Vegetative Insecticidal Protein Research Articles

Unraveling the abundance of vip3-type genes in Indian Bacillus thuringiensis across the agroclimatic landscape and impact of amino acid substitutions for safer agriculture

Vegetative insecticidal protein (vip) genes of Bacillus thuringiensis (Bt) are candidates for gene pyramiding in the resistance management of pests. The prevalence of vip genes in Bt isolates is relatively under-explored. Bt isolates recovered from 29 diverse sources in nine agro-climatic zones of India were screened for the presence of vip3-type genes by PCR with 4 sets of oligonucleotide primers. Out of 155 Bt isolates, 70.32 % (109) and 44.52 % (69) isolates were positive for amplification of partial vip3-type genes with primer sets 1 and 4, respectively. The primer set-2 was found to be more efficient for amplifying full-length genes (29.03 % /45 isolates) as compared with primer set-3 (3.23 %/ 5 isolates), also corroborated in the amplification of full-length vip3 genes in ten Bt BGSC strains used as reference. Frequency analysis revealed presence of vip3 genes in Bt isolates across all agro-climatic zones. Thus, Indian Bt isolates from diverse sources have a rich repertoire of vip3-type genes. Our study reports the highest number (45) of full-length vip3-type genes detected in a native Bt isolates collection, demonstrating enrichment of Indian Bt isolates for vip3 genes. Twelve of these genes have been cloned, sequenced, and out of these, six were found to be effective against Helicoverpa armigera in our laboratory previously. Comparison of substitutions in deduced amino acids sequence of these genes and expression of Vip3 proteins in SDS-PAGE analysis of selected native Bt isolates positive for full-length vip3-type genes indicated their biopesticidal potential.

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Genome-wide DNA methylation profile and its function in regulating Vip3Aa tolerance in fall armyworm (Spodoptera frugiperda).

Vegetative insecticidal proteins (Vips) are widely used in pest management, but Vip tolerance poses a significant threat. DNA methylation plays important roles in regulating the response of biological organisms to environmental stress, and it may also regulate fall armyworm (FAW, Spodoptera frugiperda) Vip3Aa tolerance. In this study, a DNA methylation map was developed for FAW, and its function in regulating FAW Vip3Aa tolerance was explored. The FAW genome-wide DNA methylation map showed that exons were preferred regions for DNA methylation and housekeeping genes were highly methylated. FAW was screened using Vip3Aa for ten generations, and bioassays indicated that Vip3Aa tolerance increased trans-generationally. A comparison of DNA methylation maps between Vip3Aa-tolerant and -susceptible strains showed that gene body methylation was positively correlated with gene expression level. FAW exhibits significant variation in DNA methylation among individuals, and Vip3Aa screening induces epigenetic variation based on DNA methylation. Moreover, the study demonstrated that a reduction in methylation density within the gene body of a 3'5'-cyclic nucleotide phosphodiesterase gene resulted in decreased expression and increased tolerance of FAW to Vip3Aa, which was validated through RNA interference experiments. The DNA methylation map and mechanism of Vip3Aa tolerance improve our understanding of DNA methylation and its function in Lepidoptera and provide a new perspective for developing pest management strategies. © 2024 Society of Chemical Industry.

Positive selection analysis of Cyt proteins from Bacillus thuringiensis: A conservative trend driven by negative (purifying) selection

Bacillus thuringiensis is a Gram-positive entomopathogenic bacterium that produces different pesticidal proteins: vegetative insecticidal proteins (Vpb1/Vpa2, Vip3, and Vpb4) during vegetative growth, which are secreted to the culture medium, and δ–endotoxins (Cry and Cyt) during sporulation, which accumulate into parasporal crystals. Cyt proteins are the smaller subset of δ–endotoxins targeting Diptera species. While Cry and Vip3 proteins undergo positive selection, our analysis suggests that Cyt proteins evolve following a conservative trend driven negative (purifying) selection.

Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

Physical Factors Affecting the Scale-Up of Vegetative Insecticidal Protein (Vip3A) Production by Bacillus thuringiensis Bt294

Vip3A (vegetative insecticidal protein) is a representative member of the Vip3 family, which is widely used for lepidopteran pest control. This Vip3A protein, a non-growth-associated protein, is an effective bioinsecticide against insect pests, but there is relatively little information about its production processes at large scales. Hence, the effects of environmental factors on Vip3A production by Bacillus thuringiensis Bt294 (antifoam agents, shaking speeds, agitation and aeration rates), as well as controlling physical conditions such as the lowest point of dissolved oxygen and controlling of culture pH, were observed in shaking flasks and bioreactors. The results showed that antifoam agents, flask types and shaking speeds had significant effects on Vip3A and biomass production. Cultivation without pH control and DO control in 5 L bioreactors at lower agitation and aeration rates, which was not favorable for biomass production, resulted in a high Vip3A protein production of 5645.67 mg/L. The scale-up studies of the Vip3A protein production in a pilot-scale 750 L bioreactor gave 3750.0 mg/L. Therefore, this study demonstrated the significant effects of agitation, aeration rates and culture pH on Vip3A production by B. thuringiensis Bt294. Balancing of physical conditions was necessary for obtaining the highest yield of Vip3A by slowing down the production rate of biomass. Moreover, this Vip3A protein has high potential as a bioinsecticide for lepidopteran pest control in organic crops. This information will be important for significantly increasing the Vip3A protein concentration by the bacterium and will be useful for field application at a lower cost.

Screening and characterization of Bacillus thuringiensis isolates for high production of Vip3A and Cry proteins and high thermostability to control Spodoptera spp

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces crystalline (Cry and Cyt) and soluble (vegetative insecticidal proteins or Vips) proteins during the sporulation and vegetative growth phases, respectively. Combining Cry and Vip proteins could delay insect resistance development and exhibit synergistic activity against various insect pests. This study aims to screen Bt isolates collected from Thailand for high Vip3A and Cry protein production levels and high thermostability to control Spodoptera spp. Among the selected Bt isolates with high target protein synthesis, Bt isolate 506 was found to be safe for further biopesticide formulation due to the absence of non-specific metabolite, as determined by the detection of thermo-stable β-exotoxin I based on biological assays and PCR analysis. Bt isolate 506 showed the presence of Cry1A, Cry2A, and Vip3A-type proteins identified as Cry1Aa45, Cry2Aa22, and Vip3A87, respectively. The insecticidal activity of whole culture extracts containing Vip3A and Cry mixtures and culture supernatants containing secreted Vip3A protein was evaluated against the second-instar larvae of S. exigua and S. frugiperda. The Bt isolate 506 showed high toxicity against both insects, and the insecticidal proteins produced by this isolate retained their activity after heating at 50 °C. This Bt isolate is a promising candidate for further development as a biopesticide against lepidopteran pests.

Downregulation of a transcription factor associated with resistance to Bt toxin Vip3Aa in the invasive fall armyworm.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm (Spodoptera frugiperda), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda. Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb, the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

Analysis of Synergism between Extracellular Polysaccharide from Bacillus thuringensis subsp. kurstaki HD270 and Insecticidal Proteins.

Bacillus thuringiensis (Bt) is the most widely used biopesticide worldwide and can produce several insecticidal crystal proteins and vegetative insecticidal proteins (Vips) at different growth stages. In our previous study, extracellular polysaccharides (EPSs) of Bt strain HD270 were found to enhance the insecticidal activity of Cry1Ac protoxin against Plutella xylostella (L.) and promote the binding of Cry1Ac to the intestinal brush border membrane vesicles (BBMVs). Whether the synergistic activity of Bt EPSs is common to other Cry1-type or Vip proteins is unclear, as is the potential synergistic mechanism. In this study, crude EPS-HD270 was found to increase the toxicity of Cry1-type toxins and Vip3Aa11 against different lepidopteran pests by approximately 2-fold. The purified EPS-HD270 also possessed synergistic activity against the toxicity of Cry1Ac and Vip3Aa11 against Spodoptera frugiperda (J.E. Smith) and Helicoverpa armigera (Hübner). Furthermore, we found that EPS-HD270 had a strong binding ability with Vip3Aa11 and promoted the binding of Vip3Aa11 to the BBMVs of H. armigera and S. frugiperda. Bt EPS-HD270 also protected Vip3Aa11 from proteolytic processing in larval midgut juice. Bt EPSs had universal synergistic effects on Cry1-type or Vip toxins against S. frugiperda and H. armigera. Bt EPS-HD270 exhibited synergistic activity with Vip3Aa through promotion of binding to BBMVs and protection from digestion by midgut protease. The results indicated that synergistic activity with Bt toxins was an important function of Bt EPSs, which was very different from other Bacillus spp.

