苏云金杆菌--毒素及其应用.ppt

苏云金杆菌（BT）毒素及其应用,1 What is Bacillus thuringiensis,1 苏云金杆菌是什么？,A rod-shaped(1.0-1.2 by 3-5 micron),gram-positive,facultative anaerobic,spore forming bacterium,杆状，大小为1.0-1.2 3-5 m，革兰氏阳性，兼性嫌氧，产孢的细菌,菌体照片,A facultative pathogen of some insect species commonly used as a biopesticide.,一些昆虫的兼性病原物，常用作生物杀虫剂。,More than 150 insects belonging to the orders Lepidoptera,Diptera,and Coleoptera are known to be susceptible to Bt.To be effective,Bt must be eaten by insects during their feeding stage of development,when they are larvae.Bt is ineffective against adult insects.,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。,During sporulation,it produces protein inclusions(parasporal inclusions)adjacent to the endospore,产芽孢期间，BT在邻近芽孢处形成蛋白内含体。,Electron Micrograph of a Sporulating Bt Cell,产孢BT菌体的电子显微照片,The parasporal inclusions consist of one or more insecticidal proteins in the form of a crystal or crystal-complex.These insecticidal proteins are commonly known as Insecticidal Crystal Proteins(ICP)or delta endotoxin.The primary insecticidal activity of Bt is due to ICP.It is the active ingredient of most(90%)of the microbial insecticides produced in the world.,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。,The endospore,highly resistant to environmental stress,provides a mechanism for long-term survival of Bt.,内孢子高抗环境压力，提供了BT长期生存的机制。,芽孢,Besides ICP and endospore,some Bt subspecies produce Beta Exotoxin which is toxic to all forms of life including humans,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。,An insect must have a strongly alkaline mid-gut,specific proteolytic enzymes and suitable tissue receptor-sites,to be susceptible.,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。,Bt can be produced easily on solid media or under submerged fermentation conditions-a key factor in its successful development as a biopesticide.,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。,Biotechnology and advances in the fermentation industry over the past three decades have improved the quality of Bt products.,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,Bt is key source of genes for transgenic expression to provide pest resistance in plants.,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,2 History,2 研发历史,Discovered by Dr.Ishiwatari(a Japanese bacteriologist)in 1901 in silkworm farms as the causal agent of the sotto bacillus disease.,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,Insecticidal activity was discovered in 1911 by Berliner(Germany)He named it Bacillus thuringiensis,after the German town Thuringia where the moth was found.,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。,Existance of parasporal inclusions in Bt was noted in 1915 by Berliner.,Berliner还于1915年发现BT类似于孢子的内含体的存在。,First time used as insecticides in late1920s,1920年代后期首次用作杀虫剂。,Spore-based formulations were commercially available in late 1950s,1950年代后期市场上有孢子态的制剂销售。,First registered as a pesticides in the U.S.in 1961,1961年在美国作为杀虫剂首次登记。,In 1960s,experiments with spore-based formulations produced erratic(inconsistent)results,1960年代，用孢子态制剂做的试验得到的药效结果不一。,By the 1970s it was demonstrated that the primary insecticidal activity against lepidoteran insects was due to delta(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。,Until 1977,only 13 Bt subspecies had been described;all were toxic to lepidopteran larvae only.,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。,Discovery of subspecies toxic to dipteran and coleopteran insects in 1977 and 1983,respectively.,1977年和1983年分别发现BT亚种对双翅目和鞘翅目的毒性。,3 Natural Habitat,天然生境,Plant surfaces-Bt has been found extensively in the phylloplane.Numerous subspecies have been isolated from coniferous trees,deciduous trees,and vegetables.It has also been recovered from stored products.,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-spores persist in soil,and vegetative growth occurs when nutrients are available,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。