RESUMO
The fall armyworm (Spodoptera frugiperda) is a major global pest causing severe damage to various crops, especially corn. Transgenic corn producing the Cry1F pesticidal protein from the bacterium Bacillus thuringiensis (Cry1F corn) showed effectiveness in controlling this pest until S. frugiperda populations at locations in North and South America evolved practical resistance. The mechanism for practical resistance involved disruptive mutations in an ATP binding cassette transporter subfamily C2 gene (SfABCC2), which serves as a functional Cry1F receptor in the midgut cells of susceptible S. frugiperda. The SfABCC2 protein contains two transmembrane domains (TMD1 and TMD2), each with a cytosolic nucleotide (ATP) binding domain (NBD1 and NBD2, respectively). Previous reports have demonstrated that disruptive mutations in TMD2 were linked with resistance to Cry1F, yet whether the complete SfABCC2 structure is needed for receptor functionality or if a single TMD-NBD protein can serve as functional Cry1F receptor remains unknown. In the present study, we separately expressed TMD1 and TMD2 with their corresponding NBDs in cultured insect cells and tested their Cry1F receptor functionality. Our results show that the complete SfABCC2 structure is required for Cry1F receptor functionality. Moreover, binding competition assays revealed that Cry1F specifically bound to SfABCC2, whereas neither SfTMD1-NBD1 nor SfTMD2-NBD2 exhibited any significant binding. These results provide insights into the molecular mechanism of Cry1F recognition by SfABCC2 in S. frugiperda, which could facilitate the development of more effective insecticidal proteins.
Assuntos
Bacillus thuringiensis , Endotoxinas , Animais , Spodoptera , Endotoxinas/genética , Resistência a Inseticidas/genética , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacillus thuringiensis/metabolismo , Zea mays , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
IMPORTANCE: Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant-incorporated protectant in Bt crops that consistently provides excellent control of H. zea. The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.
Assuntos
Bacillus thuringiensis , Mariposas , Animais , Estados Unidos , Zea mays/metabolismo , Endotoxinas/metabolismo , Resistência a Inseticidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Hemolisinas/genética , Mariposas/genética , Bacillus thuringiensis/genética , Larva/metabolismoRESUMO
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.
Assuntos
Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Insetos/genética , Alelos , Animais , Controle de Insetos , Resistência a Inseticidas/genéticaRESUMO
BACKGROUND: The fall armyworm (Spodoptera frugiperda (J.E. Smith)) is a highly polyphagous agricultural pest with long-distance migratory behavior threatening food security worldwide. This pest has a host range of > 80 plant species, but two host strains are recognized based on their association with corn (C-strain) or rice and smaller grasses (R-strain). The population genomics of the United States (USA) fall armyworm remains poorly characterized to date despite its agricultural threat. RESULTS: In this study, the population structure and genetic diversity in 55 S. frugiperda samples from Argentina, Brazil, Kenya, Puerto Rico and USA were surveyed to further our understanding of whole genome nuclear diversity. Comparisons at the genomic level suggest a panmictic S. frugiperda population, with only a minor reduction in gene flow between the two overwintering populations in the continental USA, also corresponding to distinct host strains at the mitochondrial level. Two maternal lines were detected from analysis of mitochondrial genomes. We found members from the Eastern Hemisphere interspersed within both continental USA overwintering subpopulations, suggesting multiple individuals were likely introduced to Africa. CONCLUSIONS: Our research is the largest diverse collection of United States S. frugiperda whole genome sequences characterized to date, covering eight continental states and a USA territory (Puerto Rico). The genomic resources presented provide foundational information to understand gene flow at the whole genome level among S. frugiperda populations. Based on the genomic similarities found between host strains and laboratory vs. field samples, our findings validate the experimental use of laboratory strains and the host strain differentiation based on mitochondria and sex-linked genetic markers extends to minor genome wide differences with some exceptions showing mixture between host strains is likely occurring in field populations.
