The interaction between arbuscular mycorrhizal fungi and j.scienta.2015.09.032

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Contents lists available atScienceDirect

Scientia Horticulturae

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i

The interaction between arbuscular mycorrhizal fungi and

Piriformospora indica improves the growth and nutrient uptake in micropropagation-derived pineapple plantlets

B.C. Moreira

^a

, F.C. Mendes

^a

, I.R. Mendes

^a

, T.A. Paula

^a

, P. Prates Junior

^a

, L.C.C. Salomão

^b

, S.L. Stürmer

^c

, W.C. Otoni

^d

, A. Guarc¸ oni M.

^e

, M.C.M. Kasuya

^a,∗

aLaboratóriodeAssociaçõesMicorrízicas/BIOAGRO,DepartamentodeMicrobiologia,UniversidadeFederaldeViçosa,36570-900,Viçosa,MG,Brazil

bDepartamentodeFitotecnia,UniversidadeFederaldeVic¸osa,36570-900,Vic¸osa,MG,Brazil

cDepartamentodeCiênciasNaturais,Fundac¸ãoUniversidadeRegionaldeBlumenau,89012-900Blumenau,SC,Brazil

dLaboratóriodeCulturadeTecidos(LCTII)/BIOAGRO,DepartamentodeBiologiaVegetal,UniversidadeFederaldeVic¸osa,36570-900,Vic¸osa,MG,Brazil

eInstitutoCapixabadePesquisa,AssistênciaTécnicaeExtensãoRural,29375-000,VendaNovadoImigrante,ES,Brazil

a r t i c l e i n f o

Articlehistory:

Received14April2015 Receivedinrevisedform 22September2015 Accepted23September2015 Availableonlinexxx

Keywords:

Ananascomosus AMF

P.indica Phosphoruslevels

a b s t r a c t

Arbuscularmycorrhizalfungi(AMF)andPiriformosporaindicaarewellknownforpromotinggrowth, development,andnutrientuptakeandforimprovingplantphotosynthesis.Thesefungirepresentpromis- ingtoolssupportingmicropropagatedplantsduringtheacclimatizationstage,andtheirusecanreduce theapplicationofphosphatefertilizers,providingeconomicandenvironmentalbenefits.Therefore,this studyaimedtoevaluatethebenefitsofinoculationwithAMFandP.indicaforthegrowthofplantletsof theImperialcultivarofpineappleinoculatedduringtheacclimatizationstageandgrownwithdifferent levelsofphosphorus(P).TheexperimentconsistedofsixPlevels(0,20,40,80,160and320mgkg⁻¹soil) withinoculationofClaroideoglomusetunicatum,Dentiscutataheterogama,Rhizophagusclarus,P.indica,a mixtureofallfungi(Mix),orcontrol(noinoculation).Theparametersvegetativegrowth,thenutrient contentsintheplants,photosyntheticefficiency,andthecomponentsofdependenceandcolonization byfungiwereassessed.Thefungalinoculationwaseffectiveforplantletgrowth,especiallyuptoaPdose of40mgkg⁻¹,increasingbothplantbiomassandtheabsorptionofallevaluatednutrients.WithPat 80mgkg⁻¹,onlythetreatmentswithC.etunicatumandMixproducedplantletsofbetterqualitythanthe non-inoculatedcontrol.ThecolonizationbyAMFandP.indicawasnotaffectedbytheadditionofPtothe soil,althoughfungaldependencedecreasedundertheseconditionsandcouldbeconsideredmoderate evenat40mgkg⁻¹forplantsinoculatedwithC.etunicatum,R.clarus,P.indicaorMix.Theinoculation ofpineappleplantletsisapromisingmethodthatcanbeemployedtoproducehigh-qualitypropagative materialforthemarket.

1. Introduction

Pineapple(Ananascomosus(L.)Merrill;Bromeliaceae)isnative to the southern and southeastern regions of Brazil, Argentina and Uruguay(Melo etal., 2006).It is a cropof great economic importancein manytropical countries(Beand Debergh, 2006), andapproximately23.34milliontonsofthisfruitwereproduced worldwidein2012(FAOSTAT,2014).Brazilianpineappleproduc-

∗ Correspondingauthorat:LaboratóriodeAssociaçõesMicorrízicas/BIOAGRO, DepartamentodeMicrobiologia,UniversidadeFederaldeViçosa,CampusUniver- sitário,AvenidaPeterHenryRolfss/n,UniversidadeFederaldeViçosa,36570-900, Viçosa,MG,Brazil.Fax:+553138992573.

E-mailaddresses:[email protected],[email protected] (M.C.M.Kasuya).

tioncorrespondsto10.6%oftotalworldwideproduction,occupying the equivalentof 6% of thetotal areaof pineapple plantations worldwide. Brazil ranks third worldwide in the production of pineapplefruit,behindThailandandCostaRica(FAOSTAT,2014).

Pineapple is vegetatively propagated,and thequality of the propagationmaterialsigniﬁcantinﬂuencesplanthealth,develop- mentandyield(BeandDebergh,2006;Kapooretal.,2008;Souza et al., 2013).A wide variety of plant material can beused for propagationofpineapple,includingfruitcrown,lateralbranches (suckersandslips),andseedlingsgrownfromstemsectionsorvia micropropagation(Hepton,2003).Researchhasbeenconducted to enhance themultiplication of pineappleby means of tissue culturetechniques(Smithetal.,2003;Souzaetal.,2013).Pineap- ple explantscan be multiplied in vitro onsolid and liquid MS medium(MurashigeandSkoog,1962)(BeandDebergh,2006;Silva http://dx.doi.org/10.1016/j.scienta.2015.09.032

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etal.,2007).Thismediumcanbesupplementedwithsucroseand cytokininsorauxins(dependingonthepurpose)asa meansto initiateculture,proliferationorgrowthoftheshoots,orevenas a meansof rooting(Smithet al., 2003;Be and Debergh, 2006;

Silvaetal.,2007).Toachievelarge-scaleproductionofpineapple plantletsatareducedcost,Escalonaetal.(1999)proposedmicro- propagationusingtemporaryimmersionsystemsinabioreactor, andthisapproachhasbeenwidelyusedsince.

