Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Isolation

and

identification

by

16S

rRNA

sequence

analysis

of

plant

growth-promoting

azospirilla

from

the

rhizosphere

of

wheat

Khadija

Ayyaz,

Ahmad

Zaheer,

Ghulam

Rasul,

Muhammad

Sajjad

Mirza

NationalInstituteforBiotechnologyandGeneticEngineering(NIBGE),JhangRoad,Faisalabad,Pakistan

a

r

t

i

c

l

e

i

n

f

o

Received23July2014 Accepted24November2015 Availableonline19April2016 AssociateEditor:IêdadeCarvalho Mendes

Keywords:

Azospirillumbrasilense Azospirillumzeae BOX-PCR

Indole-3-aceticacid(IAA) production

Phosphatesolubilization

a

b

s

t

r

a

c

t

The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizingstrainsofAzospirillumfromwheattobeusedasinoculantsforplant growthpromotion. FiveAzospirillum strainswereisolatedfromtherhizosphereof field-grownwheat(TriticumaestivumL.),anditwasconfirmedbyBOX-polymerasechainreaction (PCR)thattheisolatesweredifferentandnotre-isolatesofthesamestrain.Sequence anal-ysisofthePCR-amplified16SrRNAgeneindicatedthatfourisolatesshowedmaximum similaritytoAzospirillumbrasilenseandoneisolateshowedmaximumsimilarityto Azospir-illumzeae.ThisisthefirstreportindicatingthepresenceofanA.zeaelikeisolateinthe wheatrhizosphereinPakistan.Thebacterialisolateswerecharacterizedfortheirplant growth-promotingtraits,phosphatesolubilization,andindole-3-aceticacid(IAA) produc-tion.Noneoftheisolatesshowedphosphatesolubilizationactivityinthecommonlyused Pikovskayamedium.However,allstrains(exceptAzoK4)exhibitedabilitytosolubilize tri-calciumphosphate(TCP)inmodifiedPikovskayamediuminwhichsucrosewasreplaced byNa-malate,aswellasinTCP-supplementedLuria-Bertani(LB)medium.Organicacids, suchasacetic,citric,lactic,malic,andsuccinicacids,weredetectedinculturesupernatants ofthetestedAzospirillumstrains.AllstrainsexhibitedabilitytoproduceIAAinthegrowth medium,exceptAzospirillumsp.AzoK1.Amongthestrainstested,themaximumIAA pro-duction(30.49±1.04mgL−1)andphosphatesolubilization(105.50±4.93mgL−1)wereshown byapurecultureofAzospirillumsp.AzoK2.Inpotexperiments,single-straininoculaof Azospirillumsp.AzoK1andAzoK2improvedwheatplantgrowth.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:[email protected](M.S.Mirza).

http://dx.doi.org/10.1016/j.bjm.2015.11.035

1517-8382/©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Plant growth-promoting rhizobacteria (PGPR) isolated from therhizosphereorrootsurfacescompriseanactive compo-nentofbiofertilizers.Severaldirectorindirectmechanisms areadoptedbythesemicroorganismstoenhancethe plant growth,includingbiologicalnitrogenfixation,solubilizationof insolubleminerals,productionofphytohormones,plantrelief fromstress,andprotectionagainstpathogens.1_AmongPGPR,

strainsofAzospirillumaremostextensivelystudiedfortheir beneficialeffectsonthegrowthandyieldofmany agronom-icallyimportantcrops.2_{Ithasbeenreportedthatinoculation}

withthesediazotrophicbacteriareducestheuseofchemical nitrogenfertilizers.3_{Indole-3-aceticacid(IAA)productionby}

Azospirillumspp.hasbeenproposedtoplayamajorroleinroot proliferationandconsequent plantgrowth promotion.4 _IAA

isproducedbyAzospirillumduringallgrowthstages,andits synthesiscontinueswellafterthestationaryphase.5_This

fea-turemakesthebacteriaespeciallyqualifiedforplantgrowth promotionwhentheeffectlastsweeksormonthsafter inoc-ulation.TheamountofIAAproducedvaries greatlyamong different species and is also influenced by culture condi-tions,thegrowthstage,andavailabilityofasubstrate(s).6 _It

wasreportedthatwheatinoculationwithAzospirillumstrains resultedinayieldincreaseof31%.7_{Inoculationwith}

