INTERNATIONALJOURNALOFENVIRONMENTALSCIENCESVolume2,No1,2011 Copyright2010AllrightsreservedIntegratedPublishingAssociation Researcharticle ISSN0976 4402

1 2 3 4 NitaB.Patil ,MilindGajbhiye ,SangitaS.Ahiwale ,AparnaB.Gunjal , BalasahebP. 5 Kapadnis 1,3,4 DepartmentofEnvironmentalScience,UniversityofPune,Ganeshkhind,Pune 411007,Maharashtra,India 2,5 DepartmentofMicrobiology,UniversityofPune,Ganeshkhind,Pune411007, Maharashtra,India patil.nita1@gmail.com

OptimizationofIndole3aceticacid(IAA)productionbyAcetobacter diazotrophicusL1isolatedfromSugarcane

ABSTRACT Basedonmorphological,culturalandbiochemicalcharacteristics,anewlyisolatedbacterium from the stem of the Indian sugarcane variety Co 740 was identified as Acetobacter diazotrophicusandwasdesignatedasL1.Culturalandnutritionalconditionswereoptimized forindole3aceticacid(IAA)production.Theeffectofincreasingsucroseconcentrationand differentnitrogensourcesonIAAproductionwerestudied.Shakingincreasedtheproduction ofIAA.TheisolateproducedhighamountofIAA(26.28g/ml)whenbasalmedium(pH6) wassupplementedwithsucrose(12%w/v),yeastextract(0.05g/l),Ltryptophan(1.2g/l)and NH4Cl (0.1% w/v) at 200 rpm. IAA production increased up to 2.6 fold over control. InoculationwithA.diazotrophicusL1increasedrootandshootbiomassaswellasIAAlevel of the fresh root tissue of maize plant. The results indicated that A. diazotrophicus L1 producedhighamountofIAAthanreportedearlier. Keywords: Acetobacter diazotrophicus, endophytes, indole acetic acid, plant growth promotion,auxinproduction 1.Introduction Endophytic bacteria can be defined as those bacteria that colonize the internal tissue of the plant showing no external sign of infection or negative effect on their host (Ryan, et al., 2008). These bacteria significantly affect plant growth by increasing nutrient cycling, suppressing pathogens by producing antibiotics and siderophores or bacterial and fungal antagonistic substances and/or by producing biologically active substances such as auxins and other plant hormones (Khalid, et al., 2004). Within a few years several species of endophytic diazotrophs were discovered including A. diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia sp., Enterobacter sp. and Klebsiellasp.insugarcaneplants.Workhascontinuedontheseendophyteswithinsugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed biological nitrogen fixation and other plant growth promoting traits(Boddey,etal.,2003).Numerousendophytesareactively involved inthesynthesisof auxinsinpurecultureandinplants.IAAisaphytohormonewhichisknowntobeinvolvedin root initiation, cell division and cell enlargement. IAA producing bacteria are believed to increaserootgrowthandrootlength,resultingingreaterrootsurfaceareawhichenablesthe planttoaccessmorenutrientsfromsoil(Boiero,etal.,2007).Recently,itwasalsofoundthat IAA induces an increased level of protection in plants against external stress conditions (Bianco and Defez, 2009). Ltryptophan (Trp), an amino acid serves as a physiological 295

ReceivedonJune2011PublishedonSeptember2011

OptimizationofIndole3aceticacid(IAA)productionbyAcetobacterdiazotrophicusL1isolatedfrom Sugarcane

