Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth. - Portal Embrapa

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Environmental

Microbiology

Mangrove

endophyte

promotes

reforestation

tree

(Acacia

polyphylla

)

growth

Renata

Assis

Castro

a,b

,

Manuella

Nóbrega

Dourado

c,∗

,

Jaqueline

Raquel

de

Almeida

a

,

Paulo

Teixeira

Lacava

d

_,

_André

_Nave

e

_,

_Itamar

_Soares

_de

_Melo

f

_,

_João

_Lucio

_de

_Azevedo

a,b

,

Maria

Carolina

Quecine

a

a_University_of_São_Paulo,_Escola_Superior_de_Agricultura_“Luiz_de_Queiroz”,_Department_of_Genetics,_Piracicaba,_SP,_Brazil b_University_of_São_Paulo,_Center_for_Nuclear_Energy_in_Agriculture_(CENA),_Piracicaba,_SP,_Brazil

c_University_of_São_Paulo,_Biomedical_Science_Institute,_Department_of_{Microbiology,}_São_Paulo,_SP,_Brazil

d_Federal_University_of_São_Carlos_(UFSCar),_Center_for_Biological_and_Health_Sciences,_Department_of_Morphology_and_Pathology,_São

Carlos,SP,Brazil

e_BIOFLORA_Comercial_LTDA,_Piracicaba,_SP,_Brazil

f_EMBRAPA_Environment,_Laboratory_of_{Environmental}_{Microbiology,}_Jaguariuna,_SP,_Brazil

a

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t

i

c

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o

Articlehistory:

Received23August2016 Accepted19April2017 Availableonline19July2017 AssociateEditor:WelingtonAraújo

Keywords:

Endophyticbacteria IAA

Phosphorusandplantgrowth promotingbacteria(PGPB)

a

b

s

t

r

a

c

t

Mangrovesareecosystemslocatedinthetransitionzonebetweenlandandseathatserve asapotentialsourceofbiotechnologicalresources.Brazil’sextensivecoastcontainsoneof thelargestmangroveforestsintheworld(encompassinganareaof25,000km2_along_all_the

coast).Endophyticbacteriawereisolatedfromthefollowingthreeplantspecies:Rhizophora mangle,LagunculariaracemosaandAvicennianitida.Alargenumberoftheseisolates,115in total,wereevaluatedfortheirabilitytofixnitrogenandsolubilizephosphorous.Bacteria thattestedpositiveforbothofthesetestswereexaminedfurthertodeterminetheirlevel ofindoleaceticacidproduction.Twostrainswithhighindoleaceticacidproductionwere selectedforuseasinoculantsforreforestationtrees,andthenthegrowthoftheplantswas evaluatedunderfieldconditions.ThebacteriumPseudomonasfluorescens(strainMCR1.10) hadalowphosphorussolubilizationindex,whilethisindexwashigherintheotherstrain used,Enterobactersp.(strainMCR1.48).WeusedthereforestationtreeAcaciapolyphylla.The resultsindicatethatinoculationwiththeMCR1.48endophyteincreasesAcaciapolyphylla

shootdrymass,demonstratingthatthisstraineffectivelypromotestheplant’sgrowthand fitness,whichcanbeusedintheseedlingproductionofthistree.Therefore,wesuccessfully screenedthebiotechnologicalpotentialofendophyteisolatesfrommangrove,withafocus onplantgrowthpromotion,andselectedastrainabletoprovidelimitednutrientsand hormonesforinplantgrowth.

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

∗ _{Corresponding}_author_at:_Department_of_{Microbiology,}_Institute_of_Biomedical_Science,_University_of_São_Paulo,_Av._Prof._Lineu_Prestes,

1374–Ed.BiomédicasII,05508-900Piracicaba,SP,Brazil. E-mail:[email protected](M.N.Dourado).

https://doi.org/10.1016/j.bjm.2017.04.002

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Introduction

Mangrovesareanimportantecosystemintropicalbiomesthat occupyseveralmillion hectaresofcoastal areaworldwide.1

Brazilpossessesoneofthelargestmangroveforests,covering an area of 25.000km2 _all _along _the _coast. _This

ecosys-tem is located in the transition zone between land and sea2 _and _is _{characterized} _by _periodic _flooding, _resulting

in a unique environment with few plant species. Brazil-ianmangrovesprimarilycomprise the followingthree tree species: Rhizophora mangle, Laguncularia racemosa and Avi-cennia sp.3 _Furthermore, _the _mangroves _harbor _a _diverse

groupofmicroorganisms.4,5 _Several _studies_have_examined

the microbial community of mangroves by using metage-nomic approaches to access the microorganisms involved in carbon,6 _nitrogen7 _and _sulfer8 _metabolism. _Despite _the

high microbial diversity of mangroves, estimates suggest thatlessthan5%ofspeciesinthisenvironmenthavebeen described.5

