Bacillus Thuringiensis Proteins Research Articles

Knockdown of an ATP-binding cassette transporter in resistant western corn rootworm larvae partially reverses resistance to eCry3.1Ab protein

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, has evolved resistance to nearly every management tactic utilized in the field. This study investigated the resistance mechanisms in a WCR strain resistant to the Bacillus thuringiensis (Bt) protein eCry3.1Ab using dsRNA to knockdown WCR midgut genes previously documented to be associated with the resistance. ATP-binding cassette transporter (ABCC4), aminopeptidase-N, cadherin, and cathepsin-B were previously found to be differentially expressed in eCry3.1Ab-resistant WCR larvae when compared to susceptible larvae after feeding on maize expressing eCry3.1Ab and its near-isoline. Here we compared the susceptibility of resistant and susceptible WCR larvae to eCry3.1Ab protein in presence or absence of dsRNA targeting the above genes using 10-day diet overlay toxicity assays. Combining ABCC4 dsRNA with eCry3.1Ab protein increased susceptibility to Bt protein in WCR-resistant larvae, but the other three genes had no such effect. Among 65 ABC transport genes identified, several were expressed differently in resistant or susceptible WCR larvae, fed on eCry3.1Ab-expressing maize versus its isoline, that may be involved in Bt resistance. Our findings provide strong evidence that ABCC4 is indirectly involved in WCR resistance to eCry3.1Ab protein by enhancing the effects of Bt-induced toxicity.

A Novel Polymerase Chain Reaction (PCR)-Based Method for the Rapid Identification of Chrysodeixis includens and Rachiplusia nu.

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers.

Inheritance of Cry2Ab2 resistance in two Helicoverpa zea (Lepidoptera: Noctuidae) populations resistant to single- and dual-Bacillus thuringiensis proteins

Cry2Ab2 is a Bacillus thuringiensis (Bt) protein that has been pyramided with Cry1A.105 in transgenic maize and Cry1Ac in cotton to control some major lepidopteran pests including the corn earworm/bollworm, Helicoverpa zea (Boddie). However, the widespread occurrence of resistance of this pest to the pyramided Cry1A/Cry2A crops in the southern region of the United State has become a threat to the sustainability of the technology. In this study, multiple genetic crosses and backcrosses were developed to characterize the inheritance of Cry2Ab2 resistance in two H. zea populations resistant to the single protein, Cry2Ab2 (RR2Ab) and the dual proteins in Bt maize, Cry1A.105/Cry2Ab2 (RRVT2P). Diet-overlay bioassays with F1 hybrids from reciprocal crosses between a susceptible and the resistant populations showed that the Cry2Ab2 resistance in both RR2Ab and RRVT2P was inherited autosomally and non-recessively. Segregation tests in F2 and backcrossed generations indicated that the resistance was likely controlled by more than one locus. The effective dominance levels of the resistance estimated at each of three discriminating concentrations were similar between RR2Ab and RRVT2P, ranging from incompletely dominant to incompletely recessive. The similar inheritance observed in RR2Ab and RRVT2P suggests that the Cry2Ab2 resistance is independent from the Cry1A.105 resistance. The non-recessive inheritance of the resistance could be an important factor causing the widespread resistance of this insect to the Cry1A/Cry2A crops in the southern region. Implications of the observed inheritance properties in Bt crop resistance management are discussed.

V-ATPase C Acts as a Receptor for Bacillus thuringiensis Cry2Ab and Enhances Cry2Ab Toxicity to Helicoverpa armigera.

