苏云金杆菌BT毒素及其应用

苏云金杆菌（BT）毒素及其应用,1WhatisBacillusthuringiensis,1苏云金杆菌是什么？,Arod-shaped(1.0-1.2by3-5micron),gram-positive,facultativeanaerobic,sporeformingbacterium,杆状，大小为1.0-1.23-5m，革兰氏阳性，兼性嫌氧，产孢的细菌,菌体照片,Afacultativepathogenofsomeinsectspeciescommonlyusedasabiopesticide.,一些昆虫的兼性病原物，常用作生物杀虫剂。,Morethan150insectsbelongingtotheordersLepidoptera,Diptera,andColeopteraareknowntobesusceptibletoBt.Tobeeffective,Btmustbeeatenbyinsectsduringtheirfeedingstageofdevelopment,whentheyarelarvae.Btisineffectiveagainstadultinsects.,150种以上属于鳞翅目、双翅目、和鞘翅目的昆虫易感染Bt。Bt要昆虫幼虫取食后才会发生效力，对成虫无效。,Duringsporulation,itproducesproteininclusions(parasporalinclusions)adjacenttotheendospore,产芽孢期间，BT在邻近芽孢处形成蛋白内含体。,ElectronMicrographofaSporulatingBtCell,产孢BT菌体的电子显微照片,Theparasporalinclusionsconsistofoneormoreinsecticidalproteinsintheformofacrystalorcrystal-complex.TheseinsecticidalproteinsarecommonlyknownasInsecticidalCrystalProteins(ICP)ordeltaendotoxin.TheprimaryinsecticidalactivityofBtisduetoICP.Itistheactiveingredientofmost(90%)ofthemicrobialinsecticidesproducedintheworld.,这种类孢内含体由1-2个蛋白晶体或晶体复合物组成。这些杀虫蛋白通称为杀虫晶体蛋白（ICP）或-蛋白。BT的主要杀虫活性是由于ICP。是世界上生产的大多数微生物杀虫剂（90%）的活性成分。,Theendospore,highlyresistanttoenvironmentalstress,providesamechanismforlong-termsurvivalofBt.,内孢子高抗环境压力，提供了BT长期生存的机制。,芽孢,BesidesICPandendospore,someBtsubspeciesproduceBetaExotoxinwhichistoxictoallformsoflifeincludinghumans,除ICP和内孢子（即芽孢）外，有些BT还产生外毒素，这些毒素对所有生物包括人类都有毒害作用。,Aninsectmusthaveastronglyalkalinemid-gut,specificproteolyticenzymesandsuitabletissuereceptor-sites,tobesusceptible.,BT要起杀虫作用，必须满足3个条件，一是昆虫中肠液呈碱性；二是有特定的蛋白酶，三是有适合的组织受体位点。,Btcanbeproducedeasilyonsolidmediaorundersubmergedfermentationconditions-akeyfactorinitssuccessfuldevelopmentasabiopesticide.,BT容易在固体培养料上生产，也可以液体发酵生产，后一性能是作为杀虫剂成功开发的关键因子。,BiotechnologyandadvancesinthefermentationindustryoverthepastthreedecadeshaveimprovedthequalityofBtproducts.,过去30年在发酵工业和生物技术上取得的进展促进了BT产品的质量,Btiskeysourceofgenesfortransgenicexpressiontoprovidepestresistanceinplants.,BT是转基因表达，为植物提供抗虫性的关键基因来源。,转基因玉米,转基因棉花生测,2History,2研发历史,DiscoveredbyDr.Ishiwatari(aJapanesebacteriologist)in1901insilkwormfarmsasthecausalagentofthesottobacillusdisease.,日本细菌学家Ishiwatari博士1901年于一个蚕场发现BT是蚕sotto芽孢菌病的病原。,蚕,Insecticidalactivitywasdiscoveredin1911byBerliner(Germany)HenameditBacillusthuringiensis,aftertheGermantownThuringiawherethemothwasfound.,德国的Berliner于1911年发现BT有杀虫（地中海粉蛾）活性。并根据发现地德国小镇Thuringia命名。,ExistanceofparasporalinclusionsinBtwasnotedin1915byBerliner.,Berliner还于1915年发现BT类似于孢子的内含体的存在。