Assessment of Aphidicidal Effect of Entomopathogenic

Fungi against Parthenogenetic Insect, Mustard Aphid,

Lipaphis erysimi (Kalt.)
Cheng-Ju Yang1, Yu-Shin Nai1
1
Department of Entomology, National Chung Hsing University

Corresponding Author
Abstract
Yu-Shin Nai
[email protected]
The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and
transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic
Citation
fungi (EPF) are potential microbial control agents for controlling this pest. Therefore,
Yang, C.J., Nai, Y.S. Assessment of virulence screening of EPF isolates under Petri dish conditions is necessary before
Aphidicidal Effect of Entomopathogenic
Fungi against Parthenogenetic Insect, field application. However, the mustard aphid is a parthenogenetic insect, making
Mustard Aphid, Lipaphis erysimi
it difficult to record data during Petri dish experiments. A modified system for
(Kalt.). J. Vis. Exp. (197), e65312,
doi:10.3791/65312 (2023). 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
Date Published
suspension. The system maintained high relative humidity throughout the observation
July 21, 2023 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
DOI
using a painting brush was implemented. This protocol demonstrates a stable system
10.3791/65312
for evaluating the virulence of EPF isolates against mustard aphids or other aphids,

URL enabling the selection of potential isolates for aphid control.

jove.com/video/65312

Introduction

The mustard aphid (L. erysimi) is a notorious pest that infests cuticles, making it a potent agent for controlling aphids and
a variety of cruciferous crops, causing significant economic other plant-sucking insects4 . Furthermore, EPF has proven
losses1 . While several systematic insecticides have been to be a feasible and sustainable pest management technique,
recommended to combat aphid infestations, the frequent offering benefits such as plant pathogen antagonism and
use of these insecticides raises concerns about pesticide plant growth promotion5 .
resistance2 , 3 . Therefore, in terms of eco-friendly pest
EPF can be obtained through insect-soil baiting or isolated
management, entomopathogenic fungi (EPF) could serve
from insect cadavers in the field6 , 7 . However, before further
as a suitable alternative control strategy. EPF is an insect
use of fungal isolates, pathogenicity screening is necessary.
pathogen with the ability to infect hosts by penetrating their

Copyright © 2023 JoVE Journal of Visualized Experiments jove.com July 2023 • 197 • e65312 • Page 1 of 20
 Several studies have been conducted on the effectiveness humidity in the bioassay chamber for at least seven days
of EPF against aphids, which are significant crop pests without requiring additional replenishment of water18 , 19 .
that can cause severe damage8 , 9 . Mustard aphids, among Additionally, confining aphids to one surface ensures their
various species of aphids, have been tested for susceptibility exposure to conidia spraying and facilitates observations20 .
to several strains of Beauveria spp., Metarhizium spp., However, aphids may become stuck in the exposed
Lecanicillium spp., Paecilomyces spp., and even Alternaria, agar surface while moving within the inoculation chamber.
which is primarily known as a saprophytic and plant Furthermore, recording data in the Petri dish experiment with
pathogenic fungus but has shown some lethal effects against mustard aphids, which are parthenogenetic insects, can be
mustard aphids10 , 11 , 12 . challenging due to their rapid development and reproduction.
It is difficult to distinguish between inoculated adults and
To evaluate the effectiveness of EPF against aphids under
their progeny without removal. The details of how to proceed
laboratory conditions, bioassays can be divided into two main
with this step are seldom mentioned, and some inconsistent
parts: the inoculation chamber and fungal inoculation. The
factors, such as leaf consumption area, need to be optimized.
current protocol describes the construction of an inoculation
chamber, where aphids can be maintained using various This protocol demonstrates a stable system for screening the
methods such as an excised leaf with a petiole wrapped virulence of EPF isolates against mustard aphids, enabling
in moist cotton, an excised leaf disc with a Petri dish lined the selection of potential isolates against various aphid
with damped filter paper, direct maintenance on pot plants, species from an extensive EPF library. Field-collected aphids
or an excised leaf disc embedded in water agar within a can be identified, and a sufficient laboratory population
Petri dish or container10 , 11 , 13 . Common methods for fungal of mustard aphids can be established to evaluate the
inoculation include conidia spraying, aphid immersion into a aphidicidal effect of various fungal isolates using an
conidia suspension, leaf dipping into a conidia suspension, easy and feasible methodology with consistent outcomes.
and plant endophyte inoculation11 , 14 , 15 , 16 . While various Aphids have developed multiple evolutionary mechanisms in
inoculation methods exist, the bioassays should simulate field response to intense and repeated anthropogenic pressures
application conditions. For example, in the case of the leaf in agroecosystems, posing challenges to food security9 .
dipped method12 , 17 , the efficiency of EPF can be evaluated, Therefore, this described method could be extended to
but since the aphids infest the fungus-loaded leaves, the evaluate potential EPF isolates against various aphid
dorsal side of the aphid, which is a preferential penetration species.
site, does not usually get exposed to the fungus.
Protocol
To evaluate the aphidicidal effect of EPF under laboratory
NOTE: The complete flowchart is shown in Figure 1.
conditions, this protocol suggests using the detached-
leaf method described by Yokomi and Gottwald18 with
1. Mustard aphid collection and maintenance
some modifications, followed by conidia inoculation using a
micro-sprayer. This method maintains approximately 100% 1. Collection of mustard aphids

