Showing papers on "Bacillus thuringiensis published in 1985"
TL;DR: It was found that Plodia interpunctella, a major lepidopteran pest of stored grain products, can develop resistance to the insecticide within a few generations, indicating that the resistance can develop quickly in the field.
Abstract: Resistance to the spore-crystal protein complex of Bacillus thuringiensis, the most widely used and intensively studied microbial insecticide, has been presumed to be unlikely to occur. In this study it was found that Plodia interpunctella, a major lepidopteran pest of stored grain products, can develop resistance to the insecticide within a few generations. Resistance increased nearly 30-fold in two generations in a strain reared on diet treated with Bacillus thuringiensis and after 15 generations reached a plateau 100 times higher than the control level. Resistance was stable when selection was discontinued. The resistance was inherited as a recessive trait. Plodia interpunctella strains collected from treated grain bins were more resistant than strains from untreated bins, indicating that the resistance can develop quickly in the field.
425 citations
TL;DR: The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.
302 citations
TL;DR: Synergism Mosquitocidal activity Crystal protein combines with TSP to form a single substance that acts as a “spatially aggregating substance” to reprogram the TSPs into single molecules.
162 citations
TL;DR: This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis by a conjugation-like process.
Abstract: To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.
155 citations
TL;DR: It was concluded that transcription of the Bacillus thuringiensis subspecies israelensis gene is restricted to early sporulation stages and regulation signals are poorly recognized by E. coli RNA polymerase.
Abstract: The Mr 28.000 crystal protein gene of Bacillus thuringiensis subspecies israelensis has been cloned into pBR322 as part of a 9.7 kb HindIII fragment. From hybridization experiments of recombinant p425 DNA with B.t. subspecies israelensis RNA from different stages of growth it was concluded that transcription of the gene is restricted to early sporulation stages. Nucleotide sequence analysis revealed the presence of a large open reading frame with a coding capacity of 249 amino acids (Mr 27.340). Nuclease S1 mapping demonstrated that transcription starts 44 nucleotides upstream of the initiation codon. A Shine-Dalgarno sequence (AAGGAG) was found 10 nucleotides upstream of the translation startpoint. At the 3'-end of the gene a complex secondary structure was found immediately after the stop-codon. Despite the presence of these regulation signals only limited expression in E. coli was detected. This can be explained by assuming that B.t. subsp. israelensis promotor sequences are poorly recognized by E. coli RNA polymerase.
134 citations
TL;DR: From Bacillus thuringiensis subsp.
Abstract: From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.
114 citations
TL;DR: The cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp.
86 citations
TL;DR: The influence of soil pH, moisture, nutrient availability and indigenous microorganisms on the survival and growth of Bacillus thuringiensis and B. cereus spore inocula in soil was investigated and it was suggested that this demise is attributable to a failure of B. thurringiensis to germinate in soil.
Abstract: The influence of soil pH, moisture, nutrient availability and indigenous microorganisms on the survival and growth of Bacillus thuringiensis and B. cereus spore inocula in soil was investigated. The factor of greatest importance was nutrient availability. B. thuringiensis could not grow under most natural soil conditions, whilst B. cereus grew only slowly. Supplementing soil with additional nutrients, or autoclaving, stimulated growth of the Bacillus populations. Growth was reduced by a low soil pH (5.2 compared to 7.3), whilst both Bacillus species grew faster and survived better in wetter (at 0, −0.01 MPa) than drier (at −0.10, −1.00 MPa) soils. The death of B. thuringiensis in natural soil probably accounts for its rarity in the outdoor environment. It is suggested that this demise is attributable to a failure of B. thuringiensis to germinate in soil.
86 citations
TL;DR: Crystalline toxins from Bacillus thuringiensis israelensis and B.t. kurstaki were lethal in vitro to eggs of the nematode Trichostrongylus colubriformis, but over a 77,000-fold range of activity was found, based on the LD50 values.
81 citations
TL;DR: Results are in agreement with previous findings that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.
78 citations
TL;DR: Bacillus thuringiensis subsp.
TL;DR: Two isolates of Bacillus thuringiensis subsp.
Abstract: Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.
TL;DR: The authors showed that Bacillus thuringiensis var. israelensis can grow vegetatively, sporulate, and produce toxin in cadavers of Aedes aegypti and Anopheles albimanus larvae.
TL;DR: The incremental feeding of glucose with continuous pH adjustment prevented cell injury and death which results from prolonged exposure to acidity liberated at the high sugar concentrations which occur when glucose is added batchwise.
Abstract: Physiological studies on Bacillus thuringiensis var. entomocidus revealed the failure of the organism to survive or sporulate under low aeration levels, notably in the presence of high sugar concentrations. Cell counts, sporulation titers and potency of resulting endotoxin were found to vary with the level of aeration. The incremental feeding of glucose with continuous pH adjustment prevented cell injury and death which results from prolonged exposure to acidity liberated at the high sugar concentrations which occur when glucose is added batchwise. Increasing of dipotassium phosphate concentration in growth medium increased the potency of the resulting endotoxin.
