Showing papers on "Bacillus thuringiensis published in 1982"

Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus

Abstract: The recently discovered high-frequency transfer of plasmids between strains of Bacillus thuringiensis was used to study the genetic relationship between plasmids and production of the insecticidal delta-endotoxin crystal. Three strains of B. thuringiensis transmitted the Cry+ (crystal-producing) phenotype to Cry- (acrystalliferous) B. thuringiensis recipients. Agarose gel electrophoresis showed that one specific plasmid from each donor strain was always present in Cry+ "transcipients." The size of the transmissible crystal-coding plasmid varied with the donor strain, being 75 MDal (megadaltons) in size in HD-2, 50 MDal in HD-73, and 44 MDal in HD-263. Immunological analysis showed the Cry+ transcipients to be hybrid strains, having flagella of the recipient serotype and crystals of the donor serotype. These results demonstrate that the structural genes for the delta-endotoxin are plasmid borne. Crystal-coding plasmids also transferred into two strains of the related species Bacillus cereus and yielded transcipients that produced crystals of the same antigenicity as the donor strain.

Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715.

Abstract: From a clone bank of the entire genome of Bacillus thuringiensis, one clone that contains a plasmid ( pBT 15-88) harboring a sporulation gene was identified by molecular hybridization. This gene, identified as the crystal protein gene, occurs both on a large host plasmid DNA and in the chromosomal DNA in B. thuringiensis strain berliner 1715. The inserted sequence of pBT 15-88, which corresponds to the chromosomal sequence, was not expressed in Escherichia coli. In B. thuringiensis (kurstaki), the crystal gene was found only on a large host plasmid while in B. thuringiensis ( dendrolimus ), it is only on the chromosomal DNA. The plasmid crystal gene was cloned by ligation of a 14-kb BamHI fragment of a host plasmid DNA of 42 megadaltons from strain berliner 1715 into the BamHI site of the bifunctional vector pHV33 . In E. coli and in sporulating B. subtilis the plasmid pBT 42-1 coded for a polypeptide, detected by antibodies against the crystal protein, with the same electrophoretic mobility as the crystal protein of B. thuringiensis. The crystal gene was not expressed in vegetative cells of B. subtilis, suggesting that the control at the transcriptional level is the same in B. subtilis and in B. thuringiensis. Protein extracts from the clones harboring the hybrid plasmid are toxic for the larvae of Pierris brassicae and the protein antigen forms cytoplasmic inclusion bodies in E. coli and B. subtilis, which are visible under the light microscope.

Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki

Abstract: Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen.

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

Abstract: The crystal-forming proteins (δ-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (Mr of ca. 145,000–130,000) contain an N-terminal domain (Mr of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (Mr of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with Mr of 15,000–35,000.

Possible Mechanism for Synergism between Bacillus thuringiensis and the Gypsy Moth (Lepidoptera: Lymantriidae) Parasitoid, Apanteles melanoscelus (Hymenoptera: Braconidae)

Abstract: Gypsy moth larvae, Lymantria dispar L., were fed various doses of Bacillus thuringiensis Berliner and parasitized by Apanteles melanoscelus (Ratzeburg) before or after infection. The pathogen did not directly influence, nor was it influenced by, the parasitoid. Development of those host larvae which survived doses of B. thuringiensis was delayed compared to that of uninfected larvae. Hosts treated in the 2nd instar and held 10 days were attacked to a greater extent than similarly held, nontreated hosts of the same age, probably because more of the former had not yet molted to the 4th instar. The pathogen-induced developmental lag may explain the relatively high amount of parasitism by A. melanoscelus in B. thuringiensis field plots.

Bacillus thuringiensis in grain elevator dusts

Abstract: Grain dust from four large grain elevators along the Mississippi River near New Orleans, Louisiana, were analyzed for the presence of Bacillus thuringiensis, a pathogen of lepidopterous insects. Bo...

Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Abstract: Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing

Relationship of the syntheses of spore coat protein and parasporal crystal protein in Bacillus thuringiensis.

