Effect of plant growth-promoting bacteria on the growth and fructan production of Agave americana L.

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

Environmental

Microbiology

Effect

of

plant

growth-promoting

bacteria

on

the

growth

and

fructan

production

of

Agave

americana

L.

Neyser

De

La

Torre-Ruiz

_,

_Víctor

_Manuel

_{Ruiz-Valdiviezo}

_,

_Clara

_Ivette

_{Rincón-Molina}

_,

Martha

Rodríguez-Mendiola

_,

_Carlos

_Arias-Castro

_,

_Federico

_Antonio

_{Gutiérrez-Miceli}

_,

Héctor

Palomeque-Dominguez

_,

_Reiner

_{Rincón-Rosales}

a_{PlantBiotechnology,DEPIInstitutoTecnológicodeTlajomulco,CarreteraaSanMiguelCuyutlán,TlajomulcodeZú ˜niga,Jalisco,Mexico} b_{LaboratoryofBiotechnology,InstitutoTecnológicodeTuxtlaGutiérrez,TuxtlaGutiérrez,Mexico}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received4June2015 Accepted11February2016 Availableonline22April2016 AssociateEditor:IêdadeCarvalho Mendes Keywords: Agave Inoculation Bacteria Inulin 16SrRNA

a

b

s

t

r

a

c

t

Theeffectofplantgrowth-promotingbacteriainoculationonplantgrowthandthesugar contentinAgaveamericanawasassessed.ThebacterialstrainsACO-34A,ACO-40,and ACO-140,isolatedfromtheA.americanarhizosphere,wereselectedforthisstudytoevaluate theirphenotypicandgenotypiccharacteristics.Thethreebacterialstrainswereevaluated viaplantinoculationassays,andAzospirillumbrasilenseCdservedasacontrolstrain. Phylo-geneticanalysisbasedonthe16SrRNAgeneshowedthatstrainsACO-34A,ACO-40and ACO-140 wereRhizobiumdaejeonense, Acinetobactercalcoaceticus andPseudomonas mosselii, respectively.Allofthestrainswereabletosynthesizeindole-3-aceticacid(IAA), solubi-lizephosphate,andhadnitrogenaseactivity.Inoculationusingtheplantgrowth-promoting bacteriastrainshadasignificanteffect(p<0.05)onplantgrowthandthesugarcontentofA. americana,showingthatthesenativeplantgrowth-promotingbacteriaareapractical, sim-ple,andefficientalternativetopromotethegrowthofagaveplantswithproperbiological characteristicsforagroindustrialandbiotechnologicaluseandtoincreasethesugarcontent inthisagavespecies.

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

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

Introduction

Theuseofnitrogen-fixingmicroorganismsandplant growth-promoting bacteria (PGPB) is an important alternative to

∗ _{Correspondingauthor.}

E-mail:reriro61@hotmail.com(R.Rincón-Rosales).

replacechemicalfertilizersforthecultivationofagricultural plants.

ThesearchforPGPBaswellasresearchontheir biologi-calpropertiesareincreasingatarapidpacebecauseefforts arebeingmadetoexploitthemcommerciallyasinoculants.

http://dx.doi.org/10.1016/j.bjm.2016.04.010

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

Significantimprovementonthegrowthandyieldofcropsin responsetomicrobialinoculationhasbeenreportedbymany workers.1–3 _{Studiesconfirmthatinoculantsformulatedwith} PGPBhaveshownpositiveeffectsontheagriculturalyieldand cropquality.2,4_{RegardingtheeffectofPGPBonplantgrowth,} ithasbeenreportedthatGlycinemaxL.Merrillseedlings inoc-ulatedwithPGPB(Pseudomonassp.strainAK-1,and Bacillus sp.strain SJ-5) demonstrated enhancedplant biomassand that the plants had a higher proline content than control plants.5_{Inanotherstudy,theefficiencyofMesorhizobium,} Azo-tobacter,and Pseudomonasonthegrowth,yield,anddisease suppressioninchickpeaplants(CicerarietinumL.)was evalu-ated.PseudomonasshowedpositiveIAAproduction,phosphate solubilization, and antagonistic activities against Fusarium oxysporumandRhizoctoniasolanicomparedtootherstrains.6 Martínez-Rodriguezetal.7_{alsoreportedthatcultivable} endo-phyticbacteriafromtheleafbaseofAgavetequilanaWebervar. Bluehavethepotentialtoenhanceplantgrowth.

Scientificevidencesupportsthattheagavegenusincludes severalspeciesofeconomic,socialandculturalimportance forpeoplearoundtheworld.8_{Agaveplantsaregreatlyrelevant} toMexicobecausethiscountryisconsideredtobethepoint oforiginoftheevolutionanddiversificationofthisgenus.9 Approximately163speciesgrowinMexico,and123species areendemic.10

AgaveamericanaL.hassuccessfullyadaptedtoclimaticand edaphicconditionsandproliferatedinthehighlandsof Chia-pas,Mexico,whereitisanimportantsourceofnaturalfibre, medicine,fructans,andtraditionalalcoholicbeveragesforthe localcommunity. Due to the economic significanceofthis plant,severalcommercialplantationshavebeenestablished in the state of Chiapas to produce sufficient raw materi-alsforagro-industrialuse.However,whentheplantletsare transplanted tothe field,their growth and development is slow, and consequently, 5–7 years are required to obtain matureplantsforindustrialuse.11_{Analternativefor} obtain-ingmatureplantsforindustrialuseistheapplicationofplant growth-promotingbacteria,butitisnecessarytoassessthe possibleeffectsofPGPBonA.americanatoincreasethesurvival andgrowthofplantlets.PGPBarerhizospherebacteriathat enhanceplantgrowthbyawidevarietyofmechanisms,such asphosphatesolubilization,siderophoreproduction, biolog-icalnitrogenfixation,phytohormoneproduction,antifungal activity,inductionofsystemicresistance,promotionof bene-ficialplant-microbesymbioses,andsoon.12,13

Manyaspectsofthemicrobialcommunityassociatedwith agavesarestillunknownandonlyamanuscriptrelatedto14 suggests that thehypothesis that PGPBinoculation signifi-cantlyincreasesthegrowthandsugarcontent(mainlyinulin) inA.americanaistrue.Therefore,theobjectiveofthisstudy wastoevaluatetheeffectofPGPBinoculationonplantgrowth andsugaraccumulationinA.americana.

Materials

and

methods

Bacterialstrains

Thebacterial strains ACO-34A, ACO-40, and ACO-140 were chosensubsequenttoastudyofapproximately235strains

previously isolated from the rhizosphere of A. americana. These three strains were selected based on their capacity fornitrogenfixation,auxin production,P-solubilization and biosynthesisofIAA(TableS1)andwereprovidedbythe Insti-tuto Tecnológico de Tuxtla Gutiérrez, while the reference strainAzospirillumbrasilenseCdwasprovidedbytheCentro deCienciasGenómicas,Cuernavaca,México.Allstrainswere growninyeastextract-mannitol(YMA)medium15_at28◦_Cand

preservedat4◦Cuntiluse.