Azospir-illumalsoincreasestheuptakeofmineralnutrientssuchasN, P,K,andCabywheat.8

Soilsusuallycontainsufficientreservesofphosphorusto supportplantgrowthifPisinavailableforms.Unfortunately, phosphorusmaybepresentinwater-insolubleformswhen it is not readily accessible to plant roots. Therefore, there is a dire necessity to make phosphorus more available to plants.9_{Phosphorussolubilizationactivityhasbeenreported}

formanygeneraofPGPR,includingAzospirillum.PGPRproduce organicacids,whichconvertinsolublephosphorusinto avail-ableforms,i.e.,primaryandsecondaryorthophosphates.10,11

Theorganic acids produced by PGPR includemalic, acetic, citric, oxalic, lactic, formic, gluconic, and 2-keto-gluconic acids.12,13 _{Inpurebacterialcultures,organicacidproduction}

hasbeendemonstrateduponutilizationofvarioussugarsand sugaralcohols,suchassucrose,glucose,fructose,d-xylose, andmannitol.10_{Phosphate-solubilizingmicrobesare}

consid-eredimportantmembers ofPGPR, and theirapplication as biofertilizershasbeenshowntoimprovegrowthofcerealsand othercrops.13

In the present study, five Azospirillum strains were iso-lated from the rhizosphere of wheat, followed by strain

identificationusingDNA-basedtechniques[BOX-polymerase chain reaction (PCR) and analysis of 16S rRNA gene sequences]. AfterconfirmationofIAAproduction,P solubi-lizationandamplificationofthenifHgene,aseffectsof inocu-lationwithselectedAzospirillumstrainswasstudiedonwheat plants.

Materials

and

methods

Studysite

Thisstudyonisolationofplantgrowth-promotingazospirilla continuedfor16months(fromJanuary2012toApril2013)at theNationalInstituteforBiotechnologyandGenetic Engineer-ing (NIBGE),Faisalabad,Pakistan.TheNIBGEislocatedina subtropicalareaofPakistan(31◦2336.2Nand73◦0139.9E)at anelevationof184m.

IsolationofAzospirillumstrainsfromtherhizosphereof wheat

IsolationofAzospirillum-likestrainsfromtherhizosphereof wheat (Triticum aestivum L.) was carried out by an enrich-mentculturetechniqueusingsemi-solid,N-freemalate(NFM) medium.14 _{Twenty-fiverhizosphericsoil sampleswere}

col-lected from field-grownplantsatthe tilleringstage, i.e.,40 daysaftersowing.Soilsuspensionswerepreparedbyadding 1g of soil to 10mL of water, and 100␮L was transferred to 1.5mLmicrocentrifuge tubescontaining 0.9mL of semi-solidNFMmedium.TheinoculatedmicrotubesofNFMwere incubated for24–72h at 30◦C. Thegrowth of Azospirillum-likecellsshowingtypicalspiralmotilitywasdetectedunder a light microscope at 1000× magnification (Akon, Japan). Six consecutive transfers from inoculated to fresh NFM microtubesweremadetoobtainanenrichedculture.Serial dilutions(10×)oftheenrichedcultureweremade,and 100-␮Laliquotsof10−3–10−5dilutionswerespreadonNFMagar platesandincubatedfor4–6daysat30◦C.Bacterialcolonies were pickedfromthe plates,transferredtoNFM semi-solid medium,andincubatedfor24–48hat30◦C.Bacterialgrowth obtainedinNFMmediumwasstreakedonLuria-Bertani(LB) agar plates,15 _{and the plates were incubated at 30}◦_{C for}

24–72htoobtainsinglecolonies.Afterrepeatedstreakingand transferofsinglebacterialcolonies,fivepureisolateswere obtained.Forlong-termstorage,bacterialgrowthobtainedin LB broth waskept inthe mediumwith 15–20%glycerolat −80◦_C.

Table1–PCRprimersusedforamplificationof16SrRNA,nifH,andBOX-PCRinthisstudy.

Nameofprimer Primersequence References

BOXAIR 5_{-CTACGGCAAGGCGACGCTGACG-3} ₂₉ PAforward (16SrRNA) 5_{-AGACTTTGATCCTGCTCAG-3} ₄₅ PHreverse (16SrRNA) 5-AAGGAGGTGATCCAGCCGCA-3 45

PolF(nifH) 5-TGCGAYCCSAARGCBGACTC-3 46

DifferentiationofAzospirillumstrainsbyBOX-PCR

All chemicals, except the primers usedfor PCR, were pur-chasedfromFermentas,Germany.EachPCRreactionmixture (25␮L)contained0.15␮Lof5U␮L−1_{TaqDNApolymerase,5␮L}

of10×Taq polymerasebuffer, 5␮Lof25mMMgCl2,2␮Lof

5mMdeoxynucleotidetriphosphates(dNTPs),1.5␮Lof10␮M BOXAIRprimer(Table1),and25ngofDNAtemplateextracted byCTAB method.16 _{Thereactionmixturewas incubated in}

athermocycler(Eppendorf,Germany)at94◦Cfor5min, fol-lowedby30cyclesof94◦Cfor1min,42◦Cfor1min,and72◦C for1.5min,andfinalextensionat72◦Cfor10min.Amplified PCRproductsofdifferentsizeswereseparatedon1%agarose gelelectrophoresisandstainedwithethidiumbromide.Gels were viewedunderultraviolet (UV)lightand photographed using a gel documentation system (CN-1000/26MX, Vilber Lourmat,France).

