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February2016 TheUseofBactericidesinPlantAgriculturewith ReferencetoUseinCitrustoMitigateHLB StephanieL.Slinski,Ph.D.,BactericideProjectManager,CRDF ThepurposeofthiscommunicationistodiscusstheuseofbactericidesinplantagriculturetocontroldiseaseepidemicsandtheapproachesthathavebeentestedtocontrolHuanglongbing(HLB)incitrustrees. TheCitrusResearchandDevelopmentFoundation(CRDF)hasmadethistopicapriorityinrespondingto HLBinFloridacitrus.HLBisadiseasedevastatingthecitrusindustryinFloridaandthroughouttheworld. Presently,nochemicaltreatmentorresistantplantisavailablethatwillcontrolthedisease.FortheFlorida citrusindustrytosurvivethisepidemic,achemicalcontrolwillbenecessarytosuppressthedisease,keepingthetreesinproductionuntilgrovescanbereplantedwithresistantortolerantvarieties.Thisissimilar toapproachestakenduringplantdiseaseepidemicsinothercrops,whichwereeventuallycontrolledbythe plantingofresistantvarieties.Oxytetracycline,streptomycinsulfateandcopperhavebeenthemainchemicalsavailabletotreatbacterialplantdiseasesintheUS.Theuseofcopperislimitedtofoliardiseasesinregionswherecopperresistanceisnotwidespread.Streptomycinandoxytetracyclineareroutinelyusedon somecropswhencopperisinadequate.Oxytetracyclinehasalsooftenbeenusedinthepasttohelpmanageplantdiseaseepidemics.Researchusingbactericidesoncitrussuggeststhatchemicalcontrolmayimprovecitrustreehealthandcontributetosustainingthecitrusindustryinthecurrentepidemic. Introduction ThediseaseHLBhasbeenknowninmanyregionsofthe worldforoveracentury.ThisdiseasewasonlyintroducedintoFloridaadecadeago,buthassincereached epidemicproportions.HLBinFloridaispresumably causedbythebacteriumCandidatusLiberibacterasiaticus(CLas),whichistransmittedbyaninsect,theAsian citruspsyllid(ACP,DiaphorinacitriKuwayama).Thebacteriumisinjecteddirectlyintothevascularsystemofthe treebythevectorandcanspreadsystemicallydownto therootsandthroughoutthetreecanopy.Thenumber ofFloridacitrusgrovesstillinproductionhasshrunk dramaticallybecauseofHLBduetoadecreaseinmarketablefruitandsignificantlyhigherthanpreviousproductioncostsresultingfromtreedecline.Thecitrusindustryemploysaround62,000peopleandhasaneconomicimpactofapproximately10.8billiondollars(1). TheJanuary2016all-orangecropestimateofonly69 millionboxeswaslessthanhalfofthepre-epidemic numbers.Withtherisingcoststokeepthediseased treesinproduction,thesustainabilityoftheindustryis inquestion.Whatwillultimatelyensurethecontinued existenceoftheindustryareresistantcitrusvarieties, butnoresistantvarietyispresentlybeingpropagatedin nurseries.Evenifaresistantvarietywereidentifiedtoday,itwouldtakemanyyearstoreplantmorethan 500,000acresofcitrusandforthosetreestoreachmaturity.PlantdiseaseepidemicslikeHLBarerelatively common,andwhileultimatelyepidemicsarecontrolled usingresistantortolerantplantvarieties,achemical controlisoftenusedasastopgap. AgriculturalBactericides Plantdiseasescausedbybacteriaareverydifficultto control,especiallywhenabacterialikeCLasresidesin thephloemoftheplantvascularsystem,andfew bactericidesareavailabletotreatbacterialdiseases. Althoughcopperhasbeenusedinagricultureforseveral centuriesasafoliartreatmenttoprotectplantsfrom fungalandbacterialdiseases,whenusedsystemicallyat bactericidallevels,copperistoxicthus,copperhasnot beendemonstratedtobeaneffectivetreatmentfor