Phenotypicandgenotypicanalysisofstrains

ThecellmorphologiesofthestrainsisolatedfromA.americana were examined bylightmicroscopy (Zeiss® _{PS7,Germany).}

TheGramreactionwasdeterminedusingakit(Merck®_,

Ger-many),accordingtothemanufacturer’sprocedure,andcolony morphologywasdeterminedwithcellsgrownonYMAplates at28◦Cfor5days.16

Bacteriological and physiological characterization of strainsACO-34A,ACO-40,andACO-140wereperformedwith isolatesfromYMAmedium.Salttolerancewasevaluatedat 28◦Cwith0.5,1.0,2.0,3.0and5.0%(w/v)NaClandpHlevelsof 4.0,5.0,9.0and11.0.Acidoralkaliproductionwasdetermined on the same mediumsupplementedwith 25mgmL−1 bro-mothymolblueasapHindicator.16_{Antibioticresistancewas} testedonYMAplatesfollowingtheprocessrecommendedby Martínez-Romeroetal.17_{.Inaddition,theAlandCutolerance} ofthestrainsweredeterminedonsolidYMAmedium.18

16SrRNAgenesequencingandphylogeneticanalysis

The strains were grown in 2.0mL of YMA medium overnight. Total genomic DNA was extracted using a DNA Isolation Kit for Cells and Tissues (Roche®_,

Switzer-land), according to the manufacturer’s specifications. PCR was performed with the bacterial universal 16S rRNA primers fD1 (5-AGAGTTTGATCCTGGCTCAG-3) and rD1 (5 -AAGGAGGTGATCCAGCC-3), which amplified products of approximately1500bases,andprocedureswereperformedas describedbyWeisburgetal.19_{.ThePCRproductswerepurified} usingthe PCRProductPurificationSystemKitfrom Roche®

andsequenced(Macrogen®_{,Korea).Allsequenceswere}

com-pared with the reference sequences obtained by a BLAST search.20_{ThesequenceswerealignedusingtheCLUSTALX} (2.0)softwarewiththedefaultsettings.21_{Minormodifications} inthealignmentweremadeusingtheBIOEDITsequence edi-tor.Phylogeneticandmolecularevolutionaryanalyseswere performedwithMEGAv5.2.22_{Thephylogenetictreeofthe16S} rRNAgenesequencesfromtypestrainswasconstructedby Neighbour-Joining23_{andaBootstrapanalysiswith1000} pseu-doreplicates using the Tamura-Neimodel.24 _{The 16S rRNA} gene sequence of strains ACO-34A, ACO-40, and ACO-140 weredepositedintheGenBankdatabaseundertheaccession numbersKM349967,KM349968,andKM349969,respectively. Additionally, strains ACO-34A, ACO-40, and ACO-140 were depositedinDSMZ(DeutscheSammlungvon Mikroorganis-menundZellkulturen),Germanyasanopencollectionunder thedepositnumbersDSM101606,DSM01771andDSM01784, respectively.

MeasurementofPGPBefficiency QuantificationofIAAproduction

Bacteria were grown in conical flaskscontaining 50mL of YMAmediumcomposedofmannitol(0.25gL−1),K2HPO410%

(5mLL−1),MgSO410%(2mLL−1),NaCl10%(1mLL−1),CaCO3

(1gL−1) and yeast extract (3gL−1) at a pH of 6.8, supple-mentedwith100mgL−1ofl-tryptophan.Afterincubationat 30±2◦Conarotaryshakerfor48h,theculturemediumwas centrifugedat5000×gfor10min,andthesupernatantwas filteredthrougha 0.22-␮mmembranefilter. TheIAAlevels inthe filteredsupernatants of each strain were measured by high-performance liquid chromatography (HPLC) using aPerkinElmer model series 200A equippedwith aSupelco LiChrosorbRPC18column(5␮m;4.6by150mm).Themobile

phasewasacetonitrile–50mMKH2PO4(pH3)(30/70)ataflow

rateof1mLmin−1.Eluateswereanalyzedwithadiodearray detectorat220nm,andIAAwasquantifiedbyintegratingthe area underthe peak; authentic IAA(Sigma)was usedas a standard.TheIAAproducedbyeachstrainwasmeasuredin triplicate.25

Estimationofphosphatesolubilizationinbrothassayby PGPB

Theisolateswere individuallygrowninYM brothmedium overnight,and theOD600nmwasadjustedto1.0.Thecells

were washedtwicein0.85% sterileRingerssolution before inoculating in National Botanical Research Institute Phos-phate(NBRIP)growthmediumcontaininginsolubletricalcium phosphate(Ca3(PO4)2).26ThepHoftheNBRIPmediumwas

adjustedto7.0beforeautoclaving.Thestrainswereinoculated in20-mLvialscontaining NBRIPmediumand incubated at 30◦Conashaker(150rpm)for5days.Afterincubation,5.0mL ofeachstrainwastakenandcentrifuged,andthepHofthe supernatantwasrecorded.Theavailablephosphoruscontent intheculturesupernatantaswellasthecontrol(supernatant obtainedfrommediumnotinoculatedwithbacteria)was esti-matedusingthevanado-molybdatecolorimetricmethod27_by measuringthe absorbanceatawavelengthof420nm.Each treatmentwasreplicatedthreetimes.

Assayforacetylenereductionactivity(ARA)

ThenitrogenfixationabilityofthePGPBstrainswas deter-minedbytheacetylenereductionactivity.28_Thetubeswere sealedwithrubberstoppersafterinoculationandthen incu-batedfor24hinNFBmedium.29_{Tenpercentoftheairwas} removedfromeachtube,andanequalvolumeofacetylene wasinjectedusingasyringe,beforeincubatingthetubesat 30◦Cfor24h.Aparalleluninoculatedcontroltubewas pre-pared.A 100-␮L airsamplewas taken,and the amount of ethylene produced from acetylene was determined by gas chromatography (Perkin Elmer, USA). The ARA value was reportedtoas[ARAnmolC2H4percultureh−1].