Micropropagatedplantsare moreuniformand showgreater synchronyofﬂoweringandfruitingintheﬁeld(Singhetal.,2012).

Micropropagationalsofacilitatestheintroductionofplantletsin newareas(Escalonaetal.,1999),theproductionofhigh-quality materialinanyseason(Chandraetal.,2010),large-scaleproduction andpathogen-freecrops(Routetal.,2006;Souzaetal.,2013).This techniqueisusedtoobtainalargenumberofplantsthataregenet- icallyidenticaltotheparentplant.Forpineapple,thistechnique hasseveraladvantagesoverconventionalmethodsofpropagation, includingarapidandefﬁcientincreaseintheproductionofplantsof selectedvarieties(González-Olmedoetal.,2005;Farahani,2013).

Althoughmicropropagationisefﬁcient,ahighmortalityratehas beenobservedduringtheacclimatizationprocessduetophysiolog- icalchangescausedbytheinvitroenvironment,suchaschanges inthefunctionofthestomataandroots,undevelopedcuticles,and photosyntheticinefﬁciency(Pospíˇsilováetal.,1999;Hazarikaetal., 2002;Hazarika,2006;Xiaoetal.,2011;KumarandRao,2012;Singh etal.,2012).

Ifestablishedduringtheearlystageofacclimatization,anasso- ciationwithbeneﬁcialfungicanreducethestressofacclimatization andpromotethegrowthofmicropropagatedplants(Kapooretal., 2008;Singhetal.,2012;Yadavetal.,2013a,b).Inoculationwith arbuscularmycorrhizalfungi(AMF)(Glomeromycota)andPirifor- mosporaindica(rootendophyticfungus,Basidiomycota)hasproven tobeapromisingalternativefortheproductionofplantletsofsupe- riorquality(SahayandVarma,1999;Kapooretal.,2008;Yadav etal.,2013a,b).Thesefungihavealsobeenreportedtoincrease plants’nutrientuptake(Smithetal.,2010;Varmaetal.,2012),tol- erancetodroughtandsaltstresses(Augé,2001;Varmaetal.,2012), resistancetotheeffectsofheavymetals(Azcón-Aguilaretal.,1997;

Varmaetal.,2012)andphotosyntheticefﬁciency(Estrada-Lunaand Davies,2003;Achatzetal.,2010;Boldtetal.,2011;Yadavetal., 2013a,b).

Phosphorus (P) fertilization and phytosanitary control play important roles in the production of high-quality pineapple propagules.ThemanagementofPinthesoilisamajorfactorin achievingsustainableagriculturalsystems(Kahiluotoetal.,2000).

PlantsexposedtohighlevelsofPinthesoilshowreducedmycor- rhizalcolonization(Mengeetal.,1978;Kahiluotoetal.,2000;Grant etal.,2005).Thus,itishighlyimportanttoidentifytheamountof PthatwillmaximizetheeffectofbothAMFandP.indicaandallow properuptakeofthisnutrienttoprovideanadequatenutritional statusfortheplant.Likewise,itishighlyimportanttoestablishan associationwiththesefungithatwillenabletheplanttoextract themaximumbeneﬁtfromthesymbiosis.

Theaimofthisstudywastoobtainhigh-qualitypropagation materialbyevaluatingthebeneﬁtsofinoculationwithAMFand/or P.indicaattheacclimatizationstageandbyexaminingtheroles ofthesefactorsinthegrowth,nutrientuptakeandphotosynthetic efﬁciencyofpineappleplantletsunderdifferentPlevels.

2. Materialsandmethods

2.1. Invitroculture

Micropropagatedplantletsofpineapple,cultivarImperial,were subcultivated in liquid MS (Murashige and Skoog, 1962) cul-

turemediumformultiplication.Themediumwassupplemented with30gL⁻¹sucrose,1.8mgL⁻¹␣-naphthaleneaceticacid(NAA), 2mgL⁻¹indole-3-butyricacid(IBA),and2.1mgL⁻¹kinetin(KIN) atpH5.5.Theculturesweregrownin250-mLglassjarscontain- ing15mLofculturemediumandsealedwithrigidpolypropylene covers.Cultureswerekeptina growthroomat 26±2^◦Cunder a photoperiodof16hlight/8hdarkand underan irradianceof 36␮molm⁻²s⁻¹,providedbywhiteﬂuorescentlamps.Subcultures wereperformedevery40d(seeSupplementarymaterial).