Cry2Ab is a significant alternative Bacillus thuringiensis (Bt) protein utilized for managing insect resistance to Cry1 toxins and broadening the insecticidal spectrum of crops containing two or more Bt genes. Unfortunately, the identified receptors fail to fully elucidate the mechanism of action underlying Cry2Ab. Previous studies have demonstrated the involvement of vacuolar H+-ATPase subunits A, B, and E (V-ATPase A, B, and E) in Bt insecticidal activities. The present study aims to investigate the contribution of V-ATPase C to the toxicities of Cry2Ab against Helicoverpa armigera. The feeding of Cry2Ab in H. armigera larvae resulted in a significant decrease in the expression of V-ATPase C. Further investigations confirmed the interaction between V-ATPase C and activated Cry2Ab protein according to Ligand blot and homologous and heterologous competition assays. Expressing endogenous HaV-ATPase C in Sf9 cells resulted in an increase in Cry2Ab cytotoxicity, while the knockdown of V-ATPase C by double-stranded RNAs (dsRNA) in midgut cells decreased Cry2Ab cytotoxicity. Importantly, a higher toxicity of the mixture containing Cry2Ab and V-ATPase C against insects was also observed. These findings demonstrate that V-ATPase C acts as a binding receptor for Cry2Ab and is involved in its toxicity to H. armigera. Furthermore, the synergy between V-ATPase C protein and Cry2Ab protoxins provides a potential strategy for enhancing Cry2Ab toxicity or managing insect resistance.

Using Egg Parasitoids to Manage Caterpillars in Soybean and Maize: Benefits, Challenges, and Major Recommendations.

The use of egg parasitoids in Augmentative Biological Control (ABC) is a highly effective strategy within the integrated pest management (IPM) of lepidopteran defoliators. Safer than chemical insecticides, these natural antagonists have demonstrated significant efficacy. Trichogramma pretiosum and Telenomus remus, known for their high parasitism rates, are the most extensively used and studied parasitoids for controlling economically important lepidopterous in crops such as soybean and maize. Brazil, a leading adopter of crops expressing Bacillus thuringiensis (Bt) proteins, faces growing field-evolved resistance to Cry proteins in soybean and maize. This resistance, particularly of Rachiplusia nu in soybean and Spodoptera frugiperda in maize, has become more prominent in recent years, increasing insecticide use. Therefore, this article reviews the current status of egg parasitoids adoption in ABC against lepidopteran pests, emphasizing the role of Tr. pretiosum and the potential of Te. remus as sustainable alternatives to chemical insecticides to manage pests in both non-Bt and Bt crops. Additionally, we provide recommendations for using these parasitoids in ABC programs and discuss the challenges that must be addressed to optimize the adoption of biocontrol agents in ABC programs for maximum benefit.

Enhances the resistance of rice to lepidopteran pests by fusing the Cry1Ca and Cry2Aa genes with self-cleavage peptide sequence.

Accumulation of two or more Bacillus thuringiensis (Bt) proteins in plant not only improves the resistance to pests and broadens the resistance spectrum of crops, but also delays the development of pest resistance. The self-cleavage peptide sequence was used to link two codon-optimized genes, so as to achieve simultaneous accumulation of two low homologous insecticidal proteins in one plant. The rice transformants accumulating Cry1Ca and Cry2Aa proteins were fed to local lepidopteran pests and the larva mortality in 5 days were 100%. The sum of Cry1Ca and Cry2Aa proteins in leaves of transformants E1C&2A-1 and E2A&1C-18 were 10.60 and 9.55 μg g-1 fresh weight (FW), respectively, and the larva mortality of fall armyworm fed on their leaves for 5 days reached 100%. For the control transformants that expressed one Bt protein, the content of Cry1Ca in leaves of transformant E1CM031 was 14.94 μg g-1 FW, and that of Cry2Aa in leaves of transformant B2A4008S was 11.90 μg g-1 FW, but the larva mortality of fall armyworm fed on leaves of E1CM031 and B2A4008S for 5 days were 77.78% and 52.78%, respectively. Although the total Bt contents in transformants expressing one Bt protein were higher than that of transformants expressing two Bt proteins, the lethality of transformants expressing one Bt protein were obviously lower than that of transformants expressing two Bt proteins. The lethal effect of accumulating both Cry1Ca and Cry2Aa proteins in rice was stronger than that of amassing Cry1Ca or Cry2Aa protein only, which meant there was synergistic effect between Cry1Ca and Cry2Aa proteins. © 2024 Society of Chemical Industry.

