2.2. Entomopathogenic Fungi

The GHA strain of Beauveria bassiana and the IMI330189 strain of M. acridum were isolated from the wettable powders of Botanigard and Green Muscle commercial bioinsecticides, respectively. Thirty-five microliters of the respective product suspended in sterile distilled water with 0.01% Tween^® 80 were spread on potato dextrose agar (PDA) with additional chloramphenicol (0.05 g/L) to prevent bacterial contamination. Sealed plates were then placed in a light-free incubator at 25 ± 1 °C for 3 weeks. Spores were harvested by scraping the surface of the agar with a sterile L-shaped spreader and suspended in sterile distilled water with 0.01% Tween^® 80. The initial concentration of the spore suspensions was determined using a Neubauer hemocytometer cell. The final suspensions were adjusted to 10⁸ spores/mL and stored at 4 °C. Spore viability was assessed on ready-to-use suspensions by spreading three replicates of 0.1 mL of a diluted suspension at 10⁴ spores/mL on a microscopic slide overlaid with a thin slice of PDA. The germination rate was estimated by counting 100 spores using a 200× magnification microscope after incubation for 24 h at 25 ± 1 °C in the dark. Only suspensions with >96% viability were used within a maximum of 24 h.