2.2. Fungalinoculants

Isolates of AMF Dentiscutata heterogama PNB102A (= Scutel- lospora heterogama), Claroideoglomus etunicatum RJN101A (=

Glomus etunicatum) and Rhizophagus clarus RJN102A (= Glomus clarum)wereobtainedfromtheInternationalCultureCollection ofGlomeromycota(CICG,www.furb.br/cicg)attheUniversidade RegionaldeBlumenau,SantaCatarina,Brazil.Singlecultureswere establishedfollowingtheproceduresadoptedattheCICG.Brieﬂy, sporeswereextractedfromtrapcultures,separatedbymorpho- typesandinoculatedontherootsof15-day-oldSorghumbicolor seedlingsthat hadbeengrown onsterilizedsubstrate.Sorghum seedlingswerethentransplantedtocones(270cm³)inasteril- izedsand:expandedclay:soil (2:2:1v:v:v)mixandgrown for4 monthsundergreenhouseconditions.Afterthatperiod,coneswere checkedforsporulation.Plantswereallowedtodry insitu,and thecontentsofconeswerestoredinziplockplasticbagsat4^◦C for6months.TheinvitrocultureofP.indicawasobtainedfrom themicrobialcollectionoftheLaboratoryofMycorrhizalAssoci- ationsofUniversidadeFederaldeVic¸osa—MinasGeraisand was maintainedandmultipliedinKaefermedium(KM)(HillandKaefer, 2001)andstoredinthedarkat30^◦C(Kumaretal.,2011)for30d.

2.3. Characteristicsandsoilpreparation

Thesubstratewassterilizedinanautoclavefor1hat121^◦C and was composed of a mixture of soil and sand (1:1 v:v) withthefollowingcharacteristics:pH_(water)=4.9,P=1.1mgdm⁻³ (Mehlich 1),K=34mgdm⁻³, Ca=0.2cmolcdm⁻³,Mg and Al=0, sumofexchangeablebases (SB)=0.29cmolcdm⁻³,organicmat- ter (OM)=1.1dagkg⁻¹ and P_remaining=6.6mgL⁻¹. Liming was performed according toSouza et al. (1999), following therec- ommendationsforpineapplecultivationtomeettheCaandMg requirements for 2cmolcdm⁻³. The substrate was moistened, placedinplasticbagsandallowedtorestfor30datroomtempera- ture.Afterthisperiod,phosphorusfertilizationwasperformedfor eachdoseofP(0,20,40,80,160and320mgkg⁻¹ soil)usingan aqueoussolutionofKH₂PO₄justbeforetransplantationatthetime ofacclimatization.

2.4. Experimentaldesignandinoculation

Theexperimentwithpineappleplantlets(cultivarImperial)was conductedinagreenhouseandconsistedofsixdosesofP(0,20, 40,80,160and320mgkg⁻¹ofsoil)andinoculationwithC.etunicatum,D.heterogama,R.clarus,P.indica,amixtureofallfungi(Mix) oranon-inoculatedcontrol(Cont).Therewerefourreplicates,and theexperimentswereperformedfollowingacompletelyrandom- izeddesignwitha6×6factorialarrangement(seeSupplementary material).

The50-day-oldplantlets,withanaverageheightof4.4cmand 12–15leaves,weretransplantedintoplasticpotscontaining1kg ofsterilizedsubstrate.Whentheplantletsweretransplanted,the substratewasinoculatedneartheroot,usinganaverageof120 sporesoftheAMFperplantlet.InoculationwithP.indicawasper- formedwithfour1-cmdiscsofKMcontainingfungalstructures

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(hyphaeandchlamydospores).IntheMixtreatment,40sporesof eachAMFandtwo1-cmdiscswithKMcontainingP.indicawere usedforinoculation.Controlplantsreceivednofungalinoculum.

Themoistureofthesubstrateinthepotswascorrectedperiodi- callywithdistilledwater.Clark’snutrientsolution(50mL)without Pwasappliedevery20days(Clark,1975).

2.5. Plantgrowthmeasurementsandshootnutrientanalysis At210daftertransplanting,theplantheight(H),thenumber ofleaves(NL),andtheshootdrymatter(SDM)weredetermined.

The SDMwas determinedafter drying toa constant weightat 70^◦Cinanovenunderforcedventilation.Thismaterialwasthen groundinaWileymillwitha0.420-mmsieveandsubjectedto nitroperchloricdigestion(JohnsonandUlrich,1959)todetermine theconcentrationsofthenutrientsP,K,Ca,andMg.Thesenutri- ents,exceptP,weremeasuredbyopticalemissionspectrometry withinductivelycoupledplasma(ICP-OES)(Optima8300ICP-OES Spectrometer,PerkinElmer).ForNanalysisoftheshoots,themin- eralizationwasperformeddry,bydirectincinerationofthesample inmufﬂes,anditscontentwasdeterminedbytheKjeldahlmethod (EMBRAPA,1999).ThePcontentoftheshootswasdeterminedcol- orimetricallybythevitaminCmethodasmodiﬁedbyBragaand Defelipo(1974).

2.6. Photosyntheticparameters

Thevaluesofthephotosyntheticparameterswereassessedwith aPAMfluorometer[JUNIOR-PAMTeachingChlorophyllFluorome- ter(Walz,Mess-undRegeltechnik)].Thefollowingphotosynthetic parameterswereusedforanalysis:maximumphotochemicaleffi- ciency or maximum quantum yield (F_v/F_m) and quenching or non-photochemicaldissipation(qNandNPQ).Thesevalueswere obtainedfromintermediateandfullyexpandedleaves.Themin- imal fluorescence (F_o) was measured after the application of modulatedlight (<0.1␮mol photon m⁻²s⁻¹)toleavesthat had beendark-adaptedforatleast30min.Themaximalfluorescence (Fm)wasobtainedbyimposingapulsesaturationof10,000␮molm

−2s⁻¹for0.6s.