Vegetative insecticidal protein (Vip3A) production by Bacillus thuringiensis Bt294 and its efficacy against Lepidopteran pests (Spodoptera exigua)

A vegetative insecticidal protein, Vip3A, is highly active against lepidopteran pests, which are the most important pests in most tropical countries. An important aspect of the successful commercial production of this bacterial insecticide is the development of bacterial culture media that maximize the titres of this protein and cost reduction. This study aimed to investigate and optimize Vip3A production by Bacillus thuringiensis Bt294 using statistical methods and 3-step sequential approaches. The experimental design showed that the production of Vip3A was maximized to 300 mg/L when the bacterium was cultivated in medium composed of 5.05 g/L glycerol, 49.17 g/L soytone, 30.05 g/L casein hydrolysate, 1.99 g/L CaCl2.2H2O, 7.5 mg/L CuSO4, 15 mg/L MnSO4.H2O, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, 0.2 g/L MgSO4.7H2O, 5 g/L yeast extract, 2.5 mg/L NiCl2.6H2O and 3 mL/L vitamin solution. B. thuringiensis Bt294 Vip3A toxin was highly toxic to Spodoptera exigua with LC50 values of 187.1 ng/cm2 at 7 days. This result demonstrated that a high titre of Vip3A produced by B. thuringiensis Bt294 will be useful as a biological control agent. This optimization will allow production to be scaled up for commercial production in the future.

Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae).

The polyphagous pest Helicoverpa zea (Lepidoptera: Noctuidae) has evolved practical resistance to transgenic corn and cotton producing Cry1 and Cry2 crystal proteins from Bacillus thuringiensis (Bt) in several regions of the United States. However, the Bt vegetative insecticidal protein Vip3Aa produced by Bt corn and cotton remains effective against this pest. To advance knowledge of resistance to Vip3Aa, we selected a strain of H. zea for resistance to Vip3Aa in the laboratory. After 28 generations of continuous selection, the resistance ratio was 267 for the selected strain (GA-R3) relative to a strain not selected with Vip3Aa (GA). Resistance was autosomal and almost completely recessive at a concentration killing all individuals from GA. Declines in resistance in heterogeneous strains containing a mixture of susceptible and resistant individuals reared in the absence of Vip3Aa indicate a fitness cost was associated with resistance. Previously reported cases of laboratory-selected resistance to Vip3Aa in lepidopteran pests often show partially or completely recessive resistance at high concentrations and fitness costs. Abundant refuges of non-Bt host plants can maximize the benefits of such costs for sustaining the efficacy of Vip3Aa against target pests.

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis.

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

Whole genome sequencing of a novel Bacillus thuringiensis isolated from Assam soil

BackgroundBacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34′39.11''N and Longitude: 93°10′16.04''E).ResultsThe whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein.Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences.ConclusionThis is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

Transgenic maize inbred lines expressing high levels of Bacillus thuringiensis vegetative insecticidal protein (Vip3Aa86) offer effective control of maize stem borer (Chilo partellus)

Transgenic maize inbred lines expressing high levels of Bacillus thuringiensis vegetative insecticidal protein (Vip3Aa86) offer effective control of maize stem borer (Chilo partellus)

Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3's toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa's stability and toxicity inside the protease enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa's mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development.

Stability is essential for insecticidal activity of Vip3Aa toxin against Spodoptera exigua

Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines (P591, P605 and P779) located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.

Vegetative Insecticidal Protein Vip3Aa Is Transported via Membrane Vesicles in Bacillus thuringiensis BMB171.

Vegetative insecticidal protein Vip3Aa, secreted by many Bacillus thuringiensis (Bt) strains during the vegetative growth stage, represents the second-generation insecticidal toxin. In recent years, significant progress has been made on its structure and action mechanism. However, how it is translocated across the cytoplasmic membrane into the environment remains a mystery. This work demonstrates that Vip3Aa is not secreted by the General Secretion (Sec) System. To reveal the secretory pathway of Vip3A, we purified the membrane vesicles (MVs) of B. thuringiensis BMB171 and observed by TEM. The size of MVs was determined by the dynamic light scattering method, and their diameter was approximately 40–200 nm, which is consistent with the vesicles in Gram-negative bacteria. Moreover, Vip3A could be detected in the purified MVs by Western blot, and immunoelectron microscopy reveals Vip3A antibody-coated gold particles located in the MVs. After deleting its signal peptide, chitinase B (ChiB) failed to be secreted. However, the recombinant ChiB, whose signal peptide was substituted with the N-terminal 39 amino acids from Vip3A, was secreted successfully through MVs. Thus, this sequence is proposed as the signal region responsible for vesicle transport. Together, our results revealed for the first time that Vip3Aa is transported to the medium via MVs.