,Firsttimeusedasinsecticidesinlate1920s,1920年代后期首次用作杀虫剂。,Spore-basedformulationswerecommerciallyavailableinlate1950s,1950年代后期市场上有孢子态的制剂销售。,FirstregisteredasapesticidesintheU.S.in1961,1961年在美国作为杀虫剂首次登记。,In1960s,experimentswithspore-basedformulationsproducederratic(inconsistent)results,1960年代，用孢子态制剂做的试验得到的药效结果不一。,Bythe1970sitwasdemonstratedthattheprimaryinsecticidalactivityagainstlepidoteraninsectswasduetodelta(d)endotoxins,到1970年代才明白对鳞翅目害虫的主要杀虫活性是由于-毒素。,Until1977,only13Btsubspecieshadbeendescribed;allweretoxictolepidopteranlarvaeonly.,1977年以前只发现13个亚种，而且这些亚种都只对鳞翅目害虫有效。,Discoveryofsubspeciestoxictodipteranandcoleopteraninsectsin1977and1983,respectively.,1977年和1983年分别发现BT亚种对双翅目和鞘翅目的毒性。,3NaturalHabitat,天然生境,Plantsurfaces-Bthasbeenfoundextensivelyinthephylloplane.Numeroussubspecieshavebeenisolatedfromconiferoustrees,deciduoustrees,andvegetables.Ithasalsobeenrecoveredfromstoredproducts.,3.1植物表面-BT在叶表广泛发现。从针叶树、落叶树和蔬菜上分离了许多亚种。也从储藏的产品分离到。,Soil-sporespersistinsoil,andvegetativegrowthoccurswhennutrientsareavailable,3.2土壤-孢子在土壤中生存，当有营养物质时进入营养生长。,InsectHosts-variousBtsubspecieshavebeenisolatedfromdeadordyinginsectlarvae.,3.3昆虫寄主，从死虫或干虫分离到了各种各样的BT亚种。,EnvironmentalFate,4在环境中的命运,Forlepidopteranandcoleopteranpests,Btisappliedtothesurfacesoftheplantsandfordipteranpests(mosquitoesandblackflies)appliedtotheiraquatic,larvalhabitats.ManyBtinsecticidesshowpoorstabilityunderfieldconditions,andsofrequentreapplicationisrequired.,防治鳞翅目和鞘翅目的害虫，BT施在植物表面，防治双翅目害虫（蚊蝇类），施在幼虫生活的生境即水体中。许多BT杀虫剂在田间条件下稳定性差，需要反复施用。,A.TerrestrialHabitatPlantSurfaces(Phylloplanes)andSoil-Coleopteran-activeandLepidopteran-activesubspecies,A.陆生习性（植物表面和土壤）-对鞘翅目有活性的和对鳞翅目有活性的亚种。,BtICPsaredegradedquicklybysolarradiation,在阳光照射下BTICP快速降解,BtEndosporesareinactivatedrapidlywhenexposedtoUVradiation,当暴露在紫外线下时BT芽孢迅速失活。,Half-life:afewhours-10daysinmostagriculturalcrops,半衰期即活性降低一半的时间，只有几小时-在大多数农作物上10天。,Vegetativecellsandsporesmaypersistatgraduallydecreasingconcentrationsforweeks,months,oryearsasacomponentofthenaturalmicroflora.,营养菌体和孢子可作为天然微生物区系的成员生存数周、数月、数年，但菌量逐渐下降。,B.AquaticHabitats-Dipteran-activesubspecies,B.水生习性对双翅目有活性的亚种,Rapidsedimentationinallbutthefastestflowingstream,除非在快速气流中，能迅速沉降。,Sporesmaypersistforatleast22daysinsediments,孢子在沉积物中至少可存活22天。,ContactofBtsubspeciesisraelensiswithmudresultinanimmediatedisappearanceoflarvicidalactivity,BT的以色列亚种与泥浆接触时杀幼虫活性很快丧失。