Life table study of Rachiplusia nu (Lepidoptera: Noctuidae) on different food sources and artificial diet.

Rachiplusia nu Guenée is a polyphagous species able to develop on several cultivated and non-cultivated host plants. However, basic life history information about this pest on hosts is scarce. In this study, R. nu larvae did not survive on leaves of non-Bt corn, wheat, Bt cotton that expresses proteins Cry1Ac+Cry2Ab2 or on Intacta2 Xtend soybean that expresses the Cry1A.105/Cry2Ab2/Cry1Ac proteins. Rachiplusia nu showed a viable egg-to-adult biological cycle (54%-66.3%) on non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip hosts, similar to larvae raised on the artificial diet. In addition, R. nu was unable to complete larval development on non-Bt cotton, and only 45.2% of R. nu larvae reached the pupal stage when fed leaves of intacta RR2 PRO soybean that expresses the Cry1Ac protein. Larval and pupal mass of surviving insects on Cry1Ac soybean leaves were also lower (larval: 0.104 g; pupal: 0.099 g) in relation to other food sources (larval: 0.165-0.189 g; pupal: 0.173-0.192 g). The total fecundity of R. nu on Cry1Ac soybean leaves was ≈65% lower in relation to other food sources. This fact caused ≈60% the net reproductive rate (Ro) and intrinsic rate of increase (rm) when compared to other food sources. Our findings indicate that the Cry1Ac soybean negatively affects the biological parameters of R. nu. Non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip are viable food sources for the survival and development of R. nu.

Resistance of Bt and Non-Bt Soybean Cultivars Adapted to Novel Growing Regions of Brazil to Chrysodeixis includens and Spodoptera frugiperda.

Soybean is a highly valuable commodity crop for Brazil's economy. However, it faces significant threats from the attack of a complex of lepidopteran pests, particularly Chrysodeixis includens (Walker) and Spodoptera frugiperda (J. E. Smith). These pests have been managed primarily using transgenic Bt soybeans, but limited knowledge exists about the resistance levels of Bt and non-Bt cultivars adapted to novel soybean-growing areas in Brazil, such as the Minas Gerais state. This study evaluated the resistance levels of Bt and non-Bt soybean cultivars to C. includens and S. frugiperda, and whether the Bt cultivars can differentially affect these pests across larval stages. No-choice bioassays were conducted using Bt (NS6010 IPRO and P97R50 IPRO) and non-Bt soybeans (UFLA 6301 RR, P96R90 RR, and ANsc 80111 RR) at V4-stage in the laboratory with neonate (24 h) and third-instar larvae. Larvae were fed leaf discs in Petri dishes, recording the mortality, leaf consumption, and weight gain after 7 days. There was high mortality of C. includens neonates on the Bt cultivars, but this trend was not observed for older larvae. For S. frugiperda neonates, there was high mortality on the Bt cultivar NS 6010 IPRO and non-Bt cultivar UFLA 6301 RR, but only the former was effective for older larvae. Although the Bt cultivars did not kill the third instars, antinutritional effects were found, such that leaf tissue consumed was not converted to larval weight gain. These findings are important for defining regional strategies of integrated and resistance management of C. includens and S. frugiperda in expanding regions of soybean cultivation in Brazil.

Exploratory comparative transcriptomic analysis reveals potential gene targets associated with Cry1A.105 and Cry2Ab2 resistance in fall armyworm (Spodoptera frugiperda).

Genetically modified (GM) crops, expressing Bacillus thuringiensis (Bt) insecticidal toxins, have substantially transformed agriculture. Despite rapid adoption, their environmental and economic benefits face scrutiny due to unsustainable agricultural practices and the emergence of resistant pests like Spodoptera frugiperda, known as the fall armyworm (FAW). FAW's adaptation to Bt technology in corn and cotton compromises the long-term efficacy of Bt crops. To advance the understanding of the genetic foundations of resistance mechanisms, we conducted an exploratory comparative transcriptomic analysis of two divergent FAW populations. One population exhibited practical resistance to the Bt insecticidal proteins Cry1A.105 and Cry2Ab2, expressed in the genetically engineered MON-89Ø34 - 3 maize, while the other population remained susceptible to these proteins. Differential expression analysis supported that Cry1A.105 and Cry2Ab2 significantly affect the FAW physiology. A total of 247 and 254 differentially expressed genes were identified in the Cry-resistant and susceptible populations, respectively. By integrating our findings with established literature and databases, we underscored 53 gene targets potentially involved in FAW's resistance to Cry1A.105 and Cry2Ab2. In particular, we considered and discussed the potential roles of the differentially expressed genes encoding ABC transporters, G protein-coupled receptors, the P450 enzymatic system, and other Bt-related detoxification genes. Based on these findings, we emphasize the importance of exploratory transcriptomic analyses to uncover potential gene targets involved with Bt insecticidal proteins resistance, and to support the advantages of GM crops in the face of emerging challenges.

