To begin, sterilize the surface of a 24 hour starved Monochamus alternatus and Tribolium castaneum beetles with bleach, ethanol, and distilled water. To induce the fungal infection, first, immerse the pine twigs or wheat bran into the conidial suspension for 10 seconds. Then dip the beetles in the conidial suspension.
After drying, place one beetle and one twig into a sterile 50 milliliter tube. Rear the beetles in a non-humidified incubator at 25 degrees Celsius. After incubation, transfer the dead beetles into a new 50 milliliter tube, and place a piece of sterile, moist cotton within the tube.
Dissect the infected beetle bodies with fungal infections into positions like an antennae, head, thorax, abdomen, wings, and legs. Cut five millimeter disc plugs of mature fungal colonies. Place the infected beetle tissues into pre-chilled 2.5%glutaraldehyde at four degrees Celsius for two days.
Next, wash the samples thrice in 0.1%phosphate buffer for five minutes and dehydrate in increasing ethanol concentrations for 10 minutes each. Dry the samples in a vacuum freeze dryer, and then observe the fungal structures under a scanning electron microscope. Treat the wheat bran with a conidial suspension for 10 seconds and after drying, transfer them into a Petri dish.
Immersed T.castaneum beetles in the conidial suspension for 10 seconds before placing them into the Petri dish containing wheat bran. After incubation, count the dead beetles from the plate. Extract genomic DNA of parasitic entomopathogenic fungi using the fungi genomic DNA isolation kit.
Using the given sets of primers, prepare a PCR reaction mixture for gene-specific amplification. After sequencing, align the nucleotide sequence using ClustalX 2. Finally, perform phylogenetic analysis using MEGA 6 based on the maximum likelihood method.
Parasitic fungi like Aspergillus austwickii, Akanthomyces attenuatus, and Scopulariopsis alboflavescens caused significant infection symptoms in M.alternatus. SEM observations further supported the morphological characteristics of these parasitic fungi on the surface of the beetles. Entomopathogenic activity showed significantly higher mortality rates in parasitic fungi compared to the control group.
The multi-gene phylogenetic tree demonstrated genetic distances between the three parasitic fungal species and other species within their respective genera.
