Biocontrol agents play a pivotal role in managing pests and contribute to sustainable agriculture. Recent advancements in genetic engineering can facilitate the development of entomopathogenic fungi with desired traits to enhance biocontrol efficacy. In this study, a CRISPR-Cas9 ribonucleoprotein system was utilized to genetically improve the virulence of Beauveria bassiana, a broad-spectrum insect pathogen used in biocontrol of arthropod pests worldwide. CRISPR-Cas9-based disruption of the transcription factor-encoding gene Bbsmr1 led to derepression of the oosporein biosynthetic gene cluster resulting in overproduction of the red-pigmented dibenzoquinone oosporein involved in host immune evasion, thus increasing fungal virulence. Mutants defective for Bbsmr1 displayed a remarkable enhanced insecticidal activity by reducing lethal times and concentrations, while concomitantly presenting negligible or minor pleiotropic effects. In addition, these mutants displayed faster germination on the insect cuticle which correlated with higher density of free-floating blastospores in the hemolymph and accelerated mortality of the host. These findings emphasize the utility of genetic engineering in developing enhanced fungal biocontrol agents with customized phenotypic traits, and provide an efficient and versatile genetic transformation tool for application in other beneficial entomopathogenic fungi.
Fungal insecticides, while environmentally friendly, often suffer from slow action and require high doses with repeated applications to achieve effective pest control. To address these limitations, we employed CRISPR-Cas9 genome editing to enhance the virulence of the entomopathogenic fungus Beauveria bassiana. Specifically, we knocked out the Bbsmr1 gene, a transcriptional repressor that downregulates the biosynthesis of oosporein—a secondary metabolite crucial for inhibiting insect immunity and promoting fungal virulence. The resulting knockout strains exhibited significantly higher virulence compared to the wild type. Our approach demonstrates that CRISPR-Cas9 is a simple, efficient, and precise tool for editing the genome of B. bassiana, allowing for the customization of fungal biocontrol strains with desired traits such as enhanced virulence. This method holds great promise for developing more robust and effective mycoinsecticides, contributing to sustainable agriculture by reducing reliance on chemical pesticides.
Keywords: Blastospores; Filamentous fungi; Geneticin selectable marker; Genome editing; Homologous recombination; Ribonucleoprotein; Secondary metabolite.
© 2024. The Author(s).