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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+occurrence+of+entomopathogenic+fungi+(Zygomycetes:+Entomophthorales)+of+aphid+(Hemiptera:+Aphididae)+pests+of+horticultural+crops+in+Argentina.">Natural occurrence of entomopathogenic fungi (Zygomycetes: Entomophthorales) of aphid (Hemiptera: Aphididae) pests of horticultural crops in Argentina.</a> Biocontrol. 52, 641-655 (2007).</li><li>Liu, Y. C., Ni, N. T., Chang, J. C., Li, Y. H., Lee, M. R., Kim, J. S., 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>Francis, F., Fingu-Mabola, J. C., Fekih, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+endophytic+effects+of+fungal+entomopathogens+for+sustainable+aphid+control:+a+review.">Direct and endophytic effects of fungal entomopathogens for sustainable aphid control: a review.</a> Agriculture. 12 (12), 2081 (2022).</li><li>Simon, J., Peccoud, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+aphid+pests+in+agricultural+environments.">Rapid evolution of aphid pests in agricultural environments.</a> Current Opinion in Insect Science. 26, 17-24 (2018).</li><li>Ujjan, A. A., Shahzad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Entomopathogenic+Fungi+for+the+Control+of+Mustard+Aphid+(Lipaphis+erysimi)+on+canola+(Brassica+napus+L).">Use of Entomopathogenic Fungi for the Control of Mustard Aphid (Lipaphis erysimi) on canola (Brassica napus L).</a> Pakistan Journal of Botany. 44 (6), 2081-2086 (2012).</li><li>Sajid, M., Bashir, N. H., Batool, Q., Munir, I., Bilal, M., Jamal, M. A., 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=In-vitro+evaluation+of+biopesticides+(Beauveria+bassiana,+Metarhizium+anisopliae,+Bacillus+thuringiensis)+against+mustard+aphid+Lipaphis+erysimi+kalt.+(Hemiptera:+Aphididae).">In-vitro evaluation of biopesticides (Beauveria bassiana, Metarhizium anisopliae, Bacillus thuringiensis) against mustard aphid Lipaphis erysimi kalt. (Hemiptera: Aphididae).</a> Journal of Entomology and Zoology Studies. 5 (6), 331-335 (2017).</li><li>Paschapur, A. U., Subbanna, A. R. N. S., Singh, A. K., Jeevan, B., Stanley, J., Rajashekara, H., Mishra, K. K., Koti, P. S., Kant, L., Pattanayak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternaria+alternata+strain+VLH1:+a+potential+entomopathogenic+fungus+native+to+North+Western+Indian+Himalayas.">Alternaria alternata strain VLH1: a potential entomopathogenic fungus native to North Western Indian Himalayas.</a> Egyptian Journal of Biological Pest Control. 32, 138 (2022).</li><li>Miohammed, A. 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.) (Hemiptera: Aphididae) and promotes plant growth in Chinese kale under hydroponic growing conditions.</a> Biocontrol Science and Technology. 31 (10), 997-1015 (2021).</li><li>Mseddi, J., Farhat-Touzri, D. B., Azzouz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+characterization+of+thermotolerant+Beauveria+bassiana+isolates+and+with+insecticidal+activity+against+the+cotton-melon+aphid+Aphis+gossypii+(Glover)+(Hemiptera:+Aphididae).">Selection and characterization of thermotolerant Beauveria bassiana isolates and with insecticidal activity against the cotton-melon aphid Aphis gossypii (Glover) (Hemiptera: Aphididae).</a> Pest Management Science. 78 (6), 2183-2195 (2022).</li><li>Butt, T. M., Ibrahim, L., Clark, S. J., Beckett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+germination+behaviour+of+Metarhizium+anisopliae+on+the+surface+of+aphid+and+flea+beetle+cuticles.">The germination behaviour of Metarhizium anisopliae on the surface of aphid and flea beetle cuticles.</a> Mycological Research. 99 (8), 945-950 (1995).</li><li>Ullah, S., Raza, A. B. M., Alkafafy, M., Sayed, S., Hamid, M. I., Majeed, M. Z., Riaz, M. A., Gaber, N. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Verticillium+lecanii+Isolates+in+Aphids+Determined+by+Detached-leaf+Bioassay.">Virulence of Verticillium lecanii Isolates in Aphids Determined by Detached-leaf Bioassay.</a> Journal of Inbertebrate Pathology. 51, 250-258 (1988).</li><li>Vu, V. H., Hong, S. I., Kim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+entomopathogenic+fungi+for+aphid+control.">Selection of entomopathogenic fungi for aphid control.</a> Journal of Bioscience and Bioengineering. 104 (6), 498-505 (2007).</li><li>Vandenberg, J. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+conidial+dose,+moulting+and+insect+developmental+stage+on+the+susceptibility+of+cotton+aphid,+Aphis+gossypii,+to+conidia+of+Lecanicillium+attenuatum,+an+entomopathogenic+fungus.">The relationship between conidial dose, moulting and insect developmental stage on the susceptibility of cotton aphid, Aphis gossypii, to conidia of Lecanicillium attenuatum, an entomopathogenic fungus.</a> Biocontrol Science and Technology. 22 (3), 319-331 (2012).</li><li>Reingold, V., Kottakota, C., Birnbaum, N., Goldenberg, M., Lebedev, G., Ghanim, M., 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=Intraspecies+variation+ofMetarhiziumbrunneumagainst+the+green+peach+aphid,Myzus+persicae,+provides+insight+into+thecomplexity+of+disease+progression.">Intraspecies variation ofMetarhiziumbrunneumagainst the green peach aphid,Myzus persicae, provides insight into thecomplexity of disease progression.</a> Pest Management Science. 77, 2557-2567 (2021).</li><li>Ortiz-Urquiza, A., Keyhani, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+on+the+Surface:+entomopathogenic+fungi+versus+the+insect+cuticle.">Action on the Surface: entomopathogenic fungi versus the insect cuticle.</a> Insects. 4, 357-374 (2013).</li><li>Knodel, J. J., Beauzay, P., Boetel, M., Prochaska, T., Chirumamilla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2022 North Dakota Field Crop Insect Management Guide. , (2021).</li><li>Yeo, H., Pell, J. K., Alderson, P. G., Clark, S. J., Pye, B. 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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.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

