h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Dark
septate
endophyte
decreases
stress
on
rice
plants
Silvana
Gomes
dos
Santos
a,∗,
Paula
Renata
Alves
da
Silva
a,
Andres
Calderin
Garcia
a,
Jerri
Édson
Zilli
b,
Ricardo
Luis
Louro
Berbara
aaUniversidadeFederalRuraldoRiodeJaneiro(UFRRJ),Seropédica,RJ,Brazil bEmbrapaAgrobiologia,Seropédica,RJ,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received26April2016 Accepted9September2016 Availableonline27December2016 AssociateEditor:JerriZilli
Keywords: OryzasativaL. DSE
Waterstresstolerance
a
b
s
t
r
a
c
t
Abioticstressisoneofthemajorlimitingfactorsforplantdevelopmentandproductivity, whichmakesitimportanttoidentifymicroorganismscapableofincreasingplanttolerance tostress.Darkseptateendophytescanbesymbiontsofplants.Inthepresentstudy,we eval-uatedtheabilityofdarkseptateendophytesisolatestoreducetheeffectsofwaterstressin thericevarietiesNipponbareandPiauí.Theexperimentswereperformedunder gnotobi-oticconditions,andthewaterstresswasinducedwithPEG.Fourdarkseptateendophytes wereisolatedfromtherootsofwildrice(Oryzaglumaepatula)collectedfromthe Brazil-ianAmazon.Plantheightaswellasshootandrootfreshanddrymatterweremeasured. Leafproteinconcentrationsandantioxidantenzymeactivitywerealsoestimated.Thedark septateendophytesweregrown invitroinPetridishescontainingculturemedium;they exhibiteddifferentlevelsoftolerancetosalinityandwaterstress.Thetworicevarieties testedrespondeddifferentlytoinoculationwithdarkseptateendophytes.Endophytes pro-motedriceplantgrowthbothinthepresenceandintheabsenceofawaterdeficit.Decreased oxidativestressinplantsinresponsetoinoculationwasobservedinnearlyallinoculated treatments,asindicatedbythedecreaseinantioxidantenzymeactivity.Darkseptate endo-phytesfungiwereshowntoincreasethetoleranceofriceplantstostresscausedbywater deficiency.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Riceisoneofthemostconsumedcerealsintheworlddue toitsnutritionalvalueandbecauseitrepresentsan afford-ablesourceofprotein.1RicebelongstothegenusOryza,which
∗ Correspondingauthor.
E-mail:[email protected](S.G.Santos).
comprisestwocultivatedspecies(OryzaglaberrimaandOryza sativaL.)andagreatdiversityofwildspecies(notcultivated).2
Water deficiency is one of the most important causes of abiotic stress and compromises global food production, includingtheproductionofrice.Thisstresscausesirreversible oxidative damage because the activity of the plant’s
http://dx.doi.org/10.1016/j.bjm.2016.09.018
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
antioxidant system is not sufficient to limit the reactive oxygen species (ROSs) derived from byproducts of aerobic and photosynthetic metabolism to non-toxic levels. ROSs thereforeaccumulate inthe planttissues,3 wheretheycan
cause damagetocell organelles;oxidizeimportant biologi-calmoleculessuchasnucleicacids,lipidsandproteins;and compromisetheintegrityofthecellmembraneanddecrease photosynthesis.4
Plants respond to these stresses by producing catalase (CAT)andascorbateperoxidases(APX),theprimaryenzymes responsibleforthemaintenanceofH2O2atnon-toxiclevels
incellcompartments.5 However,inascenarioofprolonged stress,the antioxidant systemcannot preventcell damage causedbyoxidation,andothermechanismsbecomerelevant. Positiveeffectsofplantassociationswithbeneficial microor-ganismsunderstressconditionshavebeenreported.6–8