Abstract: Two major classes of polypeptides were extracted from the spore surface of Bacillus thuringiensis subsp. kurstaki: the 134,000-dalton protoxin that is the major component of the crystalline inclusion and spore coat polypeptides very similar to those found on Bacillus cereus spores. The quantity of spore coat polypeptides produced was reduced when compared with that produced by certain acrystalliferous mutants or by B. thuringiensis subsp. israelensis. The latter organism produced an inclusion toxic to mosquito larvae, but deposited very little of the inclusion protein on the spore surface. The reduction in spore coat protein in B. thuringiensis subsp. kurstaki was also seen in freeze-etched electron micrographs of spores. B. thuringiensis subsp. kurstaki spores germinated rather slowly when compared with related species, a property previously correlated with a deficiency or defect of the spore coat. Many mutants of B. thuringiensis subsp. kurstaki unable to form a crystalline inclusion were nontoxic and lacked a well-defined spore coat. Other mutants isolated either directly from the wild type or from coat-deficient mutants produced spores that were identical to those produced by the closely related species. Bacillus cereus, on the basis of morphology, germination rate, and the size and antigenicity of the spore coat polypeptides. Most of the protein extractable from the inclusion produced by B. thuringiensis subsp. israelensis was about 26,000 daltons, considerably smaller than the major polypeptide extractable from other inclusions. Some of the B. thuringiensis subsp. israelensis inclusion protein was found on the spore surface, but the majority of the extractable spore coat protein was the same size and antigenicity as that found on B. cereus spores. The B. thuringiensis subsp. israelensis spores germinated at a rate close to that of B. cereus, especially when the spores were formed at 37 degrees C, and the morphology of the spore surface was very similar to that of B. cereus. Images

Plasmids coding for B. Thuringiensis crystal protein, their production and their use in creating genetically engineered bacterial strains, the aforesaid strains per se and their use in producing said protein, said protein so-produced, insecticidal formulations comprising the same and a method of producing an insecticidal effect

Abstract: The crystal protein of Bacillus thuringiensis is an insecticidal substance. Unfortunately growth phase limitations are exhibited by B. thuringiensis in producing this protein. This invention, however, provides a plasmid capable of replication in a bacterial host species and containing expressible heterologous DNA coding for the crystal protein of Bacillus thuringiensis and including an expression mechanism for said heterologous DNA which is recognized by the host species system but does not exhibit substantial growth phase limitations in the bacterial host species. Genetically engineered bacterial host strains transformed by the plasmids of the invention express B. thuringiensis crystal proteins without exhibiting the aforesaid growth phase limitations.

Suppression of Heliothis spp. on Cotton by using Bacillus thuringiensis, Baculovirus heliothis, and Two Feeding Adjuvants

Abstract: The application of Bacillus thuringiensis Berliner and Baculovirus heliothis to cotton suppressed Heliothis spp. populations an average of 28% compared with the untreated check during field tests in 1980. The addition of the feeding adjuvants Coax® and Gustol® to the microbial insecticides increased the control by reducing the number of larvae by 46% in 1979 and 54% in 1980. The two feeding adjuvants Gustol® and Coax® improved efficacy of the microbial insecticides, but neither proved better than the other. In addition, spray combinations of B. heliothis + B. thuringiensis + feeding adjuvants did not prove to be superior to either pathogen alone + feeding adjuvants. For full-season use, the Heliothis spp. control in cotton using the microbials plus feeding adjuvants was not adequate compared with the insecticide standard permethrin. Significantly higher yields and lower Heliothis spp. populations were found With permethrin.

Evaluation of Bacillus thuringiensis -Spray Adjuvant-Viral Insecticide Combinations Against Heliothis spp. (Lepidoptera: Noctuidae)

Abstract: Mortality of Heliothis zea (Boddie) and Heliothis virescens (Fabricius) larvae after treatment with Bacillus thuringiensis (Berliner) was determined in bioassays with treated cotton leaves. Larval mortality depended upon dosage, species, and size of larvae tested. Bacillus thuringiensis was also found to reduce the weight of larvae surviving treatment (7 days) when compared with the untreated check. Bioassays failed to detect differences between two commercial B. thuringiensis formulations, Dipel (Abbott Laboratories, North Chicago, III.) and Thuricide (Sandoz, Inc., San Diego, Calif.). Effectiveness of B. thuringiensis was usually increased when treatments were made in combination with the commercial spray adjuvants, Coax (Trader Oil Mill Co., Ft. Worth, Tex.) and Gustol (Sandoz, Inc., San Diego, Calif.). Combinations of B. thuringiensis and nuclear polyhedrosis viruses of H. zea and Autographa californica (Speyer) usually resulted in mortality levels greater than that of B. thuringiensis alone but not greater than that of either virus alone. Advantages for combining B. thuringiensis with the nuclear polyhedrosis virus of H. zea or A. californica were not detected in this study.1

Effect of strain and medium variation on mosquito toxin production by Bacillus thuringiensis var. israelensis.