Moreover,the high diversityof culturablebacteria3 _and

culturable endophytic fungi9 _within _the _Brazilian

man-groves has not yet been explored. Few studies focus on the biotechnological potential of culturable mangrove iso-lates. Castro10 _screened _for _enzymes _for _use _in _industrial

processes, such as amylase, esterase, lipase, protease and endoglucanase.Thislargeamountofmicrobialdiversitycan beexploitedtoimprove cropscience sincethe microorgan-isms produce phytohormones, such as indole acetic acid (IAA),enzymes,andantimicrobialmolecules,andsolubilize phosphate in the host plant.11,12 _In _addition, _these

orga-nismscanfixnitrogen13 _and_increase_drought_resistance.14

More recently, the high tolerance of these microorganism to heavy metal was described15,16 _in _addition _to

char-acteristics that are important to the promotion of plant growth.

Bacteriathatexhibit thesefeatures canbeused to pro-mote the growth of different plant species such as corn, soybeans,andsugarcaneaswellasarborealspecies.17_These

beneficial characteristics of the plant–microbe interactions canbeusedinotherplants.Cross-colonizationiscommon in nature in which the same bacterium can colonize dif-ferent host plants. One example of cross-colonization is

PantoeaagglomeransisolatedfromEucalyptusgrandis,whichis ableto colonizeand promoteplant growth insugarcane.12

However, there are few studies evaluating the effects of bacterialinoculationintrees.18_The_tree_species_Acacia

poly-phylla, of the Leguminosae family, commonly known as “monjoleiro”in Brazil, iswidely used for the reforestation of degraded areas due to its ability to fix nitrogen19 _and

improve degradedsoils, thus decreasing costsand benefit-ting the environment.20 _Therefore, _the _aim _of _this _study

istoidentifyand analyze the biotechnological potentialof endophyticbacteriaisolatedfromaBrazilianmangrove envi-ronment and select strains able to promotethe growth of

A.polyphylla.

Materials

and

methods

Endophyteisolationsites

MangroveforestsampleswerepreviouslycollectedfromSão Paulo state, Brazil, as described by Castro.10 _The

follow-ingthreelocationswereassessed:(A)theBertiogalocation, whichwascontaminatedbyoilspills;(B)theuncontaminated Bertioga location, withanthropogenic impacts; and (C) the uncontaminatedCananéialocation,withlowanthropogenic impacts.Thefollowingthreemangrovespecieswereassessed: (1)R.mangle,(2)L.racemosaand(3)Avicenniasp.Theoilspill in Bertioga occurred approximately 20 years ago, and the anthropogenic impacts (domesticand industrialsewer) are stilloccurringinBertiogaatbothlocationssampled.6,10

From the whole mangrovebacterial collection, we ran-domlyselected115isolatesthatwereendophyticallyisolated fromthebranchesofmangroveplantsbelongingtotheculture collectionoftheLaboratoryofBacterialGenetics Microorgan-ism,SchoolofAgricultureLuizdeQueiroz(Esalq).3,10

Selectionofendophytes:nitrogenfixation

We started our screening by evaluating the ability of the randomly selected 115 strainsto fixatmospheric nitrogen. Qualitative assays were performed using the process of Liba.21_The_strains_were_inoculated_in_tubes_containing₁₀_mL

semi-solidNFbmedium(5gL−1_malic_acid,_0.5_g_L−1_K 2HPO4,

0.2gL−1 _MgSO

4.7H2O, 0.1gL−1 NaCl, 0.01gL−1 CaCl2·2H2O,

and 4mL 1.64% Fe-EDTA), 2mL 0.5% bromothymol blue, 2mL micronutrients (0.2gL−1 _Na

2MoO4·2H2O, 0.235gL−1

MnSO4·H2O, 0.28gL−1 H3BO3, and 0.008gL−1 CuSO4·5H2O),

and1.75gL−1_agar._Bacterial_growth_was_evaluated_after₇₂_h

ofincubationat28◦_C_in_the_dark._The_formation_of_a_growth

disc inthe culturemediumindicatedatmosphericnitrogen fixationbythebacterialstrains.Thisprocedurewasrepeated fivetimesforconfirmation.

