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Special Issue "The Insecticidal Bacterial Toxins in Modern Agriculture"

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Bacterial Toxins".

Deadline for manuscript submissions: closed (28 February 2017)

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Guest Editor
Prof. Juan Ferré

Department of Genetics, University of Valencia, Burjassot (Valencia), Spain
E-Mail
Interests: discovery of new insecticidal strains and proteins; toxicity potentiation of Cry and Vip proteins; mode of action of Cry; Vip and MTX proteins; insect genetic response to biological pesticides; bases of insect resistance to biopesticides
Guest Editor
Prof. Baltasar Escriche

Department of Genetics, University of Valencia, Burjassot (Valencia), Spain
E-Mail
Interests: discovery of new insecticidal strains and proteins; toxicity potentiation of Cry and Vip proteins; mode of action of Cry; Vip and MTX proteins; insect genetic response to biological pesticides; bases of insect resistance to biopesticides

Special Issue Information

Dear Colleagues,

Increased awareness about adverse environmental effects of human activities has prompted the use of insecticides with a low impact on systems associated with agriculture. Currently, the most successful biological products are based on protein toxins from the bacterial species Bacillus thuringiensis. Because of the remarkable properties of these proteins, their encoding genes were introduced into farming species (the so-called Bt-crops) in such a way, that these plants are self-protected against some key, insect pests. Despite the fact that a relatively large number of these toxins, with different toxicity ranges, have been described, it is still important to find new resources with novel capabilities to complement, or to replace in the future, the currently-used ones. On the other hand, it is important to continue studying their modes action in susceptible insects, and the changes occurring in resistant ones, to determine the most effective strategy for long-lasting pest control. The focus of this Special Issue of Toxins is to provide updated information on the use of B. thuringiensis and their toxins on different field crops, the interactions of these toxins with other molecules, to analyze the biochemical and molecular basis of emerging cases of resistance, and, in general, to provide information that can contribute to effective pest management with these toxins.

Baltasar Escriche
Juan Ferré
Guest Editors

Manuscript Submission Information

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Keywords

  • bacterial toxin
  • Bacillus thuringiensis
  • mode of action
  • insect resistance
  • pest management

Published Papers (12 papers)

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Editorial

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Open AccessEditorial Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture
Toxins 2017, 9(12), 396; https://doi.org/10.3390/toxins9120396
Received: 29 November 2017 / Revised: 4 December 2017 / Accepted: 4 December 2017 / Published: 9 December 2017
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Research