01 Jan 1985
Patent•
08 Oct 1985
TL;DR: In this article, a process for producing a strain of Bacillus thuringiensis having improved entomocidal properties by combining into a single strain by plasmid transfer the different entomocalidal properties of two respective starting strains was described.
Abstract: The invention relates to a strain of Bacillus thuringiensis, GC 91, a sample of which has been deposited under the accession number NCTC 118921, or a derivative or mutant thereof having entomocidal activity against lepidopterous pests. The invention also relates to a process for producing a strain of Bacillus thuringiensis having improved entomocidal properties by combining into a single strain by plasmid transfer the different entomocidal properties of two respective starting strains. The new strains thus produced are useful in entomocidol compositions.
Patent•
30 Aug 1985
TL;DR: A DNA fragment that codes for the portion of Bacillus thuringiensis crystal protein peptide that is toxic to lepidopteran insects was revealed in this article.However, it was not shown that the disclosed toxin-encoding DNA fragment is expressible in recombinant host organisms.
Abstract: A DNA fragment that codes for the portion of Bacillus thuringiensis crystal protein peptide that is toxic to lepidopteran insects. The invention also comprises the DNA and amino acid sequences for the disclosed toxin-encoding DNA fragment. In addition the invention demonstrates that the disclosed toxin-encoding DNA fragment (referred to herein as the Bacillus thuringiensis crystal protein gene toxin segment) is expressible in recombinant host organisms, and that the "toxin" protein product produced by the transformed hosts is toxic to lepidopteran insects.
TL;DR: It is concluded that the delta-endotoxins of the isolates under investigation are composed of at least two toxins and genes coding for the production of toxic factors or for their expression may be carried on both the plasmids and the chromosome.
Abstract: Mutants defective in delta-endotoxin crystal production from four closely related isolates of Bacillus thuringiensis var. aizawi with aizawi serotype crystals were as vigorous as the parents in terms of growth, extracellular protease production, sporulation and heat resistance of spores. Spores produced by mutants germinated faster than wild type spores possibly due to deficiency of protein, in the form of delta-endotoxin in the spore coat. Acrystalliferous (cry—) mutants were not active in Galleria mellonella or Pieris brassicae larvae. Mutants with small crystals (sm cry) lost activity or gained extra activity against either one or the other host, revealing the presence of different toxicity factors. Solubilized crystals of parent isolates were composed of two major polypeptides with Mr values of 130 000 and 138 000. Sm cry mutants lost either polypeptide irrespective of which insect potency had been lost. Some cry — and some sm cry mutants had the same plasmid pattern as the parent; others lost one plasmid sometimes gaining another of different size. No consistent correlation was found between plasmid loss in mutants and any loss or increase of potency indicated by bioassays. It is concluded that the delta-endotoxins of the isolates under investigation are composed of at least two toxins. The results suggest that genes coding for the production of toxic factors or for their expression may be carried on both the plasmids and the chromosome.
TL;DR: A screening of the larvicidal activity of the more than 900 strains ofBacillus thuringiensis strains, combining the Institut Pasteur collection was realized, with satisfactory results, but only a few belonging mainly to serovarsaizawai, kenyae andentomocidus showed high level of toxicity.
Abstract: A screening of the larvicidal activity of the more than 900 strains ofBacillus thuringiensis strains, combining the Institut Pasteur collection was realized. A quick bioassay using 1st instar larvae and semi-synthetic medium was developed. Many strains were toxic toSpodoptera littoralis, but only a few belonging mainly to serovarsaizawai, kenyae andentomocidus showed high level of toxicity. The profiles of strain activities differed from serovar to serovar, but within the same serovar toxicity can vary with different strains. Oneaizawai strain tested in the field gave satisfactory results, better than a commercially used strain, tested in the same experiment.
TL;DR: Intensive studies conducted in Eastern Europe to introduce and acclimatize two predatory bugs, Perillus bioculatus and Podisus maculiventris, and one fly have not given positive results so far.
Abstract: Intensive studies conducted in Eastern Europe to introduce and acclimatize two predatory bugs, Perillus bioculatus and Podisus maculiventris, and one fly, Doryphorophaga doryphorae, have not given positive results so far. Seasonal colonization with predators has provided good protection of potato and aubergine crops but the method requires release of high numbers of insects. Two microbial insecticides are produced, registered and used in the USSR for controlling the Colorado beetle, namely Boverin (Beauveria bassiana) and Bitoksybacillin (Bacillus thuringiensis var. thuringiensis 202).
TL;DR: In this paper, a procedure was described whereby both crystal and solubilized Bacillus thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity.
Abstract: The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.
TL;DR: Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity equivalent to that of the parental B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.
Abstract: A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.
TL;DR: Interaction between host plant tannins and δ-endotoxin might be a major factor where the field efficacy of B. thuringiensis preparations is unexpectedly low.
Abstract: Tannin, an important constituent of many plants, reacted strongly with the proteinaceous insecticidal metabolite of Bacillus thuringiensis. Solutions of a commercial tannin preparation stopped the activity of dissolved crystal protein and activated δ-endotoxin. Intact crystals lost their activity only partially in the presence of tannin. Interaction between host plant tannins and δ-endotoxin might be a major factor where the field efficacy of B. thuringiensis preparations is unexpectedly low.
