Bt Research 2025, Vol.16, No.5, 224-233 http://microbescipublisher.com/index.php/bt 229 reduced. Many studies use half lethal concentration (LC50) indicators to quantify this change. The intact Bt strain of plasmids are usually very low for sensitive insects. When the main virulence plasmid is lost, the LC50 is greatly increased, even reaching hundreds or thousands of times that of the wild type. In the resistant rhodopseudomoto experiment, LC50 without plasmid Bt clones was several orders of magnitude higher than the ancestral strain, showing a significant decrease in virility (Kashyap and Amla, 2007). The corrected mortality rate for pine caterpillars LM121, which has high virility to pine caterpillars, decreased from more than 90% to less than 20% after induced plasmid loss. These data intuitively prove that plasmid loss greatly reduces the lethal efficacy of Bt on target pests. Secondly, plasmid loss will narrow the virulence range of Bt strains. Bt in a complete plasmid can often poison multi-order pests at the same time, and when the plasmid carrying a specific cry gene is lost, the strain may completely lose virulence to pests of a certain order or family (Fayad et al., 2020). Figure 2 Expression of vip3Ais increased in the Bt HD73 Cry− Spo0A− (Adopted from Chen et al., 2022) Image caption: (A) Schematic representation of the constructs used to study the regulation of the vip3 and vipR genes in the sporulation mutant strain. (B) β-Galactosidase activity of the B. thuringiensis HD73− (pHT-PvipR-vipR) and HD73− Spo0A− (pHT-PvipR-vipR) cells. Data are the mean ± SEM, n = at least 3. (C) β-Galactosidase activity of the HD73− (pHT-PvipR-vipR-Pvip3) cells and HD73− Spo0A− (pHT-PvipR-vipR-Pvip3). Data are mean ± SEM, n = at least 4. (D) β-Galactosidase activity of the HD73− (pHT-PvipR) andHD73− Spo0A− (pHT-PvipR) cells. Strains were grown in LB at 37 °C. Time 0 corresponds to the entry of the bacteria into the stationary phase. Data are mean ± SEM, n = 3 (Adopted from Chen et al., 2022) 6 Methods for Detection and Analysis of Plasmid Loss 6.1 Molecular biological detection methods (PCR, Southern blot, etc.) In order to detect whether the Bt strain has plasmid loss and identify which genes are lost, commonly used molecular biological methods include PCR, nucleic acid hybridization, etc. PCR detection is the easiest and fastest method. Design specific primers based on the known Bt toxin gene sequences, and extract DNA from different generations of strains for PCR amplification. If the specific toxin gene amplification band of a certain generation of strains disappears, it indicates that the corresponding plasmid may have been lost. PCR can also be used to amplify the origin of plasmid replication or marker genes to monitor the presence of the plasmid as a whole. In addition to PCR, Southern hybridization is also a classic method: the total DNA of the strain is extracted and the Southern blot hybridization is performed using toxin genes or plasmid backbone sequence probes. If the hybridization signal of a certain generation of strains is significantly weakened or disappeared compared to wild type, it means that the plasmid DNA copy number is decreased or the plasmid is missing. This method is highly sensitive and is suitable for confirming suspicious PCR results (Gillis et al., 2017). In recent years, with the reduction of genome sequencing costs, whole genome sequencing has become a powerful tool for analyzing
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