Tricalcium phosphate solubilization and nitrogen fixation by newly isolated Aneurinibacillus aneurinilyticus CKMV1 from rhizosphere of Valeriana jatamansi and its growth promotional effect

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

Environmental

Microbiology

Tricalcium

phosphate

solubilization

and

nitrogen

ﬁxation

by

newly

isolated

Aneurinibacillus

aneurinilyticus

CKMV1

from

rhizosphere

of

Valeriana

jatamansi

and

its

growth

promotional

effect

Anjali

Chauhan

a

_,

_Shiwani

_Guleria

b

_,

_Praveen

_P.

_Balgir

c

_,

_Abhishek

_Walia

d

_,

Rishi

Mahajan

e

_,

_Preeti

_Mehta

f

_,

_Chand

_Karan

_Shirkot

g,∗

a_Dr_Y._S._Parmar_University_of_Horticulture_and_Forestry,_Department_of_Basic_Sciences_Microbiology_Section,_Nauni,_Solan,_India

b_Lovely_Professional_University,_Department_of_{Microbiology,}_Jalandhar,_Punjab,_India

c_Punjabi_University_Patiala,_Department_of_{Biotechnology,}_Patiala,_Punjab,_India

d_DAV_University,_Department_of_{Microbiology,}_Jalandhar,_Punjab,_India

e_Jaypee_University_of_Information_Technology,_Waknaghat,_Himachal_Pradesh,_India

f_DBT-IOC_Centre_for_Bioenergy_Research,_Indian_Oil_R&D_Centre,_Faridabad,_Haryana,_India

g_Dr_Y._S._Parmar_University_of_Horticulture_and_Forestry,_Department_of_Basic_Sciences,_Nauni,_Solan,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17April2015 Accepted30May2016

Availableonline24December2016 AssociateEditor:WelingtonLuizde Araújo Keywords: Valerianajatamansi Phosphatesolubilization Nitrogenﬁxation Glucosedehydrogenase Aneurinibacillusaneurinilyticus PGPR

a

b

s

t

r

a

c

t

AneurinibacillusaneurinilyticusstrainCKMV1wasisolatedfromrhizosphereofValeriana

jata-mansiandpossessedmultipleplantgrowthpromotingtraitslikeproductionofphosphate

solubilization(260mg/L),nitrogenfixation(202.91nmolethylenemL−1h−1),indole-3-acetic acid (IAA) (8.1␮g/mL), siderophores (61.60%), HCN (hydrogen cyanide) production and antifungalactivity.We investigatedthe ability ofisolate CKMV1to solubilize insoluble Pvia mechanismoforganicacidproduction.High-performance liquidchromatography (HPLC)study showed thatisolate CKMV1 producedmainly gluconic (1.34%) andoxalic acids.However,geneticevidences fornitrogenfixationandphosphate solubilizationby organicacidproductionhavebeenreportedfirsttimeforA.aneurinilyticusstrainCKMV1. Auniquecombinationofglucosedehydrogenase(gdh)geneandpyrroloquinolinequinone synthase(pqq) gene, a cofactor of gdh involved in phosphate solubilization hasbeen elucidated.Nitrogenase(nifH)genefornitrogenfixationwasreportedfromA.

aneurini-lyticus.ItwasnotablethatisolateCKMV1exhibitedhighestantifungalagainstSclerotium

rolfsii(93.58%)followedbyFusariumoxysporum(64.3%),Dematophoranecatrix(52.71%),

Rhi-zoctoniasolani(91.58%),Alternariasp.(71.08%)andPhytophthora sp.(71.37%).Remarkable

increasewasobservedin seedgermination(27.07%),shootlength(42.33%),root length (52.6%), shoot dry weight (62.01%) and root dry weight (45.7%) along with NPK (0.74, 0.36, 1.82%) content of tomato under net house condition. Isolate CKMV1 possessed

∗ _{Corresponding}_author.

E-mail:profckshirkot@gmail.com(C.K.Shirkot).

http://dx.doi.org/10.1016/j.bjm.2016.12.001

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traitsrelatedtoplantgrowthpromotion,therefore,couldbeapotentialcandidateforthe developmentofbiofertiliserorbiocontrolagentandthisistheﬁrststudytoincludethe

AneurinibacillusasPGPR.

Introduction

Soilisthereservoirforavarietyoflivingorganismscomprising microorganisms,plants,andanimals,whichdonotlivein iso-lation,butformacomplexinteractivenetwork.Averyspecial ecologicalniche,therhizosphereispresentaroundtheroots oftheplantsthatsupportagroupofmetabolicallyversatile microorganisms.1_Some_of_these_rhizospheric_bacteria_that_are

beneﬁcialtoplantsareoftenreferredasplantgrowth promot-ingrhizobacteria(PGPR).Microbialinoculantsorbiofertilizers are used tohasten biological activity toimprove availabil-ityofplantnutrientsbyﬁxingatmosphericnitrogen,making insolublephosphatesolubleanddecomposingfarmwastes, whichresultinthereleaseofnutrientsandantagonizevarious pathogenicfungibyproducing siderophore,HCN(hydrogen cyanide),Chitinase,␤-1,3-glucanaseandavarietyofdifferent antibiotics.2

