Exploring plant growth-promotion actinomycetes from vermicompost and rhizosphere soil for yield enhancement in chickpea

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ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Exploring

plant

growth-promotion

actinomycetes

from

vermicompost

and

rhizosphere

soil

for

yield

enhancement

in

chickpea

M. Sreevidya

a,b

_,

_S.

_{Gopalakrishnan}

b,∗

_,

_H.

_Kudapa

b

_,

_R.K.

_Varshney

b

a_Centre_for_{Biotechnology,}_Jawaharlal_Nehru_{Technological}_University,_Kukatpally_500072,_Telangana,_India

b_{International}_Crops_Research_Institute_for_the_Semi-Arid_Tropics,_Patancheru_502324,_Telangana,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19April2015 Accepted17August2015

AssociateEditor:JerriÉdsonZilli

Keywords: Chickpea Actinomycetes PGP Streptomyces Biocontrol Geneexpression

a

b

s

t

r

a

c

t

Themainobjectiveofthepresentstudywastoisolateandcharacterizeactinomycetesfor theirplantgrowth-promotioninchickpea.Atotalof89actinomyceteswerescreenedfor theirantagonismagainstfungalpathogensofchickpeabydualcultureandmetabolite pro-ductionassays.Fourmostpromisingactinomyceteswereevaluatedfortheirphysiological andplantgrowth-promotionpropertiesunderinvitroandinvivoconditions.Alltheisolates exhibitedgoodgrowthattemperaturesfrom20◦Cto40◦C,pHrangeof7–11andNaCl con-centrationsupto8%.ThesewerealsofoundhighlytoleranttoBavistin,slightlytolerant toThiramandCaptan(exceptVAI-7andVAI-40)butsusceptibletoBenlateandRidomilat fieldapplicationlevelsandwerefoundtoproducesiderophore,cellulase,lipase,protease, chitinase(exceptVAI-40),hydrocyanicacid(exceptVAI-7andVAI-40),indoleaceticacid and␤-1,3-glucanase.Whenthefouractinomyceteswereevaluatedfortheirplant growth-promotionpropertiesunderfieldconditionsonchickpea,allexhibitedincreaseinnodule number,shootweightandyield.TheactinomycetestreatedplotsenhancedtotalN,available PandorganicCovertheun-inoculatedcontrol.Thescanningelectronmicroscope stud-iesexhibitedextensivecolonizationbyactinomycetesontherootsurfaceofchickpea.The expressionprofilesforindoleaceticacid,siderophoreand␤-1,3-glucanasegenesexhibited up-regulationforallthreetraitsandinallfourisolates.Theactinomyceteswere

identi-ﬁedasStreptomycesbutdifferentspeciesinthe16SrDNAanalysis.Itwasconcludedthat

theselectedactinomyceteshavegoodplantgrowth-promotionandbiocontrolpotentials onchickpea.

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author.

E-mail:s.gopalakrishnan@cgiar.org(S.Gopalakrishnan).

http://dx.doi.org/10.1016/j.bjm.2015.11.030

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Introduction

Chickpea(CicerarietinumL.)isthesecondmostwidelygrown foodlegumecropaftercommonbean,withannualproduction of13.8mtworldwide.1 _Globally,_more_than _90%_of_chickpea

productionoccursinthesemi-aridtropicsofAsiaandAfrica. Asiaaccountsfor88%ofglobalchickpeaproductionwhereas India is the largest producer accounting for 75% of Asia’s chickpeaproduction.2_Several_biotic_and_abiotic_factors_were

involved in low production ofchickpea. Thebiotic factors includefungi,bacteria,viruses,nematodes,mycoplasmaand insectpests.Fungiarethelargestgroupaffectingstems,roots, leaves,ﬂowers and pods. Chickpea cropismainly affected byFusarium wilt, dry root rot, collar rot, Ascochyta blight andBotrytis graymoldcausedbyFusarium oxysporumf.sp.

ciceri(FOC),Macrophominaphaseolina,Sclerotiumrolfsii,Ascochyta

rabieiandBotrytiscinerea,respectivelyresultinginreducedcrop

yield.3,4

The microbes in rhizosphere help plants in growth-promotionandyield.Actinomycetesareoneofthemajor com-ponentsofrhizospheremicrobialpopulationsandareuseful insoilnutrientcycling5,6_as_well_as_plant_{growth-promotion}

(PGP).7_{Actinomycetes}_produce_secondary_metabolites_such_as

lyticenzymes,PGPsubstancesandantibiotics.8 _The

actino-mycetes,mainlythosebelongingtoStreptomycesspp.,make upanimportantgroupofsoilmicrobes.Streptomycesare abun-dantinsoilandhelpinthedegradationofcomplexmolecules tosimplemoleculesforplantgrowth and development.9,10

Thesearealsoreportedtodecomposeorganicmatter,promote plantgrowthandcontrolplantpathogens.11

In the present study, actinomycetes isolated from rhi-zosphereand herbalvermicompostwerecharacterizedand evaluatedforPGPpropertiesandforbiocontrol-relatedtraits. Thepromisingstrainswere alsoevaluated fortheirPGP in chickpeaunderﬁeldconditions.

