Alvesa, Sônia Regina de Souza

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

Environmental

Microbiology

Contribution

of

dark

septate

fungi

to

the

nutrient

uptake

and

growth

of

rice

plants

Carlos

Vergara

_,

_Karla

_Emanuelle

_Campos

_Araujo

_,

_Luiziene

_Soares

_Alves

_,

Sônia

Regina

de

Souza

_,

_Leandro

_Azevedo

_Santos

_,

_Claudete

_{Santa-Catarina}

_,

Krisle

da

Silva

_,

_Gilmara

_Maria

_Duarte

_Pereira

_,

_Gustavo

_Ribeiro

_Xavier

_,

Jerri

Édson

Zilli

a_{UniversidadeFederalRuraldoRiodeJaneiro,InstitutodeAgronomia,Seropédica,RJ,Brazil}

b_{UniversidadeEstadualdoNorteFluminenseDarcyRibeiro(UENF),CentrodeBiociênciaseBiotecnologia(CBB),LaboratóriodeBiologia}

CelulareTecidual(LBCT),CamposdosGoytacazes,RJ,Brazil c_{EmbrapaRoraima,BoaVista,RR,Brazil}

d_{UniversidadeFederaldeRoraima,CentrodeEstudosdaBiodiversidade,BoaVista,RR,Brazil}

e_{EmbrapaAgrobiologia,Seropédica,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6October2016 Accepted19April2017 Availableonline23August2017 AssociateEditor:WelingtonAraújo

Keywords: OryzasativaL. DSE

NO3−-N Tillering Colonization

a

b

s

t

r

a

c

t

Theuseofdarkseptatefungi(DSE)topromoteplantgrowthcanbebeneficialtoagriculture, andtheseorganismsareimportantalliesinthesearchforsustainableagriculturepractices. Thisstudyinvestigatesthecontributionofdarkseptatefungitotheabsorptionofnutrients byriceplantsandtheirensuinggrowth.Fourdarkseptatefungiisolatesthatwere identi-fiedbyInternaltranscribedspacerphylogenywereinoculatedinriceseeds(Cv.Piauí).The resultingrootcolonizationwasestimatedandthekineticparametersVmax andKmwere calculatedfromthenitratecontentsofthenutrientsolution.Themacronutrientlevelsin theshoots,andtheNO3−-N,NH4+-N,freeamino-Nandsolublesugarsintheroots,sheathes andleavesweremeasured.Thericerootsweresignificantlycolonizedbyallofthefungi,but inparticular,isolateA103increasedthefreshanddrybiomassoftheshootsandthenumber oftillersperplant,amino-N,andsolublesugarsaswellastheN,P,K,MgandScontents incomparisonwiththecontroltreatment.WheninoculatedwithisolatesA103andA101, theplantspresentedlowerKmvalues,indicatingaffinityincreasesforNO3−-Nabsorption. Therefore,theA103Pleosporalesfunguspresentedthehighestpotentialforthepromotion ofriceplantgrowth,increasingthetilleringandnutrientsuptake,especiallyN(duetoan enhancedaffinityforNuptake)andP.

PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.

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

∗ _{Correspondingauthor.}

E-mail:jerri.zilli@embrapa.br(J.É.Zilli). https://doi.org/10.1016/j.bjm.2017.04.010

Introduction

Despitetheincreasingyieldscausedbygeneticprogressover thelast30years,improvementsincropyieldshavenotbeen very evident, especially for rice,1,2 _{which is the most}

fre-quentlycultivatedandconsumedcerealintheworld.3_That

said,farmershavetofacetheincreasedcostsofseeds, chem-icals,andfertilizers.2_{Inthisscenario,increasingcropyields}

atlowercostscouldbeachievedbyincreasingtheroot sur-faceprovidedbyassociationswithfungi,culminatinginbetter nutrientuptakeandplanthealth.4,5

Dark septatefungi(DSE) are ascomycetes, and theyare characterized by having dark pigmentation, microsclerotia andmelanizedseptatehyphaethatcolonizetheroot epider-misandcortexinter-andintracellularyinthehostroots.6–11

Manyofthesefungiareabletocolonizetherootcellsofplants, promotinggrowthwithoutcausingpathologies.12,13

Thesefungiofteninhabitoligotrophicsoilsthatare asso-ciated with the roots of hundreds of plant species in all climateregionsandmajorbiometypes.12,14–20_AlthoughDSE

research is increasing, our knowledge of the diversity of thesefungiremains restricted.21 _{DSE classificationis}

grad-uallybeingimprovedand newspeciesarebeing described, but many isolates do not yet have adequate taxonomic positioning.21,22 _{For example,the three novel genera}

