RICE RESPONSE TO INOCULATION WITH P-SOLUBILIZING MICROORGANISMS FROM BRAZILIAN CERRADO

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1 Originl Article 1 RICE RESPONSE TO INOCULATION WITH P-SOLUBILIZING MICROORGANISMS FROM BRAZILIAN CERRADO RESPOSTA DE ARROZ À INOCULAÇÃO DE MICRORGANISMOS SOLUBILIZADORES DE FOSFATO DE SOLO DE CERRADO Rosely de Fátim FERREIRA 1 ; Isel Cristin Mendonç CARDOSO 2 ; Cinti Fri d SILVA 3 ; Edson Luiz SOUCHIE 4 ; Mrco Aurélio Crone CARNEIRO 5 1. Tecnólog em Produção de Grãos, Centro Federl de Educção Tecnológic de Rio Verde - CEFET Rio Verde, Rio Verde, GO, Brsil. rosely.ferreir@iest.com.r; 2. Tecnólog em Produção de Grãos, Mestrnd em Mnejo do Solo, Centro de Ciêncis Agroveterináris, Universidde do Estdo de Snt Ctrin, Lges, SC, Brsil; 3. Biólog, CEFET Rio Verde; 4. Professor Doutor, Lortório de Microiologi Agrícol, CEFET Rio Verde, Rio Verde, GO, Brsil; 5. Professor, Doutor, Centro de Ciêncis Agráris, Universidde Federl de Goiás, Cmpus Jtí, Jtí, GO, Brsil. ABSTRACT: Two experiments were done under greenhouse conditions to evlute rice growth promotion through inocultion of P-soluilizing cteri nd fungi isolted from their rhizoplne nd rhizosphere. The est inoculum concentrtion ws lso determined. In oth experiments, plnts were grown in tues (33 ml) contining sterilized soil from Cerrdo. In the first tril, ech plnt ws inoculted (1 ml with 1 6 CFU ml -1 ) with fungl nd cteril isoltes nd the mixture of oth. A completely rndomized experimentl design with 6 inocultion tretments nd 6 replictes ws used. In the second tril, plnts were inoculted with incresing concentrtions (1 6, 1 7, 1 8 nd 1 9 CFU ml -1 ) with two P-soluilizing cteril nd one P-soluilizing fungl isoltes. A completely rndomized experimentl design, s 3 x 4 fctoril (three P-soluilizing isoltes nd four incresing inoculum concentrtions), plus non inoculted tretment nd five replictes were used. In oth experiments, plnts were hrvested 24 dys fter inocultion with these microorgnisms. Plnt height, shoot nd root dry mtter/length/volume were mesured. In the first experiment, the height, root dry mtter/length/volume on rice incresed with the inocultion of the P-soluilizing fungi () s well s the mixture of nd the P-soluilizing cteri. In the second experiment, lso showed the est performnce on rice growth promotion minly t the highest inoculum concentrtion. KEYWORDS: P-soluilizing cteri nd fungi. Rhizosphere. Inoculum. Concentrtion. INTRODUCTION Phosphorus (P) is one of the mjor limiting fctors for crop production minly in tropicl soils (NORMAN et l. 1995) due to the high P fixtion prolem. This mens tht P in the soil is tightly ound to the soil prticles nd reltively unville to plnts. Thus, the minerl pool of P in the soil cn e reltively lrge ut just little prt of it is ville to plnts. Consequently, high dosges of phosphte fertilizers re required to otin high yields. Edphic P-soluilizing microorgnisms (PSMs) cn increse P vilility to plnts improving, therefore, its use efficiency. Moreover, those microorgnisms cn e inoculted in res where insolule P sources, such s Arxá, Ptos de Mins nd Ctlão ptites re used. Hence, the cost of phosphte fertiliztion cn esily decrese if these orgnisms re pplied in ssocition with low solule P sources. Moreover, these orgnisms could optimize the fixed P nturlly found in soil. Some PSMs cn lso increse plnt growth y mechnisms other thn P soluiliztion, e.g. production of phytohormones such s Indole-cetic cid (ARSHAD; FRANKENBERGAR, 1998). Severl uthors reported growth promotion through inocultion of rhizocteri on lettuce (SOTTERO et l. 26), citrus (FREITAS; VILDOSO, 24) nd whet (LUZ, 21). Others relted plnt growth promotion through simple inocultion of P- soluilizing fungi on whet (WHITELAW et l. 1997), onion (VASSILEV et l. 1997) nd soyen (ABD-ALLA; OMAR, 21). Besides tht, incresing plnt