Potential of the Bacillus thuringiensis Toxin Reservoir for the Control of Lobesia botrana (Lepidoptera: Tortricidae), a Major Pest of Grape Plants

The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.     

Use of Bacillus thuringiensis toxins for control of the cotton pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 microg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 microg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 microg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. 125I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.     

Molecular and insecticidal characterization of a Cry1I protein toxic to insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

The most notable characteristic of Bacillus thuringiensis is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel cry gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in Escherichia coli resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of Earias insulana, Lobesia botrana, Plutella xylostella, and Leptinotarsa decemlineata were 21.1, 8.6, 12.3, and 10.0 microg/ml, respectively. Binding assays with biotinylated toxins to E. insulana and L. botrana midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in B. thuringiensis-treated crops and commercial B. thuringiensis-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to B. thuringiensis toxins.     

Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

Toxicity and larval growth inhibition of 11 insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 microg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect.     

Toxicity of Bacillus thuringiensis delta-endotoxins against bean shoot borer (Epinotia aporema Wals.) larvae, a major soybean pest in Argentina

The toxicity of seven Bacillus thuringiensis Cry protoxins was tested against neonate larvae of Epinotia aporema, a major soybean pest in Argentina and South America. The most active protoxins were Cry1Ab and Cry1Ac, with LC50 values of 0.55 and 1.39 microg/ml, respectively. Cry1Aa, Cry1Ba, Cry1Ca, and Cry9Ca protoxins were equally toxic with LC50 values about 4 microg/ml, whereas Cry1Da was not toxic. The synergistic activity of different protoxin-mixtures was also analyzed, no synergistic effect between the Cry proteins was observed, with the exception of the poorly toxic Cry1Ba/Cry1Da mixture that was slightly synergistic. The binding capacity of individual Cry1 and Cry9Ca toxins to brush border membranes of E. aporema was also determined. The non-toxic Cry1Da toxin was the only toxin unable to bind to E. aporema membranes. In addition the heterologous competition experiments showed that Cry1Ab and Cry1Ac toxins share a common binding site. Based on these data, we propose that Cry1Ab and Cry1Ac toxins could be used in the biological control of E. aporema.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. ¹²⁵I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.     

Screening for Bacillus thuringiensis crystal proteins active against the cabbage looper, Trichoplusia ni

Toxicity tests were performed to find among Cry1 and Cry2 Bacillus thuringiensis crystal proteins those with high activity against the cabbage looper. Tests were performed with neonate larvae on surface-contaminated artificial diet. The crystal proteins found to be toxic were, from higher to lower toxicity: Cry1Ac, Cry1Ab, Cry1C, Cry2Aa, Cry1J, and Cry1F (LC50 of 1.14.1, 3.4-4.4, 12, 34, 87, and 250 ng/cm2, respectively). Cry1B, Cry1D, and Cry1E can be considered nontoxic (LC50 higher than 2500 ng/cm2). Cry1Aa was moderately toxic to nontoxic, depending on the source (LC50 of 420 ng/cm2 from PGS and 8100 ng/cm2 from Ecogen). In vitro binding assays with trypsin-activated 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac crystal proteins and brush border membrane vesicles from midgut larvae showed a direct correlation between toxicity and binding affinity. Heterologous competition experiments indicated that Cry1Aa and Cry1F bind, though only at very high concentrations, to the Cry1Ab/Cry1Ac shared high-affinity binding site.     

Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.     

Susceptibility of dipel-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae) to individual Bacillus thuringiensis protoxins

Dipel-resistant and -susceptible strains of Ostrinia nubilalis (Hübner) were evaluated for larval mortality and growth inhibition when fed diets containing individual Bacillus thuringiensis protoxins. Resistance ratios for four of the protoxins in Dipel (Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa) were 170-, 205-, 524-, and > 640-fold, respectively, considerably higher than the 47-fold resistance to Dipel. The Dipel-resistant strain was 36-fold resistant to Cry1Ba, a protoxin not present in Dipel. Another non-Dipel protoxin, Cry1Ca, did not cause significant mortality for either resistant or susceptible larvae with doses as high as 1.0 mg/ml. In an evaluation of larval growth inhibition, resistance to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ba was significant at concentrations of 0.054 and 0.162 microg/ml. However, growth inhibition with Cry2Aa was not significant at either dose. These data provide information on the spectrum of resistance and cross-resistance to individual Cry protoxins in this strain.     

