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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Crop Bioprotection Research » Research » Publications at this Location » Publication #414777
Research Project: Discovery and Production of Beneficial Microbes for Control of Agricultural Pests through Integration into Sustainable Agricultural Production Systems

Location: Crop Bioprotection Research

Title: Enhanced virulence through genetic engineering of Beauveria bassiana blastospores by overexpression of a cuticle-degrading endochitinase

Author
item MASCARIN, GABRIEL - Brazilian Agricultural Research Corporation (EMBRAPA)
item SHRESTHA, SOMRAJ - Auburn University
item Dunlap, Christopher
item Ramirez, Jose
item COLEMAN, JEFFREY - Auburn University

Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2025
Publication Date: 8/6/2025
Citation: Mascarin, G.M., Shrestha, S., Dunlap, C.A., Ramirez, J.L., Coleman, J. 2025. Enhanced virulence through genetic engineering of Beauveria bassiana blastospores by overexpression of a cuticle-degrading endochitinase. Journal of Invertebrate Pathology. https://doi.org/10.1016/j.jip.2025.108420.
DOI: https://doi.org/10.1016/j.jip.2025.108420

Interpretive Summary: Entomopathogenic fungi have been utilized as alternatives to chemical pesticides and are an important eco-friendly option for the sustainable management of insect pests of agricultural, veterinary, and medical importance. However, despite intensive efforts, fungi have not fulfilled expectations as biocontrol agents because of their relatively slow rate of insect control compared to chemical pesticides. In this study, we added additional copies of a gene used by the fungi to degrade the insect cuticle. The results show the modified fungus was able to kill a test insect 20% faster. The research provides insights into how to improve the efficacy of the crop protection microbial agents.

Technical Abstract: Genetic engineering of entomopathogenic fungi offers an innovative approach to improve their virulence against a broad spectrum of arthropod hosts and increase their resilience to stressful environmental factors. In this research, direct transformation of Beauveria bassiana protoplasts and recombinant plasmids containing a constitutively expressed endogenous gene encoding a chitinase (BbChit1) was conducted to improve fungal virulence. The relative gene expression and the number of gene copies in the chitinase-overexpressing mutants (OEBbChit1) were assessed, followed by phenotypic characterization of blastospores in terms of virulence, insect cellular immune response, colony morphology, growth, and tolerance to multiple chemical cell stressors. Blastospores of OEBbChit1 mutants conferred faster mortality rates and lower lethal doses when compared to the parental wild type (WT) after being topically inoculated to larvae of the model insect host Galleria mellonella. Moreover, infections by the OEBbChit1 mutant triggered an increased production of total hemocytes relative to the WT and mock control, indicating induction of the insect cellular immune response. Genomic analysis of the OEBbChit1 mutants revealed that the number of gene copies encoding the chitinase was 4 and 2 for mutants OEBbChit1-3 and OEBbCchit1-5, respectively. Furthermore, the chitinase-encoding gene was significantly upregulated by ~3-to-5-fold relative to the wildtype, confirming the overexpression of this enzyme in the mutants. Collectively, these findings provide compelling evidence of genetic improvement of B. bassiana by overexpression of chitinase-encoding gene (BbChit1) with 2-4 copies integrated into its genome leading to enhanced virulence without detrimental pleiotropic effects in fungal development.