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Enrichment of arbuscular mycorrhizal fungi in a contaminated soil after rehabilitation

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

Environmental

Microbiology

Enrichment

of

arbuscular

mycorrhizal

fungi

in

a

contaminated

soil

after

rehabilitation

Patrícia

Lopes

Leal

a,b

,

Maryeimy

Varón-López

c

,

Isabelle

Gonc¸alves

de

Oliveira

Prado

a

,

Jessé

Valentim

dos

Santos

c

,

Cláudio

Roberto

Fonsêca

Sousa

Soares

a,d

,

José

Oswaldo

Siqueira

a,e

,

Fatima

Maria

de

Souza

Moreira

a,∗

aUniversidadeFederaldeLavras,SoilScienceDepartment,GraduateProgrammeofSoilScience,Lavras,MG,Brazil bUniversidadeFederaldaBahia,MultidisciplinaryInstituteinHealth,VitóriadaConquista,BA,Brazil

cUniversidadeFederaldeLavras,ProgrammeofAgriculturalMicrobiologyBiologyDepartment,Lavras,MG,Brazil1

dUniversidadeFederaldeSantaCatarina,ImmunologyandParasitology,DepartmentofMicrobiology,Florianópolis,SC,Brazil eInstitutoValedeTecnologia,Belém,PA,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11November2014 Accepted11January2016 Availableonline24June2016 AssociateEditor:JerriÉdsonZilli

Keywords: Glomeromycota Heavymetals Soilcontamination Mycorrhiza Revegetation

a

b

s

t

r

a

c

t

Sporecounts,speciescompositionandrichnessofarbuscularmycorrhizalfungi,andsoil glomalincontentswereevaluatedinasoilcontaminatedwithZn,Cu,CdandPbafter rehabil-itationbypartialreplacementofthecontaminatedsoilwithnon-contaminatedsoil,andby

EucalyptuscamaldulensisplantingwithandwithoutBrachiariadecumbenssowing.These reha-bilitationprocedureswerecomparedwithsoilsfromcontaminatednon-rehabilitatedarea andnon-contaminatedadjacentsoils.Arbuscularmycorrhizalfungicommunitiesattributes wereassessedbydirectfieldsampling,trapculturetechnique,andbyglomalincontents esti-mate.Arbuscularmycorrhizalfungiwasmarkedlyfavoredbyrehabilitation,andatotalof 15arbuscularmycorrhizalfungimorphotypesweredetectedinthestudiedarea.Species fromtheGlomusandAcaulosporagenerawerethemostcommonmycorrhizalfungi. Num-berofsporeswasincreasedbyasmuchas300-fold,andspeciesrichnessalmostdoubled inareasrehabilitatedbyplantingEucalyptusinrowsandsowingB.decumbensininter-rows. Contentsofheavymetalsinthesoilwerenegativelycorrelatedwithbothspeciesrichness andglomalincontents.IntroductionofB.decumbenstogetherwithEucalyptuscauses enrich-mentofarbuscularmycorrhizalfungispeciesandamorebalancedcommunityofarbuscular mycorrhizalfungisporesincontaminatedsoil.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Correspondingauthorat:SetordeBiologia,MicrobiologiaeProcessosBiológicosdoSolo,DepartamentodeCiênciadoSolo,Universidade

FederaldeLavras,Lavras,MinasGerais,Brazil. E-mail:fmoreira@dcs.ufla.br(F.M.S.Moreira).

1 Presentadress:Gebiut(GeneticayBiotecnologiaVegetalyMicrobiana),UniversidaddelTolima.Ibagué,Tolima,Colombia

http://dx.doi.org/10.1016/j.bjm.2016.06.001

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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ecosystems. AMFareobligatemutualisticsymbiontsthat col-onizetherootsofmorethan80%ofplantfamilies,andwhich establish anassociation known asarbuscular mycorrhiza.2

Severalstudieshavereportedontheabundanceand occur-renceofAMFincontaminatedsoils,3–6andhavehighlighted

thehighresistanceofAMFisolatestoseveraltypesofstress, includingwaterstress,soilacidification,disaggregation,and absenceorscarcityofvegetationcover.

Mining activities produce wastes which may contain heavymetalsascontaminants.Theseresiduesaregenerally deposited on the ground, and often occupy large exten-sions. In order toprevent chemical elements contained in these residues to be exposed to leaching processes, or to the action of the winds, rehabilitation programs are usu-allyemployedin theseareas throughthe establishmentof vegetation cover. However, plants often have very limited developmentincontaminatedareas.Due totheirbeneficial effectsonplantgrowthunderstressedconditions,AMFhave been used toenhance rehabilitation ofcontaminated soils throughphytoremediation.7Besides,mycorrhizalplantsand

their associatedmicrobiota canregulate absorption, trans-formationandremovalofsoilpollutants.8Thecontribution

ofAMF to plant growth in soils contaminated with heavy metalsisrelatedtoseveralfactors,includingdiversity, abun-dance,persistence,andefficiencyofAMFpopulations,which mightvarybetweendifferentlocations,andarerelatedtothe environmentalvariablesandtothepresenceofvegetation.9