Mutation in PgABCC2 confers low-level resistance to Cry1Ac in pink bollworm.

With the increasing incidence of pest resistance to transgenic crops producing Bacillus thuringiensis (Bt) proteins in the field, elucidating the molecular basis of resistance is important for monitoring, delaying and countering pest resistance. Previous work revealed that mutation or down-regulated expression of the cadherin gene (PgCad1) is associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac, and 20 mutant PgCad1 alleles (r1-r20) were characterized. Here, we tested the hypothesis that the ABC transporter PgABCC2 is a functional receptor for the Bt toxin Cry1Ac and that a mutation is associated with resistance. We identified and characterized the first resistance allele (rC2) of PgABCC2 in the laboratory-selected Cry1Ac-resistant strain AQ-C2 of pink bollworm. The rC2 allele had a one-base deletion in exon20, resulting in a frameshift and the introduction of a premature stop codon. This resulting PgABCC2 protein had a truncated C-terminus, including the loss of the NBD2 domain. AQ-C2 exhibited 20.2-fold greater resistance to Cry1Ac than the susceptible strain, and its inheritance of Cry1Ac resistance was recessive and genetically linked to PgABCC2. When produced in cultured insect cells, recombinant wild-type and rC2 mutant PgABCC2 proteins localized within the cell plasma membrane, although substantial cytoplasmic retention was also observed for the mutant protein, while the mutant PgABCC2 caused a 13.9-fold decrease in Cry1Ac toxicity versus the wild-type PgABCC2. PgABCC2 is a functional receptor of Cry1Ac and the loss of its carboxyl terminus (including its NBD2 domain) confers low-level resistance to Cry1Ac in both larvae and in cultured cells. © 2024 Society of Chemical Industry.

Optimizing the Bacillus thuringiensis (Bt) protein concentration in cotton: Coordinated application of exogenous amino acids and EDTA to reduce spatiotemporal variability in boll and leaf toxins

During the boll formation stage, cotton bolls exhibit the lowest expression of Bacillus thuringiensis (Bt) insecticidal proteins. Resistance to insects varies notably among different organs, which poses challenges for controlling cotton bollworms. Consequently, an experimental strategy was designed in the 2020–2021 cotton growing season to coordinate the enhancement of protein synthesis and the attenuation of degradation. Two Bt cultivars of Gossypium hirsutum, namely the hybrid Sikang 3 and the conventional Sikang 1, were used as test materials. Three treatments were applied at the peak flowering period: CK (the control), T1 (amino acids), and T2 (amino acids and EDTA). The results show that, in comparison to the CK group, the Bt protein contents were significantly increased in both cotton bolls and their subtending leaves under the T1 and T2 treatments. The maximum levels of increase observed were 67.5% in cotton bolls and 21.7% in leaves. Moreover, the disparity in Bt protein content between cotton bolls and their subtending leaves notably decreased by 31.2%. Correlation analysis suggested that the primary physiological mechanisms for augmenting Bt protein content involve increased protein synthesis and reduced protein catabolism, which are independent of Bt gene expression levels. Stepwise regression and path analysis revealed that elevating the soluble protein content and transaminase activity, while reducing the catabolic enzyme activities, are instrumental in enhancing the Bt protein content. Consequently, the coordinated application of amino acids and EDTA emerges as a strategy that can improve the overall resistance of Bt cotton and mitigate the spatiotemporal variations in Bt toxin concentrations in both cotton bolls and leaves.

Diverse genetic basis of Vip3Aa resistance in five independent field-derived strains of Helicoverpa zea in the US.

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). 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. 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.

Response of the Propylea japonica Microbiota to Treatment with Cry1B Protein.