,4ClassificationofBtSubspecies,4BT亚种的分类,Basedontheserotype(serologicalanalysisofflagellaantigens)supplementedbymorphologicalandbiochemicalcriteria.,分亚种依据：根据血清型（对鞭毛抗原的血清学分析）结合形态特征和生化特征,Over67subspecieshavebeenidentified;subspeciescommonlyusedinagriculture/publichealth,67种以上的亚种已鉴定出来；亚种常用于农业和公共卫生杀虫。,Btsubspecieskurstaki(Btk)-controlsvarioustypesoflepidopterousinsects,BT的库期塔克亚种防治各种鳞翅目害虫,Btsubspeciesisraelensis(Bti)-effectiveagainstmosquitoes,blackflies.,BT的以色列亚种对蚊子、黑蝇有效。,Btsubspeciestenebrionis(alsoformerlyassubsp.sandiego)(Btte)-effectiveagainstcertainbeetle(chrysomelids)speciesandthebollweevil.,BT的拟步甲亚种（曾用名圣地亚哥亚种）对某些甲虫（叶甲）和棉铃象甲有效。,BtsubspeciesJaponensis(Btj)-effectiveagainstmanyspeciesofscarabidbeetles,BT的日本亚种对某些金龟子有效。,Btsubspeciesaizawai(Bta)-usedagainstwaxmothlarvaeinhoneycombs,BT的aizawai亚种用来防治蜂巢中的蜡蛾幼虫。,Pathogenicity/Toxicity致病性/毒性,ToxicityisduetoICP,endosporeorBetaExotoxin毒性是由于ICP、内孢子或-外毒素。InsecticidalCrystalProteins(ICP)杀虫晶体蛋白（ICP）,Alsocalledasdelta-endotoxin.Theyareprotoxin(itmustbeactivatedbeforeithasanyeffect).,ICP也称-内毒素，是原蛋白（必须要活化才有效）。,CommonlydesignatedasCryproteinsencodedbycrygenes.,ICP通常指名为Cry蛋白，由cry基因编码。,ThegenesthatencodeICParemostlyonplasmids;eachICPistheproductofasinglegene.,编码ICP的基因主要在质粒上；每种ICP是单个基因的产物。,AsubspeciescansynthesizemorethatonetypeofICP.,一个亚种可合成一种以上的ICP。,ICPshavevariousforms(bipyramidal,cuboidal,flaterhomboid,oracompositewithtwoormorecrystaltypes).,ICP有各种各样的晶形，如双金字塔形，扁菱形，或兼有两种或两种以上的晶体形状。,菱形晶体,Size:Protoxin-Alargeproteinofupto230kiloDaltons(kDa),ActiveToxin-65kDa.,大小：原毒素是一种大蛋白，达到230kDa。活性蛋白为65kDa。,Extremelypotentandtoxictotargetinsectlarvaeatpicomoleconcentrations,极毒，在pmloe浓度对靶标幼虫有毒。,MostsusceptiblespeciesbelongtotheordersLepidoptera,Diptera,andColeoptera.,大多数感染的虫种属于鳞翅目、双翅目和鞘翅目。,Mostsusceptibleinsectsarekilledbyingestionofthecrystalsalone;amixtureofsporesandcrystalsarerequiredforatoxiceffectinonlyasmallnumberofinsects.,大多数易感虫种只要摄取晶体蛋白即可被杀死；只有一小部分昆虫要求孢子和晶体同时存在。,Cryproteinsareclassifiedaccordingtotheirinsectspecificity(Hostspectrumisdesignatedinromannumerals):,Cry蛋白根据对昆虫的专化性分类（寄主范围以罗马数字表示）,CryILepidoptera（鳞翅目）-specificCryIILepidoptera-andDiptera（双翅目）-specificCryIIIColeoptera（鞘翅目）-specificCryIVDiptera-specificCryVColeoptera-andLepidoptera-specific,AmixtureofdifferentCrytoxinsresultsinasynergismofinsecticidalactivity.,将不同Cry毒素混合导致增效杀虫作用。,AmixtureofCrytoxinsandBtsporesresultsinasynergismofinsecticidalactivityinseveralinsectpests,Cry毒素与BT孢子混合在几种害虫中也导致了增效杀虫作用。,Highlyinsolubleinnormaldigestivesystemconditions-safetohumans,higheranimals,andmostinsects.,在正常消化系统状态下，毒素呈不溶状态，对人、高等动物和大多数昆虫安全。