Plantinoculationassay

The PGPB strains ACO-34A, ACO-40, and ACO-140 isolated fromtheA.americanarhizosphereandthereferencestrainA. brasilenseCdwereevaluatedinabiofertilizationtest.A. amer-icanaplantletsobtainedbymicropropagationwereplantedin polystyrenetraysthatcontainedpeatasthesubstrate(thepH

ofthepeatwas adjustedto6.7using35gNa2CO3 foreach

100gofmaterial) andcoveredwithapolyethylenesheetto maintainthehumidityandpreventdehydration.Plantswere maintained inagrowth chamberat28◦Cand 38% relative humidity(RH)witha14hlight/10hdarkphotoperiod.Lighting wasprovidedbyfluorescentlampswith50␮Em−2s−1.30

Aftertwomonths(60daysaftertransplantation),theplants weretransferredtopotscontainingpeatmoistenedwithfree NFahraeusmedium[CaCl2,0.1g;MgSO4·7H2O,0.12g;KH2PO4,

0.1g;Na2HPO4·2H2O,0.15g;Fecitrate,0.005g;Mn,Cu,Zn,B,

Motraces;dist.water,1000mL;pH6.5]asanutrientsource31 and placed in a greenhouse at 25±2◦C (natural illumina-tion).Theplantswereinoculatedwith2mLofasuspensionof eachPGPBstrainataconcentrationof1×106_UFCmL−1_.30,32 Uninoculatedplantsandotherstreatedwith30mgofKNO3

-Nperplantservedascontrols.Fourreplicateswereusedper treatment,andtheplantswerearrangedinacompletely ran-domized design.Theplantswere grownunder greenhouse conditionsfor90days.Measurementsoftheplants,thedry weight,thediameterofthestem,thenumberofleaves,and thelengthoftherootsweremadeontheplantsduringthe transplantation phase (m1) and after 90 days (m2) in the

greenhouse. Thedatausedforstatisticalanalysiswere the differencebetweenthem2-m1measurements.

Extractionandquantificationofcarbohydrates

Thecarbohydrates were extracted and quantified from the driedleaves and roots, which were washed withultrapure water.Thesampleswerecrushedandplacedinawaterbath at80◦Cfor40min,thesupernatantwasremovedby centrifu-gationat16,000×gfor15minatroomtemperature,andthe precipitatewasremoved.SampleswereadjustedtopH7.0, fil-teredthrough20-and45-␮mnylonmembranes,andstoredat 8◦Cuntiluse.Carbohydrateswereidentifiedwithapreviously described modifiedmethod.33_{Briefly,thinlayer} chromatog-raphywasperformedusingpropanol–butanol–water(12:3:4) forthe mobile phase and Merck® _{F254silica gelplates for}

thestationaryphase.Spotswere developedwithasolution containing 45% (v/v) aniline (4% v/v in acetone), 45% (v/v) diphenylamine (4% (v/v) inacetone) and 9.1% (v/v)of 85% phosphoricacid.Thedevelopingsolutionwasappliedtothe platesandallowedtodry.Theplateswereheatedto80◦Cfor 5,10,and15minuntilaclearlydefinedcolourappeared.The aldoseshadablue-greycolour,andtheketosesandsucrose wereredoramixtureofbothcolours.

For the quantification of carbohydrates, samples were adjusted to pH 7.0 and filtered through 20- and 45-␮m membranesbeforebeingplacedin2-mLvials.A10-␮L sam-ple of plant extract was injected into a HPLC-IR (Thermo Finnigan®_{, Ontario, Canada) equipped with a Rezex}

RCM-monosaccharide Ca2+ _{column, with water as the mobile}

phaseatatemperatureof85◦C,aflowrateof0.3mLmin−1, and apressureof300psi.Inulinfromchicoryroot,fructose (Sigma®_{,USA),sucrose,andglucose(Baker}®_{,USA)wereused}

asstandards.33

Statisticalanalysis

All of the data obtained in the tests for inoculation, P-solubilization, IAA production, acetylene reductionactivity

(ARA),andcarbohydratequantificationusingdifferentPGPB strains were statistically analyzed by analysis of variance (ANOVA), and the means were compared by Tukey’s test (p<0.05).34

Results

and

discussion

InvestigationofPGPBstrainsfromdifferentplantswiththe potentialto beused asinoculants has increasedinrecent years,andmanyarecommerciallyavailable.12,13,35_Taxonomic polyphasicstudies arecommonlyusedtoanalyze bacterial diversityandincludemorphological,physiological, biochem-ical,metabolic,andprincipally,phylogeneticstudies.36–38_The morphologicalandphysiologicalcharacteristicsofthe bacte-rial strainsisolated fromthe A. americanarhizospherethat have potential as inoculants are shown in Table S2. The strainsevaluated were aerobic,Gram-negative,rod-shaped, and grew rapidlyin YMAmedium.The cells grew at37◦C and had anacidic reaction. The ACO-40strain formed cir-cular,lightly opaque yellowcoloniesfrom 0.5to1.5mm in diameterwithamucoidappearance.Ontheotherhand,the ACO-140strainformedcircular,creamybeigecolonieswith regularborders,measuredapproximately3–4mmindiameter, andhadnoobservablepigmentation.Finally,strainACO-34A wascharacterizedbytheformationofcircular, semitranslu-centcreamycoloniesthatwere1.5–3.0mmindiameterwith amucoid appearance. These threestrains could growin a pH range from 4.0to 9.0. Asfor NaCl tolerance, the ACO-140straingrewin5.0%NaCl,but theACO-34Aand ACO-40 strains tolerated no more than 2.0% NaCl. The ACO-34A strainwasresistanttoampicillin(100␮gmL−1),carbenicillin (20␮gmL−1),chloramphenicol(100␮gmL−1),andkanamycin (100␮gmL−1).TheACO-40strainshowedagreatersensitivity totheantibioticsassessed.Thestrainsgrewinthepresence ofAl3+ _(500␮gmL−1_{) and Cu}2+ _(100␮gmL−1_{). These results}

indicated that someof the biological qualities that distin-guished these strains included their capacity togrow in a wide pHrange, aswell astheir abilitytotoleratedifferent NaClconcentrations andheavymetals, suchasAlandCu. Forexample,strainACO-140managedtogrowin5.0%NaCl. Thisoutcomeisofgreatsignificancebecausesalinityisoneof theharshestenvironmentalfactorsthatpreventsahighcrop yield,andmostagriculturalplantsaresensitivetoahighsalt concentrationinthesoil.Therefore,plantgrowthpromoting bacteria(PGPB)actasoneofthemosteffectivetoolsforthe

alleviationofsaltstress.AzospirillumlipoferumJA4, a genet-ically tagged strain, was capable of colonizing the roots of wheat seedlings in the presence of a high NaCl concentration.39 _{Puente et al.}40 _{reported that strains of} AzospirillumhalopraeferensandA.brasilensesurvivedin seawa-terandwerecapableofcolonizingtherootsurfacesofblack mangroveseedlings.Inlightofthis,theACO-140straincould beanalternativetopromoteA.americanagrowthundersalt stressconditions,ashasbeenpreviouslydemonstratedwith barley and oats.41,42 _{Additionally, this study demonstrated} that thesethreestrains couldgrow atlowpHand tolerate highconcentrationsofaluminiumandcopper.PGPB,suchas Pseudomona fluorescens, canproduceoxalic acid,whichalso mightbeapossiblemechanismforreducingAltoxicity.43 Rhi-zobiumsp.strainBICC651producedathreefoldhigherlevel ofsiderophoreinthepresenceof100␮MAl3+_{, whichcould}

beamechanismstorelieveAlstress,44 _{suggestingthatthe} applicationofsuchbacteriamaybeanefficientapproachto alleviatealuminiumtoxicityandeventuallyimprovesoil fertil-ity.StrainACO-34Awasresistanttovariousantibiotics,which couldbeimportantbecauseitcouldprotecttheplantagainst phytopathogensandestablishanareaintherhizospherewith agreatercapacitytoassimilatesoilnutrients.45

Weused apolyphasic approachinthis studythat com-binedthephenotypiccharacteristicsofthebacterialstrains withaphylogeneticanalysisoftheir16SrRNAgenesequences todeterminethe taxonomicstatusofstrainsisolated from the A. americana rhizosphere (Fig. 1). Strain ACO-40 had a sequencesizeof1381bp,wasaffiliatedwithmembersofthe genusAcinetobacter,andshoweda99.1%geneticidentitywith thetypestrainAcinetobactercalcoaceticusNCCB22016T_(Table1).