Dark-sidea,AquilomycesandFlavomycesbelongingtoLentitheciaceae, Morosphaeriaceae and, Massarinaceae family were recently documented.21 _{Todate,approximately40 DSEspecieshave}

beendescribed.6,21,23–26

Theability ofthese fungito promote plantgrowth has attractedattention because oftheir potentialuse in differ-entspecies,suchasconifers,grasses,andcabbages,among others.6,22,27–29 _{Furthermore, because they readily grow in}

culture media and are not biotrophic, DSEs have advan-tages over other fungi because their inoculant production canbeeasier.6,30 _Newsham13 _{performedameta-analysisof}

18 independent studies to assess the inoculation of DSE in differentcrops, concludingthat this practice raisedthe nitrogen (N) and phosphorus (P) contents as well as the plantbiomassby26–103%.Conversely,anothermeta-analysis suggestednegativetoneutraleffectsfrominoculating with non-clavicipitaceousrootfungalendophytes(includingDSE) ontheplantbiomassandNcontent.31_{Althoughtheinfluence}

ofDSEonitshostisstillunderdebate,theidentificationof DSEwithbiotechnologicalpotentialexpandsthehorizonsfor itsuseinagriculture,asisalsothe casefornitrogen-fixing bacteriaandotherbeneficialmicroorganisms.

InBrazil,recentstudieshaveshownthepresenceofDSE intherootsofhealthyOryzaglumaepatulaplantsinthe natu-ralenvironment.32_{Inthisstudy,someisolateswereableto}

colonizeand contributetothedevelopmentofOryza sativa L.withoutcausingdiseasesymptoms.32–34 _{Inthesameway,}

basedonintergenicspaces,Bonfimetal.35_{identified35DSE}

groupsrepresenting27speciesthatwereisolatedfrom7native trees,indicatingthehigh diversityofthesefungi.Likewise, anewdarkseptate fungusspeciesfrom China(Harpophora oryzae)wasrecentlyidentifiedinthehealthyrootsofOryza granulata,whichwasabletoincreasethebiomassofO.sativa, alsowithouttriggeringdiseasesymptoms.22

ThewayinwhichDSEestablishtheirassociationwiththe hostisnotfullyunderstood,butstudieshaveindicatedthat growthpromotioncanbeperformeddirectly,byenhancingthe nutrientuptake;orindirectly,byprotectingtheplantagainst abiotic stresses (drought,salinity, and high concentrations of heavymetals)and the productionof phytohormonesor analogoussubstances.27,36–38_{InBrassicacampestrisL.,a}

mutua-listic association was found with the fungus Heteroconium chaetospira,inwhichthefungussuppliesNtotheplantand theplantprovidescarbontothefungus,resultinginsignificant increasesinplantbiomass.27

ThebestgrowthpromotionresponsesfromDSEusehave beenobservedwhenorganicsourcesofNareused.12,13,27,28

However,becauseofthegeneralizedoccurrenceofdark sep-tatefungiinawiderangeofenvironments,20,39_{therearelikely}

DSEspeciesthatarealsoabletoassistinnutrientuptakefrom inorganicsources.27

DSEs have been shown to form intraradical structures resemblingthoseformedduringmycorrhizalsymbioses.For example,Acephalamacrosclerotiorumformedectomycorrhizae inpineandspruce29_{andH.chaetospiraformedloose}

intracel-lularhyphalloopsthatmorphologicallyresembledtheericoid mycorrhizaeinRhododendronobtusumvar.kaempferi.40

How-ever,therolesoftheseintraradicalhyphalstructuresarestill atthehypothesislevel.

Inapreviousstudy,thecompositionaldiversityoftheDSE that colonized native rice(O.glumaepatula)inthe Brazilian Amazonwasinvestigated.33 _{Basedoninvitrotests,32}

non-pathogenicisolateswereconsideredasDSEfungi.33_Thisstudy

wasperformedtoaddress thephylogeneticpositionandto assessthecontributionofdarkseptatefungalisolates(A101, A103,A104andA105)obtainedfromO.glumaepatula33tothe absorptionofnutrientsandthegrowthofriceplants(O.sativa) undercontrolledconditions.

Materials

and

methods

Fungalisolates,DNAextraction,andphylogenetic analyses

All of the isolates investigated here are deposited in the CRB-JD(CentrodeRecursosBiológicosJoannaDöbereinerat EmbrapaAgrobiologia–www.embrapa.br/agrobiologia/crb-jd) (A101, A103, A104, and A105). For the phylogenetic analy-ses,genomicDNAwasextractedfromeight-day-oldcultures growninPDAmedium.Fivediscsof6mmeachwere trans-ferredfromtheplateto2mLsterilemicrotubes,towhich0.2g ofglassbeads(106␮m;Sigma)and1mLofsterilizedultrapure waterwereadded.Thesuspensionwasvigorouslyagitatedby vortexandcentrifuged(1.5min;13000rpm).Thesupernatant was transferredtocleanmicrotubes, and 600␮Lofthe cell lysissolutionfromthekitWizard®GenomicDNAPurification (Promega)wasadded.Thesampleswerevigorouslyagitated byvortex(10min)andsubjectedtothreecyclesof95◦_C/ice, with10minforeach step.Thesecycleswere followedbya Wizard® GenomicDNAPurificationprotocol.The ITS1-5.8S-ITS2regionoftherDNAfragmentwasamplifiedusingprimers ITS1andITS4.41_{PCRwasperformedwith25␮Lreactions}

