Development of a Safe and Effective Bacillus thuringiensis-Based Nanobiopesticide for Controlling Tea Pests.

Mg(OH)2 was used as the nanocarrier of the Bacillus thuringiensis (Bt) Cry1Ac protein, and the synthesized Cry1Ac-Mg(OH)2 composites were regular and uniform nanosheets. Nano-Mg(OH)2 could effectively improve the insecticidal effect of the Cry1Ac protein toward Ectropis obliqua. It could enhance the damage degree of the Cry1Ac protein to intestinal epithelial cells and microvilli, induce and enrich the production of reactive oxygen species (ROS) in the midgut, and enhance the degradation of the Cry1Ac protein into active fragments. Furthermore, an anti-rinsing assay showed that the Cry1Ac-Mg(OH)2 composites were bound to the notch structure of the tea leaf surface. The retention of the Cry1Ac protein increased by 11.45%, and sprayed nano-Mg(OH)2 was rapidly absorbed by different tissues of tea plants. Moreover, nano-Mg(OH)2 and composites did not significantly affect non-target organisms. These results show that nano-Mg(OH)2 can serve as a safe and effective biopesticide carrier, which provides a new approach for stable and efficient Bt preparation.

Bacillus thuringiensis pesticidal toxins: A global analysis based on a scientometric study (1980-2021).

Several studies have been conducted on Bacillus thuringiensis (Bt) pesticidal toxins due to their successful environmentally friendly biopesticide activity against various insect pest orders, protozoa, mites, and nematodes. However, no existing study has systematically examined the trends and evolution of research on Bt pesticidal toxins from a scientometric perspective. This study aimed to analyze the trends and hotspots of global research in this field. 5757 publications on Bt pesticidal toxins were extracted from the Web of Science Core Collection (WoS) from 1980 to 2021. Statistical and scientometric analyses were performed using Excel, CiteSpace, and VOSviewer visualization tools to evaluate research evolution, journal contribution and subject categories, contributing countries and institutions, highly influential references, and most used author keywords. The 5757 publications featured in 917 journals spanning 116 subject categories. The top 5 subject categories ranked as Entomology, Biotechnology & Applied Microbiology, Microbiology, Biochemistry & Molecular Biology, and Agriculture. Out of these publications, the USA contributed the most, with 1562 publications, 72,754 citations, and 46.58 average citations per paper (ACPP); however, Belgium had the highest (106.43) ACPP among the top 20 contributing countries. The Chinese Academy of Agricultural Sciences is the leading institution with 298 publications and 21.20 ACPP. The Pasteur Institute is ranked first (90.04) in terms of ACPP. Keywords analyses revealed that recent studies are inclined toward the evolution of insect resistance against Bt toxins. In future, studies related to the development of resistance mechanisms by insects against Bt pesticidal toxins and ways to overcome them will likely receive more attention. This study highlights the past and current situations and prospective directions of Bt pesticidal toxins-related research.

Relative activity of 15 bacterial strains against the larvae of Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura (Lepidoptera: Noctuidae).

Crystal toxins produced by different strains of entomopathogenic Bacillus thuringiensis (Bt) have been characterized and widely applied as commercial biological pesticides owing to their excellent insecticidal properties. This study aimed to identify novel bacterial strains effective in controlling Spodoptera exigua Hübner, Helicoverpa armigera Hübner, and Spodoptera litura Fabricius. Fifteen culturable bacterial strains were isolated from 60 dead larvae (H. armigera and S. exigua) collected in the field. The biochemical characteristics and 16S rRNA sequences of these strains indicated that one strain (B7) was Lysinibacillus sp., 12 strains (B1, B3, B4, B5, B6, B8, P2, P3, P4, P5, P6, and DW) were Bt kurstaki, and P2-2 and B2 were Bacillus velezensis subsp. Laboratory bioassays indicated that strains B3, P6, B6, and P4 showed high toxicity to second-instar larvae of S. exigua, with LC50 values of 5.11, 6.74, 205.82, and 595.93 µg/ml, respectively; while the strains P5, B5, B6, and P6, were the most efficient against second-instar larvae of H. armigera with LC50 values of 725.82, 11,022.72, 1,282.90, 2,005.28, respectively, and strains DW, P3, P2, and B4 had high insecticidal activity against second-instar larvae of S. litura with LC50 values of 576.69, 1,660.96, 6,309.42, and 5,486.10 µg/ml, respectively. In conclusion, several Bt kurstaki strains with good toxicity potential were isolated and identified in this study. These strains are expected to be useful for biointensive integrated pest management programs to reduce the use of synthetic insecticides.