Materials

and

methods

Actinomycetesisolation

Actinomycetes were isolated from herbal vermicompost (Jatrophacurcas,Annonasquamosa,Partheniumhysterophorus,

Gli-ricidiasepiumandAzadirachtaindica)andchickpearhizosphere

soils.Tengramsofeachvermicompostandrhizospheresoils weresuspendedin90mLofsterilephysiologicalsaline(0.85% NaClindistilledwater)inabottleandkeptforshakingonan orbitalshaker(at100rpm)at28±2◦Cfor1h.Attheendof shaking,thesampleswereseriallydilutedupto105_dilutions andsamplesfrom104 _and₁₀5_dilutions_were_spread_plated (0.1mL)onactinomycetesisolation(AIA)agar(HiMedia Labo-ratories,Mumbai,India)andincubatedat28±2◦Cfor7days. ProminentcolonieswereisolatedandstoredonAIagarslants.

Selectionofantagonisticactinomycetesagainstfungal pathogensofchickpea

Atotalof89actinomyceteswerescreenedfortheir antago-nisticactivityagainstS.rolfsii,M.phaseolina(threestrainsviz.

MP-6,MP-24 andMP-115) and FOC(acquiredfrom legumes pathology, ICRISAT, Patancheru) by dual culture assay as per the protocols of Gopalakrishnan et al.12 _on _glucose

casaminoacidyeastextractagarplates.Thecultureﬁltratesof thepromisingisolates,basedonthedualcultureassay,were extractedbypartitioningagainstethylacetate(EtOAc)andthe resultantorganic(EtOAc)andaqueousfractionswere evapo-ratedonarotaryevaporatorandcollectedinaminimalvolume of methanol. Both the fractions were evaluated for their antagonisticpotentialagainstthethreefungalpathogensof chickpea(M.phaseolina,FOCandS.rolfsii).Forthis,afungaldisc of6mmdiameterwasboredandkeptatcenterofthepotato dextroseagarplateamendedwitheitherorganicoraqueous fractions(ataconcentrationof0.5%).Controlplatescontained onlymethanol.Theplateswereincubated at28±2◦Cfor5 days andinhibitionofthe pathogenwas recordedforboth dualcultureandmetaboliteproductionassaysonascaleof0–4 asfollows:0=noinhibition;1=slightinhibition;2=moderate inhibition;3=goodinhibitionand4=excellentinhibition. Colonymorphologyofselectedactinomycetes

SelectedisolatesofactinomyceteswerestreakedonAIagar by quadrant streakingtechnique and incubated for 5days at28±2◦C.Atthe end ofincubation, the isolatedcolonies wereobservedfortheirmorphology.Gramstainingwasalso performed13andobservedunderlightmicroscope.

Molecularidentiﬁcationoftheselectedactinomycetes The selected actinomycetes were sent to Macrogen Inc., Seoul, Korea for identiﬁcation by 16S rDNA analysis. The sequencesobtainedfromMacrogenwerecomparedwith sim-ilar sequences from GenBank, compared using the BLAST program,14 _aligned_using _the_Clustal _W_software15 _and _the

dendrograminferredbyneighbor-joiningmethod.16_Bootstrap

analysiswasperformedusingtheMEGAversion 4program toestimate thestatistical stabilityofthe branchesin clus-terwith1000replications.Thesequences(1460bpforSAI-13, 1474bpforSAI-291,475bpforVAI-7and 1472bpforVAI-40) weresubmittedtoNCBIandaccessionnumbersobtained. Invitroevaluationofactinomycetesfortheirphysiological traitsandfungicidetolerance

PhysiologicalpropertiessuchastolerancetopH,temperature, salinityandfungicideswerestudiedfortheselected actino-mycetes.TheactinomyceteswerestreakedonBennett’sagar (HiMediaLaboratories,Mumbai,India),adjustedtodifferent pH (5, 7, 9and 11) and saline concentrations (0–12% NaCl attheinterval of2%)andincubated at28±2◦Cfor5days. Fortemperature,theBennett’sagarplateswerestreakedwith the actinomycetesand incubated atdifferenttemperatures (20◦C,30◦C,and40◦C)for5days.Fortestat50◦C,theisolates were inoculated in Bennett’s broth and incubated at50◦C. Thefungicidetolerancewasevaluatedasperthe protocols ofGopalakrishnanetal.17_The_{actinomycetes}_were_streaked

onAIagarplatesamendedwithfungicidesBavistin,Thiram, Benlate,CaptanandRidomilatﬁeldapplicationlevels(2500, 3000,4000,3000and3000ppm)andincubatedat28±2◦Cfor

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5days.Attheendof5daysincubationthegrowthofthe acti-nomyceteswererated.TheratingscaleforpH,temperature andsalinitywererecordedonascaleof0–3asfollows:0=no growth;1=slight growth;2=moderate growth and 3=good growth.