Selectionofendophytephosphatesolubilization

Strainsthatcouldsolubilizeinorganicphosphatewere iden-tifiedbyaquantitativetest.Thistestinvolvedobservingthe presenceofahaloafterbacterialcultivationonmedium sup-plemented withCa3(PO4)2 aftersevendaysofincubationat

28◦_C._The_results_were_quantified_by_estimating_the_halo_size

(cm)anddividingitbythecolonysize(cm)togeneratea sol-ubilizationindex(SI).22

SelectionofendophytesthatproduceIAA

Thestrainsthattestedpositiveforphosphatesolubilization andnitrogenfixationweretestedfortheirabilitytoproduce IAA.ThequantitativeIAAproductionwasevaluatedusingthe PattenandGlick23_method_with_{modifications.}_The_bacterial

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(Difco,Livonia,USA)supplementedwithl-tryptophan(5mM) andincubatedat28◦_C_for₄₈_h_in_the_dark._Triplicate_cultures

werecentrifuged(5min,10,000×g,atroomtemperature),and 1.5mLSalkowskireagent24_was_added_to_1.5_mL_of_the

super-natant.Thismixturewasincubatedfor20mininthedarkat roomtemperatureandanalyzedusingaspectrophotometer (520nm;Ultrospec3000,Amersham-PharmaciaBiotech).The absorbancevaluesobtainedwereinterpolatedinastandard curvetodeterminetheIAAconcentration.

Identificationofstrainsbypartialsequencingofthe16S rDNA

The identification of 38 bacterial strains able to fix nitro-gen,solubilizephosphorusandproduceIAAwasperformed bypartialsequencingof16SrDNA.Theamplificationof16S rDNAwasperformeddirectlyfrombacterialcoloniesgrown onsolid TSAmedium(10%)(Difco,Livonia, USA)usingthe primers R1387 (5′_{-CGGTGTGTACAAGGCCCGGGAACG-3}′₎ _and

PO27F(GAGAGTTTGATCCTGGCTCAG-5′_–3′_).25

The16SrDNAgenePCRproducts(approximately1500bp) werepurifiedbythepolyethyleneglycolmethodofLis26_and

sequencedattheInstituteoftheHumanGenome(USP,São Paulo,Brazil).ThesequenceswereevaluatedwithBLASTn27

againstthedatabaseoftheGenBankDevelopmentNational CenterforBiotechnologyInformationwebsite.

Sequences were deposited in GenBank under the fol-lowing access numbers: KF356429–KF356431, KF356438, KF356439, KF356444, KF356453, KF356454, KF356457, KF356459,KF356462,KF356465andKM438481–KM438506. Growthpromotionoftheseedlingsofthereforestation angiospermtreeA.polyphylla

Weselectedtwostrainsthatpresentedpositiveresultsforall ofthetestsperformed,includingphosphorussolubilization, nitrogenfixationandIAAproduction.Bothstrainsproduced thehighestamountofIAAofthebacteriastudied.However, onestrainhadahighphosphoroussolubilizationindexvalue whiletheotherhadalowvalueforthisindex.Theselected bacteriaweregrowninliquidTSBculturemediumand incu-batedfor24hat28◦_C_at₁₅₀_rpm._The_optical_density₍₆₀₀_nm)

wasadjustedto108_cells_mL−1_._The_plant_growth_assays_were

performedinthereforestationBiofloraCompanylocatedin Piracicaba,SãoPaulo,Brazil.Theexperimentwasconducted inaplantnurserywhoseinternaltemperaturesrangedfrom 20to30◦_C_during_this_period._The_treatments_were_as_follows:

I,seedlingsofA.polyphyllauninoculatedandunfertilized;II, seedlingsuninoculatedandfertilized(usingForthSolúveis Ini-cial;ForthAquaMicrosandForthAquaCalcio,Tiete,Brazil); III,seedlingsinoculatedwithPseudomonasfluorescens(strain MCR1.10);IV,seedlingsinoculatedwithEnterobactersp.(strain MCR1.48); V, seedlings inoculated withP. fluorescens (strain MCR1.10) and Enterobacter sp. (strainMCR1.48) consortium. The treatments were performed in a completely random-izeddesignwith25repetitions(1plantperpot).Treatments inoculated withbacteria (III, IVand V) were notfertilized. Eachinoculatedsamplereceiveda1.0mLsuspensionof108

cellsmL−1_,_and_the_{un-inoculated}_controls_were_treated_with

1.0mLofsterilewateronly.Theinoculationwithbacteriawas

performedbyaddingthebacterialsuspensiontothesubstrate. Afterinoculation,theseedlingsweremaintainedintheplant nurseryfor60days.Afterthisperiod,theseedlingswere col-lected and washed in water. Then, the rootsystems were separatedfromtheshoot,andthedrymassofrootsandshoots fromtheseedlingsweremeasuredtoevaluateplantgrowth promotion.