Phosphorus(P)isoneofthemajoressentialmacronutrients forbiologicalgrowthanddevelopmentbutitiscommonly deﬁ-cientinmostnaturalsoilssinceitisﬁxedasinsolubleironand aluminiumphosphatesinacidicsoilsorcalciumphosphates inalkalinesoils.Itisgenerallyacceptedthatthemajor mech-anismofmineralphosphate solubilizationisbythe action oforganicacidssynthesizedbysoilmicroorganismsyetthe exactbiochemicalbasisofthesetransformationsarenot com-pletelyunderstood but canbeexplored.Theconcentration ofsolublePinsoilisusuallyverylow,normallyatlevelsof 1ppmorless(10MH2PO4−).ThecellmighttakeupseveralP

formsbutthegreatestpartisabsorbedintheformsofHPO42−

H2PO4−.3,4

Thegeneticbasisofmineralphosphatesolubilization phe-notypeofbacteriaisnotwellunderstood.Itisbelievedthat organic acids produced by the action of glucose dehydro-genase, which require pyrroloquinoline quinone (PQQ) as cofactorand it isaprimary mechanism behindphosphate solubilization; this assumptionhas been conﬁrmed bythe cloningofgeneinvolvedingluconicacidproductionviz.gdh

and PQQ. PQQ-dependent glucose dehydrogenase (GDH) is capableofoxidizingglucosetogluconate.Gluconateisfurther oxidizedto2-ketogluconicacidbygluconatedehydrogenase (GADH).Glucosedehydrogenase(GDH)isamemberof quino-proteins,catalysingtheoxidationofglucosetogluconicacid, requiringPQQandalsometalionssuchasCa2+(orMg2+invitro)

foritsactivity.Glucose,gluconate,manitol,andglycerolare amongthepossibleinducersofthehaloenzymeactivity,but theinformationregardingthesynthesisofthisholoenzyme (GDH-PQQ)isnotavailable.5

Microbial communities are a main component of ecosystems that play critical roles in the biochemical

transformations of elements including nitrogen ﬁxation.6

Therefore,nitrogenthatisavailabletoplantsgrownformany yearswithoutNfertilizersisconsideredtobeduetobiological ﬁxation.7Thisprocesscatalysedbynitrogenaseenzymesis essentialformaintainingfertilityinmanyecosystems.8_The

ability to ﬁx nitrogen is widely distributed among diverse groups of Bacteria and Archae in different ecosystems. A substantialmoleculardiversityofN2ﬁxingbacteriahasbeen

detectedinﬁeldgrownriceandmaizebasedonretrievalof

nifHornifDgenefragmentsfromrootDNA.9,10

Therefore,thepresentstudyaimstounderstandthe pos-sible mechanism and to characterize the genes involved in the phosphate solubilization and nitrogen ﬁxation by

Aneurinibacillusaneurinilyticusisolatedfromtherhizosphereof

medicinalplantValerianajatamansi.Informationaboutgenus

A.aneurinilyticuswithplantgrowthpromotingattributesfrom

anymedicinalplantsislimited.Hence,theresultscouldbe usefulforbetterunderstandingofmechanismofplantgrowth promotingtraits,whichcanhelpustoengineerthisPGPRfor agronomicinterest.

Material

and

methods

IsolationandcharacterizationofPGPRfromValeriana jatamansi

Sixty rhizobacteriawith multiple PGP traits were obtained by using serial dilution technique from the rhizospheric soilsofV. jatamansi,locatedinChamba,Himachal Pradesh, India.Apotentialisolateswerescreenedandselectedonthe basis ofhalozone produced inPikovskaya’s(PVK) agar for phosphate-solubilization and chromeazurol S mediumfor siderophoreproductionandgrowthonnitrogenfreemedium (NFb) for N2-ﬁxing ability. Among sixty isolated strains,

CKMV1showedmaximumeffectivenessofmultifariousplant growth-promoting attributes, i.e. phosphate-solubilization, IAA production, nitrogen ﬁxing ability, HCN production, siderophoreproductionand broadspectrumofantagonistic activityagainstcommonphytopathogenicfungi,i.e.Sclerotium

rolfsii,Rhizoctoniasolani,Phytopthorasp.,Alternariasp.,Fusarium

oxysporumandDematophoranecatrix.

Phenotypic characterizationof the bacterial isolate was donebasedontheircolonymorphology,microscopic obser-vations, andbiochemicaltests.11 _The_isolate_was_tested_for

the utilization ofdifferent carbon sources using KB009 Hi carbohydrateTM_kit_(Himedia,_Mumbai)._The_isolate_was

iden-tiﬁed basedonwholecell fattyacids derivatized tomethyl esters and analysed by gas chromatography at IMTECH, Chandigarhandby16SrDNAsequenceanalysis.

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P-solubilizationandorganicacidproduction

For qualitativeestimation ofP solubilization, isolateswere spot inoculated on PVK agar plates for 72h. The P-solubilizationwasexhibitedwithaclearzoneformedaround thecolony.Furtherquantitativeestimationofphosphoruswas doneinPVKbrothamendedwith5.0g/Ltricalciumphosphate (TCP)bythevanadomolybdatemethod.12

For the analysis of organic acids, bacterial culture was filtratedthrough0.2␮mfilter (Millipore,GTBP) and20␮lof filtrateswereinjected toHPLC(Waters996HPLC)equipped withphotodiodearraydetector.Theorganicacidseparation was carried out on RP-18 column (Merck, Germany) with 0.1%orthophosphoricacid(Merck,Germany)asmobilephase. Retentiontimeofeachsignalwasrecordedatawavelength of 210nm and compared with the three standard organic acids[gluconicacid(Sigma–Aldrich,USA),2-ketogluconicacid (Sigma,USA)andformicacid(Supelco,USA)].

Nitrogenaseactivity

Thediazotrophiccharacterwasﬁrstevaluatedbythe capa-bility of strain to grow on nitrogen free medium and by determiningthenitrogenaseactivityusingacetylene reduc-tionassay.13_Strain_was_cultivated_in_duplicate_vials_for₅_days

at28◦C.Thevials contained15mLofgasphase each and weresealedwithsiliconerubbercaps.Thisleakage presum-ablygavethepropertensionrequiredforacetylene-reducing activity.14_Duplicate_cultures_were_exposed_to_atmospheres_of

eitherairorargoncontaining10%acetylene.After2and5days ofexposuretoacetylene,1mLsampleswerewithdrawnfrom theculturevialsandanalysedbygaschromatographywith ahydrogenﬂameionizationdetector.Reductionofacetylene toethylenewasdeterminedbythepeakheightrelativetoa standardof50nmolofethylene.