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) is a dominant natural enemy of insect pests in farmland ecosystems. It also serves as an important non-target insect for environmental safety evaluations of transgenic crops. Widespread planting of transgenic crops may result in direct or indirect exposure of P. japonica to recombinant Bacillus thuringiensis (Bt) protein, which may in turn affect the biological performance of this natural enemy by affecting the P. japonica microflora. However, the effects of Bt proteins (such as Cry1B) on the P. japonica microbiota are currently unclear. Here, we used a high-throughput sequencing method to investigate differences in the P. japonica microbiota resulting from treatment with Cry1B compared to a sucrose control. The results demonstrated that the P. japonica microbiome was dominated by Firmicutes at the phylum level and by Staphylococcus at the genus level. Within-sample (α) diversity indices demonstrated a high degree of consistency between the microbial communities of P. japonica treated with the sucrose control and those treated with 0.25 or 0.5 mg/mL Cry1B. Furthermore, there were no significant differences in the abundance of any taxa after treatment with 0.25 mg/mL Cry1B for 24 or 48 h, and treatment with 0.5 mg/mL Cry1B for 24 or 48 h led to changes only in Staphylococcus, a member of the phylum Firmicutes. Treatment with a high Cry1B concentration (1.0 mg/mL) for 24 or 48 h caused significant changes in the abundance of specific taxa (e.g., Gemmatimonades, Patescibacteria, Thauera, and Microbacterium). However, compared with the control, most taxa remained unchanged. The statistically significant differences may have been due to the stimulatory effects of treatment with a high concentration of Cry1B. Overall, the results showed that Cry1B protein could alter endophytic bacterial community abundance, but not composition, in P. japonica. The effects of Bt proteins on endophytes and other parameters in non-target insects require further study. This study provides data support for the safety evaluation of transgenic plants.

Transgenic sugarcane expressing Bacillus thuringiensis Cry1Ac protein offers new possibilities for controlling the giant borer, Telchin licus (Drury), a pest difficult to control

The giant borer, Telchin licus (Drury), is an important insect pest of sugarcane in Central and South America. This pest is difficult to control with chemical or biological control approaches due to the larvae behavior of hiding in the deepest parts of the plants. Manual scavenging and skewer killing have been the main approaches for monitoring and control of T. licus in Brazil, the world's largest sugarcane producer. Recently, Brazil launched the first commercial use of transgenic sugarcane expressing Bacillus thuringiensis (Bt) proteins to control the sugarcane borer, Diatraea saccharalis (F.), a primary insect pest of sugarcane in the Americas. In this study, five independent field/greenhouse tests were conducted to evaluate the larval survival of and plant injury by T. licus on five non-Bt and five Bt sugarcane varieties containing a single cry1Ac gene. The multiple field/greenhouse tests showed that Cry1Ac sugarcane was highly effective to control T. licus; and the high control efficiency was consistent across varieties, from immature to mature plant stages, as well as for F0 generation plants and F1 ratoons. In addition, diet-incorporated bioassays were performed to determine the susceptibility of three field-collected T. licus populations in Brazil to diet treated with purified Cry1Ac protein or with a 25-fold dilution of Cry1Ac sugarcane stalk tissue. The diet bioassays exhibited that the field T. licus populations were also susceptible to the purified Cry1Ac protein, as well as to diet treated with the 25-fold dilution of stalk tissue. The results of this study provide compelling evidence that the single-protein Cry1Ac sugarcane varieties offer new possibilities for managing the T. licus, a pest that is difficult to control with other current available technologies.

