Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

We explore entomopathogenic fungal resources from wild Monochamus alternatus populations. Taking this species as an example, we could enrich the diversity of entomopathogenic fungi of other forest wood borers for future bio control applications. We have recently obtained three fungal species certified as parasitic entomopathogenic fungi of Monochamus alternatus, two of which are not well known but have proven to be potent pathogens of insects.

This protocol helps the discovery of more EPFs of Monochamus alternatus, and performs a substitute way to evaluate their entomopathogenic activities, which can also provide insights into entomopathogenic fungal exploration for other forest wood borers. 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.

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 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.

Immerse 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 ClustalX2. Finally, perform phylogenetic analysis using MEGA6, based on the maximum likelihood method.

Parasitic fungi like Aspergillus oryzae, 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.