Productionofindoleaceticacid(IAA)andACCdeaminase

For the production of auxins, each isolate was grown in Luria–Bertani broth (amended with 5mM l-tryptophan, 0.065%sodiumdodecylsulphateand1%glycerol)for72hat 37◦Cundershakeconditions.Colorimetricestimationof IAA-likeauxinswasdoneusingSalkowskireagent.15 _The_ability

ofthestraintoshowACCdeaminaseactivitywascarriedby growingthestrainsinarichmedium(TS)13_and_then

transfer-ringtoDF(DworkinandFoster)16_salt_minimal_medium_with

ACCassolenitrogensource.15

Siderophore,hydrocyanicacidandantifungalmetabolites production

Siderophore activity was determined on Chrome-Azurol S (CAS)medium.17_CAS_plates_were_{spot-inoculated}_with

bac-terialstrainsand observedfordevelopmentoforange halo againstdarkbluebackgroundaroundthecoloniesafter48h of incubation at 28◦C. A change in colour from blue to orange(hydroxamate-typesiderophore)orpurple (catechol-typesiderophore)wasconsideredasapositivereaction.The productionofsiderophoreincell-freeculturesupernatantwas determinedusingspectrophotometricmethodasdescribedby

SchwynandNeilands.17_HCN_production_was_inferred_by_the

qualitativemethodofBakerandSchipper.18_The_change_in_the

colouroftheﬁlterpaperpreviouslydippedin2%sodium car-bonatepreparedin0.05%picricacidfromyellowtodarkbrown wasratedvisuallydependingontheintensityofthecolour change.Therhizobacterialisolatewastestedfortheirability toinhibitthegrowthofdiversesoil-bornefungalpathogens usingthe invitrodual-cultureanalysis.Invitroscreeningof biocontrolPGPRwasdonebydeterminingtheinhibitoryeffect ofisolatesagainstfungususingagardiffusiontechnique,i.e.

dualplatemethod.Percentgrowthinhibitionwascalculated usingtheformulagivenbyVincent.19

I=C−_CT ×100

whereIisthepercentageofgrowthinhibition;C,thegrowth offungusincontrol;T,thegrowthoffungusintreatment.

The antifungal activity of cell-free supernatants of the selected isolate was evaluated against the target fungi F.

oxysporum,Phytophthorasp.,D.necatrix,S.rolfsiiandPhythium

aphanidermatum.

Cloningofgdh,pqqgenesresponsibleforphosphate solubilizationandnifHgenefornitrogenﬁxationfrom genomicDNAofAneurinibacillusaneurinilyticusCKMV1

Primersforamplifyingthepartialsequencesofproteingenes weredesignedaccordingtotheconservedproteinsequences (GHGTHVAGT and TATNTISGTSMA) of gdh, pqq and nif H alreadypublished. Apair ofuniversaldegenerateprimers20

Zehr-nifHf(5-TGYGAYCCNAARGCNGA-3)andZehr-nifHr(5 -ANDGCCATCATYTCNCC-3)wereusedtoamplifyPartialnifH genefromgenomicDNA.Thepairofcompleteand degener-atedesignedprimersaslistedinTable1forpqqandgdhgenes wasusedforampliﬁcation.

Brieﬂy,the genomic DNAwasisolated byusing conven-tionalmethod.21_The_genomic_DNA_were_ampliﬁed_in_a_PCR

mixturecontaining0.2mMofeachdeoxynucleoside triphos-phate,20pmolofeachprimer,2.5UofTaqDNApolymerase with1×bufferina50␮lreactionvolumeswithinitial dena-turationat94◦Cfor2min,40cyclesofdenaturationat94◦Cfor 1min,primerannealingof54◦Cfor45s(forgdh),58◦Cfor45s (forpqqsynthase)and50◦Cfor2min(fornifH)andextension of72◦Cfor2minonPCR-SystemThermalCycler(LabNet).The PCRproductsweresubjectedtoagarose(2.0%,w/v)gel elec-trophoresisinTBEbuffer,followedbyexcisingfromthegel andpurifyingbyusinggelextractionkit(Realgenomics,RBC). ThepuriﬁedfragmentswereligatedintopGEM-Tcloning vec-torbeforetransformationintochemicallycompetentcellsof

EscherichiacolistrainDH5␣.Transformantsweregrownat37◦C

on Luria Broth (LB)agar containingampicillin (100␮g/mL), IPTG (50mM)andX-gal(80␮g/mL) forblue/whitescreening of recombinant colonies. Transformed E. coli strains were conﬁrmed for the presenceofinsert of gdh, pqq and nif H using respectivedesigned primers followed by sequencing (Xceleris labs, Ahmedabad). The obtained sequences were then subjected to BLAST and ORF Finder programme at NCBItoassemblethewholesequence ofthetargetedgene. Thesequences thusobtainedwerealsosubmittedtoEMBL database. Sequences of ampliﬁed genes were aligned and

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Table1–PCRprimersfordetectionofgenes,i.e.gdh,pqq,andnifHinAneurinibacillusaneurinilyticusCKMV1.

GenesforPGP

traits

Forwardprimersequence Tm(◦C) Reverseprimersequence Tm(◦C) Size(bp) Reference

Genesforphosphatesolubilization

gdh 5-ATGTATCCGGATTTAAAAGG-3 54 5-TTAACCGCGGCCTGCCTGG-3 64 800 Selfdesigned

pqq 5-GGCTGCTGGCCGAACTGACTT-3 64 5GGCCGCAAGAAGCATTATTAG-3 60 1000 Selfdesigned

Genesfornitrogenﬁxation

nifH 5-TGYGAYCCNAARGCNGA-3 52 5-ANDGCCATCATYTCNCC-3 54 350 ZehrandMcReynolds20

bootstrappedwiththehelpofPHYLIPandphylogenetictree wasconstructedusingNeighbour-Joining(J)methodof math-ematicalaveragesandviewedinTREEVIEWsoftware.