Activity of Bacillus thuringiensis delta-endotoxins against codling moth (Cydia pomonella L.) larvae

Solubilized protoxins of nine Cry1 and one hybrid Cry1 delta-endotoxin from Bacillus thuringiensis were tested for their activity against larvae of the codling moth (Cydia pomonella L). Cry1Da was the most toxic, followed by Cry1Ab, Cry1Ba, and Cry1Ac, while Cry1Aa, Cry1Fa, Cry1Ia, and SN19 were still less active. Cry1Ca and Cry1Cb showed no activity. In vitro trypsin activation increased activity of all eight active delta-endotoxins, and dramatically enhanced toxicity of hybrid SN19, Cry1Aa, Cry1Ac, and Cry1Fa. The differences between toxicity of proteins before and after trypsin digestion suggests that proteolytic activation in the C. pomonella digestive tract plays a critical role for the activity of Cry proteins against this insect.     

Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella)

Evolution of resistance by pests could cut short the success of transgenic plants producing toxins from Bacillus thuringiensis, such as Bt cotton. The most common mechanism of insect resistance to B. thuringiensis is reduced binding of toxins to target sites in the brush border membrane of the larval midgut. We compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide. Using Cry1Ab and Cry1Ac labeled with (125)I and brush border membrane vesicles (BBMV), competition experiments were performed with unlabeled Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Ja, Cry2Aa, and Cry9Ca. In the susceptible strain, Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ja bound to a common binding site that was not shared by the other toxins tested. Reciprocal competition experiments with Cry1Ab, Cry1Ac, and Cry1Ja showed that these toxins do not bind to any additional binding sites. In the resistant strain, binding of (125)I-Cry1Ac was not significantly affected; however, (125)I-Cry1Ab did not bind to the BBMV. This result, along with previous data from this strain, shows that the resistance fits the "mode 1" pattern of resistance described previously in Plutella xylostella, Plodia interpunctella, and Heliothis virescens.     

A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai

A population (SERD3) of the diamondback moth (Plutella xylostella L.) with field-evolved resistance to Bacillus thuringiensis subsp. kurstaki HD-1 (Dipel) and B. thuringiensis subsp. aizawai (Florbac) was collected. Laboratory-based selection of two subpopulations of SERD3 with B. thuringiensis subsp. kurstaki (Btk-Sel) or B. thuringiensis subsp. aizawai (Bta-Sel) increased resistance to the selecting agent with little apparent cross-resistance. This result suggested the presence of independent resistance mechanisms. Reversal of resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai was observed in the unselected SERD3 subpopulation. Binding to midgut brush border membrane vesicles was examined for insecticidal crystal proteins specific to B. thuringiensis subsp. kurstaki (Cry1Ac), B. thuringiensis subsp. aizawai (Cry1Ca), or both (Cry1Aa and Cry1Ab). In the unselected SERD3 subpopulation (ca. 50- and 30-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai), specific binding of Cry1Aa, Cry1Ac, and Cry1Ca was similar to that for a susceptible population (ROTH), but binding of Cry1Ab was minimal. The Btk-Sel (ca. 600-and 60-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) and Bta-Sel (ca. 80-and 300-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) subpopulations also showed reduced binding to Cry1Ab. Binding of Cry1Ca was not affected in the Bta-Sel subpopulation. The results suggest that reduced binding of Cry1Ab can partly explain resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. However, the binding of Cry1Aa, Cry1Ac, and Cry1Ca and the lack of cross-resistance between the Btk-Sel and Bta-Sel subpopulations also suggest that additional resistance mechanisms are present.     

A binding site for Bacillus thuringiensis Cry1Ab toxin is lost during larval development in two forest pests

The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/microl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with (125)I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.     

