2.2.2. Isolation of Fungi from Soil Samples

In the laboratory, the 5 soil samples per site were thoroughly mixed to produce 12 composite pooled soil samples. Soils were kept at 4 °C until processing but never for longer than 5 days. All soil samples were sieved through a 2 mm mesh sieve to remove debris. Dry soil samples were slightly moistened with sterile water while wet soils were first air-dried to remove excess water and reduce the incidence of nematodes. The widely and best-known method for selecting entomopathogenic fungi, the Galleria mellonella bait method described by Zimmermann [33], was used to isolate entomopathogenic fungi from soil samples. Before being used as baits, 4–5-week-old G. mellonella larvae were heat-conditioned as described by Woodring and Kaya [34] by immersion in 56 °C sterile water for 15 s, followed by pouring cold water on top of the larvae for 30 s and then letting the larvae rest for 1 h to recover. This was done to reduce the ability of the larvae to produce webbing while in the soil. Five live heat-conditioned G. mellonella were then added to a 350 mL plastic container with an aerated lid containing 300 g of the sifted soil sample and incubated for 14 days in the dark at 25 ± 5 °C. The plastic containers were inverted once every 2 days to promote larval movement through the soil.

Containers with soil samples were checked daily, and dead larvae were removed, surface sterilized by immersing them in 70% alcohol for 10 s, rinsed thrice in sterile water for 10 s, and left to dry on a sterile paper towel. They were then individually placed in a moist chamber and incubated for 14 days at 25 ± 5 °C. Dead larvae were observed every 2 days for fungal growth, and mycelia were isolated by placing them on SDA with 0.1% antibiotics and incubated as described above. The number of dead larvae exhibiting mycosis was recorded. Fungal growth obtained from each mycosed larva was considered an isolate. Fungal isolates were stored in silica gel until morphological and molecular characterization.