Does co-inoculation of Lactuca serriola with endophytic and arbuscular mycorrhizal fungi improve plant growth in a polluted environment?

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Mycorrhiza(2018)28:235–246

https://doi.org/10.1007/s00572-018-0819-y ORIGINALARTICLE

Doesco-

inoculationofLactucaserriolawithendophyticandarbuscularmycorrhizalf ungiimproveplantg rowthinapollutedenvironment?

RafałW ażny¹^&PiotrR ozpądek¹^&RomanJ . Jędrzejczyk¹^&MartaŚliwa²^&AnnaS tojakowska³^&TeresaA nielska²^&

KatarzynaTurnau²

Received:24August2017/Accepted:3January2018/Publishedonline:23January2018

#TheAuthor(s)2018.Thisarticleisanopenaccesspublication

Abstract

Phytoremediationofpollutedsitescanbeimprovedbyco-

inoculationwithmycorrhizalandendophyticfungi.Inthisstudy,theeffectsofsingle-andco-

inoculationofLactucaserriolawithanarbuscularmycorrhizal(AM)fungus,Rhizoglomusintraradices,andendophyticfungi,Muco rsp.orTrichodermaasperellum,onplantgrowth,vitality,toxicmetalaccumulation,sesquiterpenelactoneproductionandflavonoid concentrationinthepresenceoftoxicmetalswereevaluated.InoculationwiththeAMfungusincreasedbiomassyieldoftheplantsgro wnonnon-pollutedandpollutedsubstrate.Co-

inoculationwiththeAMfungusandMucorsp.resultedinincreasedbiomassyieldofplantscultivatedonthepollutedsubstra te,whereasco-inoculationwith

T.asperellumandtheAMfungusincreasedplantbiomassonthenon-pollutedsubstrate.InthepresenceofMucorsp.,mycor- rhizalcolonizationandarbusculerichnesswereincreasedinthenon-pollutedsubstrate.Co-

inoculationwiththeAMfungusandMucorsp.increasedZnconcentrationinleavesandroots.Theconcentrationofsesquiterpenel actonesinplantleaveswasdecreasedbyAMfungusinoculationinbothsubstrates.Despiteenhancedhostplantcostscausedbymai ntainingsymbiosiswithnumerousmicroorganisms,interactionofwildlettucewithbothmycorrhizalandendophyticfungiwasmore beneficialthanthatwithasinglefungus.Thestudyshowsthepotentialofdoubleinoculationinunfavourableenvironments,including agricul-turalareasandtoxicmetal-pollutedareas.

KeywordsLactucaserriola.Mucor.Trichoderma.Arbuscularmycorrhiza.Endophyticfungi.Co-inoculation

Introduction

Innature,vegetationisalmostalwaysaccompaniedbyfungiand bacteriawhichoftenareinvisibletoobserversbutcansignific antlyinfluenceplantbiology.Indegradedenviron- ments,thediversityoftheplantandsoilmicrobiomeusually

isseverelylimited;thus,restorationattemptsrequireutiliza- tionofcarefullyselectedmicroorganisms.Themajorityofthestu diesconcerningtheroleoffungiinconferringplanttoxicmetal(

TM)stresstolerancewereconductedwitharbuscularmycorrhi zalfungi(AMF).Thisgroupofmicroorganismscanimprovepla ntgrowthandadaptationtounfavourablehabitatssuchasindus trialwastes,areassurroundingthemorthose

undercontinuousinfluenceofanthropogenicpressure

ElectronicsupplementarymaterialTheonlineversionofthisarticle(http s://doi.org/10.1007/s00572-018-0819-

y)containssupplementarymaterial,whichisavailabletoauthorizedusers .

*RafałWażnyrafal.wazn y@uj.edu.pl

1 MałopolskaCentreofBiotechnology,JagiellonianUniversity, Gronostajowa7a,30-387Kraków,Poland

2 InstituteofEnvironmentalSciences,JagiellonianUniversity,Gro nostajowa7,30-387Kraków,Poland

3 DepartmentofPhytochemistry,InstituteofPharmacology,PolishAca demyofSciences,Smętna12,31-343Kraków,Poland

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232 Mycorrhiza(2018)28:235–246

(Orłowskaetal.2005;Turnauetal.2010).Theirimportancei nnon-

pollutedenvironmentsisjustassignificant(Jeffriesetal.20 03).Althoughlaboratoryexperimentsoftenconfirmtheeffe ctofthesefungionplantgrowth,theresultsoffieldstudiesare notalwaysunequivocal.

Co-

inoculationstudies,whereplantsareinoculatedbymoretha nonetypeofmicroorganism,area rarity.Investigationsi ncludingtheroleoftheabioticenvironmentinsuchcasesare evenlesscommon.Descriptionsofmulti-

organismalassociationsalmostexclusivelyconcernco- inoculationwithRhizobiumandplantgrowthpromotingbac -teria(PGPB)(Remansetal.2008;Ahmadetal.2011),

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ectomycorrhizalfungalspeciesandmycorrhizalhelperbacte- ria(MHB)(Frey-

Klettetal.2007)orAMFandbacteria(Liuetal.2012;Bonaetal.

2016).Theeffectofco-

inoculationusuallyisbeneficialforplantgrowth(Remanseta l.2008;Liuetal.2012),butFlor-Peregrínetal.

(2014),whileinvesti-gatingco-

inoculationwithAMFandendophyticbacteria,foundthatco- inoculationhadanegativeeffectonplantscom-

paredtosingleinoculationwithAMForendophytes.Thus,inord ertogainacomprehensiveunderstandingoftheroleofsymbio ticmicroorganismsinplantbiology,studiesthatin-

cludedifferenttypesofmicroorganismsinhabitingtheplantho stinmulti-

microbesetupsarenecessary.Thelackofsuchstudiesmakesitdi fficulttounderstandthecomplexityofthesymbiosisbetweenpl antsandfungi(Omacinietal.2006).

Theuseofmycorrhizalfungiandrhizosphericbacteriainphy toremediationhasbeenreportedpreviouslyasreviewedbyRo zpądeketal.(2017)andMartinetal.