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Open AccessArticle Characterization of Asian Corn Borer Resistance to Bt Toxin Cry1Ie
Received: 24 February 2017 / Revised: 21 May 2017 / Accepted: 1 June 2017 / Published: 7 June 2017
Cited by 1 | PDF Full-text (289 KB) | HTML Full-text | XML Full-text
Abstract
A strain of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), has evolved >800-fold resistance to Cry1Ie (ACB-IeR) after 49 generations of selection. The inheritance pattern of resistance to Cry1Ie in ACB-IeR strain and its cross-resistance to other Bt toxins were determined through [...] Read more.
A strain of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), has evolved >800-fold resistance to Cry1Ie (ACB-IeR) after 49 generations of selection. The inheritance pattern of resistance to Cry1Ie in ACB-IeR strain and its cross-resistance to other Bt toxins were determined through bioassay by exposing neonates from genetic-crosses to toxins incorporated into the diet. The response of progenies from reciprocal F1 crosses were similar (LC50s: 76.07 vs. 74.32 μg/g), which suggested the resistance was autosomal. The effective dominance (h) decreased as concentration of Cry1Ie increased. h was nearly recessive or incompletely recessive on Cry1Ie maize leaf tissue (h = 0.02), but nearly dominant or incompletely dominant (h = 0.98) on Cry1Ie maize silk. Bioassay of the backcross suggested that the resistance was controlled by more than one locus. In addition, the resistant strain did not perform cross-resistance to Cry1Ab (0.8-fold), Cry1Ac (0.8-fold), Cry1F (0.9-fold), and Cry1Ah (1.0-fold). The present study not only offers the manifestation for resistance management, but also recommends that Cry1Ie will be an appropriate candidate for expression with Cry1Ab, Cry1Ac, Cry1F, or Cry1Ah for the development of Bt maize. Full article
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Open AccessArticle Assessment of Inheritance and Fitness Costs Associated with Field-Evolved Resistance to Cry3Bb1 Maize by Western Corn Rootworm
Received: 31 March 2017 / Revised: 4 May 2017 / Accepted: 5 May 2017 / Published: 11 May 2017
Cited by 3 | PDF Full-text (2117 KB) | HTML Full-text | XML Full-text
Abstract
The western corn rootworm, Diabrotica virgifera virgifera LeConte, is among the most serious insect pests of maize in North America. One strategy used to manage this pest is transgenic maize that produces one or more crystalline (Cry) toxins derived from the bacterium Bacillus [...] Read more.
The western corn rootworm, Diabrotica virgifera virgifera LeConte, is among the most serious insect pests of maize in North America. One strategy used to manage this pest is transgenic maize that produces one or more crystalline (Cry) toxins derived from the bacterium Bacillus thuringiensis (Bt). To delay Bt resistance by insect pests, refuges of non-Bt maize are grown in conjunction with Bt maize. Two factors influencing the success of the refuge strategy to delay resistance are the inheritance of resistance and fitness costs, with greater delays in resistance expected when inheritance of resistance is recessive and fitness costs are present. We measured inheritance and fitness costs of resistance for two strains of western corn rootworm with field-evolved resistance to Cry3Bb1 maize. Plant-based and diet-based bioassays revealed that the inheritance of resistance was non-recessive. In a greenhouse experiment, in which larvae were reared on whole maize plants in field soil, no fitness costs of resistance were detected. In a laboratory experiment, in which larvae experienced intraspecific and interspecific competition for food, a fitness cost of delayed larval development was identified, however, no other fitness costs were found. These findings of non-recessive inheritance of resistance and minimal fitness costs, highlight the potential for the rapid evolution of resistance to Cry3Bb1 maize by western corn rootworm, and may help to improve resistance management strategies for this pest. Full article
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Open AccessArticle Histopathological Effects of Bt and TcdA Insecticidal Proteins on the Midgut Epithelium of Western Corn Rootworm Larvae (Diabrotica virgifera virgifera)
Received: 22 March 2017 / Revised: 20 April 2017 / Accepted: 28 April 2017 / Published: 8 May 2017
Cited by 4 | PDF Full-text (5748 KB) | HTML Full-text | XML Full-text
Abstract
Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is a major corn pest in the United States, causing annual losses of over $1 billion. One approach to protect against crop loss by this insect is the use of transgenic corn hybrids expressing one [...] Read more.
Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is a major corn pest in the United States, causing annual losses of over $1 billion. One approach to protect against crop loss by this insect is the use of transgenic corn hybrids expressing one or more crystal (Cry) proteins derived from Bacillus thuringiensis. Cry34Ab1 and Cry35Ab1 together comprise a binary insecticidal toxin with specific activity against WCR. These proteins have been developed as insect resistance traits in commercialized corn hybrids resistant to WCR feeding damage. Cry34/35Ab1 is a pore forming toxin, but the specific effects of Cry34/35Ab1 on WCR cells and tissues have not been well characterized microscopically, and the overall histopathology is poorly understood. Using high-resolution resin-based histopathology methods, the effects of Cry34/35Ab1 as well as Cry3Aa1, Cry6Aa1, and the Photorhabdus toxin complex protein TcdA have been directly visualized and documented. Clear symptoms of intoxication were observed for all insecticidal proteins tested, including swelling and sloughing of enterocytes, constriction of midgut circular muscles, stem cell activation, and obstruction of the midgut lumen. These data demonstrate the effects of these insecticidal proteins on WCR midgut cells, and the collective response of the midgut to intoxication. Taken together, these results advance our understanding of the insect cell biology and pathology of these insecticidal proteins, which should further the field of insect resistance traits and corn rootworm management. Full article
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Open AccessArticle Assessment of the Antimicrobial Activity and the Entomocidal Potential of Bacillus thuringiensis Isolates from Algeria
Received: 14 March 2017 / Revised: 29 March 2017 / Accepted: 11 April 2017 / Published: 13 April 2017
Cited by 8 | PDF Full-text (3448 KB) | HTML Full-text | XML Full-text
Abstract
This work represents the first initiative to analyze the distribution of B. thuringiensis in Algeria and to evaluate the biological potential of the isolates. A total of 157 isolates were recovered, with at least one isolate in 94.4% of the samples. The highest [...] Read more.
This work represents the first initiative to analyze the distribution of B. thuringiensis in Algeria and to evaluate the biological potential of the isolates. A total of 157 isolates were recovered, with at least one isolate in 94.4% of the samples. The highest Bt index was found in samples from rhizospheric soil (0.48) and from the Mediterranean area (0.44). Most isolates showed antifungal activity (98.5%), in contrast to the few that had antibacterial activity (29.9%). A high genetic diversity was made evident by the finding of many different crystal shapes and various combinations of shapes within a single isolate (in 58.4% of the isolates). Also, over 50% of the isolates harbored cry1, cry2, or cry9 genes, and 69.3% contained a vip3 gene. A good correlation between the presence of chitinase genes and antifungal activity was observed. More than half of the isolates with a broad spectrum of antifungal activity harbored both endochitinase and exochitinase genes. Interestingly, 15 isolates contained the two chitinase genes and all of the above cry family genes, with some of them harboring a vip3 gene as well. The combination of this large number of genes coding for entomopathogenic proteins suggests a putative wide range of entomotoxic activity. Full article
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Open AccessFeature PaperEditor’s ChoiceArticle Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis
Received: 14 March 2017 / Revised: 28 March 2017 / Accepted: 4 April 2017 / Published: 7 April 2017
Cited by 13 | PDF Full-text (1780 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its [...] Read more.
Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices. Full article