Oviposition patterns of primary lepidopteran defoliators in soybean and the impact on structured refuge recommendations.

BACKGROUND: Transgenic Bt technology in soybean, with plants expressing Cry1Ac, has been adopted as an insect pest management tool. It was first adopted in large areas of South America and Asia in 2013. The risk of resistance in target pests to this technology demands insect resistance management (IRM) programs. In Brazil, a structured refuge (area of non-Bt soybean) planted adjacent to the Bt soybean crop has been an important IRM recommendation, particularly for the primary lepidopteran defoliators Anticarsia gemmatalis (Lepidoptera: Erebidae) and Chrysodeixis includens (Lepidoptera: Noctuidae). The overall goal of this study was to validate IRM recommendations to Bt soybean. The objectives were to document the impact of soybean phenology, cultivar choice and non-Bt soybean defoliation on moth oviposition. In addition, a mark-release-recapture study estimated the dispersal capacity of these species. Five field experiments per species were performed for 3 years. RESULTS: Our results revealed an increase in A. gemmatalis and C. includens oviposition, respectively, on Bt plants as a consequence of the difference in plant growth stage at the time of oviposition. Defoliation of non-Bt plants significantly increased the oviposition preference of both moth species for Bt plants. The mark-release-recapture experiment indicated an average dispersal distance of ~300 m from the release point for A. gemmatalis, with maximum recapture at 1000 m. CONCLUSION: Overall, our findings emphasize the importance of planting synchronization of Bt soybean and the structured refuge. In addition, when operational aspects in large soybean areas challenge this recommendation, the priority should be for planting the refuge area first. This approach will minimize the impact of selective oviposition of A. gemmatalis and C. includens. © 2024 Society of Chemical Industry.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Plant characterization of insect-protected soybean.

Insect-protected soybean (SIP) that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal proteins has been developed to provide protection from feeding damage caused by targeted lepidopteran insect pests. Typically, as part of environmental risk assessment (ERA), plant characterization is conducted, and the data submitted to regulatory agencies prior to commercialization of genetically modified (GM) crops. The objectives of this research were to: (a) compare soybean with and without the SIP trait in plant characterization field trials designed to fulfill requirements for submissions to global regulatory agencies and address China-specific considerations and (b) compare risk assessment conclusions across regions and the methodologies used in the field trials. The soybean with and without the SIP trait in temperate, tropical, and subtropical germplasm were planted in replicated multi-location trials in the USA (in 2012 and 2018) and Brazil (in 2013/2014 and 2017/2018). Agronomic, phenotypic, plant competitiveness, and survival characteristics were assessed for soybean entries with and without the SIP trait. Regardless of genetic background, growing region, season, or testing methodology, the risk assessment conclusions were the same: the evaluated insect-protected soybean did not differ from conventional soybean in evaluated agronomic, phenotypic, competitiveness, and survival characteristics indicating no change in plant pest/weed potential. These results reinforce the concept of data transportability across global regions, different seasons, germplasm, and methodologies that should be considered when assessing environmental risks of GM crops.

Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105†+†Cry2Ab and Cry1F Bt maize.

In the Americas, transgenic crops producing insecticidal proteins from Bacillus thuringiensis Berliner (Bt, Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.) rapidly evolved in some FAW populations, pyramided Bt maize hybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Effects of previous infestation with Bemisia tabaci MEAM1 on the resistance to Chrysodeixis includens in Bt and non-Bt soybean plants.