Copyright © 2023 JoVE Journal of Visualized Experiments jove.com July 2023 • 197 • e65312 • Page 2 of 20
 fixed, do not change it. In this study, Komatsuna
1. Flip the leaves and visually check for infestation of
(Brassica rapa var. perviridis) at 6-7-leaf stage was
mustard aphids on cruciferous crops in the field.
used (see Table of Materials). Additionally, dispose
2. Record the sampling site information (i.e., GPS) and
of the 2-3 oldest leaves.
host plant(s), and confirm the history of insecticide
2. Add water before the filter paper at the bottom is
applications with the farmers.
completely dry.
3. Use an insect aspirator or a fine painting brush (see
3. Observe the population density of the mustard
Table of Materials) to collect about 50 mustard
aphids daily. The population should grow to 2-3-fold
aphids into a 50 mL centrifuge tube from cruciferous
after 7 days.
crops in the field and bring the sample to the
laboratory within 3 h. 4. When the number of aphids increases, cut the
originally excised leaves with mustard aphids into
4. Prepare a temporary maintenance Petri dish with
4-6 smaller pieces and put each small leaf piece with
excised cruciferous leaves and water-infiltrated filter
mustard aphids into one Petri dish with fresh excised
paper at the bottom.
leaf and water-infiltrated filter paper.
5. Place the five aphids on the temporary maintenance
5. Place the Petri dish in an incubator at 25 °C with
Petri dish under room temperature (25 ± 2 °C) with
a 12:12 h (light:dark) photoperiod and observe the
a relative humidity of 70% and 12:12 h (light:dark)
population density of mustard aphids daily.
photoperiod for 14 days to confirm that aphid spare
natural enemy-free before further rearing. 6. Remove the original excised leaves after most of the
NOTE: To ensure that field-collected aphids are free aphids migrate to fresh excised leaves before the
from natural enemies such as parasitoid wasps or original leaves rot.
entomopathogenic fungi, it is crucial to confirm the
2. Molecular identification of mustard aphid
absence of these organisms before proceeding with
further rearing. NOTE: To confirm the species of field-collected mustard
aphid, molecular identification was performed using two
2. Maintainance of mustard aphids
molecular markers: the sequence characterized amplified
NOTE: To maintain mustard aphids, use pesticide-free
region (SCAR) based A05Le designed by Lu et al.21 , and
(including biopesticide) cruciferous leaves.
mustard aphid cytochrome oxidase subunit 1 (COI) region of
1. Provide a stable and sufficient supply of cruciferous
the mustard aphid.
leaves (about 25 cm2 ).
NOTE: Obtain the cruciferous leaves either from 1. Extraction of mustard aphid genomic DNA
the market or grow the plants. Before long-term,
1. Collect approximately 50 aphids in a 1.5 mL
massive rearing of mustard aphids, several different
centrifuge tube.
kinds of crucifer could be tested to find a suitable
crucifer. Once the species of the cruciferous leaf is