Amplificationof16SrRNAandnifHgenes

Foramplificationofthe16SrRNAandnifHgenes,areaction mixtureof25␮L,containing15ngoftemplateDNA,0.12␮Lof 5U␮L−1 _{TaqDNApolymerase,2.5␮Lof10×Taqpolymerase}

buffer,1␮Lof5mMdNTPs,2␮Lof25mMMgCl2,and1␮Lof

each10␮Mforwardandreverseprimers,wasprepared.The primer set PH/PA was used foramplification of 16S rDNA, and theprimer set PolF/PolRwasusedfor amplificationof

nifH(Table1).TheprogramusedforPCRamplificationof16S

rDNAincludedfollowingsteps:initialdenaturationat94◦C for5min,followed by30 cyclesat94◦C for1min,54◦Cfor 1min,and72◦Cfor2min,andfinalextensionfor10minat 72◦C.The same thermalconditions were usedfor amplifi-cationof nifH,except that the annealing temperature was 48◦C.

Identificationofisolatesby16SrRNAsequenceanalysis ThePCR productsofthe 16S rRNAgene were eluted from an agarose gel using the Gene JETTM _{Extraction Kit}

(Fer-mentas, Germany), and the purified PCR products were sequenced commercially by Macrogen, South Korea. The sequencedatawereassembledandanalyzedusingthe Laser-gene sequence analysis software package (DNA Star, Inc., Madison,WI,USA). Sequence similaritysearches usingthe BasicLocalAlignmentSearch Tool(BLAST)wereperformed by comparing the sequences obtained to other microbial sequencesintheNationalCenterforBiotechnology Informa-tion (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST/). For construction of a phylogenetic tree, the 16S rRNA gene sequences of type strains and other strains closely related to our five isolates were obtained from the NCBI database. Multiple sequence alignments were performed by the ClustalX program.17 _{Phylogenetic trees were}

con-structedbythe maximum-likelihood methodbased onthe Jukes–Cantormodel18 _{usingMEGAversion6.}19 _Allpositions

containing gaps and missing data were eliminated. There were a total of 816-base pair (bp) positions in the final dataset.

QuantificationofIAAproducedbyAzospirillumstrains For detection and quantification of IAA produced by the bacterialisolates,culturesweregrowninErlenmeyerflasks containing 40mL of LB medium supplemented with l-tryptophan(100mgL−1)at30◦Cfor5days.Thesupernatants ofthecultureswereobtainedbycentrifugationof stationary-phaseculturesat12,000×gfor15min,andthealkaline(pH 8.3) supernatants were adjusted to pH 2.8 with 1N HCl. Auxins were extracted from the acidified culture medium with equalvolumes of ethyl acetate,20 _{evaporated to}

dry-ness,andre-suspendedin1.5mLofmethanol.Thesamples werepassedthrough0.2␮mfilters(OrangeScientificGyroDisc CA-PC, Belgium) and analyzed by high-performance liquid chromatography (HPLC, Varian Prostar), with the system equippedwithaUVdetectorandaC-18column.Amixture ofmethanol:aceticacid:water(30:1:70,v/v/v)wasusedasa mobilephaseatarateof0.6mLmin−1.21_{PureIAAwasusedas}

astandard.TheIAAinthesampleswasidentifiedand quanti-fiedbycomparingtheretentiontimeandpeakareawiththose ofthestandardusingcomputersoftware(Varian Chromatog-raphySystems,WalnutCreek,CA,USA).