Dark septate endophytes (DSEs) are conidial or sterile ascomycetousfungithatcolonizelivingplantrootswithout causingany apparent negativeeffects.9,10 They are
charac-terizedbyintensedarkpigmentationand the formationof septatehyphae and occasionally microsclerotia, aswell as variousarbuscularmycorrhizalfungi(AMF).Thesefungican begrowninculturemediumandcancolonizeseveralplant species.Theycanbefoundinplantcorticalcellsinter-and intracellularlyandarepresentinseveralenvironments,even underdroughtconditions,inthe presenceofheavymetals andinoligotrophicsoils.11–16Thehypothesespresentedthus
farforthedominanceofDSEsinstressedenvironmentsand their effectson hostplant protectionare primarilyrelated tothe presenceofthepigmentmelaninatthe endophytes’ hyphaeandmicrosclerotia.12Melanincanactasan
antioxi-dantagentandalsobindheavymetalions,therebyprotecting cell structuresfrom the oxidativedamageproduced under such conditions.17 However, the DSE action mechanisms
involvedinplantprotectionhavenotyetbeenelucidated,18
but likely involve the presenceofextraradical hyphae and extracellularenzymes thatcan improvesoilexploration by roots.13
Thegoalsofthepresentstudyweretoassesstheability ofDSEfungi,originating fromtropicalsoils,togrowunder stressconditionsandtoinducestresstoleranceinriceplants (O.sativaL.).
Material
and
methods
DSEisolatesA,B,CandD(ERR01,ERR04,ERR16andERR 46,respectively)obtainedfromtherootsofwild rice(Oryza
glumaepatula)intheBrazilianAmazonbyRibeiroetal.19were
tested.TheseisolatesarestoredatCOFMEA(Embrapa Agrobi-ologyCultureCollectionofMicorrhizalFungi),andtheywere onlypartiallytaxonomicallydefineduptonow.19These
iso-lateswerepreviouslycharacterizedthroughamplificationand sequencingoftheinternaltranscribedspace(ITS1-5S-ITS2), anditwaspossibletopositiontheisolatesattheorderlevel.19
Following this analysis, A101is a member of Calosphaeri-ales,A102ismemberofCapnodialesandA103andA106are membersofPleosporales.TheITS1-5S-ITS2sequencesarealso depositedattheNCBIGenBank,accessionnumbersKR817246, KR817247,KR817248andKT780724.19
Nipponbare,animprovedvarietycommonlyusedinrice studies,andPiauí,awildvarietygrowninadrylandsystem, wereusedinallassaysinvolvingplant–fungusinteractions.
ThecapacityoftheDSEisolatestogrowunderstress con-ditionswas testedin twopreliminarytests inPetridishes. Bothwereplacedinaphytotron,usinggrowthmediumwith sodiumchloride(NaCl)orpolyethyleneglycol(PEG6000)for theinductionofsaltandwaterstress,respectively.ThePEG 6000isawidelyusedpolymertosimulatetheeffectofdrought instudiesinvolvingorganisms,primarilybecauseitis chemi-callyinertandnon-toxic.20
Forsaltstress,0.2,0.4,0.7or1molL−1NaClwasaddedto the culturemediumat26◦Ctoobtain anosmoticpotential of−0.49,−0.99,−1.73,and−2.43MPa,respectively.Acontrol treatmentwasincludedwithnosaltaddition.Thesalt con-centrationsneededtoobtainthedifferentosmoticpotentials werecalculatedusingtheVan’tHoffequation21:
os=−RTC,
where, os=osmotic potential (atm); R=ideal gas
con-stant (0.082atm. 1mol−1K−1); T=temperature (◦K);
C=concentration(molL−1).
Fungal mycelialdiscs 7mm indiameter, grown fortwo weeksinpotato-dextrose-agar(PDA)growth medium, were placed in Petri dishes containing 39gL−1 commercial PDA medium(Sigma–Aldrich,St.Louis,MO,USA)withthe addi-tionof0,0.2,0.4,0.7or1molL−1NaCl.Threereplicatesofeach treatmentwereperformed.Thedishesweregrownfor9days at26◦C.Colonydiameterwasmeasuredafter9daysusinga caliperandexpressedinmm.
