Name: Entomopathogenic Fungi (EPF) Preparation and Virulence Screening Against Mustard Aphids
Uploaded: 2023-07-21T14:20:00
Description: Watch this Scientific Journal Video about Entomopathogenic Fungi EPF Preparation and Virulence Screening Against Mustard Aphids at JoVE.com

entomopathogenic fungi (epf) preparation and virulence screening against mustard aphids

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

The mustard aphid (L. erysimi) is a notorious pest that infests a variety of cruciferous crops, causing significant economic losses1. While several systematic insecticides have been recommended to combat aphid infestations, the frequent use of these insecticides raises concerns about pesticide resistance2,3. Therefore, in terms of eco-friendly pest management, entomopathogenic fungi (EPF) could serve as a suitable alternative control strategy. EPF is an insect pathogen with the ability to infect hosts by penetrating their cuticles, making it a potent agent for controlling aphids and other plant-sucking insects4. Furthermore, EPF has proven to be a feasible and sustainable pest management technique, offering benefits such as plant pathogen antagonism and plant growth promotion5.
EPF can be obtained through insect-soil baiting or isolated from insect cadavers in the field6,7. However, before further use of fungal isolates, pathogenicity screening is necessary. Several studies have been conducted on the effectiveness of EPF against aphids, which are significant crop pests that can cause severe damage8,9. Mustard aphids, among various species of aphids, have been tested for susceptibility to several strains of Beauveria spp., Metarhizium spp., Lecanicillium spp., Paecilomyces spp., and even Alternaria, which is primarily known as a saprophytic and plant pathogenic fungus but has shown some lethal effects against mustard aphids10,11,12.
To evaluate the effectiveness of EPF against aphids under laboratory conditions, bioassays can be divided into two main parts: the inoculation chamber and fungal inoculation. The current protocol describes the construction of an inoculation chamber, where aphids can be maintained using various methods such as an excised leaf with a petiole wrapped in moist cotton, an excised leaf disc with a Petri dish lined with damped filter paper, direct maintenance on pot plants, or an excised leaf disc embedded in water agar within a Petri dish or container10,11,13. Common methods for fungal inoculation include conidia spraying, aphid immersion into a conidia suspension, leaf dipping into a conidia suspension, and plant endophyte inoculation11,14,15,16. While various inoculation methods exist, the bioassays should simulate field application conditions. For example, in the case of the leaf dipped method12,17, the efficiency of EPF can be evaluated, but since the aphids infest the fungus-loaded leaves, the dorsal side of the aphid, which is a preferential penetration site, does not usually get exposed to the fungus.
To evaluate the aphidicidal effect of EPF under laboratory conditions, this protocol suggests using the detached-leaf method described by Yokomi and Gottwald18 with some modifications, followed by conidia inoculation using a micro-sprayer. This method maintains approximately 100% humidity in the bioassay chamber for at least seven days without requiring additional replenishment of water18,19. Additionally, confining aphids to one surface ensures their exposure to conidia spraying and facilitates observations20. However, aphids may become stuck in the exposed agar surface while moving within the inoculation chamber. Furthermore, recording data in the Petri dish experiment with mustard aphids, which are parthenogenetic insects, can be challenging due to their rapid development and reproduction. It is difficult to distinguish between inoculated adults and their progeny without removal. The details of how to proceed with this step are seldom mentioned, and some inconsistent factors, such as leaf consumption area, need to be optimized.
This protocol demonstrates a stable system for screening the virulence of EPF isolates against mustard aphids, enabling the selection of potential isolates against various aphid species from an extensive EPF library. Field-collected aphids can be identified, and a sufficient laboratory population of mustard aphids can be established to evaluate the aphidicidal effect of various fungal isolates using an easy and feasible methodology with consistent outcomes. Aphids have developed multiple evolutionary mechanisms in response to intense and repeated anthropogenic pressures in agroecosystems, posing challenges to food security9. Therefore, this described method could be extended to evaluate potential EPF isolates against various aphid species.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

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

Watch this Scientific Journal Video about Entomopathogenic Fungi EPF Preparation and Virulence Screening Against Mustard Aphids at JoVE.com

Entomopathogenic Fungi (EPF) Preparation and Virulence Screening Against Mustard Aphids

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.

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Research

JoVE Journal

Biology

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

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.

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...

Collection, Maintenance, and Molecular Identification of Mustard Aphids

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 Gene-Spin 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.

Aphicidal Assessment of Entomopathogenic Fungi by the Statistical Analysis of Corrected Mortality in Mustard Aphids

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 OK 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 Probit 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.