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

Summary

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.

Abstract

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.

Introduction

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 resistance^2,3. 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 field^6,7. 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 damage^8,9. 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 aphids^10,11,12.

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 container^10,11,13. Common methods for fungal inoculation include conidia spraying, aphid immersion into a conidia suspension, leaf dipping into a conidia suspension, and plant endophyte inoculation^11,14,15,16. While various inoculation methods exist, the bioassays should simulate field application conditions. For example, in the case of the leaf dipped method^12,17, 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 water^18,19. 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.

Protocol

NOTE: The complete flowchart is shown in Figure 1.

1. Mustard aphid collection and maintenance

1. Collection of mustard aphids
   1. Flip the leaves and visually check for infestation of mustard aphids on cruciferous crops in the field.
   2. Record the sampling site information (i.e., GPS) and host plant(s), and confirm the history of insecticide applications with the farmers.
   3. Use an insect aspirator or a fine painting brush (see Table of Materials) to collect about 50 mustard aphids into a 50 mL centrifuge tube from cruciferous crops in the field and bring the sample to the laboratory within 3 h.
   4. Prepare a temporary maintenance Petri dish with excised cruciferous leaves and water-infiltrated filter paper at the bottom.
   5. Place the five aphids on the temporary maintenance Petri dish under room temperature (25 ± 2 °C) with a relative humidity of 70% and 12:12 h (light:dark) photoperiod for 14 days to confirm that aphid spare natural enemy-free before further rearing.
      NOTE: To ensure that field-collected aphids are free from natural enemies such as parasitoid wasps or entomopathogenic fungi, it is crucial to confirm the absence of these organisms before proceeding with further rearing.
2. Maintainance of mustard aphids
   NOTE: To maintain mustard aphids, use pesticide-free (including biopesticide) cruciferous leaves.
   1. Provide a stable and sufficient supply of cruciferous leaves (about 25 cm²).
      NOTE: Obtain the cruciferous leaves either from the market or grow the plants. Before long-term, massive rearing of mustard aphids, several different kinds of crucifer could be tested to find a suitable crucifer. Once the species of the cruciferous leaf is fixed, do not change it. In this study, Komatsuna (Brassica rapa var. perviridis) at 6-7-leaf stage was used (see Table of Materials). Additionally, dispose of the 2-3 oldest leaves.
   2. Add water before the filter paper at the bottom is completely dry.
   3. Observe the population density of the mustard aphids daily. The population should grow to 2-3-fold after 7 days.
   4. When the number of aphids increases, cut the originally excised leaves with mustard aphids into 4-6 smaller pieces and put each small leaf piece with mustard aphids into one Petri dish with fresh excised leaf and water-infiltrated filter paper.
   5. Place the Petri dish in an incubator at 25 °C with a 12:12 h (light:dark) photoperiod and observe the population density of mustard aphids daily.
   6. Remove the original excised leaves after most of the aphids migrate to fresh excised leaves before the original leaves rot.

2. Molecular identification of mustard aphid

NOTE: To confirm the species of field-collected mustard aphid, molecular identification was performed using two molecular markers: the sequence characterized amplified region (SCAR) based A05Le designed by Lu et al.²¹, and mustard aphid cytochrome oxidase subunit 1 (COI) region of the mustard aphid.

