The interaction between arbuscular mycorrhizal fungi and Piriformospora indica improves the growth and nutrient uptake in micropropagation-derived pineapple plantlets.

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ContentslistsavailableatScienceDirect

Scientia

Horticulturae

j o u r n a l ho me 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,∗

a_Laboratório_de_Associaç_ões_{Micorrízicas/BIOAGRO,}_Departamento_de_{Microbiologia,}_Universidade_Federal_de_Viç_osa,_36570-900,_Viç_osa,_MG,_Brazil b_Departamento_de_Fitotecnia,_Universidade_Federal_de_Viç_osa,_36570-900,_Viç_osa,_MG,_Brazil

c_Departamento_de_Ciências_Naturais,_Fundac¸_ão_Universidade_Regional_de_Blumenau,_89012-900_Blumenau,_SC,_Brazil

d_Laboratório_de_Cultura_de_Tecidos_{(LCTII)/BIOAGRO,}_Departamento_de_Biologia_Vegetal,_Universidade_Federal_de_Vic¸_osa,_36570-900,_Vic¸_osa,_MG,_Brazil e_Instituto_Capixaba_de_Pesquisa,_Assistência_Técnica_e_Extensão_Rural,_29375-000,_Venda_Nova_do_Imigrante,_ES,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received14April2015 Receivedinrevisedform 22September2015 Accepted23September2015 Availableonline9October2015 Keywords: Ananascomosus AMF P.indica Phosphoruslevels

a

b

s

t

r

a

c

t

Arbuscularmycorrhizalfungi(AMF)andPiriformosporaindicaarewellknownforpromotinggrowth, development,andnutrientuptakeandforimprovingplantphotosynthesis.Thesefungirepresent promis-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_,_increasing_both_plant_biomass_and_the_absorption_of_all_evaluated_nutrients._With_P_at 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).Brazilianpineapple

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

E-mailaddresses:mkasuya@ufv.br,catarinakasuya@gmail.com

(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)proposed

micro-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

techniqueisusedtoobtainalargenumberofplantsthatare

genet-icallyidenticaltotheparentplant.Forpineapple,thistechnique

hasseveraladvantagesoverconventionalmethodsofpropagation,

includingarapidandefﬁcientincreaseintheproductionofplantsof

selectedvarieties(González-Olmedoetal.,2005;Farahani,2013).

Althoughmicropropagationisefﬁcient,ahighmortalityratehas

beenobservedduringtheacclimatizationprocessdueto

physiolog-icalchangescausedbytheinvitroenvironment,suchaschanges

inthefunctionofthestomataandroots,undevelopedcuticles,and

photosyntheticinefﬁciency(Pospíˇsilováetal.,1999;Hazarikaetal.,

2002;Hazarika,2006;Xiaoetal.,2011;KumarandRao,2012;Singh etal.,2012).

Ifestablishedduringtheearlystageofacclimatization,an

asso-ciationwithbeneﬁcialfungicanreducethestressofacclimatization

andpromotethegrowthofmicropropagatedplants(Kapooretal.,

2008;Singhetal.,2012;Yadavetal.,2013a,b).Inoculationwith

arbuscularmycorrhizalfungi(AMF)(Glomeromycota)and

Pirifor-mosporaindica(rootendophyticfungus,Basidiomycota)hasproven

tobeapromisingalternativefortheproductionofplantletsof

supe-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).

PlantsexposedtohighlevelsofPinthesoilshowreduced

mycor-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-mLglassjars

contain-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,separatedby

morpho-typesandinoculatedontherootsof15-day-oldSorghumbicolor

seedlingsthat hadbeengrown onsterilizedsubstrate.Sorghum

seedlingswerethentransplantedtocones(270cm3₎_in_a

steril-izedsand:expandedclay:soil (2:2:1v:v:v)mixandgrown for4

monthsundergreenhouseconditions.Afterthatperiod,coneswere

checkedforsporulation.Plantswereallowedtodry insitu,and

thecontentsofconeswerestoredinziplockplasticbagsat4◦C

for6months.TheinvitrocultureofP.indicawasobtainedfrom

themicrobialcollectionoftheLaboratoryofMycorrhizal

Associ-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,organic

mat-ter (OM)=1.1dagkg−1 and Premaining=6.6mgL−1. Liming was

performed according toSouza et al. (1999), following the

rec-ommendationsforpineapplecultivationtomeettheCaandMg

requirements for 2cmolcdm−3. The substrate was moistened,

placedinplasticbagsandallowedtorestfor30datroom

tempera-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.

etuni-catum,D.heterogama,R.clarus,P.indica,amixtureofallfungi(Mix)

oranon-inoculatedcontrol(Cont).Therewerefourreplicates,and

theexperimentswereperformedfollowingacompletely

random-izeddesignwitha6×6factorialarrangement(seeSupplementary

material).

