*2.3. Bioassays*

#### 2.3.1. The E fficacy of *I. fumosorosea* against *C. perspectalis*

Five concentrations of *I. fumosorosea* ranging from 1 × 10<sup>4</sup> to 1 × 10<sup>8</sup> spores per 1 mL were used in this experiment. The last-instar larvae of BTM in treated groups were individually immersed in the suspension of conidiospores of the fungus for five seconds (dip-test). All specimens in a control group were immersed in sterile solution of 0.05% Tween ® 80 only. Then, the larvae were placed into polystyrene Petri dishes (vented, inner diameter 90 mm, height 15 mm, Gosselin ™, Borre, France) lined with moist filter paper (KA 0, Papírna Perštejn, Ltd., Perštejn, Czech Republic) and kept under constant conditions (25 ± 1 ◦C and 16L:8D photoperiod). Larvae were fed with *B. sempervirens* leaves, which were replaced daily until larva developed into pupa or died. The filter paper was also daily moistened by distilled water to maintain optimal humidity inside the Petri dishes. The insects were monitored daily for a period of three weeks to record insect development, mortality, and the development of mycosis on cadavers until all individuals died or adults emerged.

All bioassays described above were repeated twice; each replication tested 15 insect individuals. Mycosis on cadavers and emerged adults were documented by digital cameras Olympus SP-510 (Olympus Optical Co., Ltd., Tokyo, Japan) and Nikon Coolpix 4500 (Nikon Corporation, Tokyo, Japan) mounted on a tripod and using macro mode.

#### 2.3.2. Scanning Electron Miscroscopy of *I. fumosorosea* Conidia Germination on Cuticle of *C. perspectalis* Larvae

In vivo germination of fungal conidia on the insect cuticle was examined by low-temperature scanning electron microscopy (LT-SEM). BTM larvae were treated by immersing in suspension of *I. fumosorosea* conidia (concentration 5 × 10<sup>7</sup> spores mL−1) and incubated for 0, 24, and 48 h at the temperature of 25 ◦C. The larvae were mounted on an aluminum stub using Tissue-Tek (C.C.T.D. Compound, The Netherlands). The samples were extremely fast (<10−<sup>3</sup> K/s) frozen in vapor of liquid nitrogen. After freezing, the samples were transfered into a GATAN ALTO-2500 high vacuum cryo-preparation chamber (Gatan Inc., Abingdon, UK). The surface of the sample was sublimated (freeze-etched) for 5 min at the temperature of −95 ◦C and at −130 ◦C. After sublimation, the samples were sputter-coated with gold at the temperature of −130 ◦C. Coated samples were inserted into the chamber of a JEOL JSM-7401F Field Emission Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan). Images were obtained by the secondary electron signal at an accelerating voltage of 4 kV and current 10 μA using an Everhart–Thornley Detector (ETD).

#### 2.3.3. The E ffect of *B. sempervirens* Extract on *I. fumosorosea* Germination and Growth

Plant material was collected from untreated *B. sempervirens* trees grown in the Biology Centre garden. The extract used for the study was prepared at the concentration of 20% ( *w*/*v*) by grinding 2 g of fresh leaves in 10 mL of solvent (water–ethanol 1:1 mixture). Analytic grade ethanol (Penta Ltd., Czech Republic) and distilled water were used. The mixture was filtered through filter paper (KA 0, Papírna Perštejn, Ltd., Czech Republic) to remove particulate materials, and one milliliter of fresh extract was spread on the surface of 2% water agar in Petri dish and left to dry for 24 h. Then, a suspension of *I. fumosorosea* conidia was applied using an inoculation loop on the surface. Germination was evaluated in 100 spores after 24 h of incubation at 25 ± 1 ◦C as described above. The control plate was treated with solvent only. The experiment was conducted in three replicates. Spore germination was documented by NIS-Elements Imaging Software and a Nikon Eclipse E200 microscope equipped with Nikon DS-Fi3 color camera (Nikon Corporation, Tokyo, Japan).

The e ffect *B. sempervirens* extract on fungus growth was measured by a modified inhibition zone assay [23]. A half mL of conidia suspension in 0.05% Tween ® 80 at a concentration 1 × 10<sup>4</sup> spores mL−<sup>1</sup> was spread evenly on potato dextrose agar (PDA) medium in a plastic Petri dish (diameter 90 mm). A hydroalcoholic extract of *B. sempervirens* leaves prepared as described above was applied on filter paper discs (diameter 14 mm) in a dose 150 μL per disc. Control discs were treated with 150 μL of the pure solvent. The solvent was allowed to evaporate, and paper discs were placed carefully in the center of PDA plates. After 7 days incubation at 25 ◦C, the plates were photographed by a digital camera Olympus SP-510 (Olympus Optical Co., Ltd., Tokyo, Japan) mounted on a tripod to document differences in fungus growth. Then, the area of plate in the center not covered by *I. fumosorosea* mycelium was measured using ImageJ, a Java-based image analysis software [24]. The assay was conducted using 10 dishes (replications) for both treatment and control.