Profitability of cotton, Gossypium hirsutum (L.), when controlling bollworm, Helicoverpa zea (Boddie), with variable treatment thresholds

Crops expressing Bacillus thuringiensis proteins have offered valuable crop protection benefits including reduced foliar insecticide use and crop yield protection. Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) has developed resistance to one or more Bt proteins, which consequently threatens these benefits. This has resulted in a renewed emphasis on the implementation of action thresholds for H. zea management decisions in cotton. Field experiments were conducted in College Station, TX; Stoneville, MS; and Blackville, SC, during 2020–2021 to evaluate current H. zea threshold recommendations for Texas, Mississippi, and South Carolina. Three Bt cotton technologies and five thresholds were evaluated. The Bt cotton technologies evaluated included non-Bt (NBT), Bollgard® 2 (BG2; Cry1Ac + Cry2Ab), and Bollgard® 3 (BG3; Cry1Ac + Cry2Ab + Vip3Aa). Across years and locations, within NBT cotton, all thresholds, including 20% oviposition, 6% fruiting forms injury, ≥3% large larvae, and preventive sprays, resulted in greater control of H. zea and higher yield and profitability relative to non-treated NBT cotton. The threshold using 6% injury to squares and bolls was the most effective in terms of yield relative to the preventive spray. Across years and locations, the BG2 cotton sprayed based on 20% oviposition or preventively resulted in greater control of H. zea, and all treatments resulted in a yield or profitability equivalent to that of the non-treated BG2 cotton. For BG3 cotton, spraying based on any of the thresholds was not beneficial. For NBT and BG2 cotton, thresholds of 20% oviposition, 6% injury to fruiting forms, or ≥3% large larvae provided a reduction in insecticide applications relative to the preventive spray treatment, suggesting that these thresholds were effective triggers for controlling H. zea.

The Role of Glycoconjugates as Receptors for Insecticidal Proteins.

Bacillus thuringiensis (Bt) proteins are an environmentally safe and effective alternative to chemical pesticides and have been used as biopesticides, with great commercial success, for over 50 years. Global agricultural production is predicted to require a 70% increase until 2050 to provide for an increasing population. In addition to agriculture, Bt proteins are utilised to control human vectors of disease - namely mosquitoes - which account for >700,000 deaths annually. The evolution of resistance to Bt pesticial toxins threatens the progression of sustainable agriculture. Whilst Bt protein toxins are heavily utilised, the exact mechanisms behind receptor binding and toxicity are unknown. It is critical to gain a better understanding of these mechanisms in order to engineer novel toxin variants and to predict, and prevent, future resistance evolution. This review focuses on the role of carbohydrate binding in the toxicity of the most utilised group of Bt pesticidal proteins - three domain Cry (3D-Cry) toxins.

Fitness Costs and Incomplete Resistance Associated with Delayed Evolution of Practical Resistance to Bt Crops

Insect pests are increasingly evolving practical resistance to insecticidal transgenic crops that produce Bacillus thuringiensis (Bt) proteins. Here, we analyzed data from the literature to evaluate the association between practical resistance to Bt crops and two pest traits: fitness costs and incomplete resistance. Fitness costs are negative effects of resistance alleles on fitness in the absence of Bt toxins. Incomplete resistance entails a lower fitness of resistant individuals on a Bt crop relative to a comparable non-Bt crop. In 66 studies evaluating strains of nine pest species from six countries, costs in resistant strains were lower in cases with practical resistance (14%) than without practical resistance (30%). Costs in F1 progeny from crosses between resistant and susceptible strains did not differ between cases with and without practical resistance. In 24 studies examining seven pest species from four countries, survival on the Bt crop relative to its non-Bt crop counterpart was higher in cases with practical resistance (0.76) than without practical resistance (0.43). Together with previous findings showing that the nonrecessive inheritance of resistance is associated with practical resistance, these results identify a syndrome associated with practical resistance to Bt crops. Further research on this resistance syndrome could help sustain the efficacy of Bt crops.

Resistance Allele Frequency of Helicoverpa zea to Vip3Aa Bacillus thuringiensis Protein in the Southeastern U.S.