Transgenic Bt soybean plants have been developed to control insect pests, such as Anticarsia gemmatalis and Chrysodeixis includens. This objective has been achieved successfully; however, recently, some authors claimed that Bt soybean plants have been more susceptible than non-Bt soybean to Bemisia tabaci MEAM1. In addition, it is unknown whether Bt soybean plants infested by B. tabaci become less resistant to target pests. Therefore, this study aimed to evaluate: (i) whether the previous infestation with B. tabaci can compromise Bt and non-Bt soybean resistance to C. includens; (ii) the effects of B. tabaci infestations on Bt and non-Bt soybean plant growth; and (iii) whether B. tabaci feeding reduces contents of chlorophyll and carotenoids of soybean plants. Bt and non-Bt soybean plants pre-infested with B. tabaci showed no changes in resistance to C. includens. Bt soybean plants infested with B. tabaci showed a lower plant height than uninfested plants. Differently, non-Bt soybean plants exhibited no reduction in plant growth due to B. tabaci feeding. Bt soybean plants suffered a reduction in dry matter only under double infestation (B. tabaci and C. includens), while non-Bt soybean plants experienced reduction in dry matter when infested with B. tabaci and C. includens or by C. includens only. B. tabaci feeding did not alter contents of chlorophyll and carotenoids, and perhaps the reduction in plant growth was related to salivary toxins. Concluding, both Bt and non-Bt soybean plants were susceptible to B. tabaci feeding, evidencing necessity of developing soybean cultivars resistant to B. tabaci.

A soybean trypsin inhibitor reduces the resistance to transgenic maize in a population of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.

Distribution and Genetic Diversity of Genes from Brazilian Bacillus thuringiensis Strains Toxic to Agricultural Insect Pests Revealed by Real-Time PCR.

Bacillus thuringiensis is a Gram-positive aerobic bacterium and the most used biopesticide worldwide. Given the importance of B. thuringiensis strain characterization for the development of new bioinsecticides or transgenic events and the identification and classification of new B. thuringiensis genes and strains to understand its distribution and diversity, this work is aimed at creating a gene identification system based on qPCR reactions utilizing core B. thuringiensis genes cry1, cry2, cry3, cry4, cry5, app6, cry7, cry8, cry9, cry10, cry11, vpb1, vpa2, vip3, cyt1, and cyt2 for the characterization of 257 strains of B. thuringiensis. This system was based on the Invertebrate Bacteria Collection from Embrapa Genetic Resources and Biotechnology and analyzed (a) the degree of correlation between the distribution of these strains and the origin of the substrate from which the strain was isolated and (b) between its distribution and geoclimatic conditions. This study made it possible to observe that the cry1, cry2, and vip3A/B genes occur homogeneously in the Brazilian territory, and some genes are found in specific regions. The biggest reservoir of variability is within B. thuringiensis strains in each region, and it is suggested that both geoclimatic conditions and regional crops interfere with the genetic diversity of the B. thuringiensis strains present in the region, and B. thuringiensis strains can constantly exchange genetic information.

Performance of cotton expressing Cry1Ac, Cry1F and Vip3Aa19 insecticidal proteins against Helicoverpa armigera, H. zea and their hybrid progeny, and evidence of reduced susceptibility of a field population of H. zea to Cry1 and Vip3Aa in Brazil.

The genetically modified cotton DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 expressing Cry1Ac, Cry1F and Vip3Aa19 from Bacillus thuringiensis Berliner (Bt) has been cultivated in Brazil since the 2020/2021 season. Here, we assessed the performance of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton expressing Cry1Ac, Cry1F and Vip3Aa19 against Helicoverpa armigera (Hübner), Helicoverpa zea (Boddie), and their hybrid progeny. We also carried out evaluations with DAS-21023-5 × DAS-24236-5 cotton containing Cry1Ac and Cry1F. In leaf-disk bioassays, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 was effective in controlling neonates from laboratory colonies of H. armigera, H. zea and the hybrid progeny (71.9%-100% mortality). On floral bud bioassays using L2 larvae, H. zea presented complete mortality, whereas H. armigera and the hybrid progeny showed <55% mortality. On DAS-21023-5 × DAS-24236-5 cotton, the mortality of H. armigera on leaf-disk and floral buds ranged from 60% to 73%, whereas mortality of hybrids was <46%. This Bt cotton caused complete mortality of H. zea larvae from a laboratory colony in the early growth stages, but mortalities were <55% on advanced growth stages and on floral buds. In field studies conducted from 2014 to 2019, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton was also effective at protecting plants against H. armigera. In contrast, a population of H. zea collected in western Bahia in 2021/2022 on Bt cotton expressing Cry1 and Vip3Aa proteins, showed 63% mortality after 30 d, with insects developing into fifth and sixth instars, on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton. We conclude that H. armigera, H. zea, and their hybrid progeny can be managed with DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton; however we found the first evidence in Brazil of a significant reduction in the susceptibility to DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton of a population of H. zea collected from Bt cotton in Bahia in 2021/2022.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Feeding injury of major lepidopteran soybean pests in South America.