(2017).Recently,thefitnessofVerbascumlychnitisgrowninZ n-Pbindustrialsubstratewasshowntobeimprovedbyco- inoculationwithanarbuscularmycorrhizal(AM)fungusandfu ngalendophytes(Wężowiczetal.2017).Endophytes,whichc olonizeplanttissueswithoutcausinganynegativeeffec ts(HirschandBraun1992;Rodriguezetal.2009),canincreaser ootandshootbiomass(Varmaetal.1999;Omacinietal.2006;So leimanietal.2010)andcanprotectplantsagainstpathogensand unfavourableenvironmentalconditionssuchashightemperatu resandsalinity(Redmanetal.2002;Rodriguezetal.2008).

LactucaserriolaL.(wildlettuce)isacommonweed,con- sideredapioneerofopenhabitats(Lebedaetal.2004)becauseofit shightoleranceofpoorwaterandnutrientavailability(Gallar doetal.1996).Auniquefeatureofthisspeciesisitsabilitytoorie ntitsleavesinthenorth-

southdirection,therebylimitingwaterloss(WerkandEhlering er1985).Wildlettuceisabundantlyfoundalongroadsides,aban donedfields,fieldmarginsandforestclearings(WeaverandDo wns2003),andinterestingly,alsoonZn-

Pbtailings(Turnauetal.2012).

L.serriolarecentlyhasbeenproposedtobeusedformonitor- ingsoilpollution(LeGuédardetal.2012).ThegenusLactucahas beenshowntoproducecharacteristicsecondarymetabo- lites,sesquiterpenelactones,whichaccumulateinlatexcom- ponentscalledlaticifers(Michalskaetal.2009).Theselac- tonesarethesourceofthebitternessofwildlettuceleavesandi ncreasetheirrepellencetoherbivores(ReesandHarborne1 985).

Theaimofthisresearchwastobroadenourviewregardingthe responseofplantsassociatedwithmultiplemicroorgan- ismsinatoxicmetalenrichedenvironment.Weinvestigatedthei nteractionbetweenL.serriolaandanarbuscularmycor- rhizal(AM)fungusandfungalendophytesthatwereisolatedfro mplantsgrowingonindustrialwastesinSouthernPoland.Recen

tly,Rozpądeketal.

(2018)haveshowntheimportanceofaMucorstrainforplantfitne ssandmetalhomeostasis.This

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fungusisanendophytethatcolonizesboththerootsandthesho otsofArabidopsisarenosa.Thestrainwasselectedforuseinthe currentresearchinordertoverifyitspotentialtocolo-

nizeandexertitsbeneficialeffectonspeciesotherthan A.arenosa.L.serriola,beingamycorrhizalplant,wasinoc- ulatedwithMucorsp.andtheeffectwascomparedtoanotheren dophyticfungus,TrichodermaasperellumSamuels,Lieckf.

&Nirenberg,ofknownbehaviourandgrowth-improvingpo- tential(Viterboetal.2010).

Method s

Plant,fungiandsubstrate

SeedsofL.serriola(collectedfromplantsinthevicinityofKra ków,Poland)weresurfacesterilizedin8%sodiumhypochlor itefor5 min,followedby96%ethanolfor1minand75%etha nolfor3minandwashedfivetimeswithsteriledeionizedwate randthengerminatedinsterileconditionsonMurashigeandS kooq(MS)mediumdilutedfourtimesandwithaddedsucrose.

Thegerminationwasconductedat4 °Cindarknessfor2 days, followedby14hphotoperiodat21/17°C.Twoweekslater,see dlingsweretransferredintoMSmediumand,after2 daysofa daptation,theywereinoculatedwiththeendophyticfun- giMucorsp.

(NCBIaccessionnumberKU234656;strainUNIJAG.PL.5 0fromArabidopsisarenosa(L.)Hayekseeds)orTrichoder maasperellum(NCBIaccessionnum-

berMG571529;strainUNIJAG.PL.6fromDeschampsiaces pitosa(L.)P.B.leaves).Fivedaysafterinoculation,theplants weretransferredtopotcultureswithpolluted(P)ornon- polluted(NP)substrateandwithorwithoutAMfun- gusinoculum.

TheNPsubstratewasa mixtureofgardensoil(sup- pliedbyARO,Poland;pH5–6.5;N-NO3,100–

300mg/L;P,80–300mg/L;K,150–

450mg/L),sandandclayinequalvolumes.TheP substratewa smadebyaddinganadditionalvolumeofthesubstratecollecte dfromthein-

dustrialwastesite,Trzebionka(Poland50°09′34.5″N,19°25′

17.2″E)

(Orłowskaetal.2005),totheAROsoilsandandclaymixture(1 :1:1:1;v/v/v/v).Bothsubstratesweresupplementedwith1 00g/Lrockphosphate(Siarkopol,Poland).Available P(Colwell1963),KjeldahlNandorganicmatterconcentrat ionsinthesub-

stratesweremeasuredaccordingtoWilke(2005).Zn,Cd,Pb, FeandK concentrationsinthesubstratesalsowereinvestigat ed.Thewatercontentinsamples(at105°C)wasdeterminedb yamoistureanalyser,andthenthesam-

plesweredigestedin65%nitricacid(5ml)for2h(roomtemper ature—1h,atboilingpoint—1h).Aftercooling,

1.65cm^3of30%H2O2wasaddedandthesuspensionwas

heatedtotheboilingpoint.Thesuspensionwas

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Mycorrhiza(2018)28:235–246 237

1−

φ P0

ABS

P0

centrifugedfor15minat3000rpm,andthesupernatantwastran sferredtoa graduatedflask.Theprecipitate(ifobserved)wastr eatedwithdeionizedwaterandshakenuntilasuspensionforme danew;thissuspensionwascen-

trifuged.Thesupernatantwastransferredtoa graduatedflaskc ontainingthesolutionafterthefirstcentrifugation.Thisproced urewasrepeatedfivetimes.Theprecipitatewasdriedandthepo ssiblemetalcontentwasevaluatedbyX-

rayfluorescencespectroscopy.Thesolutionintheflaskwasma deupto25cm^3withdeionizedwater.Thismethodinvolvesacidd igestionthatdissolvesalltheele-

mentspresentinthematerial(Huguetetal.2015).Todetermin emetalconcentrations,atomicabsorptionspec-

trometry(flameatomicabsorptionspectrometry[FAAS]orgr aphitefurnaceatomicabsorptionspectrometry[GF-

AAS],equippedwithZeemanEffectbackgroundcorrec- tionandaCSX260auto-sampler[ThermoScientific,iC 3000])wasused.Detailedcharacteristicsofthesubstrates

leafChlorophylla andflavonoidindicesfromlighttransmi ssion.