Evaluationforproductionofextracellularenzymes, siderophore,indoleaceticacid(IAA)andhydrocyanicacid (HCN)

Selected actinomycetes were evaluated for their PGP and biocontroltraits including siderophore, chitinase,cellulase, lipase,protease, HCN,␤-1,3-glucanase and IAA production. Siderophoreproductionwasestimatedaspertheprotocolof SchwynandNeilands.18_Chitinase_production_was_estimated

byamendingagarplateswithcolloidalchitinsuspensionand mineralsaltsaccordingtothestandardprotocolsofHsuand Lockwood.19 _Production _of_cellulase _was _detected_by_using

standard protocols of Hendricks et al.20 _The_protease _and

lipaseproductions were estimatedas per the protocols of Bhattacharyaetal.21_HCN_was_{qualitatively}_assessed_by_the

methoddescribedbyLorck.22_Estimation_of_IAA_was_done_as

pertheprotocolsofPattenandGlick.23_The_rating_scales_for

siderophore,chitinase,cellulase,lipaseandproteasewereas follows:0=nohalozone; 1=halozoneof1–10mm; 2=halo zoneof11–20mm;3=halozoneof21–30mm;4=halozoneof 31–40mm;and5=41–50mm.ForHCNproduction,the follow-ingratingscalewasused:0=nocolorchange,1=lightreddish brown,2=mediumreddishbrownand3=darkreddishbrown. EvaluationofPGPpotentialofactinomycetesby“ragdoll” method

Growth-promoting activity was evaluated by the ‘ragdoll’ methodasdescribedbyChambleeandGreen.24_In_brief,

chick-pea seeds (ICCV 2) were surface sterilized by using 2.5% sodium hypochlorite solution for 5min and washed thor-oughlywithsterilized distilled water.Sterilized seedswere soakedinindividualactinomycetes(108_colony_forming_units (CFU)mL−1)for1handplacedinonehalfofawetpapertowel, foldedandrolledintoamoderatelytighttubeandplacedin aplasticbag.Thistubewaskeptat(28±2◦C)for5days.At theendofincubation,%germination,rootandshootlengths weremeasured.

EvaluationofPGPpotentialofactinomycetesunderﬁeld conditions

Theselected actinomycetes were evaluatedfor PGP poten-tialonchickpeaunderon-stationﬁeldconditionsin2013–14 post-rainyseasonatICRISATPatancheru (17◦30N;78◦16E; altitude=549m),Hyderabad,Telangana,India.Chickpea vari-etyICCV2wasusedforthistrial.Soilsattheexperimentalsite wereofvertisolwhichcontained25%sand,21%siltand52% claywithalkalinepHof7.5–8.5.TheorganicCcontentofthis ﬁeldwas 0.56%.Therhizospheresoil(top15cm)contained 642ppmoftotal Nand 9.03ppmofavailableP.The experi-mentwaslaidoutinarandomizedcompleteblockdesignwith threereplicateswithaplotsizeof4m×3ridges.The acti-nomyceteculturesweregrowninSCBat28±2◦Cfor5days.

Chickpeaseedsweretreatedwiththeactinomycetecultures (108_CFU_mL−1₎_for₅₀_min_and_sown_on_2nd_November₂₀₁₃_at arow-torowspacingof60cmandaplant-to-plantspacing of10cm.Theactinomycetecultures(1000mL;108_CFU_mL−1₎ werealsoappliedoncein15daystothesoilclosetotheplant until ﬂowering stage. The control plots containedno acti-nomycetestrains.Noseriousphytopathogensorinsectpest attackswereobservedduringthecroppingperiod.Irrigation wasdoneon23daysaftersowing(DAS)and51DASwhereas weedingon22DASand49DAS.At60DAS,thenodule num-ber, stemweight,podnumber,podweight,leafweightand leafareawerenotedandcomparedwithun-inoculated con-trol.Thecropwasharvestedmanuallyon4thFeb2014and atharvest,stoveryield,grainyieldandtotaldrymatterwere noted.Rhizospheresoilsampleswerecollectedfromadepth of0–15cmatbothﬂowering(60DAS)andcropmaturitystages andanalyzedfortotalN(ppm),availableP(ppm)andorganicC %accordingtotheprotocolsofNovozamskyetal.,25_Olsen_and

Sommers,26_and_Nelson_and_Sommers,27_respectively_and_soil

biologicalpropertiesincludingmicrobialbiomassC,microbial biomassNanddehydrogenaseactivitiesaspertheprotocolsof AndersonandDomsch,28_Brooks_et_al.,29_and_Casida,30

respec-tively.

Colonizationstudies

Chickpea roots were examined for colonization by actino-mycetesunderscanningelectronmicroscope(SEM)atRUSKA lab,CollegeofVeterinaryScience,Rajendranagar,Hyderabad, Telangana,India,aspertheprotocolsofBozzolaandRussell.31

For this,the seeds ofchickpea (ICCV2) were surface ster-ilized as explainedearlier and allowed tosproutovernight. Thesproutedseedswere soakedinselected actinomycetes for50minandtransferredcarefullyintosandtubes contain-ingsterilizedcoarsesand(50g).Boosterdoseofactinomycete (1mL; 108_CFU_mL−1₎ _was _applied _after ₇ _days. _The _tubes wereincubatedat24±2◦Cinalightchamberwithan aver-age illuminationof9600lxand photosynthetic photonflux of 350␮Em−2s−1. After two weeksof incubation, chickpea seedlingsweretakenoutandtherootswerewashedin0.1M phosphate buffer.Roottipsof4–5mmlengthwere cutand fixedinglutaraldehyde(2.5%)inphosphatebufferfor24hat 4◦C.At the endof 24h incubation, the rootsampleswere againwashed withphosphate buffer,postfixedinosmium tetraoxide(2%)for4handdehydratedusingagradedseriesof ethanol.Thedehydratedsamplesweredried,witha critical-pointliquidcarbondioxideasatransitionfluid,andadhered ontoaluminumspecimenmountswithdoublestickadhesive tape.Themountedsampleswerecoatedwithgold-palladium inanautomatedsputtercoater(JEOLJFC-1600)andexamined underSEM(JOEL-JSM5600).