The insoluble glycoprotein glomalin, which is abundantly producedbythehyphaeofsomeAMF,isknownfor accumu-latinginthesoilwhereitcanretainlargeamountsofheavy metals.10Ithasbeensuggestedthatsoilglomalincontentis

relatedtochanges associatedwithlanduseand rehabilita-tionofdegradedsoils.11 Thesechangesinvegetationcover

maydirectlyinfluenceAMFcommunitiesandthequantityand qualityofcompoundsproducedbythem,suchasglomalin.4 Therefore,abundanceandrichnessofAMForglomalin con-tentmaybeusefulindicatorsofrehabilitation.Studiesshowed that these attributes of AMF are inversely related to con-centrationofheavymetalsinthesoil.3Therefore,improved

knowledgeonthedynamicsofthesefungiincontaminated rehabilitated soilsishighly relevant, consideringthat AMF maycontributetorevegetationandphytostabilization.12The

aimofthisstudywastoevaluatetheoccurrenceandAMF com-munitycompositioninsoilsfrom areascontaminated with zinc(Zn),cadmium(Cd),copper(Cu),andlead(Pb),andsubject torehabilitation.

Materials

and

methods

Studyareasandsoilsampling

Soilsampleswerecollectedinthesummerof2009(February) fromcontaminatedsitesunderrehabilitation,inaZnsmelter unitofthe MetalMiningCompany (CompanhiaMineirade

19◦Cand22◦C,andtheannualaveragerainfallvariesbetween 1100and1420mm.Theareasarelocatedatanaverage eleva-tionof539minaflatreliefwithoriginalcerradovegetation.

By the year of 2001/2002, two sites in the dumping area designated as industrial waste deposit (System 1 – 18◦1125.70 S and 45◦1410.90 W)and ustulation (System 2 – 18◦1110.50 S and 45◦148.00 W), both contaminated by waste disposal, were sampled, and revealed high con-tentofheavymetals:Zn=17,167and1844mgkg−1;Cu=882 and 145mgkg−1; Cd=584 and 29mgkg−1; and Pb=573 and 278mgkg−1, respectively. Two pilot rehabilitationprograms weresetupbytheresearchteamfromtheDepartmentofSoil ScienceoftheFederalUniversityofLavras(UFLA)andbythe CMVstaff.Rehabilitationstrategyprojectincludedexcavation of the contaminated soil and its replacement with non-contaminated soil. Eucalyptus camaldulensis seedlings were planted in rows in 1m×1m×10m trenches, where non-contaminatedsoilreplacedthecontaminatedone.Spaceinter rowsreceiveda20cmlayerofnon-contaminatedsoil,inwhich

Brachiariadecumbenswereplantedornot(Fig.1).Thesoilused toreplace(row/trenches)orcover (interrows)the contami-natedonewastakenfrom aCerradoareaadjacent toCMV. Beforereplacement,soilwassubjectedtoliming,andreceived organicmatter(cowmanure)andfertilizers,inordertoensure adequateplantgrowth.14FortheassessmentofAMF,10

com-positesoilsamples(5sub-sampleseach)weretakenat0–20cm depth (one compositesampleper samplingpoint. The dis-tance betweensampling pointswas200m), fromrows and inter-rows ofbothrehabilitationprograms.Forcomparison, non-contaminatedsampleswerealsotakenfromCerrado(C – 18◦1055.80 S and45◦1338.50 W)andgrass pasture(P– 18◦1128.30Sand45◦1355.20W),aswellasfromanadjacent non-rehabilitatedarea(NR–18◦1109.50 Sand 45◦1434.80

W) (Table 1).Five compositesamples were taken from the

referenceareas(C,PandNR). Analysisofheavymetalsinsoils

Chemical analyses ofsoil sampleswere carried out in the Laboratory of SoilScience ofUFLA. Heavy metal contents, includingZn,Cu,Cd,andPb,wereextractedbyaquaregia solu-tion,asrecommendedbytheStateCouncilonEnvironmental Policy(ConselhoEstadualdePoliticaAmbiental–COPAM).15

TotalheavymetalcontentwasdeterminedusingtheUSEPA 3051 method, and soluble heavy metalcontent was deter-minedbytheMehlich-1method.

Occurrenceandrichnessofarbuscularmycorrhizalfungi (AMF)

The occurrence of AMF was assessed by direct extrac-tion of spores in 50mL of soil, using the wet sieving technique,16 followed by centrifugation in 50% sucrose

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Contaminated

Rehabilitated

Non-contaminated

Non-rehabilitated

Original cerrado Pasture

Rehabilitated with

E.camaldulensis and B. decumbes

R

IR

Fig.1–Partialviewandrepresentationofthesoilsamplingsites(R–plantationrows;IR–inter-rows)inareas contaminatedwithheavymetalsaftertheuseofrehabilitationsystems,non-rehabilitatedcontaminatedsites,and non-contaminatedsites(CerradoandPasture).