EfﬁcacyofAneurinibacillusaneurinilyticusCKMV1on plantgrowthpromotion

Tomato plant (Solanum lycopersicum variety Him sona) was usedasmodelsystemtomonitortheplantgrowth promo-tionandnutrientuptakebystrainCKMV1innethouse.The sand,soilandfarmyardmanure(FYM)wasmixedinaratio of1:1:1inorder tomakethepotting mixture.Themixture wasthenﬁlledinthepotsandmoistenedtoonethird sat-urationcapacity.Twenty-ﬁveseedsuntreatedortreatedwith cellsuspensionof1.5O.Dweresownatequidistanceand cov-eredwith moss grass tillemergence of plumule. After3–4 daysofseedlingsemergencethinningwasdoneand3plants per potwere maintained. Six replicatepots per treatment, with3plantsineachpotwereplacedinarandomizedblock designinnethouse.Thecontrolsweredesignedinthesame waybutinthiscase,thetomatoseedsweremock-inoculated with sterilized water. Tomato seedlings obtained from net house were analysed for shoot and root characteristics, root–shootratio, and nitrogen, phosphorus and potassium (NPK)analysisofplantbiomass.Thevigourindexwas cal-culatedusingthefollowingformulaasdescribedbyBabiand Anderson.22

Vigourindex=(Meanrootlengh+meanshootlength) ×germination(%)

Germinationenergyindex(GEI)wascomputedusingthe formula:

GEI= A1+(A1+A2)+(A1+A2+A3)+···+(A1+···+An) N+n

×100

whereA1,A2,A3isnumber ofseedsnewlygerminatedup tonth dayrespectively,Nistotalnumberofseedsusedfor treatment,nisnumberofdaysofobservations.

Statistical

analysis

Datawerestatisticallyanalysedbyanalysisofvarianceusing thegenerallinearmodeldevelopedbytheSASInstitute (ver-sion9.1;Cary,NC),andmeanswerecomparedusingtheleast

signiﬁcantdifference(LSD)method;P≤0.05wasconsidered signiﬁcant.

Results

Isolationandcharacterizationoftherhizobacterialisolate

Sixty bacterial isolates in total; thirty-eight from rhizo-sphere; and twenty two from endo rhizosphere of V.

jatamansiwere screenedformultipleplantgrowth

promot-ing (PGP) traits. Amongst them isolate CKMV1 exhibited multifariousplantgrowthpromotingtraits,viz.,HCN produc-tion;P-solubilization(260mg/L);IAAproduction(8.1␮g/mL); siderophore production(61.6%)and broad antifungal

activ-ity,i.e.F.oxysporum(64.3%),R.solani(91.6%),S.rolfsii(93.6%),

Phytophthoraspp.(71.4%)andAlternariaspp.(71.1%)(Table2).

Isolate CKMV1wasGram’spositive, motilerods,Cream, irregular,raisedelevationanderosemargin.Theisolatewas positive for catalase, nitrate reduction, arginine dehydro-lase, esculin hydrolysis, tyrosine utilization and acid from fructose,sucrose,inositol,l-malate, l-serine,glutarate, sor-bitol and negativeforcitrate utilization,Voges-Proskauer’s, indole production, starch hydrolysis and acid production fromlactose,xylose,dextrosegalactose,rafﬁnose,trehalose, melibosesucrose,sodiumglucanate,dulcitol,arabitol, man-nitol,erythritol,␣-methyl-d-glucoside,rhamnose,cellobiose, melezilose-d-mannoside, xylitole, d-arabinose, utilization, ONPG,nitratereduction,argininedehydrolase,starch hydrol-ysis,esculinhydrolysis.

Table2–ScreeningofselectedP-solubilizingbacterial

isolateAneurinibacillusaneurinilyticusCKMV1for

multifariousplantgrowthpromotingtraits.

PGPtraits Activity

P-solubilizationindex 3.1

P-solubilization(mg/L) 260

Siderophoreunit(%) 61.6

IAA(g/mL) 8.1

ACCdeaminaseactivities(nmg−1h−1) +

HCNproduction +

Nitrogenaseactivity(nmolethylenemL−1h−1) 202.91

Antifungalactivity(%)against

F.oxysporum 64.3

R.solani 91.6

S.rolfsii 93.6

Alternariasp. 71.1

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Table3–CellularfattyacidcompositionofA. aneurinilyticusstrainCKMV1byFAMEanalysis.

Fattyacids CKMV1(%) Iso-C14:0 2.2 C14:0 1.4 Iso-C15:1F 0.8 Iso-C15:0 55.6 Anteiso-C15:0 4.2 C16:1w7calcohol 0.9 Iso-C16:0 2.9 C16:1w11c 11.2 SuminFeature3a _5.2 C16:0 5.2 Iso-C17:1w10c 5.1 SuminFeature4b _2.1 Iso-C17:0 3.3

StrainCKMV1isshowingsimilaritywithAneurinibacillussp.asper MIDI.

a _Summed_Feature₃_comprised_16:1_w7c/15_iso_2OH.

b _Summed_Feature₄_comprised_17:1_ISO_I/ANTEI_B_which_could_not

beresolvedbyMIDI.

The fatty acids Iso-C14:0, C14:0, Iso-C15:1F, Iso-C15:0, Anteiso-C15:0,C16:1w7calcohol,Iso-C16:0,Iso-C17:0,Sumin Feature3,C16:0,Iso-C17:1w10c,SuminFeature4andC16:1 w11cwerepresentintheisolate(Table3).Theanalysisshowed thehighestsimilarityoftheisolatewithA.aneurinilyticusas perMIDIsystem(MicrobialIdentiﬁcationSystem,Inc.).

Theidentificationofisolatewasfurtherconfirmedby com-parisonof16S rDNAsequencesintheNCBIdatabaseusing aBLASTn analysis. Anamplicon ofsize1241bp represent-ing16S rDNAsequenceofstrainCKMV1wasobtained.16S rDNAsequenceofisolateCKMV1showedmaximumsequence similaritywithAneurinibacillus(AB271755).Thus,bysequence comparisonwithavailabledataintheGenBankusingBlast confirmedthestrainasA.aneurinilyticus.Thesequencewere depositedtoEMBLdatabaseandassignedaccessionnumber,

i.e.AB271755for16SrRNAsequencefortheisolateCKMV1.