,Insect Hosts-various Bt subspecies have been isolated from dead or dying insect larvae.,3.3昆虫寄主，从死虫或干虫分离到了各种各样的BT亚种。,Environmental Fate,4 在环境中的命运,For lepidopteran and coleopteran pests,Bt is applied to the surfaces of the plants and for dipteran pests(mosquitoes and blackflies)applied to their aquatic,larval habitats.Many Bt insecticides show poor stability under field conditions,and so frequent reapplication is required.,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.Terrestrial Habitat Plant Surfaces(Phylloplanes)and Soil-Coleopteran-active and Lepidopteran-active subspecies,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。,Bt ICPs are degraded quickly by solar radiation,在阳光照射下BT ICP快速降解,Bt Endospores are inactivated rapidly when exposed to UV radiation,当暴露在紫外线下时BT芽孢迅速失活。,Half-life:a few hours-10 days in most agricultural crops,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。,Vegetative cells and spores may persist at gradually decreasing concentrations for weeks,months,or years as a component of the natural microflora.,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.Aquatic Habitats-Dipteran-active subspecies,B.水生习性对双翅目有活性的亚种,Rapid sedimentation in all but the fastest flowing stream,除非在快速气流中，能迅速沉降。,Spores may persist for at least 22 days in sediments,孢子在沉积物中至少可存活22天。,Contact of Bt subspecies israelensis with mud result in an immediate disappearance of larvicidal activity,BT的以色列亚种与泥浆接触时杀幼虫活性很快丧失。,4 Classification of Bt Subspecies,4 BT亚种的分类,Based on the serotype(serological analysis of flagella antigens)supplemented by morphological and biochemical criteria.,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,Over 67 subspecies have been identified;subspecies commonly used in agriculture/public health,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。,Bt subspecies kurstaki(Bt k)-controls various types of lepidopterous insects,BT的库期塔克亚种防治各种鳞翅目害虫,Bt subspecies israelensis(Bt i)-effective against mosquitoes,blackflies.,BT的以色列亚种对蚊子、黑蝇有效。,Bt subspecies tenebrionis(also formerly as subsp.san diego)(Bt te)-effective against certain beetle(chrysomelids)species and the boll weevil.,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。,Bt subspecies Japonensis(Bt j)-effective against many species of scarabid beetles,BT的日本亚种对某些金龟子有效。,Bt subspecies aizawai(Bt a)-used against wax moth larvae in honeycombs,BT的aizawai亚种用来防治蜂巢中的蜡蛾幼虫。,Pathogenicity/Toxicity 致病性/毒性,Toxicity is due to ICP,endospore or Beta Exotoxin毒性是由于ICP、内孢子或-外毒素。Insecticidal Crystal Proteins(ICP)杀虫晶体蛋白（ICP）,Also called as delta-endotoxin.They are protoxin(it must be activated before it has any effect).,ICP也称-内毒素，是原蛋白（必须要活化才有效）。,Commonly designated as Cry proteins encoded by cry genes.,ICP通常指名为Cry蛋白，由cry基因编码。,The genes that encode ICP are mostly on plasmids;each ICP is the product of a single gene.,编码ICP的基因主要在质粒上；每种ICP是单个基因的产物。,A subspecies can synthesize more that one type of ICP.,一个亚种可合成一种以上的ICP。,ICPs have various forms(bipyramidal,cuboidal,flate rhomboid,or a composite with two or more crystal types).,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-A large protein of up to 230 kilo Daltons(kDa),Active Toxin-65 kDa.,大小：原毒素是一种大蛋白，达到230 kDa。活性蛋白为65 kDa。,Extremely potent and toxic to target insect larvae at picomole concentrations,极毒，在p mloe 浓度对靶标幼虫有毒。,Most susceptible species belong to the orders Lepidoptera,Diptera,and Coleoptera.,大多数感染的虫种属于鳞翅目、双翅目和鞘翅目。,Most susceptible insects are killed by ingestion of the crystals alone;a mixture of spores and crystals are required for a toxic effect in only a small number of insects.,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。