Lepidopteran pests are major factors limiting soybean productivity in South America. In some cases, effective management of these species requires the use of foliar insecticides. For sustainable use of these insecticides, they should only be applied when insect population size exceeds an economic threshold. Since this estimation requires to determine the consumption of different species, this work aimed to integrate all these factors, studying the consumption of small (less than 1 cm long) and medium (1 to 1.5 cm long) size larvae of major lepidopteran pests to vegetative and reproductive tissues on Bt (M7739IPRO variety, containing the event MON87701 which expresses the Cry1Ac protein from Bacillus thuringiensis) and non-Bt (BMX Desafio RR variety) soybeans. The feeding injury to vegetative tissues was tested in detached-leaf assays in grow chambers, and for reproductive structures the study was conducted in greenhouse with infestations at early (flowering) and mid reproductive (mid grain filling) stages. Based on the feeding behavior of the species tested, they were cast in four groups: a) Anticarsia gemmatalis and Chrysodeixis includens, defoliating only the RR variety with the lowest consumption of foliar area; b) Spodoptera eridania, defoliating both RR and IPRO varieties, consuming twice than the species mentioned above; c) Helicoverpa armigera, defoliating and being the most damaging species to pods in the RR variety; and d) S. cosmioides and S. frugiperda, defoliating and damaging pods in both varieties. The species differed in their ability to feed on IPRO varieties, so a different economic threshold should be considered. Consequently, in cases where more than one species are found simultaneously, the species composition should be considered in estimating the economic threshold. Additionally, our findings may contribute to a better decision-making to control insect feeding injury in IPRO varieties, because a slower larval growth provides more time to ensure the need of control with insecticides. In summary, this clasification contributes to an improved recommendation of sustainable insecticide use, taking into account the behavior of each species that are major soybeans pests in South America.

Toxicity of Bacillus thuringiensis Strains to Six Lepidopteran Pests of Brazilian Agricultural Landscape.

The first step for biological insecticide production is the evaluating of the pathogenicity and strains virulence of entomopathogens against the target pests. Bacillus thuringiensis (Bt) is widespread in nature, lethal pathogen of a range of orders and the most widely used entomopathogenic biological control agent. Therefore, our objective was to study the susceptibility of six important lepidopteran pests in Brazilian agricultural production systems, Anticarsia gemmatalis (Hübner), Chrysodeixis includens (Walker) Helicoverpa armigera (Hübner), Spodoptera cosmioides (Walker), S. eridania (Cramer), and S. frugiperda (Smith), to Bt strains. In vitro bioassays were conducted with neonate larvae exposed to two Bt strains, 1680A and 775E, in seven concentrations of spores and protein crystals. After 7 days, the mortality was evaluated and indicated that lethal concentration (LC50) for 775E strain ranged from 7.72 × 104 to 2.45 × 106 spores mL-1 and for 1608A strain from 5.63 × 103 to 1.21 × 106 spores mL-1. The strain 1608A was more toxic for A. gemmatalis and H. armigera than 775E strain, and then was further evaluated in greenhouse bioassays with maize and soybean plants infested separately with target insects. The strain 1608A showed an efficient control for most insect species studied in the greenhouse bioassays, promoting considerable reduction in leaf injury and demonstrating a high potential in biological control of important lepidopteran species in crop production systems in Brazil.

Varied frequencies of resistance alleles to Cry1Ab and Cry1Ac among Brazilian populations of the sugarcane borer, Diatraea saccharalis (F.).