TL;DR: In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae was observed, demonstrating spore germination in living larvae, moribunds, and in cadavers.
TL;DR: Biochemical studies suggested that different protein fragments within the crystal δ-endotoxin may be responsible for the majority of the mosquito larvacidal activity and the neurotoxic symptoms observed in larvae of Trichoplusia ni.
TL;DR: Sublethal doses of Bti appeared to retard development of late instars, and B. kurstaki had minimal activity on this insect when compared to Bti, which was also toxic to larvae of B. coprophila in the greenhouse.
Abstract: The effects of Bacillus thuringiensis var. israelensis (H-14) (Bti) were tested against the fungus gnat, Bradysia coprophila , in the laboratory and the greenhouse. The LC50 of Bti to 9-day-old larvae was 50.9 IU/cm2. When insects were exposed to Bti (50.9 IU/cm2) during the period from egg to pupa, only 8% of the insects survived compared to 84% survival of insects treated only with water. Sublethal doses of Bti appeared to retard development of late instars. B. thuringiensis var. kurstaki had minimal activity on this insect when compared to Bti. In the greenhouse, Bti was also toxic to larvae of B. coprophila .
01 Jan 1985
TL;DR: Data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp.
Abstract: Thecrystal 8-endotoxin ofBacillus thuringiensis subsp. israelensis islesstoxic tolarvae ofAnopheles freeborni thantolarvae ofAedesaegypti. However, whensolubilized crystal wasused, larvae frombothspecies showed similar sensitivities. Thiseffect presumably was duetothedifferences infeeding behavior between the twomosquito larvae whencrystal preparations areused. A procedure isdescribed whereby bothcrystal and solubilized B.thuringiensis subsp. israelensis toxin were emulsified withFreundincomplete adjuvant, with retention oftoxicity. TheuseofFreundincomplete adjuvant also allowed onetoassaythesolubilized toxinat alownanogramlevel. Furthermore, coating thetoxin withlipophilic material altered thebuoyancy ofthetoxin andreversed thesensitivities ofthetwomosquito larvae towardtheB.thuringiensis subsp. israelensis toxin. Thisdifference inbuoyancy was determined byusing anenzyme-linked immunosorbent assaythat was specific forthetoxic peptides. Thesedataindicate thateconomically feasible buoyant formulations fortheB. thuringiensis subsp. israelensis crystal can bedeveloped. Theisolation ofthebacterium Bacillus thuringiensis subsp. israelensis byGoldberg andMargalit (9)hasgenerateda tremendous amountofenthusiasm forusing this organism forbiological control ofmosquito andblack-fly larvae. Subsequent studies haveestablished that acrystalline toxin (8-endotoxin) isproduced andreleased during sporulation ofthebacterium (5,13,18). After ingestion by susceptible hosts, thecrystal isactivated bythealkaline insect gutandexerts its effects onthegutepithelial cells (11, 12). Thespecificity andpotency ofthis toxin toward the mosquito andblack-fly larvae haveresulted inits recommendation bytheWorldHealthOrganization forindustrial production asabiological agentforthecontrol ofinsect vectors formanytropical diseases (1).
TL;DR: The Bacillus thuringiensis subsp.
Abstract: The Bacillus thuringiensis subsp. israelensis mosquito larvicidal toxin is not a sulfhydryl-activated toxin. The protein disulfide bonds were cleaved and blocked without loss of toxicity. In contrast, modification of the lysine side chains eliminated toxicity. Additionally, the toxin was resistant to high concentrations of salt (8 M NaBr), organic solvents (40% methanol), denaturants (4 M urea), and neutral detergents (10% Triton X-100). However, it was inactivated by both positively and negatively charged detergents and by guanidine hydrochloride.
Patent•
22 Mar 1985
TL;DR: In this article, a novel and useful insecticide with activity against insect pests of the order Coleoptera is described, which is a B. thuringiensis microbe given the specie designation M-7.
Abstract: The subject invention concerns a novel and useful insecticide with activity against insect pests of the order Coleoptera. Pests in this order do heavy damage to crops, e.g., corn. The insecticide of the subject invention is a novel B. thuringiensis microbe given the specie designation M-7. The spores or crystal of this microbe are useful to control Coleoptera pests in various environments.
TL;DR: Bacillus thuringiensis is a gram-positive, sporeforming bacterium distinguished by its ability to produce parasporal protein crystals which are toxic to a number of lepidopteran insects of economic importance as well as to certain dipterans which are important vectors of human disease.
Abstract: Bacillus thuringiensis (BT) is a gram-positive, sporeforming bacterium distinguished by its ability to produce parasporal protein crystals. These proteins, collectively known as δ-endotoxins, are toxic to a number of lepidopteran insects of economic importance as well as to certain dipterans which are important vectors of human disease (7). Over 20 different varieties of B. thuringiensis are currently recognized on the basis of flagellar antigens (6) and immunological (19) and toxicity (8) properties of the parasporal crystals.