No bioaccumulation of Cry protein in the aphidophagous predator Harmonia axyridis.

The uptake of insecticidal Cry1Ab from genetically engineered (GE) maize, via herbivore Rhopalosiphum padi, to a predator Harmonia axyridis and its potential intergenerational transfer were investigated. Cry1Ab concentration was found to be 400-fold lower in R. padi compared to GE maize, and more than two-fold lower in H. axyridis. For 62% of H. axyridis samples, Cry1Ab was under the limit of detection (LOD), for another 13% were under the limit of quantification (LOQ). The concentration of Cry1Ab was similar between H. axyridis exposed short-term and long-term with the exception of adults after long-term. There was no correlation between Cry1Ab in females and eggs and neonates. The performance of H. axyridis was comparable between Cry1Ab and control. Histological investigation did not show any pathological changes in the digestive and reproductive systems. The detected route of exposure is unlikely to be important for functional biological control by H. axyridis in agroecosystem.

Molecular and Toxicological Characterization of a Bacillus thuringiensis Strain Expressing a Vip3 Protein Highly Toxic to Spodoptera frugiperda (Lepidoptera: Noctuidae).

The characterization of the Bacillus thuringiensis (Berliner) LBIT-418 strain was based on a previous work which indicated its high insecticidal potential. Therefore, toxicological, molecular, and biochemical characterizations were conducted in this work to identify its unique features and its potential to be developed as a bioinsecticide. This strain, originally isolated from a healthy mosquito larva, was identified within the subspecies kenyae by sequencing of the hag gene and by the multilocus sequence typing (MLST) technique. Genes cry1Ac2, cry1Ea3, cry2Aa1 and cry2Ab4, and a cry1Ia were detected in its genome, in addition to a vip3Aa gene. In this research, the latter protein was successfully cloned, expressed, and purified and showed high toxicity towards the fall armyworm, Spodoptera frugiperda (J.E. Smith), fourth instar larvae in bioassays using the microdroplet ingestion technique, estimating an LD50 of 21.38 ng/larva. Additional bioassays were performed using the diet surface inoculation technique of the strain's spore-crystal complex against diamondback moth larvae, Plutella xylostella (Linnaeus), estimating an LC50 of 10.22 ng/cm2. Its inability to produce ß-exotoxin was demonstrated by bioassays against the nematode Caenorhabditis elegans Maupas and by HPLC analysis. These results support the high potential of this strain to be developed as a bioinsecticide.

Molecular and Kinetic Models for Pore Formation of Bacillus thuringiensis Cry Toxin.

Cry proteins from Bacillus thuringiensis (Bt) and other bacteria are pesticidal pore-forming toxins. Since 2010, when the ABC transporter C2 (ABCC2) was identified as a Cry1Ac protein resistant gene, our understanding of the mode of action of Cry protein has progressed substantially. ABCC2 mediates high Cry1A toxicity because of its high activity for helping pore formation. With the discovery of ABCC2, the classical killing model based on pore formation and osmotic lysis became nearly conclusive. Nevertheless, we are still far from a complete understanding of how Cry proteins form pores in the cell membrane through interactions with their host gut membrane proteins, known as receptors. Why does ABCC2 mediate pore formation with high efficiency unlike other Cry1A-binding proteins? Is the "prepore" formation indispensable for pore formation? What is the mechanism underlying the synergism between ABCC2 and the 12-cadherin domain protein? We examine potential mechanisms of pore formation via receptor interactions in this paper by merging findings from prior studies on the Cry mode of action before and after the discovery of ABC transporters as Cry protein receptors. We also attempt to explain Cry toxicity using Cry-receptor binding affinities, which successfully predicts actual Cry toxicity toward cultured cells coexpressing ABC transporters and cadherin.