precursorforbiosynthesisofauxinsinplantsandmicrobes.Auxinsofmicrobialorigininthe interiorofplantscouldevokeaphysiologicalresponseinthehostplant.Therefore,screening oftheendophytesfortheirinvitropotentialofauxinproductioncouldprovideareliablebase forselectionofeffectiveplantgrowthpromotingbacteria.Literaturesurveyindicatedlotsof research work on the diazotrophic nature of A. diazotrophicus. However, very few reports described its ability to produce IAA. Considering this, the present investigation was conducted to demonstrate the IAA production in culture of A. diazotrophicus L1 isolated fromsugarcanestemandoptimizationofvariousfactorssuchaspH,shaking,concentration oftryptophanandsucrosealongwithdifferentnitrogensources. 2. MaterialsandMethods 2.1IsolationandscreeningforIAAproduction Severalbacterialstrainswereisolatedfromthestemofthedifferentvarietiesofsugarcaneon LGIagarmediumusingstandardprocedures(CavalcanteandDobereiner,1988).Theywere maintainedonLGIagarslantsat4Candin20%glycerolat80C.Identificationwascarried out by subjecting the isolates to cultural, morphological (colony morphology and pigmentation), microscopic (Gram staining and motility), biochemical (utilization of carbon sources and enzymatic activity) and physiological characteristics (temperature, pH, salt and sugartolerance).Oxidationofacetateandlactate,productionofaceticacidonethanolCaCO3 mediumandthegrowthon21%sucrose(w/v)and0.35%aceticacidwerealsotested.Thirty fivebacterialisolatesobtainedwerescreenedforIAAproductioninvitro.Auxinproduction, bothinthepresenceandabsenceoftryptophanwasdetermined.Forthispurpose,Erlenmeyer flask(250ml)containing50mlofLGIbrothsupplementedwithLtryptophan(0.5g/l)was 6 inoculatedwith1 mlof bacterialsuspension(9x10 CFU/ml)and incubatedinthedarkat 30Cfor7daysonshakerat200rpm.TheproductionofIAAinbrothculturewasassayedby Salkowskyscolorimetricmethod(GlickmannandDessaux,1995). 2.2OptimizationofculturalandnutritionalconditionsforIAAproduction EffectofdifferentlevelsoftryptophanonthetimecourseofIAAproductionwasdetermined usingLGI mediumsupplementedwithtryptophanin varyingconcentrations(0.2to1.6g/l). ThepHwasadjustedtodifferentvaluesrangingfrom4to8andtheeffectofvaryingpHon IAAproductionwasstudied.Impactofincreasingsucroseconcentration(6to20%w/v)and different nitrogen sources on IAA production was investigated separately. Various nitrogenous chemicals were subsequently added to the tryptophan supplemented basal medium having the most suitable concentration of sucrose. Individual effect of each inorganicnitrogenouschemicalonIAAproductionwascheckedcolorimetrically. 2.3FractionationandQuantitationbyHPLC The ethyl acetate extract of the culture supernatant was analyzed by HPLC. HPLC chromatogramswereproducedby injecting20lofthefilteredextractontoa5mreverse phase column (Waters Associates Bondapak C18, 250mm x 4mm) in a Waters Associates liquidchromatographequippedwithanultravioletdetectorabsorbingat208nm.Thesolvent system used to separate IAA was water: acetonitrile [76:24 (v/v)], flow rate was 1 ml/min (Tien,etal.,1979).Retentiontimesforpeakswerecomparedtothoseofauthenticstandards (IAAandIAAequivalents)addedtothemediumandextractedbythesameproceduresused withbacterialcultures.Quantitationwasdonebycomparisonofpeakheights.
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OptimizationofIndole3aceticacid(IAA)productionbyAcetobacterdiazotrophicusL1isolatedfrom Sugarcane

2.4Plantresponsetoinoculation Theeffectof inoculationofA.diazotrophicusL1wasstudiedonmaizebecause itisoneof the economical agricultural crops. Before this bioassay, an endophytic colonization of this isolateinmaizewastested.Itwasfoundthatthisorganismcolonizedinlargenumberswithin vascularsystem. Seedsofmaize(Zeamays)weresurfacesterilizedwith95%ethanolfor5 minandwashedwithsterilewater.Theyweregrown inaplasticcontainer(10cm x7cm) 9 containingsterile soil. Attwodays,afterseedgermination2 mlof bacterialculture(~10 cfu/ml) was added to the soil, around each seedling. Plants were uprooted 30 days after sowing the seeds. Fresh weight measurements of roots were seemed to be affected by the laboratory environment. Environmental variables cause negligible variability to dry weight valuesofrootandshootbiomass(BashananddeBashan,2005).Therefore,onlydryweight ofrootsandshootswererecorded.TheresultswerepooledforonewayANOVAstest. 3.ResultsandDiscussion 3.1IsolationandscreeningforIAAproduction Based on the morphological, cultural and biochemical characteristics, the isolates were identified as Acetobacter diazotrophicus. The identification of the bacteria was done according to the characteristics reported earlier (Cavalcante and Dobereiner, 1988 Muthukumarasamy, et al., 1999) and also following Bergeys Manual (Holt, 1994). Production of bluegreen colored colonies on yeast extractethanolCaCO3 agar and dark brown colonies on potato sucrose agar are important cultural characteristics indicating their closeidentitywithA.diazotrophicus.ThirtyfivedifferentisolatesofA.diazotrophicuswere obtained from different varieties of sugarcane. All isolates were screened for the ability to produceIAA.Growthmediumof mostoftheisolatescontainedsubstantialamountofIAA. The isolates, L1, L14, L31, L32 and L36 produced more than 15 g/ml of IAA in LGI mediumsupplementedwithtryptophan(0.5g/l).Basalmediumwithouttryptophansupported veryslowgrowthandlowerproductionofIAA.Inthismedium,themaximumconcentration ofIAAwasachievedafter1112daysofincubationonshakerat200rpm(Figure1).

