Streptomyces

JournaloyEnvironmentalSciencesVo1.17.No.1.PP.123—125,2005[SSNl00l—o742

;CNI1—262q,X

;ArtideID:1001—0742(2005)01—0123—03CLCnumber:X17Documentcode:A ;Streptomycesavermitilisfrommarine

;XIONGLi—xia,LIJian—zhong,WANGHui—li

;fResearchCenterforEco—EnvironmentalSciences,ChineseAcademyofSciences,Beijing100085,C

hina.E-mail:1ook99@263.net)

;Abstract:Theinsecticidalstrain173wasisolatedfrommarinesourceanditsactivitywasexploredbytheb

ioassayofbdneshrimpand

;Helicoverpaarmigera.Basedonmorphological,physiologicalandmolecularproperties,theinsecticid

alstrain173wasidentifiedas

;Streptomycetesavermitilis,whichisthebestinsecticidalmicroorganismfoundintheterrestrialenviron

ment.Thetaxonomyofthestrain

;173,insecticidalspectrumandpropertiesofthecorrespondinginsecticidalantibioticsarereported.

;Keywords:insecticidalmicroorganism;marine;taxonomy;Streptomycesavermitilis

;Introduction

;Theintensiveuseofchemicalpesticidesisoneofthe ;factorscontributingtohighcropyieldsandlowercommodity ;prices.Itisestimatedthat2.5milliontonsofpesticidesare ;appliedtoagriculturalcropsworldwideannually.However, ;inmostcases,theamountofpesticidecomingindirect ;contactwithtargetpestsisanextremelysmallpercentageof ;theamountapplied,usuallylessthan0.3%,andthus ;99.7%pesticideswent”somewhereelse”intheenvironment

;(Pimentel,1995).Contaminationbychemicalpesticideshas ;affectedthequalityofsoil,waterandairand,consequently, ;mayposearisktohuman,floraandfauna(Laura,2004). ;Fortunately,biologicalpesticidesaredistinguishedfrom ;conventionalchemicalpesticidesbytheirnaturaloccurrence, ;non—toxicmodeofactiontothetargetpests,safetyforhuman ;andothernon—targetorganisms,reductionofpesticide

;residuesinfoodandenvironment,preservationofother ;naturalenemies,andincreasedbiodiversityinmanaged ;ecosystems(Mike,2000;Lacey,2001).

;Thepasttwodecadeshaveseensignificantresearch ;interestinattemptstodiscoveranddevelopenvironmental ;friendlypesticidesfromnatureorigin(Prem,2000).Sofar, ;mostoftheinsecticidalmicroorganismshavebeenisolated ;fromentomopathogensandterrestrialenvironment(Leonard,

;2000).Oceanisahugedrugresource,nearly6000kindsof ;novelchemicalcompoundshavebeenidentifiedfrommarine ;livingorganisms,amongwhichabout10000compoundsexert ;biologicallyactivities,suchasanti—tumor,anti—microbeand

;anti-virus,etc.(Fenical,1993;Davidson,l995). ;However,themarineenvironmentisstillrarelyexploredfor ;thedevelopmentofbiologicalpesticidesandlittleinformation ;aboutmarinemicroorganismspossessinginsecticidalactivities ;hasbeenreported(Takahashi,1989;Hu,2000).

;Inthecourseofscreeningfornewinsecticidal

;metabolitesfrommarinemicroorganisms,thestrain173was ;foundtohavestronginsecticidalactivityagainstbothbrine ;shrimpandHelicoverpaarmigra,aswepreviouslyreported ;(Xiong,2004).Inthispaper,thetaxonomyofthestrain ;173,insecticidalspectrumandpropertiesofthe ;correspondinginsecticidalantibioticsarereportedinthis ;paper.

;1Materialsandmethods

;1.1Microorganism

;Thestrain173wasisolatedandmaintainedaspreviously ;described(Xiong,2004).