Table1–Characterizationofsoilsamplingsitesinareas

contaminatedwithheavymetalsaftertheuseofthe

rehabilitationsystems[S1(Ec-RandEc-IR)andS2(EcB-R

andEcB-IR)],innon-rehabilitatedcontaminatedarea

(NR,)andinnon-contaminatedareas[Cerrado(C)and

Pasture(P)].

Areas–soilvegetationcoveratthe samplingtime

Samplingsites

1–S1

AreaplantedwithEucalyptus

camaldulensisinrowsandwith

spontaneousvegetationbetweenrowsof

E.camaldulensis.Anthropizedsoil.

Ec-R Row Ec-IR Inter-row

2–S2AreawithE.camaldulensis

plantationinrowsandcompletecoverby

Brachiariadecumbens(sown)betweenthe

rowsofE.camaldulensisplantation.

Anthropizedsoil.

EcB-R Row EcB-IR Inter-row

3–Non-rehabilitatedcontaminated area-Areaofwastedisposalwithhighsoil contaminationandwithoutvegetation cover.Anthropizedsoil.

NR

4–OriginalCerrado–Cerradovegetation withregionaltypicalspecies.Latosols typicalcerrado.

C

5–Pasture-Brachiariadecumbensgrass plantedinLatosolstypicalcerrado.

P

with polyvinyl lactoglycerol and Melzer’s reagent. After that, phenotypic characteristicswere observedinthe com-pound microscope for taxonomic classification, according to Schenck and Pérez,17 to the International Culture

Col-lectionof(Vesicular)ArbuscularMycorrhizal Fungi(INVAM)

(http://www.invam.caf.wvu.edu), and to the Department of

Plant Pathology of the University of Agriculture, Szczecin, Poland(http://www.agro.ar.szczecin.pl/∼jblaszkowski/).Traps wereestablishedintriplicates,byplacing50gofsoilsamples withrootpieces,andmixedwith450gsterilizedsoil(121◦C, for1h, withintervals of24hbetweentwoautoclavings),in 1kgpots.Brachiariadecumbesseedsweresurfacedisinfected with0.5%sodiumhypochloritefor15min,andtheirdormancy wasinterruptedbyimmersingtheminconcentratedsulfuric acid.Ineachpot,5–10Brachiariaseedsweresown.After germi-nation,seedlingsweretrimmed,remainingthreeplantsper pot.Onceamonth,potswerewateredasneededand fertil-izedwith20mLperpotofHoagland’ssolution.18Attheendof

tenmonthsgrowingcycle,inagreenhouse,50gsoilwas sam-pled fromeach potforAMFsporeextraction,counting and identification,asdescribedabove.

The frequency of occurrence (FO) of each AMF species detectedinthedirectfieldsoilsamplesandintrapculturewas calculatedbyusingtheequationFi=Ji/K,whereFi=frequency ofthespeciesI;Ji=numberofsoilsamplesinwhichspeciesi

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P(mgdm−3)a 5.8 11.2 8.8 2.7 6.3 0.7 1.0 K(mgdm−3)a 52.6 56.8 80.8 72.7 40.8 123.4 80.4 Ca+2(cmol cdm−3)b 1.6 1.6 2.2 2.5 81.3 0.4 0.7 Mg+2(cmol cdm−3)b 1.7 0.7 1.0 1.2 22.0 0.6 0.3 Al+3(cmol cdm−3)b 0.0 0.0 0.1 0.1 1.4 1.6 0.2 H+Al(cmolcdm−3)c 1.5 1.3 2.0 2.0 14.4 4.9 2.7 Corg(gkg−1) 3.8 3.4 5.3 4.8 2.4 3.6 5.6 Mn(mgdm−3)a 46.8 56.6 55.8 83.5 1446 24.4 30.9 Zntotal(mgkg−1)a 3805 8673 574 581 47,910 23 17 Znsoluble(mgkg−1)d 2866 6680 258 332 13,695 1.8 3.6 Cutotal(mgkg−1)a 189 511 10 23 738 18 14 Cusoluble(mgkg−1)d 139 456 7.2 8 343 1.4 2.6 Pbtotal(mgkg−1)a 77 139 32 50 6488 15 7 Pbsoluble(mgkg−1)d 35 77.3 15.6 16.6 32 <2.0 <2.0 Cdtotal(mgkg−1)a 19 37 6 10 1207 0.06 0.03 Cdsoluble(mgkg−1)d 18.9 37 4.8 5.8 565 <0.02 <0.02 a USEPA3051. b KCl1molL−1. c SMP. dMehlich-1.

