Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12.

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.     

Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1.

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.     

Inheritance of resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F(1) reciprocal crosses and backcrosses between F(1) larvae and resistant (P(R)) and susceptible (P(S)) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F(1) crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F(1) larvae was slightly higher than that for P(S), suggesting that resistance is partially recessive. The responses of both backcross progeny (F(1) x P(R), F(1) x P(S)) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F(1) x P(R) backcross but decreased the correspondence with the F(1) x P(S) backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.     

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F₁ reciprocal crosses and backcrosses between F₁ larvae and resistant (P_R) and susceptible (P_S) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F₁ crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F₁ larvae was slightly higher than that for P_S, suggesting that resistance is partially recessive. The responses of both backcross progeny (F₁ × P_R, F₁ × P_S) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F₁ × P_R backcross but decreased the correspondence with the F₁ × P_S backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.     

Comparative toxicity of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki to defoliating forest Lepidoptera.

Insecticidal activities of sporulated cultures of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki were compared against four species of defoliating forest lepidopterans in diet-incorporation assays. There was no difference in LC50 between the two strains to larvae of spruce budworm (Choristoneura fumiferana), gypsy moth (Lymantria dispar), eastern hemlock looper (Lambdina fiscellaria fiscellaria), and whitemarked tussock moth (Orgyia leucostigma), whether expressed as total alkaline soluble protein, activated toxin protein, or International Units as determined by bioassay against Trichoplusia ni. Both strains were consistently more toxic than HD-1-S-1980 when compared on the basis of alkali-soluble protein, but not on the basis of activated toxin or International Units. Hybridization of genomic DNA after restriction with HindIII revealed the presence of all three cryIA toxin genes in each of the isolates used in this study, including HD-1-S-1980, which was previously reported to have lost the cryIA(b) gene.     

The utility of camptothecin as a synergist of Bacillus thuringiensis var. kurstaki and nucleopolyhedroviruses against Trichoplusia ni and Spodoptera exigua

We studied the effect of combining microbial pesticides with camptothecin (CPT) on the mortality of two lepidopteran insects: Trichoplusia ni (Hübner) and Spodoptera exigua (Hübner). CPT is an alkaloid that is often used as an anticancer agent. Here, CPT was evaluated as a microbial pesticide synergist of Bacillus thuringiensis (Bt) and insect baculovirus. The toxicity of CPT and its synergistic effects on two microbial pesticides were studied using the diet overlay method. Bioassay results showed that CPT significantly enhances the toxicity of Bt variety kurstaki to S. exigua and T ni. In addition, CPT strongly enhanced the infectivity of Autographa californica (Speyer) multinucleocapsid nucleopolyhedrovirus (AcMNPV) and S. exigua nucleopolyhedrovirus (SeMNPV). Using light microscopy, we found that CPT disrupts the peritrophic membrane of T. ni larvae and severely affects the structure of the midgut, resulting in an abnormal gut lumen morphology. We speculate that CPT increases toxicity by affecting the permeability of the peritrophic membrane.     

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.     

Mode of Action of Bipyramidal delta-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped delta-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.     

Rapid Identification of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 with Proteomic Analysis

This study was carried out to identify rapidly δ-endotoxin from Bacillus thuringiensis (Bt) subsp. kurstaki HD-1 with proteomic analysis. Protoxin was isolated from sporulated cells and purified by ultracen-trifugation using 40-70% sucrose density gradient. Protoxin was treated with trypsin to analyze digested peptides by liquid chromatographytandem mass spectrometry. The proteomic analysis for detected peptides revealed that this methodology is available for discriminating similar Bt strains by identifying Bt subsp. kurstaki HD-1-specific peptides, suggesting that proteomic analysis can be used for rapid identification of Bt strains.     

