Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

Today, eco-friendly pest management is widely preferred. Prior to implementation, however, the entomophathogenic fungi must undergo thorough testing. Our study showcases the effectiveness of an entomophathogenic fungal library in combating the mustard aphid, serving us as a prime example of its potential.

Reduction of the high mortality rate of the entomophathogenic fungi pre-treatment and the prevention of path storming in the store suspension is challenging. We have successfully developed a stable system that yielded the the expected result. This involved the use of a proper system, or bioassay chamber, to effectively maintain this tiny path during the testing process.

This protocol successfully demonstrates a feasible way to set up the detachment method without the need of measuring agar temperature, thereby simplifying the establishment of the experimental chamber. In addition, we proposed the importance of minimizing the exposure of the agar surface to prevent the aphids from getting stuck while roaming inside the chamber. We are intrigued by the immune response of the mustard aphid when confronted with entomophathogenic fungal infection prior to aphid mortality.

By studying gene expression data, we can identify critical genes that could serve as potential targets for gene knockdown in combination of entomophathogenic fungal application. Start by preparing a temporary maintenance Petri dish with excised cruciferous leaves and water-infiltrated filter paper at the bottom. Next, place five aphids on the prepared Petri dish.

When the aphids increase in number, cut the originally excised leaves into four to six smaller pieces. Transfer each small leaf piece with the aphids into separate Petri dishes containing fresh leaves and wet filter paper. To collect the aphid genomic DNA, first, homogenize the aphids using a pellet pestle.

Next, extract the DNA using a GENESpin genomic DNA isolation kit as per the manufacturer's guidelines. Finish the extraction process by eluting the genomic DNA with 50 microliters of preheated nuclease-free water. Perform PCR amplification and DNA sequencing by adding the DNA sample to the PCR master mix with primer pairs.

Analyze the PCR product using 1%agarose gel electrophoresis to confirm the identity of the mustard aphid. The field-collected aphids were confirmed as mustard aphids using molecular markers, including the PCR amplicon size and Lipaphis erysimi CO1 sequencing. 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 to 14 days before conidia harvest. Following this, pipette two to three milliliters 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 till desired concentration is obtained. To prepare the inoculation chamber, first, cut a nine centimeter diameter leaf disc 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 milliliters of the cooled water agar into the Petri dish and let it solidify in a laminar flow hood. When the agar surface is semi-solid, place the leaf disc with its abaxial surface facing up, then press it slightly to embed it in the agar.

Now, place 20 apterous adults on the leaf disc. Open the cover of the inoculation chamber to spray 0.3 milliliters of conidia suspension directly onto the aphids and the leaf disc. 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 amount of humidity, Place the chamber in an incubator set at a temperature of 25 degrees Celsius, with a light and dark photo period of 12 hours each. Use a stereo microscope to count the mortality every 12 hours for five days. The virulent screening revealed a consistent survival rate for mustard aphids, with the control group exhibiting an 85%survival rate.

Cordyceps cateniannulata demonstrated the fastest aphid-killing ability, resulting in 50%and 90%mortality at three 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 five 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. Begin by launching the SPSS software platform.

Create variables termed treatment and corrected mortality, then input the calculated results for different isolates inoculated at the same time point with the same concentration. Navigate to analyze, compare means and proportions, then independent samples T-test in SPSS for independent T-test analysis. Next, input treatment into the grouping variable box and corrected mortality into the test variables box.

Classify groups according to different fungal isolates and press okay to begin the analysis. To calculate the median lethal time and median lethal concentration, create the variables defined as total, response, and either duration or concentration. Next, enter the recorded result into the spreadsheet.

Finally, choose analyze, followed by regression, and then probe it to compute the median lethal time and median lethal concentration. Inoculations of 10 to the seven conidia per milliliter resulted in significantly different corrected mortalities for Metarhizium strain 197 and Cordyceps strain 213 at three and four DPI. The Cordyceps strain 213 exhibited a significantly shorter lethal duration compared to other treatments.

It also showed a lower lethal dose value relative to the Metarhizium 213 strain.