Extractableglomalincontents

Soil glomalin was classified as “soil glomalin related pro-tein”(SGRP).TwofractionsofSGRP(easilyextractableglomalin and total glomalin) were distinguished depending on the extractionconditionsandonthequantificationmethod.19,20

Quantification for both fractions was carried out using the Bradford’s method,21 and therefore was denominated

“Bradford-reactivesoilprotein”(BRSP)and“easilyextractable Bradford-reactivesoilprotein”(EE-BRSP).ForEE-BRSP extrac-tion,20mmol−1sodiumcitrateatpH7.0wasused,whiletotal glomalin(BRSP)wasextractedby50mmol−1sodiumcitrateat pH8.0,followedbycyclesofautoclaving(121◦Cfor1h)and centrifugation(3300rpmfor30min).CoomassieBrilliantBlue G-250andbovineserumalbumin(BSA)wereusedtogenerate the standard curve. Readings were carried out on a spec-trophotometerat595nm (values were expressedinmgg−1 soil).

Statisticalanalysis

Differencesinspores abundanceand inEE-BSRPand BRSP contents were evaluatedbymeans of analysisofvariance, usingthe Scott–Knotttest (5%)usingthe SISVARstatistical software.22Pearson correlationsbetweenmetals

concentra-tions insoil and speciesdiversity, EE-BRSPand BRSP were carriedoutbythettest(5%),usingtheSAEGstatistical pro-gram.

Results

Chemicalcharacteristicsofthesoil

Ingeneral,thereferencesites(CandP)presentedacidandlow fertilitysoilswhilethecontaminatedsitespresentedhigher

pH,CaandMgduetolimeapplicationthatwasusedinorder toreducetheactivityofmetalionsinthesoil.Chemical analy-sisofsoilsamplesrevealedvariablecontentsofmetals,being very high in contaminated samples and very low in non-contaminatedreferencesoils(Table2).NRsiteexhibitedhigh contentsofheavymetalscomparedtothecontrolsitesCandP, especiallyforZnandCd(Table2).NRsitepresentedthehighest concentrationofallthemetalselements,followedby reha-bilitatedsites.Amongtherehabilitatedsites,theoneplanted withEucalyptus(EcB-R)andBrachiaria(EcB-IR)presentedmuch lowercontentofallmetals.

AMFsporecount

Sporecountswerehigherintrapculturesthaninfieldsamples

(Table3).Atotalof5451sporeswerefoundintrapcultures,

and 3355sporeswere obtainedinthe directfield sampling technique.Thehighest(p<0.05)sporenumberinthefieldwas foundinEcB-R,followedbyEcB-IR.InNRandCsites, sporu-lation andspeciesrichness wereverylow infield samples. Intrapcultures,sporulationwashigherinplotsrevegetated withEucalyptusandBrachiaria(EcB-IR),andmuchhigherthan indirectfieldsamplesfromNRandnon-contaminatedsoils. Similar trendwasfoundforspeciesrichness.Theseresults indicate that trap culture was more appropriate to detect modificationsinAMFcommunityinducedbysoil rehabilita-tion.Theuseoftrapculturesalloweddetectingsignificantly highertotalsporeabundancevaluesforallthesampledand assessedsites,whencomparedtodirectfieldsampling,except forEcB-R,wheretherewasnosignificantdifference(p<0.05) betweenthetechniques.Sporeabundancevaluesdetectedin soilsamplesfromEcB-RandEcB-IRbyusingthetrapculture techniquewere1222and1660,respectively,whichis signifi-cantlydifferentfromthesporeabundancevaluesoftheother

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Table3–Totalnumberofspores(TS)andspeciesrichness(S)ofAMFdetectedinthedirectfieldsoilsamples(50mlsoil)

andintrapculturepots(50mlsoil)correspondingtocontaminatedandrehabilitatedareas(Ec-R,Ec-IR,EcB-R,and

EcB-IR),tonon-rehabilitatedareas(NR),andtonon-contaminatedareas[Cerrado(C)andPasture(P)].

Areaofstudy Field Trapculture

TS S TS S Ec-R 120Bb 7 885Ab 8 EC-IR 26Bb 7 470Ab 6 EcB-R 2718Aa 6 1222Aa 6 EcB-IR 452Bb 9 1660Aa 5 NR 5Bb 3 484Ab 4 C 5Bb 1 355Ab 3 P 29Bb 3 375Ab 4

Uppercaseletters=comparisonsofTSwithinrows;lowercaseletters=comparisonswithincolumns.Valuesfollowedbythesameletterdonot differbytheScott–Knotttest(p<0.05).

investigatedsites.Amongthedirectfieldsamples,spore num-berwasremarkableintheEcB-Rsite(2718spores)(Table3).