2.7. Fungalcolonization

Approximately0.5gofrootsystemperplantwasdiaphanized in10%KOH(w:v)for12h,followedbythreesuccessivewashes intapwater.Afterwashing,therootsystemwasplacedinHCl2%

(w:w)for5minandthenwasstainedwith0.05%trypanbluein lactoglycerol(w:v)at70^◦Cfor30–40min.Thesamplewasthen storedinlactoglycerol(PhillipsandHayman,1970;Brundrettetal., 1996).Rootcolonizationwasquantiﬁedbyusingthegridlineinter- sectmethod(Giovannettiand Mosse,1980)withastereoscopic microscope.

The mycorrhizal fungidependence was determined according to Plenchette et al. (1983) with modiﬁcations, using the equation:(FD)={(SDMofinoculatedplants−SDMnon-inoculated plants)/SDMof inoculatedplants}×100. Thesameformulawas usedtocalculatetheP.indicadependence.So,wearedenominating fungaldependence(FD)toreferatbothfungi.

2.8. Statisticalanalysis

Thedata weresubjected toanalysisof variance (ANOVA)at an˛ levelof 5%.Themeanswerecompared usingTukey’s test (p≤0.05).Quantitativedataweresubjectedtoaregressionanal- ysis,andtheregressioncoefﬁcientswereanalyzedusingStudent’s t-test.Thedatarelatingtofungalcolonizationwereﬁrstnormal- izedviaanarcsin√(x/100)transformationforasubsequentANOVA.

TheFD datawere classiﬁedaccordingtoHabte andManjunath (1991):>75%=excessivedependence;50–75%=highdependence;

25–50%=moderatedependence;<25%=marginaldependence;and noresponsetoinoculation=independence.

3. Results

3.1. Plantgrowthmeasurements

ThepineappleplantletsrespondedtoinoculationwithbothAMF and P. indica. A quadratic regression model successfully ﬁt the responseofthegrowthparameterstoincreasesintheapplication ofPtothesubstrate(Fig.1).

Ingeneral,theinoculatedplantletsgrewbetterthantheirnon- inoculated counterparts at doses of P up to40mgkg⁻¹ for all treatmentswithfungi(Fig.1).However,withincreasingamountsof Pinthesubstrate,thisgrowthwaslesspronounced.At80mgkg⁻¹ P, only the C. etunicatum and Mix treatments showed positive effects onSDMafterinoculationcompared withthecontrol. At 160mgkg⁻¹,theSDMinthecontroltreatmentwassuperiortothe correspondingvaluesintheothertreatments(Fig.1).

AtlowerdosesofP(0–40mgkg⁻¹P),dependingontheinoc- ulatedfungi,pineappleplantletsshowedasigniﬁcantincreasein thegrowthparametersdependingonfungalidentity.Atthedoseof 0mgkg⁻¹,therewereincreasesinHof70.8,53.4,49.5,52.4and65%

inplantletsinoculatedwithP.indica,C.etunicatum,R.clarus,D.het- erogamaandMix,respectively,whereasNLincreasedby24.7,41.1, 51.3,31.4and60.2%,respectively,forthesamefungi.Forthissame doseofP,SDMincreasedby2079.6,1310.4,1456.1,2003.8and 1889.2inresponsetoP.indica,C.etunicatum,R.clarus,D.heterogama andMix,respectively.

At40mgkg⁻¹,thisbeneﬁtwaslower,butincreasesof25.4,27.9, 18.5,1.1and29.4%inHandof10.2,24.9,24.1,4.9and31.5%inNL wereobservedforP.indica,C.etunicatum,R.clarus,D.heterogama andMix,respectively.Further,theSDMincreasedby27.1,43.2,10.7 and45.4%forP.indica,C.etunicatum,R.clarusandMix,respectively.

Atthe40mgkg⁻¹dose,theplantletsinoculatedwithD.heterogama showedadecreaseof17.71%inSDM(Fig.1).Ingeneral,thebest resultsforgrowthparameterswereobservedwhenplants were treatedwiththemixedfungalinoculum,C.etunicatumorR.clarus.

Theplantletsthatreceived80mgkg⁻¹Pandwereinoculated withR.clarusorP.indicashowed19.53%and13.99%decreasesin SDM,respectively.Atthesamedose, theC.etunicatumandMix treatmentsforthesameparametersproducedincreasesinSDMof 4.50%and13.48%,respectively(Fig.1).

3.2. Nutritionalcontentofplants

Thefungal inoculation(AMFand/or P.indica)wasbeneﬁcial (p≤0.05)forthenutritionalstatusofthepineappleplantlets.The increasedPinthesubstrateinﬂuencedthecontent(mgplant⁻¹) ofPandoftheothermacronutrients,N,K,CaandMg(Fig.2).The levelsofthesenutrientsincreasedwiththeapplicationofhigher dosesofPforalltreatments.Atthelowestdoses(0–40mgkg⁻¹), thetreatmentswithfungishowedhighervaluesthanthosefound inthecontrolplants.AtthehigherdosesofP,increasescompared tothecontrolswereobservedinthecurvesforonlyafewfungal treatments(Fig.2).