;1.2Taxonomy

;Theinsecticidalstrain173wascharacterizedbyits ;morphologicalandphysiologicalproperties(Yan,1975),as ;wellas16SrDNAsequencing.ExtractionofgenomicDNA, ;PCRamplificationof16SrDNAandpurificationofthePCR ;productswasperformedfollowingtheprocedureofChun& ;Goodfellow(Chun,1995),Kim(Kim,1996)andLu(Lu, ;2001)respectively.The16SrDNAofthestrainwasaligned ;manuallywiththeavailableStreptomycesnucleotidesequence ;thatretrievedfromEMBL/GenebankandRDP(Ribosomal ;Databaseeroject;Maidak,1997)usingClustal*1.8 ;program(Thomposon,1997).

;1.3Laboratorybioassayofsomeimportantinsectpests ;Helicoverpaarm@era,Plutella”lostllaL.,aphidsand

;Spodopteraex@Ha,whichareseriouspestsofmanyimportant ;cropsandclaimmajorshareincroplossesevery)’ear,are

;usedasthetestpests.

;Testpests:3-instarlarvaeofHelicoverpaarmigera, ;PlutellaxylostllaL.,aphidsand5podopteraex@ua; ;Positivecontrol:1ppmand10ppmavermectinB1.

;;Test

;solution:thesupernatantoffermentationbrothofthestrain ;173;Thebioassaymethodwasthesameaspreviously

;described(Xiong,2004).

;1.4Propertiesoftheinsecticidalsubstancesofthe ;strain

;

;l24X10NGLi.xiaetalVo1.17

;ThepHofelevenportionsofthefermentationbrothwere ;adiustedto2,3,4,5,6,7,8,9,10,11and12

;respectivelyforthetestofpHsensitivity.Thefermentation ;brothwereheatedat40oC,60oC,80oC,100oC,121cI二for

;30rninrespectivelyforthetestofthermo-stability.The ;insecticidalactivitiesforthesamplesobtainedfromtheabove ;treatmentswereexaminedbybrineshrimpbioassay(Xiong, ;2004).

;1.5HPLCanalysis

;Thesupernatantandmyceliaoffermentationbrothwere ;extractedrespectivelywithethylacetateforthreetimes.The ;ethylacetateextractsofthesupernatantandthemyceliawere ;concentratedunderdiminishedpressuretodryness.,rIletwo ;residuesweredissolvedinmethano1.TheavermectinBl. ;(92%)wasusedasauthenticsample.HPLCanalysiswas ;carriedoutonShimadzuLC-3Aaccordingtothemethodof ;Thomas(Thomas,1979).

;2Results

;2.1Taxonomicstudiesofthestrain173

;Theculturalandphysiologicalcharacteristicsofstrain ;173grownonvariousmediaat2oCfor14daleshownin ;Table1andTable2respectively.Theorganismisaerobic, ;mesophilicandgram-positive.Thesporechainswere ;rectiflexibilesandsporesweresubsperiealtoovalinshape ;andsmooth.

;Table1Culturold嵋racteristiesofstrainl73

;Table2Physiologicalpropertiesofstrain173The16SrDNAsequenceofthestrain173showed100%

;similaritytothatofthetypestrainStreptomycesavermitilis ;andthestIain173wasidentifiedasStreptomycesavermitilis? ;2.2Bioassayofsomeimportantinsectpests

;ThebioassayresultsareshowninTable3.Thestrain ;173sho,vedstrongactivityagainstHeliothisarmigeralarvae, ;PlutellaxylostllaL.,Spodopteraexiguaandaphids.The ;inseeticidalactivityoffermentationbrothofthestrain173was ;similartothatof10ppmavermeetinBlinviewofthedeath ;timeandmortality,indicatingthatthestrain173isavery ;promisingmarineinsecticidalStreptomycesavermitilis. ;Table3Labbioas~yof3-instarlarvae