The50-day-oldplantlets,withanaverageheightof4.4cmand

12–15leaves,weretransplantedintoplasticpotscontaining1kg

ofsterilizedsubstrate.Whentheplantletsweretransplanted,the

substratewasinoculatedneartheroot,usinganaverageof120

sporesoftheAMFperplantlet.InoculationwithP.indicawas

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(hyphaeandchlamydospores).IntheMixtreatment,40sporesof

eachAMFandtwo1-cmdiscswithKMcontainingP.indicawere

usedforinoculation.Controlplantsreceivednofungalinoculum.

Themoistureofthesubstrateinthepotswascorrected

periodi-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.These

nutri-ents,exceptP,weremeasuredbyopticalemissionspectrometry

withinductivelycoupledplasma(ICP-OES)(Optima8300ICP-OES

Spectrometer,PerkinElmer).ForNanalysisoftheshoots,the

min-eralizationwasperformeddry,bydirectincinerationofthesample

inmufﬂes,anditscontentwasdeterminedbytheKjeldahlmethod

(EMBRAPA,1999).ThePcontentoftheshootswasdetermined

col-orimetricallybythevitaminCmethodasmodiﬁedbyBragaand

Defelipo(1974).

2.6. Photosyntheticparameters

Thevaluesofthephotosyntheticparameterswereassessedwith

aPAMﬂuorometer[JUNIOR-PAMTeachingChlorophyll

Fluorome-ter(Walz,Mess-undRegeltechnik)].Thefollowingphotosynthetic

parameterswereusedforanalysis:maximumphotochemical

efﬁ-ciency or maximum quantum yield (Fv/Fm) and quenching or

non-photochemicaldissipation(qNandNPQ).Thesevalueswere

obtainedfromintermediateandfullyexpandedleaves.The

min-imal ﬂuorescence (Fo) was measured after the application of

modulatedlight (<0.1␮molphoton m−2s−1)toleavesthat had

beendark-adaptedforatleast30min.Themaximalﬂuorescence

(Fm)wasobtainedbyimposingapulsesaturationof10,000␮molm

−2_s−1_for_0.6_s.

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).Rootcolonizationwasquantiﬁedbyusingthegridline

inter-sectmethod(Giovannettiand Mosse,1980)withastereoscopic

microscope.

The mycorrhizal fungidependence was determined

accord-ing 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).Quantitativedataweresubjectedtoaregression

anal-ysis,andtheregressioncoefﬁcientswereanalyzedusingStudent’s

t-test.Thedatarelatingtofungalcolonizationwereﬁrst

normal-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,theinoculatedplantletsgrewbetterthantheir

non-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),dependingonthe

inoc-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

variedinrelationtothecontroldependingontheinoculated

fun-gus.Atthedoseof 20mgkg−1,the plantletsinoculated withP.

indicashowedanincreaseofapproximately260%overthecontrol,

whereasC.etunicatum,R.clarus,D.heterogamaandMixtreatments

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Fig.1.Numberofleaves(NL),plantheight(H)andshootdrymatter(SDM)ofpineappleplantletsmicropropagatedandinoculatedwithC.etunicatum,D.heterogama,R. clarus,P.indica,Mixorcontrol.Measurementswereobtained210daysafterinoculationinthegreenhousewithdifferentPlevelsinthesoil.◦_,_*_and_**,_signiﬁcant_at_10,₅

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.

etuni-catum,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,

respec-tively.ThesevalueswerealsolowerwithincreasingdosesofP,

showinggainsinNcontentof13,19,0.8,−0.88and36%,

respec-tively,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.

ThelevelsofKandMgwerealsoinﬂuencedbyfungal

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Fig.2.Content(mgplant−1₎_of_P,_N,_K,_Mg_and_Ca_in_{micropropagated}_pineapple_plantlets_inoculated_with_C._etunicatum,_D._heterogama,_R._clarus,_P._indica,_Mix_or_control.

Measurementswereobtained210daysafterinoculationinthegreenhousewithdifferentPlevelsinthesoil.◦_,_*_and_**,_signiﬁcant_at_10,₅_and_1%_probability,_{respectively.}

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

clarus,D.heterogamaandMixshowedincreasesof105,161,138,85

and181%inKcontentandof157,211,214,315and374%inMg

con-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.indicaandAMFpositivelyinﬂuencedthephotosynthetic

efﬁ-ciencyoftheplants,especiallyatlowdosesofP(P≤40mgkg−1)

(Fig. 3).The Fv/Fm parameter, which indicatestheefﬁciency at

whichlightenergycapturedinPSIIistransferredtoother

devel-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

inoc-ulated with AMF and for those inoculated with P. indica, with

structurescharacteristicofintraradicularcolonizationobservedin

treatedplantsandnocolonizationinthecontrolplants(Fig.4).

