rise to n increse in plnt growth. Inocultion with VA mycorrhiz lso, significntly, incresed the root length up to 68% nd 74% in rock-p nd super-p mended soil, respectively, s compred to [27] non mycorrhizl infected soil. Hung et l., reported root length increment of up to 80% when Leucen leucocephl ws inoculted with vesiculr-rbusculr [43] mycorrhize. Levy nd Syvertsen while working on the effect of drought stress on citrus, reported tht, lthough plnt to plnt vritions obscured significnt differences, VA mycorrhiz plnts did tend to hve greter totl feeder root length per plnt thn control plnts. In ddition to the mycorrhiz inocultion enhncing the plnts bsorption of more nutrients, especilly phosphorus, vi n increse in the bsorbing [48] surfce re, mycorrhiz coloniztion could hve [56] protected roots from soil pthogen, nd therefore could hve led to n increse the root growth nd nutrients cquisition of penut plnts. The highest significntly increse in height nd root length ws recorded with inocultion of Brdyrhizobium nd VA mycorrhiz for both P- [61] mended soils. Rbie et l., indictes tht dul inocultion with Glomus clrum nd Azospirillum brsilense cn increse the plnt height, dry weight nd root shoot rtio of cowpe plnts more thn single inocultion with Am fungi or NFB s well s control t ll slinity levels. The higher root to shoot rtio of the inoculted plnts could be ttributed to the effect of mycorrhiz nd/or Brdyrhizobium infection, which could hve incresed nutrients bsorption, giving rise to higher root length nd height increment with [18] uniform growth. Clpperton nd Reid reported tht s the coloniztion by vesiculr-rbusculr mycorrhizl fungi incresed, so did root to shoot rtios. In ll experimentl interctions, rock-p mended soil gve more vlues thn super-p one. This result ws in [23] greement with El-Ghndour et l.,. Grin Yield: The results obtined from Tble 4 indicted tht the grin yield nd fresh weight of one pod of penut plnts increse with the inocultion of Brdyrhizobium, compring with uninoculted plnts, in both P-mended soils. In other words, N2-fixtion process mximize the yield of inoculted plnts s [69,68] compred with uninoculted tretments. Infection with VA mycorrhizl fungi, significntly, enhnce grin yield of penut plnts, either in rock or super-p mended soil, of up to 265% nd 255%, respectively. [31] Jckson nd Mson found positive reltionships mong (P) vilbility, VA mycorrhizl infection nd pod yield in groundnut (Archis hypoge L.). [7] Alloush et l., found tht chickpe plnts inoculted with mycorrhizl fungus Glomus versiforme hd higher number of nodules, shoot phosphorus content, shoot dry weight nd grin yield thn uninoculted chickpe plnts. Further grin yield were hrvested with the inocultion of both Brdyrhizobium nd VA mycorrhizl fungi in both P-mended soils. Also solely 190

5 Tble 3: Height nd root length of penut plnts, inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium, grown in soil mended with rock-p or super-p. Inocultion Height (cm) Root length (cm) R /S rtio Height (cm) Root length (cm) R /S rtio Uninoculted Plnt+Rh 47.67*** 23.92** ** 22.60** 0.52 Plnt+VAM 54.63*** 28.93*** *** 27.03*** 0.53 Plnt+VAM+Rh 68.06*** 40.09*** *** 37.33*** 0.56 Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P Tble 4: Grin yield of penut plnts, inoculted with VA mycorrhizl Fungi nd /or Brdyrhizobium, grown in soil mended with either rock- P or super-p. Inocultion Number of pods /plnt Fresh weight of one pod (g) Number of pods /plnt Fresh weight of one pod (g) Uninoculted Plnt+Rh *** *** 3.01** Plnt+VAM *** *** 3.79** Plnt+VAM+Rh *** 5.17** *** 4.98*** Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P VA mycorrhizl infection incresed the fresh weight of one pod up to 1.5 nd 1.3 fold, compred with the non-inoculted control, in rock-p nd super-p mended soil, respectively. While the dul combintion of VA mycorrhiz with