Sequence of a lepidopteran toxin gene of bacillus thuringiensis subsp kurstaki NRD-12

Three classes of HindIII hybridizing fragments of 4.5, 5.3 and 6.6 kb in size have been identified in the Bacillus thuringiensis (B.t.) kurstaki NRD-12 strain, a situation similar to the one found in the well-studied B.t. kurstaki HD-1 strain. The probes used were short, synthetic oligodeoxynucleotides derived from the published HD-1 4.5 class delta-endotoxin gene sequence. We have cloned the NRD-12 5.3 class gene in the Escherichia coli plasmid, pUC8, and determined its nucleotide sequence. An open reading frame of 1155 amino acid residues was identified as the delta-endotoxin coding sequence based on its overall homology with the deduced amino acid sequences from the HD-1 4.5 gene and a 6.6 gene from the related kurstaki HD-73 strain. The NRD-12 5.3 gene appears to be a hybrid of the former two genes but displays a unique 78 by deletion and a 12 by insertion in the 3′-coding region. These characteristics have also been reported in two other recently reported genes isolated from B.t. berliner 1715 and kurstaki HD-1. Potential transmembrane segments have been predicted for the three classes of toxins.     

Intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki and their role in protoxin activation

The intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki were studied to identify the endogenous proteases involved in the activation of protoxin. The proteases obtained with 30% ammonium sulfate saturation were analysed by both gelatin zymography and azocasein hydrolysis. Three proteases with molecular mass 92 kDa, 78 kDa and 69 kDa were identified on gelatin gel and their gelatinolytic activity was inhibited by ethylenediamine tetraacetic acid. Significantly, 1,10-phenanthroline caused an inhibition of the azocasein hydrolytic activity by 98% and ethylenediamine tetraacetic acid by 28%. The three proteases were heat-stable at 65 °C, while the 69-kDa protease was active up to 75 °C. Intracellular protease-deficient mutants (ethyl methanesulfonate mutagenesis) could not generate the active toxin suggesting the existence of a specific enzyme affecting the conversion of protoxin to toxin.     

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% -helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% -sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both -helical and -sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

Processing of delta-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae.

Midgut juices were prepared from Adoxophyes sp., smaller tea tortrix (STT); Bombyx mori, silkworm (SW); Spodoptera litura, common cutworm (CCW); Plutella xylostella, diamondback moth (DBM); and Musca domestica, housefly (HF) and immobilized onto Sepharose 4B. delta-Endotoxins (ICPs) from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 were digested by these immobilized gut juice proteases. All gut juices tested derived relatively proteolytic resistant cores from ICP. The molecular sizes of these cores, about 55 kDa in SDS-PAGE, were resulted. In the case of CCW, however, digestion was very strong and only 1/20 concentration of core protein remained relative to other digests. The N-terminal amino acid sequencing of the core proteins showed that they were truncated at the very end of the N-terminus of protoxin, CryIA, at different sites. Although housefly larvae were completely insensitive to active toxin, the gut juice produced the core, suggesting that the housefly may lack the binding sites for the core-active toxin.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Mode of Action of Bipyramidal δ-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped δ-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.     

Isolation and Characterization of a Bacillus thuringiensis ssp. kurstaki Strain Toxic to Spodoptera exigua and Culex pipiens

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species. Received: 19 January 2001 / Accepted: 21 February 2001     

Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis

The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp. kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin. The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting. Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin. The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6). Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change. Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis.     

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.     

Enhancement of Bacillus thuringiensis Toxicity to Lepidopterous Species with the Enhancin from Trichoplusia ni Granulovirus

The enhancin from the Trichoplusia ni (Hübner) granulovirus (TnGV) is a 104-kDa viral protein that can significantly increase the virulence of several baculoviruses. Enhancin can destroy the structural integrity of the peritrophic membrane (PM) of lepidopterous larvae, resulting in more efficient penetration of the PM by virions and avoidance of inactivation by larval midgut factors. In this study we demonstrate that TnGV enhancin can also enhance toxicity of Bacillus thuringiensis (Berliner) (Bt) to several noctuid species. Addition of enhancin significantly increased the toxicity of Dipel, a commercial Bt formulation, to six species (T. ni, Helicoverpa zea (Boddie), Heliothis virescens (Fabricius), Spodoptera exigua (Hübner), Pseudoplusia includens (Walker), and Anticarsia gemmatalis (Hübner)). Furthermore, the effectiveness of four commercial Bt-based products (Cutlass, Javelin, Biobit, and Dipel) against T. ni was enhanced three- to sixfold by adding purified enhancin. We hypothesize that the PM of larvae can impede the movement of the BT toxin protein to the midgut brush border and that enhancin can increase the toxicity by affecting the permeability of the PM.