Quantificationofsolublephosphorusandorganicacidsin thegrowthmedium

For the estimation of soluble phosphorus, bacterial cul-tures were grown in threedifferent media, i.e., Pikovskaya medium,22 _{modified Pikovskaya medium, and LB medium}

supplemented with tricalcium phosphate (TCP, 5gL−1). In modified Pikovskaya medium, sucrose was replaced with sodium malate(10gL−1), which isapreferred C-sourcefor azospirilla.14 _{Thebacterial cultureswere grown in conical}

flasks containing 40mL of the media at 30◦C for 7 days. Cell-free supernatants were obtained by centrifugation of stationary-phaseculturesat12,000×gfor15min.The super-natantswereanalyzedtoestimatesolublephosphorusbythe molybdatebluecolormethod.23

Organicacidswereextractedfromcell-freeculturemedia by mixing the supernatant with an equalvolume ofethyl acetate.Theethylacetatephasewas separatedand evapo-ratedtodryness.Organicacidswerere-suspendedin1.5mL ofmethanolandfilteredthrough0.2␮mfilters(Orange Scien-tificGyroDiscCA-PC,Belgium).Thesampleswereanalyzedon aPerkinElmer(PE)series200HPLCsystemwith20␮L auto-sampler PENELSON900 seriesinterface, a PENELSON600 serieslink,andPENCI900Network Chromatography inter-face, followed by detectionusing a diode-arraydetectorat 210nmandUVspectra(190–400nm).ACationHMicro-Guard pre-column,followedbyanAminexHPX-87Hanalytical col-umn,were usedforseparation.Sulfuricacid(0.002molL−1) wasusedasamobilephasewithaflowrateof0.6mLmin−1. Citric acid, malic acid, acetic acid, succinic acid, gluconic acid, lactic acid, and oxalic acid (100ppm solutions) were used as standards. The peak areas and retention timesof the samples were compared with those of the standards forquantificationoforganicacidsproducedbythebacterial strains.

InoculationofwheatplantswithAzospirillumstrains ThreeAzospirillumstrains,AzoK1,AzoK2,andWB3,wereused as inoculants for wheat plants (variety Sehar-2006) grown in non-sterile soil pots (0.02m3_{). Thepots were kept in a}

net houseunder naturallightand temperature conditions. Azospirillum strain WB3 (previously isolated from the rhi-zosphere of wheat)24_{; was used as a positive control. The}

potswerefilled with10kgofsoil(about27cmdeep)ofthe Hafizabadseries(Aridisol-coarse-loamy,mixed,hyperthermic Ustalfic,HaplargidintheUSDepartmentofAgriculture clas-sification).Thesoilwas collectedfrom the top30cm ofan experimentalfieldoftheNIBGE.ThepHofthisclayloamwas 8.3,totalNwas0.61gkg−1,organicmatterwas6.2gkg−1,and availablePwas 68mgkg−1.Tenhealthy seedsofequalsize weresowninfivedifferentplacesatequaldistancesineach pot.Eachtreatmentwasreplicatedfivetimes.Anitrogen fer-tilizerat80%oftherecommendeddose(1.2gofurea/pot)was appliedtothe uninoculatedcontrolaswell astoall inocu-latedtreatmentsintwoequalsplitdoses,i.e.,atthetillering(30 daysaftersowing)andflowering(75daysaftersowing)stages. Beforesowing,80%ofthe recommendeddoseofP(0.6gof diammoniumphosphate/pot)wasappliedtothesoil.Bacterial culturesforplantinoculationweregrownin100mLconical flaskscontaining40mLofLBmediumat30◦Cfor24h.Cell pel-letswereobtainedbycentrifugationat12,000×gfor10min, andthepelletswerere-suspendedin40mLofsterilizedwater foruseasinocula.Onemilliliterofinoculum(1×108_cells)was

addedtoeachplantafter1weekofgermination,and1mL ofsterilizedwaterwasappliedtotheuninoculatedcontrols. Theplantswereharvestedatmaturity(180daysafter sow-ing).Plantdata(dryweightandgrainweight)for10plantsfrom eachtreatmentweremeasuredafterdryingtheplantmaterial at65◦Cfor2days.

Estimationofphosphorusandnitrogencontentsinplant material

Forestimation ofnitrogencontentsinshootsandgrains, a methoddescribedbyVanSchouwenbergandWalinge25_was

followed.Measurementofphosphoruscontentsinshootsand grainswascarriedoutbytheammoniumvanadate–molybdate method.26

Statisticalanalysis

Effectsofthebacterialisolatesongrowthparametersofwheat weredeterminedbyacompletelyrandomizeddesignanalysis ofvariance(ANOVA)usingtheStatistix8.1software.Mean val-uesandstandarderrorswerealsocalculated.Themeanswere comparedbyFisher’sleastsignificantdifferencetestat˛=0.05 forallparameters.

