For the EPF recovery from the fungal library, start by smearing the fungal isolates on a quarter strength SDA plate. Incubate it in darkness at 25 degrees Celsius for 10-14 days before conidia harvest. Following this, pipette 2-3 mL of 0.03%Tween 80 onto a cultured plate.
Then use an inoculation loop to scrape the conidia by. Next, transfer the fungal suspension to a centrifuge tube. Vigorously vortex the suspension at the highest speed, then pipette out a sample into a hemocytometer to count the number of conidia.
Dilute the conidia within the suspension using 0.03%Tween 80 til desired concentration is obtained. To prepare the inoculation chamber, first cut a 9 centimeter diameter leaf disk using the bottom Petri dish as a mold or with scissors. Next, prepare 1.5%water agar for each dish using a microwave.
Pour 30 mL of the cooled water agar into the Petri dish and let it solidify in a laminar flow hood. When the agar surface is semisolid, place the leaf disk with its abaxial surface facing up, then press it slightly to imbed it in the agar. Now, place 20 apterous adults on the leaf disk.
Open the cover of the inoculation chamber to spray 0.3 mL of conidia suspension directly onto the aphids and the leaf disk. Once the suspension has dried, close the cover to prevent the drowning of the mustard aphids. Subsequently, seal the inoculation chamber using paraffin film to maintain a high mount of humidity.
Place the chamber in an incubator set at a temperature of 25 degrees Celsius with a light and dark photoperiod of 12 hours each. Use a stereomicroscope to count the mortality every 12 hours for 5 days. The virulence screening revealed a consistent survival rate for mustard aphids with the control group exhibiting an 85%survival rate.
Cordyceps cateninnulata demonstrated the fastest aphid-killing ability, resulting in 50%and 90%moralities at 3 days and 4.5 days post-inoculation, or DPI. Beauveria strains 141, 143, and 153 showed slow aphid-killing abilities with only 5%mortality at 3 DPI. Apart from Purpureocillium, most EPF isolates exhibited corrected mortality rates higher than 70%within 5 DPI.
Beauveria bassiana demonstrated the highest mortality rate of 100%among EPF isolates. EPF mycosis was observed on cadavers of mustard aphids infected with Metarhizium species, Beauveria species, Purpureocillium lilacinum, and Cordyceps cateniannulata during the virulence screening.