BACKGROUND: Brazil is the largest grower of the world's 26 million ha of sugarcane, Saccharum officinarum. Pest damage mainly by the sugarcane borer, Diatraea saccharalis (F.), is a great challenge to the sugarcane industry. To control D. saccharalis, Brazil launched the world's first commercial use of Bt sugarcane in 2017. As part of the resistance management programs for Bt sugarcane planting, 535 F2 isoline families of D. saccharalis collected from three major sugarcane planting states (Goiás, Minas Gerais and São Paulo) in Brazil during 2019-2020 were screened for resistance to two Bt sugarcane varieties: CTC20BT expressing Cry1Ab and CTC9001BT expressing Cry1Ac. Here we report the results of the first study related to Bt resistance in a sugarcane cropping system. RESULTS: Larval survivorships of these families in an F2 screen on CTC20BT were highly correlated with their survival on CTC9001BT, whereas the Cry1Ac tissues exhibited greater insecticidal activities than Cry1Ab. Resistance allele frequencies (RAFs) for populations from Goiás and Minas Gerais were relatively low at 0.0034 for Cry1Ab and 0.0045 to Cry1Ac. By contrast, RAFs for São Paulo populations were considerably greater (0.0393 to Cry1Ab, 0.0245 to Cry1Ac). CONCLUSIONS: RAFs to Cry1Ab and Cry1Ac varied among Brazilian D. saccharalis populations. Prior selection resulting from an intensive use of single-gene Bt maize under low compliance of refuge planting could be a main factor contributing to the high RAF in São Paulo. The results suggest that mitigation measures including sufficient non-Bt maize refuge planting, effective resistance monitoring, and use of pyramided Bt sugarcane traits should be implemented promptly to prevent further increase in the RAF to ensure the sustainable use of Bt sugarcane in Brazil. MINI ABSTRACT: To control Diatraea saccharalis, Brazil launched the world's first commercial use of Bt sugarcane in 2017. As part of the resistance management programs for Bt sugarcane planting in Brazil, 535 F2 isoline families of D. saccharalis collected from three major sugarcane planting states (Goiás, Minas Gerais and São Paulo) in Brazil during 2019-2020 were screened for resistance to Cry1Ab and Cry1Ac sugarcane plants Resistance allele frequencies (RAFs) for the populations from Goiás and Minas Gerais were relatively low at 0.0034 for Cry1Ab and 0.0045 to Cry1Ac. By contrast, RAFs for the São Paulo populations were considerably greater (0.0393 to Cry1Ab, 0.0245 to Cry1Ac). Prior selection resulting from an intensive use of single-gene Bt maize under low compliance of non-Bt maize refuge planting could be a main factor contributing to the high RAF in São Paulo. The results suggest that effective mitigation measures including sufficient non-Bt maize refuge planting, effective resistance monitoring and use of pyramided Bt sugarcane traits should be implemented promptly to prevent further increase in the RAF to ensure the sustainable use of Bt sugarcane in Brazil. © 2022 Society of Chemical Industry.

MON 95379 Bt maize as a new tool to manage sugarcane borer (Diatraea saccharalis) in South America.

BACKGROUND: The sugarcane borer (SCB), Diatraea saccharalis (Lepidoptera: Crambidae), is a key pest of maize in Argentina, and genetically modified maize, producing Bacillus thuringiensis (Bt) proteins, has revolutionized the management of this insect in South America. However, field-evolved resistance to some Bt technologies has been observed in SCB in Argentina. Here we assessed a new Bt technology, MON 95379, in the laboratory, greenhouse and field for efficacy against SCB. RESULTS: In a laboratory leaf disc bioassay, both MON 95379 (producing Cry1B.868 and Cry1Da_7) and Cry1B.868_single maize (producing only Cry1B.868) resulted in 100% mortality of SCB. The level of Cry1B.868 in the Cry1B.868_single maize is comparable to that in MON 95379 maize. However, the Cry1Da_7 protein does not have high efficacy against SCB, as evidenced by < 20% mortality on Cry1Da_7_single leaf tissue. Total (100%) mortality of SCB in a Cry1B.868_single tissue dilution bioassay indicated that Cry1B.868_single maize meets the criteria to be classified as a high dose. Similar median lethal concentration (LC50 ) values were observed for MON 89034-R and susceptible SCB strains exposed to Cry1B.868 protein. MON 95379 also controlled SCB strains resistant to MON 89034 (Cry1A.105/Cry2Ab2) and Cry1Ab. Under field conditions in Brazil and Argentina, MON 95379 maize plants were consistently protected from SCB damage. CONCLUSION: MON 95379 maize will bring value to maize growers in South America by effectively managing SCB even in locations where resistance to other Bt-containing maize technologies has been reported. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Recombinant Expression of ABCC2 Variants Confirms the Importance of Mutations in Extracellular Loop 4 for Cry1F Resistance in Fall Armyworm.

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOXTM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.