Showing papers on "Bacillus thuringiensis published in 1992"

The mode of action of Bacillus thuringiensis endotoxins.

Abstract: The use of synthetic organic insecticides developed during the last half of this century may pose risks to human health and can cause environmental prob­ lems. Consequently, interest has developed in using alternative strategies for insect pest management. One contemporary approach that has received atten­ tion is the development of Bacillus thuringiensis (Bt) toxins as insecticides. B. thuringiensis, a gram-positive bacteria, produces a proteinaceous paraspor­ al crystalline inclusion during sporulation. Upon ingestion by insects, this crystalline inclusion is solubilized in the midgut, releasing proteins called S-endotoxins. These proteins (protoxins) are activated by midgut proteases, and the activated toxins interact with the larval midgut epithelium causing a disruption in membrane integrity and ultimately leading to insect death. In spite of significant efforts directed towards the study of Bt, its use in pest control is restricted, in part because of the selectivity of Bt and in part because of its moderate efficacy. Attempts to improve these two qualities have met with only moderate success because inadequate effort has been devoted to understanding the molecular basis of selectivity and insecticidal properties of these toxins.

Synthetic dna sequence having enhanced insecticidal activity in maize

Abstract: DNA sequences optimized for expression in plants are disclosed. The DNA sequences preferably encode for an insecticidal polypeptides, particularly insecticidal proteins from Bacillus thuringiensis. Plant promoters, particular tissue-specific and tissue-preferred promoters are also provided. Additionally disclosed are transformation vectors comprising said DNA sequences. The transformation vectors demonstrate high levels of insecticidal activity when transformed into maize.

Managing Insect Resistance to Bacillus thuringiensis Toxins

Abstract: Bacillus thuringiensis (B.t.) δ-endotoxins provide an alternative to chemical insecticides for controlling many species of pest insects. Recent biotechnological developments offer the promise of even greater use of B.t. toxins in genetically transformed pest-resistant crops. However, the discovery that insects can adapt to these toxins raises concerns about the long-term usefulness of B.t. toxins. Several methods for managing the development of resistance to B.t. toxins have been suggested, but none of these approaches offer clear advantages in all situations.

Broad-spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens.

Abstract: Evolution of pest resistance to insecticidal proteins produced by Bacillus thuringiensis (Bt) would decrease our ability to control agricultural pests with genetically engineered crops designed to express genes coding for these proteins. Previous genetic and biochemical analyses of insect strains with resistance to Bt toxins indicate that (i) resistance is restricted to single groups of related Bt toxins, (ii) decreased toxin sensitivity is associated with changes in Bt-toxin binding to sites in brush-border membrane vesicles of the larval midgut, and (iii) resistance is inherited as a partially or fully recessive trait. If these three characteristics were common to all resistant insects, specific crop-variety deployment strategies could significantly diminish problems associated with resistance in field populations of pests. We present data on Bt-toxin resistance in Heliothis virescens, a major agricultural pest targeted for control with Bt-toxin-producing crops. A laboratory strain of H. virescens developed resistance in response to selection with the Bt toxin CryIA(c). In contrast to other cases of Bt-toxin resistance, this H. virescens strain exhibits cross-resistance to Bt toxins that differ significantly in structure and activity. Furthermore, the resistance in this strain is not accompanied by significant changes in toxin binding, and resistance is inherited as an additive trait when larvae are treated with high doses of CryIA(c) toxin. These findings have important implications for Bt-toxin-based pest control.

Insecticidal Promise of Bacillus thuringiensis Facts and mysteries about a successful biopesticide

Abstract: T he future looks bright for Bacillus thuringiensis as an object of scientific study and as a commercial opportunity. As an insecticide, it has been valued for its specificity for target insects, its low development cost, and its environmental compatibility. B. thuringiensis also constitutes a gene pool for engineering insecticidal activity into other organisms, such as crop plants. Although B. thuringiensis is a successful bioinsecticide, little is known about its ecology.

Evaluation of synergism among bacillus thuringiensis toxins

Abstract: A simple test for synergism among toxins is described and applied to previously reported data on independent and joint toxicities of insecticidal proteins from Bacillus thuringiensis. The analysis shows synergism between a 27-kDa (CytA) toxin and 130- or 65-kDa (CryIV) toxins from B. thuringiensis subsp. israelensis against Aedes aegypti larvae. No positive synergism between 130- and 65-kDa toxins or among three CryIA toxins tested against seven species of Lepidoptera occurred. Comparisons with the original interpretations of these data show one case in which synergism occurred but was reported previously as absent and two cases that were not synergistic but were reported previously as suggestive of synergism. These results show that lack of an appropriate test for synergism can produce misleading conclusions. The methods described here can be used to test for synergistic effects of any poisons.

