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 aphid....

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....

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

List of materials used in this article

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