The16SrRNAgenesequenceofstrainACO-140was1387bp and wasclassifiedinthegenusPseudomonas,witha100.0% genetic similarity to Pseudomonas mosselii CIP 105259T_.46 Finally, thestrainACO-34Asequencewas1333bpand clus-teredwithmembersofthegenusRhizobium;its16SrRNAgene sequenceshada96.1%similaritywithRhizobiumdaejeonense L61T_.36

Thehighestamount ofIAAwasproducedbytheP. mos-seliistrainACO-140(15.7mgL−1),followedbytheR.daejeonense strainACO-34AandA.calcoaceticusstrainACO-40(Table2).The reference strainA.brasilense Cdalsoproduced asignificant amountofIAA(10.4mgL−1),accordingtoTukey’stest(p<0.05). Thecapacityoftherhizobialstrainstosolubilizephosphate wasevaluatedwithacolorimetricmethodusingNBRIPgrowth mediumthatcontainedinsolubletricalciumphosphate. All

Table1–MolecularidentificationofthePGPBstrainsisolatedfromAgaveamericanaL.

Strain Closestpartial16SrRNA

genesequence

Accession No.

16SrRNA

seq.(bp)

ClosestNCBImatch/speciesidentity Reference

ACO-34A Rhizobiumdaejeonense KM349967 1333 R.daejeonenseL61T_(AY341343)

96.1%

Zhe-Xueetal.36

ACO-40 Acinetobactercalcoaceticus KM349968 1381 A.calcoaceticusNCCB22016T_(AJ888983)

99.1%

Unpublished

ACO-140 Pseudomonasmosselii KM349969 1387 P.mosselii

CIP105259T_(AF072688)

100%

ACO-40 (KM349968)

Acinetobacter calcoaceticus D10 (JQ031270) Acinetobacter calcoaceticus B9 (JQ579640) Acinetobacter calcoaceticus NCCB 22016 (AJ888983)

Acinetobacter baylyi B2 (AF509820) Acinetobacter soli B1 (EU290155) Acinetobacter radioresistens DSM6976 (X81666) Acinetobacter baumannii DSM30007 (X81660)

Acinetobacter junii DSM6964 (X816649)

Acinetobacter gerneri 9A01 (AF509829) Acinetobacter lwoffii DSM2403 (X81665)

Acinetobacter parvus LUH4616 (AJ293691) Acinetobacter beijerinckii LUH 4759 (AJ626712)

Acinetobacter haemolyticus DSM6962 (X81662) Acinetobacter johnsonii ATCC 17909 (Z93440)

Acinetobacter schindleri LUH5832 (AJ278311) Acinetobacter bouvetii 4B02 (AF509827) 100 76 100 100 61 72 86 61 70 89 64 71 76 84 0.005 ACO-140 (KM349969 )

Pseudomonas mosselii CIP 105259 (AF072688)

Pseudomonas taiwanensis BCRC 17751 (EU103629) Pseudomonas monteilii CIP104883 (AF064458)

Pseudomonas plecoglossicida FPC951 (AB009457) Pseudomonas japonica IAM 15071 (AB126621) Pseudomonas putida IAM1236 (D84020) Pseudomonas oryzihabitans IAM1568 (D84004) Pseudomonas asplenii ATCC 23835 (AB021397)

Pseudomonas fluorescens CCM 2115 (DQ207731)

Pseudomonas mohnii IpA-2 (AM293567) Pseudomonas jessenii CIP 105274 (AF068259)

Pseudomonas koreensis Ps 9-14 (AF468452) Pseudomonas reinekei MT1 (AM293565)

Pseudomonas aeruginosa ATCC 10145 (X06684) 59 96 89 89 97 71 82 68 79 72 67 90 0.005

Rhizobium loessense CCBAU 7190B (AF364069) Rhizobium gallicum R602sp (U86343)

Rhizobium mongolense USDA 1844 (U89817) Rhizobium sullae IS123 (Y10170)

Rhizobium etli CFN 42 (U28916)

Rhizobium indigoferae CCBAU 71042 (AY034027) Rhizobium tropici CIAT 899 (U89832) Rhizobium hainanense I66 (U71078) Rhizobium huautlense SO2 (AF025852)

Rhizobium galegae ATCC 43677 (D11343)

Rhizobium undicola LMG11875 (Y17047) Rhizobium larrymoorei AF3-10 (Z30542) Rhizobium giardinii H152 (U86344)

ACO-34A (KM349967 )

Rhizobium daejeonenseR2-60 (JQ659582) Rhizobium daejeonense NBRC 102494 (AB681831) Rhizobium daejeonense L61 (AY341343)

100 99 76 100 95 100 67 99 92 94 99 75 97 54 0.005

B

A

C

Fig.1–Neighbour-joiningphylogenetictreesbasedon16SrRNAgenesequences.(A)Acinetobactercalcoaceticusstrain ACO-40(1381bp),(B)PseudomonasmosseliistrainACO-140(1387bp)and(C)RhizobiumdaejeonensestrainACO-34A(1333bp). Onlybootstrapvalues>50%areshown.TypestrainsareindicatedbythesuperscriptT.Theaccessionnumbersforthe sequencesareindicatedwithintheparentheses.Thosegeneratedinthisworkareshowninbold.

four strains had the capacityto solubilize phosphate after 2–4days ofincubation.Strain ACO-140 exhibitedthe high-estphosphatesolubilizingactivity(37.8mgL−1),followedby strainACO-40(29.8mgL−1)andstrainACO-34A(24.7mgL−1)

(Table2).ThepHvalueoftheculturemediumdecreasedfrom

an initial pH of6.8 to 4.5 as bacterial growth progressed, suggestingthat the bacteriamight secreteorganicacids to solubilizetheinsolublephosphorus.

Recently, endophytic bacteria belonging to the genera Acinetobacter, Bacillus and Pseudomonas with a capacity for

Table2–Phosphatesolubilization,IAAproduction,andacetylenereductionactivity(ARA)inthePGPBstrainsisolated fromAgaveamericanaL.