Geneexpressionstudies

The selectedactinomycetes were grown inBennett’s broth for 72h. RNA was extracted from actinomycetes by con-ventionalTrizolmethod.32_The_quality_and_quantity_of_RNA

was estimated by Nanodrop (Thermo Scientiﬁc, USA) and RNA integrity by 2100 Bioanalyzer (Agilent, USA). Quan-titative real-time polymerase chain reaction (qRT-PCR)

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Table1–Antagonisticpotentialofthefouractinomycetesagainstfungalpathogensofchickpea.

Isolate Dualcultureassay Metaboliteproductionassay

MP-6 MP-24 MP-115 FOC S.rolfsii RB-6 RB-24 RB-115 FOC S.rolfsii

SAI-13 3 2 1 1 3 2 1 1 1 1 SAI-29 2 3 2 2 0 3 4 2 1 1 VAI-7 2 2 1 3 0 2 3 0 1 0 VAI-40 3 3 4 3 1 2 4 0 1 0 Control 0 0 0 0 0 0 0 0 0 0 SE± 0.05*** _0.08 _0.07*** _0.07*** _0.04*** _0.6*** _0.8*** _3.2*** ₀ ₀ LSD(5%) 0 0 0 0 0 0 0 0 0 0

MP-6,MP-24andMP-115–threestrainsofMacrophominaphaseolina;FOC,Fusariumoxysporumf.sp.ciceri;S.rolfsii,Sclerotiumrolfsii.Therating scale0=noinhibition,1=slightinhibition,2=moderateinhibition,3=goodinhibition,4=excellentinhibition.LSD,leastsigniﬁcantdifference; SE,standarderror.

∗∗∗_{Statistically}_signiﬁcant_at_0.001.

was performed as per the manufacturer’s instructions using Applied Biosystems 7500 Real Time PCR System with the SYBR green chemistry (Applied Biosystems, USA). Gene speciﬁc primers for IAA, siderophore and ␤-1,3-glucanase were designed using Primer3 software.33

Primers for IAA (F: GTCACCGGGATCTTCTTCAAC; R: GATGTCGGTGTTCTTGTCCAG); siderophore (F: ATCCT-CAACACCCTGGTCTG; R: TCCTTGTACTGGTACGGGACTT) and ␤-1,3-glucanase (F: CCGAACACCACCTACTCCAC; R: CCAGGTTGAGGATCAGGAAG) were designed from the sequences collected from UniProtKB database

(http://www.uniprot.org/uniprot) as described in

Gopalakr-ishnanetal.34 _RNA_polymerase_principal_sigma_factor_HrdB

(SCO5820)(F:GGTCGAGGTCATCAACAAGC;R: CTCGATGAGGT-CACCGAACT)wasusedastheendogenouscontrol.qRT-PCR reactions and conditions were conducted as described earlier.34 _PCR_reactions _were _carried _out _in ₁₀_␮L_reactions

containing30ngofﬁrststrandcDNA,1XPCRbuffer,125mM dNTPs,1.5mMMgCl2,0.2mMprimersand1UTaqpolymerase and PCR cycle was programmed as, 50◦C for 2min and denaturation at 95◦C for 10min followed by 40 cycles of denaturationat95◦Cfor15sandannealingandextensionat 60◦Cfor1min.ThedataobtainedfromdifferentPCRrunsor cDNAsampleswasanalyzedusingthemeanoftheCTvalues ofthethreebiologicalreplicatesthatwerenormalizedtothe mean CT values of the endogenous gene. The expression ratioswerecalculatedusingthe2−Ctmethodandrelative transcriptionlevelswereplottedgraphically.

Statisticalanalysis

Data were analyzedbyusing analysisofvariance (ANOVA) technique,bySASGLM(GeneralLinearModel)procedure(SAS Institute2002-08, SASversion 9.3) consideringisolatesand

replicationasfixedinrandomizedcompleteblockdesign. Iso-latemeansweretestedforsignificanceandcomparedusing Fisher’sprotectedleastsignificantdifference.

Results

Screeningforpotentialactinomycetes

Of the 89 isolates screened for their antagonistic poten-tial againstimportantpathogens of chickpeasuchas FOC,

M. phaseolina and S.rolfsii by dual cultureassay, four

acti-nomycetes viz. SAI-13 and SAI-29 (isolated from chickpea rhizosphere), VAI-7 (from A. squamosa vermicompost) and VAI-40(fromJ.curcasvermicompost)werefoundtobemost promising. Whenthe cultureﬁltratesofthesefourisolates werepartitionedagainstEtOAc,bysolventextractionmethod, onlyorganicfractionswerefoundactive.Amongthefour iso-lates,theorganicfractionsofSAI-13andSAI-29werefoundto bepotentialagainstallthefungalpathogens(Table1).

