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

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.     

An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subspp. kurstaki and israelensis

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki . The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis .     

Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.     

An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subsp. kurstaki and israelensis

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki. The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis.     

Microbial Utilization of Free and Clay-Bound Insecticidal Toxins from Bacillus thuringiensis and Their Retention of Insecticidal Activity after Incubation with Microbes

The insecticidal toxins produced by Bacillus thuringiensis subspp. kurstaki and tenebrionis were resistant when bound on clays, but not when free, to utilization by pure and mixed cultures of microbes as sources of carbon and carbon plus nitrogen, and their availability as a nitrogen source was reduced. The bound toxins retained insecticidal activity both before and after exposure to microbes or pronase. The insecticidal activity of the toxins persisted for 40 days (the longest time evaluated) in nonsterile soil continuously maintained at the -33-kPa water tension and room temperature, alternately air dried and rewetted to the -33-kPa water tension, or alternately frozen and thawed, although alternate drying and wetting reduced the activity.     

Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.     

Activated insecticidal crystal proteins from Bacillus thuringiensis serovars killed adult house flies

Insecticidal crystal proteins (ICP) from Bacillus thuringiensis serovar kurstaki HD-1 and HD-73 were activated by immobilized trypsin or chymotrypsin. The activated toxins (10 μ g or more) as well as unactivated ICP killed adult house flies but not larvae. Bacillus thuringiensis strain son diego did not kill house flies. In this experimental system, the average life span of the adult house fly was 8 days and the activated toxins reduced it to 2 days. The unactivated insecticidal crystal protein also reduced it to 4 days at the same concentration as the activated toxin.     

The bacterium, Lysinibacillus sphaericus, as an insect pathogen

Since the first bacteria with insecticidal activity against mosquito larvae were reported in the 1960s, many have been described, with the most potent being isolates of Bacillus thuringiensis or Lysinibacillus sphaericus (formerly and best known as Bacillus sphaericus). Given environmental concerns over the use of broad spectrum synthetic chemical insecticides and the evolution of resistance to these, industry placed emphasis on the development of bacteria as alternative control agents. To date, numerous commercial formulations of B. thuringiensis subsp. israelensis (Bti) are available in many countries for control of nuisance and vector mosquitoes. Within the past few years, commercial formulations of L. sphaericus (Ls) have become available. Because Bti has been in use for more than 30 years, its properties are well know, more so than those of Ls. Thus, the purpose of this review is to summarise the most critical aspects of Ls and the various proteins that account for its insecticidal properties, especially the mosquitocidal activity of the most common isolates studied. Data are reviewed for the binary toxin, which accounts for the activity of sporulated cells, as well as for other toxins produced during vegetative growth, including sphaericolysin (active against cockroaches and caterpillars) and the different mosquitocidal Mtx and Cry toxins. Future studies of these could well lead to novel potent and environmentally compatible insecticidal products for controlling a range of insect pests and vectors of disease.     

Dot Blot Enzyme-Linked Immunosorbent Assay for Monitoring the Fate of Insecticidal Toxins from Bacillus thuringiensis in Soil

The release of transgenic plants and microorganisms expressing truncated genes from Bacillus thuringiensis that code for active insecticidal toxins rather than for the inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clay minerals and other soil particles. To monitor the fate of these toxins in soil, a dot blot enzyme-linked immunosorbent assay (ELISA) that detects free and particle-bound toxins from B. thuringiensis subsp. kurstaki and subsp. tenebrionis was developed. The lower limit of detection of the toxins, either free or adsorbed or bound on the clay minerals montmorillonite (M) or kaolinite (K) or on the clay-particle-size fraction separated from soil (by sedimentation according to Stokes' Law), was approximately 3 ng. Antibodies (Ab) to the toxins from B. thuringiensis subsp. kurstaki and from B. thuringiensis subsp. thuringiensis were raised in goats and rabbits, respectively, and each Ab was rendered specific by adsorption onto CNBr-activated Sepharose coupled with the other toxin. The preadsorbed Ab were specific for the toxins from both subspecies, both free and bound on M, K, or the clay-particle-size fraction of soil. The toxins that were added to sterile and nonsterile soil amended with M or K or not amended were detected on the clay-particle-size fraction of the soil after various periods of incubation by the dot blot ELISA. No toxins were detected on the silt- and sand-particle-size fractions. Each dot blot, containing various amounts of toxins and/or clays, was applied to a polyvinylidene difluoride membrane in a dot blot vacuum system. The toxins were still detectable on the clay-particle-size fraction of nonsterile soil after 40 days. This agreed with preliminary results of other studies in this laboratory that when these toxins bind on clay minerals, they become resistant to utilization by microorganisms.     