,Cry proteins are classified according to their insect specificity(Host spectrum is designated in roman numerals):,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）,CryI Lepidoptera（鳞翅目）-specificCryII Lepidoptera-and Diptera（双翅目）-specificCryIII Coleoptera（鞘翅目）-specific CryIV Diptera-specific CryV Coleoptera-and Lepidoptera-specific,A mixture of different Cry toxins results in a synergism of insecticidal activity.,将不同Cry毒素混合导致增效杀虫作用。,A mixture of Cry toxins and Bt spores results in a synergism of insecticidal activity in several insect pests,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。,Highly insoluble in normal digestive system conditions-safe to humans,higher animals,and most insects.,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。,It is soluble in reducing conditions of high pH(above pH 9.5)-a condition commonly found in the mid-gut of lepidopteran larvae.For this reason,Bt is a highly specific insecticidal agent.,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。,The midgut of susceptible insects contains the correct pH and enzymes to transform the crystal into an active toxin.,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。,Different domains of the ICP are responsible for host susceptibility(receptor recognition)and toxicity(pore formation).,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,Nomenclature for crystal protein gene of Bacillus thuringiensis(Bt)and activity spectrum against insects,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,Endospores are resistant to inactivation by heat and desiccation and persist in the environment under adverse conditions.Provide a mechanism for long-term survival of Bt.Are pathogenic for some insects,particularly when combined with ICP.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。,Beta Exotoxin,外毒素,During vegetative growth,some Bt subspecies produce Beta Exotoxin,an ATP analogue,water soluble,heat stable secondary metabolite.,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-exotoxin is an inhibitor of RNA polymerase and acts competitively with ATP in various biological processes.,-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。,It is toxic to almost all forms of life including humans.It has broad-spectrum insecticidal activity.,-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。,Products with Beta Exotoxin are prohibited in the USA.Manufacturing process includes monitoring to prevent Beta Exotoxin from appearing in products,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。,Mode of Action 作用方式,Bioactivity of Bt is dominated by the ICPs and can be summarized in the following stages。BT的生物活性是由ICP决定的。其作用方式如下。,1.Ingestion of spores and ICP by a susceptible insect larva;感染型昆虫幼虫摄入孢子和ICP。2.ICP dissolve in the alkaline midgut;ICP在碱性中肠中解离。3.Activation of the ICP by proteases;ICP被蛋白酶活化。4.Irreversible binding of the C-terminal domain of activated ICP to specific receptors in the midgut cell membrane;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.Insertion of the N-terminal domain of activated toxin in the cell membrane and formation of pores and channels in the gut membrane,followed by destruction of epithelial cells and paralysis of the digestive system;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。6 Spore germination and septicemia;孢子萌发，虫体患败血病。7 Larval death(within an hour to a few days)from starvation or septicemia.幼虫因饥饿或而败血病死亡（1小时内到几天）。,Effects on Insects 对昆虫的影响,Immediate cessation of feeding and general paralysis of the larval midgut1、立即停止取食，中肠瘫痪Subsequent symptoms include vomiting,diarrhea,general sluggishness,and general paralysis.2、其后的症状包括呕吐、腹泻、行动迟缓和瘫痪Death may follow immediately or in an hour to a few days.3、立即或1小时内到几天死亡。Sublethal effects may include:reduced feeding,reduced larval and adult longevity,reduced fecundity,reduced larval and adult body weight.4、亚致死作用可能包括：减少取食、虫体短，体重轻。,Using Bt in Integrated Pest Management BT在害虫综合治理中的应用,A.Host Range Larvae of the insect orders Lepidoptera,Diptera,and Coleoptera 寄主范围 鳞翅目、双翅目和鞘翅目昆虫的幼虫。