,ItissolubleinreducingconditionsofhighpH(abovepH9.5)-aconditioncommonlyfoundinthemid-gutoflepidopteranlarvae.Forthisreason,Btisahighlyspecificinsecticidalagent.,在高pH的还原条件下毒素是可溶的，在鳞翅目昆虫中肠内常具备此条件。因此BT是高度专化的杀虫剂。,ThemidgutofsusceptibleinsectscontainsthecorrectpHandenzymestotransformthecrystalintoanactivetoxin.,感染性昆虫的中肠含有合适的pH和酶，将晶体毒素转化为活化态毒素。,DifferentdomainsoftheICPareresponsibleforhostsusceptibility(receptorrecognition)andtoxicity(poreformation).,ICP的不同域负责寄主感染性（受体识别）和毒性（孔道形成形成）,内毒素的三个域,NomenclatureforcrystalproteingeneofBacillusthuringiensis(Bt)andactivityspectrumagainstinsects,ICP基因和蛋白的命名，cry表示基因，C大写表示蛋白，A表示氨基酸序列差异大，（a）表示氨基酸序列有小差异。罗马数字表示主要寄主谱。,Endospores芽孢,Endosporesareresistanttoinactivationbyheatanddesiccationandpersistintheenvironmentunderadverseconditions.Provideamechanismforlong-termsurvivalofBt.Arepathogenicforsomeinsects,particularlywhencombinedwithICP.芽孢抗热、抗干燥，在逆境中可存活，为BT提供了长期生存的机制，对某些昆虫致病，尤其是当与ICP组合时。,BetaExotoxin,外毒素,Duringvegetativegrowth,someBtsubspeciesproduceBetaExotoxin,anATPanalogue,watersoluble,heatstablesecondarymetabolite.,一些BT亚种在营养生长期间产生-外毒素，这是一种ATP类似物，水溶性，热稳定的次生代谢物。,Beta-exotoxinisaninhibitorofRNApolymeraseandactscompetitivelywithATPinvariousbiologicalprocesses.,-外毒素是RNA聚合酶的抑制剂，在各个生化过程中与ATP起竞争作用。,Itistoxictoalmostallformsoflifeincludinghumans.Ithasbroad-spectruminsecticidalactivity.,-外毒素对几乎所有生命形式包括人有毒。具有广谱杀虫活性。,ProductswithBetaExotoxinareprohibitedintheUSA.ManufacturingprocessincludesmonitoringtopreventBetaExotoxinfromappearinginproducts,用-外毒素做成的产品在美国被禁止。加工过程包括监测，以防止产品中出现-外毒素。,ModeofAction作用方式,BioactivityofBtisdominatedbytheICPsandcanbesummarizedinthefollowingstages。BT的生物活性是由ICP决定的。其作用方式如下。,1.IngestionofsporesandICPbyasusceptibleinsectlarva;感染型昆虫幼虫摄入孢子和ICP。2.ICPdissolveinthealkalinemidgut;ICP在碱性中肠中解离。3.ActivationoftheICPbyproteases;ICP被蛋白酶活化。4.IrreversiblebindingoftheC-terminaldomainofactivatedICPtospecificreceptorsinthemidgutcellmembrane;活化了的ICP的C-端区域与中肠细胞膜的专化受体不可逆结合。,5.InsertionoftheN-terminaldomainofactivatedtoxininthecellmembraneandformationofporesandchannelsinthegutmembrane,followedbydestructionofepithelialcellsandparalysisofthedigestivesystem;活化的毒素的N-端区域插入到细胞膜中，肠膜中形成孔道，接下来上皮破坏，消化系统瘫痪。6Sporegerminationandsepticemia;孢子萌发，虫体患败血病。7Larvaldeath(withinanhourtoafewdays)fromstarvationorsepticemia.幼虫因饥饿或而败血病死亡（1小时内到几天）。,EffectsonInsects对昆虫的影响,Immediatecessationoffeedingandgeneralparalysisofthelarvalmidgut1、立即停止取食，中肠瘫痪Subsequentsymptomsincludevomiting,diarrhea,generalsluggishness,andgeneralparalysis.2、其后的症状包括呕吐、腹泻、行动迟缓和瘫痪Deathmayfollowimmediatelyorinanhourtoafewdays.3、立即或1小时内到几天死亡。