AllthefourisolateswerefoundtobeGrampositiveand thecoloniesshowedsporulatingaerialmycelium.Thecolony morphology description of the four isolates is presented

(Table2).Analysisof16SrDNAsequencesbyneighbor-joining

methodrevealedthatallfourisolatesmatchedtoStreptomyces

spp.(100%).Thenucleotidesequencesweresubmittedto Gen-BankandNCBIaccessionnumberswereobtainedasfollows: SAI-13: KM220609,SAI-29:KM220608,VAI-7:KM220610;and VAI-40:KM220611(Fig.1).

Physiologicalandbiochemicaltraitsofselected actinomycetes

AllthefouractinomycetesgrewbetweenpH7and11,but max-imumsporulationwasobservedatpH9whilenogrowthwas

Table2–IndividualcolonymorphologyoffouractinomycetesonAIA.

Isolate Size Shape Margin Elevation Surface Opacity Color

SAI-13 Medium Circular Undulate Umbonate Rough,dry Opaque Dustwhite

SAI-29 Small Circular Lobate Raised Smooth,dry Opaque Dustwhite

VAI-7 Medium Circular Lobate Raised Rough,dry Opaque White

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Streptomyces flavolimosus sj32

Streptomyces cyaneofuscatus NBRC 13190

Streptomyces globisporus NBRC12867

Streptomyces sporovirgulis M5

SAI-29

Streptomyces griseobrunneus NBRC 12775

VA

I-7

SAI-13

Streptomyces cavourensis NBRC13026

Streptomyces albo

longus J

CM 4716

Streptomyces flavo

fungini

NBRC

13371

VA

I-40

Streptomyces koya

ngensis VK

-A60

Streptomyces limosus strain NBRC 12790

Streptomyces sampsonii

Streptomyces griseo

planus (NRR

L B-3064)

Streptomyces microflavus NBRC 13

062

100 100 92 100 100 96 84 82 52 96 100 84 62 38

Fig.1–PhylogeneticrelationshipbetweenVAI-7,VAI-40,SAI-13andSAI-29andrepresentativespeciesbasedonfulllength 16SrDNAsequencesconstructedusingtheneighbor-joiningmethod.

observedatpH5.Theactinomyceteisolatesalsogrewwell attemperaturesbetween20◦Cand40◦Cbutunabletogrow at50◦Cand allisolatestoleratedNaClconcentrationof8% (SAI-13toleratedupto10%).Whentheisolatesweretestedfor fungicidetolerance,allthefourisolateswerefoundtolerantto BavistinandsusceptibletoBenlateandRidomilwhereastwo ofthem,SAI-13andSAI-29,werefoundmoderatelytolerant toThiramandslightlytoleranttoCaptan(Table3).

Underinvitroconditions,allthefouractinomyceteswere foundtoproducesiderophore,cellulase,lipase,protease,IAA, ␤-1,3-glucanase, chitinase(except VAI-40) andHCN (except VAI-40andVAI-7).Ofthefourisolates,SAI-29producedhigher amountsofHCN,IAA,and␤-1,3-glucanasewhileSAI-13 pro-ducedhigheramountsoflipaseandprotease(Table4).

PGPofchickpeabyselectedactinomycetes

Theeffectofactinomycetesonchickpeagrowthwas demon-strated by“ragdoll”method inwhich all isolatesexhibited increase in root (17%) and shoot (3%) lengths when com-paredwiththecontrol.Ofthefouractinomycetestested,VAI-7 signiﬁcantlyenhancedbothrootandshootlengthswhereas VAI-40andSAI-29 signiﬁcantlyenhancedonlyrootlengths whencomparedtocontrol(Fig.2).

Under ﬁeld conditions, a considerable increase in agro-nomicandsoilmineralpropertieswereobservedin actino-mycetestreatedplotsovercontrolplots.At60DAS,therewas anincrease innodulenumber (upto19%), leafarea (upto 27%),podnumber(upto26%),leafweight(upto16%),plant height(upto3%)andstemweight(upto16%).Whileatﬁnal harvest,theactinomycetestreatedplotsexhibitedenhanced thestoveryield(upto23%),grainyield(upto20%),totaldry matter(upto17%),seedweight(upto16%)andseednumber

(upto22%)(Table5).Therhizospheresoilfromactinomycetes

treatedplotsenhancedupto11%oftotalN,27%ofavailable P,8%oforganicC,33%ofdehydrogenaseactivityand22%of microbialbiomasscarbon,atﬂoweringstageand3%oftotal N,19%ofavailableP,11%oforganicC,26%ofdehydrogenase activityand29%ofmicrobialbiomasscarbonatharvestwhen comparedwiththecontrol(Table6).

Colonizationandgeneexpressionstudies

Colonization ofactinomycetes on chickpea roots was con-ﬁrmedbyimagesofSEMinactinomycetestreatedroots.Allthe isolatesexhibitedgoodcolonizationonchickpearoots with-outanydamagetorootsurface.ThehyphaeofStreptomyces

werealsoseentogrowandadheretothesurfaceoftheroot (Fig.3).

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Table3–Effectoftemperature,pH,salinityand fungicidesonthegrowthoffouractinomycetes.