HLB.AtypeofbactericidesthathasbeenfoundtoprovidealevelofcontrolofHLBandmanyotherbacterial diseasesisantibiotics.Thetwomainantibioticslabeled foruseinagriculturearestreptomycinsulfateandoxytetracycline.Twoformsofoxytetracyclineareused,oxy- tetracyclinehydrochlorideandoxytetracyclinecalcium, butonceinsolutionthemoleculesareidentical.These materialshavebeenusedsincethe1950sandarealso importantinhumanandanimalmedicine,butpresently onlyafractionofonepercentoftotaldomesticantibioticuseisforplantagriculture(2). Themainusesforbothoxytetracyclineandstreptomycinsulfateinagricultureareforpreventionoffireblight (Erwiniaamylovera)inapplesandpears.Streptomycinis thepreferredbactericide,sinceitiskillsthebacteriaon contact(bactericidal),whereasoxytetracyclineisbacteriostatic,suppressingbacterialgrowth.Bacteriostatic bactericidesrequirealongerdurationofexposureto suppressgrowthlongenoughtoreducethebacterial titer.Alternatingchemicalswithdifferentmodesofactionistheprimarystrategyforresistancemanagement. Streptomycin-resistantE.amyloverastrainsnowoccur insomeareasbecausestreptomycinwastheonlychemicalavailableinthepast.Oxytetracyclinehasbeenlabeledforuseagainstfireblighttomanageresistancein someregionsandrecently,kasugamycinhasbeenapprovedforuseinmostUSstatesforthesamepurpose. Oxytetracyclineisalsousedagainstbacterialspotof peachandnectarine(Xanthomonasarboricolapv.pruni) andinnon-bearingplants,whereitismainlyappliedby injectiontopreventinfectionortotreatimportantlandscapeorspecimenplants.Oxytetracyclinehasbeen usedhistoricallytocontrolimportantbacterialandphytoplasmadiseasesinemergencysituations,suchascoconutlethalyellowingdisease,X-diseaseofpeachesand cherries,peachyellowleafrollandpeardecline.These diseasesnowaremainlycontrolledusingtolerantor resistantvarietiescombinedwithgoodmanagement practices. BactericideResearchAgainstHLB Theuseofoxytetracyclinehasbeenevaluatedinthe pasttocontrolHLBandhasbeenshowntohavesome efficacy.MostofthestudiesusingtetracyclinesandsimilarbactericidestotreatthesymptomsofHLBwere conductedinthe1960sthroughthe1980sinareasof long-establishedinfectionbythedisease.Severalmembersofthetetracyclinegroupwereinvestigatedincludingtetracyclinehydrochloride,chlortetracycline,dimethylchlortetracyclineandoxytetracycline.Thesestudies mainlyevaluatedperformanceofbactericidesapplied 2 byfoliarapplicationorbytrunkinjection,althoughsome earlierstudiesimmersedbudwoodinabactericidesolutionbeforegraftingorimmersedtherootsbeforetransplanting(3,4).Molecularmethodsfordetectionofthe HLBbacteriumwerenotavailablewhentheseearly studieswereperformedandthusthestudiesevaluated thetimingofvisiblesymptomdevelopmentasameasureofefficacy.Resultsvariedbyexperiment,buttetracyclinewasfoundtobeeffectiveinmostcaseswiththe suppressionofsymptomslastingfrommonthstoyears (3-16).Inoneexperimentevaluatingbothinjectionand foliarapplicationsoftetracycline,moretreesrecovered andsymptomsweresuppressedforalongerperiodof timewhentetracyclinewasinjectedcomparedwithfoliarapplications(8).Otherstudiesfocusedoneithertrunk injectionorfoliarapplications.Someofthetetracyclines usedintheseexperimentswerefoundtobetoxictothe plant,causingsymptomssuchasyellowing,smaller leavesanddiscolorationattrunkinjectionsites.Dimethylchlortetracyclineandoxytetracyclinedidnotcause thesamesymptoms,possiblybecauseofthegreater solubilityofthesematerials.Onestudyalsofoundthat