StressduetoPEGadditionwasmeasuredbyplacingfungal mycelialdiscs,also7mmindiameter,inPetridishes contain-ing HoaglandsolutionsolidifiedwithPhytagel (2.5gL−1),to whichPEG-6000hadbeenaddedatdifferentconcentrations (0,79.791,121.139,180.231and264.246gL−1)toobtain0,−0.1, −0.2,−0.4,and−0.8MPawaterresistances,respectively.20The
incubationconditionswerethesameastheabove.
GrowthpromotionofriceplantsbyDSEunderwater
deficit
Theexperimentwasmaintainedinthe phytotronina ran-domized complete block designin afactorial arrangement 5×2×5(fourinoculatedfungiandanuninoculatedcontrol), tworicevarieties(NipponbareandPiauí),and5PEG concentra-tions(0,79.791,121.139,180.231and264.246gL−1),withfour replicates(twoplants=oneexperimentalunit).Thericeplants weregrowninHoaglandsolutionsolidifiedwith2.5gL−1 Phy-tagelatameantemperatureof26◦C.
Theseedsofthetwotestedricevarietiesweresterilized with2.5%sodiumhypochlorideand70%ethanolfor3minand inoculatedwithfungalmyceliagrownfortwoweeksinPDA, followedbypre-germinationfor5daysinPetridishes contain-ingHoaglandsolutionsolidifiedwith1%agar.Fungalmycelial discs 7mmindiameterwereplacedonthe dishes’surface closeto theseedlings. Thecontroltreatmentsconsistedof non-inoculatedseedswithouttheadditionoffungalmycelial discstothegrowthmediumwithonePDAdiscadded(7mm indiameter).
Followingseedpre-germination,theseedlingswere trans-ferred into pots containing sterile half-strength Hoagland solution supplemented with 0.3gL−1 MgSO4 and solidified
with 2.5gL−1 Phytagel.22 The pots were kept under a 12h
light/12hdarkphotoperiodata380Mmolm−2s−1 photosyn-theticphotonfluxdensity,70%mean airrelativehumidity, andtemperatureof28/24◦C(day/night).Plantheightaswell asshootandrootfreshanddryweightsweremeasuredat30 daysaftersowing.
Proteinconcentrationandantioxidantenzymeactivity
Acompletelyrandomizedexperimentaldesignwasusedina factorialarrangementof2×2×3(inoculatedfungi,isolateA anduninoculatedcontrol),tworicevarieties(Nipponbareand Piauí),andthreePEGconcentrations(0,35and70gL−1)infour replicates(twoplants=oneexperimentalunit). Thegrowth conditionshavebeendescribedintheaboveexperiments.
Proteinconcentrationsand CATandAPX activitieswere measuredinriceplantsharvested at30daysaftersowing. LeaveswerefrozeninliquidN2andstoredat−70◦Cfor
sub-sequentquantificationofproteinconcentrationsandenzyme activitiesaccordingtothefollowingmethods.
(a) Enzymeextractionanddeterminationofprotein concen-tration
Enzyme extraction was performed by homogenizing 200mgofleafinliquidN2andadding1.5mLofthe
extrac-tionbuffer100mMpotassiumphosphatebuffer(pH7.0), with1mMEDTA,2mMDTT,0.8mMPMSF,1%PVPPand 1mM ascorbic acid (ASC). The extract was centrifuged at14,000rpmand 4◦C for30min.Thesupernatantwas collectedandstoredat−70◦Cduringtheperiodof
analy-sis.Thesupernatantscollectedwereusedforallenzyme determinations.Catalaseandperoxidaseactivitieswere expressedasMH2O2andMascorbate(AsA)per
mil-ligram (mg) protein, respectively. Theprotein contents weredeterminedaccordingtothemethodofBradford23
usingabovineserumalbumin(BSA)standardcurve. (b) DeterminationofCATactivity
The CAT activity was determined (according to Havir andMchale24)bymeasuringtheconsumptionofH
2O2at
240nmfor1min.Thereactionmixtureconsistedof20L
enzymeextractand0.980mLof50mMpotassium phos-phatebuffer,pH7.0,with20mMH2O2andwasincubated
at28◦C.Themolarabsorbancecoefficientadoptedwas 36mM−1cm−1at240nm.