disese resistnce (LUZ, 21; PAUL; SHARMA, 25) nd the potentil of reducing the use of solule P sources nd, consequently, decresing chnces of environmentl contmintions (FREITAS; VILDOSO, 24) re implicted when these orgnisms re used. Therefore, P-soluilizing microorgnisms cn e considered gret option to mximize plnt growth promotion, incresing the sustinility of the groecosystems. However, few studies focusing this group of microorgnisms hve een done in tropicl res, minly in Brzil. Nturlly, this type of reserch requires more ttention. The direct effect of PSMs nd plnt growth promoters re normlly oserved y incresing on root length, shoot dry mtter nd improvement on Received: 22/4/8 Accepted: 12/8/8 Biosci. J., Uerlândi, v. 25, n. 5, p. 1-7, Sept./Oct. 29

2 Rice response... FERREIRA, R. F. et l. 2 plnt nutrition (SABINO et l. 2). NARLOCH et l. (22) oserved greter yield on rdish through inocultion of Aspergillus sp. nd Penicillium sp., two types of P-soluilizing fungi. Sino et l. (2) lso found improvement on rice growth promotion, reporting tht the density of inoculted microorgnisms ssocited to the inocultion method is crucil to gurntee the estlishment of these orgnisms in the soil. Then, studies focusing the inoculum concentrtion of PSMs in order to mximize plnt growth promotion re required. This work evluted rice growth promotion through inocultion of P-soluilizing cteri nd fungi isolted from their rhizoplne nd rhizosphere. The effect of incresing PSMs concentrtions on plnt growth promotion ws lso evluted. MATERIALS AND METHODS This work ws done under greenhouse conditions t Centro Federl de Educção Tecnológic de Rio Verde - GO (CEFET - Rio Verde). The first step of this work ws to isolte P- soluilizing cteri (PSB) nd fungi (PSF) from the rice rhizoplne nd rhizosphere. Rice (Primver vriety) ws sown in tues (33 ml) contining soil clssified s Ltosol collected from 1 4 cm lyer t experimentl re of CEFET - Rio Verde. This soil ws used for Experiments 1 nd 2. Chemicl nd texturl nlysis were done (EMBRAPA, 1997) otining the following results: ph (in wter) = 5.6; P (Mehlich 1) =1.5 mg dm -3 ; K =.8 mmol dm -3 ; C = 1.4 mmol dm -3 ; Mg = 3.2 mmol dm -3 ; Al =. mmol dm -3 ; Orgnic mtter = 22.8 g dm -3 ; snd = 39%; silt=11% nd cly=5%. Three dys fter emergence, seedlings were thinned to two plnts per tue. Twenty two dys fter plnt emergence, PSMs isoltion ws done. Twenty plnts were used. Soil smples (1g) from the rhizoplne nd rhizosphere of ech plnt were mixed in flsks (125 ml) contining 9 ml of sline solution (.85%) following the successive dilution technique till 1-4. The dilutions 1-3 nd 1-4 were chosen for plting (triplictes), using the GL solid medium (glucose, 1g; yest extrct, 2g nd gr-gr, 15g L -1 ). Before plting, this medium ws mintined t 45 C while 1 ml of CCl 2 (1%) nd 5 ml of K 2 HPO 4 (1%) L -1 were sterilized seprtely, nd then dded to the GL medium to form CHPO 4 (1%), ccording to Sylvester- Brdley et l. (1982). After incution (four dys t 32 ºC, drkness) of the Petri dishes, direct counting of PSB nd PSF ws done. Microorgnisms le to soluilize this phosphte showed trnsprent hlo round their colonies, ccording to DI SIMINI et l. (1998). These orgnisms were purified nd stored in tues (8 ml) contining GL medium plus glycerol, under room temperture. Experiment 1: Inocultion of P-soluilizing cteri nd fungi isoltes in rice grown in soil from Cerrdo This experiment ws done to evlute rice growth promotion through inocultion of P- soluilizing cteri nd fungi isolted from their rhizoplne nd rhizosphere. Rice seeds (Primver vriety) were sterilized ccording to Rumjnek (1999) nd sown in tues (33 ml) lso sterilized (immersion in sodium hypochlorite, 1% for 1 min). Plnts were grown in utoclved soil (12 ºC for 15 min) nd irrigted every other dy. Three dys fter emergence, seedlings were thinned to one plnt per tue. A