Withinthegroupsoftreatedplantsthatreceivedeachindividual

AMF,therewasnodifference(p_≥0.05)inthepercentageof

fun-galcolonization,evenwhenhigherdosesofPwereappliedtothe

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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.indicadidnotvarywith

increas-ingdosesofP,andremainedlowerthanthatforAMF-inoculated

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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,theplantsdidnotdependonfungal

col-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).

Strongresponsesinthegrowthanddevelopmentofplants

inoc-ulatedwithAMFhavebeenwelldocumentedforseveralspeciesof

plants,e.g.,maize(ZeamaysL.)(Cozzolinoetal.,2013),oregano

(Ori-ganumonites),mint(Mentharequienii)(Karagiannidisetal.,2011),

papaya(Caricapapaya)andpineapple(Rodríguez-Romeroetal.,

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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(Brassica rapa)(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).This

character-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,CaandMgcomparedwiththe

non-inoculatedcontrols.However,theseedlingsinoculatedwithMix

andC.etunicatum,followedbyR.clarus,presentedbetter

absorp-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 the

syn-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

decreaseinrootcolonizationmayberelatedtotheincreased

con-centrationofPpergramofdrymatterintheplant(Liuetal.,2000).

However,here,inpineappleplantletsexposedtothesame

treat-ments,thepercentagesoffungalcolonizationremainedhigh,even

athighdosesofP(Table1).Similarresultswerenotedinthe

stud-iesofXavierandGermida(1997),Cozzolinoetal.(2013)andTong etal.(2013),andthecolonizationpercentagesweremuchhigher

thanthatfoundbyRodríguez-Romeroetal.(2011)inpineapple.

Thismaybeduetothedilutioneffectofthenutrientsinplant

tis-sue,inwhichincreasedabsorptionproducesahigheramountof

biomassratherthanagreaterconcentrationofnutrients,increasing

theexudationofcarbohydratesandthusstimulatingmycorrhizal

colonization(Liuetal.,2000;Azcónetal.,2003).

Foralmostallevaluatedparameters,thetreatmentcontaining

amixtureofallfungiwasmoreeffectiveinpromotingthegrowth

oftheplantlets,whichhasbeenobservedinthestudiesofYadav

(9)

ofdifferentspeciesallocatedindifferentorders(Glomeralesand

Diversisporales—Redeckeretal.,2013)hasbeenshowntohavea

positiverelationshipwithbiomassproductioninPlantago

lanceo-lata(MaheraliandKlironomos,2007).BecausedifferentAMFmay

transmitdifferentamountsofPtotheplant,theireffectsonplant

growthmaybedifferent(Shuklaetal.,2012).However,because

theplantscanbecolonizedsimultaneouslybyfungifrom

differ-enttaxa(Smithetal.,2011),thebeneﬁcialcharacteristicsofeach

funguscouldpotentiallybeexploitedbytheplant.Thispatternis

observedunderﬁeldconditions,inwhichthesumofallthe

bene-ﬁtsofcolonizationbydifferentfungicontributestothesuccessof

mycorrhizaeinfacilitatinggrowthandreproduction(Smithetal.,

2011).

ThedecreaseobservedinfungaldependencewithincreasingP

levelssuggeststhattheeffectoffungalinoculationismore

pro-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-responsiveto

phos-phatefertilizationintermsoffruitproduction(Spironelloetal.,

2004; Guarc¸oni M. and Ventura, 2011; Caetano et al., 2013),

withmycorrhizalcolonizationconsideredoneofthemainfactors

responsibleforthesupplyofPtoplants(Guarc¸oniM.andVentura,

2011).Therefore,inviewoftheimportanceofPnutritioninthe

earlystagesofcropdevelopment,theroleofmycorrhizal

associa-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),inoculatedplantletsarebetternourishedand

vigor-ous,especiallywheninoculatedwithC.etunicatumorMix.Further

ﬁeldevaluationsareneededtodeterminewhetherinoculation

dur-ingtheseedlingstageisreﬂectedinproductivityorcanserveasa

meanstoreducechemicalfertilization.Suchoutcomeswouldresult

notonlyintheoptimaluseoffertilizerbutalsoineconomicbeneﬁts

fortheproducers.

Acknowledgments

TheauthorsthanktheConselhoNacionaldeDesenvolvimento

CientíficoeTecnológico(CNPq),CoordenaçãodeAperfeiçoamento

de Pessoal de Nível Superior (CAPES), Fundac¸ão de Amparo à

PesquisadoEstadodeMinasGerais(FAPEMIG)andFundac¸ãode

AmparoàPesquisaeInovac¸ãodoEstadodeSantaCatarina(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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