;ltellcoverpa

;arrmgera

;Plutella

;ylostllaL

;Spodoptera

;extgua

;Aphids

;1ppmavermectin ;10ppmavermectin ;Strain173

;18.O5

;76.39

;84.72

;15.27

;76.38

;79.16

;9.73

;40.27

;66.66

;44.44

;93.O5

;70.82

;20.83

;79.17

;87.50

;18.O5

;79.16

;81.94

;l2.5l

;43.O5

;69.44

;47.22

;95.83

;73.60

;40.27

;81.94

;90.27 ;44.44 ;91.66 ;84.7l ;22.22 ;51.39 ;79.16 ;90.27 ;94.44 ;90.27 ;45.83 ;87.5O ;95.83 ;50.0o ;97.22 ;90.27 ;27.78 ;56.95 ;84.72 ;95.83 ;l0o

;95.83 ;79.17 ;91.67 ;91.67 ;81.95 ;91.67 ;90.28 ;37.5 ;69.48 ;83.34 ;91.67 ;91.67 ;91.67 ;87.5O ;1O0

;1O0

;90.28 ;1o0

;9861 ;45.83 ;77.81 ;91.67 ;l0o

;J00

;l0o

;

;No.1Streptomycesavermitilisfrommarine125

;2.3Propertiesoftheir~ecticidalantibioticsofthe ;strain

;Theinsecticidalsubstancesshowedagoodstabilityina ;broadpHrangefrom3to12,andgoodthermo-stabilityfrom ;40~Cto80~(2,sotheyalegoodcandidatesforindustrial ;applications.

;2.4HPLCresults

;HPLCanalysisindicatedthatboththesupernatantand ;myceliaoffermentationbrothcontainavermectinBl.Thisis ;differentfromtherelatedreport(Richard,1979)thatclaimed ;avermectinBlmainlyexistsinmycelia.Itiswellknownthat ;avermectinBl,themostpowerfulavermectin,isbroadly ;effectiveagainstmostimportantagriculturalpests(Mrozik, ;1989).

;Bycomparisonwiththetaxonomicresultsof

;Streptomycesavermitilis(Richard,1979),itwasfoundthat ;thereweremanyobviousdifferencesbetweenthemarine ;insecticidalstrain173andStreptomycesavermitilisfrom ;terrestrialenvironment,suchasmorphologyofsporophores, ;solublepigment,utilizationofraffinoseandSUCroSe,andH2S ;productionetc.Itcanbededucedthatmanydissimilarities ;canbemadebytheStreptomycesavermitilisfrommarineand ;theStreptomycesavermitilisfromterrestrialenvironment. ;Avermectins,isolatedoriginallyfromtheculturebroth ;ofStreptomycesavermitilisfromsoil,areaseriesofunique ;16-memberedmacrolides(Egfrton,1979).Owingtohigh ;efficiency,broadspectrum(Mrozik,1989),lowresidue ;(Forbes,1993),rapiddegradationintheenvironment ;(Bruce,1993)andsafefornon-targetorganisms(Payne, ;1993),theavermectinnaturalproductsarethebest ;biologicalpesticideeverfoundandtheycanreplacethe ;highlytoxicchemicals.Thus,theproducingstrain173from ;marinesourceisaverypromisingandlesspollutedbio- ;pesticidewithsignificantimpo~anceforenvironmental ;protection.

;References:

;BruceAH,WilliamJA.VandenHeta1..1993.EnvironmentaleffeCIsofthe ;usageofavermectinsinlivestock【Jj.VetParasitol,48:l09—125

;ChunJ,GoodfellowM.1995.AphylogeneticanalysisofthegenusNoeardiawith

;16SrRNAgenesequence[Jj.IntJSystBacteriol,49:l395—1402.

;DavidsonBS,1995.Newdimensionsinnaturalproductsresea~h:cultured ;marinemicroorganismslJJ.CurtOpinBiotech,(6):284—291.

;EgfrtonJR,OstlindDA,BlairLSeta1.,1979.Avermectins,newfamilyof ;potentanth-elmintie8genL~:efficacyoftheB1.component[J].InIJSyst ;Baeteriol,15(3):372—378.