Abstract: The effect of strain variation and culture medium on production of toxin lethal to mosquito larvae by Bacillus thuringiensis var. israelensis serotype H-14 was investigated. Shake flask culture of B. thuringiensis H-14 strains showed varied ability to produce toxins lethal to mosquito larvae dependent upon the particular strain and growth medium used. Buffered media demonstrated no better mosquito toxicity than did unbuffered media that ranged in pH from 5.7 to 8.1 at harvest. Although toxin production is associated with sporulation, spore count was generally not proportional to toxin produced for those strains and media evaluated.

Influence of Suspended Particulates on the Activity of Bacillus thuringiensis Serotype H-14 Against Mosquito Larvae

Abstract: The efficacy of spore-crystal formulations of Bacillus thuringiensis Berliner serotype H-14 (var. israelensis de Barjac) decreased against mosquito larvae when in aqueous environments containing a concentration of soil or clay particles ≥ 0.5 mg/ml. Fresh whole-culture B. thuringiensis var. israelensis was similarly affected by the soil and clay particles. Sand grains did not have a strong effect on efficacy except when finely ground into particles of 147 μ or less. Settling out of the particulates before the addition of B. thuringiensis var. israelensis did reduce their suppressive effect on the bacterium.

Asporogenous Bacillus thuringiensis Mutant Producing High Yields of δ-Endotoxin

Abstract: An asporogenous Bacillus thuringiensis subsp kurstaki strain IK mutant, strain 290-1, which produced high yields of δ-endotoxin, was obtained by ethyl methane sulfonate treatment of a spore suspension The mutant strain produced about the same amount of δ-endotoxin as that produced by the parent strain, but 1010 to 1011 cells did not form any detectable dormant spores

Effects of Bacillus thuringiensis var. kurstaki on tobacco budworm (Lepidoptera: Noctuidae) adult and egg stages

Abstract: Efficacy of Bacillus thuringiensis Berliner var. kurstaki against the adult and egg stages of Heliothis virescens (F.) was studied under laboratory conditions. The longevity and fecundity of adults were significantly reduced when a 5% sucrose solution containing 32,000 IUs of B. thuringiensis per ml was provided as a food source. Tobacco budworm eggs sprayed directly with B. thuringiensis hatched normally, but larval survival was significantly affected, indicating that some larvae had consumed a lethal dose during eclosion. Mortality was directly related to dosage level and age of egg.

Enzyme-linked immunosorbent assays for detection and quantitation of the entomocidal parasporal crystalline protein of Bacillus thuringiensis subspp. kurstaki and israelensis

Abstract: An enzyme-linked immunosorbent assay was used to detect and quantitate the parasporal crystal toxins of Bacillus thuringiensis subspp. kurstaki and israelensis. The assay method described is extremely sensitive, accurate, and highly specific. With this technique, crystalline insecticidal proteins from several subspecies of B. thuringiensis were compared. The dipteran crystal toxin produced by B. thuringiensis subsp. israelensis was shown to share few epitopes with the lepidopteran toxin from B. thuringiensis subspp. kurstaki, tolworthi, berliner, and alesti.

Comparative bioassays of Bacillus thuringiensis H-14 formulations against four species of mosquitoes in Malaysia.

Abstract: Comparative laboratory bioassays of three formulations of Bacillus thuringiensis H-14 (IPS-78, San 402-I and Bactimos) were conducted against late 3rd/early 4th instar larvae of four species of mosquito, viz., Aedes aegypti, Culex quinquefasciatus, Anopheles balabacensis and Mansonia (Mansonioides) indiana, in Malaysia. From the average response of the mosquito larvae to the three formulations of B. thuringiensis H-14, Ae. aegypti was found to be most susceptible, followed by Cx. quinquefasciatus, An. balabacensis and M. (M.) indiana in decreasing order. The LC50 values for Ae. aegypti, Cx. quinquefasciatus, An. balabacensis and M. (M.) indiana after a 48-hour exposure to IPS-78 formulation were 50.9, 129.3, 117.8 and 169.6 International Toxic Unit (ITU) Ae. ae./l; to San 402-I formulation were 54.6, 223.1, 405.1 and 177.6 ITU Ae. ae/l and to Bactimos formulation were 57.2, 175.7, 35.6 and 514.5 ITU Ae. ae./l respectively. The efficacy of the bacterial product was also found to be determined by its formulation in relation to the feeding and resting habits of the mosquito larvae. No delayed pupation or emergence was observed on the larvae exposed to B. thuringiensis H-14 at sub-lethal concentrations.