dNTP,0.2␮Mofeachprimer(ITS1andITS4),1.25UofTaqDNA polymerase(5U␮L−1),and 1.0␮LofDNAtemplate(approx. 50ng).Theamplificationconditionsfrom Maharachchikum-buraetal.42_{wereused.DNAsequencingwasperformedfor}

bothDNAstrands,andthesequenceswereassembledwith Bionumericssoftwarev.7.1.

Thedatasetusedforthephylogeneticanalysiscontained taxonbelongingtodothideomyceteordersandonly Pleospo-raleswasrepresentedbytheirknownfamilies.Theorder-level dataset was used to gain information about the phyloge-neticpositionofA101,A104andA105isolatesinPleosporales (Fig.2).Alignmentsofoursequencestogetherwithsequences from GenBank were performed using MUSCLE in MEGA v. 7.43 _{The sequences were edited using MEGA v. 7. Aligned}

datasetofITSwasanalyzedusingMaximumlikelihood(ML). Thebest model forML was selected based on the Akaike InformationCriterion(AIC), whichwascalculated inMEGA v. 7. ML analysis was done by calculating an initial tree usingBioNJandthesubsequentHeuristicsearchdonewith the Nearest-Neighbour-Interchange (NNI) option. Distance matriceswerecalculatedusingtheKimurathree-parameter substitutionmodel43 _{and the robustnessof the treenodes}

wasevaluatedbybootstrapanalysis44_{using1000replicates.}

The software default parameters were considered in all analyses.TheITSregionsequenceswere depositedin Gen-Bank (KR817246=A101, KR817248=A103, KR817249=A104, andKR817250=A105).

Experimentaldesign,treatments,andconditionsofthe incubatorroom

This experiment was performed in an incubator room at EmbrapaAgrobiologia,inSeropédica,RiodeJaneirostate.The experimentaldesignwascompletely randomized,withrice planttreatmentsgrownwithout(control)andwiththe inocu-lationofdarkseptateendophyticfungi(DSE).Eachtreatment includingthecontrolhad4replications,andeachreplication hadfourplants.ThePiauíricevariety,agenotypeadaptedto thenaturallow-fertilityofBraziliansoilwasused,alongwith thefungalisolatesA101,A103,A104andA105.Theplantswere grownwitha13h/11h(light/dark)photoperiod,aluminosity of384␮molm−2s−1(photosyntheticallyactivephotonflux),a relativehumidityof50–60%andatemperatureof28◦_C/24◦_C (day/night).

Inoculationandgrowthconditions

Thericeplants were grownindisposable Petriplates con-taining sterilized agar-water (1% concentration), and they were adaptedtoallowfor themycelial growth.Two plates (oneinverted) andonelidcontainedfour equidistantholes (approximately8mmindiameter),andtheholesonthe bot-tomweresealed withadhesivetapetoholdthe previously autoclavedagarwater(Fig. 1). Theset ofplates wassealed withhigh-resistanceadhesivetape(Fig.1)andthenexposed toultravioletlightfor20mininalaminarflowhoodtoprevent contamination.

ThefungalisolateswerepreviouslygrownonPDAmedium for seven days at 28◦_{C. The rice seeds were washed in} 70% alcohol for 5min and disinfested with 2.5% sodium

hypochlorite for10min, followed bysuccessivewashing in autoclaveddistilledwater.Theseedswereplacedonthe agar-watermediumatthespotswhereeachplatewasperforated. Additionally,onediskofPDAmedium(approximately8mm) indiametercontainingmyceliawasplacedalongsideofeach seed.ThePetriplatesforthecontrolplantswereinoculated withPDAplugswithoutfungalmycelium.Aftertheirsowing, theplateswereincubatedforthreedaysat28◦_{Ctofavorfungal} germinationandestablishment.

Aftertheseedshadgerminated,fourplateswithuniformly sizedseedlingswereselectedfortransfertoglasspots contain-ingonlyautoclaved,distilledwater(120◦_{Cfor1h).Afterfive} days,thewatercontainedinthepotswasreplacedbynutrient solutionthatwasformulatedaccordingto45_{at1/4oftheionic}

strength,whichwasmodifiedwith1.5mmolL−1 _ofNO 3−-N (KNO3assource).Afterthreedays,thissolutionwasreplaced byanothersolutionat1/2ionicstrength,with2.0mmolL−1 ofNO3−-N and0.5mmolL−1 ofNH4+-N, withCa(NO3)2 and (NH4)2SO4, respectively.Thisstrategywasadoptedtoavoid causingsaltstressintheplantlets.46_{Thereafter,the1/2ionic}

strengthsolutionwasreplacedeverythreedays.

