Producing Bacillus Thuringiensis Research Articles

Negative association between host plant suitability and the fitness cost of resistance to Bacillus thuringiensis (Bacillales:Bacillaceae).

Transgenic crops producing Bacillus thuringiensis (Bt) toxins are commonly used for controlling insect pests. Nearby refuges of non-Bt host plants play a central role in delaying the evolution of resistance to Bt toxins by pests. Pervasive fitness costs associated with resistance, which entail lower fitness of resistant than susceptible individuals in refuges, can increase the ability of refuges to delay resistance. Moreover, these costs are affected by environmental factors such as host plant suitability, implying that manipulating refuge plant suitability could improve the success of the refuge strategy. Based on results from a previous study of Trichoplusia ni resistant to Bt sprays, it was proposed that low-suitability host plants could magnify costs. To test this hypothesis, we investigated the association between host plant suitability and fitness costs for 80 observations from 30 cases reported in 18 studies of 8 pest species from 5 countries. Consistent with the hypothesis, the association between plant suitability and fitness cost was negative. With plant suitability scaled to range from 0 (low) to 1 (high), the expected cost was 20.7% with a suitability of 1 and the fitness cost increased 2.5% for each 0.1 decrease in suitability. The most common type of resistance to Bt toxins involves mutations affecting a few types of midgut proteins to which Bt toxins bind to kill insects. A better understanding of how such mutations interact with host plant suitability to generate fitness costs could be useful for enhancing the refuge strategy and sustaining the efficacy of Bt crops.

Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

Mutation in PgABCC2 confers low-level resistance to Cry1Ac in pink bollworm.

With the increasing incidence of pest resistance to transgenic crops producing Bacillus thuringiensis (Bt) proteins in the field, elucidating the molecular basis of resistance is important for monitoring, delaying and countering pest resistance. Previous work revealed that mutation or down-regulated expression of the cadherin gene (PgCad1) is associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac, and 20 mutant PgCad1 alleles (r1-r20) were characterized. Here, we tested the hypothesis that the ABC transporter PgABCC2 is a functional receptor for the Bt toxin Cry1Ac and that a mutation is associated with resistance. We identified and characterized the first resistance allele (rC2) of PgABCC2 in the laboratory-selected Cry1Ac-resistant strain AQ-C2 of pink bollworm. The rC2 allele had a one-base deletion in exon20, resulting in a frameshift and the introduction of a premature stop codon. This resulting PgABCC2 protein had a truncated C-terminus, including the loss of the NBD2 domain. AQ-C2 exhibited 20.2-fold greater resistance to Cry1Ac than the susceptible strain, and its inheritance of Cry1Ac resistance was recessive and genetically linked to PgABCC2. When produced in cultured insect cells, recombinant wild-type and rC2 mutant PgABCC2 proteins localized within the cell plasma membrane, although substantial cytoplasmic retention was also observed for the mutant protein, while the mutant PgABCC2 caused a 13.9-fold decrease in Cry1Ac toxicity versus the wild-type PgABCC2. PgABCC2 is a functional receptor of Cry1Ac and the loss of its carboxyl terminus (including its NBD2 domain) confers low-level resistance to Cry1Ac in both larvae and in cultured cells. © 2024 Society of Chemical Industry.

A New Cypovirus-1 Strain as a Promising Agent for Lepidopteran Pest Control.