Invitation paper (c.p. alexander fund): history of bacillus thuringiensis berliner research and development

Abstract: This review article starts with the discovery of Bacillus thuringiensis Berliner in Japan at the turn of the century and notes that the observations of the early Japanese workers clearly show that they were aware of the toxin-mediated nature of the activity of B. thuringiensis toward insect larvae. The early work in Europe with B. thuringiensis against Ostrinia nubilalis (Hubner) showed that the bacterium had promise as a microbial control agent. The commercial development of B. thuringiensis in France in the late 1930s, and in Eastern Europe and the United States in the 1950s, is traced.

Identification and characterization of a novel Bacillus thuringiensis δ‐endotoxin entomocidal to coleopteran and lepidopteran larvae

Abstract: A new class of Bacillus thuringiensis delta-endotoxins, or insecticidal control proteins (ICPs), is defined by an apparently cryptic protein with a unique primary structure and novel entomocidal specificity for certain coleopteran and lepidopteran species. The discovery of a new group of ICPs will extend the use of this natural insecticide in integrated pest-management systems.

Distribution, Frequency, and Diversity of Bacillus thuringiensis in an Animal Feed Mill.

Abstract: Bacillus thuringiensis was isolated from 36 of 50 residue samples obtained from an animal feed mill (a stored-product environment). Of 710 selected colonies having Bacillus cereus-B. thuringiensis morphology isolated from the samples, 477 were classified as B. thuringiensis because of production of parasporal delta-endotoxin crystals. There was a diverse population of B. thuringiensis, as revealed by differentiation of the isolates into 36 subgroups by using (i) their spectra of toxicity to the lepidopterans Heliothis virescens, Pieris brassicae, and Spodoptera littoralis and the dipteran Aedes aegypti and (ii) their parasporal crystal morphology. A total of 55% of the isolates were not toxic to any of these insects at the concentrations used in the bioassays; 40% of all isolates were toxic to one or more of the Lepidoptera; and 20, 1, and 1% of the isolates were toxic to only P. brassicae, H. virescens, and S. littoralis, respectively. The most frequent toxicity was toxicity to P. brassicae (36% of all isolates); 18% of the isolates were toxic to A. aegypti (5% exclusively), 10% were toxic to H. virescens, and 4% were toxic to S. littoralis. Toxicity to P. brassicae was more often linked with toxicity to H. virescens than with toxicity to S. littoralis. The frequency of toxicity was significantly greater in isolates that produced bipyramidal crystals than in isolates that produced irregular pointed, irregular spherical, rectangular, or spherical crystals.

Resistance of cotton lines containing a Bacillus thuringiensis toxin to pink bollworm (Lepidoptera : Gelechiidae) and other insects

Abstract: Three transgenic lines of cotton, Gossypium hirsutum L., carrying a modified insect-control protein from Bacillus thuringiensis subsp. kurstaki (Berliner), were evaluated for resistance to severallepidopterous insects. These three lines, along with the explant source cultivar, ‘Coker 312’, and a locally adapted control, ‘MD 51 ne’, were grown in a field experiment at Maricopa, Arizona. Early in the season, before bolls were available for infestation, the number of rosetted blooms caused by pink bollworm, Pectinophora gossypiella (Saunders), was 95% lower in the transgenic lines than in the control cultivars. The pink bollworm larvae penetrated bolls of the transgenic lines readily; however, live larvae recovered from incubated bolls, and percent seed damage were reduced 97-99% in the transgenic lines compared with the control cultivars. No live larvae were recovered from bolls of two transgenic lines, 62 and 65. The transgenic lines were highly resistant to cotton leaf perforator, Bucculatrix thurberiella Busck, as shown by an absence of larval populations and of leaf mining and feeding. The transgenic lines sustained very little leaf damage from saltmarsh caterpillar, Estigmene acrea (Drury), and beet armyworm, Spodoptera exigua (Hubner), whereas the control cultivars were almost defoliated by season’s end. As expected, the transgenic lines were not resistant to several nonlepidopterous insect pests, and they did not affect populations of beneficial insects. Higher populations of sweetpotato whitefly, Bemisia tabaci (Gennadius), on the transgenic lines than on the control cultivars may have been a consequence of reduced leaf feeding damage (by lepidopterous insects) rather than increased whitefly susceptibility of the transgenic lines. A breeding strategy to increase the insect resistance of cotton plants would be to combine the bacterial toxin trait with other resistance traits such as nectariless, okra leaf, and early maturity, known to reduce pink bollworm and other pest insects.