Helicoverpa zea is a major target pest of Bt crops expressing Cry and/or Vip3Aa proteins in the U.S.A. Widespread practical resistance of H. zea to the Cry1 and Cry2 proteins makes Vip3Aa the only effective Bt protein against this pest. Understanding the frequency of resistance alleles against Vip3Aa in field populations of H. zea is crucial for resistance management and the sustainability of Vip3Aa technology. Using a modified F2 screen method by crossing susceptible laboratory female moth with feral male moth of H. zea, we successfully screened a total of 24,576 neonates from 192 F2 families of H. zea collected from Arkansas, Louisiana, Mississippi, and Tennessee during 2019-2020. We found five F2 families containing ≥3rd instar survivors on the diagnostic concentration of 3.0 µg/cm2 Vip3Aa39. Dose-response bioassays confirmed the high levels of Vip3Aa resistance in these F2 families, with an estimated resistance ratio of >909.1-fold relative to the susceptible strain. The estimated resistance allele frequency against Vip3Aa in H. zea for these four southern states is 0.0155 with a 95% CI of 0.0057-0.0297. These data should provide critical information for understanding the risks of Vip3Aa resistance in H. zea and help design appropriate resistance management strategies for the sustainability of the Vip3Aa technology.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

Selection, effective dominance, and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

In the U.S., Helicoverpa zea (Boddie) is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins. Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S. In this study, two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant (VT2P-RR), a susceptible, and an F1 heterozygous (VT2P-RS) populations of H. zea on ears of nine Bt and three non-Bt maize hybrids. The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105, Cry1Ab, Cry1F, Cry2Ab2, and Vip3Aa20 proteins. In the laboratory, neonates of the three H. zea populations were inoculated on silks of ears collected from maize at R1–R2 plant stages; and larval survivorship was checked 10 d after neonate release. All three insect populations survived normally on non-Bt maize ears. Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize, while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize. The results demonstrated that the dual-protein resistant H. zea was not cross-resistant to Vip3Aa20-expressing maize, and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H. zea. The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize. The effective dominance levels varied greatly, from recessive to incompletely dominant, depending on maize hybrids and trials, suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance. The data generated should aid in modeling multiple-protein Bt resistance in H. zea.

Effects of Insect-Resistant Maize 2A-7 Expressing mCry1Ab and mCry2Ab on the Soil Ecosystem.

Transgenic maize 2A-7 expressing mCry1Ab and mCry2Ab has excellent resistance to lepidopteran pests. Previous studies have investigated the effects of several Bacillus thuringiensis (Bt) proteins on the soil. However, the effects of artificially modified Bt proteins on soil ecosystems are still unclear. To evaluate the effects of transgenic maize 2A-7 on soil, the physicochemical properties, enzyme activities and functional diversities of the microbial communities in rhizosphere soils from 2A-7 and its near-isogenic non-transgenic control Dongdan 6531 were analyzed at different developmental stages under field conditions. The alteration of six physicochemical properties (pH, total nitrogen, total phosphorus, organic matter, available phosphorus and alkali-hydrolyzed nitrogen) and six functional enzymes (catalase, alkaline phosphatase, sucrase, acid phosphatase, urease and alkaline protease) activities in the rhizosphere soils between the two maize cultivars were drastically correlated with plant growth stage, but not affected by the artificially modified Bt transgenes. An analysis of time-course Biolog data revealed that the functional diversity of microbial communities in the rhizosphere soil of 2A-7 and its control were similar at each developmental stage. The results suggest that transgenic maize 2A-7 has no significant impact on the soil ecosystem and provide valuable information on scientific safety assessments of 2A-7 and its commercial applications.

