revised manuscript.docx

revised manuscript.docx

《revised manuscript.docx》由会员分享，可在线阅读，更多相关《revised manuscript.docx（21页珍藏版）》请在冰豆网上搜索。

revisedmanuscript

Anewfunctionofgrapheneoxideemerge:

inactivatingphytopathogenicbacteriumXanthomonasoryzaepv.oryzae

JuanniChen,XiupingWang,HeyouHan

StateKeyLaboratoryofAgriculturalMicrobiology,CollegeofScience,HuazhongAgriculturalUniversity,Wuhan,430070,People’sRepublicofChina

Correspondingauthor.Tel:

+86-27-87288246;Fax:

+86-27-87288246

E-mail:

hyhan@

Abstract:

Xanthomonasoryzaepv.oryzae（Xoo）isonerepresentativephytopathogenicbacteriumcausingbacteriainfectionsinrice.TheantibacterialactivityofgraphenesuspendedindifferentdispersantsagainstXoowasfirstlyinvestigated.Bacteriologicaltestdata,fluorescencemicroscopeandtransmissionelectronmicroscopy（TEM）imagesareprovided,whichyieldinsightintotheantibacterialactionofthenanoscalematerials.Surprisingly,theresultsshowedgrapheneoxide（GO）exhibitssuperiorbactericidaleffectevenatextremelylowdoseinwater（250μg/mL）,almostkilling94.48%cells,incomparisontocommonbactericidebismerthiazolwithonly13.3%mortality.Thehighefficiencyininactivatingthebacteriaonaccountofconsiderablechangesinthecellmembranescausedbytheextremelysharpedgesofgrapheneoxideandgenerationofreactiveoxygenspecies（ROS）,whichmaybethefatalfactorforbacterialinactivation.GiventhesuperiorantibacterialeffectofGOandthefactthatGOcanbemass-producedwithlowcost,weexpectanewapplicationcouldbedevelopedasbacteriacideforcontrollingplantdisease,whichmaybeamatterofgreatimportanceforagriculturaldevelopment.

Keywords:

Xanthomonasoryzaepv.oryzae,graphene,antibacterialactivity,mechanisms,bacteriacide,agriculture

Introduction

Xanthomonasoryzaepv.oryzae（Xoo）,arod-shaped,Gram-negativespecies,representoneofthemostcommonpathogenswhicharethecausativeagentofbacteriainfectionscausingbacterialleafblightofrice（Oryzasativa）（Ryba-Whiteetal.1995）.BacterialleafblightisrecognizedasoneofthemostdevastatingcropbacteriadiseasesintropicalAsiancountriesandcancausesevereyieldlossofupto50%inrice（Mewetal.1987）.Protectionofcropsplantfrombacterialdiseasecansubstantiallyimproveagriculturalproduction.Commonly,theeffectiveantibacterialagentsappliedtocontrollingtheplantdiseasesareconfinedtosyntheticorfumigantchemicalsincludinginorganicbactericide,triazoles,antibiotic,metalliccompoundbiocides（organosulfurs,nitrogencompoundsetal.）,whichaccompanywithadverseimpactontheenvironment（Monrocetal.2006）.Moreover,antimicrobialresistanceinplantpathogenictargetbacteriaalsobroughtabouttheaccumulationofthecompound，whichcanresultinunimaginablylong-termconsequences（Imfeldaetal.2012）.Anotherapproachforpreventingthediseaseistoimprovethediseaseresistanceofricebymeansofthenewtransgenictechnique,yetitishardandmoney-consuming（Guetal.2005）. Consequently,itisveryurgenttodevelopalternativestogreenantibacterialtopreventcropfrompathogensinfectionstoavoidtheabove-mentionedadverseeffects（Vidaveretal.2002）.

Recently,carbonnanotubes（CNTs）,nanoscalegrapheneoxide（GO）andreducedgrapheneoxide（rGO）havebeenexhibitedextremelyantibacterialactionagainstmajorfoodbornepathogenslikeEscherichiacoli（E.coli）andStaphylococcusaureus（S.aureus）（Kangetal.2008;Akhavanetal.2010;Liuetal.2009）.Amongthese,LiuetalalsodiscoveredthatGOwasmoretoxictobacteriathanrGOandprovedthatGOkillapproximately90%bacteriaevenatsuchlowconcentrationsof80μg/mL（Liuetal.2011）.ThesestudiesondifferentspeciesofmicroorganismsdemonstratethatGOperformawidetargetofantibacterialactivity.Inaddition,theyarebelievedtobemildcytotoxicitytowardsmammaliancellsandhavebeenalsousedformedicaldisinfection,cellularimaginganddrugdelivery（Balandinetal.2008;Bolotinetal.2008;Jinetal.2010;Yangetal.2008）.Theeffectivenessofgrapheneasantimicrobialagentsmaybeattributedtoitsextraordinaryproperties,suchasgoodthermalstability,highsurfacearea,exceptionalphysiochemicalproperties,highelectronicconductivityandexcellentmechanicalstrength（Baietal.2012;Huetal.2010;Vilaetal.2012）.SeveralstudiesspecificallyhaveproposedthatsuspensionsofsharpGOnanosheetsproduceeitherdisorganizationofcellmembraneoroxidativestresswhichthoughttoberesponsibleforhighactivityofnanoparticles（Akhavanetal.2010;Bolotinetal.2008）.