Now more than ever researchers provide more and more evidence that it is necessary to develop an ecologically friendly approach to pest control. This is reflected in a sharp increase in the value of the biological insecticide market in recent decades. In our study, we found a virus strain belonging to the genus Cypovirus (Reoviridae); the strain was isolated from Dendrolimus sibiricus, possessing attractive features as a candidate for mass production of biological agents for lepidopteran-pest control. We describe the morphological, molecular, and ecological features of the new Cypovirus strain. This strain was found to be highly virulent to D. sibiricus (the half-lethal dose is 25 occlusion bodies per second-instar larva) and to have a relatively wide host range (infecting representatives of five families of Lepidoptera: Erebidae, Sphingidae, Pieridae, Noctuidae, and Lasiocampidae). The virus strain showed a strong interaction with a nontoxic adjuvant (optical brightener), which decreased the lethal dose for both main and alternative hosts, decreased lethal time, and may expand the host range. Moreover, we demonstrated that the insecticidal features were preserved after passaging through the most economically suitable host. By providing strong arguments for the possible use of this strain in pest control, we call on virologists, pest control specialists, and molecular biologists to give more attention to the Cypovirus genus, which may lead to new insights in the field of pest control research and may provide significant advantages to compare with baculoviruses and Bacillus thuringiensis products which are nowadays main source of bioinsecticides. IMPORTANCE In this article, we describe a newly discovered cypovirus strain that displays features ideally suited for the development of a modern biological insecticide: high potency, relatively broad host range, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, and ecologically friendly. Based on an alignment of CPV genomes, we suggest that the enhanced host range of this new strain is the sequence of evolutionary events that occurred after coinfections involving different CPV species within the same host. These findings suggest that we need to positively reconsider CPVs as prospective agents as biocontrol products.

Fitness Costs and Incomplete Resistance Associated with Delayed Evolution of Practical Resistance to Bt Crops

Insect pests are increasingly evolving practical resistance to insecticidal transgenic crops that produce Bacillus thuringiensis (Bt) proteins. Here, we analyzed data from the literature to evaluate the association between practical resistance to Bt crops and two pest traits: fitness costs and incomplete resistance. Fitness costs are negative effects of resistance alleles on fitness in the absence of Bt toxins. Incomplete resistance entails a lower fitness of resistant individuals on a Bt crop relative to a comparable non-Bt crop. In 66 studies evaluating strains of nine pest species from six countries, costs in resistant strains were lower in cases with practical resistance (14%) than without practical resistance (30%). Costs in F1 progeny from crosses between resistant and susceptible strains did not differ between cases with and without practical resistance. In 24 studies examining seven pest species from four countries, survival on the Bt crop relative to its non-Bt crop counterpart was higher in cases with practical resistance (0.76) than without practical resistance (0.43). Together with previous findings showing that the nonrecessive inheritance of resistance is associated with practical resistance, these results identify a syndrome associated with practical resistance to Bt crops. Further research on this resistance syndrome could help sustain the efficacy of Bt crops.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

MON 95379 Bt maize as a new tool to manage sugarcane borer (Diatraea saccharalis) in South America.

BackgroundThe sugarcane borer (SCB), Diatraea saccharalis (Lepidoptera: Crambidae), is a key pest of maize in Argentina, and genetically modified maize, producing Bacillus thuringiensis (Bt) proteins, has revolutionized the management of this insect in South America. However, field‐evolved resistance to some Bt technologies has been observed in SCB in Argentina. Here we assessed a new Bt technology, MON 95379, in the laboratory, greenhouse and field for efficacy against SCB.ResultsIn a laboratory leaf disc bioassay, both MON 95379 (producing Cry1B.868 and Cry1Da_7) and Cry1B.868_single maize (producing only Cry1B.868) resulted in 100% mortality of SCB. The level of Cry1B.868 in the Cry1B.868_single maize is comparable to that in MON 95379 maize. However, the Cry1Da_7 protein does not have high efficacy against SCB, as evidenced by < 20% mortality on Cry1Da_7_single leaf tissue. Total (100%) mortality of SCB in a Cry1B.868_single tissue dilution bioassay indicated that Cry1B.868_single maize meets the criteria to be classified as a high dose. Similar median lethal concentration (LC50) values were observed for MON 89034‐R and susceptible SCB strains exposed to Cry1B.868 protein. MON 95379 also controlled SCB strains resistant to MON 89034 (Cry1A.105/Cry2Ab2) and Cry1Ab. Under field conditions in Brazil and Argentina, MON 95379 maize plants were consistently protected from SCB damage.ConclusionMON 95379 maize will bring value to maize growers in South America by effectively managing SCB even in locations where resistance to other Bt‐containing maize technologies has been reported. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transcriptional Analysis of Cotton Bollworm Strains with Different Genetic Mechanisms of Resistance and Their Response to Bacillus thuringiensis Cry1Ac Toxin.