Brdyrhizobium, significntly, incresed the fresh weight of pod, in both soils, of up to 2 fold. The beneficil effect of VA mycorrhizl symbiosis, s well s interction with rhizobi, in generl, gretly increse growth biomss, dry mtter, nd grin yield of inoculted plnts s compred with the uninoculted ones. This indicted tht N nd P supply ws influenced by VA mycorrhizl nd by Brdyrhizobium inocultion. Rock-P soil produced more grin yield nd fresh weight of pod thn the super-p mended soil. Nodule Number nd Nitrogense Activity: Dt from Tble 5 indicted tht nodultion of the roots of penut plnts ws significntly too much improved with the ppliction of Brdyrhizobium biopreprtion either singly or combined with Am fungi, in both P-mended soils, compring with the uninoculted plnts. Gret nodule numbers (347.4 & nodules/plnt) were observed in inoculted with Brdyrhizobium lone, wheres, considerble numbers of nodules (299.8 & nodules/plnt) were noticed with the infected mycorrhize plnts s compred with non-infected plnts in both soils. The results (Tble 5) lso reveled tht nitrogense ctivity incresed within penut plnts inoculted with single Brdyrhizobium (82.4 & 79.2 nmol C2H 5/g plnt/h) or with Am fungi inocultion (75.0 & 67.5 nmol C2H 5/g plnt/h) in rock-p nd super- P mended soils, respectively. Generlly, rock- P mended soil resulted, significntly, higher vlues of nodule number nd nitrogense ctivity compring with super-p mended soil. This result ws in gree with El- [23] Ghndour et l., who reported tht inocultion with Brdyrhizobium (strin 3456 & 3339) incresed nodultion nd nitrogen content either rock-p or super- P were dded, in the sme time these prmeters were higher in cse of rock-p thn super-p ppliction. [54] Pcovsky et l., explined tht in lkline soil nd low P-deficient, rock-p enhnced nodultion nd nitrogen fixtion of mycorrhizl plnts. Also, nodulted roots cn enhnce the production of root exudtes which my ffect the growth of soil microflor. Rhizobi my lso increse the permebility of root cell to the fungus. Reports lso stted tht the presence of Am fungi is known to enhnce nodultion nd [9,36,62] nitrogen fixtion by legumes. The incresed phosphorus uptke conferred by the Am symbiosis is beneficil for the functioning of the nitrogense enzyme of the bcteril symbionts, leding to incresed nitrogen fixtion nd consequently promotion of root nd 191

6 Tble 5: Nodule numbers nd nitrogense ctivity (nmol C2H 5/g plnt/h) of penut plnts inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium, grown in soil mended with either rock-p or super-p. Inocultion Nodule number / plnt N 2 ctivity (nmol C2H 5/g plnt/h) Nodule number / plnt N 2 ctivity (nmol C2H 5/g plnt/h) Uninoculted Plnt+Rh 347.4*** 82.4*** 338.0*** 79.2*** Plnt+VAM 299.8*** 75.0*** 289.4*** 67.5*** b Plnt+VAM+Rh 421.2*** 96.9*** 411.3*** 84.9*** Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P [28] mycorrhizl development. Ishc et l., reported tht the positive effect of AM on groundnut growth, nodultion, N 2 fixtion nd N &P uptke my be ttributed to the mycorrhizl hyphe which incresed the root mss spred in the soil nd consequently incresed the nutrient uptke. It is of interest to show tht during the course of this experiment, nitrogense ctivity responses followed nodule responses to different inoculnts in both P-mended soils, this result ws in gree with tht recorded by Rbie nd [61] Huminy. Highest significnt nodule number nd nitrogense ctivity within penut plnts were recorded in the presence of dul inoculnts of Brdyrhizobium nd Am fungi. The synergistic effects of dul inoculnts of Am fungi nd nitrifying bcteri on nodule formtion nd nitrogense ctivity were [42,60,66] previously proved for different legumes plnts. In fct, it is esy to understnd tht the nodules cn fix tmospheric nitrogen, but its efficiency is mostly determined by the phosphorus nutrient condition of the host plnt