Bt Research 2025, Vol.16, No.2, 47-54 http://microbescipublisher.com/index.php/bt 51 of insecticidal crystals, the type spectrum of δ -endotoxin proteins, and the type spectrum of plasmids. For instance, in soil samples from Qatar, researchers observed various crystal forms from 700 Bt isolates through scanning electron microscopy. By combining protein spectroscopy and plasmid profiling analysis, they ultimately determined seven plasmid profiles (Plas 1 to Plas 7). These type spectra include Btk type, Bti type and five non-Bti type plasmids. Each plasmid profile is associated with a specific combination of insecticidal protein genes, indicating that the plasmid diversity of Bt strains is the genetic basis for their ecological adaptation and extended insecticidal profile (Nair et al., 2018). 6.2 Experimental evidence of plasmid-driven adaptation The experimental results show that the plasmid diversity of Bt strains will directly affect their adaptability. For instance, 19 Bt strains carrying BTi-type plasmids can effectively kill Culex mosquito larvae. Although some plasmid regions are unstable, such as the deletion of cry10 and cyt1C genes, the insecticidal activity still exists. This indicates that the gene combinations and structural changes on the plasmid can regulate the host range and adaptability of the strain. Furthermore, the diversity of plasmid type spectra and insecticidal protein genes jointly determines the targeting effect of Bt strains on different insect groups (Nair et al., 2018). Broader studies have also found that plasmids often carry insertion sequences (such as IS1), which can promote gene inactivation and rapid adaptation, thereby enhancing the survival and evolutionary potential of strains in complex environments (Sastre-Dominguez et al., 2024). 6.3 Implications for field application and strain improvement Plasmid diversity also provides rich genetic resources for the field application of Bt and the improvement of strains. Strains with different plasmid type profiles can target a variety of pests, achieving more precise biological control and reducing the risk of resistance development. The structural variation and gene recombination capabilities of plasmids provide a theoretical basis for the screening and cultivation of highly efficient, broad-spectrum or specific Bt strains. Meanwhile, understanding the role of plasmids in adaptation can also assist molecular breeding and synthetic biology, and improve the stability and efficacy of Bt preparations in the field (Nair et al., 2018; Sastre-Dominguez et al., 2024). 7 Challenges and Research Gaps 7.1 Limitations in plasmid sequencing and annotation technologies Although whole-genome sequencing and various "omics" techniques have promoted research on the diversity of Bt plasmids, traditional methods still have limitations. Methods like PCR, DNA hybridization and microarrays can only detect known genes and cannot discover new or very few plasmid elements. Proteomics can reveal the expressed products, but the overall analysis of plasmid structure and function is still insufficient. High-throughput sequencing has improved the efficiency of discovering new genes and plasmids, but data analysis and functional annotation still rely on the refinement of databases and algorithms. Therefore, there is still a gap in understanding the diversity and function of Bt plasmids (Nair et al., 2018; Pacheco et al., 2021). 7.2 Unresolved questions on plasmid stability and evolutionary trade-offs The plasmid diversity of Bt strains stems from frequent horizontal gene transfer and recombination. However, the stability of plasmids in the host, their maintenance costs, and their long-term impact on the adaptability of strains have not been fully clarified. For instance, some plasmid regions are unstable, which may cause the loss of key toxin genes or alter their expression, thereby affecting the insecticidal effect. Furthermore, how the incompatibility among different plasmids, the immune mechanism of the host, and trade-offs in evolution jointly affect the ecological adaptability and virulence spectrum of Bt strains requires more mechanism studies and theoretical modeling (Nair et al., 2018; Pacheco et al., 2021; Pilosof, 2023). 7.3 Ecological risks and biosafety considerations in deploying diverse Bt strains The diversity of Bt strains and plasmids provides a wealth of resources for biological control, but it may also bring ecological risks and biosecurity issues. Horizontal transfer of plasmids may cause toxin genes to spread to other microorganisms in the environment, thereby affecting non-target organisms or causing resistance problems. In
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