GreatereffectsoffungalinoculationonPuptakebyplantswere observedatlowerdosesofP,andthemagnitudeoftheseeffects variedinrelationtothecontroldependingontheinoculatedfun- gus.Atthedoseof 20mgkg⁻¹,the plantletsinoculated withP.

indicashowedanincreaseofapproximately260%overthecontrol, whereasC.etunicatum,R.clarus,D.heterogamaandMixtreatments presentedincreasesof1125,1621,845and2054%,respectively,in

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Fig.1.Numberofleaves(NL),plantheight(H)andshootdrymatter(SDM)ofpineappleplantletsmicropropagatedandinoculatedwithC.etunicatum,D.heterogama,R.

clarus,P.indica,Mixorcontrol.Measurementswereobtained210daysafterinoculationinthegreenhousewithdifferentPlevelsinthesoil.^◦,*and**,signiﬁcantat10,5 and1%probability,respectively.

thePcontentperplant.TheincreasedPuptakewasconsiderably lesspronouncedatthehigherPdoseof40mgkg⁻¹.Thesevalues were−2,192,194,4and228%forP.indica,C.etunicatum,R.clarus, D.heterogamaandMix,respectively.Atdosesabove80mgkg⁻¹, onlythefungiC.etunicatum,R.clarus,andMixwereeffectivefor increasingthecontentofP.Atthisdose(80mgkg⁻¹),theC.etunicatum,R.clarus,andMixtreatmentsproducedgainsof114,80and 139%,respectively,relativetothecontrol(Fig.2).

TheeffectsofthetreatmentsonNcontentweresimilartotheir effectsonPuptake.Atthedoseof0mgkg⁻¹,increasesinNcontent of110,58,84,126and140%wereobservedforthetreatmentswith

P.indica,C.etunicatum,R.clarus,D.heterogamaandMix,respectively.ThesevalueswerealsolowerwithincreasingdosesofP, showinggainsinNcontentof13,19,0.8,−0.88and36%,respectively,forthesametreatmentsatthedoseof40mgkg⁻¹(Fig.2).

At80mgkg⁻¹,onlytheC.etunicatumandMixtreatmentsshowed signiﬁcantgainsintheNcontentperplant,namely7.7%forC.etuni- catumand36.6%forMix.Fortheothertreatmentsatthe80mgkg⁻¹ doseand foralltreatments atgreater doses,thecontrol plants showedahighermeanNcontent.

ThelevelsofKandMgwerealsoinﬂuencedbyfungalinocula- tionandwerehigheratlowerdosesofP.WhenPwasappliedat

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Fig.2.Content(mgplant⁻¹)ofP,N,K,MgandCainmicropropagatedpineappleplantletsinoculatedwithC.etunicatum,D.heterogama,R.clarus,P.indica,Mixorcontrol.

Measurementswereobtained210daysafterinoculationinthegreenhousewithdifferentPlevelsinthesoil.^◦,*and**,signiﬁcantat10,5and1%probability,respectively.

20mgkg⁻¹,theplantletsinoculatedwithP.indica,C.etunicatum,R.

clarus,D.heterogamaandMixshowedincreasesof105,161,138,85 and181%inKcontentandof157,211,214,315and374%inMgcon- tent,respectively.At80mgkg⁻¹,mostoftheinoculatedplantlets stillshowedrelativeincreasesintheabsorptionofKof8,36,10, and29%forP.indica,C.etunicatum,R.clarus,andMix,respectively.

OnlytheplantletsinoculatedwithC.etunicatum,D.heterogamaand MixshowedahigherMgcontentthanthecontrols,withgainsof 9,21and38%,respectively,whenPwasappliedat80mgkg⁻¹soil (Fig.2).

TheCacontentwasalsoaffectedbytheinoculationofpineapple plantletswiththesefungi.TheabsorptionofCaincreasedmainly underexposuretolowerdosesofP,reaching91%(P.indica),126%(C.

etunicatum),113%(R.clarus),133%(D.heterogama)and253%(Mix) ofthecontrollevelatadoseof20mgkg⁻¹.ThishighCacontent comparedwiththecontrolalsodecreasedwithincreasingPdoses.

ForCa,thestrongestpositiveresponsesat80mgkg⁻¹werethose forC.etunicatumandMix(Fig.2).

3.3. Measurementsofphotosyntheticparameters

P.indicaandAMFpositivelyinfluencedthephotosyntheticeffi- ciencyoftheplants,especiallyatlowdosesofP(P≤40mgkg⁻¹) (Fig. 3).The Fv/Fm parameter, which indicatestheefficiency at

whichlightenergycapturedinPSIIistransferredtootherdevel- opingphotochemicalreactions,waslowerinthecontrolsthanin theothertreatmentsuptoadoseof40mgkg⁻¹(oruptoadose of320mgkg⁻¹inthecaseoftheP.indicaandMixtreatments).At 80and160mgkg⁻¹,nodifferencesinFv/Fmwerefoundamongthe treatments.Fortheparametersmeasuringtheamountofenergy capturedbyPSIIthatwasdissipatedasheat(qNandNPQ),the controlsweresuperiortomosttreatmentsatvariousdosesofP.

TheMixtreatmentshowedthelowestqNandNPQatvirtuallyallP levels,followedbytheremainingAMF,C.etunicatum,R.clarusand D.heterogama,whichbehavedsimilarly.ThefungusP.indicawas lessefﬁcientthantheAMFinreducingqNandNPQbutwasmore efﬁcientthanthecontrolatdosesof40and80mgkg⁻¹(Fig.3).

3.4. Fungalcolonization

Thefungal colonizationwassuccessful bothfor plants inoculated with AMF and for those inoculated with P. indica, with structurescharacteristicofintraradicularcolonizationobservedin treatedplantsandnocolonizationinthecontrolplants(Fig.4).

Withinthegroupsoftreatedplantsthatreceivedeachindividual AMF,therewasnodifference(p≥0.05)inthepercentageoffun- galcolonization,evenwhenhigherdosesofPwereappliedtothe soil.WhennoPwasadded,MixshowedalowerpercentageofAMF

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Fig.3.Maximumphotochemicalefﬁciency(Fv/Fm)andquenching(qN)ornon-photochemicaldissipation(NPQ)inmicropropagatedpineappleplantletsinoculatedwithC.

etunicatum,D.heterogama,R.clarus,P.indica,Mixorcontrol(notinoculated).Measurementswereobtainedafter210daysofgrowthinthegreenhousewithdifferentPlevels inthesoil.MeansfollowedbythesameletterwithinthesamedoseofPdonotdiffer(Tukey’stest,5%probability).