(c) DeterminationofAPXactivity
APXactivitywasdeterminedbymonitoringascorbate oxi-dationat290nmfor1min.Thereaction mixconsisted of 35Lenzyme extract and 0.965mLof 50mM potas-siumphosphatebuffer,pH7.0,with1mMH2O2,0.8mM
l-ascorbicacidanddistilledwater.Themolarabsorbance coefficientadoptedwas2.8mM−1cm−1at290nm.25
(d) Statisticalanalysis
The data obtained were analyzed using ASSISTAT 7.6 beta (pt) software to assess the normality, homogene-ityandvarianceofthedatausingtheLilliefors,Cochran and Bartlett tests, respectively. Ananalysis ofvariance (ANOVA)wasperformed,followedbyTukey’stestorthe Scott-Knott test for comparison of means at p<0.05. Regressionanalyseswereperformedtoanalyzetheeffect ofdifferentPEG6000levelsonthegrowthofDSEisolates and/orriceplants.
Results
StresscausedbytheadditionofNaClandPEG-6000affected DSEgrowth(Tables1and2).ThetestedDSEisolatesexhibited differenttolerancestobothstresses.Ingeneral,DSEisolates weremoresensitivetothewaterdeficitcausedbyNaClthan to the addition of PEG-6000 to the growth medium. Both stressesarerelatedtowaterdeficitinthegrowthmedium.The increaseinthewaterresistanceoftheculturemediumfavored the growthofisolateB,which exhibitedstatistically signif-icant differences betweendifferent levels of PEG 6000 and NaCl.
Isolate Bwas the mosttolerantto bothstressestested. The addition of larger amounts of NaCl to the culture medium,correspondingtoanosmoticpotentialof−0.49MPa, resultedinonlya10%decreaseinthefungalcolony’sradial growth. Moreover, the higher water deficit in the culture medium caused by the addition of PEG 6000 (−0.8MPa) increasedthegrowthofthisendophytebyapproximately50%
(Table1).
Table1–Fungalcolonydiameter(CD)ofdarkseptateendophytesat9daysofgrowthinHoaglandsolutionsolidified withPhytagelandwithPEG6000addition.Thevaluesarethemeansofthreereplicates.
Isolates Waterdeficit(MPa)
0 0.1 0.2 0.4 0.8 CD(mm) A 57.66Aa 47.84Ab 44.11Ab 30.27Ac 24.55Bc B 22.85Cc 28.28Cb 30.54Bb 31.74Ab 48.30Aa C 32.55Bab 34.93Ba 34.6Aab 34.59Aa 24.74Bb D 39.22Ba 40.21Aab 38.34Aab 36.90Aa 26.83Bb CV% 16.82
Valuesfollowedbythesameuppercaseletterswithinthesamecolumnorlowercaseletterswithinthesamerowarenotsignificantlydifferent fromeachotheraccordingtoTukey’stestatp<0.05.
Table2–Fungalcolonydiameter(CD)ofdarkseptateendophyticisolatesat9daysofgrowthinPDAmediumwithNaCl addition.Thevaluesarethemeansofthreereplicates.
Isolates Waterdeficit(MPa)
0 0.49 0.99 1.73 2.48 CD(mm) A 85.75Aa 32.28Cb 10.96Dc 7.00Cd 7.00Cd B 49.76Ca 48.09Aa 41.33Aa 43.78Aa 44.54Aa C 59.81Ba 39.31Bb 31.29Bb 23.96Bc 10.91BCd D 36.87Da 26.91Db 24.85Cb 20.26Bb 12.02Bc CV% 5.87
Valuesfollowedbythesameuppercaseletterswithinthesamecolumnorlowercaseletterswithinthesamerowarenotsignificantlydifferent fromeachotheraccordingtoTukey’stestatp<0.05.