thepHofthetetracyclinesolutioninfluencedthe amountoftoxicity(15),providingfurtherevidencethat theformulationchemistrycanbeadjustedtoprevent toxicity. Penicillinwasincludedinafewoftheearlystudies,and resultsrangedfromhavinglittletonoeffectonsymptoms(5,10,13),havingalesser,butmoresustainedeffectovertimewhencomparedtooxytetracycline(9),to havingasimilareffecttotetracyclineHCl(6).Amore recentstudyconductedinFloridatestedacombination ofahighdoseofpenicillinGandstreptomycinalong withseveralotherchemicalsincludingahighdoseof oxytetracyclineandkasugamycin,usingabudwoodimmersionfollowedbygraftingtechnique(17).Theresults wereinconclusiveforoxytetracyclinebecauseitwas phytotoxicatthehighrateandthegraftswereunsuccessful.Thecombinationofpenicillinandstreptomycin successfullyreducedthenumberofbacteriatoalow, butdetectablelevel,buthowthiscomparedwithoxytetracyclinewasnotdetermined,norwastheefficacyof theindividualcompounds.Thisstudydemonstratedthat kasugamycinwasineffectiveagainstCLas.InastudyexaminingthesetreatmentsappliedtothefoliageofperiwinkleplantsinfectedwithCLas,bothoxytetracycline andthecombinationtreatmentreducedtheamountof thebacteriatoastatisticallysimilarlevel(18).Penicillin doesnotcurrentlyhaveanyregistereduseinplantagri- culture.Thetimelineforregisteringthisnewactiveingredientwouldbemanyyearsaswithanynewactive ingredient.This,alongwiththehealthrisksandlackof evidenceofsuperioritytootherchemicalscurrently usedinplantagriculture,suggestthatpenicillinisnota viabletreatmentforHLB. Discussion Despiteshowingsomeefficacy,oxytetracyclinewasnot widelyusedinthepasttotreatHLB.Thisispartiallybecauseofthephytotoxicityofearlyformulationsandalso becausethemostefficientmethodforintroducingthe materialintotheplantwasbytrunkinjection.Whileeffectivetosomedegree,trunkinjectionhistoricallyhas notbeenfoundtobecosteffectivefortreatingmany acresofcitrustrees.AuthorsofareviewofcitrusgreeninginSouthAfricaexplainedthattrunkinjectionsoftetracyclineinSouthAfricadidnotbecomecommonbecauseofphytotoxicity,residuesinthefruit,production ofsmallfruitandbecausediseasesuppressionwasnot sustained(19).Anadditionalexplanationfornotrelying heavilyonantibioticsthatwasnotdiscussedinthereviewwasthattheLiberibacterspeciesfoundinAfrica (CandidatusLiberibacterafricanus),alongwiththevectorpsyllidspecies,haveagreatersensitivitytoheat thanCLasandACP,andshiftingtheindustrytoamore tropicalregioninSouthAfricahasreducedthedisease incidence.Thismaybewhymethodsfortrunkinjection treatmentswereneveroptimized.InothercitrusgrowingregionseffectedbyHLB,relocatingthecitrusproductiontoareaswithperiodsofcoolerand/ordrier weatherthatallowforadormancyperiod,orgeographicallyisolatedareas,havehelpedtoreducethepsyllid pressureandslowthespreadofthedisease. Nothavingtheoptionsavailabletootherregionswith thisdisease,theFloridacitrusindustryhasreliedon otheraspectsofcitruscultureincludingnutrition,soil chemistryandpruninginordertomanageHLB.While thesemethodscancontributetokeepingtreesineconomicalproduction,theydonotreducetheamountof infectiononceatreedevelopsthedisease.Goodpsyllid controlwillalwaysbeanimportantaspectofdisease management.Psyllidcontrolwillonlyhelptoprevent newinfections,butifachemicalisfoundtosuppress thebacteriainthetree,psyllidcontrolwillbeimportant tomaintainthissuppression.Fewmethodsareavailable