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 NOTE: Aphids used for molecular identification as mustard aphid21 . The primer pairs are listed in
should be descendants of one single aphid, and Table 2.
various stages could also be pooled in the same tube
4. Purify the PCR product of mustard aphid COI
for DNA extraction.
amplicon using a commercially available gel/PCR kit
2. Homogenize the aphids with a pellet pestle and following the manufacturer's instructions (see Table
extract the DNA using Gene-Spin Genomic DNA of Materials), and sequence the PCR product using
Isolation Kit following the manufacturer's instructions a commercial sequencing service.
(see Table of Materials).
3. Sequence analysis
3. Elute the genomic DNA with 50 µL of preheated
NOTE: According to Lu et al.21 , the DNA bending
nuclease-free water.
of A05Le is a mustard aphid-specific DNA fragment;
2. PCR amplification and DNA sequencing therefore, the A05Le PCR product does not need to be

1. Amplify the target DNA sequences from aphid further sequenced.

genomic DNA using PCR Master Mix (2x) with 1. Check the read quality by Chromas software (see
primer pairs A05LeF/A05LeR and LeCO1F/LeCO1R Table of Materials) after obtaining the LeCO1

(Table 1) with different PCR programs21 . sequence.

NOTE: The PCR reaction with a total volume of 2. Trim upstream and downstream low-quality reads by
20 µL is composed of 2 µL aphid genomic DNA opening a fasta (or txt) file and directly removing the
template, 1 µL forward and 1 µL reversed primers, low-quality reads.
10 µL PCR Master Mix (see Table of Materials), and
3. Submit the trimmed sequence to NCBI
6 µL ddH2O.
web BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
2. Perform the PCR in a thermal cycler (see Table of using default parameters.
Materials) with the following program: 94 °C for 5 NOTE: If the amplicon sizes of A05Le and LeCO1
min, 25 cycles of 94 °C for 45 s, 61 °C (for A05LeF/ primer sets are correct, theoretically, the sequence
A05LeR) and 58 °C (for LeCO1F/LeCO1R) for 45 s, blast search against NCBI standard databases must
72 °C for 1 min, followed by a final extension of 72 match mustard aphid.
°C for 5 min.
3. Preparation of Entomopathogenic fungi
3. Analyze the PCR product by 1% agarose gel
electrophoresis to confirm the identity of the mustard NOTE: The EPF used in this study is listed in Table 1.

aphid (Figure 2).

1. Recovery of EPF from fungal library
NOTE: If primer pair A05LeF/A05LeR successfully
amplifies the correct size of the DNA fragment, it 1. Thaw the preserved fungi stock (conidia suspended

has convincingly identified the field-collected aphid in 30% glycerin) from the -80 °C freezer.

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 radiation in a laminar flow hood for 30 min before
2. Transfer a small amount (approximately 10 µL)
use.
of the conidia suspension to 6 cm ¼ Sabouraud
dextrose agar (SDA) plate (0.75 g Sabouraud
4. Virulence screening against mustard aphid
dextrose broth with 1.5 g agar per 100 mL ddH2O)

and spread it evenly using a cell spreader in a 1. Preparation of newly emerged adults

laminar flow hood. 1. Move apterous (without wing) and alate (winged)

3. Seal the Petri dish with paraffin film, and incubate it adults from the rearing Petri dish to freshly excised

in darkness at 25 °C for 10-14 days. leaves to reproduce new progenies of the same
age. Remove the adults after 24 h to avoid
4. Re-culture the fungal isolates by streaking fungal
overcrowding and minimize the production of alated
mass on a ¼ SDA plate and incubating fungi in
darkness at 25 °C for 10-14 days before harvesting progenies23 , 24 . Only apterous adults will be used in
further experiments.
the conidia22 .
NOTE: The fungal culture should be used 2. Rear progenies to the adult stage and remove

approximately 10 days before inoculating the the progenies that do not develop into the adult

aphids. EPF culture that is more than 14 days old is stage after 48 h (Figure 3) to prevent age variation