FluorescenceofchlorophyllaandtheJIPtest

Chlorophyllfluorescencemeasurementswereperformedwith aHandyPeafluorimeter(HansatechInstruments,UK).Beforet hemeasurement,twomatureleaves,withoutremovingthem,of eachplant(9weeksold)weredark-

adaptedfor20mininaspecialclips.Datawereprocessedwiththe BIOLYZERsoft-

ware(LaboratoryofBioenergetics,Geneva,Switzerland).Eac hfluorescenttransientwascalculatedaccordingtotheJIP test(Tsimilli-MichaelandStrasser2008).Thefollowingmulti- parametricindiceswereusedtoassessplantvitality(Strasser etal.2000):

PIABSi stheperformanceofthephotosynthesisapparatusexpr essedinrelationtoabsorption:

RC

φ_P0 ψ₀ areshowninTable1.

Mycorrhizalinoculumwaspreparedinpotculturesof PlantagolanceolataL.withRhizoglomusintraradices(N.C.

PI_ABS¼A BSx1

−φ where^RC

x1−ψ0

Schenck& G.S.Sm.)Sieverd.,G.A.Silva& Oehl.Approx imately5mloftheinoculum,containingspores,my-

celiumandcolonizedrootfragmentswasmixedwiththeup- perlayeroftheexperimentsubstrates.Onlysandwithclay(AM fungusinoculumcarrier)wasaddedtocontrolplants.

ABSisameasureofthefractionofreactioncentrechlo- rophyll(ChlRC)perchlorophylloftheantennae(ChlAntenna).

1−φP0indicatesthecontributionoflightreactionsforprimaryphoto chemistryaccordingtotheJIPtest.Electrontransport

beyondQa(primaryquinoneacceptor)isquantifiedas^ψ^0.

Theexperimentwasafullycrossed,threefactordesignwith 0

mainfactorsofsubstrate(NPandP)×AMfungus(+and−) ×endo phyte(none,Mucorsp.orT.asperellum)performedin3replicate Sunbags×5pots/Sunbag(i.e.,180plantsi n36Sunbags;Sigma- Aldrich,USA)inagreenhouseat22°Cinnaturallightfor9week sfromMarchtoMay.Eachplant(oneperpot)wasprovidedwith 0.6Lofsubstrateandirrigatedonceaweekwith8mllongAshton nutrientsolution(0.08MKNO3;0.008μM(NH4)6Mo7O24·4H2

O;0.01MCa(NO3)2).

Chlorophyllandflavonoidmeasurement

Chlorophyllaandflavonoidconcentrationsweremeasuredintw oleaves(withoutremovingthem)ofeach8-week-

oldplant(N=15/treatment)withaDualexScientificfluorom eter

Performanceindex(PItotal):

RE PItotal¼PIABSx

ABS

where^REindicatesthecontributionofthereductionofendequiv alents.

Plantb iomass Nine-week-

oldplantswerecollectedandevaluatedforthefreshanddry weight.Fordryweightmeasurement,plantswereairdried atroomtemperaturefor2weeks.Dryweightwasusedfordeter minationofmycorrhizaldependency(MD),calculatedaccordi ngtothePlenchetteetal.(1983)index:

DWM−DWNM

(Force-A,France)accordingtothemanufacturers’instruc-

tions.Thisinstrumentenablesnon-destructiveassessmentof MD¼

DWM x100ð%Þ

Table1 Chemicalpropertiesofthenon-

polluted(NP)andpolluted(P)substratesusedintheexperiment.DifferentletterswithineachcolumnindicatestatisticallysignificantdifferencesatP≤0.05(n

=3)byttest

pH(inKCl) Pavailable( mg/kg) K(g/kg) Organicmatter(%) N(%) Zn(mg/kg) Cd(mg/kg) Pb(mg/kg) Fe(g/kg) NP 6.6±0.1a 19.3±4.1b 2.5±0.3a 6.4±0.1a 0.3±0.04a 52.0±2.1b 7.2±1.9b 0.4±0.1b 2.5±0.4b P 7.3±0.2a 34.7±10.2a 2.6±0.1a 6.0±0.1a 0.1±0.05a 917.3±21.9a 33.1±2.0a 6.3±0.1a 6.3±0.2a

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236 Mycorrhiza(2018)28:235–246 Datapresentmean±standarderror

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whereDWM—dryweightofmycorrhizalplants;DWNM— dryweightofnon-mycorrhizalplants.Thismycorrhizadepen- dencyformulaconsiderstheplantresponsetomycorrhizas.Pla ntswithamycorrhizaldependencycloseto100%arecon- sideredasfullydependentonAM.

Fungalcolonization

EndophyticcolonisationinplanttissuesstainedaccordingtoAt sattandWhiteside(2014)wasobservedwithlightfieldmi croscopy(OlympusBX53).Fortheestimationofmycor- rhizalcolonization,rootswerepreparedaccordingtothemod- ifiedPhillipsandHayman(1970)method.Therootswere washedintapwater,clearedin10%ofKOHfor24h,washedagain ,acidifiedin5%lacticacidfor2handstainedin0.01%anilineblue inpurelacticacidfor24hatroomtemperature.Stainedrootswer estoredinpurelacticacid,cutinto1cmpiecesandmountedingl ycerolonmicroscopicslides.Atleast45rootpiecesperplantwer eanalysed.Mycorrhizalfrequency(F

%),absolutemycorrhizalcolonization(m

%)andabsolutearbuscularrichness(a

%)wereassessed(Trouvelotetal.1986;http://www2.dijon.in ra.fr/mychintec/Mycocalc-prg/download.html).