Sublethaleffectsmayinclude:reducedfeeding,reducedlarvalandadultlongevity,reducedfecundity,reducedlarvalandadultbodyweight.4、亚致死作用可能包括：减少取食、虫体短，体重轻。,UsingBtinIntegratedPestManagementBT在害虫综合治理中的应用,A.HostRangeLarvaeoftheinsectordersLepidoptera,Diptera,andColeoptera寄主范围鳞翅目、双翅目和鞘翅目昆虫的幼虫。,B.UseProfile使用范围,Btisthemostversatilebiopesticideforuseinpestmanagement.BT是用于害虫治理的最通用的杀虫剂。Itisusedonagriculturalcrops,harvestedcropsinstorage,ornamentals,bodiesofwater,andaroundthehometocontrolvariousgroupsofinsects,dependingonthetypeoftoxinproducedbythespecificisolateofBt.用在农作物上、储藏期产品上、观赏植物上、水体中、和家居周围，防治不同种类的昆虫，依BT特定菌株产生的毒素类别而异。Currently,over180BtproductsareregisteredinUSA.目前有180种BT产品在美国登记。,Btisprimarilyapathogenofinsectlarvalstages.Youngerlarvaearemoresusceptiblethanolderlarvae.BT主要是一种幼虫期的病原物。TherearedifferentstrainsorvarietiesofBtavailablethathavebeenselectedforthecontrolofspecificinsects.有不同的菌系或变种用于防治特定昆虫。Itiscompatiblewithmanypesticidesandcanbeusedmixedwithchemicalinsecticides.与许多杀虫剂兼容，可与化学杀虫剂混用。,C.InsectsControlledbyBacillusthuringiensis,C.由苏云金杆菌防治的昆虫,1.Subspecieskurstaki(Biobit,Dipel,MVP,Steward,Thuricide),1、库斯塔克亚种。括号内是商品名称。,Larvalstagesofthefollowing:如下害虫的幼虫期,VegetableInsects（蔬菜害虫）-Cabbage“worms”(甘蓝上的小菜蛾、菜青虫、夜蛾等）.FieldandForageCropInsects农作物和饲草害虫（棉铃虫等）;FruitCropInsects果树害虫（桃蛀螟等）,菜青虫成虫、,斜纹夜蛾,2.Subspeciestenebrionis(拟步甲亚种)(Trident,M-One,M-Trak,Foil,Novodor,etc.),Larvalstagesofthefollowing:如下害虫的幼虫期：Coloradopotatobeetle,elmleafbeetle,cottonwoodleafbeetle科罗拉多马铃薯甲虫，榆叶甲、三叶杨叶甲,象甲,3.Subspeciesisraelensis以色列亚种(Aquabac,Vectobac,Skeetal,Gnatrol,Bactimos),Larvalstagesofthefollowing:如下昆虫的幼虫期mosquitoes,blackflies.蚊、黑蝇,MethodofApplication施用方法,Aerialspraying(forestcrops).气雾Watertreatmentbyaerialorgroundequipment(vectorcontrol).水处理Soilapplicationbydriporoverheadirrigationsystems(field,fruit,andvegetablecrops).土施Foliarapplicationbysprayingfromavehicle,backpackorhand-heldsprayer(field,vegetable,andfruitcrops).叶面喷Note:Wettingagents,surfactants,andstickersmaybeaddedtothetankmixtoenhanceeffectivenessundersomeconditions.注：湿润剂、表面活性剂和粘着剂可加到喷雾器药液中提高药效。,Ecotoxicity/Non-TargetEffects生态毒性/非靶标效应,Todate,thereisnoknownmammalianhealtheffect.迄今不知对哺乳动物的毒性AtthelabeluseratesofregisteredBtactiveingredients,non-targeteffectsareminimaltononexistentforbirds,andaquaticorganismssuchasfishandinvertebratesBT按有效成分登记剂量使用，对鸟、水生动物如鱼和节肢动物低毒或无毒。