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Open AccessArticle Baseline Susceptibility of Field Populations of Helicoverpa armigera to Bacillus thuringiensis Vip3Aa Toxin and Lack of Cross-Resistance between Vip3Aa and Cry Toxins
Received: 25 February 2017 / Revised: 23 March 2017 / Accepted: 27 March 2017 / Published: 5 April 2017
Cited by 11 | PDF Full-text (248 KB) | HTML Full-text | XML Full-text
Abstract
The cotton bollworm Helicoverpa armigera (Hübner) is one of the most damaging cotton pests worldwide. In China, control of this pest has been dependent on transgenic cotton producing a single Bacillus thuringiensis (Bt) protein Cry1Ac since 1997. A small, but significant, increase in [...] Read more.
The cotton bollworm Helicoverpa armigera (Hübner) is one of the most damaging cotton pests worldwide. In China, control of this pest has been dependent on transgenic cotton producing a single Bacillus thuringiensis (Bt) protein Cry1Ac since 1997. A small, but significant, increase in H. armigera resistance to Cry1Ac was detected in field populations from Northern China. Since Vip3Aa has a different structure and mode of action than Cry proteins, Bt cotton pyramids containing Vip3Aa are considered as ideal successors of Cry1Ac cotton in China. In this study, baseline susceptibility of H. armigera to Vip3Aa was evaluated in geographic field populations collected in 2014 from major cotton-producing areas of China. The LC50 values of 12 field populations ranged from 0.053 to 1.311 μg/cm2, representing a 25-fold range of natural variation among populations. It is also demonstrated that four laboratory strains of H. armigera with high levels of resistance to Cry1Ac or Cry2Ab have no cross-resistance to Vip3Aa protein. The baseline susceptibility data established here will serve as a comparative reference for detection of field-evolved resistance to Vip3Aa in H. armigera after future deployment of Bt cotton pyramids in China. Full article
Open AccessArticle Patterns of Gene Expression in Western Corn Rootworm (Diabrotica virgifera virgifera) Neonates, Challenged with Cry34Ab1, Cry35Ab1 and Cry34/35Ab1, Based on Next-Generation Sequencing
Received: 26 February 2017 / Revised: 22 March 2017 / Accepted: 27 March 2017 / Published: 30 March 2017
Cited by 3 | PDF Full-text (1440 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
With Next Generation Sequencing technologies, high-throughput RNA sequencing (RNAseq) was conducted to examine gene expression in neonates of Diabrotica virgifera virgifera (LeConte) (Western Corn Rootworm, WCR) challenged with individual proteins of the binary Bacillus thuringiensis insecticidal proteins, Cry34Ab1 and Cry35Ab1, and the combination [...] Read more.
With Next Generation Sequencing technologies, high-throughput RNA sequencing (RNAseq) was conducted to examine gene expression in neonates of Diabrotica virgifera virgifera (LeConte) (Western Corn Rootworm, WCR) challenged with individual proteins of the binary Bacillus thuringiensis insecticidal proteins, Cry34Ab1 and Cry35Ab1, and the combination of Cry34/Cry35Ab1, which together are active against rootworm larvae. Integrated results of three different statistical comparisons identified 114 and 1300 differentially expressed transcripts (DETs) in the Cry34Ab1 and Cry34/35Ab1 treatment, respectively, as compared to the control. No DETs were identified in the Cry35Ab1 treatment. Putative Bt binding receptors previously identified in other insect species were not identified in DETs in this study. The majority of DETs (75% with Cry34Ab1 and 68.3% with Cry34/35Ab1 treatments) had no significant hits in the NCBI nr database. In addition, 92 DETs were shared between Cry34Ab1 and Cry34/35Ab1 treatments. Further analysis revealed that the most abundant DETs in both Cry34Ab1 and Cry34/35Ab1 treatments were associated with binding and catalytic activity. Results from this study confirmed the nature of these binary toxins against WCR larvae and provide a fundamental profile of expression pattern of genes in response to challenge of the Cry34/35Ab1 toxin, which may provide insight into potential resistance mechanisms. Full article
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Open AccessArticle Use of Carabids for the Post-Market Environmental Monitoring of Genetically Modified Crops
Received: 4 January 2017 / Revised: 23 March 2017 / Accepted: 27 March 2017 / Published: 29 March 2017
Cited by 2 | PDF Full-text (1354 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Post-market environmental monitoring (PMEM) of genetically modified (GM) crops is required by EU legislation and has been a subject of debate for many years; however, no consensus on the methodology to be used has been reached. We explored the suitability of carabid beetles [...] Read more.
Post-market environmental monitoring (PMEM) of genetically modified (GM) crops is required by EU legislation and has been a subject of debate for many years; however, no consensus on the methodology to be used has been reached. We explored the suitability of carabid beetles as surrogates for the detection of unintended effects of GM crops in general PMEM surveillance. Our study combines data on carabid communities from five maize field trials in Central Europe. Altogether, 86 species and 58,304 individuals were collected. Modeling based on the gradual elimination of the least abundant species, or of the fewest categories of functional traits, showed that a trait-based analysis of the most common species may be suitable for PMEM. Species represented by fewer than 230 individuals (all localities combined) should be excluded and species with an abundance higher than 600 should be preserved for statistical analyses. Sixteen species, representing 15 categories of functional traits fulfill these criteria, are typical dominant inhabitants of agroecocoenoses in Central Europe, are easy to determine, and their functional classification is well known. The effect of sampling year is negligible when at least four samples are collected during maize development beginning from 1 April. The recommended methodology fulfills PMEM requirements, including applicability to large-scale use. However, suggested thresholds of carabid comparability should be verified before definitive conclusions are drawn. Full article
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Open AccessArticle Consumption of Bt Maize Pollen Containing Cry1Ie Does Not Negatively Affect Propylea japonica (Thunberg) (Coleoptera: Coccinellidae)
Received: 8 February 2017 / Revised: 3 March 2017 / Accepted: 11 March 2017 / Published: 16 March 2017
Cited by 6 | PDF Full-text (261 KB) | HTML Full-text | XML Full-text
Abstract
Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) are prevalent predators and pollen feeders in East Asian maize fields. They are therefore indirectly (via prey) and directly (via pollen) exposed to Cry proteins within Bt-transgenic maize fields. The effects of Cry1Ie-producing transgenic maize pollen on [...] Read more.
Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) are prevalent predators and pollen feeders in East Asian maize fields. They are therefore indirectly (via prey) and directly (via pollen) exposed to Cry proteins within Bt-transgenic maize fields. The effects of Cry1Ie-producing transgenic maize pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, survival, larval developmental time, adult fresh weight, and fecundity did not differ between ladybirds consuming Bt or non-Bt maize pollen. In the second experiment, none of the tested lethal and sublethal parameters of P. japonica were negatively affected when fed a rapeseed pollen-based diet containing Cry1Ie protein at 200 μg/g dry weight of diet. In contrast, the larval developmental time, adult fresh weight, and fecundity of P. japonica were significantly adversely affected when fed diet containing the positive control compound E-64. In both experiments, the bioactivity of the Cry1Ie protein in the food sources was confirmed by bioassays with a Cry1Ie-sensitive lepidopteran species. These results indicated that P. japonica are not affected by the consumption of Cry1Ie-expressing maize pollen and are not sensitive to the Cry1Ie protein, suggesting that the growing of Bt maize expressing Cry1Ie protein will pose a negligible risk to P. japonica. Full article
Open AccessArticle A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle
Toxins 2016, 8(12), 362; https://doi.org/10.3390/toxins8120362
Received: 16 September 2016 / Revised: 21 November 2016 / Accepted: 25 November 2016 / Published: 5 December 2016
Cited by 14 | PDF Full-text (2686 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can [...] Read more.
Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well. Full article
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Review