Toxicmetalconcentrations

Zn,CdandPbconcentrationsinleavesandrootsweremea- suredaccordingtothemethoddescribedforthesubstrates(

BPlant,fungiandsubstrate^subsection).

Sesquiterpenelactonecontent

Dry,pulverizedplantshoots(0.1g)weretreatedtwicewith10 mlofCH3OHatroomtemperature.Thepooledextractswereev aporatedinvacuoandtheresiduewasdissolvedin70%CH3CN (1ml),lefttostandovernightat4 °C,centrifuged(11.340×g,5 min)andanalyzedbyRP-

HPLC/DADmethodaccordingtoStojakowskaetal.

(2012).Samples(5μl)wereinjectedintoaPurospherRP- 18e(3×125mm,particlesize5 μm)column(Merck,Darmsta dt,Germany)andelutedwitha mobilephaseconsistingofwa terandCH3CN,ataflowrateof

1mlmin⁻¹,at40°C.Gradientelutionconditionsde- scribedbyGrassetal.(2006)wereapplied.Typicalre-

tentiontimesoftheanalyzedsesquiterpenelactoneswereasfol lows:lactucin(LC)—9.3min,lactucopicrin(L C P i kr )

—3 0 . 8m i nan d8 - de o xyl actuci n( 8 -DeoxyLC)—

25.5min.Quantificationwasperformedbymeasurementofpe akareasat260nmwithguaianolidecrepidiasideAasthereferen ce.

Statistics

StatisticalcomparisonswereperformedusingStatistica 12.5(StatSoft)andwereconsideredsignificantatP ≤

0.05.Datanormaldistributionandvariancehomogeneitywere assessedwithShapiro-

Wilk’sandLevene’stests,respectively.Ifnecessary,data(chl orophylla fluores-

cence)werenormalizedwitha log10transformation.Differ encesweretestedbytwo-way(mycorrhizalcoloni-

zation,toxicmetalconcentration)andthree-way(chloro- phyllandflavonoidindex,chlorophylla fluorescence,plantf reshanddrybiomass,lactonesconcentrations)analysisofva riance(ANOVA)followedbytheTukey’spost-

hoctest(SupplementaryTable1).Non-

pollutedandpollutedsubstrateswerecomparedbyttest.Thiste stwasalsoappliedforcomparisonofJIPtestcomponentsbe- tweentestedtreatmentsandthecontroltreatment.

Result s

Chlorophyllaandflavonoids

AMfungusinoculationdecreasedChlorophyllaconcen- trationinplantsgrownonNPandPsubstrates(Fig.1a).Endoph yticMucorincreasedchlorophyllaconcentrationwhenco- inoculatedwithAMfungusonP,whereas

T.asperellumdidnotaffectit.Totalflavonoidconcentra- tionwassignificantlyhigherinplantsgrownwithoutAMfungu sinoculum(Fig.1b).EndophyticfungiMucorand

T.asperellumhadnoe ffectontotalf lavonoid concentration.

Plantvitality

InNPsoil,PIABSandPItotalweresignificantlyhigherinAMfungu sinoculatedandAMfungus/Trichodermaco-

inoculatedplantsincomparisontonon-

inoculatedplants(Fig.2a,b).ForAMfungusandMucorco- inoculatedplants,asimilartrendwasobservedbutstatistically sig-nificantdifferenceswerenotfound.Thecontributionof lightreactionsforprimaryphotochemistry^φ^P0wassig-

ð1−φP0Þ

nificantlyhigherforalloftheAMfungustreatments

grownintheNPsoil(Fig.2c).Electrontransportbeyondprimar yquinoneacceptor(Qa)significantlydecreasedbyendophytea ndAMfungus/Mucorco-

inoculatedplantsintheNPsubstrate(Fig.2e).PIABSandPItotalwer enotsig-nificantlyincreasedbyAMfungus-

and/orendophyte-

inoculationoftheplantscultivatedinthePsubstratenorwereth efractionofreactioncentrechlorophyllperchlo-

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rophylloftheantennae(RC/ABS)andthecontributionofther eductionofendequivalents(RE/ABS;Fig.2a,b,d,

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Mycorrhiza(2018)28:235–246 239

Flavonoids[a.u.]Chlorophylla[a.u.]

f).Co-

inoculationenhancedthecontributionofthelightreactionsfor primaryphotochemistry(Fig.2c)andde-

creasedelectrontransportbeyondQaincomparisontonon- inoculatedplantsinthePsubstrate(Fig.2e).

Plantb iomass

InoculationwiththeAMfungussignificantlyincreasedthefre shweightofplantsinboththesubstrates(Fig.3a;Supplementar yFig.1).InoculationwithMucorsp.didnotaffectplantfreshw eight.T.asperellumincreaseditonNP,butnotonP (Fig.3a).Co -

inoculationwithAMfungusandMucorsp.resultedinsignific antlyhigherfreshbiomassyieldoftheplantscultivatedonPsub strate(Fig.3a).DryweightsofplantsgrownonNPandPsub- strateswerepositivelyaffectedbyAMfungus-

inoculation(Fig.3b).Trichodermainoculationincreasedpla ntdryweightonNP,butco-inoculationdidnotaffectthispa- rameter(Fig.3b).

Themycorrhizaldependency(MD)indexofL.serriolainoc ulatedwithMucorsp.andT.asperellumgrownontheNPsoilrea ched50and10%,respectively,andwaslowerthanforplantsnot inoculatedwithendophytes(70%).OnPsubstrate,MDofMuc or-inoculatedplantswassimilar

tonon-inoculatedplants(51%)andT.asperellumin- creasedplantdependencyonmycorrhizato67%.

Fungalcolonization

Endophyticfungiwereeasilyvisibleinafewdayoldseed- lingscultivatedinvitro.Theycolonizedplantrootseithert hroughroothairs(mostlynearthetipsofroothairs,wherethecel lwallisthinnest)orclosetothemeristematic,apicalregionofyo ungroots,closetotheareawhereabundantroothairswereforme d.

