*2.2. Entomopathogenic Fungus*

*Isaria fumosorosea* strain CCM 8367 was used in this study. The strain was isolated from the pupa of the horse chestnut leaf miner, *Cameraria ohridella*, Deschka & Dimi´c (Lepidoptera: Gracillariidae) collected in the Czech Republic [19] and deposited in the Czech Collection of Microorganisms in Brno as a patent culture [20,21].

The fungus was grown on PDA medium (Sigma-Aldrich, Darmstadt, Germany) at 25 ± 1 ◦C and a 16L:8D photoperiod. After 10 days of incubation, the spore suspensions were prepared from each strain by scraping o ff conidia into the sterile solution of 0.05% (*v*/*v*) Tween ® 80 (Sigma-Aldrich, Darmstadt, Germany). The suspension was filtered through sterile gauze to separate the mycelium and clusters of spores. In uniform suspension, the spores were counted with a Neubauer improved counting chamber (Sigma-Aldrich, Darmstadt, Germany), and subsequently, the suspension was adjusted to the required concentration.

The viability of spores was verified using a standard germination test [22]. Ten drops from suspension were applied using a 1 μL inoculation loop on the surface of 2% water agar, which was poured in a thin layer onto the surface of a sterile slide. After the drops had dried, the slides were moved into a wet chamber and incubated at temperature 25 ± 1 ◦C for 24 h. The percentage of germinating spores was determined using an Olympus CH20 light microscope (Olympus Optical Co., Ltd., Tokyo, Japan); bright field, 400× magnification. The spore germination in all tests was 100%.