Speciescomposition

Considering all the samples analyzed by both techniques, a total of 15 morphotypes were detected. These morpho-types corresponded to eight genera (Acaulospora, Glomus, Rhizophagus,Paraglomus,Gigaspora, Dentiscutata, Scutellospora

and Cetraspora), and six families (Acaulosporaceae, Glom-eraceae, Paraglomeraceae, Dentiscutataceae, Racocetraceae and Gigasporaceae), and most of the morphotypes (66.6%) belongedtotheAcaulosporaceaeandGlomeraceaefamilies

(Table4).ThespeciesAcaulosporafoveata,Acaulospora

colom-biana,andGlomussp.(sporocarp)weredetectedonlyintrap culturepots,whereasCetrasporapellucidaandAcaulosporasp. weredetectedonlyinthefieldsamples.

Bothdirectfieldsamplingandtrapculturetechnique pre-sentedsimilar resultsinregardtospeciesrichness.Twelve AMFspeciesweredetectedindirectfieldsamples,andahigh numberofspeciesrichness(9)wasfoundinsamplesfromthe EcB-IRsite(Table4).Trapculturetechniquealloweddetecting 13AMFspecies,andthegreatestspeciesrichness(8)occurred

intheEc-Rsite.IntrapculturesfromCerrado,andinNRsite, AMFrichnesswasverylow.

Theonlyspeciescommontoallthestudiedsiteswas Glo-mussp.,whichisconsideredageneralistspecies.Acaulospora

sp. and Glomus sp. (sporocarp) were detected in only one sampling site, and thus were rated as exclusive species

(Table 4). Glomus sp. presented the highest frequency of

occurrence(40%)intheassessmentofthedirectfield sam-ples (Fig. 2). Among the AMF species detected by the trap culture technique, Paraglomus occultum presented the highest frequency of occurrence (42.9%); however, in the directfield samples,its frequencyofoccurrencewas 12.9% (Fig. 2). Species richness was affected by the contents of heavy metals in the soil. Linear correlations were: Zn(y=7.5651−0.00009x;R2=−0.8717),Cu(y=7.86140.005x; R2=−0.72531), Pb(y=7.29950.0007x, R2=−0.8677),and Cd (y=7.3148−0.0036x;R2=−0.9630)(p<0.05).

Analysis of fungal community structure in non-rehabilitated and rehabilitated sites, as revealed by trap cultures,showsincreaseinAMF(sporenumbers)andspecies richness(Fig.3).SoilreplacementandEucalyptusplantingwas successful inrevegetating the area and inincreasing AMF. However,whenB.decumbenswassimultaneouslysowninthe inter-row, the benefitsto increaseAMF were muchhigher.

Table4–Speciesofarbuscularmycorrhizalfungi(AMF)detectedinthedirectfieldsoilsamplesandintrapculturepots

correspondingtocontaminatedandrehabilitatedareas(Ec-R,Ec-IR,EcB-R,andEcB-IR),tonon-rehabilitatedarea(NR),

andtonon-contaminatedareas[Cerrado(C)andPasture(P)].

Samplingsites Field Trapculture

Ec-R Acaulosporalongula,A.morrowiae,Rhizophagusdiaphanus,

R.intraradices,Glomussp.,Paraglomusoccultum,Gigasporasp.

A.longula,A.morrowiae,R.diaphanus,R.intraradices,Glomussp.,

P.occultum,Gigasporasp.,Dentiscutataheterogama

Ec-IR Acaulosporascrobiculata,A.morrowiae,Rhizophagusdiaphanus,

R.intraradices,Glomussp.,Paraglomusoccultum,Scutellosporasp.

A.longula,A.morrowiae,R.diaphanus,R.intraradices,Glomussp.,

Scutellosporasp.

EcB-R Acaulosporascrobiculata,A.morrowiae,Glomussp.,Paraglomus

occultum,Dentiscutataheterogama,Cetrasporapellucida

A.longula,A.scrobiculata,A.foveata,A.morrowiae,A.colombiana,

P.occultum

EcB-IR Acaulosporalongula,A.scrobiculata,A.morrowiae,A.sp.,

Rhizophagusdiaphanus,R.intraradices,Glomussp.,P.occultum,

Dentiscutataheterogama

A.longula,A.foveata,A.morrowiae,Glomussp.,Paraglomus

occultum

NR Glomussp.,Dentiscutataheterogama,Rhizophagusdiaphanus Glomusspp.(sporacarp),Paraglomusoccultum,Gigasporasp.,

Dentiscutataheterogama

C Glomussp. Acaulosporacolombiana,Glomussp.,Paraglomusoccultum

P Acaulosporascrobiculata,Glomussp.,Scutellosporasp. A.scrobiculata,Rhizophagusdiaphanus,Glomussp.,Paraglomus

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Frequency of occurrence, % 50 40 30 20 10 0 Dentiscutata heterogama Cetraspora pellucida Scutellospora sp. Gigaspora sp. Glomus sp. sporocarp Paraglomus occultum Glomus sp. Rhizophagus intraradices Rhizophagus diaphanus Acaulospora sp.

Field Trap culture

Fig.2–Speciesofarbuscularmycorrhizalfungi(AMF)detectedinsamplesofsoilcontaminatedwithheavymetalsandtheir frequencyofoccurrenceinallofthesamplingsites.