Assuntos
Fluxo Gênico , Zea mays , Animais , Brasil , Humanos , Quênia , Spodoptera , Zea mays/genéticaRESUMO
Extensive planting of crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has suppressed some major pests, reduced insecticide sprays, enhanced pest control by natural enemies, and increased grower profits. However, rapid evolution of resistance in pests is reducing these benefits. Better understanding of the genetic basis of resistance to Bt crops is urgently needed to monitor, delay, and counter pest resistance. We discovered that a point mutation in a previously unknown tetraspanin gene in the cotton bollworm (Helicoverpa armigera), a devastating global pest, confers dominant resistance to Cry1Ac, the sole Bt protein produced by transgenic cotton planted in China. We found the mutation using a genome-wide association study, followed by fine-scale genetic mapping and DNA sequence comparisons between resistant and susceptible strains. CRISPR/Cas9 knockout of the tetraspanin gene restored susceptibility to a resistant strain, whereas inserting the mutation conferred 125-fold resistance in a susceptible strain. DNA screening of moths captured from 23 field sites in six provinces of northern China revealed a 100-fold increase in the frequency of this mutation, from 0.001 in 2006 to 0.10 in 2016. The correspondence between the observed trajectory of the mutation and the trajectory predicted from simulation modeling shows that the dominance of the mutation accelerated adaptation. Proactive identification and tracking of the tetraspanin mutation demonstrate the potential for genomic analysis, gene editing, and molecular monitoring to improve management of resistance.
Assuntos
Resistência a Inseticidas/genética , Mariposas/genética , Tetraspaninas/genética , Animais , Animais Geneticamente Modificados/genética , Bacillus thuringiensis/genética , Proteínas de Bactérias/metabolismo , China , Evolução Molecular , Estudo de Associação Genômica Ampla , Gossypium/genética , Inseticidas/metabolismo , Larva/genética , Larva/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Mutação Puntual/genéticaRESUMO
The Cry1Ac protein is the most active insecticidal toxin from the bacterium Bacillus thuringiensis (Bt) to members of the heliothinae subfamily in Lepidoptera, which includes some of the most devastating pests of corn and cotton worldwide. However, there are wide discrepancies in susceptibility among members of this subfamily in the US. Specifically, susceptibility to Cry1Ac in Helicoverpa zea (Hz) is >100-fold lower when compared to Heliothis virescens (Hv) larvae. The biochemical properties and Cry1Ac protoxin processing activity of gut digestive fluids from larvae of Hz and Hv were compared to test their role in differential susceptibility to Cry1Ac. Comparatively lower protease activity, associated with slower Cry1Ac proteolytic processing, was detected in digestive fluids of Hz compared to Hv. Moreover, Cry1Ac toxin processed by Hz digestive fluids displayed significantly lower toxicity in vitro against cultured insect cells compared to toxin activated by Hv proteases. These data support a contributing role for gut proteases in differential susceptibility to Cry1Ac in heliothine larvae.
Assuntos
Proteínas de Bactérias/toxicidade , Agentes de Controle Biológico/toxicidade , Endotoxinas/toxicidade , Trato Gastrointestinal/metabolismo , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/metabolismo , Inseticidas/toxicidade , Larva/enzimologia , Mariposas/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Controle Biológico de Vetores , ProteóliseRESUMO
Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/farmacologia , Agentes de Controle Biológico/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Sistema de Sinalização das MAP Quinases , Transportadores de Cassetes de Ligação de ATP/genética , Fosfatase Alcalina/genética , Animais , Bacillus/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Agentes de Controle Biológico/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/genética , Mucosa Intestinal/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/metabolismo , Ligação ProteicaRESUMO
Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used as active components of biopesticides and as plant incorporated protectants in transgenic crops. One of the most relevant attributes of these Bt protein-based insecticidal technologies is their high specificity, which assures lack of detrimental effects on non-target insects, vertebrates and the environment. The identification of specificity determinants in Bt insecticidal proteins could guide risk assessment for novel insecticidal proteins currently considered for commercialization. In this work we review the available data on specificity determinants of crystal (Cry) insecticidal proteins as the Bt toxins most well characterized and used in transgenic crops. The multi-step mode of action of the Cry insecticidal proteins allows various factors to potentially affect specificity determination and here we define seven levels that could influence specificity. The relative relevance of each of these determinants on efficacy of transgenic crops producing Cry insecticidal proteins is also discussed.