Treatment IAAproduction

(mgL−1)

P-solubilization

(mgL−1)

ARAb

nmolC2H4percultureh−1

Pseudomonasmosselii ACO-140 15.7Aa _{37.8A 167.1B} Acinetobactercalcoaceticus ACO-40 8.5C 29.8B 106.1B Rhizobiumdaejeonense ACO-34A 10.3B 24.7B 627.4A Azospirillumbrasilense Cd 10.4B 24.2B 821.3A MSDc_{(p<0.05) 1.65 7.63 224.39}

a _{Meanvaluesoffourreplicates.Meansfollowedbythesameletterdonotshowanysignificantdifferences(p<0.05).} b _{ARA,acetylenereductionassay(␮molC}

2H4perculturefreshweighh−1). c _{MSD,minimumsignificantdifference.}

Table3–GrowthparametersforAgaveamericanaplantsinoculatedwithplantgrowth-promotingbacteria.

Treatment Plantdryweight(g) Stemdiameter(cm) Numberofleaves Rootlength(cm)

Pseudomonasmosselii ACO-140 2.44B* _{0.48A 3.0A 11.3CD} Acinetobactercalcoaceticus ACO-40 1.23D 0.27CD 2.7AB 11.3CD Rhizobiumdaejeonense ACO-34A 3.41A 0.37B 4.0A 26.6A Azospirillumbrasilense Cd 1.65C 0.34BC 1.5BC 16.2B KNO3-N 1.07D 0.23D 1.5BC 14.3BC Uninoculated 0.76E 0.18D 1.2C 9.8D MSD(p<0.05) 0.2975 0.0994 1.4510 3.1339

∗ _{Meanvaluesoffourreplicates.Meansfollowedbythesameletterdonotshowasignificantdifference(p<0.05).}  _{MSD,minimumsignificantdifference.}

nitrogen fixation, auxin production and phosphate solubi-lization were isolated from blue agave plants (A.tequilana) from Nayarit, Mexico.7 _{Pseudomonas putidaM5TSA, isolated} from an endemic cactus Mammillaria fraileana that grows inthe SonoranDesert, solubilizedinorganicphosphateand demonstratedrock-weatheringcapacity.47_These pseudomon-adsarecharacterizedbybiochemicalmechanismsandspecific enzymesthatfacilitaterockdegradation,aswellasthe biosyn-thesisofimportantsecondarymetabolites,undervariedbiotic andabioticstressconditions.48

InoculationofplantswithPGPBenhancestheassimilation ofessentialnutrientsandplant-associatedbiologicalnitrogen fixation.13,49 _{Inthisstudy,nitrogenaseactivitywasassessed} withthe acetylenereductionassay (ARA)todeterminethe abilityofPGPBtofixnitrogen.A.brasilenseCdandR.daejeonense ACO-34Astrainsshowedmaximumnitrogenaseactivity(821.3 and627.4nmolC2H4percultureh−1,respectively)compared

totheotherstrainsevaluated(Table2).Thus,thethreestrains showedthepotentialtoproduceIAAandsolubilizephosphate, aswellasnitrogen-fixationcapacity.Theseresultsare impor-tantbecausenitrogenandphosphorusarekeyelementsfor thegrowthandmetabolismofagaveplants.

Furthermore,inoculationwithPGPBhadapositiveeffect onthegrowthofA.americanaplants(Table3).A.calcoaceticus strainACO-40,P. mosseliistrainACO-140,and R.daejeonense strainACO-34Aallhadpositiveeffectsontheplantdryweight, stemdiameter,numberofleaves,androotlengthcompared touninoculatedcontrolplantsandtothosewithaddedKNO3.

Onaverage,plantsinoculatedwithR.daejeonensestrain ACO-34Aweighed2.65gmorethanuninoculatedplantsat90days postinoculation.Thestemdiameterofplantsinoculatedwith P.mosseliistrainACO-140differedsignificantly(p<0.05)from thatobtainedintheothertreatments.Plantstreatedwiththe PGPBstrainsACO-40,ACO-140,andACO-34Aexperienced sim-ilareffectsforthenumberofleavescomparedtouninoculated plantsaccordingtoanalysisbyTukey’stest(p<0.05).Plants inoculatedwithstrainACO-34Ashowedasignificantlyhigher rootlengthcomparedtoothertreatments(p<0.05).Bashan et al.,50 _{reported that the inoculation of pachycereid cacti} species (Pachycereus pringlei, Stenocereus thurberi and Lopho-cereusschottii)enhancedtheestablishmentanddevelopment ofthecactithatwereinoculatedwithA.brasilenseand trans-planted into a disturbedurban desertsoil. Puenteet al.,51

demonstratedthatanassociationbetweenthegiantcardon cactusP.pringleiandendophyticbacteriahelpedtheseedlings becomeestablishedandgrowinasoillessenvironment.Ithas also beenreported thatEnterobacter sakazakiiM2PFe, Azoto-bactervinelandiiM2PerandP.putidaM5TSA,isolatedfromthe rock-dwellingcactusM.fraileana,affected plantgrowth and themobilizationofelementsfromrocks.52_Inthisstudy, simi-larresultswerefoundforR.daejeonensestrainACO-34A,which significantlyinfluencedA. americanagrowth,demonstrating itspotentialasaPGPBduetoitscapacityfornitrogenfixation, phosphatesolubilization,andIAAbiosynthesis.PGBPstrains suchasA.brasilenseandR.daejeonensearebiologicalmodels thatmaypotentiallycontributetotherevegetationoferoded soil.Similarresultsconcerningtheoccurrenceanddiversity ofdiazotrophicbacteriainrhizospheresoilandinrootand leaf tissuesofAgavesisalanaplants havebeen reportedby Santosetal.,53 _{aswell asatestoftheir potentialforplant} growthpromotion.Therefore,PGPBstrainsinvestigatedinthis studycouldbealternativeA.americanainoculantsthatwould improveitsgrowthanddevelopment.

Thesugarcontentintheleaves,stems,andplantrootswas measuredwithaqualitativeandquantitativeanalysis.Thin layerchromatography(Fig.2)showedthatfructansynthesis wasdifferentindifferentplanttissuesandthatbacterial inoc-ulationinfluenced thedegreeofpolymerization(PD) ofthe fructans. Fructoseand sucrosewere detectedintheleaves, stems, and roots ofA. americana plantlets inoculated with thefourbacteria.However,kestosewasdetectedinA. amer-icana plantlets inoculated with all of the bacterial strains, exceptinplantletsinoculatedwithA.calcoaceticusstrain ACO-40(Fig.2B).Spotscorrespondingtosucroseweredetectedwith greaterintensityintheleavesandthestemscomparedtoroots (Fig.2BandC).Nystoseandotherspotscorrespondingto fruc-tans withPD>4weredetectedinthestemsofA.americana plantletsinoculatedwithP.mosseliistrainACO-140(Fig.2A). KestosewasonlydetectedinplantsinoculatedwithP. mos-selii. Thisresultmayindicate thattheinductionofkestose andnystosebiosynthesisisspecificanddependsonthe inocu-latedmicrobialspecies.Therefore,thisphenomenonrequires furtherinvestigation.

In addition, the inulin concentration varied from 0.23 to 1.09mgg−1 in the leaves and was higher in plantlets inoculated with R. daejeonense strain ACO-34A (Table 4).