Areas

S2 S1

NR

Number of spores / 50 mL soil

0 500 1000 1500 2000 2500 3000 Paraglomus occultum Glomus spp. (sporcarp) Rhizophagus intraradices Glomus sp. Rhizophagus diaphanus Acaulospora morrowiae Dentiscutata heterogama Acaulospora longula Gigaspora sp. Acaulospora foveata Acaulospora scrobiculata Acaulospora colombiana 13%

*

**

86% 53% 23% 18% 16% 13% 12% 11% 7% 10% 10% 8% 7% 5%

Fig.3–Numberandproportion(%)ofeachAMFspeciesrecoveredbytrapcultureforthefollowingareas:Non-rehabilitated contaminatedarea(NR);Systemwitheucalyptusplantedinrowsandwithoutbrachiariaplantedininter-rows(S1),and Systemwitheucalyptusplantedinrowsandwithbrachiariaplantedininter-rows(S2).Sporestotalamountwas484forNR, 1355doS1,and2882forS2.*AMFwithlowproportioninthesoilinNR:DentiscutataheterogamaandGigasporasp.;**AMF withlowproportioninthesoilinSystem1:Acaulosporalongula,Gigasporasp.andScutellosporasp.

Besidesincreasingthenumberofspores,thecommunitywas morebalanced,withlessspeciesdominance,whencompared tonon-rehabilitatedsite,oronlywithEucalyptus.

Glomalincontents

Revegetationincreased glomalin contents,which was very lowinthenon-rehabilitatedsite(Fig.4).Glomalinwashigher (p<0.05)inplotswithEucalyptusandBrachiaria,wherespore countswasalsohigh.EE-BRSPcontentofthedirectfield sam-plessignificantlydecreased(p<0.05)inthefollowingorder:

C>EcB-R,EcB-IR,Ec-RandP>Ec-IR>NR.RegardingtheBRSP contentsofthedirectfieldsamples,EcB-R,EcB-IR,andCsites didnotdifferfromeachotherandhadthehighestvalues, fol-lowedbyEc-RandP,whichwerealsosimilartoeachother.NR siteexhibitedthelowestBRSP(Fig.4).

Glomalin contents also negatively correlated (p<0.05) with heavy metals in the soil. EE-BRSP values were lin-earlyreducedwithconcentrationsofZn(y=1.3463−0.00003x;

R2=−0.92731), Cu (y=1.53450.0019x; R2=−0.94244); Pb (y=1.2511−0.0002x;R2=−0.97267)andCd(y=1.25650.001x; R2=−0.96303). A similar trend was observed for BRSP,

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b c b b d b a B C A A D B A 0 0.5 1 1.5 2 2.5 3 C P NR EcB-IR EcB-R Ec-IR Ec-R Glomalin (mg . g –1 soil) Sites of Study EE-BRSP BRSP

Fig.4–TotalBRSPandeasilyextractableglomalin (EE-BRSP)contentsinsamplesfromthedirectfieldsoil samplescorrespondingtothecontaminatedand rehabilitatedareas(Ec-R,Ec-IR,EcB-R,andEcB-IR),tothe non-rehabilitatedarea(NR),andtothenon-contaminated areas[Cerrado(C)andPasture(P)].Uppercaseletters– comparisonofBRSPvalues;lowercaseletters–comparison ofEE-BRSPvalues.Barswiththesameletterdonotdiffer bytheScott–Knotttest(p<0.05).

whose values were linearly reduced with contents of Zn (y=1.8846−0.00004x; R2=−0.84895), Cu (y=2.265900031x; R2=−0.97891),Pb(y=1.72870.0003x;R2=−0.76431),andCd (y=1.7371−0.0014x;R2=−0.76948)(p<0.05).

Discussion

Replacementofthecontaminatedsoilwithtreeplantationin rowswithorwithoutB.decumbenssowingintheinter-rows allowed successful rehabilitation of the soil. These reha-bilitation programspromote reductionof totaland soluble contaminantmetals (Zn,Cu, Pb,and Cd) inthe soil. Com-paredtothe concentrations found atthe beginning ofthe rehabilitationprocess,thesemetalswerereducedby90%,and allowedtheestablishmentandthegrowthoftheintroduced plants.Decreaseinmetalcontentsvariedaccordingtothe ele-ment,andwerehigherinrowsthanininter-rowsduetothe amountofsoilreplacedandtothegrowthofnewseedlings. However,ZnandCdconcentrationsexceededabitthe lim-its set by COPAM for the agricultural use ofsoil (450 and 38mgkg−1 ofdrysoilofZnandCd,respectively).15 Inturn,

NRsitepresentedthehighestconcentrationofheavymetals. Thus,itdemonstratedthatrehabilitationbysoilexcavation andreplacementwithnon-contaminatedsoilwaseffectiveto createanadequateenvironmentforplantinitialgrowth.As shownintheFig.1,plantswereabletodevelopwellinthe con-taminatedarea,andconsequentlyshowedcontinuousgrowth whichpromotedAMFmultiplication.Infact,itwasfounda totala15differentAMFmorphotypesinthearea,andmostof thesespecieshadbeenpreviouslyreportedinstudiesofAMF occurrenceandecologyinthesesitesbeforerehabilitation.3

However,differentlyfromtheseauthors,inrehabilitatedarea, itwasfoundAcaulospora longulaandRhizophagusdiaphanus,

which indicates that rehabilitation allowed sporulation of

other species unable to sporulate in the non-rehabilitated soils.