since pproprite phosphorus nutrient support is indispensble for the process of nitrogen fixtion. Mycorrhizl infection could contribute to proper phosphorus uptke in Brdyrhizobium nd ensure the ctivity of nitrogen fixtion enzyme. In this connection, [34] [72] Jh et l., ; Vldes nd Snnchez-Frnci nd [36] Johnsson et l.,, showed tht dul inocultion with mycorrhize nd nitrifying bcteri cn support both the needs for N nd P nd increse the growth of host plnt. Dt lso reveled significnt nd high significnt vlues in cse of soil fertilized with rock-p thn tht fertilized with super-p. Totl Chlorophyll Content nd Totl Crbohydrte: The results obtined from Tble 6 reveled the totl pigments content nd totl crbohydrte of leves of penut plnts. Dt showed tht inocultion with Brdyrhizobium, significntly, increses the totl pigment content of penut plnts s compring to noninfected plnts. Infection with single VA mycorrhizl fungi lso, significntly, increses the totl chlorophyll nd b, higher thn those of both bcteril nd noninfected plnts, in both soils. With this respect, [26] Hymn stted tht mycorrhizl ssocitions in terrestril plnts hve been shown to increse chlorophyll production compred with plnts without fungl ssocitions. The totl crbohydrte lso, significntly, increses with inoculted penut plnts with Brdyrhizobium singly, then, significnt, higher increse ws tbulted with the infected VAmycorrhizl plnts. However, the increse in totl pigment content ws positively correlted with the increse in totl crbohydrte content. Severl reserchers hve been proposed tht AMF symbiosis [64] incresed lef gs exchnge nd photosynthetic rte, enhnced wter uptke through improved hydrulic conductivity nd incresing lef conductnce nd [39,20] photosynthetic ctivity. The vlues recorded in soil mended with rock-p were, significntly, still higher thn tht in super-p mended soil. The most effective co-inocultion ws observed in the combined tretment with Brdyrhizobium nd VA mycorrhizl fungi, which synergisticlly incresed totl chlorophyll, b nd crbohydrte compred with singly inoculted, s well s the control plnts, in ech P-mended soil. The enhncement in chlorophyll nd crbohydrte content cn be ttributed to the increse of bsorption nd trnsloction of essentil metl ions, due to VA mycorrhizl infection, which in turn ccelerte the metbolic rtes relted to the synthesis of such constituents. The higher height increment registered with inoculted plnts could be s result of enhnced [19] inorgnic nutrient bsorption nd greter rtes of [5] photosynthesis, which obviously could hve given rise to n increse in plnt growth. Vm Infection Percentge, Protein Content nd Totl Lipid: The results presented in Tble 7 reveled VA mycorrhizl infection percent, protein nd lipid content of penut plnts grown in soil mended with either rock or super-p inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium. Slightly, non-significnt, increse in percentge of VA mycorrhizl infection in penut plnts infected with Am fungi nd Brdyrhizobium thn those with VA solely in either P fertilized soils. With this respect, we cn conclude tht VA mycorrhizl fungi influenced the competitiveness of Brdyrhizobium strins, nd tht Brdyrhizobium 192

7 Tble 6: Totl Chlorophyll content (mg /g fresh weight), nd totl crbohydrte (mg/g dry weight) of leves of penut plnts inoculted with VA mycorrhizl fungi, nd /or Brdyrhizobium, grown in soil mended with either rock-p or super-p. Inocultion Chloro- Chloro- Totl pigm- Totl crboh- Chloro- Chloro- Totl pigm- Totl crbohphyll " " phyll " b " ents (mg/g) ydrte (mg/g) phyll " " phyll " b " ents (mg/g) ydrte (mg/g) Uninoculted b 1.32 b b Plnt+Rh 1.79*** 0.59** * 1.54* * b Plnt+VAM 2.24*** 0.74*** *** 1.96*** 0.64** ** b b Plnt+VAM+Rh 2.54*** 0.84*** *** 2.25*** 0.74*** *** Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P Tble 7: Percentge of mycorrhizl infection, protein content (mg/g), nd totl lipid (mg/g) of dry seeds of penut plnts inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium, grown in soil mended with either rock-p or super-p. Inocultion % 0f VAM protein content lipid content % 0f VAM protein content lipid content infection (mg/g) (mg/g) infection (mg/g) (mg/g) Uninoculted Plnt+Rh ** 51.7** ** 47.8 Plnt+VAM * 51.3** ** 49.0* Plnt+VAM+Rh ** 53.7** *** 52.6** Compringuninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P lso ffected the root coloniztion by VA mycorrhizl [52] fungi. In ddition, the prmeters of the plnt grown in soil mended with rock phosphte were found to be more beneficil thn tht grown in super-p mended [23] soil. El-Ghndour et l., noticed tht nodultion nd micorrhizl infection were higher in soil mended with rock thn those mended with superphosphte. Inoculting plnts with Brdyrhizobium solely, high significntly, incresed their protein content to gret extent compring to non-inoculting plnts in both P pplicble soils. In ddition, inoculting with Am fungi hd significnt slightly increment compring to noninfected plnts, but still lower thn tht of bcteril inocultion. The highest significnt content ws obtined in presence of Brdyrhizobium nd Am fungi, in rock-p mended soil, which exhibited high significnt vlues thn super-p mended soil. From the previous dt, bcteril-am fungllegume triprtite symbiosis showed better nitrogen fixtion (nodule number, nitrogen nd protein contents s well s nitrogense ctivities) thn tht of bcterillegume symbiosis. These results were consistent [50] with Minerdi et l., who demonstrted the presence of genes for fixtion in endosymbiotic Burkholderi bcteri in AM mycorrhizl hyphe nd suggested tht there my be potentil for improving N supply to mycorrhizl plnts through fixtion of [62] tmospheric N. Lipid content ws notbly influenced by inocultion nd fertiliztion of soil. Penut plnts showed high content with the inocultion of Brdyrhizobium, followed by mycorrhiz symbiosis. The highest ws produced, significntly, with the dul inocultion of both inoculnts. Tble 7 lso reveled tht rock-p mended to the soil showed n effect on the lipid content of penut plnts. This result ws in [62] gree with those obtined by Rbie who reported tht rock phosphte mended to the soil showed significnt effect on the lipid content of red kidney nd whet plnts, where the lipid content of the plnts grown in soil with rock-p ws still higher thn tht without rock-p especilly in the presence of Am fungi. Nutrient Uptke: Tble 8 shows tht nitrogen plnt tissue, phosphorus nd potssium concentrtion ws much higher in the inoculted plnts thn non inoculted ones, either in rock-p or super-p mended soil. Brdyrhizobium inocultion hd incresed N, P nd K uptke by penut plnts, in both P mended soils, comprble to the uninoculted plnts. It is of interest to mention tht the Brdyrhizobium strin hs [1] the bility to solubilize rock-p, this ws proved from 193

8 -1 Tble 8: Nitrogen, phosphorus, nd potssium (NPK) content (mg g ) of dry leves of penut plnts inoculted with VA mycorrhizl fungi nd /or Brdyrhizobium, grown in soil mended with either rock-p or super-p. Inocultion N P K N P K Uninoculted Plnt+Rh *** 2.04*** 3.46** 0.39*** 2.00*** Plnt+VAM *** * 0.43*** 1.98*** b Plnt+VAM+Rh 4.01** 0.53*** 2.13*** 3.93** 0.58*** 2.11*** Compring uninoculted with other tretment in the sme column: * = significnt t P 0.05, ** = high significnt t P 0.01, *** = highly significnt t P Compring the sme tretment in rock-p with super-p: = significnt t P 0.05, b = high significnt t P 0.01, c = highly significnt t P the highly significnt dt of P uptke from Brdyrhizobium inoculted plnts in rock-p nd super-p mended soil, s compred to uninoculted plnts. Dt lso revel higher N, P nd K concentrtion in the mycorrhizl inoculted plnts, in both phosphte fertilized soils. The incresed efficiency of mycorrhizl plnts versus