Name: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids (Video) | JoVE
Uploaded: 2023-07-21T14:20:00
Description: Watch this Scientific Journal Video about Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids at JoVE.com

This research was supported by 109-2313-B-005 -048 -MY3 from the Ministry of Science and Technology (MOST).

NOTE: The complete flowchart is shown in Figure 1.
1. Mustard aphid collection and maintenance
<ol>
	<li>Collection of mustard aphids
	<ol>
		<li>Flip the leaves and visually check for infestation of mustard aphids on cruciferous crops in the field.</li>
		<li>Record the sampling site information (i.e., GPS) and host plant(s), and confirm the history of insecticide applications with the farmers.</li>
		<li>Use an insect aspirator or a f...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

<ol>
	<li>Ghosh, S., Roy, A., Chatterjee, A., Sikdar, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+regional+wind+circulation+and+meteorological+factors+on+long-range+migration+of+mustard+aphids+over+indo-gangetic+plain.">Effect of regional wind circulation and meteorological factors on long-range migration of mustard aphids over indo-gangetic plain.</a> Scientific Reports. 9, 5626 (2019).</li><li>Dhillon, M. K., Singh, N., Yadava, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventable+yield+losses+and+management+of+mustard+aphid,+Lipaphis+erysimi+(Kaltenbach)+in+different+cultivars+of+Brassica+juncea(L.)+Czern+&amp;+Coss.">Preventable yield losses and management of mustard aphid, Lipaphis erysimi (Kaltenbach) in different cultivars of Brassica juncea(L.) Czern &amp; Coss.</a> Crop Protection. 161, 106070 (2022).</li><li>Huang, F., Hao, Z., Yan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+oilseed+rape+seed+treatment+with+imidacloprid+on+survival,+feeding+behavior,+and+detoxifying+enzymes+of+mustard+aphid,+lipaphis+erysimi.">Influence of oilseed rape seed treatment with imidacloprid on survival, feeding behavior, and detoxifying enzymes of mustard aphid, lipaphis erysimi.</a> Insects. 10 (5), 144 (2019).</li><li>Mannino, M. C., Huarte-Bonnet, C., Davyt-Colo, B., Pedrini, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+insect+cuticle+the+only+entry+gate+for+fungal+infection?+insights+into+alternative+modes+of+action+of+entomopathogenic+fungi.">Is the insect cuticle the only entry gate for fungal infection? insights into alternative modes of action of entomopathogenic fungi.</a> Journal of Fungi. 5 (2), 33 (2019).</li><li>Bamisile, B. S., Akutse, K. S., Siddiqui, J. A., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+application+of+entomopathogenic+fungi+as+alternatives+to+chemical+pesticides:+prospects,+challenges,+and+insights+for+next-generation+sustainable+agriculture.">Model application of entomopathogenic fungi as alternatives to chemical pesticides: prospects, challenges, and insights for next-generation sustainable agriculture.</a> Frontiers in Plant Science. 12, 741804 (2021).</li><li>Scorsetti, A. C., Humber, R. A., Garcia, J. J., Lopez Lastra, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lecanicillium+muscarium+and+Adalia+bipunctata+combination+for+the+control+of+black+bean+aphid,+Aphis+fabae.">Lecanicillium muscarium and Adalia bipunctata combination for the control of black bean aphid, Aphis fabae.</a> Biocontrol. 63, 277-287 (2018).</li><li>Thaochan, N., Ngampongsai, A., Prabhakar, C. S., Hu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beauveria+bassiana+PSUB01+simultaneously+displays+biocontrol+activity+against+Lipaphis+erysimi+(Kalt.)+(Hemiptera:+Aphididae)+and+promotes+plant+growth+in+Chinese+kale+under+hydroponic+growing+conditions.">Beauveria bassiana PSUB01 simultaneously displays biocontrol activity against Lipaphis erysimi (Kalt.) 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+bioassay+and+screening+of+beauveria+bassiana+and+paecilomyces+fumosoroseus+against+the+russian+wheat+aphid+(homoptera:+aphididae).">Standardized bioassay and screening of beauveria bassiana and paecilomyces fumosoroseus against the russian wheat aphid (homoptera: aphididae).</a> Journal of Economic Entomology. 89 (6), 1418-1423 (1996).</li><li>Lu, W. N., Wu, Y. T., Kuo, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+species-specific+primers+for+the+identification+of+aphids+in+Taiwan.">Development of species-specific primers for the identification of aphids in Taiwan.</a> Applied Entomology and Zoology. 43 (1), 91-96 (2008).</li><li>Liu, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+selection+of+entomopathogenic+fungi+from+soil+samples+and+evaluation+of+fungal+virulence+against+insect+pests.">Isolation and selection of entomopathogenic fungi from soil samples and evaluation of fungal virulence against insect pests.</a> Journal of Visualized Experiments. 175, e62882 (2021).</li><li>Menger, J., Beauzay, P., Chirumamilla, A., Dierks, C., Gavloski, J., Glogoza, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+diagnostic-concentration+bioassay+for+detection+of+susceptibility+to+pyrethroids+in+soybean+aphid+(hemiptera:+aphididae).">Implementation of a diagnostic-concentration bioassay for detection of susceptibility to pyrethroids in soybean aphid (hemiptera: aphididae).