1. Extraction of mustard aphid genomic DNA
   1. Collect approximately 50 aphids in a 1.5 mL centrifuge tube.
      NOTE: Aphids used for molecular identification should be descendants of one single aphid, and various stages could also be pooled in the same tube for DNA extraction.
   2. Homogenize the aphids with a pellet pestle and extract the DNA using Gene-Spin Genomic DNA Isolation Kit following the manufacturer's instructions (see Table of Materials).
   3. Elute the genomic DNA with 50 µL of preheated nuclease-free water.
2. PCR amplification and DNA sequencing
   1. Amplify the target DNA sequences from aphid genomic DNA using PCR Master Mix (2x) with primer pairs A05LeF/A05LeR and LeCO1F/LeCO1R (Table 1) with different PCR programs²¹.
      NOTE: The PCR reaction with a total volume of 20 µL is composed of 2 µL aphid genomic DNA template, 1 µL forward and 1 µL reversed primers, 10 µL PCR Master Mix (see Table of Materials), and 6 µL ddH₂O.
   2. Perform the PCR in a thermal cycler (see Table of Materials) with the following program: 94 °C for 5 min, 25 cycles of 94 °C for 45 s, 61 °C (for A05LeF/A05LeR) and 58 °C (for LeCO1F/LeCO1R) for 45 s, 72 °C for 1 min, followed by a final extension of 72 °C for 5 min.
   3. Analyze the PCR product by 1% agarose gel electrophoresis to confirm the identity of the mustard aphid (Figure 2).
      ​NOTE: If primer pair A05LeF/A05LeR successfully amplifies the correct size of the DNA fragment, it has convincingly identified the field-collected aphid as mustard aphid²¹. The primer pairs are listed in Table 2.
   4. Purify the PCR product of mustard aphid COI amplicon using a commercially available gel/PCR kit following the manufacturer's instructions (see Table of Materials), and sequence the PCR product using a commercial sequencing service.
3. Sequence analysis
   NOTE: According to Lu et al.²¹, the DNA bending of A05Le is a mustard aphid-specific DNA fragment; therefore, the A05Le PCR product does not need to be further sequenced.
   1. Check the read quality by Chromas software (see Table of Materials) after obtaining the LeCO1 sequence.
   2. Trim upstream and downstream low-quality reads by opening a fasta (or txt) file and directly removing the low-quality reads.
   3. Submit the trimmed sequence to NCBI web BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using default parameters. 
      ​NOTE: If the amplicon sizes of A05Le and LeCO1 primer sets are correct, theoretically, the sequence blast search against NCBI standard databases must match mustard aphid.

3. Preparation of Entomopathogenic fungi

NOTE: The EPF used in this study is listed in Table 1.

1. Recovery of EPF from fungal library
   1. Thaw the preserved fungi stock (conidia suspended in 30% glycerin) from the -80 °C freezer.
   2. Transfer a small amount (approximately 10 µL) of the conidia suspension to 6 cm ¼ Sabouraud dextrose agar (SDA) plate (0.75 g Sabouraud dextrose broth with 1.5 g agar per 100 mL ddH₂O) and spread it evenly using a cell spreader in a laminar flow hood.
   3. Seal the Petri dish with paraffin film, and incubate it in darkness at 25 °C for 10-14 days.
   4. Re-culture the fungal isolates by streaking fungal mass on a ¼ SDA plate and incubating fungi in darkness at 25 °C for 10-14 days before harvesting the conidia²².
      NOTE: The fungal culture should be used approximately 10 days before inoculating the aphids. EPF culture that is more than 14 days old is not recommended for use in the virulence test.
2. Preparation of conidia suspension
   1. Scrape the conidia from the 10-14 days old fungal culture on ¼ SDA plate by inoculating loop after adding 2-3 mL 0.03% Tween 80 (see Table of Materials).
   2. Collect the fungal suspension into a centrifuge tube.
   3. Vortex the solution at the highest speed, count the number of conidia using a hemocytometer under a light microscope, and adjust the concentration of the conidia suspension to 10⁸ conidia/mL.
   4. Transfer the conidia suspension into a UV-sterilized micro-sprayer.
      ​NOTE: Vortex the conidia suspension again before transferring it, as the conidia are prone to precipitate. The micro-sprayer should be exposed to ultraviolet radiation in a laminar flow hood for 30 min before use.