STD20 mM STD20 mM STD20 mM STD20 mM STD20 mM STD20 mM Fructose

B

A

D

C

Pseudomonas mosselii Acinetobacter calcoaceticus ACO-40

Stem Root Leaves Stem Root

Leaves

Rhizobium daejeonense ACO-34A Azospirillum brasilense Cd

Stem Root Leaves Stem Root

ACO-140

Fructose Sucrose Kestose Nystose

Fig.2–Thinlayerchromatography(TLC)analysisoffructanexohydrolaseandfructan:fructan1-fructosyltransferases(1-FF1) enzymaticactivityinextractsfromleaves,stems,androottissueofinvitro-culturedAgaveamericanaplantletsinoculated with(A)PseudomonasmossellistrainACO-140,(B)AcinetobactercalcoaceticusstrainACO-40,(C)Rhizobiumdaejeonensestrain ACO-34A,and(D)AzospirillumbrasilenseCd.Themarkersrepresentthemobilityoffructose,sucrose,kestose,andnystose.

Nevertheless,Tukey’stestshowedthattheinulin concentra-tionwasnotsignificantlydifferentbetweentheuninoculated plantsandKNO3fertilizedplants.Thisdemonstratesthe

bio-logicalpotentialoftheagavespeciestobiosynthesizevarious types of metabolites such as sugars despite unfavourable growthconditions.54_{Intheroots,theuninoculatedplantshad} agreaterinulinconcentrationthantheplantsinoculatedwith thevariousPGPB.

Significantdifferences(p<0.05)inthesucrosecontentof A.americanaleaveswereobservedamongtreatments.Plants inoculatedwiththePGPBstrainshadagreatersucrose con-centrationthan the uninoculated plantsand those treated withKNO3.Intheplantroots,nosignificantdifferencewas

observedamongtreatmentsforsucroseconcentration. Similarly,theconcentrationofglucoseandfructoseinthe leaves washigher inplantstreated withthe R.daejeonense strainACO-34Aand P.mosseliistrain ACO-140,indicatedby

Tukey’stest(p<0.05).Theglucoseconcentrationintheroots wassignificantlyhigherinplantsinoculatedwiththeR. dae-jeonensestrainACO-34A,andthefructoseconcentrationwas higher in plants inoculated withthe A. calcoaceticus strain ACO-40comparedtotherestofthetreatments.Theseresults indicatethatfructansynthesisisdifferentindifferenttissues, andwithrespecttotheinoculatedbacteria(Fig.2).Thesugar contentinagaveplantswasdifferentamountsforplantsof differentphysiologicalages.55_{Cede ˜no}11_{reportedthatyoung} A.tequilanaplantshadhigherlevelsoffreemonosaccharides (glucose,fructose),thanadultplantsthataccumulatedfructan from8to12years.Theevaluationofsucrose,fructoseand glu-coseisimportantbecauseTrevisanetal.56_{reportedthatthese} sugars act onthe metabolismoffructans, suchas kestose andnystose.Theconcentrationoffructoseandglucoseinthe stemsresultsfromtheactivehydrolysisoffructansby fruc-tanexohydrolase.Sucroseisbiosynthesizedviacrassulacean

Table4–Effectofinoculationwithplantgrowth-promotingbacteriaonsugaraccumulationinleavesandrootsofAgave americanaL.

Treatment Inulin Sucrose Glucose Fructose Inulin Sucrose Glucose Fructose

(mgg−1)Leaves (mgg−1)Root Pseudomonasmosselii ACO-140 0.99A* _{1.04AB 1.97A 0.561A 0.35D 0.17A 0.02D 0.031B} Acinetobactercalcoaceticus ACO-40 0.23C 1.50A 0.78BC 0.054C 0.84B 0.16A 0.19B 0.040A Rhizobiumdaejeonense ACO-34A 1.09A 1.59A 1.43A 0.856A 0.76BC 0.23A 0.35A 0.033B Azospirillumbrasilense Cd 0.28B 0.77BC 0.91BC 0.217BC 0.43E 0.15A 0.08C 0.032B KNO3-N 0.44AB 0.68BC 0.63C 0.131BC 0.28E 0.14A 0.03D 0.033B Uninoculated 0.63AB 0.75BC 0.84BC 0.140BC 0.93A 0.27A 0.08C 0.012C MSD£_{(p<0.05) 0.740 0.726 0.628 0.318 0.022 0.146 0.108 0.045}

∗ _{Meanvaluesoffourreplicates.Meansfollowedbythesameletterdonotshowsignificantdifference(p<0.05).}

£ MSD,minimumsignificantdifference.

acidmetabolisminA.americanaplants.Thisdisaccharideis aprecursor to the synthesisof fructansand is hydrolysed bytheactionofvacuolar invertase,generatingglucoseand fructose.57,58 _{The1-FFT enzymesubsequently converts the} fructosemoietyto1-kestose,whichissynthesizedbythe 1-SSTenzyme, causingglucose tobereleased.59 _1-kestoseis theprecursorofallfructans.33Detectionofneo-kestose indi-cated6G-FFTenzymeactivity.60,61 _{Fructanswithdegreesof} polymerizationDP>4thatwerefoundinstemsamplesfrom plantletsinoculatedwiththeP.mosseliistrainACO-140could betheresultofmicrobialfructosyltransferaseactivityinvolved inmicrobial fructanlevan,inulin, orfructo-oligosaccharide biosynthesis.

Itis also worth noting that the concentration of inulin andothersugarswasincreasedinplantletsinoculatedwith theR.daejeonensestrainACO-34AandP.mosseliistrain ACO-140primarilyintheleavesbutnotintheroots(Table4).In another study,bacterial endophytesisolated from A. tequi-lanaleavesshowedthecapacityfornitrogenfixation,auxin production,andphosphatesolubilizationandalsoincreased inulinproduction.7

Conclusions

TheR.daejeonensestrainACO-34A,A.calcoaceticusstrain ACO-40,andP. mosseliistrainACO-140showedpotentialasPGPB duetotheirphenotypiccharacteristicsaswellastheir capac-ity fornitrogenfixation, phosphate solubilization, and IAA biosynthesis.Additionalstudiesarenecessarytoevaluatethe useofthesebacteriaforcommercialapplicationinA. ameri-canaculturetoimproveitsgrowthanddevelopmentand,most importantly,toincreaseitsfructanandinulincontents.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethanktheLaboratoryofEcologicalGenomics(CCG-UNAM) fortheirtechnicalassistance.Theprojectwasfundedby Tec-nológico Nacionalde México(TNM,Mexico)(5665.15-P and 5663.15-P)fromtheproject‘Infraestructura251805’fromthe ConsejoNacionaldeCienciayTecnología(CONACyT,Mexico).

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.04.010.

r

e

f

e

r

e

n

c

e

s

1.BashanY,HolguinG,de-BashanLE.Azospirillum–plant relationships:physiological,molecular,agricultural,and environmentaladvances(1997–2003).CanJMicrobiol. 2004;50:521–577,http://dx.doi.org/10.1139/W04-035.

2.BashanY,de-BashanLE.Bacteria/PlantGrowth-Promotion.