Transgenic crops producing Bacillus thuringiensis (Bt) insecticidal proteins are grown widely for pest control, but the evolution of resistance in target pests could reduce their efficacy. Mutations in genes encoding cadherin, ABC transporter or tetraspanin were linked with resistance to Cry1Ac in several lepidopteran insects, including the cotton bollworm (Helicoverpa armigera), a worldwide agricultural pest. However, the detailed molecular mechanisms by which these mutations confer insect resistance to Cry1Ac remain largely unknown. In this study, we analyzed the midgut transcriptomes of a susceptible SCD strain and three SCD-derived Cry1Ac-resistant strains of H. armigera (SCD-r1, with a naturally occurring deletion mutation of cadherin; SCD-KI, with a knock-in T92C point mutation in tetraspanin; and C2/3-KO, with both ABCC2 and ABCC3 knocked out). Evaluation of midgut transcript profiles of the four strains without Cry1Ac exposure identified many constitutively differentially expressed genes (DEGs) in the resistant SCD-r1 (n = 1355), SCD-KI (n = 1254) and C2/3-KO (n = 2055) strains. Analysis of DEGs in the midguts of each strain after Cry1Ac exposure revealed similar patterns of response to Cry1Ac in the SCD and SCD-r1 strains, but unique responses in the SCD-KI and C2/3-KO strains. Expression of midgut epithelium healing and defense-related genes was strongly induced by Cry1Ac intoxication in the SCD and SCD-r1 strains, while immune-related pattern recognition receptor and effector genes were highly expressed in the SCD-KI strain after Cry1Ac exposure. This study advances our knowledge of the transcriptomic basis for insect resistance to Bt toxins and provides a valuable resource for further molecular characterization of insect response to Cry1Ac toxin in H. armigera and other pest species.

Biodegradation of Malachite Green by Extracellular Laccase Producing Bacillus thuringiensis RUN1

A bacteria strain Bacillus thuringiensis RUN1identifiedby 16s RNA gene phylogenetic analysiswas used to decolorize malachite green, a triphenylmethane dye in a simulated wastewater. The ability of the organism to produce extracellular laccase and degrade the dye were also investigated. Results showed that the organism decolorized (84.67 ± 1.19 %) malachite green at 40 mgl-1 within 6 h; and the decolorization was associated to laccase production by the organism experimentally. Laccase activity increased as the decolorization process progressed, with the highest activity value of 0.1043 ± 0.02 U/min/ mg protein recorded after 24 h of incubation using ABTS as substrate. In addition, FTIR analysis showed that the strain actually degraded the dye. It was therefore concluded that this strain of Bacillus thuringiensis will be relevant in the biotreatment of industrial effluent containing malachite green and in the production of laccase, an industrially important enzyme.

Benefits of Baculovirus Use in IPM Strategies for Open Field and Protected Vegetables

Widespread use of synthetic chemical insecticides causes growing concern regarding the risks to human health and negative impacts on the environment. At the same time, many insects have become resistant toward synthetic chemical insecticides. Baculoviruses bring many benefits and allow reduced use of synthetic insecticides when included in integrated pest management (IPM). They are specific to their target pest and thus do not have any negative effects on plants, mammals, and non-target insects including natural enemies and pollinators. In addition, their unique mode of action makes them suitable for resistance management. We provide examples of how baculoviruses can be applied in IPM strategies for open field and protected vegetable crops. In field trials, baculovirus products were applied in rotations and tankmixes with chlorantraniliprole, Bacillus thuringiensis products, and spinosad and provided promising control of target pests. The conducted trials in open field and protected vegetables show that baculoviruses can offer an effective solution to control pests in open field and protected vegetable production. Including baculoviruses gave results as good as other application strategies tested while conferring important benefits in reducing residues and improving the resistance management strategy.