not recommended for use in the virulence test. between aphids for the experiment. Additionally,
remove alated adults.
2. Preparation of conidia suspension
2. Preparation of inoculation chamber
1. Scrape the conidia from the 10-14 days old fungal
NOTE: It is recommended to prepare 9 cm diameter
culture on ¼ SDA plate by inoculating loop after
cruciferous leaves first, so that the leaf disc can be
adding 2-3 mL 0.03% Tween 80 (see Table of
embedded into the water agar before solidification.
Materials).
1. Clean the cruciferous leaves with tap water, remove
2. Collect the fungal suspension into a centrifuge tube.
dirt and residue, and any other arthropod if present.
3. Vortex the solution at the highest speed, count the
2. Wipe away any excess water with a paper towel
number of conidia using a hemocytometer under a
and check for any other arthropods on the surface
light microscope, and adjust the concentration of the
of the cruciferous leaves using a stereomicroscope.
conidia suspension to 108 conidia/mL.
Remove any arthropods if present.
4. Transfer the conidia suspension into a UV-sterilized
3. Cut a 9 cm diameter leaf disc either by directly
micro-sprayer.
pressing the bottom Petri dish on the leaf as a mold
NOTE: Vortex the conidia suspension again before
or by using scissors.
transferring it, as the conidia are prone to precipitate.
NOTE: If the leaves are smaller than 9 cm in
The micro-sprayer should be exposed to ultraviolet
diameter, combine a couple of excised leaves.

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 should cover the entire leaf disc, and a thin layer
4. Prepare 1.5% water agar for each Petri dish by
of conidia suspension should cover the leaf disc. If
heating and dissolving 450 mg of agar (see Table of
droplets converge into standing water on the leaf,
Materials) in 30 mL ddH2O using microwave.
the inoculation volume should be decreased. Clean
NOTE: The amount of 1.5% water agar can be
the table with 70% ethanol before inoculation and
adjusted depending on the experiment scale.
between using different isolates.
5. Pour 30 mL of 1.5% water agar in the 9 cm Petri
2. Wait for the conidia suspension to dry, and then
dish before solidification in the laminar flow hood,
close the cover to prevent mustard aphids from
and then wait for the agar to cool down to ~40 °C
drowning.
until the surface of the agar is semi-solidified.
NOTE: Aphids seldom roam around during the
NOTE: Check if the surface is semi-solidified by
experiment; however, ensuring that the aphids do
gently horizontally shaking the Petri dish.
not escape is still necessary.
6. Place the leaf disc, abaxial surface up, on top of the
3. Seal the inoculation chamber with paraffin film
water agar, embedding the leaf disc in the agar.
to maintain high relative humidity and place the
NOTE: Minimize the exposure of the agar surface.
inoculation chamber in an incubator at 25 °C with
7. After the water agar has solidified completely, close
12:12 h (light:dark) photoperiod.
the cover of the Petri dish.
4. Open the cover of the inoculation chamber to count
NOTE: Open the cover for water vaporization
mortality under a stereomicroscope every 12 h for 5
if lots of droplets are condensing on the cover
days.
immediately.
5. Wipe out the honeydew adhering to the cover with a
8. Place 20 apterous adults on the leaf disc.
paper towel and remove the newly emerged aphids
NOTE: It is recommended to operate under a
with a fine painting brush. Clean the cover with tap
stereomicroscope to prevent damage to their
water every 24 h.
mouthparts.
NOTE: The observation period might vary for
3. EPF inoculation and observation
different species of aphids based on adult longevity.
1. Open the cover of the inoculation chamber and spray
6. Keep the cadaver on the leaf disc for mycosis to
0.3 mL of conidia suspension (from step 3.2) directly
confirm successful inoculation.
onto the aphids and leaf disc from about 15 cm
4. Confirmation of conidia germination rate
above the leaf disc.
NOTE: This step is optional. To confirm the conidia
NOTE: Vortex the conidia suspension again before
germination rate pairwise when performing the virulence
spraying. The spraying areas should be tested
screening to ensure the activity of conidia.
before the experiment as they can vary between
different sprayers. In this case, 0.3 mL with 15 1. Drop one droplet of 5 µL conidia suspension on ¼

cm distance is recommended. The spraying area SDA for three replicates.

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 Corrected mortality (%) = [(MT− MC) × 100%] / (100−
2. After 18 h, calculate the germination rate with
the light microscope. Count at least 100 spores M C)

randomly in a single droplet to confirm the fungal MT represents the mortality of the treatment group,

germination rate. and MC represents the mortality of the control group.

NOTE: Normally, the germination rate of the isolates NOTE: The mortality of the control group should not
used in the experiment should be higher than 90%. be higher than 20%.