Isolate A exhibited lower tolerance to salinityresulting from NaCl addition, exhibiting no growth at the osmotic potentials−0.99,−1.73and−2.48MPaandapproximately63% decreased growth at −0.49MPa (Table 2). At the −0.8MPa waterdeficit levelresulting fromthe addition ofPEG 6000, this isolate exhibited a 50% decrease in growth compared withthecontroltreatment.IsolatesCandDexhibited sim-ilar growth under water deficits due to increasing levels of both NaCl and PEG 6000 in the growth medium. The colony diameter of these two isolates graduallydecreased with increasingNaCl or PEG 6000 additions to the culture medium.
GrowthpromotionofriceplantsbyDSEunderwater
deficitconditions
DSEs were observed to promote the growth of rice plants (Fig.1).IntheabsenceofPEG6000,Nipponbareplants inoc-ulated with all isolates exhibited a greater plant height than thecontroltreatment, and isolateDwas significantly different from other treatments for this variable. Isolates A and B had a stronger beneficial effect on Piauí plants than the remainingtreatments. Under conditions ofwater deficit(−0.2MPa),theNipponbareplantsinoculateswithall isolates exhibited a greater plant height than the control treatment.
Underconditionsofwaterdeficit,significantdifferences inSDWwereobservedwith−0.1MPaforvarietyNipponbare andweresignificantlyhigherwithisolatesAandCthanthe remainingtreatments.Theadditionof79.791gL−1PEG6000 (−0.1MPa)tothegrowthmediumresultedina43%decrease in SDW compared with the control treatment, whereas the decrease observedfor plantsinoculated withisolate A was approximately 23%. Isolates Aand Bexhibited signif-icantly higher SDW under the same conditions for variety Piauí.
InoculationwithisolatesA,CandDresultedina signif-icant differencein RDWbetween isolateBand the control treatmentfor varietyPiauíin the culturemediumwithout PEGaddition121.139gL−1 (−0.2MPa).IntheabsenceofPEG 6000,thePiauíplantsinoculatedwithallisolatesexhibiteda greaterRDWthanthecontroltreatment,andisolatesAand BweresignificantlydifferentbetweentreatmentsCandDfor thisvariable.
TotalproteinconcentrationandCATandAPXactivitiesin
theleavesofriceplants
Forinoculatedplantsofbothricevarieties,theleaftotal pro-teinconcentrationsweresignificantlylowerwith79gL−1PEG 6000andhigherintheabsenceofPEG6000thanwith35gL−1 PEG 6000. Protein concentrations were approximately 50% higherinthetreatmentswith35gL−1PEG6000ofinoculated Nipponbareplantsandmorethan50%higherinvarietyPiauí inthe treatmentswith0and 35gL−1 PEG 6000than inthe treatmentcontrol(Fig.2).
Enzymeactivitiesweredifferentlyaffectedbywaterdeficit andDSEinoculation(Figs.3and4).
Non-inoculatedplants(controltreatment)ofbothrice vari-etiesexhibitedsignificantlylowerCATactivityat79gL−1PEG 6000thanat0and35gL−1(p<0.05).ForDSE-inoculatedplants, CATactivitywasstatisticallyhigherat79gL−1PEG6000for varietyNipponbareandat35gL−1forvarietyPiauíthanfor theremainingtestedPEG6000levels.