Resistance Risk Assessment: Realized Heritability of Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae), Tobacco Budworm (Lepidoptera: Noctuidae), and Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Abstract: A new method for estimating realized heritability ( h 2) of resistance to insecticides from laboratory selection experiments is described. A major advantage of this method for estimating h 2, the proportion of total phenotypic variation attributable to additive genetic variation, is that it requires only data that are collected routinely in selection studies (i.e., LC50 and slope before and after selection, and average mortality caused by selection each generation). Estimates of h 2 of resistance calculated with this method were virtually identical to two previously reported estimates calculated by a regression method that requires estimates of LCso for every generation. Estimates of h2 of resistance to Bacillus thuringiensis in Plutella xylostella (L.) were 0.14, 0.17, and 0.18 in three strains initiated from a moderately resistant field population in Hawaii compared with 0.047 in a laboratory strain from France. Estimated h 2 of resistance to B. thuringiensis was 0.17 in Helicoverpa virescens (F.) and 0.09 in Leptinotarsa decemlineata (Say). Extrapolation to field populations is problematic. However, assuming that these h 2 estimates can be extrapolated to the field and that populations receive prolonged and uniform exposure to B. thuringiensis causing >90% mortality each generation, substantially increased resistance would be expected after 150 previously reported studies. This approach can improve evaluation of results from selection experiments and may enhance their integration into systems of resistance risk assessment.

Gene encoding a coleopteran-active toxin

Abstract: The subject invention concerns a novel microbe and gene encoding a novel toxin protein with activity against insect pests of the order Coleoptera. Pests in the order Coleoptera do heavy damage to crops, e.g., corn. The novel Bacillus thuringiensis microbe of the invention is referred to as B.t. PS50C. The spores or crystals of this microbe, or mutants thereof, are useful to control coleopteran pests in various environments. The novel gene of the invention can be used to transform various hosts wherein the novel toxic protein can be expressed.

Inheritance of Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae)

Abstract: We tested the offspring of various crosses of Plutella xylostella (L.) to determine the mode of inheritance of resistance to a commercial formulation of the microbial insecticide Bacillus thuringiensis . The progeny of reciprocal F 1 crosses (resistant male ° susceptible female and resistant female ° susceptible male) responded alike in bioassays, indicating autosomal inheritance. The LCso of F 1 progeny was not significantly greater than the LCso of a susceptible strain, showing that resistance was recessive. Responses of progeny from a backcross (F1 ° resistant strain) corresponded more closely to predictions from a single-locus model than to predictions from models with 2, 5, or 10 loci. Deviations between observed and expected outcomes increased as the number of loci in the model increased. Estimation of the minimum number of effective factors ( sensu Lande 1981) also suggests that the number of loci influencing resistance was not large. In summary, our results suggest that resistance was controlled primarily by one or a few loci.

Interaction of Genetically Engineered Host Plant Resistance and Natural Enemies of Heliothis virescens (Lepidoptera: Noctuidae) in Tobacco

Abstract: Field experiments were conducted to examine interactions of Heliothis virescens (F.), its natural enemies, and tobacco plants engineered to express low levels of Bacillus thuringiensis Berliner toxin. Survival of H. virescens larvae was measured in response to four treatments: (1) toxin-producing plants exposed to natural enemies, (2) toxin-producing plants caged to exclude enemies, (3) toxin-free plants exposed to enemies, and (4) toxin-free plants caged to exclude enemies. B. thuringiensis toxin mediated resistance caused a significant decrease in first-instar survival, and natural enemies caused a significant decrease in third-instar survival. Survival of uncaged first instars as a proportion of survival of caged first instars was significantly lower on toxic plants than control plants, indicating synergism of resistance and natural enemies. Among collections of artificially infested larvae, parasitism by Campoletis sonorensis (Cameron) was significantly higher on toxic plants than on control plants, another indication of synergism. Among collections of wild larvae and larvae censused in the field, parasitism usually did not differ between plant lines. Larval development of H. virescens was significantly slower on toxic plants than on control plants in two out of five trials. Prolonged vulnerability to natural enemies appeared to provide a mechanism for synergism. However, synergistic increases in mortality and parasitism were detected in two trials when development rates on toxic plants and control plants were equal, indicating existence of another mechanism. B. thuringiensis toxin-mediated partial resistance appeared compatible with natural enemies for control of H. virescens . However, a simulation using a theoretical population genetic model suggested that synergism of the level measured in this study could accelerate pest adaptation to resistant plants.

Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species.

Abstract: Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2. Images

Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae.

Abstract: A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.

Field Evaluation of Transgenic Tobacco Containing Genes Encoding Bacillus thuringiensis -Endotoxin or Cowpea Trypsin Inhibitor: Efficacy Against Helicoverpa zea (Lepidoptera: Noctuidae)

Abstract: Transgenic tobacco plants expressing Bacillus thuringiensis var. kurstaki HD-73 delta-endotoxin or cowpea, Vigna unguiculata , trypsin inhibitor (CpTI) were evaluated for efficacy against artificial infestations of Helicoverpa zea (Boddie) under field conditions. Mortality of H. zea larvae was significantly higher and leaf damage significantly lower for the genotype containing Bacillus thuringiensis gene compared with nontransgenic control. Few H. zea larvae survived beyond first instar on the Bacillus thuringiensis genotype. Larval mortality was also higher and leaf damage lower on the CpT1 genotype than its corresponding control, but the effect was less consistent and less pronounced than that produced by the Bacillus thuringiensis genotype. Neither transgenic genotype had a significant effect on natural infestations of the other phytophagous or predacious insects that were monitored.