(SupplementaryFig.2a,c).Themyceliumalsowasvisiblewhen branchrootswereformed.Mucorhy-

phaewerefoundmainlyinassociationwithroothairs(Supple mentaryFig.2b).T.asperellumdevelopedvisiblemy-

celiumontherootsurface(SupplementaryFig.2d).Inolderroot sofplantscultivatedinpots,bothfungiwerevisiblegrowingbet weencorticalcellsandcausingirregularityofplantcells.

Thefrequencyofmycorrhiza(F

%)inplantrootswasveryhighandreachednearly100%ineachA Mfungustreatment(datanotshown).Mycorrhizalcolonizatio nintensity(m

%)rangedbetween29and58%dependingonthetreatment.Mu corsp.significantlyincreasedmycorrhizalcolonizationonly inNPsubstrate(Fig.4a).T.asperellumdidnotinfluence

Fig.1C hlorophylla(a)andflavon oid(b)concentrationsintheleaves of8-week-

oldLactucaserriolaplantsinocula tedwiththeAMfungus(Rhizoglo mus

a

16

ababc 12 abc

Control Mucor Trichoderma

ab a

abc abc

intraradices)andendophytic fungi(Mucorsp.orTrichodermaa sperellum)andcultivatedinnon-

polluted(NP)andpolluted(P) ⁸

substrate.Barstoppedbythes ameletterdonotdiffer

significantlyatP≤0.05by ⁴

Tukey’stest;errorbarsshowstan darderror;a.u.arbitraryunit

0

cd cd

d

bcd d

NP NP+AM P P+AM

b

0.8

a a

a a a a

0.6

0.4 b b

b b b b

0.2

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Mycorrhiza(2018)28:235–246 240

0

NP NP+AM P P+AM

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c

^ϕ^po^/(1–ϕ^po)response(relative)

d

RC/ABSr esponse(relative)

0.8 0.91 1 . 1 1.21.31.4 1.5 0.8 0.9 1 1.1 1.2

mycorrhizalcolonizationoftherootsineithersubstrate(Fig.4a) .Mucoralsosignificantlyincreasedarbusculeabundance(a

%)onlyinplantsgrowninNPsoil( Fig.4b).In plantscultivated withoutAMfungusinoculum,mycorrhizalstruc-

tureswerenotobserved.

Toxicmetalconcentrationinplanttissues Znconcentrationinnon-

inoculatedL.serriolarootsreached403mg/kg.Singleinoculati onwithAMfungusandwithen-

dophyticfungididnotaffectit(Fig.5a).Co-inoculationwith T.asperellumandAMfungussignificantlyincreasedZncon- centrationincomparisontosingleinoculationwith T.asperellum(3.3-

fold;Fig.5a).Znconcentrationinshootswasnotaffectedbysi ngleinoculationwithAMfungusorendophyte.Co- inoculationwithMucorsp.andAMfungus

increasedthisparameterincomparisontosingleinoculationwit hendophyte(1.7-

fold;Fig.5b).Zntranslocationfromroottoshootwasthemosteff ectiveinplantsinoculatedsinglywith

T.asperellum(49%)andMucor(39%).Inthecaseofentirelynon -inoculatedplants,Zntranslocationwas26%.

TheconcentrationofCdinleavesofAMfungusanden- dophyticfungiinoculatedL.serriolarangedfrom13to20mg/k ganddidnotdifferfromcontrols(Fig.5c).Pbcon-

centrationinleavesrangedfrom3to11mg/kg,dependingonthetr eatment(Fig.5d).

Sesquiterpenelactonecontent Becauseofthelowdrybiomassofthenon-

inoculatedplantsinNPsubstrate,analysisofthesecondarymeta boliteswasnotpossible.InNPsubstrate,however,co- inoculationof

Fig.2PSIIefficiencyofplantsino culatedw ithA M (Rhizoglomusi ntraradices)andendophytic(Muc ors p.o r Trichodermaasperellum )fungi

a PI_ABSresponse(relative) 0.80.91 1 . 1 1.21.31.41.5

NP

b PI_totalresponse(relative)

0.80.9 1 1.11 .21 . 3 1 . 4 1 . 5

NP

comparedtonon-

inoculatedplantscultivatedonnon -

polluted(NP)andpolluted(P)subs trates.JIPtestparameters:PIabs— absorbanceperformanceindex(a) ,PItotal—

totalperformaceindex(b),φP0/ (1−φP0)—

contributionoflightreactionsforp rimaryphotochemistry(c),RC/A BS—

fractionofreactioncentrechlorop hyllperchlorophylloftheantennae (d),Ψ0/(1−Ψ0)—

electrontransportbeyondprimary quinoneacceptor(e)andRE/ABS

—

contributionofthereductionofend equivalents(f)arepresentedrelati vetoentirelynon-

inoculatedplants;

statisticallysignificantdifference sbetweenparticulartreatmentsan dthoseentirelynon-

inoculatedplantsareindicatedbya sterisk(ttest,P≤0.05)

NP+AM

P

P+AM

NP

NP+AM

P

P+AM

e

*

**

*

**

Ψo/(1–Ψo)response(relative)

NP+AM

P

P+AM

NP

NP+AM

P

P+AM

f

*

RE/ABSresponse(relative)

0.8 0.9 1 1.1 1.2

NP *

*

NP+AM

*P

*

NP

NP+AM

P

P+AM

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0.8 0.9 1 1.1 1.2

Control Mucor Trichoderma Control Mucor Trichoderma

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Mycorrhiza(2018)28:235–246 241

Plantdryweight[g] Plantfreshweight[g]

Fig.3F resh(a)anddry(b)weight ofLactucaserriolaplantsinoculat edwithAM(Rhizoglomusintrara dices)andendophytic(Mucorsp.o rTrichodermaasperellum)fungia ndcultivatedonnon-polluted

a

^Control ^Mucor

Trichoderma3

2 a a a

(NP)andpolluted(P)substrates. ab

Barstoppedbythesameletterdono tdiffersignificantlyatP≤0.05byT ukey’stest;errorbarsshowstandar derror

ab bc

1 cd

d

bc

d cdc d

0

b

0.2

NP NP+AM P P+AM

Control Mucor

Trichodermaa

0.1 _bcd

d abc

a a

ab a

ab

cd cd cd

0.0

NP NP+AM P P+AM

endophyte-

inoculatedplantswithAMfungusdecreasedtheconcentration oflactucin(LC;Fig.6a),lactucopicrin(LCPikr;Fig.6b),and8- deoxylactucopicrin(8-

deoxyLC;Fig.6c)incomparisonwiththeplantsinoculatedeith erwithMucororwithT.asperellumasasoleendophyte.