tosuppressoreliminatethebacteriuminthetree.Heat 3 therapyisoneoftheearliestmethodsusedinthehistoryofplantpathologytoeliminatepathogensfromplant material.Thismethodhasbeenadaptedforlarger-scale useincitrusgrovesandprovidessomediseasesuppression,butisstillinexperimentaltrials. Recently,industryinteresthasgrownforthepotential useofantibioticstotreatHLB.ThisisbasedonpastresearchandalsofromtheSection18foremergencyuse ofstreptomycinsulfatetotreatanotherimportantdiseaseincitrus,citruscanker(Xanthomonascitrisubsp. citri).Fromthisinterest,severalstudiesoftheefficacy offoliarapplicationsofoxytetracyclineandstreptomycinsulfateagainstHLBhavebeenfundedbyCRDFsince mid-2014.Resultsfromthesestudies,alongwithresults producedindependentlybytheregistrants,arepartof the2015petitiontoEPAforaSection18emergency exemptionallowinguseofthesematerialsoncitrusin Florida.Thedecisiononthispetitionisforthcoming.ApprovaloftheSection18petitionwillallowgrowersto applyfoliarapplicationsofoxytetracyclineandstreptomycinsulfate. Foliarapplicationisthemostcommonmethodofapplyingpesticidestocitrusgroves.Thisisamainreasonwhy theresearchfortheSection18applicationhasinitially focusedonthismethod.Adjuvantsareavailablethat increasetheabilityofthebactericidestomovethrough theleafsurface.However,theremaybemoreefficient methodstointroducethematerialintothetreeand haveagreaterimpactonthebacterialpopulationinthe phloem.Trunkinjectionisanexampleofanalternative method.Trunkinjectionmaybeeconomicallyviableif equipmentandmethodsweretobedevelopedtoallow growerstoinjectalargenumberoftreesinashortperiodoftimesimilartohowinjectionsareconductedto preventinfectionofavocadobythelaurelwiltpathogen insouthernFlorida.Aproof-of-conceptexperimentis currentlyinplace,fundedbyCRDF,toevaluatetheeffectoftrunkinjectionsofoxytetracyclineandstreptomycinontreehealthandfruitquality.Ifsuccessful,this studymayleadtothedevelopmentofcost-effective injectiontechniquesforgrowers.Althoughtheworkhas beeninitiatedtomakethistechniqueanoptionfor growers,expansionofcurrentlabelswillberequiredto includetrunkapplication. Theuseofchemicalsalonetocontrolplantdiseasesis notsustainable,butastopgapsolution.Whenconfrontinganepidemic,chemicalcontrolisoftenthefirstline ofdefense,followedbythedevelopmentofresistant plants.Citrusplantbreedersareworkingtowardsdevelopingtolerantandresistantcitrusvarietiestosustain theindustryoverthelongterm,butuntilthesevarieties areavailable,short-termsolutionsarenecessary.The useofoxytetracyclineandstreptomycinwitheffective adjuvantsmaysuppressthebacteriainthetree,allowingthetreetoproduceastablecropofadequatequantityandqualitytosustaincurrentproduction.Further advancesinapplication,coupledwithadditionalbactericides,maybeavitalsteptowardsmanagingtheHLB epidemicinFlorida. References 1. HodgesAW,SpreenTH.(2012).Economicimpactsof citrusgreening(HLB)inFlorida2006/07-2010/11. ElectronicDataInformationSource(EDIS)FE802. UniversityofFlorida,Gainesville,FL. http://edis.ifas.ufl.edu/fe903. 2. Http://www.nass.usda.gov,UnitedStatesGovernmentAccountabilityOffice(2011) 3. Martinez,A.L.,Nora,D.M.,&Armedilla,A.L. (1970).Suppressionofsymptomsofcitrusgreening diseaseinthePhilippinesbytreatmentwithtetracyclineantibiotics.PlantDiseaseReporter,54,1007-9. 4. Martinez,A.L.(1975).Responseoftheetiologic agentofcitrusgreeningdiseaseinthePhilippinesto treatmentwithbroadspectrumantibiotics.PhilippinePhytopathology,11,58-61 5. Aubert,B.,&Bové,J.M.