Database of non-target invertebrates recorded in field experiments of genetically engineered Bt maize and corresponding non-Bt maize.

OBJECTIVES: To assess potential non-target effects of genetically engineered/modified (GM) maize that produces insecticidal proteins from Bacillus thuringiensis (Bt), numerous field experiments have been conducted worldwide. Field data are often variable and influenced by uncontrolled factors and meta-analyses can recognize general effects with increased statistical power compared to individual studies. This database represents a comprehensive collection of experimental field data on non-target invertebrates in Bt and non-Bt maize. It was created for a systematic review with the question if growing Bt maize changes abundance or ecological function of non-target animals compared to growing of non-GM maize. Systematic literature searches identified relevant data. Authors were contacted for additional information or raw data if needed and a critical appraisal scheme was developed and applied to each data record. DATA DESCRIPTION: The database contains 7279 records of non-target invertebrate abundance, activity density, or predation or parasitism extracted from 120 articles. Records for individual species and life stages, but also aggregated data are available. Each record represents a comparison of invertebrates in Bt and non-Bt maize and includes means, standard deviations and sample sizes. Additional variables characterize publication details, experimental setup, cultivars, Bt proteins, geographic location, field management, insecticide treatments, sampling details, and taxonomy.

Addition of proteinase K during the culture alter the physiology of Bacillus thuringiensis culture and the cry1Ac, nprX, nprA, and spo0A gene transcription.

Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the cost-productivity relation. The neutral protease A (NprA) is the major extracellular protein massively produced during the stationary phase by this bacterium, contributing to the Cry proteins' degradation. Also, the deletion of aprA and nprA genes enhanced the yield of Cry protein, stabilizing it. Therefore, to increase Cry production, one possibility is to degrade the NprA protease in the culture media. In the present study, proteinase K was used to hydrolyze the NprA to increase Cry production. Proteinase K was added during the exponential growth of B. thuringiensis culture. The bacilli and endospores were measured along all culture, while the Cry protein was measured at the end of the culture. The addition of PK affects the bacilli and spore kinetics positively but negatively to the Cry protein (there is no Cry protein detection). Therefore, the gene expression of the cry1Ac, nprX, nprA, and spo0A was measured. The expression of each gene was followed along all culture. Results demonstrated that PK alters both the transcriptional levels and the expression order of the genes.

Renal and hepatic changes in the offspring of rats that received biological insecticides during pregnancy and lactation.

Bacillus thuringiensis insecticides have been considered safe, being an alternative to the use of synthetic insecticides. However, studies have shown the effects of Bt Cry toxins on various organs, compromising their functions. The objective of this work was to test whether the administration of biological insecticides based on B. thuringiensis in pregnant rats will cause histopathological changes in the liver and kidneys, as well as in the levels of toxicity biomarkers, of their puppies in adulthood. Twenty rats, 90 days old, were used, divided into four groups: GC - Pregnant rats, GX - Pregnant rats that received XenTari®, GDi - Pregnant rats that received Dipel® and GDe - Pregnant rats that received deltamethrin. Insecticides were administered by gavage at a dosage of 1 mg/100 g/day (GX and GDi), and 2 mg/Kg/day (GDe) during pregnancy and lactation. In the animals of the groups whose matrices received the insecticides, there was a reduction in the levels of the biomarkers of toxicity alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine, about the control group. The biological insecticides promoted histopathological changes in the liver, with the presence of portal vein, centrilobular and sinusoidal capillaries congestion, and in the kidney, presence of cortical congestion and reduction of the subcapsular space. Histochemical evaluation in the liver demonstrated a significant reduction in glycogen in the groups that received insecticides when compared to the control group, whereas for collagen fibers in both the liver and the kidneys, no differences were observed between the experimental groups. The morphometry of the liver revealed a significant reduction in the lobular parenchyma and an increase in the non-lobular parenchyma, and in the kidney, there was a reduction in the diameter and volume of the glomerulus and Bowman's capsule of the animals whose matrices received both biological and synthetic insecticides. Thus, it is concluded that the biological insecticides XenTari® and Dipel® in sublethal doses in pregnant rats promote changes in the liver and kidney of the offspring similar to the insecticide deltamethrin, which extend into adulthood.