Statisticalanalysis

Dataweresubjectedtoanalysisofvariance,andmeanswere comparedbytheScottKnotttestforIAAproductionand phos-phorussolubilizationindex(<0.05)andtheTukeyTestforplant experiments(<0.05).Statisticalanalyseswereperformedwith Rsoftware(version3.0.2).

Results

Selectionofendophytes:nitrogenfixationandPhosphate solubilization

In the nitrogenfixation test, 33%ofthe 115 strains exam-ined(38)wereabletogrowinthe mediumfreeofnitrogen withatypicalsub-surfacegrowth,indicatingtheirabilityto fixnitrogen.Thismicroaerophilicgrowthbehaviorresultedin achangeofcoloroftheculturemediumfrombluetogreen toyellow. Thiscolorchange wascausedbychanges inthe mediumpHprobablyduetoacidicmoleculesreleasedbythe isolatetested.

All115strainsexaminedproducedahaloduringthe phos-phatesolubilization test,indicating that theywere all able to solubilize inorganic phosphate. The highest rates were observed in the genera Pantoea (MCL2.66), and the lowest rateswereobservedinthegeneraSphingosinicella(MCL2.68),

Xanthomonas (MCA2.20), Ochrobactrum (MBR2.28) and Bacil-lus(MBIA2.43,MCR2.51,MBIL2.38andMCA2.42)withindices between1.2and2.6.

QuantitativeandqualitativeassayofIAAproduction The IAA production assay was performed in 38 strains that were able to solubilize phosphate and fix nitro-gen. All of the strains tested produced IAA ranging from 16.4 to 601.7␮gmL−1_. _Pantoea _(MCL2.66 _and _MBIL2.47),

Enterobacter(MCL2.65,MCR1.48and MCR1.23),Pectobacterium

(MCR2.29), Bacillus (MCR2.51 and MBI2.63), Pseudomonas

(MBR2.7, MCR1.10) and Stenotrophomonas (MBR2.29) genera showed thehighestyields(Table1).Thelowestproduction wasapproximately16␮gmL−1_and_was_produced_by_strains

MBR2.1andMBR2.22,correspondingtothegeneraBacillusand

Curtobacterium,respectively(Table1).

Plantgrowthpromotingreforestationbyendophytic bacteria

TwohighIAAproducingstrainswereselected.Onestrainhad thehighestphosphoroussolubilizationindex,strainMCR1.48 (Enterobactersp.),whilestrainMCR1.10(P.fluorescens)hadone ofthelowestphosphoroussolubilizationindices.Bothstains

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Table1–Identificationandevaluationofinvitrotestsforplantgrowthpromotingbacteriaisolatedfrombranchesof mangroveplants.

Isolate Planthost Location Identification IAA(␮gmL−1₎a _Phosphate_(P)

solubilizationindex (SI)a

Nitrogen fixation

MBA2.21 Aviceniasp. Industrial

contaminated (Bertioga)

Bacilluspumilus 31.2d 8.0c +

Bacilluspumilus 27.6d 4.2e +

Alcaligenessp. 33.7d 3.4e +

Alcaligenesfaecalis 32.8d 3.5e +

MBR2.45 Rhizophoramangle Industrial contaminated (Bertioga)

Bacilluspumilus 24.4d 7.4d +

Ochrobactrumsp. 100.3d 1.6f +

Pseudomonassp. 406.8b 8.2c +

Curtobacterium flaccumfaciens

16.4d 8.2c +

Stenotrophomonassp. 443.2b 6.5d +

MBIA2.42 Aviceniasp. OilSpill

contamination (Bertioga)

Bacillus amyloliquefaciens

55.4d 3.6e +

48.7d 3.8e +

Pantoeaagglomerans 204.2c 10.2b +

Bacilluspumilus 27.8d 8.5c +

55.6d 2.6f +

MBIL2.38 Lagunculariaracemosa OilSpill contamination (Bertioga)