completely rndomized design with six inocultion tretments (, PSB 36,, +, + PSB 36 nd non inoculted tretment) nd six replictes were used. Four dys fter emergence, seedlings received 1 ml of GL liquid medium (glucose, 1g nd yest extrct, 2g L -1 ) contining 1 6 CFU ml -1 of ech inoculum tretment. PSB isoltes were inoculted in flsks (125 ml Erlenmeyers) contining 2 ml of GL liquid medium nd incuted (24 h, 28 C, drkness). From this suspension, 1 ml ws pipetted, quntified using successive dilution nd direct counting techniques nd stndrdized to 1 6 CFU ml -1. In reltion to the PSF isoltes, spore suspension ws prepred y the following procedure: ech isolte ws incuted in Petri dishes (four dys, 28 ºC, drkness), in GL solid medium, until the Petri dishes were covered. The spores were suspended in wter plus tween (1%), using Driglsky sptul following the sme techniques to stndrdize the inoculum to 1 6 CFU ml -1, s used with PSB isoltes. The non inoculted tretment consisted of inocultion of 1 ml of sterilized GL liquid medium without ny PSMs. After 24 dys of inocultion, plnts were hrvested nd height, shoot nd root dry mtter, root length nd volume were evluted. The root length ws clculted ccording to Tennnt (1975) while the root volume ws mesured fter root immersion in grdute cylinder contining wter, nd determining the volume difference. The dt were sumitted to ANOVA nd mens compred y Scott-Knott test (p.5), using the SISVAR sttisticl pckge (FERREIRA, 1999). Biosci. J., Uerlândi, v. 25, n. 5, p. 1-7, Sept./Oct. 29

3 Rice response... FERREIRA, R. F. et l. 3 Experiment 2: Inocultion of incresing concentrtions of P-soluilizing cteri nd fungi in rice grown in soil from Cerrdo This experiment ws done to identify the est P-soluilizing cteri nd fungi inoculum concentrtion in order to improve plnt growth. Rice seeds (Primver vriety) were sterilized ccording to Rumjnek (1999) nd sown in tues (33 ml) lso sterilized (immersion in sodium hypochlorite, 1% for 1 min). Plnts were grown in utoclved soil (12 ºC for 15 min) nd irrigted every other dy. Three dys fter emergence, seedlings were thinned to one plnt per tue. The experimentl design ws disposed in completely rndomized design, in 3 x 4 fctoril (three types of PSMs nd four incresing inoculum concentrtion), plus n dditionl tretment (sence of ny PSMs inocultion) nd five replictes. The inocultion tretments were:, PSB 36 nd. After four dys of emergence, seedlings received 1 ml of GL liquid medium contining 1 6, 1 7, 1 8 or 1 9 CFU ml -1 of ech inoculum tretment. The procedure for stndrdiztion of the PSB nd PSF incresing inoculum tretments ws the sme descried for Experiment 1. After 24 dys of inocultion, plnts were hrvested nd height, shoot nd root dry mtter, root length nd volume were evluted. The root length ws clculted ccording to Tennnt (1975) while the root volume ws mesured fter root immersion in grdute cylinder contining wter, determining the volume difference. The dt were sumitted to ANOVA nd mens compred y Scott-Knott test (p.5) nd regression nlysis (p.5) using the SISVAR sttisticl pckge (FERREIRA, 1999). RESULTS AND DISCUSSION In the first prt of this work, four PSB isoltes nd only one PSF were identified s phosphte soluilizers due to their ility to form trnsprent hlo round their colonies. Sylvester- Brdley et l. (1982) suggested the possiility of inhiition of fungl growth due to the metolites produced y cteril isoltes under in vitro conditions. These results re in greement with those uthors, who lso detected smller numer of PSF compred to PSB in soil from the Brzilin Amzon. Similr result ws lso found y Souchie et l. (26) who detected greter numer of PSB using the GL nd GELP medi for soil from n re of Secondry Forest in Prty, Rio de Jneiro. These uthors suggested tht it might e due to the presence of yest extrct in the growth medium composition. The PSMs