What type of Bt corn is suitable for a region with diverse lepidopteran pests: A laboratory evaluation

ABSTRACT Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are effective tools for controlling lepidopteran pests. However, the degree of susceptibility to Bt toxins differs among various pest species due to relatively narrow spectrum and high selectivity of such toxins. Bt corn hybrids for Chinese market were designed to target Asian corn borer Ostrinia furnacalis (Guenée), while their efficacy against other lepidopteran pests are not well defined, such as Conogethes punctiferalis (Guenée), Helicoverpa armigera (Hübner), Agrotis ypsilon (Rottemberg), and Mythimna separata (Walker), which are also important lepidopteran pests on corn in the Huang-Huai-Hai Summer Corn Region of China. To determine what type of Bt corn is suitable for this region, the efficacy of five Bt toxins, i.e., Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and Vip3A, to these five lepidopteran species was evaluated in laboratory. Both O. furnacalis and C. punctiferalis showed similar high susceptibility to all five Bt toxins. A. ypsilon and M. separate were less sensitive to Cry1Ab and Cry1Ac than the other species. H. armigera, A. ypsilon and M. separate were less sensitive to Cry1F than O. furnacalis and C. punctiferalis. H. armigera was more sensitive to Cry2Ab than other tested species. All five species were equally sensitive to Vip3A, though their LC50s were all relatively higher. These findings suggest that the first generation Bt corn expressing single Cry1 toxin should not be the first choice because of the potential risk of control failure or less efficacy against H. armigera, A. ypsilon or M. separate. The second-generation Bt corn expressing Cry1 and Cry2 toxins, or the third generation Bt corn expressing Cry1, Cry2 and Vip3A toxins might produce better protection of corn in the Huang-Huai-Hai Summer Corn Region of China.

Gene Flow Between Bt and Non-Bt Plants in a Seed Mixture Increases Dominance of Resistance to Pyramided Bt Corn in Helicoverpa zea (Lepidoptera: Noctuidae).

For delaying evolution of pest resistance to transgenic corn producing Bacillus thuringiensis (Bt) toxins, limited data are available to compare the effectiveness of refuges of non-Bt corn planted in seed mixtures versus blocks. Here we addressed this issue in the ear-feeding pest Helicoverpa zea Boddie by measuring its survival and development in the laboratory on ears from field plots with 90% Cry1A.105 + Cry2Ab corn and 10% non-Bt corn planted in a seed mixture or blocks. We compared a strain of H. zea selected for resistance to Cry1Ac in the laboratory, its parent strain not selected in the laboratory, and their F1 progeny. The relative survival of the F1 progeny and dominance of resistance were higher on ears from Bt plants in the seed mixture than the block. Half of the kernels in ears from non-Bt plants in the seed mixture produced both Cry1A.105 and Cry2Ab. However, survival on ears from non-Bt plants did not differ between the block and seed mixture. In simulations based on the observed survival, resistance to Cry1A.105 + Cry2Ab corn evolved faster with the seed mixture than the blocks, because of the higher dominance of resistance in the seed mixture. Increasing the refuge percentage improved durability of Cry1A.105 + Cry2Ab corn more for the blocks than the seed mixture. These findings imply that, for a given percentage of non-Bt corn, resistance of H. zea and other ear-feeding pests to multi-toxin Bt corn is likely to evolve faster for seed mixtures than blocks.