4. Virulence screening against mustard aphid

1. Preparation of newly emerged adults
   1. Move apterous (without wing) and alate (winged) adults from the rearing Petri dish to freshly excised leaves to reproduce new progenies of the same age. Remove the adults after 24 h to avoid overcrowding and minimize the production of alated progenies^23,24. Only apterous adults will be used in further experiments.
   2. Rear progenies to the adult stage and remove the progenies that do not develop into the adult stage after 48 h (Figure 3) to prevent age variation between aphids for the experiment. Additionally, remove alated adults.
2. Preparation of inoculation chamber
   NOTE: It is recommended to prepare 9 cm diameter cruciferous leaves first, so that the leaf disc can be embedded into the water agar before solidification.
   1. Clean the cruciferous leaves with tap water, remove dirt and residue, and any other arthropod if present.
   2. Wipe away any excess water with a paper towel and check for any other arthropods on the surface of the cruciferous leaves using a stereomicroscope. Remove any arthropods if present.
   3. Cut a 9 cm diameter leaf disc either by directly pressing the bottom Petri dish on the leaf as a mold or by using scissors.
      NOTE: If the leaves are smaller than 9 cm in diameter, combine a couple of excised leaves.
   4. Prepare 1.5% water agar for each Petri dish by heating and dissolving 450 mg of agar (see Table of Materials) in 30 mL ddH₂O using microwave.
      NOTE: The amount of 1.5% water agar can be adjusted depending on the experiment scale.
   5. Pour 30 mL of 1.5% water agar in the 9 cm Petri dish before solidification in the laminar flow hood, and then wait for the agar to cool down to ~40 °C until the surface of the agar is semi-solidified.
      NOTE: Check if the surface is semi-solidified by gently horizontally shaking the Petri dish.
   6. Place the leaf disc, abaxial surface up, on top of the water agar, embedding the leaf disc in the agar.
      NOTE: Minimize the exposure of the agar surface.
   7. After the water agar has solidified completely, close the cover of the Petri dish.
      NOTE: Open the cover for water vaporization if lots of droplets are condensing on the cover immediately.
   8. Place 20 apterous adults on the leaf disc.
      NOTE: It is recommended to operate under a stereomicroscope to prevent damage to their mouthparts.
3. EPF inoculation and observation
   1. Open the cover of the inoculation chamber and spray 0.3 mL of conidia suspension (from step 3.2) directly onto the aphids and leaf disc from about 15 cm above the leaf disc.
      NOTE: Vortex the conidia suspension again before spraying. The spraying areas should be tested before the experiment as they can vary between different sprayers. In this case, 0.3 mL with 15 cm distance is recommended. The spraying area should cover the entire leaf disc, and a thin layer of conidia suspension should cover the leaf disc. If droplets converge into standing water on the leaf, the inoculation volume should be decreased. Clean the table with 70% ethanol before inoculation and between using different isolates.
   2. Wait for the conidia suspension to dry, and then close the cover to prevent mustard aphids from drowning.
      NOTE: Aphids seldom roam around during the experiment; however, ensuring that the aphids do not escape is still necessary.
   3. Seal the inoculation chamber with paraffin film to maintain high relative humidity and place the inoculation chamber in an incubator at 25 °C with 12:12 h (light:dark) photoperiod.
   4. Open the cover of the inoculation chamber to count mortality under a stereomicroscope every 12 h for 5 days.
   5. Wipe out the honeydew adhering to the cover with a paper towel and remove the newly emerged aphids with a fine painting brush. Clean the cover with tap water every 24 h.
      NOTE: The observation period might vary for different species of aphids based on adult longevity.
   6. Keep the cadaver on the leaf disc for mycosis to confirm successful inoculation.
4. Confirmation of conidia germination rate
   NOTE: This step is optional. To confirm the conidia germination rate pairwise when performing the virulence screening to ensure the activity of conidia.
   1. Drop one droplet of 5 µL conidia suspension on ¼ SDA for three replicates.
   2. After 18 h, calculate the germination rate with the light microscope. Count at least 100 spores randomly in a single droplet to confirm the fungal germination rate.
      ​NOTE: Normally, the germination rate of the isolates used in the experiment should be higher than 90%.

5. Bioassay of selected EPF isolates

NOTE: EPF isolates that showed high virulence, which was selected from step 4, were subjected to a bioassay against mustard aphids using four concentrations of conidia suspensions (ranging from 10⁴ to 10⁷ conidia/mL).

1. Preparation of conidia suspension
   1. Repeat step 3.1 to recover the selected EPF isolates.
   2. Repeat step 3.2 to prepare four concentrations of conidia suspension, including 10⁴, 10⁵,10^6, and 10⁷ conidia/mL.
2. Preparation of newly emerged adults
   1. Repeat step 4.1 to prepare newly emerged adults.
3. Preparation of inoculation chamber
   1. Repeat step 4.1. to prepare the inoculation chamber.
4. EPF inoculation and observation
   1. Repeat step 4.3. to perform the EPF inoculation and observation with the four concentrations of conidia suspension, respectively.