Influence of Exposure to Single versus Multiple Toxins of Bacillus thuringiensis subsp. israelensis on Development of Resistance in the Mosquito Culex quinquefasciatus (Diptera: Culicidae)

The impending widespread use of transgenic crop plants encoding a single insecticidal toxin protein of Bacillus thuringiensis has focused attention on the perceived risk of rapid selection of resistance in target insects. We have used Bacillus thuringiensis subsp. israelensis toxins as a model system and determined the speed and magnitude of evolution of resistance in colonies of the mosquito Culex quinquefasciatus during selection for 28 consecutive generations with single or multiple toxins. The parental strain was synthesized by combining approximately 500 larvae from each of 19 field collections obtained from the states of California, Oregon, Louisiana, and Tennessee. At least 10,000 larvae were selected in each generation of each line at an average mortality level of 84%. The susceptibilities of the parental and selected lines were compared in parallel tests in every third generation by using fresh suspensions of toxin powders. The normal toxin complement of B. thuringiensis subsp. israelensis consists of four toxins, CryIVA, CryIVB, CryIVD, and CytA. Resistance became evident first in the line that was selected with a single toxin (CryIVD), attaining the highest level (resistance ratio [RR], >913 at 95% lethal concentration) by generation F(inf28) when the study was completed. Resistance evolved more slowly and to a lower level (RR, >122 by F(inf25)) in the line selected with two toxins (CryIVA+CryIVB) and lower still (RR, 91 by F(inf28)) in the line selected with three toxins (CryIVA+CryIVB+ CryIVD). Resistance was remarkably low (RR, 3.2) in the line selected with all four toxins. The results reveal the importance of the full complement of toxins found in natural populations of B. thuringiensis subsp. israelensis as an effective approach to resistance management.     

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.     

Bacillus spp. toxicity against Haemonchus contortus larvae in sheep fecal cultures

The gastrointestinal nematode Haemonchus contortus is a major productivity constraint in sheep. In this study, the nematicidal effects of Bacillus circulans, Bacillus cereus, Bacillus thuringiensis var. israelensis, Bt. var. osvaldocruzi, Bt. var. morrisoni, and Bt. var. kurstaki were assessed in free-living larval stages of H. contortus. A spore-crystal suspension containing approximately 2×10(8)UFC/mL of each strain was added to sheep feces that were naturally infected with H. contortus eggs, and the presence of larvae was then evaluated. We observed a significant (p>0.05) reduction in larval development when using B. circulans, B. thuringiensis var. israelensis, Bt. var. osvaldocruzi and Bt. var. kurstaki, and these effects were proportional with the amount of bacteria added to the feces. However, no effect was observed when Bt. var. morrisoni or B. cereus was added. These observations suggest that these bacteria might be effective as nematicides and may allow for the development of integrated biological control of zooparasitic nematodes.     

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Activation of insect cell adenylate cyclase by Bacillus thuringiensis delta-endotoxins and melittin. Toxicity is independent of cyclic AMP.

Insecticidal Bacillus thuringiensis (Bt) delta-endotoxins are cytolytic to a range of insect cell lines in vitro. Addition of Bt var. aizawai or var. israelensis toxins to Mamestra brassicae (cabbage moth) cells in vitro increased intracellular cyclic AMP, which was paralleled by activation of adenylate cyclase in isolated membranes. Var. kurstaki toxin, which does not lyse M. brassicae cells, had no effect on cyclic AMP concentrations in intact cells, but was able to stimulate adenylate cyclase in membrane preparations. In contrast, the bee-venom toxin melittin, which is also cytolytic, increased intracellular cyclic AMP in whole cells, but inhibited adenylate cyclase in isolated membranes. Octopamine and forskolin also increased cyclic AMP in cells, but were not cytolytic. When added to cells at concentrations exceeding their LC90 (concentration causing 90% cell death), melittin and var. israelensis toxins caused cell lysis without a concomitant increase in intracellular cyclic AMP. Taken together, these results suggest that activation of adenylate cyclase by cytolytic toxins is a secondary effect (related perhaps to interactions of these toxins with membrane lipids) and is neither necessary nor sufficient for cytolysis.     

Formation of Crystalline delta-Endotoxin or Poly-beta-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

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 相似文献   

Susceptibility of Plutella xylostella (L.) (Lepidoptera: Plutellidae) populations in Mexico to commercial formulations of Bacillus thuringiensis

Populations of diamondback moth, Plutella xylostella (L.), sampled from commercial fields of crucifers in three states of Mexico, were tested for susceptibility to commercial formulations of Bacillus thuringiensis subsp. kurstaki (Berliner) (Dipel 2X), B. thuringiensis subsp. aizawai (XenTari), delta endotoxin Cry 1C (MC), and CryIA(c) (MVP), and a mixture of B. thuringiensis subsp. kurstaki and subsp. aizawai (Agree). Leaf-dip bioassays confirmed variation in susceptibility of up to 13-fold for MVP, 12-fold for Dipel 2X, sevenfold for XenTari, fivefold for Agree, and less than fivefold for MC. Comparisons with previously published data indicate that at least the 12-fold variation in Dipel 2X would result in significant differences in control in the field. Based on the LC99 values observed for the products, we propose discriminating concentrations for each product. To ensure continued performance in the field we suggest that a resistance monitoring program be implemented to detect any changes in susceptibility to B. thuringiensis products and specific toxins and that their use be restricted to one generation per crop and that they be rotated with other groups of insecticides. Furthermore, we suggest enforcement of a crucifer host-free period and the development and implementation of cultural and biological control strategies to reduce overall population pressure so that fewer insecticidal treatments will be needed.