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The 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.In 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 (LCMON 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 WileySons Ltd on behalf of Society of Chemical Industry.

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

Insects feeding on roots of crops can damage the plant roots, resulting in yield loss due to poor water and nutrient uptake and plant lodging. In maize, the western corn rootworm (WCR) can cause severe damage to the roots, resulting in significant economic loss for farmers., This study describes three closely related proteins cloned from Brevibacillus laterosporus strains that are lethal upon feeding to Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR). Mpp75Aa1, Mpp75Aa2, and Mpp75Aa3 were toxic to WCR larvae when the larvae were fed purified protein. Transgenic plants expressing each mMpp75Aa protein were protected from feeding damage and showed a significant reduction in adult emergence from infested plants by both susceptible Cry3Bb1 and Cry34Ab1/Cry35Ab1-resistant WCR. These results demonstrate that proteins from B. laterosporus are as efficacious as the well-known Bacillus thuringiensis insecticidal proteins in controlling major insect pests such as WCR. The deployment of transgenic maize expressing mMpp75Aa, along with other active molecules lacking cross-resistance, has the potential to be a useful tool for control of WCR populations resistant to current B. thuringiensis traits. IMPORTANCE Insects feeding on roots of crops can damage the plant roots, resulting in yield loss due to poor water and nutrient uptake and plant lodging. In maize, the western corn rootworm (WCR) can cause severe damage to the roots, resulting in significant economic loss for farmers. Genetically modified (GM) plants expressing Bacillus thuringiensis insect control proteins have provided a solution for control of these pests. In recent years, populations of WCR resistant to the B. thuringiensis proteins in commercial GM maize have emerged. There is a need to develop new insecticidal traits for the control of WCR populations resistant to current commercial traits. New proteins with commercial-level efficacy on WCR from sources other than B. thuringiensis are becoming more critical. The Mpp75Aa proteins from B. laterosporus, when expressed in maize, are efficacious against the resistant populations of WCR and have the potential to provide solutions for control of resistant WCR.

Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties that could potentially reduce or eliminate insecticide application for control of Lygus and the associated environmental impacts.

There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture., Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest. IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

The use of dsRNA to control insect pests via the RNA interference (RNAi) pathway is being explored by researchers globally. However, with every new class of insect control compounds, the evolution of insect resistance needs to be considered, and understanding resistance mechanisms is essential in designing durable technologies and effective resistance management strategies. To gain insight into insect resistance to dsRNA, a field screen with subsequent laboratory selection was used to establish a population of DvSnf7 dsRNA-resistant western corn rootworm, Diabrotica virgifera virgifera, a major maize insect pest. WCR resistant to ingested DvSnf7 dsRNA had impaired luminal uptake and resistance was not DvSnf7 dsRNA-specific, as indicated by cross resistance to all other dsRNAs tested. No resistance to the Bacillus thuringiensis Cry3Bb1 protein was observed. DvSnf7 dsRNA resistance was inherited recessively, located on a single locus, and autosomal. Together these findings will provide insights for dsRNA deployment for insect pest control.

Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by >200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties that could potentially reduce or eliminate insecticide application for control of Lygus and the associated environmental impacts., Plant-feeding insects of the Lygus genus have emerged as a major pest effecting cotton crops in the USA. Here the authors optimize the insecticidal activity of a Bacillus thuringiensis crystal protein and produce transgenic plants that are resistant to feeding damage by Lygus species.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.

The Western Corn Rootworm, Diabrotica virgifera (WCR; Coleoptera: Chrysomelidae), is a serious pest of maize, causing approximately one billion dollars a year in losses and control costs. The insect has become resistant to several chemical controls and growers have asked for a control solution based on genetically-enhanced resistant varieties. Key to the production of genetically-enhanced germ plasm is the ability to use artificial diets for bioassays to identify proteins with activity against the target insect. However, artificial diets currently used for WCR bioassay do not support good WCR larval development because they were developed and optimized for the Southern Corn Rootworm (SCR). The present study was conducted to understand the nutritional physiology of WCR by altering the dietary constituents of a SCR diet and measuring the subsequent effects on WCR growth. Alteration of most dietary constituents had surprisingly little effect on WCR larval growth. The most significant factors affecting WCR growth on artificial diet were removal of formalin, addition of plant adjuvant, and adjustment of dietary pH to 9.0. These dietary changes resulted in larval WCR growth rates that were nearly double those in standard SCR rootworm diets. In some cases, WCR larvae fed on the improved diet obtained larval masses approaching those of larvae fed on maize roots.

Background and Methodology There is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination. Principal Findings and Conclusions The Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P