Table1

PercentageofcolonizationbyP.indicaandmycorrhizalfungiinmicropropagation-derivedpineappleplantletsunderdifferentphosphorus(P)levelsafter210daysof acclimatizationinthegreenhouse.

P(mgkg⁻¹) P.indica R.clarus C.etunicatum D.heterogama Mix

0 20.94±9.54 bA 67.40±10.41 aA 37.64±27.10 abA 57.75±30.66 abA 59.50±11.39 AB

20 22.78±10.12 cA 72.88±7.03 aA 26.11±22.31 cA 45.49±19.06 bcA 67.82±7.58 abAB

40 28.11±6.32 bA 69.41±7.89 aA 46.95±20.54 abA 42.72±27.27 abA 76.50±15.59 aAB

80 27.31±5.81 bA 74.35±3.58 aA 69.08±15.79 aA 37.45±25.82 abA 74.19±9.01 aAB

160 21.13±5.36 dA 71.07±5.76 abA 46.53±13.24 bcA 39.67±23.02 cdA 75.50±3.70 aAB

320 18.71±6.79 bA 69.95±7.43 abA 41.49±31.66 abA 40.56±22.38 bA 81.50±6.56 aA

Meansfollowedbythesamelowercaseletterinthesamerowandmeansfollowedbythesamecapitalletter,inthesamecolumn,donotdifferbyTukeytestat5%probability.

colonizationbutdidnotdifferfromtheotherfungaltreatments underthesameconditionsofPfertilization(Table1).FortheAMF treatmentswiththesamedoseofP,thetreatmentsinoculatedwith MixorR.clarushadthehighestaveragepercentageofmycorrhizal colonizationatthehigherdosesofP(Table1).Furthermore,the

percentageofcolonizationwithP.indicadidnotvarywithincreas- ingdosesofP,andremainedlowerthanthatforAMF-inoculated plants(Table1).

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Fig.4. TypicalstructuresobservedinassociationwitharbuscularmycorrhizalfungiandPiriformosporaindica.(A)Controlwithoutfungalcolonization;(B,DandF)colonization witharbuscularmycorrhizalfungi,withthepresenceofarbuscules,vesiclesandhyphae;(C)colonizationwithP.indicaintherootcortex;and(E)colonizationbyP.indica intheroothairs,withhyphaeandchlamydospores.Theassessmentwasperformed210daysafterinoculationinagreenhouseinthepresenceofvariousPlevelsinthesoil.

3.5. Fungaldependence

Thefungaldependenceofthepineappleplantletswasexcessive at0mgkg⁻¹andwasmoderatetohighatdosesof20,40mgkg⁻¹ P in the soil for all fungal treatments (Fig. 5). At 80, 160 and 320mgkg⁻¹Pinthesoil,theplantsdidnotdependonfungalcol- onizationforgrowthanddevelopmentintermsoftheresponseof shootbiomass.

4. Discussion

StudiesontheeffectofAMFontissueculture-derivedpineapple plantletsarescarce.However,paststudiesonthistopichaveused variousAMFspecies,includingClaroideoglomusclaroideum(=Glo- musclaroideum),Rhizophagusfasciculatus(=Glomusfasciculatum) (Gutiérrez-Olivaetal.,2009)andFunneliformismosseae(=Glomus

mosseae)(Rodríguez-Romeroetal.,2011),whichwerenotusedin thecurrentstudy.

Ingeneral,plantscolonizedbyAMFandP.indicashowedbetter growthandincreasedabsorptionofnutrients,especiallyifallfungi wereinoculatedtogether(Mix)andatlowerdosesofP.Positive responsestocolonizationbyAMFhavealreadybeenreportedin pineapple(Gutiérrez-Olivaetal.,2009;Rodríguez-Romeroetal., 2011).Note,however,thatthisstudyistheﬁrstreportevaluating thecolonizationofP.indicaanditseffectsonpineappleplantlets andshowingroothaircolonization(Fig.4)asnotedbyWalleretal.

(2005).

Strongresponsesinthegrowthanddevelopmentofplantsinoc- ulatedwithAMFhavebeenwelldocumentedforseveralspeciesof plants,e.g.,maize(ZeamaysL.)(Cozzolinoetal.,2013),oregano(Ori- ganumonites),mint(Mentharequienii)(Karagiannidisetal.,2011), papaya(Caricapapaya)andpineapple(Rodríguez-Romeroetal., 2011),includinginmicropropagatedplantssuchasAcoruscalamus

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Fig.5. FungaldependenceinpineappleplantletsinoculatedwithC.etunicatum,D.

heterogama,R.clarus,P.indicaandMix.Measurementswereobtained210daysafter inoculationinthegreenhousewithdifferentPlevels.>75%=excessivedependence;

50–75%=high dependence; 25–50%=moderate dependence; <25%=marginal dependence;noresponsetoinoculation=independence.

L.(Yadavetal.,2011),GlycyrrhizaglabraL.(Yadavetal.,2013a)and GloriosasuperbaL.(Yadavetal.,2013b).P.indicainoculationhas beenreportedtobeneﬁtbarley(Hordeumvulgare)(Walleretal., 2005), cabbage(Brassicarapa)(Sunet al.,2010)and beet(Beta vulgaris)(Varmaetal.,2012),particularlyifthesoilPlevelsare low.