inoculted in the Experiment 1 showed different ehvior in reltion to rice growth promotion (Figure 1). For instnce, height nd root dry mtter were incresed when, +, + PSB 36 nd PSB 36 were inoculted, surpssing the non inoculted tretment (Figure 1A nd B). Similr results were found y Freits e Vildoso (24), who detected incresed citrus growth through inocultion of Bcillus nd Pseudomons rhizocteri. Otherwise, no effect of these inocul were found on the shoot dry mtter, where plnts inoculted with recorded highest vlues (32 mg plnt -1 ), not differing, however, of the non inoculted tretment (22 mg plnt -1 ). The inocultion with, + nd + PSB 36 incresed root length (Figure 1C). It could e lso due to the possiility of phytohormones production y these isoltes. For instnce, the inocultion with Azospirillum rsiliensis incresed the root length nd formtion of lterl roots in whet y indol cetic cid production (SABINO et l. 2). Similrly, the root volume in rice ws incresed when nd + were inoculted (Figur 1D). It lso could hve occurred due to the sme reson reported ove. According to Bre et l. (1997) nd Bowen e Rovir (1999), mechnisms s phytohormone, vitmin nd mino cid production cn e involved on plnt growth promotion y PSMs. Hence, nd + certinly ehved s plnt growth promoters since height, root dry mtter/length/volume were incresed with their inocultion. Rice growth promotion lso might hve occurred due to the soluiliztion of the ntive P from the soil. Nturlly, if plnts hd more P vilility, etter growth would e found. In Experiment 2, no effect of incresing inoculum concentrtion on height nd shoot dry mtter ws found. Nevertheless, n effect of PSMs inocultion ws detected where showed the est performnce, incresing the vlues of these vriles (Figure 2A nd B). An interction etween PSMs nd incresing inoculum concentrtion ws oserved in reltion to root dry mtter production. A liner effect of the incresing concentrtion of ws oserved (Figure 3A). There ws no effect of the inocultion with nd PSB 36. As to the root dry mtter, induced similr results when root length/volume ws mesured (Figure 3B nd C). Biosci. J., Uerlândi, v. 25, n. 5, p. 1-7, Sept./Oct. 29

4 Rice response... FERREIRA, R. F. et l. 4 A) PSB 36 PSB Control B) C) 2.5 D) Inocultion tretments Figure 1. Height (A), root dry mtter (B), root length (C) nd root volume (D) of rice plnts inoculted with P- soluilizing cteril (PSB) nd fungl (PSF) isoltes, fter 24 dys grown in soil from Cerrdo. Mens followed y the sme letter do not differ y the Scott-Knott test (p.5). Heigth (cm plnt -1 ) 18 A) B) Shoot dry mtter (mg plnt -1 ) PSB 36 Figure 2. Height (A) nd shoot dry mtter (B) of rice plnts inoculted with P-soluilizing cteril (PSB) nd fungl (PSF) isoltes. Mens followed y the sme letter do not differ y the Scott-Knott test (p.5). Biosci. J., Uerlândi, v. 25, n. 5, p. 1-7, Sept./Oct. 29

5 Rice response... FERREIRA, R. F. et l. 5 r (mg plnt -1 ) A) Root dry mtte (y= x R 2 =.92) Root length (cm plnt -1 ) B) (y= x R 2 =.88) PSB 36 (y= x+78.97x 2 R 2 =.9) (y= x R 2 =.96) PSB 36 Root volume (cm 3 plnt -1 ) C) (y= x R 2 =.97) (y= x R 2 =.89) Inoculum concentrtion (CFU ml -1 ) Figure 3. Root dry mtter (A), root length (B) nd root volume (C) of rice plnts inoculted with incresing concentrtions of P-soluilizing cteril (PSB) nd fungl (PSF) isoltes. The isolte showed the est performnce incresing ll plnt growth vriles mesured. The estlishment ility of fungl isoltes is normlly higher thn cteril isoltes especilly due to their cpcity to produce spores. In ddition, due to their ility to produce higher iomss, greter P-soluiliztion nd, consequently, higher plnt growth promotion cpcity would e encountered. However, ccording to Wkelin et l. (24), P-soluilizing ctivity is relted not only to iomss production, ut the physiologicl stte of the iomss. Moreover, since P-soluilizing cteri generlly out-numer P- soluilizing fungi in soil y 2-15 fold (KUCEY, Biosci. J., Uerlândi, v. 25, n. 5, p. 1-7, Sept./Oct. 29