NitrogenﬁxationandACCdeaminasedetermination

Thediazotrophiccharacterwasfirstevaluatedbythe capabil-ityoftheisolatetogrowinthenitrogenfreeselectivemedium. ThestrainCKMV1wastestedforitscapacitytofixnitrogen byassayingitsnitrogenaseactivity.A.aneurinilyticusCKMV1 wasconfirmed asauthentic nitrogenfixing bacteriabythe effectivelyreductionofacetyleneapproxto202.91nmol

ethy-lenemL−1h−1.A.aneurinilyticusCKMV1wasalsofoundtobe

positivefortheACCdeaminaseactivitybyitsabilitytogrow onDFsaltmedium(Table2).

InvitrodeterminationofP-solubilizingactivityofA. aneurinilyticusCKMV1anddetectionoforganicacidsby gaschromatography

InordertostudytherelationshipsbetweenpH,organicacid productionandinsolubleP-solubilization,wegrewA.

aneurini-lyticusCKMV1inthePVKmediumupto120h. Theeffectof

timecourseonﬁnalpHandsolublePinthe PVKbrothare showninFig.1.P-solubilizationwasincreasedwithincrease

300 250 200 150 100 50 0 8 7 6 4 5 2 3 1 0 0 10 20 30 40 50 60 70 Incubation time (h) P-Solubilization (mg/L) Final pH Solub le P (mg/L) Final pH 80 90 100 110 120 130

Fig.1–ChangesinP-solubilizationandpHofmediumwith timebyA.aneurinilyticus(CKMV1).

ofculturetime,reachingamaximum(260mg/L)at72h.Onthe otherhand,theculturepHdecreasedfrom7to4.97duringthe periodofthelinearincreaseofP-solubilization(Fig.1).

TheorganicacidswereidentifiedbyHPLCinthecultural filtratesafter72hofincubation.HPLCanalysisoftheculture filtraterevealedthreemajorpeaksandrestofthepeaksmight represent the unknown organic acids for which standards werenotused(datanotshown).Thethreemajorpeakswere identified as gluconic acid, formicacid and 2-ketogluconic acid bycomparingtheirretention timeswiththe authentic standards. Asshown in Table 4, gluconic acid (1.34%) was detectedasthemajororganicacidswithsmallpercentages of2-ketogluconicacid(0.24%)andformicacid(0.10%)along withunknownorganicacidsinminorconcentrationsduring TCPsolubilizationbythetestisolate.Theproductionof glu-conicacidandsubsequentdecreaseinpHofthemediumbyA.

aneurinilyticusCKMV1strainstatesthatpossiblemechanismof

phosphatesolubilizationbyA.aneurinilyticusCKMV1couldbe bytheproductionofgluconicacid.

RelationshipofTCPsolubilization(Y)withﬁnalpH,IAA (X),%siderophoreunit(X),%growthinhibitionagainstF. oxysporum,Phytophthorasp.,D.necatrix,S.rolfsii,P. aphanidermatum(X)inPVKmedium

ToknowtheoverallimpactofP-solubilization(Y)onﬁnalpH (X1),IAA(X2),percentsiderophoreunit(X3),percentgrowth

inhibitionagainstD.necatrix(X4),anon-linearregression

anal-ysiswerecarriedout.ItwasfoundthatrYX1,rYX2,rYX3andrYX4

weresignificantat5percentlevelofsignificance.Tofindout

Table4–OrganicacidproductionbyAneurinibacillus

aneurinilyticusCKMV1inPikovskaya’sbrothafter72hof incubation.

Organicacid Retentiontime(min) Percentagea

Gluconicacid 7.48 1.34 2-Ketogluconicacid 14.76 0.24 Formicacid 9.81 0.10

a Areatheunknownsample

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4.00 4.50 5.00 Final pH of supernatant

A

(i)

Y=921 X

1–3.05

; r

2

=–0.45

Observed Power

Y=68.55 X

₂0.673

_{; r}

2

_=0.39

Y=23.25 X

₄0.981

_{; r}

2

_=0.59

Y=6.61 X

₃0.250

_{; r}

2

_=0.77

B

(ii)

C

(iii)

D

(iv)

IAA production (µg/mL)

% growth inhibition against D. necatrix % siderophore unit P-solubilization 300.00 400.00 500.00 600.00 700.00 800.00 900.00 P-solubilization 300.00 400.00 500.00 600.00 700.00 800.00 900.00 P-solubilization 300.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 20.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 30.00 40.00 50.00 60.00 70.00 400.00 500.00 600.00 700.00 800.00 900.00 P-solubilization 300.00 400.00 500.00 600.00 700.00 800.00 900.00 5.50 6.00 6.50

Fig.2–Relationshipbetweenphosphatesolubilizationand(A)ﬁnalpH,(i)Y=921X−3.05

1 ; r2=−0.45(S.E.oftheregression coefﬁcient=0.267);(B)IAAproduction,(ii)Y=68.55X0.673

2 ; r2=0.39(S.E.oftheregressioncoefﬁcient=0.245);(C)percent% siderophoreunit,(iii)Y=6.61X0.250

3 ; r2=0.77(S.E.oftheregressioncoefﬁcient=0.156);(D)percent(%)growthinhibitionof

Dematophoranecatrix,(iv)Y=23.25X0.981

4 ; r2=0.59(S.E.oftheregressioncoefﬁcient=0.278).

theprobablevalueofP-solubilization(Y) forgivenvalueof ﬁnalpH(X1),IAA(X2),percentsiderophoreunit(X3),percent

growthinhibitionagainstD.necatrix(X4);the dataobtained

onthevariableweresubjectedtotheﬁttingofvarious math-ematicalfunctions,viz.,linear,quadratic,cubic, powerand inversefunctions.Onthebasisofobservedandexpected fre-quenciesandﬁnallyonthebasisofr2_values,_it_was_observed

thatpowerfunctioninallthecasesgavethebestfitted equa-tion significant at 5%level of significance.To find out the probablevalueofP-solubilization(Y)forgivenfitted regres-sionequationsalongwithstandarderror(S.E.)ofregression coefficientsandr2_values_have_been_presented_below._Thus,

presentresultsrevealedstrongnegativerelationbetween P-solubilizationandﬁnalpHofPVKmedium.Ontheotherhand, positivecorrelationofP-solubilizationwithIAA,siderophore productionandpercentgrowthinhibitionagainstD.necatrix

wasobserved.Theirgraphicalrepresentationshavealsobeen showninFig.2A–D.