2. Open the SPSS software platform (see Table of

5. Bioassay of selected EPF isolates
Materials), create variables including "treatment"
NOTE: EPF isolates that showed high virulence, which
and "cor_mor" (representing corrected mortality),
was selected from step 4, were subjected to a bioassay
and enter the calculated results for the inoculation
against mustard aphids using four concentrations of conidia
of different isolates at the same time point with the
suspensions (ranging from 104 to 107 conidia/mL).
same inoculated concentration.

1. Preparation of conidia suspension 3. Select Analyze, Compare Means and

Proportions, Independent-Samples T Test in
1. Repeat step 3.1 to recover the selected EPF
SPSS for independent t-test analysis.
isolates.
4. In SPSS, input treatment into the "Grouping
2. Repeat step 3.2 to prepare four concentrations of
Variable" box, and cor_mor into the "Test
conidia suspension, including 104 , 105 ,106, and 107
Variable(s)". Define groups by different fungal
conidia/mL.
isolates. Press OK to analyze.
2. Preparation of newly emerged adults
2. Calculation of median lethal time (LT50) and median
1. Repeat step 4.1 to prepare newly emerged adults.
lethal concentration (LC50)
3. Preparation of inoculation chamber
1. Open the SPSS, create variables "total", "response"
1. Repeat step 4.1. to prepare the inoculation chamber. and "duration"/"concentration", and input the

4. EPF inoculation and observation recorded result into the spreadsheet.

1. Repeat step 4.3. to perform the EPF inoculation and 2. Select Analyze, Regression, Probit in SPSS to

observation with the four concentrations of conidia calculate LT50 and/or LC50.

suspension, respectively. NOTE: If cumulative mortality does not exceed 50%

eventually, LT50 and/or LC50 cannot be estimated.
6. Statistical analysis
3. Input total into the "Total Observed" box, response
1. Calculation of corrected mortality into "Response Frequency" box, duration/

1. Calculate corrected mortality using Abbott's concentration into "Covariate(s)" box. Press OK to
analyze.
formula25 , as shown below:

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 NOTE: Generally, press Options and set the formula was employed to normalize the control mortality. Most
"Significance level for use of heterogeneity factor" to EPF isolates against mustard aphids exhibited corrected
0.05. mortality rates higher than 70% within 5 d.p.i., except for
Pl-NCHU-152 (Table 3). Among these EPF isolates, Bb-
Representative Results NCHU-286 demonstrated the highest mortality rate of 100%

The presented flowchart illustrates the stable condition of the (Table 3). Additionally, EPF mycosis was observed on

mustard aphids from field collection to virulence screening. cadavers of mustard aphids infected with Metarhizium spp.,

The maintenance of aphids from field collection ensured Beauveria spp., Purpureocillium lilacinum, and Cordyceps

a stable increase in aphid colonies with an adequate cateniannulata during the virulence screening, indicating the

food supply. The field-collected aphids were confirmed as effectiveness of this system (Figure 5).

mustard aphids through the use of molecular markers,

Based on the results of the virulence screening, two
including PCR amplicon size and LeCO1 sequencing. The
EPF isolates, namely Mb-NCHU-197 and Cc-NCHU-213,
virulence screening, conducted using the detached-leaf
exhibiting rapid insect-killing activity (40% and 50% mortality
method, revealed a consistent survival rate for mustard
at 3 d.p.i., respectively), were selected for bioassay against
aphids, with the control group exhibiting an 85% survival rate
mustard aphids. The results demonstrated significantly
(Figure 4).
different corrected mortalities for Mb-NCHU-197 and Cc-

During the virulence screening, Cc-NCHU-213 demonstrated NCHU-213 at 3 and 4 d.p.i. with an inoculation of 107

the fastest aphid-killing ability, resulting in 50% and 90% conidia/mL (Figure 6). In the LT50 assay, the treatment with

mortalities at 3 days and 4.5 days post-inoculation (d.p.i.), 107 conidia/mL of Cc-NCHU-213 exhibited a significantly

respectively (Figure 4). However, varying aphid-killing shorter duration compared to other treatments (Table 4).

abilities were observed among the five B. bassiana isolates. Furthermore, the LC50 value of Cc-NCHU-213 (9.32 ×

Bb-NCHU-141, -143, and -153 exhibited slow aphid-killing 104) was lower than that of Mb-NCHU-213 (2.30 × 105 ),

abilities, with only 5% mortality at 3 d.p.i., even when indicating that Cc-NCHU-213 possesses greater virulence

excluding the effects of 0.03% Tween 80 spraying or other against mustard aphids (Table 5).

lethal factors (Figure 4). Hence, the corrected mortality

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 Figure 1: Experimental flowchart for screening EPF virulence against mustard aphids. (A) Establishment of a mustard
aphid-rearing system. (B) Preparation of EPF. (C) Fungal inoculation. (D) Statistical analysis. Please click here to view a
larger version of this figure.