In:HillelD,editor.EncyclopediaofSoilsintheEnvironment.vol.

1.Oxford,UK:Elsevier;2005:103–115.

3.LugtenbergBJJ,KamilovaF.Plant-growth-promoting

rhizobacteria.AnnuRevMicrobiol.2009;63:541–556.

4.ZahirZA,ArshadM,FrankenbergerWT.Plantgrowth

promotingbacteria:applicationsandperspectivesin

agriculture.AdvAgron.2003;81:97–168.

5.KumariS,VaishnavA,JainS,VarmaA,ChoudharyDK.

Bacterial-mediatedinductionofsystemictoleranceto

salinitywithexpressionofstressalleviatingenzymesin

Soybean(GlycinemaxL.Merrill).JPlantGrowthRegul.

2015;34:558–573.

6.VermaJP,YadavI,TiwariKN,JaiswalDK.Evaluationofplant

growthpromotingactivitiesofmicrobialstrainsandtheir

effectongrowthandyieldofchickpea(CicerarietinumL.)in

India.SoilBiolBiochem.2014;70:33–37.

7.Martínez-RodriguezJD,DelaMora-AmutioM,

Plascencia-CorreaLA,etal.Cultivableendophyticbacteria

fromleafbasesofAgavetequilanaandtheirroleasplant

8.Ahumada-SantosYP,Montes-AvilaJ,UribeMD,etal.

Chemicalcharacterization,antioxidantandantibacterial

activitiesofsixAgavespeciesfromSinaloa,Mexico.IndCrop

Prod.2013;49:143–149.

9.García-MendozaAJ.DistributionofthegenusAgave

(Agavaceae)anditsendemicspeciesinMexico.CactSuccJ.

2002;74:177–187.

10.Delgado-LemusA,TorresI,BlancasJ,CasasA.Vulnerability

andriskmanagementofAgavespeciesintheTehuacán

Valley,México.JEthnobiolEthnomed.2014;10:1–15.

11.Cede ˜noMC.Tequilaproduction.CritRevBiotechnol.

1995;15:1–11.

12.BhattacharyyaPN,JhaDK.Plantgrowth-promotingbacteria

(PGPB):emergenceinagriculture.WorldJMicrobiolBiotechnol.

2013;28:1327–1350.

13.BashanY,de-BashanLE,PrabhuSR,HernandezJP.Advances

inplantgrowth-promotingbacterialinoculanttechnology:

formulationsandpracticalperspectives(1998–2013).Plant

Soil.2014;378:1–33.

14.DesgarennesD,GarridoE,Torres-GomezMJ,Pena-Cabriales

JJ,Partida-MartinezLP.Diazotrophicpotentialamong

bacterialcommunitiesassociatedwithwildandcultivated

Agavespecies.FEMSMicrobiolEcol.2014;90:844–857.

15.VincentJM.AManualforthePracticalStudyofRootNodule

Bacteria.Oxford,UK:BlackwellScientific;1970:176–189.

16.ToledoI,LloretL,Martínez-RomeroE.Sinorhizobium

americanumsp.nov.,anewSinorhizobiumspeciesnodulating

nativeAcaciaspp.inMexico.SystApplMicrobiol.

2003;26:54–64.

17.Martínez-RomeroE,SegoviaL,MercanteFM,FrancoAA,

GrahamP,PardoMA.Rhizobiumtropici,anovelspecies

nodulatingPhaseolusvulgarisL.beansandLeucaenasp.trees.

IntJSystBacteriol.1991;41:417–426.

18.ZhangX,HarperR,KarsistoM,LindströmK.Diversityof

Rhizobiumbacteriaisolatedfromtherootnodulesof

leguminoustrees.IntJSystBacteriol.1991;41:104–113.

19.WeisburgWG,BarnsSM,PelletierDA,LaneDJ.16Sribosomal

amplicationforphylogeneticstudy.JBacteriol.

1991;73:697–703.

20.AltschulSF,GishW,MillerW,MyersEW,LipmanDJ.Basic

localalignmentsearchtool.JMolBiol.1990;215:403–410.

21.LarkinMA,Black-ShieldsG,BrownNP,etal.ClustalWand

ClustalXversion2.0.Bioinformatics.2007;23:2947–2948.

22.TamuraK,PetersonD,PetersonN,StecherG,NeiM,Kumar

S.MEGA5:molecularevolutionarygeneticsanalysisusing

maximumlikelihood,evolutionarydistance,andmaximum

parsimonymethods.MolBiolEvol.2011;28:2731–2739.

23.SaitouN,NeiM.Theneighbor-joiningmethod:anew

methodforreconstructingphylogenetictrees.MolBiolEvol.

1987;4:406–425.

24.TamuraK,NeiM.Estimationofthenumberofnucleotide

substitutionsinthecontrolregionofmitochondrialDNAin

humansandchimpanzees.MolBiolEvol.1993;10:512–526.

25.ZakharovaEA,ShcherbakovAA,BrudnikVV,SkripkoNG,

BulkhinNS,IgnatovVV.Biosynthesisofindole-3-aceticacid

inAzospirillumbrasilenseInsightsfromquantumchemistry.

EurJBiochem.1999;259:572–576.

26.NautiyalCS.Anefficientmicrobiologicalgrowthmediumfor

screeningphosphatesolubilizingmicroorganisms.FEMS

MicrobiolLett.1999;170:265–270.

27.FiskeCH,SubbarowY.Acolorimetricdeterminationof

phosphorus.JBiolChem.1925;66:375–400.

28.BurrisRH.Nitrogenfixationassay–methodsand

techniques.MethodsEnzymol.1972;24:415–431.

29.DöbereinerJ,MarrielI,NeryM.Ecologicaldistributionof

SpirillumlipoferumBeijerinck.CanJMicrobiol.

1976;22:1464–1473.

30.Ruíz-ValdiviezoVM,Ventura-CansecoLMC,CastilloSuárez

LA,Gutiérrez-MiceliFA,DendoovenL,Rincón-RosalesR.

Symbioticpotentialandsurvivalofnativerhizobiakepton

differentcarriers.BrazJMicrobiol.2015;46:735–742.

31.FahraeusG.Theinfectionofcloverroothairbynodule

bacteriastudiedbyasingleglassslidetechnique.JGen

Microbiol.1957;16:374–381.

32.BashanY.Significanceoftimingandlevelofinoculation

withrhizospherebacteriaonwheatplants.SoilBiolBiochem.

1986;18:297–301.

33.Vizcaíno-RodríguezLA,Rodríguez-MendiolaMA,

Mancilla-MargalliNA,Ávila-MirandaME,Osuna-CastroJA,

Arias-CastroC.Biosíntesisinvitrodeoligofructanos:inulinas

yneoinulinasporfructosiltransferasasdeAgavetequilana

Webervar.AzulyA.inaequidenssubsp.InaequidensKoch.

GayanaBot.2012;69:66–74.

34.SASInstituteInc.SAS/STATUser’sGuide.Version6.0.4thed.