Results

Five Azospirillum-like strains were isolated from the rhizo-sphere of wheat. All the isolates formed pinkish colonies on LB agar plates. Thecells were rod-shaped and showed spiral motility typical of azospirilla. To further strengthen

1

1000bp 500bp

250bp 360bp (Partial_{nifH gene)}

2 3 4 5 6 7

Fig.1–PCR-amplificationofpartialnifHfromAzospirillum

strainsisolatedfromwheatrhizosphere.Lane1:1kbladder (Fermentas,Germany);Lane2:isolateAzoK1;Lane3: isolateAzoK2;Lane4:isolateAzoK3;Lane5:isolateAzoK4; Lane6:isolateAzoK5andLane6:negativecontrol.

this observation based on morphological characteristics, a partialsequenceofthenitrogenase-encodinggenenifHwas amplified, whichispresent inallnitrogen fixers,including azospirilla. A DNA band ofthe expected size (360bp) was observedinallstrains(Fig.1).

TheBOX-PCRanalysisconfirmedthatthebacterialisolates weredifferentstrainsandnotre-isolatesofthesamestrain. All fivestrainsshowedunique bandingpatterns ofthePCR products(Fig.2).ThebandingpatternofisolateAzoK1showed thatitwastotallydifferentfromtheotherstrains.Thesizes ofthebandswerebetween250and1500bp.OnestrongDNA

2000bp 1500bp 1000bp 750bp 500bp 250bp 2000bp 1500bp 1000bp 750bp 500bp 250bp 1 2 3 4 5 6 7

Fig.2–DifferentiationofAzospirillumstrainsisolatedfrom wheatrhizospherebyBOX-PCR.Lane1:1kbladder (Fermentas,Germany);Lane2:isolateAzoK1;Lane3: isolateAzoK2;Lane4:isolateAzoK3;Lane5:isolateAzoK4; Lane6:isolateAzoK5andLane7:1kbladder(Fermentas, Germany).

Azospirillum sp. AzoK2 (HE977585) Azospirillum sp. AzoK5 (HE977588) Azospirillum sp. AzoK3 (HE977586)

Azospirillum brasilense Az24 (KC920687) Azospirillum brasilense LH-S4 (GU737689)

Azospirillum sp. AzoK4 (HE977587)

Azospirillum brasilense Gr29 (FR667883) Azospirillum brasilense ATCC 29145T _(AY324110)

Azospirillum doebereinerae GSF71T _(AJ238567)

Azospirillum thiophilum BV-ST _(EU678791)

Azospirillum zeae Gr35 (FR667885) Azospirillum zeae Gr61 (FR667897)

Azospirillum sp. AzoK1 (HE977584)

Azospirillum zeae N7T _(DQ682470)

Escherichia coli ATCC 11775T _(X80725)

74 94 54 76 100 70 94

Fig.3–Phylogenetictreebasedon16SrRNAgene sequences.Thetreewasconstructedbymaximum likelihoodmethod.Bootstrapvaluesover50%(basedon 100replications)areshownateachnode.Accession numbersaregiveninparentheses.Theisolatesobtainedin thepresentstudyaregiveninboldletters.

band(approximately300bp)waspresentinfourstrainsbut absentfromAzoK1.Manyminorbandsofdifferentsizeswere alsoamplifiedfromthefourstrains.

Azospirillum strain AzoK1 showed maximum 16S rRNA sequencehomology(99%)toAzospirillumzeae,andtheother fourisolatessharedhighersequencehomologywith Azospiril-lumbrasilensestrains.Theconstructedphylogenetictreebased onthe16SrRNAgenesequencesshowedthatisolateAzoK1 sharedaclusterwithA.zeae(Fig.3).Threeisolates,AzoK2, AzoK3,andAzoK5,formedaclusterwithA.brasilensestrains, andisolateAzoK4fellinaseparateclusterwithinthemain groupofA.brasilensestrains.

IAAproductionwasestimatedinLBbrothsupplemented withl-tryptophan(Table2).Themaximum amount ofIAA wasproducedbyAzospirillumsp.AzoK2,followedbystrains AzoK3andAzoK4.OnlyaminoramountofIAAwasdetected inthecultureofAzospirillumsp.AzoK1.Thequantityof phos-phate solubilized by the bacterial strains was determined in three different growth media, i.e., Pikovskaya medium, modified Pikovskaya medium, and TCP-supplemented LB medium. In Pikovskaya medium, phosphate solubilization was not detected for any strain after 7 days of

incuba-tion(Table2).However,phosphate-solubilizingactivityofthe

isolateswas detectedin modified Pikovskaya mediumand TCP-supplementedLB.InmodifiedPikovskaya medium,the maximumamountofphosphatewassolubilizedby

Azospir-illum sp. AzoK2.Phosphatesolubilizationwas notdetected inmodifiedPikovskayamediumforAzospirillumsp.WB3and AzoK4.All theAzospirillumstrainssolubilizedphosphatein TCP-supplementedLB,exceptAzospirillumsp.AzoK4(Table2). The maximum amount of phosphate was solubilized by Azospirillumsp.AzoK2.