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Geographical variation in larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis spore-crystal mixtures and purified crystal proteins and associated resistance development in India

The susceptibility of larvae of the diamondback moth, Plutella xylostella Linnaeus to purified crystal proteins and spore-crystal preparations of Bacillus thuringiensis was investigated for 13 populations from seven states in India. The LC50 (microg ml(-1), 48 h) values of Cry proteins for different populations of P. xylostella ranged from 0.14-3.74 (Cry1Aa), 0.007-1.25 (Cry1Ab), 0.18-2.47 (Cry1Ac) and 0.12-3.0 (Cry1C). The LC50 (mg (ai) l(-1), 48 h) of spore-crystal preparations ranged from 0.02-0.98 (HD-1) and 0.06-2.14 (HD-73). Significantly higher LC50 values for all tested toxins and strains were obtained with populations collected from Iruttupallam and Ottanchathiram in the southern state of Tamil Nadu, whereas some of the populations collected from the northern part of India were more susceptible than the susceptible IARI 17-65 population. The high levels of resistance in the Iruttupallam and Ottanchathiram populations to Cry1Ab suggested selection pressure by Cry1Ab, which is the predominant toxin in B. thuringiensis formulations used in India. Cry1Ab was found to be more toxic than the other toxins. The population from Iruttupallam showed increased resistance following selection with Cry1Ab in the laboratory (LC50 from 1.25 to 4.31 microg ml(-1) over two generations) and also showed cross resistance to CrylAa and CrylAc. The resistance to Biobit in the field population from Iruttupallam declined slowly; requiring c. 33 generations for an overall 10-fold decline in LC50 when the insects were reared in the laboratory without exposure to B. thuringensis.     

Cross-resistance and stability of resistance to Bacillus thuringiensis toxin Cry1C in diamondback moth.

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.     

Extent of variation of the Bacillus thuringiensis toxin reservoir: the case of the geranium bronze,Cacyreus marshalli butler (Lepidoptera: Lycaenidae)

Despite the fact that around 200 cry genes from Bacillus thuringiensis have already been cloned, only a few Cry proteins are toxic towards a given pest. A crucial step in the mode of action of Cry proteins is binding to specific sites in the midgut of susceptible insects. Binding studies in insects that have developed cross-resistance discourage the combined use of Cry proteins sharing the same binding site. If resistance management strategies are to be implemented, the arsenal of Cry proteins suitable to control a given pest may be not so vast as it might seem at first. The present study evaluates the potential of B. thuringiensis for the control of a new pest, the geranium bronze (Cacyreus marshalli Butler), a butterfly that is threatening the popularity of geraniums in Spain. Eleven of the most common Cry proteins from the three lepidopteran-active Cry families (Cry1, Cry2, and Cry9) were tested against the geranium bronze for their toxicity and binding site relationships. Using 125I-labeled Cry1A proteins we found that, of the seven most active Cry proteins, six competed for binding to the same site. For the long-term control of the geranium bronze with B. thuringiensis-based insecticides it would be advisable to combine any of the Cry proteins sharing the binding site (preferably Cry1Ab, since it is the most toxic) with those not competing for the same site. Cry1Ba would be the best choice of these proteins, since it is significantly more toxic than the others not binding to the common site.     

Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China

The composition and distribution of insecticidal crystal proteins (Cry proteins) and their genotypes of Bacillus thuringiensis isolates from warehouses were evaluated through SDS-PAGE and PCR techniques. The results showed that the electrophoretic patterns of delta-endotoxin crystal preparations were divided into five types. The isolates containing approximately 135 kDa with a 65-kDa protein or only a approximately 135-kDa protein, which amounted to 55.74 and 35.25% of all isolates respectively, were the two major profiles of Cry protein isolated. The distribution of cry genes of B. thuringiensis from warehouses was highly variable. Cry protein genotypes detected in B. thuringiensis isolates included cry1Aa5, cry1Ab9, cry1Ac5, cry1Ba, cry1Ca1, cry1Da1, cry1Ea3, cry2, and cry3 genes, but not cry1Fa2. Among them, cry2, cry1Ac5, and cry1Ab9 genes were the most common in our B. thuringiensis isolates. Most B. thuringiensis isolates contained several cry genes in a total of 18 profiles. Among them, cry1Ac5 with cry1Ea3; cry1Aa5, cry1Ab9, cry1Ac5 with cry1Ea3; and cry1Aa5, cry1Ab9 with cry1Ac5 were the three principal profiles. The distribution of the Cry proteins and cry genes in isolates depended on geography and type of warehouses. Gene profiles may be used as markers for insecticidal activity of B. thuringiensis strains, but they did not directly reflect the toxic level of B. thuringiensis strains. The serotype of B. thuringiensis strains did not directly reflect the specific cry gene profiles in the strains, but certain relationships can be established between the serotype and cry genotype.