,B.Use Profile 使用范围,Bt is the most versatile biopesticide for use in pest management.BT是用于害虫治理的最通用的杀虫剂。It is used on agricultural crops,harvested crops in storage,ornamentals,bodies of water,and around the home to control various groups of insects,depending on the type of toxin produced by the specific isolate of Bt.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,over 180 Bt products are registered in USA.目前有180种BT产品在美国登记。,Bt is primarily a pathogen of insect larval stages.Younger larvae are more susceptible than older larvae.BT主要是一种幼虫期的病原物。There are different strains or varieties of Bt available that have been selected for the control of specific insects.有不同的菌系或变种用于防治特定昆虫。It is compatible with many pesticides and can be used mixed with chemical insecticides.与许多杀虫剂兼容，可与化学杀虫剂混用。,C.Insects Controlled by Bacillus thuringiensis,C.由苏云金杆菌防治的昆虫,1.Subspecies kurstaki(Biobit,Dipel,MVP,Steward,Thuricide),1、库斯塔克亚种。括号内是商品名称。,Larval stages of the following:如下害虫的幼虫期,Vegetable Insects（蔬菜害虫）-Cabbage“worms”(甘蓝上的小菜蛾、菜青虫、夜蛾等）.Field and Forage Crop Insects 农作物和饲草害虫（棉铃虫等）;Fruit Crop Insects 果树害虫（桃蛀螟等）,菜青虫成虫、,斜纹夜蛾,2.Subspecies tenebrionis(拟步甲亚种)(Trident,M-One,M-Trak,Foil,Novodor,etc.),Larval stages of the following:如下害虫的幼虫期：Colorado potato beetle,elm leaf beetle,cottonwood leaf beetle 科罗拉多马铃薯甲虫，榆叶甲、三叶杨叶甲,象甲,3.Subspecies israelensis以色列亚种(Aquabac,Vectobac,Skeetal,Gnatrol,Bactimos),Larval stages of the following:如下昆虫的幼虫期 mosquitoes,blackflies.蚊、黑蝇,Method of Application 施用方法,Aerial spraying(forest crops).气雾Water treatment by aerial or ground equipment(vector control).水处理Soil application by drip or overhead irrigation systems(field,fruit,and vegetable crops).土施Foliar application by spraying from a vehicle,backpack or hand-held sprayer(field,vegetable,and fruit crops).叶面喷Note:Wetting agents,surfactants,and stickers may be added to the tank mix to enhance effectiveness under some conditions.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-Target Effects生态毒性/非靶标效应,To date,there is no known mammalian health effect.迄今不知对哺乳动物的毒性At the label use rates of registered Bt active ingredients,non-target effects are minimal to nonexistent for birds,and aquatic organisms such as fish and invertebratesBT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。,Applications of formulated（制剂化的）Bt are not toxic to most beneficial or predator（捕食性）insects.Treatment of honeycombs蜂巢（with Bt subspecies aizawai does not have a detrimental（有害的）effect upon bees,nor on the honey produced.Very high concentrations(10（8）spores/ml)of Bt subspecies tenebrionis（拟步甲亚种）,which is used against beetles（甲虫）such as the Colorado potato beetle,reduced longevity of honey bee adults but did not cause disease.Bt applied at rates used for mosquito control may cause the death of some non-target species.It did not have negative effects on frogs.,Threatened（受威胁的）and Endangered（濒临灭绝的）Species:Bt may be a hazard to endangered species if it is applied to areas where they live.Certain uses of Bt will jeopardize（危害）the continued existence of endangered moths（蛾）,butterflies（蝶）,certain beetles,and some flies(dipterans);effects vary depending on the subspecies of Bt used.,Advantages of BtBT的优点,Bt is essentially non-toxic to humans,pets and wildlife(inhalation or contact with eyes or open wounds should be avoided).对人、宠物和野生动物基本上无毒（应避免吸入或接触）。There is no waiting period from time of application before re-entering the field.施药后即可入田。,Different strains of Bt are class-specific,meaning beneficial or non-target insects are not harmed.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。The insects that ingest Bt and later die from it,are not considered dangerous to birds or other animals that may feed on the dead insects.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。Bt is not known to cause injury to plants on