Althoughthepositiveresponsesofthegrowthparameterswere stronglyrelatedtothecontrolsatsmallerdosesofP,thegrowth ofinoculatedplantletsatthedoseof80mgkg⁻¹wassuperiorto theirgrowthatthedoseof40mgkg⁻¹.Thisresultindicatesthat bothofthesedoses,whichareabovethatrecommended(approx- imately36mgkg⁻¹)forcultureunderconditionsoflowsoilPin theﬁeld(Souzaetal.,1999),aregoodoptionsfortheproduction ofplantletsinoculatedwiththesefungi.TheincreasesinNL,Hand SDMresultingfromassociationswithAMFand/orP.indica,espe- ciallyatrelativelylowdosesofP,arecloselylinkedwiththefungal colonizationandmorphologyoftheroots.Thepineapplehasashort andcompactrootsystemnearthestem,withmanythickrootsand limitedbranching(d’EeckenbruggeandLeal,2003).Thischaracter- isticindicatesthatfungalcolonizationplaysanimportantrolein theuptakeofwaterandnutrients.Colonizedrootscanexplorea greatervolumeofsoil(SmithandRead,1997)andshowmorethan 100timesthecoverageareaoftherootsofanon-colonizedplant (Sieverding,1991).Moreover,thesmaller-diameterhyphalfungi allowaccesstothesoilmicropores(Grantetal.,2005),andthe broadgrowthofthecolonizedrootsresultsinawell-developed networkofseveralcentimetersinextent,whichcanreachareas wherePisnotdepleted(Smithetal.,2011).Incontrast,plantsnot colonizedwiththesesymbioticfungicannotexploretheseareas.

NutrientabsorptionwasconsiderablyinﬂuencedbyAMFand P.indica,especiallyuptoaPdoseof40mgkg⁻¹(Fig.2).Thefungi increasedthecontentsofP,N,K,CaandMgcomparedwiththenon- inoculatedcontrols.However,theseedlingsinoculatedwithMix andC.etunicatum,followedbyR.clarus,presentedbetterabsorp- tionformanyofthesenutrients,evenataPdoseof80mgkg⁻¹. Thesebeneﬁts observedin colonized plantlets have oftenbeen describedfortheuptakeofP(Kahiluotoetal.,2000;Yadavetal., 2010;Rodríguez-Romeroetal.,2011;Tongetal.,2013;Xieetal., 2014),ofN(Azcónetal.,2008;Oelmülleretal.,2009;Rodríguez- Romeroetal.,2011;Tongetal.,2013),ofK(Rodríguez-Romero etal.,2011),ofS(Oelmülleretal.,2009),ofCuandZn(Kahiluoto

etal.,2000;Karagiannidisetal.,2011;Tongetal.,2013),andofCa, FeandMn(Karagiannidisetal.,2011).Theseﬁndingsincludethe resultsofstudiesofpineappleplantlets(Rodríguez-Romeroetal., 2011)andmayvarybasedonthespeciesofplant-colonizingAMF incombinationwiththeconcentrationofPinthesoil(Gutiérrez- Olivaetal.,2009).Inaddition,differencesinthesebeneﬁtsmay dependontheplantvarietystudied(Karagiannidisetal.,2011)or thehosttissueanalyzed(Tongetal.,2013).

For the pineapple plantlets that received more than 160mgPkg⁻¹, the controls presented better growth parame- ters and nutrient contents per plant, indicating that the fungi tend tohave deleteriouseffects onthese characteristicsat this highlevelofPinthesoil.TheincreasingavailabilityofPinsoil, incombinationwiththehighrateofcolonization,canreducethe growthoftheplantduetothehighercostofcarbonsynthesizedby theplanttosupplythefungianddecreasedbeneﬁtsfornutrient absorptionbecausetheiravailability isgreater(Kahiluotoetal., 2000).

AtlowerdosesofP,pineappleplantletsinoculatedwithAMF showedhigher efﬁciencyof PSIIin capturingenergy, which is senttootherphotosyntheticreactions.However,thecontrolslost moreenergyasheat,showinghigherqNandNPQvaluesthanthe inoculatedplants,particularlyatdosesof20,40and80mgkg⁻¹ (Fig.3).Asaresult,thephotosyntheticrateofinoculatedplantlets washigherthanthatofthecontrols.Thisbehaviorwasalsofound intomatoplantsinoculatedwithF.mosseae(Boldtetal.,2011).In barleyseedlings,ahigherphotosyntheticratewasobservedunder lowlightconditionswhenplantswereinoculatedwithP.indica (Achatzetal.,2010).Investmentsofplantresourcesin thesyn- thesisofphotoassimilatestobetransferredtofungisuchasAMF andP.indicaareoffset,inpart,bythehigherphotosyntheticrates (Balotaetal.,2011;Smithetal.,2011)and/orbysavingsresulting fromthereductioninrootproduction (Smithetal.,2011).Fac- torsthat enhancephotosynthesisbymycorrhizalplants include increasesinthetransportofinorganicelementsfromthesoilto theplant(Soleimanzadeh,2010),inchlorophyllcontent(Estrada- LunaandDavies,2003;Yadavetal.,2013a,b),andintheratesof photosyntheticstorageandexport(Augé,2001).

Thesecharacteristicsofgrowthpromotion,improvednutrient contentandenhancedphotosyntheticefﬁciencyhavemotivated increasingresearchactivityontheinoculationofplantlets with AMF(Estrada-LunaandDavies,2003;Kapooretal.,2008;Singh etal.,2012;Yadavetal.,2013a,b)andwithP.indica(Sahayand Varma,1999)toaidin theacclimatizationstage.Forpineapple, theinoculated plantletsweremorevigorous andshowedbetter developmentduringthisstage.

