# The Effect of Exposure to an Electromagnetic Field on Entomopathogenic Fungi
## 2 Materials and Methods
### The Effect of EMFs on Fungi  
The fungi were inoculated on PDA with the addition of chloramphenicol (reducing the risk of bacterial contamination) in Petri dishes (diameter 90 mm). A disc (5 mm) with the inoculum of the respective fungus was placed in the central part of the plate. The Petri dishes were sealed with parafilm. The experiment was carried out in 4 replications. The diameter of the mycelium was measured 7 days (the end of exposure to EMFs) and 14 days after the experiment began. The effect of EMFs on the growth of fungi was evaluated on the basis of an inhibition/stimulation coefficient calculated according to [PERSON]'s formula: I = [(C \(-\) E)/C] \(\times\) 100%, where I--inhibition index of fungus linear growth (%); C--diameter of fungus colony in control; and E--diameter of fungus colony exposed to the EMF [35]. The coefficient of the linear growth rate was also calculated using the formula described by [PERSON] et al. [36]: T = (A/D) + (b\({}_{1}\)/d\({}_{1}\)) + \(\ldots\) + (b\({}_{\rm x}\)/d\({}_{\rm x}\)), where T--linear growth coefficient; A -colony diameter measured at the end of the experiment [mm]; D--number of days since the start of the experiment; b\({}_{1}\), b\({}_{\rm x}\)--increase in colony diameter since the last measurement [mm]; and d\({}_{1}\), d\({}_{\rm x}\)--number of days since the last measurement. The growth inhibition/stimulation coefficient (I) described above was used to evaluate the inhibitory effect of the EMF on EPF sporulation, spore germination ability, and germ tube length.  
Sporulation was assessed 14 days after the start of the experiment by sampling a fragment of mycelium on the edge of the colony. The mycelium was placed in a tube with 10 cm\({}^{3}\) of distilled water with Tween 80 (0.05% concentration) (Sigma-Aldrich, London, UK) and shaken for 10 min. The spore concentration in the suspension was determined in a Burker chamber under a light microscope [33; 37].  
The fungi's spore germination ability was also assessed; to do this, a small amount (50 \(\mu\)L) of spore suspension was transferred by micropipette into a cavity on basic glass (so-called glass with a tear). The sheets of glass were placed in Petri dishes into which a small amount of distilled water was poured to prevent the spore suspension on the basic glass from drying out. After 24 h, the sheets of glass with a tear were placed under a scaled microscope to check the number of germinating spores. Germinated conidia were identified as those with germ tubes longer than the width of an ungerminated conidium [38; 39]. To evaluate the effect of the EMF on the growth on the germ tubes, their length was measured 48 h after the start of the experiment. In total, 200 hundred conidia were examined for each germination and germ tube length test. Tests were performed in four replications.  
The pathogenicity of the entomopathogenic fungi was assessed on the basis of the mortality of test insects treated with spores of the respective fungi. The test insects were _Tenebrio molitor_ master larvae of similar weight (ca. 140 mg). A total of 10 insects were used for each repetition. Insects were placed in sterile Petri dishes with moistened filter paper. The insects were then sprayed with 1 mL of a spore suspension of the tested fungus at a concentration of 1 \(\times\) 10\({}^{7}\) mL\({}^{-1}\). The control insects were sprayed with distilled water. All dishes containing insects were kept in the same conditions: in the dark, with a temperature of 22 \({}^{\circ}\)C. The experiments were carried out in 4 replications. Insect mortality was checked every 24 h for 10 days. Dead larvae were put into Petri dishes lined with filter paper moistened with sterile distilled water. The mycelium development of EPFs on their bodies was the confirmed cause of their mortality.