Theseﬁttedregressionlinescanbesuccessfullyemployed towork out the valueofP-solubilization for agiven value ofﬁnalpH,IAA,%siderophoreunitand%growthinhibition againstinhibitionagainsttestedfungalpathogens,andhence, theseregressionlinescanberegardedasanestimation equa-tion.

Cloningofphosphatesolubilizingandnitrogenﬁxinggene ofA.aneurinilyticusCKMV1

PCR ampliﬁcation produced a reliable fragment of about ∼800bpforcomplete gdh(Fig. 3A),∼1000bpforpartialpqq

(Fig.3B)and∼350bpfornifH(Fig.3C).Sequencingofcloned completegdhandpartialnifHconcludedthatthecompletegdh

genefromA.aneurinilyticusCKMV1was786bplongencoding

261aminoacidproteinsandpartiallyampliﬁednifHis324bp longencodinga108aminoacidscorrespondingtonifHgene. SequenceofcompletegdhgeneofA.aneurinilyticusCKMV1 wasanalysed withcorresponding sequencesof15different (gdh) gene of Bacillus spp. reported from different parts of the world. Sequence analysisrevealed that gdhgene of A.

aneurinilyticusCKMV1showedmaximumhomology(99%)with

gdhgeneofBacillus subtilis(Acc. No.AP011541andAcc.No.

NC000964)(Fig.4).

SequenceofpartialnifHgeneofA.aneurinilyticusCKMV1 wasalsoanalysedwithcorrespondingsequencesof13

differ-ent(nifH)geneofBacillusspp.reportedfromdifferentparts

oftheworld.Sequence analysisrevealed thatnifHgeneof

A.aneurinilyticusCKMV1showedmaximumhomology(97.5%)

withnifHgeneofBacillussp.strainBT97(Acc.No.DQ36803)

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M

A

1 2

B

2 1 M

C

2 1 M 1000 bp 1000 bp 1000 bp 750 bp 500 bp 500 bp 500 bp 300 bp ~350 bp ~1000 bp ~800 bp

Fig.3–Agarosegelelectrophoresisshowingtheamplificationofcompleteglucosedehydrogenase(gdh),partialpqq synthaseandpartialnifHgenefromA.aneurinilyticus(CKMV1).LaneM:100bpMarker.(A)Lanes1and2:showing∼800bp amplificationcorrespondingtocompleteglucosedehydrogenase(gdh)genefromA.aneurinilyticus(CKMV1).(B)Lanes1and 2:showing∼1000bpamplificationcorrespondingtoPQQsynthasegenefromA.aneurinilyticus(CKMV1).(C)Lanes1and2: showing∼350bpamplificationcorrespondingtonifHgenefromA.aneurinilyticus(CKMV1).

NC 006270B NC 009848B NZ ACMR010 NZ ACLT010 NZ ACMN010 CP0030178Bm NC 014019B NC 014103B NC 014551B SampleO CKMV1 AP 011541Bs NC 000964B NZ AFDH010 NZ AGIP010 CP 002906.1 NC 009725B 100 _10 _10 EF471734CC EF471735CC 93 AY373371w5 AY373366c6 AY373365c5 AY373369T1 BAU23649Ba 65 HE655434Bp 73 AY373367g1 99 100 100 100 60 DQ776751AS JX154865SN JX154760SN

A

B

CKMV1 DQ364803BT 100 55 62 47 100 45 100 79 71 98 100 100 100 100 98 75 100

Fig.4–Neighbour-joiningtreebasedoncompleteglucosedehydrogenasegenesequences(A)andpartialnifHgene sequences(B)showingthephylogeneticrelationshipofstrainCKMV1.Thenumbersatthenodesindicatethelevelsof bootstrapsupportbasedondatafor1000replicates;valuesinferredgreaterthan50%areonlypresented.Thescalebar indicates100substitutionspernucleotideposition.

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Table5–Effectofinoculationofplantgrowthpromoting

rhizobacteriaAneurinibacillusaneurinilyticusCKMV1on

theplantgrowthpromotingparametersandnutrient

uptakeoftomatoseedlings.

Control CKMV1 T(cal)

Growthparameters

Shootlength(cm) 18.82 21.82 53.03

Shootdryweight(g) 2.89 3.95 48.49

Lengthofroots(cm) 7.04 9.10 46.89

Rootdryweight(g) 0.62 0.70 5.11

Nutrientuptake

NPKcontentofsoil(kg/ha) N=206.98 N=382.59 67.87

P=123.20 P=123.63 27.50 K=215.00 K=262.36 52.89 NPKcontentofshoot biomass(%) N=0.64 N=0.74 6.39 P=0.33 P=0.36 2.91 K=1.53 K=1.82 18.54 T(tab)=2.353.

geneofBacillusazotoﬁxans(AccNo.BAU23649)reportedfrom USA.Itwasalsoconﬁrmedbyphylogeneticanalysis(Fig.4)as

nifHgeneofA.aneurinilyticusCKMV1clusteredcloselywithnif

HgeneofotherreportedisolatesofBacillussp.

The sequences were deposited to EMBL database and assignedwithaccessionnumberforcompletegdh(HG515387) andpartialnifH(HG515388).