Figure1:ProductionofIAAindifferentculturalmediaby A.diazotrophicusL1.Medium1 LGImediumwithLtryptophan(0.5%w/v),Medium2LGImediumwithNH4Cl(0.1%

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w/v)andMedium3NitrogenfreeLGImedium.Theexperimentswereperformedin triplicatesandthebarsindicatestandarddeviations. 3.2OptimizationofIAAproduction IAA concentration was increased when the concentration of tryptophan in the medium was increasedfrom0.2to1.0g/l,remainedstaticupto1.2g/landthendeclinedwhenitwasfrom 1.2to1.6g/ltryptophanafter6daysofincubationonshakerat200rpm.Thus,Ltryptophan at a concentration of 1.2 g/l was best for IAA production whereas higher concentration of tryptophanexertedtheadverseeffectsonIAAproduction(Figure2).

Figure2:EffectofdifferentlevelsoftryptophanonIAAproductionby A.diazotrophicusL1. Theexperimentswereperformedintriplicatesandthebarsindicatestandarddeviations. Results indicated that Ltryptophan was more active for IAA production, though bacteria wereabletoproduceIAAinabsenceoftryptophan.Inpresenceoftryptophan,themicrobes release greater quantities of IAA and related compounds (Lee, et al., 2004). There was maximum IAA production at pH 6 by A. diazotrophicus L1 (Figure 3). High acidic and alkalinepHwasnotsuitableforIAAproduction.

Figure3:EffectofpHonIAAproductionby A.diazotrophicusL1.Theexperimentswere performedin triplicatesandthebarsindicatestandarddeviations. Among the carbon sources, sucrose is the best carbon source for the growth of A. diazotrophicus.Itisthemaincarbonsourcepresentinitsnaturalhabitat,thesugarcaneplant. Itcangrowinpresenceofupto30%(w/v)sucrose.IAAproductionwasstudiedatvarious
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levelsofsucroseanditwasfoundthatmaximumproductionoccurredat12%(w/v)sucrose (datanotshown).ItwasfoundthatIAAsynthesisrequiresdepletionofcarbonsourcefrom thegrowthmedium(Bastian,etal.,1998).Itisrelevanttoourobservation.Nitrogensourceis anothergrowth factoraffectingIAAproduction.Amongthetestednitrogensources,NH4Cl wasfoundtobethemostsuitablenitrogensourceforIAAproduction(Figure4).0.1%(w/v) NH4ClwastheoptimumforhighestIAAproduction.

Figure4:Effectofdifferentnitrogensources(eachat0.1%(w/v)level)onIAAproduction by A.diazotrophicusL1.ANH4Cl,BNaNO3,CKNO3 andD(NH4)2SO4. Theexperiments wereperformedintriplicatesandthebarsindicatestandarddeviations. ThebacteriumstartedtoproduceIAAatthebeginningofitsgrowthandreachedmaximum at starting of the stationary phase (Figure 5). Thus, the optimum conditions for maximum productionofIAAbyA.diazotrophicusL1werefoundtobetheLGImediumsupplemented withtryptophan(1.2g/l),sucrose(12%w/v),NH4Cl(0.1%w/v)andpH6at30Conshaker at200rpmindarkafter6daysofincubation.

Figure5:TimecourseofIAAproductionby A.diazotrophicusL1inoptimizedLGImedium. Theexperimentswereperformedintriplicatesandthebarsindicatestandarddeviations.

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TheconcentrationofIAAinculturebrothwasalsoanalyzedbyHPLC.Organismproduced gibberellic acid and indole 3propionic acid as the additional products (Figure 6). In this study, A. diazotrophicus L1 produced IAA (26.28 g/ml) which is higher than the reports madeearlier(Lee,etal.,2004Muthukumarasamy,etal.,2002).

Figure6:HPLCanalysisoftheethylacetateextractsoftheculturebrothinoculatedwith A. diazotrophicusL1 3.3Effectofinoculationonmaizegrowth Roots of plants showed a striking growth response when maize seedlings were inoculated with A. diazotrophicus L1. Roots were densely covered by root hairs. Very few were developed on uninoculated control plants. Both root hairs and the number of roots were denseronplantsgrownwiththisisolate.Alsorootsandroothairswerelongeroninoculated plants.Thebacterialinoculationsignificantlyincreasedrootandshootbiomassofmaizeafter 30 days of growth. The fresh roots of maize plant inoculated with the bacterium contained highamountofIAAthannormaluninoculatedmaizeroots(Table1). Table1:Effectofinoculationwith A.diazotrophicusL1onthegrowthofmaizeplant
Parameter a Rootbiomass(g) a Shootbiomass(g) IAAcontentinroots a (g/gfreshtissue)
a

