苏芸金杆菌的一个新血清型

从土壤中分离出一株产生伴孢晶体的芽孢杆菌。该菌株具有苏芸金杆菌的典型特征,严生不规则型晶体,鞭毛抗原及生化反应与已知的苏芸金杆菌21个血清型不㈤,不产生β-外毒素。对棉铃虫、粘虫、大蜡螟及尖音库蚊幼虫均无毒性,是一个新血清型(H22),定名为苏芸金杆菌山东变种(Bacillus thuringiensis serovar shandongiensis, H22)。     

苏云金杆菌δ-内毒素、芽孢及苏云金素A对棉铃虫毒性及拒食性的比较

苏云金杆菌库斯塔克变种HD-1的晶体蛋白与芽孢、HD-1无晶体突变株(Cry-)的芽孢以及苏云金素A对棉铃虫Helicoverpa armigera毒性和拒食性的比较研究显示,HD-1晶体蛋白对棉铃虫的杀虫毒力高, 拒食作用强; HD-1芽孢对棉铃虫具有一定的杀虫活性和生长抑制作用, 并有很强的拒食作用; HD-1无晶体突变株(Cry-)芽孢对棉铃虫无毒也无拒食作用;苏云金素A对棉铃虫的生长发育有极显著的抑制作用, 但对棉铃虫无拒食作用,由此证明晶体蛋白是苏云金杆菌杀虫活性和拒食作用的主要来源。苏云金素A与苏云金杆菌芽孢晶体混合物一起使用, 可使棉铃虫的死亡率显著提高。     

昆虫病原菌苏云金芽孢杆菌群(Bacillus thuringiensis Group)的分类

采用形态、生化、血清学的方法对国外引种的15株巳知菌和本国51株未知菌进行了变种的分型研究。结果表明：苏云金芽孢杆菌(Bacillus thuringiensis)作为有益的细菌资源在藏国分布较广。51株菌中属于血清型H1苏云金芽孢杆菌苏云金变种(Baeillus thuringiensisvar. Thuringiensis)有11株,血清型H50-5b苏云金芽孢杆菌蜡螟变种(B．Thur．Var．Galleriae)31株,血清型H4-4b苏云金芽孢杆菌松蜩变种(B.thur. Var. dengrolimut)3株,血清型H4-4a。苏云金芽孢杆菌肯尼亚变种(B. thur. Var kenyae) 5株,同时发现血清型H．苏云金芽孢杆菌玉米螟变种(B. thur var. ostrinia,)一株。玉米螟变种不同于该菌群的巳知菌株,认为是一个新变种。实践证明,利用血清学抗原抗体具有高度特异{生的凝集反应来鉴别苏云垒芽孢杆菌变种,简便,快速,准确。而该群噬菌体却不能作为区分变种的标准。     

抗夜蛾科苏云金芽孢杆菌WY-197菌株的生物学特性研究

苏云金芽孢杆菌 (Bacillusthuringiensis,Bt)WY 197菌株是从 4 0 0多株野生型Bt菌中筛选分离的1株对甜菜夜蛾和棉铃虫都具有高效杀虫活性的菌株。初步比较WY -197菌株和HD 1的形态特征、生理生化特性和伴孢晶体蛋白 ,结果表明 ,WY -197属于H3a3b3c血清型 (subsp .kurstaki) ;伴孢晶体形态主要为大小不一的菱形、小正方形。SDS -PAGE电泳图谱表明主要由 135ku和 6 5ku两种蛋白成分组成 ,在菌株生长形态、发酵培养特征、生理生化特性方面与生产菌株HD-1差别不大。采用生物测定的方法 ,进一步鉴定比较它们对甜菜夜蛾初孵、二龄、三龄幼虫和棉铃虫的毒效差异。结果表明 ,BtWY 197菌株发酵液对甜菜夜蛾初孵、二龄、三龄幼虫的LC50 分别为 0 .31、0 .6 1、2 .5 7μL/mL ,其中对甜菜夜蛾初孵幼虫的毒效是对照菌HD 1发酵液的 7倍 ,表明BtWY 197菌株对甜菜夜蛾三龄以下幼虫具有高毒力。对棉铃虫的毒力效价为 30 0 0IU/ μL ,略低于HD- 1菌株。     