Trait SAI-13 SAI-29 VAI-7 VAI-40

Temperature 20◦C 2 2 2 2 30◦C 3 3 3 3 40◦C 3 3 3 3 50◦_C ₀ ₀ ₀ ₀ pH 5 0 0 0 0 7 3 3 3 3 9 3 3 3 3 11 2 2 2 2 13 0 0 0 0 Salinity(%NaCl) 0 3 3 3 3 2 3 3 3 3 4 3 3 3 3 6 2 2 3 3 8 2 2 2 3 10 2 0 0 0 12 0 0 0 0 Fungicidetolerancea Bavistin(2500ppm) 3 3 3 3 Thiram(3000ppm) 1 2 0 0 Benlate(4000ppm) 0 0 0 0 Captan(3000ppm) 1 1 0 0 Ridomil(3000ppm) 0 0 0 0 0=no growth; 1=slight growth; 2=moderate growth; 3=good growth.

a _At_ﬁeld_application_level.

InPGPgeneexpressionproﬁlestudies,goodqualityRNA wasisolatedfromallthefouractinomycetestrains.qRT-PCR analysis on selected PGP genes of actinomycetes revealed up-regulationofallthethreegenesandonallthefour acti-nomycetes.␤-1,3-Glucanasegenewashighlyup-regulatedin SAI-29(5 folds)followedbyVAI-7,VAI-40and SAI-13while IAAgenewashighlyup-regulatedinSAI-13(4folds)followed byVAI-7, VAI-40and SAI-29. Siderophore gene was highly

up-regulatedinSAI-13(1fold)followedbySAI-29,VAI-7and VAI-40(Fig.4).

Discussion

Actinomycetes are used for PGP and biocontrol of plant pathogensinagriculture.Forinstance,Streptomycesspp.were reportedtoenhancePGPofcropssuchastea,35_wheat,36_rice,34

bean,11 _tomato37 _and_pea.38 _In_the_present_study,_when_the

89 actinomycetesscreenedfortheir antagonisticpotentials againstimportantfungal pathogens ofchickpea. Ofwhich, fourisolates(SAI-13,SAI-29,VAI-7andVAI-40)werefoundto bepromising.TherewasnoinhibitionofS.rolfsiibySAI-29in dualcultureassayhowever,whenthepathogenreachedthe antagonistthegrowthofthepathogenwasarrested.Hence, SAI-29wasfurtherselectedformetaboliteproductionassay inwhichtheorganicfractionwasfoundtobeeffectivein con-trollingS.rolfsii.Similarly,VAI-40wasfoundtobeeffectivein dualcultureassaywhilenoteffectiveinmetabolite produc-tionassay,perhapsthemetaboliteproducedwasofvolatilein nature.Ofthefourisolates,SAI-13andSAI-29werefoundto havebroadspectruminhibitionagainstalltestedpathogens ofchickpea.Thesefourisolatesfoundtovaryintheirshape, elevation,opacity,margins,colorandsize.Molecular identiﬁ-cationofthefourisolates,basedontheir16SrDNAsequence analysis, revealed that all actinomycetes belong to

Strepto-mycesspp.SimilarobservationswerenotedbyAraújo39_where

heobservedStreptomycesasdry,smoothorhairycolonieswith airbornemyceliumofdifferentcolors.However,these pheno-typiccharacteristicsofStreptomycesvarywiththecomposition oftheculturemedium.40

In the present investigation,all the Streptomyces strains grew well attemperatures 20◦C to40◦C,analkalinepHof 11 and salineconcentrationsofup to8%.Hence,it canbe concluded thatthe Streptomycesisolates usedin this study can beused insemi-arid tropics, alkaline and salinesoils. The distributionofStreptomycesspp. indiverse ecosystems is one of the mainreasons for ability to adapt to a wide range of environmental conditions, allowing them to sur-viveinhightemperatures,saline,acidicandalkalinesoils.39

ThesetraitshelptheStreptomycesspp.incompetingwiththe

Table4–Productionofextracellularenzymesandplantgrowthpromotingtraitsbyfouractinomycetes.

Isolate Productionscorefor IAA ␤-1,3-Glucanase

Siderophore Chitinase Cellulase Lipase Protease HCN (␮g/mL) (mg/mL)

SAI-13 1 2 4 3 6.0 1 12.4 0.25 SAI-29 2 2 4 2 5.5 2 14.6 0.32 VAI-7 2 2 4 2 4.3 0 6.6 0.16 VAI-40 1 0 4 2 5.7 0 3.6 0.12 SE± 0 0 0 0 0.24* ₀ _0.32*** _0.012*** LSD(5%) 0 0 0 0.3 1.2 0 1.51 0.038

HCN,hydrocyanicacid;IAA,indoleaceticacid.Theratingscalesforsiderophore,chitinase,cellulase,lipaseandproteasewereasfollows:0=no halozone,1=halozoneof1–10mm,2=halozoneof11–20mm,3=halozoneof21–30mm,4=halozoneof31–40mm;5=41–50mm.ForHCN production,thefollowingratingscalewasused:0=nocolorchange,1=lightreddishbrown,2=mediumreddishbrown,3=darkreddishbrown. LSD,leastsigniﬁcantdifference;SE,standarderror.