Disregarding thecontrolsites (PandC), 15AMFspecies were foundin thepresent study.Thisnumber was similar to,orhigherthantheonesreportedbyotherstudiescarried outinheavymetalscontaminatedsoils.StudycarriedinIndia foundonlyfourAMFspeciesinasoilcontaminatedwithheavy metals(Cu,Zn,Cr,CdandPb)collectedfromatannery treat-mentplant.23Inotherstudiescarriedinbauxiteminingareas

inBrazil,andinsewagesludgedepositionareasthatwere con-taminatedwithZn,Cd,Cu,NiandPbinGermany,researchers foundsixAMFspecies.4,24 Fifteenspeciesweredetectedin soils contaminatedwith Zn, Cu, Pb,Ni, and Cdin India.25

ThepresentresultsshowedthatrevegetationwithEucalyptus

andBrachiariaassociatedwithreplacementofcontaminated soilbyuncontaminatedsoilmaycontributedtoacontinuous revegetationprocessofthearea.

It was foundpredominance ofspecies from the Glomus

and Acaulospora genera, which confirms that theyare bet-teradaptedtostressfulconditionsofsoilcontaminatedwith heavymetals.26 Infact,Glomussp.wasageneralistspecies

sinceitwastheonlyonefoundinallthesites.ThespeciesP. occultumwasalsoconsideredageneralist,sinceitwasdetected bythetrapculturetechniqueinmostplots.Aspointedout byotherauthors,27–29AcaulosporaandGlomusgeneraarewell

adaptedtoexcessofheavymetals,andfewreportsrevealthe occurrenceofP.occultumincontaminatedsoils,asobserved inthepresentstudy.However,Melloni,SiqueiraandMoreira4 reported theoccurrenceofP.occultum inminingsoilunder rehabilitation.Rhizophagusintraradicesrecordedinthisstudy hadonlybeenpreviouslyrecordedinsoilscontaminatedwith Zn,Cu,CdandPbinbauxiteminingareasinthenorthofBrazil andingypsumminingareas.3,28,30,31

AmongtheAMFspeciesdetectedindirectfieldsamples,

Acaulospora sp.and C. pelucida were not found in the trap culture pots. In turn,the species A. foveata,A. colombiana,

andGlomussp.(sporocarp)wereexclusivelydetectedbythis method.

ExceptforA.colombiana,whichwasfoundonlyinthetrap culturefromCerrado,itissuggestedthatthechangeinthe AMFcommunity structuremay berelatedto alterationsin thestresslevels.Inthisstudy,stresslevelswerehigherinthe field(higherheavymetalconcentrations),whencomparedto trapcultures(lowerheavymetalconcentrationspromotedby dilutingtheoriginalsoilinthepreparationoftraps),whichis supportedbyRydlováandVosátka.32Theseauthors

empha-sizedthatAMFmaylosetheiradaptationandstresstolerance whensub-culturedwithoutinitialstress.

TheseresultshighlighttheimportanceofdetectingAMF diversitybymultiplyingthemintrapculturepots,inaddition tothesurveyofsporesdirectlycollectedfromthefield,inorder topromoteaccesstoalargernumberofspeciesinstudieson diversityandtoaffordamorecompletepictureofthebiology, ecology, anddiversityofAMFcommunities.33 Nevertheless,

severalauthorssuggestthatthetrapculturemethodmight selectforspeciesthateasilysporulate, andmightomit the onesthatcolonizeroots,althoughtheydonotsporulate.34

Aftertheevaluationofthedirectfieldsamples,itwasfound greaterAMFspeciesrichnessinEcB-IR,whereasforthetrap cultures,thegreatestspeciesrichnesswasdetectedinEc-R.