Growthresponseofseedtreatmentwithliquid formulationofbacterialisolateCKMV1ontomato seedlingsinnethouse

The plant growth promotion potential of A. aneurinilyticus

strain CKMV1 was determined in tomato seeds. A signiﬁ-cantinﬂuenceongrowthwasresultedwithtreatmentofA.

aneurinilyticusstrainCKMV1intomatoseedlingsgrowninpots

undernethouseconditions(Table5).Higherpercent germina-tion(88.7%),andvigourindex(2768.57)wererecordedforseeds treatedwith isolateCKMV1 whichwas signiﬁcantlyhigher thanthatofuntreatedseeds.Itwasobservedthatthe bac-terizedseedlingsrecorded22.6%and13.8%higherrootand shootlengths,respectively, comparedtouninoculated con-trol.Seedbacterizationresultedingreaterenhancementof therootgrowth,ascomparedtotheshootgrowth.Increasein shootdryweight(26.8%)androotdryweight(12.9%)wasalso observed.

Impactofseedtreatmentonnitrogen,phosphorusand potassiumcontentofsoilinnethouse

Effectofseedtreatmentwithbacterialstrainsinnethouse onnitrogen(N),phosphorus(P)andpotassiumcontent(K)of soilwasdeterminedfromtwomonthsoldtomatoseedlings. NPKcontent ofthe soilbeforesowingoftomato seedwas 194.4kg/ha, 108.0kg/ha and 201.9kg/ha, respectively. The results for NPK content of soil before and after experi-ment are given in Table 5. In net house, higher nitrogen (382.59kg/ha),phosphorus(123.63kg/ha)andpotassium con-tent(262.36kg/ha)was observed incaseof CKMV1treated

soil than the untreated soil which was kept as a control (206.98kg/ha;123.20kg/ha;215.0kg/ha),respectively.

Impactofseedtreatmentonplantnutrientuptakeinnet house

ItwasobservedthatNPKcontentofwholeshootsystemof treated seedlings with A. aneurinilyticusstrain CKMV1 was signiﬁcantlyhigherthantheuntreatedseedlings(Table5). Per-cent nitrogen(0.74), percentphosphorus (0.36)andpercent potassiumcontent(1.82)washigherincaseofCKMV1seedling ascomparedtountreatedcontrol.

Discussion

Many soil bacteria and especiallyrhizosphere bacteria can stimulate plant growth through a number of direct and indirect pathways. The present investigation was carried out on a strain of A. aneurinilyticus (CKMV1) isolated from rhizosphereofmedicinalplantV.jatamansirevealedits mul-tipleplantgrowthpromotingproperties.Thiscomprisedits ability for solubilization of complex insoluble phosphate, production of IAA, and siderophore production, nitrogen fixationand antifungalactivity.Significantphosphate solu-bilization (260.0mg/L) and decrease in pH from 7.0to 4.96 was obtained for isolate CKMV1, after 72h of incubation. Different bacterial isolates have different capabilities for phosphatesolubilization,PseudomonasfluorescensandBacillus

megatariumwasreportedtosolubilize400mg/Lofphosphate,

with reduction in pH from 7 to 4.23 ₆₉₈_mg/L, ₃₆₀_mg/L,

230mg/Lofsolublephosphatewasalsoreportedtobe pro-ducedbyPantoeaagglomerans24_;_Penicillium_radicum_and_Rahnella

aquatilis,25 _{respectively.} _{Signiﬁcance} _of _IAA _production _by

PGPR isolateshasbeen realizedand workedout byseveral researchersinrecentyears.26–28_Strain_CKMV1_maximum

pro-ducedIAA(8.1␮g/mL)after72hofincubation.IAAproduction bymicrobespromotestherootgrowthbydirectlystimulating plantcellelongationorcelldivision(Table2).15

Biocontrol potentialofaPGPR wasassociatedwith pro-ductionofnumberofsecondarymetabolitesandantibiotics, siderophore productionand HCNproductionwere someof them.29,30 _Isolate _CKMV1 _showed _siderophore _production

(61.60%)alongwithHCNproduction.Alongwiththisitalso showedantifungalactivityagainstdifferentfungalpathogens oftomato,viz.,F.oxysporum(64.3%),R.solani(91.6%),S.rolfsii

(93.6%),Phytophthoraspp.(71.4%),Alternariaspp.(71.1%)andD.

necatrix(52.7%)(Table2).Thismightbeduetotheproduction

ofsiderophorebyPGPRasitmakesironavailabletotheplant anunavailabletopathogensthuseffectiveincontrollingplant pathogens.31,32

Biochemicalcharacterization,FAMEanalysis(Table3),16S rDNAampliﬁcationandsequencingwassuccessfullyusedto identifytheCKMV1uptospecieslevel,andsequencingdata validatedthestrainCKMV1asA.aneurinilyticus,ﬁrsttime iso-lated from medicinal plant rhizosphere with multiple PGP traits.EarliertherewasareportofisolationofA.aneurinilyticus

fromsludgeofpulpandpapermill.33

Nitrogen ﬁxation was also recorded for the ﬁrst time in PGPR belonging to genus Aneurinibacillus by performing

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acetylenereductionassayforestimationofnitrogenase activ-ity. PCR amplification of nif H along with estimation of nitrogenaseactivityofRhizobiaandBacillushasbeen exten-sivelyusedtoconfirmthenitrogenfixingabilityofdiazotrophs

andBacillussp.34,35_In_{nitrogen-ﬁxing}_bacteria,_nitrogenase_is

encodedbyasetofoperonswhichincludesregulatorygenes (suchasnifLA),structuralgenes(suchasnifHDK)andother supplementarygenes.NifDKencodesfordinitrogenase reduc-taseandnifHforFeproteinsubunit.36_Nif_genes_are_found_in

manybacteriabesidesrhizobiaandareapartofgenomethat areconsideredaspartofthenormalbacterialgenome.37

Phosphatesolubilizationwassigniﬁcantlycorrelatedwith ﬁnalpHofsupernatant, percentsiderophoreunit, IAAand percentgrowthinhibitionagainstD.necatrix.Severalauthors attributes P solubilization of inorganic phosphate to the production and release of organic acids.4 _However, _others

suggestedsomeadditionalmechanismsasisconﬁrmed by weakorevenlackinglinearcorrelationbetweenpHandthe amountofP-solubilized.Strongnegativecorrelationbetween pHandPsolubilized(r=−0.45)inthepresentstudy(Fig.2)is similartonegativecorrelationbetweenpHandP-solubilized asreportedbyAlametal.38_The_inverse_correlation_between

thepHvalueofthecultureandreleasedofPsuggestedthat the solubility of phosphate isdirectly correlated with acid production but are not the only possible mechanisms for P-solubilization.