我国部分地区土壤中的苏芸金芽孢杆菌和球形芽孢杆菌

从云南、贵州、四川和陕西4省的土壤中分离到大量苏芸金芽孢杆菌(Bacillus thuringie—nsis)和球形芽孢杆菌(Bacillus sphaeticus)菌株。血清型分析表明,苏芸金芽孢杆菌分离株分属于23个血清型中的13个血清型,另有近20％的自凝型菌株及部分与所有标准菌抗血清无反应的菌株。对该两种昆虫病原细菌的生态分布规律进行了分析。研究了全部苏芸金芽孢杆菌分离株对鳞翅目、鞘翅目及双翅目的6种昆虫的毒力特性、伴孢晶体与芽孢的形态,以及晶体蛋白质成分。观察和测定了球形芽孢杆菌分离株的形态和毒力,并分析了部分菌株的晶体蛋白质成分。得到22株高效苏芸金芽孢杆菌和2株高效球形芽孢杆菌。证明苏芸金芽孢杆菌是典型的土壤微生物类群,我国西南地区土壤中的苏芸金芽孢杆菌资源十分丰富。     

杀鞘翅目苏云金芽孢杆菌新菌株及其杀虫剂的研究

从中国土壤中分离出2株杀鞘翅目昆虫的苏云金芽孢杆菌(Bacillus thuringiensis) YM03及SHQ11-10。YM03的血清型为H8a8b,SHQ1110的H血清型未知。二菌株皆产近菱形的薄扁伴孢晶体,分别含68～70kD和65kD的晶体蛋白质。毒力生物测定证明对柳蓝叶甲(Plagiodera versicolora)及马铃薯甲虫(Leptinotarsa decemlineata)有高毒效。发酵性能良好。YM03粉剂田间防治马铃薯甲虫有高效。稀释400倍喷雾,防治效果达94.6%。     

一株对棉铃虫高效的苏云金杆菌

１９７９年从舍蝇（Ｍｕｓｃａｖｉｃｉｎａ）幼虫中分离出一株能够形成伴孢晶体的芽孢杆菌７９００７。该菌具有苏云金杆菌天门变种（７２１６）的典型特征。血清型属Ｈ_{３ａ－３ｂ},但培养特征,生化特征与知的Ｈ_{３ａ－３ｂ}的戈尔斯德变种（ＨＤ－１）、天门变种（７２１６）略有不同,特别是对棉铃虫的毒力大大高于巳知的菌株,预示着将成为我国防治棉铃虫的一株高效菌。     

苏芸金杆菌的质粒检测及载晶体蛋白合成基因的质粒在不同菌株间的传递

分析了苏芸金杆菌(Bacillus thuringiensis)7个变种19个野生菌株及无晶体(sp+cr-)和无芽孢(sp-cr+)突变株的质粒图型,证实了苏芸金杆菌的质粒组成既有变种的特异性,亦有菌株的特异性。载有晶体蛋白质合成基因的质粒,B. thuringiensis var．Kurstaki HD-191 菌株可能为42和50Mdal,B．Thuringiensis var. aizawai HD-282菌株为47Mdal, B．Thuringiensis var．Israelensis IPS-82菌株为57Mdal的质粒,而B．Thuringiensis var. wuhanensis 140 菌株的此种基因则可能不在质粒上而在染色体中。HD-191载晶体蛋白合成基因的质粒以很高频率被传递给其无晶体突变株HD-1并在后者细胞中表达。B. thuringiensis var. israelensisIPS-82菌株和 B．Thuringiensis var. kurstaki 无晶体突变株HD—1之间载晶体蛋白合成基因质粒的传递未能成功,提示可能存在着不相容性的障碍。     

苏云金芽孢杆菌蜡螟变种伴孢晶体蛋白基因的克隆及表达

用改进的碱解法分析基因供体菌苏云金芽孢杆菌蜡螟变种81-6(Bacillus thuringiensis var.galleriae 81-6)株有两种小质粒和一种大质粒。 将碱解法抽提到的质粒用Pstl完全酶解,与同样用PstI完全酶解的载体质粒pcAMPUC连接,以此重组质粒转化苏云金杆菌无晶体蛋白突变株 Bacillus thuringiensis var,kurstaki HD-1 Cry—B株的原生质体。用ELlSA法筛选到一株阳性转化菌落,分析了其质粒组成和插入片段的大小。毒性试验显示阳性转化子对昆虫幼虫有毒杀作用。     

苏云金杆菌的一个新生物型—SD—5菌株

从霜天蛾病死幼虫中分离到1株能形成伴孢晶体毒素的芽孢杆菌,杀虫效力和发酵性能优良,经生理生化特性测定和血清学研究,SD-5菌株的晶体蛋白特性不同于7216和HD-1菌株,属于库斯塔克亚种(H303b)的1个新生物型.     