Organic acids weredetectedincell-freesupernatantsof modifiedPikovskayamediumandTCP-supplementedLBafter 7daysofincubation.Acetic,citric,lactic,malic,andsuccinic acidsweredetectedinalmostallAzospirillumcultures.In mod-ifiedPikovskayamedium,aceticandmalicacidsweredetected inhigheramountsinthecell-freesupernatantsofall Azospir-illumstrains,butrelativelyminoramountsoflacticacidwere present in this medium(Fig. 4A). In TCP-supplemented LB medium,aceticacidwasproducedbyallstrainsinsignificant amounts(Fig.4B).Themaximumamountsofcitricacidwere producedbythreeisolates,exceptAzoK1andAzoK4,inthis medium.Asignificantamountofsuccinicacidwasonly pro-ducedbyAzoK2,andmalicacidwasoneofthemajoracids detectedinthesupernatantsoftwostrains,WB3andAzoK5. Themaximumplantshootdry weight(8.95±0.20g) and grainweight(6.17±0.11g)wererecordedinthestrain AzoK1-inoculatedtreatment,andtheincreasesintheplantshootand grainweightsovertheuninoculatedcontrolwere19and15%, respectively(Table3).Thenitrogenandphosphoruscontents inthegrainsandshootsoftheinoculatedplantswerenot sig-nificantlydifferentfromthoseofthecontrolplants(Table4).

Discussion

Inthepresentstudy,therhizospheresoiloffield-grownwheat plants wascollected and usedforisolation of Azospirillum-like strains, which are known to improve the growth and productivity ofmanyagriculturalcropspecies.27_Azospirilla

havebeenisolatedfromrootsofnumerouswildandcultivated grasses,cereals,andlegumesandfromtropical,subtropical, andtemperatesoils.28IntheN-freegrowthmedium,whichis selectiveforAzospirillum,14_{alltheisolatesshowedexcellent}

growth andcharacteristicspiralmotility.OnLBagarplates, theyformedpinkish,wrinkledcolonies.Furthermore,apartial nifHsequencewassuccessfullyamplifiedfromalltheisolates, indicatingthat theyare potentialnitrogenfixers,similarto azospirilla. Thecell morphologyandcolonycolorofall the Azospirillum-likeisolatesobtainedinthepresentstudywere similar.

BOX-PCR polymorphism patterns have been effec-tively used for differentiation of bacterial strains.29 _All

the Azospirillum isolates included in this study exhib-ited unique, strain-specific banding patterns obtained by BOX-PCR. Azospirillum strain AzoK1 showed a BOX-PCR bandingpatternsignificantlydifferentfromthoseoftheother strains. OneDNAband ofabout 300bpwasamplifiedfrom fourstrainsbutwasabsentfromtheAzospirillumstrainAzoK1 profile,indicatingthatthelatterstrainbelongstoadifferent grouporspecies.

Onthebasisofthesequenceanalysisofthe16SrRNAgene, isolatesAzoK2,AzoK3,AzoK4,andAzoK5were96–99%similar toA.brasilensestrains,andthefifthstrain,AzoK1,showedhigh sequencehomology(99%)withA.zeae.IsolateAzoK1shareda

Table2–Phosphatesolubilizationandindoleaceticacid(IAA)productionbyAzospirillumstrains(Mean±SD).

Nameoftheisolates Indoleaceticacid (mgL−1)with tryptophan SolubilizedP (mgL−1)in Pikovskayamedium SolubilizedP (mgL−1)inmodified Pikovskayamedium SolubilizedP (mgL−1)in TCP-supplemented LBmedium Azospirillumsp.AzoK1 0.84±0.75 ND 52.40±3.42 62.10±1.65 Azospirillumsp.AzoK2 30.49±1.04 ND 72.00±2.94 105.50±4.93 Azospirillumsp.AzoK3 24.35±0.91 ND 26.55±2.01 44.60±2.91 Azospirillumsp.AzoK4 24.33±3.03 ND ND ND Azospirillumsp.AzoK5 19.15±1.34 ND 39.45±3.24 60.50±2.16 Azospirillumsp.WB3 14.15±1.48 ND ND 43.65±2.15

ND,notdetected;SD,standarddeviation.

clusterwiththeA.zeaetypestrain,andtheotherfourisolates formedaclusterwithA.brasilensestrains.