InPsubstrate,Mucorsp.increasedlactucopicrinconcen- trationinleaves(Fig.6b).SingleinoculationwithAMfungussig nificantlydecreasedLCandLCPikrconcentration(Fig.6a,b).

WhenMucorsp.inoculatedplantswereco- inoculatedwithAMfungus,LCPikrand8- deoxyLCconcentrationssignifi- cantlydecreased(Fig.6b,c).Co-

inoculationwithAMfungusandT.asperellumdecreasedLCa ndLCPikrconcentration(Fig.6a,b).

Discussion

Inthisstudy,wehaveshownthatallthreeconsideredfactors(ino culationwithmycorrhizalandendophyticfungiandsup- plementationofthesubstratewithTM)hadvariouseffectson L.serriolaplants.ThegrowthresponseoftheplanttotheAM funguswasthestrongestandwasindependentofthesubstrateco ndition:allAMplantsyieldedmorebiomassthantheirre-

spectivecontrols.Additionally,theconcentrationofflavo- noidswassignificantlylower,indicatingstressattenuation

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241

4 Mycorrhiza(2018)28:235–246

arbusculerichness[%]mycorrhizalcolonization[%] Znconcentration[mg/kg]inshootZnconcentration[mg/kg]inrootCdconcentration[mg/kg]inshoot Pbconcentration[mg/kg]inshoot

b

a

d

forAMplants.Co-inoculationwithendophyticfungiampli- fiedthegrowthresponsebutdifferedbetweenfungusspeciesa ndsubstrate.ThebeneficialeffectsofTrichodermawerere- strictedtoNPsoil.In thepresenceofTM,nosignificantgro wthdifferenceinTrichodermainoculatedplantswasfound,i ndependentlyofthepresence/absenceoftheAMfun-

gus,suggestingthatthesefungicannotbebeneficialtothepl ant(intermsofgrowthacceleration)undermetaltoxicity.Mu corsp.,ontheotherhand,exerteditseffectonlyunderthepresen ceofTMincombinationwiththeAMfungus(Mucorsp.co- inoculatedplantsyieldedmorebiomassthanAMfun- gusaloneplants).Thisindicatesthatundermetaltoxicity,thisf ungusspeciescancomplementtheAMfungus.Mostimpor-

tantly,ourstudyshowsthattheeffectsofmycorrhizascanbeamp lifiedbyco-

inoculation.Studyingtheresponseofplantstotoxicmetalsbyin cludingdiversefungiintheexperimentrevealspotentiallyimp ortantsynergismamongthefungi.

EndophyticfungisuchasthoseinthegeneraCryptosporio psis,PhialocephalaandStagonospora(Schultz2006andrefere ncestherein)andmycorrhizalfungisharethesamenicheinsider oottissues,andbothdependonthesamecarbonsource.Thissug geststhatthesetwogroupsoffungimightcompetewitheachoth erforspaceandresources.Inourexperiment,however,mycorrhi zalcolonizationonlywasaf-

fectedbyMucorsp.inNPsubstrate.Althoughsimilartrends

a Non-polluted+AM Polluted+AM

70 a

60 ab ab

50 abc

bc

40 c

30

a

800

600

400

200

0

Polluted Polluted+AM

a ab

ab ab

b b

20 Control Mucor Trichoderma

10 Polluted Polluted+AMa

0

b Non-polluted+AM Polluted+AM

250

200 ab ab

150 b

ab ab

100

80

60

a

ab

c

bc bc

100 50 0

25

Polluted Polluted+AM

a

40 bc

c2 0

a a

20

a

15 a

10 0

Control Mucor Trichoderma 5

Fig.4EffectofendophytesMucorsp.andTrichodermaasperellumon 0

theabsolutemycorrhizalcolonization(a)andarbusculeabundance(b)of plantsinoculatedwithRhizoglomusintraradicesandcultivatedonnon- polluted(NP)andpolluted(P)substrates;barstoppedbythesameletter

donotdiffersignificantlyatP≤0.05byTukey’stest;errorbarsshow ¹⁵ standarderror

10

Polluted Polluted+AMa ab

ab

b

wereobservedinPsubstrate,thedifferenceswerenotstatis- 5 b

ticallysignificant.Theseresultsindicatethatco-inoculation

withanendophyticfungus,suchasMucor,canimprovecol- ⁰ onisationbyanAMfungus,andthus,itcouldhavepotentialinthe

productionofinoculum.Whatisimportantisthatnodifference swerefoundundertheresource-

limitedconditionsoftheminedumpsubstrate.Insuchanenviro nment,severenutrientandwaterlimitationmayhaveanegativei mpactonthesymbiosis.Here,wefoundthatthepresenceoftheen do-

phytedoesnotaffectmycorrhizalcolonisationandarbuscularri

chnessinthepollutedsubstratum.Aninterestingcomple- mentaryinvestigationwouldbethebehaviourofthefungalendo phyteinsuchconditions.This,however,requiresfurtherresearch.

