Bt Research 2025, Vol.16, No.2, 63-69 http://microbescipublisher.com/index.php/bt 67 sequencing and molecular markers, which can accurately determine the plasmid transfer pathways and receptor strains (Wang et al., 2025). Acman et al. 's research in 2019 proposed that network analysis and high-throughput sequencing have also been used to track the flow of plasmids in bacterial populations on a large scale, demonstrating that plasmids can spread widely among different strains, species, and even genera. 6.3 Outcomes and implications for biocontrol efficacy and risk assessment Plasmid-mediated HGT enhances the biocontrol diversity and adaptability of Bt strains. Wang et al. (2025) found that it enables Bt strains to rapidly acquire new insecticide toxin genes, enhancing the effectiveness of biological control. However, plasmids may also carry risk genes such as antibiotic resistance, increasing the risk of drug resistance spreading in environmental and agricultural systems. Tracking and evaluating plasmid transfer events is of great significance for the safe and sustainable application of Bt biocontrol agents (Acman et al., 2019; Wang et al., 2025). 7 Challenges and Research Gaps 7.1 Limitations of current plasmidome sequencing and annotation technologies High-throughput sequencing and omics technologies have advanced the research on Bt plasmid diversity and HGT mechanisms, but there are still many problems with the existing plasmid sequencing and annotation methods. Firstly, there are many complex repetitive sequences and highly homologous regions on plasmids, which makes plasmid assembly and differentiation very difficult and also affects the accurate identification of complete plasmid structure and functional genes. Secondly, the functions of many genes carried by plasmids are unknown, and the existing annotation databases are not perfect, making it difficult to explain the ecological and evolutionary roles of many "recessive" or "presumed" genes (Scholar et al., 2024). In addition, existing technologies also have difficulty distinguishing gene transfers between plasmids and chromosomes, which limits the comprehensive tracking of HGT dynamics (Domingues et al., 2025). 7.2 Unresolved questions about stability of transferred plasmids There are still many uncertainties regarding the stability of plasmids in new hosts at present. Plasmids can diffuse rapidly through HGT, but their long-term presence in recipient strains is affected by multiple factors, such as metabolic burden, copy number regulation, compatibility between the host and the plasmid, and environmental selection pressure (Domingues et al., 2025). Studies have found that if plasmids bring adaptive advantages, they may be retained for a long time, but in the absence of selective pressure, they may also be lost due to increased metabolic burden (Lee et al., 2021). It is still unclear which mechanisms determine the persistence of plasmids in the Bt population, nor the dynamic patterns of plasmid retention or loss in different ecological environments. 7.3 Potential ecological risks of plasmid dissemination beyond Bt Plasmid-mediated HGT not only occurs within Bt strains but may also spread across species or genera, posing ecological and biosafety risks. Scholar et al. (2024) and Domingues et al. (2025) demonstrated that functional genes such as resistance genes and virulence factors can spread among environmental microorganisms through plasmids, which may lead to the spread of antibiotic resistance or affect non-target organisms. Scholar et al. (2024) and Domingues et al. (2025) also hold that there is still a lack of systematic assessment of the diffusion range and frequency of plasmids in the natural environment, as well as their impact on the structure and ecological functions of microbial communities. Therefore, it is highly necessary to enhance risk monitoring and mechanism research. 8 Concluding Remarks Plasmids are mobile genetic elements and the main vectors for horizontal gene transfer (HGT) between Bt strains. They rapidly spread insecticidal toxins, resistance and adaptability genes among different strains through mechanisms such as conjugation and natural transformation. This enables Bt strains to evolve more rapidly and also helps them adapt to different ecological environments. Plasmids not only carry multiple functional genes but also evolve along with the host, promoting the diversity of microbiota and maintaining the stability of the ecosystem.
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