Bacillussubtilis 56.8d 2.4f +

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Table1– (Continued)

Isolate Planthost Location Identification IAA(␮gmL−1₎a _Phosphate_(P)

solubilizationindex (SI)a

Nitrogen fixation

Pantoeadispersa 422.7b 11.2b +

Pantoeaagglomerans 188.6c 8.4c +

Pantoeasp. 220.4c 9.8b +

MBIR2.24 Rhizophoramangle OilSpill contamination (Bertioga)

MBIR2.2 Rhizophoramangle OilSpill contamination (Bertioga)

MCA2.42 Aviceniasp. Uncontaminated

(Cananéias)

Bacillussp. 38.5d 2.6f +

MCL2.66 Lagunculariaracemosa Uncontaminated (Cananéias)

Pantoeaagglomerans 513.9a 13.7a +

Enterobacterludwigii 573.9a 11.2b +

Sphingosinicellasp 75.4d 1.2f +

MCA2.20 Lagunculariaracemosa Uncontaminated (Cananéias)

Xanthomonas campestris

86.1d 1.2f +

MCR1.48 Rhizophoramangle Uncontaminated (Cananéias)

Enterobactersp. 540a 11.1b +

Enterobactersp. 477.1b 9.0lc +

Pseudomonas fluorescens

441.8b 6.5d +

Peptobacteriumsp. 601.7a 8.2c +

Bacillussafensis 402.5b 2.1f +

Pantoeaagglomerans 124.8c 6.8d +

a _In_a_column,_values_with_the_same_letter_do_not_differ_at_a_5%_significance_level_(Scott_Knott_test).

(withhighandlowphosphoroussolubilization)were inocu-latedinA.polyphylla plants.Theseedlingstesteddisplayed differentresultswhencomparedtothecontrol(Table2,Fig.1). ThetreatmentwithMCR1.10(thelowphosphorous solu-bilizationstrain)aswellastheconsortiumtreatment(V)was statisticallysimilartothecontrol(notinoculatedandnot fer-tilized),indicatingalackofabilitytopromoteplantgrowth.In contrast,seedlingsinoculatedwithMCR1.48(high phospho-roussolubilizationstrain)causedanincreaseindryrootand shootbiomass.Someofthetreatedseedlingshadanincrease inthenumberofroothairsintheiradventitiousrootsalong withrootpagethickening whencomparedwiththecontrol

(Table2,Fig.1).TheseedlingswithMCR1.48(highphosphorous

solubilizationstrain)inoculumandassociatedwith fertiliza-tionwerestatisticallysimilar.

Discussion

Theabilityofendophyticbacteria topromoteplantgrowth isattributedtodirectmechanismssuchasnitrogenfixation, planthormoneproduction(mainlyIAA),28_phosphate

solubi-lization,andsiderophoreproduction29_as_well_as_the_increased

absorptionofwaterandnutrientsandthesuppressionof dele-teriousmicroorganismsbymetaboliteproduction.30

Phosphatesolubilizationbymicroorganismshasan impor-tant functionin supplyingphosphorus (P) toplants31 _with

thepotentialtobeusedasinoculants.Manyauthors32_report

that the abilityof microorganisms to solubilize phosphate correlates withthe ability toproduce organic acids and/or extracellular polysaccharide.33–35 _In _this _study, _all _of _the

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Table2–TheeffectofsoilinoculationofendophyticmangrovebacteriaonA.polyphyllaafter60daysfollowing inoculation.

Treatment Isolate Shootdrymass(g)a _Root_dry_mass_(g)a

Treatment1(addingonly water) – 0.38±0.04b 0.61±0.17ab Treatment2(adding fertilizer) – 0.43±0.09ab 0.58±0.25b Treatment3(inoculating MCR1.10) Pseudomonasfluorescens (MCR1.10)–lowP solubilization 0.32±0.05b 0.49±0.16b Treatment4(inoculating MCR1.48) Enterobactersp.(MCR1.48)– highPsolubilization 0.50±0.06a 0.82±0.53a Treatment5(inoculating MCR1.10+MCR1.48)

MCR1.10andMCR1.48 0.43±0.07ab 0.65±0.20ab

Average – 0.41±0.13 0.62±0.25

a _In_a_column,_values_with_the_same_letter_do_not_differ_at_a_5%_significance_level_(Tukey_test).