Cary,NC:SASInstituteInc.;1989:3471–3487.

35.SanchezAC,GutierrezRT,SantanaRC,UrrutiaAR,MichielsJ,

VanderleydenJ.Effectsofco-inoculationofnativeRhizobium

andPseudomonasstrainsongrowthparametersandyieldof

twocontrastingPhaseolusvulgarisL.genotypesunderCuban

soilconditions.EurJSoilBiol.2014;62:105–112.

36.Zhe-XueQ,Hee-SungB,Jong-HwanB,Wen-FengC,

Wan-TaekI,Sung-TaikL.Rhizobiumdaejeonensesp.nov.

isolatedfromacyanidetreatmentbioreactor.IntJSystEvol

Microbiol.2005;55:2543–2549.

37.MuletM,GomilaM,LemaitreB,LalucatJ,García-ValdésE.

TaxonomiccharacterisationofPseudomonasstrainL48and

formalproposalofPseudomonasentomophilasp.nov.SystAppl

Microbiol.2012;35:145–149.

38.Rincón-RosalesR,Villalobos-EscobedoJM,RogelMA,

MartínezJ,Orme ˜no-OrrilloE,Martínez-RomeroE.Rhizobium

calliandraesp.nov.,Rhizobiummayensesp.nov.and

Rhizobiumjaguarissp.nov.,rhizobialspeciesnodulatingthe

medicinallegumeCalliandragrandiflora.IntJSystEvol

Microbiol.2013;63:3423–3429.

39.BacilioM,RodriguezH,MorenoM,HernandezJP,BashanY.

Mitigationofsaltstressinwheatseedlingsbyagfp-tagged

Azospirillumlipoferum.BiolFertilSoils.2004;40:188–193.

40.PuenteME,HolguinG,GlickBR,BashanY.Rootsurface

colonizationofblackmangroveseedlingsbyAzospirillum

halopraeferensandAzospirillumbrasilenseinseawater.FEMS MicrobiolEcol.1999;29:283–292.

41.BacilioM,MendozaA,BashanY.Endophyticbacteriaofthe

rock-dwellingcactusMammillariafraileanaaffectplant

growthandmobilizationofelementsfromrocks.EnvironExp

Bot.2012;81:26–36.

42.ChangP,GerhardtKE,HuangXD,etal.Plant

growth-promotingbacteriafacilitatethegrowthofbarley

andoatsinsalt-impactedsoil:implicationsfor

phytoremediationofsalinesoils.IntJPhytoremediation.

2014;16:1133–1147.

43.AppannaVD,HamelRD,LévasseurR.Themetabolismof

aluminumcitrateandbiosynthesisofoxalicacidin

Pseudomonasfluorescens.CurrMicrobiol.2003;47:32–39.

44.RoyN,ChakrabarttyPK.Effectofaluminumonthe

productionofsiderophorebyRhizobiumsp.(Cicerarietinum).

CurrMicrobiol.2000;41:5–10.

45.BeneduziA,AmbrosiniA,PassagliaLMP.Plant

Growth-PromotingBacteria(PGPB):theirpotentialas

antagonistsandbiocontrolagents.GenetMolBiol.

2012;35:1044–1051.

46.DabboussiF,HamzeM,SingerE,GeoffroyV,MeyerJM,Izard

D.Pseudomonasmosseliisp.nov.,anovelspeciesisolatedfrom

clinicalspecimens.IntJSystEvolMicrobiol.2002;52:

363–376.

47.LopezBR,BashanY,BacilioM.Endophyticbacteriaof

Mammillariafraileana,anendemicrock-colonizingcactusof

thesouthernSonoranDesert.ArchMicrobiol.

48.AliSZ,SandhyaV,RaoVL.Isolationandcharacterizationof

drought-tolerantACCdeaminaseand

exopolysaccharide-producingfluorescentPseudomonassp.

AnnMicrobiol.2014;64:493–502.

49.CalvoP,NelsonL,KloepperJW.Agriculturalusesofplant

biostimulants.PlantSoil.2014;383:3–41.

50.BashanY,RojasA,PuenteME.Improvedestablishmentand

developmentofthreecactispeciesinoculatedwith

Azospirillumbrasilensetransplantedintodisturbedurban

desertsoil.CanJMicrobiol.1999;45:441–451.

51.PuenteME,LiCY,BashanY.Endophyticbacteriaincacti

seedscanimprovethedevelopmentofcactusseedlings.

EnvironExpBot.2009;66:402–408.

52.LopezBR,Tinoco-OjangurenC,BacilioM,MendozaA,

BashanY.Endophyticbacteriaoftherock-dwellingcactus

Mammillariafraileanaaffectplantgrowthandmobilizationof

elementsfromrocks.EnvironExpBot.2012;81:26–36.

53.SantosAFD,MartinsCYS,SantosPO,etal.Diazotrophic

bacteriaassociatedwithsisal(AgavesisalanaPerrineex

Engelm):potentialforplantgrowthpromotion.PlantSoil.

2014;385:37–48.

54.GobeilleA,YavittJ,StalcupP,ValenzuelaA.Effectsofsoil

managementpracticesonsoilfertilitymeasurementson

AgavetequilanaplantationsinWesternCentralMexico.Soil TillageRes.2006;87:80–88.

55.ArrizonJ,MorelS,GschaedlerA,MonsanP.Comparisonof

thewater-solublecarbohydratecompositionandfructan

structuresofAgavetequilanaplantsofdifferentages.Food

Chem.2010;122:123–130.

56.TrevisanF,OliveiraVF,CarvalhoMAM,GasparM.Effectsof differentcarbohydratesourcesonfructanmetabolismin plantsofChrysolaenaobovatagrowninvitro.FrontPlantSci. 2015;6:681,http://dx.doi.org/10.3389/fpls.2015.00681.

57.VargasWA,PontisHG,SalernoGL.Differentialexpressionof

alkalineandneutralinvertasesinresponseto

environmentalstresses:characterizationofalkalineisoform

asstressleaves.Planta.2007;226:1535–1545.

58.ChenTH,HuangYC,YangCS,YangCC,WangAY,SungHY.

Insightsintothecatalyticpropertiesofbamboovacuolar

invertasethroughmutationalanalysisofactivesiteresidues.

Phytochemistry.2009;70:25–31.

59.LüscherM,HochstrasserU,BollerT,WiemkenA.Isolationof

sucrose:sucrose1-fructosyltransferase(1-SST)frombarley

(Hordeumvulgare).NewPhytol.2000;145:225–232.

60.FujishimaM,SakaiH,UenoK,etal.Purificationand

characterizationofafructosyltransferasefromonionbulbs

anditskeyroleinthesynthesisoffructo-oligosaccharidesin

vivo.NewPhytol.2005;165:513–524.

61.UenoK,OnoderaS,KawakamiA,YoshidaM,ShiomiN.

MolecularcharacterizationandexpressionofacDNA

encodingfructan:fructan6G-fructosyltransferasefrom

asparagus(Asparagusofficinalis).NewPhytol.