Abstract

Entomopathogenic fungi of the genus Metarhizium can degrade and penetrate the insect cuticle through the coordinated action of hydrolytic enzymes, mainly lipases, proteases, and chitinases, whose production varies according to the fungal species and fermentation conditions. These enzymes can be generated via submerged fermentation and subsequently employed to enhance the insecticidal activity of fungal conidia. This study aimed to increase protease production from Metarhizium robertsii Mt015 to strengthen biological control agents based solely on fungal biomass. The culture medium composition and physicochemical parameters were optimized using a statistical design approach. Biological activity assays were then performed using Tuta absoluta larvae as the target insect and Beauveria bassiana as the reference control, tested both alone and in combination with the protease extract. Optimization identified wheat bran, casein, and an initial pH of 8–10 as the most influential variables, achieving a 5.5-fold increase in protease activity compared to the basal medium. When the protease extract was combined with B. bassiana conidia, the mortality rate reached 78.2%, significantly higher than the 55.6% achieved with B. bassiana conidia alone. Bliss independence analysis indicated that the observed larval mortality exceeded the additive expectation (Δ = 23.3 percentage points; 95% CI: 16.7–30.0), supporting a synergistic interaction between B. bassiana and the protease extract at 0.68 U/mL. These results demonstrate that enzymatic supplementation markedly improves the insecticidal performance of entomopathogenic fungi, supporting the use of enzyme-enriched formulations as a complementary strategy to strengthen biological control agents and advance the development of next-generation biopesticides.

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