For Nipponbareplants, inoculationwithDSEresultedin approximately 60% lower CATactivity at35gL−1 PEG 6000 andsignificantly(approximately100%)higherCATactivityat 79gL−1PEGthanforthenon-inoculatedplants.ForthePiauí plants,inoculationdecreasedCATactivityinalltreatments (28.66,8.36and22.34%at0,35and79gL−1PEG6000levels, respectively);however,thisdifferencewasstatistically signif-icantforonlythetreatmentwithoutthePEG6000addition. Non-inoculated(control)plantsexhibitedhigherAPXactivity at35gL−1PEG(p<0.05)thanattheremainingtestedPEG6000 levelsfortheNipponbarevarietyandhigherAPXactivityboth withoutandwith35gL−1PEG6000thanwith79gL−1PEG6000 levelforthePiauívariety.
InoculatedNipponbareplantsexhibitedthesame behav-iorasnon-inoculatedcontrolplants,withhigherAPXactivity observedat35gL−1PEG6000.PEG6000additionhadnoeffect ontheAPXactivityofinoculatedPiauíplants.
Discussion
The symbiotic efficiency of DSE for rice plants has been demonstratedwithdifferentisolatesanditsspecificityunder stressconditions.Aswithothersymbionts,suchas nitrogen-fixingbacteriaandmycorrhizae,notallisolateswereobserved
Concentrations of PEG-6000 (Mpa) 0 -0.1 -0.2 -0.4 -0.8 Plant height (cm) 0 10 20 30 40 50 CONTROL A B C D 0 -0.1 -0.2 -0.4 -0.8 0 10 20 30 40 50 0 -0.1 -0.2 -0.4 -0.8
Shoot dry weight (g)
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0 -0.1 -0.2 -0.4 -0.8 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0 -0.1 -0.2 -0.4 -0.8
Root dry weight (g)
0.00 0.01 0.02 0.03 0.04 0.05
A
B
C
D
E
Nipponbare Piaui b b b a c b a bb a b aaaa a aaa a aaaaa b a a b b b a a bb aa a a a a aaaa aaaaa b a b a a b b b a a a a aa a aa a a a aaaaa b a a bb a a aa a a a a a a a a aaa aaaaa b a b aa b a bb a aaa a a bb aa a aa aaa 0 -0.1 -0.2 -0.4 -0.8 0.00 0.01 0.02 0.03 0.04 0.05F
c a a bb a aaaa b a b a a a aaaa a aaa aFig.1–Plantheight,shootsdryweightandrootsdryweightofNipponbare(a,c,e)andPiauí(b,d,f)riceplantsinresponse todifferentlevelsofPEG6000at30daysfollowinginoculationwithdarkseptatefungi(A,B,C,D).Thevaluesarethemeans offourreplicates.
topositivelyinfluencetheplant.26Thus,ourresultsindicate
thatsomeDSEstimulateimprovementsinplantsunderstress whereasothersdonot.Forexample,DinducedNipponbare growth underbothnormal andstress conditions; however, forPiauívariety,isolateAwasmoreprominent.Furthermore, isolateCpromotedincreasesinleafdrymatterfor Nippon-bareonlywhensubjectedtostress.ForPiauí,isolatedCalso promotedincreases.TheseresultssuggestthatDSEisolates responddifferently dependingonstressconditionsandthe plant–fungusgenotypes.27
Theeffects of inoculationwith endophytic microorgan-ismssuch as DSE on plantgrowth have been observed to
varyandcanbepositive,negativeornull.Theoutcomeofthe associationdepends,amongotherfactors,onthespecificity betweensymbionts,themicroorganisms’abilitytoestablish associations,andtheinteractionbetweentheendophyteand hostplantatlevelsfromthemolecularsignalinginvolvedin thecolonizationprocessuptothehostplant’sphysiological response.28 In this study, onlypositive and null effects of
DSEinoculationwereobservedonthemeasuredparameters, and no negative effects were observed. Null effects were observed at the highest levels of water deficit although the two ricevarieties responded differently to inoculation. According to Redman et al.,28 a singlefungal isolatefrom
PEG 6000 (g L-1) PEG 6000 (g L-1) 0 35 79 Total protein (mg g -1 FW) 0 10 20 30 40 Aa Ba Bb Bb Cb Aa 0 35 79 0 10 20 30 40 Aa Aa Bb Bb Bb Aa
A
B
Inoculated ControlFig.2–Proteinconcentrationsperfreshweight(FW)ofleavesofNipponbare(a)andPiauí(b)riceplantsinoculatedwithDSE isolateA(Err01)ornotinoculated(control)andunderdifferentPEG6000levels;differentlowercaseletterswithinthesame PEG6000leveloruppercaselettersbetweendifferentPEGlevelsindicatesignificantdifferencesaccordingtotheScott-Knott testatp<0.05.