Ingeneral,increasesintheavailabilityofsoilPtendtodecrease mycorrhizalcolonization(Kahiluotoetal.,2000;Soleimanzadeh, 2010;Balotaetal.,2011;Smithetal.,2011;Shuklaetal.,2012).This decreaseinrootcolonizationmayberelatedtotheincreasedcon- centrationofPpergramofdrymatterintheplant(Liuetal.,2000).

However,here,inpineappleplantletsexposedtothesametreat- ments,thepercentagesoffungalcolonizationremainedhigh,even athighdosesofP(Table1).Similarresultswerenotedinthestud- iesofXavierandGermida(1997),Cozzolinoetal.(2013)andTong etal.(2013),andthecolonizationpercentagesweremuchhigher thanthatfoundbyRodríguez-Romeroetal.(2011)inpineapple.

Thismaybeduetothedilutioneffectofthenutrientsinplanttis- sue,inwhichincreasedabsorptionproducesahigheramountof biomassratherthanagreaterconcentrationofnutrients,increasing theexudationofcarbohydratesandthusstimulatingmycorrhizal colonization(Liuetal.,2000;Azcónetal.,2003).

Foralmostallevaluatedparameters,thetreatmentcontaining amixtureofallfungiwasmoreeffectiveinpromotingthegrowth oftheplantlets,whichhasbeenobservedinthestudiesofYadav etal.(2011)andYadavetal.(2013a,b).Thecomplementaryeffect

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ofdifferentspeciesallocatedindifferentorders(Glomeralesand Diversisporales—Redeckeretal.,2013)hasbeenshowntohavea positiverelationshipwithbiomassproductioninPlantagolanceo- lata(MaheraliandKlironomos,2007).BecausedifferentAMFmay transmitdifferentamountsofPtotheplant,theireffectsonplant growthmaybedifferent(Shuklaetal.,2012).However,because theplantscanbecolonizedsimultaneouslybyfungifromdiffer- enttaxa(Smithetal.,2011),thebeneficialcharacteristicsofeach funguscouldpotentiallybeexploitedbytheplant.Thispatternis observedunderfieldconditions,inwhichthesumofallthebene- fitsofcolonizationbydifferentfungicontributestothesuccessof mycorrhizaeinfacilitatinggrowthandreproduction(Smithetal., 2011).

ThedecreaseobservedinfungaldependencewithincreasingP levelssuggeststhattheeffectoffungalinoculationismorepro- nouncedatlowerdosesofP(Kahiluotoetal.,2000;Balotaetal., 2011).However,evenwithnodependenceoftheplantletsatthe 80mgkg⁻¹doseandmoderatedependenceatthe40mgkg⁻¹dose forC.etunicatumandMix,thehighrateofcolonizationunderthese conditionsishighlysignificantforpineapplecultivationbecause therecommendedsoilPlevelforthiscultureinsoilswithalowP levelisapproximately35mgkg⁻¹ (Souzaetal.,1999).Thiseffect maybemoresignificantunderfieldconditions,wherethesoilisless homogeneousandmycorrhizalassociationcanimprovenutrient absorption.

Inthefield,pineappleis considerednon-responsivetophos- phatefertilizationintermsoffruitproduction(Spironelloetal., 2004; Guarçoni M. and Ventura, 2011; Caetano et al., 2013), withmycorrhizalcolonizationconsideredoneofthemainfactors responsibleforthesupplyofPtoplants(GuarçoniM.andVentura, 2011).Therefore,inviewoftheimportanceofPnutritioninthe earlystagesofcropdevelopment,theroleofmycorrhizalassocia- tionattheseedlingstageremainstobeestablishedinthecontextof theexplorationoftheoverallproductivepotentialofplants(Grant etal.,2005).

For pineapple plantlets, inoculation withAMF and P. indica confersmanybeneﬁtsanddoesnotinterferewiththeprocessof producingpropagativematerialovertime.Inaddition,itdoesnot addalabor-intensivecomponenttotheproductionchainbecause theinoculationmethodissimpleandplantletsmustremaininthe greenhouseforapproximatelysixmonths(Farahani,2013).

5. Conclusions

MicropropagatedpineappleplantletscolonizedbyAMFandP.

indicashowenhancedgrowth,nutrientuptakeandphotosynthetic efficiency,particularlyatlow soilPlevels(≤40mgkg⁻¹).AtaP levelequivalenttotherecommendedfertilizationrate(35mgkg⁻¹ inthefield),inoculatedplantletsarebetternourishedandvigor- ous,especiallywheninoculatedwithC.etunicatumorMix.Further fieldevaluationsareneededtodeterminewhetherinoculationdur- ingtheseedlingstageisreflectedinproductivityorcanserveasa meanstoreducechemicalfertilization.Suchoutcomeswouldresult notonlyintheoptimaluseoffertilizerbutalsoineconomicbenefits fortheproducers.

Acknowledgments

TheauthorsthanktheConselhoNacionaldeDesenvolvimento CientíficoeTecnológico(CNPq),CoordenaçãodeAperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à PesquisadoEstadodeMinasGerais(FAPEMIG)andFundaçãode AmparoàPesquisaeInovaçãodoEstadodeSantaCatarina(FAPESC) forfinancialsupport.TheauthorsalsothankUniversidadeRegional de Blumenau-Santa Catarinafor providing theinoculum main-

tainedattheInternationalCultureCollectionofGlomeromycota (CICG).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.scienta.2015.

09.032.

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