Thirty-twodaysaftergermination(DAG),theplantswere deprivedofnutrientsolutionNO3−-Nforaperiodof72hto increasetheroots’capacitytoabsorbN47_afterwhicha

nutri-entsolutioncontaining0.5mmolL−1_ofNO

3−,againbyusing KNO3asthesource,wassupplied.During11h,samplesofthe solutionwerethencollectedevery30minbyremoving0.5mL aliquotsfromeachpottoplotthedepletioncurvesand deter-minethekineticparametersVmax andKm.48,49Thesamples wereplacedinmicrotubesandtheNO3−concentrationwas measuredaccordingto50_{asadjustedfollowingAlvesetal.}51

TheVmaxandKmvaluesweredeterminedbyusingthe math-ematicalgraphingmethodproposedbyComettietal.52_with

theCineticawin1.0program(UniversidadeFederaldeVic¸osa, MinasGerais,Brazil).

Solublefractionandcolonizationmeasurement

Followingthecollectionofthelastnutrientsolutionsample, the plantswere cutandtheheightsandnumbers oftillers fromeachplantweredetermined.Thefreshanddrymasses oftherootsand shootswere measured,inthesecondcase afterbeingdriedinaforced-airchamberat65◦_Cuntil reach-ingaconstantweight.Thefreshsamples(onegramofroot, sheath and leaftissue)were homogenized in80% ethanol, and after partitioning the samples with chloroform,53 _the

levels of NO3−-N,50 free amino-N,54 NH4+-N,55 and soluble sugars56_{inthesolublefractionweremeasured.Theremaining}

shoot material was used to determine the macronutrient concentrations.57_{Afterremovingtheshoots,afreshrootwas}

removedfromeachpotandkeptonalcohol50%toobservethe colonized roots.Thediafanizationand stainingoftheroots were performed according to.58 _{The rootsections (approx.}

A

B

D

C

E

F

G

Lid

Inverted plate

Holes

Plate

P

etri plates

Pot 0.07

1.3

1.3

ø 9.00cm

ø 8.50cm

ø 8.50cm

8.50

12.00

Fig.1–VesselusedtostudytheNO3−-Nuptakekineticsinriceplants(var.Piauí)thatwereinoculatedwithdarkseptate

fungiandsubjectedto0.5mMofNO3−-Nafter72hofNnutrientdeprivation.(A)Petridishescontainingagar-waterwith

seedsandmyceliumdiscs;(B)fungalbiofilmofA103(10DAG);(C)fungalcharacteristicsatthecollectiontime(35DAG);(D) non-inoculatedplantat41DAG;(E)thestartofA103establishment(3DAG);(F)Potused;(G)overviewofvessel.

lightmicroscopy,the samplesweredehydratedtwiceinan ethanolseriesof70,90and100%for1heach.After dehydra-tion,thecolonizedrootswereinfiltratedwithhistoresin(Leica, Wetzlar,Germany)and100%ethanol(1:1,v/v)for12handthen with100%historesinfor24hbeforebeingembeddedin his-toresin.Sections(thatwereapproximately5␮mthick)were slicedusing a rotary microtome (Leica).The sampleswere observedandtheimageswereanalyzedasdescribedabove.

Statisticalanalyses

Ananalysisofvariance and at-test forthe comparisonof meanswereappliedtothedata(p<0.05)withAssistat61_and

Sisvarsoftware.62

Results

and

discussion

PhylogeneticsoftheisolatesbasedonITS

The sequences obtained from the isolated fungi were first subjected to a BLAST search (http://www.ncbi.nlm.

nih.gov) and to pairwise sequence alignment (http://www.

fungalbarcoding.org).FortheA101fungus,onlymatcheswith

lessthan 88% similaritywere found,indicating thatit rep-resentedanunknowntaxon.Fortheother threeisolates,a similaritytoafungusbelongingtothePleosporalesorderwas

found;thisisthelargestorderintheDothideomycetesclassand encompassessomerecognizeddarkseptatefungalfamilies.21

According to the results of the phylogenetic analyses, the three isolates (A103, A104 and A105) belonged to the Pleosporalesorderandarenestedinfamiliesofthesuborder Massarineae,neartheMorosphaeriaceae(Fig.2).Additionally, the three new isolates are closely related to some genera that were treated as incertae sedis (a genus notyet placed withinaspecificfamily/genus),suchasMassarinaigniaria(CBS 845.96),Periconiamacrospinosa (CBS135663),Noosiabankssiae (CPC17282),Flavomycesfulophazii(CBS135761)andFlavomyces fulophazii(CBS135664)(Fig.2).21_{Therefore,theITSsequence}

analysisindicatedthatourisolatesrepresentnewtaxathat willrequireaproperpolyphasicanalysisinthefuture,butat leastthreeisolatesbelongtotheclassicaldarkseptatefungal group,i.e.,thePleosporalesorder.