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 Figure 2: Electrophoresis of mustard aphid genomic DNA amplified with A05Le and Le CO1 primer sets.
Electrophoresis was performed on a 1% agarose gel. M = 100 bp DNA ladder; bp = base pairs.The red asterisk indicates the
target bend of PCR amplification. Please click here to view a larger version of this figure.

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 Figure 3: Differences between apterous fourth-instar nymph and adult mustard aphids. (A) Fourth-instar nymph. (B)
Adult. The tibiae of the hind legs of the fourth-instar nymph are whitish (marked with red arrow). Newly emerged aphids were
removed by fine camel brush during virulence tests. Scale bar = 1 mm. Please click here to view a larger version of this
figure.

Figure 4: Mortality heat map of 13 EPF isolates against mustard aphids through the detached-leaf method. Please
click here to view a larger version of this figure.

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 Figure 5: Observation of fungal mycosis of 13 EPF isolates. Mepe-NCHU-2 = Metarhizium pemphigi; Mp-NCHU-11 =
Metarhizium pinghaense; Mb = Metarhizium baoshanense;Cc = Cordyceps cateniannulata; Ba = Beauveria australis;Bb =
Beauveriabassiana; Pl = Purpureocillium lilacinum. Scale bar = 1 mm. Please click here to view a larger version of this figure.

Figure 6: Corrected mortality of fungal isolates Mb-NCHU-197 and Cc-NCHU-213 against mustard aphid. The error
bars represent the standard deviation (SD). The mortalities of Mb-NCHU-197 and Cc-NCHU-213 at the same time point with
the same inoculated concentration were compared using independent t-test, and mortalities marked with an asterisk were
found to be significantly different (p < 0.05). Please click here to view a larger version of this figure.

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 Isolate Species Host or source* Location

Mp-NCHU-2 Metarhizium pemphigi soil Yilan

Ml-NCHU-9 Metarhizium lepidiotae soil Yilan

Mp-NCHU-11 Metarhizium pinghaense soil Yilan

Ba-NCHU-113 Beauveria australis soil Taichung

Bb-NCHU-141 Beauveria bassiana Hypothenemus hampei Chiayi

Bb-NCHU-143 Beauveria bassiana Hypothenemus hampei Chiayi

Pl-NCHU-152 Purpureocillium lilacinum Tessaratoma papillosa Chiayi

Bb-NCHU-153 Beauveria bassiana Rhynchophorus ferrugineus Chunghua

Bb-NCHU-157 Beauveria bassiana Rhynchophorus ferrugineus Chunghua

Mb-NCHU-196 Metarhizium baoshanense soil Taichung

Mb-NCHU-197 Metarhizium baoshanense soil Taichung

Cc-NCHU-213 Cordyceps cateniannulata soil Taichung

Bb-NCHU-286 Beauveria bassiana Cerambycidae Taichung

Table 1: EPF isolates used in this study.

Primer name Sequence (5' - 3') Product size (bp) Reference

A05Le F-GGGTCTTGGATGGTGTGGTG 953 Lu et al. [21]

R-AGGGGTCTTGTCGCCATTTT

LeCO1 F-CTTTTCCCATGATCAATTTT 593 This study

R-ACGTAGTGGAAATGAGCAAC

Table 2: Primer pairs used for molecular identification of mustard aphids.

