Published: July 21st, 2023
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, 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.
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 aphid....
NOTE: The complete flowchart is shown in Figure 1.
1. Mustard aphid collection and maintenance
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.......
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.......
|10 μL Inoculating Loop
|100 bp DNA Ladder III
|2x SuperRed PCR Master Mix
|50 mL centrifuge tube
|6 cm Petri dish
|Alpha Plus Scientific
|6 mm insect aspirator
|70 mm filter paper NO.1
|Toyo Roshi Kaisha
|9 cm Petri dish
|Alpha Plus Scientific
|BioGreen Safe DNA Gel Buffer
|GenepHlow Gel/PCR Kit
|Gene-Spin Genomic DNA Isolation Kit
|Komatsuna leaves (Brassica rapa var. perviridis)
|Tai Cheng Farm
|MiniAmp Thermal Cycler
|Thermo Fisher Scientific
|Mustard aphid (Lipaphis erysimi)
|Tian Cheng brush company
|Sabouraud Dextrose Broth
|TAE buffer 50x
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