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Open AccessReview In Vivo Crystallization of Three-Domain Cry Toxins
Received: 13 January 2017 / Revised: 10 February 2017 / Accepted: 23 February 2017 / Published: 9 March 2017
Cited by 4 | PDF Full-text (798 KB) | HTML Full-text | XML Full-text
Abstract
Bacillus thuringiensis (Bt) is the most successful, environmentally-friendly, and intensively studied microbial insecticide. The major characteristic of Bt is the production of proteinaceous crystals containing toxins with specific activity against many pests including dipteran, lepidopteran, and coleopteran insects, as well as nematodes, protozoa, [...] Read more.
Bacillus thuringiensis (Bt) is the most successful, environmentally-friendly, and intensively studied microbial insecticide. The major characteristic of Bt is the production of proteinaceous crystals containing toxins with specific activity against many pests including dipteran, lepidopteran, and coleopteran insects, as well as nematodes, protozoa, flukes, and mites. These crystals allow large quantities of the protein toxins to remain stable in the environment until ingested by a susceptible host. It has been previously established that 135 kDa Cry proteins have a crystallization domain at their C-terminal end. In the absence of this domain, Cry proteins often need helper proteins or other factors for crystallization. In this review, we classify the Cry proteins based on their requirements for crystallization. Full article
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