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 Isolate Species Corrected mortality (%)

Mp-NCHU-2 Metarhizium pemphigi 76.47

Ml-NCHU-9 Metarhizium lepidiotae 82.35

Mp-NCHU-11 Metarhizium pinghaense 76.47

Ba-NCHU-113 Beauveria australis 82.35

Bb-NCHU-141 Beauveria bassiana 88.24

Bb-NCHU-143 Beauveria bassiana 88.24

Pl-NCHU-152 Purpureocillium lilacinum 35.29

Bb-NCHU-153 Beauveria bassiana 82.35

Bb-NCHU-157 Beauveria bassiana 88.24

Mb-NCHU-196 Metarhizium baoshanense 76.47

Mb-NCHU-197 Metarhizium baoshanense 88.24

Cc-NCHU-213 Cordyceps cateniannulata 94.12

Bb-NCHU-286 Beauveria bassiana 100.00

Table 3: Corrected mortality rates of 13 EPF isolates against mustard aphids at 5 days post inoculation (d.p.i.).

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 Isolate Species Conc. N* LT50 (days)† 95% Slope (SE) X2 (df)‡
(conidia/mL) confidence
limits

Mb- Metarhizium 104 60 3.816 a 3.61–4.05 0.60 (0.05) 15.64 (28)

NCHU-197 baoshanense

105 60 3.112 bd 2.95–3.28 0.72 (0.05) 14.61 (28)

106 60 2.908 b 2.76–3.06 0.85 (0.06) 15.04 (28)

107 60 2.549 c 2.40–2.69 0.90 (0.06) 24.31 (28)

Cc- Cordyceps 104 60 3.948 a 3.71–4.23 0.53 (0.05) 8.81 (28)

NCHU-213 cateniannulata

105 60 3.237 d 3.07–3.41 0.72 (0.05) 22.86 (28)

106 60 2.414 c 2.28–2.54 1.08 (0.07) 28.30 (28)

107 60 2.132 e 2.02–2.25 1.41 (0.10) 28.96 (28)

*Number of insects observed.

†LT50 values marked with different letters were considered

significantly different as their 95% confidence limits did not overlap.

‡X2 , Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom

Table 4: LT50 values of fungal isolates Mb-NCHU-197 and Cc-NCHU-213 against mustard aphids under different
conidia concentrations. *Number of insects observed. †LT50 values marked with different letters were considered
significantly different as their 95% confidence limits did not overlap. ‡X2 , Chi-square value in Pearson's goodness-of-fit test;
df, degrees of freedom.

Copyright © 2023 JoVE Journal of Visualized Experiments jove.com July 2023 • 197 • e65312 • Page 15 of 20
 Isolate Species N* LC50 (conidia/ 95% confidence Slpoe (SE) X2 (df)‡

mL)† limits

Mb-NCHU-197 Metarhizium 240 2.30 × 105 a 8.63 × 104 – 0.43 (0.08) 10.14 (10)
baoshanense
5.70 × 105

Cc-NCHU-213 Cordyceps 240 9.32 × 104 a 4.97 × 104 – 0.76 (0.09) 4.33 (10)
cateniannulata
1.62 × 105

*Number of insects observed.

†LC50 values marked with different letters were considered

significantly different as their 95% confidence limits did not overlap.

‡X2 , Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom

Table 5: LC50 values of fungal isolates Mb-NCHU-197, and Cc-NCHU-213 against mustard aphids. *Number of insects
observed. †LC50 values marked with different letters were considered significantly different as their 95% confidence limits did
not overlap. ‡X2 , Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom.

Discussion instar nymphs but not in adults (Figure 3). Previous studies
on L. attenuatum, B. bassiana against cotton aphids, and M.
Crucifers, a group of vegetables, are frequently infested by
brunneum against green peach aphids have demonstrated
multiple aphid species, including mustard aphid (L. erysimi)
that molting can be a strategy to avoid infection28 , 29 , 30 .
and cabbage aphid (Brevicoryne brassicae)26 . Both species
Therefore, misidentifying aphid stages can lead to errors in
have been reported in Taiwan27 , and it is possible for
virulence assessment, as molting can affect the effectiveness
them to coexist at the collection site. To distinguish closely
of EPF28 , 29 , 31 . To address this issue and ensure greater
related aphid species, this study employed a molecular
accuracy and precision in virulence evaluation, exclude
identification technique using a multiplex primer set21 . By
aphids with whitish portions on their hind leg tibiae that have
designing a molecular marker from the mustard aphid COI
not reached the adult stage unless intentionally using aphid
gene fragment, we successfully identified the mustard aphid,
nymphs. These aphids do not fully mature and may undergo
thus confirming the reliability of the molecular marker A05Le
molting, which could allow them to escape the infection
for this purpose21 .
process of EPF. Additionally, a time course of observation
The observations revealed that distinguishing between and data recording every 12 h is recommended. The 12 h
apterous fourth-instar nymphs and adult mustard aphids interval is preferable for demonstrating differences between
based solely on their size or morphology is challenging multiple promising isolates with similar aphid-killing abilities
without experience. However, a crucial distinguishing feature and facilitates the precise calculation of LT50. However, the
is the tibia of the hind legs, which appears white in fourth-