Benefitsimposeduponfungalcolonisationofbothmycor- rhizalandendophyticfungimaybeassociatedwithimprovedphot osynthesisefficiency(Ruiz-

Lozanoetal.1996;Shengetal.2008;Rozpądeketal.2014,2015 ,2016).Hereweshowanadditiveeffectofco-

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Mycorrhiza(2018)28:235–246 241

inoculation,intermsofplantgrowthaccompaniedbyimproved PSIIefficiency.AMfungusinoc-

ulationimprovedthetwomainphotosynthesisindicesPIabsand PItotali nNPsubstrate.Theeffectwassustaineduponco-

inoculationwithTrichodermabutnotundermetaltoxicity(no

Fig.5ToxicmetalconcentrationofLactucaserriolainoculatedwithAM(R hizoglomusintraradices)andendophytic(Mucorsp.andTrichodermaasp erellum)fungiandcultivatedonpollutedsubstrate:Zninroot(a),Zninshoot (b),Cdinshoot(c),Pbinshoot(d).Barstoppedbythesameletterdonotdiffe rsignificantlyatP≤0.05byTukey’stest;errorbarsshowstandarderror

growthresponse),providingfurtherevidencefortheroleofthe abioticenvironmentindeterminingthethree-wayinterac- tionbetweenlettuce,TrichodermaandtheAMfungus.Interes tingly,inoculationwithTrichodermawithoutAMfun-

gushadapositiveeffectonplantgrowthbutdidnotaffectphoto synthesis,suggestingthatthefungusimpactonelectrontranspor tefficiencyisconferredbythemycorrhizalfungus.PSIIefficie ncyinplantsgrownonP,incontrasttoNPsub-

strate,wasnotchangeduponsinglemycorrhizalorendophytein oculationnorco-inoculation,althoughco-inoculationen- hancedthecontributionofthelightreactionsforprimarypho- tochemistryincomparisontonon-

inoculatedplants.PreviousstudiescarriedoutonVerbascuml ychnitiswithasimilarly

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Mycorrhiza(2018)28:235–246 24 3

%ofdryweight%ofdryweight%ofdryweight

Fig.6Concentrationofsesquiterp enelactonesinleavesofLactucase rriolainoculatedwithAM(Rhizog lomusintraradices)andendophyti c(Mucorsp.andTrichodermaaspe rellum)fungiandcultivatedonnon -

polluted(NP)andpolluted(P)subs trates:>Lactucin(LC)

(a),Lactucopicrin

a

LC

0.04 0.03 0.02 0.01 0

bcb c

a aba b

bc

cd cd

de de

e

(LCPikr)(b),8-Deoxylactucin (8-DeoxyLC)

(c).Barstoppedbythesameletterd onotdiffersignificantlyatP≤0.05 byTukey’stest;errorbarsshowsta ndarderror

b

^LCPik

r

0.05

NP NP+AM P P+AM

a 0.04

0.03 0.02 0.01 0

b b

cd dee

b ab

c dec d e

NP NP+AM P P+AM

c

8-deoxyLC

0.03

a 0.02

0.01

abc d bcda b a b c

e e

d db c d

0

NP NP+AM P P+AM

pollutedsubstrate(30kmfrompresentlyinvestigatedarea,W ężowiczetal.2015)haveshownthatthepresenceofmy- corrhizadidnotchangePSIIefficiency;however,AMfungusin oculationwasabletoimprovethephotosynthesisrateofplan t–

endophyteconsortiumwhichwasnegativelyaffectedbyinocu lationwiththesingleendophyticfungus,Diaporthesp.

(Wężowiczetal.2017).Thequantumyieldofprimaryphotoc hemistryandtheabilitytotransferelectronsfromPSIItoPSIwe reupregulatedbyco-

inoculationwithanAMfungusandtheendophyticfungusDiap orthesp.(Wężowiczetal.2017)similarlytoourAMfungus—

Mucorsp.andAMfungus—

Trichodermamodelsinthepresentstudy.

Accordingtotheliterature,inoculationwithAMFusuallyresult edinincreasingchlorophyllcontentinhostplants(Abdel-

FattahandMohamedin2000;Zuccarini2007;Vafadaretal.201 4).Baslametal.

(2011,2013b)reportedthatthesymbiosisoflettucewithAMFincr

easedtheamountofchlorophyllinleaves.Accordingtothesameaut hors(Baslametal.2013a),however,inoculationalsocanhavetheop positeeffect.Inour

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242

44 Mycorrhiza(2018)28:235–246

study,mycorrhizalplantsyielded3–4- foldhigherbiomassthantheirrespectivenon- mycorrhizalcontrols,butchlorophyllacon-

centrationwasdecreasedby20%.Thisindicatesthatphotosyn- thesiswasimprovedbecauseofanupregulationoftheefficiency ofelectrontransportwithinthephotosystemsandnotbyincreas- ingthenumberoffunctionalreactioncentres.

Plantsingeneraldevelopedtwostrategiesallowingthento withstandhighquantitiesofTMinthesubstrate(Baker1981).T hefirstoneisthedevelopmentofasophisticatednetworkofintri nsicdetoxificationmechanismsallowingplantstoaccu- mulateTMinhighconcentrations.Thesecondstrategyallowsp lantstopreventTMuptake(avoidance).Cultivatedcroplet- tuceaswellasitsrelatives,wildlettucespecies,areknownforth eirabilitytoaccumulaterelativelyhighamountsoftoxicmet als(Pb,Cd,As,Zn)intheirleavesandroots(Cobbetal.2000).T hecontributionofmycorrhizalfungitotoxicmetaluptakebyt heplantisdependentonmetalconcentration(Leyvaletal.199 7).Athighmetalconcentrationinsoil,my-

corrhizasreducedZnandCdaccumulation,butatlow

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concentrationincreasedZnanddecreasedCdaccumulationinle ttuceshoots(Schüeppetal.1987).Inthepresentstudy,myc orrhizasalonedidnotaffecttheaccumulationoftoxicmetals.