Assuntos
Proteínas de Bactérias/química , Endotoxinas/química , Proteínas Hemolisinas/química , Controle Biológico de Vetores/métodos , Animais , Toxinas de Bacillus thuringiensis , Plantas Geneticamente Modificadas , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Transgenic soybean producing the Cry1Ac insecticidal protein from the bacterium Bacillus thuringiensis is used to control larvae of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) and the soybean looper [Chrysodeixis includens (Walker)]. The main threat to the sustainability of this technology is the development of resistance, which could be delayed by using pyramiding of diverse Bt insecticidal genes. We report high activity of Cry2Ac7 and Vip3Aa11 but not Cry1Ie2 against larvae of A. gemmatalis and C. includens. In addition, we also report anti-feeding activity of Cry1Ie2 and Cry7Ab3 in adults of the bean leaf beetle [Ceratoma trifurcata (Foster)], an alternative pest of soybean.
Assuntos
Bacillus thuringiensis/metabolismo , Besouros , Proteínas Hemolisinas/metabolismo , Larva , Mariposas , Controle Biológico de Vetores/métodos , Animais , Bacillus thuringiensis/genética , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas , Glycine maxRESUMO
Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with (125)I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution.
Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Lepidópteros/efeitos dos fármacos , Animais , Bacillus/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Larva/efeitos dos fármacos , Peso Molecular , Ligação Proteica , Análise de Sobrevida , Tenebrio/efeitos dos fármacos , Zea mays/parasitologiaRESUMO
Insect resistance threatens sustainability of insecticides based on Cry proteins from the bacterium Bacillus thuringiensis (Bt). Since high levels of resistance to Cry proteins involve alterations in Cry-binding midgut receptors, their identification is needed to develop resistance management strategies. Through Illumina sequencing we generated a transcriptome containing 16,161 annotated unigenes for the Oriental leafworm (Spodoptera litura). Transcriptome mining identified 6 contigs with identity to reported lepidopteran Cry toxin receptors. Using PCR we confirmed their expression during the larval stage and compared their quantitative expression in larvae from susceptible and a field-derived Cry1Ca resistant strain of S. litura. Among reduced transcript levels detected for most tested contigs in the Cry1Ca-resistant S. litura larvae, the most dramatic reduction (up to 99%) was detected for alkaline phosphatase contigs. This study significantly expands S. litura transcriptomic resources and provides preliminary identification of putative receptor genes with altered expression in S. litura resistant to Cry1Ca toxin.
Assuntos
Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Spodoptera/genética , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Feminino , Regulação da Expressão Gênica , Proteínas Hemolisinas/metabolismo , Larva , Masculino , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , TranscriptomaRESUMO
Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.
