Transgenic crops producing insecticidal proteins are effective to manage lepidopteran pests. Development of insect-resistance is the major threat to Bacillus thuringiensis (Bt) crops such as Cry1Ah-Maize. Laboratory selection with Bt-Cry1Ah toxin incorporated in artificial diet, during 48 generations of Asian corn borer (ACB) Ostrinia furnacalis produced 200-fold resistance. This resistant colony ACB-AhR readily consumed and survived on Cry1Ah-expressing Bt-maize. Cross-resistance analysis showed high cross-resistance to Cry1F (464-fold), moderate cross-resistance to Cry1Ab (28.38-fold), Cry1Ac (22.11-fold) and no cross-resistance to Cry1Ie toxin. This ACB-AhR cross-resistant phenotype is different from ACB-Cry1Fa resistant population that showed no cross resistance to Cry1Ah, suggesting that different mechanisms of resistance were selected in these two populations. Bioassays of reciprocal F₁ crosses-progeny suggested autosomal inheritance of Cry1Ah resistance with no maternal effects. The dominance of resistance increased as concentration decreased. In Cry1Ah-maize tissues the progeny of reciprocal F₁ crosses behaved as functionally recessive. Progenies analysis from backcrosses (F_1 ×resistant strain) suggested polygenic contribution to Cry1Ah- resistance in ACB-AhR. The use of multiple toxins is an imperative factor for delaying evolution of resistance to Cry1Ah-corn in ACB. However, the fact that ACB-AhR showed cross resistance to Cry1Fa indicates that selection of toxins for pyramided plants should be carefully done.

The oriental armyworm (OAW), Mythimna separata (Walker), is a destructive pest of agricultural crops in Asia and Australia. Commercialized Bt crops have performed very well against their target pests; however, very few studies have been done on the susceptibility of OAW to Bt toxins in either sprays or expressed in Bt crops. In this work, we evaluated the toxicities of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, Cry2Aa, Cry2Ab, Cry1Ie, Vip3Aa19, Vip3Aa16, and Vip3Ca against OAW neonate larvae, as well as the interaction between Cry and Vip toxins. The results from bioassays revealed that LC₅₀ (lethal concentration for 50% mortality) values ranged from 1.6 to 78.6 μg/g (toxin/diet) for those toxins. Among them, Vip3 proteins, along with Cry1A proteins and Cry2Aa, were the ones with the highest potency, with LC₅₀ values ranging from 1.6 to 7.4 μg/g. Synergism between Cry and Vip toxins was observed, being high in the combination of Vip3Aa16 with Cry1 toxins, with synergetic factors ranging from 2.2 to 9.2. The Vip3Ca toxin did not show any synergistic effect with any of the toxins tested. These results can help in designing new combinations of pyramiding genes in Bt crops, as well as in recombinant bacteria, for the control of OAW as well as for resistance management programs.

Background: Seed Myco-priming based on consortium of entomopathogenic fungi is very effective seed treatment against Ostrinia furnacalis herbivory. Maize regulates defense responses against herbivory by the production of defense-related enzymatic and non-enzymatic antioxidants, phytohormones, and their corresponding genes. Jasmonic acid (JA) plays a key role in plant-entomopathogenic fungi-herbivore interaction. Results: To understand how a consortium of the entomopathogenic fungi Beauveria bassiana and Trichoderma asperellum induce changes in the response of maize to herbivory and increase the crop yield, 2-year field experiment, antioxidant enzymes, leaf transcriptome, and phytohormone were performed. Fungal inoculation enhanced the production of antioxidant enzymes and JA signaling pathway more than the normal herbivory. The comparison between single inoculated, consortium inoculated, and non-inoculated plants resulted in distinct transcriptome profiles representing a considerable difference in expression of antioxidant- and JA- responsive genes identified through Weighted gene co-expression network analysis (WGCNA) and expression analysis, respectively. Seed priming with a consortium of B. bassiana and T. asperellum significantly enhanced the expression of genes involved in antioxidants production and JA biosynthesis cascade, with the highest expression recorded at 24-h post O. furnacalis larval infestation. They reduced the larval nutritional indices and survival up to 87% and enhancing crop yield and gross return up to 82-96% over the year 2018 and 2019. Conclusion: From our results we suggest that a consortium of B. bassiana and T. asperellum can be used synergistically against O. furnacalis in maize under field condition and can mediate antioxidants- and JA- associated maize defense response by boosting up the expression of their responsive genes, thereby enhancing crop yield.

Background: Asian corn borer (ACB), Ostrinia furnacalis can develop resistance to transgenic Bacillus thuringiensis (Bt) maize expressing Cry1Ah-toxin. However, the mechanisms that regulate the resistance of ACB to Cry1Ah-toxin are unknown. Objective: In order to understand the molecular basis of the Cry1Ah-toxin resistance in ACB, “omics” analyses were performed to examine the difference between Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains of ACB at both transcriptional and translational levels. Results: A total of 7,007 differentially expressed genes (DEGs) and 182 differentially expressed proteins (DEPs) were identified between ACB-AhR and ACB-BtS and 90 genes had simultaneous transcription and translation profiles. Down-regulated genes associated with Cry1Ah resistance included aminopeptidase N, ABCC3, DIMBOA-induced cytochrome P450, alkaline phosphatase, glutathione S-transferase, cadherin-like protein, and V-ATPase. Whereas, anti-stress genes, such as heat shock protein 70 and carboxylesterase were up-regulated in ACB-AhR, displaying that a higher proportion of genes/proteins related to resistance was down-regulated compared to up-regulated. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 578 and 29 DEGs and DEPs, to 27 and 10 pathways, respectively (P < 0.05). Furthermore, real-time quantitative (qRT-PCR) results based on relative expression levels of randomly selected genes confirmed the “omics” response. Conclusion: Despite the previous studies, this is the first combination of a study using RNA-Seq and iTRAQ approaches on Cry1Ah-toxin binding, which led to the identification of longer length of unigenes in ACB. The DEGs and DEPs results are valuable for further clarifying Cry1Ah-mediated resistance.

Evolution of insect resistance to Bt toxins challenges the use of Cry toxins to control agricultural pests. In lepidopterans, Cry toxin affinity towards multiple midgut epithelial receptors has become a matter of dispute. Cry1Ah toxin-binding proteins were identified in the larval midgut of susceptible (ACB-BtS) and resistant (ACB-AhR) strains of the Asian corn borer (ACB). A pull-down assay was performed using biotinylated Cry1Ah toxin, and the binding proteins were identified by employing liquid chromatography–tandem mass spectrometry (LC-MS/MS). This study aimed to find the binding consistency of the midgut epithelial protein to the Cry1Ah toxin. The binding proteins from different fractions of SDS-PAGE showed a different pattern. We observed an isoform of prophenoloxidase PPO1b (UniProt Acc No. A0A1Q1MKI0), which was found only in the ACB-AhR fractions. Prophenoloxidase (proPO) is an extraordinary defense molecule activated in insect species during pathogen invasion and the wound healing process. Importantly, this prophenoloxidase might have direct/indirect interaction with the Cry1Ah toxin. Our data also suggest that factors like techniques, enrichment of binding proteins in the sample and the reversible and irreversible nature of the brush border membrane vesicles (BBMVs) to Cry toxins could cause the inconsistency in the protein–protein interactions. Moreover, inside the larva midgut, the influence of the Cry toxins under physiological conditions might be different from the laboratory procedures.