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ofother AMFspecies atsampling time. However,byusing trapping, it was possible to recover two other species (A.

colombianaandP.occultum).Itshouldalsobeconsideredthat samplingoccurredintherainyseason,andatthiscondition, lowamountofsporesisusuallyfound.35,36Ontheotherhand,

AMFtotaldiversityfoundinCerradowaslower,when com-paredtorehabilitationsystemsandpasture,possiblybecause Cerradoismorestablethantheotherenvironmentsregarding thevariationinsoilcharacteristics.Theseconditionscouldact asselectionpressure on fungal communitieswithreduced sporulation, or produce spores with low capacity toresist adverseconditions.37,38Thisisconsistentwithfindingsoflow

sporedensityinclimaxecosystems.39Thus,althoughseveral

studieshavedemonstratedhighdiversityofAMFinCerrado, edaphicconditionsandvegetationcovermightalsopromote changesinsporedensity.4

Inastudyonsoilsfromabauxiteminingareaunder reha-bilitation,it was foundthat miningaffected spore density, whichincreasedafterthe beginning ofrehabilitation.4 The

lower spore density inthe Cand P, mightbeattributed to thenaturalstabilityoftheseecosystems,especiallyduetothe constantpresenceofhosts,andtotheabsenceofabrupt vari-ationsinsoilfertility.Thesecharacteristicsmightensurethe survivaloffungalspecieswithlownaturalsporulation abil-ity,orwhichproducesporeswithpoorresistancetoadverse conditions.4,40 Similar effectsmightalso befoundinareas

subjectedtoagriculturalcultivation,suchaspasture,which are influenced bysoil handling techniques, suchas tilling andfertilization,extensivemonoculture,andtheuseof agri-culturaltoxins. According tosome authors these handling techniques might reduce the incidence of some AMF and sporulation.40

Thenumberofsporescountedinthedirectfieldsamples waslower,whencomparedtothecountinthetrapculture potsin allthe investigatedsites. Ithasbeen reportedthat thenumber ofsporesinthedirectfield samplesisusually low, andthey are parasitized.41 Besides that, usuallyall of

thesubcellularstructuresneededforaccurateidentification ofthespeciesarenotintact.Therefore, AMFmultiplication systemin hostplantsunder controlledconditions isused. Inthe presentstudy,the use ofthetrap culturetechnique allowedtheobservationthatAMFspeciesfrom siteshighly contaminatedwithheavymetals,suchasNR,presentedhigh sporulationcapacityunderlesscontaminatedconditions,as wellasinthepresenceofamycotrophichostplant,B. decum-bens,whichwasusedasabaitplantinthetrapculturepots. Inaddition,thegreatersporulationfoundinEcB-IRmightbe associatedwiththepresenceofB.decumbensintheinter-row areas,whichprobablyfavoredsporulation.Studiesthat eval-uatedtheeffectofB.decumbensinrevegetationofdegraded soils,showed400%increaseinsoilspores,whichconfirmed thatthisgrassspeciesiseffectiveinmultiplyingAMF.42

Soilrestorationsystem2(Table1)providedbetterbalance betweenAMFpopulations,sincenopredominanceofanyAMF

In the present study, BRSP was related to soil rehabili-tation, sinceNR,wherethe soilpresentedlowervegetation coverandhigherconcentrationsofheavymetals,presented lowerEE-BRSPandBRSPcontents,assuggestedfordegraded areasingeneral.11Inconditionswithhighconcentrationsof

heavymetals,AMFusuallypresentsprotectivemechanisms, suchasgreaterproductionandrenewalofexternalmycelium, which contributestoglomalin synthesis.43,44 However,it is

worth noting that regardless ofthe rehabilitationprogram (systems 1 and 2),EE-BRSP and BRSP values found in the contaminated areas were higher than, or similar to those foundbyotherauthorsinareaswithothertypeof contam-inants.Studies showed valuesofEE-BRSPthat variedfrom 0.4to0.07mggsoil−1,together withanincreaseinthelevel ofcontamination withCr.45 Thehigh levels ofglomalinin Cerradosoilwereprobablyrepresentedbyoldstocksofthis glycoprotein,sinceitsstabilityandpersistenceinpreserved environmentscanreach42yearsintropicalsoils.46However,

noneofthe treatmentsreachedBRSPvaluessimilar tothe 60mggsoil−1foundinforestsoilsoftemperateareas.46

Conclusion

Theconcentration ofheavymetalsinthe soilofthe areas under rehabilitationaffected the diversityand the compo-sition of AMF communities. The rehabilitation system 2, whichincludedplantingofEucalyptusinrowsandBrachiaria

in the inter-rows, presentedthe mostfavorable conditions forthedevelopmentandoccurrenceofAMF,withpotential applicationinrehabilitationprogramofcontaminatedareas. Althoughatalesserdegree,positiveeffectswerealsoobserved insystem1.ItwasevidentthatGlomussp.isadaptedtothe stress caused by different concentrations ofheavy metals, sinceitwasdetectedinallthesitescontaminatedwithZn,Cu, Cd,andPb.DiversityofAMFandglomalincontentscanbe con-sideredgoodindicatorsofrehabilitationofsoilscontaminated withZn,Cu,PbandCd.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthanktheCoordinationofHigherEducation Per-sonnelTraining(Capes)andSupportFoundationtoResearch ofMinasGerais(Fapemig),forfinancialsupportofthisproject, andforthefellowships.TheauthorsalsothanktheNational CouncilofScientificandTechnologicDevelopment(CNPq)for financialsupportandfellowships.

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