6. Statistical analysis

1. Calculation of corrected mortality
   1. Calculate corrected mortality using Abbott's formula²⁵, as shown below:
      Corrected mortality (%) = [(M_T− M_C) × 100%] / (100− M_C)
      M_T represents the mortality of the treatment group, and M_C represents the mortality of the control group.
      ​NOTE: The mortality of the control group should not be higher than 20%.
   2. Open the SPSS software platform (see Table of Materials), create variables including "treatment" and "cor_mor" (representing corrected mortality), and enter the calculated results for the inoculation of different isolates at the same time point with the same inoculated concentration.
   3. Select Analyze, Compare Means and Proportions, Independent-Samples T Test in SPSS for independent t-test analysis.
   4. In SPSS, input treatment into the "Grouping Variable" box, and cor_mor into the "Test Variable(s)". Define groups by different fungal isolates. Press OK to analyze.
2. Calculation of median lethal time (LT₅₀) and median lethal concentration (LC₅₀)
   1. Open the SPSS, create variables "total", "response" and "duration"/"concentration", and input the recorded result into the spreadsheet.
   2. Select Analyze, Regression, Probit in SPSS to calculate LT₅₀ and/or LC₅₀.
      NOTE: If cumulative mortality does not exceed 50% eventually, LT₅₀ and/or LC₅₀ cannot be estimated.
   3. Input total into the "Total Observed" box, response into "Response Frequency" box, duration/concentration into "Covariate(s)" box. Press OK to analyze.
      NOTE: Generally, press Options and set the "Significance level for use of heterogeneity factor" to 0.05.

Results

The presented flowchart illustrates the stable condition of the mustard aphids from field collection to virulence screening. The maintenance of aphids from field collection ensured a stable increase in aphid colonies with an adequate food supply. The field-collected aphids were confirmed as mustard aphids through the use of molecular markers, including PCR amplicon size and LeCO1 sequencing. The virulence screening, conducted using the detached-leaf method, revealed a consistent survival rate for mustard aphids, with the...

Discussion

Crucifers, a group of vegetables, are frequently infested by multiple aphid species, including mustard aphid (L. erysimi) and cabbage aphid (Brevicoryne brassicae)²⁶. Both species have been reported in Taiwan²⁷, and it is possible for them to coexist at the collection site. To distinguish closely related aphid species, this study employed a molecular identification technique using a multiplex primer set²¹. By designing a molecular m...

Materials

Name	Company	Catalog Number	Comments
10 μL Inoculating Loop	NEST Scientific	718201
100 bp DNA Ladder III	Geneaid	DL007
2x SuperRed PCR Master Mix	Biotools	TE-SR01
50 mL centrifuge tube	Bioman Scientific	ET5050-12
6 cm Petri dish	Alpha Plus Scientific	16021
6 mm insect aspirator	MegaView Science	BA6001
70 mm filter paper NO.1	Toyo Roshi Kaisha
70% ethanol
9 cm Petri dish	Alpha Plus Scientific	16001
Agar	Bioman Scientific	AGR001.1	Microbiology grade
Agarose	Bioman Scientific	PB1200
BioGreen Safe DNA Gel Buffer	Bioman Scientific	SDB001T
Chromas	Technelysium
GeneDoc
GenepHlow Gel/PCR Kit	Geneaid	DFH300	https://www.geneaid.com/data/files/1605861013102532959.pdf
Gene-Spin Genomic DNA Isolation Kit	Protech Technology	PT-GD112-V3	http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf
Hemocytometer	Paul Marienfeld	640030
Komatsuna leaves (Brassica rapa var. perviridis)	Tai Cheng Farm	1-010-300410
Microsprayer
MiniAmp Thermal Cycler	Thermo Fisher Scientific	A37834
Mustard aphid (Lipaphis erysimi)
Painting brush	Tian Cheng brush company	4716608400352
Parafilm M	Bemis	PM-996
Pellet pestle	Bioman Scientific	GT100R
Sabouraud Dextrose Broth	HiMedia	MH033-500G
SPSS Statistics	IBM
TAE buffer 50x	Bioman Scientific	TAE501000
Tween 80	PanReac AppliChem	142050.1661