Virtual Evaluation of Insecticidal Potential of Fused Vip3Aa and Garlic Lectin against Fall Armyworm (Spodoptera frugiperda)

The use of transgenic crops to combat insect infestation has been proven to be an effective and environment-friendly approach. However, insecticidal proteins with a single mode of action are prone to the development of insect resistance. Researchers are now interested in combining insecticidal proteins to acquire multiple modes of action to address that problem. Here, we have performed a virtual evaluation of a fusion protein that was in silico constructed by combining the vegetative insecticidal protein (Vip3Aa) of Bacillus thurengensis and the garlic leaf lectin of Allium sativum (ASAL) with a fl exible peptide linker (GGGGS)3. Two fusion modes were conducted, one with ASAL as the N-terminus (AV) and the other where Vip3Aa is the N-terminus (VA) of the fusion construct. Molecular dynamics simulation of the fusion constructs (AV and VA) revealed their differences in conformational changes and compactness, with VA being more stable and favorable. Protein-protein docking studies against the fall armyworm aminopeptidase-N of S. frugiperda resulted in the identifi cation of interacting residues at the interface. A novel fusion protein proposed in this study would be valuable for rational design of potent insecticidal proteins with multiple modes of action.

Responses to Bt toxin Vip3Aa by pink bollworm larvae resistant or susceptible to Cry toxins.

Transgenic crops that make insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized management of some pests. However, evolution of resistance to Bt toxins by pests diminishes the efficacy of Bt crops. Resistance to crystalline (Cry) Bt toxins has spurred adoption of crops genetically engineered to produce the Bt vegetative insecticidal protein Vip3Aa. Here we used laboratory diet bioassays to evaluate responses to Vip3Aa by pink bollworm (Pectinophora gossypiella), one of the world's most damaging pests of cotton. Against pink bollworm larvae susceptible to Cry toxins, Vip3Aa was less potent than Cry1Ac or Cry2Ab. Conversely, Vip3Aa was more potent than Cry1Ac or Cry2Ab against laboratory strains highly resistant to those Cry toxins. Five Cry-susceptible field populations were less susceptible to Vip3Aa than a Cry-susceptible laboratory strain (APHIS-S). Relative to APHIS-S, significant resistance to Vip3Aa did not occur in strains selected in the laboratory for > 700-fold resistance to Cry1Ac or both Cry1Ac and Cry2Ab. Resistance to Cry1Ac and Cry2Ab did not cause strong cross-resistance to Vip3Aa in pink bollworm, which is consistent with predictions based on the lack of shared midgut receptors between these toxins and previous results from other lepidopterans. Comparison of the Bt toxin concentration in plants relative to the median lethal concentration (LC50 ) from bioassays may be useful for estimating efficacy. The moderate potency of Vip3Aa against Cry1Ac- and Cry2Ab-resistant and susceptible pink bollworm larvae suggests that Bt cotton producing this toxin together with novel Cry toxins might be useful as one component of integrated pest management. © 2022 Society of Chemical Industry.

Isolation and molecular characterization of Bacillus thuringiensis subsp. kurstaki toxic to lepidopteran pests Spodoptera spp. and Plutella xylostella.

Bacillus thuringiensis (Bt) has been widely used as a biological control agent for lepidopteran pests. However, resistance to Bt is a major concern associated with Spodoptera spp. (Noctuidae) and Plutella xylostella (Plutellidae). For efficient control of Noctuidae and Plutellidae, novel Bt strains which have high toxicity and a broad host range are needed. To develop novel Bt strains as used for bio-insecticides, the Bt IMBL-B9 with high toxicity against Spodoptera exigua, Spodoptera frugiperda and P. xylostella was isolated and characterized. The Bt kurstaki IMBL-B9 strain produced bipyramidal and cuboidal crystals consisting of cry toxins with molecular weights of 130 and 65 kDa, respectively. This strain harbors eight crystal protein genes in total, including cry1Ea and one vegetative insecticidal protein gene. The median lethal concentration (LC50 ) values of IMBL-B9 against S. exigua and S. frugiperda were 21.8- and 19.3-fold lower than those of the Bt kusrstaki strain, and 5.6- and 4.9-fold lower than those of Bt aizawai strain, respectively. To evaluate the insecticidal activity of Cry proteins from IMBL-B9, cry gene-sourced recombinant Bt strains were constructed. These strains have insecticidal activity and synergic action against lepidopteran pests. In this study, a novel Bt kurstaki IMBL-B9 strain was isolated and this could be useful for the development of new bio-insecticide or cry gene-based recombinant products as an alternative solution against lepidopterans, including Noctuidae and Plutellidae. © 2022 Society of Chemical Industry.