To develop a new generation of eco-friendly biopesticides, this study used an optimized random mutagenesis approach to generates oligosporogenic mutants fr

Introduction: The persistence of Bacillus thuringiensis ( B. thuringiensis ) spores in the environment can lead to ecological and safety concerns, despite the widespread use of this microbial biopesticide. Developing oligosporogenic strains that retain high insecticidal activity represents a promising key strategy for sustainable pest management. Objectives: The aim of this study was to generate and characterize novel oligosporogenic mutants of the B. thuringiensis BLB1 strain through an iterative UV-mutagenesis approach, combined with Whole Genome Sequencing (WGS) to resolve the genetic basis of their phenotype. Methods: Successive sequential rounds of UV irradiation (254 nm) were applied, and mutants were screened for reduced sporulation while maintaining crystal production. Two promising candidates, T3 and T8, were selected for kinetic studies, protein quantification, and bioassays against five major lepidopteran pests. Their genomes were sequenced using Illumina technology and subjected to comparative genomic analysis. Results: Both mutants exhibited a significant delay in sporulation and altered glucose consumption patterns compared to the wild-type BLB1 strain. Despite the reduction in spore counts (5 × 10 ⁷ and 3 × 10 ⁷ spores/ml, respectively), T3 and T8 retained stable Cry protein profiles (Cry1 and Cry2). Bioassays revealed that both mutants maintained high toxicity, reflected by low LC50, particularly against Grapholita molesta (17 ng/cm ² for both mutants) and Ostrinia nubilalis (32 and 26 ng/cm ² , respectively), compared to the wild-type strain BLB1 (25 and 18 ng/cm ² , respectively). WGS confirmed that none of the detected cry genes were affected by mutagenesis, explaining the preserved insecticidal activity. These findings demonstrate that iterative UV mutagenesis effectively decoupled sporulation from insecticidal activity. Conclusion: These findings demonstrate that iterative UV mutagenesis effectively decoupled sporulation from insecticidal activity. The resulting mutants, T3 and T8, represent promising candidates for safer and more effective commercial biopesticide applications, combining reduced environmental persistence with strong biocontrol efficacy.