Entomopathogenic Fungi Isolation from Soil

The soil sample mentioned above was subjected to insect baiting to isolate entomopathogenic fungi, which were subsequently identified using our earlier methodologies and others (Vivekanandhan et al., 2021 (link); Mathulwe et al., 2023 ). The bait method employing third-instar larvae of Tenebrio molitor is an exceptionally sensitive technique used to isolate insect pathogenic fungi from soil. Fifteen T. molitor larvae in their third instar were transferred to a plastic container with 300 g of soil, measuring 15 cm (L) × 10 cm (W) × 10 cm (H). After securing the container with a lid, it was placed in an incubator set at 26 ± 1°C and 85% relative humidity. For 15 days, plastic containers were observed twice daily. After collecting the larval cadavers and sterilizing them for 2–3 min with 70% ethanol, the sterile cadavers were transferred to Petri plates (90 mm × 15 mm) containing pre-prepared potato dextrose agar medium PDA (HiMedia, India) (Perumal et al., 2023a (link)). Plates were kept at a relative humidity of 85% and a temperature of 26 ± 2°C for 7 to 10 days. The pure fungal cultures were isolated from the dead larval cadaver according to Balumahendhiran et al. (2019) (link) and Vivekanandhan et al. (2023) (link) and preserved in a biochemical oxygen demand incubator (BOD) (Smartscience, India) at 26 ± 2°C for future experiments.