After collecting the pine sawyer, Monochamus alternatus beetles, place the beetle into a sterile tube containing fresh twigs. Rear the beetles in a non-humidified incubator at 25 degrees Celsius. After incubation, record beetles showing decreased feeding and mobility.
Transfer the stiff dead beetles to wet chambers. Dissect the beetles with fungal infections into main positions such as antennae, head, thorax, abdomen, wings, and legs. Using scissors, cut the integuments of each position into small pieces.
Gently press the outer surface of these pieces onto the PDA plate containing antibiotics. Seal the plate with parafilm and incubate at 25 degrees Celsius until the tissues are entirely covered by mycelium. Then transfer the single colony according to phenotypic properties onto a new PDA plate.
Extract the genomic DNA using the fungi genomic DNA isolation kit. Prepare PCR mixture with primers ITS1 and ITS4 to amplify the rDNA-ITS region of the DNA. Use a camera to capture mature, fungal pure colony morphology on the front and back sides of the PDA plate.
Pluck conidia from the pure fungal colonies with an inoculation needle and transfer them to a glass slide with a drop of sterile water. Then place a cover slip over the specimen without introducing bubbles. Cut a five millimeter agar block with a scalpel from the edge of the fungal colony and transfer it to a clean glass slide with a drop of sterile water.
Using a fine needle, carefully separate the fungi from the agar to observe specific structures. Under an optical microscope, observe the asexual morph of the fungi, including the posture of the hyphae, conidiophores, phialides, and conidia.