Ourfindingsrevealedthatthewheatrhizosphereis colo-nizedbyA.brasilense-aswellasA.zeae-relatedisolates.This isthefirstreportindicating thepresenceofanA.zeae-like

isolateinthewheatrhizosphereinPakistan.TheA.zeaetype strainwasoriginallyisolatedfromrootsofmaizeinCanada.30

Originally,thisisolatewasidentifiedandreportedas

Azospir-illum lipoferum31 _{but was later re-identified on the basisof}

a polyphasictaxonomic approach,including morphological characterization, Biolog analysis, DNA–DNA hybridization, and16SrRNA,cpn60,andnifHsequenceanalysis.30_Venieraki

etal.32_{isolatedthreeAzospirillumstrainsfromtherhizosphere}

ofwheatgrowninGreeceandshowedthattheisolates recov-eredwereA.zeaebyphylogeneticanalysisbasedon16SrRNA genesequences. Acetic acid mmolL –1 mmolL –1

WB3 AzoK1 AzoK2 AzoK3 AzoK4 AzoK5

WB3 AzoK1 AzoK2 AzoK3 AzoK4 AzoK5 Citric acid Succinic acid Malic acid Lactic acid

A

B

Fig.4–ProductionoforganicacidsbybacterialisolatesgrowninmodifiedPikovskayamedium(A)andTCP-supplemented LB(B).AzospirillumstrainsWB3,AzoK1,AzoK2,AzoK3,AzoK4andAzoK5wereused.

Table3–Effectofbacterialinoculationongrowthofwheatplantsinthesoilpotexperiment.

S.No. Nameoftreatments Shootdryweight(gplant−1)a _{Grainweight(gplant}−1₎a

1 Azospirillumsp.AzoK1 8.95A 6.17A

2 Azospirillumsp.AzoK2 8.89A 6.02B

3 Azospirillumsp.WB3 8.22B 5.97B

4 Non-inoculatedcontrol 7.52C 5.35C

LSD(5%) 0.37 0.13

LSD,leastsignificantdifference.

a _{Tenplantsfromeachtreatmentwereusedfortheanalysis.}

AlltheAzospirillumstrainstestedinthepresentstudyfailed togrowandsolubilize Pinthe commonlyusedPikovskaya medium.Ithasbeenreportedthatazospirillacannotutilize disaccharides,suchassucrose,whichisaddedtoPikovskaya mediumasasoleC-source.33,34_{Asaresult,theAzospirillum}

strainsdidnotsolubilizehardlysolublephosphorus,suchas TCP.Azospirilla use organic acids, suchas malic,succinic, a-ketoglutaric,gluconic, andlactic acids,astheir preferred carbonsources.33 Therefore,Pikovskayamediumwas modi-fiedbyreplacingsucrosewithNa-malateandusedtostudyP solubilizationbytheAzospirillumstrains.Thetested Azospir-illumstrainsproducedorganicacidsinthegrowthmedium. Acetic acid was produced by all strains, including AzoK4, butthelattershowednoP-solubilizingactivity.Averysmall amountofmalicacidwasdetectedinallculturesupernatants comparedtothe control,showingthat mostofthe malate addedtothemodifiedPikovskayamediumwasconsumedby thebacteriaduringincubation.

Phosphate-solubilizingactivityoftheAzospirillumstrains wasalsostudiedinTCP-supplementedLB medium.Allthe strainssolubilizedphosphate,exceptAzospirillumsp.AzoK4. Theamount ofsolublephosphate, determined inthe TCP-supplemented LB medium, was higher compared to the modified Pikovskaya medium. Azospirillum sp. AzoK2 pro-ducedthemaximumamountofsolublephosphorus,followed byAzospirillumsp.AzoK1.Azospirillumsp.AzoK4failedtoshow phosphate solubilization and produced only acetic acid in theTCP-supplementedLBmedium.Inthisgrowthmedium, organic acids, e.g., acetic,citric, malic, lactic, and succinic acids, were produced by the other strains tested in the presentstudy,butgluconicand␣-ketoglutaricacidswerenot detected.GoebelandKrieg35 _{showedthatgluconicacidwas}

notformed during growth of A. lipoferum and A. brasilense strainsonfructoseandwasonlydetectedinanA.lipoferum

cultureduringgrowthonglucose.Inthesamestudy,itwas reportedthatA.brasilenseshowedonlyslightuptakeofglucose andextremelyslowgrowthonthiscarbonsource.Rodriguez etal.10 _{reportedthatAzospirillumstrainscouldproduce}

glu-conic acid in vitro when grownon fructose amended with glucose asaninducer ofgluconic acidproductionandalso showedinvitrophosphate-solubilizingcapabilities.Thishas suggested thatphosphate solubilizationbythese strainsis mediatedbyglucoseorgluconicacidmetabolism.Since sol-ubilizationofphosphateprecededthedetectionofgluconic acid in the medium, it ispossible that even low levels of theacid(belowtheHPLCdetectionlimit)startedtodissolve sparingly solublephosphate. Alternatively, consumptionof gluconic acidbygrowing cellscould havetakenplace. Fur-thermore,ithasbeenreportedthatAzospirillumhalopraeferens, abacteriumthatdoesnotuseglucoseandconsequentlydoes notproduceacid,cansolubilizeinsolubleinorganicphosphate invitrobyanunknownmechanism.36