(1980).Effectofpenicillin ortetracyclineinjectionsofcitrustreesaffectedby greeningdiseaseunderfieldconditionsinReunion Island.InProceedingsoftheconferenceInternationalOrganizationofCitrusVirologists. 6. Bové,J.M.,Bonnet,P.,Garnier,M.,&Aubert,B. (1980).Penicillinandtetracyclinetreatmentsof greeningdisease-affectedcitrusplantsintheglasshouse,andthebacterialnatureoftheprocaryote associatedwithgreening[Spiroplasmacitri].InProceedingsoftheconferenceInternationalOrganizationofCitrusVirologists. 4 7. Buitendag,C.H.,&Bronkhorst,G.J.(1983).MicroinjectionofcitrustreeswithN-pyrrolidinomethyl tetracycline(PMT)forthecontrolofgreeningdisease.CitrusandSubtropicalFruitJournal,592,8-10. 8. Capoor,S.P.,Thirumalachar,M.J.,Pandey,P.K.,& Chakraborty,N.K.(1974).Controlofthegreening diseaseofcitrusbyBP-101:anewchemotherapeutant,27,50-52. 9. Cheema,S.S.,Kapur,S.P.,&Bansal,R.D.(1985). Efficacyofvarioustherapeuticagentsagainstgreeningdiseaseofcitrus.JournalofResearch-PunjabAgriculturalUniversity(India). 10. Kapur,S.,Kapur,S.P.,&Kang,S.S.(1996).Chemotrunkinjectionforthecontrolofcitrusgreening.IndianJournalofVirology,12,55-57. 11. Nariani,T.K.,Raychaudhuri,S.P.,&Viswanath,S. M.(1971).Responseofgreeningpathogenofcitrus tocertaintetracyclineantibiotics.CurSci. 12. Schwarz,R.E.,&VanVuuren,S.P.(1971).Decrease infruitgreeningofsweetorangebytrunkinjection oftetracyclines.PlantDiseaseReporter. 13. Schwarz,R.E.,Moll,J.N.,&VanVuuren,S.P. (1974).Controlofcitrusgreeninganditspsyllavectorbytrunkinjectionsoftetracyclinesandinsecticides.InProceedingsofthe6thConferenceofthe InternationalOrganizationofCitrusVirologists(pp. 26-29).Riverside:Univ.Calif. 14. Tourabally,D.,Lallmahomed,G.M.(1977).Tetracyclinetreatmentforcitrusgreeningdisease.Annual Reportfortheyear1974,38-39 15. VanVuuren,S.P.(1977).Determinationofoptimal concentrationandpHoftetracyclinehydrochloride fortrunkinjectionofgreeninginfectedcitrustrees. Phytophylactica. 16. VanVuuren,S.P.,Moll,J.N.,&DaGraca,J.V. (1977).Preliminaryreportonextendedtreatmentof citrusgreeningwithtetracyclinehydrochlorideby trunkinjection[Bacterium-likeorganisms].Plant DiseaseReporter(USA). 17. Zhang,M.,Powell,C.A.,Guo,Y.,Doud,M.S.,& Duan,Y.(2012).Agraft-basedchemotherapymethodforscreeningeffectivemoleculesandrescuing huanglongbing-affectedcitrusplants.Phytopathology,102(6),567-574. 18. Zhang,M.,Powell,C.A.,Zhou,L.,He,Z.,Stover,E., &Duan,Y.(2011).Chemicalcompoundseffective againstthecitrushuanglongbingbacterium'CandidatusLiberibacterasiaticus'inplanta.Phytopathology,101(9),1097-1103. 19. Buitendag,C.H.,&VonBroembsen,L.A.(1993). LivingwithcitrusgreeninginSouthAfrica.InProc. 12thConferenceoftheInternationalOrganizationof CitrusVirologists.UniversityofCalifornia,Riverside (pp.269-273). OtherRelevantLiterature Stockwell,V.O.,&Duffy,B.(2012).Useofantibioticsinplantagriculture. McManus,P.S.(2014).Doesadropinthebucket makeasplash?Assessingtheimpactofantibiotic useonplants.Currentopinioninmicrobiology,19, 76-82. McManus,P.S.,Stockwell,V.O.,Sundin,G.W.,& Jones,A.L.(2002).Antibioticuseinplantagriculture.AnnualReviewofPhytopathology,40(1),443465. daGraça,J.V.,Douhan,G.W.,Halbert,S.E.,Keremane,M.L.,Lee,R.F.,Vidalakis,G.,&Zhao,H.(2015). Huanglongbing:Anoverviewofacomplexpathosystemravagingtheworld'scitrus.Journalofintegrativeplantbiology. 5