Identification and characterization of a new cry-like gene found in a Bacillus cereus strain.

Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture.

In the last ten years, ABC transporters have emerged as unexpected yet significant contributors to pest resistance to insecticidal pore-forming proteins from Bacillus thuringiensis (Bt). Evidence includes the presence of mutations in resistant insects, heterologous expression to probe interactions with the three-domain Cry toxins, and CRISPR/Cas9 knockouts. Yet the mechanisms by which ABC transporters facilitate pore formation remain obscure. The three major classes of Cry toxins used in agriculture have been found to target the three major classes of ABC transporters, which requires a mechanistic explanation. Many other families of bacterial pore-forming toxins exhibit conformational changes in their mode of action, which are not yet described for the Cry toxins. Three-dimensional structures of the relevant ABC transporters, the multimeric pore in the membrane, and other proteins that assist in the process are required to test the hypothesis that the ATP-switch mechanism provides a motive force that drives Cry toxins into the membrane. Knowledge of the mechanism of pore insertion will be required to combat the resistance that is now evolving in field populations of insects, including noctuids.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system.

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1.

Insect pests are a threat to agriculture as they cause a loss of 15-22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal 'Cry' proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.

Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis.

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used in sprayable formulations or produced in transgenic crops as the most successful alternatives to synthetic pesticides. The most relevant threat to sustainability of Bt insecticidal proteins (toxins) is the evolution of resistance in target pests. To date, high-level resistance to Bt sprays has been limited to one species in the field and another in commercial greenhouses. In contrast, there are currently seven lepidopteran and one coleopteran species that have evolved practical resistance to transgenic plants producing insecticidal Bt proteins. In this article, we present a review of the current knowledge on mechanisms of resistance to Bt toxins, with emphasis on key resistance genes and field-evolved resistance, to support improvement of Bt technology and its sustainability.

Meeting technical challenges for protein characterization and surrogate equivalence studies that resulted from insecticidal protein co-expression in maize event MZIR098.

Safety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.1Ab and mCry3A proteins as derived from Event MZIR098 maize was challenging because of the difficulty in purifying/isolating these proteins that are of similar molecular weight and have considerable shared sequence and immunogenicity. This also applies to establishing the biochemical equivalence to the microbially produced surrogate proteins, as highly-purified plant protein is required. While use of crude plant extracts facilitated functional equivalence testing with the surrogate proteins, a separate technical challenge had to be met. The eCry3.1Ab and mCry3A proteins display differentiated modes of action toward CRW pests, however, with the same overall target pest spectrum, no differential test organism existed to allow equivalence testing for one insecticidal protein in the presence of the other. To establish that the microbially produced proteins are suitable surrogates for the plant-produced proteins, the challenges in the protein purification and bioactivity testing had to be addressed. This article describes technical solutions to assess and characterize the insecticidal proteins in this new event and thereby confirm equivalence/suitability of the microbially produced protein surrogates.

The compatibility of Bacillus thuringiensis Cry protein-solubilizing buffers with the droplet feeding method in fall armyworm larvae.

The volumes of alkaline (pH > 10), Bacillus thuringiensis Cry protein-solubilizing buffers imbibed by fall armyworm larvae in droplet feeding assays were determined. The buffers differed in the presence or concentration of key ingredients, including buffering agents, chelating agents, reducing agents, and protease inhibitors. For both first and second instar larvae, the buffer used had a significant effect on the volume imbibed. The study showed that the droplet feeding method is compatible with Cry protein-solubilizing buffers, but that it is important to determine the volume imbibed for every buffer used in dose-dependent bioassays in order to reduce dose errors.

Field-Evolved Resistance of Northern and Western Corn Rootworm (Coleoptera: Chrysomelidae) Populations to Corn Hybrids Expressing Single and Pyramided Cry3Bb1 and Cry34/35Ab1 Bt Proteins in North Dakota.

