Abstract: The sweet chestnut (Castanea sativa Mill.) is a crop of significant socioeconomic and ecological importance in Portugal. However, its production is heavily affected by several pests, notably chestnut weevils and moths (locally known as bichados), including Cydia fagiglandana, C. splendana, Curculio elephas, and Pammene fasciana. The continuous use of chemical insecticides to control these pests raises environmental and public health concerns, highlighting the urgent need to develop sustainable and eco-friendly alternatives. Entomopathogenic fungi (EPF) represent a promising biological tool capable of selectively reducing pest populations, in line with European Union policies aimed at decreasing agrochemical inputs. Herein, this study aimed to isolate, identify, and assess the diversity of EPF from chestnut grove soils in experimental plots located in Trancoso, as well as to evaluate their field efficacy against chestnut pests compared with the commercial insecticides Affirm Opti® and Spintor 480 SC®. EPF were isolated using the insect-bait method with Tenebrio molitor larvae and identified based on morphological traits and molecular analysis of the ITS and TEF1 regions, followed by phylogenetic analyses. The results revealed a high diversity and abundance of EPF in the chestnut grove soils, with six species isolated. Metarhizium robertsii was the most frequently isolated species, followed by Beauveria bassiana. In field trials, strains of these two predominant species significantly reduced chestnut fruit infestation compared with the chemical treatment. Notably, B. bassiana achieved 100% efficacy against all evaluated pests, followed by M. robertsii, whereas the chemical treatment (Affirm Opti®) exhibited the lowest efficacy. Overall, the native EPF isolates demonstrated strong potential in controlling chestnut pests, particularly B. bassiana, supporting their integration into sustainable and eco-friendly pest management strategies. Further studies are underway to optimize formulation and application methods to facilitate the development of effective commercial bioinsecticides.

Acknowledgments: This work was supported by the project 101074466 — LIFE21-CCA-IT-LIFE FAGESOS and by National Funds through the Fundação para a Ciência e a Tecnologia (FCT) under the scope of the projects UID/04033/2025 (CITAB) (https://doi.org/10.54499/UID/04033/2025) and LA/P/0126/2020 (Inov4Agro) (https://doi.org/10.54499/LA/P/0126/2020). EHE thanks the PRR and the European Funds NextGeneration EU for the research grant BIPD/UTAD/3/2023.