Fig.1–Monjoleirorootsunderdifferenttreatments:(A)Treatment1(additionofonlywater);(B)Treatment2(additionof fertilizer);(C)Treatment3(inoculationofPseudomonasfluorescensMCR1.10);(D)Treatment4(inoculationofEnterobactersp. MCR1.48).

endophytestestedcould solubilizePin vitro,althoughtheir solubilizationindicesvaried.

IntheIAAproductionassay, strainsproducedsimilaror higheramounts ofIAAthan previousreportsindicated36,37

demonstratingtheirpotentialtoimproveplantgrowth.Some strainsofthesamespecieshaddifferentvalues,possiblydue tothedifferentamountsoftryptophaninthestrainswitheach celllinehavinganoptimalconcentrationforinterferingwith thesynthesisofIAA.38,39

Severalstudiesreportthatendophyticmicroorganismsare abletopromoteplantgrowthindifferentplants,17,40_such_as

corn(ZeaMays), peppermint(Menthapiperita),41Vitis vinifera

L.42_and_pineapple_(Ananas_comosus).43_However,_there_are_few

reports on the use of endophytic bacteria on growth pro-motion in woody plants. Previousstudies were performed mainlyontreesimportantinthepaperandcelluloseindustry, suchaspinesandeucalyptus.18_Burns_and_Schwarz44_found

thatanunidentifiedbacteriuminduced90%ofrootexplant inPinuselliottiiwhencomparedtothecontrol.44 _Other _tree

speciesstudiedincludedpoplar(Populus),whichwasusedas amodelplant,andthebacteriumBurkholderiamultivorans,a nonpathogenicstrain,reportedtobeablecolonizetheroots

and significantlypromotethegrowth ofpoplarseedlings.45

Moreover,Bashan46_reported_that_Azospirillum_brasilense _and

Bacilluspumiluspromotedthegrowthoftwotreesusedin refor-estation(Prosopisarticulataand Parkinsoniamicrophylla),thus increasingplantdevelopmentandsurvival.

Two highIAAproducingstrains, P.fluorescensand Entero-bactersp.,wereselectedtoinoculateplantsandevaluatetheir growth infield conditions sinceboth the Pseudomonas and

Enterobactergenerahavebeenpreviouslyreportedtopromote the growthofplants.47–49 _The_main_difference_between_the

strainsselectedisthatEnterobactersp.(MCR1.48)hasahighP solubilizationindex,whileP.fluorescens(MCR1.10)hasoneof thelowestPsolubilizationindices.Weselectedthecommonly usedreforestationtreeA.polyphylla,whichhasfewpublished studiesinvolvinginoculationbythebacteriaofinterest.The presentwork demonstratesthattheinoculationofahighP solubilizationstrainMCR1.48(Enterobactersp.)increasedthe shootdrymassofmonjoleiro.Thisresultindicatesthat phos-phoroussolubilizationplaysakeyroleintreeplantgrowth. Therefore,thisstudyisthefirstknownreportofthegrowth promotionofA.polyphyllafrom theinoculationofan endo-phyticbacterium.

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Inreforestation,therotationtimeislong,sothe inocula-tionofmicroorganismstoacceleratethetrees’development should help them grow more quickly over the long term. Wereportanincreaseintherateofseedlingsurvival,early establishmentinthefieldafterplantingalongwith improve-mentinqualityandcharacteristicsoftherootsystem,i.e.,all advantagesdesirableforbetterproduction.Therefore,we suc-cessfullyscreenedthebiotechnologicalpotentialofendophyte isolatesfrommangroves,focusingonplantgrowthpromotion. Weselectedastrainabletoprovidelimitednutrientsbyfixing nitrogensincenotallformsofnitrogeninthesoilareavailable totheplant;bysolubilizingphosphorus,whichispresentin soilbutisalsonotavailabletoplants;andbyproducingplant hormonesinvolvedinplantgrowthsuchasauxin(IAA).

Theuseofanendophyticmicroorganisminthesubstrate asinoculumfortheproductionofseedlingsofA.polyphyllais ahighlysuccessfulstrategythatcanbeutilizedinthe refor-estationnursery.TheinoculanteffectoftheEnterobactersp. endophyticbacteriainA.polyphyllaseedlingsaffectthe qual-ityofseedlingproduction,improving thedevelopmentand possiblyreducingthecostofchemicalfertilizationperformed bythenurserycompany.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This work was supported by a grant from FAPESP (Proc.2004/15414-6)toR.A.andbyafellowshipfromCNPqto M.N.D.(Proc150228/2017-1).WethankBiofloraforproviding thestructureandstafftodevelopnurseryexperiments.

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