Northern, Diabrotica barberi Smith & Lawrence, and western, D. virgifera virgifera LeConte, corn rootworms (Coleoptera: Chrysomelidae) are major economic pests of corn, Zea mays L., in North America. Corn hybrids expressing Bacillus thuringiensis Berliner (Bt) toxins are commonly used by growers to manage these pests. Several cases of field-evolved resistance to insecticidal proteins expressed by Bt corn hybrids have been documented in many corn-producing areas of North America, but only for D. v. virgifera. In 2016, beetles of both species were collected from five eastern North Dakota corn fields and reared in a growth chamber. In 2017, larvae reared from those populations were subjected to single-plant bioassays to screen for potential resistance to Cry3Bb1, Cry34/35Ab1, and pyramided Cry3Bb1 + Cry34/35Ab1 Bt toxins. Our results provide the first documented report of field-evolved resistance in D. barberi to corn hybrids expressing Cry3Bb1 (Arthur problem population) and Cry34/35Ab1 (Arthur and Page problem populations, and the Ransom and Sargent populations) proteins in North America. Resistance to Cry3Bb1 was also observed in the Ransom population of D. v. virgifera. Increased larval survival on the pyramided Cry3Bb1 + Cry34/35Ab1 hybrid was observed in both species. No cross-resistance was evident between Cry3Bb1 and Cry34/35Ab1 in any of the D. barberi populations tested. Our experiments identified field-evolved resistance to Bt toxins in some North Dakota populations of D. barberi and D. v. virgifera. Thus, more effective control tools and improved resistance management strategies are needed to prolong the durability of this technology for managing these important pests.

Efficacy of Bt Maize for Control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in South Africa.

Spodoptera frugiperda (J.E. Smith) is an invasive pest species that threatens maize production by small holder farmers in Africa. Bt maize that express Cry proteins have been used effectively for control of this pest in the United States, Canada, and several countries in South America. Spodoptera frugiperda has evolved resistance to Cry1F Bt maize in Puerto Rico, Brazil and United States, and Cry1Ab Bt maize in Brazil. This study provides the first data on the efficacy of Bt maize for the control of S. frugiperda in Africa. Susceptibility levels of nine S. frugiperda populations were evaluated between January 2018 and May 2018, including a laboratory reared reference population. Larval feeding bioassays were conducted in which plant tissue of maize expressing Cry1Ab (single-toxin event - designated Bt1) or Cry1A.105 + Cry2Ab2 (pyramid-toxin event - designated Bt2), were fed to larvae. Survival and different life history parameters were recorded. Results indicate moderate survival on Bt1 maize, which supports field observations of commercial level control provided by this event. Very high levels of S. frugiperda larval mortality occurred on Bt2 maize (<1% survival). The moderate susceptibility of S. frugiperda to Cry1Ab could be ascribed to the latter being a low-dose event for this pest, as well as the fact that the individuals which initially arrived on the continent may have carried alleles with resistance to this protein. Moderate overall survivorship (4-35%) of S. frugiperda on Cry1Ab maize in South Africa reflects the possible presence of alleles resistant to this toxin, indicating the importance of future resistance monitoring.

Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm.

A novel Bacillus thuringiensis Cry protein, Cry8Hb, active against Diabrotica virgifera virgifera (Western corn rootworm, WCRW) was discovered. Unexpectedly, the anti-rootworm activity of the Cry8Hb toxin was enhanced significantly by fusing Escherichia coli maltose binding protein (MBP) to this Cry toxin. While the exact mechanism of the activity enhancement remains indefinite, it is probable that the enhancement is a result of increased solubility of the MBP-Cry8Hb fusion in the rootworm midgut. This hypothesis was examined using a synthetic Cry3 protein called IP3-1, which was not soluble at a neutral pH like Cry8Hb and marginally active to WCRW. When IP3-1 was fused to MBP, its anti-WCRW activity was enhanced 13-fold. To further test the hypothesis, DNA shuffling was performed on IP3-1 to increase the solubility without MBP. Screening of shuffled libraries found six new IP3 variants showing very high anti-WCRW activity without MBP. Sequence and 3D structure analysis of those highly active, shuffled IP3 variants revealed several charge-altering mutations such as Lys to Glu on the putative MBP-attaching side of the IP3 molecule. It is likely that those mutations make the protein acidic to substitute the functions of MBP including enhancing the solubility of IP3 at a neutral pH.