The Effect of Bacillus Thuringiensis Var. Israelensis on Toxorhynchites Rutilus Rutilus (Diptera: Culicidae) in the Presence and Absence of Prey

Abstract: The effect of Bacillus thuringiensis var. Israelensis (serotype 14; IPS-78) on larvae of Toxorhynchites rutilus rutilus was determined in the laboratory. The survival of 4th-instar larvae exposed to 10 ppm B. thuringiensis (H-14) in the absence of prey was unaffected after 10 days of continuous exposure. Fourth instars exposed to 1.0, 5.0 and 10 ppm of the bacteria in the presence of excess prey (20 Aedes aegypti larvae) responded with 23, 62 and 95% mortality, respectively, after 10 days. One ppm B. thuringiensis (H-14) in the presence of 5, 10 and 20 Ae. aegypti larvae produced 9, 21 and 23% mortality, respectively, after 10 days. First instars were considerably more susceptible to B. thuringiensis (H-14) than were older instars. In the presence of excess prey, 98% mortality was observed 10 days after exposure to 0.5 ppm; those larvae fed on a nonliving diet at the same concentration responded with 80% mortality. No significant difference in mortality was observed between 4th instars exposed to excess prey at 1.0 ppm (IPS-78) or to cadavers of Ae. aegypti that had fed on 1.0 ppm B. thuringiensis (H-14) 24 h earlier. Neither blended cadavers that had died as a result of consuming B. thuringiensis (H-14) nor water in which cadavers had set overnight produced mortality in 4th instars of Tx. r. rutilus .

Survival of Tobacco Budworm (Lepidoptera: Noctuidae) Larvae after Short-Term Feeding Periods on Cotton Treated with Bacillus thuringiensis

Abstract: Studies were conducted to determine if any adverse effects ensued when larvae of Heliothis virescens (F.) survived various feeding periods on cotton treated with Bacillus thuringiensis Berliner. Some of the 2nd instars that were fed B. thuringiensis -treated terminals for 6, 18, or 30 h and then transferred to an untreated diet survived. The ability of larvae to recover decreased with increases in dosage rate or exposure time. After feeding periods of 18 or 30 h, duration of the larval stage was significantly increased. Effects on pupae were similar. No effects on adult longevity, fecundity, egg viability, or F1 larval survival were detected.

Efficacy of Dipel and Geocoris punctipes (Hemiptera: Lygaeidae) against the Tobacco budworm (Lepidoptera: Noctuidae) on Cotton

Abstract: The efficacy of Bacillus thuringiensis Berliner in combination with Geocoris punctipes (Say) for control of Heliothis virescens (F.) on cotton was studied under greenhouse and field cage conditions. Various rates of B. thuringiensis (141, 282,561, and 1,121 g/ha) were evaluated in combination with predator densities ranging from one G. punctipes nymph per plant to one per four plants. Results indicated potential for good control with rates as low as 282 g of B. thuringiensis per ha and one Geocoris nymph per four plants.

High-Temperature Sensitivity of Formulations of Bacillus thuringiensis var. israelensis

Abstract: Formulations of the bacterium Bacillus thuringiensis var. israelensis , but not Bacillus thuringiensis var. kurstaki , lost most of their insecticidal activities during 28 days of exposure at 50°C. The half-lives of one liquid-flowable and two wettable-powder formulations of B. thuringiensis var. israelensis were 18.0, 7.2, and 6.0 days, respectively. No loss in activity was detected for the wettable-powder formulation of B. thuringiensis var. kurstaki . On the basis of viable spore counts, half-lives were 6.0 days for the liquid-flowable formulation, and 18.5 and 10.5 days for the two wettable-powder formulations, of B. thuringiensis var. israelensis . Differences in thermal sensitivity were attributed to relative differences in the ultrastructure of endocrystals of B. thuringiensis var. israelensis and B. thuringiensis var. kurstaki .

Monoclonal antibodies against functionally distinct sites on the delta-endotoxin of Bacillus thuringiensis var. thuringiensis

Abstract: Monoclonal antibodies against the toxic units ofBacillus thuringiensis delta-endotoxin were raised by the hybridoma technique and detected by an indirect enzyme-linked immunosorbent assay (ELISA). Out of 5 positive clones, I was found to secrete antibodies which inactivate the toxin.