Colonization

The inoculation of the rice plants withfour different DSE isolatesdidnotcauseanydiseasesymptoms,indicatingthe compatibilityoftheassociation.Otherstudiesalsoindicated thatDSEcancolonizetherootcortexofplantswithout caus-inganypathologies.13_{Inthesameway,therootsofallofthe}

Table1–Heightsoftheshoots,numberofriceplant tillers(Piauívariety)at35DAG,withandwithout inoculationwithdifferentdarkseptatefungalisolates andthepercentagefungalcolonizationofriceroots.

Treatment Heightof shoots(cm)

Numberof tillers(pot−1₎

Colonization(%)

Control 67.1±1.6b 2.0±0.00c – A101 67.6±0.9b 3.13±0.06b 33.3±6.0b A103 72.4±1.1a 4.06±0.26a 60.3±10.6a A104 62.88±1.0c 3.25±0.20b 63.6±9.0a A105 67.7±1.0b 3.44±0.05b 77.2±5.4a

CV(%) 3.90 10.85 23.79

Means±SE(n=4)followedbythesamelower-caselettersinthe columndonotdiffersignificantly(t-test(LSD),p<0.05).DAG,days aftergermination.Themeansoffourcomposedrepetitions(each repetitionhadfourplantsperpot).SE,standarderror.

microsclerotiainthecortexroot(Fig.3E,D,F,K,L,P,QandR). A104andA105formedintracellularhyphaethatwerestained withmethyl blue inthe roots ofall of the inoculated rice plants(Fig.3I,M,and N).A103andA101formed intracellu-larmelanizedseptatehyphae (Fig.3GandS),andthe A103 fungusalsoformedchlamydospore-likestructuresthat sur-roundedthevascularbundleregion(Fig.3H)aswellasaloose intracellularhyphalloop(Fig.3C)andA101formed microscle-rotia(Fig.3S)intherootsofalloftheinoculatedriceplants.In addition,duringtheexperiment,thefunguswasfoundtobe growingabundantlyalloverthewater-agarinthePetridish thathadbeentossedintothepotplantedwithrice(Fig.1C). Moreover,abundantfungalgrowthwasdirectlyobservedover therootsurface,evenwhentherootsweresubmergedinthe nutrientsolution(Fig.1B).Thecontrolplantsdidnotdisplay anyfungalcolonization(Figs.1Dand3A).However,inallofthe treatmentswithdarkseptateinoculation,abundant coloniza-tionswereobserved,varyingfrom33to77.2%(Table1).The percentageofcolonizedrootsintheplantsinoculated with A103,A104,A105wassignificantlyhigherthanthatofA101 fungus(Table1).Theseresultsaresimilartoresultsobtained recentlybyLukeˇsováetal.,29_{inwhichtheauthorsobserved}

alooseintracellularhyphalloopformedbyA. macrosclerotio-rum in birchroots, with colonization varyingfrom 51–61% when the birches were inoculated with Acephala applanata andA.macrosclerotiorumand 50–80%whenblueberrieswere inoculatedwithPhialocephalafortiniiss.l.–A.applanataspecies complex(PAC).

Plantbiomass,growthandnutrientaccumulation

Anevaluationofthedrymattershowedthattheplantsthat wereinoculatedwithisolateA103hadhighervaluesforthe roots,sheathes,leavesandshoots,withincreasesof44%,43%, 25%and 32%,respectively,incomparisonwiththecontrol, indicatingthatthisfunguscontributestoriceplantgrowth (Fig.4A).However,theinoculationwithA104caused reduc-tionsof24%,11%and22%inthedrymatterofthesheaths, leavesandshoots,respectively(Fig.4A),andisolatesA101and A105didnotinfluencetheplantdrymatter(Fig. 4A). Like-wise,theheightsoftheplantswereinfluencedpositivelyby

A103inoculation.However,A104causedanegativeeffect,and therewasnoeffectfromA101andA105incomparisonwith thecontrol(Table1).Thehigherrootmassprobablyindicates thegreaterpresenceoflateralandsecondaryrootsandroot hairs,whichisacharacteristicofrootsthatare morphologi-callyadaptedforamoreefficientuptakeofnutrients.Thistrait isdesirable,primarilybecauseitisanimportantattributefor the absorptionofnutrientssuchasNand Punderlowsoil fertilityconditions.48,63,64