aspecific geographicallocation can behaveasa pathogen, mutualistorcommensalistdependingonthe physiologyof the host plant and genetic differences between cultivars, whichcanaltertheoutcomeofthesymbiosis.29studiedthe
effectofDSEinoculationongrowthpromotionofthegrasses
Psathyrostachys juncea, Agropyron cristatum and Bouteloua gracillisunderwaterstressconditionsandobservedpositive effectsonA. cristatumand P. junceabutnegativeeffectson
B.gracillis.Theauthorconsideredthedifferentresponsesto berelatedtothe factthatB.gracillisisaC4plant,whereas
A.cristatumandP.junceaareC3plants.C4plantsarebetter adapted to dry environments because they can maintain the efficiency of the photosynthetic apparatus when the stomatacloseduringlong dryperiods,thus avoidingwater loss under conditions of high temperature and low water availability.
Similarlytothepresentstudy,increasesinrice(O.sativa
L.) dry weight were observed byYuan et al.10 in response
toinoculationwiththe DSEHarpophoraoryzaesp.ininvitro
experimentsperformedinChina,withtheinoculatedplants exhibiting57%higherdryweightthannon-inoculatedplants. TheDSEpromotedbothshootandrootgrowthininoculated plants.However,largerincreaseswereobservedinrootthanin shootbiomass.30Inthisstudy,alackofresponsetoDSE
inoc-ulationwasobservedforallmeasuredparameterswiththe exceptionofRDWinbothtestedricevarietiesunder condi-tionsofhigherstress.Thislackofresponsemaybeassociated withthelowerfungalgrowthobservedundertheseconditions. WiththeexceptionofisolateB,theremainingisolates exhib-itedsignificantlylowergrowthatthislevel.Theonlyresponses observedatthehighestlevelofwaterdeficit(−0.4MPa)were forRDW;thismayberelatedtoahigherinvestmentinroot
0 35 79 C AT ac ti v ity ( m o l H 2 02 m in -1 mg -1 pro tei n) 0 100 200 300 400 Ba Bb Aa Aa Aa Bb 0 35 79 0 100 200 300 400 Bb Aa Ba Aa Aa Ba
A
B
PEG 6000 (g L-1) PEG 6000 (g L-1) Inoculated ControlFig.3–Catalase(CAT)activityintheleavesofNipponbare(a)andPiauí(b)riceplantsinoculatedwithDSEisolateA(Err01) ornotinoculated(control)atdifferentlevelsofPEG6000;thevaluesarethemeans±standarderror(n=4);different
lowercaseletterswithinthesamePEG6000leveloruppercaselettersbetweendifferentPEG6000levelsindicatesignificant differencesaccordingtoTukey’stestatp<0.05.
0 35 79
APX activity
(
mol ASA min
-1 mg -1 protein) 0 100 200 300 400 500 600 Inoculated Control Bb Ab Ba Ba Aa Ba
A
0 35 79 0 100 200 300 400 500 600B
Ab Ab Aa Aa Aa Ba PEG 6000 (g L-1) PEG 6000 (g L-1)Fig.4–Ascorbateperoxidase(APX)activityintheleavesofNipponbare(a)andPiauí(b)riceplantsinoculatedwithDSE isolateA(Err01)ornotinoculated(control)withdifferentadditionsofPEG6000;thevaluesarethemeans±standarderror (n=4);differentlowercaseletterswithinthesamePEG6000leveloruppercaselettersbetweendifferentPEG6000levels indicatesignificantdifferencesaccordingtoTukey’stestatp<0.05.