Bacterialisolatesobtainedfromtherhizosphereofvarious plantshavebeenshowntoproduceIAAinpureculture.37_In

thepresentstudy,allthebacterialstrainsproducedsignificant amounts of IAA in the culture medium, except Azospiril-lumsp.AzoK1.Undernaturalconditions,plantrootsexcrete organic compounds, including l-tryptophan, which can be utilizedbyrhizobacteriaforIAAbiosynthesis.6_Perrigetal.38

reportedIAAproductionrangingfrom2.9to10.8mgL−1bytwo agronomicallyimportantAzospirillumstrains.Fatimaetal.39

also detectedIAA inthe range from 19.4 to30.2mgL−1 in A. brasilensecultures. Venierakietal.40 _{isolated twoA.zeae}

strainsfromtherhizosphereofwheatandfoundthatboth pro-ducedIAA(29.8and194.8mgL−1,respectively).Inthepresent study,theamountofIAAproducedbyAzospirillumsp.AzoK1 in tryptophan-amended LB was 0.84mgL−1, which is very lowcomparedtopreviousreports.ThehigherlevelsofIAA

Table4–EffectofAzospirilluminoculationonthenitrogenandphosphoruscontentsofwheatplants(shootandgrain)in

thesoilpotexperiment(mean±SD).

Treatments Nitrogencontents(gkg−1)a _{Phosphoruscontents(gkg}−1₎a

Shoot Grain Shoot Grain

1.Azospirillumsp.AzoK1 4.46±0.40 23.33±1.00 0.59±0.02 3.16±0.20

2.Azospirillumsp.AzoK2 4.45±0.30 23.34±1.00 0.60±0.01 3.17±0.07

3.Azospirillumsp.WB3 4.40±0.70 23.30±1.50 0.59±0.01 3.16±0.04

4.Non-inoculatedcontrol 4.44±0.50 23.31±1.00 0.59±0.03 3.16±0.03 SD,standarddeviation.

productionreportedbyVenierakietal.40_{couldbeduetoa}

dif-ferentIAAdetectionmethod(spectrophotometryratherthan HPLC)ortothefactthatadifferentgrowthmedium(NFb)was usedforbacterialgrowth.Azospirillumisarhizospheric bac-teriumthathasbeenreportedtoincreasetheyieldofwheat plantsbyproducing phytohormonesandgrowth regulators andbyprovidingsaltstressrelief.7 _{Basedonthestudieson}

maizeandwheat,Hungriaetal.41 _{proposedthattheuseof}

inoculantscontainingAzospirillummighthelpreachthegoalof reducingtheuseofchemicalfertilizersglobally.Inthepresent study,alltheinoculatedstrainsshowedapositiveeffecton plantgrowthcomparedtotheuninoculatedcontrol.The max-imumincreasesinshootdryweight(19%)andgrainweight (15%)over the uninoculated control were recorded for the plantsinoculated withAzospirillumsp.AzoK1.Thenitrogen andphosphoruscontentsofthegrainsandshootsofthe inoc-ulatedplantswerenotsignificantlydifferentfromthoseofthe controlplants,suggestingthatthegrowthpromotionmight havebeen mainlyduetophytohormoneproductionbythe inoculatedstrains.Prinsenetal.42_{reportedthatinoculationof}

wheatwithAzospirillumresultedinanincreaseofseedyield by7.4–10.31%infieldtrialswherenoNfertilizationhadbeen applied.Díaz-ZoritaandFernández-Canigia43 demonstrated thataliquidformulationofA.brasilenseincreasedthe num-berofharvestedgrainsby6.1%andthegrainyieldofwheat by260kgha−1(8.0%).InoculationwithAzospirillumhasbeen foundtoaffectearlyplantandrootdevelopment,shootand rootdryweight,grainyield,andthenitrogenuptakeefficiency ofplants.44

Inthepresentstudy,alltheisolatesexhibitedplant growth-promoting traits in pure culture and also improved plant growthinpotexperiments.Therefore,thesestrainsqualifyas potentialcandidatestobeusedforproductionofbiofertilizers inPakistanandshouldbefurtherevaluatedasinoculantsin fieldtrials.

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