Large-Scale Monitoring of the Frequency of Ryanodine Receptor Target-Site Mutations Conferring Diamide Resistance in Brazilian Field Populations of Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

Simple SummaryFall armyworm (FAW), Spodoptera frugiperda, is a destructive moth pest species on various crops, particularly corn. It is native to the tropical regions of the Western Hemisphere such as Brazil, but recently invaded Africa, India, China, and Australia. Its larval stages damage crops by feeding, and to keep them under damage thresholds, insecticide applications are common. Due to frequent insecticide applications, FAW evolved resistance to different chemical classes of insecticides, including diamides. Field relevant levels of diamide resistance are usually conferred by ryanodine receptor (RyR) mutations and compromising recommended label rates. Diamide resistance in FAW so far remained restricted to laboratory-selected strains. Here, we investigated the frequency of specific resistance mutations in field-collected Brazilian populations of FAW by an F2 screen, selected two populations (BA-R and TF-R) for high levels of diamide resistance, deciphered the genetics of resistance, and employed a molecular genotyping assay to correlate resistance levels with the presence of RyR mutations. Crossin studies indicated that resistance is autosomal and (incompletely) recessive in both strains. F1 backcrosses suggested monogenic resistance, supported by the identification of an I4734M/K target-site mutation in the RyR. Our results will help to sustainably manage diamide resistance in FAW in Brazil.Fall armyworm (FAW), Spodoptera frugiperda, is an important lepidopteran pest in the Americas, and recently invaded the Eastern Hemisphere. In Brazil, FAW is considered the most destructive pest of corn and cotton. FAW has evolved resistance to many insecticides and Bacillus thuringiensis (Bt) proteins. Here, a large-scale monitoring was performed between 2019 and 2021 to assess diamide insecticide susceptibility in more than 65 FAW populations sampled in corn and cotton. We did not detect a significant shift in FAW susceptibility to flubendiamide, but a few populations were less affected by a discriminating rate. F2 screen results of 31 selected FAW populations across regions confirmed that the frequency of diamide resistance alleles remained rather stable. Two laboratory-selected strains exhibited high resistance ratios against flubendiamide, and cross-resistance to anthranilic diamides. Reciprocal crosses indicated that resistance is autosomal and (incompletely) recessive in both strains. F1 backcrosses suggested monogenic resistance, supported by the identification of an I4734M/K target-site mutation in the ryanodine receptor (RyR). Subsequent genotyping of field-collected samples employing a TaqMan-based allelic discrimination assay, revealed a low frequency of RyR I4790M/K mutations significantly correlated with phenotypic diamide resistance. Our findings will help to sustainably employ diamides in FAW resistance management strategies across crops.

Estimation of resistance allele frequencies to Cry1A.105 and Cry2Ab2 in the corn earworm (Lepidoptera: Noctuidae) with F2 isolines generated from a mass-mating method

The corn earworm/bollworm, Helicoverpa zea (Boddie), in the United States is targeted by transgenic corn and cotton expressing Bacillus thuringiensis (Bt) proteins. Previous studies have shown that resistance to Cry1A/Cry2A Bt corn has occurred in the field. In this study, we established 103 F2 isolines by mass-mating of H. zea populations collected during 2018 and 2019 in four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. Dissection of mated females showed that at least 92.7% of the isolines were originated from single matings, suggesting that the applied mass-mating method is a suitable approach to establish H. zea F2 isolines. F2 screen showed that resistance allele frequencies in these isolines were high to the two Bt proteins expressed in MON 89034 corn hybrids, averaging 0.405 for Cry1A.105 and 0.330 for Cry2Ab2. The observed high resistance allele frequencies confirm that resistance to Cry1A/Cry2A in the insect is common in the southeastern region. In addition, the F2 screen showed that resistance allele frequencies (0.490 for Cry1A.105 and 0.541 for Cry2Ab2) in the populations collected in 2019 were significantly greater than the frequencies (0.312 for Cry1A.105 and 0.081 for Cry2Ab2) in the populations collected in 2018, indicating that selection for the Cry resistance was likely still ongoing in the field. Information generated from this study is useful to assess resistance risk, refine resistance modeling, and improve IRM programs to mitigate Cry resistance in H. zea for the sustainable use of Bt crop technology in the United States.