;FeniealW,1993.ChemicalstudiesofInarinebacteria:developinganewresource ;[J].ChemRev,93:1673--1683.

;ForbesB,1993.Areviewofregionalandtemporaluseofavermeetinsincattle ;andhorsesworldwide[J].VetParasitol,48:l9—28.

;HuZY,LiouSZ,HuangYJeta1.,2000.Arapidscreeningassayfor ;insecticidalantibioticsproducedbymarineactinomycetes[J].MarsciBuu, ;19(4):36—41.

;KimD,ChunJ,SahinNeta1.,1996.Aanlysisofthermophilieeladeswithinthe ;genusStreptomyeesby16SribosomalDNAsequencecomparisons[J].IntJ ;SystBaeleriol,46:581--587.

;LaeeyLA,FrutosR,KayaHKeta1.,2001.Insectpathogensasbiological ;controlagents:dotheyhaveafuture?【J].BioloControl,21:23(}’--248.

;LaumP,MarcoT,EttoreC,2004.Acalculationproceduretoas$~spotential ;envirenmentalriskofpesticidesatthefarmlevel【J].EcolIndicat,4:

;Ill一123.

;LeonardGC,JuliusJM,2O00.Reviewbio.pesticides:areviewoftheiraction, ;applicationsandefficacy【Jj.PestManageSci,56:65l一676.

;LuzH,LiuzH,WangLMeta1.,2001.S~charopolyspora伽msp.nov.

;andSaccharopolysporathermophilasp.nov.,novelaetinomycetesfromsoil ;[Jj.IntJ0fSystEnvolBaeteriol,5l:3l9—325.

;MaidakBL,OlsenGL,Lal~nNeta1.,1997.Theribosomealdatabasepmject ;(RDP)[J].NueleicAcidsRes,25:l09一Il1.

;MikeN,2000.neregulationofnaturalproductsascroppmteetiortagentS[J]. ;PestManageSci,56:677—680.

;MrozikH,EskolaP,LinnB0eta1.,1989.Discoveryofnovelavermectilmwith ;unprecedentedinsecticidalactivity[J].Experientia,45:3l5—3l6.

;PayneGT,SoderlundDM,1993.Actionsofavermectinanalogson7- ;aminobutyrieacid(GABA)一sensitiveandGABA—insensitivechloridechannels

;inmousebrain【JJ.PestleBiochemPhys,47:l78—184.

;PimentelD,1995.Amountsofpesticidesreachingtargetpests:environmental ;impactsandethics【J].JAsrieEnvironEthics,8:l7—29.

;PremW,2000.Livingsystemsasnaturalcropprotectionagents[J].PestManage ;Sci.56:68l一687

;RichardWB,BrintonMM,EdwardEB,1979.Avermectins,newfamilyof ;potentanthelmintieagents:producingorganismandfermentation[J]. ;AntimicrobAgentsChemother,15:36l一367

;TakahashiA,KurasawaS,IkedaD,1989.Ahemicidin,Anewacarieidaland ;anti—tumorsustaneeI.Taxonomy,fermentation.isolationandphysical—

;chemicalandbiologicalpropeaies[J].JAntibiotics,42:1556--1561.

;ThomasWM,LousC,DouglasJCeta1.,1979.Avermectins.newfamilyof ;potentanthelminticagents:isolationandchromatographicprope~ies[J]. ;AntimicrobAgentsChemother,15:368—37l

;ThompsonJD,GibsonTJ,P1ewniakFeta1.,1997TheClustalXwindows ;interface:flexiblestrategiesformultiplesequencealignmentaidedbyquality ;analysistools[J].NucleicAcidRes,25:4876--4882.

;XiongLX,LiJZ,KongFZ,2004.Streptom)sp.173,aninsecticidal ;microorganismfrommarine【JJ.LettApplMicrobiol,38:32—37.

;YanXC,1975.IdentificationhandbookofStreptomyces[M].Beijing:Science ;Press.

;(ReceivedforreviewMarch31,2004.AcceptedMay14,2OO4)

;

DOCSFORD

Streptomyces