Inoculated 0.490.08 1.360.2 9.811.76

Control 0.320.06 0.770.1 4.461.21

Eachvaluerepresentsmean ofreplicate(n=5)SD

The ability of A. diazotrophicus L1 to produce a high amount of IAA in culture might correlateforhighlevelofIAAinfreshroots.Thus,theendophyteenhancedtheIAAlevelin roots which is responsible for development of root hairs and the number of roots that are denser on plants grown with this isolate. Interactions between IAAproducing bacteria and plants lead to diverse outcomes on the plant side, varying from pathogenesis to phytostimulation(Spaepen,etal.,2007).Thus,the isolateA.diazotrophicusL1showedthe ability to produce higher amountof IAA that might have physiological implications in root developmentoftheplants.
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4.Acknowledgements Financial assistance tothe corresponding author from University Grants Commission, New Delhiisgratefullyacknowledged. 5.References 1. Bashan,Y.anddeBashan,L.E.(2005),freshweightmeasurementsofrootsprovide inaccurateestimatesoftheeffectsofplantgrowthpromotingbacteriaonrootgrowth: acriticalexamination.Soil.Biol.Biochem.,37:pp17951804. 2. Bastian, F., Cohen, A., Piccoli, P., Luna, V., Bottini, R., Baraldi, R. and Bottini, R. (1998),productionofIAAandgibberellinsA1andA3by Acetobacterdiazotrophicus and Herbaspirillum seropedicae in chemicallydefined culture media. Plant growth regulation,24(1):pp711. 3. Bianco,C.andDefez,R.(2009),Medicagotruncatulaimprovessalttolerancewhen nodulated by an indole3acetic acidoverproducing Sinorhizobium meliloti strain. J. Exp.Bot.,60(11):pp30973107. 4. Boddey,R.M.,Urquiaga,S.,Alves,B.J.R.and Reis,V.(2003),endophytic nitrogen fixationinsugarcane:presentknowledgeandfutureapplications.PlantSoil,252:pp 139149. 5. Boiero,L.,Perrig,D.,Masciarelli,O.etal.(2007), phytohormoneproductionbythree strains of Bradyrhizobium japonicum and possible physiological and technological implications.Appl.Microbiol.Biotechnol.,74:pp874880. 6. Cavalcante, V.A. and Dobereiner, J. (1988), a new acidtolerant nitrogenfixing bacteriumassociatedwithsugarcane.PlantSoil, 108:pp2331. 7. Glickmann,E.andDessaux,Y.(1995),acriticalexaminationoftheSpecificityofthe Salkowski reagent for indolic compounds produced by phytopathogenic Bacteria. Appl.Environ.Microbiol.,61(2):pp793796. 8. Holt, J.G. (1994), group 4 Gram negative aerobic/microaerophilic rods and cocci. th BergeysManualofDeterminativeBacteriology9 Ed.LippincottWilliamsWilkins, pp71126. 9. Khalid, A., Arshad, M. and Zahir, Z.A. (2004), screening plant growthpromoting rhizobacteria for improving growth and yield of wheat. J. Appl. Microbiol., 96: pp 473480. 10. Lee,S.,FloresEncarnacion,M.,ContrerasZentalla,M.,GarciaFlores,L.,Escamilla, J.E. and Kennedy, C. (2004), Indole3acetic acid biosynthesis is deficient in Gluconacetobacterdiazotrophicusstrainswithmutationsincytochromecbiogenesis genes.J.Bacteriol.,186(16):pp53845391. 11. Muthukumarasamy,R.,Revathi,G.,Seshadri,S.andLaxshminarasimhan,C.(2002), Gluconacetobacter diazotrophicus (Syn. Acetobacter diazotrophicus), a promising diazotrophicendophyteintropics.Curr.Science,83:pp137145.
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12. Muthukumarswamy,R.,Revathi,G.andLakshminarasimhan,C.(1999),influenceof nitrogen fertilization on the isolation of A. diazotrophicus and Herbaspirillum spp. fromIndiansugarcanevarieties.Biol.Fert.Soils,29:pp157164. 13. Ryan,R.P.,Germaine,K.,Franks,A.,Ryan,D.J.andDowling,D.N.(2008),bacterial endophytes:recentdevelopmentsandapplications.FEMS.Microbiol.Lett,278:pp1 9. 14. Spaepen, S., Vanderleyden, J. and Remans, R. (2007), indole3acetic acid in microbialandmicroorganismplantsignaling.FEMS.Microbiol.Rev.,pp124. 15. Tien,T.M.,Gaskins,H.andHubbell,D.H.(1979),plantgrowthsubstancesproduced byAzospirillumbrasilenseandtheireffectonthegrowthofPearlMillet(Pennisetum americanumL.).Appl.Environ.Microbiol,37(5):pp10161024.

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