non-mycorrhizl is cused by the ctive uptke nd trnsport of nutrients by mycorrhize. The highest significnt vlues of N, P nd K were recorded with the pir inoculnts combintions, where the vlues were higher under rock-p thn super-p mended soil. [11] With this respect, Azcón-Aguilr et l., recorded tht inocultion with Glomus mossee not only ffected plnt growth nd nutrition in Medicgo stiv, but lso enhnced the ctivity of Rhizobium meliloti when it ws pplied s n inoculnt. In legume plnts the importnce of AMF symbiosis hs been ttributed to high P requirements on the nodultion nd N 2 fixtion process which requires [12] enhnced P uptke. Thus, AMF hve been shown to [33] improve productivity in soils of low fertility nd re prticulrly importnt for incresing the uptke of 3-[32] slowly diffusing ions such s PO 4. Plnt roots lone my be incpble of tking up phosphte ions tht re immobilized, for exmple, in soils with bsic ph. The mycelium of the mycorrhizl fungus cn however ccess these phosphorus sources, [44] nd mke them vilble to the plnts they colonize. The mechnisms of incresed bsorption re both physicl nd chemicl. Mycorrhizl myceli re much smller in dimeter thn the smllest root hir. For this reson they re ble to explore greter volume of soil nd hve much lrger surfce re for bsorption. Also, the cell membrne chemistry of fungi is different from tht of plnts. Mycorrhize re especilly beneficil for the plnt prtner in nutrient-poor soils. Mycorrhizl plnts re often more resistnt to diseses, such s those cused by microbil soil-borne [21,59] pthogens, nd re lso more resistnt to the [67,65] effects of drought. These effects re perhps due to the improved wter nd minerl uptke in mycorrhizl [60] plnts. Quilmbo reported tht inocultion with n indigenous inoculnt resulted in incresed lef nd root growth nd prevented the expected increse in root to shoot rtio nd root-weight rtio tht re normlly observed under phosphorus deficient nd drought stress conditions in penut. Penut (Archis hypoge L.) plnts grown in sterilized soil without VA-fungi inocultion developed visible symptoms of phosphorus [47] [40] nd zinc deficiency. Krishn nd Bgyrj stted tht inocultion of penut plnts with mycorrhizl fungus Glomus fscicultum incresed uptke of phosphorus nd micronutrients such s zinc, copper, mngnese nd iron. The higher plnt tissue nitrogen content in inoculted plnts could be ttributed to hyphe uptke. It hs been reported tht the existence of extr-rdicl hyphl bridges between individul plnts permits [48] trnsfer of nutrients such s nitrogen. They reported tht bout 24% of the totl nitrogen uptke in mycorrhizl plnts could be ttributed to uptke nd delivry by the externl hyphe. There is lso evidence tht nitrogen is tken up by vesiculr-rbusculr mycorrhiz hyphe from inorgnic sources of mmonium nd therefore, the higher nitrogen concentrtion in mycorrhizl plnts could be ttributed to the hyphe uptke. The sme could be sid of the higher potssium concentrtion in inoculted plnts. In other words, mycorrhizl fungi cn improve bsorption + of N from NH 4 nitrogen minerl fertilizers, [8,35] trnsporting it to the host plnt. Its trnsport nd bsorption cn lso increse biomss production in [46] soils with low potssium. In comprtment pots [45] experiment, Li et l., demonstrted tht bout 10% of the totl potssium uptke in mycorrhizl coch grss ws due to hyphl uptke nd trnsport. [38] Kohler et l., reported tht the folir P nd K contents incresed significntly with the Bcillus subtilis or Glomus intrrdices inocultion, lone or in combintion. Conclusion: We conclude tht nturl microbil inoculnts enrich the nutritionl vlue of the soil nd it is dvisble to use these microorgnisms s biofertilizers in the presence of low dose of N nd P fertilizers in low soil fertility. Also, rock-p could be considered s vilble nd chep source of P fertiliztion. 194

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