,Applicationsofformulated（制剂化的）Btarenottoxictomostbeneficialorpredator（捕食性）insects.Treatmentofhoneycombs蜂巢（withBtsubspeciesaizawaidoesnothaveadetrimental（有害的）effectuponbees,noronthehoneyproduced.Veryhighconcentrations(10（8）spores/ml)ofBtsubspeciestenebrionis（拟步甲亚种）,whichisusedagainstbeetles（甲虫）suchastheColoradopotatobeetle,reducedlongevityofhoneybeeadultsbutdidnotcausedisease.Btappliedatratesusedformosquitocontrolmaycausethedeathofsomenon-targetspecies.Itdidnothavenegativeeffectsonfrogs.,Threatened（受威胁的）andEndangered（濒临灭绝的）Species:Btmaybeahazardtoendangeredspeciesifitisappliedtoareaswheretheylive.CertainusesofBtwilljeopardize（危害）thecontinuedexistenceofendangeredmoths（蛾）,butterflies（蝶）,certainbeetles,andsomeflies(dipterans);effectsvarydependingonthesubspeciesofBtused.,AdvantagesofBtBT的优点,Btisessentiallynon-toxictohumans,petsandwildlife(inhalationorcontactwitheyesoropenwoundsshouldbeavoided).对人、宠物和野生动物基本上无毒（应避免吸入或接触）。Thereisnowaitingperiodfromtimeofapplicationbeforere-enteringthefield.施药后即可入田。,DifferentstrainsofBtareclass-specific,meaningbeneficialornon-targetinsectsarenotharmed.不同的BT菌系有不同的寄主范围，对益虫和非靶标昆虫无害。TheinsectsthatingestBtandlaterdiefromit,arenotconsidereddangeroustobirdsorotheranimalsthatmayfeedonthedeadinsects.摄入BT而后死亡的虫体不会对取食死虫的鸟和其它动物造成危害。Btisnotknowntocauseinjurytoplantsonwhichithasbeenappliedandisnotconsideredharmfultotheenvironment.未发现对植物无药害，对环境可认为无害。,TherehavbeenonlyafewofcasesofBtresistancereportedtodate;overusemustbeavoided.Relyingonanyonepesticidecanleadtothebuildupofresistanceinthepestpopulation.Pesticiderotationandculturalcontrolmethodsshouldbeusedtoslowdownoreliminatethepossibilityofresistancedevelopment.迄今为止对BT产生抗性的例子不多。要避免过量施用。要轮换用药并结合栽培防治以减慢或消除抗性发展的可能性。,Btusecanintegratewellwithothernaturalcontrolswhentryingtocontrolapestinsect.Forexample,theuseofBtproductsintheappleandpearecosystemdoesnotcausespidermiteproblems;miteoutbreakscommonlyresultfollowingdestructionoftheirnaturalenemiesbylessselectivetreatments.BT防治害虫时可与天然防治措施兼容性好。如在苹果和梨园用药不会造成螨的猖獗，因为BT对螨的天敌没有什么影响。,DisadvantagesofBt缺点,Btactsasastomachpoison;consequentlygoodcoverageisessential;Btmustbeeatentobeeffective.BT是一种胃毒剂，害虫吃了才有效，因此施药时要喷撒均匀。ItisonlyeffectiveagainstimmaturestagesofLepidoptera,Diptera,andColeopterafeedingonexposedplantsurfaces.Thislimitsitsusefulnessagainstinsectpeststhattunnelintotheplantsorplantparts.Examplesincludecodlingmothandcornearworm,whicharesusceptibletoBtbutrarelyhaveanopportunitytoeatitinfielduse.只对植物体表的未成年阶段有效，因而对钻蛀到植物体内的虫没有什么效。,Itlackstransmissionandspreadingfacilities,itmustbeappliedlikeachemi