我国南北方土壤中苏云金芽孢杆菌的分布及杀虫特性

从我国南部和北部地区12省的1491份土样中分离出苏云金芽孢杆菌(Bacillus thuringiensis,以下简称为 Bt)221株。经鉴定,属于已有血清型(H_1～H_(45))中的24个血清型。此外尚有部分自凝型和未知血清型菌。东北和内蒙古地区的出菌土样率为12.6％,出菌率为17.2％,优势血清型为H_4、H_(10)、H_3、H_(13)、H_5及H_(29),其中以黑龙江省土壤Bt菌最丰富,出菌土样率和出菌率分别达21.4％和29.4％,还分离出H_(43)型菌2株;西北地区的出菌土样率和出菌率分别为6.6％和7.1％,远低于东北、内蒙古地区,优势血清型为H_4、H_5、H_(19)、H_(10)及H_3;南部4省土壤中的优势血清型为H_3及H_5,Bt菌的平均分出率高,但地区分布极不均衡,湖南、江苏的出菌土样及出菌率均在4.8％～4.9％,广东珠江三角洲土壤中分别高达77.8％和129.6％,湖北长江沿岸冲积土中则未分离出Bt菌。毒力初测结果表明:北部地区的Bt分离株中仅有1.6％对棉铃虫(Heliothis armigera)高效,1.1％对柳蓝叶甲(Plagwdera versicolo…     

一株对苹果树鳞翅目害虫高毒力的苏云金芽胞杆菌YX-1

苏云金芽胞杆菌Bacillus thuringiensis(Bt)YX-1是从土壤中分离的对多种鳞翅目害虫具有杀虫活性的新菌株。为了探索该菌株在果树上应用的可行性,本研究测定了Bt YX-1菌株对苹果树上6种鳞翅目害虫的杀虫毒力,同时对该菌株的晶体形态特征、蛋白型、生长特性、基因型等进行了分析。结果表明,Bt YX-1菌株产生菱形伴胞晶体,SDS-PAGE分析表明该菌株表达的主要蛋白条带分子量约为130ku和60ku;基因型鉴定表明,Bt YX-1菌株含有cry1Ac、cry2Ac、cry1I、vip3Aa和cry34-35基因;生物活性测定表明,Bt YX-1菌株的孢晶混合物对美国白蛾Hlyphantria cunea、棉铃虫Helicoverpa armigera、斜纹夜蛾Prodenia litura、梨小食心虫Grapholitha molesta、苹小卷叶蛾Adoxophyes orana以及苹掌舟蛾Phalera flavescens的LC50分别为14.48、2.72×103、6.24×104、1.01×102、3.52×104、4.73×103mg/L,均低于标准菌株Bt HD-1的LC50。发酵上清液的杀虫活性很低,2龄棉铃虫幼虫的死亡率仅为8.33%,但是该上清液能显著提高孢晶混合物的毒力,说明上清液中含有增效物质。研究结果表明该菌株具有进一步开发为商品制剂的潜力。     

从湖北神农架原始森林土壤中分离的高毒力苏云金芽孢杆菌

从神农架原始森林土壤中分离出苏云金芽孢杆菌 9株。经过生理生化和血清学鉴定 ,此 9株苏云金芽孢杆菌分属于H7、H6和H14。生物测定结果表明 :两株H7型菌株对棉铃虫幼虫有较高的毒力 ;另两株对致倦库蚊幼虫和白纹伊蚊幼虫有很强的毒杀作用 ,此两株属苏云金芽孢杆菌H14。     

31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明：25株含cry1基因,表达蛋白130～150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明：cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。     

Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae)

In Australia, transgenic cotton plants expressing the cry1Ac gene from Bacillus thuringiensis Berliner variety kurstaki are less toxic to first-instar Helicoverpa armigera (Hübner) after the plant is producing fruit. We developed two bioassay methods (leaf mush, leaf disk) to test if the physiological state of the plants explained changes in toxicity and a third method (diet incorporation) was developed to quantify the toxicity of Bt leaves when mixed in chickpea diet. Cry1Ac protein was less toxic to H. armigera larvae when the protein was mixed with leaves from fruiting versus presquare conventional cotton. Differences in LC50 varied from 2.4- to 726-fold, depending on the source of toxin and conventional plant material. These results suggest that plant-toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein. The possible role of tannins in these changes is discussed.     

