Potencial biopesticida de enzimas extracelulares de Beauveria bassiana

Abstract

Entomopathogenic fungi have shown great potential for the control of insects and other parasites. Beauveria bassiana produces diverse extracellular hydrolytic enzymes, including proteases and chitinases. In this context, the objective of this project was to evaluate the in vitro biopestic potential of extracellular enzymes (proteases and chitinases) produced by B. bassiana against animal parasites, plant parasites, and insect pests, as well as the in silico characterization of the proteases produced by the fungus from phylogenetic analyzes and three-dimensional (3D) structure prediction. The first step was to evaluate different culture media for the induction of proteases and chitinases using the commercial isolates of B. bassiana: IBCB 66 and ESALQ PL63, as well as the isolate IP 361. The action of the crude extract and the precipitate in the in vitro control of nematodes and trematodes parasitic to animals and plants was determined, as well as the effect of the precipitate on pupae of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). The second step was carried out from the in silico assay of B. bassiana proteases in relation to phylogenetic analysis with other genera of entomopathogenic fungi. To achieve this objective, the enzymes were produced under solid and liquid fermentation conditions, in order to determine the highest chitinolytic and proteolytic activity of commercial isolates of B. bassiana. The results demonstrated that the best culture medium (p < 0.01) for protease and chitinase was the solid rice medium with milk serum. Regarding the nematicidal activity of the enzymatic crude extract, the percentage of reduction was 58% in the case of nematodes parasitic to animals and 19% for plant parasites, presenting a significant difference compared to the control treatment. However, the in vitro assay with the precipitated extract of B. bassiana IP 361 applied to the eggs of Eurytrema pancreaticum caused a 47% reduction compared to the denatured extract (p < 0.01). In the test with S. frugiperda, the active precipitate of B. bassiana IP 361 reduced the number of pupae by 100% compared to the control treatment. On the other hand, there was no significant difference (p> 0.01) between the active group and the group with synthetic insecticide. The active precipitate revealed five bands with molecular weights of 25.6 to 66.9 kDa in the zymogram. In silico analysis identified the lineage of proteases Pr1 and Pr2 and their three-dimensional structure. These results indicate that B. bassiana proteases and chitinases can be useful in the control of parasitic diseases and pests, presenting potential as a sustainable alternative.