6 Rice response... FERREIRA, R. F. et l ) their contriution to the plnt P vilility cn not e minimized. The isolte pprently showed low functionl comptiility with rice. Considering the incresing inoculum concentrtion, decresing root length / volume ws detected (Figure 3B nd C). Souchie et l. (25) reported certin incomptiility etween P-soluilizing cteri nd Enteroloium contortisiliquum. According to these uthors, lower shoot nd root dry mtter were found when mix of three P-soluilizing isoltes were inoculted. Hence, efforts re required to elucidte the specific mechnisms from these microorgnisms in order to mximize their plnt growth promotion potentil. Moreover, further studies in order to testing the efficiency of these isoltes under nturl soil conditions re lso interesting. CONCLUSIONS Height, root dry mtter/length/volume of rice plnts were incresed in the presence of nd +. The single inocultion with the ws not efficient, showing performnce similr to the non inoculted tretment. The isolte showed the est performnce on rice growth promotion in the highest inoculum concentrtion. RESUMO: Dois experimentos form conduzidos em condições de cs de vegetção pr vlir cpcidde promotor de crescimento de rroz (Oryz stiv L.) por ctéris e fungos soluilizdores de fosfto isoldos de su rizosfer, lém de identificr melhor concentrção de inoculnte que mximizsse o crescimento vegetl. Em mos os experimentos, s plnts form cultivds em tuetes (33 ml) contendo solo de Cerrdo utoclvdo. No primeiro experimento, cd plnt foi inoculd com 1 ml contendo 1 6 UFC ml -1 com isoldos fúngicos, cterinos e mistur de mos. Utilizou-se um delinemento inteirmente csulizdo com 6 trtmentos de inoculção e 6 repetições. No segundo experimento, s plnts form inoculds com concentrções crescentes (1 6, 1 7, 1 8 e 1 9 UFC ml -1 ) com dois isoldos cterinos e um fúngico, utilizndo-se um delinemento inteirmente csulizdo, esquem ftoril 3 x 4 (três tipos de microrgnismos inoculdos e qutro concentrções de inoculnte), mis um trtmento não inoculdo e 5 repetições. Em mos os experimentos, s plnts form colhids 24 dis pós inoculção com os soluilizdores, vlindo-se ltur, mtéri sec de prte ére, mtéri sec de riz, extensão e volume rdiculr. No primeiro experimento, ltur de prte ére, mtéri sec, extensão e volume ds rízes ds plnts de rroz form incrementds com inoculção do fungo soluilizdor de fosfto FSF 8 e su mistur com ctéri soluilizdor de fosfto BSF 24. No segundo experimento, o FSF 8 demonstrou mior potencil de estímulo o crescimento de rroz que os isoldos cterinos, principlmente ns concentrções de inóculo mis lts. PALAVRAS-CHAVE: Bctéris e fungos soluilizdores de fosfto. Rizosfer. Concentrção de inoculnte. REFERENCES ARSHAD, M.; FRANKENBERGER, W. T. Plnt growth-regulting sustnces in the rhizosphere: Microil production nd functions. Advnces in Agronomy, Orlndo, v. 62, , BAREA, J. M. et l. Interctions etween mycorrhizl fungi nd rhizosphere microorgnisms within the context of sustinle soil-plnt systems. In: GANGE, A. C.; BROWN, V. K. Multitrophic interctions in terrestril systems. Cmridge: Blckwell Science, 1997, p BOWEN, G. D.; ROVIRA, A. D. The rhizosphere nd its mngement to improve plnt growth. Advnces in Agronomy, Orlndo, v. 66, p. 1-12, DI SIMINI, C. D. et l. Soluiliztion of zinc phosphte y strin of Pseudomons fluorescences isolted from forest soil. Biology nd Fertility of Soils, New York, v. 28, n. 1, p , EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA. Mnul de métodos de nálise de solo. Brsíli: Emrp Solos, 1997, 212 p. FERREIRA, D. F. Progrm Sisvr Versão 4.6 (Build 61). Cpturdo em 11 out. 25. Online. Disponível n Internet Biosci. J., Uerlândi, v. 25, n. 5, p. 1-7, Sept./Oct. 29

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