∗ _{Statistically}_signiﬁcant_at_0.05. ∗∗∗_{Statistically}_signiﬁcant_at_0.001.

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Fig.2–Effectofactinomycetesstrainsonrootandshootlengthsofchickpeaseedlings.

nativemicroﬂorawhenintroducedintotherhizosphereofthe host.41_The_salinity_and_pH_levels_of_the_rhizosphere_soil_are

importantfactorsforthemicrobestocompeteandsurvivein rhizosphere.Therefore,thetoleranceforhighsalinityandpH shouldbethecriteriaforselectionofmicroorganisms.42

Inthisstudy,all thefourStreptomycesspp.weretolerant toBavistinwhereasSAI-13andSAI-29weretolerantto Thi-ramandCaptanatﬁeldapplicationlevels.Thesefungicides were generallyusedto controlsoilborne fungal pathogens. Hence,itisconcludedthatthefourStreptomycesspp.are com-patiblewithBavistin,ThiramandCaptanandhencecanbe treated together on the seeds tocontrol soil borne fungal pathogensandthusreducingtheuseoffungicidesin control-lingpathogens.

TheselectedfourStreptomycesspp.possessbiocontrolas wellasPGPpropertiessuchasproductionofIAA,siderophore, ␤-1,3-glucanase,lipase,protease,cellulase,chitinase(except VAI-40) and HCN (except VAI-7and VAI-40).IAA is a phy-tohormone which comes under auxins group and helps in improving plant growth by stimulating cell elongation, root initiation, seed germination and seedling growth.43

Siderophoresareusuallyproducedbyvarioussoilmicrobes including Streptomyces spp. to bind Fe3+ _from _the environ-mentand makeit availableforits owngrowth;plantsalso utilizetheseasanironsource.44_Streptomyces_spp._from

rhizo-spheresoilhasbeen reportedtoproducesiderophoresand inhibitthe growthofphytopathogens.38 _HCN_production_is

alsoreportedtoplayarole indiseasesuppression.45 _Some

of the recent studies indicated that metabolites produced by microbes such as HCN may enhance plant establish-ment in rhizosphere.46 _The _isolates _from _the _rhizosphere

soilof chickpea were observed to exhibit HCN production

potentialwhichpromotedplantgrowthdirectlyorindirectly or synergistically.47 _Suwan _et _al.48 _proposed _that

actino-mycetesactaspotentialasbiologicalcontrolagentsbecauseof itsintenseantagonisticactivityviatheproductionofvarious antifungalsubstancessuchaschitinaseand␤-1,3-glucanase. Inthepresentinvestigation,theeffectofthefour

Strepto-mycesspp.onthegrowthofchickpeaseedlingswasstudied

by “ragdoll” method, in which VAI-7 exhibited maximum increaseinshootandrootlengths.Thisobservationwas fur-therconfirmedinthefieldtrials.Underfieldconditions,the

Streptomycesspp.increasednodulenumberoverun-inoculated

controldemonstratingadirectproofforenhancingnitrogen ﬁxation.TheStreptomycesstrainsusedinthisstudyexhibited increaseinagronomicpropertiessuchastheshootweight, leafweight,leafarea,plantheight,grainyieldandstoveryield overtheun-inoculatedcontrol.Theyalsoincreasedthetotal N,available P,organic C%, microbial biomassC, microbial biomassNanddehydrogenasepropertiesinplotstreatedwith

Streptomycesspp.Thephosphatemineralizationismainly

pro-videdbyphosphatasesorphosphohydrolases.Theseenzymes converttheunavailablephosphatetoavailableformbyslow mineralizationofphyticacid andpolyphosphates.49 _In_the

presentstudyalltheactinomycetesproducedlyticenzymes indicatingtheirabilitytoconvertcomplexmoleculesto sim-pleravailableforms,whichmaybeattributedtotheincreased levelsoftotalNandavailablePintheinoculatedplots.The substrate usedin measuringthe dehydrogenase activity is 2,3,5triphenyltetrazoliumchloride(TTC)andwasconverted totriphenylformazan(TPF)bytheactionofdehydrogenase, whichisdirectlyproportionaltothenumberof microorgan-isms insoil.28 _In _the _present _study,_all _the _plots _exhibited

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Fig.3–SEMimagesofchickpearootscolonizedwithactinomycetesstrains.

un-inoculated control plots indicates that the Streptomyces

spp.applied inthe ﬁeld were able tosurviveinthe rhizo-sphereandconferPGP.AmongthefourstrainsofStreptomyces

usedinthisstudy,VAI-7wasfoundtoenhancehighestgrain yieldandtotaldrymatter(by20%and17%,respectively).The enhancementinyieldbyVAI-7couldbeitsbothdirectand indirectPGPtraitsincludingIAA,siderophore,chitinase, cellu-lase,lipase,␤-1,3-glucanaseandprotease(Table4).Further,as mentionedearlier,VAI-7alsoenhancedhighestshootaswell asrootlengthsofchickpeaseedlings(Fig.2).VAI-7wasalso foundtoenhancegreatlysoilbiologicalandmineralnutrient properties including microbial biomass carbon, dehydroge-nase,totalN,availablePandorganiccarbon(Table6).Hence,

theStreptomycesstrainVAI-7couldbeexploitedforitsPGPand

yieldenhancementtraits.

