The method for entomopathogenic fungi isolation is important for developing microbial control agents. This protocol can be used to isolate and select high virulence entomopathogenic fungal isolates from the soil samples. This technique includes three prongs of pathogenicity screening and combines the thermotolerance and conidia production assay to make the effective conidia number ranking.
The method can help select high virulence and promising entomopathogenic new fungal isolates for commercializing. This protocol focuses on biological control, it could also be used to discover the entomopathogenic fungal isolates to study the interaction between pests and entomopathogenic fungi. Demonstrating the procedure will be Yao-Chia Liu and Nian-Tong Ni, master student from my laboratory.
Start with the collection of the soil sample by removing one centimeter of the surface soil and then collecting the soil within the five to 10 centimeter depth from each sampling site using a shovel. After recording the details of each sampling site, collect and maintain 100 grams of soil sample into a plastic bag at room temperature to perform the fungal isolation protocol within three hours. To isolate the potential entomopathogenic fungi, or EPF, add 100 grams of the soil sample in a plastic cup and place five Tenebrio molitor worms on the surface of the soil at room temperature in the dark for two weeks with observing and recording the larva mortality and mycosis daily.
Keep the dead larva in the cup until two weeks for fungal isolation. After two weeks, transfer the dead insects to a clean bench and use a sterile toothpick to collect the conidia. To obtain the primary culture of fungi, streak the collected conidia on a quarter-strength Sabouraud dextrose agar medium or SDA in a 55-millimeter plate to incubate at 25 degree Celsius for seven days.
On the eighth day, restreak each primary culture fungi on one 55-millimeter quarter-strength SDA plate in a laminar flow hood before incubating the culture at 25 degree Celsius for seven days to obtain single colonies of fungi. In the first pathogenicity screening, place five Tenebrio molitor larvae directly on the surface of each pure fungal culture plate at 25 degrees Celsius. After observing and recording the mycosis and mortality for 10 days, select the fungal isolates for further analysis.
To perform the second virulence test, harvest the conidia of each fungal isolate by vortexing for one minute and count the number of conidia using a hemocytometer. When done, adjust the conidia suspension to a concentration of one times 10 to the 1/7 conidia per milliliter in a 0.03%surfactant solution before spreading 10 microliters of fungal suspension onto a 55-millimeter quarter-strength SDA plate to grow for seven days at 25 degrees Celsius in the dark. On the eighth day, place five Tenebrio molitor larvae directly on the surface of each pure fungal culture plate and seal the plates with Parafilm film to incubate while observing the mycosis and mortality as described before.
After repeating the second virulence test in triplicate for each fungal isolate, select the isolates to perform third virulence test for the target pest like Spodoptera litura. Collect circa one square millimeter EPF from the seven-day quarter-strength SDA plate to extract the fungal genomic DNA using a fungal genomic DNA extraction kit. Next, amplify the fungal internal transcribed spacer or ITS region by PCR of the DNA sample following the PCR program described in the text manuscript.
After sequencing the PCR-amplified product by commercial sequencing service, use the NCBI BLAST to search for similar fungi in the NCBI database and select the relative fungal species for phylogenetic analysis. Align the multiple sequences and trim the conserved sequence region manually with GeneDoc. Perform the phylogenetic analysis based on the minimum evolution, neighbor joining, and maximum likelihood methods.
To study the morphology of the fungi, capture the fungal culture colony growth for seven days with a camera and record the growth, the form:fluffy or firm, and color of the colonies. To observe the conidia in conidiophores, scrape conidia from the pure culture fungal colony with an inoculation loop and transfer the spores to a glass slide containing 0.1%Tween 80 solution. Use a scalpel to cut a agar block of the fungal colony and then transfer the agar block to a glass slide and add the 0.1%Tween 80 solution to wash off most of the excess conidia on agar.
Next, cover the slide with a cover slip for light microscopic observation of the conidia. Measure and record the width and length of the conidia and conidiophores to compare the difference between different fungal isolates. Arrange a conidial production assay by culturing the selected fungal isolate on quarter-strength SDA medium at around 25 degrees Celsius in the dark for 10 days.
Then prepare one milliliter conidial suspension of the fungal isolate in 0.03%surfactant solution followed by adjusting the concentration to one times 10 to the 1/7 conidia per milliliter as described before. After adding three droplets of 10 microliters of conidial suspension on a quarter-strength SDA plate, incubate the culture at 25 degrees Celsius in the dark for seven, 10, and 14 days to count the sporulation of fungi. At each time point, detach a five-millimeter agar block from the center of the colony with a cork borer and transfer the block into a 1.5-milliliter microcentrifuge tube containing one milliliter of 0.03%surfactant solution.
After vortexing the tube at 3, 000 rotations per minute at room temperature for 15 minutes, use a hemocytometer to count the number of conidia. For the thermotolerance assay, culture the selected fungal isolate and prepare one times 10 to the 1/7 conidia per milliliter suspension as illustrated earlier. After vortexing, heat the conidial suspension in a dry bath at 45 degree Celsius for different time intervals.
Post heat exposure, add three droplets of five microliters of conidial suspension on a 55-millimeter quarter-strength SDA plate at each time point and then incubate the plates at around 25 degree Celsius for 18 hours. To determine the germination rate, count the number of germinated conidia spores with five randomly selected fields under the light microscope at 200 times magnification. Calculate the total effective conidia number or ECN using the formula, and then calculate principle component analysis or PCA of fungal strains as described in the manuscript.
Select the best performing fungal strains based on ECN or PCA to perform the virulence test of target pests. In the second virulence test, the virulence of the 26 fungal isolates against Tenebrio molitor mealworms was assessed. 12 fungal isolates with high pathogenicity were selected for the virulence test against Spodoptera litura.
To better understand the fungal taxonomic positions, 26 isolates from the first pathogenicity screening were subjected to molecular analysis based on the ITS region. Through the cleaning method of fungi morphological observations, the structures of conidiophores could be seen clearly with 0.1%Tween 80 solution. The conidia's color, shape, and arrangement were studied through microscopic observations of the conidia colony.
The ECN combines conidia production and thermotolerance data of each EPF. The high viability of a fungal strain was observed when the ECN value was high. Also, great coordination between the PCA and the ECN was revealed, suggesting that the ECN could be used to evaluate the hierarchy of viability-related parameters.
The pathogenicity screening extracting the fungal DNA and culturing the fungal isolate are important in the procedure. By using the ECN calculation formula, the ideal internal pathogenic fungi can be selected. Combination of different insect species, such as greater wax moth and mealworm together to bait the entomopathogenic fungi from the soil samples might increase the diversity of entomopathogenic fungi.
The microorganism diversity of soil is a very interesting topic during the operation of this protocol. It could be also further investigated in the future via this protocol.