Copyright © 2023 JoVE Journal of Visualized Experiments jove.com July 2023 • 197 • e65312 • Page 16 of 20
 time interval can be adjusted based on the different life cycles aphids became stuck upside down with their legs upward and
of other insect species or determined through a small-scale had to be rescued back to the leaf disc. This may be due to the
pretest. differences in aphid size or mobility, indicating that increasing
the agar concentration alone may not solve the problem.
Although the detached-leaf method may leave minimal
Therefore, ensure that the leaf disc fully covers the agar
honeydew on the leaf disc, most of the honeydew adheres to
surface by using a leaf that fits more snugly to the Petri dish
the Petri dish cover since the leaf disc is in close proximity. It
size, which can help alleviate this issue. In comparison to the
may be wiped or washed away during the observation period.
detached-leaf method described by Yokomi and Gottwald18 ,
However, honeydew is undoubtedly present in the practical
this study has improved upon one aspect by using a leaf size
environment of crop cultivation when aphids invade32 . The
that is approximately a good fit for the Petri dish. This allows
honeydew present in the inoculation chamber may somewhat
for relative quantification of food consumption and minimizes
simulate the situation when EPF is inoculated into aphids
the exposed agar surface. Additionally, the leaf needs to be
in the field. Aphid cuticles mostly contain honeydew, which
"embedded" in the semi-solidified water agar, whereas in the
serves as a source of nutrients for the fungus and may
original reference, it was described as "placed" on the water
stimulate the germination of EPF16 . Thus, the combination of
agar18 . Embedding the leaf disc into the water agar reduces
EPF application and honeydew production by aphids can be
the likelihood of aphids getting stuck to the agar surface and
advantageous.
facilitates observation, as the aphids are confined to one side.

To confirm that aphid death is due to infection, cadavers

This protocol provides detailed instructions on how to proceed
are typically transferred from the EPF inoculation chamber to
with the detached-leaf method and EPF inoculation, along
damp filter paper or a culture medium for observing fungal
with some modifications that have facilitated operations,
outgrowth11 , 14 , 33 , 34 . However, in this study, water agar was
observations, and consistent results. It also establishes a
used in the inoculation chamber to maintain high humidity,
standardized method for selecting EPF isolates against plant-
allowing the aphid cadavers to remain on the leaves without
sucking pests under laboratory conditions. Furthermore,
needing to be moved for fungal outgrowth observation.
conducting a standard check with known effective or
Although the detached-leaf method provides information on
commercially available products can be performed for the
the aphidicidal effect of EPF isolates, it still has limitations.
future commercialization of EPF isolates.
The water agar used to maintain the leaf disc condition can
cause aphids to become stuck while moving in the inoculation Disclosures
chamber. To address this issue, the percentage of water agar
The authors declare there is no conflict of interest involved in
was increased to 3%, providing a firm enough surface for
this work.
Russian wheat aphids (Diuraphis noxia) to walk on20 . In this
experiment, mustard aphids were able to move comfortably
on water agar surfaces with concentrations ranging from 1.5%
to 3%. However, as the observation period progressed, a few

Copyright © 2023 JoVE Journal of Visualized Experiments jove.com July 2023 • 197 • e65312 • Page 17 of 20
 (Hemiptera: Aphididae) pests of horticultural crops in
Acknowledgments Argentina. Biocontrol. 52, 641-655 (2007).

This research was supported by 109-2313-B-005 -048 -MY3 7. Liu, Y. C., Ni, N. T. , Chang, J. C., Li, Y. H.,
from the Ministry of Science and Technology (MOST). Lee, M. R., Kim, J. S., et al. Isolation and selection
of entomopathogenic fungi from soil samples and
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