Theresultsofthedrymatterandplantheightevaluations were widely variable among the fungal isolates. The A103 isolatepromotedthegreatestaccumulationofbiomassand the greatestelongationofthe shoot,which waspreviously observed.Forexample,inDendrobiumnobile65_{and Saussurea}

involucrata,66 _{DSE inoculation also promoted greater plant}

heightsandbiomassesintheshootsandrootsincomparison withthecontrolplants.Moreover,theinoculationofthehost plants(blueberries)andbirchseedlingswithA.applanataand A.macrosclerotiorumshowedincreasingdryshootweightsand freshrootweights.29_{However,thelatterauthorobserved}

neg-ativeeffectswhentheblueberrieswereinoculatedwithPAC, whichisinaccordancewith.31

Forthedrymattervaluesandheightsoftheplantstreated withA103,therewasalargernumberoftillersintheplants inoculatedwiththefungalisolates,withthestandoutagain being the plants that were inoculated with A103, with an increase of100% comparedwiththecontrol(Table1).This more intensetillering likely resultsfrom the greater plant vigorthatoccurswheninoculatingwiththisisolatebecause other studies have shown a high correlation between the nutritionalstateandsupplementationofcarbohydrates.67,68

Newsham69 _{foundthattheDSEfungusP.graminicola,which}

iscurrentlycalledHarpophoraradicicola,wasbeneficialtothe grassVulpiaciliatabecauseinoculatedgrasseshadincreased tillernumbers,rootlengths,anddrybiomassesfortheshoots androots,whencomparedwithnon-inoculatedseedlingsin agrowthroomandaglasshouseexperiment.

Anassessmentofthemacronutrientcontentsintheshoots showedthattheA103treatmentincreasedtheN,MgandS contentsinrelationtothecontrol(by29,19and30%, respec-tively), andthose levelswerelower intheinoculationwith A104;therewerenodifferenceswithA101andA105(Table2). Therefore,A103inoculationpromotedagreateraccumulation ofthesenutrientswhiletheotherfungalisolatestendednot toproduceeffects.

The 30% higher accumulation ofN foundin this study intheA103-inoculatedplantswhenammoniumnitratewas appliedas theNsourceappearstoindicate that greaterN accumulationdoesnotnecessarilyoccurwhentheNsourceis organic.Thisfindingmightresultfromthelowavailabilityof thistypeofnitrogensourceorthespecializationofthisfungus fortheabsorptionofmineralNintropicalsoils,aspreviously observedformycorrhizalfungi.70–73

b a

B

A

D

C

a _a a

a a a a a a a a a a a a a a a a a ab ab ab ab ab ab b a c c c c c c c c c ab c c c

b bb b b bc b b b ab b b b b b b d b b b b b bc bc bc bc cd d b b b b b b Control A101 A103 A104 A105

Dry mass(g pot

-1)

NH

4

+-N (µmol pot -1)

Free amino-N (µmol pot

-1)

Soluble sugar (mg pot

-1) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 300 60 40

Root Sheath Leaf Shoot Root Sheath Leaf Shoot

20 0 250 200 150 100 50 0 140 120 100 80 60 40 20 0

Fig.4–Drymass(A),thecontentsoffreeamino-N(B),freeNH4+-N(C)andsolublesugars(D)asdeterminedat35DAGin

riceplants(Piauívariety)withoutinoculation(whitebar)andthoseinoculatedwiththefollowingdifferentdarkseptate fungalisolates:A101(checkeredbars),A103(closedbars),A104(greybars)andA105(stripedbars).Differentletterswithina givenplanttissue(rootorsheathorleaforshoot)indicatesignificantdifferencesamongthetreatmentatp<0.05(t-test (LSD)).Theerrorbarsarestandarderror(n=4).Themeansoffourcomposedrepetitions(eachrepetitionhadfourplantsper pot).Theshootbiomassisthesumofthesheathesandleaves.

inorganicsourceofP.12,13_{TheCalevelwasequalforallofthe}

treatmentsincomparisonwiththecontrol,eventhoughthere wasatendency forhigheraccumulationintheplantsthat wereinoculatedwithA103andA105(Table2).

Nitrogenabsorptionkinetics

In evaluating the NO3− exhaustion rate obtainedfrom the nutrientsolution,inoculatingwithA101andA103increased NO3−consumptionincomparisonwiththecontroltreatment

(Fig.5AandB).Ingeneral,theabsorptionspeedwashigher duringthefirsthourswithA101,butintheplantsinoculated withA103,thehighestabsorptionspeedwasobserved approx-imately 7hafter inoculation,and the NO3− content ofthe nutrientsolutionwasdepleted11hafterthestartofthe exper-iment(Fig.5B).However,inoculatingwithA104andA105did notcauseanychangeinrelationtothecontrol.

