The isolation of entomopathogenic fungi is significant for developing a biopesticide and can be helpful to study the diversity, distribution, and the ecology of this particular group. Using insect baits to isolate entomopathogenic fungi is considered a low cost and highly efficient method. chloramphenicol, thiabendazole, cycloheximide, artificial media does not use dodine and requires less space in the soil sample process.
Critical steps include investigating CTC plates after one and two weeks to prevent other fungi from narrowing and entomopathogenic fungi in development, and then identifying entomopathogenic fungi colonies based on their macro morphology and micro morphology. Begin by collecting 800 grams of soil to a depth of 10 centimeters using a small shovel. Store the samples in polypropylene bags at room temperature until the start of the experiment.
Small roots can also be collected since entomopathogenic fungi are known to have rhizosphere competence. For isolation of fungi using chloramphenicol, thiabendazole, cycloheximide, or CTC selective artificial medium, weigh 0.35 grams of the soil sample with or without roots. Place this sample in a 1.5 milliliter microtube.
While working in a biosafety cabinet, add one milliliter of sterile 0.01 volume percent polyoxyethylene sorbitan monooleate aqueous suspension to the microtube containing soil and vortex the tube for 30 seconds. When done remove and pipette 50 microliters of the supernatant onto the center of Petri plates with CTC medium. Then use a sterile Drigalski spatula of a six millimeter diameter to homogeneously disperse the suspensions onto the surface of the medium.
Incubate the plates in climate chambers at 25 degrees Celsius and a minimum of 80%relative humidity in the dark. After seven, 14, and 21 days of incubation, observe the plates for the growth of fungal colonies. To isolate the fungi using surface disinfected Galleria mellonella and Tenebrio molitor late stage larvae as insect baits.
Immerse the larva into 0.5%sodium hypochlorite for one minute for sterilization. Then wash the larvae twice with sterile water. Assemble the baits in plastic pots with 10 small holes of 10 millimeter diameter in the lid containing 250 grams of the collected soil.
Homogenize the soil every other day to allow maximum contact of larvae with soil and analyze the pots daily, seeking dead insects. After removing dead insects from the pot, superficially sterilize insects with 0.5%sodium hypochlorite for one minute. To favor the mycosis, place the sterile insects in a humid chamber at 80%relative humidity and 25 degrees Celsius for seven days.
On mycosis, harvest the conidia from the insect surface and with the help of the microbiologic loop, place the conidia on potato dextrose agar medium containing 0.05%chloramphenicol or PDAC medium under a stereoscopic microscope. As an alternative, place the whole infected larvae on the PDAC medium. And incubate the culture plates in a climate chamber as explained earlier.
After 14 days of incubation, identify the EPF species by analyzing the macro morphological characteristics of the fungal cultures on the plates as described in the manuscript. Next transfer the aerial conidia to slide cultures for three days at 80%relative humidity and 25 degrees Celsius. After three days stain the slide with lactophenol blue to observe the microscopic features.
Under an optical microscope, observe the fungal structures such as arrangement, conidiophores, shape, and size of conidia at 400 times and confirm the EPF identification. In the study, 51 EPF strains of Metarhizium and Beaveria species were isolated from 524 soil samples. The representative analysis shows the micromorphological characteristics of a few isolates of Metarhizium species and Beaveria species.
The occurrence of EPF in the 24 soil samples was studied using three different methods of isolation. Galleria mellonella bait proved to be more efficient in the isolation, followed by Tenebrio molitor bait and CTC medium. The same 24 soil samples showed no recovery of Beaveria species, but only Metarhizium.
The most important things to remember are transferring the aerial conidia to slide coat observing the microscope features and confirming the entomopathogenic fungi identification using morphological keys. Additional methods include the use of other selective artificial media with a specific chemicals to reduce the growth of contaminants such as dodine. Seeking new fungal isolates with outstanding bio control traits is crucial to increasing fungi effectiveness in arthropod-pest control and exploring further questions in the invertebrate pathology field.