Thoughgrapheneanditsderivativeswaspreviouslyshowntobeabroadspectrumbactericidalagentactiveinvitroagainstbothgrampositiveandgramnegativepathogenicbacterium（Akhavanetal.2010）,theiractionagainstplantpathogenicbacteriumhavenotbeenstudied.Onlyrecently,onereportwasputforwardthatCNTsandGOhadsuperiorinactivationeffectsoncopper-resistantRalstoniasolanacearumandcellmembranedamageisthecausativefactor（Wangetal.2013）.Practically,breakdownofresistanceisgreatlyessentialforcontrollingplantpathogenXoo.Though,biotechnologyhasbeenusedtoovercometheproblembutthewayisrestrictedtothegeneticengineeringofpathogen-resistantplants（Leachetal.1995）.GOhasneverreportedtocausebacterialresistanceforcomplicatingbacterialinfectionstherapy.Besides,itsgermicidalactivityandlowcostcausedbythecheaprawmaterialhavemuchmoreadvantagesovertraditionalbactericidesorotherchemicalcompound（Bolotinetal.2008;Hummersetal.1958;Liuetal.2008）.Thereby,itisindispensabletoclarifytheircytotoxicityonphytopathogenicbacteriumandextenditsgeneralfunctiontobepotentiallyappliedtodesignstrategiesforplantbacterialdiseasemanagement.

Towardsthispurpose,asystematicevaluationofgrapheneoxideandreducedgrapheneoxide（rGO）againstselectedplant-pathogenicbacteriumXoowasinvestigatedforthefirsttime.Specifically,howthematerialaffectscellgrowthandthemechanismsofcontactinteractionofgraphenewiththebacteriaareexamined.TheresultsfoundGOcouldcreatelethaleffectsonbacteria,notonlyduetophysicallyinjurythecellstructurebutalsochemicallyinduceharmoxidationaction.Theuniqueadvantageofphysicalinjury,whichcurrentchemicalpesticidecannotbebeyondthereachof,guaranteethisnanoparticletobeefficaciousantibacterialagentforeliminatingorcompletelykillingsurroundingdrug-resistantandmultidrug-resistantplantsinfectedbacterium.Itisanticipatedthattheresearchtoacertainextentwillpromotethebetterapplicationinthebiologyfieldandthedevelopmentofnanoscienceandnanotechnology（Hussainetal.2009）.

MethodsandMaterials

SysthesisofGOandrGO

GOwaspreparedfromnaturegraphitepowdersbythemodifiedHummersmethod（Hummersetal.1958）.Firstly,thenaturegraphitepowders（99.99%;Sigma-Aldrich）wereoxidizedtoproducegraphiteoxide（GtO）.Afterbeingwashedusingdeionizedwatertoremovechemicalresidues,theproducedGtOdispersedindeionizedwaterwassonicated（Elamsonic,S60H）for2htoexfoliatetheGO.TheobtainedbrownGOwasreducedtorGObyhydrazinehydrate（Liuetal.2011）,andthereducedgrapheneoxidewasdispersedbybathsonicationat200Wfor2h.Bismerthiazol（93.7%）wasboughtfromHubei provincial seedcompany.

Thetwotypesofmaterialswerecharacterizedbyseveraltechniques.TheparticlestabilityandsizeofGOandrGOdispersionswasevaluatedwithdynamiclightscattering（DLS）onMalvernZetasizerNanoseriesa（Malvern,England）.Adropofdispersionwerespreadedonafreshlycutmicasurfaceandletthesamplesair-driedforAFManalysiswhichabtainedon（Agilent5500 ）andtheultravioletabsorptionspectrawereacquiredontheNicoletEvolution300UV-visspectrometer.ThemorphologyofgraphenewasinspectedandobtainedbyTEM（HitachiH-7650,Japan）.TheRamanspectrawereobtainedviaRamanspectrometer（Renishaw,UK）equippedwithaconfocalmicroscope（Leica,DMLM/P/11888500,Germany）.Fouriertransforminfrared（FT-IR）spectrawereperformedonaNicoletAvatar-330spectrometerwith2cm-1resolutionusingtheKBrpellettechnique.

Bacterialcellpreparations

Xanthomonasoryzaepv.oryzae（Xoo）waspurchasedfromtheStateKeyLaboratoryofAgriculturalMicrobiologyofHuazhongAgriculturalUniversity.XooweregrowninLB（Luria-Bertani）brothmediuminahumidifiedincubatorat30℃withconstantagitationovernight.Thebacteriacultureswereharvestedinthemidexponentialgrowthphaseandcentrifugedat6000rpmfor5mintocollectthecells,andthenthebacteriabreadwaswashedthreetimeswithdeionizedwaterinordertowipeoffmediumconstituentsandotherchemicalmacromolecules.Afterthat,cellswereresuspendedindeionizedwater,0.9%NaCland0.1Mphosphatebuffersolution（pH7.0）andthesuspensionsweredilutedtodesiredconcentrationof107-108colonyformingunits（CFU/mL）respectively.WewillutilizeDIwater,0.9%NaClandPBSasabbreviationsthroughoutthisarticle.Allexperimentswerecarriedoutatroomtemperature.

Bacterialcellsgrowthcurve

Toexaminethebacterialgrowthrate,andthegrowthcurveofbacteriawasdetermined.200μLofthedilutedcellssuspensionsmixedwith20μLofdifferenttestconcentrations（50,100,150,200,250μg/mL）ofGO,rGOorbismerthiazolwasincubatedat30℃for2hwithgentleshaking.Thecontrolsamplecontained200μLofthecellsuspensionsaddedto20μLofDIwater.Themixturewasthentransferredto5mLtubescontaining2mLLBmediumandthetubeswerekeptrotatingonashakerat120rpmat30℃.Alltreatmentswerepreparedintriplicate.Cellsgrowthwasdetectedbymeasuringtheopticaldensity（OD）a