InoculationwiththeendophyticMucorsp.alonede-

creasedtheZnconcentrationinplantroots.Surprisingly,inco ntrast,co-inoculationwithMucorsp.increasedtheZncon- centrationinrootsandshoots.Theseresultsindicatethatvar- iousfungiandtheircombinationsplaydifferent,oftenoppo- site,rolesininfluencingahost’sstrategytoTMstress.Thismigh tbeimportantincontrollingplantmetalhomeostasisinphytore mediationapplications.Theresultspresentedhereconfirmobs ervationsreportedrecently(Rozpądeketal.2018).InArabid opsisarenosa,growninpollutedsubstratefromtheBBolesła w^minedump,inoculationwithMucorsp.affectedplantgrow thandmetalhomeostasis.Inoculated

A.arenosaaccumulatedlessZnandtranslocatedCdfromroottos hootsmoreefficientlythaninnon-

inoculatedplants.Additionally,Mucorsp.activatedroottosho otmetaltranslo-

cationwhichwasaccompaniedbyupregulationofsever almetaltransportergenes(Rozpądeketal.2018).Thisindicates theimportanceofendophytesinadaptationofplantstotoxicenvi ronments.

Plantssynthesizeawidevarietyofphytochemicalsthatarerequ iredforbasicmetabolismandareessentialfortheinterac- tionbetweentheplantandtheenvironmentinprocessesasso- ciatedwithdefenceandsignalling.Plantpolyphenolsand monophenolicsareagroupofphytochemicalswhosepotentialfor amelioratingenvironmentalstressinplantshasbeenespe- ciallywelldocumented(Quideauetal.2011;Giovannettietal.20 13;Sbranaetal.2014).Evenmildenvironmentalstressessuchash eatshock,chillingandhighlightintensityinduced2–3-

foldphenoliccompoundconcentrationincreasesincultivatedlett uce(Ohetal.2009).Here,wequantifiedtheabundanceofthephen ols:caftaricacid,chlorogenicacid,cichoricacid,coniferin,3,5 -dicaffeoylquinicacid,4,5-

dicaffeoylquinicacid,aswellsomeunidentifiedcaffeicacidderiv ativesandfoundthattheirconcentrationsweredecreasedbymyco rrhizalinoc-

ulation(datanotshown).Additionally,AMfungusinoculationdec reasedtotalflavonoidconcentrationinleaves,whereasen- dophyticfungi,MucorandT.asperellum,didnotaffectit.The seobservationsledustospeculatethatmycorrhizasde- creasedTMstress.Inthiscontext,theAMfungusseemstobemor eimportantforL.serriolaindecreasingTM-

inducedplantstressthantheendophytes.

Anothergroupofsecondarymetabolitesfrequentlyreportedin plantsoftheLactucagenusissesquiterpenelactones,partic- ularly8-

deoxylactucin,jacquinelin,crepidiasideB,lactucin,11β,13- dihydrolactucin,lactucopicrinandlactusideA (Michalskae tal.2009).Theselactonesareaccumulatedwithinlaticifersasaco nstitutivecomponentoflatexandhaveanti-

herbivoreproperties.Wedetectedlactucin,lactucopicrinand8-

deoxylactucininL.serriolaleaves.Thesamesesquiterpenelacto nesweredetectedinCichoriumintybusleaves,wherethey

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providedasignificantbarrieragainstherbivory(ReesandH arborne1985).Theconcentrationofthesesquiterpenelac- tonesanalysedinplantleaveswasdecreasedbyAMfungusinoc ulationinplantsgrowninbothsoiltreatments,butnotbyfungale ndophytesinsingleinoculationexperiments.Thisim-

pliesthatthesynthesisofthesecompoundsmaybedownreg- ulatedtofacilitatecolonisationbyAMF,butdownregulationisn otrequiredduringtheplant-endophyteinteraction.Thisim- pliesvariationinmechanismsofAMfungusandendophyteinter action.Co-

inoculationwithendophyticfungiincreasedsesquiterpenelac toneconcentrationinsomecases(comparedtoAMfungusinocu latedplants)butdidnotrestoreittolevelsfoundinplantslacking mycorrhizas,suggestingthatco-

inoculationwithendophyticfungitendedtorestoretheplantses quiterpenelactonephenotype.TherewasnodifferenceintheA Mfunguscolonisationbetweensingleandco-

inoculationtreatments,sotheobservedeffectdidnotresultinlow erAMfunguscolonisationcausedbytheendophyte.Intheavail ableliterature,theeffectofAMFonsesquiterpenelactoneprodu c-

tioninplantsalsoisnotclear.InthecaseofArnicamontana,only aminorityofAMfungusinoculainvestigatedwasabletoincreas etheconcentrationofthesecompoundsinshoots,whiletheresto ftheAMFtesteddidnotaffectit(Jurkiewiczetal.2010).InC.int ybusshoots,mycorrhizasdidnotaffectsesqui-

terpenelactoneproduction(Rozpądeketal.2014).

Inconclusion,theresultspresentedhereindicatethatinoc- ulationofL.serriolawitharbuscularmycorrhizalfungisig- nificantlyimprovedplantbiomassinpollutedandnon- pollutedsubstrates.Additionalinoculationwithendophytic fungiMucorsp.orT.asperellumenhancedthisbenefici aleffect.Co-

inoculationoftheplantswithanAMfungusandMucoralsoin creasedZnconcentrationinleavesofLactucaandimprovedm ycorrhizalcolonisation.Despitethatin-

creasedhostplantcostswerepotentiallycausedbymaintain- ingsymbiosiswithmultiplemicroorganisms,interactionof wildlettucewithbothmycorrhizalandendophyticfungiwasm orebeneficialthanwithasinglefungalpartner.Hightoler- anceofthisplantspeciestodroughtsupportedbymycorrhizasa ndendophytesimprovingbiomassandaffectingtoxicmetalac cumulationshowsthepotentialofapplicationofthismodelinu nfavourableenvironments.

AcknowledgementsTheauthorswouldliketothankWeronikaJanasan dMartynaJanicka(JagiellonianUniversity,Poland)fortheirassistanc ewithplantinoculation,cultivationandharvesting.

FundinginformationThisworkwasfundedbyTheNationalScienceCe ntre,MaestroProject,DEC-2011/02/A/NZ9/00137.

OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo mmo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativeco mmons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distributi on,andreproductioninanymedium,providedyougiveappropriatecred ittotheoriginalauthor(s)andthesource,providealinktotheCreativeCom monslicense,andindicateifchangesweremade.

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