growthbyplantsexposedtotheseconditionsinanattemptto increasewateruptake.31
ThepresenceofDSEfungienvelopingrootcellsinter-and intracellularlymayberelatedtohigherprotectionagainstthe oxidativedamageresulting fromthe −0.4MPawaterdeficit andthefactthattheonlyeffectsobservedatthislevelofstress wereonRDW.Theproductionofmelanizedhyphaemaybea survivalstrategyofDSEfungiinstressedenvironmentsand mayprotectplantsfromfreeradicals.32Themelaninproduced
byDSEalsohashighantioxidantpowerandmaybeimportant forplantsurvivalinstressenvironments.33
Several genes and proteins respond to stresses caused by different environmental stimuli, such as salinity, low temperatures and drought.34 Under these conditions, the
overexpression of cell protection-related proteins, such as antioxidantenzymes,mayoccur.35,36Additionally,plant
pro-tein concentrations can be influenced by several factors includingincreasesinthestressintensityand/ordurationand theefficiencyoftheplant’santioxidantsystem.37
CATand APXsare theprimary enzymesinvolvedinthe antioxidantsystem ofplantsand havesimilar roles inthe eliminationofROSbecausebothactbyreducingH2O2intoO2
andH2O.However,APXusesascorbateasthereducingagent38
andhasahighersubstrateaffinitythanCAT.3Therefore,even
lowH2O2concentrationsaresufficienttoinduceAPX
activ-ityintheprocessofcellulardetoxification.39,40Therearefew
reportsofchangesintheactivityofantioxidantenzymesasa resultofinoculationwithendophyticfungiunderstress con-ditions,suchasthose presentedinthiswork.Weobserved stressreductionsinplantsinoculatedwithisolateA,as indi-catedbythedecrease inCATandAPXactivity atnearlyall stresslevelstested.
AnothermechanismbywhichDSEmayhavecontributedto highergrowthofinoculatedplantsunderwaterstress condi-tionsinthepresentstudyistheproductionofafungalnetwork extendingbeyondtherootdepletionzone.12,13,41
Theantioxidantpowerofthemelanininthefungal struc-tures may also have functioned as an antioxidant agent, controllingthefreeradicalsformedduetooxidativestressand preventingcelldamage,therebycontributingtohigherplant growthundertheseconditions.12,18,42–44
Ourresultssuggestthatendophyticmicroorganisms,such asDSEs,arecapableofpromotinghostplantgrowththrough mechanisms other than those relatedtoplant nutritionor nutrientsolubilization.45,46SomeDSEisolatesobtainedfrom
tropicalenvironmentsexhibitdifferentlevelsofstress toler-anceinvitro.Moreover,someisolatecancolonizeandpromote thegrowthofthetworicevarietiesbothwithandwithoutthe presenceofastressor.
Conflicts
of
interest
Theauthorsdeclarethattheyhavenoconflictsofinterest.
Acknowledgments
The authors thank the Brazilian Federal Agency for Sup-portandEvaluationofGraduateEducation(Coordenac¸ãode Aperfeic¸oamento dePessoalde NívelSuperior –CAPES)for the scholarship granted to the author. The Brazilian Agri-cultural Research Corporation (Embrapa, Carbioma project no.02.11.05.001),Fundac¸ãodeAmparoàPesquisanoEstado doRiode Janeiro(FAPERJ-E – 26/103.242/2011)theNational CouncilforScientificandTechnologicalDevelopment(CNPq– 562.601/2010-4;563304/2010-3and562955/2010-0),Fundac¸ão deAmparoàPesquisadeMinasGerais(FAPEMIGCRA– APQ-00001-11),andtheInter-AmericanInstituteforGlobalChange Research(IAI-CRNII-021)areacknowledgedforsupportingthe researchactivities.
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