Bt Research 2025, Vol.16, No.2, 55-62 http://microbescipublisher.com/index.php/bt 57 3 Challenges in Current Bt Applications 3.1 Field-evolved resistance in target insect populations The evolution of resistance of field pests to Bt toxins is one of the most serious problems at present. Nowadays, worldwide, many important pests have shown resistance to Bt toxin under field or laboratory conditions, especially lepidopteran pests such as Helicoverpa armigera and Spodoptera litura (Bravo and Soberón, 2008; Afzal et al., 2024). The main mechanism of resistance is mutation or reduced expression of target receptors, such as cadherin and aminopeptidase N in the intestine, which reduces the binding ability of toxins (Tiewsiri and Wang, 2011). Although the spread of resistance may be slower when recessive inheritance or the initial resistance level is low, resistance can spread rapidly under intense selection pressure and mismanagement (Afzal et al., 2024; Aswathi et al., 2024). The emergence of these resistant pests directly affects the persistence and economic benefits of Bt crops in the field. 3.2 Limited activity spectrum across insect orders The insecticidal range of Bt toxin is limited and it is mainly effective against pests such as Lepidoptera, Coleoptera and Diptera. For other pests, such as Hemiptera and mites, the effect is very weak or almost ineffective. Some important agricultural pests are inherently insensitive to existing Bt toxins because their intestinal receptors are different, and the toxins cannot bind effectively and thus cannot be lethal (Deist et al., 2014; Aswathi et al., 2024). Although methods such as protein engineering and hybrid toxins have broadened the activity spectrum of some toxins, it is still very difficult to achieve broad-spectrum control of all major pests. 3.3 Environmental persistence, formulation, and delivery constraints The stability and persistence of Bt toxins in the field are also affected by the environment. For instance, ultraviolet radiation, rainfall erosion and soil adsorption can all reduce the efficacy of toxins, thereby leading to unstable field control effects. In addition, the delivery methods of Bt preparations also vary. Spraying is susceptible to environmental influences. Although genetically modified crops are more stable, they may encounter problems such as insufficient gene expression, non-target effects and ecological security (Aswathi et al., 2024). New delivery methods, such as microcapsules and microparticles, have shown some advantages in experiments, but further improvements are needed in terms of cost, field application and regulation. 4 Engineering Strategies for Improved Efficacy 4.1 Domain shuffling and protein engineering for expanded host range Through domain recombination and protein engineering, the target of action of Bt toxin can be significantly expanded. Some studies insert specific binding peptides or non-Bt toxin fragments into the key regions of Bt toxins, which can make the toxins more effective against new pests. For instance, after inserting the pea aphid intestinal binding peptide into the ring structure of Cyt2Aa toxin, the killing power of the toxin against aphids was greatly enhanced, providing a new method for the application of Bt toxin to Hemiptera pests (Chougule et al., 2013). In addition, by using fragment replacement technology and inserting specific peptide segments into the ring region of Cry toxin, engineered strains with strong toxicity to rice pests can also be obtained (Deist et al., 2014). 4.2 Mutagenesis and directed evolution approaches to enhance potency Site-directed mutagenesis, saturation mutagenesis and directed evolution and other methods are often used to enhance the activity of Bt toxins and solve the problem of pest resistance. Through high-throughput screening methods such as phage display and ribosome display, toxin variants that can bind to new receptors with high affinity can be quickly identified. For instance, by using phage assisted continuous evolution (PACE) technology, scientists have obtained a modified version of Cry1Ac. This variant can efficiently bind to the novel receptor of resistant insects, and the insecticidal power against resistant pests has increased by 335 times (Deist et al., 2014; Badran et al., 2016; Dovrat and Aharoni, 2016). In addition, site-directed mutagenesis can also help study the key action sites of toxins and receptors, providing a basis for subsequent rational design (Deist et al., 2014; Pacheco et al., 2015).
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