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

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, ...

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1.2K Views.  National Chung Hsing University. 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.

Video: Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

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...

entomopathogenic fungi (epf) preparation and virulence screening against mustard aphids

Entomopathogenic Fungi (EPF) Preparation and Virulence Screening Against Mustard Aphids

aphicidal assessment of entomopathogenic fungi by the statistical analysis of corrected mortality in mustard aphids

Aphicidal Assessment of Entomopathogenic Fungi by the Statistical Analysis of Corrected Mortality in Mustard Aphids

mass production of entomopathogenic fungi, metarhizium robertsii and metarhizium pinghaense, for commercial application against insect pests

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain...

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

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

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the...

comparison of methods for isolating entomopathogenic fungi from soil samples

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC...

collection, maintenance, and molecular identification of mustard aphids

Collection, Maintenance, and Molecular Identification of Mustard Aphids

isolation, behavioral identification, and pathogenicity assessment of entomopathogenic fungi from a forest wood borer

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild &lt;em&gt;Monochamus alternatus&lt;/em&gt; Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural...

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

microwave-assisted extraction of phenolic compounds and antioxidants for cosmetic applications using polyol-based technology

Author Spotlight: Eco-Friendly Extraction of Bioactive Compounds Using Polyol-Based Microwave-Assisted Techniques

This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use...

Polyol-Based Microwave-Assisted Extraction for Phenolic and Antioxidant Yields

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Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

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a facile and eco-friendly route to fabricate poly(lactic acid) scaffolds with graded pore size

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

enhanced photoluminescence of curcuma longa extracts via chitosan-mediated energy transfer for textile authentication applications

Author Spotlight: Eco-friendly Photoluminescent Textile Authentication with Curcumin

Photoluminescence is one of the most effective authentication mechanisms being used today. Utilizing and enhancing naturally sourced materials with inherent photoluminescent properties and incorporating them into fabric substrates can lead to development of green, sustainable, and functional textiles for smart applications.

Use of Chitosan to Enhance Photoluminescence of Curcumin for Textile Authentication

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inoculating and observing arbuscular mycorrhizal cultures on superabsorbent polymer-based autotrophic systems

Author Spotlight: Enhancing AM Fungi Research with SAP-AS &amp;#8212; A Novel Technique for Single-Spore Cultures

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic...

Culturing Arbuscular Mycorrhizal Fungi Using SAP-AS for Enhanced Observation

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developing a feeding assay system for evaluating the insecticidal effect of phytochemicals on helicoverpa armigera

Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly...

Assessment of Phytochemical Effects on &lt;em&gt;Helicoverpa armigera&lt;/em&gt; Through Feeding Assay

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testing nicotine tolerance in aphids using an artificial diet experiment

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

regenerative peripheral nerve interface: surgical protocol for a randomized controlled trial in postamputation pain

Author Spotlight: Regenerative Peripheral Nerve Interface (RPNI) Surgery in Postamputation Pain Management

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular...