The toxicity of Bacillus thuringiensis 9816C to Spodoptera exigua related to the binding sites on BBMV

Abstract The binding and pore formation properties of toxins derived form Bacillus thuringiensis 9816C were analyzed by using brush border membrane vesicles (BBMV) of Spodoptera exigua and Helicoverpa armigera , and the results were compared to the results of toxicity bioassays. The strain 9816C is highly toxic to both S. exigua and H. armigera , whereas HΔ-73, which only produces Cry1Ac, is merely effective for H. armigera. Ligand blot experiment performed with peroxidase-labeled toxins revealed that the toxins of the two strains had the same binding sites as H. armigera BBMV and different binding sites from S. exigua BBMV. The toxins of Bt 9816C bind to a 210-kDa protein of S. exigua BBMV, while Cry1Ac cannot recognize this binding site. Both toxins were tested for the ability to alter the permeability of S. exigua BBMV, as measured by a light scattering assay. The toxins of Bt 9816C, which is toxic to S. exigua , permeabilized BBMV, whereas Cry1Ac did not. These results suggest that the specific binding site recognized by Bt 9816C toxins is responsible for its high toxicity against Spodoptera exigua.     

粘虫颗粒体病毒与苏云金杆菌联合作用对甜菜夜蛾的影响

以甜菜夜蛾为试虫,测定了粘虫颗粒体病毒（PuGV-Ps）对苏云金杆菌（Bt）毒力的增效作用。结果表明PuGV对甜菜夜蛾没有致毒作用,但Bt中加入PuGV后可以提高Bt对甜菜夜蛾的毒力,甜菜夜蛾致死中量LC50由Bt单剂的1．094mg／mL下降到0．862mg／mL,共毒系数达127。亚致死剂量Bt处理甜菜夜蛾影响了幼虫的生长发育,表现为幼虫生长量相对减少、蛹重下降、化蛹率降低和化蛹历期延长,添加了PuGV-Ps后进一步增强了Bt对甜菜夜蛾的生长发育的抑制作用。甜菜夜蛾中肠蛋白酶活性测定结果表明,PuGV-Ps对甜菜夜蛾中肠酶活性具有抑制作用;昆虫同时取食PuGV-Ps和Bt后,中肠酶液总蛋白酶活力都有所下降,在中肠酶液最适pH范围内蛋白酶活力抑制作用最明显。     

Clostridium bifermentans serovar malaysia, a new anaerobic bacterium pathogen to mosquito and blackfly larvae

A strain of Clostridium bifermentans individualized as serovar malaysia (C.b.m.) according to its specific H antigen is toxic to mosquito and blackfly larvae when given orally. The toxicity occurs in sporulated cells which contain, in addition to spores, proteinic parasporal inclusion bodies and feather-like appendages; the amino acid content of the inclusion bodies is similar to that of Bacillus thuringiensis serovar israelensis (B.t.i.) and B. sphaericus crystals. The toxicity to Anopheles stephensi is as high as that of B.t.i. and the best strains of B. sphaericus. Culex pipiens is somewhat less susceptible, and Aedes aegypti much less. Pure parasporal inclusion bodies, isolated by ultracentrifugation on sucrose gradients, are highly toxic to mosquito larvae. The larvicidal power is destroyed by heating at 80 degrees C or by treatment with 50 mM NaOH. It is preserved by freeze-drying. The innocuity to mice of the sporulated cells is shown by different routes of administration: force-feeding, percutaneous, subcutaneous, intraperitoneal or intravenous injections. The potential for the biological control of mosquito and blackfly larvae is suggested.     

Disruption of a cadherin gene associated with resistance to Cry1Ac {delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera

A laboratory strain (GY) of Helicoverpa armigera (Hubner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to "mode 1," the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.