Effect of Bt toxin Cry1Ab on two freshwater caddisfly shredders \u2013 an attempt to establish dose-effect relationships through food-spiking

Genetically modified organisms (GMOs), which produce Bacillus thuringiensis (Bt) toxins, are widely used in agriculture in some parts of the world. Despite this, ecotoxicological methods, tailored to GMOs, are lacking to assess effects on aquatic environments. With the objective to investigate a food-related exposure pathway for aquatic shredders, we used a new food-spiking method while caddisfly larvae (Chaetopteryx spec., Sericostoma spec.) served as test species. Pure Cry1Ab toxins were spiked on black alder leaf discs and subsequently used in a feeding experiment. The toxin did not influence larval mortality compared to the control. The results, however, showed significant effects on larval lipid content (Chaetopteryx spec.) and development (Sericostoma spec.) at concentrations of 17.2 and 132.4 ng Cry1Ab/mg leaf, respectively. These changes are indicative for impacts on the fitness of the specimen and thus relevant in a risk assessment context. Ultimately, the food-spiking method allowed applying different Bt toxin concentrations leading to the establishment of dose-response relationships for various response variables. The use of long test durations and sublethal endpoints (consumption, lipid content, growth, larval instars) is, moreover, advisable when testing GMO effects.

Experimental and theoretical landscape influences on Spodoptera frugiperda movement and resistance evolution in contaminated refuge areas of Bt cotton

Transgenic cotton plants producing Bacillus thuringiensis (Bt) (Berliner) insecticidal proteins have contributed to the management of key lepidopteran pests. Larval high dispersal rates between non-Bt and Bt plants in landscapes with seed contamination can speed the evolution of insect resistance to Bt plants. We evaluated the effect of Bt and non-Bt cotton plants on the larval dispersal pattern and survival of susceptible, heterozygous and Cry1F-resistant Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) genotypes in pure and contaminated artificial micro-landscape. A computer model was used to analyse the consequences of S. frugiperda larval dispersal behaviour on resistance evolution in refuge areas with different contamination levels and migration of different adult genotype combinations. The biological data from artificial micro-landscape experiments were used in simulations of macro-landscape scales. The Cry1F-resistant genotype avoided non-Bt cotton. The heterozygote had a similar larval dispersal behaviour as the susceptible genotype when non-Bt cotton was the central plant. Our simulations provide evidence that in refuge areas contaminated with Bt cotton plants, the evolution of resistance may be > 75-fold faster in relation to a contamination-free refuge. In conclusion, S. frugiperda resistance management practices on regional scales with contamination-free refuges are important to prevent loss in different crops.

First record of Peridroma saucia Hübner (Lepidoptera: Noctuidae) in transgenic soybeans

The widespread cultivation of transgenic soybeans has caused significant changes in the spectrum of Lepidoptera larvae, both in the number of species as well as on their densities in the field. Transgenic crops producing Bacillus thuringiensis (Bt) insecticidal proteins have successfully reduced the incidence of the most common caterpillars infesting soybeans, namely Anticarsia gemmatalis (Lepidoptera: Erebidae) and Chrysodeixis includens (Lepidoptera: Noctuidae). However, lepidopteran species not previously recorded on the crop have been recently found, and are of concern due to the possibility of adaptation to the genetically modified cultivars. The occurrence of Peridroma saucia Hübner (Lepidoptera: Noctuidae) is described for the first time in Brazil feeding on genetically modified soybean cultivars.

ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.

CRISPR/Cas9-based mutation reveals Argonaute 1 is essential for pigmentation in Ostrinia furnacalis.

Ostrinia furnacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. furnacalis due to a lack in genomic information. In this report, we identified only one transcript of O. furnacalis Argonaute 1 (OfAgo1) gene from the genome and cloned the open reading frame. OfAgo1 presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgo1 mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgo1 disrupted cuticle pigmentation by down-regulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgo1, revealing a role of OfAgo1 in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. furnacalis, providing a new insight for pest management.

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control.

SummaryThe coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP‐47Aa, from an isolate of Pseudomonas mosselii. PIP‐47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP‐47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP‐47Aa show significant protection from root damage by WCR. PIP‐47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP‐1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP‐47Aa is a novel insecticidal protein for controlling the corn rootworm pests.

A Single Point Mutation Resulting in Cadherin Mislocalization Underpins Resistance against Bacillus thuringiensis Toxin in Cotton Bollworm

Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism.

A selective insecticidal protein from Pseudomonas for controlling corn rootworms.

The coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.