Assuntos
Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas/efeitos dos fármacos , Oryza/parasitologia , Plantas Geneticamente Modificadas/parasitologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , China , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Cinética , Mariposas/química , Mariposas/metabolismo , Oryza/genética , Oryza/metabolismo , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
BACKGROUND: Implementation of resistance management tools is crucial for the continued efficacy of insect control technologies. An important aspect of insect resistance management (IRM) is the combined or sequential use of different modes-of-action to reduce selection pressure and delay evolution of resistance. This is especially important for insect pests with established ability to develop resistance to insecticides, such as the Colorado potato beetle (Leptinotarsa decemlineata, CPB). A new class of insecticides, based on double-stranded RNA (dsRNA) activating the gene silencing RNA-interference (RNAi) pathway, are currently under review for regulatory approval and commercial use in the USA against CPB. However, there is no information available on the potential for cross-resistance between RNAi insecticides and other classes of insecticides used against CPB. Herein, we aim to fill this knowledge gap by capitalizing on the availability of a CPB strain highly resistant to dsRNAs and test its susceptibility to diverse small-molecule insecticide classes compared to reference dsRNA-susceptible CPB strains. RESULTS: Differences in activity were observed among the four insecticides tested, with abamectin demonstrating highest activity against all three strains of CPB. However, no differences were observed among the dsRNA-resistant and susceptible CPB strains for any of the tested compounds. Overall, these results demonstrate lack of cross-resistance to commonly used chemical insecticides in the dsRNA-resistant strain of CPB. CONCLUSION: These data support the use of these different insecticide classes along with RNAi-based insecticides as part of an effective insect resistance management framework aimed at delaying resistance in CPB. © 2023 Society of Chemical Industry.
Assuntos
Besouros , Inseticidas , Praguicidas , Solanum tuberosum , Animais , Besouros/genética , Larva , Inseticidas/farmacologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/farmacologia , Praguicidas/farmacologia , Solanum tuberosum/genética , Interferência de RNARESUMO
The bacterium Bacillus thuringiensis (Bt) is the most economically successful biopesticide to date, and Bt insecticidal proteins are produced in transgenic crops for pest control. However, relevant details in the Bt-mediated killing process remain undefined. In our previous research, we observed reduced larval susceptibility to Bt Cry1Ca in Chilo suppressalis, a major rice pest in China, after gut microbiota elimination. Here, we tested the hypothesis that gut microbiota, particularly abundant Enterococcus spp., influences C. suppressalis susceptibility to Cry1Ca. We isolated and identified four Enterococcus spp. from C. suppressalis gut microbiota and evaluated their impact on Cry1Ca toxicity. Among the four Enterococcus spp. identified, three of them (E. casseliflavus, E. faecalis, and E. mundtii) dramatically increased larval mortality when introduced in axenic C. suppressalis challenged with Cry1Ca. Gut epithelial damage by Cry1Ca promoted the translocation of Enterococcus spp. from the gut lumen into the hemocoel, where they proliferated and induced larval melanization and hemocyte apoptosis. Our combined findings demonstrate that the presence of specific gut microbiota can greatly affect susceptibility to Cry1Ca through melanization and apoptosis of hemocytes. Better understanding of the Bt intoxication process guides the development of bio-enhancers for Bt-based microbial biopesticides and potential improvement of transgenic crops.
Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Oryza , Animais , Enterococcus , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas , Proteínas Hemolisinas/metabolismo , Mariposas/genética , Larva , Inseticidas/farmacologia , Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/metabolismo , Animais Geneticamente Modificados , Oryza/genéticaRESUMO
Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides.
Assuntos
Controle Biológico de Vetores , Interferência de RNA , Animais , Solanum tuberosum/genética , Controle de Insetos/métodos , Resistência a Inseticidas/genética , Insetos/genéticaRESUMO
BACKGROUND: Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long-term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field-derived H. zea strains (M1-RR, AC4-RR, R2-RR and R15-RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70-RR). RESULTS: Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 µg cm-2, in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1-RR and LT#70-RR; another shared by R2-RR and R15-RR; and a distinct one for AC4-RR. CONCLUSION: Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Proteínas de Bactérias , Resistência a Medicamentos , Mariposas , Mariposas/efeitos dos fármacos , Mariposas/genética , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Resistência a Medicamentos/genética , Controle de Pragas/métodos , Dose Letal Mediana , Teste de Complementação Genética , Genes Recessivos/genética , AnimaisRESUMO
Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.