OrganicacidproﬁlingofA.aneurinilyticusCKMV1revealed theproductionoffourorganicacidswithgluconicacidontop with1.34%(Table4).39 _The_presence_of_organic _acid_in

cul-tureﬁlterateprovidedﬁrstcluethatthepossiblemechanism ofphosphatesolubilizationbyA.aneurinilyticusCKMV1isvia productionoforganicacidsinthemedium.Productionof mul-tiple organic acids hasalso been demonstrated forBacillus

species.40,41 _However,_there_was_no_previous _report_in

liter-atureongluconicacidproductionbythebacteriabelonging togenusAneurinibacillustowhichCKMV1belongs.Sincethe productionoforganic acids is considered tobethe princi-palmechanismformineralphosphatesolubilization,itcould beassumedthatanygeneinvolvedinorganicacidsynthesis mighthaveaneffectonthischaracter.42_Gluconic_acid

pro-ductioniscatalyzedbyperiplasmicoxidationofglucoseby membraneboundglucosedehydrogenase(GDH).42_In

Gram-negative bacteria, the synthesisof gluconic acid has been showntobedependentonpyrroloquinolinequinine(PQQ)as anenzymaticcofactorofthegdh.43,44_However,_studies

con-cerningwithdetectionofGDHencodinggene(gdh)involved inP-solubilizationhasbeenconductedinBacillussp.isolated fromrhizosphereapplebutnotinAneurinibacillus.Geneticsof phosphatesolubilizationandmineralizationcouldbestudied oncethebiochemicalmechanismofsolubilizationisknown.

A.aneurinilyticusCKMV1accumulate260mg/Lofphosphorus

fromTCP(Table2).Theresultsalsoshowedanegative rela-tion between pH of supernatant and P-solubilization from tri-calciumphosphate which further conﬁrmsthat organic acidshasbeenproducedinliquidmediathatlowersthepH ofmedia.Therefore,(gdh)geneencodingtheenzymeglucose dehydrogenaseresponsibleforgluconicacidproductionand (pqq) gene encoding the enzyme PQQ synthase were char-acterized from A. aneurinilyticus CKMV1 byusing designed primers.ThecompletegdhgenefromCKMV1wasalsocloned

andsequencedandsequenceanalysisrevealedthatthe com-pletegdhgenewas786bpinlengthencoding261aminoacids with99%homologytogdhgenesequencefromBacillus

sub-tilis(NC00964andAP011541)(Figs.3and4).Theconservation

of the gdh gene across the Bacillus spp. indicated the sig-nificance ofenzyme in sugar metabolism. But in order to confirm the role ofglucosedehydrogenase inacid produc-tion,itwasimportanttoconfirmquinoproteinnatureofgdh

from A.aneurinilyticuswhichwasconﬁrmedbythe

ampliﬁ-cationofpqqsynthasegeneof∼1000bpfromstrainCKMV1 by using designed primers. The presence of pqq synthase along withglucosedehydrogenase(gdh)inA. aneurinilyticus

strainCKMV1confirmedthequinoproteinnatureofgdhand alsoconfirmeditsroleinacidproductionfollowedby phos-phatesolubilizationwhichwasalsoconfirmedthroughHPLC data.

Inpresentstudy,weinvestigatedtheeffectivenessofPGPR isolateCKMV1onincreaseinseedgerminationrateaswell as growth of seedlings. The evaluation of results of the plantgrowthpromotionoftomatoseedlingundernethouse indicatedthat CKMV1asPGPRstrain capableofpromoting growthoftomato.CKMV1significantlyincreasedplantheight, root length and production ofdry matter from shoot and root oftomato seedlings(Table5).Germination percentage and vigourindex inCKMV1treatedseedswassignificantly higherthanthatofcontrol.Plantinoculationwithphosphate solubilizingmicroorganismssuchasPseudomonaswas bene-ficial toproducethelateral radicles.45PGPRs enhancedthe growthofseedlingsrootbyinducingtheproductionof phy-tohormonessuchasauxins(IAA).Thereweremanyreports regardingtheenhancementofrootandplantgrowthby apply-ingbacteria.6,28

Conclusion

In conclusion, the present study is the ﬁrst report on A.

aneurinilyticus strain CKMV1 isolated from the rhizosphere

of V. jatamansi, showing P-solubilization, N-ﬁxation, IAA,

siderophore, HCN production,ACC deaminase activity and biocontrol traitsagainst phytopathogenicfungi, i.e.F.

oxys-porum,Phytophthorasp.,D.necatrix,S.rolfsii.Thisreportalso

proved that decrease in final pHwas occurred during sol-ubilization of phosphorus in the medium which may be due to production of different organic acid which were simultaneouslydetectedbyHPLCduringP-solubilizationand their production was further confirmed by amplification and sequencing ofgenes such as gdh and pqq responsible fororganic acid production.Identificationofgenes forPGP traits can open new avenues for further manipulation of this strain. Further the inoculation of bacterial strain not only improves growth of seedlings but also increases the uptakeofshootNPKcontentswhichweresignificantlymore in caseof inoculated seedlingsthan the untreatedcontrol and the increase may be caused by nitrogen fixation and phosphoroussolubilizationabilityofPGPR.Hence,itis con-cludedthattheA.aneurinilyticusstrainCKMV1,isolatedfrom rhizosphereofV.jatamansi,canbeusedaspotential biofer-tilizerfortomatoandvariousvegetablecropsnotbeingcrop specific.

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Conﬂicts

of

interest

Theauthordeclaresthattheresearchwasconductedinthe absence of any commercial or ﬁnancial relationships that couldbeconstruedasapotentialconﬂictofinterest.

Acknowledgements

Wethankthe AllIndiaNetworkProjectonSoilBiodiversity andBiofertilizer(ICAR)forprovidingnecessaryfundstocarry outthisresearchwork.

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