Chemical Regulation of Bacillus Thuringiensis ∂ -Endotoxin Expression in Transgenic Plants

Abstract: It has been suggested that widespread cultivation of transgenic crops that express high constitutive levels of the lepidopteran–specific Bacillus thuringiensis ∂–endotoxin will lead to selection for insects resistant to the toxin. As a possible method for managing the development of resistance, we have devised a system for temporally controlling the expression of the endotoxin. Tobacco plants harboring a toxin gene driven by a chemically–responsive promoter were treated with a chemical regulator. Chemical induction resulted in accumulation of toxin mRNA, and caused the plants to become insect tolerant. Such controllable gene expression in plants may find broad application in agriculture.

Construction of Novel Bacillus thuringiensis Strains with Different Insecticidal Activities by Transduction and Transformation.

Abstract: The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a cryIA(a) δ-endotoxin gene, were transferred into several Bacillus thuringiensis subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 × 10-8 to 2 × 10-6 transductant per CFU, depending on the strain and on the plasmid. In Cry- and Cry+ native recipients, the introduction of the cryIA(a) gene resulted in the formation of large bipyramidal crystals that were active against the insect Plutella xylostella (order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into B. thuringiensis subsp. entomocidus, aizawai, or israelensis by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene cryIIIA, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various B. thuringiensis recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned δ-endotoxin genes in the different recipients. Images

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

Abstract: When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident δ-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.

Factors influencing the activity of Bacillus thuringiensis var. israelensis treatments.

Abstract: Environmental factors influence the effectiveness of microbial control agents in mosquito control programs. Four of these factors (water temperature, larval density, sunlight and the effect of associated filter feeders) were studied with Bacillus thuringiensis var. israelensis under laboratory and semifield conditions in Europe using different instars of Aedes vexans, Ae. aegypti and Culex pipiens. Bioassays conducted at a low temperature (5 degrees C) yielded 10-fold higher LC50 and LC90 values compared with those conducted at a high temperature (25 degrees C). The efficacy of B.t.i. decreased in a linear manner with increasing larval density. Sunlight can reduce the effectiveness of B.t.i. by several times. Competition in food intake by filter feeding Daphnia resulted in lower mortality of mosquito larvae after B.t.i. applications.

Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination.

Abstract: We describe a novel approach for the insertion of an insecticidal toxin gene into a resident plasmid in Bacillus thuringiensis (Bt). A gene encoding a coleopteran-specific toxin was cloned within a fragment of IS232 and inserted into a plasmid thermosensitive for replication in Bt. The plasmid was used to transform a Bt strain toxic to lepidoptera, and the transformants were then selected at non-permissive temperature for clones in which the vector had integrated into a copy of IS232 present on a resident plasmid. A second recombination event was selected such that the vector was eliminated and the newly introduced toxin gene was conserved. The resulting strain contained only DNA of Bt origin, and displayed insecticidal activity against both lepidoptera and coleoptera.

Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis.

Abstract: Summary The Bacillus thuringiensis cryllAδ-endotoxin gene is found as the third-gene in a three-gene operon, with a sporulation-dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Naturally occurring Bacillus thuringiensis in Indonesia

Abstract: S. HASTOWO, B.W. LAY AND M. OHBA. 1992. A total of 135 strains of Bacillus thuringiensis were isolated from soils of sericultural and natural environments of various regions in Indonesia: Sumatra, Java, Bali, Timor, Sulawesi and Kalimantan. Serologically, 63 strains were assigned to flagella (H) serotypes 3abc, 3ade, 4ac, 5ac, 6ab, 8ab, 9, 11, 17, 20ac, and 24, indicating a varied flora of B. thuringiensis in Indonesia. Of these, the serotype 3ade predominated, followed by the serotypes 3abc and 6ab. The other 72 strains (53·3%) were not sero-reactive against any of the H antisera to B. thuringiensis H serotypes 1–24. In toxicity tests, 34 strains belonging to serotypes 3abc, 3ade, 4ac, and 8ab showed larvicidal activity to the silkworm, Bombyx mori, while 74·8% did not exhibit insecticidal activity against larvae of B. mori and the mosquitoes, Aedes aegypti and Culex quinquefasciatus. Morphotypes of parasporal inclusions were not correlated with insecticidal activities.