RESUMO
The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a highly polyphagous pest native to the tropical Americas that has recently spread to become a global super-pest threatening food and fiber production. Transgenic crops producing insecticidal Cry and Vip3Aa proteins from Bacillus thuringiensis (Bt) are used for control of this pest in its native range. The evolution of practical resistance represents the greatest threat to sustainability of this technology and its potential efficacy in the S. frugiperda invasive range. Monitoring for resistance is vital to management approaches delaying S. frugiperda resistance to Bt crops. DNA-based resistance screening provides higher sensitivity and cost-effectiveness than currently used bioassay-based monitoring. So far, practical S. frugiperda resistance to Bt corn-producing Cry1F has been genetically linked to mutations in the SfABCC2 gene, providing a model to develop and test monitoring tools. In this study, we performed targeted SfABCC2 sequencing followed by Sanger sequencing to confirm the detection of known and candidate resistance alleles to Cry1F corn in field-collected S. frugiperda from continental USA, Puerto Rico, Africa (Ghana, Togo, and South Africa), and Southeast Asia (Myanmar). Results confirm that the distribution of a previously characterized resistance allele (SfABCC2mut) is limited to Puerto Rico and identify 2 new candidate SfABCC2 alleles for resistance to Cry1F, one of them potentially spreading along the S. frugiperda migratory route in North America. No candidate resistance alleles were found in samples from the invasive S. frugiperda range. These results provide support for the potential use of targeted sequencing in Bt resistance monitoring programs.
Assuntos
Bacillus thuringiensis , Heterópteros , Animais , Spodoptera/genética , Alelos , Produtos AgrícolasRESUMO
The fall armyworm (JE Smith) (Spodoptera frugiperda) is a polyphagous pest targeted by selected Cry and Vip3A insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) that are produced in transgenic Bt corn and cotton. Available evidence suggests that sublethal larval exposure to Cry1Ac increases flight activity in adult Spodoptera spp. However, it is not known whether this effect is also observed in survivors from generally lethal exposure to Cry1Ac. Moreover, while multiple cases of field-evolved resistance to Bt proteins have been described in the native range of S. frugiperda, the effect of resistance on flight behavior has not been examined. Long-distance migratory flight capacity of S. frugiperda is of concern given its ongoing global spread and the possibility that migrants may be carrying resistance alleles against pesticides and Bt crops. In this study, we used rotational flight mills to test the effects of generally lethal exposure to Cry1Ac in susceptible and sublethal exposure in Cry1F-resistant S. frugiperda strains. The results detected altered pupal weight after larval feeding on diet containing Cry proteins, which only translated in significantly increased tendency for longer flights in female moths from the susceptible strain. This information has relevant implications when considering current models and assumptions for resistance management of Bt crops.
RESUMO
The genus Diatraea (Lepidoptera: Crambidae) includes stem borers representing the most critical sugarcane pests in the Americas. Colombia's most widely distributed and damaging Diatraea species include Diatraea saccharalis, D. indigenella, D. busckella, and D. tabernella. The reduced efficacy of biological tools commonly used in controlling several species highlights the importance of evaluating alternative management strategies, such as transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The selection of optimal Bt insecticidal proteins for Diatraea control depends on bioassays with purified Bt proteins. Because there is no described artificial diet for borer species other than D. saccharalis and availability of most purified Bt toxins is restricted, this study aimed at developing a bioassay method using fresh corn tissue and providing proof of concept by testing susceptibility to the Cry1Ac insecticidal protein from Bt. Toxicity was evaluated with a single Cry1Ac dose applied directly to corn discs. Stem borer mortality after seven days was higher than 90% for all four tested Diatraea species, while control mortality was below 8%. In addition, we observed that Cry1Ac caused more than 90% weight inhibition in all survivors and delayed development. These results validate the use of this method to determine mortality and growth inhibition due to the consumption of the Cry1Ac protein in each of the Diatraea species. Furthermore, this method could be used to assess other entomopathogenic substances to control these insect pests.