Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Association analysis between serotype, cry gene content, and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates native to Spain

Serotyping, cry gene content, and toxicity to Helicoverpa armigera were determined for 178 isolates of Bacillus thuringiensis native to Spain. A total of 13 different cry1 and cry2 genes were detected when isolates were screened by PCR analysis. Results showed that cry2 and cry1Ia were the most frequent cry genes in the collection (74 and 57%, respectively); whereas cry1D, cry1Aa, cry1Ab, and cry1C were only moderately abundant (49, 48, 47, and 36%, respectively). The most uncommon cry genes were cry1Ac, cry1E, cry1B, cry1Ib, cry1Ad, cry1F, and cry1G, with frequencies of 24, 14, 13, 8, 5, 5, and 1%, respectively. The distribution of some cry genes was somewhat associated with particular serovars. For example, genes cry1C and cry1D were especially frequent in the serovar aizawai, while cry1B was very frequent in the serovar thuringiensis. Bioassays against H. armigera larvae showed a wide variation in the insecticidal potency, even among strains sharing the same set of cry genes and within the same serotype.     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

Toxicity and larval growth inhibition of 11 insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 microg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect.     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

Cross-resistance of pink bollworm (Pectinophora gossypiella) to Bacillus thuringiensis toxins

Two strains of pink bollworm (Pectinophora gossypiella) selected in the laboratory for resistance to Bacillus thuringiensis toxin Cry1Ac had substantial cross-resistance to Cry1Aa and Cry1Ab but not to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa, Cry9Ca, H04, or H205. The narrow spectrum of resistance and the cross-resistance to activated toxin Cry1Ab suggest that reduced binding of toxin to midgut target sites could be an important mechanism of resistance.     

Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis.

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.     

土壤中对甲虫有活性苏云金芽胞杆菌的筛选

近几年来,甲虫已成为十字花科蔬菜的头号害虫。本文采集了湖北省各地区192份土壤样品,分离到苏云金芽胞杆菌(简称Bt)菌株74株,以甲虫代表——黄粉虫为靶标昆虫筛选了15株典型Bt,得到一株对甲虫有活性的Bt L1;生物测定该菌的半致死浓度(LC50)为221.20μg·g-1;聚丙烯酰胺凝胶电泳(SDS-PAGE)显示L1至少具有3个杀虫晶体蛋白;PCR扩增杀虫晶体蛋白基因,测序发现Bt L1中含有cry1Ac,cry1Aa,cry1Ia基因,推测Bt L1中对甲虫活性的杀虫晶体蛋白基因为cry1Ia。     

Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera:Noctuidae)

AIMS: To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. METHODS AND RESULTS: The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. CONCLUSIONS: It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.     

Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.     

Toxic activity of Bacillus Thuringiensis isolates to Aedes Aegypti (L.) (Diptera: Culicidae) larvae

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2%) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3%) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100% mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.     

Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.     

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。     

A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai

A population (SERD3) of the diamondback moth (Plutella xylostella L.) with field-evolved resistance to Bacillus thuringiensis subsp. kurstaki HD-1 (Dipel) and B. thuringiensis subsp. aizawai (Florbac) was collected. Laboratory-based selection of two subpopulations of SERD3 with B. thuringiensis subsp. kurstaki (Btk-Sel) or B. thuringiensis subsp. aizawai (Bta-Sel) increased resistance to the selecting agent with little apparent cross-resistance. This result suggested the presence of independent resistance mechanisms. Reversal of resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai was observed in the unselected SERD3 subpopulation. Binding to midgut brush border membrane vesicles was examined for insecticidal crystal proteins specific to B. thuringiensis subsp. kurstaki (Cry1Ac), B. thuringiensis subsp. aizawai (Cry1Ca), or both (Cry1Aa and Cry1Ab). In the unselected SERD3 subpopulation (ca. 50- and 30-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai), specific binding of Cry1Aa, Cry1Ac, and Cry1Ca was similar to that for a susceptible population (ROTH), but binding of Cry1Ab was minimal. The Btk-Sel (ca. 600-and 60-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) and Bta-Sel (ca. 80-and 300-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) subpopulations also showed reduced binding to Cry1Ab. Binding of Cry1Ca was not affected in the Bta-Sel subpopulation. The results suggest that reduced binding of Cry1Ab can partly explain resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. However, the binding of Cry1Aa, Cry1Ac, and Cry1Ca and the lack of cross-resistance between the Btk-Sel and Bta-Sel subpopulations also suggest that additional resistance mechanisms are present.     

Use of a Cry1Ac-Resistant Line of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) to Detect Novel Insecticidal Toxin Genes in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

This paper describes a screening strategy incorporating resistant insect lines for discovery of new Bacillus thuringiensis toxins against a background of known genes that would normally mask the activity of additional genes and the application of that strategy. A line of Helicoverpa armigera with resistance to Cry1Ac (line ISOC) was used to screen Cry1Ac-expressing strains of B. thuringiensis for additional toxins with activity against H. armigera. Using this approach, a number of Cry1Ac-producing strains with significant toxicity toward Cry1Ac-resistant H. armigera were identified. When the insecticidal protein complement of one of these strains, C81, was examined in detail, a novel cry2 gene (cry2Af1) was detected.     

苏云金芽胞杆菌YBT1520杀虫晶体蛋白基因的属性

通过Ｓｏｕｔｈｅｒｎ杂交发现高毒力苏云金芽胞杆菌（Ｂａｃｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ）ＴＢＴ－１５２０菌株含有两个杀虫晶体蛋白基因片段,其５’＝末端所在ＨｉｎｄⅢ片段分别为６．８ｋｂ和４．６ｋｂ,它们对应的基因分别命名为ｃｒｙ２１８和ｃｒｙ４．６。经ＰＣＲ鉴定,该菌含有ｃｒｙ１Ａａ、ｃｒｙ１Ａｂ和ｃｒｙ１Ａｃ基因,以及ｃｒｙ２基因,其中ｃｒｙ２１８属于ｃｒｙ１Ａｃ。分析了ｃｒｙ１Ａｃ基因     

Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

Characterization of Cry34/Cry35 binary insecticidal proteins from diverse Bacillus thuringiensis strain collections

Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.