</a> Journal of Economic Entomology. 113 (2), 932-939 (2020).</li><li>Zhang, R., Chen, J., Jiang, L., Qiao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genes+expression+difference+between+winged+and+wingless+bird+cherry-oat+aphid+Rhopalosiphum+padi+based+on+transcriptomic+data.">The genes expression difference between winged and wingless bird cherry-oat aphid Rhopalosiphum padi based on transcriptomic data.</a> Scientific Reports. 9, 4754 (2019).</li><li>Abbott, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+computing+the+effectiveness+of+an+insecticide.">A method of computing the effectiveness of an insecticide.</a> Journal of Economic Entomology. 18, 265-267 (1925).</li><li>Liu, T. X., Sparks, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphids on Cruciferous Crops: Identification and Management. , 9-11 (2001).</li><li>Kuo, M., Chianglin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependent+life+table+of+brevicoryne+brassicae+(l.)(hemiptera:+aphididae)+on+radish.">Temperature dependent life table of brevicoryne brassicae (l.)(hemiptera: aphididae) on radish.</a> Formosan Entomologist. 27, 293-302 (2007).</li><li>Im, Y., Park, S., Lee, S. Y., Kim, J., Kim, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-Stage+defense+mechanism+of+the+cotton+aphid+aphis+gossypii+against+infection+with+the+insect-killing+fungus+beauveria+bassiana+JEF-544.">Early-Stage defense mechanism of the cotton aphid aphis gossypii against infection with the insect-killing fungus beauveria bassiana JEF-544.</a> Frontiers in Immunology. 13, 907088 (2022).</li><li>Kim, J. J., Roberts, D. 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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;L Inoculating Loop</td>
			<td>NEST Scientific</td>
			<td>718201</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder III</td>
			<td>Geneaid</td>
			<td>DL007</td>
			<td></td>
		</tr>
		<tr>
			<td>2x SuperRed PCR Master Mix</td>
			<td>Biotools</td>
			<td>TE-SR01</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Bioman Scientific</td>
			<td>ET5050-12</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Alpha Plus Scientific</td>
			<td>16021</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mm insect aspirator</td>
			<td>MegaView Science</td>
			<td>BA6001</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mm filter paper NO.1</td>
			<td>Toyo Roshi Kaisha</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>9 cm Petri dish</td>
			<td>Alpha Plus Scientific</td>
			<td>16001</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Bioman Scientific</td>
			<td>AGR001.1</td>
			<td>Microbiology grade</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioman Scientific</td>
			<td>PB1200</td>
			<td></td>
		</tr>
		<tr>
			<td>BioGreen Safe DNA Gel Buffer</td>
			<td>Bioman Scientific</td>
			<td>SDB001T</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromas</td>
			<td>Technelysium</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GeneDoc</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GenepHlow Gel/PCR Kit</td>
			<td>Geneaid</td>
			<td>DFH300</td>
			<td>https://www.geneaid.com/data/files/1605861013102532959.pdf</td>
		</tr>
		<tr>
			<td>Gene-Spin Genomic DNA Isolation Kit</td>
			<td>Protech Technology</td>
			<td>PT-GD112-V3</td>
			<td>http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Paul Marienfeld</td>
			<td>640030</td>
			<td></td>
		</tr>
		<tr>
			<td>Komatsuna leaves (Brassica rapa var. perviridis)</td>
			<td>Tai Cheng Farm</td>
			<td>1-010-300410</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsprayer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniAmp Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr>
			<td>Mustard aphid (Lipaphis erysimi)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Painting brush</td>
			<td>Tian Cheng brush company</td>
			<td>4716608400352</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle</td>
			<td>Bioman Scientific</td>
			<td>GT100R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud Dextrose Broth</td>
			<td>HiMedia</td>
			<td>MH033-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistics</td>
			<td>IBM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer 50x</td>
			<td>Bioman Scientific</td>
			<td>TAE501000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>PanReac AppliChem</td>
			<td>142050.1661</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of aphidicidal effect of entomopathogenic fungi against parthenogenetic insect, mustard aphid, lipaphis erysimi (kalt.)

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.

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

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...

Watch this Scientific Journal Video about Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids at JoVE.com

Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids (Video) | JoVE

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, ...