RPNI Surgery for Treating Postamputation Pain

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testing the physiological barriers to viral transmission in aphids using microinjection

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Aphids are effective transmitters of plant viruses.  Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for...

green synthesis, characterization, encapsulation, and measurement of the release potential of novel alkali lignin micro-/submicron particles

Author Spotlight: Development and Characterization of Eco-Friendly Lignin-Based Microparticles for Enhanced Delivery of Bioflavonoids

We describe novel, simple methodologies of synthesis and characterization of biocompatible lignin micro- and submicron particles. These formulations provide a facile approach for the utilization of the heteropolymer, as well as an alternative for the rational design of multifunctional carrier matrices with potential applicability in biomedicine, pharmaceutical technology, and the food...

Lignin Micro-/Submicron Particle Synthesis for Bioflavonoid Release

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culturing and genetically manipulating entomopathogenic nematodes

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are...

Start by preparing a temporary maintenance Petri dish with excised cruciferous leaves and water infiltrated filter paper at the bottom. Next place five aphids on the prepared Petri dish. When the aphids increase in number, cut the originally excised leaves into four to six smaller pieces.Transfer each small leaf piece with the aphids into separate Petri dishes containing fresh leaves and wet filter paper. To collect the aphid genomic DNA, first homogenize the aphids using a pellet pestle. Next, extract the DNA using a Gene-Spin genomic DNA isolation kit as per the manufacturer's guidelines.Finish the extraction process by eluting the genomic DNA with 50 microliters of preheated nuclease-free water. Perform PCR amplification and DNA sequencing by adding the DNA sample to the PCR master mix with primer pairs. Analyze the PCR product using 1%agarose gel electrophoresis to confirm the identity of the mustard aphid.The field collected aphids were confirmed as mustard aphids using molecular markers, including the PCR amplicon size and Lipaphis erysimi CO1 sequencing.

For the EPF recovery from the fungal library, start by smearing the fungal isolates on a quarter strength SDA plate. Incubate it in darkness at 25 degrees Celsius for 10-14 days before conidia harvest. Following this, pipette 2-3 mL of 0.03%Tween 80 onto a cultured plate.Then use an inoculation loop to scrape the conidia by. Next, transfer the fungal suspension to a centrifuge tube. Vigorously vortex the suspension at the highest speed, then pipette out a sample into a hemocytometer to count the number of conidia.Dilute the conidia within the suspension using 0.03%Tween 80 til desired concentration is obtained. To prepare the inoculation chamber, first cut a 9 centimeter diameter leaf disk using the bottom Petri dish as a mold or with scissors. Next, prepare 1.5%water agar for each dish using a microwave.Pour 30 mL of the cooled water agar into the Petri dish and let it solidify in a laminar flow hood. When the agar surface is semisolid, place the leaf disk with its abaxial surface facing up, then press it slightly to imbed it in the agar. Now, place 20 apterous adults on the leaf disk.Open the cover of the inoculation chamber to spray 0.3 mL of conidia suspension directly onto the aphids and the leaf disk. Once the suspension has dried, close the cover to prevent the drowning of the mustard aphids. Subsequently, seal the inoculation chamber using paraffin film to maintain a high mount of humidity.Place the chamber in an incubator set at a temperature of 25 degrees Celsius with a light and dark photoperiod of 12 hours each. Use a stereomicroscope to count the mortality every 12 hours for 5 days. The virulence screening revealed a consistent survival rate for mustard aphids with the control group exhibiting an 85%survival rate.Cordyceps cateninnulata demonstrated the fastest aphid-killing ability, resulting in 50%and 90%moralities at 3 days and 4.5 days post-inoculation, or DPI. Beauveria strains 141, 143, and 153 showed slow aphid-killing abilities with only 5%mortality at 3 DPI. Apart from Purpureocillium, most EPF isolates exhibited corrected mortality rates higher than 70%within 5 DPI.Beauveria bassiana demonstrated the highest mortality rate of 100%among EPF isolates. EPF mycosis was observed on cadavers of mustard aphids infected with Metarhizium species, Beauveria species, Purpureocillium lilacinum, and Cordyceps cateniannulata during the virulence screening.

Begin by launching the SPSS software platform. Create variables termed treatment and corrected mortality. Then input the calculated results for different isolates inoculated at the same time point with the same concentration.Navigate to Analyze, Compare Means and Proportions, then Independent Samples T Test in SPSS for independent T-test analysis. Next, input treatment into the grouping variable box and corrected mortality into the Test Variables box. Classify groups according to different fungal isolates and press OK to begin the analysis.To calculate the median lethal time and median lethal concentration, create the variables defined as Total, Response, and either Duration or Concentration. Next, enter the recorded result into the spreadsheet. Finally, choose Analyze, followed by Regression, and then Probit to compute the median lethal time and median lethal concentration.Inoculations of 10 to the seven conidia per milliliter resulted in significantly different corrected mortalities for Metarhizium strain 197 and Cordyceps strain 213 at three and four DPI. The Cordyceps strain 213 exhibited a significantly shorter lethal duration compared to other treatments. It also showed a lower lethal dose value relative to the Metarhizium 213 strain.