AmongthekineticparametersofNO3−absorption,there wasonlyasignificanteffectforKm (Table3).TheA101and A103inoculationledtolowerKmvaluesthanthecontrol.

Table2–Macronutrientcontentsinthericeplantshoots(Piauívariety),at35DAG,withandwithoutinoculationwith differentdarkseptatefungalisolates.

Treatment N P K Ca Mg S

mgpot−1

Control 56.3±3.1b 13.1±0.3cd 49.2±3.8bc 7.9±0.6ab 8.0±0.3b 11.4±0.6b

A101 53.2±1.7b 13.7±0.7c 40.5±1.3c 6.4±0.2b 8.0±0.2b 10.3±0.3b

A103 72.8±1.1a 19.4±0.4a 65.0±4.9a 9.7±1.2a 9.5±0.3a 14.9±0.5a

A104 42.7±3.2c 11.6±0.6d 43.8±2.3c 6.8±0.5b 5.6±0.3c 8.6±0.5c

A105 53.9±1.0b 15.6±0.4b 54.6±1.2ab 9.6±0.3a 7.2±0.2b 11.4±0.2b

CV(%) 9.22 7.47 13.94 18.41 7.44 8.73

Controly=0.571055+0.012101x-0.012916x2+0.000715x3 R2=0.99*

Controly=0.571055+0.012101x-0.012916x2+0.000715x3 R2=0.99* Controly=0.571055+0.012101x-0.012916x2+0.000715x3 R2=0.99*

Controly=0.571055+0.012101x-0.012916x2+0.000715x3 R2=0.99*

A101y=0.567379-0.021279x-0.006336x2+0.000353x3 R2=0.99*

A104y=0.580133-0.022386x-0.006526x2+0.00039x3 R2=0.99* A105y=0.555153+0.031065x-0.013024x2+0.000573x3 R2=0.96*

A103y=0.557485+0.058437x-0.022065x2+0.00109x3 R2=0.98*

0.8

B

A

D

C

0.6

0.4

0.2

0.0

0.8

0.6

0.4

0.2

0.0 0

Time (h)

NO

3

- - N (mM)

NO

3

- - N (mM)

Time (h) 6

4

2 8 10 0 2 4 6 8 10

Fig.5–DepletionofN-NO3−inthenutrientsolutionforeachriceplant(Piauívariety)inoculatedwithdarkseptatefungi

A101(A),A103(B),A104(C)andA105(D),thensubjectedto0.5mMofN-NO3−after72hofNdeprivation.*Significant

accordingtotheF-testat5%probability.Theerrorbarsarestandarderror(n=4).

Table3–KineticparametersKmandVmaxforNO3− absorptionasdeterminedat35DAGinriceplants(Piauí variety)withandwithoutinoculationwithdifferent darkseptatefungalisolates.

Treatment Vmax(␮molg−1h−1) Km(␮molL−1)

Control 56.5±4.4a 88.6±4.1a

A101 55.1±3.0a 49.7±10.8bc

A103 46.8±3.2a 22.0±3.4c

A104 60.6±16.5a 76.4±14.8ab

A105 48.4±2.7a 96.1±11.9a

CV(%) 25.81 26.21

Means±SE(n=4)followedbythesamelower-caselettersinthe columndonotdiffersignificantly(t-test(LSD),p<0.05).Themeans offourcomposedrepetitions(eachrepetitionhadfourplantsper pot).SE,standarderror.

LowerKm valueswere estimatedforthe A101and A103

treatments(44and75%lowerthanthecontrol,respectively), indicatinggreater affinity fortransporting theNO3− ofthe

plantsthatwerecolonizedbythesetwoisolatesincomparison withtheothertreatments.Inthesameway,thebetternitrate uptakeefficiencyofthecolonizedtomatorootbyarbuscular mycorrhizalfungi(AMF)comparedwithanon-inoculated con-trolwaspreferentiallymediatedbyahigherexpressionofNRT 2.3,amemberofthehigh-affinitytransportsystem.74_Another

studyshowedthat soilinoculationwithAMFincreasedthe plantgrowthandNuptakeofwheatwhencomparedwitha non-inoculatedcontrol,anditwasattributedtoupregulation bytheAMFofnitratetransportergenes.75

Morphology, physiology and root system development, amongotheraspects,determinethevariationsinthe absorp-tionkineticsfactors(Km,VmaxandCmin)ofplantspecies.This variationisassociatedinturnwiththeadaptationofplantsto differentecosystems.48 _{Thesemorphophysiologicaltraitsof}

therootsystemareinfluencedbythemicroorganismsthatare presentintherhizosphere.75,76_{Therefore,itcanbeassumed}

thatthegreater NO3− absorptionefficiencyobservedinthe A103-colonized plantsisexplained bythelower Km values causedbythepresenceofthisfungus.Thisgreaterefficiency inacquiringNO3−whentherearelowconcentrationsin solu-tioncouldbeanindicationofplantadaptationstonutritional stressconditionsintropicalregions,whicharecausedbythe nitrogendynamicoftheenvironment.49