TheSEMimages, inthisstudy,showedthat allthe four

Streptomyces strains colonized the roots of chickpea

with-outcausinganydamage.Compoundssuchasaromaticand phenoliccompounds,sugars,aminoacids,organicacidsand carbohydratesfromrootexudatesactsaschemoattractants that helps in root colonization.50,51 _Colonization _accounts

to the proliferation of microbes but not their movement.1

Weller52reportedthatamicroorganismthatcolonizesrootis idealforuseasbiocontrolaswellasplantgrowth-promoting agents.53 _{Actinomycetes} _strains _like _{Micromonospora} _spp.,

Streptomyces spp., Streptosporangium spp., and Thermobiﬁda

spp., were reported as best to colonize the plant rhizo-sphere,showinganimmensepotentialityasbiocontrolagent against wide range ofroot pathogenic fungi.54 _Association

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 85–95

93

Nodule number (plant−1) Leafarea (cm−2plant−1) Leafweight (gplant−1) Podnumber (plant−1) Shootweight (gplant−1) Plantheight (cm) Seedweight (gplant−1) Seednumber (plant−1) Stoveryield (tha−1) Grainyield (tha−1) Totaldry matter (tha−1) SAI-13 50 660 4.13 62 4.17 48 196 77 1.70 1.70 3.40 SAI-29 59 665 3.82 64 3.99 49 199 80 2.17 1.85 4.01 VAI-7 60 692 4.41 79 4.69 49 202 70 1.89 2.09 3.98 VAI-40 61 855 4.09 61 4.05 48 196 85 1.72 1.82 3.53 Control 49 626 3.71 59 3.96 47 195 66 1.68 1.67 3.34 SE± 2.1** _36.4** _0.134* _3.2** _0.159* _0.4* _0.448** _1.6*** _0.059*** _0.089* _0.129** LSD(5%) 6.7 118.8 0.436 10.4 0.490 0.6 3.7 5.2 0.175 0.249 0.346

LSD,leastsigniﬁcantdifference;SE,standarderror.

∗ _{Statistically}_significant_at_0.05. ∗∗ _{Statistically}_significant_at_0.01. ∗∗∗_{Statistically}_significant_at_0.001.

Table6–Effectofthefouractinomycetesonrhizospheresoilmineralpropertiesofchickpeaunderﬁeldconditions(2013–2014)–atﬂoweringandcropmaturity.

Isolate Atﬂowering Atcropmaturity

TotalN (ppm) AvailableP (ppm) Organic carbon (%) Dehydrogenase activity (␮gTPFg−1 soil24h−1) Microbial carbon (␮gg−1soil) TotalN (ppm) AvailableP (ppm) Organic carbon (%) Dehydrogenase activity (␮gTPFg−1 soil24h−1) Microbial carbon (␮gg−1soil) SAI-13 759 9.6 0.56 61.5 634 725 7.1 0.56 76.0 584 SAI-29 685 9.3 0.56 43.5 649 729 7.1 0.56 71.0 778 VAI-7 742 12.6 0.59 50.5 777 736 8.5 0.59 74.5 608 VAI-40 726 9.5 0.57 60.0 782 747 7.5 0.62 68.0 568 Control 675 9.2 0.54 41.0 612 721 6.9 0.55 55.0 551 SE± 11.9* _0.26** _0.005** _2.37** _29.7* _4.6* _0.14** _0.01* _2.51* _24.9** LSD(5%) 46.8 1.03 0.018 9.31 116.7 15.3 0.52 0.038 9.87 97.9

LSD,leastsigniﬁcantdifference;SE,standarderror;TPF:triphenylformazan.

∗ _{Statistically}_signiﬁcant_at_0.05. ∗∗ _{Statistically}_signiﬁcant_at_0.01.

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0 1 2 3 4 5 6 SAI-29 VAI-7 VAI-40 SAI-13

Associate editor: Jerri Édson Zilli

β-1.3 glucanase IAA Siderophore

Fig.4–ExpressionproﬁleofIAA,␤-1,3-glucanaseand siderophoregenesoffouractinomycetesstrains.

productionof antibiotics, extracellular enzymes, phytohor-mones,siderophoresandphosphatesolubilization,protects plantagainstbioticandabioticstress.55_It_is_concluded_that_all

thetestedStreptomycesstrainsnotonlycolonizedontheroots butalsoproliferatedand enhancedPGP inchickpea.Inthe presentstudy,geneexpressionstudiesforIAA,siderophore and ␤-1,3-glucanase genes exhibited up-regulation of the threegenes,supportingthebiochemicalresults.The impor-tanceofgeneexpressionhasbeenwidelyrecognizedingene function.56

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

WethankCouncilofScientificandIndustrialResearch,New Delhi,India,forthe financialsupportprovidedtoM Sreev-idyaduringherPhD.Thiswork was undertakenaspart of theCGIAR ResearchProgramonGrainLegumes. ICRISATis amemberofCGIARConsortium.Wewouldalsoliketothank ICRISATandallofthestaffmembersofthebiocontrolunit, includingAlekhyaG,SatyaA,PrasadPVS,ManoharP,Nagappa B,BharathDandJabbarAfortheirsignificantinputsinthe laboratoryandfieldexperiments.

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