Plantsolublefraction

Thenitratecontentsoftheroots,sheathes,leavesandshoots ofthericeplantswerenotinfluencedbythetreatments, con-firmingtheabilityofthePiauívarietytoaccumulatenitrate, alongwithamino-Ninthesheathes,asalreadyobservedin previousstudies.49,64,77–79_{However,thefreeamino-Ncontent}

andshootspresentedlowercontentsofamino-Nthanthe con-trolplants,withrespectivereductionsof18%and 24%.The amino-Ndecreaseintheshootscouldberelatedtothe trans-portofaminoacidsfromthisparttotheroot,tosupplythe metabolicdemandsoftheA104andA105fungibecausethere arereportsthatsomedarkseptatefungiusefreeaminoacids astheirsolesourceofcarbonandnitrogen.27_Otherstudies

haveshownthatthemetabolicactivityofmycorrhizalfungi cancauseconsiderablechangesintheNmetabolism,witha reducedconcentrationoffreeamino-N.80–82

TheNH4+-N content wasgenerally higher inthe leaves thanintherootsandsheathes(Fig.4C).Similarresultshave beenobservedinthisvarietybyotherauthors.64,78,79,83 _The

inoculationdidnotinfluencetheNH4+-Ncontentsintheroots and sheathes(Fig. 4C). In contrast,there was lower NH4+ -NaccumulationintheleavesforA104andA105treatments comparedwiththecontrol(Fig.4C).Intheshoots,withthe exceptionoftheA105treatment,whichpresentedareduction of30%intheNH4+-Ncontent,allofthetreatmentspresented NH4+-Ncontentsequaltothatofthecontrol.Anotherstudy foundan NH4+-N reduction intransgeniccarrot rootsthat wereinoculatedwithAMF.82

Ingeneral,the solublesugar contentsofthe rootswere lower thanthose ofthesheathes, whichinturn contained a lower content than the leaves (Fig. 4D). Similar results were obtained by49 _{for the same rice variety. The soluble}

sugarcontent oftherootswas notinfluencedbythe pres-enceofthefungi(Fig.4D).Bycontrast,therewasadifference betweenthetreatmentsinthesheathes,leavesandshoots, withtheA103inoculationcausingthehighestsolublesugar contents,atanincreaseof46%comparedwiththecontrol. Thisresultindicatesthatinoculatingriceplants(Piauívariety) withA103canenhance maintenancerespirationand plant growth.Thehigherobservedsugarcontentisanindicationof moreefficientphotosynthesisintheA103-inoculatedplants. Thisfindingcanbeexplainedbythefactthatplants associ-atedwithDSEcanpresenthigher levelsofchlorophylland betterefficiencyofphotosystemIIphotochemistry.84 _Inthis

case,theestablishmentoftheassociationbetweenthehost plantandDSEshouldbesimilartotheeffectofmycorrhizae formation,whichresultsinchangesinthehostplant’s phys-iology,withincreasesinthechlorophylllevelsintheleaves and a consequently higher productionof photoassimilates andcarbohydrates.85,86

Theincreasesintheroot,sheath,leafandshootmasses along withthe greater heightand number of tillersinthe plantstreatedwiththeA103fungal isolateisanindication ofthebetternutritionalstateoftheseplantsaspromotedby anassociationwiththisfungus.Thistrendwasalsoevidenced bythehigheraccumulationsofsolublesugarsandthemore efficientuptakeofnutrients,especiallynitrogen(asindicated bythelowervalueofKm).

Concluding

remarks

Thereareimportantdifferencesinriceplantbenefits, depend-ingontheassociatedfungiandrangingfromthepromotion ofplantgrowthtonoeffect.ThePleosporalesfungusA103 pre-sentedhighpotentialforuseinthepromotionofriceplant

growth,increasingthetilleringandabsorptionofnutrients, especiallyN(withanenhancedaffinityforabsorbingthis ele-ment)andP.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WewouldliketothanktheUniversidadeFederalRuraldoRio deJaneiro(UFRRJ),particularlyfortheLaboratoryNutritionof PlantsandBiochemicalPlant,theUniversidadeEstadualdo NorteFluminenseDarcyRibeiro(UENF),especiallyforLBCT, theEmpresaBrasileiradePesquisaAgropecuária(Embrapa), the Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estadodo Riode Janeiro (FAPERJ) and the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) forfinancial support.Weare gratefulto MarcusSperandio, MarcelaJacques,OrlandoHuertas,PeterSoaresdeMedeiros andGuilhermeGomesfortheirsupport.

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